VZV antigen variant, nucleic acid, pharmaceutical composition and application thereof

Abstract

The invention discloses a VZV antigen variant, nucleic acid, a pharmaceutical composition and application of the VZV antigen variant. The VZV antigen variant has a Y582G difference compared with an amino acid sequence as shown in SEQ ID NO: 1; and / or the VZV antigen variant has the deletion of 561 to 623 sites, 569 to 623 sites or 574 to 623 sites compared with the amino acid sequence as shown in SEQ ID NO: 1, and / or the VZV antigen variant has the deletion of 561 to 623 sites, 569 to 623 sites or 574 to 623 sites; and / or the VZV antigen variant is a modification having a protein transmembrane region and an intracellular region compared with the amino acid sequence as shown in SEQ ID NO: 1, and the modification of the protein transmembrane region and the intracellular region is that the transmembrane region and the intracellular region of the original VZV antigen are replaced by a transmembrane region of SARS-CoV-2Spike protein or a transmembrane region of influenza H protein. Compared with the VZV antigen in the prior art, the VZV antigen variant has stronger immunogenicity, can obtain higher binding antibody titer, and has higher protective efficacy.

Description

[0001] This application claims the priority of Chinese patent application No. 202311552335X filed on November 20, 2023. This application cites the full text of the above Chinese patent application. Technical Field

[0002] The present invention belongs to the field of vaccines, and specifically relates to a VZV antigen variant, nucleic acid, pharmaceutical composition and application thereof. Background Technology

[0003] Varicella zoster virus (VZV) is a subfamily of herpesvirus, namely human herpesvirus type 3. The virus particles are 150nm to 200nm. It is a linear double-stranded DNA virus. There are 9 glycoproteins embedded in the viral envelope, namely gE, gI, gC, gH, gL, gB, gK, gM and gN. Among them, gE is the main viral structural protein, the key protein for viral replication and assembly, the most important glycoprotein recognized by the host immune system, and the main target protein for vaccine design.

[0004] The first infection of VZV usually occurs in early childhood, with chickenpox as the main clinical manifestation. After infection, the virus will lurk in the neurons of the human ganglia, presenting a latent infection. When the body's immunity decreases, the virus will be reactivated and replicated in large quantities, causing an immune response in the peripheral sensory nerves and the unilateral dermatomes innervated by the nerves, causing herpes zoster with erythema, clustered chickenpox and neuralgia as the main characteristics.

[0005] Postherpetic neuralgia (PHN) is the most complex and common adverse complication of herpes zoster, manifested as burning pain or electric shock pain. Some patients also experience itching pain, mainly persistent pain and throbbing pain, and even accompanied by hyperalgesia or abnormal pain, which seriously affects the patient's quality of life in terms of physical, psychological, functional and social aspects.

[0006] The main methods of prevention and treatment of herpes zoster are active prevention with vaccines, antiviral drug treatment, and treatment of neuralgia caused by complications.

[0007] The incidence of herpes zoster increases with age and gradually tends to be younger; complications such as neuralgia seriously affect the patient's life, but there is a lack of rapid and effective treatment methods. Vaccination can prevent herpes zoster. Taking all the above factors into consideration, the demand for herpes zoster vaccine is also increasing year by year.

[0008] At present, only 4 herpes zoster vaccines have been approved for marketing globally, namely Zostavax of Merck & Co., SkyZoster of SK Chemicals Co., Ltd., Ganwei of Changsheng Bioengineering Co., Ltd., and Shingrix of GlaxoSmithKline. SkyZoster is only sold in South Korea, with a market share of about 1.0%; Ganwei was launched in 2023 and is only sold in China; Zostavax has been discontinued; Shingrix was approved by the FDA for marketing in 2017 and conditionally approved by the National Medical Products Administration in 2019. It is the first herpes zoster vaccine launched in China and was officially launched for sale in China in June 2020. According to statistics, the market share of Shingrix is almost 100%. Both Zostavax and Ganwei are live attenuated vaccines. The former is applicable to people aged 50 and above, with a protection rate of about 50%, and one dose is administered; the latter is applicable to people aged 40 and above, and only one dose is required. Shingrix is a recombinant protein vaccine, with the gE protein combined with the AS01B adjuvant, applicable to people aged 60 and above, with a protection rate of over 90%, and two doses are required.

[0009] Currently, the R & D technical routes of herpes zoster vaccines are mostly live attenuated vaccines, protein recombinant vaccines, mRNA vaccines, and adenovirus vaccines. In addition to the above-mentioned marketed products, several domestic companies have entered the clinical stage for live attenuated vaccines. For example, the freeze-dried herpes zoster vaccine (VZV-7D) of Beijing Wantai Biological Pharmacy Co., Ltd. and the live attenuated herpes zoster vaccine of the Shanghai Institute of Biological Products have both entered the clinical phase II, and the live attenuated herpes zoster vaccine of Changchun Qijian Biological Co., Ltd. has obtained approval; for recombinant vaccines, in addition to the marketed Shingrix, the recombinant vaccine of Green Valley (Beijing) Biotech Co., Ltd. in China adopts an innovative tetramer molecular structure and has advanced rapidly, and has entered the clinical phase II; the recombinant herpes zoster vaccine of Yidao Biotech Co., Ltd. has also entered the clinical phase II; mRNA vaccines are currently all in the R & D stage and there are no marketed products; the chimpanzee adenovirus vector herpes zoster vaccine developed by CanSino Biologics Inc. in the UK has entered the preclinical stage. Summary of the Invention

[0010] To solve the technical defects of the existing herpes zoster mRNA vaccine products, such as fewer varieties and poor immune effects, the present invention provides a VZV antigen variant, nucleic acid, pharmaceutical composition, and their applications. The VZV antigen variant can induce a higher immune response, such as a stronger T cell immune response, induce a stronger cellular immune response, and induce a higher level of cytokines (such as TNF-α), and the nucleic acid can achieve a higher antigen expression level.

[0011] One technical solution provided by the present invention is that the VZV antigen variant has a difference of Y582G compared with the amino acid sequence shown in SEQ ID NO:1;

[0012] and / or, the VZV antigen variant has a deletion at positions 561 - 623, 569 - 623, or 574 - 623 compared to the amino acid sequence shown in SEQ ID NO:1;

[0013] and / or, the VZV antigen variant has a modification in the protein transmembrane region and intracellular region compared to the amino acid sequence shown in SEQ ID NO:1, and the modification in the protein transmembrane region and intracellular region is replacing the transmembrane region and intracellular region of the VZV antigen (such as the wild - type VZV antigen) with the transmembrane region of the SARS - CoV - 2 Spike protein or the transmembrane region of the influenza H protein.

[0014] In the present invention, the VZV antigen variant is also referred to as the VZV antigen, except that the amino acid sequence is defined as the wild - type VZV antigen shown in SEQ ID NO:1.

[0015] One technical solution provided by the present invention is: a VZV antigen, wherein the VZV antigen has the amino acid residue at position 582 replaced by G based on SEQ ID NO:1;

[0016] and / or, the VZV antigen has a deletion at positions 561 - 623, 569 - 623, or 574 - 623 based on SEQ ID NO:1;

[0017] and / or, the VZV antigen has a modification in the protein transmembrane region and intracellular region based on SEQ ID NO:1, and the modification in the protein transmembrane region and intracellular region is replacing the transmembrane region and intracellular region of the wild - type VZV antigen with the transmembrane region of the SARS - CoV - 2 Spike protein or the transmembrane region of the influenza H protein.

[0018] In some preferred embodiments, the VZV antigen variant further has one or more of the differences of Y569A, S593A, S595A, T596A, and T598A compared to the amino acid sequence shown in SEQ ID NO:1; preferably, the VZV antigen variant further has the differences of Y569A, S593A, S595A, T596A, and T598A compared to the amino acid sequence shown in SEQ ID NO:1;

[0019] and / or, the modification in the protein transmembrane region and intracellular region is replacing the transmembrane region and intracellular region of the VZV antigen (such as the wild - type VZV antigen) with the transmembrane region of the SARS - CoV - 2 Spike protein; the transmembrane region and intracellular region of the VZV antigen are positions 538 - 647 of SEQ ID NO:1.

[0020] Preferably, the VZV antigen is one or more of the following substitutions: substituting the amino acid residue at position 569 with A, substituting the amino acid residue at position 593 with A, substituting the amino acid residue at position 595 with A, substituting the amino acid residue at position 596 with A, and substituting the amino acid residue at position 598 with A based on SEQ ID NO:1; preferably, the VZV antigen is further substituted with A at the amino acid residue at position 569, substituted with A at the amino acid residue at position 593, substituted with A at the amino acid residue at position 595, substituted with A at the amino acid residue at position 596, and substituted with A at the amino acid residue at position 598 based on SEQ ID NO:1;

[0021] And / or, the modification of the transmembrane region and intracellular region of the protein is to replace the transmembrane region and intracellular region of the wild-type VZV antigen with the transmembrane region of the SARS-CoV-2 Spike protein.

[0022] In a preferred embodiment of the present invention, the VZV antigen contains a Hibit-HA-tag, and its sequence is, for example, SEQ ID NO:37.

[0023] In a preferred embodiment of the present invention, the transmembrane region and intracellular region of the VZV antigen are positions 538-647 of SEQ ID NO:1.

[0024] In a preferred embodiment of the present invention, the amino acid sequence of the transmembrane region of the SARS-CoV-2 Spike protein is as shown in SEQ ID NO:10.

[0025] In a preferred embodiment of the present invention, the amino acid sequence of the transmembrane region of the influenza H protein is as shown in SEQ ID NO:11.

[0026] In a preferred embodiment of the present invention, the amino acid sequence of the VZV antigen contains the amino acid sequence shown in any one of SEQ ID NOs: 2-9.

[0027] In the present invention, capital letters represent amino acids in single-letter representation, and their meanings are well-known to those skilled in the art. In the present invention, numbers represent the positions of amino acids in the amino acid sequence before deletion or substitution. The single capital letter before the number usually represents the amino acid before deletion or substitution at that position, and the single capital letter after the number usually represents the amino acid after substitution at that position. Those skilled in the art can easily infer the position represented by the number in the amino acid sequence such as the VZVgE antigen amino acid sequence.

[0028] To solve the above technical problems, another technical solution provided by the present invention is: an isolated nucleic acid, and the isolated nucleic acid contains a nucleotide sequence encoding the VZV antigen as described in the present invention.

[0029] In a preferred embodiment of the present invention, the isolated nucleic acid is mRNA. Preferably, the mRNA comprises one or more of Cap1, 5'UTR, and 3'-UTR-polyA.

[0030] In a specific embodiment of the present invention, the nucleotide sequence encoding the VZV antigen comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 15-25, 27-36.

[0031] To solve the above technical problems, another technical solution provided by the present invention is: an expression element, which comprises a starting plasmid and the isolated nucleic acid as described in the present invention.

[0032] In a preferred embodiment of the present invention, the starting plasmid is pCDNA3.1.

[0033] To solve the above technical problems, another technical solution provided by the present invention is: a method for preparing the isolated nucleic acid as described in the present invention, which is to perform in vitro transcription on the expression element as described in the present invention. The in vitro transcription is a conventional in vitro transcription in the art.

[0034] To solve the above technical problems, another technical solution provided by the present invention is: a pharmaceutical composition, which comprises the VZV antigen as described in the present invention, the isolated nucleic acid or expression element as described in the present invention, and a lipid.

[0035] In a preferred embodiment of the present invention, the lipid is a composition composed of an ionizable lipid compound, DSPC (distearoyl phosphatidylcholine), cholesterol, and DMG-PEG2000 (dimyristoyl glycerol-polyethylene glycol 2000). Preferably, the ratio of the ionizable lipid compound, DSPC, cholesterol, and DMG-PEG2000 is 50:10:38.5:1.5.

[0036] In a preferred embodiment of the present invention, the composition is prepared according to the following steps:

[0037] The firefly luciferase (Fluc) mRNA was diluted in 50 mM citrate buffer (pH 4.0) to obtain an mRNA solution. An ethanol solution of lipids and the mRNA solution were mixed at a flow rate of 12 mL / min and a volume ratio of 1:3 by using a microfluidic device (such as the NanoAssemblr (manufacturer: Precision Nanosystems) microfluidic mixing system) to prepare lipid nanoparticles with an ionizable lipid to mRNA nitrogen-phosphorus ratio of 3-15:1. Ethanol was removed by dialysis against 0.01 M phosphate buffer (PBS) for 12-24 h. Finally, the lipid nanoparticle solution was filtered through a 0.22 μm sterile filter and concentrated by ultrafiltration (Amicon-Ultra, MWCO 10KDa) to obtain an LNP formulation encapsulating Fluc mRNA with ionizable lipid / DSPC / cholesterol / DMG PEG2000.

[0038] In a preferred embodiment of the present invention, the pharmaceutical composition comprises SEQ ID NO:5 or The amino acid sequence shown in SEQ ID NO:8 and the lipids include LQ104 or E16b2.

[0039] In a preferred embodiment of the present invention, the pharmaceutical composition is a vaccine formulation. Preferably, the vaccine formulation further includes an adjuvant.

[0040] To solve the above technical problems, another technical solution provided by the present invention is: the application of the VZV antigen as described in the present invention, the isolated nucleic acid as described in the present invention, or the pharmaceutical composition as described in the present invention in the preparation of a drug for preventing VZV infection. Preferably, the VZV infection is chickenpox or herpes zoster such as postherpetic neuralgia.

[0041] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0042] The reagents and raw materials used in the present invention are all commercially available.

[0043] The positive and progressive effects of the present invention are as follows:

[0044] The VZV antigen provided by the present invention has stronger immunogenicity than the existing technology VZV, can obtain a higher binding antibody titer, and has higher protective efficacy. The vaccine prepared from the VZV antigen nucleotide sequence of the present invention has a humoral immune response and a cellular immune response that are not inferior to the VZV vaccine of Shingrix (and some effects are even better), and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 For in vitro expression of the varicella-zoster virus vaccine antigen.

[0046] Figure 2Packaging LNP with alternative antigen sequences for in vitro expression.

[0047] Figure 3 Immunization protocol for C57BL / 6J mice with the primary screening antigen.

[0048] Figure 4 Inducing humoral immune response in mice with alternative vaccines.

[0049] Figure 5 Inducing cellular immune response in mice with alternative vaccines. Part A is a bar graph showing the cytokine levels of IL-2, IFN-γ, and TNF-α produced by CD4+ T cells and CD8+ T cells. Part B is a stacked bar graph showing the cytokine levels of IL-2, IFN-γ, and TNF-α produced by CD4+ T cells and CD8+ T cells.

[0050] Figure 6 In vitro expression of varicella-zoster virus vaccine antigen iteration.

[0051] Figure 7 In vitro expression after packaging LNP with iterative antigen sequences.

[0052] Figure 8 Immunization protocol for C57BL / 6J mice with iterative antigen.

[0053] Figure 9 Inducing humoral immune response in mice with iterative alternative vaccines. Part A shows the antibody levels induced in mice at a dosing dose of 1 μg. Part B shows the antibody levels induced in mice at a dosing dose of 5 μg.

[0054] Figure 10 Inducing cellular immune response in mice with 1 μg of iterative alternative vaccine; Part A shows the IFN-γ factor level in mice; Part B shows the IL-2 factor level in mice.

[0055] Figure 11 Inducing cellular immune response in mice with 5 μg of iterative alternative vaccine; Part A shows the IFN-γ factor level in mice; Part B shows the IL-2 factor level in mice.

[0056] Figure 12 Immunization protocol for guinea pigs with iterative antigen.

[0057] Figure 13 Inducing humoral immune response in guinea pigs with iterative alternative vaccines. Part A shows the antibody levels induced in guinea pigs at a dosing dose of 5 μg. Part B shows the antibody levels induced in guinea pigs at a dosing dose of 25 μg.

[0058] Figure 14 Inducing cellular immune response in guinea pigs with iterative alternative vaccines.

[0059] Figure 15 The structure of mRNA. All the mRNA structures shown in the figure contain the structural elements in "VZVE", and VZVE-1, 2, 3, 4, 5, 6, and 7 are not shown repeatedly. Detailed implementation mode

[0060] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0061] Example 1 Screening of RQ3400 - Varicella - zoster vaccine antigen

[0062] Different mutants or truncated forms of the VZVgE protein were screened through in vitro antigen expression. The antigen sequences of VZVE, VZVE - 1, VZVE - 2, VZVE - 3, VZVE - 4, and VZVE - 5 were synthesized respectively by chemical synthesis. The antigen sequences were codon - humanized, and three copies were synthesized respectively as templates for in vitro transcription. UTP in the mRNAs (the structure of mRNA is as shown) was replaced with Pseudo - UTP. The mRNAs were transfected into 293T cells (Thermo fisher, catalog number: K1538), and the expression of each antigen sequence was verified by Western blot. The specific steps are as follows: Figure 15 shown) and the sequences were verified by Western blot. The specific steps are as follows:

[0063] I. In vitro transcription and mRNA transfection

[0064] 1. Obtain the linearized target fragment by PCR

[0065] According to the requirements of the PCR reaction, add 8.7 μL of ddH 2 O, 0.5 μL of template DNA (source: GenScript), 0.4 μL of forward primer and 0.4 μL of reverse primer (source: GenScript), and 10 μL of PrimerSTAR MAX polymerase (source: TAKARA, catalog number: R450A) into a 1.5 mL centrifuge tube, and mix well.

[0066] As shown in Table 1 below:

[0067] Table 1 PCR system

[0068]

[0069] The final PCR reaction conditions are shown in Table 2 below:

[0070] Table 2 PCR conditions

[0071]

[0072] After the PCR reaction is completed, perform gel electrophoresis verification. If the nucleic acid bands are uniform, the next IVT reaction can be carried out. If not satisfied, it needs to be redone.

[0073] 2. In Vitro Transcription (IVT)

[0074] A small amount of IVT test is carried out according to the ratio in Table 3 below (20 μL system):

[0075] Table 3 IVT System

[0076]

[0077]

[0078] After preparation, place the entire reaction system in a 37 °C water bath and incubate for 2 h. After the reaction is completed, perform DNA template digestion. At this time, add 1 μL DNase I (source: Vazymy, catalog number: EN401 - 01) (35 μL for 1 mL IVT reaction), digest at 37 °C for 15 min, and then perform IVT recovery. The recovery steps are as follows:

[0079] I. Add 80 μL ddH2O to 20 μL of the digestion product to make up to 100 μL.

[0080] II. Add 350 μL of Solution D and 250 μL of absolute ethanol (source: Hushi, catalog number: 801769722), mix well, transfer to a nucleic acid purification column (source: Solarbio, catalog number: N1012), and centrifuge at 10000 g for 1 min.

[0081] III. Add 500 μL of 70% ethanol, centrifuge at 10000 g for 1 min, repeat once, and then centrifuge at 10000 g for 2 min.

[0082] IV. Add 70 μL of sodium citrate (source: sigma, catalog number: C8532) to the purification column, let it stand at room temperature for 1 min, then centrifuge at 10000 g for 2 min, and collect the flow - through liquid, which is the target RNA.

[0083] Prepare a 1% agarose gel according to the above ratio and perform RNA electrophoresis at 160 V for 20 min. After electrophoresis, observe it in a gel imaging system (source: Tianneng, catalog number: Tanon 4600SF). If the band is single, the concentration can be measured.

[0084] 3. Cell Transfection

[0085] After incubating 1 μg of the mRNA sample with 2 μL of lipo2000 at room temperature for 20 min, the above mixed sample was added to 1×10⁶ 293T cells and cultured at 37 °C in 5% CO 2 for 18 - 20 h, and then the cells were collected.

[0086] The in vitro expression results of the varicella-zoster virus vaccine antigen are as Figure 1 shown. The sequences marked with arrows, namely VZVE-1-1, VZVE-3-1, VZVE-4-1, and VZVE-5-2, were well expressed in 293T cells. NC was the negative control, and no corresponding bands were detected. These 4 antigen sequences were used as alternative sequences for LNP packaging. In addition, the VZVE-4-3 mRNA sequence was mutated at Y581G on the basis of VZVE (the mRNA sequence is shown in SEQ ID NO: 26) and could be used as a comparison. The amino acid sequence of VZVE-4-3 is shown in SEQ ID NO: 38.

[0087] Using the above alternative antigen sequences VZVE-1-1, VZVE-3-1, VZVE-4-1, and VZVE-5-2 as templates for in vitro transcription, UTP in the mRNAs sequences was replaced with Pseudo-UTP. The lipids used for packaging were independently developed by Blue Magpie, including LQ007, LQ025, and LQ104, with SM102 lipid as a control. The preparation method of the lipids used was as follows: A series of ionizable lipid compounds, distearoyl phosphatidylcholine (DSPC, Nippon Fine Chemical Co., Ltd., product number: S01005), cholesterol (Nippon Fine Chemical Co., Ltd., product number: O01001), and dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000, Guobang Pharmaceutical Co., Ltd., product number: O02005) were respectively dissolved in ethanol solution, and the ratios of the four lipid components were adjusted (ionizable lipid compound: cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5) and mixed to obtain an ethanol solution of the mixed lipids (total lipid concentration was 12.5 mM). The above 4 alternative antigen mRNAs were respectively diluted in 50 mM citrate buffer (pH 4.0) to obtain mRNA solutions. By using a NanoAssemblr (manufacturer: Precision Nanosystems) microfluidic mixing system to mix the ethanol solution of the lipids and the mRNA solution at a flow rate of 12 mL / min and a volume ratio of 1:3, lipid nanoparticles were prepared with a nitrogen-phosphorus ratio of ionizable lipid to mRNA of 3-15:1. Ethanol was removed by dialysis with 0.01 M phosphate buffer (PBS) for 12-24 h. Finally, the lipid nanoparticle solution was filtered through a 0.22 μm sterile filter and concentrated by ultrafiltration (Amicon-Ultra, MWCO 10KDa) to obtain an LNP preparation encapsulating the alternative antigen mRNA with ionizable lipid / DSPC / cholesterol / DMG PEG2000.

[0088] The packaged LNP-mRNA (i.e., mRNA encapsulated with lipids), LNP-empty (indicating empty lipids without mRNA), and the corresponding mRNAs were transfected into 293T cells, and their in vitro expression was verified by Western blot (Anti-VZVgE: sc-56995, Santa Cruz; Anti-GAPDH: 60004-1-1g, proteintech). Figure 2 It can be seen that both mRNAs (positive control) and LNP-mRNAs were expressed. The expression effect was the best after packaging with LQ104 lipid, the expression effect after packaging with SM102 lipid was inferior to that of LQ104, the expression effects after packaging with SM102 and LQ025 lipids were similar, the expression effect was the worst after packaging with LQ007 lipid, and no corresponding bands were detected for LNP-empty as a negative control.

[0089] Example 2: Evaluate the immunogenicity of the candidate vaccine in C57BL / 6J mice

[0090] In this example, female C57BL / 6J mice aged 6 - 8 weeks (Shanghai Lingchang Biotechnology Co., Ltd., Spf (Suzhou) Biotechnology Co., Ltd.) were selected, with 8 or 16 animals in each group. The mice in each group were administered drugs at week 0 and week 4, including placebo (PBS, P1010 - 100*2L, Solarbio) and the packaged candidate vaccine. By intramuscular injection, the dosage was 1 μg / mouse (that is, each mouse was injected with a vaccine preparation containing 1 μg of mRNA); blood was collected and serum was separated for standby at week 2 and week 8 after the second administration. At week 14 after the second administration, the spleens of the mice were removed (see the experimental strategy in Figure 3 ), and the mouse spleen cells were separated for standby.

[0091] The indirect ELISA method (coated antigen: Varicella zoster virus (strain Oka vaccine) Envelope Glycoprotein E (gE), His Tag VZV gE, product number: Acro biosystems, GLE - V52H3; secondary antibody: Goat Anti - mouse IgG(H + L), product number: C030205, Biotong; chromogenic solution: 1×TMB substrate solution (APExBio, cat#: K1131), product number: Thermo Fisher, 002023; ELISA 96 - well plate: 514201, NEST) was used to detect the antigen (VZV gE) - specific antibody titers in the serum at week 2 and week 8 after the second administration. The results are as Figure 4 shown. At week 2 and week 8, the packaged candidate vaccine could induce the production of VZV gE - specific antibodies in mice, and LQ104 - VZVE - 4 - 1 had the best effect.

[0092] According to the results of the antigen - specific antibody levels induced by the candidate vaccine in mice, at week 14 after the second administration, the spleens of the mice in the LQ104 - VZVE - 4 - 1 and SM102 - VZVE - 4 - 1 groups were taken, and flow cytometry and ELISpot methods were used to detect the cytokine levels of IL - 2, IFN - γ, and TNF - α produced by CD4+ T cells and CD8+ T cells.

[0093] 1. Detection of antigen - specific T cell responses

[0094] Mouse spleen antigen-specific T cell responses were measured by intracellular cytokine staining (ICS). Briefly, a gE peptide library (covering the full length of the gE protein, 15mers for each peptide, with 11aa overlap between peptide segments, synthesized by: GenScript) or a medium containing an equal amount of DMSO as a negative control was added to a 96-well plate. Mouse spleen cells resuspended in RPMI 1640 complete medium (product number: 6016011, purchased from: Dalio) were added, and after incubation at 37 °C for 1 hour, a protein transport inhibitor (product number: 554724, purchased from: BD Bioscience) was added and incubated again for 5 hours. The cells were washed once with PBS and stained with Fixable Viability Stain 510 (product number: 564406, purchased from: BD Bioscience). After incubation for 10 minutes, the cells were washed and anti-mouse CD16 / CD32 (product number: 553142, purchased from: BD Bioscience) was added and incubated at 4 °C for 10 minutes. A mixture of anti-mouse CD3-FITC, CD4-APC, and CD8-Percp-cy5.5 antibodies against surface molecules (product numbers were: 553061, 553051, 551162 respectively, purchased from: BD Bioscience) was added for staining. After incubation for 30 minutes, the cells were washed twice, fixed and permeabilized for 20 minutes, then washed once, and stained with a mixture of anti-cytokine anti-mouse IFN-γ-Pe-Cy7, IL-2-BV605, TNF-α-BV650, IL-4-BV711, and IL-5-PE (product numbers were: 557649, 563943, 563911, 564005, and 562049 respectively, all purchased from: BD Bioscience) antibody solutions. After incubation for 30 minutes, the cells were washed twice and resuspended in 200 μL of PBS. Fluorescence signals were analyzed by a CYTEK Aurora / NL flow cytometer (model: NL–CLC V16B14R8, purchased from Cytek Biosciences).

[0095] 2. ELISPOT

[0096] Mouse spleen antigen-specific IFN-γ and IL-2 responses were measured by enzyme-linked immunosorbent spot assay (ELISPOT). Briefly, mouse spleen cells were isolated and added to the plate, a gE peptide library (the same as ICS) or a medium containing an equal amount of DMSO as a negative control was added, and after culturing for 22 h, the subsequent operations were carried out according to the instructions of the mouse IFN-γ ELISPOT kit (product number: 2210006, purchased from: Dalio) and the mouse IL-2 ELISPOT kit (product number: 3441-4APW-10, purchased from: MabTech). An ELISPOT spot counter (model: AT-Spot 2100, purchased from SINSAGE) was used to count the spots.

[0097] From Figure 5 Parts A and B, it can be seen that compared with SM102-VZVE-4-1, LQ104-VZVE-4-1 can induce a stronger T cell immune response.

[0098] Example 3 performs antigen sequence iteration on the screened VZVE-4 antigen

[0099] After initially screening out the VZVE-4 antigen in this example, the Hibit-HA-tag (YPYDVPDYAGSSGVSGWRLFKKIS, SEQ ID NO:37) fused during screening was deleted from the VZVE-4 antigen to obtain the VZVE-4-deltag antigen sequence. Additionally, 3 sequences (VZVE-4-deltag-1-1, VZVE-4-deltag-1-2, VZVE-4-deltag-1-3) were synthesized by codon optimization, using Pseudo-UTP. Then, the transmembrane region and intracellular region of the original VZVgE protein were replaced with the transmembrane region of the SARS-CoV-2 Spike protein (VZVE-7) or the transmembrane region of the influenza H protein (VZVE-6). After codon humanization, 3 sequences were synthesized as templates for in vitro transcription, also using Pseudo-UTP. The mRNAs of VZVE-4-deltag, VZVE-4-deltag-1, VZVE-6, and VZVE-7 antigens were transfected into 293T cells, and the protein expression levels were detected by Western blot, as Figure 6 shown. The expression level of VZVE-7-2 was the highest (i.e., the expression effect of the 2nd sequence in the codon-optimized sequence of VZVE-7 was the best), followed by VZVE-4-deltag, then VZVE-4-deltag-1, and the expression levels of VZVE-6 were all relatively poor.

[0100] Based on the comprehensive Western blot results, the VZVE-7-2 and VZVE-4-deltag antigen sequences were selected, and the new delivery lipid E16b2 and LQ104 were used to package these two sequences. The results were as Figure 7 shown. LNP-mRNAs were all expressed in 293T cells, and the antigen expression level of LQ104-mRNAs was slightly higher than that of E16b2-mRNAs.

[0101] Example 4 evaluates the immunogenicity of the shingles vaccine iterative antigen in C57BL / 6J mice

[0102] In this example, female mice aged 6 - 8 weeks were selected and divided into a placebo group, a positive control group (GSK's marketed recombinant vaccine Shingrix, catalog number: HT4D4), and 4 groups of iterative alternative vaccine groups (experimental groups). There were 8 or 16 animals in each group. The mice in each group were administered drugs at week 0 and week 3 via intramuscular injection, with the administered doses being 1 μg mRNA / mouse and 5 μg mRNA / mouse respectively; blood was collected and serum was separated at week 2 after the first administration, and at week 2 and week 4 after the second administration for standby; the spleens of the mice were taken at week 4 after the second administration, and the splenocytes of the mice were separated for standby (see the experimental strategy in Figure 8 ).

[0103] The indirect ELISA method (using the same reagents as in Example 2) was used to detect the antigen (VZVgE) - specific antibody titers in the serum at week 2 after the first administration, and at week 2 and week 4 after the second administration. The results are as shown in Figure 9 . The results showed that the iterative alternative vaccines could all induce the production of VZVgE - specific antibodies in mice. At the 1 μg dose, the antibody level induced by E16b2 - VZVE - 7 - 2 was 1.5 - 2.5 times that of Shingrix ( Figure 9 , part A); at the 5 μg dose, the antibody level induced by E16b2 - VZVE - 7 - 2 was comparable to that of Shingrix ( Figure 9 , part B).

[0104] At week 4 after the second administration, the spleens of the mice were taken, and flow cytometry and ELISpot methods (the experimental methods and steps were the same as in Example 2) were used to detect the levels of cytokines IL - 2 and IFN - γ produced by antigen - induced CD4+ T cells. Figure 10 The results showed that under the immunization condition of the 1 μg dose, the CD4+ T cell immune responses induced by the iterative alternative vaccines were all superior to Shingrix. For the IFN - γ cytokine, the level of LQ104 - VZVE - 7 - 2 was the highest ( Figure 10 , part A), while for the IL - 2 cytokine, the level of E16b2 - VZVE - 7 - 2 was the highest and was significantly increased compared to Shingrix, indicating that E16b2 - VZVE - 7 - 2 induced a stronger cellular immune response ( Figure 10 , part B). As shown in parts A and B of Figure 11 , under the immunization condition of the 5 μg dose, the CD4+ T cell immune responses induced by the iterative alternative vaccines were non - inferior to Shingrix, and considering the levels of the two cytokines, it can be seen that regardless of which lipid packaging was selected, compared with VZVE - 4 - deltag, VZVE - 7 - 2 induced a stronger cellular immune response.

[0105] Example 5: Evaluation of the immunogenicity of iterative antigens of herpes zoster vaccines in guinea pigs

[0106] In this example, the immunogenicity of the iterative antigen of the herpes zoster vaccine was further evaluated in guinea pigs (Hartley strain guinea pigs, Jiashan Jintutu Industry Professional Cooperative). Female guinea pigs at 6 - 8 weeks of age were selected and divided into a placebo group, a positive control group (GSK's marketed recombinant vaccine Shingrix), and an iterative alternative vaccine group (4 experimental groups). There were 8 or 16 animals in each group. The mice in each group were administered drugs at week 0 and week 4 by intramuscular injection, and the dosage for both administrations was 5 μg mRNA / guinea pig and 25 μg mRNA / guinea pig; blood was collected and serum was separated at week 2 after the first administration, week 2 and week 4 after the second administration for standby; the spleens of guinea pigs were taken at week 4 after the second administration, and guinea pig splenocytes were separated for standby (see the experimental strategy in Figure 12 ).

[0107] The indirect ELISA method (using the same reagents as in Example 2) was used to detect the antigen (VZVgE) - specific antibody titers in the serum at week 2 after the first administration, week 2 and week 4 after the second administration, as shown in Figure 13 . Figure 13 It shows that the iterative alternative vaccines can all induce the production of VZVgE - specific antibodies in guinea pigs, and among the doses of 5 μg ( Figure 13 part A) and 25 μg ( Figure 13 part B), the antibody level induced by E16b2 - VZVE - 7 - 2 is comparable to that of Shingrix. Figure 14 It shows that at week 4 after the second administration, the spleens of guinea pigs were taken, and the ELISA method was used to detect the antigen - induced T - cell immune response. Under the immunization condition of 5 μg dose, the TNF - α induced by the experimental alternative vaccine LQ104 - VZVE - 4 - deltag was the highest, followed by E16b2 - VZVE - 7 - 2, and the TNF - α induced by both alternative vaccines was higher than that of Shingrix; under the immunization condition of 25 μg dose, the TNF - α induced by the experimental alternative vaccine E16b2 - VZVE - 7 - 2 was the highest.

[0108] The sequence information involved in this application is shown in Table 4 below.

[0109] Table 4 Antibody Amino Acid Sequences and mRNA Sequences

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

Claims

1. A VZV antigen variant, characterized in that: The VZV antigen variant has a difference of Y582G compared to the amino acid sequence shown in SEQ ID NO: 1; and / or, the VZV antigen variant has a deletion of positions 561-623, 569-623 or 574-623 compared to the amino acid sequence shown in SEQ ID NO: 1; And / or, the VZV antigen variant has a modified protein transmembrane region and an intracellular region compared to the amino acid sequence shown in SEQ ID NO: 1, and the modification of the protein transmembrane region and the intracellular region is to replace the transmembrane region and the intracellular region of the VZV antigen with the transmembrane region of the SARS-CoV-2 Spike protein or the transmembrane region of the influenza H protein.

2. The VZV antigen variant according to claim 1, characterized in that The VZV antigen variant further has one or more of the differences among Y569 A, S593A, S595A, T596A and T598A compared with the amino acid sequence shown in SEQ ID NO: 1; preferably, the VZV antigen variant further has the differences among Y569 A, S593 A, S595 A, T596 A and T598A compared with the amino acid sequence shown in SEQ ID NO: 1; And / or, the modification of the transmembrane region and intracellular region of the protein is to replace the transmembrane region and intracellular region of the VZV antigen with the transmembrane region of the SARS-CoV-2 Spike protein; the transmembrane region and intracellular region of the VZV antigen are positions 538-647 of SEQ ID NO:

1.

3. The VZV antigen variant according to claim 2, characterized in that The amino acid sequence of the transmembrane region of the SARS-CoV-2 Spike protein is shown in SEQ ID NO: 10; the amino acid sequence of the transmembrane region of the influenza H protein is shown in SEQ ID NO:

11.

4. The VZV antigen variant according to claim 1, characterized in that The amino acid sequence of the VZV antigen variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-9.

5. An isolated nucleic acid, characterized in that The isolated nucleic acid comprises a nucleotide sequence encoding the VZV antigenic variant according to any one of claims 1-4.

6. The isolated nucleic acid according to claim 5, characterized in that The isolated nucleic acid is mRNA; preferably, the mRNA comprises one or more of Cap1, 5'UTR and 3'UTR-polyA; more preferably, the nucleotide sequence encoding the VZV antigen variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 15-25, 27-36.

7. An expression element, characterized in that The expression element comprises a starting plasmid and the isolated nucleic acid according to claim 5 or 6; the starting plasmid is pCDNA3.

1.

8. A method for preparing the isolated nucleic acid according to claim 6, characterized in that: The method comprises in vitro transcription of the expression element according to claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the VZV antigen variant according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5 or 6, and the expression element according to claim 7, and a lipid; Preferably, the lipid is a composition consisting of an ionizable lipid compound, DSPC (distearoylphosphatidylcholine), cholesterol and DMG-PEG2000 (dimyristoylglycerol-polyethylene glycol 2000); More preferably, the pharmaceutical composition is a vaccine preparation; preferably, the pharmaceutical composition further comprises an adjuvant.

10. Use of the VZV antigen variant according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5 or 6, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing VZV infection; preferably, the VZV infection is varicella or herpes zoster, such as postherpetic neuralgia.

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