CN1434831A - 对hiv免疫应答的改进或与其相关的改进 - Google Patents
对hiv免疫应答的改进或与其相关的改进 Download PDFInfo
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- CN1434831A CN1434831A CN00817677A CN00817677A CN1434831A CN 1434831 A CN1434831 A CN 1434831A CN 00817677 A CN00817677 A CN 00817677A CN 00817677 A CN00817677 A CN 00817677A CN 1434831 A CN1434831 A CN 1434831A
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Abstract
公开了一种无病原体形式的适于给予人类受试者的免疫原,该免疫原包含:至少一部分HIV的gag蛋白,所述gag蛋白来源于一种HIV分化体或与一种或多种HIV分化体具有共有序列,并至少包含部分p17和p24;以及包含多个氨基酸序列的合成多肽,每个序列包含一种HIV蛋白的人CTL表位,并且其中许多HIV蛋白表现为合成多肽形式,所述CTL表位的选择是为了刺激对一种或更多感兴趣的HIV分化体的免疫应答。
Description
发明领域
本发明涉及一种免疫原,该免疫原被设计为在人类受试者中引发抗HIV免疫应答(特别是细胞介导的应答),一种编码该免疫原的核酸分子,包含所述免疫原和/或所述核酸分子的组合物,以及在人类受试者中诱导抗HIV免疫应答(特别是细胞介导的应答)的方法。
发明背景
开发有效的人类免疫缺陷病毒(HIV)疫苗是当前对获得性免疫缺陷综合征(AIDS)进行研究的主要目的之一。尽管HIV感染的预防和强效药物结合治疗取得了进展,但估计每天仍有16,000人受到感染。90%以上的新感染发生在发展中国家,在这些国家还无法获得或负担最新的医学进展。这些国家的最大希望是开发有效的,可获得的HIV疫苗。目前科学家们对开发AIDS疫苗的可能性越来越乐观(McMichael和Hanke 1999 Nat.Med.5,612-614;Gold 1999 IAVI Report 4,pp1-2,8-9,15-16&18)。
一种理想的疫苗应该诱导无病原体的免疫,因此在暴露后,病毒应该在体内检测不到。然而,这可能是一个不实际的目标。而一个能达到的目标可能是导致有限和短暂病毒复制的疫苗诱导的免疫,此后病毒不能被检测到,不产生疾病症状和传播到其它个体。替代地,一种有可能成功的疫苗可以诱导至少使被检测病毒保持低水平的免疫应答,从而完全或基本预防进展为AIDS和传播。
为了诱导无病原体的免疫,可能需要用预防性疫苗来引发体液和细胞介导的免疫应答。由于HIV被分离和测序,已经付出了相当多的努力来开发诱导中和性抗体(nAb)的基于被膜的疫苗。然而,已经证明这是极端困难的(Heilman和Baltimore 1998 Nat.Med.4(4Suppl.)532-534)。尽管有人报道在诱导抗实验室HIV株的nAb上取得了一些成功(Berman等,1990 Nature 345,622-625;Fultz等,1992 Science 256,1687-1690),但中和初级分离物极为困难(Trkola等,1998 J.Virol.72,1876-1885;Haynes 1996 Lancet34,933-937)。核心gp120的晶体结构为最初15年的相对失败提供了解释,它揭示了HIV防止有效诱导nAb的多种机制(Wyatt等,1998Nature 393,705-711;Kwong等,1998 Nature 393,638-659)。由于这些困难,许多疫苗设计者的重点转移到了诱导细胞介导的免疫应答,这是(主要)由细胞毒T淋巴细胞介导的。
细胞毒T淋巴细胞(CTL)通常是CD8+细胞,并且以至少两种不同的方式参与生物体的防御:它们杀灭感染病毒的细胞;而且它们分泌各种直接或间接参与抑制病毒复制的细胞因子和趋化因子。接种后的CTL介导的保护可能取决于循环中的CTL水平,并可能特异性针对复制循环中早期表达(调节蛋白)而不是晚期表达(结构蛋白)的蛋白。
CD8+T细胞应答的诱导和维持需要CD4+T淋巴细胞(辅助T细胞)提供的“辅助”。在一些HIV感染的个体中,检测到了高水平的HIV特异性辅助应答。
诱导强CD8+T细胞应答的方法的鉴定将提供研究它们在影响HIV感染过程中的作用的工具,并可能刺激研究有效HIV疫苗的进展。以前曾经将原型的HIV疫苗构建为一串部分重叠的可以被鼠、猕猴和人CTL识别的表位,它是通过对人类为安全和可接受的疫苗载体而递送的,即DNA载体和修饰的痘苗病毒Ankara(MVA)载体(Hanke等,1998Vaccine 16,426-435;Hanke等,1998J.Gen.Virol.79,83-90)。在小鼠中,发现最有效的CTL诱导方案是用DNA激发,然后用MVA加强(Hanke等,1998 Vaccine 16,439-445;Schneider等,1998 Nat Med.4,397-402),即用编码相关多肽的核酸激发小鼠,然后通过接种表达相关表位的修饰痘苗病毒Ankara(“MVA”)载体而加强。
WO 98/56919公开了一种“激发-加强”接种策略,包括(i)用包含靶抗原的一种或更多T细胞表位来源和可药用载体的组合物激发,和(ii)用包含靶抗原的一种或更多T细胞表位来源的组合物加强,该组合物中包括至少一种与激发组合物中的T细胞表位相同的T细胞表位。
本发明的目的包括,但不限于,提供可以用于引发人类HIV特异性应答的免疫原。在此引入说明书中提到的所有文件和公开文献作为参考。
发明概述
本发明的第一方面提供了以适于给予人类受试者的无病原体形式存在的免疫原,该免疫原包含:至少一部分HIV的gag蛋白,所述gag蛋白来源于HIV分化体或与一种或更多HIV分化体具有共有序列,并至少包含部分p17和p24;以及包含多个氨基酸序列的合成多肽,每个序列包含一种HIV蛋白的人CTL表位,并且其中许多HIV蛋白表现为合成多肽形式,所述CTL表位的选择是为了刺激对感兴趣的一种或更多HIV分化体的免疫应答。
这里提到的“无病原体的”是指一般不存在病毒、细菌、真菌、酵母、衣原体、支原体和前述微生物的孢子(尤其是在人类中致病的微生物)。然而,免疫原可以包含一种或更多用于在人类受试者中表达gag蛋白和/或合成多肽的特异性已知微生物(如病毒或细菌)载体。这些载体是本领域技术人员熟知的,并包括,例如,腺病毒和痘病毒等在人类中本身不致病或者经遗传操作或其它修饰使其在人类中不致病的病毒。特别优选的是痘苗病毒,尤其是修饰的痘苗病毒Ankara(MVA)。其它特别优选的病毒载体包括Semliki Forest病毒(SFV)和Sindbis病毒(见Smerdon和Liljestrom 2000,Gene Ther.Regul.1,1-31)。合适的细菌载体包括BCG,以及沙门氏菌的减毒株(特别是开发为腹泻疾病疫苗的沙门氏菌“双aro”突变体),和志贺氏菌(见Shata等,2000 Mol.Med.Today 6,66-71)。
其它可以用于产生免疫原的表达系统包括烟草花叶病毒(TMV)表达载体(Palmer等,1999 Arch.Virol.144,1345-60)和蓝舌病毒的NS1小管(Adler等,1998 Med.Microbiol.Immunol.(Berl.)187,91-96)。
这里用到的名词“合成的”是指其整体不存在于天然存在的HIV分离物中的多肽。名词“合成的”不准备指必须由固相肽合成的常规化学技术合成的多肽。尽管这代表一种可能性,多肽一般优选通过编码合成多肽的适当核酸分子的转录和/或翻译而合成。一些合成方法是本领域技术人员熟知的,不构成本发明的一部分。
gag蛋白(或其部分)和合成多肽优选以一定的方式连接为单个整体。例如,病毒载体可以将gag蛋白和合成多肽表达为免疫原中分开的成分。替代地,免疫原的这两种成分可以彼此共价偶联或结合,或与共同的载体实体(如脂质体、ISCOM或分子)共价偶联或结合。在优选实施方案中,免疫原的gag蛋白和合成多肽成分存在于单个多肽或融合蛋白。在这种融合蛋白中,gag蛋白和合成多肽可以基本是存在的仅有成分。替代地,融合蛋白可以包含可以与其它HIV抗原(或其部分)相关或来自于其它来源的其它成分。这样,在一些实施方案中,多肽可以包含基本邻接于(即小于10个间插氨基酸残基)合成多肽的gag蛋白的一部分。在其它实施方案中,gag蛋白部分和合成多肽可以由一种或更多间插成分(即有10个或更多间插氨基酸残基)分开,间插成分一般包括一种或更多其它HIV抗原。
免疫原一般也优选不包含完整gag蛋白氨基酸序列。一般存在55%-95%的gag蛋白,优选65%-85%,最优选约75%。
已知野生型HIV gag蛋白由三个部分,即p17,p24和p15组成。这些部分在感染细胞中合成为单个多蛋白,p17在N端。一般地,在HIV感染的细胞中,p17的N端是豆蔻酰化的。
理想的状况是免疫原的gag部分至少包含部分p17和p24,但一般优选p17成分将以一定的方式被修饰,以便预防豆蔻酰化。通过反转免疫原中p17和p24的顺序可以方便地达到这一目的,这样p17不再处于游离N端并且因此不能被豆蔻酰化。本发明者确信这可以改进向受试者免疫系统呈递来自于免疫原的肽的效率。
免疫原的gag蛋白成分一般包含至少一个T辅助细胞的II类HLA限制性肽表位,并优选包含多个这样的表位(优选使许多不同的II类HLA限制性等位基因表现在gag成分中)。gag蛋白成分一般也将包含一种或更多CTL I类HLA限制性肽表位。
免疫原的合成多肽成分将方便地采用一串CTL表位的形式,每一个分别表现为约8-12个氨基酸的序列,或含有约8-12个氨基酸的序列。理想的情况是至少一些(优选大多数)表位将部分重叠(使一种表位的一个或更多氨基酸也包含在邻接表位的序列中)。一些“非表位”氨基酸序列可以出现在邻近表位之间,但一般要避免这种情况。
邻近表位之间的非表位氨基酸优选少于20个氨基酸残基,更优选少于10个残基,最优选1-5个氨基酸残基。很明显这些非表位氨基酸序列可以包含优化合成多肽表达水平或优化其免疫原性的接头、间隔区等。
因此在一种极端的实施方案中,合成多肽中的所有人CTL表位都可以重叠。在另一个极端,每一个表位都可以由至少一些非表位氨基酸将其与邻近表位隔开。一般优选至少50%的人CTL表位是重叠的。
除了重叠表位,合成多肽可以至少包含一些“邻接表位”。邻接表位是指不重叠但不由间插非表位氨基酸序列分开的表位。
因此在优选实施方案中,合成多肽包含不同HIV蛋白的小片段(一般约10-20个氨基酸残基)一起拼凑成的嵌合型,这些片段一般将包含一个、两个或三个已知邻接和/或重叠的人CTL表位。合成多肽中存在相同HIV蛋白的许多片段,这些片段一般选自蛋白的不连续部分,因此合成肽一般不会包含对应于特定HIV蛋白的超过20-25个连续氨基酸残基的序列。通常合成肽的设计基本省略了已知不含人CTL表位的HIV蛋白部分。
许多不同的HIV蛋白优选表现为合成肽。合成肽可以包含存在于任何HIV抗原的表位,但优选包含至少一种存在于下列一种或多种蛋白的表位:p25;pol;gp41;gp120和nef。在一种优选的实施方案中,合成多肽包含至少一种存在于前面提到的每一种HIV蛋白的CTL表位。合成多肽可以额外包含至少一种存在于每一种下列HIV蛋白的CTL表位:vpr,vpu,vif(特别是)tat和rev。
应该理解这里用到的术语“人CTL表位”不是指得到的表位的蛋白的来源,而是指由至少一部分人群的CTL识别并应答的表位。一般地说,人CTL表位将由世界人口的至少0.01%、优选0.1%以及更优选至少1%识别。
在一种特定实施方案中,免疫原特别排除了来自于一般由人免疫系统识别的env蛋白的任何表位。大多数当前使用的诊断实验是基于HIV env特异性免疫应答的检测,因此通过将env成分排除在免疫原外,可以区分由病毒感染和接种免疫原导致的免疫应答。
在一种实施方案中,存在于合成多肽中的CTL表位的选择将导致产生对HIV分化体A的免疫应答。然而合成肽优选足够大,并适当选择CTL表位,从而刺激对不同HIV分化体的交叉反应性免疫应答。这可以通过引入一种或更多不同HIV分化体中的保守CTL表位而方便地达到。一些这样的表位是本领域技术人员已知的(见下面的表1)。
在一种优选实施方案中,免疫原包含至少一个由一种或多种实验室检验哺乳动物(如小鼠和/或猴)识别的表位(方便为CTL表位)。这种表位可以容易插入合成多肽。引入此类表位允许在使用实验室检验哺乳动物(如小鼠或猕猴)进行的效力测定中测定不同批免疫原的质量、再生性和/或稳定性。这种表位的例子包括氨基酸序列ACTPYDINQML(Seq.ID No.1;含有来自于猿免疫缺陷病毒SIV,gagp27的显性表位,由猕猴CTLs识别)和RGPGRAFVTI((Seq.ID No.2;一种来自于HIV env蛋白的H-2Dd限制性CTL小鼠表位)。
一系列共23个适于引入合成多肽的人CTL表位见表1。该列表不是完全的。优选的免疫原将包含至少10个这种人CTL表位,更优选至少15个,最优选至少20个。在一种特别优选的实施方案中,列于表1中的23个人CTL表位的每一种将以合成多肽的形式出现,可选择与同样列于表1的猕猴和鼠表位一起。
理想的免疫原可以额外包含小分子标志或标记。这种标志或标记应尽可能小,从而尽量减小存在于免疫原中的外源物质量。方便的标志或标记允许检验免疫原表达的检验和/或免疫原的定量。适当的标志包括由单克隆抗体Pk识别的表位(Hanke等,1992 J.Gen.Virol.73,653-660)。
本发明的免疫原将方便地与其它物质混合,从而提供疫苗组合物。例如,疫苗组合物可以额外包含一种或更多下列成分:佐剂(如明矾)、脂质体、或免疫刺激复合物(ISCOM)。疫苗一般包含无病原体稀释液、赋形剂或载体,如生理可接受的液体(如盐水或磷酸缓冲液)。疫苗可以以液体或更优选以冻干固体存在,在给药前冻干固体悬浮或溶解于生理可接受的液体。
将免疫原给予人类受试者的方法对本领域的技术人员是很明确的。具体地说,这些方法包括肌内注射、皮下注射、或通过无针头注射设备经皮肤递送。另外,也可通过口服、鼻内或任何其它的适当途径给予疫苗(特别是包含细菌载体如减毒沙门氏菌或志贺氏菌载体)。适当的免疫原剂量一般可以是1-500mg,一般10-100mg,取决于免疫原的大小和接受者的体重等。如果需要,适当的剂量可以通过接受不同剂量免疫原的不同受试者组的常规试验和误差确定,以及测定受试者对免疫原的免疫应答以判断最优剂量大小。受试者的免疫应答可以通过常规免疫技术测定(如铬释放测定,使用从受试者血样中获得的外周血淋巴细胞,作用于肽脉冲激发的或病毒感染的铬标记靶细胞)
第二方面,本发明提供了一种编码融合蛋白的核酸分子,该融合蛋白包含本发明第一方面的免疫原。
核酸分子优选为分离的无病原体形式,适于给予人类受试者。核酸分子优选为“人源化的”,即使用经常用于高度表达的人基因中的密码子来编码特定氨基酸(Andre等,1998 J.Virol.72 1497-1503),而不是由HIV使用的密码子。
理想状况是将核酸分子包含在载体中,编码免疫原的序列与人细胞中有活性的启动子序列可操作性连接。便利的条件是启动子为强病毒启动子,如人巨细胞病毒(CMV)启动子。载体优选也包括在人细胞中起作用的增强子和聚腺苷酸信号。理想地,为了最大的安全性,载体不应该含有在人细胞中有功能的复制起点,以便预防不希望的载体复制。
载体可以以分离物形式给予受试者,可以作为基本“裸露”的核酸(优选DNA),或可以装配在病毒、细菌、脂质体或金包被的颗粒等递送设备中。一种合适的递送设备为修饰的痘苗病毒Ankara(MVA):免疫原基因或开放读码框架(ORF)可以插入MVA,例如,在胸腺嘧啶激酶位点。
本领域的技术人员应该理解,载体可以包含编码免疫原的核酸(此核酸因此符合本发明的第二方面),载体也可以在其表面或内部具有本发明第一方面的多肽免疫原。
不论是作为裸核酸还是装配在递送设备中给药,至少一些给予的核酸进入了受试者细胞并被转录(如果需要)或翻译,导致受试者中免疫原的原位合成,受试者然后产生对免疫原的免疫应答。
对于免疫原制剂本身,可以通过皮下或肌内注射、口服递送或通过无针头注射器的方式经皮肤给药等许多已知的途径给予人类受试者编码免疫原的核酸。通过无针头注射设备给药的具体实施例公开于WO97/34652和WO 97/48485。核酸的合适剂量可以是10μg-10mg,一般为100μg-1mg,取决于核酸分子大小、给药途径、接受者体重等。同样,常规试验和误差(具有本教导的益处)将使本领域的技术人员可以判断核酸的最优剂量。
已经用以下方法成功将DNA递送到动物组织,阳离子脂质体[Watanabe等,Mol.Reprod.Dev.38:268-274(1994);Sharkey等,WO 96/20013],直接注射裸DNA至动物肌肉组织[Robinson等,Vacc.11:957-960(1993);Hoffman等,Vacc.12:1529-1533;(1994);Xiang等,Virol.199:132-140(1994);Webster等,Vacc.12:1495-1498(1994);Davis等,Vacc.12:1503-1509(1994);以及Davis等,Hum.Molec.Gen.2:1847-1851(1993)]和胚胎[Naito等,Mol.Reprod.Dev.39:153-161(1994);和Burdon等,Mol.Reprod.Dev.33:436-442(1992)],或用“基因枪”技术皮内注射DNA[Johnston等,Meth.Cell Biol.43:353-365(1994)]。
对于基于DNA的疫苗,通过注射裸质粒DNA而递送已经在小鼠模型表现出诱导体液和细胞免疫应答的潜能。然而,在更大的动物中,已经证明利用DNA递送而接种受到了阻碍,因为这需要大量DNA,或者会诱导抗原的持续表达,从而有可能发展为对抗原耐受。Berglund报道了通过用含有甲病毒DNA表达载体的质粒DNA注射给小鼠而诱导或增强免疫应答的策略,该载体在真核表达盒中具有重组SemlikiForest病毒(SFV)复制子[Berglund等,Nature Biotechnol.16:562-565(1998)]。该真核表达盒控制SFV复制子的初级核酸转录物的表达。将这种编码异源抗原的SFV复制子转录物运送到胞浆中并通过自我编码的SFV复制酶复合物扩增。扩增的RNA复制子导致高水平产生编码异源抗原的mRNA。相似的结果由Polo和他的研究小组描述过[Polo等,Nature Biotechnol.16:517-518(1998);Hariharan等,J.Virol.72:950-958(1998)]。两组都发现可以用小量注入质粒DNA诱导强免疫应答。
另外,一种克服了常规递送方法的缺点而向动物递送DNA的方法是给予减毒的、含有细菌DNA载体的侵袭性细菌,该载体具有编码所表达基因的真核表达盒。例如,授权于Powell等的美国专利5,877,159描述了可以侵入动物细胞而不建立复制性感染或导致疾病的活细菌,从而引入了编码可以由动物细胞表达的抗原的真核表达盒。
本发明的第三方面提供了一种刺激人受试者的抗HIV免疫应答的方法,该方法包含制备本发明第一方面的免疫原,或本发明第二方面的核酸分子;以及给予受试者所述免疫原或核酸分子。
方便地,该方法既包含给予免疫原,也包含给予核酸分子。具体地说,该方法优选包含一次或多次给予核酸分子(“激发”),然后经过一段适当的间隔(如1周至4个月),再一次或多次给予免疫原(“加强”)。可以通过例如给予包含免疫原和/或编码抗原的核酸分子的有能力复制的(即减毒病毒或细菌)或不复制载体而进行加强。加强优选通过给予作为MVA病毒颗粒一部分的免疫原而完成,该颗粒可以有利地包含编码免疫原的核酸。
在一种优选实施方案中,该方法的进行将导致在受试者中产生保护性免疫应答,因此当受试者其后暴露于HIV感染时,受试者不会产生与HIV感染相关的AIDS症状。
本发明的第四方面提供了本发明第一方面的免疫原和/或本发明第二方面的核酸在制备预防或治疗人类受试者HIV感染的药物中的用途。
第五方面,本发明提供了一种编码图8A所示氨基酸序列的核酸序列。方便地,核酸序列包含或基本由图8B所示核苷酸序列组成。
第六方面,本发明提供了一种包含图8A所示氨基酸序列的多肽。
第五方面的核酸和/或第六方面的多肽可以用于所描述的与本发明其它方面相关的免疫原/疫苗组合物,因此这些免疫原和疫苗也考虑在本发明的范围内,并且可以包含前面所述的载体等(特别是MVA)。本发明在第七方面进一步提供了一种刺激人类受试者中抗HIV免疫应答的方法,该方法包含给予受试者本发明第五方面的核酸和/或本发明第六方面的多肽。最后,本发明提供了本发明第五方面的核酸和/或本发明第六方面的多肽在制备预防或治疗人类受试者HIV感染的药物中的用途。
下面将通过说明性的实施例并参考附图进一步描述本发明,在附图中:
图1A是本发明的免疫原HIVA,HIVTA和HIV AeT;以及免疫原PPA的示意图;
图1B表示HIVA免疫原的氨基酸序列(Seq.ID No.26);
图2A表示编码HIV A免疫原的核酸(称作“HIVA基因”)的核苷酸序列(Seq.ID No.27);
图2B是用于构建HIVA基因的方法的示意图;
图3是本发明的DNA载体分子(pTHr.HIVA)以及其构建方法的示意图;
图4是表示pTHr.HIVA转染后的小鼠细胞表达的HIVA的免疫荧光检测的显微照片。
图5A,B和C表示使用从接种本发明的DNA分子或免疫原的小鼠获得的脾细胞进行的铬释放测定的结果。
图6A表示HIV TA免疫原的氨基酸序列(Seq.ID No.28);
图6B表示编码HIV TA免疫原的核酸分子的核苷酸序列(Seq.IDNo.29);
图7A表示存在于HIV AeT免疫原中的tat多肽的氨基酸序列(Seq.ID No.30);
图7B表示编码HIV AeT免疫原的核酸分子的核苷酸序列(Seq.IDNo.31);
图8A表示本发明第六方面的PPA免疫原的氨基酸序列(Seq.IDNo.32);
图8B表示编码PPA免疫原的核酸分子(符合本发明的第五方面)的核苷酸序列(Seq.ID No.33)。
实施例实施例1
本实施例涉及一种用于疫苗的免疫原,该疫苗主要用于诱导由CD4+辅助T淋巴细胞和CD8+效应T淋巴细胞的协同作用介导的细胞免疫应答。已经设计将命名为HIVA(Hanke & McMichael Nat.Med.6,951-955)的免疫原用于在肯尼亚内罗毕进行的一项III期有效性临床试验。图1A是包括HIVA的一些免疫原的示意图。HIVA来源于HIV-1分化体A的序列,该分化体是内罗毕地区的最主要HIV分化体并且由大约73%的gag蛋白与一串25个部分重叠的CTL表位融合组成。HIVA的gag结构域含有p24和p17,与病毒gag p17-p24-p15多蛋白的顺序相反。这种重排防止p17 N端的豆蔻酰化,p17 N端的豆蔻酰化可以将重组蛋白导向到细胞膜,因此防止有效降解为I类主要组织相容复合物(MHC)呈递所必需的肽。
图1B表示HIVA免疫原的氨基酸序列(Seq.ID No.26)。对应于用于装配基因的限制性核酸内切酶位点的氨基酸表示为粗体(GS对应于Bam HI,GT对应于kpnI,EF对应于EcoRI)。
gag结构域的氨基酸序列来源于HIV-1分化体A的共有序列蛋白数据库(Korber等,″Human retroviruses and AIDS;a compilationand analysis of nucleicacid and aminoacid sequences″1997)。当没有肯尼亚株序列时,没有强氨基酸分化体A的区域优选偏向于乌干达分离体。HIV-1 gag蛋白不仅含有重要的I类MHC,也含有刺激CD4+T辅助细胞的II类限制性表位。
HIVA蛋白的C端命名为多CTL表位合成多肽。包括在HIVA中的CTL表位被感染在肯尼亚流行的HIV分化体A株的患者的CTL识别,该表位为8-10个氨基酸长,来源于gag,pol,nef或env蛋白(Rowland-Jones等,1998 J.Clin.Invest.102,1758-1765;Dorrell等,1999 J.Virol.73,1708-1714)。这些表位中的许多是优势免疫表位,并且在其它HIV-1分化体中相对保守(表1)(因此应该可以引发与分化体A以外的其它HIV病毒分化体交叉反应的免疫应答)。它们由17种不同的HLA等位基因呈现,包括常见非洲等位基因和在大多数种族人群中常见的等位基因。据估计由9种最常见的HLA等位基因呈现的优选表位应该覆盖普通人群,而不考虑种族血统(Sydney等,1996 Immunol.Today
17,261-266)。因此,只要大多数HIV感染的供体对gag p17/p24产生良好的CTL应答,每种疫苗应该具有应答于至少两种或三种存在于HIVA蛋白中的CTL表位的潜能。
HIVA合成多肽也包含分别由猕猴和鼠CTL识别的SIV gag和HIVenv表位,因此容易在小鼠(如果需要也可以是猕猴)效力测定中评估临床应用的批量产品的质量、再生性和稳定性。为便于检测全长蛋白和估计表达水平,将单克隆抗体表位Pk(Hanke et al,1992 J.Gen.Virol.73,653-660)添加到HIVA的C端。没有理由相信三种非HLA表位会对接种个体产生健康威胁。
表1.包括在HIVA合成多肽多表位区的CD8+细胞表位
a-按出现在多表位中的顺序列出各表位(Seq.ID Nos.3-25,1,2)。b-存在于约50%(小写字母)或90%(大写字母)被测序的HIV分化体分离物中的特定表位序列。c-“=”表示该表位存在于分化体A gag结构域N端。 d-一种来源于SIV gag27的显性表位,其侧翼分别为N端的Ala
表位a | I型MHC限制 | 来源 | HIV分化体b |
ALKHRAYEL | HLA-A*0201 | nef | a |
PPIPVGEIY | HLA-B35 | p24 | a/B/c/D/F/G |
GEIYKRWII | HLA-B8 | p24 | a/B/c/D/F/G |
KRWIILGLNK | HLA-B*2705 | p24 | A/B/C/D/F/G/H |
FRDYVDRFYK | HLA-B18 | p24 | BD(A=C/F/G/H)c |
RDYVDRFYKTL | HLA-B44 | P24 | B/D(A=C/F/G/H)c |
DRFYKTLRA | HLA-B14 | p24 | B/D(A=C/F/G/H) |
AIFQSSMTK | HLA-A*0301,A11,A33 | pol | a/B/c/D/G/H |
ITLWQRPLV | HLA-A*6802 | pol | a/b/C/D/F/G/H |
ERYLKDQQL | HLA-B14 | gp41 | a/b/C/D |
YLKDQQLL | HLA-A24,B8 | gp41 | a/b/C/D |
TVYYGVPVWK | HLA-A*0301 | gp120 | A/B/C/D/g |
RPQVPLRPMTY | HLA-B51 | nef | A/b/D/E/F/G |
QVPLRPMTYK | HLA-A*0301,A11 | nef | A/b/D/E/F/G |
VPLRPMTY | HLA-B35 | nef | A/b/D/E/F/G |
AVDLSHFLK | HLA-A11 | nef | a/B/d/f |
DLSHFLKEK | HLA-A*0301 | nef | A/B/D/F |
FLKEKGGL | HLA-B8 | nef | A/B/C/D/E/F/G |
ILKEPVHGV | HLA-A*0201 | pol | A/B/C/D/G |
ILKEPVHGVY | HLA-Bw62 | pol | A/B/D |
HPDIVIYQY | HLA-B35 | pol | a |
VIYQYMDDL | HLA-A*0201 | pol | A/B/C/D/F/G/H |
TPGPGVRYPL | HLA-B7 | nef | b/c |
ACTPYDINQMLd | Mamu-A*01 | p27 | SIV |
RGPGRAFVTIe | H-2Dd | env | HIV |
和C端的Leu,由猕猴CTL识别,可以用于在猕猴中进行的效
力研究。E-由I类鼠MHC呈递的CTL表位,用于小鼠中的效力测定。
由于准备采纳“激发/加强”方案,其中激发通过给予核酸而完成,因此需要设计编码HIVA免疫原的核酸序列,以便增加HIVA在人细胞中的表达。首先,为保证有效起始从第一个甲硫氨酸密码子开始的翻译,在HIVA开放读码框架(ORF)前引入12个核苷酸长的Kozak共有序列(Kozak 1987 Nucl.Acids res.
15,8125-8148)。其次,通过用高度表达的人类基因中常用的密码子替代大多数HIV-1来源的密码子而优化产生的mRNA的翻译(Andre等,1998,前面已经引用)。HIVA ORF是1608个碱基对长的双链DNA HindIII-XbaI片段中的一部分。(图2A表示含有HIVA ORF的HindIII-XbaI插入序列的核苷酸序列;Seq.ID No.27)。在图2A中,包括了用于装配部分PCR产物的核酸内切酶位点(核苷酸1-6=HindIII;319-324=BamHI;712-717=KpnI;1135-1140=EcoRI;和1603-1608=XbaI)。
质粒DNA是用标准方案(Sambrook等,″Molecular Cloning.ALaboratory Manual″2nd Edition;Cold Spring Harbor)制备和处理的。在体外将HIVA基因构建为四部分(如图2B所示)。每一部分通过70-90个碱基长的重叠正链和负链寡脱氧核苷酸的装配而制备。根据生产商的说明用EconoPureTM试剂盒(Perkin Elmer)纯化合成的寡脱氧核苷酸,退火并连接,然后进行PCR装配。在每个步骤后凝胶纯化PCR产物,直到获得具有期望的、唯一的末端限制性核酸内切酶位点的片段。克隆并测序这四个产物,将其连接在一起而产生完整的HIVA基因。然后将HIVA基因插入下面所述的pTHr构建体和MVA疫苗载体。
PTHr载体。设计了一种用于直接基因转移的pTHr载体,其目的是尽量减小功能元件的数目,从而尽量减小需要给予的DNA量。
以前已经描述过pTH的构建(Hanke等,1998 Vaccine
16,426-435)。它包含人巨细胞病毒(hCMV)AD169株基因组的有效的表达增强子/启动子/内含子盒(Whittle等,1987 Protein Eng.1,499-505;Bebbington 1991 Methods 2,136-145)。启动子区后为pRc/CMV(Invitrogen)来源的多接头和牛生长激素基因的聚腺苷酸化信号。赋予被转化细菌氨苄青霉素抗性的β内酰胺酶基因和双链DNA原核复制起点ColE1均来自质粒pUC19。pTH不含哺乳动物细胞复制起点。在将HIVA DNA插入pTH多接头后,去除MluI和DraI位点间的β内酰胺酶基因片段,产生的构建体pTHr.HIVA(图3)在细菌中利用Cobra制药有限公司(Keele,UK)开发的阻抑物-滴定系统繁殖,该系统选择携带质粒的细菌,而不需要在质粒上存在抗生素抗性基因(Williams等,1998 Nucl.Acids Res.26,2120-2124)。所以,DNA接种不将大量抗生素抗性基因拷贝引入人类。pTHr.HIVA的构建如图3所示(CMV e/p/i=人CMV增强子/启动子/内含子A盒;BGHpA=牛生长激素聚腺苷酸化信号;ColE1=细菌中dsDNA复制起点;箭头符号表示阻抑物结合序列)。
在补充了10%胎牛血清(FBS;Gibco)、2nM L-谷氨酰胺和青霉素/链霉素的Dulbecco改良Eagle培养基(DMEM;Gibco)中维持293T细胞和鸡胚胎成纤维细胞(CEF)。在湿化的孵育器中,5%CO2和37℃的条件下培养细胞。利用DEAE-右旋糖苷一氯奎法(Hanke等,1998Vaccine 16,426-435)用pTHr.HIVA瞬时转染293T细胞。简言之,在6孔组织培养板中的盖玻片上使2.5×105个293T细胞生长过夜。第二天,用每孔5g DNA转染细胞。48小时后,固定转染细胞,透化处理它们的细胞膜,用SV5-P-k mAb并随后用抗鼠FITC结合的抗体检测所表达的重组蛋白。
说明该标本的显微照片见图4。该图表示三个转染细胞和一个作为本底的未转染细胞(左上)。
通过给予表达HIVA免疫原的MVA载体而完成加强。MVA是用于临床应用的安全减毒痘苗病毒,几乎失去其在人类细胞中复制的能力(Mayr等,1975 Infection 105,6-14)。已经广泛描述过MVA作为疫苗载体的用途以及使其成为在减毒痘病毒载体中成为一种引起人们兴趣的选择的特征(见如Sutter等,1994 Vaccine12,1032-1040;和Moss等,1996 Adv.Exp.Med.Biol.397,7-13)。
将HIVA基因连接到质粒pSC11中,该质粒将基因定位到亲代MVA胸腺嘧啶激酶位点(Carroll和Moss1995 Biotechniques19,352-256)。在从特定不含病原体的禽类卵中获得的初级CEF上培养大量重组MVA原种。在Beckman SW28转子上以13,500rpm的转速将经过36%(W/V)的蔗糖缓冲液提取的胞浆提取物离心80分钟以便纯化MVA。利用共同插入的β半乳糖苷酶基因,在用适当底物孵育感染的细胞单层后从蓝斑的数目判断病毒原种滴度。
疫苗效力测定。利用H-2Dd限制性表位的存在,在几组Balb/c小鼠中检测DNA和MVA载体的效力(Takahashi等,1993Int.Immunol.5,849-857)。对于pTHr.HIVA疫苗,小鼠可以用针头肌内注射100μgDNA或使用PowderJect疫苗公司(Madison,WI,USA)的皮肤XR基因递送设备,将总共2μgDNA隔3周进行两次皮内免疫。在最后一次免疫后10或21天处死小鼠。去除免疫小鼠的脾,用2ml注射器橡皮柱塞逐个按压其通过细胞粗滤器(Falcon)。洗涤脾细胞并将其分成两半,一半冻冷用于四聚物分析,另一半悬于5ml淋巴细胞培养基中(R10,20mM HEPES和15mMβ巯基乙醇)并通过在5%CO2,37℃下的湿化孵育器中用2μg/ml RGPGRAFVTI(Seq.ID No.2)肽孵育5天而进行体外重新刺激。
在U型底加样孔(96孔板;Costar)中2倍稀释效应细胞,起始效应细胞与靶细胞比例为100∶1。然后将没有补充或补充10-6M肽的培养基中的5000个铬标记的P815靶细胞加入效应细胞并在37℃下孵育5小时。计算孔中的自发和总铬释放,其中分别将靶细胞单独或与5%的Triton X-100一起放置在培养基中。特异性裂解的百分比计算为[(样品释放-自发释放)/(总释放-自发释放)]×100。自发释放低于总c.p.m的5%。
典型测定的结果见图5A-C,5A-C为特异性裂解百分比与效应细胞:靶细胞比例关系的图示。图5A表示从用100μg pTHr.HIVA DNA免疫一次的小鼠中获得的脾细胞的结果。图5B表示从用pTHr.HIVA DNA皮内免疫两次的小鼠得到的结果。图5C表示从用107pfu的MVA.HIVA肌内免疫的小鼠得到的结果。在图5A到5C中,每条线代表一个小鼠。肽脉冲激发靶细胞的裂解由涂黑的符号表示,未脉冲激发的靶细胞由空心符号表示。
两种pTHr.HIVA载体递送方式都是高免疫原性的,并且在所有被免疫动物中诱导高细胞裂解活性(见图5A和B)。同样,单次肌内注射107个MVA.HIVA的斑形成单位在所有疫苗中都引发强效肽特异性裂解活性,这是在培养物再刺激后测得的(图5C)。实施例2
制备编码基于HIVA的多蛋白免疫原的其它核酸构建体。这些其它的构建体和由它们编码的免疫原被称作HIVTA和HIVAeT,如图1A所示。图中也表示了称作PPA的构建体/免疫原。相关的DNA/氨基酸序列分别如图6A/B-8A/B所示,尽管图7A仅表示HIVAeT中氨基酸序列的一部分(可归于tat),即HIVTA中序列的额外部分。(注意DNA序列的5’和3’端的HindIII和XbaI位点的序列没有表示于图6B,7B和8B)。该构建体是通过与前面所描述的构建HIVA的方法相似的方法制备的。
HIVTA和HIVAeT的设计原理与HIVA相同,但额外包括了HIV-1分化体A tat序列,该序列表达为与gag和多表位合成多肽融合的蛋白的一部分(在HIVTA中,tat序列在gag和合成多肽之间),或存在于同样的构建体,但表达为分开的多肽(在HIVAeT中),这是由于引入了内部核糖体进入位点(IRES)。
每一种核酸分子都可以用于免疫受试者,其方法类似于上述与HIVA DNA相关的方法。同样,这些分子可以被引入适当的载体,特别是MVA,如实施例1中所描述,并将得到的载体用于免疫受试者。
个体HIV分离体中遗传多样性的意义和它对于疫苗设计的启示已经有长期的争论。HIV-1分化体在欧洲占大多数,而在美国,占大多数并且研究最多的是分化体B。在中非和东非,占大多数的流行HIV-1株是分化体A,而分化体C在南非、印度和中国占大多数。一般说来,相对于CTL,分化体特异性疫苗设计要求更细心考虑nAb的诱导。尽管在CTL表位中有一些重要的分化体间差异,许多表位在各种分化体中是保守的,这部分是由于结构/功能限制。然而,为促进效力研究的解释,应该试图使疫苗与在试验人群中占大多数的地区流行株相匹配,其目的在于如果没有达到交叉保护,可以使任何成功的方法适于其它分化体。
序列表<110>医疗研究局
国际艾滋病疫苗行动组织
内罗毕大学<120>对HIV免疫应答的改进或与其相关的改进<130>MJL/C1248′1/M<140><141><160>33<170>PatentIn Ver.2.1<210>1<211>11<212>PRT<213>猿免疫缺陷病毒<400>1Ala Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu1 5 10<210>2<211>10<212>PRT<213>人免疫缺陷病毒<400>2Arg Gly Pro Gly Arg Ala Phe Val Thr Ile1 5 10<210>3<211>9<212>PRT<213>人免疫缺陷病毒<400>3Ala Leu Lys His Arg Ala Tyr Glu Leu1 5<210>4<211>9<212>PRT<213>人免疫缺陷病毒<400>4Pro Pro Ile Pro Val Gly Glu Ile Tyr1 5<210>5<211>9<212>PRT<213>人免疫缺陷病毒<400>5Gly Glu Ile Tyr Lys Arg Trp Ile Ile1 5<210>6<211>10<212>PRT<213>人免疫缺陷病毒<400>6Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys1 5 10<210>7<211>10<212>PRT<213>人免疫缺陷病毒<400>7Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys1 5 10<210>8<211>11<212>PRT<213>人免疫缺陷病毒<400>8Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu1 5 10<210>9<211>9<212>PRT<213>人免疫缺陷病毒<400>9Asp Arg Phe Tyr Lys Thr Leu Arg Ala1 5<210>10<211>9<212>PRT<213>人免疫缺陷病毒<400>10Ala Ile Phe Gln Ser Ser Met Thr Lys1 5<210>11<211>9<212>PRT<213>人免疫缺陷病毒<400>11Ile Thr Leu Trp Gln Arg Pro Leu Val1 5<210>12<211>9<212>PRT<213>人免疫缺陷病毒<400>12Glu Arg Tyr Leu Lys Asp Gln Gln Leu1 5<210>13<211>8<212>PRT<213>人免疫缺陷病毒<400>13Tyr Leu Lys Asp Gln Gln Leu Leu1 5<210>14<211>10<212>PRT<213>人免疫缺陷病毒<400>14Thr Val Tyr Tyr Gly Val Pro Val Trp Lys1 5 10<210>15<211>11<212>PRT<213>人免疫缺陷病毒<400>15Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr1 5 10<210>16<211>10<212>PRT<213>人免疫缺陷病毒<400>16Gln Val Pro Leu Arg Pro Met Thr Tyr Lys1 5 10<210>17<211>8<212>PRT<213>人免疫缺陷病毒<400>17Val Pro Leu Arg Pro Met Thr Tyr1 5<210>18<211>9<212>PRT<213>人免疫缺陷病毒<400>18Ala Val Asp Leu Ser His Phe Leu Lys1 5<210>19<211>9<212>PRT<213>人免疫缺陷病毒<400>19Asp Leu Ser His Phe Leu Lys Glu Lys1 5<210>20<211>8<212>PRT<213>人免疫缺陷病毒<400>20Phe Leu Lys Glu Lys Gly Gly Leu1 5<210>21<211>9<212>PRT<213>人免疫缺陷病毒<400>21Ile Leu Lys Glu Pro Val His Gly Val1 5<210>22<211>10<212>PRT<213>人免疫缺陷病毒<400>22Ile Leu Lys Glu Pro Val His Gly Val Tyr1 5 10<210>23<211>9<212>PRT<213>人免疫缺陷病毒<400>23His Pro Asp Ile Val Ile Tyr Gln Tyr1 5<210>24<211>9<212>PRT<213>人免疫缺陷病毒<400>24Val Ile Tyr Gln Tyr Met Asp Asp Leu1 5<210>25<211>10<212>PRT<213>人免疫缺陷病毒<400>25Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu1 5 10<210>26<211>527<212>PRT<213>人工序列<220><223>人工序列说明:嵌合多肽<400>26Met Pro Ile Val Gln Asn Ala Gln Gly Gln Met His Gln Ala Leu Ser1 5 10 15Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe
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35 40 45Pro Gln Asp Leu Asn Met Met Leu Asn Ile Val Gly Gly His Gln Ala
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85 90 95Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln
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115 120 125Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met
130 135 140Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro145 150 155 160Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu Gln
165 170 175Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln
180 185 190Asn Ala Asn Pro Asp Cys Lys Ser Ile Leu Arg Ala Leu Gly Pro Gly
195 200 205Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro
210 215 220Gly His Lys Ala Arg Val Leu Gly Thr Gly Ala Arg Ala Ser Val Leu225 230 235 240Ser Gly Gly Lys Leu Asp Ala Trp Glu Lys Ile Arg Leu Arg Pro Gly
245 250 255Gly Lys Lys Lys Tyr Arg Leu Lys His Leu Val Trp Ala Ser Arg Glu
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275 280 285Cys Gln Gln Ile Met Glu Gln Leu Gln Ser Ala Leu Lys Thr Ser Glu
290 295 300Glu Leu Lys Ser Leu Phe Asn Thr Val Ala Thr Leu Tyr Cys Val His305 310 315 320Gln Arg Ile Asp Val Lys Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu
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405 410 415Pro Leu Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Thr Val Tyr
420 425 430Tyr Gly Val Pro Val Trp Lys Arg Pro Gln Val Pro Leu Arg Pro Met
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500 505 510Gly Arg Ala Phe Val Thr Ile Pro Asn Pro Leu Leu Gly Leu Asp
515 520 525<210>27<211>1608<212>DNA<213>人工序列<220><223>人工序列说明:嵌合多肽<400>27aagcttcccg ccgccaccat gcccatcgtg cagaacgccc agggccagat gcaccaggcc 60ctgtcccccc gcaccctgaa cgcctgggtg aaggtgatcg aggagaaggc cttctccccc 120gaggtgatcc ccatgttctc cgccctgtcc gagggcgcca ccccccagga cctgaacatg 180atgctgaaca tcgtgggcgg ccaccaggcc gccatgcaga tgctgaagga caccatcaac 240gaggaggccg ccgagtggga ccgcctgcac cccgtgcacg ccggccccat cccccccggc 300cagatgcgcg agccccgcgg atccgacatc gccggcacca cctccaccct gcaggagcag 360atcggctgga tgacctccaa cccccccatc cccgtgggcg acatctacaa gcgctggatc 420atcctgggcc tgaacaagat cgtacgcatg tactcccccg tgtccatcct ggacatccgc 480cagggcccca aggagccctt ccgcgactac gtggaccgct tcttcaagac cctgcgcgcc 540gagcaggcca cccaggaggt gaagaactgg atgaccgaga ccctgctggt gcagaacgcc 600aaccccgact gcaagtccat cctgcgcgcc ctgggccccg gcgccaccct ggaggagatg 660atgaccgcct gccagggcgt gggcggcccc ggccacaagg cccgcgtgct gggtaccggc 720gcccgcgcct ccgtgctgtc cggcggcaag ctggacgcct gggagaagat ccgcctgcgc 780cccggcggca agaagaagta ccgcctgaag cacctggtgt gggcctcccg cgagctggag 840cgcttcgccc tgaacccctc cctgctggag accgccgagg gctgccagca gatcatggag 900cagctgcagt ccgccctgaa gacctccgag gagctgaagt ccctgttcaa caccgtggcc 960accctgtact gcgtgcacca gcgcatcgac gtgaaggaca ccaaggaggc cctggacaag 1020atcgaggaga tccagaacaa gtccaagcag aagacccagc aggccgccgc cgacacccag 1080tcctcctcca aggtgtccca gaactacgcc ctgaagcacc gcgcctacga gctggaattc 1140cctccaattc ctgtcgggga gatttataaa cggtggatca tttttaggga ttatgtcgat 1200aggttttata aaacgctcag ggccatcttc cagtcctcca tgaccaagat caccctgtgg 1260cagcgccccc tggtggagcg ctacctgaag gaccagcagc tgctgaccgt gtactacggc 1320gtgcccgtgt ggaagcgccc ccaggtgccc ctgcgcccca tgacctacaa ggccgtggac 1380ctgtcccact tcctgaagga gaagggcggc ctgatcctga aggagcccgt gcacggcgtg 1440taccaccccg acatcgtgat ctaccagtac atggacgacc tgacccccgg ccccggcgtg 1500cgctaccccc tggcctgcac cccctacgac atcaaccaga tgctgcgcgg ccccggccgc 1560gccttcgtga ccatccccaa ccccctgctg ggcctggact gatctaga 1608<210>28<211>633<212>PRT<213>人工序列<220><223>人工序列说明:嵌合多肽<400>28Met Pro Ile Val Gln Asn Ala Gln Gly Gln Met His Gln Ala Leu Ser1 5 10 15Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe
20 25 30Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr
35 40 45Pro Gln Asp Leu Asn Met Met Leu Asn Ile Val Gly Gly His Gln Ala
50 55 60Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp65 70 75 80Asp Arg Leu His Pro Val His Ala Gly Pro Ile Pro Pro Gly Gln Met
85 90 95Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln
100 105 110Glu Gln Ile Gly Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly Asp
115 120 125Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met
130 135 140Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro145 150 155 160Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu Gln
165 170 175Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln
180 185 190Asn Ala Asn Pro Asp Cys Lys Ser Ile Leu Arg Ala Leu Gly Pro Gly
195 200 205Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro
210 215 220Gly His Lys Ala Arg Val Leu Gly Thr Gly Ala Arg Ala Ser Val Leu225 230 235 240Ser Gly Gly Lys Leu Asp Ala Trp Glu Lys Ile Arg Leu Arg Pro Gly
245 250 255Gly Lys Lys Lys Tyr Arg Leu Lys His Leu Val Trp Ala Ser Arg Glu
260 265 270Leu Glu Arg Phe Ala Leu Asn Pro Ser Leu Leu Glu Thr Ala Glu Gly
275 280 285Cys Gln Gln Ile Met Glu Gln Leu Gln Ser Ala Leu Lys Thr Ser Glu
290 295 300Glu Leu Lys Ser Leu Phe Asn Thr Val Ala Thr Leu Tyr Cys Val His305 310 315 320Gln Arg Ile Asp Val Lys Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu
325 330 335Glu Ile Gln Asn Lys Ser Lys Gln Lys Thr Gln Gln Ala Ala Ala Asp
340 345 350Thr Gln Ser Ser Ser Lys Val Ser Gln Asn Tyr Ala Leu Lys His Arg
355 360 365Ala Tyr Glu Leu Glu Phe Met Ala Thr Thr Met Asp Pro Val Asp Pro
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405 410 415Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln
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580 585 590Asp Leu Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Ala Cys Thr Pro
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20 25 30Val Cys Cys Tyr His Cys Pro Val Cys Phe Leu Asn Lys Gly Leu Gly
35 40 45Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Gly Thr Pro Gln
50 55 60Ser Asn Lys Asp His Gln Asn Pro Ile Pro Lys Gln Pro Ile Pro Gln65 70 75 80Thr Gln Gly Ile Ser Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu
85 90 95Ser Lys Thr Glu Thr Asp Pro Glu
100<210>31<211>2493<212>DNA<213>人工序列<220><223>人工序列说明:嵌合多肽<400>31cccgccgcca ccatgcccat cgtgcagaac gcccagggcc agatgcacca ggccctgtcc 60ccccgcaccc tgaacgcctg ggtgaaggtg atcgaggaga aggccttctc ccccgaggtg 120atccccatgt tctccgccct gtccgagggc gccacccccc aggacctgaa catgatgctg 180aacatcgtgg gcggccacca ggccgccatg cagatgctga aggacaccat caacgaggag 240gccgccgagt gggaccgcct gcaccccgtg cacgccggcc ccatcccccc cggccagatg 300cgcgagcccc gcggatccga catcgccggc accacctcca ccctgcagga gcagatcggc 360tggatgacct ccaacccccc catccccgtg ggcgacatct acaagcgctg gatcatcctg 420ggcctgaaca agatcgtgcg catgtactcc cccgtgtcca tcctggacat ccgccagggc 480cccaaggagc ccttccgcga ctacgtggac cgcttcttca agaccctgcg cgccgagcag 540gccacccagg aggtgaagaa ctggatgacc gagaccctgc tggtgcagaa cgccaacccc 600gactgcaagt ccatcctgcg cgccctgggc cccggcgcca ccctggagga gatgatgacc 660gcctgccagg gcgtgggcgg ccccggccac aaggcccgcg tgctgggtac cggcgcccgc 720gcctccgtgc tgtccggcgg caagctggac gcctgggaga agatccgcct gcgccccggc 780ggcaagaaga agtaccgcct gaagcacctg gtgtgggcct cccgcgagct ggagcgcttc 840gccctgaacc cctccctgct ggagaccgcc gagggctgcc agcagatcat ggagcagctg 900cagtccgccc tgaagacctc cgaggagctg aagtccctgt tcaacaccgt ggccaccctg 960tactgcgtgc accagcgcat cgacgtgaag gacaccaagg aggccctgga caagatcgag 1020gagatccaga acaagtccaa gcagaagacc cagcaggccg ccgccgacac ccagtcctcc 1080tccaaggtgt cccagaacta cgccctgaag caccgcgcct acgagctgga attccctcca 1140attcctgtcg gggagattta taaacggtgg atcattttta gggattatgt cgataggttt 1200tataaaacgc tcagggccat cttccagtcc tccatgacca agatcaccct gtggcagcgc 1260cccctggtgg agcgctacct gaaggaccag cagctgctga ccgtgtacta cggcgtgccc 1320gtgtggaagc gcccccaggt gcccctgcgc cccatgacct acaaggccgt ggacctgtcc 1380cacttcctga aggagaaggg cggcctgatc ctgaaggagc ccgtgcacgg cgtgtaccac 1440cccgacatcg tgatctacca gtacatggac gacctgaccc ccggccccgg cgtgcgctac 1500cccctggcct gcacccccta cgacatcaac cagatgctgc gcggccccgg ccgcgccttc 1560gtgaccatcc ccaaccccct gctgggcctg gactgagcgg ccgcccctct ccctcccccc 1620cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt 1680tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct 1740tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga 1800atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga 1860ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac 1920gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag 1980ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc 2040agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg 2100tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt 2160gaaaaacacg atgataatat ggccacaacc atggaccccg tggaccccaa cctggagccc 2220tggaaccacc ccggctccca gcccaccacc cccggctcca agtgctactg caaggtgtgc 2280tgctaccact gccccgtgtg cttcctgaac aagggcctgg gcatctccta cggccgcaag 2340aagcgccgcc agcgccgcgg caccccccag tccaacaagg accaccagaa ccccatcccc 2400aagcagccca tcccccagac ccagggcatc tccaccggcc ccaaggagtc caagaagaag 2460gtggagtcca agaccgagac cgaccccgag taa 2493<210>32<211>1445<212>PRT<213>人工序列<220><223>人工序列说明:嵌合多肽<400>32Met Pro Ile Val Gln Asn Ala Gln Gly Gln Met His Gln Ala Leu Ser1 5 10 15Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe
20 25 30Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr
35 40 45Pro Gln Asp Leu Asn Met Met Leu Asn Ile Val Gly Gly His Gln Ala
50 55 60Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp65 70 75 80Asp Arg Leu His Pro Val His Ala Gly Pro Ile Pro Pro Gly Gln Met
85 90 95Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln
100 105 110Glu Gln Ile Gly Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly Asp
115 120 125Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met
130 135 140Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro145 150 155 160Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu Gln
165 170 175Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln
180 185 190Asn Ala Asn Pro Asp Cys Lys Ser Ile Leu Arg Ala Leu Gly Pro Gly
195 200 205Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro
210 215 220Gly His Lys Ala Arg Val Leu Gly Thr Gly Ala Arg Ala Ser Val Leu225 230 235 240Ser Gly Gly Lys Leu Asp Ala Trp Glu Lys Ile Arg Leu Arg Pro Gly
245 250 255Gly Lys Lys Lys Tyr Arg Leu Lys His Leu Val Trp Ala Ser Arg Glu
260 265 270Leu Glu Arg Phe Ala Leu Asn Pro Ser Leu Leu Glu Thr Ala Glu Gly
275 280 285Cys Gln Gln Ile Met Glu Gln Leu Gln Ser Ala Leu Lys Thr Ser Glu
290 295 300Glu Leu Lys Ser Leu Phe Asn Thr Val Ala Thr Leu Tyr Cys Val His305 310 315 320Gln Arg Ile Asp Val Lys Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu
325 330 335Glu Ile Gln Asn Lys Ser Lys Gln Lys Thr Gln Gln Ala Ala Ala Asp
340 345 350Thr Gln Ser Ser Ser Lys Val Ser Gln Asn Tyr Ala Leu Lys His Arg
355 360 365Ala Tyr Glu Leu Glu Phe Gly Ile Lys Val Lys Gln Leu Cys Lys Leu
370 375 380Leu Arg Gly Ala Lys Ala Leu Thr Asp Ile Val Thr Leu Thr Glu Glu385 390 395 400Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Asp Pro Val
405 410 415His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln
420 425 430Lys Gln Gly Gln Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe
435 440 445Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Lys Arg Ser Ala Gln Thr
450 455 460Asn Asp Val Lys Gln Leu Ala Glu Val Val Gln Lys Val Val Met Glu465 470 475 480Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile Gln
485 490 495Lys Glu Thr Trp Glu Thr Trp Trp Met Asp Tyr Trp Gln Ala Thr Trp
500 505 510Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp
515 520 525Tyr Gln Leu Glu Lys Asp Pro Ile Ala Gly Ala Glu Thr Phe Tyr Val
530 535 540Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly Lys Ala Gly Tyr Val545 550 555 560Thr Asp Arg Gly Arg Gln Lys Val Val Ser Leu Thr Glu Thr Thr Asn
565 570 575Gln Lys Thr Glu Leu His Val Ile His Leu Ala Leu Gln Asp Ser Gly
580 585 590Ser Glu Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile
595 600 605Gln Ala Gln Pro Asp Arg Ser Asp Pro Val Asp Pro Asn Leu Glu Pro
610 615 620Trp Asn His Pro Gly Ser Gln Pro Thr Thr Pro Gly Ser Lys Cys Tyr625 630 635 640Cys Lys Val Cys Cys Tyr His Cys Pro Val Cys Phe Leu Asn Lys Gly
645 650 655Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Gly Thr
660 665 670Pro Gln Ser Asn Lys Asp His Gln Asn Pro Ile Pro Lys Gln Pro Ile
675 680 685Pro Gln Thr Gln Gly Ile Ser Thr Gly Pro Lys Glu Ser Lys Lys Lys
690 695 700Val Glu Ser Lys Thr Glu Thr Asp Pro Glu Asp Ala Gly Arg Ser Gly705 710 715 720Asn Ser Asp Glu Glu Leu Leu Lys Ala Ile Arg Ile Ile Lys Ile Leu
725 730 735Tyr Gln Ser Asn Pro Tyr Pro Lys Pro Lys Gly Ser Arg Gln Ala Arg
740 745 750Lys Asn Arg Arg Arg Arg Trp Arg Ala Gly Gln Arg Gln Ile Asp Ser
755 760 765Leu Ser Glu Arg Ile Leu Ser Thr Cys Leu Gly Arg Pro Ala Glu Pro
770 775 780Val Pro Leu Gln Leu Pro Pro Leu Glu Leu Asp Cys Ser Glu Asp Cys785 790 795 800Gly Thr Ser Gly Thr Gln Gln Ser Gln Gly Ala Glu Thr Gly Val Gly
805 810 815Arg Pro Gln Val Ser Val Glu Ser Ser Ala Val Leu Gly Ser Gly Thr
820 825 830Lys Glu Gly Thr Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
835 840 845Lys Ala Ala Phe Asp Leu Ser Phe Phe Leu Lys Glu Lys Gly Gly Leu
850 855 860Asp Gly Leu Ile Tyr Ser Lys Lys Arg Gln Glu Ile Leu Asp Leu Trp865 870 875 880Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro
885 890 895Gly Pro Gly Ile Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu
900 905 910Val Pro Val Asp Pro Asp Glu Val Glu Glu Ala Thr Gly Gly Glu Asn
915 920 925Asn Ser Leu Leu His Pro Ile Cys Gln His Gly Met Asp Asp Glu Glu
930 935 940Lys Glu Thr Leu Arg Trp Lys Phe Asp Ser Ser Leu Ala Leu Lys His945 950 955 960Arg Ala Arg Glu Leu His Pro Glu Ser Tyr Lys Asp Cys Pro Gln Ile
965 970 975Thr Leu Trp Gln Arg Pro Leu Val Thr Lys Ile Gly Gly Gln Lys Thr
980 985 990Arg Gly Gly Lys Trp Ser Lys Ser Ser Ile Val Gly Trp Pro Glu Val
995 1000 1005Arg Glu Arg Ile Arg Gln Thr Pro Thr Ala Ala Arg Glu Arg Thr Arg1010 1015 1020Gln Ala Pro Thr Ala Ala Lys Val Gly Ala Val Ser Gln Asp Leu Asp1025 1030 1035 1040Lys His Gly Ala Val Ser Ser Asn Val Asn His Pro Ser Cys Ala Trp
1045 1050 1055Leu Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Glu Leu Leu Asp
1060 1065 1070Thr Gly Ala Asp Asp Thr Val Leu Glu Asp Ile Asn Leu Pro Gly Lys
1075 1080 1085Trp Lys Pro Lys Met Ile Gly Gly Ile Gly Gly Leu Ile Lys Val Lys1090 1095 1100Gln Tyr Asp Gln Ile Leu Ile Glu Ile Cys Gly Lys Lys Ala Ile Gly1105 1110 1115 1120Thr Val Leu Val Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met
1125 1130 1135Leu Thr Gln Ile Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu
1140 1145 1150Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Lys
1155 1160 1165Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Thr Glu Ile Cys1170 1175 1180Ala Asp Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu Asn1185 1190 1195 1200Pro Tyr Asn Thr Pro Ile Phe Ala Ile Lys Lys Lys Gln Ser Thr Lys
1205 1210 1215Trp Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp
1220 1225 1230Phe Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys Lys
1235 1240 1245Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val1250 1255 1260Pro Leu Asp Glu Ser Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser1265 1270 1275 1280Thr Asn Asn Glu Thr Pro Gly Val Arg Tyr Gln Tyr Asn Val Leu Pro
1285 1290 1295Gln Gly Trp Lys Gly Ser Pro Ile Phe Gln Ser Ser Met Thr Lys Ile
1300 1305 1310Leu Glu Pro Phe Arg Ser Lys Asn Pro Asp Ile Val Ile Tyr Gln Tyr
1315 1320 1325Met Asp Asp Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg1330 1335 1340Thr Lys Ile Glu Glu Leu Arg Ala His Leu Leu Ser Trp Gly Phe Ile1345 1350 1355 1360Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly
1365 1370 1375Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Glu Leu Pro
1380 1385 1390Glu Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys
1395 1400 1405Leu Asn Trp Ala Ser Gln Ile Tyr Ala Cys Thr Pro Tyr Asp Ile Asn1410 1415 1420Gln Met Leu Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Pro Asn Pro1425 1430 1435 1440Leu Leu Gly Leu Asp
1445<210>33<211>4350<212>DNA<213>人工序列<220><223>人工序列说明:嵌合多肽<400>33cccgccgcca ccatgcccat cgtgcagaac gcccagggcc agatgcacca ggccctgtcc 60ccccgcaccc tgaacgcctg ggtgaaggtg atcgaggaga aggccttctc ccccgaggtg 120atccccatgt tctccgccct gtccgagggc gccacccccc aggacctgaa catgatgctg 180aacatcgtgg gcggccacca ggccgccatg cagatgctga aggacaccat caacgaggag 240gccgccgagt gggaccgcct gcaccccgtg cacgccggcc ccatcccccc cggccagatg 300cgcgagcccc gcggatccga catcgccggc accacctcca ccctgcagga gcagatcggc 360tggatgacct ccaacccccc catccccgtg ggcgacatct acaagcgctg gatcatcctg 420ggcctgaaca agatcgtgcg catgtactcc cccgtgtcca tcctggacat ccgccagggc 480cccaaggagc ccttccgcga ctacgtggac cgcttcttca agaccctgcg cgccgagcag 540gccacccagg aggtgaagaa ctggatgacc gagaccctgc tggtgcagaa cgccaacccc 600gactgcaagt ccatcctgcg cgccctgggc cccggcgcca ccctggagga gatgatgacc 660gcctgccagg gcgtgggcgg ccccggccac aaggcccgcg tgctgggtac cggcgcccgc 720gcctccgtgc tgtccggcgg caagctggac gcctgggaga agatccgcct gcgccccggc 780ggcaagaaga agtaccgcct gaagcacctg gtgtgggcct cccgcgagct ggagcgcttc 840gccctgaacc cctccctgct ggagaccgcc gagggctgcc agcagatcat ggagcagctg 900cagtccgccc tgaagacctc cgaggagctg aagtccctgt tcaacaccgt ggccaccctg 960tactgcgtgc accagcgcat cgacgtgaag gacaccaagg aggccctgga caagatcgag 1020gagatccaga acaagtccaa gcagaagacc cagcaggccg ccgccgacac ccagtcctcc 1080tccaaggtgt cccagaacta cgccctgaag caccgcgcct acgagctgga attcggcatc 1140aaggtgaagc agctgtgcaa gctgctgcgc ggcgccaagg ccctgaccga catcgtgacc 1200ctgaccgagg aggccgagct ggagctggcc gagaaccgcg agatcctgaa ggaccccgtg 1260cacggcgtgt actacgaccc ctccaaggac ctgatcgccg agatccagaa gcagggccag 1320gaccagtgga cctaccaaat ctaccaggag cccttcaaga acctgaagac cggcaagtac 1380gcccgcaagc gctccgccca gaccaacgac gtgaagcagc tggccgaggt ggtgcagaag 1440gtggtgatgg agtccatcgt gatctggggc aagaccccca agttccgcct gcccatccag 1500aaggagacct gggagacctg gtggatggac tactggcagg ccacctggat tcccgagtgg 1560gagttcgtga acaccccacc cctggtgaag ctgtggtatc agctggagaa ggaccccatc 1620gccggcgccg agaccttcta cgtggacggc gccgccaacc gcgagaccaa gctgggcaag 1680gccggctacg tgaccgaccg gggccgccag aaggtggtgt ccctgaccga gaccaccaac 1740cagaagaccg agctgcacgt catccacctg gccctgcagg actccggctc cgaggtgaac 1800atcgtgaccg actcccagta cgccctgggc atcatccagg cccagcccga cagatctgac 1860cccgtggacc ccaacctgga gccctggaac caccccggct cccagcccac cacccccggc 1920tccaagtgct actgcaaggt gtgctgctac cactgccccg tgtgcttcct gaacaagggc 1980ctgggcatct cctacggccg caagaagcgc cgccagcgcc gcggcacccc ccagtccaac 2040aaggaccacc agaaccccat ccccaagcag cccatccccc agacccaggg catctccacc 2100ggccccaagg agtccaagaa gaaggtggag tccaagaccg agaccgaccc cgaggacgcc 2160ggccgctccg gcaactccga cgaggagctg ctgaaggcca tccgcatcat caagatcctg 2220taccagtcca acccctaccc caagcccaag ggctcccgcc aggcccgcaa gaaccgccgc 2280cgccgctggc gcgccggcca gcgccagatc gactccctgt ccgagcgcat cctgtccacc 2340tgcctgggcc gccccgccga gcccgtgccc ctgcagctgc cccccctgga gctggactgc 2400tccgaggact gcggcacctc cggcacccag cagtcccagg gcgccgagac cggcgtgggc 2460cgcccccagg tgtccgtgga gtcctccgcc gtgctgggct ccggcaccaa ggagggtacc 2520gtgcgccccc aggtgcccct gcgccccatg acctacaagg ccgccttcga cctgtccttc 2580tttctgaagg agaagggcgg cctggacggc ctgatctact ccaagaagcg ccaggagatc 2640ctggacctgt gggtgtacca cacccagggc tacttccccg actggcagaa ctacaccccc 2700ggccccggca tccgctaccc cctgaccttc ggctggtgct tcaagctggt gcccgtggac 2760cccgacgagg tggaggaggc caccggcggc gagaacaact ccctgctgca ccccatctgc 2820cagcacggca tggacgacga ggagaaggag accctgcgct ggaagttcga ctcctccctg 2880gccctgaagc accgcgcccg cgaactccac cccgagtcct acaaggactg cccccagatc 2940accctgtggc agcgccccct ggtgaccaag atcggcggcc agaagacgcg tggcggcaag 3000tggtccaagt cctccatcgt gggctggccc gaggtgcgcg agcgcatccg ccagaccccc 3060accgccgccc gcgagcgcac ccgccaggcc cccaccgccg ccaaggtggg cgccgtgtcc 3120caggacctgg acaagcacgg cgccgtgtcc tccaacgtga accacccctc ctgcgcctgg 3180ctggaggccc aggaagagga agaggtgggc ttccccgagc tcctggacac cggcgccgac 3240gacaccgtgc tggaggacat caacctgccc ggcaagtgga agcccaagat gatcggcggc 3300atcggcggct tgatcaaggt gaagcagtac gaccagatcc tgatcgaaat ctgcggcaag 3360aaggccatcg gcaccgtgct ggtgggcccc acccccgtga acatcatcgg ccgcaacatg 3420ctgacccaga tcggctgcac cctgaacttc cccatctccc ccatcgagac cgtgcccgtg 3480aagctgaagc ccggcatgga cggccccaag gtgaagcagt ggcccctgac cgaggagaag 3540atcaaggccc tgaccgaaat ctgcgccgac atggagaagg agggcaagat cagtaagatc 3600ggccccgaga acccctacaa cacccccatc ttcgccatca agaagaagca gtccaccaag 3660tggcgcaagc tggtggactt ccgcgagctg aacaagcgca cccaggactt ctgggaggtg 3720cagctgggca tcccccaccc cgccggcctg aagaagaaaa agtccgtgac cgtgctggac 3780gtgggcgacg cctacttctc cgtgcccctg gacgagtcct tccgcaagta caccgccttc 3840accatcccct ccaccaacaa cgagaccccc ggcgtgcgct accagtacaa cgtgctgccc 3900cagggctgga agggatcccc catcttccag tcctccatga ccaagatcct ggagcccttc 3960cgctccaaga accccgacat cgtgatctac cagtacatgg acgacctgta cgtgggctcc 4020gacctggaga tcggccagca ccgcaccaag atcgaggagc tgcgcgccca cctgctgtcc 4080tggggcttca tcacccccga caagaagcac cagaaggagc cccccttcct gtggatgggc 4140tacgagctgc accccgacaa gtggaccgtg cagcccatcg agctgcccga gaaggactcc 4200tggaccgtga acgacatcca gaagctggtg ggcaagctga actgggcctc ccaaatctac 4260gcctgcaccc cctacgacat caaccagatg ctgcgcggcc ccggccgcgc cttcgtgacc 4320atccccaacc ccctgctggg cctggactag 4350
Claims (47)
1.一种适于给予人类受试者的无病原体形式的免疫原,该免疫原包含:至少一部分HIV的gag蛋白,所述gag蛋白来源于一种HIV分化体或与一种或更多HIV分化体具有共有序列,并至少包含部分p17和p24;以及包含多个氨基酸序列的合成多肽,每个序列包含一种HIV蛋白的人CTL表位,并且其中许多HIV蛋白表现为合成多肽形式,所述CTL表位的选择是为了刺激对一种或更多感兴趣的HIV分化体的免疫应答。
2.权利要求1的免疫原,其中所述至少一部分gag蛋白和合成多肽作为融合蛋白存在。
3.权利要求1或2的免疫原,其中gag蛋白55-95%的氨基酸序列存在于gag的所述部分。
4.权利要求1、2或3的任意一项的免疫原,其中gag蛋白65-85%的氨基酸序列存在于gag的所述部分。
5.前面任意一项权利要求的免疫原,其中修饰了所述至少部分p17以防止N端豆蔻酰化。
6.前面任意一项权利要求的免疫原,其中至少一些存在于合成多肽中的人CTL表位是重叠的。
7.权利要求6的免疫原,其中至少50%存在于合成多肽中的人CTL表位是重叠的。
8.前面任意一项权利要求的免疫原,其中至少一些存在于合成多肽中的人CTL表位是邻接的。
9.前面任意一项权利要求的免疫原,其中至少一些人CTL表位是由非表位氨基酸序列分开的。
10.前面任意一项权利要求的免疫原,包含至少一个由一种或更多实验室检验哺乳动物识别的表位。
11.权利要求10的免疫原,其中所述由一种或更多实验室检验哺乳动物识别的表位是CTL表位。
12.前面任意一项权利要求的免疫原,其中合成多肽包含至少10个表1所指出的人CTL表位。
13.前面任意一项权利要求的免疫原,其中合成多肽包含至少15个表1所指出的人CTL表位。
14.前面任意一项权利要求的免疫原,其中合成多肽包含至少20个表1所指出的人CTL表位。
15.前面任意一项权利要求的免疫原,其中合成多肽包含所有23个表1所指出的人CTL表位。
16.一种用于人类受试者的HIV免疫原,该免疫原包含:至少一部分gag蛋白,所述gag蛋白来源于一种感兴趣的HIV分化体或与一种感兴趣的HIV分化体具有共有序列,其中所述部分至少包含部分p17和p24,并且经修饰而防止N端豆蔻酰化;以及包含来自于多种不同HIV蛋白的多个人CTL表位的合成多肽,每个CTL表位具有8-12个氨基酸,所述CTL表位的选择是为了刺激对感兴趣的分化体的免疫应答。
17.一种用于人类受试者的HIV免疫原,该免疫原包含:至少一部分gag蛋白,所述gag蛋白来源于一种感兴趣的HIV分化体或与一种感兴趣的HIV分化体具有共有序列,其中所述部分至少包含部分p17和p24,并且经修饰而防止N端豆蔻酰化;以及包含来自于多种不同HIV蛋白的多个人CTL表位的合成多肽,每个CTL表位具有8-12个氨基酸,所述CTL表位的选择是为了刺激对所述感兴趣的分化体的免疫应答;并且所述合成多肽可选择具有至少一个由实验室动物识别的免疫原性表位。
18.前面任意一项权利要求的免疫原,包含图1B所示的氨基酸序列。
19.权利要求18的免疫原,基本由图1B所示的氨基酸序列组成。
20.前面任意一项权利要求的免疫原,其中所述感兴趣的分化体选自HIV分化体A,B,C,D,E,F,G和H。
21.权利要求20的免疫原,其中所述感兴趣的分化体为HIV分化体A。
22.权利要求20的免疫原,其中所述感兴趣的分化体为HIV分化体B。
23.权利要求20的免疫原,其中所述感兴趣的分化体为HIV分化体C。
24.前面任意一项权利要求的免疫原,其中所述人CTL表位包括至少一种来自于HIV蛋白nef,p24,p17,pol,gp41和gp120的每一种的表位。
25.权利要求24的免疫原,其中所述人CTL表位包括至少一种来自于HIV蛋白nef,p24,p17,pol,gp41,gp120,tat和rev的每一种的表位。
26.权利要求24的免疫原,其中所述人CTL表位包括至少一种来自于HIV蛋白nef,p24,p17,pol,gp41,gp120,tat,rev,vpr,vpu和vif的每一种的表位。
27.编码前面任意一项权利要求的免疫原的核酸分子。
28.权利要求27的核酸分子,其中将免疫原编码为融合蛋白。
29.权利要求27或28的核酸分子,包含图2A所示核苷酸序列。
30.包含权利要求27、28或29的任意一项的核酸分子的载体。
31.权利要求30的一种特殊载体,进一步包含权利要求1-26的任意一项的免疫原。
32.一种刺激人类受试者中抗HIV免疫应答的方法,该方法包括以下步骤:制备权利要求1-26的任意一项的免疫原和/或权利要求27-29的任意一项的核酸分子;并将该免疫原和/或该核酸分子给予受试者。
33.权利要求32的方法,其中通过一次或多次给予该核酸分子激发受试者;并随后通过一次或多次给予免疫原而加强。
34.权利要求1-26的任意一项的免疫原和/或权利要求27-29的任意一项的核酸分子在制备预防或治疗人类受试者中HIV感染的药物中的用途。
35.一种刺激人类受试者中抗HIV免疫应答的方法,包括一次或多次以足够激发对所述免疫原的免疫应答的含量给予所述受试者编码权利要求1-26的任意一项的免疫原的核酸分子,并一次或多次以足够加强对所述免疫原常见部分的免疫应答的含量给予所述受试者编码和/或含有权利要求1-26的任意一项的免疫原的修饰痘苗病毒Ankara(MYA)颗粒。
36.一种细菌,含有权利要求1-26的任意一项的免疫原,或含有权利要求27-29的任意一项的核酸分子。
37.权利要求36的细菌,该细菌为减毒病原体,适于给予人类受试者。
38.一种编码包含图8A所示氨基酸序列的多肽的核酸分子。
39.一种编码基本由图8A所示氨基酸序列组成的多肽的核酸分子。
40.权利要求38或39的核酸分子,包含图8B所示核苷酸序列。
41.一种包含图8A所示氨基酸序列的多肽。
42.一种基本由图8A所示氨基酸序列组成的多肽。
43.一种特殊的载体,包含权利要求38或39的核酸序列和/或权利要求41或42的多肽。
44.一种刺激人类受试者中抗HIV免疫应答的方法,该方法包括以下步骤:制备权利要求38或39的核酸分子和/或权利要求41或42的多肽,并将该核酸分子和/或该多肽给予受试者。
45.权利要求38或39的核酸分子和/或权利要求41或42的多肽在制备预防或治疗人类受试者中HIV感染的药物中的用途。
46.一种基本如此前所描述并参考附图的免疫原。
47.一种基本如此前所描述并参考附图的刺激人类受试者中抗HIV免疫应答的方法。
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GBGB9930294.5A GB9930294D0 (en) | 1999-12-23 | 1999-12-23 | Improvements in or relating to immune responses to HIV |
GB0025234A GB0025234D0 (en) | 2000-10-14 | 2000-10-14 | Improvements in or relating to immune responses to HIV |
GB0025234.6 | 2000-10-14 |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN1968710B (zh) * | 2004-06-01 | 2013-12-25 | 默沙东公司 | HIV gp41融合中间体的稳定的肽模拟物 |
CN102656271A (zh) * | 2009-10-08 | 2012-09-05 | 巴法里安诺迪克有限公司 | 人体内产生对hiv的广泛t细胞应答 |
CN110372798A (zh) * | 2012-01-27 | 2019-10-25 | 埃斯蒂维制药有限公司 | 用于hiv疫苗接种的免疫原 |
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ATE457996T1 (de) | 2010-03-15 |
EP1240186A2 (en) | 2002-09-18 |
DE60043856D1 (de) | 2010-04-01 |
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