WO2020259609A1 - 一种重组带状疱疹疫苗组合物及其用途 - Google Patents

一种重组带状疱疹疫苗组合物及其用途 Download PDF

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WO2020259609A1
WO2020259609A1 PCT/CN2020/098179 CN2020098179W WO2020259609A1 WO 2020259609 A1 WO2020259609 A1 WO 2020259609A1 CN 2020098179 W CN2020098179 W CN 2020098179W WO 2020259609 A1 WO2020259609 A1 WO 2020259609A1
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vaccine composition
adjuvant
protein
vaccine
content
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PCT/CN2020/098179
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French (fr)
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周晨亮
王紫琰
刘海涛
刁美君
魏健
刘革
史力
莫呈钧
张智
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怡道生物科技(苏州)有限公司
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Priority to AU2020308378A priority Critical patent/AU2020308378A1/en
Priority to US17/623,525 priority patent/US20220347293A1/en
Publication of WO2020259609A1 publication Critical patent/WO2020259609A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A61K39/25—Varicella-zoster virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16741—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16743—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention belongs to the field of biomedicine. Specifically, the present invention relates to a recombinant shingles vaccine composition and use.
  • VZV Varicella zoster virus
  • HZ herpes zoster
  • Chickenpox is usually seen in childhood, while herpes zoster does not develop until adulthood. After the primary infection with chickenpox, the virus can be latent in the host's ganglia. With age, immune function impairment or immunosuppression, VZV can be reactivated and cause shingles.
  • herpes zoster The clinical manifestation of herpes zoster is a unilateral vesicular rash.
  • the obvious feature is that it is limited to a single skin segment, usually accompanied by radicular pain.
  • Patients may have obvious pain and discomfort. Symptoms can last for several weeks or months. In severely ill patients, they can even last for several years, resulting in a decline in the quality of life. In rare cases, shingles may not appear as a rash. Complications occur in about 25% of people with shingles, and they increase with age.
  • the most common serious complication is post-herpetic neuralgia (PHN), that is, pain that persists after the acute phase of herpes.
  • PPN post-herpetic neuralgia
  • the incidence of herpes zoster patients is 10%-30%. The pain can be It lasts for several months or even years, seriously affecting the quality of life of patients.
  • the risk factors that affect the onset of shingles are age, immune deficiency, gender
  • VZV Most primary VZV infections occur in childhood, then VZV is latent in the ganglia, and VZV can be reactivated in adulthood. Studies have shown that about 99% of Americans aged 40 and over have serological evidence of VZV infection; 90% of Europeans aged 20-29 years are seropositive for anti-VZV; in some countries in South America, Australia and Asia The primary VZV infection may occur later, but 90% of people over 40 years old will have VZV seropositivity. Therefore, on a global scale, most adults are at risk of developing shingles and its related complications.
  • the global incidence of herpes zoster is (3 ⁇ 5)/1000 person-years, and the Asia- Pacific region is (3 ⁇ 10)/1000 person-years, and it is increasing by 2.5% ⁇ 5.0% year by year, and the hospitalization rate is (2 ⁇ 25)/100,000
  • the mortality rate is (0.017 ⁇ 0.465) per 100,000 person-years, and the recurrence rate is 1% ⁇ 6%.
  • my country is in a state of high-degree aging, and the social and economic burden brought by HZ is increasing year by year.
  • HZ has brought a huge negative impact on the quality of life of patients, especially elderly patients.
  • According to data released by the National Bureau of Statistics it is estimated that the population over 40 years old in 2017 was about 650 million. If the HZ incidence rate is 2.5/1000 person-years, it is conservatively estimated that there are about 1.6 million new cases of HZ in my country each year.
  • HZ live attenuated vaccine Zostavax and HZ subunit vaccine Shingrix are on the market worldwide (HZ live attenuated vaccine Zostavax and HZ subunit vaccine Shingrix).
  • Merck’s Zostavax is a live attenuated vaccine, which contains the same virus strain as the VZV Oka strain used in the varicella vaccine.
  • the vaccine formulation uses a minimum potency of 19400 PFU. It was approved by the FDA in 2006 and has been approved in more than 60 countries.
  • One dose can be subcutaneously administered to people over 50 years of age.
  • Shingrix developed by GlaxoSmithKline is a subunit vaccine based on recombinant gE protein supplemented with a new adjuvant AS01 B.
  • VZV-specific immune response is essential to reduce the incidence of herpes zoster and post-herpetic neuralgia.
  • Shingrix developed by GlaxoSmithKline is a subunit vaccine based on the new adjuvant AS01 B , which has been proven to be effective To prevent herpes zoster and its neuralgia, this new adjuvant plays a key role in this vaccine. Therefore, choosing a suitable adjuvant to develop a vaccine can improve the cellular and humoral immune response in the target population, which can effectively prevent herpes zoster.
  • the AS01 B adjuvant system of GlaxoSmithKline contains liposomes, MPL and QS-21, which can cause a strong cellular immune response.
  • CpG ODN can be produced on a large scale by chemical synthesis, which is easy for quality control.
  • CpG ODN is an agonist of Toll-like receptor 9 (TLR9), which can stimulate cells expressing TLR9 and activate downstream natural immune response pathways. On the one hand, it induces the expression of type I interferon and inflammatory factors. Make plasmacytoid dendritic cells mature, thereby enhancing humoral immunity and cellular immune response.
  • TLR9 Toll-like receptor 9
  • Aluminum adjuvant is by far the most widely used human vaccine adjuvant, and the combined adjuvant composed of other adjuvants also has a wide range of applications.
  • the second phase clinical trials of hepatitis B and malaria preventive vaccines have all used aluminum adjuvant. And CpG 7909 combined adjuvant.
  • One aspect of the present invention is to provide a recombinant herpes zoster preventive vaccine, which contains an antigen against varicella-zoster virus (VZV). More specifically, the above-mentioned antigen may be the gE protein of the VZV virus.
  • VZV varicella-zoster virus
  • the VZV described in the present invention is a double-stranded DNA virus with only one serotype.
  • the genome is about 125,000 bp in length, encoding 71 genes, and finally expressing 67 different proteins, including 7 glycoproteins (gp I ⁇ gp VI). ), respectively named gE, gB, gH, gI, gC, gK and gL.
  • Glycoprotein E (Glycoprotein E, gE protein) is the main surface structural protein of VZV and the most important neutralizing antigen, with high conservation Sex.
  • the VZV-gE glycoprotein is encoded by ORF68 and consists of 623 amino acids. It is a type I transmembrane glycoprotein.
  • VZV-gE The gE glycoprotein is most abundantly expressed on the surface of viruses and infected cells, and has both T and B cell epitopes. Therefore, the research on VZV-gE is the most extensive, and it is expected to become the antigen in a new generation of subunit varicella or shingles vaccines. .
  • VZV gE protein shows that the protein molecule of wild-type VZV gE includes four regions, namely signal peptide, extracellular region, transmembrane region, and intracellular region.
  • the three antigenic determinants of VZV gE are all distributed In the extracellular region (for example, you can refer to the literature Grose C. Glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation, and intracellular trafficking. Annu Rev Microbil. 1990, 44: 59-80.).
  • gE protein sequences for example, Sequence IDs Q9J3M8.1, AQT34120.1, AGY33616.1, AEW88548.1 amino acid sequences
  • the inventors of the present invention retrieved 25 full-length sequences of VZV gE protein from NCBI, which were derived from different virus strains.
  • the results of amino acid sequence analysis and comparison showed that the protein sequences of 7 virus strains were identical (AAK19946 .1, AHB80298.1, Q9J3M8.1, ABE67176.1, AGL51024.1, AFO85645.1, ABF22079.1), and each point is the most conservative. Therefore, these sequences can be used as ideal basis for gE antigen protein selection.
  • the modification methods for gE protein are also mature.
  • the transmembrane region (hydrophobic region) and intracellular are generally removed. Area (for example patent CN102517302A).
  • the signal peptide region is usually cut by signal peptidase (for example, patents CN102548578A, CN102711812B, and documents GSK References).
  • the transmembrane region, intracellular region and signal peptide can be predicted by related application software, such as SignalP (http://www.cbs.dtu.dk/services/SignalP/)
  • SignalP http://www.cbs.dtu.dk/services/SignalP/
  • TMHMM Server V.2.0 software For predictive analysis of whether the protein has a signal peptide, use TMHMM Server V.2.0 software to predict the transmembrane region of the protein, and use PSORT software to determine the accuracy of the secreted signal peptide and whether the signal peptide cleavage site can be identified and cleaved.
  • a typical method includes: expressing the above-mentioned gE protein in CHO cells, which includes the following steps:
  • step (3) Use the cell line obtained in step (3) for expression to obtain VZV gE protein.
  • an antigen protein with a purity of more than 95% can be obtained.
  • the method of using CHO cell lines to stably express the VZV gE recombinant protein is a well-known method in the art.
  • Molecular Cloning Test Guide and other documents, such as Haumont M, et al., Virus Research 40 (1996 ), 199-204, Purification, characterization and immunogenicity of recombinant varicella-zoster virus glycoprotein gE secreted by Chinese hamster ovary cells.
  • Those skilled in the art can also choose other expression methods such as Escherichia coli, Pichia yeast, etc. to obtain gE protein (for example, refer to patent CN108315344A or CN107022559A for further introduction on gE protein preparation).
  • the vaccine composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is an aluminum adjuvant combined with a CpG ODN adjuvant.
  • the aluminum adjuvant of the present invention is currently the most widely used type of adjuvant in vaccines. It has a history of more than 80 years of application and a record of tens of billions of people. It was once approved by the U.S. Food and Drug Administration (FDA). The only adjuvant approved for use in human vaccines. There have been many studies on the use of aluminum adjuvants in vaccines.
  • CpG ODN adjuvant is a class of immunostimulatory oligopeptides known to have adjuvant properties, which can activate B cells, NK, DC dendritic cells and induce the release of IL-12 and IFN- ⁇ , thereby inducing a strong Th1-type response And cellular immunity.
  • This kind of adjuvant is also well known to those skilled in the art, and has been described in the prior art, for example, in international applications such as WO 96/02555 and WO 99/33488.
  • CpG ODN adjuvants such as CpG 7909 (Coley) or CpG 1018 (Dynavax).
  • CpG 7909 Coley
  • CpG 1018 Dynavax
  • the preparation method provided by the present invention is to mix the aforementioned antigen protein of the present invention with the aforementioned pharmaceutically acceptable carrier.
  • the gE protein should best be adsorbed on the aluminum adjuvant.
  • CpG ODN adjuvant is also best adsorbed on aluminum adjuvant.
  • the vaccine preparation provided by the present invention has an antigen protein content between about 10-100 ⁇ g, more preferably between 50-100 ⁇ g, and an aluminum adjuvant content between about 100-600 ⁇ g, and more preferably, the content Between 225-600 ⁇ g, the content of CpG ODN adjuvant is about 50-1200 ⁇ g, more preferably, between 300-900 ⁇ g.
  • the use of the aforementioned vaccine composition is provided to prevent or treat diseases or disorders related to herpes zoster virus infection.
  • the third aspect of the present invention is to provide a kit containing a vaccine administration device, including but not limited to a needle device, a liquid injection device, a powder device and a spray device.
  • the selection of the device is mainly based on different modes of administration. Common modes of administration include intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, or oral/digestive tract, respiratory tract and urogenital mucosal administration.
  • the vaccine of the present invention can be injected intramuscularly, and the common delivery device is a needle device. Usually, it can be administered as a single dose, and its components can also be administered in combination at the same time or at different times.
  • the inventors of the present invention surprisingly discovered that the combination of the above two adjuvants and gE protein will show surprisingly high immunological activity. Therefore, the vaccine composition of the present invention becomes a new generation of varicella-zoster vaccine.
  • Example 1 Preparation of vaccine composition containing VZV gE antigen protein
  • the inventors of the present invention have prepared the following vaccine composition preparations (0.5ml/dose), each of which contains VZV gE protein, aluminum adjuvant and CpG ODN adjuvant.
  • the specific preparation method is as follows: first adsorb the gE antigen stock solution onto an aluminum adjuvant (aluminum phosphate adjuvant) to prepare adsorbed samples with different ratios of gE antigen/aluminum adjuvant (w/w) (here, the aluminum adjuvant) The content is essentially aluminum content); then CpG ODN samples (CpG 7909) of different concentrations are added to the gE antigen/aluminum adsorption sample. After the above formulations are fully mixed, if they are not applied immediately, they should be stored at 4°C.
  • the specific ratios are as follows:
  • Example 1 For obtaining the recombinant shingles vaccine composition to be evaluated in Example 1.
  • the inventors carried out immunogenicity studies using C57BL/6 mice as animal models.
  • the immunogenicity of the vaccine composition provided by the present invention is studied by using gE protein as the antigen and aluminum adjuvant and CpG ODN as adjuvants. 6-8 weeks old C57BL/6 mice were randomly divided into groups, 10 mice in each group, the vaccine prepared by intramuscular injection of gE protein combined with adjuvant, the vaccine group and the adjuvant group were set up, 0,3 weeks of immunization, and the fifth week of blood sampling Take the spleen.
  • the ELISA method was used to detect the anti-VZV gE protein binding antibody titer in the serum, namely total IgG, and the ELISPOT method was used to detect the cellular immunity level in spleen cells, mainly the expression of IFN- ⁇ .
  • the invention of this application has also undergone parallel experiments with the same ratio to verify the effect of the vaccine composition composed of 1018 in CpG ODN, VZV gE protein, and aluminum adjuvant. , Other parameters and test procedures remain unchanged.
  • the final data shows that its IgG (GMT) value is basically the same as when CpG 7909 is used, and its IFN- ⁇ value is slightly lower than CpG 1018, but there is no significant difference.
  • IgG (GMT) value is basically the same as when CpG 7909 is used, and its IFN- ⁇ value is slightly lower than CpG 1018, but there is no significant difference.
  • the immunogenicity evaluation method is a professional technical method in the field.
  • the more specific operation method is as follows:
  • mice aged 6-8 weeks were selected to be randomly divided into groups, with 10 mice in each group.
  • Vaccines of different doses were injected intraperitoneally (Table 1) with an injection volume of 0.05ml; 0 and 3 weeks of immunization, blood was collected from the spleen at week 5, serum was separated for ELISA to detect binding antibody titer, splenic lymphocytes were separated for ELISPOT analysis.
  • Serum was collected 2 weeks after the second immunization of mice, and the antibody titer of anti-gE protein binding was detected.
  • the antigen gE stock solution is diluted with PBS to 1 ⁇ g/ml, and 100 ⁇ l of the diluted stock solution is added to each well of the ELISA plate. 4°C overnight. Washing machine.
  • Coated ELISPOT plate (aseptic operation, performed one day before taking the spleen)
  • mice were sacrificed and immersed in 75% ethanol. Take out the mouse spleen in a clean bench. Put a burned 200-mesh copper mesh in a 35mm petri dish, add 1ml of lymphocyte separation solution, and grind with the plunger of a 1ml syringe. After filtering the suspension with spleen cells through a burned 200-mesh copper mesh, transfer to a 15ml centrifuge tube, add the lymphocyte separation solution to 4ml, and cover the surface with 0.5ml of RPMI1640 basic medium. Room temperature, 800g, speed 3, centrifugation for 30 minutes. Aspirate the lymphocyte layer, add 10ml RPMI1640 basal medium, wash, and centrifuge at 250g at room temperature for 10 minutes. Discard the supernatant, add 2ml RPMI1640 complete medium to resuspend the cells, and count.
  • Add cells Dilute the cells with complete medium to 6 ⁇ 10 6 /ml according to the results of the cell count, and add mAb CD28-A to the cell suspension diluted 1000 times. Add 100 ⁇ l/well to the ELISPOT plate. Positive control: 1 ⁇ l ConA stimulant was added, and the stimulation concentration was 5 ⁇ g/ml. Sample to be tested: add stimulus gE protein peptide library diluted with serum-free medium, final concentration 2 ⁇ g/ml; negative control: no ConA stimulus, nor stimulus short peptide. Incubate at 37°C with 5% CO 2 for 24 hours, during which time the culture plate cannot be moved to avoid changes in cell position and blur of ELISPOT spots.
  • Discard the cell suspension add 200 ⁇ l/well of sterile PBS and wash the plate 5 times. Take 50 ⁇ l of biotin-labeled detection antibody and add it to 10 ml of diluent (PBS+0.1% BSA), mix well, and filter with 0.2 ⁇ m filter. Add 100 ⁇ l to each well and incubate at 37°C for 2 hours. Discard the diluent of the biotin-labeled detection antibody, add 200 ⁇ l/well of sterile PBS and wash the plate 5 times. Dilute the antibody with diluent (PBS+0.1% BSA), take 50 ⁇ l and add 10ml of diluent, mix well, and filter with 0.2 ⁇ m filter.
  • the vaccine composition prepared according to the specific components and proportions provided by the present invention has good immune activity, especially when the content of gE protein is between 50-100 ⁇ g, aluminum adjuvant
  • the content of CpG ODN adjuvant is between 225-600 ⁇ g
  • the content of CpG ODN adjuvant is between 300-900 ⁇ g

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Abstract

本发明公开了一种重组带状疱疹疫苗组合物及其用途。本发明提供的新型疫苗组合物，相较其他抗原及佐剂的组合，具有更有益的免疫效果。

Description

一种重组带状疱疹疫苗组合物及其用途 技术领域
本发明属于生物医药领域，具体而言，本发明涉及一种重组带状疱疹疫苗组合物和用途。
背景技术
水痘带状疱疹病毒(varicella zoster virus，VZV)是八种人类疱疹病毒之一，也叫做3型人疱疹病毒。水痘-带状疱疹病毒遍布全球，具有很强的传染性，迄今只发现一种血清型，在自然界VZV仅感染人类。既可引发水痘，也可引发带状疱疹(herpes zoster，HZ)，水痘通常见于儿童期，带状疱症则在成年以后才会发病。在原发感染水痘后，病毒可潜伏在宿主的神经节中，随着年龄增长、免疫功能受损或免疫抑制，VZV可以被重新激活并引发带状疱疹。带状疱疹临床表现为单侧性水泡样皮疹，明显的特征是仅局限于单一皮肤节段，通常伴神经根性疼痛。患者可有明显疼痛和不适，症状可持续数周或数月，在重症患者中甚至可持续数年，导致生活质量下降，极少数情况下带状疱疹也可无皮疹出现。约25％患带状疱疹的人会发生并发症，并且随着年龄的增长而增加。最常见的严重并发症是带状疱疹后神经痛(post-herpetic neuralgia，PHN)，即疱疹急性期后仍持续存在的疼痛，在带状疱疹患者中发生率为10％-30％，疼痛可持续数月甚至数年，严重影响患者生活质量。影响带状疱疹发病的危险因素有年龄、免疫功能缺陷、性别以及其他潜在因素。
大多数原发的VZV感染发生在童年，然后VZV潜伏在神经节中，成年后VZV可以被再激活。研究表明，约99％的40岁及以上的美国人有感染过VZV的血清学证据；90％的20-29岁的欧洲人抗VZV血清反应为阳性；在南美洲、澳大利亚和亚洲的一些国家，原发性VZV感染可能会发生得晚一些，但90％的40岁以上人群会出现VZV血清阳性反应。因此在全球范围内，绝大多数成年人都有发生带状疱疹及其相关并发症的危险。全球带状疱疹的发病率为(3～5)/1000人年，亚太地区为(3～10)/1000人年，并逐年递增2.5％～5.0％，住院率(2～25)/10万人年，死亡率(0.017～0.465)/10万人年，复发率1％～6％。目前我国属 于高程度老年化状态，HZ带来的社会经济负担也在逐年加重，对个体而言，HZ给患者尤其是老年患者生活质量带来了巨大的负面影响。据国家统计局公布的数据估计2017年40岁以上人口约6.5亿，若按照HZ发病率为2.5/1000人年保守估计我国每年新发HZ约160万例。
由于药物治疗只能缓解症状，接种疫苗是预防HZ及其并发症的最佳策略。目前全球仅有2款HZ疫苗上市销售(HZ减毒活疫苗Zostavax和HZ亚单位疫苗Shingrix)。默沙东的Zostavax为减毒活疫苗，所含病毒株与水痘疫苗所用的VZV Oka株相同，该疫苗配方所采用的最低效力为19400PFU，于2006年获得FDA批准上市，迄今已在60多个国家获准可在50岁以上人群中皮下接种1剂。葛兰素史克开发的Shingrix是一种基于重组gE蛋白辅以新型佐剂AS01 B的亚单位疫苗，三期临床试验数据显示该亚单位疫苗在老年人中有着优于Zostavax的免疫原性和有效性，并于2017年获FDA批准上市，适用于50岁及以上人群，需接种2剂。目前Shingrix已被条件性批准在中国上市，但国内在研疫苗均为减毒活疫苗，其保护效力和免疫持久性会低于Shingrix，而Shingrix因产能等问题全球缺货，因此国内急需一种自主研发的亚单位疫苗帮助减少由带状疱疹及其并发症引起的疾病负担。
VZV特异性的免疫反应对减少带状疱疹发病率和带状疱疹后神经痛至关重要，葛兰素史克开发的Shingrix是一种基于新型佐剂AS01 B的亚单位疫苗，已被证实可有效预防带状疱疹及其神经痛，该新型佐剂在此疫苗中起到了关键作用。因此，选择合适的佐剂来开发一种疫苗，从而提高目标人群中的细胞免疫和体液免疫反应，可有效预防带状疱疹。葛兰素史克的AS01 B佐剂系统包含脂质体、MPL和QS-21，能引起很强的细胞免疫反应，但目前MPL和QS-21受来源限制生产规模有限，并且在国内规模化生产条件不成熟，无法满足后续临床使用需求。而CpG ODN可通过化学合成大规模生产，易于质控。CpG ODN是Toll样受体9(toll-like receptor 9，TLR9)的激动剂，可以刺激表达TLR9的细胞并激活下游天然免疫反应通路，一方面诱导I型干扰素和炎症因子表达，另一方面使浆细胞样树突状细胞成熟，从而增强体液免疫和细胞免疫反应。铝佐剂是迄今为止使用最为广泛的人用疫苗佐剂，与其他佐剂组成的联合佐剂也有着广泛的应用，已完成临床二期试验的乙肝和疟疾预防性疫苗中均使用了铝佐剂和CpG 7909的联合佐 剂。
尽管现有技术中已经开发了一些针对VZV的疫苗，然而还存在VZV蛋白表达效率低下，表达获得的蛋白活性低，免疫效果不理想等问题。因此，本领域还有必要开发改进的VZV疫苗产品。且从策略上来说，总是需要具有改进的免疫原性的新组合物或疫苗，例如从佐剂或制剂组合方面进行尝试和改进以提高免疫应答。
发明内容
本发明的一方面在于提供一种重组带状疱疹预防性疫苗，该疫苗中包含针对水痘带状疱疹病毒(VZV)的抗原。更具体地，上述抗原可为VZV病毒的gE蛋白。
本发明所述之VZV为双链DNA病毒，仅有一个血清型，基因组全长约125000bp，编码71个基因，最终表达67个不同的蛋白质，包括7种糖蛋白(Glycoprotein，gp I～gp VI)，分别命名为gE、gB、gH、gI、gC、gK和gL，其中糖蛋白E(Glycoprotein E，gE蛋白)是VZV主要的表面结构蛋白以及最重要的中和抗原，具有较高的保守性。具体来说，VZV-gE糖蛋白由0RF68编码，有623个氨基酸组成，为I型跨膜糖蛋白。gE糖蛋白在病毒和感染细胞表面表达最为丰富，并兼有T、B细胞表位，所以针对VZV-gE的研究最为广泛，期望其成为新一代的亚单位水痘或带状疱疹疫苗中的抗原。
现有的VZV gE蛋白的研究可知，野生型VZV gE的蛋白分子包括有四个区域，即信号肽、胞外区、跨膜区和胞内区，而VZV gE的3个抗原决定簇均分布在胞外区(例如可参考文献Grose C.Glycoproteins encoded by varicella-zoster virus：biosynthesis，phosphorylation，and intracellular trafficking.Annu Rev Microbil.1990，44：59-80.)。在NCBI数据库中，已有许多现有的gE蛋白序列(例如Sequence ID为Q9J3M8.1、AQT34120.1、AGY33616.1、AEW88548.1的氨基酸序列)可供本领域技术人员进行选择。本发明的发明人从NCBI上共检索到25条VZV gE蛋白的全长序列，分别来源于不同的病毒株，氨基酸序列分析和比对结果显示：其中7个病毒株的蛋白序列完全一致(AAK19946.1，AHB80298.1，Q9J3M8.1，ABE67176.1，AGL51024.1，AFO85645.1，ABF22079.1)，且各位点保守性最高。故而这些序列均可以作为gE抗 原蛋白的理想选择基础。
同时，考虑到疏水性跨膜区域对外源蛋白质表达的影响等因素，针对gE蛋白的改造手段也是成熟的，例如，为了提高蛋白的表达水平，一般将去除跨膜区(疏水区)和胞内区(例如专利CN102517302A)。同时，在利用载体细胞(宿主细胞)内的翻译机构翻译突变体蛋白前体并转移至细胞膜分泌到细胞外时，信号肽区域通常会被信号肽酶切断(例如专利CN102548578A，CN102711812B，以及文献GSK引用的文献)。作为本领域的常规技术手段，跨膜区、胞内区及信号肽均可通过相关应用软件进行预测，如可利用SignalP(http：//www.cbs.dtu.dk/services/SignalP/)对蛋白质是否有信号肽进行预测分析，利用TMHMM Server V.2.0软件预测蛋白的跨膜区域，利用PSORT软件则可确定分泌型信号肽的准确性以及该信号肽切割位点是否可被识别并切割。
为了开发本发明的目的，在本发明的实施例中，使用在NCBI上ID＝Q9J3M8.1的作为基础，并截短C端77个氨基酸(去除开跨膜区和胞内区)，作为模型抗原(N端1-30个氨基酸为信号肽)。
利用现代分子生物的常规技术手段可轻易地获得上述抗原，典型的方法包括：在CHO细胞中表达上述gE蛋白的方法，包括下述步骤：
(1)将本发明的gE基因克隆入表达载体中；
(2)将步骤(1)所得的表达载体转化至CHO细胞中；
(3)通过迷你细胞群的筛选和单克隆筛选，获得稳定表达gE蛋白的细胞株；
(4)使用步骤(3)所得的细胞株进行表达，获得VZV gE蛋白。
将上述获得的蛋白，经常规的处理方式，例如疏水层析、阴离子交换层析、羟基磷灰石层析、超滤和纳滤后可获得纯度为95％以上的抗原蛋白。
需要说明的是，使用CHO细胞株稳定表达VZV gE重组蛋白的方法是本领域众所周知的方法，具体可参照《分子克隆试验指南》及其他一些文献，例如Haumont M，等人，Virus Research 40(1996)，199-204，Purification，characterization and immunogenicity of recombinant varicella-zoster virus glycoprotein gE secreted by Chinese hamster ovary cells。本领域技术人员也可以选择例如大肠杆菌、毕赤酵 母等在内的其他表达方式获得gE蛋白(例如可参考专利CN108315344A或CN107022559A中关于gE蛋白制备的进一步介绍)。
在优选的实施方式中，所述疫苗组合物进一步包含药学上可接受的载体。在一个优选的实施方式中，所述药学上可接受的载体为铝佐剂联合CpG ODN佐剂。本发明的发明人惊讶地发现，上述两种佐剂和gE蛋白的联合将显示出令人惊奇的高免疫活性。从而使得本发明的疫苗组合物成为新一代的水痘带状疱疹疫苗。
本发明所述之铝佐剂是目前在疫苗中应用最为广泛的一类佐剂，有着80多年的应用历史和数百亿人次的使用记录，一度也是被美国食品和药品监督管理局(FDA)批准用于人类疫苗的唯一佐剂。目前已经有非常多将铝佐剂用于疫苗的研究，比较系统的研究可见于例如《Vaccine Design：The Subunit and Adjuvant Approach》一书(ISBN：0-306-44867-X)中的例如第八章《铝化合物及钙化合物的佐剂特性》以及第九章《铝佐剂的结构及特性》，或者《Vaccine Adjuvants：Preparation Methods and Research Protocols》一书(ISBN：1-59259-083-7)中的例如第四章《铝化合物作为疫苗佐剂的使用》。目前已有一些商业化的铝佐剂可供使用于疫苗中，包括但不限于
Figure PCTCN2020098179-appb-000001
(氢氧化铝)以及
Figure PCTCN2020098179-appb-000002
(磷酸铝)。前述研究中也已经说明了如何制备铝佐剂的制备方式，本领域技术人员可根据需要制备所需的铝佐剂应用于疫苗制剂中。
CpG ODN佐剂为一类已知具有佐剂特性的免疫刺激性寡肽，能激活B细胞、NK、DC树突状等细胞，诱导释放IL-12、IFN-γ，从而诱导强Th1型应答和细胞免疫。该种佐剂也是本领域技术人员所熟知的，已经在现有技术，例如国际申请已经在例如WO 96/02555、WO 99/33488中描述过。目前已有一些商业化的CpG ODN佐剂，例如CpG 7909(Coley公司)或CpG 1018(Dynavax公司)。当然，本领域技术人员也可以根据需要设计人工合成序列。
本发明所提供的疫苗制剂的具体制备方式可参考例如《Vaccine Design：The Subunit and Adjuvant Approach》一书(ISBN：0-306-44867-X)。作为最常规的方式之一，本发明所提供的制备方法为将本发明前述之抗原蛋白与前述药学上可接受的载体相混合。作为更具体的举例，在优选的一个实施例中，gE蛋白应最好吸附至铝 佐剂上。同时，CpG ODN佐剂也最好吸附在铝佐剂上。
关于疫苗制剂剂量选择的一般原则，每一剂疫苗的量应能够在受接种者体内引起免疫保护性反应同时没有显著的毒副作用。一般而言，不同抗原蛋白的剂量均略有不同，可通过观察抗体滴度和受试者的其他反应来确定某一特定疫苗的最适剂量。本发明所提供的疫苗制剂，其抗原蛋白的含量约在10-100μg之间，更优选地在50-100μg之间，铝佐剂的含量约在100-600μg之间，更优选地，该含量在225-600μg之间，CpG ODN佐剂的含量约50-1200μg之间，更有选地，在300-900μg之间。
在本发明的第二方面，在于提供前述疫苗组合物的用途，用于预防或治疗带状疱疹病毒感染相关疾病或病症。
本发明的第三方面，在于提供一种试剂盒，该试剂盒中包含疫苗给予装置，包括但不限制于针头装置，液体喷射装置，粉末装置和喷雾装置。装置的选择主要基于不同的给药方式，常见的给药方式包括肌肉注射、腹膜内注射、皮内注射或皮下注射，或口腔/消化道、呼吸道和泌尿生殖道粘膜给药。本发明的疫苗常见的可通过肌肉注射的方式，常见的给予装置则是针头装置。常见地，可以单剂给药，其组分也可以同时或不同时联合施用。
本发明的发明人惊讶地发现，上述两种佐剂和gE蛋白的联合将显示出令人惊奇的高免疫活性。从而使得本发明的疫苗组合物成为新一代的水痘带状疱疹疫苗。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
具体实施方式
实施例1：含有VZV gE抗原蛋白的疫苗组合物的制备
为了研究本发明所提供的疫苗组合物之技术效果。本发明的发明人制成了以下多种疫苗组合物制剂(0.5ml/剂)，每种组合物均含有VZV gE蛋白、铝佐剂以及CpG ODN佐剂。具体配制方法为：先将gE抗原原液吸附到铝佐剂(磷酸铝佐剂)上，制备成不同比例的gE抗原/铝佐剂(w/w)比例的吸附样品(此处的铝佐剂含量实质铝元素的含量)；然后向该gE抗原/铝吸附样品中加入不同浓度的CpG ODN样品 (CpG 7909)。上述制剂充分混合后，如果不立即施用，则储存于4℃，具体比例如下：
表1
编号 gE(μg) 铝佐剂(μg) CpG ODN佐剂(μg) 体积(μl)
1 10 600 300 500
2 10 600 600 500
3 10 600 900 500
4 10 600 1200 500
5 50 600 300 500
6 50 600 600 500
7 50 600 900 500
8 50 600 1200 500
9 100 600 300 500
10 100 600 600 500
11 100 600 900 500
12 100 600 1200 500
13 0 600 1200 500
14 10 225 600 500
15 50 225 600 500
16 100 225 600 500
实施例2：
针对获得实施例1中待评价的重组带状疱疹疫苗组合物。发明人以C57BL/6小鼠为动物模型开展了免疫原性研究。以gE蛋白为抗原，以铝佐剂和CpG ODN为佐剂研究通过本发明提供的疫苗组合物之免疫原性。选择6-8周龄C57BL/6小鼠随机分组，每组10只小鼠，肌肉注射用gE蛋白联合佐剂制备的疫苗，设置疫苗组和佐剂组，0、3周免疫，第5周采血取脾脏。采用ELISA法检测血清中的抗VZV gE蛋白的结合抗体滴度即total IgG，采用ELISPOT法检测脾细胞中的细胞免疫水平，主要为IFN-γ的表达。同时，需要说明的是，除上述16组试 验外，本申请的发明还进行过相同配比的平行试验，用于验证CpG ODN中1018与VZV gE蛋白、铝佐剂组成疫苗组合物后的效果，其他参数及试验步骤均不变。最终获得的数据显示，其IgG(GMT)值与使用CpG 7909时基本相同，其IFN-γ值略低于CpG 1018，但无明显差距，例如对应组3，当CpG ODN为CpG 7909时，IFN-γ值＝24，对应组4，为30，对应组9-11，为120、125、102。
免疫原性的评价方法为本领域的行规技术手段，作为举例，更具体的操作方式如下：
1、重组带状疱疹疫苗的动物实验
选择6～8周龄C57BL/6小鼠随机分组，每组10只。腹腔注射不同剂量的疫苗(表1)，注射体积为0.05ml；0、3周免疫，第5周取血取脾脏，分离血清进行ELISA检测结合抗体滴度，分离脾淋巴细胞进行ELISPOT分析。
2、结合抗体滴度检测
取小鼠第二次免疫后2周血清，检测抗gE蛋白的结合抗体滴度。
(1)抗原gE原液用PBS稀释至1μg/ml，ELISA板每孔加入100μl稀释好的原液。4℃过夜。洗板机清洗。
(2)用PBS配置5％脱脂奶，ELISA板每孔加入100μl脱脂奶。37℃保温2小时。洗板机清洗。
(3)PBS配置2％脱脂奶，将待测血清梯度稀释，ELISA板每孔加入稀释血清100μl，37℃保温1小时。洗板机清洗。
(4)用PBS配置的2％脱脂奶将羊抗鼠按1∶10000比例稀释，ELISA板每孔加入100μl脱脂奶稀释的二抗。37℃保温1小时。洗板机清洗。
(5)按显色缓冲液9ml，TMB 1ml，3％H 2O 210μl的比例配制显色液。ELISA板每孔加入100μl显色液。37℃保温10min。ELISA板每孔加入50μl终止液。
(6)450nm/620nm读数。
3、细胞免疫检测
小鼠第二次免疫后2周，每组小鼠取脾，分离淋巴细胞。通过ELISPOT测定小鼠脾淋巴细胞(n＝2)表达IFN-γ的水平。
(1)包被ELISPOT板(无菌操作，取脾脏前一天进行)
35％的酒精润湿ELISPOT板，按15μl/孔的量加入96孔ELISPOT 板中，保留时间不超过1分钟。加入200μl/孔无菌水洗板5次。取150μl的IFN-γ包被抗体，加入10mlPBS中，混匀，0.2μm滤膜过滤。取包被抗体稀释液，100μl/孔加入96孔ELISPOT板中，4℃过夜。
(2)ELISPOT板封闭(无菌操作)
弃掉包被抗体，加入200μl/孔无菌PBS洗板5次。按200μl/孔的量将1640完全培养基(含10％FBS)加入到96孔ELISPOT板中，室温封闭30分钟以上。弃去液体，在灭菌的纱布上尽量扣干水分，以免下步加入培养基时产生气泡。
(3)淋巴细胞准备(无菌操作)
处死小鼠，浸泡于75％乙醇中。在超净台中取出小鼠脾脏。在35mm培养皿中放入一块儿烧过的200目铜网，加入1ml淋巴细胞分离液，用1ml注射器的推杆研磨。把悬有脾细胞的分离液经烧过的200目铜网过滤后，转移到15ml离心管中，加入淋巴细胞分离液至4ml，液面上覆盖0.5ml的RPMI1640基础培养基。室温、800g、升降速3、离心30分钟。吸出淋巴细胞层，再加入10ml RPMI1640基础培养基，洗涤，室温、250g、离心10分钟。弃去上清液，加入2ml RPMI1640完全培养基重悬细胞，计数。
(4)加样(无菌操作)
加细胞：根据细胞计数结果用完全培养基稀释细胞至6×10 6/ml，同时mAb CD28-A稀释1000倍加入到细胞悬液中。100μl/孔加入ELISPOT板内。阳性对照：加入ConA刺激物1μl，刺激浓度为5μg/ml。待测样品：加入用无血清培养基进行稀释的刺激物gE蛋白肽库，终浓度2μg/ml；阴性对照：不加ConA刺激物，也不加刺激物短肽。37℃5％CO 2培养24小时，期间不可移动培养板以免造成细胞位置的变动进而ELISPOT斑点的变模糊。
(5)斑点检测
弃掉细胞悬液，加入200μl/孔无菌PBS洗板5次。取50μl生物素标记检测抗体加入到10ml稀释液(PBS+0.1％BSA)中，混匀，0.2μm滤膜过滤。每孔加入100μl，37℃孵育2小时。弃掉生物素标记检测抗体稀释液，加入200μl/孔无菌PBS洗板5次。用稀释液(PBS+0.1％BSA)稀释抗体，取50μl加稀释液10ml，混匀，0.2μm滤膜过滤。每孔加入100μl，37℃孵育1小时。从此步骤开始避光操作。弃掉抗体稀释液， 加入200μl/孔无菌PBS洗板5次。按50μl/孔的量加入Fluorescence enhancer-II到96孔ELISPOT板中，37℃孵育15分钟。弃掉板内液体，将板倒扣在吸水纸上，拍干细小的水珠。取下保护层，放在电热恒温培养箱中，37℃避光将膜烘干。将ELISPOT板置于
Figure PCTCN2020098179-appb-000003
S5 VersC CnClyzer酶联斑点图像自动分析仪内，调节好合适的参数，进行斑点计数。
具体结果如下表2所示：
表2抗原、铝佐剂和CpG ODN佐剂配比研究结果汇总
Figure PCTCN2020098179-appb-000004
表3抗原、铝佐剂和CpG1018配比研究结果汇总
Figure PCTCN2020098179-appb-000005
从表2-3中可知，根据本发明所提供的特定组分及比例制备而得的疫苗组合物，具有良好的免疫活性，尤其是当gE蛋白的含量在50-100μg之间，铝佐剂的含量在225-600μg之间，CpG ODN佐剂的含量在300-900μg之间时，其具有优异的细胞免疫效果，可作为新一代疫苗组合物。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种重组带状疱疹疫苗组合物，其特征在于，所述重组带状疱疹疫苗组合物包含VZV病毒的gE蛋白。
  2. 如权利要求1所述的疫苗组合物，其特征在于，所述疫苗组合物进一步包含药学上可接受的载体。
  3. 如权利要求2所述的疫苗组合物，其特征在于，所述药学上可接受的载体为铝佐剂联合CpG佐剂。
  4. 如权利要求3所述的疫苗组合物，其特征在于，所述gE蛋白的含量在10-100μg之间，更优选地在50-100μg之间。
  5. 如权利要求3所述的疫苗组合物，其特征在于，所述铝佐剂的含量约在100-600μg之间，更优选地，在225-600μg之间。
  6. 如权利要求3所述的疫苗组合物，其特征在于，所述CpG佐剂的含量约50-1200μg之间，更有选地，在300-900μg之间。
  7. 如权利要求3所述的疫苗组合物，其特征在于，所述gE蛋白的含量在10-100μg之间，所述铝佐剂的含量约在100-600μg之间，所述CpG佐剂的含量约50-1200μg之间。
  8. 如权利要求3所述的疫苗组合物，其特征在于，所述gE蛋白的含量在50-100μg之间，所述铝佐剂的含量约在225-600μg之间，所述CpG佐剂的含量约300-900μg之间。
  9. 如以上任一权利要求所述的疫苗组合物在用于预防带状疱疹病毒感染相关疾病中的用途。
  10. 一种试剂盒，其包含如以上任一权利要求所述的疫苗组合物，其特征在于，所述试剂盒包含疫苗给予装置，所述给予装置包括针头装置，液体喷射装置，粉末装置和喷雾装置。
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