CN103977394B - 包含脂质体包胶的寡核苷酸和表位的免疫刺激性组合物 - Google Patents
包含脂质体包胶的寡核苷酸和表位的免疫刺激性组合物 Download PDFInfo
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Abstract
本发明涉及用于增强免疫应答的组合物、具有免疫原性的表位、其筛选和制备方法、针对肽抗原的抗体及其筛选和制备方法。本发明的组合物可用于通过增强免疫应答来预防或治疗各种免疫缺陷疾病,例如癌症、流感病毒、丙肝病毒和RSV(呼吸道合胞病毒)。
Description
本申请是申请日为2010年6月16日、申请号为201080032046.1的同名申请的分案申请。
发明背景
发明领域
本发明涉及包含脂质体包胶的寡核苷酸和表位的免疫刺激性组合物。
相关技术描述
对基于表位的肽类疫苗已进行了大规模研究,通过它们与作为B-细胞表位和T-细胞表位的B细胞受体(BCR)和MHC的结合能力,这些疫苗对于诱导和调节免疫应答以防止传染性和恶性疾病来说是至关重要的(1-3)。化学失活的疫苗被广泛用于临床,但是这些疫苗具有缺点,例如病毒重新激活的风险、用于维持疫苗稳定性的成本、引起自体免疫疾病以及某些疫苗中不支持足够的保护作用(1,3,4)。为了克服这些缺点,最近30年中开发了合成肽类,通过使用被设计用于刺激特定淋巴细胞亚群的表位来操控免疫应答,以引起选择性的B-和T-细胞瘾大。因此,肽类疫苗作为用于抗癌疫苗(5,6)和传染病例如流感病毒(3)、疟疾(7)、乙型肝炎(8)和HIV(9)的潜在有用的预防和治疗方法,受到关注。尽管肽类疫苗在各种动物模型中被活跃地研究,但它们的功效仅限于治疗人类。为了提高肽类疫苗的效能,评估了将脂质体用于疫苗递送(10),并使用佐剂例如鞭毛(11)和CpG-DNA(12)进行配制以增加免疫应答的强度。
在开发增强细胞毒性T淋巴细胞(CTL)应答的疫苗中,对脂质体作为递送介质以进行了大规模评估(10,13)。包胶的脂质体能够针对环境提供保护并递送到靶细胞。pH敏感性脂质体例如磷脂酰-β-油酰-γ-棕榈酰乙醇胺(DOPE,二油酰磷脂酰乙醇胺)/胆甾醇半琥珀酸酯(CHEMS)的特征在于在低pH(5.0)下将内含物释放到胞质溶胶中并发生脂类掺混(14)。研究人员已显示,pH敏感性脂质体提高了抗原向胞质溶胶的递送和CTL应答的诱导(15)。此外,在用包胶在pH敏感性脂质体中的从汉坦病毒核衣壳蛋白(M6)或人乳头状瘤病毒E7合成的CTL表位免疫的小鼠中,报道了有效的抗原特异性CTL应答(16)。为了增加抗原被巨噬细胞和树突状细胞的摄取,使用阳离子型脂质体作为递送介质。通过包胶的阳离子型脂质体例如lipofectamine、DC-Chol、DC-Chol/DOPE、EPC/SA/C等,增强了CTL应答和抗体生产(10)。
尽管脂质体是用于将抗原递送至APC的强有力载体,但仍对其进行了调查以增强免疫原性和佐剂活性。CpG-DNA与其他免疫刺激剂例如鞭毛、脂A、细胞因子等相比,作为用于免疫佐剂的潜在有用的治疗形式,其免疫刺激活性受到关注(17,18)。几项调查显示,CpG-DNA上调抗原呈递细胞活性、Th1免疫应答、免疫球蛋白(Ig)同种型转换(19-21)。通过用脂质体包胶CpG-DNA,增强了作为强有力佐剂的免疫刺激活性。Suzuki等显示,包胶在阳离子型脂质体中的CpG-DNA诱导了IL-12和IFN-γ的表达,并且共包胶有卵清蛋白(OVA)的CpG-DNA-脂质体引起OVA特异性CTL的诱导,其表现出对抗表达OVA的肿瘤的强细胞毒性(22)。此外,SSCL增加了被B细胞、树突状细胞和巨噬细胞的摄取,并且CpG-DNA与OVA的共包胶增加了抗原特异性IFN-γ和IgG生产(23)。此外,Li等的调查显示,共包胶在DSPC/Chol脂质体中的CpG-DNA和HER-2/neu衍生肽增强了CTL应答和IgG生产(24)。
硫代磷酸酯改性的CpG-DNA(PS-DNA)已被用于临床应用(25),所述改性用硫取代骨架中非桥接的氧,为其提供了核酸酶抗性和向细胞中的高效摄入。然而,几项研究指出,PS-DNA引起骨架相关的副作用,例如暂时性脾肿大、淋巴滤泡破坏和关节炎(26-28)。因此,调查人员开发了磷酸二酯键CpG-DNA(PO-DNA)的天然对应物以诱导没有严重副作用的最优先天免疫应答。与PS-DNA相反,在人类细胞中没有观察到PO-DNA的效应。然而,报道了在用包胶在脂质体(DOTAP,lipofectin)中的PO-DNA和非CpG-DNA刺激的人类细胞中,诱导了有效的免疫应答(29,30)。
在以前的研究中,我们通过牛分枝杆菌(M.bovis)基因组DNA的计算机辅助分析鉴定了天然磷酸二酯键CpG-DNA(PO-DNA),并筛选到具有免疫刺激活性的牛分枝杆菌基因组DNA序列(31)。我们的实验分析证实了含有CpG基序的有效PO-DNA、即MB-ODN4531(O),具有作为强有力佐剂诱导抗原驱动的Th1应答而没有严重副作用的功能效应(31,32)。在本研究中,我们比较了包胶在几种脂质体中的PO-DNA(MB-ODN4531(O))在人类和小鼠细胞中刺激免疫应答的能力。此外,我们显示,包胶在DOPE/CHEMS脂质体中的PO-DNA(MB-ODN4531(O))和几种肽显著增加了肽特异性IgG生产。这些结果表明,通过用DOPE/CHEMS脂质体递送以及使用MB-ODN4531(O)的佐剂效应,增进了肽疫苗的效能,其可以被迅捷地用于传染病大流行、治疗性抗体的开发和暴露于生物恐怖主义药剂的应用中。
在整个本申请中参考了各种出版物和专利,其引用被提供在括号中。为了充分描述本发明以及本发明所属技术领域的现状,在此将这些出版物和专利的公开内容以其全文引为本申请的参考。
发明概述
本发明人进行了深入研究,以开发能够预防和治疗各种癌症和传染病的新的免疫佐剂。结果,他们鉴定到了蛋白抗原的肽表位,并发现将所述表位和寡核苷酸包胶在具有特定组成的脂质体中提供了极大增强的免疫刺激活性。
因此,本发明的目的是提供用于增强免疫应答的组合物。
本发明的另一个目的是提供用于具有免疫原性的表位的筛选方法。
本发明的另一个目的是提供用于针对蛋白抗原的抗体的筛选方法。
本发明的另一个目的是提供针对蛋白抗原的抗体的制备方法。
本发明的另一个目的是提供对抗甲型流感病毒、癌症、丙肝病毒或RSV(呼吸道合胞病毒)的肽类疫苗组合物。
从下面的详细描述以及随附的权利要求书和附图,本发明的其他目的和优点将变得明显。
附图简述
图1a-1c显示了用脂质体(DOPE:CHEMS(1:1))和HEL(鸡卵溶菌酶)复合物腹膜内免疫的BALB/c小鼠的体液应答。以10日的时间间隔三次注射HEL-MB-ODN4531(O)-脂质体复合物。然后分析IgG(图1a)、IgG1(图1b)和IgG2a(图1c)的生产,以证实总IgG的生产和与Th1免疫应答相关的IgG2a的生产增加。我们将包胶在DOPE:CHEMS复合物中的MB-ODN4531(O)定义为Lipoplex(O)。
图2a-2e显示了考虑到亲水性、疏水性、二级结构、抗原性和两亲性,从甲型禽流感(H5N1)/Vietnam/2004毒株的HA(血细胞凝集素)选择用于制备肽-PO-DNA(MB-ODN4531(O))-脂质体复合物的表位。将所选的10个候选表位(hH5N1HA58、hH5N1HA113、hH5N1HA233、hH5N1HA336、hH5N1HA363、hH5N1HA370、hH5N1HA377、hH5N1HA384、hH5N1HA387和hH5N1HA394)制备成肽-PO-DNA(MB-ODN4531(O))-脂质体复合物,然后三次腹膜内给药于BALB/c小鼠,并收集血清。作为分析IgG量的结果,证实了每种肽特异性总IgG的量(图2a)和每种肽特异性总IgG的滴度(图2d)以及与Th1免疫应答相关的IgG2a的生产(图2c)增加了。此外,我们还检查到在二次和三次应答中产生了更大量的hH5N1HA370肽特异性IgG(IgG2a)(图2e)。A/Vietnam/1203/2004hH5N1HA蛋白的氨基酸序列根据与人类H3序列(A/Aichi/2/68)的比对进行编号。
图3a-3b显示了从收集到的血清得到的结果,所述血清通过向BLAB/c小鼠三次腹膜内给药与各种脂质体(DOPE:CHEMS(比例为6:4)、DOPE:CHEMS(比例为1:1)、DOPE:CHEMS(比例为1:0)、DOPE:CHEMS(比例为0:1)、lipofectin、lipofectamine、DOTAP或泊洛沙姆407)复合的PO-DNA(MB-ODN4531(O))和肽(H5N1HA233)来获得。结果表明,当DOPE:CHEMS的摩尔比为1:1时,抗H5N1HA233肽的总IgG的量(图3a)和总IgG的滴度(图3b)最高。
图4显示了从收集到的血清得到的结果,所述血清通过向BLAB/c小鼠三次腹膜内给药与PO-DNA、PS-DNA或各种非CpG-DNA复合的肽(H5N1HA233)和脂质体(DOPE:CHEMS(1:1))来获得。结果表明,与非CpG-DNA相比,当与PO-DNA4531(O)或PS-DNA4531(S)复合时,抗H5N1HA233肽的总IgG的量最高。MB-ODN4531在H5N1HA233肽特异性IgG生产中的佐剂效应是CG序列依赖性和骨架修饰不依赖性的。
图5显示了对应于H1N1毒株和H5N1毒株中的hH5N1HA370表位的保守序列,以及保守序列特异性IgG的生产。按照表3和表4中的描述合成了H1N1毒株和H5N1毒株中对应于A/Vietnam/1203/2004毒株的hH5N1HA370表位的长为17个氨基酸的保守序列。用共包胶在DOPE:CHEMS(比例为1:1)复合物中的50μg每种肽(hH1N1-NYHA370、hH1N1-OHHA370、hH1N1-WSNHA370、A/H1N1-TXHA370、hH5N1HA370和hH5N1-HKHA370)和50μgMB-ODN4531(O)(用Lipoplex(O)+肽表示),以10日的时间间隔对5只BALB/c小鼠进行三次腹膜内免疫接种。在最后一次免疫后10天收集抗血清,然后通过ELISA测定抗每种肽的特异性总IgG的量(图5a)、抗每种肽的特异性IgG1的量(图5b)、抗每种肽的特异性IgG2a的量(图5c)以及抗每种肽的特异性IgG的滴度(图5d)。
图6显示了使用Lipoplex(O)与对应于hH5N1HA233表位的保守序列的复合物的免疫接种对IgG生产的影响。合成了甲型流感病毒H5N1毒株(表5)和各种甲型流感病毒亚型(表6)中对应于A/VietNam/1203/2004毒株的hH5N1HA233表位的长为14个氨基酸的保守序列。用共包胶在DOPE:CHEMS(比例为1:1)复合物中的50μg每种肽和50μgMB-ODN4531(O)(用Lipoplex(O)+肽表示),以10日的时间间隔对3只BALB/c小鼠进行三次腹膜内免疫接种。在最后一次免疫后10天收集抗血清,然后通过ELISA测定抗每种肽的特异性总IgG的量(图6a,图6d)、抗每种肽的特异性IgG1的量(图6b)、抗每种肽的特异性IgG2a的量(图6c)。
图7显示了共包胶在DOPE:CHEMS复合物中的对应于hH5N1HA370(或hH5N1HA233)的保守序列和MB-ODN4531(O)对IgG生产的影响。合成了甲型流感病毒亚型(H7和H9)(表6,表9)中对应于A/VietNam/1203/2004毒株的hH5N1HA370(或hH5N1HA233)表位的长为17个氨基酸的保守序列。用共包胶在DOPE:CHEMS(比例为1:1)复合物中的50μg每种肽和50μgMB-ODN4531(O)(用Lipoplex(O)+肽表示),以10日的时间间隔对3只BALB/c小鼠进行三次腹膜内免疫接种。在最后一次免疫后10天收集抗血清,然后通过ELISA测定抗每种肽的特异性总IgG的量。
图8a-8f显示了血凝抑制和病毒中和的分析,证实了H5N1HA蛋白和H1N1HA蛋白被通过PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233、hH1N1-HKHA233)-DOPE:CHEMS复合物所产生的抗血清的特异性识别。图8a显示,由PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233、hH1N1-HKHA233)-DOPE:CHEMS复合物所产生的每种抗血清抑制由重组H5N1病毒(rH5N1病毒PR8/H5Lo)和A/WSN/1993病毒诱导的鸡红细胞的血凝。图8b-f显示,由PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233、hH1N1-HKHA233)-DOPE:CHEMS复合物所产生的每种抗血清抑制rH5N1病毒PR8/H5Lo和A/WSN/1993病毒对MDCK细胞的感染。
图9a-9d显示了在rH5N1病毒激惹的小鼠中用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-DOPE:CHEMS复合物预防接种的预防效能。以10日的时间间隔两次用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后鼻部给药10LD50rH5N1病毒(PR8/H5Lo)。图9a显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后存活。图9b表明,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后体重重新增加。图9c显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后具有正常的肺部组织。图9d显示,在用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠中,在鼻部给药10LD50rH5N1病毒后3日内和6日内肺部组织中的病毒减少。
图10a-10d显示了在小鼠适应性A/WSN/1933H1N1病毒(maA/WSN/1933病毒)激惹的小鼠中用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-DOPE:CHEMS复合物预防接种的预防效能。以10日的时间间隔两次用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后鼻部给药10LD50maA/WSN/1933病毒。图10a显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1993病毒后存活。图10b表明,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1993病毒后体重重新增加。图10c显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1993病毒后具有正常的肺部组织。图10d显示,在用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠中,在鼻部给药10LD50maA/WSN/1993病毒后3日内和6日内肺部组织中的病毒减少。
图11a-11c显示了在rH5N1病毒激惹的小鼠中用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-DOPE:CHEMS复合物预防接种的预防效能。以10日的时间间隔两次用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后鼻部给药10LD50rH5N1病毒(PR8/H5Lo)。图11a显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后存活。图11b表明,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后恢复其体重。图11c显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后具有正常的肺部组织。
图12a-12b显示了在maA/WSN/1933病毒激惹的小鼠中用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-DOPE:CHEMS复合物预防接种的预防效能。以10日的时间间隔两次用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后鼻部给药10LD50maA/WSN/1933病毒。图12a显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1993病毒后存活。图12b表明,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233)-脂质体给药的小鼠在鼻部给药10LD50maA/WSN/1993病毒后恢复其体重。
图13a-13c显示了在maA/WSN/1933病毒或rH5N1病毒(PR8/H5Lo)激惹的小鼠中用PO-DNA(MB-ODN4531(O))-肽(hH1N1-WSNHA233)-DOPE:CHEMS复合物预防接种的预防效能。以10日的时间间隔两次用PO-DNA(MB-ODN4531(O))-肽(hH1N1-WSNHA233)-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后鼻部给药10LD50maA/WSN/1933病毒或10LD50rH5N1病毒PR8/H5Lo。图13a显示,用PO-DNA(MB-ODN4531(O))-肽(hH1N1-WSNHA233)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1933病毒或10LD50rH5N1病毒后存活。图13b表明,用PO-DNA(MB-ODN4531(O))-肽(hH1N1-WSNHA233)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1933病毒或10LD50rH5N1病毒后恢复其体重。图13c显示,用PO-DNA(MB-ODN4531(O))-肽(hH1N1-WSNHA233)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1933病毒或10LD50rH5N1病毒后具有正常的肺部组织。
图14a-14c显示了在maA/WSN/1933病毒激惹的小鼠中用PO-DNA(MB-ODN4531(O))-肽(hH1N1-HKNHA233)-DOPE:CHEMS复合物预防接种的预防效能。以10日的时间间隔两次用PO-DNA(MB-ODN4531(O))-肽(hH1N1-HKHA233)-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后鼻部给药10LD50maA/WSN/1933病毒。图14a显示,用PO-DNA(MB-ODN4531(O))-肽(hH1N1-HKHA233)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1933病毒后存活。图14b表明,用PO-DNA(MB-ODN4531(O))-肽(hH1N1-HKHA233)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1933病毒后恢复其体重。图13c显示,用PO-DNA(MB-ODN4531(O))-肽(hH1N1-HKHA233)-脂质体复合物给药的小鼠在鼻部给药10LD50maA/WSN/1933病毒后具有正常的肺部组织。
图15a-15b显示了在PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233、hH1N1-HKHA233)-DOPE:CHEMS复合物给药的小鼠中,通过在鼻部给药10LD50rH5N1病毒(PR8/H5Lo)(图15a)和10LD50maA/WSN/1993病毒(图15b)后产生的抗血清进行的血凝抑制。
图16a-16f显示,在用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233、hH1N1-HKHA233)-DOPE:CHEMS复合物给药、然后鼻部给药10LD50rH5N1病毒或10LD50maA/WSN/1993病毒的小鼠的血清和BALF(支气管肺泡灌洗液)中,针对每种表位的IgG和IgA抗体的生产显著增加。
图17a-17d显示了在rH5N1病毒激惹的小鼠中用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-DOPE:CHEMS复合物预防接种的预防效能。以10日的时间间隔两次用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后在接种后2个月内鼻部给药10LD50rH5N1病毒。图17a显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后存活。图17b表明,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后恢复其体重。图17c显示,用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠在鼻部给药10LD50rH5N1病毒后具有正常的肺部组织。图17d显示,在用PO-DNA(MB-ODN4531(O))-肽(hH5N1HA370)-脂质体复合物给药的小鼠中,在鼻部给药10LD50rH5N1病毒后3日或6日内肺部组织中的病毒减少。
图18a-18d表明,使用PO-DNA(MB-ODN4531(O))-表位-DOPE:CHEMS复合物进行表位特异性抗体生产比使用病毒更有效。图18a显示,在以10日的时间间隔三次腹膜内给药PO-DNA(MB-ODN4531(O))-UV灭活的rH5N1病毒-DOPE:CHEMS复合物(Lipoplex(O)+灭活的PR8/H5Lo)的小鼠中,与病毒特异性结合的总IgG和与Th1免疫应答相关的IgG2a的生产增加。图18b显示,在以10日的时间间隔三次腹膜内给药PO-DNA(MB-ODN4531(O))-UV灭活的rH5N1病毒-DOPE:CHEMS复合物的小鼠中,总IgG的滴度增加。图18c-18d显示,在使用PO-DNA(MB-ODN4531(O))-表位(hH5N1HA233或hH5N1HA370)-DOPE:CHEMS复合物预防接种的血清中,与每种肽结合的总IgG的滴度增加。
图19a-19d显示了考虑到亲水性、疏水性、二级结构、抗原性和两亲性,从HCV-E1蛋白的三个候选表位(表10)中选择用于开发基于PO-DNA(MB-ODN4531(O))-肽-DOPE:CHEMS复合物的疫苗的表位。将所选的3个候选表位(HCV-E157、HCV-E1202、HCV-E1269)制备成PO-DNA(MB-ODN4531(O))-每种肽-脂质体复合物,然后三次腹膜内给药于BALB/c小鼠,并收集血清。证实了HCV-E157肽特异性总IgG的生产(图19a)增加。此外,在用HCV-E1202肽预防接种的血清中,总IgG的量(图19a)以及与Th1免疫应答相关的IgG2a的量和滴度(图19b和19c)增加。此外,本发明人还从用与各种脂质体复合的MB-ODN4531(O)和肽(HCV-E1202)三次腹膜内给药的BALB/c小鼠收集血清。当DOPE:CHEMS的摩尔比为1:1时(图19d),HCV-E1202肽特异性总IgG的滴度最高。
图20a-20c显示了考虑到亲水性、疏水性、二级结构、抗原性和两亲性,从HSRV(人类呼吸道合胞病毒)G和F蛋白中的3个候选表位(表10)中选择用于开发基于PO-DNA(MB-ODN4531(O))-肽-DOPE:CHEMS复合物的疫苗的表位。将所选的3个候选表位(HSRV-G1、HSRV-G150、HSRV-F99)制备成MB-ODN4531(O)-每种肽-脂质体复合物,然后三次腹膜内给药于BALB/c小鼠,并收集血清。证实了HSRV-G1肽特异性总IgG(图20a)和IgG2a(图20b)的生产增加。此外,在用HSRV-G1肽肽预防接种的血清中,HSRV-G1肽特异性总IgG的滴度(图20c)和与Th1免疫应答相关的IgG2a的生产(图20b和20c)增加。
图21a-21d显示了考虑到亲水性、疏水性、二级结构、抗原性和两亲性,从HSRV(人类呼吸道合胞病毒)F蛋白中的17个候选表位(表11和12)中选择用于开发基于PO-DNA-肽-脂质体复合物的疫苗的表位。将每种所选的17个候选表位制备成MB-ODN4531(O)-每种肽-脂质体复合物,然后三或四次腹膜内给药于BALB/c小鼠,并收集血清。证实了与4个候选(HSRV-F3a、HSRV-F3a-2、HSRV-F7、HSRV-F9)肽特异性结合的总IgG(图21a、21b、21c和21d)和IgG2a(图21b、21c和21d)的生产增加。
图22a-22b显示了考虑到亲水性、疏水性、二级结构、抗原性和两亲性,从人类整合蛋白β4(在大多数癌细胞中表达)中的6个候选表位(表13)中选择用于开发基于PO-DNA-肽-脂质体复合物的疫苗的表位。将每个所选的6个候选表位制备成MB-ODN4531(O)-每种肽-脂质体复合物,然后四次腹膜内给药于BALB/c小鼠,并收集血清。证实了与4个候选表位(hIB4-VWA-1-2、hIB4-VWA-1-3、hIB4-VWA-2、hIB4-VWA-3、hIB4-EGF-1)肽特异性结合的总IgG的量(图22a)和总IgG的滴度(图22b)增加。
图23a-23f显示了考虑到亲水性、疏水性、二级结构、抗原性和两亲性,从已知在肝癌中特异性表达的hTM4SF5(人类四次跨膜蛋白4超家族成员5)蛋白中的6个候选表位(表10)中选择表位。将所选的6个候选表位(hTM4SF5R1、hTM4SF5R2-1、hTM4SF5R2-2、hTM4SF5R2-3、hTM4SF5R2-4、hTM4SF5R2-5)制备成MB-ODN4531(O)-每种肽-脂质体复合物,然后三次腹膜内给药于BALB/c小鼠,并收集血清。证实了hTM4SF5R2-3和hTM4SF5R2-5肽特异性总IgG(图23a)以及IgG2a(图23b)的生产增加。此外,在用hTM4SF5R2-3肽预防接种的血清中,总IgG的滴度(图23b)和与Th1免疫应答相关的IgG2a的生产(图23b)增加。此外,本发明人还从用与各种脂质体复合的MB-ODN4531(O)和肽(hTM4SF5R2-3)三次腹膜内给药的BALB/c小鼠收集血清。当DOPE:CHEMS的摩尔比为1:1时(图23c),hTM4SF5R2-3肽特异性总IgG的滴度最高。本发明人从用与脂质体(DOPE:CHEMS(1:1))复合的hTM4SF5R2-3-CpG-DNA(MB-ODN4531(O)、MB-ODN4531GC(O)或MB-ODN4531(S))给药的小鼠收集血清(图23d)。在MB-ODN4531(O)中hTM4SF5R2-3肽特异性总IgG的滴度最高。本发明人从用PO-DNA(MB-ODN4531(O))-hTM4SFR2-3肽-DOPE:CHEMS复合物三次腹膜内给药的TLR9敲除的BALB/c小鼠收集血清(图23e-23f)。TLR9敲除的BALB/c小鼠没有显示出总hTM4SF5R2-3肽特异性IgG的任何滴度,表明通过PO-DNA(MB-ODN4531(O))-hTM4SFR2-3肽-DOPE:CHEMS复合物产生抗体依赖于TLR9。
图24a-24f显示了II类MHC介导的呈递和Th1分化对通过用共包胶在DOPE:CHEMS复合物中的表位和MB-ODN4531(O)免疫接种引起的IgG生产的影响。图24a显示了对共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)做出响应的IgG生产的动力学。以10日的时间间隔三次用共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)对3只BALB/c小鼠进行腹膜内注射。在相对于免疫接种时间的第1天四次腹膜内收集血清,并通过ELISA测定肽特异性总IgG、IgG1、IgG2a和IgM的量。图24b显示,CD4+细胞的瞬时贫化阻止了由共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)的免疫接种所引起的IgG生产。在相对于免疫接种时间的第-3、-1、1和3日,四次向每只小鼠腹膜内注射100μgGK1.5(抗CD4抗体)。从第0日起,以10日的间隔时间三次向3只小鼠腹膜内注射共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)(用Lipoplex(O)+TM4SF5R2-3表示)。收集血清并通过ELISA测定肽特异性总IgG的量。使用正常IgG作为对照。(图24c和24d)。由共包胶在DOPE:CHEMS复合物中的HCVE2-202肽和MB-ODN4531(O)的免疫接种所引起的IgG生产需要II类MHC和II类MHC限制的T细胞活化。以10日的时间间隔,用共包胶在DOPE:CHEMS复合物中的50μgHCVE2-202肽和50μgMB-ODN4531(O)(用Lipoplex(O)+HCVE2-202表示)三次腹膜内注射C57BL/6小鼠、C57BL/6II类MHC敲除小鼠(MHC-IIKO)(图23c)或C57BL/6OT-II转基因小鼠(OT-IITG)(图23d)(n=3)。收集血清并通过ELISA测定HCVE2-202肽特异性总IgG、IgG1、IgG2a的量(图24eand24f)。由共包胶在DOPE:CHEMS复合物中的50μghTM4SF5R2-3肽和50μgMB-ODN4531(O)的免疫接种所引起的IgG生产需要STAT4而不是STAT6。以10日的时间间隔,用共包胶在DOPE:CHEMS(比例为1:1)复合物中的50μghTM4SF5R2-3肽和50μgMB-ODN4531(O)(用Lipoplex(O)+TM4SF5R2-3表示)三次腹膜内注射BALB/c小鼠、BALB/cSTAT4敲除小鼠(STAT4KO)(图23e)或BALB/cSTAT6敲除小鼠(STAT6KO)(图23f)(n=3)。收集血清并使用ELISA测定hTM4SF5R2-3肽特异性总IgG、IgG1、IgG2a的量。这些实验执行了2或3次,获得相同的结果。
图25a-25e显示出hTM4SF5R2-3肽特异性抗体识别肝癌细胞的TM4SF5蛋白并调节肝癌细胞的功能。在Huh-7和SNU-761中通过RT-PCR观察到TM4SF5的表达(图25a)。通过FACS证实了hTM4SF5R2-3肽特异性单克隆抗体识别Huh-7和SNU-761细胞的TM4SF5蛋白(图25b)。图25c通过MTT测定法显示,当用hTM4SF5R2-3肽特异性单克隆抗体处理肝癌细胞时,表达TM4SF5的Huh-7细胞的生长被抑制。图25d显示,通过hTM4SF5R2-3肽特异性单克隆抗体的处理,Huh-7细胞的S-期减少。图25e显示,当用hTM4SF5R2-3肽特异性单克隆抗体处理表达TM4SF5的肝癌细胞(Huh-7)时,肌动蛋白具有独特的应力纤维外形,支撑着与不表达TM4SF5的细胞中相同的外延的多边形形状。尽管已知在表达TM4SF5的肝癌细胞中能够观察到肌动蛋白的异常集束,但通过肌动蛋白染色在不表达TM4SF5的细胞中检测到了具有支撑外延的多边形形状的独特外形的应力纤维。用抗hTM4SF5R2-3抗体处理的表达TM4SF5的细胞(Huh-7),强烈诱导与不表达TM4SF5的细胞的肌动蛋白类似的外形独特的应力纤维。这些结果表明抗体靶向表达hTM4SF5的细胞。
图26a-26b显示出hTM4SF5R2-3肽特异性抗体在体内抑制人类肝癌细胞生长。从肿瘤移植后第7日起,以3日的时间间隔5次给药10mg/KghTM4SF5R2-3肽特异性抗体,对带有Huh-7细胞异体移植物的无胸腺裸鼠进行处理(图26a)。当肿瘤尺寸达到±2000mm3的体积时,将小鼠处死并评估肿瘤的重量(图26b)。
图27a-27b显示了在用BNL-HCC细胞激惹的同种异体移植肝癌模型中,用PO-DNA(MB-ODN4531(O))-hTM4SF5R2-3肽-DOPE:CHEMS复合物进行预防接种的预防效能。以10日的时间间隔三次用PO-DNA(MB-ODN4531(O))-hTM4SF5R2-3肽-脂质体复合物对BALB/c小鼠进行腹膜内免疫接种,然后将含有50%基质胶的5x106个BNL-HCC细胞皮下接种到背部右侧中。在肿瘤细胞抑制后7周将小鼠处死,并评估肿瘤的重量。
发明详述
在本发明的一个方面,提供了用于增强免疫应答的组合物,所述组合物作为活性成分包含包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中的(a)免疫刺激性寡核苷酸和(b)表位。本发明人为了开发能够预防和治疗各种癌症和传染病的的新免疫佐剂已进行了深入研究。结果,它们鉴定了蛋白抗原的肽表位,并发现包胶在特定组成的脂质体中的表位和寡核苷酸提供了极大增强的免疫刺激活性。
当在本文中使用时,术语“阴离子型表面活性剂”是指由具有疏水和亲水两部分并在整个分子上带有负电荷的两亲分子构成的试剂。优选情况下,本发明的阴离子型表面活性剂包括但不限于磷脂酰甘油、心磷脂、磷脂酰丝氨酸、二酰基磷脂酰丝氨酸、磷酸二鲸蜡酯、磷脂酸、二酰基磷脂酸、油酸、N-十二烷酰磷脂酰乙醇胺、NSPE(N-琥珀酰磷脂酰乙醇胺)、NGPE(N-戊二酰磷脂酰乙醇胺)、LPG(赖氨酰磷脂酰甘油)和CHEMS(胆甾醇半琥珀酸酯)。更优选情况下,本发明的阴离子型表面活性剂是CHEMS。
当在本文中使用时,术语“中性磷脂”是指类似两性离子,在即使在一部分原子上带电荷,但在整个分子上具有零正电荷的磷脂,以及其中每个原子都不带电荷的磷脂。优选情况下,本发明的中性磷脂包括但不限于磷脂酰胆碱、DPPC(二棕榈酰磷脂酰胆碱)、DSPC(1,2-二硬脂酰-sn-甘油-3-磷酸胆碱)、DMPC(二肉豆蔻酰磷脂酰胆碱)、胆甾醇、PEG-PE(聚乙二醇磷脂酰乙醇胺)、DOPC(二油酰磷脂酰胆碱)和DOPE(二油烯基磷脂酰乙醇胺)。更优选情况下,本发明的中性磷脂是DOPE。
当在本文中使用时,术语“脂质体”是指通过形成脂双层而制备的脂类载体。脂质体通常是生物相容的,并且由于其两亲性能够通过疏水质膜。取决于制备方法和所递送的核苷酸的长度,脂质体的直径一般为20-2000nm。
根据优选实施方案,本发明的脂质体是CHEMS与DOPE的混合物。
本发明的DOPE:CHEMS的摩尔比优选为7:3-3:7,更优选为4.5:5.5-5.5:4.5,最优选为5.0:5.0。
本发明的脂质体的制备可以通过本技术领域的专业人员已知的各种方法来进行,并优选通过有机溶液混合法或去污剂混合法(US5705385;US08/660,025)来制备。更优选情况下,可以通过将DOPE与CHEMS混合,用氮气蒸发成无溶剂脂质膜的形式,溶解在醇溶液中,最后与水溶性核苷酸混合物混合,来制造脂质体。
在通过混合有机溶剂制备本发明的脂质体的情况下,所述有机溶剂包括氯仿、甲醇、乙醇、正丙醇或丁醇。优选情况下,所述有机溶剂是乙醇。
当在本文中使用时,术语“包胶”是指将待递送材料包封在相对稳定的壳中,用于在体内高效递送。
当在本文中使用时,术语“免疫刺激性”是指以可测量的程度诱导针对抗原的初始免疫应答或增加已有免疫应答。
本发明的免疫刺激性寡核苷酸包含本技术领域的专业人员已知的任何免疫刺激性寡核苷酸。例如,所述免疫刺激性寡核苷酸可以是形成发夹结构的回文序列、CpG基序、CpT基序、多个G的结构域或其他已知ISS(免疫刺激性序列)。例如,本发明的免疫刺激性寡核苷酸包含在US20080045473、WO2006/063152或WO1998/18810中公开的寡核苷酸。
所述CpG寡核苷酸包括在WO2006/080596中公开的由本发明人开发的那些CpG寡核苷酸。例如,可以使用由下式表示的CpG寡核苷酸:HKCGTTCRTGCSGM(其中R是A或G;S是C或G;H是A、T或C;K是G或T;M是C或A)。
本发明的免疫刺激性寡核苷酸包括天然存在的核苷酸、骨架修饰的核苷酸(例如肽核酸(PNA)(M.Egholm等,Nature,365:566-568(1993))、硫代磷酸酯DNA、二硫代磷酸酯DNA、氨基磷酸酯DNA、酰胺连接的DNA、MMI连接的DNA、2’-O-甲基RNA、α-DNA和膦酸甲酯DNA)、糖修饰的核苷酸(例如2'-O-甲基RNA、2'-氟代RNA、2'-氨基RNA、2'-O-烷基DNA、2'-O-烯丙基DNA、2'-O-炔基DNA、己糖DNA、吡喃糖基RNA和失水己糖醇DNA)以及碱基修饰的核苷酸(例如C-5取代的嘧啶(取代基包括氟-、溴-、氯-、碘-、甲基-、乙基-、乙烯基-、甲酰基-、乙炔基-、丙炔基-、炔基-、噻唑基-、咪唑基-和吡啶基)、具有C-7取代基的7-脱氮杂嘌呤(取代基包括氟-、溴-、氯-、碘-、甲基-、乙基-、乙烯基-、甲酰基-、炔基-、烯基-、噻唑基-、咪唑基-和吡啶基)、次黄苷和二氨基嘌呤)。优选情况下,本发明的寡核苷酸是天然存在的核苷酸。
根据优选实施方案,本发明的免疫刺激性寡核苷酸具有磷酸二酯骨架或硫代磷酸酯骨架。
本发明的免疫刺激性寡核苷酸的长度是优选为8-100个核苷酸,更优选为15-50个核苷酸,最优选为13-25个核苷酸,但不限于此。
优选情况下,本发明的免疫刺激性寡核苷酸选自SEQIDNO:14至SEQIDNO:18。更优选情况下,本发明的免疫刺激性寡核苷酸是SEQIDNO:14。
本发明人以前的实验分析证实了源于牛分枝杆菌(Mycobacteriumbovis)基因组DNA的潜在的CpG-DNA作为Th1应答性佐剂发挥效力,并且它激活转录因子NF-κB(31)。
具体来说,本发明的寡核苷酸是位于牛分枝杆菌基因组DNA上4531位的20bp的寡核苷酸MB-ODN4531(SEQIDNO:14)、MB-ODN4531上的一个CG二核苷酸被GC二核苷酸代替的MB-ODN4531(GCO)(SEQIDNO:15)、MB-ODN4531的3’-末端的7个碱基被删除并且骨架中的桥接氧原子被硫原子代替的MB-ODN4531(S)T13(SEQIDNO:16)、一个CG二核苷酸被CT二核苷酸代替并且骨架中的桥接氧原子被硫原子代替的MB-ODN4531(S)CT寡核苷酸(SEQIDNO:17)和具有与MB-ODN4531互补的序列并且骨架中的桥接氧原子被硫原子代替的MB-ODN4531(S)CS寡核苷酸(SEQIDNO:18)。本发明人的实验分析揭示,在各具有不同寡核苷酸的所有复合物中,给药MB-ODN4531寡核苷酸/肽/脂质体复合物诱导最高的总IgG水平。
当在本文中使用时,术语“表位”是指抗原与抗体相互作用的部分。更具体来说,术语表位包括能够与免疫球蛋白或T-细胞受体特异性结合的任何蛋白决定簇。此外,本发明的表位包括能够增强免疫应答的任何分子或材料。例如,本发明的表位包括但不限于肽、编码所述肽的核酸和糖蛋白。
当在本文中使用时,术语“肽”是指通过氨基酸残基之间的肽键形成的线性分子,术语“肽表位”是指包含能够诱导B细胞和/或T细胞的特异性应答的表位的肽。
本发明的肽表位的长度优选为7-30个氨基酸,更优选为10-25个氨基酸,最优选为10-17个氨基酸,但不限于此。
根据优选实施方案,本发明的表位是具有选自SEQIDNO:1至SEQIDNO:13和SEQIDNO:19至SEQIDNO:46的氨基酸序列的肽表位。
本发明人合成了源于甲型禽流感病毒的HA蛋白、甲型猪流感病毒的HA蛋白、H1N1甲型流感病毒的HA蛋白、H7甲型流感病毒的HA蛋白、H9甲型流感病毒的HA蛋白、肝癌的hTM4SF5(人类四次跨膜蛋白4超家族成员5)蛋白、人类整合蛋白β4(hIB4)、丙肝病毒的包膜蛋白、RSV(呼吸道合胞病毒)的黏附(G)糖蛋白(HRSV-G)和RSV的融合蛋白(HRSV-F)的肽。然后通过筛选在给药时增加免疫应答的肽,获得了SEQIDNO:1至SEQIDNO:13和SEQIDNO:19至SEQIDNO:46。因此,包含SEQIDNO:1至SEQIDNO:9和SEQIDNO:19至SEQIDNO:37的任一氨基酸序列的肽都具有针对甲型流感病毒的免疫刺激效应,包含SEQIDNO:10或SEQIDNO:11的氨基酸序列的肽都具有针对肝癌的免疫刺激效应,包含SEQIDNO:42至SEQIDNO46的氨基酸序列的肽都具有对人类整合蛋白β4的免疫刺激效应,包含SEQIDNO:12的氨基酸序列的肽具有针对丙肝病毒的免疫刺激效应,包含SEQIDNO:13和SEQIDNO:38至SEQIDNO41的氨基酸序列的肽都具有针对RSV的免疫刺激效应。
本发明的组合物可以包含其他药物或免疫佐剂以提供附加的免疫刺激效应。免疫佐剂的类型对于本技术领域的专业人员来说是已知的(“疫苗设计——亚基和佐剂方法”(VaccineDesign-TheSubunitandAdjuvantApproach),1995,《药物生物技术》(PharmaceuticalBiotechnology),第6卷,Powell,M.F.和Newman,M.J.主编,PlenumPress,NewYorkandLondon,ISBN0-306-44867-X)。优选情况下,本发明的免疫佐剂包括铝盐或钙盐(例如氢氧化物或磷酸盐)。
优选的免疫佐剂的实例包括但不限于:铝盐,钙盐(例如氢氧化物或磷酸盐),颗粒载体(WO96/33739)如水包油乳液(WO95/17210,EP0399843)或脂质体,源于石碱木(QuillajaSaponariaMolina)的免疫活性皂角苷提取物(例如QuilA),3-O-脱乙酰单磷酰脂A,胞壁酰二肽,3D-MPL(3-O-脱酰基单磷酰脂A)。
可以使用本发明的组合物作为治疗方法的病症或疾病的实例包括但不限于:
(i)癌症(例如胃癌、肺癌、乳腺癌、卵巢癌、肝癌、支气管癌、鼻咽癌、喉癌、胰腺癌、膀胱癌、结肠直肠癌、结肠癌、宫颈癌、脑癌、前列腺癌、骨癌、皮肤癌、甲状腺癌、甲状旁腺癌和输尿管癌);
(ii)病毒性疾病,例如由下列病毒的感染所引起的疾病:腺病毒、疱疹病毒(例如HSV-I、HSV-II、CMV或VZV)、痘病毒(例如正痘病毒,如天花或牛痘或传染性软疣)、细小核糖核酸病毒(例如鼻病毒或肠道病毒)、正粘病毒(例如流感病毒包括H5N1禽流感病毒)、副粘病毒(例如5-副流感病毒、流行性腮腺炎病毒、麻疹病毒和呼吸道合胞病毒(RSV))、冠状病毒(例如SARS)、乳多空病毒(例如乳头状瘤病毒,例如引起生殖器疣、寻常疣或跖疣的乳头状瘤病毒)、嗜肝DNA病毒(例如乙肝病毒)、黄病毒(例如丙肝病毒或登革病毒)或反转录病毒(例如慢病毒如HIV);
(iii)细菌性疾病,例如由例如下列细菌的感染引起的疾病:埃希式杆菌属(Escherichia)、肠杆菌属(Enterobacter)、沙门氏菌属(Salmonella)、葡萄球菌属(Staphylococcus)、志贺菌属(Shigella)、李斯特菌属(Listeria)、气杆菌属(Aerobacter)、螺旋杆菌属(Helicobacter)、克雷伯氏菌属(Klebsiella)、变形杆菌属(Proteus)、假单胞菌属(Pseudomonas)、奈瑟菌属(Neisseria)、梭状芽胞杆菌属(Clostridium)、芽孢杆菌属(Bacillus)、棒状杆菌属(Corynebacterium)、分枝杆菌属(Mycobacterium)、弯曲杆菌属(Campylobacter)、弧菌属(Vibrio)、沙雷氏菌属(Serratia)、普罗威登斯菌属(Providencia)、色杆菌属(Chromobacterium)、布鲁氏杆菌属(Brucella)、耶尔森氏菌属(Yersinia)、嗜血杆菌属(Haemophilus)或包特菌属(Bordetella);
(iv)其他传染病,例如衣原体、真菌性疾病包括但不限于念珠菌病、曲霉病、组织胞浆菌病、隐球菌脑膜炎,或寄生性疾病包括但不限于疟疾、卡氏肺囊虫肺炎、利什曼原虫病、隐孢子虫病、弓形体病和锥虫感染;
(v)TH2介导的特异反应性疾病,例如特异反应性皮炎或湿疹、嗜酸性细胞增多症、哮喘、过敏症、过敏性鼻炎和Ommen综合征;
(vi)某些自体免疫疾病,例如alopeciagreata、关节强硬性脊椎炎、抗磷脂综合征、自体免疫性阿狄森病、自体免疫性肾上腺炎、自体免疫性溶血性贫血、自体免疫性肝炎、自体免疫性卵巢炎和睾丸炎、自体免疫性血小板减少症、Behset病、大疱性类天疱疮、心肌病、乳糜泻-皮肤炎、慢性疲劳免疫功能障碍综合征、慢性炎性脱髓鞘性多发性神经病、Churg-Strauss综合征、瘢痕性类天疱疮、CREST综合征、冷血凝素病、克罗恩病、盘状红斑狼疮、原发性混合型冷球蛋白血症、纤维肌痛-纤维肌炎、肾小球肾炎、突眼性甲状腺肿、Guillain-Barre综合征并发桥本甲状腺炎、特发性肺纤维化、特发性血小板减少性紫癜、IgA神经病、溃疡性结肠炎引起的幼年型关节炎、扁平苔癣、红斑狼疮、美尼尔氏病、混合型结缔组织病、多发性硬化症、I型或免疫介导的糖尿病、重症肌无力、寻常性天疱疮、恶性贫血、结节性多动脉炎、多软骨炎、自身免疫性多内分泌腺综合征、风湿性多肌痛、多肌炎和皮肌炎、原发性无丙种球蛋白血症、原发性胆汁性肝硬变、牛皮癣、牛皮癣关节炎、雷诺现象、赖特综合征、类风湿性关节炎、肉样瘤病、硬皮病、僵人综合征、系统性红斑狼疮、红斑狼疮、多发性大动脉炎、颞动脉炎、巨细胞动脉炎、溃疡性结肠炎、眼色素层炎、白癜风和韦格纳肉芽肿;
(vii)炎性疾病包括哮喘、脑炎、炎性结肠炎、慢性阻塞性肺病、过敏症、脓毒性休克、肺纤维变性、未分化脊柱关节病、未分化关节病、关节炎、炎性骨质溶解和由慢性病毒或细菌感染引起的慢性炎症;以及
(viii)与伤口修复相关的疾病,例如瘢痕瘤形成和其他瘢痕类型的抑制,以及增强伤口愈合、包括慢性伤口。
优选情况下,本发明的组合物用于癌症、病毒性疾病、细菌性疾病、传染病或自体免疫疾病。
本发明的组合物可以提供成药物组合物。本发明的药物组合物除了活性成分化合物之外还包括可药用载体。本发明的药物组合物中包含的常用于药物制剂的可药用载体,包括但不限于乳糖、右旋糖、蔗糖、山梨糖醇、甘露糖醇、淀粉、阿拉伯胶、磷酸钾、藻酸盐、明胶、硅酸钾、微晶体纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、甲基羟基苯甲酸酯、滑石粉、硬脂酸镁和矿物油。本发明的药物组合物还可以包含润滑剂、保湿剂、甜味剂、调味剂、乳化剂、悬浮剂和防腐剂。适合的可药用载体和制剂的详细情况,可以在《Remington药物学》(Remington'sPharmaceuticalSciences)(第19版,1995)中发现。
本发明的药物组合物可以口服或肠胃外给药,并且优选通过肠胃外给药。对于肠胃外给药来说,它可以静脉内、皮下、肌肉内、腹膜内或透皮给药。
本发明的药物组合物的适合剂量,可以随着药物制剂方法、给药方法、患者的年龄、体重、性别、病原体状态、饮食、给药时间、给药路线、排泄率和对所使用的药物组合物的敏感性而变。优选情况下,本发明的药物组合物可以以0.001-10000mg/kg(体重)的每日剂量给药。
可以按照本技术领域的专业人员已知的常规技术,用如上所述的可药用载体和/或介质配制本发明的药物组合物,最终提供几种形式,包括单位药剂形式和多剂形式。制剂的非限制性实例包括但不限于在油性或水性介质中的溶液、悬液或乳液、酏剂、粉剂、颗粒剂、片剂和胶囊,并且还可以包含分散剂或稳定剂。
在本发明的另一方面,提供了用于具有免疫原性的表位的筛选方法,所述方法包含下列步骤:
(a)将(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中;
(b)用所述包胶有(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽的脂质体免疫非人类动物;
(c)分析所述被免疫非人类动物的免疫应答。
由于本发明的表位、肽、免疫刺激性寡核苷酸和脂质体已在上文描述,因此在这里为了避免过度重复而将其省略。
脂质体包胶的免疫刺激性寡核苷酸或肽的免疫接种方法包括本技术领域的专业人员已知的方法,优选为肠胃外给药。对于肠胃外给药来说,它可以静脉内、皮下、肌肉内、腹膜内或透皮给药。
在本发明中使用的“非人类动物”包括通常在本技术领域中使用的各种动物,优选为哺乳动物,最优选为小鼠、兔或大鼠。
被免疫动物中免疫应答的测量,通过分析用所选脂质体包胶的肽给药的对象动物的血清中抗肽抗体(总IgG、IgG1和IgG2a)的滴度来进行。优选情况下,用于测量抗体滴度的方法包括但不限于ELISA(酶联免疫吸附测定法)、侧向流试验、MIA(磁免疫测定法)、免疫沉淀。更优选情况下,可以使用ELISA分析。
当特定肽序列增加抗肽抗体的滴度时,所述特定肽被确定为表位或肽疫苗。
在本发明的另一方面,提供了针对蛋白抗原的抗体的筛选方法,所述方法包含下列步骤:
(a)将(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的蛋白抗原的肽包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中;
(b)用所述包胶有(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽的脂质体免疫非人类动物;以及
(c)通过分析所述被免疫非人类动物的免疫应答来筛选具有免疫原性的肽表位;
(d)将所述筛选到的肽表位与待分析的目标抗体相接触;
(e)将步骤(d)的结果与所述蛋白抗原相接触;以及
(f)分析所述蛋白抗原与目标抗体的结合。
本发明的筛选方法可以通过各种方法来执行,特别是通过本技术领域的专业人员已知的各种结合测定法进行的高通量方法。
本发明的蛋白抗原或候选抗体可以用可检测标记物标记。例如,所述可检测标记物包括但不限于化学标记物(例如生物素)、酶标记物(例如辣根过氧化物酶、碱性磷酸酶、过氧化物酶、萤光素酶、β-半乳糖苷酶和β-葡萄糖苷酶)、放射活性标记物(例如C14、I125、P32和S35)、荧光标记物(例如香豆素、荧光素、FITC(荧光素异硫氰酸酯)、罗丹明6G、罗丹明B、TAMRA(6-羧基-四甲基-罗丹明)、Cy-3、Cy-5、德克萨斯红、AlexaFluor、DAPI(4,6-二咪基-2-苯基吲哚)、HEX、TET、Dabsyl和FAM)、发光标记物、化学发光标记物、FRET(荧光共振能量转移)标记物或金属标记物(例如金和银)。
为了使用可检测标记的蛋白抗原或候选抗体,可以通过由标记物产生的信号来分析蛋白抗原与抗体的结合。当使用碱性磷酸酶时,可以使用溴氯吲哚磷酸(BCIP)、硝基四氮唑蓝(NBT)、萘酚-AS-B1-磷酸盐和ECF(增强化学发光物)作为底物;在使用辣根过氧化物酶的情况下,可以使用氯萘酚、氨基乙基咔唑、二氨基联苯胺、D-萤光素、光泽精(双-N-甲基吖啶硝酸酯)、试卤灵苯甲基醚、发光氨、Amplex红试剂(10-乙酰基-3,7-二羟基吩噁嗪,Pierce)、HYR(对苯二胺-HCL和邻苯二酚)、TMB(3,3,5,5-四甲基联苯胺)、ABTS(2,2-吖嗪-二[3-乙基苯并噻唑啉磺酸酯])、OPD(邻苯二胺)和萘酚/派洛宁作为底物。
可选地,蛋白抗原与抗体的结合可以不需标记相互作用物来测量。例如,可以使用微生理记录仪来分析抗体与抗原的结合。微生理记录仪是使用LAPS(光可寻址电位测定传感器)测定细胞的环境酸化率的装置。酸化率的改变可用作候选抗体与蛋白抗原结合的指示物(33)。
候选抗体与蛋白抗原的结合能力可以通过实时BIA(生物分子相互作用分析)来测定(34,35)。BIA是不需标记相互作用物的特异性相互作用实时分析技术(例如BIAcoreTM)。使用SPR(表面等离子体共振)的变化作为分子间实时反应的指示物。
在本发明的另一方面,提供了用于制备针对蛋白抗原的抗体的方法,所述方法包含下列步骤:
(a)将(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的蛋白抗原的肽包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中;
(b)用所述包胶有(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽的脂质体免疫非人类动物;以及
(c)通过分析所述被免疫非人类动物的免疫应答来筛选具有免疫原性的肽表位;
(d)通过用所述筛选到的肽表位免疫非人类动物来生产抗体。
根据本发明,从被免疫动物获得抗体的步骤通过本技术领域的专业人员已知的各种不同方法来剂型,所述方法包括乙醇沉淀方法、离子交换吸附层析或蛋白A或蛋白G柱层析。可选地,可以使用结合有特异性抗原的琼脂糖珠通过吸附层析从哺乳动物血浆分离纯的免疫球蛋白。可选地,还可以从具有从外周血淋巴结或B细胞获得的抗体蛋白的遗传信息的cDNA文库获取免疫球蛋白信息。根据所述信息,可以通过遗传工程方式制备免疫球蛋白。通过上述方法制备的遗传重组免疫球蛋白含有哺乳动物免疫球蛋白氨基酸或人源化遗传重组免疫球蛋白的基本序列及其部分突变(VaughanTJ等,“人类抗体设计”(Humanantibodiesdesign),NatureBiotech16:535-539(1998))。纯化的抗体在使用前可以储存在冰上。此外,本发明的方法可以另外包含通过从被免疫动物捕获B细胞来产生单克隆抗体、人源化抗体或亲和成熟抗体的步骤。
在本发明的另一方面,提供了针对甲型流感病毒的肽类疫苗组合物,其包含选自SEQIDNO:1至SEQIDNO:9和SEQIDNO:19至SEQIDNO:37的氨基酸序列。
在本发明的另一方面,提供了针对人类整合蛋白β4的肽类疫苗组合物,其包含选自SEQIDNO:42至SEQIDNO:46的氨基酸序列。
在本发明的另一方面,提供了针对肝癌的肽类疫苗组合物,其包含选自SEQIDNO:10和SEQIDNO:11的氨基酸序列。
在本发明的另一方面,提供了针对丙肝病毒的肽类疫苗组合物,其包含SEQIDNO:12的氨基酸序列。
在本发明的另一方面,提供了针对RSV(呼吸道合胞病毒)的肽类疫苗组合物,其包含选自SEQIDNO:13和SEQIDNO:38至SEQIDNO:41的氨基酸序列。
由于本发明的肽类疫苗在描述免疫刺激性组合物中已经提到过,因此在这里为了避免过度重复而将其省略。
在本发明的另一方面,提供了用于预防或治疗甲型流感病毒传染病、癌症、肝癌、丙型肝炎或RSV(呼吸道合胞病毒)传染病的方法。
本发明的特点和优点概述如下:
(a)本发明提供了用于增强免疫应答的组合物、具有免疫原性的表位、其筛选和制备方法、针对肽抗原的抗体及其筛选和制备方法。
(b)本发明可以有效用于通过增强免疫应答来预防或治疗各种免疫缺陷疾病,例如癌症、流感病毒、丙肝病毒和RSV(呼吸道合胞病毒)。
现在将通过实施例对本发明进行进一步详细描述。对于本技术领域的专业人员来说,显然这些实施例的目的在于更具体地说明,而在随附的权利要求书中提出的本发明的范围不限于实施例或受实施例所限。
实施例
实施例1:寡脱氧核苷酸和试剂
ODN(寡脱氧核苷酸)从SamchullyPharm(Seoul,韩国)合成。MB-ODN4531由20的碱基构成,含有三个CpG基序(下划线):AGCAGCGTTCGTGTCGGCCT。在本研究中使用的MB-ODN4531序列是磷酸二酯(O)或硫代磷酸酯(S)修饰的。MB-ODN4531(O)的硫代磷酸酯形式是MB-ODN4531(S)。MB-ODN4531GC是MB-ODN4531的衍生物,其中一个CG序列被倒置成GC(下划线):AGCAGGCTTCGTGTCGGCCT。将荧光或生物素标签偶联到每个ODN的3’末端。ODN的内毒素含量当通过Limulusamebocyte测定法(WhittakerBioproducts,Walkersville,MD,USA)测量时,小于1ng/mgODN。
表1.合成的ODN衍生物
CG二核苷酸向GC或CT的改变用下划线的粗体字母标出。MB-ODN4531(S)CS是MB-ODN4531的互补序列。无,磷酸二酯骨架连接;
S,硫代磷酸酯骨架修饰
实施例2:候选表位的选择和肽的合成
根据亲水性、疏水性、二级结构、抗原性指数、两亲性来选择肽序列。为了鉴定基于表位的肽的效应,我们从几个甲型流感病毒毒株的HA蛋白(表2、3、4、5、6、7、8和9)、肝癌的hTM4SF5(人类四次跨膜蛋白4超家族成员5)蛋白、丙肝病毒的包膜蛋白、RSV(呼吸道合胞病毒)的黏附(G)糖蛋白(G(hRSV-G))(表10)、RSV的融合蛋白(HRSV-F)(表11和12)和人类整合蛋白β4(hIB4)(表13)合成了长为14或17个氨基酸的肽。甲型流感病毒HA蛋白的氨基酸序列根据与人类H3序列(A/Aichi/2/68)的比对进行编号。肽通过Fmoc固相方法,使用自动肽合成仪(PeptronIII-R24,Peptron,Daejeon,韩国)来合成。在将合成的肽从树脂上去保护后,通过反相HPLC(Waters2690SeparationsModule,Waters,Milford,USA),使用VydacC8分析型RP柱对肽进行纯化和分析,达到纯度>90%。使用质谱仪(HP1100SeriesLC/MSD,Hewlett-Packard,Roseville,USA)对肽进行鉴定。
表2.A/VietNam/1203/2004hH5N1HA蛋白的候选表位
根据亲水性、疏水性、二级结构、抗原性指数、两亲性,从A/Vietnam/1203/2004H5N1HA蛋白(NCBI数据库AAW80717)中选择用于表位筛选的肽序列(http://tools.immuneepitpoe.org/main/index.html)。A/Vietnam/1203/2004hH5N1HA蛋白的氨基酸序列根据与人类H3序列(A/Aichi/2/68)的比对进行编号。
表3.对应于hH5N1HA370表位的序列在甲型流感病毒H5N1和H1N1毒株中的保守性
甲型流感病毒HA蛋白的氨基酸序列根据与人类H3序列(A/Aichi/2/68)的比对进行编号。
表4.对应于hH5N1HA370表位的序列在猪来源的甲型流感病毒H1N1毒株中的保守性
(分离编号)*表示含有特定序列的毒株在迄今为止分离到的猪来源的甲型流感H1N1病毒的1750个不同毒株中的编号。
表5.来自A/Vietnam/1203/2004的hH5N1HA233表位与来自其他H5N1毒株的相应序列的序列比对
(分离编号)*表示含有特定序列的毒株在迄今为止分离到的人类H5N1病毒的279个不同毒株中的编号。
表6.对应于H5N1HA233表位的序列在甲型流感病毒亚型中的保守性
表7.对应于H5N1HA233表位的序列在迄今为止报道的H1N1毒株中的保守性
(分离编号)*表示含有对应于H5N1HA233表位的特定序列的毒株在迄今为止分离到的人类H1N1病毒的2209个不同毒株中的编号。
表8.对应于H5N1HA233表位的序列在迄今为止的猪来源的甲型流感病毒H1N1毒株中的保守性
(分离编号)*表示含有对应于H5N1HA233表位的特定序列的毒株在迄今为止分离到的猪来源的甲型流感H1N1病毒的1751个不同毒株中的编号。
表9.对应于A/H1N1HA370表位的序列在迄今为止报道的甲型流感病毒亚型中的保守性
表10.人类肝癌的人类四次跨膜蛋白4超家族成员5(hTM4SF5)、HCV(HCV-E)的包膜蛋白(E蛋白)、人类RSV中的黏附糖蛋白G和融合蛋白(F蛋白)(HRSV-G,HRSV-F)的候选表位
表11.hRSVA长毒株F蛋白的候选表位
根据hRSVA长毒株F蛋白的亲水性、疏水性、二级结构、抗原性指数、两亲性来选择用于表位筛选的肽序列。
表12.hRSVA长毒株中候选表位的序列变异
表13.人类整合蛋白β4(hIB4)的候选表位
实施例3:CpG-DNA-肽(或蛋白)-脂质体复合物的制备
在本发明中使用了下列脂质体:CHEMS、Chol、DOPE和DSPC购自Sigma。DC-Chol和PEG-PE从Avanti-PolarLipids(Alabaster,AL,USA)获得。CpG-DNA和蛋白(或肽)与DOTAP(Roche,Indianapolis,IN,USA)、lipofectamine(Invitrogen,Carlsbad,CA,USA)或lipofectin(Invitrogen,Carlsbad,CA,USA)的复合物按照制造商的详细说明来制备。用DOPE/CHEMS、DSPC/Chol、DSPC/CHEMS/PEG-PE、Chol/DOPE/PEG-PE或Dc-Chol/DOPE/PEG-PE包胶CpG-DNA和蛋白(或肽)所构成的脂质体复合物的制备按照以前的报道(14,23)并进行了修改。简单来说,将DOPE和CHEMS以1:1的摩尔比混合,然后将混合物用氮气蒸发以制造无溶剂的脂质薄膜,并混合在乙醇中(终浓度为10%),然后重悬浮在等体积的水溶性CpG-DNA和蛋白(或肽)混合物中,在室温下剧烈搅拌30分钟。在将pH调整到7.0后,使用超声波发生器将lipoplex溶液轻微超声30秒,并用0.22μm滤膜过滤,然后在液氮中重复冻融三次(14,23)。
实施例4:通过CpG-DNA-肽(或蛋白)-脂质体复合物诱导体液免疫应答
<4-1>免疫接种
小鼠维持在无特定病原体条件下。4周龄雄性BALB/c(H-2b)小鼠购自CentralLab.AnimalInc.(Seoul,韩国)。本发明人的动物研究得到Hallym大学动物护理和使用管理委员会(InstitutionalAnimalCareandUseCommitteeofHallymUniversity)的批准。
以10日的时间间隔三次向4周龄BALB/c小鼠腹膜内注射HEL(鸡卵溶菌酶)(50μg/小鼠)和MB-ODN4531(O)(50μg/小鼠)复合物或HEL-MB-ODN4531-脂质体复合物。10日后,通过心脏穿刺方法获得血液,并通过离心沉淀血细胞来收集血清。从收集到的血清,通过ELISA分析抗HEL抗体(总IgG、IgG1、IgG2a)的滴度。
<4-2>ELISA
在注射后10天将小鼠处死。从小鼠收集血清并将其储存在-70℃。为了测量IgG、IgG1和IgG2a滴度,我们用10μg/ml的HEL包被96孔免疫板(NalgenNuncInternational),然后用含1%BSA的PBST阻断它们。将血清添加到每块板的顶行,然后将PBST的连续1:3稀释液置于下面的行中。我们将板在室温下温育4小时,然后用PBST清洗它们。接下来,我们加入辣根过氧化物酶偶联的山羊抗小鼠IgG抗体、抗小鼠IgG1抗体或抗小鼠IgG2a抗体,并将板温育2小时。比色测定法使用一步ABTS(PierceBiotechnologyInc.,Rockford,IL,USA)显色,我们使用LabsystemsMultiskan微孔板读板器(GMIInc.,Ramsey,MI,USA)测量405nm处的吸收值(19)。
调查了用HEL-MB-ODN4531和脂质体复合物腹膜内免疫接种的BALB/c小鼠的体液免疫应答。注射HEL-MB-ODN4531-脂质体复合物与注射单独的HEL、HEL-MB-ODN4531混合物或HEL-脂质体复合物相比增加了抗体量,证实了MB-ODN4531-脂质体复合物在体液免疫应答中的免疫佐剂效应。不完全弗氏佐剂是从60年前就已使用的代表性免疫佐剂。但它的局限性在于它不诱导细胞免疫刺激效应,并且不能应用于人类。MB-ODN4531-脂质体复合物被证明起到增加体液免疫力以及通过刺激免疫细胞诱导细胞免疫应答的免疫佐剂的作用。此外,MB-ODN4531-脂质体复合物在Th1-免疫应答特异性IgG2a抗体的生产中有效。
<4-3>小鼠和免疫接种
小鼠维持在无特定病原体条件下。4周龄雄性BALB/c(H-2b)小鼠购自CentralLab.AnimalInc.(Seoul,韩国)。本发明人的动物研究得到Hallym大学动物护理和使用管理委员会(InstitutionalAnimalCareandUseCommitteeofHallymUniversity)的批准。
以10日的时间间隔三或四次向小鼠腹膜内注射肽(50μg/小鼠)-CpG-DNA(MB-ODN4531)-脂质体复合物。
<4-4>抗原特异性IgELISA
在注射后10天将小鼠处死。从小鼠收集血清并将其储存在-70℃。将获得的血清用PBS/0.2%叠氮钠进行1:10稀释,然后储存在-20℃。为了测量IgG、IgG1和IgG2a的总量,我们用10μg/ml的每种肽包被96孔免疫板(NalgenNuncInternational,Rochester,NY,USA),然后用含有1%BSA的含0.05%Tween20的PBS(PBST)阻断它们。将血清用PBS进行1:400稀释,并添加到每块板的孔中。
为了检测总IgG、IgG1和IgG2a,我们使用了1:5,000稀释的生物素偶联的大鼠抗小鼠IgG抗体、大鼠抗小鼠IgG1抗体和大鼠抗小鼠IgG2a抗体(BDPharmingen,SanDiego,CA,USA)。
为了测量IgG、IgG1和IgG2a的滴度,我们用10μg/ml每种蛋白或肽包被96孔免疫板(NalgenNuncInternational,Rochester,NY,USA),然后用含有1%BSA的PBST阻断它们。将血清添加到每块板的顶行,然后将PBST的1:3连续稀释液置于下面的行中。我们将板在室温下温育2小时,然后用PBST清洗它们。接下来,我们加入生物素偶联的大鼠抗小鼠IgG抗体、大鼠抗小鼠IgG1抗体或大鼠抗小鼠IgG2a抗体,并将板温育2小时。在清洗三次后,向板加入HRP(辣根过氧化物酶)偶联的链亲和素,然后温育1小时。使用TMB溶液(KPL,Gaithersburg,MD,USA)执行比色测定分析,并使用分光光度计(SpectraMax250,MolecularDevices,Downingtown,PA,USA)分析450nm处的吸收值。
实施例5:使用CpG-DNA-H5N1(或其他流感病毒毒株)HA肽-脂质体复合物筛选表位
<5-1>CpG-DNA-H5N1(或其他流感病毒毒株)HA肽-脂质体复合物的免疫接种
如实施例3中所述,根据亲水性、疏水性、二级结构、抗原性和两亲性从各种甲型流感病毒的HA(血细胞凝集素)蛋白中选择候选表位(表2-表9),来制备CpG-DNA-肽-脂质体复合物。将制备的CpG-DNA-肽-脂质体复合物(50μg/小鼠)以10日的时间间隔三次腹膜内注射到BALB/c小鼠中。10日后,通过心脏穿刺方法获得血液,并通过离心沉淀血细胞收集血清。从收集的血清,通过ELISA分析抗肽抗体(总IgG、IgG1、IgG2a)的滴度和量。
<5-2>ELISA
在注射后10日将小鼠处死。将获得的血清用PBS/0.2%叠氮钠进行1:10稀释,然后储存在-20℃。向96孔免疫板(NalgenNuncInternational,Rochester,NY,USA)添加每种所选的肽(10μg/ml,碳酸氢钠缓冲液,pH9.6),并在4℃留置16小时。然后按照实施例<4-4>中所述分析与每种肽特异性结合的总IgG(图2a、2d、2e、5a、5d、6a、6d和7)、IgG1(图2b、2e、5b、5b和7)、IgG2a(图2c、2e、5c、6c和7)的量和滴度。
在选自甲型禽流感(H5N1)/Vietnam/2004毒株HA蛋白的肽中,hH5N1HA58、hH5N1HA233、hH5N1HA336和hH5N1HA370增加了总IgG(图2a、2d)和IgG2a(图2c)的量和滴度。对应于hH5N1HA370肽的H1N1肽(hH1N1-NYHA370、hH1N1-OHHA370、hH1N1-WSNHA370、A/H1N1-TXHA370)和H5N1病毒中存在的hH5N1-HKHA370肽(表3、表4)增加了总IgG的量和滴度(图5a、5d)。此外,与Th1免疫应答相关的IgG2a的生产也增加了(图5c)。此外,H5N1病毒毒株中存在的对应于hH5N1HA233肽的hH5N1HA233-1、hH5N1HA233-2、hH5N1HA233-3、hH5N1HA233-4、hH5N1HA233-6、hH5N1HA233-7、hH5N1HA233-9和hH5N1HA233-11肽(表5),以及各种甲型流感病毒毒株中存在的对应于hH5N1HA233肽的hH1N1-WSNHA233、hH1N1-HKHA233、hH1N1-ThaiHA233、A/H1N1-TXHA233、mH2N5HA233、hH7N7HA233、hH9N2HA233、sH15N2HA233肽(表6),增加了总IgG的量(图6d)。
另外,H7N7和H9N2病毒中存在的对应于hH5N1HA370肽的hH7N7HA370、hH9N2-STHA370和hH9N2-HKHA370肽(表9),增加了总IgG的量(图7a)。H7N7和H9N2病毒中存在的对应于hH5N1HA233肽的hH7N7HA233和hH9N2HA233肽(表6)增加了总IgG的量(图7b)。
实施例6:CpG-DNA-肽-脂质体复合物对体液免疫应答的诱导依赖于脂质体和CpG-DNA的种类
如实施例3中所述将MB-ODN4531-肽(hH5N1HA233)与各种脂质体(DOPE:CHEMS(6:4、1:1、1:0和0:1)、lipofectin、lipofectamine、DOTAP和泊洛沙姆407)复合,如实施例<4-3>中所述三次腹膜内注射到BALB/c小鼠并收集血清。作为测量hH5N1HA233肽特异性总IgG的量的结果(图3a),它在DOPE:CHEMS摩尔比为1:1时最高。H5N1HA233肽特异性总IgG的滴度也在DOPE:CHEMS摩尔比为1:1时最高(图3b)。
如实施例3中所述将肽(hH5N1HA233)-脂质体(DOPE:CHEMS(1:1))与表1中显示的各种PO-DNA或PS-DNA进行复合,如实施例<4-3>中所述三次腹膜内注射到BALB/c小鼠,然后收集血清。作为测量H5N1HA233肽特异性总IgG的量的结果,当使用PO-DNAMB-ODN4531(O)和PS-DNAMB-ODN4531(S)复合物时量最高(图4)。这些结果表明PO-DNA或PS-DNA的CG序列发挥了重要作用。
实施例7:由CpG-DNA-肽-脂质体复合物产生的抗体的血凝抑制和病毒中和的分析
<7-1>重组H5N1病毒
通过八质粒反向遗传学方法(36),将A/Vietnam/1203/2004(H5N1)和A/PuertoRico/8/34(PR8)(H1N1)流感病毒的基因区段克隆到用于病毒挽救和基因重配的质粒中。将如此获得的病毒在10日龄孵化鸡卵的尿囊腔中繁殖。这些重配病毒包括“PR8/H5Lo”,其带有禽谱系A/Vietnam/1203/2004的HA基因区段和PR8的7个互补基因区段。
<7-2>血凝抑制测定法
血凝抑制测定法按照以前的描述进行(37)。简单来说,将病毒稀释至4HA单位,并与等体积的受体破坏酶处理过的血清样品的两倍连续稀释液在室温下温育1小时。向孔中加入等体积的0.5%鸡红细胞并温育30分钟,以测量HI滴度。
<7-3>病毒中和测定法
在MDCK细胞中进行的病毒中和测定法按照以前的描述进行(38)。将约100PFU/ml的流感病毒(rH5N1病毒和A/WSN/1933)与等体积的热灭活的两倍连续稀释的血清样品在37℃温育1小时。温育后,向增补有10%FBS和TPCK((L-甲苯磺酰基氨酰-2-苯基)乙基氯甲基酮,1μg/ml)处理过的胰蛋白酶的最低必需培养基中的MDCK细胞合生单层加入该混合物。将细胞培养72小时,然后测定细胞病理效应。通过下列方程计算中和百分率:中和(%,抑制百分数)=[(只用病毒处理的噬斑数–与病毒混合的连续稀释血清的噬斑数)/只用病毒处理的噬斑数]x100。
如实施例3中所述将PO-DNA-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233或hH1N1-HKHA233)与脂质体(DOPE:CHEMS)复合,如实施例<4-3>中所述三次腹膜内注射到BALB/c小鼠并收集血清。证实了hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233或hH1N1-HKHA233肽特异性抗体抑制了由rH5N1病毒PR8/H5Lo和A/WSN/1933病毒所诱导的血凝(图8a)。
如实施例3中所述将PO-DNA-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233、hH1N1-HKHA233或A/H1N1-TXHA233)与脂质体(DOPE:CHEMS)复合,如实施例<4-3>中所述三次腹膜内注射到BALB/c小鼠并收集血清。将hH5N1HA233肽特异性抗体预先用rH5N1病毒PR8/H5Lo和A/WSN/1933处理(图8b),然后感染MDCK细胞。证实了病毒的滴度低,并且hH5N1HA233肽特异性抗体中和病毒。
将hH5N1HA370肽特异性抗体预先用rH5N1病毒PR8/H5Lo和A/WSN/1933处理(图8c),然后感染MDCK细胞。证实了病毒的滴度低,并且hH5N1HA370肽特异性抗体中和病毒。
将给药PO-DNA-肽(hH1N1-WSNHA233或hH1N1-HKHA233)-脂质体(DOPE:CHEMS)的血清预先用rH5N1病毒PR8/H5Lo和A/WSN/1933处理,然后感染MDCK细胞。证实了病毒的滴度低,并且每种肽特异性抗体中和病毒(图8d和8e)。
此外,将给药PO-DNA-肽(A/H1N1-TXHA233)-脂质体(DOPE:CHEMS)的血清预先用rH5N1病毒PR8/H5Lo和A/WSN/1933处理,然后感染MDCK细胞。证实了病毒的滴度低,并且A/H1N1-TXHA233肽特异性抗体中和病毒(图8f)。
实施例8:CpG-DNA-肽-脂质体复合物预防接种的效能
<8-1>预防接种和病毒激惹实验
以10日的时间间隔两次向4周龄BALB/c小鼠腹膜内注射包胶在DOPE/CHEMS脂质体中的增补有50μgMB-ODN4531(O)的50μg肽。第二次免疫接种后10日,用10LD50maA/WSN/1933或10LD50rH5N1病毒鼻内接种小鼠进行激惹。
<8-2>病毒激惹后的体重和存活率测量
感染后,每日观察小鼠的临床征兆并称量体重。10LD50rH5N1病毒鼻内感染后3日,小鼠开始失去体重并在12日内死亡。但是用PO-DNA-肽(hH5N1HA370、hH5N1HA233或hH1N1-WSNHA230)-脂质体(DOPE:CHEMS)接种两次然后用10LD50rH5N1病毒鼻内感染的小鼠直到第9日才失去体重,并且逐渐恢复并存活(图9a、9b、11a、11b、13a和13b)。
用PO-DNA-肽(hH5N1HA370、hH5N1HA233、hH1N1-WSNHA233或hH1N1-HKHA233)-脂质体(DOPE:CHEMS)接种两次然后用10LD50maA/WSN/1933病毒鼻内感染的小鼠起先失去体重,然后逐渐恢复并存活(图10a、10b、12a、12b、13a、13b、14a和14b)。
用PO-DNA-肽(hH5N1HA233)-脂质体(DOPE:CHEMS)接种两次然后用maA/WSN/1933病毒鼻内感染的小鼠显示出50%的存活率(图12a)。
用PO-DNA-肽(hH1N1-WSNHA233)-脂质体(DOPE:CHEMS)接种两次然后用rH5N1病毒PR8/H5Lo鼻内感染的的小鼠显示出38%的存活率(图13a)。
<8-3>肺组织染色
在指定时间点通过吸入二乙醚麻醉剂处死小鼠并取出整个肺组织。为了进行组织病理学检查,将肺固定在4%的缓冲甲醛溶液中,通过常规方法包埋在石蜡中,并切成4μm厚的切片。将样本用苏木精和曙红染色。在用PO-DNA-肽(hH5N1HA233或hH5N1HA370)-脂质体(DOPE:CHEMS)接种两次然后用10LD50rH5N1病毒或10LD50maA/WSN/1933病毒鼻内感染的小鼠中,肺组织恢复到正常状况(图9c、10c、11c)。
此外,在用PO-DNA-肽(hH5N1-WSNHA233或hH5N1-HKHA233)-脂质体复合物以10日的时间间隔接种两次然后用10LD50rH5N1病毒或10LD50maA/WSN/1933病毒鼻内感染的小鼠中,肺组织恢复到正常状况(图13c和14c)。
<8-4>小鼠组织中病毒滴度的测量
在用10LD50rH5N1病毒或10LD50maA/WSN/19333鼻内感染3日和6日后,分离肺脏并在1mlPBS中匀浆,以分析病毒的滴度。将病毒储用液或肺组织匀浆液的10倍连续稀释悬液添加到6孔板中MDCK细胞的合生单层上,并在室温下温育1小时进行吸附,每10分钟振摇一次。取出悬液,并用含有2%oxoid琼脂、5%NaHCO3、1%DEAE葡聚糖和(L-甲苯磺酰基氨酰-2-苯基)乙基氯甲基酮(TPCK,1μg/ml)处理过的胰蛋白酶的MEM覆盖细胞。在37℃温育3天后,将板用1ml结晶紫染色15分钟,以使我们能够观察到噬斑。对噬斑数进行计数以确定滴度。在用PO-DNA-肽(hH5N1HA370)-脂质体(DOPE:CHEMS)接种两次然后用10LD50rH5N1病毒或10LD50maA/WSN/1933病毒鼻内感染的小鼠中,肺组织的病毒滴度减少了(图9d和10d)。
<8-5>血凝抑制测定法
在用PO-DNA-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233或hH1N1-HKHA233)-脂质体(DOPE:CHEMS)接种两次然后用10LD50rH5N1病毒鼻内感染的小鼠中,由所产生的抗体诱导的凝血的抑制显著增加(图15a)。
在用PO-DNA-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233或hH1N1-HKHA233)-脂质体(DOPE:CHEMS)接种两次然后用maA/WSN/1933病毒鼻内感染的小鼠中,由所产生的抗体诱导的凝血的抑制显著增加(图15b)。
<8-6>抗体的测量
将小鼠用PO-DNA-肽(hH5N1HA233、hH5N1HA370、hH1N1-WSNHA233或hH1N1-HKHA233)-脂质体(DOPE:CHEMS)接种两次,然后用10LD50rH5N1病毒或10LD50maA/WSN/1933病毒鼻内感染。在感染后6天分离血清。也分离了BALF(支气管肺泡灌洗液)以测量hH5N1HA229或hH5N1HA371肽特异性抗体(总IgG和IgA)的量。当用10LD50rH5N1病毒感染时,血清中的总IgG和BALF中的IgA显著增加(图16a、16c和16f)。当用10LD50maA/WSN/1933病毒感染时,血清中的总IgG和BALF中的IgA显著增加(图16b、16d、16e和16f)。
实施例9:CpG-DNA-肽-脂质体复合物预防接种的记忆效能
<9-1>预防接种和病毒激惹试验
以10日的时间间隔两次向4周龄BALB/c小鼠腹膜内注射包胶在DOPE:CHEMS脂质体中的增补有50μgMB-ODN4531(O)的50μg肽。第二次免疫接种后两个月,用10LD50rH5N1病毒鼻内接种小鼠进行激惹。
<9-2>病毒激惹后的体重和存活率测量
感染后,每日观察小鼠的临床征兆并称量体重。用10LD50rH5N1病毒鼻内感染后3天,小鼠开始失去体重,并在14天内死亡。但是用PO-DNA-肽(hH5N1HA370)-脂质体(DOPE:CHEMS)接种两次然后在两个月内用10LD50rH5N1病毒鼻内感染的小鼠直到11天才失去体重,并且逐渐恢复并存活(图17a和17b)。
<9-3>肺组织染色
在指定时间点通过吸入二乙醚麻醉剂处死小鼠并取出整个肺组织。为了进行组织病理学检查,将肺固定在4%的缓冲甲醛溶液中,通过常规方法包埋在石蜡中,并切成4μm厚的切片。将样本用苏木精和曙红染色。在用PO-DNA-肽(hH5N1HA370)-脂质体(DOPE:CHEMS)以10日的时间间隔接种两次然后用10LD50rH5N1病毒鼻内感染的小鼠中,肺组织恢复到正常状态(图17c)。
<9-4>小鼠组织中病毒滴度的测量
在用10LD50rH5N1病毒鼻内感染3日或6日后,分离肺组织并在1mlPBS中匀浆,以分析病毒的滴度。病毒滴度通过实施例<8-4>中所描述的噬斑测定法测量。在用PO-ODN-肽(hH5N1HA370)-脂质体(DOPE:CHEMS)接种两次然后用10LD50rH5N1病毒鼻内感染的小鼠中,在感染后6日内肺组织的病毒滴度显著降低(图17d)。
实施例10:使用CpG-DNA-肽-脂质体复合物的表位筛选效能
<10-1>重组甲型禽流感病毒的灭活
将rH5N1病毒(PR8/H5Lo)暴露于紫外光(UV)以便灭活病毒,波长为254nm,暴露时间为5分钟,病毒粒子与UV光之间的距离为5cm。通过噬斑测定法验证病毒感染性的灭活。
<10-2>免疫接种
用灭活的rH5N1病毒或灭活的rH5N1病毒与脂质体的复合物或灭活的rH5N1病毒-MB-ODN4531(50μg/小鼠)-脂质体复合物腹膜内注射4周龄BALB/c小鼠。以10日的时间间隔三次注射同样量的灭活rH5N1病毒与MB-ODN4531的混合物。10日后,通过心脏穿刺方法获得血液,并通过离心沉淀血细胞来收集血清。从收集的血清,按照实施例<4-4>中的描述通过ELISA分析抗重组H5N1病毒抗体(总IgG、IgG1、IgG2a)的量和滴度。
<10-3>ELISA
在注射后10日将小鼠处死。将获得的血清用PBS/0.2%叠氮钠进行1:10稀释,然后储存在-20℃。向96孔免疫板(NalgenNuncInternational,Rochester,NY,USA)添加每种所选的肽(hH5N1HA233、hH5N1HA370)或灭活的rH5N1病毒(10μg/ml,碳酸氢钠缓冲液,pH9.6),并在4℃留置16小时。然后按照实施例<4-4>中所述分析与H5N1病毒或每种肽特异性结合的总IgG、IgG1和IgG2a的生产(图18)。
证实了在用灭活rH5N1病毒接种的血清中,总IgG的量和滴度(图18a和18b)以及与Th1免疫应答相关的IgG2a的生产增加了。
在甲型禽流感/H5N1/Vietnam/2004毒株的HA蛋白中,hH5N1HA233和hH5N1HA370肽增加每种肽特异性总IgG的滴度,而用灭活rH5N1病毒免疫接种的血清不显示hH5N1HA233或hH5N1HA370肽特异性抗体的任何滴度(图18c和18d),表明在用MB-ODN4531(O)-每种肽-脂质体(DOPE:CHEMS)复合物免疫接种的血清中产生了hH5N1HA233或hH5N1HA370肽特异性抗体。
实施例11:使用CpG-DNA-肽-脂质体复合物筛选HCV表位
<11-1>CpG-DNA-HCV肽-脂质体复合物的免疫接种
在HCVE1和E2蛋白中,如实施例3中所述根据亲水性、疏水性、二级结构、抗原性和两亲性选择了三个表位(表10),用于制备CpG-DNA-HCV肽-脂质体复合物。如实施例<4-3>中所述,将制备的CpG-DNA-HCV肽-脂质体复合物(50μg/小鼠)以10日的时间间隔三次腹膜内注射到BALB/c小鼠中。10日后,通过心脏穿刺方法获得血液,并通过离心沉淀血细胞收集血清。从收集的血清,通过ELISA分析抗肽抗体(总IgG、IgG1、IgG2a)的滴度。
<11-2>ELISA
在注射后10日将小鼠处死。将获得的血清用PBS/0.2%叠氮钠进行1:10稀释,然后储存在-20℃。向96孔免疫板(NalgenNuncInternational,Rochester,NY,USA)添加每种所选的肽(10μg/ml,碳酸氢钠缓冲液,pH9.6),并在4℃留置16小时。然后按照实施例<4-4>中所述分析与每种肽特异性结合的总IgG(图19a)、IgG1和IgG2a(图19b和19c)的生产。
证实了在用选自HCVE2蛋白的HCV-E2202肽免疫接种的血清中,总IgG的滴度(图19c)和与Th1免疫应答相关的IgG2a的生产增加了。
<11-3>脂质体类型对CpG-DNA-肽-脂质体诱导的体液免疫应答的影响
如实施例3中所述将MB-ODN4531-肽(HCV-E1202)与各种类型的脂质体(DOPE:CHEMS(1:1)、DSPC:Chol(1:1)、DSPC:CHEMS:PEG-PE(1:1:1)、Chol:DOPE:PEG-PE(1:1:1)、Dc-Chol:DOPE:PEG-PE(1:1))复合,并如实施例<4-4>中所述三次腹膜内注射到BALB/c小鼠。作为测量HCV-E2202肽特异性总IgG的滴度的结果,在DOPE:CHEMS的摩尔比为1:1时滴度最高(图19d)。
实施例12:使用CpG-DNA-肽-脂质体复合物筛选hRSV表位
<12-1>CpG-DNA-hRSV肽-脂质体复合物的免疫接种
在hRSVG和F蛋白中,如实施例3中所述根据亲水性、疏水性、二级结构、抗原性和两亲性选择了20个表位(表10、11和12),用于制备CpG-DNA-每种hRSV肽-脂质体(DOPE:CHEMS)复合物。如实施例<4-3>中所述,将制备的CpG-DNA-每种hRSV肽-脂质体复合物(50μg/小鼠)以10日的时间间隔三次或四次腹膜内注射到BALB/c小鼠中。10日后,通过心脏穿刺方法获得血液,并通过离心沉淀血细胞收集血清。从收集的血清,通过ELISA分析抗肽抗体(总IgG、IgG1、IgG2a)的滴度。
<12-2>ELISA
在注射后10日将小鼠处死。将获得的血清用PBS/0.2%叠氮钠进行1:10稀释,然后储存在-20℃。向96孔免疫板(NalgenNuncInternational,Rochester,NY,USA)添加每种所选的肽(10μg/ml,碳酸氢钠缓冲液,pH9.6),并在4℃留置16小时。然后按照实施例<4-4>中所述分析与每种肽特异性结合的总IgG(图20a和21)、IgG1和IgG2a(图21b和21c)的生产。
证实了在用来自hRSVG和F蛋白的HRSV-G1、HRSV-Fa3、HRSV-Fa3-2、HRSV-F7和HRSV-F9肽免疫接种的血清中,总IgG的量(图20a、21a和21d)和与Th1免疫应答相关的IgG2a的生产(图20b、20c、21b、21c和21d)增加了。
实施例13:使用CpG-DNA-肽-脂质体复合物筛选人类整合蛋白β4表位<13-1>CpG-DNA-hIB4肽-脂质体复合物的免疫接种
在大多数癌细胞中表达的人类整合蛋白β4蛋白(hIB4)中,如实施例3中所述根据亲水性、疏水性、二级结构、抗原性和两亲性选择了6个表位(表13),用于制备CpG-DNA-每种hIB4肽-脂质体(DOPE:CHEMS)复合物。如实施例<4-3>中所述,将制备的CpG-DNA-每种hIB4肽-脂质体复合物(50μg/小鼠)以10日的时间间隔三次或四次腹膜内注射到BALB/c小鼠中。10日后,通过心脏穿刺方法获得血液,并通过离心沉淀血细胞收集血清。从收集的血清,通过ELISA分析抗肽抗体(总IgG)的滴度。
<13-2>ELISA
在注射后10日将小鼠处死。将获得的血清用PBS/0.2%叠氮钠进行1:10稀释,然后储存在-20℃。向96孔免疫板(NalgenNuncInternational,Rochester,NY,USA)添加每种所选的肽(10μg/ml,碳酸氢钠缓冲液,pH9.6),并在4℃留置16小时。然后按照实施例<4-4>中所述分析总IgG的量(图22a)和总IgG的滴度(图22b)。
证实了在用选自hIB4蛋白的hIB4-VWA-1-2、hIB4-VWA-1-3、hIB4-VWA-2、hIB4-VWA-3和hIB4-EGF-1肽免疫接种的血清中,总IgG的量(图22a)和总IgG的滴度(图22b)增加了。
实施例14:使用CpG-DNA-肽-脂质体复合物筛选肝癌特异性TM4SF5蛋白表位
<14-1>CpG-DNA-TM4SF5肽-脂质体复合物的免疫接种
在TM4SF5蛋白中,如实施例3中所述根据亲水性、疏水性、二级结构、抗原性和两亲性选择了6个表位(表10),用于制备CpG-DNA-TM4SF5肽-脂质体(DOPE:CHEMS)复合物。如实施例<4-3>中所述,将制备的CpG-DNA-每种TM4SF5肽-脂质体复合物(50μg/小鼠)以10日的时间间隔三次腹膜内注射到BALB/c小鼠中。10日后,通过心脏穿刺方法获得血液,并通过离心沉淀血细胞收集血清。从收集的血清,通过ELISA分析抗每种肽抗体(总IgG、IgG1、IgG2a)的滴度。
<14-2>ELISA
在注射后10日将小鼠处死。将获得的血清用PBS/0.2%叠氮钠进行1:10稀释,然后储存在-20℃。向96孔免疫板(NalgenNuncInternational,Rochester,NY,USA)添加每种所选的肽(10μg/ml,碳酸氢钠缓冲液,pH9.6),并在4℃留置16小时。然后按照实施例<4-4>中所述分析与每种肽特异性结合的总IgG(图23a)、IgG1和IgG2a(图23b)的生产。
证实了在用选自TM4SF5蛋白的TM4SF5R2-3或TM4SF5R2-5肽免疫接种的血清中,总IgG的滴度(图23a)和与Th1免疫应答相关的IgG2a的生产(图23b)增加了。
<14-3>脂质体类型对CpG-DNA-肽-脂质体诱导的体液免疫应答的影响
如实施例3中所述将MB-ODN4531-肽(TM4SF5R2-3)与各种类型的脂质体(DOPE:CHEMS(1:1)、DSPC:Chol(1:1)、DSPC:CHEMS:PEG-PE(1:1:1)、Chol:DOPE:PEG-PE(1:1:1)、Dc-Chol:DOPE:PEG-PE(1:1))复合,并如实施例<4-4>中所述三次腹膜内注射到BALB/c小鼠。作为测量TM4SF5R2-3肽特异性总IgG的滴度的结果,在DOPE:CHEMS的摩尔比为1:1时滴度最高(图23c)。
<14-4>CpG-DNA类型对CpG-DNA-肽-脂质体诱导的体液免疫应答的影响
如实施例3中所述将TM4SF5R2-3肽-脂质体DOPE:CHEMS(1:1)与表1中显示的各种类型的PO-DNA(MB-ODN4531(O)、MB-ODN4531(GCO)或PS-DNA(MB-ODN4531(S))复合,并如实施例<4-4>中所述三次腹膜内注射到BALB/c小鼠。作为测量TM4SF5R2-3肽特异性总IgG的滴度的结果,在使用PO-DNA(MB-ODN4531(O))或PS-DNA(MB-ODN4531(S))时滴度最高(图23d)。
<14-5>TLR9对CpG-DNA-肽-脂质体诱导的免疫应答的影响
为了调查TLR9在用共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)注射的小鼠的抗体生产中的作用,我们使用BALB/cTLR9敲除小鼠和野生型小鼠评估了IgG生产。正如所预期的,共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)的IgG生产能力绝对依赖于TLR9,因为当用共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)注射时,TLR9敲除小鼠不产生IgG(图23e)。相反,发现用HEL与IFA的组合注射小鼠增加了野生型小鼠和TLR9敲除小鼠的IgG生产(图23f)。
<14-5>II类MHC介导的呈递和Th1分化对CpG-DNA-肽-脂质体诱导的体液免疫应答的影响
为了评估对表位和MB-ODN4531(O)共包胶DOPE:CHEMS复合物免疫接种作出响应的IgG生产的动力学,我们用共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)以10日的时间间隔三次腹膜内注射BALB/c小鼠。BALB/c小鼠在二次和三次应答中产生较大量肽特异性IgG(IgG2a)(图24a)。接下来,我们调查了对共包胶在DOPE:HEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)作出响应的IgG生产对II类MHC介导的呈递的需要性。如图24b中所示,通过用抗CD4抗体腹膜内注射BALB/c小鼠以贫化CD4+细胞,极大降低了肽特异性IgG的生产。此外,我们还观察到在II类MHC敲除小鼠和OT-II转基因小鼠中,对共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)的免疫接种作出响应的IgG和IgG2a生产的减少(图24c、24d)。我们还研究了在对共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)的免疫接种作出响应的IgG生产中Th1分化的参与性。在STAT4敲除小鼠中,检测到对共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)的免疫接种作出响应的IgG和IgG2a生产的降低,所述STAT4促进T细胞分化成Th1细胞(图24e)。然而,如图24f中所示,没有观察到STAT6敲除对共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)诱导的IgG生产的影响。这些结果表明,在CD4+细胞中,来自共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽和MB-ODN4531(O)的免疫接种的IgG(IgG2a)的生产,需要II类MHC介导的呈递和Th1分化。
实施例15:小鼠抗hTM4SF5单克隆抗体的生产
在用共包胶在DOPE:CHEMS复合物中的hTM4SF5R2-3肽(50μg)和MB-ODN4531(O)(50μg)以10日的时间间隔三次腹膜内注射BALB/c小鼠后,按照标准杂交瘤技术筛选产抗hTM4SF5R2-3肽特异性mAb的杂交瘤细胞(39)。通过蛋白A柱层析(AmershamPharmaciaBiotech,Piscataway,NJ,USA)从腹水液纯化抗hTM4SF5R2-3肽mAb(IgG2a)。
实施例16:通过由CpG-DNA-TM4SF5肽-脂质体复合物产生的单克隆抗体识别TM4SF5蛋白
<16-1>TM4SF5在人类肝细胞瘤细胞中的表达分析
人类肝癌细胞系(Huh-7)购自美国典型培养物保藏中心(AmericanTypeCultureCollection)(ATCC,Manassas,VA,USA)。人类肝细胞系(SNU-398、SNU-423、SNU-739和SNU-761)购自韩国细胞系库(KoreanCellLineBank)(Seoul,韩国)。Huh-7、SNU-398、SNU-423、SNU-739和SNU-761细胞培养在含有10%FBS(胎牛血清;Hyclone,Logan,UT,USA)的RPMI1640培养基中。人类正常肝细胞(PromoCell,Heidelberg,德国)按照制造商的详细说明进行培养。所有细胞在5%CO2和95%空气条件下在37℃温育。为了调查hTM4SF5mRNA的表达,进行RT-PCR。使用RNeasy小提试剂盒(Qiagen,Germantown,MD,USA)分离总RNA,并通过以前描述的方法进行cDNA的制备(40)。使用下列引物组执行标准的25个PCR循环:人类β-肌动蛋白,5’-GGGTCAGAAGGATTCCTATG-3’和5’-CCTTAATGTCACGCACGATTT-3’(500bp);hTM4SF5。Huh-7和SNU-761细胞显示出高的TM4SF5mRNA表达水平(图25a)。
<16-2>通过识别TM4SF5R2-3肽的单克隆抗体识别TM4SF5蛋白(FACS分析)
为了调查抗体的结合,将肝癌细胞在含0.1%BSA的PBS中清洗,并在4℃下与10μg/ml人类IgG(sigma)温育20分钟,用于阻断Fc受体结合。在阻断后,将细胞与纯化的抗TM4SF5R2-3肽抗体(实施例15)培养1小时。然后将细胞在含0.1%BSA的PBS中清洗,并与FITC偶联的山羊抗小鼠IgG抗体(BDBiosciences)在4℃培养30分钟。证实了纯化的抗TM4SF5R2-3肽抗体与被证明表达TM4SF5mRNA的Huh-7和SNU-761细胞结合(图25c)。
实施例17:通过由CpG-DNA-TM4SF5肽-脂质体复合物产生的抗体抑制肝癌细胞生长
<17-1>通过抗抗TM4SF5R2-3肽单克隆抗体抑制细胞生长
在用抗TM4SF5R2-3肽抗体(10μg/ml)处理72小时后,通过MTT测定法分析人类肝癌细胞(Huh-7和SNU-739)的生长。
将肝癌细胞在48孔板中温育72小时,向每块板加入3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑(MTT,Sigma-Aldrich)溶液,并在37℃继续温育4小时。在除去培养基后,将甲腊晶体溶解在DMSO中。使用分光光度计(SpectraMax250,MolecularDevices,Downingtown,PA,USA)在570nm处监测显色,参比波长为650nm。通过MTT测定法证实了表达TM4SF5的Huh-7细胞当用抗TM4SF5R2-3肽抗体处理时,其生长被抑制(图25d)。相反,不表达TM4SF5的细胞(SNU-739)不受影响(图25d)。
<17-2>通过抗TM4SF5R2-3肽抗体调控细胞周期
将肝癌细胞(Huh-7和SNU-739)用抗TM4SF5R2-3肽抗体(10μg/ml)处理72小时,并观察细胞周期。通过在溶解于含RNase(200μg/ml)的PBS的PI(碘化丙啶)(20μg/ml)中进行温育,来测量DNA含量。将细胞在室温下染色30分钟,并通过FACScan流式细胞仪(BDbiosciences)进行分析。通过ModFitLT3.0软件分析数据以获得细胞周期分布。
对每个群体中细胞周期阶段的分布进行比较。观察到表达TM4SF5的Huh-7细胞当用抗TM4SF5R2-3肽抗体处理时,其S期被停止。相反,不表达TM4SF5的SNU-739的细胞周期不受影响(图25d)。
<17-3>通过抗TM4SF5R2-3肽抗体抑制肝癌细胞
已经发现,表达TM4SF5的肝癌细胞失去细胞-细胞接触抑制能力,从而发育成肝癌细胞(41)。尽管表达TM4SF5的肝癌细胞显示出肌动蛋白的异常集束,但在不表达TM4SF5的细胞中,通过肌动蛋白染色检测到了支撑外延的多边形形状的应力纤维的独特外形(41)。因此,我们用抗TM4SF5R2-3肽抗体(10μg/ml)对肝癌细胞(Huh-7和SNU-739)处理72小时,并对肌动蛋白进行观察。
在抗hTM4SF5抗体(10μg/ml)处理前,将细胞在12孔板中的玻璃盖片上培养1天。在用抗体处理细胞72小时后,将细胞用4%聚甲醛固定,用0.1%TritonX-100通透化,并用偶联有罗丹明的鬼笔环肽(MolecularProbes,Eugene,OR,USA)染色。细胞核用HoechstNo.33258染色。用LSM510METANLO(CarlZeiss,Jena,德国)对固定后的样品进行扫描。
当用抗hTM4SF5R2-3肽特异性抗体处理表达TM4SF5的肝细胞瘤细胞(Huh-7)时,肌动蛋白具有与不表达TM4SF5的细胞中相同的支撑外延的多边形形状的应力纤维的独特外形,表明抗体靶向表达hTM4SF5的细胞(图25f)。这些结果表明,由共包胶在脂质体(DOPE:CHEMS)中的MB-ODN4531(O)和hTM4SF5R2-3所产生的抗体可以有效地用于检测hTM4SF5蛋白,并且抗体介导的hTM4SF5定向与肝细胞瘤细胞的功能变化相关,其能够使细胞生长降低,以改变细胞功能的多样性。
实施例18:在体内通过hTM4SF5R2-3肽特异性抗体抑制肝癌细胞生长
<18-1>裸鼠中的异种移植实验
将5x106个肝癌细胞(Huh-7)皮下注射到裸鼠的右胁。在癌症生长至直径为5mm后,将样品分成三组,即PBS、正常IgG和抗TM4SF5R2-3肽抗体组(n=5/组)。每个组以3日的时间间隔5次注射PBS和每种抗体(100mg/Kg小鼠)。在5周的观察期间使用卡尺测量癌症的总体积。异种移植实验的分析揭示,靶向肝癌细胞的抗TM4SF5R2-3肽抗体足以在体内降低肿瘤生长(图26a和26b)。
实施例19:通过用MB-ODN4531(O)-hTM4SF5R2-3肽-脂质体复合物预防接种在体内抑制肝癌细胞生长
<19-1>小鼠肿瘤同种异体移植实验
以10日的时间间隔三次用MB-ODN4531(O)-TM4SF5R2-3肽-脂质体(DOPE:CHEMS)复合物腹膜内注射4周龄BALB/c小鼠。在第三次免疫接种后10日,将含有5x106个BNL-HCC细胞的50%基质胶皮下注射到BALB/c小鼠的右胁中。在7周的观察期间使用卡尺测量癌症的总体积。用MB-ODN4531(O)-TM4SF5R2-3肽-脂质体(DOPE:CHEMS)复合物免疫接种的小鼠与未处理的小鼠相比,肿瘤体积明显减小(图27a和27b).
尽管已经描述了本发明的优选实施方案,但应该理解,在本发明的精神范围之内对其进行改变和修改,对于本技术领域的专业人员来说可能变得显而易见,本发明的范围将由随附的权利要求书及其等效物确定。
本申请还涉及下列实施方案:
1.一种用于增强免疫应答的组合物,所述组合物作为活性成分包含了包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中的(a)免疫刺激性寡核苷酸和(b)表位。
2.项目1的组合物,其中阴离子型表面活性剂选自磷脂酰甘油、心磷脂、磷脂酰丝氨酸、二酰基磷脂酰丝氨酸、磷酸二鲸蜡酯、磷脂酸、二酰基磷脂酸、油酸、N-十二烷酰磷脂酰乙醇胺、NSPE(N-琥珀酰磷脂酰乙醇胺)、NGPE(N-戊二酰磷脂酰乙醇胺)、LPG(赖氨酰磷脂酰甘油)和CHEMS(胆甾醇半琥珀酸酯)。
3.项目2的组合物,其中阴离子型表面活性剂是CHEMS(胆甾醇半琥珀酸酯)。
4.项目1的组合物,其中中性磷脂选自磷脂酰胆碱、DPPC(二棕榈酰磷脂酰胆碱)、DSPC(1,2-二硬脂酰-sn-甘油-3-磷酸胆碱)、DMPC(二肉豆蔻酰磷脂酰胆碱)、胆甾醇、PEG-PE(聚乙二醇磷脂酰乙醇胺)、DOPC(二油酰磷脂酰胆碱)和DOPE(二油烯基磷脂酰乙醇胺)。
5.项目4的组合物,其中中性磷脂是DOPE(二油烯基磷脂酰乙醇胺)。
6.项目1的组合物,其中脂质体是CHEMS和DOPE的混合物。
7.项目6的组合物,其中脂质体含有摩尔比为4.5:5.5至5.5:4.5的DOPE和CHEMS。
8.项目7的组合物,其中DOPE与CHEMS的摩尔比为5.0:5.0。
9.项目1的组合物,其中免疫刺激性寡核苷酸具有磷酸二酯骨架或硫代磷酸酯骨架。
10.项目9的组合物,其中免疫刺激性寡核苷酸含有CpG基序、CpT基序、多个G的结构域或形成发夹二级结构的回文序列。
11.项目1的组合物,其中表位是具有选自SEQIDNO:1至SEQIDNO:13和SEQIDNO:19至SEQIDNO:46的氨基酸序列的肽表位。
12.一种用于具有免疫原性的表位的筛选方法,所述方法包含下列步骤:
(a)将(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中;
(b)用所述包胶有(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽的脂质体免疫非人类动物;
(c)分析所述被免疫非人类动物的免疫应答。
13.一种用于针对蛋白抗原的抗体的筛选方法,所述方法包含下列步骤:
(a)将(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的蛋白抗原的肽包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中;
(b)用所述包胶有(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽的脂质体免疫非人类动物;以及
(c)通过分析所述被免疫非人类动物的免疫应答来筛选具有免疫原性的肽表位;
(d)将所述筛选到的肽表位与待分析的目标抗体相接触;
(e)将步骤(d)的结果与所述蛋白抗原相接触;以及
(f)分析所述蛋白抗原与目标抗体的结合。
14.一种用于制备针对蛋白抗原的抗体的方法,所述方法包含下列步骤:
(a)将(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的蛋白抗原的肽包胶在含有阴离子型表面活性剂和中性磷脂的脂质体中;
(b)用所述包胶有(i)免疫刺激性寡核苷酸和(ii)作为表位候选材料的肽的脂质体免疫非人类动物;以及
(c)通过分析所述被免疫非人类动物的免疫应答来筛选具有免疫原性的肽表位;
(d)通过用所述筛选到的肽表位免疫非人类动物来生产抗体。
15.项目12至14任一项的方法,其中阴离子型表面活性剂是CHEMS(胆甾醇半琥珀酸酯)。
16.项目12至14任一项的方法,其中中性磷脂是DOPE(二油烯基磷脂酰乙醇胺)。
17.项目12至14任一项的方法,其中脂质体是CHEMS和DOPE的混合物。
18.项目12至14任一项的方法,其中脂质体含有摩尔比为4.5:5.5至5.5:4.5的DOPE和CHEMS。
19.项目12至14任一项的方法,其中DOPE与CHEMS的摩尔比为5.0:5.0。
20.项目12至14任一项的方法,其中免疫刺激性寡核苷酸含有CpG基序、CpT基序、多个G的结构域或形成发夹二级结构的回文序列。
21.一种针对甲型流感病毒的肽类疫苗组合物,其包含选自SEQIDNO:1至SEQIDNO:9和SEQIDNO:19至SEQIDNO:37的氨基酸序列。
22.一种针对人类整合蛋白β4的肽类疫苗组合物,其包含选自SEQIDNO:4至SEQIDNO:46的氨基酸序列。
23.一种针对肝癌的肽类疫苗组合物,其包含SEQIDNO:10和SEQIDNO:11的氨基酸序列。
24.一种针对丙肝病毒的肽类疫苗组合物,其包含SEQIDNO:12的氨基酸序列。
25.一种针对RSV(呼吸道合胞病毒)的肽类疫苗组合物,其包含SEQIDNO:13和SEQIDNO:38至SEQIDNO:41的氨基酸序列。
26.一种用于预防或治疗甲型流感病毒传染病的方法,所述方法包含向对象给药项目21的组合物。
27.一种用于预防或治疗癌症的方法,所述方法包含向对象给药项目22的组合物。
28.一种用于预防或治疗肝癌的方法,所述方法包含向对象给药项目23的组合物。
29.一种用于预防或治疗丙型肝炎的方法,所述方法包含向对象给药项目24的组合物。
30.一种用于预防或治疗RSV(呼吸道合胞病毒)传染病的方法,所述方法包含向对象给药项目25的组合物。
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Claims (2)
1.一种针对呼吸道合胞病毒的肽类疫苗组合物,其包含由SEQIDNO:41的氨基酸序列组成的肽。
2.一种肽,其由SEQIDNO:41的氨基酸序列组成。
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