Varicella zoster vaccine composition and application thereof

Abstract

The invention discloses a varicella zoster vaccine composition and application thereof, and belongs to the technical field of biological vaccines, the composition comprises a VZV gE antigen, a CpG ODN adjuvant and an aluminum adjuvant, and the nucleotide sequence of the CpG ODN adjuvant is shown as SEQ ID NO: 1. According to the invention, the CpG ODN adjuvant with a specific nucleotide sequence is selected, so that the immunostimulatory activity is better; the varicella zoster vaccine composition provided by the invention can generate high humoral immune response earlier, and induces protective immunity for at least one month in advance; the antibody level of twice immunization of a Shigrix vaccine can be reached or close to once immunization, the stable high humoral immune level for a long time can be kept, and meanwhile cellular immunity can be generated.

Description

Technical Field

[0001] The present invention relates to the technical field of biological vaccines, and more particularly to a varicella-zoster vaccine composition and uses thereof. Background Art

[0002] Varicella-zoster virus (VZV), or human herpesvirus 3, is a neurotropic human alpha herpes virus that causes chickenpox and herpes zoster (HZ). Shingles is an acute skin disease caused by the reactivation of latent VZV and primarily affects the elderly and other immunocompromised individuals. Approximately 1.56 million new cases of HZ occur annually in people over 50 years of age in China, with older age increasing the risk and severity of the disease.

[0003] Herpes zoster is a self-limiting disease characterized by a rash that is often flaky and often spread across the skin. Postherpetic neuralgia (PHN) is a chronic painful condition and one of the most common complications of the disease. Herpes zoster vasculitis is associated with morbidity and mortality. Renal and gastrointestinal complications have also been reported. The primary treatment is early intervention with acyclovir or brivudine. Vaccination is the primary strategy for preventing and treating herpes zoster in the elderly and immunocompromised patients.

[0004] Currently, there are four shingles vaccines approved for marketing worldwide, namely Zostavax from Merck, SkyZoster from SK Chemical Co., Ltd., Ganwei from Biopharmaceuticals, and Shingrix from GlaxoSmithKline.

[0005] Merck's Zostavax shingles vaccine is a live-attenuated vaccine primarily intended for use in people over 50 years of age. Its effectiveness decreases with age. Furthermore, people receiving immunosuppressive therapy or those with preexisting immune disorders, such as HIV infection, are not suitable for vaccination due to the presence of some viral activity in the live-attenuated vaccine. Similar issues exist with SkyZoster and Beike Biopharmaceuticals' Ganwei, both live-attenuated vaccines.

[0006] To overcome the population limitations of attenuated shingles vaccines, further research and development has resulted in inactivated and recombinant protein vaccines. Recombinant protein vaccines utilize genetic engineering techniques to create immunologically active protein or peptide fragments, which serve as the vaccine's active ingredient. However, compared to attenuated shingles vaccines, inactivated and recombinant protein vaccines have lower immunogenicity and are therefore less effective.

[0007] To enhance vaccine immune activity, adjuvants have been found to play a crucial role in the prevention and treatment of herpes zoster (shingles) vaccines. Selecting an appropriate adjuvant to develop a vaccine can enhance both cellular and humoral immune responses in the target population, effectively preventing herpes zoster. Aluminum adjuvants can induce immune enhancement and help macrophages build memory immune responses. For example, Green Bamboo Bio's recombinant herpes zoster vaccine, LZ901, contains recombinant herpes zoster virus gE protein and an aluminum adjuvant per 0.5 mL dose.

[0008] To further enhance the immune activity of vaccines, new adjuvants have been gradually developed. For example, GlaxoSmithKline's Shingrix is ​​a recombinant herpes zoster vaccine (CHO cells) that uses the novel adjuvant AS01B (MPL+QS-21). CpG ODN adjuvants are a new class of immunostimulants discovered in recent years. Their chemical structure consists of oligodeoxynucleotides containing an unmethylated cytidine-phosphate-guanosine (CpG) motif. They exhibit immune responses similar to those of natural CpG pattern recognition receptors, can bind to Toll-like receptors on cell membranes, and effectively trigger mammalian immune responses through the TLR9 signaling pathway. The immune response induced by CpG ODN is primarily Th1-type, inducing a shift from a Th2-type immune response to a Th1-type response, thereby stimulating cellular immunity. By activating immune-competent cells such as T cells, B cells, and NK cells, they produce a large number of various cytokines, thereby enhancing the body's specific and nonspecific immune responses. They are a crucial link between innate and acquired immunity.

[0009] Given the limited availability of currently available shingles vaccines, there is an urgent need to develop new VZV vaccines to meet the vaccination needs of the aging population. Therefore, developing new vaccines with adjuvants or formulation combinations to improve and enhance the immune response is a very challenging strategy. Summary of the Invention

[0010] In view of this, the main purpose of the present invention is to provide a varicella-zoster vaccine composition that can further enhance the immune activity of the varicella-zoster vaccine.

[0011] To achieve the above object, the technical solution of the present invention is as follows:

[0012] A varicella-zoster vaccine composition comprises a VZV gE antigen and a CpG ODN adjuvant, wherein the nucleotide sequence of the CpG ODN adjuvant is shown in SEQ ID NO: 1.

[0013] Wherein, at least one nucleotide in the CpG ODN adjuvant is phosphorothioated, and preferably, all nucleotides are phosphorothioated.

[0014] Furthermore, in the composition, the content of the VZV gE antigen is 20-150 μg, preferably 40-100 μg.

[0015] Furthermore, in the composition, the content of the CpG ODN adjuvant is 10-3000 μg, preferably 20-2000 μg, and more preferably 50-500 μg.

[0016] It should be noted that, with respect to vaccine dosage, the dosage per dose can induce an immune protective response in the human body without significant toxic side effects. In the prior art, for varicella-zoster vaccines, the dosage per dose is generally 0.5 ml. That is, the above-mentioned contents of VZV gE antigen and CpG ODN adjuvant are based on the dosage per dose (0.5 ml). Of course, in animal experiments, such as mouse experiments, the mouse dose is generally one-tenth of the human dose (which can also be converted based on other methods, such as combining body surface area data). According to this standard mouse experiment, the dosage per dose is 0.05 ml. In this case, the content of VZV gE antigen in each dose is approximately 2-15 μg, the content of CpG ODN adjuvant is approximately 1-300 μg, and the subsequent content of aluminum adjuvant is approximately 0.03-0.05 mg.

[0017] Furthermore, the composition further comprises any one or a combination of aluminum adjuvant, MPL, liposome, and QS21.

[0018] Wherein, the aluminum adjuvant is any one or a combination of aluminum hydroxide, aluminum phosphate, and amorphous aluminum hydroxyphosphate. MPL is a type of lipopolysaccharide, which is a dephosphorylated derivative of lipopolysaccharide A (LPS). Lipopolysaccharide is a complex molecule present in the outer membrane of bacterial cells and is one of the key components that cause immune responses during bacterial infections. MPL is a modified form of LPS. By dephosphorylation, the part of the original LPS molecule that causes a strong inflammatory response is reduced, which enables MPL to retain the ability to activate the immune system while reducing its potential harmful effects. Its main function is to activate antigen-presenting cells and induce adaptive immune responses of the immune system, thereby enhancing the efficacy of the vaccine. Liposomes are tiny vesicles wrapped by a lipid bilayer. These liposomes can surround and encapsulate drugs, vaccine antigens or other active ingredients to form a structure called a liposome carrier. They can be used to deliver drugs or vaccines. In vaccine preparation, liposomes can encapsulate and carry vaccine antigens, enhance their stability, and improve the immune system's response to antigens. Liposomes can also be used as immune adjuvants to help stimulate the immune system. QS21 is a glycoside extracted from tree bark, commonly used as an immune adjuvant for vaccines. It can activate antigen-presenting cells, thereby enhancing the immune response to vaccine antigens. It also helps promote the immune system's memory response, enabling it to better cope with possible future infections. QS21 is also commonly used with other adjuvants and antigens to enhance the immunogenicity of vaccines.

[0019] Furthermore, in the composition, the content of the aluminum adjuvant is 300-500 μg, preferably 350-450 μg.

[0020] Furthermore, in the composition, the content of VZV gE antigen is preferably 80-200 μg / mL (40-100 μg), the content of CpGODN adjuvant is preferably 100-1000 μg / mL (50-500 μg), and the content of aluminum adjuvant is 600-1000 μg / mL (300-500 μg).

[0021] Furthermore, the amino acid sequence of the VZV gE antigen is selected from one of SEQ ID NOs: 2-6.

[0022] The present invention also provides a method for preparing any of the above-mentioned varicella-zoster vaccine compositions, comprising the following steps:

[0023] 1) Preparing VZV gE protein: synthesizing a gene encoding the VZV gE protein and constructing an expression vector; transfecting the constructed vector into CHO cells; creating a stable transfected cell pool; screening a cell line stably expressing the VZV gE protein from the pool; and finally expressing and purifying the protein to produce a VZV gE antigen having an amino acid sequence of any one of SEQ ID NOs: 2-6;

[0024] Among them, protein purification usually includes chromatography, centrifugation, filtration and other methods to purify VZV gE protein from the collected cell culture supernatant;

[0025] 2) Preparation of a single CpG ODN adjuvant vaccine composition: Before injection, add CpG ODN to the VZV gE protein obtained in step 11) and mix thoroughly;

[0026] 3) Preparation of a single aluminum adjuvant vaccine composition: Add aluminum adjuvant (Alum) to the VZV gE protein obtained in step 1), mix thoroughly, and incubate at 4°C overnight;

[0027] 4) Preparation of a dual-adjuvant vaccine composition: Add aluminum adjuvant (Alum) to the VZV gE protein obtained in step 1, mix thoroughly, and incubate at 4° C. overnight; then, add CpG ODN before injection and mix thoroughly.

[0028] The present invention also provides use of any of the above-mentioned varicella-zoster vaccine compositions in the preparation of a medicament for preventing and / or treating varicella-zoster virus infection and / or varicella-zoster virus-mediated diseases.

[0029] The present invention also provides use of any of the above-mentioned herpes zoster vaccine compositions in the preparation of a medicament for generating humoral and / or cellular immune responses against varicella-zoster virus in a subject.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] In the present invention, by selecting a CpG ODN adjuvant with a specific nucleotide sequence, it has better immunostimulatory activity; the CpG ODN adjuvant combined with an aluminum adjuvant dual-adjuvant vaccine can produce a higher humoral immune response earlier and induce protective immunity at least one month in advance; a single immunization can reach or approach the antibody level of two immunizations with the Shingrix vaccine, and can maintain a stable high humoral immunity level for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the test results of the effects of different doses of CpG HP007 on the immunogenicity of the herpes zoster vaccine composition in Example 2.

[0033] Figure 2 This is a graph showing the effects of different adjuvants on the immunogenicity of the herpes zoster vaccine composition in Example 3 (humoral immune response).

[0034] Figure 3 This is a graph showing the effects of different adjuvants on the immunogenicity of the herpes zoster vaccine composition in Example 3 (cellular immune response).

[0035] Figure 4A This is a graph showing the immunogenicity test results comparing the double-adjuvant herpes zoster vaccine compositions containing CpG HP007 and CpG 684 in Example 4.

[0036] Figure 4B This is a graph comparing the immunogenicity test results of the double-adjuvant herpes zoster vaccine compositions containing CpG HP007 and CpG 7909 in Example 4.

[0037] Figure 5 This is a graph showing the immunogenicity test results of different herpes zoster vaccines in Example 5. DETAILED DESCRIPTION

[0038] The following description sets forth numerous specific details to facilitate a thorough understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific implementations disclosed below.

[0039] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] General Technical and Definitional Notes

[0041] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0042] As used herein, the phrase "comprising" is open ended, indicating that such embodiments may include additional elements. In contrast, the phrase "consisting of is closed ended, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of is partially closed ended, indicating that such embodiments may further include elements that do not materially change the basic characteristics of such embodiments.

[0043] As used herein, the term "about" with respect to a value encompasses 90% to 110% of that value (eg, about 100 μg of HP007 refers to 90 μg to 110 μg of HP007).

[0044] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to achieve a beneficial or desired result, including a clinical result, and thus, an "effective amount" depends on the context in which it is used. In the context of administering an immunogenic composition, an effective amount contains enough antigen and TLR9 agonist to stimulate an immune response (preferably a seroprotective level of antibody to the antigen).

[0045] The terms "individual" and "subject" refer to mammals. "Mammals" include, but are not limited to, humans, non-human primates (e.g., monkeys), livestock, sports animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0046] The term "dose" as used herein with respect to a vaccine composition refers to the measured portion of the vaccine composition that a subject takes (administered or receives) at any one time.

[0047] As used herein, the terms "isolated" and "purified" refer to material that is removed from at least one component with which the material is naturally associated (e.g., removed from its original environment). When used with recombinant proteins, the term "isolated" refers to a protein that has been removed from the culture medium of the host cells in which the protein was produced.

[0048] As used herein, the term "vaccine" refers to any biological product, including proteins, polysaccharides, nucleic acids, live vectors, or infectious agents, that, after injection or mucosal administration, can induce the body to produce specific antibodies and / or cellular immunity against a specific pathogen, thereby protecting the body from or eliminating the pathogen. Vaccines are autoimmune preparations for preventing infectious diseases, made by artificially attenuating, inactivating, or genetically engineering pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolites. Vaccines retain the pathogen's ability to stimulate the animal's immune system. When an animal comes into contact with a harmless pathogen, its immune system produces certain protective substances, such as immune hormones, active physiological substances, and specific antibodies. When the animal is exposed to the pathogen again, its immune system, relying on its existing memory, produces more protective substances to prevent the pathogen from causing harm.

[0049] As used herein, the term "immunization" refers to the process of increasing a mammalian subject's response to an antigen and thereby improving its ability to resist or overcome infection and / or resist disease.

[0050] The term "vaccination" as used herein refers to the introduction of a vaccine into the body of a mammalian subject.

[0051] "Adjuvant" refers to a substance that, when added to a composition comprising an antigen, enhances or strengthens the immune response of a mammalian recipient to the antigen upon exposure. Adjuvants include, but are not limited to, alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles such as poly(lactide-co-glycolide) microparticles.

[0052] As used interchangeably herein, the terms "polynucleotide" and "oligonucleotide" are molecules formed by linking mononucleotides composed of sugars (e.g., deoxyribose or ribose), phosphate groups, and bases. The sugars and bases are linked to form nucleosides, which are linked by phosphate groups to form nucleotides. The bases forming nucleosides include pyrimidines, which include thymine (abbreviated as T or t), cytosine (abbreviated as C or c), uracil (abbreviated as U or c), or their derivatives, and purines, which include adenine (abbreviated as A or a), guanine (abbreviated as G or g), or their derivatives. "Polynucleotide" and "oligonucleotide" include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), modified oligonucleotides and oligonucleosides, or combinations thereof. The configuration of an oligonucleotide can be linear or circular, or an oligonucleotide can contain linear and circular segments. Oligonucleotides are polymers of nucleosides typically linked by phosphodiester bonds, although alternative linkages, such as phosphorothioate, can also be used in oligonucleotides.

[0053] As used herein, the terms "CpG," "CpG motif," and "cytosine-phosphate-guanosine" refer to unmethylated cytidine-phosphate-guanosine dinucleotides that, when present in an oligonucleotide, contribute to a measurable immune response in vitro, in vivo, and / or ex vivo. Examples of measurable immune responses include, but are not limited to, antigen-specific antibody production, cytokine secretion, lymphocyte populations (e.g., NK cells, CD4 + T lymphocytes, CD8 + Preferably, the CpG oligonucleotide preferentially activates a Th1 type response.

[0054] As used herein, the term "CpG ODN" refers to a synthetic CpG-containing oligodeoxynucleotide that can enhance antibody production and stimulate T helper 1 (Th1) cell responses.

[0055] The present invention is described in detail below through specific examples.

[0056] In the following examples, the materials used are from the following sources:

[0057] PBS phosphate buffered saline dry powder was purchased from Solarbio; CpG HP007, CpG 684, and CpG 7909 were provided by Jiangsu Taipuri Biotechnology Co., Ltd.

[0058] Aluminum hydroxide adjuvant was purchased from Thermo;

[0059] Herpes zoster virus gE protein was purchased from SinoBiological;

[0060] BALB / c mice were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd.

[0061] The nucleotide sequence of CpG HP007 is shown in SEQ ID NO: 1, specifically: 5'-tcgcgaacgttcgccgcgtacgtacgcgg-3';

[0062] The nucleotide sequence of CpG 684 is shown in SEQ ID NO: 7, specifically: 5′-tcgacgttcgtcgttcgtcgtcgttc-3′;

[0063] The nucleotide sequence of CpG 7909 is shown in SEQ ID NO: 8, specifically: 5'-tcgtcgttttgtcgttttgtcgtt-3'.

[0064] Example 1 Preparation of Herpes Zoster Vaccine Composition

[0065] To investigate the technical effects of the herpes zoster vaccine compositions provided by the present invention, the following multiple herpes zoster vaccine compositions were prepared in this example, each containing VZV gE protein, aluminum adjuvant, and / or CpG ODN adjuvant. The specific preparation methods are as follows:

[0066] 1. Preparation of VZV gE protein (CHO cell expression), including the following steps: (1) molecular cloning: gene synthesis, expression vector construction; (2) establishment of expression cell lines: stable cell pool, monoclonal cell line; (3) protein expression and purification. The five VZV gE proteins prepared are:

[0067] SEQ ID NO: 2

[0068] (MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLIRYAAWTGGLA)、

[0069] SEQ ID NO:3

[0070] (MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAA)、

[0071] SEQ ID NO:4

[0072] (MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHF)、

[0073] SEQ ID NO:5

[0074] (MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHV)、

[0075] SEQ ID NO:6

[0076] ().

[0077] 2. Preparation of a single CpG ODN adjuvant vaccine composition: Before injection, add CpG ODN to the VZVgE protein obtained in step 1 and mix thoroughly.

[0078] 3. Prepare a single aluminum adjuvant vaccine composition: Add aluminum adjuvant (Alum) to the VZV gE protein obtained in step 1, mix thoroughly, and incubate at 4°C overnight.

[0079] 4. Prepare a multi-adjuvant vaccine composition: Add aluminum adjuvant (Alum) to the VZV gE protein obtained in step 1, mix thoroughly, and incubate at 4°C overnight. Then, add CpG ODN before injection and mix thoroughly.

[0080] Example 2 Effect of CpG HP007 Dosage on the Immunogenicity of Herpes Zoster Vaccine Compositions in Mice

[0081] In order to evaluate the effect of the CpG HP007 dose on the immunogenicity of the herpes zoster vaccine composition prepared in Example 1, this example conducted an immunogenicity study using C57BL / 6J mice as an animal model. The immunogenicity evaluation method is as follows:

[0082] 1. Animal experiment design

[0083] Eight-week-old female C57BL / 6J mice were randomly divided into seven groups of 12 mice each. Each group of mice was immunized once on days 0 and 28 with different herpes zoster vaccine compositions via intramuscular injection in a 50 μL injection volume. Orbital blood was drawn on days 21, 28 (before the second immunization), and 31, with 200-300 μL of blood drawn from each mouse. The serum was separated and assayed for binding antibody titers using ELISA. The grouping of mice in this example is shown in Table 1.

[0084] Table 1: Mouse grouping

[0085] Grouping Vaccine composition and dosage 1 PBS 2 5 μg gE 3 5μg gE+1μg HP007 4 5μg gE+8μg HP007 5 5μg gE+24μg HP007 6 5μg gE+72μg HP007 7 5μg gE+8μg HP007+40μg Alum

[0086] Wherein, gE refers to the sequence prepared in Example 1, such as the VZV gE antigen described in SEQ ID NO: 2; HP007 refers to the purchased sequence, such as CpG HP007 described in SEQ ID NO: 1.

[0087] 2. Binding antibody titer detection

[0088] The gE protein-specific antibody titers in the sera of mice on D21, D28 (before the second immunization), and D31 were determined by ELISA.

[0089] (1) Assay method: The antigen gE protein (CHO) is coated on an ELISA plate to capture anti-gE protein-specific antibodies in mouse serum. Unbound sites are blocked with incubation buffer, and diluted mouse serum, a blank control, and a standard are added. Then, a goat anti-mouse IgG secondary antibody labeled with horseradish peroxidase (HRP) is added to bind to the antibodies in the mouse serum to be tested. A color development solution is added to develop the color, and a stop solution is added to terminate the reaction. The color reaction is observed, and the A450 value is measured using an ELISA reader.

[0090] (2) Result determination: Cut-off value (COV) = blank control group OD value × 2.1. Antibody titer is defined as the highest serum dilution that results in an absorbance value greater than the cut-off value. Antibody titer is expressed as geometric mean titer (GMT).

[0091] (3) Test results such as Figure 1 shown.

[0092] Depend on Figure 1CpG HP007 enhanced the immunogenicity of the antigen in a dose-dependent manner within the 1-72 μg dose range. Furthermore, the CpG HP007+Alum dual-adjuvant vaccine significantly enhanced VZV gE-specific antibody titers (more than 100-fold) compared to the CpG HP007 vaccine alone.

[0093] Example 3 Comparison of immunogenicity of herpes zoster vaccine compositions with different adjuvants in mice

[0094] In order to evaluate the effects of different adjuvants on the immunogenicity of herpes zoster vaccine compositions, this example conducted an immunogenicity study using C57BL / 6J mice as an animal model. The immunogenicity evaluation method is as follows:

[0095] 1. Animal experiment design

[0096] 8-week-old female C57BL / 6J mice were randomly divided into 6 groups of 12 mice per group. At D0 and D28, each group of mice was immunized once, and different herpes zoster vaccine compositions were immunized by intramuscular injection with an injection volume of 50 μL. Orbital blood was collected at D21, D28 (before the second immunization), and D31, and 200-300 μL of blood was collected from each mouse at each time. The serum was separated and the binding antibody titer was detected by ELISA. At D35, some mice in the 1st, 3rd, 4th, and 6th groups were euthanized, spleen tissue was taken, and spleen lymphocytes were separated and subjected to ELISPOT analysis. The grouping of mice in the present embodiment is shown in Table 2.

[0097] Table 2: Mouse grouping

[0098] Grouping Vaccine composition and dosage 1 PBS 2 5 μg gE 3 5μg gE+8μg HP007 4 Shingrix 5 5μg gE+MF59 6 5μg gE+8μg HP007+40μg Alum

[0099] Among them, in Group 4, each mouse was vaccinated with GSK's Shingrix containing 5 μg of gE protein + 50 μL of AS01B; except Shingrix, which is a commercially available shingles vaccine, the VZV gE proteins in the other groups were all self-produced gE proteins, that is, the sequence prepared in Example 1, taking the VZV gE antigen described in SEQ ID NO: 3 as an example; HP007 represents the purchased sequence such as CpG HP007 described in SEQ ID NO: 1; Shingrix is ​​a recombinant shingles vaccine using the new adjuvant AS01B (MPL + QS-21) in the prior art; MF59 is also a commonly used vaccine adjuvant in the prior art, and its main components include an oil phase, an aqueous phase and a surfactant.

[0100] 2. Binding antibody titer detection

[0101] The gE protein-specific antibody titers in the sera of mice on D21, D28 (before the second immunization), and D31 were determined by ELISA.

[0102] The determination method and result determination are as described in Example 2.

[0103] Test results such as Figure 2 shown.

[0104] Depend on Figure 2 As shown, antibody titers in the CpG HP007+Alum dual-adjuvant vaccine group increased more than 10-fold compared to Shingrix 21 and 28 days after the first immunization (D21 and D28). Antibody titers in the CpG HP007+Alum dual-adjuvant vaccine group remained at high levels across the three serum samples, with no significant changes.

[0105] This suggests that the CpG HP007+Alum dual-adjuvant vaccine can generate a high humoral immune response early, inducing protective immunity at least one month in advance. Furthermore, a single immunization with the CpG HP007+Alum dual-adjuvant vaccine can achieve antibody levels comparable to or approaching those achieved with two immunizations with the Shingrix vaccine, and can maintain high and stable humoral immunity levels for an extended period.

[0106] 3. Cellular immune response detection

[0107] The gE-specific T cells in the mouse spleen were determined by ELISPOT. Specifically, the number of IFN-γ secreting cells in the mouse spleen was determined using IFN-γ ELISPOT (IFN-γ ELISPOT kit, BD Bioscience).

[0108] (1) Assay method: spleen cells (3×10 5 Cells / well) were seeded in a 96-well plate, gE protein was added at a final concentration of 5 μg / mL, and culture medium was used as a negative control. The cells were then incubated at 37°C and 5% CO2 for 24 hours. The incubation solution was discarded, and after washing, the detection antibody (biotin-labeled anti-IFN-γ antibody) was added and incubated at room temperature for 2 hours. After washing, Streptavidin-HRP was added and incubated at room temperature for 1 hour. After washing, substrate was added for color development. The reaction was terminated with deionized water and dried. The spots were counted using an ELISPOT reader.

[0109] (2) Test results such as Figure 3 shown.

[0110] Figure 3 The results of ELISOT assay of D35 are shown. Figure 3 It can be seen that the gE-specific cellular immunity level in the HP007+Alum dual-adjuvant vaccine group was significantly higher than that in the Shingrix group and the HP007 single-adjuvant vaccine group (P value < 0.01).

[0111] Conclusion: HP007+Alum dual-adjuvant vaccine can produce stronger cellular immune effect, which is significantly better than Shingrix.

[0112] Example 4 Comparison of the effects of different CpGs on the immunogenicity of herpes zoster vaccines

[0113] In order to evaluate the effects of different adjuvants on the immunogenicity of herpes zoster vaccine compositions, this example conducted an immunogenicity study using BALB / c mice as an animal model. The immunogenicity evaluation method is as follows:

[0114] 1. Animal experiment design

[0115] Eight-week-old BALB / c mice were randomly divided into nine groups of eight mice each. Each group was immunized intramuscularly on day 0, with 0.1 mL injected intramuscularly. Blood was collected from the tail vein on day 7, and serum was isolated. Anti-gE protein titers in the serum were measured by ELISA, and assessed by measuring OD450 values, using the same method as above. The specific groupings for this example are shown in Table 3.

[0116] Table 3: Mouse grouping

[0117] Grouping Vaccine composition and dosage 1 PBS 2 10 μg gE 3 <![CDATA[10μg gE+40μg Al(OH)3]]> 4 <![CDATA[2.5μg CpG 684+10μg gE+40μg Al(OH)3]]> 5 <![CDATA[5μg CpG 684+10μg gE+40μg Al(OH)3]]> 6 <![CDATA[2.5μg CpG 7909+10μg gE+40μg Al(OH)3]]> 7 <![CDATA[5μg CpG 7909+10μg gE+40μg Al(OH)3]]> 8 <![CDATA[2.5μg CpG HP007+10μg gE+40μg Al(OH)3]]> 9 <![CDATA[5μg CpG HP007+10μg gE+40μg Al(OH)3]]>

[0118] Among them, gE is also the sequence prepared in Example 1, taking the VZV gE antigen described in SEQ ID NO: 4 as an example; the sequence of CpG 684 is shown in SEQ ID NO: 7; the sequence of CpG 7909 is shown in SEQ ID NO: 8; and the sequence of CpG HP007 is shown in SEQ ID NO: 1.

[0119] 2. Test results such as Figure 4A and Figure 4B shown.

[0120] Depend on Figure 4A The results showed that the dual-adjuvant vaccine group significantly increased anti-gE protein titers compared to the single-aluminum adjuvant vaccine group. Furthermore, the antibody titers in the CpG HP007 + aluminum hydroxide dual-adjuvant vaccine group were higher than those in the CpG 684 + aluminum hydroxide dual-adjuvant vaccine group, indicating that CpG HP007 in the shingles vaccine has better immunostimulatory activity than CpG 684.

[0121] Depend on Figure 4BThe results showed that the dual-adjuvant vaccine group significantly increased anti-gE protein titers compared to the single-aluminum adjuvant vaccine group. Furthermore, the antibody titers in the CpG HP007 + aluminum hydroxide dual-adjuvant vaccine group were higher than those in the CpG 7909 + aluminum hydroxide dual-adjuvant vaccine group, indicating that CpG HP007 in the shingles vaccine has better immunostimulatory activity than CpG 7909.

[0122] Example 5 Comparison of the Immunogenicity of Different Herpes Zoster Vaccines

[0123] To compare the immunogenicity of different herpes zoster vaccine compositions, this study used C57BL / 6J mice as an animal model to conduct immunogenicity studies. The immunogenicity evaluation methods are as follows:

[0124] 1. Animal experiment design

[0125] Eight-week-old female C57BL / 6J mice were randomly divided into five groups of 12 mice each. Each group of mice was immunized once on days 0 and 28 by intramuscular injection with different herpes zoster vaccine compositions in a 50 μL injection volume. Orbital blood was collected on days 21, 28 (before the second immunization), 31, and 56, with 200-300 μL of blood drawn from each mouse. The serum was separated and tested for binding antibody titers using ELISA. The grouping of mice in this example is shown in Table 4.

[0126] Table 4: Mouse grouping

[0127]

[0128]

[0129] Wherein, gE is also the sequence prepared in Example 1, taking the VZV gE antigen described in SEQ ID NO: 5 as an example.

[0130] 2. Binding antibody titer detection

[0131] The gE protein-specific antibody titers in the sera of mice on D21, D28 (before the second immunization), D31, and D56 were determined by ELISA.

[0132] The determination method and result determination are as described in Example 2.

[0133] Test results such as Figure 5 shown.

[0134] Depend on Figure 5It can be seen that the immunogenicity of the 5μg gE+40μg Alum+8μg CpG HP007 vaccine group is stronger than that of the 10μg gE+40μg Alum+5μg CpG 684 vaccine group, proving the superiority of the former vaccine composition in components and ratios.

[0135] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A varicella zoster vaccine composition, characterized in that, it comprises VZV gE antigen and CpG ODN adjuvant, and the nucleotide sequence of the CpG ODN adjuvant is as shown in SEQ ID NO:

1.

2. The varicella zoster vaccine composition according to claim 1, characterized in that, in the composition, the content of the VZV gE antigen is 20 - 150 μg.

3. The varicella zoster vaccine composition according to claim 1, characterized in that, in the composition, the content of the CpG ODN adjuvant is 10 - 3000 μg.

4. The varicella zoster vaccine composition according to claim 1, characterized in that, it further comprises any one or a combination of several of aluminum adjuvant, MPL, liposome, QS21.

5. The varicella zoster vaccine composition according to claim 4, characterized in that, in the composition, the content of the aluminum adjuvant is 300 - 500 μg.

6. The varicella zoster vaccine composition according to claim 4, characterized in that, in the composition, the aluminum adjuvant is any one or a combination of several of aluminum hydroxide, aluminum phosphate, amorphous hydroxyaluminum phosphate.

7. The varicella zoster vaccine composition according to claim 1, characterized in that, the amino acid sequence of the VZV gE antigen is selected from one of SEQ ID NO: 2 - 6.

8. Use of the varicella zoster vaccine composition according to any one of claims 1 - 7 in the preparation of a medicament for preventing and / or treating varicella - zoster virus infection and / or varicella - zoster virus - mediated diseases.

9. Use of the varicella zoster vaccine composition according to any one of claims 1 - 7 in the preparation of a medicament for generating a humoral immune and / or cellular immune response against varicella - zoster virus in a subject to be treated.

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