Varicella zoster virus vaccine and methods of making and using same

Abstract

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

Description

Technical Field

[0001] This disclosure relates to the field of gene therapy technology, and in particular to nucleic acid vaccines encoding varicella-zoster virus antigens. Background Technology

[0002] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0003] A complete varicella-zoster virus consists of a core, capsid, inner membrane, and envelope. There is only one serotype of varicella-zoster virus. Its genome contains 71 genes encoding 67 different proteins, including seven glycoproteins named gE, gB, gH, gI, gC, gK, and gL. Glycoproteins gE, gB, and gH are abundant in infected cells and are also present in the virion's cell membrane. Initial infection manifests as chickenpox symptoms. After the virus is suppressed, it remains latent in the body, causing recurrent infections when the body's immunity is weakened, resulting in shingles, hence the name varicella-zoster virus (VZV). The primary targets of infection are newborns, adults, and immunocompromised patients; postherpetic neuralgia is a common complication. Humans are its only natural host, and the skin is the virus's primary target organ.

[0004] In 2006, the U.S. Food and Drug Administration (FDA) approved Merck's live attenuated shingles virus (OKA strain) vaccine for use in people aged 60 and older. To further improve the vaccine's protective efficacy in the elderly and address potential safety concerns associated with live attenuated vaccines, GSK developed HZ / su, a subunit vaccine based on the OKA strain surface glycoprotein gE, following Merck's Zostavax. Because this vaccine contains only gE, it is considered safer and can be used in pregnant women and immunocompromised individuals.

[0005] The inventors recognized that the traditional vaccine for preventing varicella-zoster virus infection and recurrence is mainly the live attenuated varicella vaccine (OKA strain). This vaccine has a certain protective effect, but it has disadvantages such as safety and difficulty in storage and transportation. Moreover, its immunization effect is not ideal in the elderly, with a protection rate of only about 50%. At the same time, traditional vaccines lack cellular immune response, which leads to a high recurrence rate of VZV. Summary of the Invention

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

[0007] Some aspects of this disclosure provide a varicella-zoster virus vaccine that contains or encodes varicella-zoster virus (VZV) antigen, the varicella-zoster virus vaccine being selected from nucleic acid vaccines, peptide vaccines, or viral vaccines.

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

[0009] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0010] (a) At least one consecutive or spaced amino acid deletion of amino acids from position 569 to 623.

[0011] (b) A polymerizing element is attached to the C-terminus, the amino acid sequence of which is shown in Seq ID NO.20 to Seq ID NO.24.

[0012] (c) An enhancement element is attached to the N-terminus of the sequence, the amino acid sequence of which is shown in Seq ID NO.45-46.

[0013] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0014] (a) At least one of the following: deletion of at least one consecutive or spaced amino acid at positions 569–623; deletion of at least one consecutive or spaced amino acid at positions 569–579; deletion of at least one consecutive or spaced amino acid at positions 574–623; deletion of at least one consecutive or spaced amino acid at positions 588–623.

[0015] (b) A polymerizing element is attached to the C-terminus, the amino acid sequence of which is shown in Seq ID NO.20 to Seq ID NO.24.

[0016] (c) An enhancement element is attached to the N-terminus of the sequence, the amino acid sequence of which is shown in Seq ID NO.45-46.

[0017] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 569 to 579.

[0018] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or spaced amino acid deletion from amino acid positions 574 to 623.

[0019] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 574 to 623.

[0020] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or spaced amino acid deletion at positions 588 to 623.

[0021] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 588 to 623.

[0022] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 569–579 and 588–623.

[0023] 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 contains an amino acid sequence that is at least 80% identical to Seq ID NO.7 or Seq ID NO.34.

[0024] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is as shown in Seq ID NO.34, or contains an amino acid sequence that is at least 80% identical to or identical to Seq ID NO.34.

[0025] In some embodiments, 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. In some embodiments, the DNA molecules include stranded DNA molecules and / or circular DNA molecules. In some embodiments, the RNA molecules include mRNA or circular RNA.

[0026] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0027] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8-17 or SEQ ID No. 35-44;

[0028] (b) mRNA molecules derived from (a) that have one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and have the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0029] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0030] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.93.

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

[0032] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8-12 or SEQ ID No. 35-39;

[0033] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0034] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0035] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in either SEQ ID No. 8 or SEQ ID No. 35;

[0036] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0037] In some embodiments, the nucleic acid molecule has the nucleotide sequence encoding VZV glycoprotein gE shown in any of SEQ ID No. 8, or has at least 80% identity with Seq ID No. 8. In some embodiments, the mRNA molecule has a GC base pair percentage of 55% to 58% and a CAI value of 0.79 to 0.93.

[0038] In some embodiments, the nucleic acid molecule has the nucleotide sequence encoding VZV glycoprotein gE shown in any of SEQ ID No. 35, or has at least 80% identity with Seq ID No. 35. In some embodiments, the mRNA molecule has a GC base pair percentage of 55% to 58% and a CAI value of 0.90 to 0.93.

[0039] In some embodiments, the mRNA includes at least one of a nucleotide sequence encoding an enhancing element, a nucleotide sequence encoding a cis-regulatory element, and a nucleotide sequence encoding a polymerizing element.

[0040] In some implementations, the nucleotide sequence encoding the cis-regulatory element is shown in Seq ID NO.19.

[0041] In some implementations, the nucleotide sequence encoding the multiplying element may also be as shown in Seq ID NO.25–29.

[0042] In some implementations, the nucleotide sequence encoding the enhancement element is shown in Seq ID NO.49–50.

[0043] In some implementations, the mRNA consists of a sequence comprising 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.

[0044] In some implementations, 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.

[0045] In some implementations, the 5' end cap is m7Gppp(5')(2'-OMeA)pG.

[0046] 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.

[0047] In some implementations, the 3'UTR sequence is as shown in Seq ID NO. 4 to 6. In some implementations, the 3'UTR sequence is as shown in Seq ID NO. 4.

[0048] In some embodiments, one or more uridines in the mRNA are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0049] In some embodiments, the varicella-zoster virus vaccine includes a delivery formulation. In some embodiments, the delivery formulation includes lipid nanoparticles or cationic liposomes.

[0050] In some embodiments, the lipid nanoparticles comprise at least one of protonable cationic lipids, structural lipids, auxiliary lipids, and surfactants.

[0051] In some embodiments, the lipid nanoparticles comprise, by weight, 40-60 parts protonable cationic lipids, 10-40 parts structural lipids, 10-30 parts auxiliary lipids, and 0.5-5 parts surfactants.

[0052] In some embodiments, the protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA. In some embodiments, the structural lipid comprises cholesterol and / or cholesterol derivatives. In some embodiments, the accessory lipid comprises at least one of DSPC, DOPE, DOPC, DOPG, and DOPS. In some embodiments, the surfactant comprises at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0053] In some embodiments, the liposome nanoparticles comprise, by molar percentage, 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-DMG.

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

[0055] In some embodiments, the method for preparing the varicella-zoster virus vaccine involves dissolving mRNA in a buffer solution to obtain an aqueous phase, measuring and dissolving each lipid component of liposome nanoparticles in an organic solvent to obtain an organic phase, mixing the aqueous phase and the organic phase, and then removing the organic phase to obtain the varicella-zoster virus vaccine.

[0056] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2 to 4, more preferably 1:3.

[0057] In some embodiments, the buffer solution comprises citrate buffer or sodium acetate, more preferably citrate buffer. In some embodiments, the pH of the buffer solution is 3 to 7, more preferably 4. In some embodiments, the concentration of mRNA in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, more preferably 0.1 mg / mL.

[0058] In some embodiments, the coding region of the mRNA encodes the varicella-zoster virus VZV antigen, which is a variant of the VZV glycoprotein gE.

[0059] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0060] (a) At least one consecutive or spaced amino acid deletion of amino acids from position 569 to 623;

[0061] (b) A polymerizing element is attached to the C-terminus, the amino acid sequence of which is shown in Seq ID NO.20 to Seq ID NO.24;

[0062] (c) An enhancement element is attached to the N-terminus of the sequence, the amino acid sequence of which is shown in Seq ID NO.45-46.

[0063] In some embodiments, the isolated mRNA includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0064] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8-17 or SEQ ID No. 35-44;

[0065] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0066] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0067] A method for inducing an immune response against VZV in a subject by administering at least one of the varicella-zoster virus vaccine as described in any of the preceding claims, or a varicella-zoster virus vaccine prepared by the method described above, or isolated mRNA as described above.

[0068] In some implementations, the immune response against VZV includes the production of cytokines in lymphocytes.

[0069] In some embodiments, the lymphocytes are CD4+ T cells and / or CD8+ T cells.

[0070] In some embodiments, the cytokine is one or more of IFN-γ, IL-2, IL-4, IL-10, and TNF-α.

[0071] In some implementations, the immune response against VZV includes an increase in the amount of cytokines produced in lymphocytes.

[0072] In some embodiments, the immune response against VZV includes generating antibodies that specifically bind to the VZV glycoprotein gE encoded by nucleic acids.

[0073] In some embodiments, the antibody is a neutralizing antibody against VZV or cells infected with VZV. In some embodiments, the serum titer of the antibody increases in the subject. Attached Figure Description

[0074] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0075] Figure 1 A schematic diagram of VZV glycoprotein gE is shown.

[0076] Figure 2A schematic diagram of the structure of one of the VZV glycoprotein gE variants is shown.

[0077] Figure 3 This diagram shows the structure of one of the mRNA molecules in a herpes zoster virus nucleic acid vaccine that encodes the VZV glycoprotein gE variant antigen.

[0078] Figure 4 The expression of the mRNA encoding the VZV glycoprotein gE in Example 4 is shown in both intracellular and membrane surface regions.

[0079] Figure 5 A schematic diagram of the mouse experimental evaluation process is shown.

[0080] Figure 6 The level of CD4+ T cell response in mice induced by the varicella-zoster virus vaccine in Example 6 is shown.

[0081] Figure 7 The results of Example 6 show how the varicella-zoster virus vaccine induced the secretion of IFN-γ and IL-2 by mouse spleen lymphocytes.

[0082] Figure 8 The example of Example 6 shows how the varicella-zoster virus vaccine induced the secretion of TNF-α and IL-4 by mouse spleen lymphocytes.

[0083] Figure 9 The results of Example 6 show the specific IgG antibody titer and neutralizing antibody titer in mouse serum induced by the varicella-zoster virus vaccine.

[0084] Figure 10 The intracellular expression of the mRNA encoding the VZV glycoprotein gE in Example 7 is shown.

[0085] Figure 11 The expression of the mRNA encoding VZV glycoprotein gE on the membrane surface is shown in Example 7.

[0086] Figure 12 The level of CD4+ T cell response in mice induced by the varicella-zoster virus vaccine in Example 8 is shown.

[0087] Figure 13 The results of Example 8 show how the varicella-zoster virus vaccine induced the secretion of IFN-γ and IL-2 by mouse spleen lymphocytes.

[0088] Figure 14 The results of Example 8 show how the varicella-zoster virus vaccine induced the secretion of TNF-α and IL-4 by mouse spleen lymphocytes.

[0089] Figure 15The results of Example 8 show the specific IgG antibody titer and neutralizing antibody titer in mouse serum induced by the varicella-zoster virus vaccine.

[0090] Figure 16 The level of mouse CD4+ T cell response induced by the varicella-zoster virus vaccine in Example 9 is shown.

[0091] Figure 17 The results of Example 9 show how the varicella-zoster virus vaccine induced the secretion of IFN-γ and IL-2 by mouse spleen lymphocytes.

[0092] Figure 18 The results of Example 9 show how the varicella-zoster virus vaccine induced the secretion of TNF-α and IL-4 by mouse spleen lymphocytes.

[0093] Figure 19 The results of Example 9 show the specific IgG antibody titer and neutralizing antibody titer in mouse serum induced by the varicella-zoster virus vaccine.

[0094] Figure 20 The results of Example 10 show how the varicella-zoster virus vaccine induced the secretion of IFN-γ and IL-4 by mouse spleen lymphocytes.

[0095] Figure 21 The results show the specific IgG antibody titer and neutralizing antibody titer in mouse serum induced by the varicella-zoster virus vaccine of Example 10. Detailed Implementation

[0096] Currently available or soon-to-be-released varicella or shingles vaccines are all developed based on the VZV-oka strain genome. This strain was isolated and attenuated by the Institute of Microbiology, Osaka University, Japan in 1992. Because of its milder clinical reactions and better immunogenicity, the VZV oka strain is currently the only attenuated strain approved by the WHO for vaccine production. The strain used in this disclosure is the VZV oka strain.

[0097] The VZV glycoprotein gE is composed of 623 amino acids encoded by the ORF68 gene and is present on the surface of viral particles, as well as on the surface and cytoplasm of infected cells. This protein molecule can be divided into an extracellular region (1–543), a transmembrane region (544–568), and an intracellular region (569–623). In the convalescent serum of herpes zoster patients, VZV antibodies primarily target three viral glycoproteins: gE, gB, and gH, with gE being the most prevalent. Figure 1This is a schematic diagram of VZV glycoprotein gE. VZV glycoprotein gE includes a 21-amino acid signal peptide sequence (“SP” in the diagram, positions 1-21), a 22-amino acid TM sequence (“TM” in the diagram, positions 544-568), and a 55-amino acid AC sequence (“AC” in the diagram, positions 569-623). VZV gE is a typical type I membrane glycoprotein. It moves from the endoplasmic reticulum to the Golgi apparatus for post-translational modification, and then is transferred to the plasma membrane on the cell surface. Subsequently, through endocytosis, gE is transported back to the TGN (trans-Golgi apparatus) for virion assembly and encapsulation.

[0098] The VZV glycoprotein gE variants described in this disclosure are artificially mutated and modified VZV glycoprotein gE. These mutations and modifications (including but not limited to amino acid residue substitution, insertion and / or addition, deletion, and covalent modification) result in an amino acid sequence conforming to VZV glycoprotein gE obtained through mutation and modification of the wild-type VZV glycoprotein gE. The mutation sites of the VZV glycoprotein gE variants described in this disclosure are mainly concentrated at the AC terminus (the domain responsible for endocytosis). These mutations prevent the endocytosis of VZV glycoprotein gE, allowing more VZV glycoprotein gE to be displayed on the membrane surface. For example, substituting amino acid Y at position 569 of VZV glycoprotein gE with another amino acid can disrupt phosphorylation at position 569, preventing the correct folding of the 568-AYRV-571 motif, thereby preventing VZV glycoprotein gE from targeting the trans-Golgi network (TGN) and disrupting endocytosis. For example, the deletion of amino acid 582 in VZV glycoprotein gE can disrupt the phosphorylation at the original amino acid 582 site, thereby disrupting endocytosis.

[0099] In some embodiments, this disclosure provides a varicella-zoster virus vaccine that contains or encodes a varicella-zoster virus (VZV) vaccine antigen that can elicit an effective neutralizing antibody response against varicella-zoster virus (VZV).

[0100] Some aspects of this disclosure provide a varicella-zoster virus vaccine that contains or encodes varicella-zoster virus (VZV) antigen, the varicella-zoster virus vaccine being selected from nucleic acid vaccines, peptide vaccines, or viral vaccines.

[0101] In some embodiments, the antigen is selected from VZV glycoprotein gE, VZV glycoprotein gB, VZV glycoprotein gH, VZV glycoprotein gI, VZV glycoprotein gC, VZV glycoprotein gK, and VZV glycoprotein gL.

[0102] In some embodiments, the antigen is VZV glycoprotein gE.

[0103] In some embodiments, the antigen is a VZV glycoprotein gE variant.

[0104] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0105] (a) At least one consecutive or spaced amino acid deletion of amino acids from position 569 to 623;

[0106] (b) A polymerizing element is attached to the C-terminus, the amino acid sequence of which is shown in Seq ID NO.20 to Seq ID NO.24;

[0107] (c) An enhancement element is attached to the N-terminus of the sequence, the amino acid sequence of which is shown in Seq ID NO.45-46.

[0108] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0109] (a) At least one of the following: deletion of at least one consecutive or spaced amino acid at positions 569–623; deletion of at least one consecutive or spaced amino acid at positions 569–579; deletion of at least one consecutive or spaced amino acid at positions 574–623; deletion of at least one consecutive or spaced amino acid at positions 588–623.

[0110] (b) A polymerizing element is attached to the C-terminus, the amino acid sequence of which is shown in Seq ID NO.20 to Seq ID NO.24;

[0111] (c) An enhancement element is attached to the N-terminus of the sequence, the amino acid sequence of which is shown in Seq ID NO.45-46.

[0112] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or spaced amino acid deletion from positions 569 to 623, for example, but not limited to, deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids from positions 569 to 623. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 consecutive or intermittent amino acid deletions.

[0113] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 569 to 623.

[0114] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or spaced amino acid deletion at positions 569 to 579, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive or spaced amino acids at positions 569 to 579.

[0115] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 569 to 579.

[0116] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or spaced amino acid deletion at positions 574 to 623. For example, it can be, but is not limited to, deletions of 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 consecutive or spaced amino acid deletions at positions 574 to 623.

[0117] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 574 to 623.

[0118] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or spaced amino acid deletion at positions 588 to 623. For example, it can be, but is not limited to, deletions of 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, or 35 consecutive or spaced amino acid deletions at positions 588 to 623.

[0119] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 588 to 623.

[0120] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 569–579 and 588–623.

[0121] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 20 to Seq ID NO. 24) linked to the C-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 569 to 623. For example, this could be, but is not limited to, deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids at positions 569 to 623. 1, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 consecutive or intermittent amino acid deletions.

[0122] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in Seq ID NO. 24) linked to the C-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 569-623. For example, this could be, but is not limited to, deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids at positions 569-623. 1, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 consecutive or intermittent amino acid deletions.

[0123] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO.20 to Seq ID NO.24) and a deletion of amino acids 569 to 623 (VE6).

[0124] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in amino acid sequence 24) attached to the C-terminus of the sequence and a deletion of amino acids 569–623 (VE6).

[0125] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 20 to Seq ID NO. 24) attached to the C-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 569 to 579. For example, it can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive or spaced amino acids at positions 569 to 579.

[0126] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 20) attached to the C-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 569-579. For example, it can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive or spaced amino acids at positions 569-579.

[0127] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO.20 to Seq ID NO.24) and a deletion of amino acids 569 to 579.

[0128] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in Seq ID NO. 24) attached to the C-terminus of the sequence and a deletion of amino acids 569–579.

[0129] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is Seq ID NO.20 to Seq ID). (As shown in NO.24) and at least one consecutive or spaced amino acid deletion at positions 574 to 623, for example, but not limited to, deletions of 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 consecutive or spaced amino acid deletions at positions 574 to 623.

[0130] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a multiplying element linked to the C-terminus of the sequence (the multiplying element amino acid sequence Seq ID). (As shown in NO.24) and at least one consecutive or spaced amino acid deletion at positions 574 to 623, for example, but not limited to, deletions of 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 consecutive or spaced amino acid deletions at positions 574 to 623.

[0131] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO.20 to Seq ID NO.24) and a deletion of amino acids 574 to 623.

[0132] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in Seq ID NO. 24) attached to the C-terminus of the sequence and a deletion of amino acids 574–623.

[0133] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 20 to Seq ID NO. 24) attached to the C-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 588 to 623. For example, it can be, but is not limited to, deletions of 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, or 35 consecutive or spaced amino acid deletions at positions 588 to 623.

[0134] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in Seq ID NO. 24) attached to the C-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 588-623. For example, it can be, but is not limited to, deletions of 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, or 35 consecutive or spaced amino acid deletions at positions 588-623.

[0135] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO.20 to Seq ID NO.24) and a deletion of amino acids 588 to 623.

[0136] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in Seq ID NO. 24) attached to the C-terminus of the sequence and a deletion of amino acids 588–623.

[0137] The positions of the amino acid sequences described in this disclosure, such as "amino acids 570-592", are located based on the full-length amino acid sequence of the wild-type VZV glycoprotein gE (as shown in Seq ID NO. 30). For example, "amino acids 570-592" refers to amino acids 570-592 of the wild-type VZV glycoprotein gE (as shown in Seq ID NO. 31).

[0138] The term "continuous or intermittent amino acid deletion" as used in this disclosure refers to either "continuous amino acid deletion" or "intermittent amino acid deletion." For example, n consecutive amino acid deletions are the (m)th, (m+1)th, ..., (m+n-1)th amino acids, where m, m+1, ..., m+n-1 are consecutive natural numbers. Another example is n intermittent amino acid deletions, where the (m1)th, (m2)th, ..., (mn)th amino acid deletions are the (m1)th, (m2)th, ..., (mn)th amino acids, where m1, m2, ..., mn are increasing natural numbers, and at least two adjacent natural numbers among m1, m2, ..., mn are not consecutive, differing by at least 2.

[0139] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancing element linked to the N-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 569-623. For example, this deletion 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, etc., from positions 569-623. The enhancement element consists of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54 consecutive or spaced amino acid deletions; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45-46.

[0140] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element attached to the N-terminus of the sequence and a deletion of amino acids 569–623; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45–46.

[0141] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element attached to the N-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 569-579. For example, it can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive or spaced amino acids at positions 569-579; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45-46.

[0142] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element attached to the N-terminus of the sequence and a deletion of amino acids at positions 569–579; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45–46.

[0143] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element linked to the N-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 574-623. For example, it can be, but is not limited to, deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 amino acids at positions 574-623. The enhancement element consists of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 consecutive or spaced amino acid deletions; the amino acid sequence of the enhancement element is shown in SeqID NO. 45-46.

[0144] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element attached to the N-terminus of the sequence and a deletion of amino acids 574–623; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45–46.

[0145] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element linked to the N-terminus of the sequence and at least one consecutive or spaced amino acid deletion at positions 588-623. For example, it can be, but is not limited to, deletions of 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, or 35 consecutive or spaced amino acid deletions at positions 588-623; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45-46.

[0146] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element linked to the N-terminus of the sequence and a deletion of amino acids 588–623; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45–46.

[0147] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes an enhancement element attached to the N-terminus of the sequence and deletions of amino acids at positions 569–579 and 588–623; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45–46.

[0148] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in Seq ID NO. 24) linked to the C-terminus of the sequence, an enhancing element linked to the N-terminus of the sequence, and at least one consecutive or spaced amino acid deletion at positions 569-623. For example, this could be, but is not limited to, deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids at positions 569-623. The enhancement element consists of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54 consecutive or spaced amino acid deletions; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45-46.

[0149] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 24), an enhancing element attached to the N-terminus of the sequence, and a deletion of amino acids at positions 569–623; the amino acid sequence of the enhancing element is shown in Seq ID NO. 45–46.

[0150] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 24), a reinforcing element attached to the N-terminus of the sequence, and at least one consecutive or spaced amino acid deletion at positions 569-579. For example, it can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive or spaced amino acids at positions 569-579; the amino acid sequence of the reinforcing element is shown in Seq ID NO. 45-46.

[0151] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 24), an enhancing element attached to the N-terminus of the sequence, and a deletion of amino acids at positions 569–579; the amino acid sequence of the enhancing element is shown in Seq ID NO. 45–46.

[0152] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element (shown in Seq ID NO. 24) linked to the C-terminus of the sequence, an enhancing element linked to the N-terminus of the sequence, and at least one consecutive or spaced amino acid deletion at positions 574-623. For example, this could be, but is not limited to, deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at positions 574-623. The enhancement element consists of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 49 consecutive or spaced amino acid deletions; the amino acid sequence of the enhancement element is shown in Seq ID NO. 45-46.

[0153] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 24), an enhancing element attached to the N-terminus of the sequence, and a deletion of amino acids at positions 574 to 623; the amino acid sequence of the enhancing element is shown in Seq ID NO. 45 to 46.

[0154] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 24), a reinforcing element attached to the N-terminus of the sequence, and at least one consecutive or spaced amino acid deletion at positions 588-623. For example, it can be, but is not limited to, deletions of 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, or 35 consecutive or spaced amino acid deletions at positions 588-623; the amino acid sequence of the reinforcing element is shown in Seq ID NO. 45-46.

[0155] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 24), an enhancing element attached to the N-terminus of the sequence, and a deletion of amino acids at positions 588–623; the amino acid sequence of the enhancing element is shown in Seq ID NO. 45–46.

[0156] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a polymerizing element attached to the C-terminus of the sequence (the amino acid sequence of the polymerizing element is shown in Seq ID NO. 24), an enhancing element attached to the N-terminus of the sequence, a deletion of amino acids at positions 569–579, and a deletion of amino acids at positions 588–623; the amino acid sequence of the enhancing element is shown in Seq ID NO. 45–46.

[0157] The “enhancing element” described in this disclosure is an amino acid sequence located at the N-terminus of the amino acid sequence of the VZV glycoprotein gE variant, which can enhance the antigen-specific immune response of the VZV glycoprotein gE variant, such as flagellin protein (FLA-2, amino acid sequence as shown in Seq ID NO. 45) or chemokine CCL19 (amino acid sequence as shown in Seq ID NO. 46).

[0158] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant further includes a linker element connected to the C-terminus of the enhancement element, the linker element being selected from the P2A sequence or the GS linker sequence. In some embodiments, the amino acid sequence of the P2A sequence is shown in Seq ID NO. 47. In some embodiments, the GS linker sequence is selected from (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, and Seq ID NO. 48; wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; and q is an integer from 1 to 5.

[0159] 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 contains an amino acid sequence that is at least 80% identical to Seq ID NO.7 or Seq ID NO.34, for example, but not limited to an amino acid sequence that contains at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO.7 or Seq ID NO.34.

[0160] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is as shown in Seq ID NO.7, or contains an amino acid sequence that is at least 80% identical to Seq ID NO.7, for example, but not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO.7.

[0161] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected sequentially as Seq ID NO.45, Seq ID NO.48, and Seq ID NO.7.

[0162] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected sequentially as Seq ID NO.46, Seq ID NO.47, and Seq ID NO.7.

[0163] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected sequentially as Seq ID NO.45, Seq ID NO.48, Seq ID NO.7, and Seq ID NO.24.

[0164] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected sequentially as Seq ID NO.46, Seq ID NO.47, Seq ID NO.7, and Seq ID NO.24.

[0165] In some embodiments, the N-terminus to C-terminus of the amino acid sequence of the VZV glycoprotein gE variant is sequentially linked as Seq ID NO.7 and Seq ID NO.24.

[0166] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is as shown in Seq ID NO.34, or contains an amino acid sequence that is at least 80% identical to or identical to Seq ID NO.34, for example, but not limited to an amino acid sequence that contains at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO.34.

[0167] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected with Seq ID NO.45, Seq ID NO.48 and Seq ID NO.34 in sequence.

[0168] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected sequentially as Seq ID NO.46, Seq ID NO.47, and Seq ID NO.34.

[0169] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected sequentially as Seq ID NO.45, Seq ID NO.48, Seq ID NO.34, and Seq ID NO.24.

[0170] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant has N-terminus to C-terminus connected sequentially as Seq ID NO.46, Seq ID NO.47, Seq ID NO.34, and Seq ID NO.24.

[0171] In some embodiments, the N-terminus to C-terminus of the amino acid sequence of the VZV glycoprotein gE variant is sequentially linked as Seq ID NO.34 and Seq ID NO.24.

[0172] Figure 2This is a schematic diagram of the structure of one of the VZV glycoprotein gE variants. In the diagram, "Enhancer" represents an enhancing element selected from flagellin protein (FLA-2, amino acid sequence as shown in Seq ID NO. 45) or chemokine CCL19 (amino acid sequence as shown in Seq ID NO. 46), or an enhancing element sequence disclosed prior to the date of this application. "Linker" represents a linking element selected from P2A sequences or GS linker sequences, or an enhancing element sequence disclosed prior to the date of this application. "VZV gE" represents the VZV glycoprotein gE variant. "PolyP" represents a polymerizing element. In some embodiments, "PolyP" can be replaced by a cis-regulatory element in the VZV glycoprotein gE variant.

[0173] In some embodiments, the varicella-zoster virus vaccine is a nucleic acid vaccine; the nucleic acid vaccine contains nucleic acid molecules; the nucleic acid molecules include DNA molecules and / or RNA molecules.

[0174] In some embodiments, the DNA molecule includes a stranded DNA molecule and / or a circular DNA molecule.

[0175] In some implementations, the RNA molecule includes mRNA or circular RNA.

[0176] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0177] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8-17 or SEQ ID No. 35-44;

[0178] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0179] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0180] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.93.

[0181] The GC base pair percentage content described in this disclosure is the percentage of GC bases in the open reading frame (ORF) of mRNA relative to the total number of bases in the ORF. For example, if the ORF of RNA [M] contains 120 bases, of which 60 are GC bases, then the GC content of the RNA [M] is 50%.

[0182] The CAI (Codon Adaptivity Index) described in this disclosure refers to the degree of consistency between the frequency of synonymous codons and the optimal codon in a coding region, and can be used to assess the expression level of a foreign gene in the host. Values ​​range from 0 to 1: a higher CAI value indicates a higher expression level of the foreign gene in the host.

[0183] In some embodiments, the nucleic acid molecule has the nucleotide sequence encoding VZV glycoprotein gE shown in any one of SEQ ID No. 8-17 or SEQ ID No. 35-44, or has at least 80% identity with Seq ID No. 8-17 or SEQ ID No. 35-44. For example, it can be, but is not limited to, an mRNA molecule containing at least 80%, 85%, 90%, 95%, or 98% identity with the nucleotide sequence encoding VZV glycoprotein gE of Seq ID No. 8-17 or SEQ ID No. 35-44.

[0184] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0185] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8-12 or SEQ ID No. 35-39;

[0186] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0187] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0188] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.93.

[0189] In some embodiments, the nucleic acid molecule has the nucleotide sequence encoding VZV glycoprotein gE shown in any one of SEQ ID No. 8-12 or SEQ ID No. 35-39, or has at least 80% identity with the nucleotide sequence encoding VZV glycoprotein gE of SEQ ID No. 8-12 or SEQ ID No. 35-39. For example, it can be, but is not limited to, an mRNA molecule containing at least 80%, 85%, 90%, 95%, or 98% identity with the nucleotide sequence encoding VZV glycoprotein gE of SEQ ID No. 8-12 or SEQ ID No. 35-39.

[0190] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0191] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in either SEQ ID No. 8 or SEQ ID No. 35;

[0192] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0193] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0194] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.93.

[0195] In some embodiments, the nucleic acid molecule has the nucleotide sequence encoding VZV glycoprotein gE shown in any of SEQ ID No. 8, or has at least 80% identity with Seq ID No. 8. For example, it can be, but is not limited to, an mRNA molecule containing a nucleotide sequence encoding VZV glycoprotein gE that has at least 80%, 85%, 90%, 95%, or 98% identity with Seq ID No. 8. In some embodiments, the GC base pair percentage of the mRNA molecule is 55% to 58%, and the CAI value of the mRNA molecule is 0.79 to 0.93.

[0196] In some embodiments, the nucleic acid molecule has the nucleotide sequence encoding VZV glycoprotein gE shown in any of SEQ ID No. 35, or has at least 80% identity with Seq ID No. 35. For example, it can be, but is not limited to, an mRNA molecule containing a nucleotide sequence encoding VZV glycoprotein gE that has at least 80%, 85%, 90%, 95%, or 98% identity with Seq ID No. 35. In some embodiments, the GC base pair percentage of the mRNA molecule is 55% to 58%, and the CAI value of the mRNA molecule is 0.90 to 0.93.

[0197] In some embodiments, the mRNA includes at least one of a nucleotide sequence encoding an enhancing element, a nucleotide sequence encoding a cis-regulatory element, and a nucleotide sequence encoding a polymerizing element.

[0198] In some embodiments, the amino acid sequence of the cis-regulatory element is shown in Seq ID NO.18.

[0199] In some implementations, the nucleotide sequence encoding the cis-regulatory element is shown in Seq ID NO.19.

[0200] The term "cis-regulatory element" as used in this 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 peptide. Cis-regulatory elements are typically located in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhance the translation level of upstream or downstream coding sequences. For example, a cis-regulatory element in the 5'-UTR of a nucleic acid molecule may be located between the promoter and start codon. Cis-regulatory element sequences disclosed prior to the date of this filing may be incorporated into this disclosure.

[0201] In some implementations, the nucleotide sequence encoding the multiplying element may also be as shown in Seq ID NO.25–29.

[0202] In some implementations, the nucleotide sequence encoding the enhancement element is shown in Seq ID NO.49–50.

[0203] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0204] (a) An mRNA molecule having a nucleotide sequence encoding a variant of VZV glycoprotein gE as shown in any one of SEQ ID No. 8 to 17 and a nucleotide sequence encoding a multiplying element as shown in SeqID No. 25 to 29 connected to the 3' end of the nucleotide sequence encoding a variant of VZV glycoprotein gE as shown in any one of SEQ ID No. 8 to 17;

[0205] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0206] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0207] In some embodiments, the mRNA molecule has a GC base pair percentage of 55% to 58% and a CAI value of 0.79 to 0.93.

[0208] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0209] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8 to 17 and the nucleotide sequence encoding the multiplying element shown in SeqID NO. 19 connected to the 3' end of the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8 to 17;

[0210] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0211] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0212] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.93.

[0213] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0214] (a) An mRNA molecule having a nucleotide sequence encoding an enhancing element as shown in any one of Seq ID NO. 49–50, a linker nucleotide sequence as shown in any one of Seq ID NO. 49–50 connected to the 3' end of the nucleotide sequence encoding an enhancing element as shown in any one of Seq ID NO. 49–50, and a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID NO. 8–17 connected to the 3' end of the linker nucleotide sequence as shown in any one of Seq ID NO. 49–50;

[0215] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0216] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0217] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.93.

[0218] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0219] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 35 to 44 and the nucleotide sequence encoding the multiplying element shown in Seq ID No. 25 to 29 connected to the 3' end of the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8 to 17;

[0220] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0221] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0222] In some embodiments, the GC base pair percentage of the mRNA molecule is 55% to 58%, and the CAI value of the mRNA molecule is 0.90 to 0.93.

[0223] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0224] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 35 to 44 and the nucleotide sequence encoding the multiplying element shown in Seq ID No. 19 connected to the 3' end of the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8 to 17;

[0225] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0226] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0227] In some embodiments, the GC base pair percentage of the mRNA molecule is 55% to 58%, and the CAI value of the mRNA molecule is 0.90 to 0.93.

[0228] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0229] (a) An mRNA molecule having a nucleotide sequence encoding an enhancing element as shown in any one of Seq ID NO. 49–50, a linker nucleotide sequence as shown in any one of Seq ID NO. 49–50 connected to the 3' end of the nucleotide sequence encoding an enhancing element as shown in any one of Seq ID NO. 49–50, and a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID NO. 35–44 connected to the 3' end of the linker nucleotide sequence as shown in any one of Seq ID NO. 49–50;

[0230] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0231] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0232] In some embodiments, the GC base pair percentage of the mRNA molecule is 55% to 58%, and the CAI value of the mRNA molecule is 0.90 to 0.93.

[0233] In some implementations, nucleic acid molecules can be sequence optimized using mRNA sequences to improve properties related to expression efficacy after in vivo administration. These improvements include: enhancing mRNA stability; increasing translational efficiency in target tissues; reducing the number of truncated proteins expressed; improving protein folding or preventing misfolding; reducing the toxicity of the expression product; reducing cell death induced by the expression product; and increasing and / or decreasing protein aggregation, resulting in mRNAs with improved properties. Sequence optimization also aims to: optimize the formulation and delivery characteristics of nucleic acid-based therapeutics while maintaining structural and functional integrity; overcome expression thresholds; increase expression rates; improve half-life and / or protein concentration; optimize protein localization; and avoid adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization techniques include: (1) codon optimization based on codon frequencies in specific organs and / or host organisms to ensure proper folding and expression; (2) adjusting G / C content to increase mRNA stability or reduce secondary structures; (3) minimizing tandem repeat codons or base runs that may impair gene construction or expression; (4) customizing transcription and translation control regions; and (5) reducing or eliminating problematic secondary structures within polynucleotides.

[0234] Sequence identity between two nucleotide sequences indicates the percentage of identical nucleotides between the sequences. Sequence identity between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0235] The term "% identity" or similar term refers to the percentage of identical nucleotides or amino acids between sequences being compared at optimal alignment. This percentage is purely statistical, and the differences between the two sequences may (but are not necessarily) be randomly distributed across the entire length of the sequences being compared. Comparison of two sequences is typically performed after optimal alignment by comparing equivalent sequences relative to fragments or a "comparison window" to identify local regions of the corresponding sequences.

[0236] In some embodiments, based on the provided mRNA open reading frame sequence, those skilled in the art can obtain the corresponding circular RNA open reading frame sequence and, in accordance with the content of published documents such as CN202180048567.4, prepare a complete circular RNA sequence encoding the same amino acid sequence. In some embodiments, based on the provided mRNA sequence, those skilled in the art can obtain the corresponding DNA sequence (e.g., uracil to thymine conversion). Similarly, based on the provided DNA sequence, those skilled in the art can obtain the corresponding RNA sequence (e.g., thymine to uracil conversion). In some embodiments, based on the provided RNA or DNA sequence, those skilled in the art can obtain the corresponding amino acid sequence.

[0237] The open reading frame (ORF) described in this disclosure is a continuous DNA or RNA segment 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).

[0238] In some embodiments, the mRNA described in this disclosure consists of a sequence comprising 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.

[0239] In some implementations, 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;

[0240] In some implementations, the 5' end cap is m7Gppp(5')(2'-OMeA)pG.

[0241] In some embodiments, the length of the 5'UTR is preferably 10 to 200 nucleotides.

[0242] In some implementations, the 5'UTR is 15 to 100 nucleotides in length.

[0243] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1-3.

[0244] In some implementations, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1.

[0245] In some implementations, the 3'UTR sequence is as shown in Seq ID NO.4-6.

[0246] In some implementations, the 3'UTR sequence is as shown in Seq ID NO.4.

[0247] In some implementations, one or more uridines in the mRNA are replaced with modified nucleosides.

[0248] In some embodiments, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0249] In some embodiments, the modified nucleoside is N1-methyl-pseuuridine (m1ψ).

[0250] The 5′ untranslated region (UTR) described in this disclosure refers to the sequence of mRNA that does not encode a polypeptide, located directly upstream (i.e., 5′) of the start codon (i.e., the first codon of the mRNA transcript translated by ribosomes). When an RNA transcript is generated, the 5′ UTR may contain a promoter sequence. Such promoter sequences are known in the art. The RNA sequence of the 5′ UTR is shown as one of Seq ID NO. 1, Seq ID NO. 2, or Seq ID NO. 3, or a 5′ UTR sequence disclosed prior to the date of this application may also be incorporated into this disclosure.

[0251] The 3' untranslated region (UTR) described in this disclosure refers to the sequence of mRNA that does not encode a polypeptide, located downstream of a stop codon. 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 prior to the date of this application may also be incorporated into this disclosure.

[0252] The poly(A) tail described in this disclosure is a downstream sequence of the 3′UTR of mRNA containing multiple consecutive adenosine monophosphates (ATPs). The poly(A) tail may contain 10 to 300 ATPs. In cells and / or in vivo, the poly(A) tail serves to protect mRNA from enzymatic degradation and to aid in transcription termination and / or mRNA export from the nucleus and translation.

[0253] Figure 3This is a schematic diagram of the structure of one of the mRNA molecules in a herpes zoster virus nucleic acid vaccine encoding the VZV glycoprotein gE variant antigen. In the diagram, "Enhancer" represents an enhancing element selected from flagellin protein (FLA-2, nucleotide sequence as shown in Seq ID NO. 49) or chemokine CCL19 (nucleotide sequence as shown in Seq ID NO. 50), or an enhancing element sequence disclosed prior to the date of this application. "Linker" represents a linker element selected from P2A sequences or GS linker sequences (nucleotide sequences as shown in Seq ID NO. 47-48), or a linker element sequence disclosed prior to the date of this application. "VZV gE" represents the VZV glycoprotein gE variant. "PolyP" represents a polymerizing element (nucleotide sequences as shown in Seq ID NO. 25-29). In some embodiments, "PolyPr" can be replaced by a cis-regulatory element in the VZV glycoprotein gE variant. The "PolyA" in the diagram represents the poly(A) tail.

[0254] In some embodiments, the mRNA molecule includes a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0255] In some embodiments, the mRNA described in this disclosure is a self-replicating mRNA that carries a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase, RdRP). Specifically, the sequence design content of Chinese Patent CN202110424124.2 regarding self-replicating mRNA is incorporated into this disclosure.

[0256] In some implementations, the varicella-zoster virus vaccine includes a delivery formulation.

[0257] In some embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0258] In some embodiments, the lipid nanoparticles comprise at least one of protonable cationic lipids, structural lipids, auxiliary lipids, and surfactants.

[0259] In some embodiments, the lipid nanoparticles comprise, by weight, 40-60 parts protonable cationic lipids, 20-40 parts structural lipids, 10-30 parts auxiliary lipids, and 0.5-5 parts surfactants.

[0260] In some embodiments, the lipid nanoparticles comprise, by weight, 40-60 parts protonable cationic lipids, 10-40 parts structural lipids, 10-30 parts auxiliary lipids, and 0.5-5 parts surfactants.

[0261] In some embodiments, the protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA.

[0262] In some embodiments, the structural lipids include cholesterol and / or cholesterol derivatives.

[0263] In some embodiments, the assisting lipids include at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0264] In some embodiments, the surfactant includes at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0265] In some embodiments, the liposome nanoparticles comprise, by molar percentage, 20% to 50% cationic lipids, such as, but not limited to, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 10% to 50% DSCP, such as, but not limited to, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 5% to 20% cholesterol, such as, but not limited to, 5%, 10%, 15%, or 20%; and 1% to 5% PEG-DMG, such as, but not limited to, 1%, 1.5%, 2%, 3%, 4%, or 5%.

[0266] In some embodiments, the liposome nanoparticles comprise, by molar percentage, 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-DMG.

[0267] In some embodiments, this disclosure provides a method for preparing a varicella-zoster virus vaccine according to any of the foregoing embodiments, 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.

[0268] In some embodiments, the method for preparing the varicella-zoster virus vaccine involves dissolving mRNA in a buffer solution to obtain an aqueous phase, measuring and dissolving each lipid component of liposome nanoparticles in an organic solvent to obtain an organic phase, mixing the aqueous phase and the organic phase, and then removing the organic phase to obtain the varicella-zoster virus vaccine.

[0269] 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 solution comprises citrate buffer or sodium acetate, preferably citrate buffer. In some embodiments, the pH of the buffer solution 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.

[0270] In some embodiments, the organic solvent is selected from C1-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, a microfluidic mixture of the aqueous and organic phases is used, and the organic solvent is filtered using tangential flow. Preferably, the flow rate of the microfluidic system is >3 ml / min, more preferably 12 ml / min.

[0271] In some embodiments, the mixture further includes a concentration step, which brings the final concentration of mRNA to 50 μg / mL to 200 μg / mL, preferably 100 μg / mL.

[0272] In some embodiments, the lipid nanoparticles have a diameter of less than about 200 nm. In some embodiments, the lipid nanoparticles have a diameter of less than about 150 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 100 nm. In some embodiments, the lipid nanoparticles have a diameter of about 55 nm to about 90 nm.

[0273] In some embodiments, the varicella-zoster virus vaccine is a peptide vaccine. The peptide vaccine is an immune composition prepared by chemical synthesis or genetic engineering techniques according to the amino acid sequence of a known or predicted antigenic epitope in the varicella-zoster virus antigen gene. In some embodiments, the peptide vaccine described in this disclosure is an immune composition prepared by chemical synthesis techniques according to the amino acid sequence of varicella-zoster virus VZV glycoprotein gE, combined with artificial mutations and modifications. In some embodiments, the peptide vaccine described in this disclosure is obtained by fermentation using genetic engineering techniques (such as constructing genetically engineered bacteria) according to the amino acid sequence of varicella-zoster virus VZV glycoprotein gE, combined with artificial mutations and modifications.

[0274] In some embodiments, the varicella-zoster virus vaccine is a viral vaccine. In some embodiments, the viral vaccine described in this disclosure delivers the varicella-zoster virus VZV glycoprotein gE gene into the human body via a harmless microorganism, inducing an immune response in the body's immune system. Important viruses used in induced cellular immunity assays include variants of bovine poliovirus and poliovirus.

[0275] In some embodiments, this disclosure provides an isolated mRNA whose coding region nucleic acid sequence encodes varicella-zoster virus (VZV) antigen, said antigen being selected from VZV glycoprotein gE, VZV glycoprotein gB, VZV glycoprotein gH, VZV glycoprotein gI, VZV glycoprotein gC, VZV glycoprotein gK, and VZV glycoprotein gL. In some embodiments, said antigen is a variant of VZV glycoprotein gE.

[0276] In some embodiments, the amino acid sequence of the isolated mRNA VZV glycoprotein gE variant includes at least one of (a) to (c):

[0277] (a) At least one consecutive or spaced amino acid deletion of amino acids from position 569 to 623;

[0278] (b) A polymerizing element is attached to the C-terminus, the amino acid sequence of which is shown in Seq ID NO.20 to Seq ID NO.24;

[0279] (c) An enhancement element is attached to the N-terminus of the sequence, the amino acid sequence of which is shown in Seq ID NO.45-46.

[0280] In some embodiments, the isolated mRNA includes an open reading frame (ORF), the nucleotide sequence of which includes any one of (a) to (b):

[0281] (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in any one of SEQ ID No. 8-17 or SEQ ID No. 35-44;

[0282] (b) A mRNA molecule derived from (a) that has one or more nucleotides substituted, deleted or added in the nucleotide sequence defined in (a) and has the same GC base pair percentage and / or CAI value as the original nucleotide sequence.

[0283] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

[0284] In some embodiments, the mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.93.

[0285] In some embodiments, this disclosure provides an isolated DNA, characterized in that the DNA is obtained by reverse transcription of the isolated mRNA or mRNA molecule nucleic acid as described in any of the preceding claims, or the DNA is a sequence or mRNA molecule capable of being transcribed to the isolated mRNA as described in any of the preceding claims.

[0286] In some embodiments, the isolated DNA is isolated strand DNA or isolated circular DNA.

[0287] In some embodiments, this 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 isolated mRNA or isolated DNA as described in any of the preceding claims.

[0288] In some embodiments, this disclosure provides a method comprising administering to a subject an effective amount of at least one of the following: the shingles vaccine described in any one of the preceding claims, the isolated mRNA described in any one of the preceding claims, the isolated DNA described in any one of the preceding claims, or the biological material described in any one of the preceding claims, to induce a neutralizing antibody response against the varicella-zoster virus in the subject.

[0289] In some embodiments, the method includes administering to a subject at least one effective amount of any of the preceding herpes zoster vaccine, any of the preceding isolated mRNA, any of the preceding isolated DNA, or any of the preceding biological materials.

[0290] In some implementations, the time interval between the first and second applications of the at least two applications is not less than 14 days.

[0291] In some embodiments, the effective amount described 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 preceding herpes zoster vaccines, any of the preceding isolated mRNAs, any of the preceding isolated DNAs, or any of the preceding biological materials.

[0292] In some embodiments, this disclosure provides a method for inducing an immune response against VZV in a subject by administering at least one of the aforementioned herpes zoster vaccine, isolated mRNA as described in any one of the preceding claims, isolated DNA as described in any one of the preceding claims, or biological material as described in any one of the preceding claims. The immune response in the subject is equivalent to the immune response in a subject vaccinated with a conventional vaccine against VZV at a dose level 2 to 100 times higher than that of an RNA vaccine.

[0293] In some implementations, the immune response against VZV includes the production of cytokines in lymphocytes.

[0294] In some embodiments, the lymphocytes are CD4+ T cells and / or CD8+ T cells.

[0295] In some embodiments, the cytokine is one or more of IFN-γ, IL-2, IL-4, IL-10, and TNF-α.

[0296] In some implementations, the immune response against VZV includes an increase in the amount of cytokines produced in lymphocytes.

[0297] In some embodiments, the immune response against VZV includes generating antibodies that specifically bind to the VZV glycoprotein gE encoded by nucleic acids.

[0298] In some embodiments, the antibody is a neutralizing antibody against VZV or cells infected with VZV. In some embodiments, the serum titer of the antibody increases in the subject.

[0299] Example 1

[0300] A lipid nanoparticle comprising RNA encoding a variant of the VZV glycoprotein gE, wherein the lipid nanoparticle comprises, by molar percentage, 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol and 1.5% PEG-DMG.

[0301] The preparation method is as follows:

[0302] (a) Dissolve the RNA in a citrate buffer at pH 4 and adjust the concentration to 0.1 mg / mL to obtain the aqueous phase.

[0303] (b) Dissolve Dlin-MC3-DMA, DSCP, cholesterol and PEG-DMG in anhydrous ethanol according to the formulation amount, and adjust the concentration of lipid components in the organic phase to 6 mg / mL to obtain the organic phase.

[0304] (c) The aqueous phase from step (a) and the organic phase from step (b) were mixed at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 ml / min. The mixture was immediately diluted 100 times with PBS solution at pH 7.4, and ethanol was removed from the solution using tangential flow filtration (TFF). The mixture was then concentrated to a concentration of 55 μg / ml of mRNA to obtain lipid nanoparticles containing RNA encoding the gE variant of the VZV glycoprotein.

[0305] Example 2

[0306] Using luciferase as a reporter gene, in vivo fluorescence imaging was employed to study the efficiency of different vaccine vector formulations (as shown in Table 1 below, "MC3" refers to Dlin-MC3-DMA, "+": luciferase expression in mice was detected by a small animal in vivo fluorescence imaging system after administration) in delivering mRNA encoding the luciferase gene in mice. The physicochemical properties of different compound formulations (preparation methods are described in Example 1) were also tested, and the results are shown in Table 1. The study found that increasing the lipid-to-mRNA mass ratio improved the encapsulation efficiency of mRNA in lipid nanoparticles, thus enhancing its stability. Furthermore, moderately increasing the polyethylene glycol (PEG) content in the formulation improved the in vivo expression efficiency of mRNA. Therefore, considering factors such as mRNA encapsulation efficiency and in vivo mRNA delivery efficiency, formulations 3 and 4 were selected for subsequent mRNA vaccine research.

[0307] Table 1

[0308]

[0309] Table 2 shows the ability of different formulations of cationic lipid nanoparticles to encapsulate the mRNA encoding luciferase and the particle size data of the formed nanoparticles. All formulations can compress luciferase mRNA into nanoparticles with a particle size of less than 100 nm and a net neutral surface potential, while also encapsulating at least 50% of the mRNA, thus all possessing a certain in vivo delivery effect. "MC3" refers to Dlin-MC3-DMA.

[0310] Table 2

[0311]

[0312] Example 4

[0313] Cells were transfected with mRNA encoding the VZV glycoprotein gE variant, and the expression of the VZV glycoprotein gE variant in cells was detected.

[0314] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.32, Seq ID NO.33, Seq ID NO.8, and Seq ID NO.35, and are designated as VE1, VE3, VE6, and VE7, respectively. In addition to the open reading frames, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the mRNA is replaced with 5' pseudouracil.

[0315] The amino acid sequence of the VZV glycoprotein gE encoded by the mRNA (VE1) shown in Seq ID NO.32 is shown in Seq ID NO.30, which is the wild-type VZV glycoprotein gE.

[0316] The open reading frame sequence, as shown in Seq ID NO.33, encodes the VZV glycoprotein gE variant with deletions at amino acid sites 574–623 and a Y569A substitution.

[0317] The open reading frame sequence is shown in Seq ID NO.8. The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE6) is shown in Seq ID NO.7. It is based on the VZV wild-type glycoprotein gE, with amino acid sites 569-623 deleted.

[0318] The open reading frame sequence is shown in Seq ID NO.35. The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE7) is shown in Seq ID NO.34. It is based on the wild-type VZV glycoprotein gE, with deletions at amino acid sites 569–579 and 588–623.

[0319] The immunoblotting assay for detecting the in vitro expression of the VZV glycoprotein gE variant was performed as follows: HEK293 cells were seeded in multi-well plates. 4 μg of mRNA was mixed with Lipofectamine RNAiMAX (Thermo Fisher Scientific) and transfected into the cells. Cells were harvested after 24 hours, treated with lysis buffer, and 20 μg of total protein was loaded onto SDS-PAGE gels. Immunoblotting was performed using anti-varicella-zoster virus glycoprotein E antibody and internal control antibody (β-actin or GAPDH) to remove the target protein. The cells were then incubated with secondary antibody, followed by chemiluminescence immunoassay. The target protein was visualized using a chemiluminescence analyzer. Cells without mRNA transfection served as a negative control (results are shown in [link to results]). Figure 4 ).

[0320] The method for detecting antigen expression on the surface of HEK293 cells by flow cytometry was as follows: HEK293 cells were seeded into multi-well plates, and 2 μg of mRNA was mixed with the transfection reagent Lipofectamine RNAiMAX (purchased from Thermo Fisher Scientific) and transfected into the cells. Specific transfection procedures were performed according to the product instructions. 24 hours after transfection, HEK293 cells were immunolabeled with anti-varicella-zoster virus glycoprotein E antibody and fluorescent secondary antibody, and then the fluorescence signal was detected by flow cytometry (results are shown in [link to results]). Figure 4 ).

[0321] In the Western blot diagram, Ne represents cells without mRNA transfection, the negative control; M represents the marker. The Western blot diagram shows that VE1, VE3, VE6, and VE7 were successfully transfected, expressing VZV glycoprotein gE in HEK293 cells.

[0322] As can be seen from the antigen map detected by flow cytometry, VE1, VE3, VE6 and VE7 all express VZV glycoprotein gE on the surface of HEK293 cell membranes, with VE6 and VE7 expressing slightly more than VE1 and VE3.

[0323] Example 5

[0324] Seven-week-old BALB / c mice were selected, and each mouse was intramuscularly injected with 10 μg of lipid nanoparticles encoding the RNA of the VZV glycoprotein gE variant, with an injection volume of 75 μL per mouse. The negative control was an equal volume of PBS injection (labeled PBS). The positive control was 5 μg of varicella-zoster virus glycoprotein E (labeled gE) encapsulated with Freund's adjuvant. All vaccine formulations were administered twice, on days 0 and 14. Fourteen days after the last administration, the serum titers of specific IgG antibodies, neutralizing antibodies, and varicella-zoster virus-specific CD4+ and CD8+ T lymphocyte signals were measured.

[0325] 1. Multifunctional detection of CD4+ and CD8+ T lymphocyte response levels

[0326] Splenic lymphocytes isolated from mouse spleens were added to wells of a cell culture plate and stimulated with a varicella-zoster virus glycoprotein E peptide library. After 48 hours of culture, Brefeldin A (purchased from Biolegend) was added for further stimulation. The cell suspension was collected and an inhibitor (purchased from BDPharmingen) was added to block the Fc receptor, followed by incubation with specific monoclonal fluorescent antibodies (CD3, CD4, and CD8). After washing twice, the cells were permeabilized. Intracellular staining was then performed using fluorescent antibodies against IFN-γ and TNF-α (purchased from Biolegend), and after washing, the fluorescence signal was detected by flow cytometry.

[0327] 2. Detection of extracellular cytokines TNF-α, IL-2, IFN-γ and IL-4

[0328] Mouse spleen lymphocytes (4 × 10⁶ cells / well) stimulated with a varicella-zoster virus glycoprotein E peptide library were collected, and the cell culture supernatant was harvested. After diluting the cell supernatant, 100 μL / well was added to a pre-coated plate and incubated at room temperature for 90 minutes. The plate was then washed with washing buffer, followed by incubation with the detection antibody diluted with diluent at room temperature, followed by washing with washing buffer again. The enzyme conjugate Streptavidin-HRP diluted with diluent was added and incubated at room temperature for 30 minutes. The plate was then washed with washing buffer, and 100 μL TMB was added. The plate was incubated in the dark for 15 minutes. After stopping the reaction, the plate was read for detection.

[0329] 3. Detection of specific IgG antibody titers in mouse serum

[0330] The titer of VZV-specific total IgG antibody in mouse serum was detected using an ELISA method. VZV gE protein was added to 96-well plates and incubated overnight at 4°C. The plates were then blocked with 2% BSA solution at room temperature. Serially diluted mouse serum samples were added and incubated at room temperature for 2 hours, followed by washing with PBST. Goat anti-mouse IgGFc(HRP) anti-mouse IgG antibody was added and incubated at room temperature. After washing, TMB was added and incubation was completed. The reaction was terminated with 2M sulfuric acid solution, and readings were taken at 450 nm / 630 nm. The cutoff value was calculated as twice the arithmetic mean of the detection signals from negative serum samples.

[0331] 4. Neutralizing activity assay for varicella-zoster virus

[0332] Serum samples were inactivated and serially diluted, then incubated with varicella-zoster virus Oka strain (purchased from ATCC) for 1 hour. The cells were then added to 96-well plates containing MRC-5 monolayer cells and incubated for 2 hours. The medium was changed, and culture medium was added to each well for further incubation. The supernatant was discarded, and the cells were fixed. Fluorescently labeled detection antibodies were added, and the plates were read using a CTL instrument. The antibody titer representing a 50% inhibition rate was calculated.

[0333] Example 6

[0334] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.32, Seq ID NO.33, Seq ID NO.8, and Seq ID NO.35. The four mRNAs were prepared into lipid nanoparticles containing the RNA encoding the VZV glycoprotein gE variant, i.e., varicella-zoster virus vaccines, according to the method described in Example 1, and were designated as VE1, VE3, VE6, and VE7, 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' polyA tail of 100 adenine nucleotides; and the uracil in the mRNA was replaced with 5' pseudouracil.

[0335] The immunogenicity of the prepared lipid nanoparticles encoding the gE variant of the VZV glycoprotein (i.e., varicella-zoster virus vaccines VE1, VE3, VE6, and VE7) was evaluated according to the method described in Example 5.

[0336] 1. Multifunctional CD4+ lymphocyte response level detection

[0337] See results Figure 6 The results showed that the varicella-zoster virus vaccine components VE1, VE3, VE6, and VE7, as well as the gE protein, could all induce CD4+ and CD8+ T cell responses in mice. The figure, from left to right, compares the levels of responses from multifunctional CD4+ lymphocytes simultaneously producing TNF-α+ and IFN-γ+, producing TNF-α+, producing IFN-γ+, and producing the sum of all three responses. The figure shows that VE7 can induce slightly higher levels of TNF-α and IFN-γ CD4+ T cell responses.

[0338] 2. Detection of extracellular cytokines TNF-α, IL-2, IFN-γ and IL-4

[0339] Results of extracellular cytokines IFN-γ and IL-2 detection are shown in the figure. Figure 7IFN-γ and IL-2 are important antiviral cytokines that reflect the level of Th1 cell responses. Results showed that varicella-zoster virus (VZV) vaccines VE1, VE3, VE6, and VE7, as well as the gE protein, could induce high levels of IFN-γ and IL-2 in mice. Among these, the VE7 VZV vaccine induced slightly higher levels of IFN-γ in mice than the other vaccine groups. Combined with the results of multifunctional CD4+ lymphocyte response level detection, varicella-zoster virus (VZV) vaccines VE1, VE3, VE6, and VE7, as well as the gE protein, could induce high levels of Th1 cell responses in mice.

[0340] Results of extracellular cytokines TNF-α and IL-4 detection are shown in the figure. Figure 8 The results showed that VE1, VE3, VE6, VE7, and gE protein could all induce mice to produce high levels of TNF-α. The IL-4 levels induced by varicella-zoster virus vaccine VE6 and VE7 in mice were slightly higher than those induced by gE protein and varicella-zoster virus vaccine VE1 and VE3.

[0341] 3. Detection of specific IgG antibody titers in mouse serum

[0342] Fourteen days after the second immunization, ELISA was used to detect VZV glycoprotein gE-specific IgG antibodies in mouse serum. Serum was diluted 100-fold to 1,000,000-fold, and the OD450 was measured by indirect ELISA. In this example, the antigen used was one that specifically recognizes the amino acid sequence of wild-type VZV glycoprotein gE. Figure 9 As shown, when the varicella-zoster virus vaccine VE1, VE3, VE6, and VE7 were diluted to 1,000,000-fold, the samples reached their final dilution titers; when the gE protein was diluted to 10,000-fold, the samples reached their final dilution titers. The results indicate that the IgG antibody titers of the varicella-zoster virus vaccine VE1, VE3, VE6, and VE7 were significantly different from those of the gE protein (P<0.01).

[0343] 4. Neutralizing activity assay for varicella-zoster virus

[0344] Fourteen days after the second immunization, the titer of VZV neutralizing antibodies in mouse serum was detected using a live VZV virus tropism reduction assay. Figure 9As shown, the geometric mean titer of neutralizing antibodies against the gE protein is less than 8; the geometric mean titer of neutralizing antibodies against VE1 in the varicella-zoster virus vaccine is 2224; the geometric mean titer of neutralizing antibodies against VE6 in the varicella-zoster virus vaccine is 1575; the geometric mean titer of neutralizing antibodies against VE7 in the varicella-zoster virus vaccine is 3139; and the geometric mean titer of neutralizing antibodies against VE3 in the varicella-zoster virus vaccine is approximately 2403. The geometric mean titer of neutralizing antibodies against VE7 in the varicella-zoster virus vaccine is higher than that of neutralizing antibodies against VE1, VE6, VE3, and gE protein in the varicella-zoster virus vaccine.

[0345] Based on the combined results of serum-specific IgG antibody titers, neutralizing antibody titers, and Th1 and Th2 cell response levels, the varicella-zoster virus vaccines VE7 and VE6 showed better overall performance than varicella-zoster virus vaccines VE1 and VE3; among them, VE7 showed the best overall performance.

[0346] Example 7

[0347] Cells were transfected with mRNA encoding the VZV glycoprotein gE variant, and the expression of the VZV glycoprotein gE variant in cells was detected.

[0348] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.33, Seq ID NO.53, Seq ID NO.35, Seq ID NO.55, and Seq ID NO.57, and are denoted as VE3, VE3E, VE7, VE7E, and VE7F, respectively. In addition to the open reading frames, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the mRNA is replaced with 5' pseudouracil.

[0349] The open reading frame sequence, as shown in Seq ID NO.53, encodes the VZV glycoprotein gE variant with a deletion of amino acid sites 574–623, a Y569A substitution, and the addition of a nucleotide encoding a cis-regulatory element at the 3' end (as shown in Seq ID NO.19).

[0350] The open reading frame sequence is shown in Seq ID NO.55. The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE7E) is shown in Seq ID NO.34. It is based on the VZV wild-type glycoprotein gE, with deletions at amino acid sites 569–579 and 588–623 (sequence shown in Seq ID NO.19).

[0351] The open reading frame sequence is shown in Seq ID NO.57. The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE7F) is shown in Seq ID NO.34. Based on the VZV wild-type glycoprotein gE, amino acid sites 569-579 and 588-623 are deleted, and a nucleotide encoding a polymerization element is added to the 3' end (amino acid sequence shown in Seq ID NO.21, nucleotide sequence shown in Seq ID NO.26).

[0352] The in vitro expression of the VZV glycoprotein gE variant was detected by immunoblotting as described in Example 4, and the expression of the antigen on the surface of HEK293 cells was detected by flow cytometry. The amounts of mRNA added in the in vitro expression experiments were 1 μg and 4 μg, respectively (see results). Figure 10 The amount of mRNA added in the cell surface expression experiment was 0.25 μg (see results). Figure 11 ).

[0353] In the Western blot diagram, Ne represents cells without mRNA transfection, the negative control; M represents the marker. The Western blot diagram shows that VE3, VE3E, VE7, and VE7E were all successfully transfected, expressing VZV glycoprotein gE in HEK293 cells. At different transfection doses, VE7 expression was the highest, while VE7F was almost undetectable.

[0354] Flow cytometry analysis of the antigen map showed that VE3, VE3E, VE7, VE7E, and VE7F all expressed VZV glycoprotein gE on the HEK293 cell membrane. At low transfection doses, VE7 showed higher expression levels on the membrane.

[0355] Example 8

[0356] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.33, Seq ID NO.53, Seq ID NO.35, Seq ID NO.55, and Seq ID NO.57. The five mRNAs were prepared into lipid nanoparticles containing the RNA encoding the VZV glycoprotein gE variant, i.e., varicella-zoster virus vaccines, according to the method described in Example 1, and were designated as VE3, VE3E, VE7, VE7E, and VE7F, 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' polyA tail of 100 adenine nucleotides; and the uracil in the mRNA was replaced with 5' pseudouracil.

[0357] The immunogenicity of the prepared lipid nanoparticles encoding the gE variant of the VZV glycoprotein (i.e., varicella-zoster virus vaccines VE3, VE3E, VE7, VE7E, and VE7F) was evaluated according to the method described in Example 5.

[0358] 1. Multifunctional CD4 T lymphocyte response level detection

[0359] See results Figure 12 The results showed that the varicella-zoster virus vaccine VE3, VE3E, VE7, VE7E and VE7F, as well as gE protein, could induce CD4+ T cell responses in mice.

[0360] Compared with gE protein, varicella-zoster virus vaccines VE3, VE3E, VE7, VE7E, and VE7F induced higher levels of TNF-α and IFN-γ CD4+ T cell responses (P<0.05). The multifunctional CD4+ T cell responses of VE7, VE7E, VE7F, and VE3 were slightly higher than those of VE3E; the multifunctional CD4+ T cell responses of VE7 and VE7E were slightly higher than those of VE3.

[0361] 2. Detection of extracellular cytokines TNF-α, IL-2, IFN-γ and IL-4

[0362] Results of extracellular cytokines IFN-γ and IL-2 detection are shown in the figure. Figure 13 IFN-γ and IL-2 are important antiviral cytokines that reflect the level of Th1 cellular responses. The results showed that, compared with gE protein, the varicella-zoster virus vaccines VE3, VE3E, VE7, VE7E, and VE7F induced higher levels of Th1 cytokines.

[0363] Results of extracellular cytokines TNF-α and IL-4 detection are shown in the figure. Figure 14 The results showed that, compared with gE protein, varicella-zoster virus vaccines VE3, VE3E, VE7, VE7E, and VE7F could all induce higher levels of TNF-α in mice. The IL-4 levels induced by varicella-zoster virus vaccines VE7, VE7E, and VE7F in mice were slightly higher than those induced by gE protein and varicella-zoster virus vaccines VE3 and VE3E.

[0364] 3. Detection of specific IgG antibody titers in mouse serum

[0365] Fourteen days after the second immunization, ELISA was used to detect VZV glycoprotein gE-specific IgG antibodies in mouse serum. Serum was diluted 100-fold to 1,000,000-fold, and the OD450 was measured by indirect ELISA. In this example, the antigen used was one that specifically recognizes the amino acid sequence of wild-type VZV glycoprotein gE. Figure 15 As shown, when the varicella-zoster virus vaccines VE3, VE3E, VE7, VE7E, and VE7F were diluted to 1 million times, the samples reached their final dilution titers; when the gE protein was diluted to 10,000 times, the samples reached their final dilution titers.

[0366] 4. Neutralizing activity assay for varicella-zoster virus

[0367] Fourteen days after the second immunization, the titer of VZV neutralizing antibodies in mouse serum was detected using a live VZV virus tropism reduction assay. Figure 15 As shown, the geometric mean titer of neutralizing antibodies against the gE protein virus is approximately 58, while the geometric mean titers of neutralizing antibodies against the varicella-zoster virus vaccine VE3E, VE3, VE7, VE7E, and VE7F virus are 1205, 643, 1405, 1170, and 771, respectively.

[0368] Based on the combined results of the specific IgG antibody titer, neutralizing antibody titer, and Th1 and Th2 cellular response levels in the serum, the varicella-zoster virus vaccine VE7 showed better overall performance than the varicella-zoster virus vaccine VE3. Compared with VE7, although VE7-E and VE7-F had low in vitro expression levels, they both showed good cellular and humoral immune response signals.

[0369] Example 9

[0370] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO. 8–17 and Seq ID NO. 33. Eleven mRNAs were prepared into lipid nanoparticles containing the RNA encoding the VZV glycoprotein gE variant, i.e., varicella-zoster virus vaccines, according to the method described in Example 1, and were designated as VE6, VE61, VE62, VE63, VE64, VE65, VE66, VE67, VE68, VE69, and VE3, respectively. In addition to the open reading frames, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in Seq ID NO. 1), a 3' UTR (as shown in Seq ID NO. 4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the mRNA was replaced with 5' pseudouracil.

[0371] The open reading frame sequences (Seq IDs) of the mRNA encoding the VZV glycoprotein gE variant had CAI values ​​of 0.79, 0.93, 0.92, 0.93, 0.93, 0.80, 0.81, 0.81, 0.82, and 0.80, respectively, and GC contents of 56.82%, 57.70%, 57.64%, 57.53%, 57.59%, 52.00%, 53.05%, 53.81%, 52.99%, and 52.17%.

[0372] The immunogenicity of the prepared lipid nanoparticles encoding the gE variant of the VZV glycoprotein (i.e., varicella-zoster virus vaccines VE6, VE61, VE62, VE63, VE64, VE65, VE66, VE67, VE68, VE69 and VE3) was evaluated according to the method described in Example 5.

[0373] Results of multifunctional CD4 T lymphocyte response level assay are shown in Figure 16 The results of the detection of extracellular cytokines IFN-γ and IL-2 are shown in the figure. Figure 17 The results of the detection of extracellular cytokines TNF-α and IL-4 are shown in the figure. Figure 18 The results of the detection of specific IgG antibody titers and varicella-zoster virus neutralizing activity in mouse serum are shown in [the table below]. Figure 19 .

[0374] Based on the detection results of CD4 T cell response, as well as extracellular cytokines IFN-γ, IL-2, IL-4 and TNF-α, the varicella-zoster virus vaccine VE6, VE61, VE62, VE63, VE64, VE65, VE66, VE67, VE68 and VE69 and gE protein can induce high levels of Th1 and Th2 cell responses in mice.

[0375] When the varicella-zoster virus vaccines VE6, VE61, VE62, VE63, VE64, VE65, VE66, VE67, VE68 and VE69 were diluted to 1 million times, the samples reached the final dilution titer, and the geometric mean titer of virus neutralizing antibodies was relatively high.

[0376] Based on the combined results of specific IgG antibody titers, neutralizing antibody titers, and Th1 and Th2 cell response levels in the serum, the varicella-zoster virus vaccines VE6, VE61, VE62, VE63, VE64, VE65, VE66, VE67, VE68, and VE69 showed superior overall performance; while the varicella-zoster virus vaccine VE6 showed the best overall performance.

[0377] Example 10

[0378] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.35-44 and Seq ID NO.33. Eleven mRNAs were prepared into lipid nanoparticles containing RNA encoding the VZV glycoprotein gE variant, i.e., varicella-zoster virus vaccines, according to the method described in Example 1, and were designated as VE7, VE71, VE72, VE73, VE74, VE75, VE76, VE77, VE78, VE79, and VE3, respectively. In addition to the open reading frames, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the mRNA was replaced with 5' pseudouracil.

[0379] The open reading frame sequences (Seq IDs) of the mRNA encoding the VZV glycoprotein gE variant had CAI values ​​of 0.93, 0.93, 0.93, 0.93, 0.92, 0.80, 0.79, 0.81, 0.81, and 0.80, respectively, and GC contents of 57.87%, 57.89%, 57.89%, 57.77%, 57.37%, 52.26%, 51.58%, 52.14%, 51.15%, and 52.49%, respectively.

[0380] The immunogenicity of the prepared lipid nanoparticles encoding the gE variant of the VZV glycoprotein (i.e., varicella-zoster virus vaccines VE7, VE71, VE72, VE73, VE74, VE75, VE76, VE77, VE78, VE79 and VE3) was evaluated according to the method described in Example 5.

[0381] Results of extracellular cytokines IFN-γ and IL-4 detection are shown in the figure. Figure 20 The results of the detection of specific IgG antibody titers and varicella-zoster virus neutralizing activity in mouse serum are shown in [the table below]. Figure 21 .

[0382] Based on the detection results of extracellular cytokines IFN-γ and IL-4, the varicella-zoster virus vaccines VE7, VE71, VE72, VE73, VE74, VE75, VE76, VE77, VE78 and VE79, as well as gE protein, can induce high levels of Th1 and Th2 cell responses in mice.

[0383] When the varicella-zoster virus vaccines VE7, VE71, VE72, VE73, VE74, VE75, VE76, VE77, VE78 and VE79 were diluted to 1 million times, the samples reached the final dilution titer, and the geometric mean titer of virus neutralizing antibodies was relatively high.

[0384] Based on the combined results of the specific IgG antibody titers, neutralizing antibody titers, and Th1 and Th2 cell response levels in the serum, the varicella-zoster virus vaccines VE7, VE71, VE72, VE73, VE74, VE75, VE76, VE77, VE78, and VE79 showed superior overall performance; while the varicella-zoster virus vaccine VE7 showed the best overall performance.

[0385] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure.

Claims

1. A varicella-zoster virus vaccine, characterized in that, The varicella-zoster virus vaccine encodes the varicella-zoster virus antigen and is a nucleic acid vaccine; the antigen is a VZV glycoprotein gE variant; the amino acid sequence of the VZV glycoprotein gE variant includes deletions of amino acids 569-579 and 588-623, and the amino acid sequence of the VZV glycoprotein gE variant is shown in Seq ID NO.

34.

2. The varicella-zoster virus vaccine as described in claim 1, characterized in that, The nucleic acid vaccine contains nucleic acid molecules; the nucleic acid molecules are mRNA.

3. The varicella-zoster virus vaccine as described in claim 2, characterized in that, The mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b): (a) An mRNA molecule having the nucleotide sequence encoding the gE variant of the VZV glycoprotein shown in any one of SEQ ID Nos. 35 to 44; (b) mRNA molecules derived from (a) that have one or more nucleotides substituted in the nucleotide sequence defined in (a) and have the same GC base pair percentage and / or CAI value as the sequence shown in (a). The mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.

97.

4. The varicella-zoster virus vaccine as described in claim 3, characterized in that, The mRNA molecule has a GC base pair percentage of 50% to 58% and a CAI value of 0.79 to 0.

93.

5. The varicella-zoster virus vaccine as described in claim 3, characterized in that, The mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b): (a) An mRNA molecule having the nucleotide sequence encoding the gE variant of the VZV glycoprotein as shown in any one of SEQ ID Nos. 35 to 39; (b) An mRNA molecule derived from (a) having one or more nucleotides substituted in the nucleotide sequence defined in (a) to have the function of encoding the gE variant of the VZV glycoprotein and having the same GC base pair percentage and / or CAI value as the sequence shown in (a).

6. The varicella-zoster virus vaccine as described in claim 5, characterized in that, The mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b): (a) An mRNA molecule having the nucleotide sequence encoding the VZV glycoprotein gE variant shown in SEQ ID No. 35; (b) An mRNA molecule derived from (a) having one or more nucleotides substituted in the nucleotide sequence defined in (a) to have the function of encoding the gE variant of the VZV glycoprotein and having the same GC base pair percentage and / or CAI value as the sequence shown in (a).

7. The varicella-zoster virus vaccine as described in claim 6, characterized in that, The nucleic acid molecule is shown in SEQ ID No.

35.

8. The varicella-zoster virus vaccine as described in claim 3, characterized in that, The mRNA consists of a sequence from the 5' end to the 3' end, including a 5' cap, a 5' UTR, an ORF, a 3' UTR, and a 3' poly(A) tail. The 5' end cap is selected from one of ARCA, mCAP, dmCAP, and tmCAP; The 5'UTR nucleotide sequence is shown in Seq ID NO.1-3; The 3'UTR sequence is shown in Seq ID NO.4 to 6; One or more uridines in the mRNA are replaced with a modified nucleoside; the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

9. The varicella-zoster virus vaccine as described in claim 8, characterized in that, The 5' end cap has the following cap analogues with the structure: m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7G(5')ppp(5')(2'OMeG)pG.

10. The varicella-zoster virus vaccine as described in claim 9, characterized in that, The 5' end cap is m7Gppp(5')(2'OMeA)pG.

11. The varicella-zoster virus vaccine as described in claim 8, characterized in that, The 5'UTR nucleotide sequence is shown in Seq ID NO.

1.

12. The varicella-zoster virus vaccine as described in claim 8, characterized in that, The 3'UTR sequence is shown in Seq IDNO.

4.

13. The varicella-zoster virus vaccine as described in claim 8, characterized in that, The modified nucleoside is N1-methyl-pseuuridine (m1ψ).

14. The varicella-zoster virus vaccine as described in claim 3, characterized in that, The varicella-zoster virus vaccine includes a delivery formulation; The delivery formulation is lipid nanoparticles; The lipid nanoparticles comprise: protonable cationic lipids, structural lipids, auxiliary lipids, and surfactants; The lipid nanoparticles, by weight, comprise: 40-60 parts protonable cationic lipids, 10-40 parts structural lipids, 10-30 parts auxiliary lipids, and 0.5-5 parts surfactants. The protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200 and DlinDMA; The structural lipid is cholesterol; The assisting lipids include at least one of DSPC, DOPE, DOPC, DOPG, and DOPS; The surfactant includes at least one of PEG-DMG, PEG-DSPE and TPGS.

15. The varicella-zoster virus vaccine as described in claim 14, characterized in that, The lipid nanoparticles comprise, by molar percentage, 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-DMG.

16. The method for preparing the varicella-zoster virus vaccine according to any one of claims 8 to 15, characterized in that, It includes: The mRNA and delivery formulation are mixed to form a varicella-zoster virus vaccine; The delivery formulation is lipid nanoparticles.

17. The method for preparing the varicella-zoster virus vaccine as described in claim 16, characterized in that, It includes: mRNA The aqueous phase was obtained by dissolving the lipid nanoparticles in a buffer solution. Each lipid component of the lipid nanoparticles was measured and dissolved in an organic solvent to obtain an organic phase. The aqueous and organic phases were mixed and the organic phase was removed to obtain the varicella-zoster virus vaccine. The volume ratio of the aqueous phase to the organic phase is 1:2 to 4, the buffer solution includes citrate buffer, the pH of the buffer solution is 3 to 7, and the concentration of mRNA in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL.

18. The method for preparing the varicella-zoster virus vaccine as described in claim 17, characterized in that, The volume ratio of the aqueous phase to the organic phase is 1:

3.

19. The method for preparing the varicella-zoster virus vaccine as described in claim 17, characterized in that, The pH of the buffer solution is 4.

20. The method for preparing the varicella-zoster virus vaccine as described in claim 17, characterized in that, The concentration of mRNA in the aqueous phase was 0.1 mg / mL.

21. An isolated mRNA, wherein the coding region of the mRNA encodes a varicella-zoster virus VZV antigen, said antigen being a VZV glycoprotein gE variant; The amino acid sequence of the VZV glycoprotein gE variant includes the deletion of amino acids 569-579 and 588-623, and the amino acid sequence of the VZV glycoprotein gE variant is shown in Seq ID NO.

34.

22. The isolated mRNA as described in claim 21, characterized in that, The isolated mRNA includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b): (a) An mRNA molecule having the nucleotide sequence encoding the gE variant of the VZV glycoprotein shown in any one of SEQ ID Nos. 35 to 44; (b) mRNA molecules derived from (a) that have one or more nucleotides substituted in the nucleotide sequence defined in (a) and have the same GC base pair percentage and / or CAI value as the sequence shown in (a). The mRNA molecule has a GC base pair percentage of 50% to 70% and a CAI value of 0.79 to 0.97.

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