A zoster vaccine and uses thereof

Abstract

The present invention provides a vaccine comprising a VZV gE protein and a complex adjuvant, wherein the VZV gE protein is amino acids 1-546 of wild-type gE, and the complex adjuvant comprises an emulsion of squalene and / or squalane and CpG. The present invention also provides methods of making the vaccine, and uses of the vaccine for preventing herpes zoster disease and its complications.

Description

Technical Field

[0001] This invention provides a compound adjuvant shingles vaccine, which relates to a varicella-zoster virus (VZV) gE recombinant protein vaccine containing compound adjuvant and its application in the prevention of shingles disease, belonging to the field of immunological prevention. Background Technology

[0002] Herpes zoster is an acute infectious skin disease caused by VZV, and humans are the only known host for VZV. Children without immunity to this virus develop chickenpox after infection. Some infected individuals become carriers without developing symptoms. Due to the virus's neurotropic properties, it can remain dormant in the neurons of the dorsal root ganglia of the spinal cord for a long period after infection. When immunity is low, or during periods of fatigue, infection, or a cold, the virus can reactivate, multiply, and migrate along nerve fibers to the skin, causing severe inflammation of the affected nerves and skin.

[0003] VZV is morphologically identical to herpes simplex virus (HSV). VZV has only one serotype, and its genome contains 71 genes encoding 67 different proteins, including six glycoproteins (gp I-gp VI), now uniformly named gE, gB, gH, gI, gC, and gL. Glycoproteins gE, gB, and gH are extremely abundant in infected cells and are also present in the virion's cell membrane.

[0004] Shingles can be prevented through vaccination. Vaccines consist of an antigen as the active ingredient and an immune adjuvant. An immune adjuvant, or nonspecific immune proliferator, refers to a substance that, when injected into the body along with or beforehand, enhances the body's immune response to the antigen or alters the type of immune response. Adjuvants can be immunogenic or non-immunogenic. There are many types of adjuvants, and there is currently no unified classification method. The immunobiological effects of adjuvants include enhancing immunogenicity, increasing antibody titers, altering the type of antibody produced, and inducing or enhancing delayed hypersensitivity reactions. However, the mechanisms of action of adjuvants are not fully understood, and different adjuvants have different mechanisms of action. Summary of the Invention

[0005] In a first aspect, the present invention provides a vaccine comprising VZV gE protein and a compound adjuvant.

[0006] In a second aspect, the present invention provides a method for preparing the vaccine of the first aspect, the method comprising:

[0007] (1) Synthesize the gene encoding VZV gE protein, clone it into an expression plasmid and transfect cells, and screen cell lines that secrete gE fusion protein;

[0008] (2) Collect the cell culture supernatant expressing gE protein, and obtain gE protein after separation and purification;

[0009] (3) The gE protein is mixed with an emulsion of squalene and / or squalane and CpG to obtain the vaccine.

[0010] Thirdly, the present invention provides the use of the vaccine of the first aspect for the prevention of herpes-associated diseases and their complications.

[0011] The advantage of this invention lies in using squalene and / or squalane emulsions with CpG as a combined adjuvant, leveraging their synergistic effect to enhance the gE-specific antibody response and T-cell immune response induced by the gE recombinant vaccine. Therefore, a vaccine formulated with squalene and / or squalane emulsions and CpG, in combination with gE protein, can be used to prevent shingles and its complications. Detailed Implementation

[0012] On one hand, the present invention provides a vaccine comprising VZV gE protein and a compound adjuvant.

[0013] Preferably, the VZV gE protein is a gE protein with the transmembrane region and intracellular region removed; more preferably, the VZV gE protein is amino acid 1-538 of wild-type gE or amino acid 1-546 of wild-type gE; most preferably, the VZV gE protein is amino acid 1-546 of wild-type gE.

[0014] Preferably, the composite adjuvant comprises an emulsion of squalene and / or squalane, and CpG.

[0015] The squalene and / or squalane emulsion is an oil-in-water emulsion. The oil phase component is squalene or squalane or a mixture of both, with squalene accounting for 0%-100% of the oil phase component; preferably, squalene accounts for 20-80% or 40-60% of the oil phase component; more preferably, squalene accounts for 50% of the oil phase component and squalane accounts for 50% of the oil phase component; even more preferably, 100% squalene is used as the oil phase. The oil phase accounts for 1%-10% (v / v) of the total volume of the emulsion, preferably 3%-5%, more preferably 5%. The aqueous phase component contains a human-tolerable pH buffer and an ionic strength regulator, wherein the buffer contains citrate and / or phosphate, and the ionic strength regulator includes sodium chloride and / or potassium chloride, and the pH value of the aqueous phase is in the range of 6.0-8.0. The oil-in-water emulsion may contain a surfactant, which may be in the aqueous phase or the oil phase, and is a nonionic surfactant or a combination thereof. A combination of Tween-80 and Span 85 is preferred, with each surfactant preferably comprising 0.2%-5% (v / v) of the total volume of the oil-in-water emulsion. Particularly preferred is that the contents of Tween-80 and Span 85 are each 0.5% (v / v) of the total volume of the oil-in-water emulsion. The oil phase component, aqueous phase component, and surfactant are mixed by stirring or shearing, and then subjected to multiple high-pressure homogenizations until a homogeneous formulation containing oil droplets of the desired particle size is obtained. In the oil-in-water emulsion, the oil droplet diameter is in the submicron range, with an average diameter of less than 1 μm; preferably, the oil droplet diameter is less than 500 nm; more preferably, the diameter is less than 300 nm; particularly preferably, the diameter is between 90-180 nm, or between 50-140 nm.

[0016] In one specific embodiment, the oil phase is a mixture of squalene and squalane, each comprising 50%. The oil phase accounts for 1% (v / v) of the total emulsion volume. The aqueous phase contains a human-tolerable pH buffer and an ionic strength regulator, wherein the pH buffer comprises citrate and / or phosphate, and the ionic strength regulator comprises sodium chloride and / or potassium chloride, and the pH of the aqueous phase is 6.0. The surfactant is a combination of Tween-80 and Span 85, at contents of 0.2% (v / v) and 5% (v / v) of the total oil-in-water emulsion volume, respectively. The oil phase, aqueous phase, and surfactant are mixed by stirring or shearing, and then subjected to multiple high-pressure homogenizations until a homogeneous formulation containing oil droplets of the desired particle size is obtained.

[0017] In another specific embodiment, the oil phase component is squalene, comprising 1% (v / v) of the total emulsion volume. The aqueous phase component contains a human-tolerable pH buffer and an ionic strength regulator, wherein the pH buffer comprises citrate, and the ionic strength regulator comprises sodium chloride and / or potassium chloride, and the pH of the aqueous phase is 6.0. The surfactant is a combination of Tween-80 and Span 85, preferably at contents of 0.5% (v / v) and 0.2% (v / v) of the total volume of the oil-in-water emulsion, respectively. The oil phase component, aqueous phase component, and surfactant are mixed by stirring or shearing, and then homogenized under high pressure multiple times until a homogeneous formulation containing oil droplets of the desired particle size is obtained.

[0018] In another specific embodiment, the oil phase component comprises squalene, wherein the oil phase accounts for 10% (v / v) of the total emulsion volume. The aqueous phase component comprises a human-tolerable pH buffer and an ionic strength regulator, wherein the pH buffer comprises phosphate, and the ionic strength regulator comprises sodium chloride and / or potassium chloride. The pH value of the aqueous phase is in the range of 8.0. The surfactant is a combination of Tween-80 and Span 85, with contents of 0.2% (v / v) and 0.5% (v / v) of the total oil-in-water emulsion volume, respectively. After mixing the oil phase component, aqueous phase component, and surfactant by stirring or shearing, the mixture is subjected to multiple high-pressure homogenizations until a homogeneous formulation containing oil droplets of the desired particle size is obtained.

[0019] In another specific embodiment, the oil phase component comprises squalene, wherein the oil phase accounts for 10% (v / v) of the total emulsion volume. The aqueous phase component comprises a human-tolerable pH buffer and an ionic strength regulator, wherein the pH buffer comprises phosphate, and the ionic strength regulator comprises sodium chloride and / or potassium chloride. The pH value of the aqueous phase is in the range of 8.0. The surfactant is a combination of Tween-80 and Span 85, each comprising 0.5% (v / v) of the total volume of the oil-in-water emulsion. The oil phase component, aqueous phase component, and surfactant are mixed by stirring or shearing, and then homogenized under high pressure multiple times until a homogeneous formulation containing oil droplets of the desired particle size is obtained.

[0020] In another specific embodiment, the oil phase component comprises squalane, wherein the oil phase accounts for 10% (v / v) of the total emulsion volume, and the aqueous phase component comprises a human-tolerable pH buffer and an ionic strength regulator, wherein the pH buffer comprises phosphate, and the ionic strength regulator comprises sodium chloride and / or potassium chloride, and the pH value of the aqueous phase is in the range of 8.0. The surfactant is a combination of Tween-80 and Span 85, each accounting for 0.5% (v / v) of the total volume of the oil-in-water emulsion. The oil phase component, aqueous phase component, and surfactant are mixed by stirring or shearing, and then subjected to multiple high-pressure homogenizations until a homogeneous formulation containing oil droplets of the desired particle size is obtained.

[0021] The CpG ODNs (CpG for short) of this invention refer to nonmethylated thiooligodeoxynucleotides with immunostimulatory activity, the number of which is between 10 and 40. CpG ODNs are classified into four types according to their structure and immunomodulatory activity. Type A CpG ODNs are composed of a mixture of natural deoxynucleotides and thiodeoxynucleotides, and usually contain a CpG motif unit linked by a natural phosphoro-oxygen bond, with palindromic sequences on both sides, and tails formed by multiple G nucleotide units at the 3' and 5' ends. Type A CpG ODNs promote pDC maturation and secretion of IFN-α, but have no effect on B cells. Due to the presence of poly-G tails, Type A CpG ODNs are prone to forming complex polymers in solution, so they are rarely used in clinical trials. However, Type A CpG ODNs can be packaged into stable virus-like particles (VLPs) and used as adjuvants in preclinical and clinical studies (J. Immunol. 172(3), 1777–1785 (2004)). Type B CpG ODNs consist entirely of unmethylated thiodeoxynucleotides and are composed of one or more CpG motif units. Type B CpG ODNs promote pDC differentiation to produce TNF-α and stimulate B cell proliferation and IgM secretion. Type C CpG ODNs are similar to Type B in nucleotide units, consisting entirely of thiophosphate nucleotides. Structurally, they contain palindromic CpG motifs similar to Type A CpG ODNs, thus allowing them to form stem-loop structures or dimers. Type C CpG ODNs stimulate B cells to secrete IL-6 and stimulate pDC cells to produce IFN-α. Type P CpG-ODNs contain double palindromes, can form hairpins at their GC-rich 3' ends, and can further form palindromic structures due to the presence of 5' palindromes. These highly ordered structures are considered to be the cause of the strongest type I IFN production in CpG-ODNs.

[0022] For the CpG of the present invention, the preferred number of nucleotide units is between 15 and 25, and the preferred CpG types are type B, type C, and type P, with type B and type C being particularly preferred. This substance can stimulate immune cells in the body to produce corresponding cytokines, thereby regulating the body's immune response against specific antigens. Preferred CpG sequences include, but are not limited to: CpG-684 (5'-TCGACGTTCGTCGTTCGTCGTTC-3'), D-SL01 (5'-TCGCGACGTTCGCCCGACGTTCGGTA-3'), CpG-1018 (5'-TGACTGTGAACGTTCGAGATGA-3'), CpG-1760 (5'-ATAATCGACGTTCAAGCAAG-3'), CpG-1862 (5'-TCCATGACGTTCCTGACGTT-3'), CpG-1960 (… 5'-TCCTGTCGTTCCTGTCGTT-3'), CpG-1965(5'-TCCTGTCGTTTTTTGTCGTT), CpG-1967(5'-TCGTCGCTGTCTGCCCTTCTT-3'), CpG-1968(5 '-TCGTCGCTGTTGTCGTTTCTT-3'), CpG-1982(5'-TCCAGGACTTCTCTCAGGTT-3'), CpG-2002(5'-TCCACGACGTTTTCGACGTT-3'), CpG-200 5(5'-TCGTCGTTGTCGTTGTCGTT-3'), CpG-2006(5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), CpG-2007(5'-TCGTCGTTGTCGTTTTGTCTGTT-3' ), CpG-2008(5'-GCGTGCGTTGTCGTTGTCGTT-3'), CpG-2010(5'-GCGGCGGGCGGCGCGCGCCC-3'), CpG-2012(5'-TGTCGTTTGTCGTTTGTCGT T-3'), CpG-2013(5'-TGTCGTTGTCGTTGTCGTT-3'), CpG-2014(5'-TGTCGTTGTCGTTGTCGTT), CpG-2015(5'-TCGTCGTCGTCGTT-3'), CpG -2016(5'-TGTCGTTGTCGTT-3'), CpG-2102(5'-TCGTCGTTTTGACGTTTTGTCGTT-3'), CpG-2103(5'-TCGTCGTTTTGACGTTTTGACGTT-3'),CpG-2116 (5'-TCGTCGTTCCCCCCCCC-3'), CpG-2216 (5'-GGgggacgatcgtcGGGGGG-3'), CpG-2336 (5'-GGGgacgacgtcgtgGGGGGG-3'), CpG-2395 (5'-TCGTCGTTTTCGGCGCGCGCCG-3'), CpG-M362 (5'-TCGTCGTCGTTCGAACGACGTTGAT-3'), CpG-D-SL03 (5'-TCGCGAACGTTCGCCGCGTTCGAACGCGG-3'). Most preferably, the composite adjuvant includes CpG-2006. The lowercase letters of the CpG ODNs represent natural phosphorus-oxygen bonds, and the uppercase letters represent phosphorus-sulfur bonds. The method for preparing CpG in this invention is well known to those skilled in the art; for example, it can be synthesized chemically using a solid-phase phosphoramidite method, which is widely used in DNA chemical synthesis due to its high efficiency and rapid coupling. Currently, the artificial synthesis of CpG is mostly outsourced to specialized gene synthesis companies.

[0023] In the compound adjuvant, the dosage of squalene and / or squalane emulsion in a single-dose vaccine can be 50 μL-1000 μL, preferably 250 μL-600 μL; the dosage of CpG in a single-dose vaccine is between 2 μg-8 mg, preferably 100 μg-3 mg.

[0024] The single-dose dosage of gE protein described in this invention is 5μg-150μg, preferably 20μg-60μg.

[0025] The gE protein of the present invention is soluble in an emulsion containing squalene and / or squalane and CpG, and exists in a liquid dosage form. The single-dose dosage of gE protein is 5 μg-150 μg, preferably 20 μg-60 μg; the single-dose dosage of squalene and / or squalane emulsion is 50 μL-1000 μL, preferably 250 μL-600 μL; and the dosage of CpG in a single-dose vaccine is between 2 μg-8 mg, preferably 100 μg-3 mg.

[0026] Alternatively, the gE protein of the present invention can also be preserved as a separate lyophilized powder and then dissolved in a squalene or squalane emulsion before injection. The preferred method for storing the gE protein is as a separate lyophilized powder, which is then dissolved in an emulsion containing squalene or squalane before injection. The lyophilized gE protein powder may contain CpG, meaning the gE protein can be lyophilized together with CpG and then dissolved in an emulsion containing squalene or squalane before injection. The lyophilized gE powder composition contains a nonionic surfactant, a buffer salt, and a lyophilization protectant. The nonionic surfactant can be Tween 20, Tween 40, Tween 60, Tween 80, or a combination thereof, preferably Tween 80; the mass ratio of the nonionic surfactant to the gE protein is between 1:1 and 3:1, preferably 1:1 to 2:1, and most preferably 1.6:1. The buffer salt can be a citrate or a phosphate, preferably a composition of sodium dihydrogen phosphate and dipotassium hydrogen phosphate. The freeze-drying protectant includes, but is not limited to, trehalose, chitosan, sucrose, and glucose, with sucrose being preferred. The mass ratio of the freeze-drying protectant to gE protein is between 1000:1 and 25:1, preferably 800:1 to 200:1, and most preferably 400:1. The method for preparing the freeze-dried powder according to this invention involves a solute concentration in the solution before freeze-drying of 0.1 mg / mL to 100 mg / mL, preferably 0.4 mg / mL to 10 mg / mL. The solution before freeze-drying is subjected to pre-freezing, low-temperature freeze-drying, and room-temperature freeze-drying to remove moisture. The preferred pre-freezing temperature is -80°C, the preferred low-temperature freeze-drying temperature is between -20°C and -60°C, and the preferred room-temperature freeze-drying temperature is between 4°C and 25°C. The freeze-drying time at each stage is determined by the total mass of the freeze-dried material, ultimately yielding a freeze-dried powder with an intact appearance and no collapse. The freeze-drying process, temperature, and time can be adjusted according to the total mass of the freeze-dried material.

[0027] The use of squalene and / or squalane emulsions in combination with CpG as described in this invention refers to the application to the human body. Before human application, CpG may be present in the squalene and / or squalane emulsion; alternatively, CpG may be lyophilized and stored together with gE protein, and then mixed with the squalene and / or squalane emulsion before human application. Specifically, in this invention, CpG may exist alone as a soluble molecule in the formulation containing squalene or squalane emulsion, or it may exist as a solid molecule in the lyophilized powder of gE protein.

[0028] Specifically, the vaccine of the present invention may comprise a liquid emulsion of VZV gE protein and CpG and squalene and / or squalane; or the vaccine may comprise a solid emulsion of VZV gE protein and CpG and a liquid emulsion of squalene and / or squalane; or the vaccine may comprise a solid emulsion of VZV gE protein and a liquid emulsion of CpG and squalene and / or squalane; or the vaccine may comprise a liquid emulsion of VZV gE protein and CpG and a liquid emulsion of squalene and / or squalane; or the vaccine may comprise a liquid emulsion of VZV gE protein and a liquid emulsion of CpG and squalene and / or squalane.

[0029] In addition, the present invention also provides a method for preparing the vaccine of the present invention, the method comprising:

[0030] (1) Synthesize the gene encoding VZV gE protein, clone it into an expression plasmid and transfect cells, and screen cell lines that secrete gE fusion protein;

[0031] (2) Collect the cell culture supernatant expressing gE protein, and obtain gE protein after separation and purification;

[0032] (3) The gE protein is mixed with an emulsion of squalene and / or squalane and CpG to obtain the vaccine.

[0033] Preferably, the VZV gE protein is a gE protein with the transmembrane region and intracellular region removed; more preferably, the VZV gE protein is amino acid 1-538 of wild-type gE or amino acid 1-546 of wild-type gE; most preferably, the VZV gE protein is amino acid 1-546 of wild-type gE.

[0034] Preferably, the composite adjuvant comprises an emulsion of squalene and / or squalane, and CpG.

[0035] Preferably, the expression plasmid is a mammalian cell expression plasmid; the transfection is liposome co-transfection; and the cell is a CHO cell.

[0036] In one specific embodiment, the separation and purification described in step (2) is performed by the following steps: (1) purification using a Capto Q anion exchange chromatography column; (2) purification using Capto Butyl ImpRes hydrophobic chromatography; (3) purification using Capto MMC chromatography; (4) tangential flow ultrafiltration; and (5) removal of potential viruses using a Planova 15N nanofilter.

[0037] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention fall within the scope of the claims of the present invention.

[0038] Example 1: Preparation of gE protein

[0039] A gene encoding a gE protein (amino acids 1-538 of the wild-type gE sequence or amino acids 1-546 of the wild-type gE sequence, denoted as gE(1-538) or gE(1-546) respectively) was synthesized (sequences can be found in GenBank: AEW88548.1 or AGY33616.1). Following standard molecular biology techniques, this gene was cloned into the mammalian cell expression plasmid pcdna3.1, co-transfected with liposomes into CHO cells, and cell lines stably secreting the gE fusion protein were selected using the selective antibiotic genicin.

[0040] Collect the cell culture supernatant expressing gE protein. After filtration, adjust the pH of the supernatant to 6.5 and perform the first purification step on a Capto Q anion exchange chromatography column: equilibrate the Capto Q anion exchange chromatography column with equilibration buffer (20 mM phosphate buffer, pH 6.5); then, load the cell supernatant onto the column and wash the column with equilibration buffer and a solution of 20 mM PB + 200 mM NaCl, pH 6.5; elute the fraction containing gE protein with a solution of 20 mM PB + 450 mM NaCl, pH 6.5. The second purification step involved Capto Butyl ImpRes hydrophobic chromatography. During the Capto Q anion exchange chromatography stage, the gE protein fraction eluted with 20 mM PB + 450 mM NaCl at pH 6.5 was diluted to 1 M with ammonium sulfate and adjusted to pH 7.0. The sample was then loaded onto a Capto Butyl ImpRes column and washed with equilibration buffers of 20 mM PB + 1 M (NH4)2SO4 at pH 7.0 and 20 mM PB + 500 mM (NH4)2SO4 at pH 7.0. The gE protein was then eluted with 20 mM PB + 200 mM (NH4)2SO4 at pH 7.0. The third purification step involved Capto MMC chromatography. The Capto MMC column was equilibrated with 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) + 300 mM NaCl at pH 7.0. A sample diluted 2-fold with water for injection was loaded onto the Capto MMC column, and the flow-through peak containing gE protein was collected. The fourth purification step involved tangential flow ultrafiltration to concentrate the sample containing gE protein from the previous step and buffer exchange. A 0.1 mM... 2 Ultrafiltration was performed using a 10 kDa polyethersulfone membrane. The membrane was then rinsed with 3 L of PBS buffer (8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, 2.68 mM KCl, pH 7.2) until the pH reached 7.2 during osmosis. The gE protein was concentrated to above 1.0 mg / mL using the same ultrafiltration system, and then permeabilized with 10 volumes of PBS buffer at a permeabilization pressure of 15 psi for 90-120 min. The fifth purification step involved removing potential viruses using a Planova 15N nanofilter: the permeabilization solution containing gE protein was first pre-filtered through a 0.22 μm filter; then, the gE solution was filtered through a Planova 15N nanofilter at a constant pressure of 0.5 bar, and the virus-free solution was collected on the other side.

[0041] Example 2: Compound Adjuvant Formulation

[0042] This embodiment uses an emulsion of 100% squalene and an emulsion of 50% squalene and 50% squalane, wherein:

[0043] The 100% squalene emulsion composition is: 5% squalene (v / v), 0.65% Tween-80 (v / v), 0.35% Span 85 (v / v); the remainder is phosphate buffer.

[0044] The emulsion composition of 50% squalene and 50% squalane is: 4.5% (v / v) squalene + squalane, 0.45% (v / v) Tween-80, 0.55% (v / v) Span 85; the remainder is citrate buffer.

[0045] The compound adjuvant formulation of the present invention may be:

[0046] Formula 1: The dosage of 100% squalene emulsion in a single dose of vaccine is 50 μL, and the dosage of CpG-2006 in a single dose of vaccine is 2 μg.

[0047] Formula 2: The dosage of 100% squalene emulsion in a single dose of vaccine is 1000 μL, and the dosage of CpG-2006 in a single dose of vaccine is 2 μg.

[0048] Formula 3: The dosage of 100% squalene emulsion in a single dose of vaccine is 50 μL, and the dosage of CpG-2006 in a single dose of vaccine is 8 mg.

[0049] Formula 4: The dosage of 100% squalene emulsion in a single dose of vaccine is 1000 μL, and the dosage of CpG-2006 in a single dose of vaccine is 8 mg.

[0050] Formula 5: The dosage of 100% squalene emulsion in a single dose of vaccine is 250 μL, and the dosage of CpG-2006 in a single dose of vaccine is 100 μg.

[0051] Formula 6: The dosage of 100% squalene emulsion in a single-dose vaccine is 250 μL, and the dosage of CpG-2006 in a single-dose vaccine is 3 mg.

[0052] Formula 7: The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-2006 in a single-dose vaccine is 100 μg.

[0053] Formula 8: The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-2006 in a single-dose vaccine is 3 mg.

[0054] Formula 9: The dosage of 100% squalene emulsion in a single dose of vaccine is 600 μL, and the dosage of CpG-2006 in a single dose of vaccine is 100 μg.

[0055] Formula 10: The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-1018 in a single-dose vaccine is 100 μg.

[0056] Formula 11: The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-1965 in a single-dose vaccine is 100 μg.

[0057] Formula 12: The emulsion of 50% squalene and 50% squalane is used in a single dose of vaccine at a dose of 600 μL, and CpG-684 is used in a single dose of vaccine at a dose of 100 μg.

[0058] Formula 13: The emulsion of 50% squalene and 50% squalane is used in a single dose of vaccine at a dose of 600 μL, and CpG-D-SL01 is used in a single dose of vaccine at a dose of 100 μg.

[0059] Formula 14: The emulsion of 50% squalene and 50% squalane is used in a single dose of vaccine at a dose of 600 μL, and CpG-2216 is used in a single dose of vaccine at a dose of 100 μg.

[0060] Formula 15: The emulsion of 50% squalene and 50% squalane is used in a single dose of vaccine at a dose of 600 μL, and CpG-2336 is used in a single dose of vaccine at a dose of 100 μg.

[0061] Formula 16: The emulsion of 50% squalene and 50% squalane is used in a single dose of vaccine at a dose of 600 μL, and CpG-2395 is used in a single dose of vaccine at a dose of 100 μg.

[0062] Formula 17: The emulsion of 50% squalene and 50% squalane is used in a single dose of vaccine at a dose of 600 μL, and CpG-M362 is used in a single dose of vaccine at a dose of 100 μg.

[0063] Formula 18: The emulsion of 50% squalene and 50% squalane is used in a single dose of vaccine at a dose of 600 μL, and CpG-D-SL03 is used in a single dose of vaccine at a dose of 100 μg.

[0064] Example 3: Preparation of Immunotherapy Agents

[0065] Preparation of single-dose liquid formulations:

[0066] Immunotherapy Agent 1 (Combined Adjuvant is Formula 1) – Take 5 μg (14 μL) of gE protein (gE(1-546))(0.372 mg / ml), add 50 μL of 100% squalene emulsion, 2 μg (0.7 μL) of CpG-2006 (3 mg / ml), and then add 35.3 μL of PBS buffer to make up to 100 μL. Mix in an EP tube, slowly invert 10 times, let stand for 5 minutes, and shake well before use.

[0067] Following the dosage described in Example 2, prepare the same immunomodulator 2-18 (compound adjuvant is formulation 2-18): wherein each gE (1-546) protein is 5 μg, and then add 100% squalene emulsion (formulation 1-11) or 50% squalene and 50% squalane emulsion (formulation 12-18) and CpG in sequence.

[0068] Example 4: Immunization of mice

[0069] Commercially available C57BL / 6 mice were used. Mice were divided into 5 groups of 5 mice each. Each group was immunized via intramuscular injection of PBS buffer (negative control), gE protein (gE(1-546)) (5 ​​μg / mouse), gE protein (5 μg / mouse) + 100% squalene emulsion (50 μL / mouse), gE protein (5 μg / mouse) + CpG-2006 (20 μg / mouse), or gE protein (5 μg / mouse) + 100% squalene emulsion (50 μL / mouse) + CpG-2006 (20 μg / mouse). Two immunizations were administered, two weeks apart, with each injection volume being 100 μL.

[0070] Example 5 Humoral Immunity Detection

[0071] Fourteen days after the second immunization, blood was collected by enucleation. The serum was separated by centrifugation at 3000 rpm for 15 min after standing at 37°C for 1 h and then at 4°C for 1 h. The antibody titer in the serum was detected by ELISA. 0.5 μg / well of gE protein was coated onto a 96-well plate and incubated overnight at 4°C. The next day, the plate was blocked with 3% BSA for 1 h. Serum samples of 10-fold serial dilutions were added sequentially to the plates and incubated at 37°C for 2 h. HRP-labeled goat anti-mouse IgG antibody or goat anti-mouse IgG1 / IgG2a antibody (1:1000 dilution) was added. For antibody typing, HRP-labeled rabbit anti-goat IgG antibody was added at the end, and the plates were incubated for 1 hour each time. The plates were washed 6 times with PBS-T solution (0.05% Tween-20), and then TMB solution was added for color development for 15 min. The color development reaction was terminated with 2M sulfuric acid solution, and the absorbance at OD 450 nm was measured using a microplate reader.

[0072] The results are shown in Table 1. Neither the PBS group nor the gE protein (gE(1-546)) group produced strong gE-specific antibodies. The gE + 100% squalene emulsion group and the gE + CpG-2006 group slightly increased antibody levels, but not significantly. The gE + 100% squalene emulsion + CpG-2006 group produced very high antibody levels. This indicates that the combined use of gE, 100% squalene emulsion, and CpG significantly synergistically induces a gE-specific humoral immune response and produces higher levels of IgG than the combined use of gE and 100% squalene emulsion, or gE and CpG alone, or the sum of gE + 100% squalene emulsion and gE + CpG. Therefore, the combined use of gE, 100% squalene emulsion, and CpG can be considered an excellent candidate vaccine for shingles.

[0073] Table 1

[0074]

[0075] The above experiments demonstrate the necessity of adjuvants. To determine whether different gE protein lengths affect the immunogenicity of recombinant vaccines, we selected two gE proteins of different lengths (i.e., gE(1-538) and gE(1-546)). For the preparation of a single-dose liquid formulation: Take 5 μg (14 μL) of gE(1-538) or gE(1-546) (0.372 mg / ml), add 50 μL of 100% squalene emulsion, 2 μg (0.7 μL) of CPG-1018 (3 mg / ml), and then add 35.3 μL of PBS buffer to bring the volume to 100 μL. Mix in an EP tube, slowly invert 10 times, let stand for 5 minutes, and shake well before use.

[0076] Mice were immunized with the prepared liquid formulation using the method described in Example 4. Following immunization, antigen-specific antibodies were detected, and the results are shown in Table 2. Both lengths of gE, when used in combination with adjuvant, elicited high antibody responses. gE(1-546) was selected as the recombinant vaccine antigen for the following experiments.

[0077] Table 2

[0078]

[0079] To investigate the effects of different adjuvant formulations on immunogenicity, experiments were conducted using different adjuvant formulations and the same dose (5 μg / animal) of gE, with the same immunization strategy. The results are shown in Table 3. All adjuvant formulations mixed with gE elicited a strong humoral immune response, indicating that the different adjuvant formulations have similar functions. However, considering both dosage savings and overall effectiveness, the strongest immune response was achieved when gE and 600 μL of 100% squalene emulsion were combined with 100 μg of CpG-2006 (formulation 9).

[0080] Table 3

[0081]

[0082] Based on the determined compound adjuvant formulation, in order to screen for suitable CpG sequences, the IgG titers after immunization with compound adjuvants including gE (5 μg / animal) and emulsions of different CpGs with 100% squalene or 50% squalene and 50% squalane were further tested. The immunization strategy was the same as above.

[0083] The results are shown in Table 4. The highest IgG levels were observed when gE was combined with 600 μL of 100% squalene emulsion and 100 μg of CpG-2006 (Formula 9), indicating that CpG-2006 is the most ideal adjuvant candidate. The combined use of gE and 100% squalene emulsion with CpG-2006 as a compound adjuvant resulted in high levels of antigen-specific antibodies. Thus, the optimal gE antigen length and compound adjuvant formulation have been determined.

[0084] Table 4

[0085]

[0086] Example 6 Cellular Immunoassay

[0087] As described in Example 4, the mice were immunized.

[0088] Twenty-one days after the second immunization, spleens were collected under aseptic conditions, and the spleens from each group of mice were mixed together. Specifically, a spleen cell suspension was prepared using the following steps:

[0089] (1) Mice were euthanized by cervical dislocation. After surface disinfection with 75% alcohol, six-well plates were prepared. 2 mL of RPMI 1640 complete culture medium was added to each well. Each group of mice was placed in one six-well plate, with 5 wells per group. Throughout the experiment, the groups were clearly marked. Spleen was removed under aseptic conditions.

[0090] (2) Wrap the spleen with gauze (200 mesh), grind it in culture medium with the handle of a 5mL syringe until no tissue is visible, then transfer the suspension from each well to a 15mL centrifuge tube, rinse the bottom of the six-well plate with 1mL of culture medium, and transfer the rinsing solution to the centrifuge tube. Centrifuge at 300×g for 10min. Discard the supernatant, add 3mL of ACK lysis buffer, mix well, and let stand at room temperature for 3min. Repeatedly invert and mix, and observe. Flocculent precipitate may appear. Add about 6mL of culture medium to stop the lysis, mix well, pass through gauze, and transfer to a new 15mL centrifuge tube. Centrifuge at 300×g for 5min. Discard the supernatant. The precipitate at the bottom of the tube will show two layers. The bottom layer is flesh-colored cell clusters with red ruptured red blood cell fragments, which is normal. Resuspend the cells with 5mL of culture medium, wash once, and centrifuge at 300×g for 5min. Discard the supernatant, add 3mL of culture medium to resuspend the cells, and mix thoroughly to obtain a spleen cell suspension.

[0091] Perform cell counting at 1×10⁻⁶. 6 Experiments were conducted using cells per well. Three stimulation conditions were set up: a negative control using culture medium, gE protein stimulation (final concentration 5 μg / mL), and a phytohemagglutinin (PHA) positive control (final concentration 5 μg / mL). Cells were stimulated under these three conditions and incubated at 37°C for 24 h. The liquid in each well was poured off, and each well was washed 5 times with 200 μL of PBS. 100 μL of diluted biotin-labeled monoclonal antibody (R4-6A2) working solution was added to each well, and the cells were incubated at room temperature for 2 h. The liquid in each well was poured off, and each well was washed 5 times with 200 μL of PBS. 100 μL of diluted streptavidin-HRP working solution was added to each well, and the cells were incubated at room temperature for 1 h. The liquid in each well was poured off, and each well was washed 5 times with 200 μL of PBS. 100 μL of ready-to-use TMB substrate solution was added to each well and developed for 5-30 min until obvious spots appeared; the development time needs to be determined through preliminary experiments. The development was stopped by washing with deionized water. If necessary, remove the plate base (the soft plastic underneath the plate) and rinse the bottom of the membrane; allow the plate to dry. Inspect and count the spots using an ELISpot reader, referring to the MABTECH IFN-γ detection kit for details.

[0092] The results are shown in Table 5. Neither the PBS group nor the gE group could induce a cellular immune response; the gE + 100% squalene emulsion group and the gE + CpG-2006 group could slightly increase the cellular immune response, but not significantly; while the gE + 100% squalene emulsion + CpG-2006 group could induce a strong cellular immune response.

[0093] Table 5

[0094]

[0095] The results showed that the combined use of gE, 100% squalene emulsion, and CpG-2006 significantly synergistically induced gE-specific cellular immune responses compared to the combination of gE and 100% squalene emulsion alone, gE and CpG-2006 alone, or gE + 100% squalene emulsion, or gE + CpG alone. Therefore, the combination of gE, 100% squalene emulsion, and CpG-2006 can be considered an excellent candidate vaccine for herpes zoster.

[0096] Similarly, to compare the advantages and disadvantages of different compound adjuvant formulations, cellular immune responses were tested using the same method as above.

[0097] The results are shown in Table 6. Consistent with the humoral immunity results, from the perspective of dose saving and immunogenicity, the highest proportion of IFN-γ positive T cells was observed when gE (5 μg / animal) and 600 μL of 100% squalene emulsion were used in combination with 100 μg CpG-2006 (i.e., formulation 9).

[0098] Table 6

[0099]

[0100] To determine suitable CpG sequences, cellular immune responses were also assessed for different combinations of CpGs with 100% squalene emulsions or 50% squalene and 50% squalane emulsions. The immunization strategy is as described above.

[0101] The results are shown in Table 7. The combination of gE and 100% squalene emulsion with CpG-2006 adjuvant (formulation 9) can induce a high antigen-specific cellular immune response. Therefore, the combination of 100% squalene emulsion and CpG-2006 adjuvant is the optimal candidate adjuvant screened in this experiment.

[0102] Table 7

[0103]

Claims

1. A vaccine, wherein the active ingredient of the vaccine comprises VZV gE protein and a complex adjuvant; wherein the VZV gE protein is a gE protein with its transmembrane and intracellular regions removed, and the complex adjuvant comprises a squalene emulsion and CpG; wherein the squalene emulsion is a 100% squalene emulsion, which is an oil-in-water emulsion, comprising: squalene, wherein squalene accounts for 100% of the oil phase, Tween-80 and Span 85, and the remainder is buffer solution; or the squalene emulsion is a 50% squalene and 50% squalane emulsion, which is an oil-in-water emulsion, comprising: squalene and squalane, wherein squalene and squalane each account for 50% of the oil phase, Tween-80 and Span 85, and the remainder is buffer solution; Squalene accounts for 1%-10% (v / v) of the total volume of the emulsion. The dosage of squalene emulsion used in a single-dose vaccine is 50 μL-1000 μL; the dosage of CpG used in a single-dose vaccine is 2 μg-8 mg. The VZV gE protein therein is amino acid 1-538 of wild-type gE or amino acid 1-546 of wild-type gE, and the amino acid sequence of wild-type gE is referenced in GenBank: AEW88548.

1.

2. The vaccine according to claim 1, wherein the VZV gE protein is amino acid 1-546 of wild-type gE.

3. The vaccine according to claim 1, wherein the CpG exists as a soluble molecule in the squalene emulsion or as a solid molecule in the lyophilized powder of gE protein.

4. The vaccine according to claim 3, wherein the active ingredient of the vaccine comprises a liquid form of VZV gE protein and an emulsion of CpG and squalene; or the active ingredient of the vaccine comprises a solid form of VZV gE protein and CpG and an emulsion of liquid squalene; or the active ingredient of the vaccine comprises a solid form of VZV gE protein and an emulsion of liquid CpG and squalene.

5. The vaccine according to claim 4, wherein the active ingredient of the vaccine comprises an emulsion of VZV gE protein and CpG in liquid form and squalene in liquid form.

6. The vaccine according to claim 1, wherein squalene accounts for 3%-5% of the total volume of the emulsion.

7. The vaccine according to claim 6, wherein squalene comprises 5% of the total volume of the emulsion.

8. The vaccine according to claim 1, wherein the CpG is type A, type B, type C or type P.

9. The vaccine according to claim 8, wherein, The CpGs are CpG-684, D-SL01, CpG-1018, CpG-1760, CpG-1862, CpG-1960, CpG-1965, CpG-1967, CpG-1968, CpG-1982, CpG-2002, CpG-2005, CpG-2006, CpG-2007, CpG- 2008, CpG-2010, CpG-2012, CpG-2013, CpG-2014, CpG-2015, CpG-2016, CpG-210 2. CpG-2103, CpG-2116, CpG-2216, CpG-2336, CpG-2395, CpG-M362, CpG-D-SL03.

10. The vaccine according to claim 9, wherein the CpG is CpG-2006.

11. The vaccine according to claim 1, wherein the squalene emulsion is used in a single-dose vaccine at a dose of 250 μL-600 μL; and the CpG is used in a single-dose vaccine at a dose of 100 μg-3 mg.

12. The vaccine according to claim 1, wherein the dosages of squalene emulsion and CpG are respectively: The dosage of 100% squalene emulsion in a single-dose vaccine is 50 μL, and the dosage of CpG-2006 in a single-dose vaccine is 2 μg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 1000 μL, and the dosage of CpG-2006 in a single-dose vaccine is 2 μg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 50 μL, and the dosage of CpG-2006 in a single-dose vaccine is 8 mg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 1000 μL, and the dosage of CpG-2006 in a single-dose vaccine is 8 mg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 250 μL, and the dosage of CpG-2006 in a single-dose vaccine is 100 μg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 250 μL, and the dosage of CpG-2006 in a single-dose vaccine is 3 mg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-2006 in a single-dose vaccine is 100 μg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-2006 in a single-dose vaccine is 3 mg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-1018 in a single-dose vaccine is 100 μg; and / or The dosage of 100% squalene emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-1965 in a single-dose vaccine is 100 μg; and / or The dosage of the 50% squalene and 50% squalane emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-684 in a single-dose vaccine is 100 μg; and / or The dosage of the 50% squalene and 50% squalane emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-D-SL01 in a single-dose vaccine is 100 μg; and / or The dosage of a 50% squalene and 50% squalane emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-CpG-2216 in a single-dose vaccine is 100 μg; and / or The dosage of 50% squalene and 50% squalane emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-CpG-2336 in a single-dose vaccine is 100 μg; and / or The dosage of a 50% squalene and 50% squalane emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-CpG-2395 in a single-dose vaccine is 100 μg; and / or The dosage of 50% squalene and 50% squalane emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-CpG-M362 in a single-dose vaccine is 100 μg; and / or The dosage of the 50% squalene and 50% squalane emulsion in a single-dose vaccine is 600 μL, and the dosage of CpG-CpG-D-SL03 in a single-dose vaccine is 100 μg.

13. The vaccine according to claim 12, wherein the dosage of the 100% squalene emulsion in a single dose of the vaccine is 600 μL, and the dosage of CpG-2006 in a single dose of the vaccine is 100 μg.

14. A method for preparing a vaccine according to any one of claims 1-13, the method comprising: (1) Synthesize the gene encoding VZV gE protein, clone it into an expression plasmid and transfect cells, and screen cell lines that secrete gE fusion protein; The VZV gE protein mentioned above is a gE protein with both transmembrane and intracellular regions removed. (2) Collect the cell culture supernatant expressing gE protein, and obtain gE protein after separation and purification; (3) The gE protein is mixed with a squalene emulsion and CpG to obtain the vaccine; The VZV gE protein therein is amino acid 1-538 of wild-type gE or amino acid 1-546 of wild-type gE, and the amino acid sequence of wild-type gE is referenced in GenBank: AEW88548.

1.

15. The method according to claim 14, wherein the expression plasmid is a mammalian cell expression plasmid; the transfection is liposome co-transfection; and the cell is a CHO cell.

16. The method of claim 14, wherein the VZV gE protein is amino acid 1-546 of wild-type gE.

17. The method according to claim 14 or 15, wherein the separation and purification in step (2) is performed by the following steps: (1) purification using a Capto Q anion exchange chromatography column; (2) purification using Capto Butyl ImpRes hydrophobic chromatography; (3) purification using Capto MMC chromatography; (4) tangential flow ultrafiltration; and (5) removal of potential viruses using a Planova 15N nanofilter.

18. Use of the vaccine of any one of claims 1-13 in the preparation of a medicament for the prevention of herpes zoster disease and its complications.

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