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

Abstract

Disclosed are a VZV antigen variant, a nucleic acid, a pharmaceutical composition and the use thereof. The VZV antigen variant has a difference of Y582G compared to an amino acid sequence as set forth in SEQ ID NO: 1; and / or the VZV antigen variant has deletions at positions 561-623, 569-623 or 574-623 compared to an amino acid sequence as set forth in SEQ ID NO: 1; and / or the VZV antigen variant has a modification of a transmembrane region and intracellular region of a protein compared to an amino acid sequence as set forth in SEQ ID NO: 1, wherein the modification of the transmembrane region and intracellular region of the protein involves the replacement of a transmembrane region and intracellular region of an original VZV antigen with a transmembrane region of a SARS-CoV-2Spike protein or a transmembrane region of an influenza H protein. The VZV antigen variant has a stronger immunogenicity than the VZV antigen in the prior art, can achieve a higher titer of binding antibodies, and has a higher protection efficacy.

Description

A VZV antigen variant, nucleic acid, pharmaceutical composition and use thereof

[0001] This application claims priority to Chinese Patent Application No. 202311552335X filed on November 20, 2023, and Chinese Patent Application No. 2024104602193 filed on April 16, 2024. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention belongs to the field of vaccines, and in particular relates to a VZV antigen variant, a nucleic acid, a pharmaceutical composition and applications thereof. Background Art

[0003] Varicella zoster virus (VZV) belongs to the herpesvirus subfamily, namely human herpesvirus type 3. The virus particles are 150nm to 200nm and are linear double-stranded DNA viruses. The viral envelope is embedded with nine glycoproteins, namely gE, gI, gC, gH, gL, gB, gK, gM and gN. Among them, gE is the main viral structural protein, a 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] Initial VZV infection typically occurs in early childhood, with varicella as the primary clinical manifestation. Following infection, the virus remains dormant within neurons in the human ganglia, presenting an insidious infection. When immunity declines, the virus reactivates and replicates in large numbers, triggering an immune response in peripheral sensory nerves and the unilateral dermatomes innervated by these nerves, causing herpes zoster, characterized by erythema, clusters of varicella, and neuralgia.

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

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

[0007] The incidence of shingles increases with age, with the disease becoming more common among younger patients. Complications such as neuralgia severely impact patients' lives, but there is a lack of effective and rapid treatment options. Vaccination can prevent shingles. These factors combined have led to a growing demand for shingles vaccines.

[0008] Currently, only four shingles vaccines are approved for marketing worldwide: Merck's Zostavax, SK Chemicals' SkyZoster, Beike Biopharmaceuticals' Ganwei, and GlaxoSmithKline's Shingrix. SkyZoster is only available in South Korea, with a market share of approximately 1.0%. Ganwei will be available in China only, launching in 2023. Zostavax has been discontinued. Shingrix, approved by the FDA in 2017 and conditionally approved by the National Medical Products Administration in 2019, is the first shingles vaccine to be marketed in China and officially launched there in June 2020. According to statistics, Shingrix holds a near-100% market share. Both Zostavax and Ganwei are live attenuated vaccines. The former is suitable for people aged 50 and older, with a protection rate of approximately 50% and a single dose. The latter is suitable for people aged 40 and older and also requires only a single dose. Shingrix is ​​a recombinant protein vaccine, which uses gE protein combined with AS01B adjuvant. It is suitable for people aged 60 and above, with a protection rate of more than 90%. Two doses are required.

[0009] Currently, the main R&D routes for shingles vaccines include attenuated vaccines, recombinant protein vaccines, mRNA vaccines, and adenovirus vaccines. In addition to the aforementioned marketed attenuated vaccines, several domestic companies have entered clinical trials. For example, Wantai Biological's freeze-dried shingles vaccine (VZV-7D) and the Shanghai Institute of Herpes Zoster's live attenuated vaccine have both entered Phase II clinical trials, and Changchun Qijian's live attenuated shingles vaccine has already received approval. In addition to the marketed Shingrix, the domestic recombinant vaccine developed by Green Bamboo Biotechnology utilizes an innovative tetrameric molecular structure and is progressing rapidly, having entered Phase II clinical trials. Yidao Bio's recombinant shingles vaccine has also entered Phase II clinical trials. mRNA vaccines are currently in the research and development stage, with no marketed products yet. CanSino Biologics' chimpanzee adenovirus-based shingles vaccine has entered the preclinical stage. Summary of the Invention

[0010] To address the technical deficiencies of existing herpes zoster mRNA vaccine products, such as limited variety and poor immune efficacy, the present invention provides a VZV antigen variant, nucleic acid, pharmaceutical composition, and its use. The VZV antigen variant can induce a stronger immune response, such as a stronger T cell immune response, a stronger cellular immune response, and higher cytokine levels (such as TNF-α), and the nucleic acid can achieve higher antigen expression.

[0011] A technical solution provided by the present invention is: 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 of 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 of the protein transmembrane region and the 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 (e.g., 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 expressed as VZV antigen, except that the amino acid sequence is limited to the wild-type VZV antigen as shown in SEQ ID NO: 1.

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

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

[0017] And / or, the VZV antigen is modified based on SEQ ID NO: 1, and the protein transmembrane region and intracellular region are modified, and the modification of the protein transmembrane region and intracellular region 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 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 following differences compared to the amino acid sequence shown in SEQ ID NO: 1: Y569 A, S593 A, S595 A, T596 A, and T598A; preferably, the VZV antigen variant further has Y569 A, S593 A, S595 A, T596 A, and T598A compared to the amino acid sequence shown in SEQ ID NO: 1;

[0019] 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 (e.g., 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 based on SEQ ID NO: 1, with the amino acid residue at position 569 replaced by A, the amino acid residue at position 593 replaced by A, the amino acid residue at position 595 replaced by A, the amino acid residue at position 596 replaced by A, and the amino acid residue at position 598 replaced by A or more; preferably, the VZV antigen is based on SEQ ID NO: 1, with the amino acid residue at position 569 replaced by A, the amino acid residue at position 593 replaced by A, the amino acid residue at position 595 replaced by A, the amino acid residue at position 596 replaced by A, and the amino acid residue at position 598 replaced by A;

[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 comprises Hibit-HA-tag, the sequence of which 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 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 shown in SEQ ID NO: 11.

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

[0027] In the present invention, capital letters represent single-letter amino acids, 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 positions represented by the numbers in the VZVgE antigen amino acid sequence based on 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, wherein the isolated nucleic acid comprises 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, the present invention provides another technical solution: 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, the present invention provides another technical solution: a method for preparing the isolated nucleic acid according to the present invention, wherein the method comprises in vitro transcription of the expression element according to the present invention. The in vitro transcription is conventional in the art.

[0034] To solve the above technical problems, the present invention provides another technical solution: a pharmaceutical composition comprising the VZV antigen as described in the present invention, the isolated nucleic acid or expression element as described in the present invention, and lipids.

[0035] In a preferred embodiment of the present invention, the lipid is a composition consisting of an ionizable lipid compound, DSPC (distearoylphosphatidylcholine), cholesterol, and DMG-PEG2000 (dimyristoylglycerol-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] Firefly luciferase (Fluc) mRNA was diluted in 50 mM citrate buffer (pH 4.0) to obtain an mRNA solution. Lipid nanoparticles were prepared by mixing an ethanolic lipid solution and an mRNA solution using a microfluidic device (e.g., NanoAssemblr (Precision Nanosystems) microfluidic mixing system) at a flow rate of 12 mL / min in a 1:3 volume ratio, with a nitrogen-phosphorus ratio of 3 to 15:1 for the ionizable lipid to mRNA. The ethanol was removed by dialysis against 0.01 M phosphate buffered saline (PBS) for 12-24 hours. Finally, the lipid nanoparticle solution was filtered through a 0.22 μm sterile filter and concentrated by ultrafiltration (Amicon-Ultra, MWCO 10 kDa) to obtain a LNP formulation encapsulating Fluc mRNA using ionizable lipid / DSPC / cholesterol / DMG PEG2000.

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

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

[0040] To address the above technical problems, the present invention provides another technical solution: use of the VZV antigen, isolated nucleic acid, or pharmaceutical composition described herein in the preparation of a medicament for preventing VZV infection. Preferably, the VZV infection is varicella or herpes zoster, such as postherpetic neuralgia.

[0041] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0043] The positive progress effect of the present invention is:

[0044] The VZV antigen provided by the present invention has stronger immunogenicity than existing VZV vaccines, can achieve higher binding antibody titers, and has higher protective efficacy. Vaccines prepared from the VZV antigen nucleotide sequence of the present invention have humoral and cellular immune responses that are non-inferior to those of the Shingrix VZV vaccine (and in some cases even superior), and have broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0046] Figure 2 shows the in vitro expression of alternative antigen sequences after packaging into LNPs.

[0047] Figure 3 shows the immunization procedure for C57BL / 6J mice using the primary screening antigen.

[0048] Figure 4 shows the humoral immune response induced by the alternative vaccine in mice.

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

[0050] Figure 6 shows the iterative in vitro expression of varicella-zoster virus vaccine antigens.

[0051] Figure 7 shows the in vitro expression of iterative antigen sequences after packaging LNPs.

[0052] Figure 8 shows the iterative antigen immunization program for C57BL / 6J mice.

[0053] Figure 9 shows the humoral immune response induced in mice by the iterative candidate vaccines, where Part A shows the antibody level induced in mice at a dose of 1 μg, and Part B shows the antibody level induced in mice at a dose of 5 μg.

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

[0055] Figure 11 shows the cellular immune response in mice induced by 5 μg of iterative candidate vaccines; Part A is the IFN-γ factor level in mice; Part B is the IL-2 factor level in mice.

[0056] Figure 12 shows the iterative antigen guinea pig immunization program.

[0057] Figure 13 shows the humoral immune response induced in guinea pigs by the iterative candidate vaccines. Part A shows the antibody level induced in guinea pigs at a dose of 5 μg, and Part B shows the antibody level induced in guinea pigs at a dose of 25 μg.

[0058] Figure 14 shows the cellular immune response induced by iterative candidate vaccines in guinea pigs.

[0059] Figure 15 shows the structure of mRNA. The mRNA structures shown in the figure all contain the structural elements in "VZVE", and VZVE-1, 2, 3, 4, 5, 6 and 7 are not displayed repeatedly. DETAILED DESCRIPTION

[0060] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0061] Example 1 RQ3400-Herpes Zoster Vaccine Antigen Screening

[0062] Different mutants or truncations of the VZVgE protein were screened by in vitro antigen expression. The antigen sequences of VZVE, VZVE-1, VZVE-2, VZVE-3, VZVE-4, and VZVE-5 were synthesized by chemical synthesis. The antigen sequences were codon-humanized, and three sequences were synthesized as templates for in vitro transcription. The UTP in the mRNA sequences (the structure of the mRNA is shown in Figure 15) was replaced with Pseudo-UTP. The mRNA was transfected into 293T cells (Thermo Fisher, Cat. No. K1538) and the expression of each antigen sequence was verified by Western blot. The specific steps are as follows:

[0063] 1. In vitro transcription and mRNA transfection

[0064] 1. PCR to obtain linearized target fragment

[0065] According to the requirements of the PCR reaction, add 8.7 μL of ddH2O, 0.5 μL of template DNA (source: GenScript synthesis), 0.4 μL of forward and 0.4 μL of reverse primers (source: GenScript synthesis), and 10 μL of PrimerSTAR MAX polymerase (source: TAKARA, product 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] The final PCR reaction conditions are shown in Table 2 below:

[0069] Table 2 PCR conditions

[0070] After the PCR reaction is completed, gel electrophoresis is performed to verify that the nucleic acid bands are uniform. If the bands are uniform, the next IVT reaction can be carried out. If not, the reaction needs to be repeated.

[0071] 2. In vitro transcription (IVT)

[0072] A small amount of IVT testing was performed according to the following ratio in Table 3 (20 μL system):

[0073] Table 3 IVT system

[0074] After the preparation is completed, the entire reaction system is placed in a 37°C water bath and incubated for 2 hours. After the reaction is completed, the DNA template is digested by adding 1 μL DNase I (source: Vazymy, product number: EN401-01) (35 μL for 1 mL IVT reaction) and digesting at 37°C for 15 minutes before IVT recovery. The recovery steps are as follows:

[0075] I. Add 80 μL of ddH2O to 20 μL of digestion product to make up to 100 μL.

[0076] II. Add 350 μL of Solution D and 250 μL of anhydrous ethanol (Source: Shanghai Test, Catalog No.: 801769722), mix thoroughly, transfer to a nucleic acid purification column (Source: Solarbio, Catalog No.: N1012), and centrifuge at 10,000 g for 1 min.

[0077] III. Add 500 μL of 70% ethanol and centrifuge at 10,000 g for 1 min. Repeat once and then centrifuge at 10,000 g for 2 min.

[0078] IV. Add 70 μL of sodium citrate (source: Sigma, catalog number: C8532) to the purification column. After incubation at room temperature for 1 minute, centrifuge at 10,000 g for 2 minutes. Collect the flow-through, which is the target RNA.

[0079] Prepare 1% agarose gel according to the above ratio and perform RNA electrophoresis at 160V for 20min. After electrophoresis, observe the gel using a gel imaging system (source: Tianneng, catalog number: Tanon 4600 SF). If a single band is present, the concentration can be determined.

[0080] 3. Cell Transfection

[0081] After 1 μg of mRNA sample was incubated with 2 μL of lipo2000 at room temperature for 20 min, the mixed sample was added to 1e6 293T cells and cultured at 37°C, 5% CO2 for 18-20 h, and then the cells were collected.

[0082] The results of in vitro expression of varicella-zoster virus vaccine antigens are shown in Figure 1. The sequences marked with arrows, namely VZVE-1-1, VZVE-3-1, VZVE-4-1, and VZVE-5-2, expressed well in 293T cells. NC, a negative control, showed no corresponding bands. These four antigen sequences were used as candidate sequences for LNP packaging. Furthermore, the VZVE-4-3 mRNA sequence, based on VZVE, had a Y581G mutation (mRNA sequence shown in SEQ ID NO: 26) for comparison. The amino acid sequence of VZVE-4-3 is shown in SEQ ID NO: 38.

[0083] The candidate antigen sequences VZVE-1-1, VZVE-3-1, VZVE-4-1, and VZVE-5-2 were used as templates for in vitro transcription, with the UTP in the mRNA sequences replaced with pseudo-UTP. The packaging lipids used were independently developed by Blue Magpie, including LQ007, LQ025, and LQ104.

[0084] Preparation of lipid compound LQ007:

[0085] Step 1: Preparation of LQ007-1

[0086] The material ratio is shown in Table 4 below:

[0087] Table 4 Material ratio

[0088] Operation process:

[0089] 6-Bromohexanoic acid, isodecyl alcohol, DCC, DMAP, and DCM were added to the reaction flask and stirred at room temperature for 12 h. TLC (EA:PE=20:1) showed that the reaction was complete.

[0090] Post-processing:

[0091] The reaction solution was filtered through celite and dried in a rotary evaporation cycle. After purification by column chromatography, 2 g of a colorless oil was obtained.

[0092] TLC results: EA:PE=20:1, product Rf=0.6.

[0093] Step 2: Preparation of LQ001-1

[0094] The material ratio is shown in Table 5 below

[0095] Table 5 Material ratio

[0096] Operation process:

[0097] 8-Bromooctanoic acid, heptadecan-9-ol, DCC, DMAP and DCM were added to the reaction flask, and the mixture was stirred at room temperature for 12 h. TLC (EA:PE=20:1) showed that the reaction was complete.

[0098] Post-processing:

[0099] The reaction solution was filtered through celite and dried in a rotary evaporation cycle. After purification by column chromatography, 40 g of a colorless oil was obtained with a yield of 85%.

[0100] 1 HNMR (500MHz, CDCl3) δ: 4.87 (p, 1H), 3.40 (t, 2H), 2.28 (t, 2H), 1.85 (p, 2H), 1.63 (p, 2H), 1.54-1.47 (m, 4H), 1.43 (dt, 2H), 1.33 (dt, 4H), 1.27 (d, 24H), 0.88 (t, 6H).

[0101] Step 3: Preparation of LQ001-2

[0102] The material ratio is shown in Table 6 below

[0103] Table 6 Material ratio

[0104] Operation process:

[0105] LQ001-1, ethanolamine, and acetonitrile were added to the reaction flask, heated to 30°C, and stirred for 12 hours. TLC (EA:PE=20:1) showed that the reaction was complete.

[0106] Post-processing:

[0107] The reaction solution was spin-dried and diluted with 500 mL of ethyl acetate, washed twice with 500 mL of water, and the organic phase was dried over anhydrous sodium sulfate and spin-dried. After purification by column chromatography, 25 g of a colorless oil was obtained with a yield of 87%.

[0108] 1 HNMR (500MHz, CDCl3) δ: 4.86 (p, 1H), 3.65-3.61 (m, 2H), 2.80-2.75 (m, 2H), 2.61 (t, 2 H), 2.27(t, 2H), 1.62(p, 3H), 1.48(dt, 7H), 1.32(s, 7H), 1.27(d, 22H), 0.87(t, 6H).

[0109] Step 4: Preparation of LQ007

[0110] The material ratio is shown in Table 7 below:

[0111] Table 7 Material ratio

[0112] Operation process:

[0113] LQ001-2, LQ007-1, K2CO3, KI, methyl tert-butyl ether, and acetonitrile were added to the reaction flask, and the mixture was heated to 80°C and stirred for 12 h. TLC (DCM:MeOH=10:1) showed that the reaction was complete.

[0114] Post-processing:

[0115] The reaction solution was filtered and dried in a rotary evaporation cycle. After purification by column chromatography, 2 g of a colorless oil was obtained with a yield of 80%.

[0116] 1 HNMR (500MHz, CDCl3) δ: 4.86 (p, 1H), 4.14-4.02 (m, 2H), 3.56 (t, 2H), 2.61 (t, 2H), 2.48 (q, 4H), 2.32-2. 25(m, 4H), 1.68-1.58(m, 6H), 1.56-1.40(m, 10H), 1.36-1.21(m, 38H), 1.19-1.03(m, 4H), 0.87(t, 12H).

[0117] LCMS: Rt: 3.83min; MS m / z(ESI): 696.7[M+H] + .

[0118] Preparation of lipid compound LQ025:

[0119] Step 1: Preparation of LQ025-1

[0120] The material ratio is shown in Table 8 below:

[0121] Table 8 Material Proportions

[0122] Operation process:

[0123] 6-Bromohexanoic acid, 3-cyclopentyl-1-propanol, DCC, DMAP, and DCM were added to the reaction flask and stirred at room temperature for 12 h. TLC (EA:PE=20:1) showed that the reaction was complete.

[0124] Post-processing:

[0125] The reaction solution was filtered through celite and dried in a rotary evaporation cycle. After purification by column chromatography, 2.8 g of a colorless oil was obtained.

[0126] TLC results: EA:PE=20:1, product Rf=0.6.

[0127] Step 2: Preparation of LQ025

[0128] The material ratio is shown in Table 9 below:

[0129] Table 9 Material Proportions

[0130] Operation process:

[0131] LQ001-2, LQ025-1, K2CO3, KI, methyl tert-butyl ether, and acetonitrile were added to the reaction flask and heated to 80°C with stirring for 12 h. TLC (DCM:MeOH = 10:1) showed that the reaction was complete, with an Rf of 0.4.

[0132] Post-processing:

[0133] The reaction solution was filtered and dried in a rotary evaporation cycle. After purification by column chromatography, 2 g of a colorless oil was obtained with a yield of 80%.

[0134] 1 HNMR (500MHz, CDCl3) δ: 4.86 (p, 1H), 4.05 (t, 2H), 3.58 (t, 2H), 2.64 (t, 2H), 2.55-2.46 (m, 4H), 2.29 (dt, 4H), 1. 75 (td, 3H), 1.69-1.56 (m, 8H), 1.55-1.43 (m, 10H), 1.37-1.28 (m, 10H), 1.25 (s, 23H), 1.06 (d, 3H), 0.87 (t, 6H).

[0135] LCMS: Rt: 3.83min; MS m / z(ESI): 666.7[M+H] + .

[0136] Preparation of lipid compound LQ104:

[0137] Step 1: Preparation of LQ001-1

[0138] The material ratio is shown in Table 10 below:

[0139] Table 10 Material ratio

[0140] Operation process:

[0141] 8-Bromooctanoic acid, heptadecan-9-ol, DCC, DMAP and DCM were added to the reaction flask, and the mixture was stirred at room temperature for 12 h. TLC (EA:PE=20:1) showed that the reaction was complete.

[0142] Post-processing:

[0143] The reaction solution was filtered through celite and dried in a rotary evaporation cycle. After purification by column chromatography, 40 g of a colorless oil was obtained with a yield of 85%.

[0144] 1 HNMR (500MHz, CDCl3) δ: 4.87 (p, 1H), 3.40 (t, 2H), 2.28 (t, 2H), 1.85 (p, 2H), 1.63 ( p, 2H), 1.54-1.47 (m, 4H), 1.43 (dt, 2H), 1.33 (dt, 4H), 1.27 (d, 24H), 0.88 (t, 6H).

[0145] Step 2: Preparation of LQ104

[0146] Preparation of LQ104

[0147] The material ratio is shown in Table 11 below:

[0148] Table 11 Material ratio

[0149] Operation process:

[0150] LQ001-1, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI, methyl tert-butyl ether, and acetonitrile were added to the reaction flask and heated to 80°C with stirring for 12 h. TLC (DCM:MeOH = 10:1) indicated the reaction was complete.

[0151] Post-processing:

[0152] The reaction solution was filtered and dried in a rotary evaporation cycle, and purified by column chromatography to obtain 1.1 g of a colorless oil.

[0153] 1 HNMR (400MHz, CDCl3) δ: 4.86 (p, 2H), 3.65-3.59 (m, 4H), 2.68-2.59 (m, 8H), 2.57-2 .49(m, 4H), 2.27(t, 4H), 1.61(p, 5H), 1.49(dt, 12H), 1.28(d, 61H), 0.87(t, 12H).

[0154] Preparation of lipid compound E16b2:

[0155] Step 1: Preparation of compound 1

[0156] Reaction formula:

[0157] The material ratio is shown in Table 12 below:

[0158] Table 12 Material Proportions

[0159] Operation process:

[0160] In a 1 L reaction flask, 6-bromohexanoic acid, DCC, DMAP, and DCM were added, followed by 9-heptadecanol. The mixture was stirred at room temperature for 12 h. TLC (PE:EA = 20:1) indicated the reaction was complete (product rf value 0.6).

[0161] Post-processing:

[0162] The reaction solution was filtered and dried in a rotary evaporation cycle, and purified by column chromatography to obtain 14.4 g of a colorless oil.

[0163] Step 2: Preparation of E16b2

[0164] Reaction formula:

[0165] The material ratio is shown in Table 13 below:

[0166] Table 13 Material Proportions

[0167] Operation process:

[0168] Compound 1, N,N'-bis(2-hydroxyethyl)ethylenediamine, K2CO3, KI, and acetonitrile were added to a reaction flask and heated to 80°C with stirring for 12 h. TLC (DCM:MeOH = 10:1) indicated the reaction was complete (rf value of the product was 0.5).

[0169] Post-processing:

[0170] The reaction solution was filtered and dried in a rotary evaporation cycle, and purified by column chromatography to obtain 770 mg of a colorless oil.

[0171] 1 H NMR (600MHz, CDCl3) δ: 4.85 (p, J=6.2Hz, 2H), 3.65 (t, J=4.8Hz, 4H), 2.73-2.65 (m, 8H), 2.61 (t, J=8.1Hz, 4H), 2.28 (t , J=7.4Hz, 4H), 1.64 (p, J=7.5Hz, 4H), 1.51 (dq, J=19.0, 7.3, 6.8Hz, 12H), 1.35-1.20 (m, 54H), 0.87 (t, J=6.9Hz, 12H).

[0172] SM102 lipid was used as a control. The lipids used were prepared by dissolving a series of ionizable lipid compounds, distearoylphosphatidylcholine (DSPC, Nippon Seika Co., Ltd., Catalog No. S01005), cholesterol (Nippon Seika Co., Ltd., Catalog No. O01001), and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000, Guobang Pharmaceutical, Catalog No. O02005) in ethanol. The four lipid components were then mixed in an adjusted ratio (ionizable lipid compound: cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5) to prepare a mixed lipid ethanol solution (total lipid concentration of 12.5 mM). The four candidate antigen mRNAs were diluted in 50 mM citrate buffer (pH 4.0) to prepare mRNA solutions. Lipid nanoparticles were prepared by mixing an ethanolic lipid solution and an mRNA solution in a 1:3 volume ratio using a NanoAssemblr (Precision Nanosystems) microfluidic mixing system at a flow rate of 12 mL / min, with a nitrogen-phosphorus ratio of 3 to 15:1 for the ionizable lipid to mRNA. The ethanol was removed by dialysis against 0.01 M phosphate buffered saline (PBS) for 12-24 hours. Finally, the lipid nanoparticle solution was filtered through a 0.22 μm sterile filter and concentrated by ultrafiltration (Amicon-Ultra, MWCO 10 kDa) to obtain a LNP formulation encapsulating the candidate antigen mRNA using ionizable lipid / DSPC / cholesterol / DMG PEG2000.

[0173] Packaged LNP-mRNA (i.e., mRNA packaged with lipids), LNP-empty (meaning empty lipids without mRNA), and 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). As shown in Figure 2, both mRNAs (positive control) and LNP-mRNAs were expressed. Expression was best after LQ104 lipid packaging, while expression was inferior to that after SM102 lipid packaging. Expression was similar between SM102 and LQ025 lipid packaging, and the worst was observed after LQ007 lipid packaging. No corresponding bands were detected in LNP-empty, a negative control.

[0174] Example 2 Evaluation of the immunogenicity of candidate vaccines in C57BL / 6J mice

[0175] In this embodiment, 6-8 week old C57BL / 6J female mice (Shanghai Lingchang Biotechnology Co., Ltd., Sibeifu (Suzhou) Biotechnology Co., Ltd.) were selected, with 8 or 16 animals in each group. Each group of mice was administered with placebo (PBS, P1010-100*2L, Solebo) and packaged alternative vaccines at week 0 and week 4, respectively. The administration dose was 1 μg / mouse by intramuscular injection (i.e., each mouse was injected with a vaccine preparation having an mRNA content of 1 μg); and blood was collected and serum was separated at week 2 and week 8 after the second administration for standby use. The spleen of the mice was removed at week 14 after the second administration (see Figure 3 for the experimental strategy), and the mouse spleen cells were separated for standby use.

[0176] The antigen (VZVgE)-specific antibody titers in serum at weeks 2 and 8 after the second administration were measured using an indirect ELISA (envelope antigen: Varicella zoster virus (strain Oka vaccine) Envelope Glycoprotein E (gE), His Tag VZV gE, Catalog No.: Acro biosystems, GLE-V52H3; secondary antibody: Goat Anti-mouse IgG (H+L), Catalog No.: C030205, Bio-Tong; developer: 1× TMB substrate solution (APExBio, cat#: K1131, Catalog No.: Thermo Fisher, 002023); ELISA 96-well plate: 514201, NEST). As shown in Figure 4, the encapsulated selection vaccine induced VZVgE-specific antibodies in mice at both weeks 2 and 8, with LQ104-VZVE-4-1 having the best effect.

[0177] Based on the results of the antigen-specific antibody levels induced by the alternative vaccines in mice, the spleens of mice in the LQ104-VZVE-4-1 and SM102-VZVE-4-1 groups were collected 14 weeks after the second administration, and the cytokine levels of IL-2, IFN-γ and TNF-α produced by CD4+T cells and CD8+T cells were detected by flow cytometry and ELISpot methods.

[0178] 1. Antigen-specific T cell response detection

[0179] Antigen-specific T cell responses in mouse spleens were assessed by intracellular cytokine staining (ICS). Briefly, a gE peptide library (covering the full length of the gE protein, 15-mer peptides with an 11-aa overlap between peptides, synthesized by GenScript) or an equal amount of DMSO-containing medium as a negative control was added to a 96-well plate. Mouse splenocytes were resuspended in RIPM1640 complete medium (Cat. No. 6016011, purchased from Dayou) and incubated at 37°C for 1 hour. Protein transport inhibitor (Cat. No. 554724, purchased from BD Bioscience) was then added and incubated again for 5 hours. Cells were washed once with PBS and stained with Fixable Viability Stain 510 (Cat. No. 564406, purchased from BD Bioscience). After a 10-minute incubation, cells were washed and anti-mouse CD16 / CD32 (Cat. No. 553142, purchased from BD Bioscience) was added and incubated at 4°C for 10 minutes. Cells were stained with a mixture of anti-mouse CD3-FITC, CD4-APC, and CD8-Percp-cy5.5 antibodies (Cat. Nos. 553061, 553051, and 551162, respectively, purchased from BD Bioscience). After a 30-minute incubation, cells were washed twice, fixed and permeabilized for 20 minutes, and washed once. Cells were then stained with a mixture of anti-cytokine antibodies against mouse IFN-γ-Pe-Cy7, IL-2-BV605, TNF-α-BV650, IL-4-BV711, and IL-5-PE (Cat. Nos. 557649, 563943, 563911, 564005, and 562049, respectively, all purchased from BD Bioscience). After a 30-minute incubation, cells were washed twice and resuspended in 200 μL of PBS. The fluorescence signal was analyzed by CYTEK Aurora / NL flow cytometer (model: NL-CLC V16B14R8, purchased from Cytek Biosciences).

[0180] 2.ELISPOT

[0181] Mouse spleen antigen-specific IFN-γ and IL-2 responses were measured using the enzyme-linked immunospot (ELISPOT) technique. Briefly, isolated mouse spleen cells were plated and incubated with a gE peptide library (same as ICS) or culture medium containing an equal amount of DMSO as a negative control. After 22 hours of incubation, subsequent procedures were performed according to the instructions for the mouse IFN-γ ELISPOT (Cat. No. 2210006, purchased from Dayou) and mouse IL-2 ELISPOT kits (Cat. No. 3441-4APW-10, purchased from MabTech). Spots were counted using an ELISPOT spot counter (Model: AT-Spot 2100, purchased from SINSAGE).

[0182] As can be seen from Parts A and B of Figure 5 , LQ104-VZVE-4-1 can induce a stronger T cell immune response than SM102-VZVE-4-1.

[0183] Example 3 Iteration of antigen sequence of the screened VZVE-4 antigen

[0184] In this example, after the VZVE-4 antigen was initially screened, 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. Three additional sequences (VZVE-4-deltag-1-1, VZVE-4-deltag-1-2, and VZVE-4-deltag-1-3) were synthesized through codon optimization using pseudo-UTP. Next, the transmembrane and intracellular regions 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, the three synthesized sequences were used 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 their protein expression levels were detected by Western blot. As shown in Figure 6, VZVE-7-2 was expressed at the highest level (i.e., the second sequence in the codon-optimized sequence of VZVE-7 had the best expression effect), followed by VZVE-4-deltag, then VZVE-4-deltag-1, and the expression levels of VZVE-6 were all poor.

[0185] Based on the Western blot results, VZVE-7-2 and VZVE-4-deltag antigen sequences were selected and packaged using the new delivery lipids E16b2 and LQ104. The results are shown in Figure 7. Both LNP-mRNAs were expressed in 293T cells, and the antigen expression level of LQ104-mRNAs was slightly higher than that of E16b2-mRNAs.

[0186] Example 4 Evaluation of the immunogenicity of herpes zoster vaccine iterative antigens in C57BL / 6J mice

[0187] 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 four groups of iterative alternative vaccine groups (experimental groups). Each group consisted of 8 or 16 animals. Each group of mice was dosed at week 0 and week 3, respectively, by intramuscular injection, with doses of 1 μg mRNA / mouse and 5 μg mRNA / mouse, respectively. Blood was collected and serum was isolated in the second week after the first dose and in the second and fourth weeks after the second dose, respectively. The spleens of the mice were collected and spleen cells were isolated in the fourth week after the second dose (see Figure 8 for the experimental strategy).

[0188] An indirect ELISA method (using the same reagents as in Example 2) was used to measure antigen (VZVgE)-specific antibody titers in serum at week 2 after the first dose and at week 2 and week 4 after the second dose. The results, as shown in Figure 9 , show that all iterative candidate vaccines induced VZVgE-specific antibodies in mice. At a 1 μg dose, E16b2-VZVE-7-2 induced antibody levels that were 1.5 to 2.5 times higher than Shingrix (Figure 9, Part A); at a 5 μg dose, E16b2-VZVE-7-2 induced antibody levels comparable to Shingrix (Figure 9, Part B).

[0189] The spleen of the mice was taken 4 weeks after the second administration, and the levels of IL-2 and IFN-γ cytokines produced by antigen-induced CD4+ T cells were detected by flow cytometry and ELISpot methods (the experimental methods and steps are the same as those in Example 2). Figure 10 shows that under the condition of 1 μg dose immunization, the CD4+ T cell immune response induced by the iterative candidate vaccine was better than Shingrix. For IFN-γ cytokines, LQ104-VZVE-7-2 had the highest level (Part A of Figure 10), while for IL-2 cytokines, E16b2-VZVE-7-2 had the highest level, and it was significantly improved compared to Shingrix, indicating that E16b2-VZVE-7-2 induced a stronger cellular immune response (Part B of Figure 10). As shown in parts A and B of Figure 11, under the 5μg dose immunization condition, the CD4+T cell immune response induced by the iterative candidate vaccine was non-inferior to Shingrix, and the comprehensive levels of the two cytokines showed that regardless of the lipid packaging selected, VZVE-7-2 induced a stronger cellular immune response than VZVE-4-deltag.

[0190] Example 5 Evaluation of the immunogenicity of iterative antigens of herpes zoster vaccines in guinea pigs

[0191] This example further evaluated the immunogenicity of the iterative antigen of the herpes zoster vaccine in guinea pigs (Hartley strain guinea pigs, Jiashan County Jintu Rabbit Industry Professional Cooperative). Female mice aged 6-8 weeks were selected and divided into a placebo group, a positive control group (GSK's marketed recombinant vaccine Shingrix), and an iterative alternative vaccine (4 experimental groups). There were 8 or 16 animals in each group, and each group of mice was administered at week 0 and week 4, respectively, by intramuscular injection. The two doses were 5 μg mRNA / guinea pig and 25 μg mRNA / guinea pig; and blood was collected and serum was separated in the second week after the first administration and in the second and fourth weeks after the second administration; the spleen of the guinea pig was taken in the fourth week after the second administration, and the guinea pig spleen cells were separated for later use (see Figure 12 for the experimental strategy).

[0192] The antigen (VZVgE)-specific antibody titers in serum were measured using an indirect ELISA method (using the same reagents as in Example 2) two weeks after the first administration and two and four weeks after the second administration, as shown in Figure 13. Figure 13 shows that all iterative candidate vaccines can induce VZVgE-specific antibodies in guinea pigs, and that the antibody levels induced by E16b2-VZVE-7-2 are comparable to those of Shingrix at doses of 5 μg (Part A of Figure 13) and 25 μg (Part B of Figure 13). Figure 14 shows that the antigen-induced T cell immune response was measured by ELISA in guinea pig spleens four weeks after the second administration. Under the 5μg dose immunization condition, the experimental group alternative vaccine LQ104-VZVE-4-deltag induced the highest TNF-α production, followed by E16b2-VZVE-7-2. The TNF-α induced by the two alternative vaccines was higher than Shingrix; under the 25μg dose immunization condition, the experimental group alternative vaccine E16b2-VZVE-7-2 induced the highest TNF-α production.

[0193] The sequence information involved in this application is shown in Table 14 below.

[0194] Table 14 Antibody amino acid sequence and mRNA sequence

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 antigen 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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