CN113058033A - 一种用于预防和治疗乙型肝炎的药物组合物及其用途 - Google Patents
一种用于预防和治疗乙型肝炎的药物组合物及其用途 Download PDFInfo
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- CN113058033A CN113058033A CN201911291638.4A CN201911291638A CN113058033A CN 113058033 A CN113058033 A CN 113058033A CN 201911291638 A CN201911291638 A CN 201911291638A CN 113058033 A CN113058033 A CN 113058033A
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Abstract
本发明涉及一种用于预防和治疗乙型肝炎的药物组合物及其用途,其包含:i)HBsAg、该抗原的片段、该抗原的变体,或者其至少两种的混合物,ii)HBcAg、该抗原的片段、该抗原或者该抗原的片段的变体,或者其至少两种的混合物,iii)CpG‑ODN,该寡聚核苷酸为全硫代修饰,其序列中具有一个或多个拷贝的5’‑AACGTT‑3’基序或5’‑GTCGTT‑3’基序。本发明还涉及所述药物组合物在制备用于治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途。
Description
技术领域
本发明涉及生物制药领域,具体地涉及一种用于预防和治疗乙型肝炎的药物组合物,其包含乙肝表面抗原、乙肝核心抗原和具有免疫刺激活性的硫代寡聚脱氧核苷酸。
背景技术
乙型肝炎病毒(HBV)感染是世界范围的严重公共卫生问题之一。HBV感染是导致慢性乙型肝炎、肝硬化和肝细胞癌的重要原因(Fattovich G.J.Hepatol.2008;48:335-352)。临床治疗慢性HBV感染的常用药物主要有核苷类似物和干扰素。核苷类似物无法完全清除肝细胞内的cccDNA,且长期使用易导致耐药突变株的出现以及停药后反弹(Kwon H,LokAS.Nat Rev Gastroenterol Hepatol.2011;8:275-284)。干扰素不适合用于无症状的HBV携带者,在慢性HBV患者中,使用半年后HBeAg血清学转换发生率仅为33%,而且干扰素副作用较大也限制其应用(Tang SX,Yu GL.Lancet 1990;335(8684):302)。
目前广泛应用的乙肝蛋白疫苗通过诱导体液免疫,产生保护性中和抗体,达到预防的目的。大量的研究发现中和抗体仅能够消除胞外病毒颗粒,而胞内感染的病毒则主要依赖特异性的细胞免疫反应,辅助性T细胞、CD4+T细胞产生的IFN-γ等Th1型细胞因子,尤其是病毒特异性的杀伤性T淋巴细胞(cytotoxic T lymphocyte,CTL)来清除(ChinR,Lacamini S.Rev Med Viorl.2003:13(4):255-72)。细胞免疫反应的强弱直接决定着乙肝的预后。因此,理想的治疗性乙肝疫苗需要同时诱导特异性的体液免疫和细胞免疫,突破乙肝的免疫耐受。例如中国专利CN104043120B提供了一种治疗性乙肝疫苗,其包括乙肝表面抗原(HBsAg)、乙肝核心抗原(HBcAg)、寡聚脱氧核苷酸(CpG),可以突破乙肝的免疫耐受,用于病毒性乙型肝炎,尤其是慢性乙型肝炎的治疗。
CPG寡聚脱氧核苷酸(CPG-ODN)佐剂具有免疫增强作用,可与细胞中的TLR9(Toll-like receptor 9)结合发挥作用,诱生依赖Thl途径的免疫应答,活化B细胞分化为分泌抗原特异性抗体的浆细胞,并刺激IFN-γ表达,从而促进细胞免疫应答,增加无免疫、低免疫应答及免疫抑制人群的免疫效应。但是,CPG佐剂具有结构多样性,主要分为A型,B型和C型,不同类型的CPG佐剂在实际应用中效果差异大,成药效果不确定性高。
CPG1018是由Dynavax公司研发的一种B型CPG佐剂,现已应用于该公司的乙肝预防性疫苗产品HEPLISAVTM(Barry M,Cooper C.Expert Opin Biol Ther,2007,7(11):1731-1737)。临床研究显示,该佐剂安全性高,但免疫增强作用尚不能满足乙肝治疗性疫苗的需求。云南沃森的一项研究结果(钱雯、王丽丽、马波,免疫学杂志,2014(10))也显示,将CPG1018佐剂配合HBsAg抗原使用,效果仅略高于铝佐剂+HBsAg抗原,但低于CpG1018佐剂+铝佐剂+HBsAg抗原的双佐剂***。CPG7909是由Coley公司研发的一种B型CPG佐剂,葛兰素史克的乙肝预防性疫苗Engerix-B即应用该佐剂(Cooper C L,Davis H L.Journal ofClinical Immunology,2004,24(6):693-701)。临床研究显示,该佐剂安全性高,但免疫增强作用尚不能满足乙肝治疗性疫苗的需求。云南沃森的一项研究(钱雯、王丽丽、马波,免疫学杂志,2014(10))显示,将CPG 7909佐剂与铝佐剂进行联合之后,方可取得较好的治疗效果。但是,接种含铝佐剂的疫苗会导致人体产生一些不良反应,如红斑、皮下结节、接触性超敏、肉芽肿、肌筋膜炎等。
发明内容
针对现有技术中存在的上述问题,本发明人进行了大量试验和研究,并出乎意料地发现,将上述两种佐剂分别应用于双抗原疫苗组合物,即使不使用铝佐剂,也能够取得显著性治疗和/或预防效果。
因此,本发明的一个目的是提供一种用于预防和/或治疗乙型肝炎的药物组合物,所述药物组合物仅使用CPG1018佐剂或CPG7909佐剂,不含铝佐剂,可在慢性HBV感染患者中产生强烈的免疫应答,诱导乙肝核心抗体发生亚型转变和/或突破乙型肝炎病毒患者的免疫耐受,其既可以用于制备乙肝预防性疫苗,又可以用于制备乙肝治疗性疫苗。
本发明的另一目的是提供该药物组合物在制备用于预防和/或治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途。
为实现上述目的,本发明提供了一种药物组合物,其包含:
i)乙肝表面抗原(HBsAg)、该抗原的片段、该抗原的变体,或者其中至少两种的混合物;
ii)乙肝核心抗原(HBcAg)、该抗原的片段、该抗原的变体,或者其中至少两种的混合物;
iii)佐剂,其中所述佐剂包含序列中具有一个或多个拷贝的5’-AACGTT-3’基序的CpG寡聚脱氧核苷酸(CpG-ODN),且不包含皂苷佐剂和/或铝佐剂;或所述佐剂包含序列中具有一个或多个拷贝的5’-GTCGTT-3’基序的CpG寡聚脱氧核苷酸,且不包含皂苷佐剂和/或铝佐剂。
在本发明的一些实施方案中,所述佐剂仅为序列中具有一个或多个拷贝的5’-AACGTT-3’基序的CpG寡聚脱氧核苷酸;优选地,所述CpG寡聚脱氧核苷酸的序列为:5’-TGACTGTGAACGTTCGAGATGA-3’(SEQ ID NO:3)。
在本发明的一些实施方案中,所述佐剂仅为序列中具有一个或多个拷贝的5’-GTCGTT-3’基序的CpG寡聚脱氧核苷酸;优选地,所述CpG寡聚脱氧核苷酸的序列为:5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’(SEQ ID NO:4)。
在本发明的又一些实施方案中,所述CpG-ODN包含硫代磷酸酯连接。特别地,所述CpG-ODN为硫代CpG寡聚脱氧核苷酸,优选地为全硫代CpG寡聚脱氧核苷酸。
在本发明的再一些实施方案中,所述HBsAg具有SEQ ID NO:1所示序列。
在本发明的另一些实施方案中,所述HBcAg具有SEQ ID NO:2所示序列。优选地,所述乙肝核心抗原片段由SEQ ID NO:2中的第149~183个连续氨基酸组成,进一步优选由第152~183个连续氨基酸组成。
在本发明的又一些实施方案中,所述药物组合物中的组分i),ii)和iii)之间的相对重量比范围是2:1:1~40;优选地,组分i),ii)和iii)之间的相对重量比为2:1:1,2:1:8,2:1:20,2:1:30或2:1:40,进一步优选为2:1:1,2:1:8,2:1:20。
在本发明的又一些实施方案中,所述药物组合物还含有:iv)可药用载体。
在一个方面,本发明涉及所述药物组合物在制备用于预防和/或治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途;优选地,所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
在又一个方面,本发明涉及所述药物组合物在制备用于在对象中产生针对乙型肝炎病毒的体液免疫和/或细胞免疫应答的药物中的用途。
在另一个方面,本发明涉及所述药物组合物在制备用于使对象中乙肝核心抗体发生亚型转变的药物中的用途。
在再一个方面,本发明涉及所述药物组合物在制备用于在对象中突破乙型肝炎病毒免疫耐受之药物中的用途。
特别地,所述药物组合物为乙肝预防性疫苗或乙肝治疗性疫苗,优选为乙肝治疗性疫苗。
本发明的药物组合物实现了出乎意料的技术效果:
本申请的发明人通过大量实验发现,发现本发明的药物组合物中采用不同序列的B型CpG作为佐剂会产生不同的免疫效果,其中,CpG1018和CpG7909作为佐剂应用在乙肝疫苗中,相对于其他序列的不同的B型CpG佐剂具有突出的免疫优势,且所述药物组合物不包含皂苷佐剂或铝佐剂,在避免与之相关的毒副作用同时,与现有技术相比也具有非常显著的数据优势。本发明另一方面也摸索出了所述药物组合物中乙肝表面抗原、乙肝核心抗原和所述佐剂的最适重量比例,在所述比例范围内,具有最佳的免疫效果。
实验结果表明,本发明所述药物组合物可以突破转基因小鼠的免疫耐受,产生高滴度的抗HBsAg抗体、抗HBcAg抗体、中和抗体,介导Th1细胞免疫应答,促进IgG2a抗体亚型的分化和成熟。同时通过对其血清中乙肝表面抗原表达水平的检测显示,该药物组合物的多次免疫可以显著清除转基因小鼠体内的乙肝病毒,使其乙肝病毒表达水平下降。另外,该药物组合物在C57BL/6(N)小鼠体内能介导强抗HBcAg的免疫应答,并且实现了抗HBcAg抗体亚型转变,即表现出的乙肝表面抗体(HBcAb)的IgG2a抗体水平呈升高趋势,与乙肝感染患者痊愈的抗体亚型关系一致。与现有技术(中国专利CN104043120B)公开的技术方案(HBcAg、HBsAg和CpG ODN的组合)相比,本发明所述药物组合物在免疫刺激方面显著优于前者,药效结果具有显著的优势。
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 MolecularBiology(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。
尽管阐明本发明的宽范围的数字范围和参数是近似值,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“1至40”的范围应认为包含最小值1和最大值40之间(包含端点)的任何和所有子范围,也就是说,包括所有以最小值1或更大起始的子范围,例如1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。
术语“或”可与术语“和/或”互换使用,除非上下文另有清楚指明。
本文中使用的术语“多肽”是指氨基酸聚合物,并且无具体的最少氨基酸数目限制。因此,其同样包括肽、寡肽、二聚体、三聚体、低聚体、颗粒等。再者,术语“多肽”不仅包括在核糖体中翻译后得到的纯氨基酸聚合物,还包括经过翻译后修饰(例如糖基化、乙酰化、磷酸化、硫代等)获得的多肽。
本文中使用的术语“乙肝表面抗原(HBsAg)”旨在涵盖天然HBsAg、HBsAg抗原性片段、HBsAg功能性变体及其任意组合。特别地,所述天然HBsAg为含有226个氨基酸的天然HBsAg多肽。更特别地,所述HBsAg为来源于现今已知HBV标准基因型A、B、C、D、E、F、G和/或H的天然HBsAg多肽。在某些实施方案中,所述HBsAg具有SEQ ID NO:1所示的序列。
本文中使用的术语“HBsAg抗原的片段”旨在表示下述多肽,即该多肽具有天然HBsAg中少于226个氨基酸的连续或不连续的片段,并且所述多肽保留天然HBsAg的抗原性。
本文中使用的术语“HBsAg抗原的变体”旨在表示下述多肽,即该多肽相对于天然HBsAg或HBsAg片段有至多30个、至多25个、至多20个、至多15个、至多10个、至多5个、至多4个、至多3个、至多2个、至多1个氨基酸缺失、***、添加或替换,并且所述多肽保留天然HBsAg的功能(例如抗原性)。
本文中使用的术语“乙肝核心抗原(HBcAg)”旨在涵盖天然HBcAg、HBcAg抗原性片段、HBcAg功能性变体及其任意组合。特别地,所述天然HBcAg为含有183个氨基酸的天然HBcAg多肽。更特别地,所述天然HBcAg为来源于现今已知HBV标准基因型A、B、C、D、E、F、G和/或H的天然HBcAg。
在一些实施方案中,所述HBcAg选自HBcAg1-X多肽,其表示天然HBcAg的1-X位氨基酸的片段,特别地,X为149至183。在另一些实施方案中,所述HBcAg选自具有下述序列的多肽:SEQ ID NO:2的1-149位氨基酸、SEQ ID NO:2的1-150位氨基酸、SEQ ID NO:2的1-151位氨基酸、SEQ ID NO:2的1-152位氨基酸、SEQ ID NO:2的1-153位氨基酸、SEQ ID NO:2的1-154位氨基酸、SEQ ID NO:2的1-155位氨基酸、SEQ ID NO:2的1-156位氨基酸、SEQ ID NO:2的1-157位氨基酸、SEQ ID NO:2的1-158位氨基酸、SEQ ID NO:2的1-159位氨基酸、SEQ IDNO:2的1-160位氨基酸、SEQ ID NO:2的1-161位氨基酸、SEQ ID NO:2的1-162位氨基酸、SEQID NO:2的1-163位氨基酸、SEQ ID NO:2的1-164位氨基酸、SEQ ID NO:2的1-165位氨基酸、SEQ ID NO:2的1-166位氨基酸、SEQ ID NO:2的1-167位氨基酸、SEQ ID NO:2的1-168位氨基酸、SEQ ID NO:2的1-169位氨基酸、SEQ ID NO:2的1-170位氨基酸、SEQ ID NO:2的1-171位氨基酸、SEQ ID NO:2的1-172位氨基酸、SEQ ID NO:2的1-173位氨基酸、SEQ ID NO:2的1-174位氨基酸、SEQ ID NO:2的1-175位氨基酸、SEQ ID NO:2的1-176位氨基酸、SEQ IDNO:2的1-177位氨基酸、SEQ ID NO:2的1-178位氨基酸、SEQ ID NO:2的1-179位氨基酸、SEQID NO:2的1-180位氨基酸、SEQ ID NO:2的1-181位氨基酸、SEQ ID NO:2的1-182位氨基酸和SEQ ID NO:2的1-183位氨基酸。在某些实施方案中,所述HBcAg具有SEQ ID NO:2所示的序列。
本文中使用的术语“HBcAg抗原的片段”旨在表示下述多肽,即该多肽具有天然HBcAg中少于183个氨基酸的连续或不连续的片段,并且所述多肽保留天然HBcAg的抗原性。
本文中使用的术语“HBcAg抗原的变体”旨在表示下述多肽,即该多肽相对于天然HBcAg或HBcAg片段有至多30个、至多25个、至多20个、至多15个、至多10个、至多5个、至多4个、至多3个、至多2个、至多1个氨基酸缺失、***、添加或替换,并且所述多肽保留天然HBcAg的功能(例如抗原性)。
优选地,在本发明中,HBcAg和HBsAg在本发明的组合物中都以颗粒形式存在。
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HBsAg、HBcAg和CpG1018或CpG7909)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CpG寡聚脱氧核苷酸(即硫代CpG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CpG寡聚脱氧核苷酸。
在另一些实施方案中,所述CpG-ODN包含一个或多个的5’-AACGTT-3’基序。在又一些实施方案中,所述CpG-ODN长度为22个核苷酸,特别地,所述CpG-ODN具有下列的序列:5’-TGACTGTGAACGTTCGAGATGA-3’(SEQ ID NO:3)。
在另一些实施方案中,所述CpG-ODN包含一个或多个的5’-GTCGTT-3’基序。在又一些实施方案中,所述CpG-ODN长度为24个核苷酸,特别地,所述CpG-ODN具有下列的序列:5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’(SEQ ID NO:4)。
本文使用的“治疗有效量”或“有效量”是指足以显示其对于所施用对象的益处的剂量。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。
本文使用的术语“乙型肝炎病毒介导的疾病”旨在表示乙肝病毒(HBV)所导致、诱发、加重、提高其发生风险和/或与其有关的疾病,例如乙型肝炎病毒携带者、乙型肝炎、肝硬化、肝腹水、肝癌等。
本文使用的术语“乙肝核心抗体发生亚型转变”旨在表示施用本发明的药物组合物后,对象中抗HBcAg抗体亚型关系转变为与乙肝治愈患者中的抗体亚型关系一致,即与乙肝感染患者痊愈的抗体亚型关系一致。特别地,在小鼠中,抗HBcAg抗体亚型由IgG1>IgG2a转变为IgG2a>IgG2b>IgG1或者IgG2b>IgG2a>IgG1,例如IgG2a>IgG1,在人中,抗HBcAg抗体亚型由IgG1>IgG3>IgG4转变为IgG3>IgG1>IgG4。
本文所使用的术语“对象”是指哺乳动物,如人类,但也可以是其它动物,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。
优选的药用载体尤其是水、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。所用药用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。
附图说明
图1显示了不同佐剂对HBsAg抗原特异性IFN-γ分泌水平的影响;
图2显示了不同佐剂对HBcAg抗原特异性IFN-γ分泌水平的影响;
图3显示了不同剂量的CpG7909对HBsAg抗原特异性IFN-γ分泌水平的影响;
图4显示了不同剂量的CpG7909对HBcAg抗原特异性IFN-γ分泌水平的影响;
图5显示了本发明的药物组合物对血清中HBsAg水平的影响;
图6显示了本发明的药物组合物对血清中HBsAb水平的影响;
图7显示了本发明的药物组合物对血清中HBV DNA水平的影响;
图8显示了本发明的药物组合物对血清中HBsAg特异性抗体水平的影响;其中,A图:各组小鼠血清的HBsAb IgG水平;B图:各组小鼠血清的HBsAb IgG1水平;C图:各组小鼠血清的HBsAb IgG2a水平;D图:各组小鼠血清的HBsAb IgG2a与IgG1的比值;
图9显示了本发明的药物组合物对血清中HBcAg特异性抗体水平的影响;其中,A图:各组小鼠血清的HBcAb IgG水平;B图:各组小鼠血清的HBcAb IgG1水平;C图:各组小鼠血清的HBcAb IgG2a水平;D图:各组小鼠血清的HBcAb IgG2a与IgG1的比值;
图10显示了本发明的药物组合物对HBsAg抗原特异性IFN-γ分泌水平的影响;
图11显示了本发明的药物组合物对HBcAg抗原特异性IFN-γ分泌水平的影响。
具体实施方式
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乙肝疫苗组合物佐剂筛选材料的制备
1、HBsAg原液:HBsAg蛋白的氨基酸序列如SEQ ID NO:1所示。
HBsAg蛋白由HBsAg基因重组酵母细胞制备,酵母细胞种类包括汉逊酵母、酿酒酵母和毕赤酵母,优选为汉逊酵母。具体制备步骤参考中国专利申请CN108330145A,HBsAg基因重组汉逊酵母细胞经发酵培养,收获菌体。经破菌处理和硅胶吸附、柱层析和TFF等步骤纯化。
2、HBcAg原液:HBcAg蛋白的氨基酸序列如SEQ ID NO:2所示。
HBcAg蛋白由HBcAg基因重组酵母细胞制备,酵母细胞种类包括汉逊酵母、酿酒酵母和毕赤酵母,优选汉逊酵母。具体制备步骤参考中国专利申请CN108047316A,HBcAg基因重组汉逊酵母细胞经发酵培养,收获菌体。经破菌处理和硫酸铵、柱层析和TFF等步骤纯化。
3、CPG寡聚脱氧核苷酸原料的制备方法:
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
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。
4、使用PBS溶液(购自Hyclone公司)将步骤1和2制得的HBsAg原液和HBcAg原液分别稀释至200μg/ml和100μg/ml;使用PBS溶液将步骤3制得的各CPG原料分别溶解并稀释至100μg/ml;使用PBS溶液将Al(OH)3(购自天坛生物)溶解并稀释至100μg/ml,供下一步使用。
实施例2乙肝疫苗组合物的佐剂筛选实验
1、实验动物:C57BL/6(N)小鼠,雄性,4周龄,117只,上海灵畅实验动物技术有限公司,
2、实验分组:见表2,每次注射量为100μL/只。其中A组为阴性对照,PBS溶液100μL/只。
表2实验动物分组
3、实验步骤: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。分别使用刺激物HBsAg特异性肽库PS4和HBcAg特异性肽库PCP刺激脾细胞;按照试剂盒说明书,使用ELISPOT试剂盒(BD公司)检测HBsAg和HBcAg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,HBsAg特异性肽库序列参考中国专利CN104043120B的实施例7;HBcAg特异性肽库序列见SEQ ID NO:13~27。
4、实验结果:ELISPOT斑点结果见图1和图2,结果显示,CpG佐剂的免疫效果整体优于Al(OH)3佐剂。不同序列的B型CpG佐剂具有不同的免疫效果,其中,CpG1018、CpG7909、CpGT和CpG 684整体优于A型CpG佐剂和C型CpG佐剂,而CpG1668和CPG D2的免疫效果较差,诱导产生HBsAg和HBcAg特异性IFN-γ水平均低于A型CpG佐剂和C型CpG佐剂。与现有技术公开的CpG T佐剂相比,CpG 684免疫效果无显著性差异,而CpG1018和CpG7909具有显著的免疫优势,且两者中,CpG7909略优于CpG1018。
实施例3乙肝疫苗组合物的剂量筛选实验
1、实验动物:C57BL/6(N)小鼠,雄性,4周龄,54只,上海灵畅实验动物技术有限公司,
2、实验分组:见表3,每次注射量为100μL/只。其中A组为阴性对照,PBS溶液100μL/只。
表3实验动物分组
3、实验步骤:同实施例2。
4、实验结果:ELISPOT斑点结果见图3和图4,结果显示,CpG7909的免疫效果与剂量呈正相关,随着佐剂剂量的增加,诱导产生的HBsAg和HBcAg特异性IFN-γ水平也有所提高,但随着剂量进一步增加至300μg/只小鼠,IFN-γ水平反而下降。分析原因主要是因为种属差异性,小鼠模型无法客观反映过高剂量CpG佐剂的临床药效。为保证后续试验的准确性,选择剂量1作为实验剂量。
实施例4乙肝模型建立及实验组设置
1、实验动物及模型建立:C57BL/6(N)小鼠,雄性,4周龄,36只,上海灵畅实验动物技术有限公司,rAAV8-HBV腺病毒,购自北京五加和分子医学研究所有限公司。在C57BL/6(N)小鼠上尾静脉注射rAAV8-HBV腺病毒,建立rAAV8-HBV持续感染C57BL/6(N)的小鼠模型。
2、试剂材料:实施例1制得。
3、实验分组:见表4,每次注射量为100μL/只。其中A组为阴性对照,PBS溶液100μL/只。
表4实验动物分组
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,共给药6次,分别于尾静脉注射rAAV8-HBV病毒后第4周、第6周、第8周、第10周、第12周、第14周给药。给药开始后每2周采1次血,采血时间分别为第4周、第6周、第8周、第10周、第12周、第14周、第16周、第18周、第20周、第22周。第22周将所有小鼠处死。
实施例5乙肝疫苗组合物对小鼠血清中HBsAg水平的影响
1、血清HBsAg的检测步骤:委托南京鼓楼医院检测。
使用两步免疫测定法,首先检测样本和包被乙型肝炎表面抗体的顺磁微粒子结合,经过洗涤,加入吖啶酯标记的乙型肝炎表面抗体结合物,再经过洗涤,加入预激发液和激发液到反应混合物中,测定检测样本的相对发光值(RLU),样本中的HBsAg含量与RLU之间成正相关性,通过产生的ARCHTITECT HBsAg标准曲线测定小鼠血清样本中HBsAg浓度,最终小鼠血清样本中HBsAg浓度为测定值的50~200倍。
2、结果分析:如图5所示,CpG1018(D组)和CpG7909(E组)对应的HBsAg水平呈明显下降趋势,在免疫过程结束后可以保持稳定的免疫效果,且免疫效果显著优于阳性对照组(C组)。其中,D组的HBsAg水平从起始>6000IU/ml降低到500IU/ml左右,该组在第二次免疫(第6周)后HBsAg水平即下降40%以上,且在第14周免疫结束后下降率均维持在85%左右。E组的HBsAg水平从起始>6000IU/ml降低到470IU/ml左右,该组在第二次免疫(第6周)后HBsAg水平即下降43%以上,且在第14周免疫结束后下降率均维持在89%左右。
实施例6乙肝疫苗组合物对小鼠血清中HBsAb水平的影响
1、血清HBsAb的检测步骤:委托南京鼓楼医院检测。
使用两步免疫测定法,即首先检测样本和重组HBsAg(rHBsAg)包被顺磁微粒子混合,经过洗涤,加入吖啶酯标记的rHBsAg结合物,再经过洗涤,加入预激发液和激发液到反应混合物中,测定检测样本的相对发光值(RLU),样本中的HBsAb含量与RLU之间成正相关性,通过产生的ARCHTITECT HBsAb校准曲线测定小鼠血清样本中HBsAb浓度,最终小鼠血清样本中HBsAb浓度为测定值的50~200倍。
2、结果分析:如图6所示,CpG1018(D组)和CpG7909(E组)在第二次免疫(第6周)后开始产生HBsAb(>10mIU/ml),且随着免疫次数的增加,HBsAb水平呈现不断增长的趋势,且增长趋势显著优于阳性对照组(C组)。其中,两组均在免疫结束后两周(第十六周)之后HBsAb水平接近饱和,D组可达4.0Log即10000mIU/ml左右的HBsAb水平,E组可达4.3Log即19953mIU/ml左右的HBsAb水平;而阴性对照组(A组)和抗原对照组(B组)均未产生HBsAb水平。
实施例7乙肝疫苗组合物对小鼠血清中HBV DNA水平的影响
1、血清HBV DNA的检测步骤:使用qPCR法检测小鼠血清中HBV DNA水平,具体参考乙型肝炎病毒核酸测定试剂盒操作说明(上海科华生物工程股份有限公司),即首先参照试剂盒中煮沸法的检验方法,提取工作标准品和待测血清样本中的HBV DNA;其次采用实时荧光定量PCR法,检测工作标准品和待测血清样本中的HBV DNA的荧光信号,通过拟合“Cp值-工作标准品浓度(Log)”线性曲线(线性相关系数∣r∣≥0.95),计算待测血清样本中HBV DNA的浓度。
2、结果分析:如图7所示,CpG1018(D组)和CpG7909(E组)对应的HBV DNA水平呈现总体下降的趋势,且在第四次免疫(第10周)至第六次免疫(第14周)期间降幅较明显,降幅显著大于阳性对照(C组),且六免后趋于较稳定趋势;而阴性对照(A组)和抗原对照(B组)对应的HBV DNA水平在免疫过程中虽有波动,但总体均无降低,且有不断增长趋势。
实施例8乙肝疫苗组合物在小鼠血清中HBsAg和HBcAg特异性抗体检测
1、检测步骤:用纯化的HBsAg和HBcAg包被96孔酶标板,形成固相抗原,经过封闭处理后,将待测血清以一定的起始稀释度作倍比稀释,设多个稀释度,在96孔酶标板中加入倍比稀释后的血清样本,再与辣根过氧化物酶(HRP)标记的抗IgG/IgG1/IgG2a抗体结合,形成抗原-抗体(血清)-酶标抗体复合物,最后加入底物,用3,3',5,5'-四甲基联苯胺(TMB)显色,并用酶标仪测定450nm波长下的吸光度(OD值),显色颜色的深浅与检测样本中的HBsAg和HBcAg特异性抗体IgG/IgG1/IgG2a水平呈正相关,通过拟合“吸光度OD值-血清样本稀释倍数(Log)”的关系曲线,进行抗体滴度的判定。
2、结果分析:
1)血清中HBsAb IgG抗体及亚型检测结果:
各组不同时间用ELISA方法检测小鼠血清中HBsAb IgG抗体及亚型水平如图8所示。CpG1018(D组)和CpG7909(E组)均产生较高滴度的抗HBsAg特异性IgG/IgG1/IgG2a抗体,且随着免疫次数的增加,抗体水平不断增长,在第六次免疫(第14周)时抗体水平接近饱和,其中,D组特异性抗体滴度可达5.1个对数值以上,E组特异性抗体滴度可达5.2个对数值以上;阴性对照(A组)未检测到特异性抗体,抗原对照(B组)和阳性对照(C组)均可产生一定的HBsAg特异性IgG/IgG1/IgG2a抗体水平,但均显著低于D组和E组;且该药物组合物更偏向于Th1通路。
2)血清中HBcAb IgG抗体及亚型检测结果:
各组不同时间用ELISA方法检测小鼠血清中HBcAb IgG抗体及亚型水平如图9所示。CpG1018(D组)和CpG7909(E组)均产生较高滴度的抗HBcAg特异性IgG/IgG1/IgG2a抗体,且随着免疫次数的增加,抗体水平不断增长,在第六次免疫(第14周)时抗体水平接近饱和,其中,D组和E组的特异性抗体滴度均可达4.4个对数值以上;阴性对照(A组)为未检测到特异性抗体,抗原对照(B组)和阳性对照(C组)均可产生一定的HBsAg特异性IgG/IgG1/IgG2a抗体水平,但均显著低于D组和E组;且该药物组合物更偏向于Th1通路,D图可见特异性抗体IgG2a呈显著上升趋势,反映出该药物组合物可以促进抗HBcAg抗体亚型转变。
实施例9乙肝疫苗组合物的ELISPOT检测分析
1、细胞免疫:
1)检测步骤同实施例2
2)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、体液免疫:
1)检测步骤:小鼠处死后采血,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HBsAb和HBcAb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。
2)评价指标:HBsAb的参考值cut-off value(COV)=阴性对照平均OD值×2.1;样本的OD值小于COV值,检测结果为阴性;样本的OD值大于或等于COV值,检测结果为阳性。
HBcAb的参考值cut-off value(COV)=阴性对照平均OD值×0.3;样本的OD值小于COV值,检测结果为阳性;样本的OD值大于或等于COV值,检测结果为阴性。
3)实验结果:
表5 CPG 1018的实验结果数据
表6 CPG 7909的实验结果数据
结果分析:ELISPOT斑点结果见图10和图11,结果显示,CpG1018(D组)和CpG7909(E组)均可诱导产生较强的HBsAg和HBcAg特异性IFN-γ水平,其中,D组分别高于500SFC/106脾细胞和高于250SFC/106脾细胞,E组分别高于550SFC/106脾细胞和高于300SFC/106脾细胞,均显著高于阳性对照(C组)。且细胞免疫中,两组的HBsAg特异性IFN-γ阳转率均为100%,HBcAg特异性IFN-γ阳转率均为100%。体液免疫中,两组的HBsAb阳转率和HBcAb阳转率均为100%。
综上所述,本发明所述用于预防或治疗乙型肝炎的药物组合物使用CPG1018佐剂或CPG7909佐剂,均可在慢性HBV感染患者中产生强烈的免疫应答,诱导乙肝核心抗体发生亚型转变和/或突破乙型肝炎病毒患者的免疫耐受,其中,CPG7909佐剂的免疫效果略优于CPG1018佐剂。该药物组合物作为慢性乙肝预防性或治疗性疫苗,具有广阔的市场前景。
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。
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<110> 南京远大赛威信生物医药有限公司
<120> 一种用于预防和治疗乙型肝炎的药物组合物及其用途
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Gln Ser Arg Glu Ser Gln Cys
180
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 3
tgactgtgaa cgttcgagat ga 22
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 4
tcgtcgtttt gtcgttttgt cgtt 24
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 5
tcgttcgttc gttcgttcgt t 21
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 6
tcgacgttcg tcgttcgtcg ttc 23
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
tccatgacgt tcctgatgct 20
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 8
tgtcgtcgtc gtttgtcgtt tgtcgtt 27
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 9
gggggacgat cgtcgggggg 20
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 10
ggggacgacg tcgtgggggg g 21
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 11
tcgtcgtttc gcgcgcgccg 20
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 12
tcgtcgttcg ttcgtcgaac gacgtttgat 30
<210> 13
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 13
Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly
1 5 10 15
<210> 14
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 14
Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Asn
1 5 10 15
<210> 15
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 15
Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Asn Leu Ala Thr Trp
1 5 10 15
<210> 16
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 16
Cys Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn
1 5 10 15
<210> 17
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 17
Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro
1 5 10 15
<210> 18
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 18
Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu
1 5 10 15
<210> 19
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 19
Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser
1 5 10 15
<210> 20
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 20
Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val
1 5 10 15
<210> 21
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 21
Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu
1 5 10 15
<210> 22
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 22
Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln
1 5 10 15
<210> 23
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 23
Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe
1 5 10 15
<210> 24
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 24
Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys
1 5 10 15
<210> 25
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 25
Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly
1 5 10 15
<210> 26
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 26
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val
1 5 10 15
<210> 27
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 27
Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu
1 5 10 15
Claims (14)
1.一种药物组合物,其包含:
i)乙肝表面抗原、该抗原的片段、该抗原的变体,或者其中至少两种的混合物,
ii)乙肝核心抗原、该抗原的片段、该抗原的变体,或者其中至少两种的混合物,
iii)佐剂,所述佐剂包含序列中具有一个或多个拷贝的5’-AACGTT-3’基序的CpG寡聚脱氧核苷酸,且不包含皂苷佐剂和/或铝佐剂;或所述佐剂包含序列中具有一个或多个拷贝的5’-GTCGTT-3’基序的CpG寡聚脱氧核苷酸,且不包含皂苷佐剂和/或铝佐剂。
2.根据权利要求1所述的药物组合物,其中,所述佐剂仅为序列中具有一个或多个拷贝的5’-AACGTT-3’基序的CpG寡聚脱氧核苷酸;优选地,所述CpG寡聚脱氧核苷酸的序列为:5’-TGACTGTGAACGTTCGAGATGA-3’。
3.根据权利要求1所述的药物组合物,其中,所述佐剂仅为序列中具有一个或多个拷贝的5’-GTCGTT-3’基序的CpG寡聚脱氧核苷酸;优选地,所述CpG寡聚脱氧核苷酸的序列为:5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’。
4.根据权利要求1至3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。
5.根据权利要求4所述的药物组合物,其中,所述CpG寡聚脱氧核苷酸为硫代CpG寡聚脱氧核苷酸;优选地,所述CpG寡聚脱氧核苷酸是全硫代CpG寡聚脱氧核苷酸。
6.根据权利要求1至5中任一项所述的药物组合物,其中,所述乙肝表面抗原为SEQ IDNO:1所示序列。
7.根据权利要求1至5中任一项所述的药物组合物,其中,所述乙肝核心抗原为SEQ IDNO:2所示序列;优选地,所述乙肝核心抗原片段由SEQ ID NO:2中的第149~183个连续氨基酸组成,进一步优选由第152~183个连续氨基酸组成。
8.根据权利要求1至7中任一项所述的药物组合物,其中,组分i),ii)和iii)之间的相对重量比范围是2:1:1~40;优选地,组分i),ii)和iii)之间的相对重量比为2:1:1,2:1:8,2:1:20,2:1:30或2:1:40,进一步优选为2:1:1,2:1:8,2:1:20。
9.根据权利要求1至8中任一项所述的药物组合物,其中,所述药物组合物还含有:
iv)可药用载体。
10.根据权利要求1至9中任一项所述的药物组合物在制备用于预防和/或治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途;优选地,所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
11.根据权利要求1至9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乙型肝炎病毒的体液免疫和/或细胞免疫应答的药物中的用途。
12.根据权利要求1至9中任一项所述的药物组合物在制备用于使对象中乙肝核心抗体发生亚型转变的药物中的用途。
13.根据权利要求1至9中任一项所述的药物组合物在制备用于在对象中突破乙型肝炎病毒免疫耐受之药物中的用途。
14.根据权利要求10至13中任一项所述的用途,其中,所述药物为乙肝预防性疫苗或乙肝治疗性疫苗,优选为乙肝治疗性疫苗。
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