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WO2024108955A1 - 乙型肝炎疫苗 - Google Patents

乙型肝炎疫苗 Download PDF

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Publication number
WO2024108955A1
WO2024108955A1 PCT/CN2023/097101 CN2023097101W WO2024108955A1 WO 2024108955 A1 WO2024108955 A1 WO 2024108955A1 CN 2023097101 W CN2023097101 W CN 2023097101W WO 2024108955 A1 WO2024108955 A1 WO 2024108955A1
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WIPO (PCT)
Prior art keywords
hepatitis
hbsag
adjuvant
cells
vaccine
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Application number
PCT/CN2023/097101
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English (en)
French (fr)
Inventor
尹丰
李子刚
张亚萍
廉晨珊
Original Assignee
深圳湾实验室坪山生物医药研发转化中心
北京大学深圳研究生院
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Publication date
Application filed by 深圳湾实验室坪山生物医药研发转化中心, 北京大学深圳研究生院 filed Critical 深圳湾实验室坪山生物医药研发转化中心
Priority to CN202380069076.7A priority Critical patent/CN119947750A/zh
Publication of WO2024108955A1 publication Critical patent/WO2024108955A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present disclosure generally relates to the field of biomedicine, and more specifically, to the field of vaccines.
  • Vaccine adjuvants are auxiliary substances added to vaccines to non-specifically enhance or change the body's specific immune response to antigens. Adjuvants can induce the body to produce long-term and efficient immune responses, while improving the body's protective ability and reducing the amount of antigens used, thereby reducing the production cost of vaccines.
  • Vaccine adjuvants currently approved by the U.S. Food and Drug Administration include aluminum adjuvants, oil emulsion adjuvants (MF59 and AS03), AS04, non-methylated CpG oligonucleotides, and AS01 (liposomes containing monophosphoryl lipid A and saponin QS-21).
  • Aluminum adjuvants are still the most widely used vaccine adjuvants. Although aluminum adjuvants have been verified to be safe and effective vaccine adjuvant formulas for nearly a hundred years, some people still experience adverse reactions at the injection site after vaccination with vaccines containing aluminum adjuvants, such as granulomas, subcutaneous nodules, contact hypersensitivity reactions, erythema, etc. Aluminum adjuvants often deliver antigens by attaching to the surface of cell membranes. They do not enter cells themselves and cannot participate in the intracellular processing and presentation of antigens. They can only induce humoral immune responses and are difficult to use in the development of therapeutic vaccines.
  • the present disclosure relates to a hepatitis B vaccine comprising hepatitis B virus surface antigen (HBsAg) and an adjuvant, wherein the adjuvant is a cyclic polypeptide represented by formula (I):
  • the present disclosure relates to the use of the cyclic polypeptide represented by formula (I) as an adjuvant in the preparation of a hepatitis B vaccine.
  • the present disclosure relates to a cyclic polypeptide of formula (I) for use as a hepatitis B vaccine adjuvant:
  • the present disclosure relates to a method for preventing or treating hepatitis B, comprising administering a preventively effective amount of a hepatitis B vaccine to an individual in need thereof, wherein the hepatitis B vaccine comprises hepatitis B virus surface antigen (HBsAg) and an adjuvant, wherein the adjuvant is a cyclic polypeptide represented by formula (I):
  • Figure 1 shows the structure and mass spectrometry identification results of the M-CP polypeptide, wherein (a) shows a schematic diagram of the preparation of M-CP from a linear polypeptide; (b) shows the structural formula of M-CP; and (c) shows the mass spectrometry identification results of M-CP.
  • FIG. 2 shows the morphology and Tyndall phenomenon of the hepatitis B antigen protein mixture to which the M-CP ring-closing polypeptide adjuvant is added.
  • FIG3 shows the particle size and potential of a mixture of hepatitis B antigen proteins to which different types of adjuvants are added, wherein (a) shows the particle size of each group; and (b) shows the potential of each group of solutions.
  • FIG. 4 shows the TEM morphology of the complex of HBsAg antigen protein and HBsAg-M-CP.
  • Figure 5 shows the long-term monitoring of the particle size and potential of the hepatitis B antigen protein mixture after adding M-CP adjuvant using DLS, wherein (a) shows the change in particle size of the complex at each time period; and (b) shows the change in potential of the complex at each time period.
  • FIG. 6 shows the cytotoxicity of the mixture of hepatitis B antigen proteins added with different adjuvants on BMDCs.
  • FIG. 7 shows the effect of adding different adjuvants to the mixture of hepatitis B antigen proteins on the growth state of BMDCs cells.
  • Figure 8 shows the effect of adding a mixture of hepatitis B antigen proteins with different adjuvants on the maturity of BMDCs cells, wherein (a) shows the proportion of CD11c-positive cells; (b) shows the proportion of CD11c and CD80 double-positive cells; and (c) shows the proportion of CD11c and CD86 double-positive cells.
  • FIG9 shows the effect of a mixture of hepatitis B antigen proteins with different adjuvants on the secretion of tumor-related factors by BMDCs, wherein (a) shows the content of the tumor-related factor TNF- ⁇ in the supernatant; (b) shows the content of interleukin IL-6 in the supernatant; and (c) shows the content of interleukin IL-12 in the supernatant.
  • FIG10 shows a schematic diagram of an animal immunization scheme and a dosing schedule, wherein (a) shows the time points of mouse immunization; and (b) shows the dosing schedules for four groups of mice.
  • Figure 11 shows the hepatitis B virus antibody titers in the sera of mice at week 1, week 2, week 4 and week 6 after immunization, wherein (a) shows the results of week 1; (b) shows the results of week 2; (c) shows the results of week 4; and (d) shows the results of week 6.
  • Figure 12 shows the content of B cells in the peripheral blood of mice at week 6 after immunization, wherein (a) shows the statistical data of the proportion of CD19 + cells in CD3- cells; and (b) shows the scattered distribution diagram of CD19 + CD3 - negative cell clusters.
  • FIG13 shows the content of CD8 + T cells in the peripheral blood of mice at week 6 after immunization, wherein (a) shows the statistical data of the proportion of CD8 + T cells in CD3 + T cells; and (b) shows the scattered distribution diagram of CD8 + CD3 + cell clusters.
  • Figure 14 shows the proportion of central memory T cells and effector memory T cells in lymphocytes after restimulation of the spleen of mice at week 6 after immunization, wherein (a) shows the distribution of CD3 + T labeled with CD44 and CD62L antibodies; (b) shows the proportion of central memory T cells (CD44 + CD62L + ) in CD3 + T cells; and (c) shows the proportion of effector memory T cells (CD44 + CD62L- ) in CD3 + T cells.
  • hepatitis B virus surface antigen refers to the small spherical particles and tubular particles contained in the outer shell of hepatitis B virus.
  • adjuvant i.e. immune adjuvant
  • immune adjuvant refers to a non-specific immunopotentiator that, when injected together with an antigen or pre-injected into the body, can enhance the body's immune response to the antigen or change the type of immune response.
  • mammal refers to animals including, for example, dogs, cats, cows, sheep, horses, and humans, etc. In certain embodiments, the mammal includes humans.
  • the present disclosure relates to a hepatitis B vaccine comprising hepatitis B virus surface antigen (HBsAg) and an adjuvant, wherein the adjuvant is a cyclic polypeptide represented by formula (I):
  • the present disclosure relates to the use of the cyclic polypeptide represented by formula (I) as an adjuvant in the preparation of a hepatitis B vaccine.
  • the present disclosure relates to a cyclic polypeptide of formula (I) for use as a hepatitis B vaccine adjuvant:
  • the adjuvant and hepatitis B virus surface antigen are in a nanoscale complex.
  • the present disclosure relates to a method for preventing or treating hepatitis B, comprising: The subject of the method is administered a preventively effective amount of a hepatitis B vaccine, wherein the hepatitis B vaccine comprises a hepatitis B virus surface antigen (HBsAg) and an adjuvant, wherein the adjuvant is a cyclic polypeptide represented by formula (I):
  • a hepatitis B vaccine comprises a hepatitis B virus surface antigen (HBsAg) and an adjuvant, wherein the adjuvant is a cyclic polypeptide represented by formula (I):
  • the subject is a mammal.
  • the subject is a human.
  • the adjuvant and hepatitis B virus surface antigen are in a nanoscale complex.
  • reagents and equipment used in the examples of the present disclosure are conventional and commercially available. For example:
  • the linear peptide (Fmoc-RRMEHRMEW) was purchased from Qiangyao Biotechnology Co., Ltd.
  • the dry powder of the "sulfonium salt ring-closure" peptide (named M-CP) was obtained by HPLC separation and purification and freeze-drying with a freeze dryer.
  • the molecular weight of the ring-closure product was identified by MS.
  • Peptide adjuvant forms stable nanoscale complex with HBsAg
  • the commercially available finished vaccine exists in the form of a suspension and needs to be mixed before use.
  • M-CP "sulfonium salt ring-closing" polypeptide adjuvant on the microscopic morphology of hepatitis B antigen (HBsAg)
  • M-CP 2 mg/mL was mixed with 20 ⁇ g/mL of hepatitis B stock solution.
  • the particle size and potential changes of hepatitis B antigen protein, hepatitis B antigen protein mixture with M-CP adjuvant, and finished hepatitis B mixed solution (containing aluminum adjuvant) were continuously monitored using a Malvern laser particle size analyzer (DLS).
  • DLS Malvern laser particle size analyzer
  • the finished hepatitis B vaccine presents a fluffy white precipitate after being placed for 24 hours, while the hepatitis B antigen solution mixed with M-CP polypeptide presents a uniform turbid state. And from its Tyndall phenomenon, it can be seen that the particle size of the antigen protein increases after adding M-CP.
  • the results of DLS ( Figure 3) also show that after adding M-CP, the particle size of the hepatitis B antigen protein increases from the original 100nm to about 230nm, while the particle size of the aluminum salt adjuvant group (i.e., the finished preparation group) reaches the micron level. From the perspective of potential, the M-CP solution is positively charged. When added to the negatively charged HBsAg solution, the overall potential of the solution is about -10 mV, which can help the stability of the system.
  • Peptide adjuvants do not affect the stability of HBsAg protein
  • the hepatitis B antigen protein mixture with the addition of M-CP adjuvant was stored at 4°C, and the particle size and potential changes of the mixture were detected on days 0, 1, 2, 4, 7, 14, 21, 28, 42, 56 and 105.
  • the particle size and potential of the HBsAg protein stock solution and the antigen mixture with the addition of M-CP increased with time. All remained unchanged, indicating that M-CP adjuvant can meet the needs of long-term stable storage of HBsAg.
  • the present disclosure first evaluated the M-CP-induced DC cell maturation and the secretion of immune factors at the cellular level.
  • the homemade BMDCs were seeded into 24-well plates at a density of 5 ⁇ 10 5 cells/well 24 hours in advance.
  • 5% RPMI1640 medium, HBsAg (2 ⁇ g/mL), HBsAg-Al (2 ⁇ g/mL), HBsAg-M-CP (HBsAg is 2 ⁇ g/mL, M-CP is 250 ⁇ g/mL) were co-incubated with BMDCs for 24 hours.
  • Microscopic observation of the cell morphology of each group showed (Figure 7) that the cells in the HBsAg-M-CP treatment group were close to the control, with no significant changes in cell morphology.
  • a lot of granular substances adhered to the cell surface of the HBsAg-Al treatment group which is consistent with the cell survival curve results in Figure 6, and also indicates that the M-CP polypeptide has better biocompatibility.
  • ELISA kits were used to measure the content of tumor-related factors TNF- ⁇ , interleukin IL-6 and interleukin IL-12 in the culture supernatant of BMDCs to evaluate the activation of immune cells.
  • TNF- ⁇ tumor-related factors
  • the ability of HBsAg-M-CP to stimulate the secretion of tumor-related factors was significantly higher than that of the HBsAg-Al group (p ⁇ 0.001).
  • similar situations were found in the test results of IL-6 and IL-12. Based on the results of Figure 8, it can be seen that the M-CP adjuvant can assist antigen proteins in activating DC cells, provide the possibility for the activation and proliferation of downstream immune cells through co-stimulatory signals, and then stimulate subsequent antigen-specific immune responses.
  • Peptide adjuvants induce immune response to antigens in animals
  • the antibody response level of the HBsAg-M-CP group was close to that of the finished hepatitis B vaccine group, which once again showed that the "sulfonium salt ring-closing" peptide has the potential to be used as an adjuvant for hepatitis B vaccine.
  • Peptide adjuvants activate both humoral and cellular immune pathways
  • Peptide adjuvants induce long-term immune response
  • the present invention further evaluates the ability of the "sulfonium salt ring-closing" polypeptide to assist antigens in achieving long-term effectiveness. Specifically, under sterile conditions, the spleens of each group of mice in the sixth week after immunization were obtained, the spleens were ground with a soft rubber tip of a syringe, and passed through a 70 ⁇ m cell sieve to obtain a single cell suspension of the spleen.
  • the cell density was adjusted to 5 ⁇ 10 6 cells/mL, 200 ⁇ L of cells were mixed with 1 mL of HBsAg solution (5 ug/mL), and after incubation in a 37°C incubator for 72 hours, the cells were collected and stained according to the operation in Example 5 (CD3e, CD44, CD62L).
  • relational terms such as first and second, etc. are used merely to distinguish one entity or operation from another entity or operation, but do not necessarily require or imply any such actual relationship or order between these entities or operations.

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Abstract

公开了乙型肝炎疫苗，其包含乙型肝炎病毒表面抗原（HBsAg）和佐剂，其中所述佐剂为式（I）所示的关环多肽：

Description

乙型肝炎疫苗
相关申请的引用
本公开要求于2022年11月25日向中华人民共和国国家知识产权局提交的申请号为202211514858.0、发明名称为乙型肝炎疫苗的中国发明专利申请的全部权益，并通过引用的方式将其全部内容整体并入本公开。
领域
本公开大体上涉及生物医药领域，更具体地，本公开涉及疫苗领域。
背景
疫苗佐剂是一种添加到疫苗中的辅助性物质，用于非特异性增强或改变机体对抗原的特异性免疫响应。佐剂能够诱发机体产生长期、高效的免疫反应，在提高机体保护能力的同时减少抗原用量，降低疫苗的生产成本。目前被美国食品药品监督管理局(FDA)批准的疫苗佐剂有铝佐剂、油乳佐剂(MF59和AS03)、AS04、非甲基化的CpG寡核苷酸和AS01(包含单磷酰脂质A和皂素QS-21的脂质体)。然而，由于临床对于疫苗佐剂安全性的严苛要求，导致铝佐剂仍是使用最广泛的疫苗佐剂。尽管经近百年的验证，铝佐剂被认为是安全有效的疫苗佐剂配方，但部分人群在接种含有铝佐剂的疫苗后，仍会出现接种部位的不良反应，如肉芽肿、皮下结节、接触性超敏反应、红斑等。铝佐剂往往通过贴附在胞膜表面的方式递送抗原，其自身不进入细胞，无法参与抗原胞内加工和递呈，只能引起体液免疫反应，很难用于治疗性疫苗的研发。且伴随新型疫苗(重组蛋白疫苗、亚单位疫苗、裂解疫苗、短肽疫苗、DNA疫苗、RNA疫苗)研究的开展，现有疫苗佐剂很难满足不同类型疫苗研发的需求。更安全、机制更明确、且具备同时诱导体液免疫和细胞免疫的新型疫苗佐剂亟待开发。
概述
一方面，本公开涉及乙型肝炎疫苗，其包含乙型肝炎病毒表面抗原(HBsAg)和佐剂，其中所述佐剂为式(I)所示的关环多肽：
另一方面，本公开涉及式(I)所示的关环多肽作为佐剂在制备乙型肝炎疫苗中的用途，
另一方面，本公开涉及用作乙型肝炎疫苗佐剂的式(I)所示的关环多肽：
再一方面，本公开涉及预防或治疗乙型肝炎的方法，其包括向需要所述方法的个体给予预防有效量的乙型肝炎疫苗，其中所述乙型肝炎疫苗包含乙型肝炎病毒表面抗原(HBsAg)和佐剂，其中所述佐剂为式(I)所示的关环多肽：
附图简要说明
图1示出了M-CP多肽的结构和质谱鉴定结果，其中(a)示出了线性多肽制备M-CP的示意图；(b)示出了M-CP的结构式；以及(c)示出了M-CP的质谱鉴定结果。
图2示出了添加M-CP关环多肽佐剂的乙肝抗原蛋白混合液的形态和丁达尔现象。
图3示出了添加不同类型佐剂的乙肝抗原蛋白混合物的粒径和电位情况，其中(a)示出了各组的粒径大小；以及(b)示出了各组溶液的电位大小。
图4示出了HBsAg抗原蛋白和HBsAg-M-CP复合的TEM形貌。
图5示出了利用DLS长期监测添加M-CP佐剂后乙肝抗原蛋白混合物的粒径和电位情况，其中(a)示出了各时间段复合物的粒径变化；以及(b)示出了各时间段复合物的电位变化。
图6示出了添加不同佐剂的乙肝抗原蛋白混合物对BMDCs的细胞毒性。
图7示出了添加不同佐剂的乙肝抗原蛋白混合物对BMDCs细胞生长状态的影响。
图8示出了添加不同佐剂的乙肝抗原蛋白混合物对BMDCs细胞成熟度的影响，其中(a)示出了CD11c阳性细胞的占比；(b)示出了CD11c和CD80双阳性细胞的占比；以及(c)示出了CD11c和CD86双阳性细胞的占比。
图9示出了不同佐剂的乙肝抗原蛋白混合物对BMDCs分泌肿瘤相关因子的影响，其中(a)示出了上清液中肿瘤相关因子TNF-α的含量； (b)示出了上清液中白介素IL-6的含量；以及(c)示出了上清液中白介素IL-12的含量。
图10示出了动物免疫方案的示意图和给药方案，其中(a)示出了小鼠免疫的时间节点；以及(b)图示出了4组小鼠的给药方案。
图11示出了小鼠免疫后第1周、第2周、第4周和第6周血清中乙肝病毒抗体滴度，其中(a)示出了第1周的结果；(b)示出了第2周的结果；(c)示出了第4周的结果；以及(d)示出了第6周的结果。
图12示出了免疫后第6周小鼠外周血中B细胞的含量，其中(a)示出了CD19+细胞在CD3-细胞中的占比统计数据；以及(b)示出了CD19+CD3-阴性细胞团的散点分布图。
图13示出了免疫后第6周小鼠外周血中CD8+T细胞的含量，其中(a)示出了CD8+T细胞在CD3+T细胞中的占比统计数据；以及(b)示出了CD8+CD3+细胞团的散点分布图。
图14示出了免疫后第6周小鼠脾脏再刺激后，淋巴细胞中中央记忆T细胞和效应记忆T细胞的占比情况，其中(a)示出了CD44、CD62L抗体标记的CD3+T的分布图；(b)示出了中央记忆T细胞(CD44+CD62L+)在CD3+T细胞中的占比；以及(c)示出了效应记忆T细胞(CD44+CD62L-)在CD3+T细胞中的占比。
详述
在以下的说明中，包括某些具体的细节以对各个公开的实施方案提供全面的理解。然而，相关领域的技术人员会认识到，不采用一个或多个这些具体的细节，而采用其它方法、部件、材料等的情况下仍实现实施方案。
除非本申请中另有要求，在整个说明书和所附的权利要求书中，词语“包括”、“包含”、“含有”和“具有”应解释为开放式的、含括式的意义，即“包括但不限于”。
在本公开和所附权利要求书中使用时，除非上下文另有明确规定，否则不带数量指示的单数指称物包括复数指称物。
在整个说明书中提到的“一实施方案”、“实施方案”、“在另一实施 方案中”或“在某些实施方案中”意指在至少一实施方案中包括与该实施方案所述的相关的具体参考要素、结构或特征。因此，在整个说明书中不同位置出现的短语“在一实施方案中”或“在实施方案中”或“在另一实施方案中”或“在某些实施方案中”不必全部指同一实施方案。此外，具体要素、结构或特征可以任何适当的方式在一个或多个实施方案中结合。
应当理解，在本公开的说明书和所附的权利要求书中用到的单数形式的冠词“一”(对应于英文“a”、“an”和“the”)包括复数的对象，除非文中另外明确地规定。因此，例如提到的包含“含有至少两个甲硫氨酸和至少两个锍盐中心的关环多肽”的组合物包括一种含有至少两个甲硫氨酸和至少两个锍盐中心的关环多肽，或两种或多种含有至少两个甲硫氨酸和至少两个锍盐中心的关环多肽。
定义
在本公开中，术语“乙型肝炎”，即乙型病毒性肝炎(viral hepatitis type B)，系指由乙型肝炎病毒引起的以肝脏病变为主的一种传染病。
在本公开中，术语“乙型肝炎病毒表面抗原(HBsAg)”系指乙型肝炎病毒外壳部分所含的小球形颗粒与管型颗粒。
在本公开中，术语“佐剂”，即免疫佐剂，系指非特异性免疫增强剂，当与抗原一起注射或预先注入机体时，可增强机体对抗原的免疫应答或改变免疫应答类型。
在本公开中，术语“哺乳动物”系指包括例如狗、猫、牛、羊、马和人类等的动物。在某些实施方案中，哺乳动物包括人类。
具体实施方式
一方面，本公开涉及乙型肝炎疫苗，其包含乙型肝炎病毒表面抗原(HBsAg)和佐剂，其中所述佐剂为式(I)所示的关环多肽：
在某些实施方案中，佐剂与乙型肝炎病毒表面抗原(HBsAg)为纳米级复合物。
另一方面，本公开涉及式(I)所示的关环多肽作为佐剂在制备乙型肝炎疫苗中的用途，
在某些实施方案中，佐剂与乙型肝炎病毒表面抗原(HBsAg)为纳米级复合物。
另一方面，本公开涉及用作乙型肝炎疫苗佐剂的式(I)所示的关环多肽：
在某些实施方案中，佐剂与乙型肝炎病毒表面抗原(HBsAg)为纳米级复合物。
再一方面，本公开涉及预防或治疗乙型肝炎的方法，其包括向需 要所述方法的个体给予预防有效量的乙型肝炎疫苗，其中所述乙型肝炎疫苗包含乙型肝炎病毒表面抗原(HBsAg)和佐剂，其中所述佐剂为式(I)所示的关环多肽：
在某些实施方案中，个体为哺乳动物。
在某些实施方案中，个体为人类。
在某些实施方案中，佐剂与乙型肝炎病毒表面抗原(HBsAg)为纳米级复合物。
下文中，本公开将通过如下实施例进行详细解释以便更好地理解本申请的各个方面及其优点。然而，应当理解，以下的实施例是非限制性的而且仅用于说明本公开的某些实施方案。
实施例
本公开的实施例中使用的试剂和设备均为常规的并且可商购的。例如：

实施例1
“锍盐关环”多肽的制备和鉴定
从强耀生物科技有限公司购买获得线性多肽(Fmoc-RRMEHRMEW)。用含有甲酸、乙腈和水(甲酸:乙腈:水(v:v:v)＝1:5:4)的混合溶液将线性多肽干粉溶解，并加入10eq的邻二溴苄。室温过夜反应后用HPLC分离纯化、冻干机冻干获得“锍盐关环”多肽的干粉(命名为M-CP)。利用MS对关环产物的分子量进行鉴定。图1结果显示，该多肽在两个甲硫氨酸位置引入两个带正电荷的锍盐 中心后，伴随多肽分子量由原来的1551.8Da增加至1655.9Da，这与理论分子量结果一致。随后将M-CP用去离子水溶解，Nanodrop测定浓度后存储在-20℃备用。
实施例2
多肽佐剂与HBsAg形成稳定的纳米级复合物
市售成品疫苗以混悬液的状态存在，使用之前需要混匀。为了评估M-CP“锍盐关环”多肽佐剂对乙肝抗原(HBsAg)微观形态的影响，将M-CP(2mg/mL)与20μg/mL的乙肝原液混合。采用马尔文激光粒度分析仪(DLS)持续监测乙肝抗原蛋白、添加M-CP佐剂的乙肝抗原蛋白混合物、成品乙肝混合液(含铝佐剂)的粒径和电位变化情况。从图2的形貌图中可以看出，成品乙肝疫苗放置24小时之后呈现蓬松的白色沉淀，而混有M-CP多肽的乙肝抗原溶液呈现均一浑浊的状态。且从其丁达尔现象可以看出，加入M-CP以后抗原蛋白的颗粒度增加。DLS的结果(图3)同样显示加入M-CP以后，乙肝抗原蛋白的粒径从原来的100nm，增加至230nm左右，而铝盐佐剂组(即成品制剂组)的粒径达到微米级别。从电位来看，M-CP溶液为正电，加入到带负电的HBsAg溶液中，溶液的整体电位为-10mV左右，这可为体系的稳定性提供帮助。
进一步地，借助透射电镜观察了加入M-CP以后复合物的微观形貌，图4的结果显示HBsAg蛋白呈现无规则的分散状态，而加入M-CP以后复合物呈现均一的球状结构，粒径大小与DLS的结果一致。
实施例3
多肽佐剂不影响HBsAg蛋白的稳定性
为了评估加入“锍盐关环”多肽以后HBsAg的存储稳定性，将添加了M-CP佐剂的乙肝抗原蛋白混合物于4℃静置存储，分别在第0天、1天、2天、4天、7天、14天、21天、28天、42天、56天和105天检测混合物的粒径和电位变化情况。从图5的结果可以看出，随着时间延长，HBsAg蛋白原液和加入M-CP的抗原混合液的粒径和电位 均维持不变，表明M-CP佐剂可满足HBsAg长时间稳定储存的需求。
实施例4
多肽佐剂具有良好的生物安全性
为了评估“锍盐关环”多肽的生物安全性，本公开选用经典的CCK8策略，评估M-CP对鼠源骨髓BMDCs细胞生长的影响。
(1)BMDCs的培养步骤如下：
取4只6-8周龄的C57BL/6小鼠，用CO2处死后，在超净台中取出小鼠后肢的股骨和胫骨，剔去周围的肌肉组织，并用剪刀剪开关节的两头，随后用灌满1mL 0％1640培养基的注射器冲洗骨髓，至骨腔变白。将获得的原代细胞吹打均匀后1500rpm离心5min。去除上清液的细胞用1×红细胞裂解液重悬，室温孵育5min后加入10％RPMI1640终止裂解。1500rpm离心5min后去除上清，随后用10％RPMI1640清洗两次后，用含有20ng/mL GM-CSF和10ng/mL IL-4因子的10％RPMI1640重悬细胞。将细胞密度调整为1×106个细胞/mL，以1mL/孔的细胞量接种到24孔板中，记为培养的第0天。随后在培养的第二天和第四天分别进行全量和半量换液，在第六天获得未成熟的鼠源BMDCs细胞。
(2)CCK8实验评估多肽的细胞毒性
将上述培养获得的鼠源BMDCs细胞以8000个/孔的密度提前24h接种到96孔板中。将HBsAg(2μg/mL)、成品疫苗制剂(记为HBsAg-Al，2μg/mL)、加入M-CP的抗原蛋白溶液(记为HBsAg-M-CP，其中HBsAg2μg/mL，M-CP为250μg/mL)，M-CP多肽(250μg/mL)四种溶液依次梯度稀释，给药处理BMDCs，24h后每孔加入10μL CCK8溶液。随后在37℃培养箱中继续培养2h。用全功能酶标仪测定各孔在450nm处的吸光值。以未做给药处理的细胞为对照组，按照公式(细胞存活率(％)＝(OD实验组-0.05)/(OD空白-0.05)×100)计算细胞的生长抑制率，评估M-CP的细胞毒性。图6的结果显示，在最大给药计量时，HBsAg-M-CP和M-CP给药组有接近100％的细胞存活率，表明M-CP具有良好的生物安全性。但是HBsAg-Al给药组中发现，随着给药浓度的增加， BMDCs的细胞存活率有逐渐下降的趋势，这可能是由于大量的铝盐佐剂贴附在细胞的表面，在一定程度上影响了细胞活度。已报道的乙肝疫苗注射导致的肉芽、疼痛等副作用也许与此有关。
实施例5
多肽佐剂辅助诱导DC细胞成熟
为了进一步评估“锍盐关环”多肽作为佐剂的有效性，本公开首先在细胞水平评估了M-CP诱导DC细胞成熟的情况以及免疫因子的分泌情况。
(1)多肽佐剂促进DC细胞成熟
将自制的BMDCs提前24h以5×105个细胞/孔的密度接种到24孔板中。分别将5％RPMI1640培养基、HBsAg(2μg/mL)、HBsAg-Al(2μg/mL)、HBsAg-M-CP(HBsAg为2μg/mL，M-CP为250μg/mL)与BMDCs共孵育24h。显微镜观察各组的细胞形态发现(图7)，HBsAg-M-CP处理组的细胞与control接近，无显著的细胞形态变化。而HBsAg-Al处理组细胞表面粘附很多颗粒状物质，这与图6中的细胞生存曲线结果相吻合，也预示着M-CP多肽有更良好的生物兼容性。
24h后收取细胞培养上清用于免疫因子测定。收取药物处理后的细胞，用PBS清洗后1000rpm，离心5min后，用100μL cell flow buffer重悬细胞。加入1μL CD16/CD32抗体，室温孵育10min后，分别加入CD11c-APC、CD80-FITC和CD86-PE室温孵育20min。随后补加1mL PBS，1000rpm室温离心5min后，去除上清。用PBS清洗一次，最后用1000μL cell flowbuffer重悬细胞，采用流式细胞仪检测各组细胞被荧光抗体标记的情况。以CD11c、CD80和CD86的含量评估DC细胞的成熟。图8中的结果显示，相较于对照组和HBsAg蛋白处理组，HBsAg-Al和HBsAg-M-CP组细胞表面的CD11c、CD80和CD86均有显著增加，并且HBsAg-M-CP组的表达量显著高于HBsAg-Al组，表明“锍盐关环”多肽具有更强的促DC细胞成熟的能力，这为后续T细胞和B细胞的活化提供了保障。
(2)多肽佐剂诱导免疫细胞分泌肿瘤相关因子
ELISA试剂盒测定BMDCs培养上清中肿瘤相关因子TNF-α、白介素IL-6和白介素IL-12的含量，用来评估免疫细胞活化的情况。以TNF-α为例(图9)，各组细胞上清中TNF-α的含量从高到底依次是HBsAg-M-CP>HBsAg-Al>HBsAg＝对照。HBsAg-M-CP刺激肿瘤相关因子分泌的能力显著高于HBsAg-Al组(p<0.001)。同样的，在IL-6和IL-12的检测结果中也发现了类似的情况。综合图8的结果，可以看出M-CP佐剂可以辅助抗原蛋白激活DC细胞，通过共刺激信号为下游免疫细胞的活化、增殖提供可能，进而激发后续抗原特异性免疫应答。
实施例6
多肽佐剂在动物水平诱导抗原的免疫应答
为了评估“锍盐关环”多肽在动物水平辅助乙肝抗原诱导免疫应答的情况，本公开选用BalB/c小鼠(雌性，6-8周)，均分为4组，每组6只。分别用PBS、HBsAg(2μg)、HBsAg-Al(2μg)、HBsAg-M-CP(HBsAg2μg，M-CP为250μg)通过肌肉注射的方式免疫小鼠。免疫方案参照图10，即每间隔两周免疫一次，共免疫三次。在第三次免疫后一周、两周、四周和六周通过小鼠眼眶静脉丛采血，分离获得血清，并用乙肝病毒抗体检测试剂盒检测血清中的抗体滴度。图11结果显示，在免疫后一周HBsAg-M-CP组血清中乙肝抗体滴度最高，显著高于HBsAg组(p<0.05)。在第二周、四周和六周的血清滴度数据中也发现了类似的结果，表明“锍盐关环”多肽可辅助抗原活化免疫系统，产生强有效的免疫应答。同时在实验中，HBsAg-M-CP组与成品乙肝疫苗组的抗体应答水平接近，再次表明“锍盐关环”多肽有作为乙肝疫苗佐剂的应用潜能。
实施例7
多肽佐剂同时激活体液免疫和细胞免疫通路
为了解析“锍盐关环”多肽诱导免疫应答的原因，在免疫结束后第六周获得小鼠的新鲜血液(含肝素钠的EP管收集，防止凝血)，分析 血液中免疫细胞的占比。这里以CD4+T细胞作为细胞免疫通路的标志物(CD3e，CD4染色)，以B细胞作为体液免疫通路活化的标志物(CD19染色)。取50μL小鼠新鲜血液，加入1mL 1×红细胞裂解液重悬，室温孵育5min后，1500rpm室温离心5min。去除上清后再次用PBS清洗一次。随后按照实施例5中的操作对淋巴细胞染色。清洗后上机检测荧光标记细胞的占比。图12的结果显示，添加了“锍盐关环”多肽的HBsAg组血液中B细胞(CD3e-CD19+)的占比为51.4％，显著高于铝盐组(39.3％)和HBsAg蛋白免疫组(28.5％)。表明M-CP佐剂的加入有助于抗原诱导B细胞响应。进一步，通过检测CD3e+CD4+阳性的细胞发现，HBsAg-M-CP组血清中CD4+T细胞的含量显著高于HBsAg-Al组(图13)。而HBsAg-Al与HBsAg组间无显著性差异，表明铝盐更倾向于辅助体液免疫通路活化。而“锍盐关环”多肽可同时激活细胞免疫通路和体液免疫通路，这为提高疫苗的长时间记忆能力提供了更多可能性。
实施例8
多肽佐剂诱导机体产生长期免疫响应
进一步地，在实施例7的基础上，本发明进一步评估了“锍盐关环”多肽辅助抗原实现长期有效性方面的能力。具体的，在无菌条件下，获取免疫后第六周的各组小鼠的脾脏，用注射器软胶头将脾脏研磨，并过70μm细胞筛，获得脾脏的单细胞悬浮液。将细胞密度调整为5×106个/mL，取200μL细胞与1mL HBsAg溶液(5ug/mL)混合，在37℃培养箱孵育72h后，收取细胞，并按照实施例5中的操作对细胞进行染色(CD3e、CD44、CD62L)。图14结果显示，加入M-CP多肽的免疫组，脾脏细胞被HBsAg抗原再刺激后，该组细胞中的中央记忆T细胞(图14b，CD3e+CD44+CD62L+)和效应记忆T细胞(图14c，CD3e+CD44+CD62L-)的占比最多，显著高于HBsAg-Al组。预示着“锍盐关环”多肽佐剂可显著提高疫苗的长期免疫响应，有望提高疫苗的长期有效性。
在本公开中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。
从前述中可以理解，尽管为了示例性说明的目的描述了本公开的具体实施方案，但是在不偏离本公开的精神和范围的条件下，本领域所述技术人员可以作出各种变形或改进。这些变形或修改都应落入本公开所附权利要求的范围。

Claims (8)

  1. 乙型肝炎疫苗，其包含乙型肝炎病毒表面抗原(HBsAg)和佐剂，其中所述佐剂为式(I)所示的关环多肽：
  2. 如权利要求1所述的乙型肝炎疫苗，其中所述佐剂与所述乙型肝炎病毒表面抗原(HBsAg)为纳米级复合物。
  3. 式(I)所示的关环多肽作为佐剂在制备乙型肝炎疫苗中的用途，
  4. 如权利要求3所述的乙型肝炎疫苗，其中所述佐剂与所述乙型肝炎病毒表面抗原(HBsAg)为纳米级复合物。
  5. 用作乙型肝炎疫苗佐剂的式(I)所示的关环多肽：
  6. 预防或治疗乙型肝炎的方法，其包括向需要所述方法的个体给予预防有效量的乙型肝炎疫苗，其中所述乙型肝炎疫苗包含乙型肝炎病毒表面抗原(HBsAg)和佐剂，其中所述佐剂为式(I)所示的关环多肽：
  7. 如权利要求6所述的方法，其中所述个体为哺乳动物，优选人类。
  8. 如权利要求6或7所述的方法，其中所述佐剂与所述乙型肝炎病毒表面抗原(HBsAg)为纳米级复合物。
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