CN1269524C - 胸腺素强化的遗传免疫方法 - Google Patents
胸腺素强化的遗传免疫方法 Download PDFInfo
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- CN1269524C CN1269524C CNB028212770A CN02821277A CN1269524C CN 1269524 C CN1269524 C CN 1269524C CN B028212770 A CNB028212770 A CN B028212770A CN 02821277 A CN02821277 A CN 02821277A CN 1269524 C CN1269524 C CN 1269524C
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Abstract
本发明描述了胸腺素对强化对丙型肝炎病毒的细胞免疫应答的用途。公开了使对丙型肝炎病毒感染易感的患者免于这种感染的免疫方法,包括与一个或多个α胸腺素结合给予所述患者一编码一个或多个丙型肝炎病毒肽的多核苷酸。还公开了适于抗丙肝病毒免疫的组合物,包括一个或多个编码一个或多个丙肝病毒肽的多核苷酸,以及一个或多个胸腺素。
Description
相关申请
本申请要求享有2001年10月26日提交的临时申请60/330,638的权益。
背景技术
慢性丙型肝炎病毒(HCV)感染是导致慢性肝病、肝硬化和肝细胞癌的主要医学难题。在大多数由输血后导致的肝炎病例中,丙型肝炎病毒被推定是主要诱因。尽管已采取了措施改进捐血人库的质量并且对所捐的血进行补测,但是在接受输血的人中急性感染的发病率依然很高。在有急性HCV感染的患者中至少一半的患者会发展成慢性肝炎(结果显示大约90%的患者是非甲非乙型肝炎(NANB)),并且在这一组患者中至少有20%会发展成肝硬化。
就阻止或延缓HCV相关疾病发展的目的而言,业界已对多种用于此项目的药物进行了评价。目前关注的标准涉及干扰素α和病毒唑的使用。然而,相当数量的个体对这种治疗无反应。遗传免疫已显示出增强对HCV结构蛋白和非结构蛋白广泛基础的细胞免疫应答。然而,按照Tokushige等人在1996年所提出的观点来看,在几个实验动物模型中各种HCV构建物(construct)的生物学活性都很弱。
遗传免疫法是一种对传统的抗原-基疫苗(例如减毒的病毒或蛋白亚单位疫苗)的用途的新的替换方法(emerging alternative)。遗传免疫法使用裸DNA免疫受体。该DNA在含义(sense)上是“裸的”,即它不需要任何为促进其进入细胞起作用的传染性输送媒介,例如病毒粒子。当给予裸DNA时,受体可表达质粒DNA编码的蛋白,然后可刺激一个由细胞毒T细胞、辅助性T细胞和抗体组成的特定的免疫应答。使用DNA疫苗不需要纯化用于接种的病原体或免疫保护的抗原,并且没有毒性逆转的可能,因为该DNA编码单一的病毒蛋白。
使用多核苷酸用于免疫具有很多优点,例如,可以使用任何由多核苷酸编码的抗原进行免疫。而且,在天然的状态下,编码抗原的该多核苷酸被表达成“纯”抗原并被正常宿主细胞修饰。而且,多核苷酸操作容易和便宜,并且其干品或其存在于一宽温度范围的溶液中都很稳定。因此,这项技术对于发展高效亚单位疫苗很有价值。
诱导出的体液和/或细胞介导的免疫应答可以对例如细菌、病毒和真核微生物(例如:寄生虫)等病原因子的感染提供保护或预防性免疫。该预防性体液和/或细胞介导的免疫应答然后与病原体的传染性或活性相互干扰,或限制其扩散或生长,导致对随后由病原体带来的免疫激发的防备性保护。免疫应答也可以抗击涉及产生特定蛋白的细胞的疾病和病症。
通过肌注的遗传免疫已在各种动物中显示出对抗许多病毒有效。例如,豚鼠抗2型单纯疱疹病毒(HSV)感染的免疫(Boume等,1996);小鼠抗流感病毒的免疫(Fu等,1997);鸡抗流感病毒的疫苗(Kodihalli等,1997);以及哺乳动物和鸟类抗轮状病毒(Herrmann等,美国专利No.5,620,896)。Daheshia等(1997)公开了将编码IL-1的裸DNA单独应用在表现出疱疹性基质角膜炎的动物的角膜上解决了这些动物受到的损害,使得损害症状缓解。
在例如美国专利No.5,830,876(Weiner等)中可以发现遗传免疫的一般讨论和其用途。
已发现得自胸腺的一族多肽免疫调节物—胸腺素,在淋巴细胞中引发成熟的(maturational)事件,增强T细胞功能并促进免疫缺陷的重建。胸腺素α1(THNα1)是一个分子量为3100的28个氨基酸的酸性多肽,具有有效的免疫活性,包括刺激α-和γ-干扰素产生、增加巨噬细胞迁移抑制因子产生、诱导T细胞标记物(markers)、IL-2受体的表达、以及提高T细胞辅助性细胞的活性。THNα1的分离、特性和用途已在例如美国专利No.4,079,127中描述。
在治疗慢性HCV的尝试中已将各种抗病毒剂用作单独的治疗剂,包括无环鸟苷、阿糖腺苷和阿糖胞苷。用这些抗病毒剂单独治疗通常不成功,由于这些药剂高毒性或者导致最初抑制某些病毒复制,但不能持续长时间抑制病毒复制。见例如,Alexander等,1988.
仍然非常需要能有效而具有较少旁效应地攻击病毒并调控免疫应答系统和降低复发率的对HCV的治疗。
发明概述
本发明描述了在一个对HCV的NS5蛋白的优选实施方案中,α胸腺素在增强CD4+和CD8+对HCV的致免疫肽的细胞免疫应答的用途。增强对病毒和细胞蛋白的细胞免疫应答是困难的。本发明描述了α胸腺素的体内用途,其可以显著地增强对HCV蛋白的抗原决定部位的细胞毒T细胞应答和增殖的T细胞应答。在一个优选实施方案中,该胸腺素是胸腺素α1。DNA-基疫苗是一个新技术,而且与先前所有类型的疫苗接种都不相同(那些疫苗接种是基于蛋白、肽、或灭活病毒,所有都引起体液、或抗体-介导的免疫,而DNA-基疫苗则引起细胞介导的免疫),因此α胸腺素增加疫苗的效力的事实对于DNA-基疫苗在HCV的治疗和预防上提供了一种新的、未预料的改进。
本发明提供了编码一个或多个丙型肝炎病毒肽的多核苷酸与一个或多个α胸腺素一起在制备药物联合免疫剂(pharmaceutical combination)上的用途,该药物联合免疫剂用于使对丙型肝炎病毒感染易感的患者免于这种感染。
本发明还提供了一种药物联合免疫剂,含有编码一个或多个丙型肝炎病毒肽的多核苷酸以及一个或多个α胸腺素。
附图简述
图1为DNA-基免疫机制的说明;
图2为丙型肝炎病毒(HCV)基因组的示意性表示;
图3显示与编码NS5的多核苷酸一起给予5μg胸腺素α1i.p.,在3个激发浓度(0.1μg、0.5μg和1μg)下对于T细胞增殖应答的影响;
图4显示在2个淋巴细胞效应细胞/靶细胞(L/T)比率上,与编码NS5的多核苷酸一起给予5μg胸腺素α1i.p.,对于细胞毒应答的影响。
发明详述
本发明描述了α胸腺素与DNA-基(或“遗传的”)免疫结合以显著增强对HCV的细胞免疫应答的用途。一个优选的实施方案提供强化的带有胸腺素α1的DNA-基疫苗(thymalfasin)。本发明在预防性疫苗和治疗性疫苗的发展上具有广泛的含义,并在对伴随DNA-基免疫的病毒和细胞蛋白的细胞免疫应答上提供了显著的改进。
将编码致免疫的HCV肽、多肽或蛋白的多核苷酸与一种或多种α胸腺素联合体内直接给药于动物。该多核苷酸编码一多肽,该多肽与一作为靶的致免疫的HCV蛋白共有至少一个表位(epitope)。通过个体的细胞表达该多核苷酸以形成致免疫的靶蛋白,该靶蛋白引起广泛基的(broad based)抗HCV的免疫应答。HCV基因组编码2个外壳蛋白(E1和E2)和6个结构蛋白(NS2、NS3、NS4A、NS4B、NS5A和NS5B)。图2。编码这些病毒蛋白的任何的多核苷酸、或其结合或其片段都可用于本发明。
本发明可应用天然的(即自然产生的)α胸腺素以及合成的α胸腺素和重组的α胸腺素,该重组的α胸腺素具有天然α胸腺素的氨基酸序列、与其基本相同的氨基酸序列、或来自于它的简并的氨基酸序列、以及它们的生物学上的活性同类物,这些同类物具有与天然α胸腺素基本相同的生物活性的取代的、缺失的、延长的、置换的和甚至修饰的序列。优选的胸腺素是胸腺素α1。
α胸腺素的分离、特性和用途已在例如美国专利No.4,079,127、美国专利No.4,353,821、美国专利No.4,148,788和美国专利No.4,116,951中描述。引起需要程度的强化效力的DNA-基疫苗所需要的α胸腺素的量可以通过常规剂量滴定实验来确定。已发现当以高至16mg/kg体重/天的剂量给药时,α胸腺素对于人类是安全的。α胸腺素的优选剂量是在0.001mg/kg体重/天至10mg/kg体重/天的范围内,最优选的剂量是大约0.02mg/kg体重/天。
本发明的多核苷酸序列为DNA或RNA序列,该序列可操作地连接于转录调节子的序列,编码抗原的/致免疫的HCV多肽。这些序列可以与编码控制这些多肽表达的调节蛋白的其他多核苷酸序列联合使用。该调节蛋白可以通过与基因组DNA结合而发挥作用,以调节其转录;或者,它可以通过与信使RNA结合而发挥作用,以增加或降低其稳定性或翻译效率。
术语“可操作地连接于转录或翻译调节序列”是指一多肽编码序列和最小的转录和翻译控制序列以这样一种方式连接:当适当的分子(例如,转录激活子蛋白)连接在该调节序列时允许多肽表达。当一多核苷酸具有由一靶细胞表达所需的所有遗传信息时,例如,启动子等等,它操作地编码一多肽。术语“启动子”或“启动子序列”这里指足以指导转录的最小的序列。通常通过一起始位点和一终止位点将一DNA多核苷酸序列结合以形成一DNA转录单位,并且该DNA多核苷酸被转录以产生一初级转录产物。
在体内输送到细胞的多核苷酸材料可以采用任何数量的型式,并且本发明不限于对任何特定的HCV多肽进行编码的任何特定的多核苷酸,尽管编码NS5蛋白的多核苷酸、或其片段是优选的。在文献中已报道了含有编码HCV抗原或免疫原的基因的质粒,并且本领域技术人员可以容易地得到(见例如,Tokushige等,1996).
多核苷酸可以编码一个或多个抗原,例如来自2个或更多的不同病毒蛋白的抗原。或者,该多核苷酸可以包括2个或更多的不同DNA序列,一个序列编码一抗原,而另一个(或几个)可以编码是或不是抗原的多肽。例如,载体(vector)可以编码2个(或多个)HCV抗原。在另一个实施方案中,另一个(或几个)多肽可以发挥增强抗HCV的免疫应答的作用(例如,辅助表位、细胞因子、载体多肽、霍乱毒素亚单位、或其他免疫刺激剂)。
可以将该多核苷酸另外插入包括用于多核苷酸的表达的序列的载体中。当2个或多个多肽编码DNA序列存在于一个载体中时,为了表达2个或多个非融合的多肽,每个抗原编码DNA序列的转录可以被来自其自身的启动子指导。或者,一个启动子可以驱动2个或多个抗原编码DNA序列的表达,这些抗原编码DNA序列互相结合在框架中以表达一融合蛋白。
在这些方法中使用的多核苷酸可以是不与宿主细胞的基因组结合的序列。这些多核苷酸可以是非复制DNA序列,或在遗传上设计成缺乏基因组结合能力的特别复制序列。可以在具有促进核酸摄取或恢复免疫系统细胞至接种位点的佐剂或其他物质存在下,将该多核苷酸给予患者。应当理解的是,该多核苷酸本身可通过由宿主细胞提供的转录因子、或由DNA转录单位提供的转录因子在宿主细胞中表达。
根据本发明的方法,可表达的DNA和mRNA都可被输送到细胞中形成其中的一多肽翻译产物。如果该核酸包含适当的控制序列,它们将指导相关地大量的被编码蛋白的合成。
致免疫的多肽的剂量可以通过临床医生或兽医在动物模型上使用或其他本领域技术人员熟知的测试系统容易地确定。制剂将包括在水溶液中的有效量的DNA。给予的DNA的量取决于例如进行疫苗接种的患者的年龄、体重和生理条件等因素。DNA的量也取决于患者的免疫系统合成抗体的能力,以及需要保护的程度。可以由本领域普通技术人员通过常规试验建立剂量反应曲线而容易地建立有效剂量。优选的剂量范围是在1μg/kg患者体重至1mg/kg患者体重之间。可以通过一次给予该DNA免疫患者,或者通过多次给药。患者可以需要多次给药以维持对特定病原体的免疫状态。
构建用于成功转化的异种多核苷酸的方法和组合物是本领域技术人员熟知的,并且可以使用相同的构建组合物和方法产生这里使用的多核苷酸。多核苷酸的具体的组合物不是本发明的重点,而且本发明不依赖于使用的具体的转化多核苷酸的组合物。多核苷酸的适当的成分包括启动子、多腺苷酸化序列、终止信号、拼接信号、可选择的标记、报道基因、增强子、病毒复制子、内含子、和细菌质粒序列都是本领域熟知的。Sambrook等(1989)提供了异种多核苷酸构建的适当的方法。
可以通过多种已知的方法产生多核苷酸。例如,可以将编码一预选的抗原的DNA插入一表达载体中(例如见,Sambrook等(1989))。随着自动核酸合成设备的应用,当核酸序列已知时可以直接合成DNA,或者通过PCR、克隆和发酵的结合合成DNA。而且,当预选的多核苷酸的序列已知时,可以推出该核苷酸合适的克隆序列。
当多核苷酸是mRNA时,可以在体外从相应的DNA中容易地制备。例如,在单独的核糖核苷三磷酸盐存在下,使用噬菌体RNA多聚酶SP6、T3或T7从DNA模板制备mRNA的传统技术。合适的噬菌体启动子,例如复制位点的T7原点位于要转录的基因的模板DNA直接上游。以这种方式使用T7的系统是公知的,并且在文献中有描述。
本发明的另一个方面涉及一免疫组合物,当其被导入一个其中能够诱导或具有诱导的免疫应答的个体时,该免疫组合物在这个个体中诱导对由此编码的HCV蛋白的免疫应答,其中该组合物包括编码并表达一个或多个HCV的抗原/免疫原的多核苷酸,与一个或多个α胸腺素结合。该免疫应答可以用于治疗或预防,并且可以以抗体免疫或细胞免疫的形式,例如由CTL或CD4+T细胞引起的免疫。
实施例1
质粒构建
作为病毒基因来源,使用包含HCV的全长开放阅读框(ORF)称为pBRTM/HCV1-3011的质粒来克隆进表达载体(Grakoui等人1993)。使用下述引物在插入设计的起始和终止密码子和限制性内切酶位点后PCR克隆构建pAp031-Ns5:对于NS5,5’T CAG TCT AGA ATG TCC GGC TCC TGG CTAAGG GA-3’(XbaI)[SEQ ID No.1]和5’-A GCT ACG CGT TCA CCG GTT GGGGAG GAG GT-3’(MluI)[SEQ ID No.2]。PCR扩增后,使用高保真PCR系统(Bochringer Mannheim,Indianapolis,IN),将cDNA片段插入含有劳氏肉瘤病毒增强子成分和CMV启动子的质粒表达载体pAp031中。将构建体转染进入DH5α细胞,接着通过用2×氯化铯离心或者用使用Endofree缓冲系统的Qiagen Giga试剂盒(Santa Clara,CA)纯化质粒DNA。通过使用标准方法的测序分析证实编码非结构蛋白的cDNA的正确插入。为建立作为CTL分析的靶细胞的稳定的NS5表达细胞系,也将非结构蛋白编码基因片段克隆入带有新霉素选择标记的pcDNA3和pcDNA3.1/Zeo(-)表达载体(Invitrogen,San Diego,CA)。将XbaI和MluI片段NS5亚克隆进Litmus-38载体的NheI/MluI位点,并且连接到pcDNA3和pcDNA3.1/Zeo(-)的EcoRI/XhoI多克隆位点。将含有NS4的HbaI和BamHI片段连接到Litmus-29(New England Biolabs),用KpnI和EcoRI重切,然后连接到pcDNA3载体。质粒定名为pcDNA3.1/Zeo(-)-NS5。将编码鼠IL-2的DNA表达质粒(pcD/3-IL2)克隆并如前述纯化(Geissler等1997a,1997b,1997c).
遗传免疫
为增强质粒DNA的细胞摄取,用总量100μl的0.25%hupivacaine在多位点注射Balb/c小鼠的四头肌。4天后,将质粒构建物以100μl 0.9%NaCl的终体积在5个不同位点注射到相同区域。2周后,在相对的腿上用质粒DNA加强小鼠。在最终免疫10天后杀死小鼠。将小鼠分成3组,每组5只动物。组1=用100μg模拟DNA免疫小鼠;组2=用50μg pAp031NS5和50μg模拟DNA免疫小鼠;组3=用50μg pAp031NS5和50μg模拟DNA免疫小鼠,并每周2次接受5μg胸腺素α1i.P。将动物隔2周免疫3次,并在最后免疫10天后进行研究。
T细胞增殖分析
用异氟烷(Aerrane,Anaquest,NJ)麻醉小鼠,通过眼球后穿刺去除血液并收获脾细胞。在37℃8.3%NH4Cl/0.17mol/LTris(pH 7.4)中保温10分钟除去红血细胞。用96孔平底板以每孔5×105细胞在含10%FCS的完全培养基(Dulbeccos Modified Eagle’s Medium,Mediatech,Washington,DC)中一式三份培养脾细胞。以不同浓度(1、5、10μg/ml)的重组NS5刺激脾细胞。最后加入2-巯基乙醇至终浓度50μmol/L。作为抗原特异性的对照,用10μg/ml的重组的人绒毛膜促性腺激素的β-亚单位刺激效应细胞,重组的人绒毛膜促性腺激素的β-亚单位由该细胞分泌并已显示其是一种强的T细胞免疫原(Geissler等,1997d)。刺激脾细胞3天。加入[3H]-胸苷(1μCi/孔)后,将细胞培养18小时。收获细胞后测定与DNA结合的[3H]-胸苷。图3。用背景活性校正结合的放射活性(Δcpm)。
细胞毒性分析
将来自免疫的小鼠的脾细胞悬浮在含10%FCS和50μmol/L2-巯基乙醇的完全DMEM中;然后体内免疫5天后将细胞用于细胞毒活性分析。用20,000rad处理后,以5U/ml的浓度加入重组鼠IL-2一次,并将应答(responder)细胞(4×107)与6.25×106稳定表达NS5的同源细胞共同培养。培养5天后,收获细胞毒效应淋巴细胞群。用靶细胞系的51Cr-标记的SP2-NS5细胞在96孔圆底板中进行4小时51Cr-释放实验。以1∶10和1∶100的淋巴细胞效应细胞:靶细胞(L/T)比例进行CTL分析。如下计算细胞毒百分数:
实验释放代表在效应细胞存在下每分钟由靶细胞释放的平均数。总释放代表用5%TritonX-100的靶细胞全部溶胞后释放的放射活性。图4。
自然释放代表存在于仅得自靶细胞的培养基中的放射活性。
统计分析
为比较不同组间的结果,我们使用了非参数的Mann0Whitney U试验。P<0.05认为具有统计学上的显著性。根据CD4+和CD8+T细胞应答的P值数目分别得自所有的用于刺激的重组NS5浓度和E:T值。
显示在图3和图4中的结果证明通过在DNA-基免疫治疗中α胸腺素的共同给予可以获得免疫应答的明显改善。共同给予胸腺素增加CD4对所有3个激发浓度的增殖应答几乎达100%。图3。通过α胸腺素的共同给予,细胞毒性百分数也增加达100%,或更多。图4。
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美国专利4079127(Goldstein,等)
美国专利4116951(Wang)
美国专利4148788(Wang)
美国专利4353821(Birr,等)
美国专利5620896(Herrmann,等)
美国专利5830876(Weiner,等)
Claims (14)
1、编码一个或多个丙型肝炎病毒肽的多核苷酸与一个或多个α胸腺素一起在制备免疫组合物上的用途,该免疫组合物用于使对丙型肝炎病毒感染易感的患者免于这种感染。
2、权利要求1所述的用途,其中所述多核苷酸编码一个或多个丙型肝炎病毒外壳蛋白。
3、权利要求1所述的用途,其中所述多核苷酸编码一个或多个选自NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的丙型肝炎病毒蛋白。
4、权利要求3所述的用途,其中所述多核苷酸编码一个或多个NS5蛋白。
5、权利要求1-4的任何一个所述的用途,其中所述α胸腺素为胸腺素α1。
6、权利要求1-4的任何一个所述的用途,其中所述胸腺素的剂量为0.001~10毫克/千克体重/天。
7、权利要求6所述的用途,其中所述胸腺素的剂量为0.02毫克/千克体重/天。
8、一种免疫组合物,含有包括一个或多个丙型肝炎病毒肽的多核苷酸以及一个或多个α胸腺素。
9、权利要求8所述的免疫组合物,其中所述多聚糖苷酸编码一个或多个丙型肝炎病毒外壳蛋白。
10、权利要求8所述的免疫组合物,其中所述多核苷酸编码一个或多个选自NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白和NS5B蛋白的丙型肝炎病毒蛋白。
11、权利要求10所述的免疫组合物,其中所述多核苷酸编码一个或多个NS5蛋白。
12、权利要求8-11的任何一个所述的免疫组合物,其中所述α胸腺素为胸腺素α1。
13、权利要求8-11的任何一个所述的免疫组合物,其中所述胸腺素的剂量为0.001~10毫克/千克体重/天。
14、权利要求13所述的免疫组合物,其中所述胸腺素的剂量为0.02毫克/千克体重/天。
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ATE382365T1 (de) | 2008-01-15 |
WO2003035010A3 (en) | 2003-12-04 |
IL161603A0 (en) | 2004-09-27 |
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