WO2021115409A1 - 药物组合物及其用途 - Google Patents

药物组合物及其用途 Download PDF

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WO2021115409A1
WO2021115409A1 PCT/CN2020/135571 CN2020135571W WO2021115409A1 WO 2021115409 A1 WO2021115409 A1 WO 2021115409A1 CN 2020135571 W CN2020135571 W CN 2020135571W WO 2021115409 A1 WO2021115409 A1 WO 2021115409A1
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pharmaceutical composition
saponins
cpg
composition according
varicella
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PCT/CN2020/135571
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English (en)
French (fr)
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葛君
李建强
孙娇娇
任苏林
周童
王世伟
王晓东
黄精俸
陈悦
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南京远大赛威信生物医药有限公司
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Priority to US17/784,086 priority Critical patent/US20230076371A1/en
Priority to KR1020227023841A priority patent/KR20220114032A/ko
Priority to JP2022535203A priority patent/JP2023506441A/ja
Priority to CA3161638A priority patent/CA3161638A1/en
Priority to EP20900086.8A priority patent/EP4074336A1/en
Priority to AU2020400234A priority patent/AU2020400234A1/en
Publication of WO2021115409A1 publication Critical patent/WO2021115409A1/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
    • A61K39/39—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • 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
    • 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
    • A61K39/29—Hepatitis virus
    • 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
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • 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
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biopharmaceuticals.
  • the present invention relates to a pharmaceutical composition comprising herpes gE protein and an immunostimulatory composition.
  • the immunostimulatory composition comprises saponins and CpG oligodeoxynucleotides, or the immunostimulatory composition is composed of an adjuvant comprising saponin and CpG oligodeoxynucleotides, wherein the CpG oligodeoxynucleotides
  • the nucleotide sequence has two or more copies of the 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif.
  • the present invention also relates to the use of the pharmaceutical composition for preventing herpes zoster, chickenpox or post-herpetic neuralgia.
  • Shingles is a viral skin disease caused by varicella-zoster virus (VZV).
  • VZV varicella-zoster virus
  • VZV enters the local lymph nodes through the respiratory mucosal epithelium during primary infection, and the virus-infected lymphocytes then enter the blood circulation through the lymphatic circulation to infect the peripheral blood mononuclear leukocytes.
  • the virus spreads to the skin with the bloodstream, and the clinical manifestation is chickenpox. After the chickenpox is cured, the virus lurks in the cranial ganglia to replicate, and migrates to the skin along the peripheral nerves.
  • the clinical manifestations are herpes zoster.
  • the most important complication of herpes zoster is post-herpetic neuralgia (PHN). Approximately 20% of people over 50 will develop PHN, and as the age increases, the likelihood of PHN appears higher. The pain lasts for months or even years.
  • PPN post-herpetic
  • Vaccination with herpes zoster vaccine is an effective means to prevent herpes zoster and relieve PHN and other complications.
  • the main shingles vaccines currently on the market include Merck’s Zostavax and GlaxoSmithKline’s Shingrix. Among them, Zostavax was launched in 2006 as a live attenuated vaccine; Shingrix was launched in October 2017 as a recombinant shingles vaccine.
  • Adjuvant oligodeoxynucleotide is a new type of immunostimulant discovered in recent years. Its chemical nature is an oligodeoxynucleotide containing cytosine and guanine dinucleotide, which has a pattern recognition effect with natural CpG.
  • the body-like immune response can bind to Toll-like receptors on the cell membrane and effectively trigger mammalian immune responses through the TLR9 signaling pathway.
  • the immune response caused by CpG is mainly Th1 type, which can induce the conversion of Th2 type immune response to Th1, thereby stimulating cellular immunity.
  • T cells, B cells, NK cells and other immunologically active cells By activating T cells, B cells, NK cells and other immunologically active cells, a large number of various cytokines are produced, thereby enhancing the body's specific and non-specific immune effects. It is an important link connecting natural and acquired immunity.
  • Saponins are glycosides in which aglycones are triterpenoids or spirostane compounds, and belong to plant-derived adjuvants.
  • the commonly used saponin adjuvants are QS series, which are saponins extracted from Quillaja saponaria. The most commonly used in this series is QS-21 adjuvant, but QS-21 can induce cell hemolysis and has certain systemic and local toxic side effects. . Alving et al. (ALVING CR, MATYAS G, BECK Z, et al. Revue Roumaine de Chimie, 2016, 61(8): 631-635.) found that ALF liposomes combined with MPLA and QS-21 as adjuvants HIVgp140 protein can effectively increase the antibody titer in serum.
  • the pharmaceutical composition provided by the present invention contains a double adjuvant, and the saponins and CpG oligodeoxynucleotides exhibit high-efficiency synergistic effects, which can mediate a stronger immune response.
  • the present invention also provides the use of the pharmaceutical composition for preventing and/or treating chickenpox, herpes zoster or post-herpetic neuralgia. Compared with the prior art, the pharmaceutical composition of the present application exhibits excellent immune preventive and therapeutic effects.
  • the present invention provides a pharmaceutical composition comprising:
  • Herpes gE protein active fragments of the protein, variants of the protein, or a mixture of at least two of them,
  • An immunostimulatory composition comprising saponin and CpG oligodeoxynucleotides, or the immunostimulatory composition consists of an adjuvant comprising saponin and CpG oligodeoxynucleotides; wherein The CpG oligodeoxynucleotide sequence has two or more copies of 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif.
  • the sequence of the CpG oligodeoxynucleotide is selected from any one of the following: CpG T1: TCG TTC GTT CGT TTC GTT (SEQ ID NO: 2), CpG T2: TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 3) and CpG T3: TCG TCG TCG TCG TCG TCG (SEQ ID NO: 4);
  • the sequence of the CpG oligodeoxynucleotide is CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 2).
  • the pharmaceutical composition according to the present invention wherein the saponins are selected from the group consisting of saponins of Quercus saponins, ginsenosides, platycodon saponins, astragalus saponins, notoginseng saponins, glycyrrhizin, albizia bark saponins, Ophiopogon japonicus saponins, saikosaponins or bamboo One or more of J.
  • the saponins are saponins of Quercus saponins, ginsenosides, platycodin or astragaloside IV, more preferably, the saponins of Quercus saponins are QS-7, QS-17, QS-18 or QS-21, further preferably, the Quillaja saponin is QS-21;
  • the ginsenoside may be ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1 or ginsenoside Re; and the platycodin may be Platycodin D, Platycodon D2 or a mixture of the two;
  • the astragalus saponins can be monomers such as astragaloside IV (astragaloside IV), astragaloside I, and astragaloside II, or two or more of them.
  • the mixture of notoginseng saponins may be notoginsenoside R1; the Ophiopogon japonicus saponins may be Ophiopogon japonicus saponins D, etc.; the saikosaponins may be saikosaponin a, saikosaponin d, or a mixture of both
  • the saponins of Albizia Julibrissin can be total saponins of Albizia Julibrissin, etc.
  • the glycyrrhizin can be total saponins of Glycyrrhiza
  • the saponins of Panax japonicus can be total saponins of Panax japonicus.
  • composition according to the present invention wherein the adjuvant containing saponin is an immunostimulatory complex adjuvant (Iscom adjuvant).
  • immunostimulatory complex adjuvant Iscom adjuvant
  • composition according to the present invention wherein the CpG oligodeoxynucleotide comprises phosphorothioate linkage; preferably, the CpG oligodeoxynucleotide is a thiooligodeoxynucleotide, More preferably, it is a full-thio oligodeoxynucleotide.
  • composition according to the present invention wherein the weight ratio of the CpG oligodeoxynucleotide to the saponin is 1-40:0.1-2, preferably 2-40:0.1-2.
  • composition according to the present invention wherein the weight ratio of the CpG oligodeoxynucleotide to the saponin is 2:1.
  • the vaccine composition according to the present invention further comprises: iii) a pharmaceutically acceptable carrier.
  • the herpes gE protein comprises or consists of the amino acid sequence shown in SEQ ID NO:1; preferably, the active fragment of the herpes gE protein comprises SEQ ID NO: The consecutive amino acids from position 1 to position X in 1 or consist of them, wherein X is an integer between 507 and 518.
  • the pharmaceutical composition according to the present invention wherein the weight ratio between components i) and ii) of the vaccine composition is 1:1.1-42, preferably 1:2.1-42.
  • the pharmaceutical composition according to the present invention wherein the weight ratio between components i) and ii) of the vaccine composition is 1:3.
  • the present invention provides a shingles vaccine or varicella vaccine, which comprises the pharmaceutical composition.
  • the present invention provides the use of the pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of varicella-zoster virus infection and/or varicella-zoster virus-mediated diseases; preferably Said varicella-zoster virus infection and/or varicella-zoster virus-mediated disease is selected from varicella, herpes zoster or post-herpetic neuralgia.
  • the present invention provides the use of the pharmaceutical composition in the preparation of a medicament for generating humoral immunity and/or cellular immune response against varicella-zoster virus in a subject.
  • the drug is a shingles vaccine or a varicella vaccine, preferably a shingles vaccine.
  • the present invention provides a method for preventing and/or treating varicella-zoster virus infection and/or varicella-zoster virus-mediated disease, the method comprising administering to a subject in need of prevention And/or a therapeutically effective amount of the pharmaceutical composition; preferably, the varicella-zoster virus infection and/or varicella-zoster virus-mediated disease is selected from varicella, herpes zoster or post-herpetic neuralgia .
  • the present invention provides a method for producing a drug for humoral immunity and/or cellular immune response against varicella-zoster virus in a subject, the method comprising administering an effective amount of the drug to the subject in need combination.
  • the immunostimulatory composition provided by the present invention achieves unexpected technical effects and can mediate a stronger immune response.
  • the immunostimulatory effect of CpG T1, CpG T2, and CpGT3 alone is weaker than that of CpG1018, CpG7909, CpG1826, etc., but when combined with QS21, the immunostimulatory composition exhibits unexpected synergistic effects, and the immune effect is significantly enhanced.
  • CpG T1, CpG T2 and CpGT3 have been verified by preliminary experiments, and the application effects in herpes zoster antigen are equivalent. Among them, CpG T1 and CpG T2 contain the same motif 5'-TTCGTT-3'. CpGT3 has been proved by experiments with other antigens (such as hepatitis B surface antigen, hepatitis B core antigen, etc.), and its immune function is equivalent to that of CpG T1, CpG T2.
  • antigens such as hepatitis B surface antigen, hepatitis B core antigen, etc.
  • the herpes zoster vaccine containing immune stimulants provided by the present invention has superior immune stimulating effects.
  • Cellular immune experiments have proved that the vaccine can induce a strong herpes gE protein-specific IFN- ⁇ level. The immune effect is significantly better than a single adjuvant.
  • Humoral immunity experiments also proved that the vaccine can produce higher levels of herpes gE protein-specific IgG/IgG1/IgG2a antibodies, and the effect is better than a single adjuvant, and significantly better than the combination of other CPG adjuvants and QS21.
  • Figure 1 shows the effect of different CPG oligodeoxynucleotides on the secretion level of herpes gE antigen-specific IFN- ⁇ ;
  • Figure 2 shows the effect of different immunostimulatory composition dosages on the secretion level of herpes gE antigen-specific IFN- ⁇ ;
  • Figure 3 shows the effect of the herpes zoster vaccine according to the present invention on the secretion level of herpes gE antigen-specific IFN- ⁇ ;
  • Figure 4 shows the effect of the shingles vaccine according to the present invention on the levels of antigen-specific IgG antibodies and their subtypes in mouse serum;
  • panel A IgG levels in the serum of mice in each group
  • panel B IgG1 levels in the serum of mice in each group
  • panel C IgG2a levels in the serum of mice in each group
  • panel D IgG2a and IgG1 in the serum of mice in each group Ratio of
  • Figure 5 shows the effects of different saponins on the secretion level of herpes gE antigen-specific IFN- ⁇ .
  • the terms “pharmaceutical composition”, “combination drug” and “drug combination” are used interchangeably, which means at least one drug and optional pharmaceutically acceptable excipients combined together to achieve a specific purpose A combination of agents or excipients.
  • the pharmaceutical composition includes combinations separated in time and/or space, as long as they can work together to achieve the purpose of the present invention.
  • the ingredients eg, gE protein, QS-21, CpG oligodeoxynucleotide
  • the ingredients contained in the pharmaceutical composition may be administered to the subject as a whole, or separately administered to the subject.
  • the ingredients contained in the pharmaceutical composition are separately administered to the subject, the ingredients may be administered to the subject simultaneously or sequentially.
  • CpG oligodeoxynucleotide or “CpG-ODN” as used herein refers to a short single-stranded synthetic DNA molecule containing one or more "CpG" units, where C represents cytosine and G represents bird Purine, p represents a phosphodiester bond.
  • C represents cytosine and G represents bird Purine
  • p represents a phosphodiester bond.
  • the CpG oligodeoxynucleotide is unmethylated.
  • the CpG-ODN comprises a phosphorothioate linkage or phosphorothioate backbone.
  • the CpG-ODN is phosphorothioate oligodeoxynucleotide (ie, thiooligodeoxynucleotide).
  • all internucleotide linkages in the CpG-ODN are phosphorothioate linkages, that is, the CpG-ODN is a full-thio-oligodeoxynucleotide.
  • the CpG-ODN contains two or more copies of the 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif.
  • the CpG-ODN has a sequence selected from the following: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 2), TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 3) or TCG TCG TCG TCG TCG TCG (SEQ ID NO: 4), preferably TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 2).
  • Ginsenosides platycodon saponins, astragalus saponins, notoginseng saponins, glycyrrhiza saponins, albizia bark saponins, Ophiopogon saponins, saikosaponins or panax japonicus saponins
  • Ginsenoside is a kind of sterol compound, which mainly exists in medicinal materials of the genus Ginseng, and is the active ingredient in ginseng.
  • the ginsenoside is preferably monomers such as ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1, ginsenoside Re, etc., or a mixture of two or more saponins monomers;
  • platycodin is preferably platycodin D.
  • Astragalus saponins are preferably monomers such as Astragaloside IV (Astragaloside IV), Astragaloside I, Astragaloside II, or a mixture of two or more of them; 3.
  • Heptasaponins are preferably notoginsenoside R1, etc.; Ophiopogon japonicus saponins are preferably Ophiopogon japonicus saponins D, etc.; Saikosaponins are preferably saikosaponin a, saikosaponin d or a mixture of both; Albizia saponins are preferably Albizia bark total Saponins and the like; glycyrrhizin are preferably total glycyrrhiza saponins and the like; and ginseng saponins are preferably total saponins of ginseng and the like.
  • Iscom adjuvant used herein is an immunostimulatory complex adjuvant, specifically Iscom matrix (ISCOM MATRIX) that does not contain an antigen, and is an adjuvant with a cage structure composed of phospholipids, saponins, and cholesterol.
  • terapéuticaally and/or prophylactically effective amount refers to a dose sufficient to show its benefit to the subject to which it is administered.
  • the actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the individual being treated.
  • the prescription of treatment (such as the decision on dosage, etc.) is ultimately the responsibility of the general practitioner and other doctors and rely on them to make decisions, usually taking into account the disease to be treated, the individual patient’s condition, the delivery site, the method of application, and what is already known to the doctor. Know other factors.
  • mammal refers to humans, but can also be other animals, such as wild animals (such as herons, storks, cranes, etc.), domestic animals (such as ducks, geese, etc.) or laboratory animals (such as orangutans, monkeys, etc.) Rats, mice, rabbits, guinea pigs, groundhogs, ground squirrels, etc.).
  • wild animals such as herons, storks, cranes, etc.
  • domestic animals such as ducks, geese, etc.
  • laboratory animals such as orangutans, monkeys, etc.
  • Rats mice, rabbits, guinea pigs, groundhogs, ground squirrels, etc.
  • composition of the present invention may also contain additional additives, such as pharmaceutically acceptable carriers or additives, especially when it is in the form of a pharmaceutical preparation.
  • Preferred pharmaceutical carriers are especially water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3 % Glycerin, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutical carrier used depends in particular on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the composition according to the invention may contain wetting agents, emulsifiers or buffer substances as additives.
  • compositions, vaccine or pharmaceutical preparation according to the present invention can be administered by any suitable route, such as oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the nucleic acid sequence is optimized according to the target protein sequence, so that its codons conform to the mammalian expression system, and the target gene is synthesized; the synthesized target gene is ligated with pcDNA3.1(+) plasmid by restriction enzyme digestion and ligation, and transformed Top 10 competent, pick the positive single clones, and sequence the positive single clones to verify; a large number of monoclonal bacteria are amplified, and a large number of endotoxin-free plasmid extraction kits are used to extract a large number of plasmids that meet the requirements of cell transfection; Transfection method The plasmid is used to transfect CHO suspension cells.
  • the supernatant of the fermentation broth is collected by centrifugation at 5000 rpm for 30 min at 4°C.
  • the fermentation broth was dialyzed into a solution containing 50 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole in a 4°C chromatography cabinet at a dialysis ratio of 1:100.
  • the dialysis was performed every 4 hours for a total of 3 times; the collected samples were passed through a nickel column For purification, perform SDS-PAGE detection on the collected target protein peak samples, combine the purer purification solution, and dialyze the solution containing 20mM phosphate and 150mM NaCl in a 4°C chromatography cabinet for 24h, with a dialysis ratio of 1:100, every 8h Change the liquid once; pass the sample through a 0.22 ⁇ m sterile filter membrane and store it in a refrigerator at 4°C for later use.
  • the oligodeoxynucleotide is a synthetically prepared oligodeoxynucleotide sequence fragment, which contains one or more CpG motifs.
  • the CPG sequence used in this example is shown in Table 1:
  • the phosphoramidite tetrazole active intermediate Enter the synthesis column to form the phosphoramidite tetrazole active intermediate, which undergoes a condensation reaction with the deprotected nucleotides on CpG; 3) Connection: the phosphoramidite tetrazole active intermediate encounters the deprotection on CpG When the nucleotide of the protective group, it will have an affinity reaction with its 5'hydroxyl group, condense and remove the tetrazole. At this time, the oligonucleotide chain will extend forward by one base; 4) Oxidation: the nucleotide is single in the condensation reaction.
  • the body is connected to the oligonucleotide connected to CpG through a phosphorous ester bond, and the phosphorous ester bond is unstable and easy to be hydrolyzed by acid or alkali.
  • a thio reagent is used to oxidize the phosphoramidite to a phosphorous double bond.
  • This terminal hydroxyl after the above five steps, a deoxynucleotide is connected to the nucleotide of CpG; repeat the above deprotection, activation, ligation, oxidation, and sealing process to obtain a crude DNA fragment; finally It can be subjected to post-synthesis processing such as cutting, deprotection, purification, and quantification.
  • mice C57BL/6(N) mice, female, 5 weeks old, 42 mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • the herpes gE stock solution obtained in Example 1 was diluted to 50 ⁇ g/mL with PBS solution (purchased from Hyclone), and the CpG obtained in Example 1 was diluted to 100 ⁇ g/mL with PBS solution.
  • the injection volume per mouse was 100 ⁇ L/mouse, and the control group was injected with 100 ⁇ L PBS solution/mouse.
  • the spleen was taken from the mouse on the 7th day after immunization, and the spleen lymphocytes were prepared according to the conventional method. The details are as follows: Take the spleen aseptically: use sterile forceps and scissors to cut the spleen, place it in a 70 ⁇ m cell strainer, and place it in a pretreatment containing 2 mL. Cold-treated 2% FBS (purchased from GIBCO)-PBS plate; grind the spleen with a grinding rod, and spleen cells enter the plate through the mesh to obtain a cell suspension. Use a Pasteur pipette to put the suspension into a 40 ⁇ m cell filter.
  • gE-specific peptide library to stimulate splenocytes; use ELISPOT kit (BD company) to detect gE antigen-specific IFN- ⁇ secretion level according to kit instructions; use ImmunoSPOT Series 3 enzyme-linked spot analyzer to read ELISPOT kit The number of spots measured (refer to Example 7 of Chinese Patent CN104043120B for specific operation steps).
  • the gE-specific peptide library sequence is shown in SEQ ID NO: 7-21.
  • the results of ELISPOT spots are shown in Figure 1.
  • the results show that the immune effects of vaccine compositions with different CPG adjuvants are similar.
  • CpG7909 has the best effect, slightly higher than CpG T1 ⁇ T3;
  • CpG1018 and CpG T1 ⁇ T3 have the same immune effect;
  • CpGT1 has the best effect, which is higher than CpGT2 and CpGT3. Therefore, the follow-up experiment mainly uses CpGT1 and CpG7909 as the main comparison.
  • mice C57BL/6(N) mice, female, 5 weeks old, 72 mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • Herpes gE protein, CpG T1, CpG 7909 are all prepared in Example 1;
  • each injection volume is 100 ⁇ L/mouse.
  • the control group was injected with 100 ⁇ L of PBS solution per mouse.
  • the results of ELISPOT spots are shown in Figure 2.
  • the results show that the dose changes of CpG T1 and QS21 have a significant impact on the therapeutic effect of the vaccine composition.
  • the herpes gE protein and immunostimulatory composition (CpG T1+QS-21) are higher than 1.
  • the vaccine composition of 2.1 induces the production of herpes gE protein-specific IFN- ⁇ levels significantly higher than that of the CPG7909 group, such as the immunostimulatory composition of dose 5 to dose 9 in Table 4.
  • the induction effect of further increase in the adjuvant dose did not increase significantly, presumably because the mice could not accurately reflect the immune strength of the adjuvant.
  • the adjuvant dose used is lower than the equivalent CPG7909 group, so it also has unexpected technical effects.
  • mice C57BL/6(N) mice, female, 5 weeks old, 48 mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • the herpes gE stock solution obtained in Example 1 was diluted to 50 ⁇ g/mL and 10 ⁇ g/mL with PBS solution (purchased from Hyclone), and QS-21 (purchased from BRENNTAG company, CAS.NO.A010-023) was diluted with PBS solution. Dilute to 50 ⁇ g/mL and 10 ⁇ g/mL, respectively, and use PBS solution to dilute the CpG obtained in Example 1 to 100 ⁇ g/mL and 20 ⁇ g/mL, respectively.
  • PBS solution purchased from Hyclone
  • QS-21 purchased from BRENNTAG company, CAS.NO.A010-023
  • mice C57BL/6(N) mice, female, 5 weeks old, 48 mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • Detection procedure Collect blood and separate serum on the 28th day after immunization (place the whole blood in a constant temperature incubator at 37°C for 40 minutes, centrifuge at 12000 rpm, 4°C for 10 minutes; aspirate the supernatant and freeze it at -20°C for later use), follow the kit Instructions, use ELISA kit (Shanghai Kehua) to detect the positive conversion rate of the herpes gE protein-specific antibody.
  • a blank control, a negative control, and a sample to be tested are set up in the test, each with two parallel wells, the negative control is negative mouse serum; in addition to the blank control, each well is added with a negative control or a sample to be tested, and then an enzyme conjugate After mixing and sealing the plate, incubate at 37°C for 30 minutes; wash each well with washing solution, add developer A and developer B to each well, mix and seal the plate and incubate at 37°C for 15 minutes; add stop solution to each well to mix Evenly; use a microplate reader to read the OD value of each well at 450nm wavelength.
  • mice C57BL/6(N) mice, female, 5 weeks old, 48 mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • Herpes gE protein, CpG T1, CpG 7909 are all prepared in Example 1;
  • QS-21 (CAS.NO.A010-023, purchased from BRENNTAG); Ginsenoside Rg1 (CAS: 22427-39-0, purchased from Nanjing Chunqiu Bioengineering Co., Ltd.), Astragaloside IV (CAS: 84687- 43-4, purchased from Nanjing Chunqiu Biological Engineering Co., Ltd.), Platycodin D (CAS: 58479-68-8, purchased from Hubei Yunmei Technology Co., Ltd.), Iscom adjuvant (purchased from Shanghai Xiyuan Biological Technology Co., Ltd.)
  • each injection volume is 100 ⁇ L/mouse.
  • the control group was injected with 100 ⁇ L of PBS solution per mouse.
  • the herpes zoster vaccine provided by the present invention has a superior immune stimulating effect.
  • Cellular immune experiments have proved that the vaccine can induce a strong herpes gE protein-specific IFN- ⁇ level, and the protein immune effect is significantly better than that of a single adjuvant. Agent.

Abstract

一种药物组合物，其包含：i)疱疹gE蛋白、所述蛋白的活性片段、所述蛋白的变体，或者其中至少两种的混合物；ii)免疫刺激组合物，所述免疫刺激组合物包含皂苷和CpG寡聚脱氧核苷酸，或者所述免疫刺激组合物由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成。所述药物组合物在制备用于预防和/或治疗水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病的药物中的用途。药物组合物达到了出乎意料的技术效果，可介导更强的免疫应答。

Description

药物组合物及其用途 技术领域
本发明属于生物制药领域。具体地，本发明涉及一种药物组合物，所述药物组合物包含疱疹gE蛋白和免疫刺激组合物。其中，所述免疫刺激组合物包含皂苷和CpG寡聚脱氧核苷酸，或者所述免疫刺激组合物由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成，其中所述CpG寡聚脱氧核苷酸序列具有两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。本发明还涉及所述药物组合物用于预防带状疱疹、水痘或带状疱疹后神经痛的用途。
背景技术
带状疱疹是由水痘-带状疱疹病毒(varicella-zoster virus，VZV)引起的病毒性皮肤病。VZV原发感染时通过呼吸道黏膜上皮进入局部淋巴结复制，感染病毒的淋巴细胞随后经淋巴循环进入血液循环感染外周血单核白细胞，病毒随血流播散至皮肤，临床表现为水痘。水痘痊愈后，病毒潜伏在颅脑神经节复制，并沿外周神经移行至皮肤，临床表现为带状疱疹。带状疱疹的最主要并发症是疱疹感染后神经痛(post-herpetic neuralgia，PHN)，50岁以上人群中约20％会发生PHN，且随着年龄增大，出现PHN的可能性越高，疼痛持续数月甚至数年。
接种带状疱疹疫苗是预防带状疱疹、减轻PHN等并发症的有效手段，目前，已上市的带状疱疹疫苗主要有默沙东的Zostavax与葛兰素史克的Shingrix。其中，Zostavax于2006年上市，为减毒活疫苗；Shingrix于2017年10月上市，为重组带状疱疹疫苗。
发明人对现有技术进行了深入研究，发现佐剂对带状疱疹疫苗的治疗作用起到重要作用。佐剂寡聚脱氧核苷酸(CpG)是近年来发现的一类新型免疫刺激剂，其化学本质为含有胞嘧啶鸟嘌呤二核苷酸的寡脱氧核苷酸，具有与天然CpG模式识别受体相似的免疫反应，能与细胞膜上的Toll样受体结合，通过TLR9信号通路有效引发哺乳动物免疫反应。CpG引起的免疫反应以Th1型为主，可诱导Th2型免疫应答向Th1转换，从而激发细胞免疫。通过激活T细胞、B细胞、NK细胞等免疫活性细胞，产生大量的多种细胞因子，从而增强机体的特异性和非特异性免疫效应，是连接天然和获得性免疫的重要纽带。
皂苷为一类苷元为三萜或螺旋甾烷类化合物的糖苷，属植物来源佐剂。 目前常用的皂苷佐剂有QS系列，是从皂树中提取得到的皂苷，该系列中最常用的是QS-21佐剂，但QS-21可诱导细胞溶血，有一定系统和局部的毒副作用。Alving等(ALVING CR,MATYAS G,BECK Z,et al.Revue Roumaine de Chimie,2016,61(8):631-635.)研究发现，以ALF脂质体结合MPLA和QS-21作为佐剂对抗HIVgp140蛋白可有效增加血清中抗体滴度。
现有技术(WO2001051083A3)曾报道关于皂苷与CpG寡聚脱氧核苷酸的免疫刺激组合物，其中CpG佐剂主要涉及CpG1826和CpG7909。然而，由于CpG寡聚脱氧核苷酸具备结构多样性，不同序列的CpG佐剂间的效果差异巨大。
因此，当前对免疫效果更强的佐剂以及药物存在需求。
发明内容
针对现有技术的不足，发明人在进行了大量研究后，发现了免疫效果更强的免疫刺激组合物，以及包含所述免疫刺激组合物的药物组合物。本发明提供的药物组合物包含双佐剂，并且其中皂苷和CpG寡聚脱氧核苷酸之间表现出高效的协同作用，可以介导更强烈的免疫应答。本发明还提供了所述药物组合物用于预防和/或治疗水痘、带状疱疹或带状疱疹后神经痛的用途。与现有技术相比，本申请的药物组合物表现出优异的免疫预防和治疗作用。
本发明的目的是通过以下技术方案实现的：
一方面，本发明提供了一种药物组合物，其包含：
i)疱疹gE蛋白、所述蛋白的活性片段、所述蛋白的变体，或者其中至少两种的混合物，
ii)免疫刺激组合物，所述免疫刺激组合物包含皂苷和CpG寡聚脱氧核苷酸，或者所述免疫刺激组合物由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成；其中，所述CpG寡聚脱氧核苷酸序列具有两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。
根据本发明所述的药物组合物，其中所述CpG寡聚脱氧核苷酸的序列选自以下中的任一种：CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:2)、CpG T2：TCG TTC GTT CGT TCG TTC GTT CGT T(SEQ ID NO:3)和CpG T3：TCG TCG TCG TCG TCG TCG TCG(SEQ ID NO:4)；
优选地，所述CpG寡聚脱氧核苷酸的序列为CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:2)。
根据本发明所述的药物组合物，其中，所述皂苷选自皂树皂苷、人参皂苷、桔梗皂苷、黄芪皂苷、三七皂苷、甘草皂苷、合欢皮皂苷、麦冬皂苷、柴胡皂苷或竹节参皂苷中的一种或多种；优选地，所述皂苷为皂树皂苷、人 参皂苷、桔梗皂苷或黄芪甲苷，更优选地，所述皂树皂苷为QS-7、QS-17、QS-18或QS-21，进一步优选地，所述皂树皂苷为QS-21；所述人参皂苷可以为人参皂苷Rg1、人参皂苷Rg3、人参皂苷Rb1或人参皂苷Re；所述桔梗皂苷可以为桔梗皂苷D、桔梗皂苷D2或其两者的混合物；所述黄芪皂苷可以为黄芪甲苷(黄芪皂苷IV)、黄芪皂苷I、黄芪皂苷II等单体或其中两种或两种以上皂苷单体的混合物；所述三七皂苷可以为三七皂苷R1；所述麦冬皂苷可以为麦冬皂苷D等；所述柴胡皂苷可以为柴胡皂苷a、柴胡皂苷d或其两者的混合物；所述合欢皮皂苷可以为合欢皮总皂苷等；所述甘草皂苷可以为甘草总皂苷；所述竹节参皂苷可以为竹节参总皂苷。
根据本发明所述的药物组合物，其中，所述包含皂苷的佐剂为免疫刺激复合物佐剂(Iscom佐剂)。
根据本发明所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸包含硫代磷酸酯连接；优选地，所述CpG寡聚脱氧核苷酸为硫代寡聚脱氧核苷酸，更优选地为全硫代寡聚脱氧核苷酸。
根据本发明所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸与所述皂苷的重量比为1～40∶0.1～2，优选为2～40∶0.1～2。
根据本发明所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸与所述皂苷的重量比为2∶1。
根据本发明所述的疫苗组合物，其还包含：iii)可药用载体。
根据本发明所述的药物组合物，其中，所述疱疹gE蛋白包含如SEQ ID NO:1所示的氨基酸序列或由其组成；优选地，所述疱疹gE蛋白的活性片段包含SEQ ID NO:1中的第1位～第X位的连续氨基酸或由其组成，其中，X为507至518之间的整数。
根据本发明所述的药物组合物，其中，所述疫苗组合物的组分i)和ii)之间的重量比为1∶1.1～42，优选为1∶2.1～42。
根据本发明所述的药物组合物，其中，所述疫苗组合物的组分i)和ii)之间的重量比为1∶3。
另一方面，本发明提供了一种带状疱疹疫苗或水痘疫苗，其包含所述药物组合物。
再一方面，本发明提供了所述药物组合物在制备用于预防和/或治疗水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病的药物中的用途；优选地，所述水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病选自水痘、带状疱疹或带状疱疹后神经痛。
再一方面，本发明提供了所述药物组合物在制备用于在对象中产生针对水痘-带状疱疹病毒的体液免疫和/或细胞免疫应答的药物中的用途。其中， 所述药物为带状疱疹疫苗或水痘疫苗，优选为带状疱疹疫苗。
再一方面，本发明提供了一种预防和/或治疗水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的药物组合物；优选地，所述水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病选自水痘、带状疱疹或带状疱疹后神经痛。
再一方面，本发明提供了一种在对象中产生针对水痘-带状疱疹病毒的体液免疫和/或细胞免疫应答的药物的方法，所述方法包括给予有需要的受试者有效量的药物组合物。
本发明提供的免疫刺激组合物达到了出乎意料的技术效果，可介导更强的免疫应答。单独使用CpG T1、CpG T2和CpGT3的免疫刺激作用弱于CpG1018、CpG7909和CpG1826等，但与QS21联合应用后，所述免疫刺激组合物表现出出人意料的协同作用，免疫效果显著增强。
CpG T1、CpG T2和CpGT3经初步实验验证，在带状疱疹抗原中的应用效果相当。其中CpG T1、CpG T2含有相同的基序5’-TTCGTT-3’，CpGT3经其他抗原(例如乙肝表面抗原、乙肝核心抗原等)实验证明，免疫作用与CpG T1、CpG T2相当。
与现有技术相比，本发明提供的含有免疫刺激物的带状疱疹疫苗具有优越的免疫刺激作用，细胞免疫实验证明该疫苗可诱导产生较强的疱疹gE蛋白特异性IFN-γ水平，蛋白免疫效果显著优于单一佐剂。体液免疫实验也证明该疫苗可产生较高的疱疹gE蛋白特异性IgG/IgG1/IgG2a抗体水平，且效果优于单一佐剂，显著优于其他CPG佐剂和QS21的组合。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了不同的CPG寡聚脱氧核苷酸对疱疹gE抗原特异性IFN-γ分泌水平的影响；
图2显示了不同的免疫刺激组合物剂量对疱疹gE抗原特异性IFN-γ分泌水平的影响；
图3显示了根据本发明的带状疱疹疫苗对疱疹gE抗原特异性IFN-γ分泌水平的影响；
图4显示了根据本发明的带状疱疹疫苗对小鼠血清中抗原特异性IgG抗体及其亚型水平的影响；
其中，A图：各组小鼠血清的IgG水平；B图：各组小鼠血清的IgG1水平；C图：各组小鼠血清的IgG2a水平；D图：各组小鼠血清的IgG2a与 IgG1的比值；
图5显示了不同皂苷对疱疹gE抗原特异性IFN-γ分泌水平的影响。
定义
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
尽管本发明以广义范围显示数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“2至40”的范围应认为包含最小值2和最大值40之间(包含端点)的任何和所有子范围，也就是说，所有以最小值2或更大起始的子范围，例如2至6.1，以及以最大值40或更小终止的子范围，例如5.5至40。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
另外应注意，如本说明书中所使用的，单数形式包括其所指对象的复数形式，除非清楚且明确的限于一个所指对象。术语“或”可与术语“和/或”互换使用，除非上下文另有清楚指明。
本文使用的术语“药物组合物”、“组合药物”和“药物组合”可互换地使用，其表示组合在一起以实现某种特定目的的至少一种药物以及任选的可药用赋形剂或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述药物组合物中所含的成分(例如gE蛋白、QS-21、CpG寡聚脱氧核苷酸)可以以整体施用于对象，或者分开施用于对象。当所述药物组合物中所含的成分分开地施用于对象时，所述成分可以同时或依次施用于对象。
本文使用的术语“CpG寡聚脱氧核苷酸”或“CpG-ODN”是指短的单链合成DNA分子，其含有一个或更多个“CpG”单元，其中C表示胞嘧啶，G表示鸟嘌呤，p表示磷酸二酯键。特别地，所述CpG寡聚脱氧核苷酸是非甲基化的。在一些实施方案中，所述CpG-ODN包含硫代磷酸酯连接或硫代磷酸酯骨架。也就是说，在一些实施方案中，所述CpG-ODN为硫代磷酸酯寡聚脱氧核苷酸(即硫代寡聚脱氧核苷酸)。优选地，所述CpG-ODN中所有核苷酸间连接均为硫代磷酸酯连接，即所述CpG-ODN为全硫代寡聚脱氧核苷酸。在另一些实施方案中，所述CpG-ODN包含两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。特别地，所述CpG-ODN具 有选自下列的序列：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:2)、TCG TTC GTT CGT TCG TTC GTT CGT T(SEQ ID NO:3)或TCG TCG TCG TCG TCG TCG TCG(SEQ ID NO:4)，优选地为TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:2)。
本文使用的“人参皂苷、桔梗皂苷、黄芪皂苷、三七皂苷、甘草皂苷、合欢皮皂苷、麦冬皂苷、柴胡皂苷或竹节参皂苷”是指是存在于对应植物中的活性成分，例如人参皂苷是一种固醇类化合物，主要存在于人参属药材中，是人参中的活性成分。在一些实施方案中，所述人参皂苷优选为人参皂苷Rg1、人参皂苷Rg3、人参皂苷Rb1、人参皂苷Re等单体或其中两个或两个以上皂苷单体的混合物；桔梗皂苷优选为桔梗皂苷D、桔梗皂苷D2或其两者的混合物；黄芪皂苷优选为黄芪甲苷(黄芪皂苷IV)、黄芪皂苷I、黄芪皂苷II等单体或其中两个或两个以上皂苷单体的混合物；三七皂苷优选为三七皂苷R1等；麦冬皂苷优选为麦冬皂苷D等；柴胡皂苷优选为柴胡皂苷a、柴胡皂苷d或其两者的混合物；合欢皮皂苷优选为合欢皮总皂苷等；甘草皂苷优选为甘草总皂苷等；竹节参皂苷优选为竹节参总皂苷等。
本文使用的“Iscom佐剂”为免疫刺激复合物佐剂，具体地为不包含抗原的Iscom基质(ISCOM MATRIX)，是由磷脂、皂苷、胆固醇组成的笼状结构的佐剂。
本文使用的“治疗和/或预防有效量”或“有效量”是指足以显示其对于所施用对象益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
本文所使用的术语“哺乳动物”是指人类，也可以是其它动物，如野生动物(如苍鹭、鹳、鹤等)，家畜(如鸭、鹅等)或实验动物(如猩猩、猴子、大鼠、小鼠、兔子、豚鼠、土拨鼠、地松鼠等)。
在另一些实施方案中，本发明组合物还可包含另外的添加剂，如可药用载体或添加剂，尤其是当它以药物制剂形式存在时。
优选的药物载体尤其是水，缓冲水溶液，优选等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用药物载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
根据本发明的药物组合物、疫苗或者药物制剂可通过任何适宜的途径施 用，例如可口服、鼻、皮内、皮下、肌内或静脉内施用。
以下结合附图通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以作出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
实施发明的最佳实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1重组带状疱疹疫苗组合物的制备方法
1.1制备疱疹gE蛋白：氨基酸序列如SEQ ID NO:1所示。
参考文献Thomsson E,Persson L等在《Journal of Virological Methods》2011，第175卷，第1期，第53-59页中的报道，具体步骤如下：
根据目的蛋白序列对其核酸序列进行优化，使其密码子符合哺乳动物表达体系，并对目的基因进行合成；将合成的目的基因通过酶切连接的方式与pcDNA3.1(+)质粒连接，转化Top 10感受态，挑取阳性单克隆，对阳性单克隆进行测序验证；大量扩增单克隆菌体，用无内毒素质粒抽提试剂盒进行大量抽提符合细胞转染的质粒；通过瞬时转染方式用质粒转染CHO悬浮细胞，CHO细胞活力低于70％或发酵时间大于7天时，在4℃下以5000rpm离心30min收集发酵液上清。将发酵液在4℃层析柜中透析至含50mM Tris-HCl、500mM NaCl、20mM咪唑的溶液中，透析比例1∶100，每4h透析一次，共透析3次；通过镍柱对收集的样品进行纯化，对收集目的蛋白峰样品进行SDS-PAGE检测，合并较纯的纯化液，用含20mM磷酸盐、150mM NaCl的溶液在4℃层析柜中透析24h，透析比例1∶100，每8h换液一次；将样品过0.22μm无菌滤膜，4℃冰箱中保存备用。
1.2制备CPG寡聚脱氧核苷酸原料：
寡聚脱氧核苷酸是合成制备的寡聚脱氧核苷酸序列片段，其含有一个或多个CpG基序，本实施例使用CPG序列见表1：
表1 CPG寡聚脱氧核苷酸的具体序列
Figure PCTCN2020135571-appb-000001
具体制备方法：使用常规固相亚磷酰胺三酯法化学合成方法制备，由3’端开始，1)脱保护基：先用三氯乙酸脱去连接在CpG上的核苷酸的保护基团DMT(二甲氧基三苯甲基)，获得游离的5’羟基，以供下一步缩合反应使用；2)活化：将亚磷酰胺保护的核苷酸单体与四氮唑活化剂混合并进入合成柱，形成亚磷酰胺四唑活性中间体，此中间体与CpG上已脱保护基的核苷酸发生缩合反应；3)连接：亚磷酰胺四唑活性中间体遇到CpG上已脱保护基的核苷酸时，将与其5’羟基发生亲和反应，缩合并脱去四唑，此时寡核苷酸链向前延长一个碱基；4)氧化：缩合反应时核苷酸单体是通过亚磷酯键与连在CpG上的寡核苷酸连接，而亚磷酯键不稳定，易被酸或碱水解，此时使用硫代试剂将亚磷酰胺氧化为硫磷双键的磷酸三酯，从而得到稳定的寡核苷酸；5)封闭：缩合反应后为了防止连在CpG上的未参与反应的5’羟基在随后的循环反应中被延伸，常通过乙酰化来封闭此端羟基；经过以上五个步骤后，一个脱氧核苷酸就连到CpG的核苷酸上；重复以上的脱保护基、活化、连接、氧化、封闭过程即可得到一个DNA片段粗品；最后对其进行切割、脱保护基、纯化、定量等合成后处理即可。
实施例2 CPG寡聚脱氧核苷酸的筛选实验
2.1实验动物：
C57BL/6(N)小鼠，雌性，5周龄，42只，购于上海斯莱克实验动物有限责任公司。
2.2试剂材料：
使用PBS溶液(购自Hyclone公司)将实施例1中获得疱疹gE原液分别稀释至50μg/mL，使用PBS溶液将实施例1中获得的CpG分别稀释至100μg/mL。
2.3实验分组：
见表2，每只小鼠每次注射量为100μL/只，对照组注射PBS溶液100μL/只。
表2实验动物分组及每组注射量
Figure PCTCN2020135571-appb-000002
2.4动物免疫：
所有组别每2周进行1次肌肉注射，接种部位为右侧后大腿，连续给药2次，即分别于第0周和第2周注射给药，第4周将所有小鼠处死。
2.5检测步骤：
小鼠免疫后第7天取脾，按常规方法制备脾淋巴细胞，具体如下：无菌操作取脾脏：用无菌镊子及剪刀剪取脾脏，放于70μm细胞滤网中，置于含有2mL预冷处理的2％FBS(购自GIBCO公司)-PBS的平皿中；用研磨棒研磨脾脏，脾脏细胞通过筛目进入平皿中，得到细胞悬液，用巴氏吸管将悬液放入经40μm细胞滤网过滤(购自BD公司)的50mL无菌离心管；500×g，4℃离心5分钟；弃去上清，加入2mL 1×破红剂(购自BD公司)重悬细胞，4℃避光静置5分钟，以破碎红细胞；加入10mL 2％FBS-PBS终止破红反应；500×g，4℃离心5分钟；弃去上清，加入5mL 2％FBS-PBS重悬细胞备用。
使用刺激物gE特异性肽库刺激脾细胞；按照试剂盒说明书，使用ELISPOT试剂盒(BD公司)检测gE抗原特异性IFN-γ分泌水平；使用ImmunoSPOT Series 3酶联斑点分析仪读取ELISPOT试剂盒测出的斑点数(具体操作步骤参考中国专利CN104043120B的实施例7)。其中gE特异性肽库序列见SEQ ID NO:7～21。
表3 gE特异性肽库
gE特异性肽库 序列
SEQ ID NO:7 SVLRYDDFHIDEDKL
SEQ ID NO:8 YDDFHIDEDKLDTNS
SEQ ID NO:9 HIDEDKLDTNSVYEP
SEQ ID NO:10 DKLDTNSVYEPYYHS
SEQ ID NO:11 TNSVYEPYYHSDHAE
SEQ ID NO:12 YEPYYHSDHAESSWV
SEQ ID NO:13 YHSDHAESSWVNRGE
SEQ ID NO:14 HAESSWVNRGESSRK
SEQ ID NO:15 SWVNRGESSRKAYDH
SEQ ID NO:16 RGESSRKAYDHNSPY
SEQ ID NO:17 SRKAYDHNSPYIWPR
SEQ ID NO:18 YDHNSPYIWPRNDYD
SEQ ID NO:19 SPYIWPRNDYDGFLE
SEQ ID NO:20 WPRNDYDGFLENAHE
SEQ ID NO:21 DYDGFLENAHEHHGV
2.6评价指标：
若对照孔斑点数≤5SCF，样品孔斑点数≥10SCF，判定为阳性；若5SFC＜对照孔斑点数≤10SCF，样品孔斑点数/对照孔斑点数≥2，判定为阳性；若对照孔斑点数＞10SFC，样品孔斑点数/对照孔斑点数≥3，判定为阳性。
2.7实验结果：
ELISPOT斑点结果见图1，结果显示：采用不同CPG佐剂的疫苗组合物免疫效果相近，其中CpG7909的效果最好，略高于CpG T1～T3；CpG1018与CpG T1～T3的免疫效果相当；本发明提供的三个序列中，CpGT1的效果最优，高于CpGT2和CpGT3。因此，后续实验主要以CpGT1和CpG7909为主要对比。
实施例3不同剂量的免疫刺激组合物对重组带状疱疹疫苗组合物的药 效影响
3.1实验动物及模型建立：
C57BL/6(N)小鼠，雌性，5周龄，72只，购于上海斯莱克实验动物有限责任公司。
3.2试剂材料：
1)疱疹gE蛋白、CpG T1、CpG 7909均由实施例1制得；
2)QS-21(CAS.NO.A010-023，购自BRENNTAG公司)；
3)使用PBS溶液(购自Hyclone公司)将疱疹gE原液稀释至50μg/mL，使用PBS溶液将QS-21分别稀释至5μg/mL、50μg/mL、100μg/mL，使用PBS溶液将CpG T1溶解并分别稀释至50μg/mL、100μg/mL、2mg/mL，使用PBS溶液将CPG7909溶解并稀释至100μg/mL，供下一步使用。
3.3实验分组：
见表4，每次注射量为100μL/只。其中对照组注射PBS溶液100μL/只。
3.4实验步骤：同实施例2.5。
3.5实验结果：
ELISPOT斑点结果见图2，结果显示，CpG T1与QS21的剂量变化对疫苗组合物的治疗效果有显著性影响，其中，疱疹gE蛋白与免疫刺激组合物(CpG T1+QS-21)高于1：2.1的疫苗组合物诱导产生疱疹gE蛋白特异性IFN-γ的水平显著高于CPG7909组，例如表4的剂量5-剂量9的免疫刺激组合物。但由于种属差异性，佐剂剂量进一步增加诱导效果并无显著性增加，推测原因为小鼠无法准确反映佐剂的免疫强度。
尽管表4中的剂量1、2、4与对照CPG7909组的免疫刺激效果相当，但是使用的佐剂剂量低于同等CPG7909组，因此也具有出乎意料的技术效果。
表4实验动物分组
Figure PCTCN2020135571-appb-000003
Figure PCTCN2020135571-appb-000004
实施例4重组带状疱疹疫苗组合物的细胞免疫药效验证
4.1实验动物：
C57BL/6(N)小鼠，雌性，5周龄，48只，购于上海斯莱克实验动物有限责任公司。
4.2试剂材料：
使用PBS溶液(购自Hyclone公司)将实施例1中获得疱疹gE原液分别稀释至50μg/mL和10μg/mL，使用PBS溶液将QS-21(购自BRENNTAG公司，CAS.NO.A010-023)分别稀释至50μg/mL和10μg/mL，使用PBS溶液将实施例1中获得的CpG分别稀释至100μg/mL和20μg/mL。
4.3实验分组：见表5，每只小鼠每次注射量为100μL/只，其中A组为阴性对照，注射PBS溶液100μL/只。
表5实验动物分组及每组注射量
Figure PCTCN2020135571-appb-000005
4.4动物免疫：所有组别每2周进行1次肌肉注射，接种部位为右侧后大腿，连续给药2次，即分别于第0周和第2周注射给药，第4周将所有小鼠处死。
4.5检测步骤：同实施例2.5。
4.6评价指标：同实施例2.6。
4.7实验结果：各组小鼠脾细胞分泌gE特异性IFN-γ的T淋巴细胞斑点数水平如图3所示，gE特异性IFN-γ阳转率结果如表6所示。结果表明：免疫高剂量的E、F组对应的脾细胞分泌gE特异性IFN-γ的T淋巴细胞斑点 数水平(>4000SFC/10 6脾细胞)均显著高于低剂量的G、H组；其中，E组、G组(CpGT1+QS-21)对应的脾细胞分泌gE特异性IFN-γ的T淋巴细胞斑点数水平高于同剂量的F组、H组(CpG7909+QS-21)，E-H组IFN-γ的转阳率均为100％。
表6脾细胞分泌的gE特异性IFN-γ的转阳率
组别 A B C D E F G H
转阳数量/只 1/6 0/6 4/6 5/6 6/6 6/6 6/6 6/6
转阳率/％ 16.7 0 66.7 83.3 100 100 100 100
实施例5重组带状疱疹疫苗组合物的体液免疫药效验证
5.1实验动物：
C57BL/6(N)小鼠，雌性，5周龄，48只，购于上海斯莱克实验动物有限责任公司。
5.2试剂材料：同实施例3.2。
5.3实验分组：同实施例3.3。
5.4动物免疫：所有组别每2周进行1次肌肉注射，接种部位为右侧后大腿，连续给药2次，即分别于第0周和第2周注射给药。
5.5检测步骤：免疫后第28天进行采血，分离血清(全血置于37℃恒温培养箱放置40min，12000rpm、4℃离心10min；吸取上清，冻存于-20℃备用)，按照试剂盒说明书，使用ELISA试剂盒(上海科华)检测产生的疱疹gE蛋白特异性抗体阳转率。检测设空白对照、阴性对照和待测样品，每种两个平行孔，其中阴性对照为阴性小鼠血清；除空白对照外，各孔分别加入阴性对照或待测样品，再加入酶结合物，混匀封板后37℃孵育30分钟；使用洗涤液洗涤各孔，每孔加入显色剂A液和显色剂B液，混匀封板后37℃孵育15分钟；每孔加入终止液混匀；使用酶标仪读取450nm波长处的各孔OD值。
5.6实验结果：ELISA检测小鼠血清中抗原特异性IgG抗体及亚型水平如图4所示，其中，A图：各组小鼠血清的IgG水平；B图：各组小鼠血清的IgG1水平；C图：各组小鼠血清的IgG2a水平；D图：各组小鼠血清的IgG2a与IgG1的比值。
结果表明：含有本发明所述免疫刺激物的E组免疫效果显著优于单独的CpG组(C组)和QS-21组(D组)和双佐剂对照(F组)，且对应的IgG和IgG2a抗体水平与两组均存在显著差异，即在QS-21中加入CpG可提高相应的体液免疫水平。
实施例6不同皂苷对重组带状疱疹疫苗组合物的药效影响
6.1实验动物及模型建立：
C57BL/6(N)小鼠，雌性，5周龄，48只，购于上海斯莱克实验动物有限责任公司。
6.2试剂材料：
1)疱疹gE蛋白、CpG T1、CpG 7909均由实施例1制得；
2)QS-21(CAS.NO.A010-023，购自BRENNTAG公司)；人参皂苷Rg1(CAS:22427-39-0，购自南京春秋生物工程有限公司)、黄芪甲苷(CAS:84687-43-4，购自南京春秋生物工程有限公司)、桔梗皂苷D(CAS:58479-68-8，购自湖北云镁科技有限公司)，Iscom佐剂(购自上海熹垣生物科技有限公司)
3)使用PBS溶液(购自Hyclone公司)将疱疹gE原液稀释至50μg/mL，使用PBS溶液将各皂苷分别稀释至50μg/mL，使用PBS溶液将CpG T1和CpG 7909分别溶解并稀释至100μg/mL，供下一步使用。
6.3实验分组：
见表7，每次注射量为100μL/只。其中对照组注射PBS溶液100μL/只。
6.4实验步骤：同实施例2.5。
6.5实验结果：
ELISPOT斑点结果见图5，结果显示，CpG T1与各皂苷联合应用后均产生了高效的协同作用，诱导产生gE特异性IFN-γ水平显著高于其他CpG和皂苷的组合物，其中，QS-21的效果最佳。
表7实验动物分组
Figure PCTCN2020135571-appb-000006
综上所述，本发明提供的带状疱疹疫苗具有优越的免疫刺激作用，细胞免疫实验证明该疫苗可诱导产生较强的疱疹gE蛋白特异性IFN-γ水平，蛋白免疫效果显著优于单一佐剂。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，所述修改和改变应归属于权利要求书的范围。

Claims (23)

  1. 一种药物组合物，其包含：
    i)疱疹gE蛋白、所述蛋白的活性片段、所述蛋白的变体，或者其中至少两种的混合物，
    ii)免疫刺激组合物，所述免疫刺激组合物包含皂苷和CpG寡聚脱氧核苷酸，或者所述免疫刺激组合物由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成；其中，所述CpG寡聚脱氧核苷酸序列具有两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。
  2. 根据权利要求1所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸的序列选自以下中的任一种：CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:2)、CpG T2：TCG TTC GTT CGT TCG TTC GTT CGT T(SEQ ID NO:3)和CpG T3：TCG TCG TCG TCG TCG TCG TCG(SEQ ID NO:4)；
    优选地，所述CpG寡聚脱氧核苷酸的序列为CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:2)。
  3. 根据权利要求1或2所述的药物组合物，其中，所述皂苷选自皂树皂苷、人参皂苷、桔梗皂苷、黄芪皂苷、三七皂苷、甘草皂苷、合欢皮皂苷、麦冬皂苷、柴胡皂苷或竹节参皂苷的一种或多种。
  4. 根据权利要求3所述的药物组合物，其中，所述皂树皂苷为QS-7、QS-17、QS-18或QS-21，优选地，所述皂苷为QS-21；所述人参皂苷为人参皂苷Rg1、人参皂苷Rg3、人参皂苷Rb1或人参皂苷Re；所述桔梗皂苷为桔梗皂苷D、桔梗皂苷D2或其两者的混合物；所述黄芪皂苷为黄芪甲苷、黄芪皂苷I、黄芪皂苷II或其中两种或两种以上皂苷单体的混合物；所述三七皂苷为三七皂苷R1；所述麦冬皂苷为麦冬皂苷D；所述柴胡皂苷为柴胡皂苷a、柴胡皂苷d或其两者的混合物；所述合欢皮皂苷为合欢皮总皂苷；所述甘草皂苷为甘草总皂苷；所述竹节参皂苷为竹节参总皂苷。
  5. 根据权利要求1至4中任一项所述的药物组合物，其中，所述包含皂苷的佐剂为Iscom佐剂。
  6. 根据权利要求1至5中任一项所述的药物组合物，其中所述CpG寡聚脱氧核苷酸包含硫代磷酸酯连接。
  7. 根据权利要求1至6中任一项所述的药物组合物，其中所述CpG寡聚脱氧核苷酸为硫代寡聚脱氧核苷酸。
  8. 根据权利要求1至7中任一项所述的药物组合物，其中所述CpG寡聚脱氧核苷酸为全硫代寡聚脱氧核苷酸。
  9. 根据权利要求1至8中任一项所述的药物组合物，其中CpG寡聚脱氧核苷酸与皂苷的重量比为1～40∶0.1～2，优选为2～40∶0.1～2。
  10. 根据权利要求1至9中任一项所述的药物组合物，其中CpG寡聚脱氧核苷酸与皂苷的重量比为2∶1。
  11. 根据权利要求1至10中任一项所述的药物组合物，其中，所述药物组合物还含有：
    iii)可药用载体。
  12. 根据权利要求1至11中任一项所述的药物组合物，其中，所述疱疹gE蛋白包含如SEQ ID NO:1所示的氨基酸序列或由其组成。
  13. 根据权利要求1至12中任一项所述的药物组合物，其中，所述疱疹gE蛋白的活性片段包含SEQ ID NO:1中的第1位～第X位的连续氨基酸或由其组成，其中，X为507至518之间的整数。
  14. 根据权利要求1至13中任一项所述的药物组合物，其中，所述组分i)和ii)之间的重量比为1∶1.1～42，优选为1∶2.1～42。
  15. 根据权利要求1至14中任一项所述的药物组合物，其中，所述组分i)和ii)之间的重量比为1∶3。
  16. 一种带状疱疹疫苗或水痘疫苗，其包含如权利要求1至15中任一项所述的药物组合物。
  17. 根据权利要求1至15中任一项所述的药物组合物在制备用于预防和/或治疗水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病的药物 中的用途。
  18. 根据权利要求17所述的用途，其中，所述水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病选自水痘、带状疱疹、带状疱疹后神经痛。
  19. 根据权利要求1至15中任一项所述的药物组合物在制备用于在对象中产生针对水痘-带状疱疹病毒的体液免疫和/或细胞免疫应答的药物中的用途。
  20. 根据权利要求19所述的用途，其中，所述药物为带状疱疹疫苗或水痘疫苗。
  21. 一种预防和/或治疗水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病的方法，所述方法包括给予有需要的受试者有效量的根据权利要求1至15中任一项所述的药物组合物。
  22. 根据权利要求21所述的方法，其中，所述水痘-带状疱疹病毒感染和/或水痘-带状疱疹病毒介导的疾病选自水痘、带状疱疹、带状疱疹后神经痛。
  23. 一种在对象中产生针对水痘-带状疱疹病毒的体液免疫和/或细胞免疫应答的药物的方法，所述方法包括给予有需要的受试者有效量的根据权利要求1至15中任一项所述的药物组合物。
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BR112022011445A2 (pt) 2022-08-30
JP2023506441A (ja) 2023-02-16
KR20220114031A (ko) 2022-08-17
US20230073321A1 (en) 2023-03-09
TW202128217A (zh) 2021-08-01
CN112972671B (zh) 2024-04-19
JP2023506440A (ja) 2023-02-16
TW202128219A (zh) 2021-08-01
AU2020400234A1 (en) 2022-07-07
US20230040021A1 (en) 2023-02-09
KR20220114032A (ko) 2022-08-17
CN112972672B (zh) 2024-04-19
CN112972671A (zh) 2021-06-18
US20230076371A1 (en) 2023-03-09
AU2020403296A1 (en) 2022-07-07
ZA202207790B (en) 2023-10-25
EP4074334A1 (en) 2022-10-19
CA3161638A1 (en) 2021-06-17
CA3161628A1 (en) 2021-06-17
CN112972670A (zh) 2021-06-18
EP4074335A1 (en) 2022-10-19
CN112972672A (zh) 2021-06-18
CN112972670B (zh) 2023-12-19
JP2023506439A (ja) 2023-02-16
EP4074336A1 (en) 2022-10-19
WO2021115410A1 (zh) 2021-06-17
WO2021115408A1 (zh) 2021-06-17

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