CN1171814A - 多核苷酸结核病疫苗 - Google Patents
多核苷酸结核病疫苗 Download PDFInfo
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- CN1171814A CN1171814A CN95197250A CN95197250A CN1171814A CN 1171814 A CN1171814 A CN 1171814A CN 95197250 A CN95197250 A CN 95197250A CN 95197250 A CN95197250 A CN 95197250A CN 1171814 A CN1171814 A CN 1171814A
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Abstract
编码结核分支杆菌(M.tb蛋白)的基因被克隆进真核表面载体中,以使其在哺乳肌肉细胞体内表达所编码的蛋白。动物免疫是通过注射这些称为多核苷酸疫苗或PNV的这些DNA构建体到动物的肌肉中而进行的。产生抗M.tb抗原的免疫抗血清。在接种小鼠的脾细胞中检测到特异的T-细胞应答,抗原应答中细胞因子分泌的类型提示有Th1型辅助T-细胞应答的出现(即高IL-2和IFN-γ)。M.tbDNA疫苗的保护效用在牛分支杆菌BCG攻击后的小鼠中得到证明,如测定M.tbDNA接种鼠的脾和肺中分支杆菌相对于对照DNA接种小鼠或天然小鼠中的初次感染增殖的降低。
Description
发明背景
抗病毒和细菌疫苗开发的主要障碍是不同分离体和株中外部蛋白的多样性,尤其抗多种血清型或高突变率的种类,需要诱发中和作用抗体和/或保护性细胞介导的免疫反应。由于小鼠和人细胞毒性T-淋巴细胞(CTLs),能识别来自内部保守的病毒蛋白的表位[J.W.Yewdell等,美国国家科学院院报82,1785(1985);A.R.M.Townsend等,细胞44,959(1986);A.J.McMichael等,J.Gen.Virol.67,719(1986);J.Bastin等,实验医学杂志165,1508(1987);A.R.M.Townsend和H.Bodmer,免疫学年评7,601(1989)1,被认为在抗病毒的免疫应答中是重要的[Y.-L.Lin和B.A.Askonas,实验医学杂志154,225(1981);I.Gardner等,欧洲免疫学杂志4,68(1974);K.L.Yap和G.L.Ada,自然273,238(1978);A.J.McMichael等,新英格兰医学杂志309,13(1983);P.M.Taylor和B.A.Askonas,免疫学58,417(1986)],许多努力方向是能提供抗不同病毒株的异源性保护的CTL疫苗的开发。
众所周知,当T细胞受体识别和MHC I类和/或II类分子相连的外来肽时,CTLs杀死病毒或细菌感染的细胞。这些肽可能衍生自内源合成的外来蛋白,而不论蛋白在病原体中的定位和功能。通过识别保守性蛋白的表位,CTLs可能提供异源性保护。在细胞内细菌存在的情况下,从细菌分泌或释放的蛋白被MHC I类和II类分子加工和呈递,因而产生了在降低或消除感染中起作用的T细胞应答。
大多数产生CTL应答的努力在于使用复制载体,以在细胞中产生蛋白抗原[J.R.Bennink等,同上,311,578(1984);J.R.Bennink和J.W.Yewdell,当今微生物免疫学论题163,153(1990);C.K.Stover等,自然351,456(1991);A.Aldovini和R.A.Young,自然351,479(1991);R.Schafer等.,J.Immunol.149,53(1992);C.S.Hahn等美国国家科学院院报89,2679(1992)],或者它们集中于把肽导入细胞溶质中[F.R.Carbone和M.J.Bevan,实验医学杂志169,603(1989);K.Deres等自然342,561(1989);H.Takahashi等,同上344,873(1990);D.S.Collins等,免疫学杂志148,3336(1992);M.J.Newman等,同上148,2357(1992)]。这二种途径的局限性可能降低了它们作为疫苗的可应用性。反转录病毒在维持重组病毒复制能力的情况下对作为融合蛋白表达的多肽的大小和结构具有限制[A.D.Miller,当今微生物免疫学论题158,1(1992)],载体如牛痘作下一步的免疫作用的有效性能被抗牛痘的免疫应答所缓和[E.L.Cooney等,柳叶刀337,567(1991)]。另外,病毒载体和修饰的病原体有内在的风险,能妨碍其在人类中应用[R.R.Redfield等,新英格兰医学杂志316,673(1987);L.Mascola等,Arch Intern.Med.149,1569(1989)]。因此,被呈递的肽段表位的选择依赖于个体MHC抗原的结构,所以肽疫苗由于远亲群体中MHC单倍型的多样性具有限的效用性。
Benvenisty,N.和Reshef.L[美国国家科学院院报83,9551-9555,(1986)]表明腹膜内(i.p.),静脉内(i.v.)或肌肉内(i.m.)导入小鼠的氯化钙沉淀的DNA能表达。DNA表达载体在鼠中肌肉内(i.m.)注射的结果说明DNA被肌肉细胞吸收,表达由DNA编码的蛋白[J.A.Wolff等,科学,247,1465(1990);G.Ascadi等,自然352,815(1991)]。表明质粒以附加体的方式被维持,没有复制。后来在大鼠、鱼和灵长目骨骼肌和大鼠心肌的i.m.注射后观察到持续的表达[H.Lin等;循环82,2217(1990);R.N.Kitsis等,美国国家科学院院报88,4138(1991);E.Hansen等,FEBS Lett.290,73(1991);S.Jiao等,人类基因治疗3,21(1992));J.A.Wolff等,人类分子遗传1,363(1992)]。用核苷酸作治疗剂的技术在WO90/11092(1990年10月4日)报道,其中用裸多核苷酸接种脊椎动物。
最近,在活化CTLs消除肿瘤中抗原呈递细胞表面表位的B7和主要组织相容性复合物(MHC)呈递的协同作用做了综述[Edgington生物技术1 1,1117-1119,1993]。一旦抗原呈递细胞(APC)表面的MHC分子呈递表位给T细胞受体(TCR),在相同APC表面表达的B7起第二信号的作用与CTLA-4或CD28结合。其结果是CD4+辅助细胞的快速分裂,其传递信号给CD8+T细胞,使其增殖和杀死APC。
对方法的成功来说,肌肉内免疫并非必要。因此,Tang等[自然,356,152-154(1992)]发现包被编码牛生长激素(BGH)的DNA的金微粒导入小鼠皮肤,结果小鼠产生了抗BGH的抗体。弗斯等[分析生物化学,205,365-368,(1992)]表明喷射(jet)注射器可用于转染活动物的皮肤、肌肉、脂肪和乳腺组织。最近综述了导入核苷酸的不同方法[Friedman,T.科学244,1275-1281(1989)]。也可见Robinson等[提交到关于新疫苗现代方法,包括AIDS预防的1992会议的论文摘要,冷泉港,p92;疫苗11,957(1993)],其中主张鸟类流感DNA im,ip和iv对鸡给药,以提供保护抗致死攻击。小鼠中DNA:阳离子脂质体复合物的静脉注射由Zhu等证明[科学261,209-211(1993年7月9日);亦见于WO93/24640,1993年12月9日],结果是克隆的转基因系统地表达。最近,Ulmer等[科学259,1745-1749,(1993)]报道注射编码流感病毒蛋白的DNA可异源性地保护抗流感病毒感染。
Wang报道[美国国家科学院院报90,4156-4160(5月,1993)]用克隆的基因组(未剪切)的HIV基因肌肉内接种,能诱发小鼠抗HIV的免疫应答。不过,所获的免疫应答水平非常低,该系统使用小鼠乳腺瘤病毒(MMTV)长末端重复(LTR)启动子部分和猴病毒40(SV40)启动子和终止子部分。已知SV40能转化细胞,可通过整合进宿主细胞DNA而起作用。因而,Wang等所描述的系统,施用于人是完全不合适的,这是本发明的目的之一。
WO93/17706描述了一种接种动物抗病毒的方法,其中载体颗粒用基因构建体包被,包被的颗粒加速进入动物细胞。
由Wolff等(同上)的研究最先证明编码报告基因的质粒DNA肌肉注射,结果是在注射位点和其附近的肌细胞内该基因的表达。最近报道表明编码流感A血凝素(Montgomery D.L等,1993,细胞生物学,12,pp777-783),或核蛋白(Montgomery D.L等,同上;Ulmer,J.B.等,1993,科学,259,pp.1745-1749)的质粒注射,成功地免疫小鼠抗流感。对疱疹病毒DNA免疫作用的首次应用已有报道(Cox等,1993,病毒学杂志,67,pp.5664-5667)。编码牛疱疹病毒(BHV-1)糖蛋白g IV的质粒注射在小鼠和小牛中产生了抗g IV的抗体。用BHV-1鼻内攻击表明免疫小牛减轻症状,排出基本上比对照少的病毒。
结核病(TB)是由病原体结核分支杆菌引起的肺慢性感染病。TB是世界范围临床上最显著的感染病之一,伴随而来的是每年300万人死亡和1千万新病例。估计多达世界人口的三分之一会被感染,在发展中国家,已报道5千5百万发病的TB病例。直到本世纪初,TB是美国主要死因。可是,随着卫生条件的改善和抗微生物药物的出现,死亡率稳定下降至预计到2000年该病将被根除的程度。不过,在大多数发达国家,自1980年代中期以来发病的TB病例数每年上升。该复发部分归因于迁移和免疫后损害的,HIV感染个体数目增加。假如趋势没有减弱,预计TB将在下一个十年要了三千万以上人的性命。象这些数学显得使人惊恐一样,甚至更为关注的是结核分支杆菌的多药抗性(MDR)株的出现。这些MDR株不被传统药物疗法所驯服,特别在城市中心引起最近几次TB的爆发。所以,长期TB控制的关键组分之一将是有效的疫苗[综述见Bloom和Murray,1993,科学257,1055]。
结核分支杆菌是感染巨噬细胞的细胞内病原菌,能在该类型细胞吞噬溶酶体的恶劣环境中存活。大多数吸入的杆菌被活化的小泡巨噬细胞破坏。可是,存活的杆菌能在巨噬细胞中增殖,靠细胞死亡而释放,发出了淋巴细胞、单核细胞和巨噬细胞向该点浸润的信号。负载有杆菌的巨噬细胞的裂解由迟发型超敏反应所介导,导致环绕感染细胞区域的紧密干酪样结核结节的出现。继续的DTH引起结节液化,因而释放所捕获的杆菌。大剂量的胞外杆菌进一步激发DTH,引起对支气管的损害和由淋巴的、血原性的以及支气管通路散播,最终使感染性的杆菌由呼吸扩散。
对TB的免疫性涉及效应细胞的几种类型。由细胞因子比如γ-干扰素对巨噬细胞的活化是把细胞内分支杆菌增殖降到最低的一种有效方法。不过,靠这种方法常未取得完全根除分支杆菌。抗TB的保护的获得需要T淋巴细胞。在这些之中,CD8+和CD4+T细胞似乎是重要的[Orme等,1993,传染病杂志,167,1481]。这些细胞类型在对分支杆菌应答中分泌γ-干扰素,显示有Th1免疫应答,和具有对分支杆菌脉冲的靶细胞的细胞毒活性。最近用β-2微球蛋白和CD8缺陷的小鼠研究中,表明CTL应答在提供抗结核分支杆菌的保护中是关键的[Flynn等,1992,美国国家科学院院报89,12013;Flynn等,1993,实验医学杂志178,2249;Cooper等,1993,实验医学杂志178,2243]。相反,B淋巴细胞似乎没有涉及,抗分支杆菌的抗体的被动输入不提供保护。所以,抗TB的有效疫苗必须产生细胞介导的免疫应答。
T细胞抗原刺激需要MHC分子的呈递作用。为了使分支杆菌抗原可进入抗原呈递通路,它们必须从细菌中释放。在感染的巨噬细胞中,这可能与分泌或细菌裂解完成。分支杆菌具有许多潜在的T细胞抗原,有几种现已确认[Andersen 1994,Dan Med.Bull 41,205]。这些抗原的一些由细菌分泌。一般认为抗TB的免疫性由直接针对这些所分泌抗原的CD8+和CD4+T细胞所介导。在TB的小鼠和豚鼠模型中,由降低体重减轻所测定的对细菌攻击的保护作用,已应用分泌的分支杆菌抗原的混合物获得。[Pal和Horowitz,1992,感染免疫60,4781;Andersen 1994,感染免疫62,2536;Collins,1994,Veterin.微生物学40,95]。
几种潜在的保护性T细胞抗原已在结核分支杆菌中确认,一些作疫苗的靶物正在研究。最近工作显示主要的T细胞抗原是它们停留在巨噬细胞中的过程中,由分支杆菌分泌的那些蛋白,例如:i)蛋白抗原85复合体(85A,85B,85C)[wiker和Harboe,1992,微生物学综述,58,648],ii)一种称作ESAT-6的6 kDa蛋白[Ardersen 1994,感染免疫62,2536],iii)一种与Phos有同源性的38 kDa脂蛋白[Young和Garbe,1991微生物学研究,142,55;Ardersen 1992,感染病杂志166,874],iv)65kDa的GroEl热休克蛋白[Siva和Lowrie,1994,免疫学82,244],v)一种富含脯氨酸和苏氨酸的55 kDa蛋白[Romain等,1993,美国国家科学院院报90,5322];和Vi)一种19 kDa脂蛋白[Faith等,1991,免疫学74,1]。
三种抗原85蛋白(A、B和C)的每一种的基因已被克隆和测序[Borremans等,1989感染免疫57,3123;Content等,感染免疫59,3205;DeWit等1994,DNA序列4,267]。此外,这些结构相关蛋白是感染和接种后强烈的T细胞应答的目标[Huygen等,Scand.J.Immunol.27,187;Launois等1991,临床实验免疫学86,286;Huygen等,1992,感染免疫60,2880;Munk等,1994,感染免疫62,726;Launois等1994,感染免疫62,3679]。因此,抗原85蛋白被认为是良好的疫苗靶物。
发明概述
为检验DNA免疫在预防M.tb疾病中的有效性,将编码M.tb蛋白的DNA序列克隆进真核表达载体中。这些DNA构建体当注射到动物中时,激发了免疫应答。免疫的动物用分支杆菌感染,以评价是否用基因(或其他M.tb基因)的直接DNA免疫能保护它们免除疾病。因而公开的是包括DNA构建体和RNA转录体的核苷酸,通过注射或其他途径直接导入动物组织,能诱导M.tb蛋白的体内表达。这些核苷酸注射能激发免疫应答,应答导致对M.tb抗原特异的细胞毒性T淋巴细胞(CTLs)的产生和对后续的攻击有保护作用的特异于M.tb的辅助T淋巴细胞应答的产生。将这些核苷酸用作诱导对M.tb免疫性的疫苗是有用的,它能预防感染和/或改善M.tb相关的疾病。
附图简述
Fig1.显示克隆M.tb基因进入表达载体的一般原理。
Fig2.显示V1Jns.tPA85A.C1的载体图谱。
Fig3.显示V1Jns.85A.C2的载体图谱。
Fig4.显示V1Jns.85A.C3的载体图谱。
Fig5.显示V1Jns.tPA85B.C1的载体图谱。
Fig6.显示V1Jns.tPA85C.C1的载体图谱
Fig7.显示构建体N末端序列的核实。
Fig8.显示在组织培养中M.tb蛋白的表达。
Fig9.显示在接种DNA的小鼠中对抗原85A特异的抗体的产生。
Fig10.显示在BALB/c小鼠中由Tb DNA疫苗而产生IL-2。
Fig11.显示在C57BL/6小鼠中由Tb DNA疫苗而产生IL-2。
Fig12.显示在BALB/c小鼠中由Tb DNA疫苗而产生IFN-γ。
Fig13.显示在C57BL/6小鼠中由Tb DNA疫苗而产生IFN-γ。
Fig14.显示在BALB/c小鼠中,Tb DNA疫苗不产生IL-4。
Fig15.显示在小鼠中,Tb DNA疫苗不产生IL-6。
Fig16.显示在小鼠中,Tb DNA疫苗不产生IL-10。
Fig17.显示用Tb DNA疫苗接种的C57BL/6小鼠的肺中BCG的增殖降低。
Fig18.显示用Tb DNA疫苗接种的BALB/c小鼠的肺中BCG的增殖降低。
Fig19.显示用Tb DNA疫苗接种的BALB/c小鼠的脾中BCG的增殖降低。
Fig20.显示用Tb DNA疫苗接种的C57BL/6小鼠的脾中BCG的增殖降低。
发明详述
本发明提供的多核苷酸,当直接导入包括诸如人类的哺乳动物的脊椎动物体内时,诱导编码蛋白在动物中表达。在此所用的多核苷酸是一种核酸,它含有依赖其导入活脊椎动物细胞中的基本调控元件,和能指导细胞机构产生包含多核苷酸基因编码的翻译产物。在本发明的一个实施方案中,多核苷酸是一种多脱氧核糖核酸,其包括有操纵性地连结于转录启动子的结核分支杆菌(M.tb)基因。在本发明的另一实施方案中,多核苷酸疫苗包括编码服从于由真核细胞机构(核糖体、tRNAs、和其他翻译因子)翻译的M.tb基因的多核糖核酸。那里由多核苷酸编码的蛋白是除在病理条件下在动物中不正常产生的一种,(即一种异源性蛋白)如与M.tb有关的蛋白,动物的免疫系统被活化发动保护性免疫应答。因为这些外源蛋白由动物自身组织产生,表达的蛋白由主要组织相容性系统(MHC)以类似于当真正的M.tb感染发生时的方式所处理。如在本公开内容所示的结果是诱导抗M.tb的免疫应答。为对一种编码蛋白产生免疫应答的目的的多核苷酸在此称作多核苷酸疫苗或PNV。
本发明有许多实施方案使本领域的技术人员能从特例中正确评价。这样,可成功地使用不同的转录启动子、终止子、穿梭载体(carriervector)或特异的基因序列。
本发明提供一种应用多核苷酸的方法,导入哺乳动物组织中时,多核苷酸诱导因而产生编码蛋白的体内多核苷酸表达。对本领域技术人员极明显的是能产生编码蛋白的核苷序列的变异或衍生序列,而改变编码蛋白的氨基酸序列。改变的表达蛋白可能具有改变的氨基酸序列,而仍能引起和分支杆菌蛋白起反应的免疫应答,改变的表达蛋白被认为是功能性等价体。另外,编码全长蛋白部分的全长基因的片段也可构建。这些片段能编码一种蛋白或肽,其可诱导出和分支杆菌蛋白起反应的抗体,这些片段被认为是功能性等价体。
在本发明的一个实施方案中,编码M.tb基因产物的一个基因被掺入到表达载体中。载体含由真核RNA聚合酶识别的转录启动子和在M.tb基因编码序列末端的转录终止子。在优选的实施方案中,启动子是具有内含子A序列的巨细胞病毒启动子(CMV-intA),尽管本领域的技术人员会认为可用许多其他的已知启动子如强免疫球蛋白或其他真核基因启动子的任一个。优选的转录终止子是牛生长激素终止子。CMVintA-BGH终止子的结合是优选的。此外,为有助于在原核细胞中多核苷酸的制备,抗生素抗性标志也选择性地包括在合适的原核启动子的转录控制下的表达载体中。可用氨苄青霉素抗性基因,新霉素抗性基因或任何其它合适的抗生素抗性标志。在本发明的优选实施方案中,抗生素抗性基因编码对新霉素/卡那霉素抗性的基因产物。更进一步,为助于生长在原核生物中多核苷酸的高水平产生,载体含原核的复制起点和具有高拷贝数是有利的。许多商品化的原核克隆载体的任一个均能提供这些元件。在本发明优选的实施方案中,这些功能体由商品化称为pUC系列的载体提供。不过移去非必需DNA序列可能是需要的。这样,可移去pUC的lacZ和lacI编码序列。也需要载体不能在真核细胞中复制。这使多核苷酸疫苗序列整合到接受体基因组的风险降低到最低限度。
在另一实施方案中,使用表达载体pnRSV,其中劳斯肉瘤病毒(RSV)的长末端重复序列(LTR)被用作启动子。仍在另一实施方案中,应用V1,一种克隆有CMV启动子和BGH转录终止子的突变pBR322载体。在本发明优选的实施方案中,结合V1和pUC19元件而产生的表达载体称作V1J。
克隆进V1J,V1JtPA或另一需要的表达载体中的是M.tb基因,例如抗原85复合体基因之一,或任何其他能诱导抗M.tb免疫应答(CTLs,辅助T淋巴细胞和抗体)的M.tb基因。在另一实施方案中,氨苄霉素抗性基因从V1J中移去,代之以新霉素抗性基因以产生V1J-neo,按照本发明,使用时可把许多不同的M.tb基因克隆到V1J-neo中。仍在另一实施方案中,载体是V1Jns,这是与V1J-neo相同,除了在V1J-neo的2114位点,唯一的Sfil限制性位点构建到单独的Kpn1位点上。在人基因组DNA中Sfil位点的出现率非常低(大约每100,000碱基1个位点)。这样,简单地把提取的基因组DNA的Sfi1消化,该载体允许对表达载体整合到宿主DNA中作仔细监测。在一进一步的实施方案中,载体是V1R。在这载体中,尽可能地“剪去”非必需DNA以产生高度紧密的载体。该载体允许用作较大的插入片段,当编码特异的病毒基因构建体被导入周围组织时,较少涉及非必需的序列被编码和细胞吸收的优化。根据本领域人员熟知的方法,可完成用于产生前面的载体修饰和发展步骤的方法。
根据本工作,本领域的技术人员会认为本发明的应用之一是提供体内和体外的检测和分析系统,结果能找出和CTL和T-细胞增殖应答以及其他参数的M.tb序列多样性的相关性。这些不同的基因的分离和克隆可按本领域技术人员熟知的方法完成。本发明进一步提供一种方法对疫苗产生的M.tb株和序列作系统鉴别。来自M.tb株的初级分离体基因的结合提供一种免疫原,它能诱导抗临床的微生物分离体的免疫应答,因而满足迄今为止该领域未遇的需求。因而,假如毒性分离体变化,免疫原可修改以反映必需的新序列。
在本发明的一个实施方案中,编码M.tb蛋白的基因直接与转录启动子相连。组织特异的启动子或增强子例如肌肉肌酸激酶(MCK)增强子元件的应用可望限制多核苷酸在一特定的组织类型中表达。例如,肌细胞是最终分化时不再分裂的细胞。外源DNA整合进染合体似乎需要细胞分裂和蛋白合成。因此,对非分裂细胞如肌细胞限制性的蛋白表达是优选的。不过,CMV启动子的应用对获得PNV导入许多组织中表达是合适的。
M.tb和其他基因优先地连接到特异地优化作多核苷酸接种的表达载体中,在此描述的元件包括转录启动子、免疫原性表位、编码免疫增强或免疫调节基因的附加顺反子和它们自身的启动子,转录终止子、细菌复制起点和抗生素抗性基因。选择性地,载体可含为多顺反子mRNA表达的内部核糖体进入位点(IRES)。本领域的技术人员会正确评价由DNA对等物编码的体外转录产生多顺反子mRNA的RNA在本发明范围之内。为该目的,必须用如强RNA聚合酶启动子作T7或SP6启动子为转录启动子,用线性化的DNA模板进行体外连续转录。这些方法在本领域中已众所周知。
抗后续攻击的多核苷酸M.tb免疫原的保护效用通过本发明的DNA免疫作用得到阐明。由于不涉及感染原,不需要细菌的组装和复制和允许决定簇选择,这是有利的。因此,因为分支杆菌基因产物的序列在M.tb的不同株中可能保守,能获得由M.tb另一菌株引起的抗后续攻击的保护作用。
编码抗原85A,B或C的DNA表达载体的注射可导致抗后续攻击的显著的保护免疫性的形成。尤其是会产生特异的CTLs和辅助T淋巴细胞应答。
因为M.tb的每一个基因产物在不同的M.tb株中高度保守和因为免疫应答能在细胞内表达和MHC处理的反应中产生,预计许多不同的M.tbPNV构建体会产生交叉反应的免疫应答。
本发明提供一种方法不需要自身复制因素或佐剂诱导异源性保护免疫。病毒蛋白和人生长激素DNA注射后[Tang等,自然356,152,1992]抗表达蛋白的高效价抗体的产生显示,这是一种制取基于抗体的疫苗的方便和高效的方法,该疫苗靶向于保守抗原分别或和细胞毒性T淋巴细胞和辅助T细胞结合。
产生和纯化DNA构建体的容易性和传统的蛋白纯化相比是有利的,便于结合疫苗的产生。因而可制备例如编码抗原85复合体基因和任何其他的M.tb基因,也包括非M.tb基因的多种构建体,混合和共施用。另外,以下的DNA注射维持了蛋白表达[H.Lin等,循环82,2217(1990);R.N.Kitsis等,美国国家科学院院报88,4138(1991);Hansen等,FEBS Lett,290,73(1991);S.Jiao等,人类基因治疗3,21(1992);J.A.Wolff等,人类分子遗传学1,363(1992)],B-和T-细胞记忆的持久性被增强[D.Gray和P.Mat Zinger,实验医学杂志,174,969(1991);S.Oehen等,同上176,1273(1992)],因而产生长期存活的体液和细胞介导的免疫性。
导入到疫苗受体的能表达的DNA或转录的RNA数量具有非常宽的剂量范围,依赖于所用的转录和翻译的启动子的强度。此外,免疫应答的强度可能依赖于蛋白表达的水平和表达的基因产物的免疫原性。一般来说,大约1ng~5mg,100ng~2.5mg,1μg~750μg为有效剂量范围,优选直接用于肌肉组织的DNA约为10μg~300μg。皮下注射、真皮导入、透皮印模(impression)和其它给药方式如腹膜内、静脉内或吸入输送也是合适的。也可考虑提供加强接种。在用M.tb多核苷酸免疫原接种后,也可考虑用M.tb蛋白免疫原比如抗原85复合体基因产物予以加强。肠道外施用如白介素-12蛋白(或其他细胞因子,例如GM-CSF)静脉内、肌肉内、皮下或其他方式的给药,同时或接着本发明的PNV的肠道外导入是有利的。
多核苷酸可以是裸露的,那就是说,与影响受体免疫系统的任何蛋白、佐剂或其他因素无关。在这种情况下,多核苷酸必须在生理学可接受的溶液中,例如但不局限于无菌的盐水或无菌的缓冲盐水中。替代方法是DNA可和脂质体作相连,如本领域熟知的卵磷脂脂质体或其他脂质体DNA-脂质体混合物,或者DNA可和本领域熟知的佐剂相连,如蛋白或其他载体以促进免疫应答。也可用帮助DNA细胞吸收的试剂,例如但并不局限于钙离子。这些试剂在此一般称作转染促进试剂和药学上可接受的载体。包被多核苷酸微粒的包被技术在本领域中已经熟知,和本发明相衔接也是有用的。对计划用于人的DNA,最终的DNA产物在药学可接受的载体或缓冲溶液中可能是有用的。药学可接受的载体或缓冲溶液在本领域中众所周知,包括各种手册如雷明顿氏药学科学所描述的那些种类。
在另一实施方案中,本发明是一种多核苷酸,它包含能表达的连续核苷酸序列,在所述的多核苷酸体内导入真核组织时产生基因产物。编码的基因产物优选地起免疫刺激物或抗原的作用,能引起免疫应答。因此,在本实施方案中核苷酸序列编码一种M.tb免疫原的表位和选择性地编码一种细胞因子或一种T-细胞共刺激元件如B7蛋白家族的一个成员。
用基因而不是其基因产物的免疫有几个优点。首先是相对的简单性,通过它天然的或接近天然的抗原呈递给免疫系统。哺乳动物蛋白在细菌、酵母或甚至哺乳动物细胞中重组表达常需要广泛的处理,以保证合适的抗原性。DNA免疫的第二个优点是有可能免疫原进入MHC I类通路而引起细胞毒性T细胞应答。用编码流感A核蛋白(NP)的DNA免疫小鼠产生CD8+对NP的应答,保护小鼠对抗异源性流感株的攻击(Montgomery D.L.等,同前;Ulmer.J.等,同前)。
有充足的证据表明细胞介导的免疫在控制M.tb感染中是重要的[Orme等,1993,传染病杂志167,1481;Cooper等1993,实验医学杂志178,2243;Flynn等,1993,实验医学杂志178,2249;Orme等,1993,免疫学杂志151,518]。由于DNA免疫能引起体液和细胞介导的免疫应答,其最大的优点是提供一种相对简单的方法,为潜在的疫苗检查大量的M.tb基因。
正如以上讨论,DNA注射免疫亦允许多组分亚单位疫苗的快速组合。和多种流感基因同时免疫已于最近报道(Donnelly J.等,1994,疫苗,pp55-59)。包含其产物活化免疫系统不同武器的基因的一个M.tb疫苗也可提供对后续攻击的完全保护。
本发明的疫苗对人类及家养或农业动物给药用是有用的。本发明的疫苗可用于预防和/或对抗任何农业动物的感染,包括但不限于奶牛,奶牛对分支杆菌感染敏感。给动物和人服用这些疫苗的技术分别对于兽医和人类健康领域的技术人员众所周知。
提供如下的实施例说明本发明,但不限于在此相同的例子。
实施例1
生产疫苗的载体
A)V1表达载体
表达载体V1构建自pCMVIE-AKI-DHFR[Y.Whang等,病毒学杂志61,1796(1987)]。用EcoRI切割载体和自连接移去AKI和DHFR基因。该载体在CMV启动子中不含内含子A,所以加一段缺乏内部SacI位点的PCR片段[在B.S.Chapman等,核苷酸研究19,3979(1991)定位在1855]。用于PCR反应的模板是pCMVintA-Lux,来自pCMV6a120[见B.S.Chapman等,同上,]包括hCMV-IE1增强子/启动子和内含子A的HindIII和Nhe I片段连接到pBL3的Hind III和Xba I位点中产生pCMVIntBL制成。来自RSV-Lux[J.R.de Wet等,分子细胞生物学,7,725,1987]的1881碱基对的虫荧光素酶基因片段(Hind III-Sma I Klenow补平)克隆到pCMVIntbL的SalI位点,它是Klenow补平和磷酸酶处理过的。
跨越内含子A的引物是:
5′引物, SEQ.ID:1:
5′-CTATATAAGCAGAG CTCGTTTAG-3′;3′引物,SEQ.ID:2:
5′-GTAGCAAAGATCTAAGGACGGTGA CTGCAG-3′
用于移去SacI位点的引物是:
有义引物,SEQ.ID:3:
5′-GTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC-3′
和反义引物,SEQ.ID:4:
5′-GTGCGAGCCCAATCTCCACGCTCATTTTCAGACACA TAC-3′
用SacI和BglII切下PCR片段,插入到用相同的酶切过的载体中。
B)V1J表达载体
创制V1J的目的是从载体V1移去启动子和转录终止元件以便在更精确的前后关系中确定它们,创造一种更紧密的载体和改善质粒纯化产率。
V1J衍生自V1和商业化的质粒pUC18。V1用Ssp I和EcoRI限制酶消化产生二个DNA片段。这些片段的较小一个,含能控制异源基因表达的CMVintA启动子和牛生长激素(BGH)转录终止元件,从琼脂糖电泳凝胶纯化。然后该DNA片段的末端用T4 DNA聚合酶“钝化”,以便于另一个“钝端”DNA片段的连接。
被选的pUC18提供表达载体的“骨架”。众所周知它能产生高的质粒产率,序列和功能被特征描述以及体积小。用Hae II限制酶部分消化从该载体中移去整个lac操纵子。质粒的剩余部分从琼脂糖电泳凝胶纯化,钝端处理用T4 DNA聚合酶和小中肠碱性磷酸酶,连接到上述的CMVintA/BGH元件上。获得的质粒显示在pUC骨架中二个可能的启动子元件方向的一种。这些质粒的一个在大肠杆菌中产生更高产量的DNA,命名为V1J。载体的结构通过连接区的序列分析证实,与V1比较,进一步表明产生相仿的或更高的异源基因表达。
C)V1Jneo表达载体
必须移去锚定在V1J上用于细菌抗生素筛选的ampr基因,因为氨苄霉素在大规模发酵中是不需要的。V1J的pUC骨架上的ampr基因用SspI和Eam1105I限制酶消化移去。质粒的其余部分用琼脂糖凝胶电泳纯化,用T4 DNA聚合酶纯化,然后用小牛肠碱性磷酸酶处理。商品化的Kanr基因,衍生自转座子903,含在pUC4K质粒中,用Pst I限制酶切下,琼脂糖凝胶电泳纯化、用T4 DNA聚合酶使末端钝化。该片段和V1J骨架连接,产生的两个方向具有Kanr基因的质粒,命名为V1Jneo#′s1和3。这些质粒的每一个用限制酶消化分析,连接区域的DNA序列测定证实,表明产生与V1J相似的质粒数量。对这些V1Jneo载体异源基因产物的表达也与V1J相当。选出的V1Jneo#3,自此后称为V1Jneo,含作为表达构建体与V1J中的ampr基因相同方向的Kanr基因。
D)VIJns表达载体
一个Sfi位点加到V1Jneo上以便于整合研究。商业来源的13个碱基对Sfi接头(新英格兰生物实验室)加到载体的BGH序列中的KpnI位点上。V1Jneo用KpnI线性化,凝胶纯化,T4 DNA聚合酶钝化,连接到钝化的SfiI接头上。通过限制性作图挑选克隆分离体,通过接头的序列分析证实。新载体命名为V1Jns。在V1Jns(有SfiI)中异源基因的表达相当于在V1Jneo(有KpnI)中相同基因的表达。
E)V1Jns-tPA
为提供异源的前导肽序列分泌和/或膜蛋白,修饰V1Jns以包含人组织特异的纤维蛋白酶原活化子(tPA)前导肽。二个合成的互补寡聚体退火,然后连接进已被Bgl II消化的V1Jn中。有义和反义寡聚体是:5′-GATC ACC ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT GTGCTG CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC AGC GA-3′SEQID:5:,和5′-GAT CTC GCT GGG CGA AAC GAA GAC TGC TCC ACACAG CAG CAG CAC ACA GCA GAG CCC TCT CTT CAT TGC ATC CATGGT-3′SEQ.ID:6。Ko zak序列在有义寡聚体划有下线。这些寡聚体有突出的碱基,与BglII酶切序列连接时兼容。连接后上游BglII位点被毁而下游BglII被保持作下一步的连接。二个连接位点及整个tPA前导序列通过DNA序列测定证实。另外,为了和统一优化的载体V1Jns(=V1Jneo和SfiI位点)一致,用T4 DNA聚合酶钝化KpnI位点接着和SfiI连接子连结(目录号1138,新英格兰生物实验室),SfiI限制点代替了V1Jn-tPA的BGH终止子区中的KpnI位点。该修饰通过限制性消化和琼脂糖凝胶电泳证实。
F)pGEM-3-X-IRES-B7
(其中X=任何抗原基因)作为提供编码免疫原基因和编码免疫刺激蛋白基因协调表达的双顺反子疫苗构建体的一个实例,鼠B7基因PCR扩增自B淋巴瘤细胞系CH1(从ATCC获得)。B7是提供在主要组织相容性复合物I和II的前后关系中的抗原必需的共激活化T细胞的蛋白家属的一个成员。CH1细胞是提供B7 mRNA良好来源,因为它们有组成性活化的表型,B7主要由活化的抗原呈递细胞如B细胞和巨噬细胞表达。这些细胞进一步在体外用cAMP或IL-4刺激,制备mRNA用标准的硫氰酸胍操作。cDNA合成的操作用该mRNA和GeneAmp RNA PCR试剂盒(Perkin-Elmer Cetus)及特异地针对B7定位在B7翻译开放阅读框的下游的引物寡聚体(5′-GTA CCT CAT GAG CCA CAT AAT ACC ATG-3′,SGQ.ID:7:)。B7被PCR扩增,分别用下列有义和反义PCR寡聚体:5′-GGT ACA AGA TCT ACC ATG GCT TGC AAT TGT CAG TTG ATG C-3′SEQ.ID:8:,和5′-CCA CAT AGA TCT CCA TGG GAA CTA AAG GAAGAC GGT CTG TTC-3′,SEQ.ID:9:。这些寡聚体供有在插入片段末端的BglII限制酶位点,和含一个NcoI限制位点及紧密地位于3′端BglII位点前的一个额外NcoI位点的Ko zak翻译起始序列。NcoI消化产生的片段适于克隆进已用NcoI消化的pGEM-3-IRES中。得到的载体,pGEM-3-IRES-B7,含一个易于转移给V1Jns-X的IRES-B7盒,其中X代表一个编码抗原的基因。
G)pGEM-3-X-IRES-GM-CSF
(其中X=任何抗原基因)该载体含的盒与上述C项载体的相似,除了用免疫刺激细胞因子GM-CSF基因而不是B7之外。GM-CSF是巨噬细胞分化和刺激的细胞因子,已表明它在体内引起强烈的抗肿瘤T细胞活性[G.Dranoff等,美国国家科学院院报,90,3539(1993)。
H)pGEM-3-X-IRE S-IL-12
(其中X=任何抗原基因)该载体含的盒与上述C项的载体相似,除了用免疫刺激细胞因子IL-12基因,而不是B7之外。已阐明IL-12在使免疫应答移向与体液应答相对的细胞的,T细胞主导的通路中有影响性作用[L.Alfonso等,科学,263,235,1994]。
实施例2
载体V1R的制备
通过努力继续优化基本的接种载体,制备了V1Jns的衍生体,称作V1R。构建该载体的目的是获得没有非必需DNA序列的最小体积的疫苗载体,它仍保留整个优化的异源基因表达特性和V1J及V1Jns赋予的高的质粒产率。据文献和实验确定:(1)包括大肠杆菌复制起点的pUC骨架之中的区域能被移去,而不影响细菌的质粒产率;(2)接着卡那霉素开放阅读框的kanr基因的3′区能移去,假如细菌的终止子插入其位的话;和(3)自BGH终止子的3′一半的~300bp能移去而不影响其调节功能(在BGH元件中接着的是原来的Kpn I限制酶位点)。
构建V1R用PCR从V1Jns分成三个DNA片段,分别代表CMVintA启动子/BGH终止子,复制起点和卡那霉素抗性元件。对每一片段唯一的限制酶(位点)用PCR寡聚体加到每一片段末端:CMVintA/BGH加入SspI和XhoI;Kanr基因加入EcoRV和BamHI;及orir加入BclI和SalI。选择这些酶位点是因为用每一位点的后来丢失,它们使来自PCR的DNA片段的每一个直接连接:EcoRV和SspI留下连接相容的钝端DNAs,而BamHI和BclI留下互补突出端与SalI和XhoI一样。通过PCR获得这些片段后,每一片段用如上所示的合适的限制酶消化,然后在含全部三种DNA片段的单一反应混合液中连接在一起。设计orir的5′端包括在该区通常所见的T2rho独立终止子序列,以能提供给卡那霉素抗性基因的终止信息。连接产物用限制酶消化(>8个酶)和连接接口的DNA序列测定证实。在V1R中DNA质粒产量和用病毒基因的异源性表达似乎与V1Jns相似。所得的载体大小的净减小数为1346bp(V1Jns=4.86kb;V1R=3.52kb)。
用于合成V1R的PCR寡聚物序列(限制酶位点已划下线,在下列序列的括号中识别):
(1)5′-GGT ACA AAT ATT GG CTA TTG GCC ATT GCA TAC G-3′[SspI],SEQ.ID:10:,
(2)5′-CCA CAT CTC GAG GAA CCG GGT CAA TTC TTC AGCACC-3′[XhoI,SEQ.ID:11:
(为CMVintA/BGH片段)
(3)5′-GGT ACA GAT ATC GGA AAG CCA CGT TGT GTC TCA AAATC-3′[EcoRV],SEQ.ID:12:
(4)5′-CCA CAT GGA TCCG TAA TGC TCT GCC AGT GTT ACAACC-3′[BamHI],SEQ.ID:13:
(为卡那霉素抗性基因片段)
(5)5′-GGT ACA TGA TCA CGT AGA AAA GAT CAA AGG ATC TTCTTG-3′[BclI],SEQ.ID:14:,
(6)5′-CCA CAT GTC GAC CC GTA AAA AGG CCG CGT TGC TGG-3′[SalI],SEQ.ID:15:
(为大肠杆菌复制起点)
实施例3
细胞培养和转染
为制备稳定表达M.tb抗原的转染细胞系,RD细胞(人横纹肌肉瘤ATCC CCL136)生长在37℃、5%CO2下,添加有10%热失活的胎牛血清,20mM HEPES,4mM L-谷氨酰胺,和青霉素及链霉素每一种为100μg/mL的Dulbecco’s修饰的Eagle’s培养基(DMEM)中。细胞以1.5×106细胞/100mm2板接种,培养18小时。用CellPhect试剂盒(Pharmacia),细胞用每板TB构建体10μg和共转染的Ca+构建体10μg转染,DNA加入细胞后甘油休克5小时(15%甘油在PBS中,pH7.22.5分)。转染后72小时,用2x-10mL冷PBS,pH7.2,加5mL冷TEN缓冲液(40mM TRIS-Cl,pH7.5,1mM EDTA,150mM NaCl)洗板,刮下,收获培养物。作蛋白表达分析,细胞沉淀在50μL的单一去垢剂裂解缓冲液(50mM Tris-Cl,pH8.0,150mM NaCl,0.029。NaN3,1%NonidetP-40,100mM PMSF,2μg/ml抑蛋白酶肽,2μg/mL亮抑蛋白酶肽和1μg/mL Pepstatin A)中裂解,在冰上超声(破裂2~15秒)。裂解物在13,000×g,4℃下离心10分钟。蛋白浓度用布莱德福德Bradford法测定,每道20μg细胞提取蛋白上样到10%TRIS-甘氨酸聚丙烯酰胺凝胶(Novex)上,然后转移到Immobilon P(Millipore)膜上。免疫印迹用1:20稀释的小鼠单克隆抗体TD 17-4[Huygen等,1994,感染免疫62,363]反应过夜,接着和1∶1000稀释的羊抗小鼠IgG Fc过氧化酶(Jackson)反应1.5小时。印迹用ECL试剂盒(Amersham)显色。
实施例4
克隆和DNA制备
1.V1Jns-tPA-85A(含成熟的Ag85A具tPA信号序列)的构建用下列引物进行:
有义85A.C1引物[SEQ.ID.NO:16]
GG AAG ATC TTT TCC CGG CCG GGC TTG CCG
BglII
反义85A引物[SEQ.ID.NO:17]
GGAAGATCTTGTCTGTTCGGAGCTAGGC
结核分支杆菌Ag85A扩增自质粒p85A.tub,它是一800bp HindIII片段与来自Borremans等,1989[感染免疫57,3123],图2的1600bp HindIII-SphI片段连接而制备的。得到的2400bp插入片段亚克隆在BlueScribeM13+的HindIII和SpnI位点中。在BlueScribe M13+(VCS/Stratagene)中的整个编码序列和侧翼区,在下列条件下用所示的引物进行PCR扩增。每100μl反应液含2.5单位克隆的Pfu DNA聚合酶(Stratagene),200mM dNTP,每一引物0.5μg和在补充有酶(Stratagene)的反应缓冲液中的250ng模板DNA。Hybaid热反应器按下列编程:94℃变性5分钟,接着25个循环(94℃1分钟,55℃2分钟和72℃3分钟),72℃延伸10分钟结束。
扩增的DNA用50μg/ml蛋白酶K(Boehringer Mannheim)37℃消化30分钟,95℃加热10分钟接着苯酚(氯仿-异戊醇)抽提二次,一倍体积的异丙醇沉淀,70%乙醇洗二次,干燥并溶解在20μl H2O中。3μg扩增的DNA用40单位的BglII(Boehringer Mannheim)消化,907bp的片段(在85A-C1的情况下)在1%琼脂糖凝胶上分离,按厂家的指导书在“Prep a Gene”(BioRad)上提取。
该片段50ng与20ng BglII消化和脱磷酸化的V1Jns.tPA载体,在含位于连接缓冲液中的2.5单位T4 DNA连接酶(Amersham)的10μl反应液中,14℃连接16小时,转化到感受态DH5大肠杆菌(BRL)中,在含卡那霉素(50μg/ml)的LB琼脂培养基上铺板。挑取转化体,其质粒DNA用BglII(证实插入片段的存在)限制性酶切和PvuII确定其方向。
2.V1Jns-85A[C2](含成熟Ag85A,不具信号序列)的构建用下列引物进行:
有义85AC2[SEQ.ID.NO.:18]
GGA AGA TCT ACC ATG GGC TTT TCC CGG CCG GGC TTG C
反义85A[SEQ.ID.NO.:17]
GGA AGA TCT TGC TGT TCG GAG CTA GGC
除克隆是在V1Jns上之外,接着进行与上面1中相同的操作。
3.V1Jns-85A[C3](含Ag85A具其自身的信号序列)的构建用引物进行:
有义85A C3[SEQ.ID.NO.:19]
GGA AGA TCT ACC ATG GCA CAG CTT GTT GAC AGG GTT
反义85A[SEQ.ID.NO.:17]
GGA AGA TCT TGC TGT TCG GAG CTA GGC
除克隆是在V1Jns上之外,接着进行与上面1中相同的操作。
4.V1Jns-tPA-85B[C1](含Ag85B,具tPA信号序列)的构建用下列引物进行:
有义85B[C1][SEQ.ID.NO.:20]
GG AAG ATC TCC TTC TCC CGG CCG GGG CTG CCG GTC GAG
反义85B[SEQ.ID.NO.:21]
GGA AGA TCT AAC CTT CGG TTG ATC CCG TCA GCC
除PCR的模板是p85B.tub外,接着进行与上面1中相同的操作。
5.V1Jns-tPA-85C[C1](含Ag85C,具tPA信号序列)的构建用下列引物进行:
有义85C[C1][SEQ.ID.NO.:22]
GG AAG ATC TCC TTC TCT AGG CCC GGT CTT CCA
反义85C[SEQ.ID.NO.:23]
GGA AGA TCT TGC CGA TGC TGG CTT GCT GGC TCA GGC
除PCR的模板是p85C.tub外,接着进行与上面1中相同的操作。
6.V1Jns-85B[C2](含Ag85B,不具信号序列)的构建用下列引物进行:
有义85B[C2][SEQ.ID.NO.:24]
GGA AGA TCT ACC ATG GGC TTC TCC CGG CCG GGG CTG C
反义85B[SEQ.ID.NO.:21]
GGA AGA TCT AAC CTC GGT TGA TCC CGT CAG CC
除PCR的模板是p85B.tub外,和克隆是V1Jns上之外,接着与上面1中相同的操作。
7.V1Jns-85C[C2](含Ag85C,不含信号序列)的构建用下列引物进行:
有义85C[C2][SEQ.ID.NO.:25]
GGA AGA TCT ACC ATG GGC TTC TCT AGG CCC GGT CTT C
反义85C[SEQ.ID.NO.:23]
GGA AGA TCT TGC CGA TGC TGG CTT GCT GGC TCA GGC
除PCR的模板是p85C.tub和克隆是在V1Jns中外,接着进行与上面1中相同的操作。
限制性分析后,所有构建体的跨载体连接部分做序列分析。大规模DNA制备基本按所述的方法(Montgomery,D.L.等,上文)。
质粒构建体的特点通过载体-插入连接部分的限制性作图和序列分析得到(见图1-6)。结果与发表的M.tb序列数据一致,表明对每个构建体来说启动子是完整的(图7)。也表明的是与M.tb Ag85无关的作为克隆的结果插入不同的附加氨基酸残基。
实施例5
来自V1Jns.tPA质粒的M.tb蛋白表达
在使用前一天,横纹肌肉瘤细胞(ATCC CCL136)在添加10%热灭活胎牛血清、2mM L-谷氨酰胺、25mM HEPEs,50U/ml青霉素和50μg/ml链霉素的高葡萄糖DMEM中,以每9.5cm2孔1.2×106细胞的密度种在六孔组织培养板中。(全部来自BRL-Gibco)苯酚:氯仿抽提氯化铯纯化的质粒DNA,用pharmacia Cellphect试剂用磷酸钙沉淀,除RD细胞每个9.5cm2孔用5-15μg之外,按试剂盒的指示使用。培养物在加磷酸钙-DNA沉淀后甘油休克6小时,重新换液后,收获前将培养物培养二天。
转染培养物的裂解液制备在添加1μm亮抑蛋白酶肽,1μm胃蛋白酶抑制剂,300nM抑蛋白酶肽和10μM TLCK的1X RIPA(0.5%SDS,1.0%TRITON X-100,1%脱氧胆酸钠,1mM EDTA,150mM NaCl,25mM TRIS-HCl pH7.4)中,短暂超声以降低粘性。裂解物通过电泳在10%Tricine胶(Novex)上分离,然后转移到硝酸纤维素膜上。免疫印迹用M.tb单克隆抗体17/4和32/15处理[Huygen等,1994,感染免疫62,363],用ECL检测试剂盒(Amersham)显色。
M.tb抗原85复合体基因的表达通过RD细胞的瞬时转染阐明。转染或模拟转染细胞的裂解液SDS PAGE分离,免疫印迹法分析。图8显示V1Jns.tPA-85A(C1)、V1Jns.tPA-85A(C2)、V1Jns.tPA-85A(C3)和V1Jns.tPA-85B(C1)转染细胞表达一种表观分子量约为30-32 kDa的免疫反应蛋白。
实施例6
用PNV免疫和抗原85蛋白在体内表达
5~6周龄的雌性BALB/c和C57BL/6小鼠腹膜(ip)内注射在盐水中的5mg盐酸氯胺酮(Aveco,Fort Dodge,IA)和0.5mg甲苯噻嗪(Mobley Corp.,Shawnee,KS.)的混合液麻醉。后腿用70%乙醇擦洗。动物用悬浮在盐水中的DNA(2mg/m1)100μl注射三次;每腿50μl。免疫后17-18天,收集血清样品,作抗-Ag85抗体存在分析。图9显示来自注射Ag85 DNA小鼠(C1)的血清有特异的免疫印迹反应,而来自接受不含有基因插入(V1J)的对照DNA的小鼠没有特异免疫印迹反应。反应性检出是血清至少1∶160稀释,抗300ng纯化的抗原85A(图96)。这说明Ag85DNA注射导致体内Ag85表达,这样在二种BALB/c和C57BL/6(B6)小鼠中抗体应答的产生是有效的。
实施例7
抗原85特异的T细胞应答
在对特异抗原重刺激如Huygen等,1992[感染免疫60,2880]所述的应答中,对来自接种小鼠的脾细胞作细胞因子分泌分析。脾细胞用来自牛分支杆菌BCG纯化抗原85A或相当于C57BL/6小鼠的一个熟知的T细胞表位(氨基酸241~260)的20-聚肽(p25)的培养滤过(CF)蛋白温育。小鼠用V1Jns.tPA85A(C1)(100μg)间隔三周免疫三次,最后一次注射1 7天后分析。细胞因子分析IL-2、干扰素-γ(IFN-γ)和IL-6用生物分析法,IL-4和IL-10用ELISA法。基本的IL-2和EFN-γ产生在两种用V1Jns.tPA85A(C1)接种的BALB/c和C57BL/6小鼠中观察到(图10-13)。此外,C57BL/6小鼠也与H-2b限制的T细胞表位有反应(图13)。在V1Jns.tPA85A接种小鼠中,IL-4、IL-6和IL-10没有增加(图14-16)。这些结果提示辅助T细胞应答的Th1型由DNA疫苗产生。
实施例8
对分支杆菌攻击的保护作用
为测试M.tb DNA疫苗的效用,小鼠用活牛分支杆菌BCG(0.5mg)的静脉内注射攻击,在脾和肺中分析BCG增殖。作为对照,BCG增殖在攻击的首次实验小鼠(第一次感染)和在DNA注射期间用BCG接种的攻击小鼠(第二次感染)中测定。与第一次感染小鼠或用对照DNA V1J接种的小鼠比较,在接种V1Jns.tPA85A(C1)小鼠的肺中,克隆形成单位(CFU)的数目基本上降低。在C57BL/6小鼠中,攻击后第8天CFU降低了83%(图17),在BALB/c小鼠中第20天CFU降低了65%(图18)。在脾中,在BALB/c小鼠中攻击后第20天(图19)和在C57BL/6小鼠中第8天(图20)CFU降低了约40%。因此,M.tb DNA疫苗注射后观察到的免疫应答在活M.bovis攻击模型中提供了保护。
序列表(1)一般信息:
(i)申请人:CONTENT,JEAN
HUYGEN,KRIS
LIU,MARGARET A.
MONTGOMERY,DONNA
ULMER,JEFFREY
(ii)发明题目:多核苷酸结核病疫苗
(iii)序列数:25
(iv)通讯地址:
(A)收信人:JACK L.TRIBBLE
(B)街道:126 E.LINCOLN AVE.,P.O.BOX 2000
(C)城市:RAHWAY
(D)州:NEW JERSEY
(E)国家:美国
(F)邮政编码:07065-0907
(v)计算机可读形式:
(A)介质类型:Floppy disk
(B)计算机:IBM PC兼容
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,Version#1.25
(vi)当前申请资料:
(A)申请号:US08/338,992
(B)提交日期:1994年11月14日
(C)分类:
(viii)委托人/代理人信息:
(A)姓名:TRIBBLE JACK L.
(B)登记号:32,633
(C)参考/备审件号:19342
(ix)电讯信息:
(A)电话:(908)594-5321
(B)传真:(908)594-4720(2)SEQ ID NO:1的信息:
(i)序列特征:
(A)长度:23个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:1:CTATATAAGC AGAGCTCGTT TAG 23(2)SEQ ID NO:2的信息:
(i)序列特征:
(A)长度:30个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:2:GTAGCAAAGA TCTAAGGACG GTGACTGCAG 30(2)SEQ ID NO:3的信息:
(i)序列特征:
(A)长度:39个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:3:GTATGTGTCT GAAAATGAGC GTGGAGATTG GGCTCGCAC 39(2)SEQ ID NO:4的信息:
(i)序列特征:
(A)长度:39个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:4:GTGCGAGCCC AATCTCCACG CTCATTTTCA GACACATAC 39(2)SEQ ID NO:5的信息:
(i)序列特征:
(A)长度:78个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:5:GATCACCATG GATGCAATGA AGAGAGGGCT CTGCTGTGTG CTGCTGCTGT GTGGAGCAGT 60CTTCGTTTCG CCCAGCGA 78(2)SEQ ID NO:6的信息:
(i)序列特征:
(A)长度:78个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:6:GATCTCGCTG GGCGAAACGA AGACTGCTCC ACACAGCAGC AGCACACAGC AGAGCCCTCT 60CTTCATTGCA TCCATGGT 78(2)SEQ ID NO:7的信息:
(i)序列特征:
(A)长度:27个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:7:GTACCTCATG AGCCACATAA TACCATG 27(2)SEQ ID NO:8的信息:
(i)序列特征:
(A)长度:40个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:8:GGTACAAGAT CTACCATGGC TTGCAATTGT CAGTTGATGC 40(2)SEQ ID NO:9的信息:
(i)序列特征:
(A)长度:42个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:9:CCACATAGAT CTCCATGGGA ACTAAAGGAA GACGGTCTGT TC 42(2)SEQ ID NO:10的信息:
(i)序列特征:
(A)长度:33个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述: SEQ ID NO:10:GGTACAAATA TTGGCTATTG GCCATTGCAT ACG 33(2)SEQ ID NO:11的信息:
(i)序列特征:
(A)长度:36个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:11:CCACATCTCG AGGAACCGGG TCAATTCTTC AGCACC 36(2)SEQ ID NO:12的信息:
(i)序列特征:
(A)长度:38个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:12:GGTACAGATA TCGGAAAGCC ACGTTGTGTC TCAAATC 38(2)SEQ ID NO:13的信息:
(i)序列特征:
(A)长度:37个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型: DNA(基因组的)
(xi)序列描述: SEQ ID NO:13:CCACATGGAT CCGTAATGCT CTGCCAGTGT TACAACC 37(2)SEQ ID NO:14的信息:
(i)序列特征:
(A)长度:39个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:14:GGTACATGAT CACGTAGAAA AGATCAAAGG ATCTTCTTG 39(2)SEQ ID NO:15的信息:
(i)序列特征:
(A)长度:34个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:15:CCACATGTCG ACCCGTAAAAA GGCCGCGTTG CTGG 35(2)SEQ ID NO:16的信息:
(i)序列特征:
(A)长度:29个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:16:GGAAGATCTT TTCCCGGCCG GGCTTGCCG 29(2)SEQ ID NO:17的信息:
(i)序列特征:
(A)长度:28个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:17:GGAAGATCTT GTCTGTTCGG AGCTAGGC 28(2)SEQ ID NO:18的信息:
(i)序列特征:
(A)长度:37个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:18:GGAAGATCTA CCATGGGCTT TTCCCGGCCG GGCTTGC 37(2)SEQ ID NO:19的信息:
(i)序列特征:
(A)长度:36个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:19:GGAAGATCTA CCATGGCACA GCTTGTTGAC AGGGTT 36(2)SEQ ID NO:20的信息:
(i)序列特征:
(A)长度:38个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:20:GGAAGATCTC CTTCTCCCGG CCGGGGCTGC CGGTCGAG 38(2)SEQ ID NO:21的信息:
(i)序列特征:
(A)长度:33个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:21:GGAAGATCTA ACCTTCGGTT GATCCCGTCA GCC 33(2)SEQ ID NO:22的信息:
(i)序列特征:
(A)长度:32个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:22:GGAAGATCTC CTTCTCTAGG CCCGGTCTTC CA 32(2)SEQ ID NO:23的信息:
(i)序列特征:
(A)长度:36个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:23:GGAAGATCTT GCCGATGCTG GCTTGCTGGC TCAGGC 36(2)SEQ ID NO:24的信息:
(i)序列特征:
(A)长度:37个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:24:GGAAGATCTA CCATGGGCTT CTCCCGGCCG GGGCTGC 37(2)SEQ ID NO:25的信息:
(i)序列特征:
(A)长度:37个碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:DNA(基因组的)
(xi)序列描述:SEQ ID NO:25:GGAAGATCTA CCATGGGCTT CTCTAGGCCC GGTCTTC 37
Claims (22)
1.一种在导入脊椎动物组织中诱导一种或多种抗分支杆菌免疫应答的多核苷酸,其免疫应答选自抗体、CTL、辅助T淋巴细胞应答和保护性免疫应答,其中所述的多核苷酸包括编码一种或多种分支杆菌蛋白或其功能性等价体的一个或多个基因,所述的基因可操作地连在转录启动子上。
2.权利要求1的多核苷酸,其中所述的基因编码一种结核分支杆菌蛋白和其功能性等价体。
3.权利要求2的多核苷酸,其中所述的基因编码一种选自抗原85A、B、和/或C的和其功能性等价体的蛋白。
4.一种在脊椎动物中诱导抗分支杆菌表位的免疫应答的方法,包括将根据权利要求1的1ng~5mg多核苷酸导入到脊椎动物组织中。
5.权利要求4的方法,其中所述的基因编码一种结核分支杆菌蛋白和其功能性等价体。
6.权利要求5的方法,其中所述的基因编码一种选自抗原85A、B和C和其功能性等价体的蛋白。
7.一种诱导抗分支杆菌抗原免疫应答的疫苗,包括权利要求1的多核苷酸和一种药学可接受的载体。
8.权利要求7的疫苗,其中所述的抗原是一种结核分支杆菌抗原和其功能性等价体。
9.权利要求8的疫苗,其中所述的抗原是一种选自抗原85A、B和C以及其功能性等价体的蛋白。
10.一种诱导抗分支杆菌抗原的免疫应答的方法,包括将一或多种分离和纯化的分支杆菌基因导入到脊椎动物的组织中,引起预防分支杆菌感染和/或减轻分支杆菌疾病的免疫应答。
11.权利要求10的方法,其中所述的基因编码一种结核分支杆菌蛋白和其功能性等价体。
12.权利要求11的方法,其中所述的基因编码一种选自抗原85A、B、和C及其功能性等价体的蛋白。
13.一种多核苷酸,其包括:
a)一种真核转录启动子;
b)一种操作性地与所述启动子连接的编码一或多种分杆菌表位的开放阅读框,和一种翻译终止信号;以及
c)选择性地含有一种或多种操作性连接的IRES,一种或多种编码一种或多种附加基因的开放阅读框,和一种或多种转录终止信号。
14.权利要求13的多核苷酸,其中所述c)的附加基因是选自GM-CSF、IL-12、干扰素、和T-细胞共刺激蛋白B7家族的一个成员的免疫调节或免疫刺激基因。
15.权利要求13的多核苷酸,其中所述的a)的分支杆菌基因编码一种结核分支杆菌蛋白和其功能性等价体。
16.权利要求15的多核苷酸,其中所述a)的分支杆菌基因编码一种选自抗原85A、B和C的和其功能性等价体的结核分支杆菌蛋白。
17.权利要求13的多核苷酸,其中所述c)的附加基因是选自抗原85A、B和C和其功能性等价体的结核分支杆菌基因。
18.一种在需要治疗时用一种多核苷酸治疗病人的方法,该多核苷酸在导入脊椎动物组织中后诱导一或多种选自抗体、CTL、辅助T淋巴细胞应答和保护性免疫应答的抗分支杆菌免疫应答,其中所述的多核苷酸包括编码一种或多种分支杆菌蛋白或其功能性等价体的基因,所述的基因可操作性地连接在一转录启动子上。
19.权利要求18的方法,其中所述的基因编码一种结核分支杆菌蛋白和其功能性等价体。
20.权利要求19的方法,其中所述的基因编码一种或多种选自抗原85A、B和C及其功能性等价体的蛋白。
21.权利要求10的方法,其中所述的患者是家养动物或家畜。
22.一种在家养或农业动物中诱导抗分支杆菌感染的免疫应答的方法,包括权利要求1的多核苷酸和一种药学上可接受的载体。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US08/338,992 | 1994-11-14 | ||
US08/338,992 US5736524A (en) | 1994-11-14 | 1994-11-14 | Polynucleotide tuberculosis vaccine |
Publications (1)
Publication Number | Publication Date |
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CN1171814A true CN1171814A (zh) | 1998-01-28 |
Family
ID=23326996
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CN95197250A Pending CN1171814A (zh) | 1994-11-14 | 1995-11-13 | 多核苷酸结核病疫苗 |
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US (2) | US5736524A (zh) |
EP (1) | EP0792358B1 (zh) |
JP (1) | JP3881014B2 (zh) |
KR (1) | KR970707281A (zh) |
CN (1) | CN1171814A (zh) |
AT (1) | ATE296882T1 (zh) |
AU (1) | AU715067B2 (zh) |
CZ (1) | CZ289383B6 (zh) |
DE (1) | DE69534250T2 (zh) |
DK (1) | DK0792358T3 (zh) |
ES (1) | ES2242193T3 (zh) |
FI (1) | FI972034A (zh) |
HU (1) | HU222369B1 (zh) |
IL (1) | IL115883A0 (zh) |
MX (1) | MX9703606A (zh) |
NO (1) | NO972196L (zh) |
NZ (1) | NZ296477A (zh) |
PL (1) | PL184839B1 (zh) |
PT (1) | PT792358E (zh) |
RU (1) | RU2186109C2 (zh) |
SK (1) | SK283254B6 (zh) |
WO (1) | WO1996015241A2 (zh) |
ZA (1) | ZA959608B (zh) |
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