黑猩猩腺病毒疫苗载运体的制作方法

本申请是申请日为2005年1月18目的中国专利申请201210056795.9“黑猩猩腺病毒疫苗载运体”的分案申请。相关申请的交叉引用本申请要求于2004年1月23日提交的美国临时申请no.60/538,799的权益，通过引用将它合并在此。本发明涉及重组载体领域，更具体地涉及重组复制缺陷性黑猩猩腺病毒载体的产生和用途，来在哺乳动物宿主中引发免疫反应。
背景技术：
：腺病毒(ads)包含在两栖动物、禽类和哺乳动物中发现的、具有无包被的二十面体衣壳结构的双链dna病毒的大家族(straus，adenovirusinfectionsinhumans.intheadenoviruses.451-498，1984；hierholzeretal.，j.infect.dis.，158：804-813，1988；schnurranddondero，intervirology.，36：79-83，1993；jongetal.，jclinmicrobiol.，37：3940-3945：1999)。与逆转录病毒相比，腺病毒可以转导几个哺乳动物物种的许多细胞类型，包括分裂和非分裂细胞，而不整合到宿主细胞的基因组中。一般而言，腺病毒dna一般是非常稳定的并保持游离(例如，染色体外)，除非发生了转化或肿瘤发生。此外，在容易进行临床级组合物的制药规模生产的良好定义的生产系统中，腺病毒载体可以繁殖到高产量。这些特征和它们被良好表征的分子遗传学，使得重组腺病毒载体成为用作疫苗载运体(carrier)的好的候选者。一般地，重组腺病毒载体的生产依靠使用能够补足腺病毒基因产物的功能的包装细胞系，所述功能已经被删除或被工程化为无功能的。目前，两种良好表征的人类c亚群腺病毒血清型(即，had2和had5)被广泛地用作大多数用于基因治疗的腺病毒载体的病毒主干的来源。也已经测试了复制缺陷性人类腺病毒载体作为用于递送各种免疫原的疫苗载体，所述免疫原来源于各种传染原(例如，病毒、寄生虫或细菌性病原体)和肿瘤细胞，包括肿瘤相关抗原(taa)。在实验动物(例如，啮齿动物、犬和非人灵长类)中进行的研究表明，携带转基因的重组复制缺陷性人类腺病毒载体引发了针对转基因产物的体液和细胞介导的免疫反应，所述转基因编码来源于人类乳头状瘤病毒(hpv-16)的e6和e7癌蛋白(he，zetal.，(2001)virology，270：3583-3590)、狂犬病病毒糖蛋白(xiang，z.etal(1996)virolgy，219：220-227)、plasmodiumfalciparum的环子孢子蛋白rodrigueze.etal.(1997)j.immunol.158：1268-1274)以及其他异源抗原的免疫原。一般而言，研究人员已经报道了，通过使用利用预计可引发免疫反应的高剂量重组腺病毒载体的免疫方案；或通过使用采用腺病毒载体的连续施用的免疫方案，所述腺病毒载体来源于不同血清型但携带相同的转基因产物用作强化免疫，在非人类实验系统中成功地使用人类腺病毒载体作为疫苗载运体(mastrangeli，etal.，humangenetherapy，7：79-87(1996)。然而，预计的是，来源于遍在的人类血清型，例如2和5型的疫苗载运体将在人类群体中遇到先存的(pre-existing)体液和细胞免疫。因而，尽管已经在采用啮齿动物、犬和非人灵长类宿主的实验系统中成功地使用复制缺陷性重组人类腺病毒作为疫苗载运体，预计人类的先天和适应性免疫会显著地限制这些血清型作为疫苗载运体的实用性。这种预期是基于这一事实，包括2型和5型的c亚群腺病毒感染在人类群体中是地方性的。结果，作为自然感染的结果，大多数人类在生命的头五年内血清转变。因而，来源于在人类中天然感染并复制的病毒的载体，可能不是用作疫苗载运体的最优候选者。与使用人类腺病毒衍生载体相关的另一个问题是，用于繁殖重组病毒的生产方法产生被有复制能力的腺病毒(rca)污染的载体成品的风险。这是起因于，来自重组载体和存在于e1补足辅助细胞系，例如人类293(graham，f.l.etal，(1977)j.gen.virol.36：59-72.)细胞中的腺病毒基因的重叠序列之间的同源重组。在为用于临床试验制备的载体成品中rca的存在构成了安全风险，因为它可能促进复制缺陷性病毒的流动和传播。缺陷病毒的传播可能加重宿主免疫应答，并引起其他有害的免疫病理后果。(fallux，f.j.，etal.humangenetherapy9：1909-1917(1998)。因此，食品和药品管理局(fda)和其他管理机构已经颁布了指导原则，其建立了对可能存在于用于临床用途的载体制品中的rca水平的限制。加以rca限制的意图是确保患者有限地暴露于用于临床试验的组合物中的复制性腺病毒。因而，存在着开发适用于哺乳动物宿主的腺病毒疫苗载运体的持续的需求，其是：易于操作、可经受制药规模的生产和长期储存、在人类补足细胞系中能以高水平复制、高度的免疫原性、没有与人类腺病毒的常见血清型交叉反应的中和b细胞表位、符合管理机构颁布的安全rca标准、并且其符合在适合人类使用的激发/强化(prime/boost)方案中使用。技术实现要素：本发明涉及来源于黑猩猩腺病毒的重组复制缺陷性腺病毒载体和用于在人类e1表达细胞系中产生黑猩猩腺病毒载体的方法。本发明还提供了产生适用于人类的临床级载体成品的方法，和使用所公开的载体作为疫苗载运体来引发保护性和/或治疗性免疫反应的方法。本发明进一步提供了使用本发明的重组腺病毒来制备疫苗组合物的方法，所述疫苗组合物被设计以递送编码免疫原的转基因和指导编码免疫原的转基因的表达。在一个实施方式中，本发明考虑使用所公开载体作为疫苗载运体用于疫苗的施用，所述疫苗包含编码来源于传染原的免疫原的转基因。在第二种实施方式中，本发明考虑使用所公开的载体来制备和施用癌症疫苗。在特定的实施方式中，本发明考虑制备和施用包含编码taa的转基因的癌症疫苗。在一个方面，本发明公开了五种黑猩猩腺病毒(chads)的完整基因组序列，在此称为chad3(seqidno：1)(附图5a-5k)、chad6(seqidno：2)(附图6a-6k)、cv32(seqidno：3)(附图7a-7k)、cv33(seqidno：4)(附图8a-8k)和cv23(seqidno：5)(附图9a-9j)。chad3和chad6是根据在此公开的方法分离的新的腺病毒。chad3和chad6的基因组长度分别是37741和36648个碱基对。chad3六邻体基因(seqidno：41)包含seqidno：1的核苷酸(nt)19086-21965(不包括终止密码子)，chad3纤维基因(seqidno：42)包含seqidno：1的nt32805-34487(不包括终止密码子)。chad6六邻体(hexon)基因包含seqidno：2的nt18266-21124(seqidno：43)(不包括终止密码子)，它的纤维基因(seqidno：44)包含seqidno：2的nt32218-33552(不包括终止密码子)。根据从全长六邻体肽的多重序列比对得出的序列同源性，chad3被分类入人类c亚群，chad6被分类入人类e亚群。cv32、cv33和cv23腺病毒的基因组的长度分别是36，606、36，535和32，020个碱基对。cv32(pan6)(atccn.vr-592)、cv33(pan7)(atccn.vr-593)和cv23(pan5)(esoterixinc.，)全都被确定为与人类ad4(had4)(e亚群)相关(wigand，retal.intervirology1989，30：1-9)。然而，根据六邻体序列比对，随后将cv32表征为与类似于had4相比更接近地类似于人类d亚群成员。在第二个方面，本发明提供了根据在此公开的方法分离的21种其他黑猩猩腺病毒(chad20、chad4、chad5、chad7、chad9、chad10、chad11、chad16、chad17、chad19、chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82)的纤维基因和六邻体基因的核苷酸序列。chad20、chad4、chad5、chad7、chad9、chad10、chad11、chad16、chad17、chad19的纤维基因核苷酸序列分别在附图10-19中列出，在此称为seqidno：6-15：(seqidno：6，chad20)；(seqidno：7，chad4)；(seqidno：8，chad5)；(seqidno：9，chad7)；(seqidno：10，chad9)；(seqidno：11，chad10)；(seqidno：12，chad11)；(seqidno：13，chad16)(seqidno：14，chad17)和(seqidno：15，chad19)chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82的纤维基因核苷酸序列在此称为：(seqidno：58，chad8)、(seqidno：60，chad22)、(seqidno：62，chad24)、(seqidno：64，chad26)、(seqidno：66，chad30)、(seqidno：68，chad31)、(seqidno：70，chad37)、(seqidno：72，chad38)、(seqbdno：74，chad44)、(seqidno：76，chad63)和(seqidno：78，chad82)，并在序列表中列出。chad20、chad4、chad5、chad7、chad9、chad10、chad11、chad16、chad17、chad19的六邻体基因核苷酸序列分别在附图21-30中列出，在此称为seqidno：16-25：(seqidno：16，chad20)；(seqidno：17，chad4)；(seqidno：18，chad5)；(seqidno：19，chad7)；(seqidno：20，chad9)；(seqidno：21，chad10)；(seqidno：22，chad11)；(seqidno：23，chad16)；(seqidno：24，chad17)和(seqidno：25，chad19)。chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82的六邻体基因核苷酸序列在此称为：(seqidno：97，chad8)、(seqidno：99，chad22)、(seqidno：101，chad24)、(seqidno：103，chad26)、(seqidno：105，chad30)、(seqidno：107，chad31)、(seqidno：109，chad37)、(seqidno：111，chad38)、(seqidno：113，chad44)、(seqidno：115，chad63)和(seqidno：117，chad82)，并在序列表中列出。在第三个方面，本发明提供了根据在此公开的方法分离的21种其他黑猩猩腺病毒(chad20、chad4、chad5、chad7、chad9、chad10、chad11、chad16、chad17、chad19、chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82)的纤维蛋白和六邻体蛋白的氨基酸序列。在此公开和要求权利的纤维蛋白称为：(seqidno：83，chad3)、(seqidno：84，chad6)、(seqidno：48，chad20)、(seqidno：49，chad4)、(seqidno：50，chad5)、(seqidno：51，chad7)、(seqidno：52，chad9)、(seqidno：53，chad10)、(seqidno：54，chad11)、(seqidno：55，chad16)、(seqidno：56，chad17)、(seqidno：57，chad19)、(seqidno：59，chad8)、(seqidno：61，chad22)、(seqidno：63，chad24)、(seqidno：65，chad26)、(seqidno：67，chad30)、(seqidno：69，chad31)、(seqidno：71，chad37)、(seqidno：73，chad38)、(seqidno：75，chad44)、(seqidno：77，chad63)和(seqidno：79，chad82)。附图20a一20g提供了比较在此公开和要求权利的纤维蛋白的氨基酸序列与以下纤维蛋白的氨基酸序列的比对：c1(seqidno：85)、cv68(seqidno：86)、pan5(作为选择称为cv23)(seqidno：80)、pan6(作为选择称为cv32)(seqidno：81)和pan7(作为选择称为cv33)(seqidno：82)。在此公开和要求权利的六邻体蛋白称为：(seqidno：122，chad3)、(seqidno：123，chad6)、(seqidno：87，chad20)、(seqidno：88，chad4)、(seqidno：89，chad5)、(seqidno：90，chad7)、(seqidno：91，chad9)、(seqidno：92，chad10)、(seqidno：93，chad11)、(seqidno：94，chad16)、(seqidno：95，chad17)、(seqidno：96，chad19)、(seqidno：98，chad8)、(seqidno：100，chad22)、(seqidno：102，chad24)、(seqidno：104，chad26)、(seqidno：106，chad30)、(seqidno：108，chad31)、(seqidno：110，chad37)、(seqidno：112，chad38)、(seqidno：114，chad44)、(seqidno：116，chad63)和(seqidno：118，chad82)。附图31a-31j提供了在此公开和要求权利的六邻体蛋白的氨基酸序列与以下六邻体蛋白的氨基酸序列的比较：c1(seqidno：124)、cv68(seqidno：125)、pan5(做为选择称为cv23)(seqidno：119)、pan6(做为选择称为cv32)(seqidno：120)和pan7(做为选择称为cv33)(seqidno：121)。六邻体蛋白的多重序列比对容许技术人员进行与人类腺病毒血清型的建议分类一致的系统发生分析(rux，j.j.，etal(2003)j.virol.77：9553-9566)。在可选择的方面，本发明进一步提供了21种其他的黑猩猩腺病毒分离物。将包含chad20、chad4、chad5、chad7、chad9、chad10、chad11、chad16、chad17、chad19、chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82的样品根据布达佩斯条约保藏在欧洲动物细胞保藏中心(ecacc，portondown，salisbury，wiltshire，sp4ojg，英国)。其保藏日、分类命名和保藏号如下表所示：保藏日期保藏编号分类命名2003年12月12日03121201chimpanzeeadenovirustype42003年12月12日03121202chimpanzeeadenovirustype52003年12月12日03121203chimpanzeeadenovirustype72003年12月12日03121204chimpanzeeadenovirustype92003年12月12日03121205chimpanzeeadenovirustype102003年12月12日03121206chimpanzeeadenovirustype112003年12月12日03121207chimpanzeeadenovirustype162003年12月12日03121208chimpanzeeadenovirustype172003年12月12日03121209chimpanzeeadenovirustype192003年12月12日03121210chimpanzeeadenovirustype202005年1月12日05011201chad82005年1月12日05011202chad222005年1月12日05011203chad242005年1月12日05011204chad262005年1月12日05011205chad302005年1月12日05011206chad312005年1月12日05011207chad372005年1月12日05011208chad382005年1月12日05011209chad442005年1月12日05011210chad632005年1月12日05011211chad82这些保藏物将根据国际承认用于专利程序的微生物保藏布达佩斯条约的条款进行维持。这些保藏仅为了方便本领域技术人员而进行，而不是认可保藏是35u.s.c.§112所需的。在授予专利之时，除了37c.f.r.§1.808(b)规定的要求之外，对保藏的材料的公众可获得性的所有限制将被不可撤销地除去。在其他方面，本发明还提供了复制缺陷性重组腺病毒载体，其能够感染哺乳动物细胞，优选的人类细胞，并指导编码的转基因产物的表达。如在此展示的，所公开的载体(vector)适合用做疫苗载运体(carrier)用于递送包含免疫原的转基因，针对所述免疫原的免疫反应是期望的。在特定实施方式中，本发明提供了能够在人类e1表达(即，包装)细胞系中高水平复制的重组复制缺陷性黑猩猩腺病毒载体。在一个实施方式中，本发明提供了能够在per.c6tm细胞中复制的重组腺病毒。一般而言，本发明包括的重组载体提供了能避开对腺病毒血清型的先存免疫的疫苗载运体，所述先存免疫是在人类群体中一般遇到的。更具体地说，本发明的重组载体包含载体主干序列，在此显示了其缺少与人类腺病毒衍生载体的常见血清型交叉反应的中和b表位。本发明进一步提供了包括腺病毒部分和质粒部分的类群特异性的穿梭载体，其中所述腺病毒部分一般地包含：a)病毒左末端(itr和包装信号)、pix基因的部分和病毒基因组右末端；和b)基因表达盒。采用了在指定为相同血清型亚群(即，a、b、c、d或e亚群)的腺病毒之间观察到的核苷酸序列同一性设计类群特异性的穿梭载体，并可用于操作在此公开的核苷酸序列，和/或用于克隆属于相同亚群的其他黑猩猩腺病毒，产生含有删除了e1区域的黑猩猩腺病毒基因组的腺病毒前质粒(pre-plasmid)。本发明的其他方面包括包含腺病毒疫苗载体和/或腺病毒前质粒载体的宿主细胞、生产所述载体的方法，包括将所述腺病毒疫苗载体导入表达腺病毒e1蛋白的宿主细胞中，并收获产生的腺病毒疫苗载体。在特定的实施方式中，本发明提供了生产复制缺陷性黑猩猩腺病毒载体的方法，包括将所公开的腺病毒载体之一导入表达腺病毒e-1的人类细胞，并收获产生的重组腺病毒。本发明的另一个方面还提供了包含本发明的腺病毒载体的疫苗组合物。包含重组黑猩猩腺病毒载体的组合物可以单独施用或与其他基于病毒或非基于病毒的dna/蛋白疫苗组合施用。它们也可以作为更广阔的治疗方式的部分来施用。这些组合物可以以预防性或治疗性的设置施用给哺乳动物宿主，优选人类宿主。如在此所示，单独地或以组合形式施用所公开的疫苗组合物，例如激发强化方式或不同血清型ad载体的多次注射，引起哺乳动物中能够特异性识别由转基因编码的免疫原的免疫反应的诱导。在此公开和要求权利的方法之一，包括向哺乳动物(是幼稚的(naive)或被激发以对目标抗原有免疫活性的)施用足够量的重组黑猩猩腺病毒载体，所述载体含有它的野生型e1基因的至少一个功能删除，带有包含启动子的序列，所述启动子能指导编码至少一个目标抗原的核苷酸序列的表达，其中施用所述重组抗体引发(或激发)抗原特异性免疫反应。在一个实施方式中，本发明提供了设计以诱导针对传染原(例如，病毒或细菌性病原体或哺乳动物寄生虫)的免疫反应(预防性或治疗性的)的方法。在第二实施方式中，本发明提供了设计以在哺乳动物中诱导免疫反应的方法，所述免疫反应将破坏对自体抗原，例如taa的耐受性。本发明的这个方面考虑使用所公开的载体作为疫苗载运体用于制备和施用癌症疫苗。根据以下对发明的详细说明，本发明的其他实施方式和益处将显而易见。附图说明附图1是概括了用于构建chad6穿梭载体(parschad6-3)的克隆策略的示意图。附图2是说明用于在e.coli菌株bj5183中通过同源重组克隆chad6病毒基因组的克隆策略的示意图。附图3是说明各种chad6穿梭质粒的元件的示意图，包括：parschad6-3gag；parschad6-3seap；parschad6-3egfp；和parschad6-3nsmut。附图4是说明用于克隆chad6δe1表达载体的同源重组方案的示意图。附图5a-5k提供了chad3的基因组核苷酸序列(seqidno：1)。附图6a-6k提供了chad6的基因组核苷酸序列(seqidno：2)。附图7a-7k提供了cv32的基因组核苷酸序列(seqidno：3)。附图8a-8k提供了cv33的基因组核苷酸序列(seqidno：4)。附图9a-9j提供了cv23的基因组核苷酸序列(seqidno：5)。附图10提供了chad20的纤维基因的核苷酸序列(seqidno：6)。附图11提供了chad4的纤维基因的核苷酸序列(seqidno：7)。附图12提供了chad5的纤维基因的核苷酸序列(seqidno：8)。附图13提供了chad7的纤维基因的核苷酸序列(seqidno：9)。附图14提供了chad9的纤维基因的核苷酸序列(seqidno：10)。附图15提供了chad10的纤维基因的核苷酸序列(seqidno：11)。附图16提供了chad11的纤维基因的核苷酸序列(seqidno：12)。附图17提供了chad16的纤维基因的核苷酸序列(seqidno：13)。附图18提供了chad17的纤维基因的核苷酸序列(seqidno：14)。附图19提供了chad19的纤维基因的核苷酸序列(seqidno：15)。附图20a-20g提供了以下的纤维蛋白的氨基酸序列：chad3、chad4、chad5、chad6、chad7、chad8、chad9、chad10、chad11、chad16、chad17、chad19、chad20、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82，与来自c1(seqidno：85)、cv68(seqidno：86)、pan5(也称为cv23)(seqidno：80)、pan6(也称为cv32)(seqidno：81)和pan7(也称为cv33)(seqidno：82)的参考纤维蛋白序列的比较。附图21提供了chad20的六邻体基因的核苷酸序列(seqidno：16)。附图22提供了chad4的六邻体基因的核苷酸序列(seqidno：17)。附图23提供了chad5的六邻体基因的核苷酸序列(seqidno：18)。附图24提供了chad7的六邻体基因的核苷酸序列(seqidno：19)。附图25提供了chad9的六邻体基因的核苷酸序列(seqidno：20)。附图26提供了chad10的六邻体基因的核苷酸序列(seqidno：21)。附图27提供了chad11的六邻体基因的核苷酸序列(seqidno：22)。附图28提供了chad16的六邻体基因的核苷酸序列(seqidno：23)。附图29提供了chad17的六邻体基因的核苷酸序列(seqidno：24)。附图30提供了chad19的六邻体基因的核苷酸序列(seqidno：25)。附图31a-31j提供了chad3、chad4、chad5、chad6、chad7、chad8、chad9、chad10、chad11、chad16、chad17、chad19、chad20、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82的六邻体蛋白的氨基酸序列，与来自c1(seqidno：124)、cv68(seqidno：125)、pan5(也称为cv23)(seqidno：119)、pan6(也称为cv32)(seqidno：120)和pan7(也称为cv33)(seqidno：121)的参考纤维蛋白序列的比较。附图32提供在此公开的人工序列seqidno：26-40和seqidno：45和46，包括寡聚物和引物的列表。附图33是属于不同血清型亚群(即，c、e和d亚群)的chad载体的免疫断点(break-point)的图示。通过在用表达gag的chad3、chad11、chad20、cv33、cv68、chad6、chad9、chad10、cv32、chad4、chad7和chad16载体免疫的小鼠中进行滴定实验，测定引发可测量免疫反应的最低剂量。附图34提供了在恒河猴中引发的cea特异性t细胞反应的图示，所述恒河猴次序地用人类腺病毒载体(mrkad5rhcea)、12周间隔后用黑猩猩腺病毒载体(cv33rhcea)免疫。通过ifn-γelispot分析评估免疫反应，数据说明了在不存在(dmso)和存在恒河猴ceac和d肽库的情况下孵化之后每百万个外周血单核细胞(pbmc)中斑点形成细胞(sfc)的数目。附图35提供了用paupsearch(wisconsinpackageversion10.3，aceelrysinc.)进行全长六邻体肽序列的多重序列比对和用treeview进行可视化和操作得出的人类和黑猩猩腺病毒的系统树。附图36是响应于向预先暴露于had5的小鼠施用chad3和had5gag载体获得的免疫结果的图示。免疫后3周通过ifn-γelispot使用纯化的脾细胞评估细胞介导的免疫。附图37是在用chad3hcea和ad5hcea免疫的人类cea转基因小鼠中引发的抗ceacmi的动力学的图示。通过用cea肽库刺激的pbmc的ics评估cmi。结果表示为ifnγ+cd8+/总pbmc的％。附图38a-d是暴露于moi50、250和1250的不同chad载体的不同人类初级细胞的感染效率的图示，所述chad载体表达egfp并属于不同的亚群(b、c、d、e)。结果表示为荧光细胞/总细胞的％。具体实施方式如在整个说明书和附随的权利要求书中使用的，采用以下定义和缩写：术语“盒”是指核酸分子，其包含至少一个要表达的核酸序列以及它的转录和翻译控制序列。改变所述盒，将使掺入了所述盒的载体指导不同序列或序列的组合的表达。在本发明的范围中，盒中存在的核酸序列通常编码免疫原。由于工程化的限制性位点存在于5′和3′末端，所述盒可以容易地插入、除去或用另一个盒替换。术语“顺式作用元件”是指调节与它们连接的基因的核苷酸序列。存在于dna中的顺式作用元件调节转录，转录成mrna的那些能够调节rna加工、转换(turnover)和蛋白质合成。术语“载体”是指一些工具，通过它们能将dna片段导入到宿主有机体或宿主组织中。存在着各种型式的载体，包括质粒、病毒(包括腺病毒)、噬菌体和粘粒。术语“启动子”是指在dna链上、rna聚合酶能与之结合的识别位点。启动子与rna聚合酶形成起始复合物来启动和驱动转录活性。通过活化序列例如增强子，或抑制序列例如沉默子，可以修饰所述复合物。术语“药学有效量”是指重组腺病毒的数量，在特定施用途径中有效转导宿主细胞并提供足够的转基因表达水平以引发免疫反应。术语“有复制能力的”重组腺病毒(adv)是指具有完整的或功能上必需的早期基因(即，e1a、e1b、e2a、e2b和e4)的腺病毒。野生型腺病毒是有复制能力的。术语“复制缺陷性”重组adv是指一种腺病毒，由于已经被工程化以具有病毒复制所必需的基因产物的至少一种功能删除或完全删除，而变得不能复制。本发明的重组黑猩猩腺病毒载体是复制缺陷性的。术语“哺乳动物”是指任何哺乳动物，包括人类。术语“序列同一性百分比”或“同一性”在核酸序列的上下文中是指当进行最大相关性比对时两个序列中的相同的残基。序列同一性比较的长度可以是在基因组的全长上(例如，约36kbp)，基因的开放阅读框的全长上，蛋白、亚基或酶上[参见，例如，提供腺病毒编码区域的框]，或至少约500到5000个核苷酸的片段上，是期望的。然而，在例如，至少约九个核苷酸、通常至少约20到24个核苷酸、至少约28到32个核苷酸、至少约36个或更多核苷酸的较小片段之间的同一性也是期望的。类似地，可以对氨基酸序列，在蛋白的全长上或其片段，容易地确定“序列同一性百分比”。适当地，片段长度至少约8个氨基酸，并可以达到约700个氨基酸。在此描述了适合的片段的实例。在默认设置下使用在此定义的这种算法和计算机程序，容易地确定同一性。优选的，这种同一性是在蛋白、酶、亚基的全长上，或在长度至少约8个氨基酸的片段上进行的。然而，同一性可以基于更短的区域，在所述区域上适合于放入同一的基因产物的用途。一般地，腺病毒构建体、基因构建体通过参考其中含有的基因来命名。例如，“pchad3δe1gag”是指包含删除了e1区域的chad3黑猩猩腺病毒基因组的质粒构建体。在这个质粒中，e1区域被免疫原表达盒替代，所述免疫原表达盒包含处在人类cmv启动子控制下之后是牛生长激素多聚腺苷酸信号的hivgag基因。类似地，pcv33de1-e3nsmut是指在此公开的第二种质粒构建体，其包含删除了e1和e3区域的cv33黑猩猩腺病毒基因组，所述e1和e3区域被免疫原表达盒替代，所述免疫原表达盒包含处在人类cmv启动子控制下之后是牛生长激素多聚腺苷酸信号的hcv非结构基因。缩写“ag”是指抗原。在整个说明书中和在附随的权利要求中使用的，单数形式“a”、“an”和“the”包括复数的引用，除非上下文中明显地另外指示了。腺病毒(ads)是已经在几种禽类和哺乳动物宿主中鉴定的无包被的二十面体病毒。人类ads(had)属于mastadenovirus属，包括所有已知的人类和动物(例如，牛、猪、犬、鼠、马、猿和绵羊)来源的许多ads。根据许多生物学、化学、免疫学和结构标准，包括大鼠和恒河猴红血球的血细胞凝集性质、dna同源性、限制性内切酶裂解模式、g+c含量百分比和致癌性，将人类腺病毒分成六个亚群(a-f)(straus，1984，intheadenoviruses，ed.h.ginsberg，pps.451-498，newyork：plenuspress，andhorwitz，1990invirology，eds.b.n.fieldsandd.m.knipe，pps.1679-1721)。迄今为止，已经鉴定了51种不同的血清型，并根据它们的血细胞凝集性质和生物物理和生物化学标准分类成亚群。腺病毒颗粒是二十面对称性的，取决于血清型，直径为60-90nm。二十面体衣壳由三种主要蛋白，六邻体(ii)、五邻体基部(iii)和隆突纤维(iv)以及许多微小蛋白(即，vi、viii、ix、iiia和iva2)组成(w.c.russel，j.gen.virol.，81：2573-2604(2000))。在人体中观察到的先存免疫性的一个方面是体液免疫，其可以引起特异于病毒蛋白的抗体的产生和存留。由腺病毒引发的体液反应主要地针对主要结构蛋白质：六邻体、五邻体和纤维。公开的报道已经证实了，包含针对多种血清型的抗体的滴度是常见的(dambrosio，e.(1982)j.hyg.(london)89：209-219)，先存滴度的重要部分具有中和活性。对腺病毒的中和免疫性是型特异性的(type-specific)，特定血清型的腺病毒的感染仅赋予对该血清型的免疫性。一些报道已经表明，针对六邻体的抗体是最强烈的和最具中和性的(toogood，c.i.a.，crompton，j.andhayr.t.(1992)j.gen.virol.73，1429-1435)。因此，合理的假定是，对型特异性中和作用负责的表位位于七个高变区内，所述高变区是通过对从不同血清型得出的六邻体序列进行比对来鉴定的。(crawford-miksza，landd.p.schnurr.(1996)j.virol.70：1836-1844)。已经通过不同的方法，包括免疫血清从处理的动物到幼稚动物的被动转移，证实了型特异性中和抗体的存在与用基于相同血清型的载体不能引发免疫反应之间的直接相关性。一般而言，对特定病毒血清型的先存体液免疫降低了载体施用的治疗效力。此外，施用基于特定病毒血清型的载体引发了针对该载体的、阻止相同血清型的重复施用的免疫反应。在特定的实施方式中，本发明提供了一种绕过所述不利影响的方法，所述不利影响与对had的常见血清型的先存免疫性的后果有关。更具体地，本发明考虑使用以不在人类中传播的血清型为特征的黑猩猩腺病毒载体。因此，本发明提供了缺少与常见人类血清型的表位发生交叉反应的中和b细胞表位的腺病毒(chad)载体作为疫苗载运体。虽然已经报道了腺病毒特异性细胞介导的免疫性(cmi)可能是交叉反应性的，基于多种血清型的重复注射的疫苗接种研究表现了比基于单个载体的免疫程序更高的效率。这些实验进一步证明了疫苗目的的载体施用的主要局限是针对该载体的体液性先存免疫性。对使用人类腺病毒作为疫苗载运体有关的难题的可能解决方案包括，施用更高剂量的预测会遭遇先存免疫反应的腺病毒(例如，c亚群血清型)，和使用基于稀有人类血清型的载体。然而，使用更高剂量的疫苗增加了疫苗的成本和不希望的副作用的风险，临床前测试的结果表明人类的另外的血清型比had5和had6有更低的免疫原性。在避免针对载体的腺病毒主干元件的宿主体液和细胞免疫反应的问题和最小化使用可能被有复制能力的腺病毒(rca)污染的人类腺病毒衍生载体成品的风险的尝试中，已经表征和开发了几种非人类腺病毒作为疫苗载运体(soudois，c.etal(2000)j.virology，74：10639-10649；farina，s.f.etal(2001)j.virology，75：11603-11613；cohen，c.j.etal(2002)j.gen.virology，83：151-155.)。使用非人类腺病毒序列来绕过与先存免疫性有关的难题的前提基于这种观察结果，即针对常见人类腺病毒血清型的中和抗体不太可能交叉中和非人类病毒。然而，病毒和细胞因素的不相容性给大多数可选择的载体系统(牛、绵羊、犬)带来了实际的局限性，其特征在于必须在非人类细胞系中繁殖的缺点。wilson等已经公开了一报告，描述了基于黑猩猩腺病毒68型(cv68)c68的复制缺陷性载体的鉴定，所述腺病毒最初是从黑猩猩的肠系膜淋巴结分离的(basnight，m.，et.al.(1971)am.j.epidemiol.94：166-171.)，cv68被完全测序并发现在整体结构上类似于人类腺病毒(farina，s.f.etal.，j.virol.75(23)：11603-11613(2001))。病毒的基因组长度是36,521碱基对，已经描述为最类似人类腺病毒的e亚群，与大多数已经测序的人类ad4开放阅读框有90％的同一性。cv68itrs的长度是130碱基对，存在所有主要的腺病毒早期和晚期基因。cv68的特征在于不在人类中传播(circulate)的血清型，其缺少与常见的人类血清型的表位交叉反应的中和b细胞表位。虽然黑猩猩腺病毒类似于人类腺病毒，但在人类中没有记录到针对黑猩猩血清型的交叉反应性中和免疫。(farina，s.f.etal.j.virol.(2001)75(23)：11603-13)。来源于cv68的重组载体被描述为足够类似于人类血清型以支持表达柯萨奇病毒(coxsackievirus)和腺病毒受体的细胞的转导(cohen，c.etal.，j.gen.virol.83：151-155(2002)。重要地，cv68的特征是与人类腺病毒足够的相似性水平以支持它在携带来自人类腺病毒5型的e1的293细胞中复制(farina，s.f.etal.，j.virol.75(23)：11603-11613(2001)。此外，根据人类血清型5e1的侧翼序列与cv68衍生载体序列的侧翼序列是非同源的这一观察结果，预计不会发生同源重组。因而，预计cv68衍生的疫苗成品(vaccinestocks)被rca污染的可能性很低。同一组研究者随后报道了，使用cv68衍生的腺病毒序列作为疫苗载运体用于在小鼠中诱导针对狂犬病病毒糖蛋白的抗体。通过用迷你基因(minigene)盒替换e1a和e1b基因产生了复制缺陷型的cv68。用含有e1删除、包含编码狂犬病病毒糖蛋白的转基因产物的腺病毒重组体(adc68rab.gp)免疫的小鼠，发展出对狂犬病病毒的保护性免疫，并且对不然就是致死剂量的狂犬病病毒的攻击保持了抗性(xiang，zetal.，j.virol.76(5)：2667-2675(2002)。近来已经报道了表达密码子优化的、截短形式的hiv-1的gag的第二种cv68构建体在小鼠中诱导强有力的gag特异性cd8+t细胞反应。疫苗诱导反应显示了能提供对牛痘gag重组病毒攻击的保护(fitzgerald，j.c.etal.，j.immunol.170：1416-1422(2003)。对用人类腺病毒血清型5预先免疫的小鼠使用cv68gag或ad5gag载体进行实验性疫苗接种展现出，与通过cv68疫苗引发的相比，通过ad5引发的更显著的gag特异性t细胞的降低和对病毒攻击的保护。c68gag疫苗效力的降低是由于ad5特异性cd8+t细胞的交叉反应性(同上)。一同考虑这个数据表明，猿衍生的复制缺陷性腺病毒载体与基于常见的人类血清型的载体相比更适合用作人类疫苗载运体。如在此所示，用人类ad5强烈免疫的小鼠(附图36)可以用chad3-gag腺病毒载体进行免疫的实验结果表明，先存的抗人类ad5免疫性不会降低chad载体引发的gag特异性cmi反应。这些结果与人类ad5交叉反应性b和t细胞表位不存在于chad3或chad6载体中的结论相一致。一般而言，非常好地表征了腺病毒基因组，尽管存在几种不同的血清型，在类似地安置了特定功能的腺病毒基因组的整体结构上存在某些普遍的保守性。在美国专利no.6,083,716(黑猩猩腺病毒载体)和pct公开的申请wo03/000851(快速筛选细菌转化体和新的猿腺病毒蛋白的方法)中提供了wilson等公开的黑猩猩腺病毒c1和cv68的核苷酸序列，以及每个病毒的e1a、e1b、e2a、e2b、e3、e4、l1、l2、l3、l4和l5基因的位置，通过引用将其中的教导合并在此。腺病毒基因组的每个末端都包含称为反向末端重复(itr)的序列，其是病毒复制必需的。病毒还包含病毒编码的蛋白酶，其是加工某些结构蛋白所需的，所述结构蛋白是产生传染性病毒颗粒所需的。根据宿主细胞转导后病毒基因的表达顺序，描述了腺病毒基因组的结构。更具体地，根据转录是在dna复制开始之前还是之后发生，将病毒基因称为早期(e)或晚期(l)基因。在转导的早期阶段，腺病毒的e1、e2、e3和e4基因被表达以使宿主细胞作好病毒复制的准备。通过删除病毒基因组的必需的早期区域1(e1)，病毒可以变为复制缺陷性的。brodyetal，1994annnyacadsci.，716：90-101。在晚期阶段期间，接通了晚期基因l1-l5的表达，它们编码病毒粒子的结构元件。所有晚期基因都处在单个启动子的控制之下，编码的蛋白包括五邻体(l2)、六邻体(l3)、100kda支架蛋白(l4)和纤维蛋白(l5)，它们形成了新的病毒粒子，腺病毒dna将被包裹在其中。取决于病毒的血清型，在单个宿主细胞中可以产生10,000-100,000个子代腺病毒粒子。最终，腺病毒复制过程引起细胞的裂解。通过从公开的黑猩猩核酸序列中删除特定的核苷酸序列并插入来自其他dna序列、对转基因的插入、表达和其他遗传操作有用的序列，构建了在此公开的复制缺陷性腺病毒载体。因此，在此描述的重组黑猩猩腺病毒可以含有来源于一种或多种黑猩猩腺病毒的腺病毒序列，或来自黑猩猩腺病毒和来自人类腺病毒的序列。可以重组地、合成地产生适合的多核苷酸序列，或可以从天然来源分离。适用于本发明的特定方面的腺病毒序列包括缺少与常见的人类血清型交叉反应的中和b细胞表位的序列。至少，本发明的重组黑猩猩腺病毒(例如，载体)含有复制和病毒颗粒封装所需的黑猩猩腺病毒顺式作用元件，与至少一个免疫原表达盒组合。一般地，顺式作用元件侧翼于包含编码至少一个抗原的转基因的表达盒。更具体地，本发明的载体含有必需的腺病毒的顺式作用5′反向末端重复(itr)序列(其起到复制起点的作用)、3′itr序列、包装/增强子结构域和编码异源分子的核苷酸序列。无论所述重组载体是仅包含最小的腺病毒序列还是包含仅具有特定基因(例如，e1和/或e3或e4区域)的功能性删除的完整腺病毒基因组，本发明的载体包含黑猩猩腺病毒衣壳。一般而言，在此公开的腺病毒载体包含复制缺陷性腺病毒基因组，其中所述腺病毒基因组不具有功能性e1基因，并包含免疫原表达盒，其包含：a)编码至少一个免疫原的核酸，针对该免疫原的免疫反应是期望的；和b)与编码所述免疫原的核酸序列可操作连接的异源(即，对于所述腺病毒序列而言)启动子；和转录终止子。更具体地，本发明提供了由重组腺病毒基因组组成的复制缺陷性载体，所述重组腺病毒基因组缺少选自由e1、e2、e3和e4构成的组的至少一个早期基因。在一个实施方式中，通过用免疫原表达盒替换、或破坏在此公开的腺病毒分离物(例如，chad3、chad6、chad4、chad5、chad7、chad9、chad10、chad11、chad16、chad17、chadl9、chad20、chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63或chad82)之一的e1基因来制备复制缺陷性载体。例如，可以通过删除/破坏chad3(seqidno：1)或cha6(seqidno：2)的e1基因来制备载体。做为选择，复制缺陷性载体可以从在此公开的其他腺病毒分离物的任何一个来制备，包括chad3、chad6、chad4、chad5、chad7、chad9、chad10、chad11、chad16、chad17、chad19、chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82或chad20。在其他实施方式中，本发明的复制缺陷性载体包含来源于已经被任选的工程化而缺少功能性e3基因的、在此公开的chads之一的腺病毒基因组。要理解的是，在此公开的黑猩猩腺病毒序列可以通过完全除去早期基因或通过使基因失效或无功能来变成复制缺陷性的。要理解的是，本发明包括了特征为具有修饰的载体，所述修饰例如破坏腺病毒表达一种或多种选定基因产物的能力的“功能性删除”。如在此使用的术语“功能性删除”宽泛地涵盖具有使特定基因产物无功能的效果的修饰。一般而言，功能性删除采取部分或全部删除腺病毒基因的形式。然而，技术人员将容易认识到，其他操作，包括但不限于进行引入移码突变的修饰也能实现功能性删除。例如，本发明的重组黑猩猩腺病毒载体可以通过导入一种修饰来成为复制缺陷性的，所述修饰被设计以干扰、或功能性删除所述病毒表达腺病毒e1a和/或e1b的能力。公知的是，可以通过导入一种修饰来获得复制缺陷性腺病毒载体，所述修饰被设计以干扰、或功能性删除e2基因的组中一个或多个基因的表达。更详细地，可以通过钝化聚合酶基因、或前末期蛋白基因或dna结合蛋白基因来构建复制缺陷性载体。此外，删除或钝化e4区域表达的基因是构建复制缺陷性黑猩猩ad载体的可选择的策略。可以组合早期基因的删除或钝化来产生更为减毒的载体。做为选择，复制缺陷性chad载体也可以包含在其他病毒基因中的另外的修饰，例如晚期基因l1到l5。此外，可以通过组合获自不同血清型的六邻体和纤维基因来产生新的腺病毒疫苗载运体。六邻体和纤维基因混移(shuffling)策略的使用也将容许研究者改变chad的生物学性质和促进具有不同的趋性或具有新的血清学特征的载体的产生。要理解的是本发明涵盖重组腺病毒载体，其包含了完整基因或其部分的删除，所述删除单独地或任意组合地有效破坏所修饰的基因的生物学活性。例如，尽管在此所示希望的是与e1基因的功能性删除相组合向载体中导入异源ad5e4序列，还是可以构建重组猿腺病毒，其具有e4区域表达的基因的功能性删除。做为选择，可以消除腺病毒延迟早期e3基因的功能；然而由于e3的功能不是重组腺病毒粒子的产生所必需的，不必将该基因产物重新放回以产生能够包装在本发明中有用的病毒的重组体。在本发明的一个实施方式中，使用的复制缺陷性腺病毒载体是含有e1以及任选的e3的删除的黑猩猩c亚群腺病毒。例如，对于chad3，可以将适合的e1删除/破坏导入从bp460到bp3542的区域(参考seqidno：1)。对于chad6，可以将适合的e1删除/破坏导入从bp457到bp3425的区域(参考seqidno：2)。对于cv32，e1删除优选的从bp456到bp3416(参考seqidno：3)；对于cv33，e1删除优选的从bp456到bp3425(参考seqidno：4)和对于cv23，e1删除优选的从bp456到bp3415(参考seqidno：5)。对于cv32和cv33，e3删除优选的分别为从bp27446到bp31911(参考seqidno：3)；从bp27146到bp31609(参考seqidno：4)。本领域技术人员可以根据序列同源性和多重序列比对容易地确定对于其他黑猩猩分离物的等同序列。本领域技术人员将容易地认识到，为了构建删除e1的腺病毒载体，必需考虑载体主干的结构和包含转基因的核酸序列的组成来进行许多决定。例如，研究者必需确定e1删除的大小是否适应转基因的大小。如果不适应，则必需向载体的主干中导入另外的删除。包含转基因的核酸序列可以是基因、或基因的功能部分，一般将以表达盒的形式存在。一般地，基因表达盒包括：(a)编码感兴趣的蛋白或抗原的核酸；(b)与编码所述蛋白的核酸可操作连接的异源启动子；和(c)转录终止信号。所述核酸可以是dna和/或rna，可以是双链或单链的。为了在期望的宿主(例如，哺乳动物宿主)中表达可以对核酸进行密码子优化。还必须考虑主干内要导入转基因的部位和转基因的方向来作出决定。更具体地，可以按e1平行(5′到3′转录)或反平行(相对于载体主干在3′到5′方向上转录)方向插入转基因。此外，需要鉴定合适的转录调节元件并与所述转基因可操作地连接，所述转录调节元件能够指导转基因在哺乳动物宿主细胞中的表达，载体是为所述哺乳动物宿主细胞准备用作疫苗载运体的。“可操作连接的”序列包括与被调节的核酸序列邻接的表达控制序列，和反式作用的、或在一定距离上来控制所调节的核酸序列的调节序列。调节序列包括：合适的表达控制序列，例如转录起始、终止、增强子和启动子序列；有效的rna加工信号，例如剪接和多聚腺苷酸信号；增强翻译效率的序列(例如，kozak共有序列)；增强蛋白质稳定性的序列和任选的促进蛋白质分泌的序列。这些和其他常见的载体元件的选择是常规的，许多适合的序列对于本领域技术人员而言是公知的。(参见，例如，sambrooketal.和其中引用的参考文献，例如，第3.18-3.26和第16.17-16.27页，和ausubeletal.，currentprotocolsinmolecularbiology，johnwiley&sons，newyork，1989)。在特定的实施方式中，所述启动子是由真核rna聚合酶识别的异源启动子(即，对于腺病毒序列而言)。在优选的实施方式中，所述启动子是“强”或“高效”启动子。强启动子的实例是即时早期人类巨细胞病毒启动子(chapmanetal，1991nucl.acidsres19：3979-3986，通过引用将其合并)。可以使用没有(cmv)或含有内含子a序列(cmv-inta)的人类cmv启动子，尽管本领域技术人员公认的是可以使用许多其他已知的启动子，例如强免疫球蛋白或其他真核基因启动子，包括ef1α启动子、鼠cmv启动子、劳氏肉瘤病毒(rsv)启动子、sv40早期/晚期启动子和β-肌动蛋白启动子。可以用于本发明的启动子的进一步实例是强免疫球蛋白启动子、ef1α启动子、鼠cmv启动子、劳氏肉瘤病毒启动子、sv40早期/晚期启动子和β肌动蛋白启动子，虽然本领域技术人员可以理解的是根据本发明的方法可以使用能实现预定宿主中的表达的任何启动子。启动子可以包含可调节序列，例如tet操纵子序列。在试图抑制基因转录的情况中，例如提供了调节转录和表达的可能性的序列是有用的。适合的基因表达盒还包含转录终止序列。优选的转录终止子是牛生长激素终止子。cmvinta-bgh终止子的启动子/转录终止组合是特别优选的，虽然也可以使用其他的启动子/终止子组合。如在此所示，50个核苷酸长度的牛生长激素终止/多聚腺苷酸信号(bghpa)或短的合成多聚a信号(spa)定义如下：aataaaagatctttattttcattagatctgtgtgttggttttttgtgtg(seqidno：26)。一般而言，例示了适合的终止序列。多聚a信号被插入到包含转基因的核酸序列之后和3′腺病毒itr序列之前。在此描述的重组腺病毒载体可以含有来源于一种或多种腺病毒株的腺病毒序列。适合的序列可以从天然的来源获得，或重组地、合成地、或通过其他遗传工程或化学方法产生。在特定的实施方式中，重组黑猩猩腺病毒是包含非黑猩猩腺病毒多核苷酸序列的嵌合重组体。适合的非黑猩猩腺病毒序列可以从人类腺病毒株获得。例如，可以用had5e4(ad5nt32816到nt35619)或ad5e4orf6(ad5nt33193到nt34077)(ad5genbank登记no：m73260)替换天然的e4区域。一般而言，由本发明的重组腺病毒载体递送的免疫原(抗原性分子)包括多肽、蛋白或酶产物，其由与一核苷酸序列组合的转基因编码，所述核苷酸序列提供了必需的调节序列来指导所述编码的产物在宿主细胞中的转录和/或翻译。转基因的组成取决于载体的预定用途。例如，如果免疫原性组合物是被设计以在哺乳动物宿主中引发特异于传染原的抗体反应或细胞介导免疫反应，则合适的是使用编码至少一个免疫原性产物的核酸序列，所述免疫原性产物预计为接受者赋予病原体特异性免疫。做为选择，如果所述组合物是制备用作癌症疫苗的，适合的转基因可以包含自体抗原例如taa的免疫原性部分，其是根据引发足够效力的保护性免疫反应的目标来选择的，来破坏对特定taa的宿主耐受性，和引发足以预防癌症的起始或预防肿瘤发展的、长期的(例如，记忆性)反应。因此，适合的免疫原性基因产物可以从感染哺乳动物宿主的多种病原体(例如，但不限于，病毒、寄生虫、细菌和真菌)获得，或从癌症或肿瘤细胞获得。虽然在此用特定集合的测试免疫原举例说明了本发明，要理解的是本发明不限于使用在此例示的抗原。更具体地，本发明考虑使用异源的和自体的抗原作为免疫原，包括但不限于taa。在一个实施方式中，本发明提供了免疫原性组合物(例如，疫苗)，用于诱导针对由传染原表达的抗原(即，免疫原)的免疫反应。例如，希望的是引发针对感染人类和/或非人类动物物种的病毒的免疫反应。病毒家族的实例包括小rna病毒科(picornaviridae)，其包括六个不同的属，例如口蹄病毒(aphtovirus)、心脏病毒(cardiovirus)、肠道病毒(enterovirus)、肝病毒(hepatovirus)、parechovirus、鼻病毒(rhinovirus)，针对所述病毒家族的预防性和/或治疗性免疫反应是希望的。小rna病毒的实例是：口蹄疫病毒(foot-and-mouthdiseaseviruses)、脑心肌炎病毒(encephalomyocarditisviruses)、脊髓灰质炎病毒(polioviruses)、coxackieviruses、人类肝炎a病毒、人类parechoviruses、鼻病毒，针对所述小rna病毒的免疫反应是希望的。杯状病毒科(caliciviridae)包括与人类中由病毒的norwalk群引起的流行性胃肠炎和在动物中的其他综合症有关的不同的属，所述其他综合症如在兔中与兔出血病病毒有关的出血性疾病，或在猫中由猫杯状病毒引起的呼吸道疾病。另一个病毒家族是星状病毒科(astroviridae)，其包含从人类以及许多不同的动物物种分离的病毒，引发针对该病毒家族的免疫反应是希望的。人类星状病毒与胃肠炎和幼儿的腹泻有关。做为选择，给哺乳动物宿主赋予对病毒的披膜病毒科(togaviridae)的成员的免疫性是希望的，其包括两个属：甲病毒属(alphavirus)和rubivirus。甲病毒属与人类和兽类疾病有关，例如关节炎(即切昆贡亚热病毒(chikungunyavirus)、辛德毕斯病毒(sindbisvirus))或脑炎(东方马脑炎病毒(easternequineencephalitisvirus)、西方马脑炎病毒(westernequineencephalitisvirus))。风疹病毒提供了可选择的病毒目标，其是rubivirus属的唯一成员，是与发烧和淋巴腺病有关的轻度皮疹疾病暴发的原因。当母亲在早期妊娠期间获得时，风疹病毒感染还与胎儿异常有关。黄病毒科(flaviviridae)是另一个病毒家族，由三个属组成：黄病毒属、pestiviruses和肝病毒属，包括重要的人类以及动物病原体。黄病毒属成员中的许多是节肢动物传播的人类病原体，引起各种疾病包括发烧、脑炎和出血热。登革热病毒、黄热病毒、日本脑炎病毒、西尼罗河热病毒、蜱传播的脑炎病毒是主要的全球性或地域性(地方性)的病原体。pestivirus属包括有主要是经济上的重要性的动物病原体，例如牛病毒性腹泻病毒、标准猪瘟病毒(classicalswinefevervirus)、边界疾病病毒(borderdiseasevirus)。肝炎c病毒是肝病毒属的唯一成员，与急性和慢性肝炎有关。重组腺病毒表达的hcv蛋白可以引发保护性的和治疗性的免疫反应，限制在世界范围内影响17000万人的病毒感染的后果。做为选择，来源于冠状病毒科(coronaviridae)的成员的抗原可以由重组腺病毒载体表达以获得对感染的保护。通过用选自包括chad3、4、5、6、7、9、10、11、16、17、19、20、chad8、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82的组的、表达一种或多种sars-cov蛋白的一种或多种黑猩猩腺病毒进行免疫，可以获得对严重急性呼吸综合症冠状病毒(sars-co病毒)的保护，所述sars-cov蛋白无限制地包括核壳(n)蛋白、聚合酶(p)蛋白、膜(m)糖蛋白、刺突(s)糖蛋白、小包膜(e)蛋白或由该病毒表达的任何其他的多肽。包括狂犬病病毒的棒状病毒科(rhabdoviridae)成员可以是表达病毒蛋白的重组疫苗的目标。其他可能的目标包括filoviridae家族，包含ebola样病毒和marburg样病毒属，与严重的出血热暴发有关；副粘病毒科(paramyxoviridae)包含在人类中已知的最流行的某些病毒，如麻疹、呼吸道合胞体病毒、副流感病毒和兽医感兴趣的病毒如新城疫和牛瘟病毒；正粘病毒科(orthomyxoviridae)包括流感a、b、c病毒；布尼安病毒科(bunyaviridae)主要由节肢动物传染到脊椎动物宿主，包含重要的人类病原体，如里夫特山谷热(riftvalleyfever)、sinnombre、hantaan、puumala病毒；沙粒病毒科(arenaviridae)包含淋巴细胞性脉络丛脑膜炎、拉沙热、阿根廷出血热(argentinehemorragicfever)、玻利维亚出血热(bolivianhemorragicfever)病毒；bornaviridae科包含主要在马和绵羊中引起中枢神经系统疾病的病毒；呼肠孤病毒科(reoviridae)包括轮状病毒，世界范围内的婴儿和幼儿中严重痢疾性疾病的最重要原因，可以感染人类和其他哺乳动物的环状病毒(蓝舌病、兽疫流行性出血病病毒)；逆转录病毒科(retroviridae)，病毒的一个大组，包含重要的人类病原体如人类免疫缺陷性病毒1和2(hiv-1和hiv-2)和人类t细胞白血病毒1型和2型(htlv1和2)，以及非人类慢病毒，例如感染绵羊和山羊的maedi/visnaviruses，感染马的马传染性贫血病毒，感染牛的牛免疫缺陷病毒，感染猫的猫免疫缺陷病毒；多瘤病毒科(polyomaviridae)包括小的dna致癌病毒，典型病毒是分别感染小鼠和恒河猴的多形瘤和sv40(从人类患者分离了与sv40近相关的bk和jc病毒)；乳头状瘤病毒科(papillomaviridae)由一组dna病毒组成，所述病毒感染高等脊椎动物包括人类，产生疣和湿疣。乳头状瘤病毒感染与人类和动物中的癌症发展有关。人类乳头状瘤病毒与子宫颈癌、阴道癌和皮肤癌有关。疱疹病毒科(herpesviridae)包括一些亚科，在其中分类了许多人类和其他哺乳动物的重要病原体。抗原的适合的来源可以是但不限于单纯性疱疹病毒1和2，水痘-带状疱疹病毒、epstein-barr病毒、细胞巨化病毒、人类疱疹病毒6a、6b和7、卡波西肉瘤相关疱疹病毒。抗原的进一步适合的来源是痘病毒科的成员，如猴痘病毒、molluscumcontagiusum病毒、天花病毒；肝炎b病毒，肝病毒科的典型成员，以及引起急性和/或慢性肝炎的其他病毒如肝炎delta病毒、肝炎e病毒。本发明的腺病毒载体还适合于制备免疫原性组合物，所述免疫原性组合物被设计以在人类或动物中刺激针对非病毒病原体表达的蛋白的免疫反应，所述病原体包括细菌、真菌、寄生虫病原体。例如在此公开的载体可以用于制备针对，但不限于：staphylococcusaureus、streptococcuspyogenes、streptococcuspneumoniae、霍乱弧菌(vibriocholerae)、破伤风杆菌(clostridiumtetani)、neisseriameningitis、corynebacteriumdiphteriae、结核分枝杆菌(mycobacteriatuberculosis)和麻风杆菌(leprae)，listeriamonocytogenes和legionellapneumofila的疫苗。希望制备预防性或治疗性疫苗的真菌和哺乳动物寄生虫的实例包括：白色念珠菌(candidaalbicans)、烟曲菌(aspergillusfumigatus)、histoplasmacapsulatum、plasmodiummalariae、leishmaniamajor、trypanosomecruzi和brucei、schistosomahaematobium、mansoni和japonicum；entamoebahistolytica，和已知与人类丝虫病有关的filaria的许多种类。癌症一般涉及编码多肽的基因的失调，所述多肽有助于维持细胞周期或控制细胞增殖(例如，生长因子、致癌基因、受体和肿瘤抑制物)。涉及癌症的许多基因的产物在各种各样的肿瘤细胞表面表达。在人类和动物癌症中已经鉴定了可被t和b淋巴细胞识别的多种肿瘤抗原。适用于抗癌疫苗试验的极大多数肿瘤相关抗原(taa)被描述为“自体抗原”，因为除了在肿瘤细胞上表达之外，它们还在正常的组织上和/或在胎儿发育期间表达。目标人群对taa的免疫耐受可以解释为什么许多癌症疫苗被证实是无效的。肿瘤抗原可以通过正常细胞基因的致癌突变体来产生，改变的原癌基因或肿瘤抑制物基因例如，ras、p53或bcr-abl蛋白是可以刺激t/b细胞反应的改变的细胞蛋白的实例。肿瘤抗原可以是在肿瘤细胞中过量表达的正常细胞蛋白(酪氨酸酶、gp100、mart通常在黑素细胞中以低水平表达，在黑素瘤中过量表达)，或在肿瘤细胞中异常表达的正常细胞蛋白(mage、bage、gage在黑素瘤和许多癌中表达，但是在睾丸和胎盘中正常表达)。肿瘤抗原可以是致癌病毒的产物：由宫颈癌表达的乳头状瘤病毒e6和e7蛋白；由ebv+淋巴瘤和鼻咽癌产生的ebvebna-1蛋白；在sv40诱导的实验肿瘤中的sv40t抗原。癌胎抗原(oncofetalantigens)在癌细胞和正常发育的(胎儿)组织中高水平表达，但不在成年人组织中表达。癌胚抗原(cea)和甲胎蛋白(afp)是良好表征的癌胎抗原的实例。新近的证据支持了能引发免疫反应的taa的存在，因而使这类抗原成为疫苗疗法的适合的免疫原。然而，作为一类抗原，taa是非常不好的免疫原，在t细胞发育期间高度特异于taa的t细胞不是被删除了就是被无反应化了。因此，预期的是，携带肿瘤的宿主对特定taa的免疫反应是极其微弱的。由于破坏对目标taa的宿主耐受性的内在需要，实验性的临床疫苗研究特别集中于开发能增强taa特异性t细胞反应的免疫策略。一般地而言，有效的癌症疫苗必须克服免疫耐受性并且增强宿主的免疫反应到预防性和/或保护性的水平。在许多实验性疫苗研究中已经将抗肿瘤效果与对taa的t细胞反应相关联。在可选择的实施方式中，本发明考虑能用于诱导针对肿瘤抗原的免疫反应的免疫原性组合物(例如，癌症疫苗)。适合的组合物将含有包含编码肿瘤抗原的核酸序列的重组黑猩猩腺病毒，和生理学上可接受的载运体。在特定的实施方式中，盒的编码序列元件可以编码单个免疫原，例如，来源于自体抗原的免疫原性肽序列，例如肿瘤相关抗原。在某些实施方式中，编码免疫原的核酸序列(例如，转基因)可以是为在特定哺乳动物物种中的表达优化了的密码子。在其他实施方式中，编码序列可以编码超过一个免疫原，例如一种或多种密码子优化的肿瘤抗原。例如，利用所公开的腺病毒载体的癌症疫苗可以编码自体抗原的组合，自体抗原例如：her2/neu、cea、hepcam、psa，psma、端粒酶、gap100、melan-a/mart-1、muc-1、ny-eso-1、存活蛋白、溶基质蛋白酶3、酪氨酸酶、mage3、cml68、cml66、oy-tes-1、ssx-2、sart-1、sart-2、sart-3、ny-co-58、ny-br-62、hklp2、vegf。有效的癌症疫苗的开发需要鉴定能在接种的患者中引发抗原特异性免疫的策略和产生在主动免疫结束后能够留存的免疫反应。策略的成功将取决于针对目标抗原的可测量免疫反应是否与针对癌症的发生或复发的保护作用相关。已经显示了细胞介导的免疫和体液免疫的效应机制都能杀死肿瘤细胞。然而，来自实验系统的数据表明，抗原特异性t细胞是消除肿瘤细胞的最强大的免疫机制。效应t细胞对肿瘤特异性抗原(例如，taas)的识别预计可容许taa象肿瘤排斥抗原一样起作用。公开的研究表明，刺激cd8+和cd4+辅助t细胞反应对于达到最优的肿瘤清除是重要的((greenberg，p.d.(1991)adv.immunol.49：281-355；pardoll，d.m.etal.(1998)curr.opin.immunol.10：588-94)。临床反应(即，效力)已经与分泌干扰素γ的细胞毒性t细胞的增加相关联。分析方法的出现，例如在此使用的elispot分析，以展现当前疫苗载运体的效力，容许研究者测量对免疫接种方式的t细胞反应，从而便于癌症疫苗的开发。癌症疫苗可以是预防性或治疗性的。癌症疫苗的预防性用途的一般假定是，在携带肿瘤的受试者中taa是极其弱的免疫原或功能上无免疫原性的。更具体地，在癌症免疫学领域，在患有癌症的患者中疫苗可以用作免疫疗法。因此，可以设计癌症疫苗来引发针对taa的免疫反应，所述taa由先存的肿瘤或恶性肿瘤表达。因而，在特定的实施方式中，治疗性癌症疫苗意图用于携带肿瘤的患者，其发展了对常规治疗方式的抗性，或在常规治疗后有发展出复发的高可能性。腺病毒的高免疫原性使得腺病毒载体对用于疫苗载运体的情况是特别好的候选者，所述疫苗载运体被设计以破坏宿主对自体抗原的耐受性。在自体免疫疾病中首先描述的表位或决定簇传播的现象，已经与mhci类和mhcii类限制性反应相关联，并与her-2/neu蛋白特异性t细胞免疫的发展相关联。表位传播代表了对免疫原性蛋白的特定部分的免疫反应的产生，随后是免疫天然传播到存在于同一蛋白上的其他抗原决定簇。例如，disis等在84％的患有过量表达her-2/neu的恶性肿瘤的患者中观察到表位传播，所述患者被施用了疫苗，所述疫苗包含与粒细胞-巨噬细胞集落刺激因子混合的、来源于her-2蛋白的可能的t辅助表位的肽(j.clin.oncol.(2002)20(11)：2624-2632)。重要地，表位传播与her-2/neu蛋白结构域反应的产生有关，并提示了有效地绕过了免疫耐受性的免疫。适用于在此公开的腺病毒载体和方法作为癌症疫苗的目标的taa应当具有许多特征。例如，目标taa必须具有有利的表达分布型，意味着与正常组织相比，在肿瘤或恶性组织中它应当优先地表达或过量表达。此外，因为在肿瘤发生中起作用的taa更有可能在癌症发展的不同阶段期间保留，适合的目标taa还应当在肿瘤发展和转移的整个阶段保持。适合的目标taa还应当在肿瘤内同质地表达。第三，适合的目标taa必需不经受绝对的免疫耐受性。更具体地，必须有某些证据，可以识别感兴趣的taa并对其进行应答的t细胞没有从宿主的t细胞库中完全地删除(berinstein，n.l.，j.clin.oncol.29(8)：2197(2002)。癌胚抗原(cea)具有许多特征，使它成为用作抗癌疫苗的目标抗原的吸引人的taa。它是ig超家族的成员，所述ig超家族的特点在于有利的表达模式。它在超过50％的所有人类癌症中表达，并与肿瘤发生过程有关，这表明它的表达在整个癌症发展过程中可能是被选择的和保守的。此外，已经证明对cea的免疫耐受性不是绝对的。公开的研究确定了，人类t细胞可以识别表达cea的癌细胞，成为对表达cea的癌细胞有活性的，并裂解表达cea的癌细胞(berinstein，n.l.，j.clin.oncol.29(8)：2197(2002)。例如，用表达cea的重组牛痘病毒免疫患者，结合随后的基于肽的体外刺激，产生了能裂解自体肿瘤的cd8+mhc限制性ctl(tsang，k.y.etal.j.natl.cancerinst.，(1995)87：982-990)。另外地，报道了手术后用重组cea免疫结肠直肠癌患者诱导了针对cea的弱的抗体和细胞反应(samanci，a.，etal.(1998)cancerimmunol.immunother.47：131-142.)。进一步的，向诊断患有结肠直肠癌的患者施用抗cea抗独特型抗体，产生了抗cea抗体和独特型特异性t细胞增殖(foon，l，a.etal.(1995)j.clin.invest.，96：334-342)。文献还表明，可以用几种不同的疫苗接种方法克服癌症患者中对cea的耐受性(即，用重组cea或重组正痘或禽痘一cea病毒疫苗接种，施用抗独特型抗体，用cea激动剂表位脉冲树突状细胞)。cea是癌胎糖蛋白(oncofetalglycoprotein)，其在正常胎儿结肠中表达，在正常的结肠粘膜中以小得多的程度表达。它还在大多数腺癌，特别是结肠、胰腺、乳房、肺、直肠和胃的腺癌中过量表达。许多结肠直肠癌症和一些癌瘤产生在血清中可测量的高水平的cea，这使之成为最广泛使用的恶性肿瘤的血清学标记，特别是在患有结肠直肠癌的患者中。提供了用于本发明的组合物和方法的适合的免疫原的第二种taa是her2/erb-2(也称为neu)原癌基因的产物。象cea一样，her2/neu具有有利的表达模式，并且不经受绝对耐受性。更具体地，在正常成年人各种组织，包括皮肤和乳房，和胃肠道、生殖道、和泌尿道的组织的上皮细胞中，检测到低水平的her2/neu转录产物表达和185kd的多肽产物；在胚胎发育期间在相应的胎儿组织中检测到更高水平的表达(pressetal.，oncogene5：953-962(1990)。几个系列的证据暗示了在人类乳房、肺、前列腺、卵巢、子宫内膜和结肠直肠肿瘤中her-2的扩增与致瘤性转化之间的联系(disisandcheever，adv.cancerresearch71：343-371(1997)。一般而言，her2/neu的过量表达与癌症患者的不良预后和较高的复发率有关(slamonetal.，science244：707-712(1989)。因而，特异于her-2/neu蛋白的疫苗在许多不同人类恶性肿瘤的疾病复发预防方面具有广泛的应用和实用性。her2/neu编码具有内在的酪氨酸激酶活性并显示了与表皮生长因子(egf)受体的广泛同源性的跨膜糖蛋白(akiyama，tetal.，(1986)science232：1644-1646)。使用her2作为癌症免疫疗法的靶点的最初的临床研究之一，采用了her-2特异性单克隆抗体herceptin用于乳腺癌的治疗(goldenbergmm(1999)clin.ther.21：309-318)。这引起了随后的努力，其集中于使用her-2作为免疫系统的t细胞支路(arm)的靶点来引发有效的抗瘤反应，包括使用包含her-2结构域的重组融合蛋白来激活自体抗原递呈细胞。公开的报道确定了，患有表达neu的乳腺和卵巢癌症的许多癌症患者产生了针对her2/neu癌基因的蛋白产物的免疫反应(例如，产生抗原特异性抗体和t细胞)。重组腺病毒序列、转基因和其他载体元件装配到各种中间质粒和穿梭载体中，使用所述质粒和载体来产生重组病毒粒子，都可以使用本领域技术人员公知的标准教科书中描述的常规技术来实现(sambrooketal，molecularcloning：alaboratorymanual，2nded.，coldspringharborpress，coldspringharbor，ny(1989)。这种技术包括但不限于，常规的cdna克隆技术、使用来源于腺病毒基因组的重叠寡核苷酸序列、同源重组、聚合酶链式反应、标准转染技术、琼脂覆层中的病毒斑块，和其他相关的方法。为了帮助在原核细胞中制备多核苷酸，常常制备腺病毒载体的质粒型式(腺病毒前质粒)。腺病毒前质粒含有腺病毒部分和质粒部分。腺病毒部分与本发明的腺病毒载体(含有无功能的或删除的e1和任选的e3区域的腺病毒序列)中包含的腺病毒部分和侧翼是方便的限制性位点的免疫原表达盒基本上相同。腺病毒前质粒的质粒部分常常含有处在原核启动子的转录控制下的抗生素抗性标记，从而抗生素的表达不会在真核细胞中出现。可以使用氨苄青霉素抗性基因、新霉素抗性基因和其他药学上可接受的抗生素抗性标记。为了有助于在原核生物中通过发酵高水平产生多核苷酸，有益的是腺病毒前质粒含有原核复制起点并且是高拷贝数的。许多商业上可获得的原核克隆载体提供了这些益处。希望的是除去非必需的dna序列。同样希望的是所述载体不能在真核细胞中复制。这将多核苷酸疫苗序列整合到接受者的基因组中的风险最小化。当希望将多核苷酸的表达限制到特定的组织类型时，可以使用组织特异性启动子或增强子。可以通过使用腺病毒主干dna和合适的穿梭载体(被设计以靶向特定的删除并将期望的限制性位点掺入产生的质粒中)进行同源重组产生腺病毒前质粒(包含具有期望的删除和插入的复制缺陷性腺病毒的基因组的质粒)。在这个过程中使用的穿梭载体可以使用本领域广泛了解和理解的一般方法来产生，例如，考虑期望的删除来对腺病毒末端进行pcr，将各个片段顺序克隆到合适的克隆质粒中。然后可以消化腺病毒前质粒并通过磷酸钙共同沉淀或其他合适的方法转染入补足细胞系。然后发生病毒复制和扩增，一种由于显著的细胞病变效果而变明显的现象。然后在病毒复制完成之后收获感染的细胞和培养基(一般地，转染后7-10天)。一般而言，在构建和装配期望的腺病毒前质粒之后，通过转染表达腺病毒e1的人类细胞系将腺病毒前质粒营救为病毒。在包装细胞系和载体的病毒基因之间的补充性允许载体中的腺病毒-转基因序列被复制并包装到病毒颗粒衣壳中，引起重组腺病毒的生产。可以通过本领域技术人员已知的各种方法的任何一种，来分离和纯化产生的病毒，用于本发明的方法。对本领域技术人员显而易见的是，当向病毒载体中导入一个或多个选定的黑猩猩腺病毒基因的删除时，可以在生产过程期间通过在生产细胞系中存在的序列来提供被删除的基因产物的功能。因而，可以通过永久转化的细胞系来提供所操作的基因的功能，所述细胞系的特征是某些或全部腺病毒功能，所述功能是包装所需的但在载体中是无功能的(例如，e1a、e1b、e2a、e2b、e4的任何一个)。做为选择，可以用包含必需基因的构建体来感染或短暂地转染适合的细胞以提供所述功能，来将必需的腺病毒功能提供给适合的包装细胞系。因此，本发明还提供了在表达e1的人类细胞系中生产黑猩猩腺病毒载体的方法。更具体地，所公开的载体可以在e1补足细胞系中增殖，包括已知的细胞系293和per.c6tm。这些细胞系都表达腺病毒e1基因产物。在1997年1月3日公开的wo97/00326中描述了per.c6tm，通过引用将其合并在此。它是用e1基因片段转导的原代人类成视网膜细胞系，所述e1基因片段补足复制缺陷性第一代腺病毒的产生，但被设计以防止通过同源重组产生有复制能力的腺病毒。在grahametal(1977)j.gen.virol36：59-72中描述了293细胞，也通过引用将其合并在此。本领域技术人员将认识到术语“第一代腺病毒”是指复制缺陷性腺病毒，其是e1区域无功能的或删除了e1区域的，并任选的是e3区域无功能的或删除了e3区域。意图用作临床试验中的疫苗组合物的复制缺陷性腺病毒载体的批次应当经证明是无rca的(fallaux，f.j.etal(1998)humangenetherapy，9：1909-1917)。实际上，这是劳动密集的过程，需要建立和使用昂贵的筛选程序。本领域技术人员将认识到，高频率的rca产生不仅引起生产的批次的高失败率，还严重地限制了规模扩大的努力。消除载体的核苷酸序列和存在于辅助生产/包装细胞系的基因组中的腺病毒序列之间的同源性，将消除生产的载体批次被同源重组产生的rca污染的可能性。一般地，重组复制缺陷性腺病毒载体在提供e1基因产物intrans的细胞系中增殖。当载体来自相同的或非常相似的血清型时，提供必需的e1基因产物intrans是非常有效的。例如，公知的是删除了e1(即，δe1)的c群血清型(ad2和ad5)载体，可以在含有并表达ad5e1区域的293或per.c6细胞中增殖。然而，已经观察到，存在于293和per.c6生产细胞中的ad5e1序列可能不总是完全地补足非c群血清型的复制。因此，c亚群以外的删除e1的血清型，例如，来自a、b、d、e和f亚群的那些，在293或per.c6细胞中可能以比相应wt病毒更低的效率复制，或可能根本不复制。这可能是由于ad5(c亚群)e1b55k基因产物不能够建立与非c群血清型的e4orf6基因产物的功能性相互作用。在表达ad5e1的细胞中复制效率的降低根据不同亚群的载体而变化。虽然来自d和e亚群腺病毒的δe1载体可以以可变的效率在293和per.c6tm细胞中被营救(rescue)和增殖，b亚群δe1载体的增殖被完全地削弱了(vogelsr，etal.(2003)aug.replication-deficienthumanadenovirustype35vectorsforgenetransferandvaccination：efficienthumancellinfectionandbypassofpreexistingadenovirusimmunity.jvirol.77(15)：8263-71).尽管ad5e1b55k和载体表达的e4orf6蛋白之间的相互作用在相同亚群的成员内是保守的，当表达非c血清型的e4orf6蛋白时它可能不是足够稳定的。e1b-55k/e4-orf6复合物的这种低效或不稳定形成引起了δe1载体的降低的增殖的缺乏。因此，已经经验性确定的是，为了成功地和有效地营救b群血清型的重组腺病毒，需要产生表达感兴趣血清型的e1区域的细胞系。在表达ad5e1的细胞如293或per.c6tm中，表达可以限于e1b55k蛋白。做为选择，可以修饰适合的ad5e1表达细胞系以便除了ad5e1之外还表达完整的ad5e4区域(或仅e4orf6)。表达ad5e1和orf6的细胞系的产生，在补足可选择的腺病毒血清型方面是有用的；参见，例如，abrahamsenetal.，1997j.virol.8946-8951。e4(orf6)掺入到ad5补足细胞系是已知的，产生intrans提供血清型特异性e1基因产物的血清型特异性细胞系也是已知的。做为选择，非c群载体增殖的效率可以通过用ad5orf6取代天然的e4区域修饰病毒主干来改善。用来自ad5或其他c亚群病毒(ad1、ad2、ad6)的orf6仅替换天然的orf6也可以得到类似的结果。美国专利no.5,849,561公开了在还表达ad5-e4基因的部分的ad5-e1补足细胞系中补足删除e1的非c群腺病毒载体。授权给vigne等的美国专利no.6,127,175公开了稳定转染的哺乳动物细胞系，其表达腺病毒的e4区域的一部分，优选的是orf6/orf6/7。这种细胞系对于补足e4区域中的重组ad基因组缺陷是有用的。包含所公开的腺病毒载体的组合物，包括疫苗组合物，是本发明的重要的方面。这些组合物可以以预防性或治疗性的设置施用给哺乳动物宿主，优选的人类宿主。可能的宿主/接种者包括但不限于灵长类和特别是人类和非人灵长类，并包括有商业或驯养的兽医学重要性的任何非人哺乳动物。包含重组黑猩猩腺病毒载体的组合物可以单独施用或与其他基于病毒或非基于病毒的dna/蛋白疫苗组合施用。它们也可以作为更广阔的治疗方式的部分来施用。在本发明的特定实施方式中，所公开的载体可以用于被设计以破坏宿主对自体抗原或肿瘤相关抗原的耐受性的免疫方案。许多taa的鉴定已经允许了开发用于癌症治疗的主动疫苗接种方法。被加工和呈递的细胞表面抗原和细胞内抗原都提供了有用的靶点。一般而言，所公开的破坏宿主对自体抗原的耐受性的方法包括：(a)通过施用包含第一chad载体或质粒载体的第一疫苗组合物来刺激对自体抗原的抗原特异性反应，所述第一chad载体或质粒载体携带编码自体抗原的核苷酸序列，针对所述自体抗原的抗原特异性免疫反应是期望的，和(b)通过施用包含不同的血清型的重组chad载体的第二疫苗组合物来维持和扩展(a)的免疫反应，所述重组chad载体含有其基因组e1基因的至少一个功能性删除，并且在e1基因的位置上含有包含启动子的序列，所述启动子能指导编码在激发步骤中释放的相同自体抗原的dna的表达，从而宿主产生具有破坏对自体抗原的耐受性的效果的免疫反应。因此，技术人员可以使用这种公开内容来设计可能适用于破坏宿主耐受性的几种不同的免疫方案。例如，可能的是使用一种方案，其中第一或激发免疫包含质粒dna，所述质粒dna编码特定的自体抗原，例如taa，任何随后的免疫包含chad载体。包含编码自体抗原的核苷酸序列的质粒dna序列可以在一次或多次注射中，有或者没有电刺激地、肌肉内地递送。例如，基于通过电穿孔多次(例如，3或4或5次)肌肉注射编码taa的质粒dna，随后一次或多次肌肉注射包含编码相同taa的转基因的chad载体的免疫方案，被在此公开和要求权利的一般方法包括。做为选择，破坏耐受性的适合的方案可以包括用包含编码自体抗原的转基因的chad或had载体进行一次或多次激发免疫，随后用相同的chad载体或已知与用于激发免疫的载体不是交叉反应性的、不同的chad载体进行一次或多次强化免疫。例如，使用chad3激发和用chad6进行强化，或用chad3激发随后用chad6和chad9进行强化的免疫方案可以用于破坏宿主耐受性。在特定的实施方式中，本发明考虑使用包含选自以下构成的组的至少一个肿瘤相关抗原的自体抗原：her2/neu、cea、epcam、psa、psma、端粒酶、gap100、melan-a/mart-l、muc-1、ny-eso-1、存活蛋白、溶基质蛋白酶3、酪氨酸酶、mage3、cml68、cml66、oy-tes-1、ssx-2、sart-1、sart-2、sart-3、ny-co-58、ny-br-62、hklp2、vegf。在特定的实施方式中，本发明提供了通过向患有癌症的哺乳动物递送编码taa的重组黑猩猩腺病毒、诱导针对特异于选定恶性肿瘤的肿瘤相关抗原的免疫反应(例如，体液或细胞介导的)的方法。在本发明的这个方面的优选实施方式中，引发的免疫反应构成了以抗原特异性cd4+和cd8+t细胞的产生为特征的免疫反应。本发明的免疫原性组合物可以以预防性或治疗性的设置(setting)施用给哺乳动物宿主，优选的人类宿主。可能的宿主/接种者包括但不限于灵长类和特别是人类和非人灵长类，并包括有商业或驯养的兽医学重要性的任何非人哺乳动物。包含重组黑猩猩腺病毒载体的组合物可以单独施用或与其他基于病毒或非基于病毒的dna/蛋白疫苗组合施用。它们也可以作为更广阔的治疗方式的部分来施用。用于本发明的方法的适合的组合物可以包含本发明的重组病毒载体，结合生理学上可接受的成分，例如缓冲液、生理盐水或磷酸盐缓冲盐水、蔗糖、其他盐和聚山梨酸酯。在肌肉注射时它不引起组织刺激。优选的是冷冻待用的。任选的，本发明的疫苗组合物可以配制为含有其他成分，例如但不限于，佐剂、稳定剂、ph值调节试剂，或防腐剂。这些成分是本领域技术人员公知的。预想的是，本发明的重组黑猩猩腺病毒以“有效量”施用给人类或兽医学上的宿主，所述“有效量”是重组病毒的数量，其是有效的以在选定的施用途径下转导宿主细胞并提供转基因的足够表达水平来引发给接受者/疫苗接种者(vaccine)赋予治疗效益或保护性免疫的免疫反应。导入疫苗接受者的疫苗组合物中病毒粒子的数量取决于使用的转录和翻译启动子的强度和表达的基因产物的免疫原性。一般地，免疫或预防有效剂量的1×107到1×1012个粒子(即，1×107、2×107、3×107、5×107、1×108、2×108、3×108、5×108或1×109、2×109、3×109、5×109)，和优选的约1×1010到1×1011个粒子直接施用到肌肉组织中。皮下注射、皮内导入、压过皮肤，和其他施用模式，例如腹膜内的、静脉内的或吸入递送也是考虑的。本发明的重组黑猩猩腺病毒载体可以单独地、作为混合形式的激发/强化疫苗接种方式的部分、或在基于不同载体血清型的多次注射的组合的接种方式中来施用。一般地，将包含至少一个免疫原的激发剂量施用给哺乳动物宿主，所述哺乳动物宿主需要针对特定病原体或自体抗原的有效免疫反应。这种剂量有效地激发免疫反应，从而在随后的抗原鉴定中，宿主能够立即产生针对该免疫原的增强的或强化的免疫反应。利用用于激发和强化的可选择制剂的混合形式疫苗接种方案可以引起增强的免疫反应。激发一强化施用一般包括激发(通过病毒载体、质粒、蛋白，等)受试者至少一次，容许经过预定长度的时间，然后强化(通过病毒载体、质粒、蛋白，等)。通常采用多次免疫，一般1-4次，尽管可以使用更多次数。激发和强化之间的时长一般可从约四个月到一年，虽然本领域技术人员理解的是可以使用其他的时间范围。在中和免疫性变得明显之前，每种载体的多次注射可以在约2周的时间范围(timeframe)内施用。在本发明的某些实施方式中，给予疫苗超过一次的腺病毒疫苗载体施用，可以以伴有质粒疫苗的施用的方式来给予。与在此公开的载体组合使用的适合的质粒疫苗包括编码至少一个免疫原的质粒，针对所述免疫原的激发的或强化的免疫反应是期望的，和异源启动子，其能够指导编码免疫原的核酸序列的表达，可操作地与免疫原编码序列连接，和转录终止序列。例如，可以使用一种剂量给药方式，其利用不同血清型的重组复制缺陷性黑猩猩腺病毒载体的多次注射。做为选择，可以给予个体第一剂量(即，激发剂量)的质粒疫苗，和第二剂量(即，强化剂量)，其包含复制缺陷性重组黑猩猩腺病毒载体，所述载体包含在质粒疫苗中递送的相同免疫原的编码序列。做为选择，可以给予个体第一剂量编码至少一个免疫原的人类腺病毒疫苗载体，随后是第二剂量，其包含在此公开的复制缺陷性重组黑猩猩腺病毒载体，所述载体包含与在激发剂量中递送的相同的免疫原的编码序列。在第二个可选择的实施方式中，可以首先施用包含本发明的载体的疫苗组合物，随后施用质粒疫苗。在这些实施方式的任何一个中，可以以病毒载体或质粒形式给予个体多剂量的相同免疫原。可以有预定的最小时间量，来分隔施用。除了通过本发明的重组复制缺陷性黑猩猩腺病毒载体递送感兴趣的单个蛋白或抗原之外，可以通过独立的运载体(vehicle)或通过相同的运载体来递送两种或多种蛋白或抗原。可以将多个基因/功能等同物连接到适当的穿梭质粒，来产生包含多个开放阅读框的腺病毒前质粒。多个基因/功能等同物的开放阅读框可以可操作地连接到不相同的启动子和转录终止序列。如在此所示，适合的免疫方式可以采用不同的腺病毒血清型。这种方案的一个实例是包含第一血清型，例如chad3或chad6的重组腺病毒载体的激发剂量，随后是包含第二血清型的重组黑猩猩腺病毒载体的强化剂量。在可选择的实施方式中，激发剂量可以包含独立的腺病毒运载体的混合物，每种腺病毒运载体包含编码不同蛋白/抗原的基因。在这种情况下，强化剂量也可以包含载体的混合物，每种载体包含编码独立的蛋白/抗原的基因，条件是强化剂量施用重组病毒载体，所述重组病毒载体包含编码与激发剂量中所递送的相同或相似组的抗原的遗传物质。可以进一步组合这些多基因/载体的施用形式。进一步处在本发明的范围内的在于，组合的用药方式，其包括来自特定抗原的多个但各不相同的成分。使用不被针对人类载体的病毒元件的先存免疫性中和的、来源于黑猩猩腺病毒的重组载体，提供了对于使用人类ad载体作为疫苗载运体的替代性选择。由于腺病毒是高度免疫原性的，腺病毒载体对用于疫苗载运体的情况是特别好的候选者，所述疫苗载运体被设计以破坏宿主对自体抗原的耐受性。此外，以能够与人类病毒相比较的效率、在人类细胞中，特别是在per.c6tm细胞系中增殖黑猩猩病毒的能力，从调节的角度和从治疗剂或疫苗的大规模产生的角度都提供了相当大的益处。因此，本发明提供了一组黑猩猩腺病毒序列、载体和质粒，容许制备重组病毒，所述重组病毒可单独地或组合地用作遗传疫苗接种的疫苗载运体。为了描述和公开可能与本发明相联系使用的方法和材料，在此提及的所有出版物通过引用合并。在此的任何内容都不能解释为承认本发明没有资格根据在先的发明在日期上早于这种公开。参考附图描述了本发明的优选的实施方式，要理解的是本发明不限于这些明确的实施方式，由本领域的技术人员可以实行各种改变和修改而不背离如附随的权利要求中所定义的本发明的范围和精神。以下实施例说明、但不限制本发明。实施例1：chads黑猩猩腺病毒分离物的分离、克隆、测序和表征在nirc(newiberiaresearchcenter4401w.admiraldoyledrivenewiberia，la′70560)，在病毒转运培养基(vtm；microtestm4-rmultimicrobetransportmedium，remelinc.)中收集大便样本，然后冷冻，或直接在-70℃冷冻。样本保持冷冻在＜-70℃，直到对它们进行处理用于接种到细胞培养物中。在那时，将样本解冻，然后在过量的冷却的病毒转运培养基中涡旋。在样本分离成悬浮液之后，在1500-1800rpm将它们离心10分钟。将上清液连续过滤通过0.8和0.2μm针筒过滤器，然后将过滤了的材料接种到细胞培养物中(200-250μl进入壳瓶(shellvial)，200-300μl进入试管培养物)。将每种处理过的样本接种到播种了293细胞或a549细胞的试管培养物和壳瓶培养物中。每次接种一组样品时制备对照(阳性和阴性)培养物。一旦已经接种了set-up中的所有壳瓶，将它们在室温下以2000rpm(900×g)离心60±10分钟。在转子停止旋转后立即从离心机取下小瓶以防止培养物中的热破坏。离心之后，从壳瓶中吸出接种物，在每个小瓶中使用新鲜的无菌pasteur吸管以防止交叉污染。每次用1.0ml新鲜培养基洗涤培养物三次。洗涤三次之后将新鲜培养基(1.0ml)移液到每个小瓶中，将壳瓶置于孵化器中35-37℃三到四天(约96hr)。在培养期结束时，从培养物吸出上清液，用免疫荧光分析(ifa)缓冲液每次使用约1.0ml缓冲液洗涤每个小瓶中的细胞层两次。通过向每个小瓶添加1.0ml冷却的丙酮(2-8℃10分钟)来固定细胞。用与壳瓶盖片相关的样本标识数字标记清除了丙酮的载玻片。使用初级小鼠抗腺病毒抗体[mab8052，chemicon]处理壳瓶盖片进行腺病毒感染的细胞的荧光标记。借助于荧光显微镜评估载玻片。使用10×物镜扫描每个制备物，记录免疫荧光覆盖加样孔的程度(1+到4+)。使用40×物镜证实特异性免疫荧光的存在或不存在。如对壳瓶所描述的按相同的顺序对试管培养物进行接种(例如，首先是阴性对照、随后是临床样本和阳性对照)。容许接种物在36-38℃吸附60-120分钟。在吸附期之后，从试管中吸出样本/对照，用新鲜培养基替代。接种后三到四天，和此后的一周一次，从培养试管中吸出培养基，用1.5ml新鲜培养基替换。接种后的至少21天内至少每隔一天视觉上监视培养试管的cpe。用黑猩猩样本接种的培养物与对照进行比较，并通过观察cpe程度评级。14天后没有显示cpe的培养物转移到新鲜的试管培养物；21天后cpe阴性的培养试管考虑为阴性的。具有3-4+cpe的培养试管进行涡旋10秒。使用无菌的1.0ml血清学吸管从试管的壁上刮下细胞，悬浮在培养物上清液中。之后用样本标识数字和日期标记5ml扣帽试管(snapcaptube)，保存在-70℃。将500μl细胞悬液从培养试管转移到扣帽试管中，在2-8℃保存至多一天，直到使用间接免疫荧光抗体技术进行处理来检测腺病毒(相当于染色壳瓶的步骤)。黑猩猩腺病毒扩增野生型黑猩猩腺病毒cv32、cv33、cv23和cv68购自atcc(atcc登记号：cv32，vr-592；cv-33，vr-593；)，或购自esoterixinc.austin，texas，原始分离物如下通过使用表达e1的人类细胞系per.c6tm或293来增殖。简要地，将细胞培养在dulbecco′smodifiedeagles培养基(dmem；gibcobrl，lifetechnologies)中，其补充有10％胎牛血清(fbsgibcobrl，lifetechnologies)、1％青霉素-链霉素、2mm谷氨酰胺和10mmmgcl2(per.c6tm)。在补充有5％马血清(gibcobrl，lifetechnologies)的dmem中进行腺病毒感染。当100％的细胞展现出病毒诱导的细胞病变效应(cpe)时，收集感染的细胞和培养基，通过三个循环的冷冻和解冻来裂解。在扩增的第一次传代之后，通过感染播种在96孔平板中的293细胞来克隆所有野生型黑猩猩腺病毒(cv)成品(stocks)。通过对在病毒扩增的第一次传代获得的细胞溶胞产物进行限制稀释，来进行病毒克隆。挑取五个分离的克隆并顺序地增殖。在扩增的3-4次连续传代之后，在种植在5个双层细胞工厂(nunc)(200百万细胞/细胞工厂)的细胞上进行大规模的腺病毒制备。通过在氯化铯密度梯度上的两个超离心步骤从细胞溶胞产物获得纯化的病毒粒子。病毒基因组dna的测序通过在1％sds-ten中用蛋白酶k(0.5mg/ml)消化(55℃2小时)，从3×1012pp的纯化的病毒制品分离基因组dna。在苯酚-氯仿萃取和乙醇沉淀之后，将基因组dna重悬浮在水中，提供用于基因组测序。为了全长ad基因组测序，将纯化的病毒dna雾化以产生随机剪切的片段。用e.colidna聚合酶和多核苷酸激酶的克列诺片段(klenowfragment)将dna片段平端化。平端片段在低熔点琼脂糖凝胶上跑动以纯化大小范围在1-3kb的片段，并克隆到puc19载体的smai位点以产生猎枪文库(shotgunlibrary)。连接物用于转化感受态xl1-bluemrf。通过在含x-gal和iptg的lb琼脂上的白色/蓝色筛选鉴定阳性菌落。从文库中分离出质粒dna的三到四个96孔板(block)，用puc正向和反向引物测序。使用phred-phrap软件包筛选所有测序读数的质量和载体序列。将通过了筛选的读数装配成邻接物(contigs)。设计引物来直接测序腺病毒dna用于封闭缺口，并确定两个末端的dna序列。对选定的病毒获得了完整的病毒基因组序列，包括chad3(seqidno：1)、chad6(seqidno：2)、cv32(seqidno：3)、cv33(seqidno：4)和cv23(seqidno：5)。表1提供了数据，总结了chad3、chad6、pan5(cv23)、pan6(cv32)、pan7(cv33)、c1和c68腺病毒基因组的核苷酸序列之间的同一性百分比。使用作为fasta程序包版本2的部分的align程序计算比对(williamr.penson，universityofvirginia；myers&miller，cabios1989，4：11-17)。表1.黑猩猩腺病毒基因组之间的核苷酸序列同一性百分比为了表征新的腺病毒分离物(例如，chad20、chad4、chad5、chad7、chad8、chad9、chad10、chad11、chad16、chad17、chad19、chad22、chad24、chad26、chad30、chad31、chad37、chad38、chad44、chad63和chad82)，也通过引物步进(primerwalking)确定了六邻体和纤维基因的核苷酸序列。纤维基因：seqidno：6-15：(seqidno：6，chad20)；(seqidno：7，chad4)；seqidno：8，chad5)；seqidno：9，chad7)；seqidno：10，chad9)；seqidno：11，chad10)；seqidno：12，chad11)；seqidno：13，chad16)seqidno：14，chad17)，seqidno：15，chad19)和(seqidno：58，chad8)，(seqidno：60，chad22)，(seqidno：62，chad24)，(seqidno：64，chad26)，(seqidno：66，chad30)，(seqidno：68，chad31)，(seqidno：70，chad37)，(seqidno：72，chad38)，(seqidno：74，chad44)，(seqidno：76，chad63)和(seqidno：78，chad82)。附图20a-20g提供了在此公开和要求权利的chad分离物的纤维蛋白的氨基酸序列的比较。在以下列出了六邻体基因序列：seqidnos：16-25：(seqidno：16，chad20)；seqidno：17，chad4)；seqidno：18，chad5)；seqidno：19，chad7)；seqidno：20，chad9)；seqidno：21，chad10)；seqidno：22，chad11)；seqidno：23，chad16)；seqidno：24，chad17)seqidno：25，chad19)，(seqidno：97，chad8)，(seqidno：99，chad22)，(seqidno：101，chad24)，(seqidno：103，chad26)，(seqidno：105，chad30)，(seqidno：107，chad31)，(seqidno：109，chad37)，(seqidno：111，chad38)，(seqidno：113，chad44)，(seqidno：115，chad63)和(seqidno：117，chad82)。附图31a-31j提供了在此公开和要求权利的chad分离物的六邻体蛋白的氨基酸序列的比较。黑猩猩腺病毒分类根据已经建议的人类腺病毒血清型(分为六个亚群)的分类(horowitz，ms(1990)adenoviridaeandtheirreplication.invirologyb.n.fieldsandd.m.knipe，eds(ravenptess，newyork)pp.1679-1740)将不同的黑猩猩腺病毒株分类，通过使用alignx程序(informax，inc)进行氨基酸和核苷酸序列比对来得出。通过查看不同限制性内切酶的病毒基因组限制性图谱和通过分析六邻体基因的高变区7(hvr7)的序列，得到新的分离物的初步分类。为此，对侧翼于hvr7的高度保守区域设计两个引物，tgtcctaccarctcttgcttga(seqidno.45)和gtggaarggcacgtagcg(seqidno.46)。使用纯化的病毒dna或粗的293溶胞产物作为模板通过pcr扩增hvr7，然后测序。根据hvr7序列分析，我们将新分离的病毒分类入对人类腺病毒所提出的亚群(a-f)(horowitz，ms(1990)adenoviridaeandtheirreplication.invirologyb.n.fieldsandd.m.knipe，eds(ravenpress，newyork)pp.1679-1740)。通过比对人类和黑猩猩腺病毒六邻体氨基酸序列获得附图35中呈现的系统树。该结果与根据使用alignx程序(informax，inc)限于六邻体hvr7的核苷酸序列比对的初步分类相一致。由采用paupsearch(wisconsinpackageversion10.3，accelrysinc.)进行的全长六邻体肽序列的多重序列比对得出该树，并用treeview进行可视化和操作。使用wisconsin程序包中安装的paupsearch程序进行bootstrap置信度分析。对于每个比对，使用“heuristicsearch”作为检索条件和maximumparsimony作为最优条件将程序运行1000次重复，报告的置信度值取自50％majority-ruleconsensus。实施例2：chad穿梭载体和表达载体的构建以及营救载体的构建和营救使用适合的穿梭载体通过同源重组将基因组病毒dna克隆到标准质粒载体中，所述穿梭载体含有来源于病毒基因组的左末端和右末端的病毒dna序列(附图2)。如以下更完整地描述的，采用在分类为相同血清型亚群的病毒之间观察到的序列同源性来开发类群特异性穿梭载体。分类为d和e亚群的黑猩猩腺病毒的基因组病毒dna结果是足够同源的，容许构建通用穿梭载体来克隆属于这两个亚群的病毒。d/e亚群穿梭载体的构建完全地测序了chad6病毒基因组(seqidno：2)，获得的信息用于构建穿梭载体以便于通过d和e亚群黑猩猩腺病毒的同源重组来进行克隆。在附图1中描述了构建chad6穿梭载体，在此称为parschad6-3。附图32提供了用于在此描述的克隆实验的寡核苷酸序列的列表(seqidno：26-40和seqidno：45-46)。简要地，通过用寡核苷酸5′-atggaattcgtttaaaccatcatcaataatatacctc-3(seqidno：27)和5′-cgctggcactcaagagtggcctc-3’(seqidno：28)通过pcr扩增来自chad6dna的左末端的457bp，用ecori和snabi消化并克隆入ecori-snabi酶切的pnebad35-2，产生pnebchad6-li。通过使用寡核苷酸5’-atgaagcttgtttaaacccatcatcaataatatacct-3’(seqidno：29)和5’-atctagacagcgtccatagcttaccg-3’(seqidno：30)通过pcr扩增右侧chad6itr(bp36222到bp36648)，用限制性内切酶hindiii和xbai消化并克隆入hindiii-xbai消化的pnebchad6-li，产生pnebchad6-rli。最后，与chad6基因组dna序列的核苷酸3426-3813相应的dna片段用寡核苷酸：5′-atgctacgtagcgatcgcgtgagtagtgtttgggggtgggtggg-3’(seqidno：31)和5′-taggcgcgccgcttctcctcgttcaggctggcg-3’(seqidno：32)扩增，用snabi和asci消化，然后与snabi-asci消化的pnebchad6-rli连接，从而产生pnebchad6-rlide1。为了改善在dh5αe.coli菌株中的重组和质粒增殖的效率，通过pmei消化从pnebchad6-rlidel切下含有chad6的左侧和右侧itr以及pix基因片段的1306bp片段，并转移到不同的质粒载体，所述质粒载体通过用寡核苷酸：5′-gatctagttagtttaaacgaattcggatctgcgacgcg-3’(seqidno：33)和5′ttcgatcatgtttaaacgaaattaagaattcggatcc-3’(seqidno：34)通过pcr扩增从pmrkad5seap获得。这个最终的连接步骤产生chad6穿梭载体parschad6-3。c亚群穿梭载体的构建完全地测序了chad3病毒基因组(seqidno：1)，获得的信息用于构建穿梭载体以便于通过c亚群黑猩猩腺病毒的同源重组来进行克隆。简要地，用于克隆c亚群黑猩猩腺病毒的穿梭载体，在此称为pchad3egfp，如下构建：用寡核苷酸5′-tattctgcgatcgctgaggtgggtgagtgggcg-3’(seqidno：35)和5′-taggcgcgcccttaaacggcatttgtgggag-3’(seqidno：36)通过pcr扩增含有pix编码区的chad3dna片段(nt3542-4105)，用sgfi-asci消化，然后克隆到用sgfi-asci消化的parscv32-3中，产生pars-chad3d。用寡核苷酸5′-cgtctagaagacccgagtcttaccagt-3’(seqidno：37)和5′-cgggatccgtttaaaccatcatcaataatataccttatt-3’(seqidno：38)通过pcr扩增chad3右末端(nt37320-37441)，用xbai和bamhi消化，然后连接到用xbai和bamhi限制的pars-chad3d，产生pars-chad3rd。用寡核苷酸5′-atggaattcgtttaaaccatcatcaataatatacctt-3’(seqidno：39)和5′-atgacgcgatcgctgatatcctataataataaaacgcagactttg-3’，(seqidno：40)通过pcr扩增chad3病毒dna左末端(nt1-460)，用ecori和sgfi消化，然后克隆到用ecori和sgfi消化的pars-chad3rd，从而产生pars-chad3rld。还设计病毒dna盒以含有位于两个itr的末端的限制性内切酶位点(pmel)，从而消化将从质粒dna释放病毒dna。b亚群穿梭载体的构建根据已经描述的c和d/e亚群穿梭体构建的策略，构建b亚群穿梭体。简言之，通过用chad30的相应片段取代左侧末端、pix区域、右侧末端来修饰pars-chad3rld。此外，用克隆在chad30e4启动子的控制下的ad5e4orf6替代chad30的e4区域。该穿梭质粒命名为pchad30egfp穿梭载体。δe1黑猩猩腺病毒载体的构建b亚群：通过在e.coli菌株bj5183中进行同源重组来构建b亚群黑猩猩腺病毒载体。用bsteii和bstl107i消化的pchad30egfp穿梭载体与chad8和chad30，纯化的病毒dna，共转化bj5183细胞。在pix基因、存在于线性化pchad30egfp穿梭体和病毒基因组dna末端的右侧itrdna序列之间的同源重组，容许它插入到质粒载体中，同时删除被egfp表达盒替换的e1区域。构建根据人类细胞巨化病毒(hcmv)启动子和牛生长激素多聚腺苷酸信号(bghpolya)的表达盒，来表达分泌的碱性磷酸酶(seap)、egfp、hivgag、hcvns区域(如附图3中对chad6穿梭载体所描述的)以及肿瘤相关抗原如来自人类和恒河猴来源的cea和her2/neu。所有表达盒都通过同源重组插入到chad30载体中。c亚群：通过在e.coli菌株bj5183中进行同源重组来构建c亚群黑猩猩腺病毒载体。用bsteii和bstl107i消化的pchad3egfp穿梭载体与chad3、chad11、chad19和chad20纯化的病毒dna共转化bj5183细胞。在pix基因、存在于线性化pchad3egfp和病毒基因组dna末端的右侧itrdna序列之间的同源重组，容许它插入到质粒载体中，同时删除被egfp表达盒替换的e1区域。构建根据人类细胞巨化病毒(hcmv)启动子和牛生长激素多聚腺苷酸信号(bghpolya)的表达盒，来表达分泌的碱性磷酸酶(seap)、egfp、hivgag、hcvns区域(如附图3中对chad6穿梭载体所描述的)以及肿瘤相关抗原如来自人类和恒河猴来源的cea和her2/neu。d和e亚群：为了根据d和e亚群黑猩猩腺病毒构建δe1载体，用asci消化穿梭载体parschad6-3，与cv32、cv33、cv68、chad4、chad5、chad6、chad7、chad9、chad10、和chad16纯化的病毒dna共转染入e.coli菌株bj5183。在来自pix基因的dna序列和存在于线性化parschad6-3和病毒基因组dna末端的右侧itrdna序列之间的同源重组，容许它插入到质粒载体中，同时删除e1区域(附图2和4)。构建根据人类细胞巨化病毒(hcmv)启动子和牛生长激素多聚腺苷酸信号(bghpolya)的表达盒，来表达分泌的碱性磷酸酶(seap)、egfp、hivgag、hcvns基因(附图3)以及肿瘤相关抗原如人类和恒河猴来源的cea和her2/neu。所有的表达盒都插入到parschad6-3载体的单个snabi位点，以通过同源重组转移到如附图4描述的δe1腺病毒前质粒中。δe1载体的营救和扩增种植在6cm细胞培养平皿上的5×106个per.c6tm细胞用通过核酸内切酶消化从质粒序列释放的10微克克隆病毒载体转染。使用lipofectamine(invitrogen)进行dna转染。转染后5-10天收集转染的细胞和培养基，通过冷冻和解冻裂解。然后通过在293或per.c6tm细胞上连续传代来扩增营救的载体。通过感染种植在5-10个细胞工厂(nunc，inc.)上总共1-2×109个细胞来进行大规模的扩增。通过两次运行超速离心机，在氯化铯梯度上获得纯化的载体制品，相对含10％甘油的pbs进行透析，以等分量保存在-70℃。实施例3：中和研究为了评估针对在此公开的黑猩猩腺病毒的中和抗体在人类血清中的流行度，进行中和分析。该分析评估了血清预孵化对携带分泌碱性磷酸酶(seap)基因的黑猩猩腺病毒转导人类293细胞的能力的影响。中和滴度被定义为给出在有单独的病毒的阳性对照中观察到的seap活性的50％降低的血清稀释度。将cv33-seap、cv32-seap和chad3-seap载体的2×106到1.5×107个物理粒子稀释在100μl完全培养基中，添加到稀释于完全培养基中的等体积的人类或黑猩猩血清中。在各种稀释度测试每种血清样品(从1/18稀释度开始的五个4倍的增加，到1：4608)。将样品在37℃预孵化一小时，然后添加到播种于96孔平板(3×104细胞/孔)中的293细胞。孵化一小时后除去接种物，用新鲜培养基重新喂养细胞，24小时后，移出50μl的培养基，通过化学发光分析测量seap活性。中和滴度被定义为给出在有单独的病毒的阳性对照中观察到的seap活性的50％降低的血清稀释度。测试一组100个人类血清的chad中和活性。并行的用ad5seap载体测试相同的组。表2.针对黑猩猩腺病毒的中和抗体的流行度表2中提供的结果表明了针对来源于黑猩猩腺病毒的载体的中和抗体在人类血清中的极低的流行度。仅四份血清显示了对于cv32载体的超过阈值200的滴度，而8份显示了对于chad3seap载体的超过200的滴度。相反地，检查的黑猩猩血清的组显示了抗黑猩猩ad免疫性的非常高的流行度。这些发现证实了如所预期的，基于黑猩猩ad的载体具有非常小的机会在人类中被中和。因此它们是对先存的抗人类ad免疫性难题的理想解决方案，所述免疫性限制了基于常见人类ad血清型例如ad5的病毒载体的施用。实施例4：chad载体向性(tropism)通过在不同组织来源的人类初级细胞的组上的egfp表达，来评定ad5和chad载体介导的基因转移效力。根据厂家的指示培养人类软骨细胞、成骨细胞、角质形成细胞、黑素细胞、骨骼肌细胞和黑素细胞。如所描述的获得人类单核细胞、不成熟的和成熟的树突状细胞(dc)(romani，n.etal.1996，j.immunol.methods，196，137.)。通过facs分析检测转导的荧光细胞。测试的人类初级细胞的组(panel)包括在心血管疾病、类风湿性关节炎、组织工程(骨骼、皮肤和软骨)和疫苗接种领域对基于体内以及体外基因转移的不同治疗策略很重要的靶细胞。附图38a-d中呈现的结果表明，如不同细胞类型感染的不同效率所展现的，不同的黑猩猩腺病毒可以识别除car以外的受体。鼠免疫研究方法和材料：免疫方案和脾细胞/pbmc制备免疫：用在0.1ml缓冲液中稀释的选定腺病毒免疫小鼠。每个载体剂量分成两个50μl的等分量，注射到小鼠的两个四头肌中。脾细胞制备：注射后3周处死小鼠，切除它们的脾脏，转移到10mlr10中(10％fcs、55mm2-巯基乙醇、1mhepes缓冲液、2mml-谷氨酰胺、rpmi培养基1640中的1x青霉素-链霉素溶液)。通过钢筛切碎脾脏，用2mlr10洗涤筛子之后，将脾细胞转移到50mlfalcon试管中，在室温(rt)以1200rpm离心10分钟。除去上清液，添加3mlack裂解缓冲液(gibcobrlformulation#79-0422dg)。室温下孵化细胞5分钟。添加45mlixpbs，如上述对试管进行离心。用30mlr10洗涤之后，将细胞重悬浮在5mlr10中，通过70m尼龙细胞过滤器(falcon2350)过滤。用990μlturk′s溶液(merck040417345)稀释10μl的细胞，进行计数。最后将细胞稀释到r10中的107细胞/ml。外周血单核细胞(pbmc)制备：将小鼠血液样品(150μl)转移到含50μlpbs/2％edta的2mleppendorf试管。向每个试管添加1mlack缓冲液。轻轻地混合并在rt孵化5分钟。样品在微离心机中以1500rpm离心5分钟。弃去上清液，组合来自相同的免疫群(immunizedcohorts)的白血球沉淀。重复ack缓冲液孵化，然后将pbmc的沉淀重悬浮在1mlr10培养基中。ifn-γelispot分析用以2.5μg/ml的浓度在pbs中稀释的纯化的大鼠抗小鼠ifn-γ单克隆抗体(pharmingen，cat.551216)以100μl/孔包被milliporemaip45平板，在4℃孵化过夜(o/n)。用无菌的pbs洗涤平板两次，通过在co2孵化器中与200μl/孔的r10孵化2小时来封闭非特异性结合位点。在表达hivgag的ad载体的免疫实验中，将9-mer的肽(amqmlketi，balb/c小鼠中定位的cd8hivgag表位)(seqidno：47)在r10中稀释到2μg/ml，以50μl/孔的数量添加到孔中。在用hcv-ns表达载体进行的免疫实验中，使用了一肽库，其覆盖了ns3解旋酶结构域和代表解旋酶结构域中包含的定位的cd8表位的9-mer肽。通过覆盖完整氨基酸序列的重叠的15-mer肽的库，来评估表达人类cea抗原的chads的免疫试验。作为对照使用dmso和伴刀豆球蛋白a。以5×105和2.5×105的数量向每个孔中添加细胞。在co2孵化器中o/n孵化之后，用0.05％tween20/pbs洗涤平板，添加在分析缓冲液(5％fbs、0.005％tween20、pbs)中稀释1/250的50μl/孔的生物素化大鼠抗小鼠ifn-γ单克隆抗体(pharmingencat.554410)。在4℃孵化平板o/n，如上进行洗涤。在分析缓冲液中将链霉抗生物素蛋白-碱性磷酸酶结合物(bd554065)稀释1/2500，以50μl/孔的数量添加，室温2小时。洗涤之后，向平板添加50μl/孔的bcip/nbt1-step溶液(pierce34042)进行显影。通过用去离子水洗涤孔停止反应。通过elispot读数器自动地对斑点计数。鼠ifn-γ细胞内染色(ics)将脾细胞稀释为2×106细胞每1mlr10，用如上所述相同的抗原以2μg/ml的浓度刺激。作为对照，使用dmso和葡萄球菌肠毒素b(seb)。在co2孵化器中过夜孵化之后，用facs缓冲液(1％fcs、0.01％nan3、pbs)洗涤细胞，将纯化的抗小鼠cd16/cd32fc片段(克隆2.4g2，pharmingencat.553142)稀释1/25，以100μl/样品的数量添加，在4℃孵化15分钟。在facs缓冲液中洗涤细胞，以100μl/样品的数量添加在facs缓冲液中1∶50稀释的结合apc的抗小鼠cd3e(克隆145-2c11，pharmingen#553066)、结合pe的抗小鼠cd4(克隆l3t4，bdpharmingencat.553142)和结合percp的抗小鼠cd8a(克隆53-6.7，pharmingencat.553036)。将细胞室温孵化30分钟，洗涤，固定并渗透化(permeabilized)(bectondickinson，facsperm2)，与在permwash(100μl/样品)中1∶50稀释的结合fitc的抗小鼠ifn-γ(pharmingencat.554411)在室温孵化30分钟。洗涤后的细胞重悬浮在500μl1％甲醛/pbs中，使用cellquest软件(bectondickinson)在facs-calibur流动细胞计数器上，进行细胞内细胞因子染色(ics)分析。实施例5：chad载体在小鼠中引发强烈的cmi反应使用表达hivgag转基因的载体在小鼠中评估在此公开的chad载体引发细胞介导的免疫反应(cmi)的能力。简要地，用从105直到1010vp/小鼠的成十倍增加剂量的不同载体注射5只balb/c小鼠的各组。注射后三周通过对脾细胞中gag特异性cd8+t细胞进行定量来确定免疫反应的强度。在体外用产生balb/c小鼠中定位的gagcd8+t细胞表位的肽进行刺激之后，通过elispot分析或通过ifn-γ细胞内染色和facs分析来测定分泌inf-γ的cd8+t细胞的数量。从5个免疫的动物获得结果，在表3中报告，表示为每106个脾细胞中的斑点形成细胞。显示的是，在与9-mercd8+gag表位或与gag肽库孵化之后每百万个脾细胞的斑点形成细胞的数量。gag肽库由重叠10aa、涵盖完整的gag序列的20aa的肽组成。阳性值以粗体报告。表3中提供的数据表明，施用在此公开和要求权利的chad载体引发了强烈的细胞介导的免疫反应，其是能够与had5引发的反应相比较的。通过察看引起阳性免疫结果的最低载体剂量(免疫断点)，我们对不同载体的效力进行了评分，c亚群chad3gag是最有效的，断点在106pp载体剂量。免疫断点的评分在附图33中示出。表3.用黑猩猩ad载体免疫的balbc小鼠中的gag特异性t细胞反应实施例6：chad3和cv33gag载体引发以gag特异性cd8+t细胞为特征的cmi反应为了表征响应于包含hivgag转基因的chad载体而引发的cmi反应，来自用不同剂量的载体免疫的五只小鼠的群组(cohorts)的脾细胞库通过细胞内ifn-γ染色进行分析。表3和表4中显示的数据是在独立的实验中采集的。将脾细胞稀释为2×106细胞每1mlr10，用如上所述相同的抗原以2μg/ml的浓度刺激。作为对照，使用dmso和seb(葡萄球菌肠毒素b)。在co2孵化器中o/n孵化之后，用facs缓冲液(1％fcs、0.01％nan3、pbs)洗涤细胞，将纯化的抗小鼠cd16/cd32fc片段(克隆2.4g2，pharmingencat.553142)稀释1/25，以100μl/样品的数量添加，在4℃孵化15分钟。在facs缓冲液中洗涤细胞，以100μl/样品的数量添加在facs缓冲液中1∶50稀释的结合apc的抗小鼠cd3e(克隆145-2c11，pharmingen#553066)、结合pe的抗小鼠cd4(克隆l3t4，bdpharmingencat.553142)和结合percp的抗小鼠cd8a(克隆53-6.7，pharmingencat.553036)。将细胞室温孵化30分钟，洗涤，固定并渗透化(permeabilized)(bectondickinson，facsperm2)，与在permwash(100μl/样品)中1∶50稀释的结合fitc的抗小鼠ifn-γ(pharmingencat.554411)在室温孵化30分钟。洗涤后的细胞重悬浮在500μl1％甲醛/pbs中，使用cellquest软件(bectondickinson)在facs-calibur流动细胞计数器上进行分析。表4提供了数据，总结了作为gag特异性cd8+或cd4+t细胞的gag特异性cd3+t细胞的百分比。阳性结果以粗体报告。在此提供的数据表明，由来源于分类为不同血清型亚群(即，c、d和e亚群)的病毒的chad载体引发的免疫反应的细胞分布型是类似的，所有gag特异性反应都主要以cd8+t细胞为特征。此外，注意到，在高的载体剂量下，在所有免疫实验中gag特异性cd4+反应都变得明显。ics分析证实了chad3载体可以以106载体剂量刺激抗gagcd8+反应。表4.用不同亚群的黑猩猩腺病毒载体免疫的小鼠中gag特异性t细胞的表征实施例7：chad载体引发hcvns特异性t细胞反应相对于mrkad6nsmut的效力，在c57/black6小鼠中评估了cv32-nsmut和cv33-nsmut载体的效力。动物注射了从107开始直到109vp/小鼠的成10倍增加的载体剂量。单次注射后3周，通过用再生ns3蛋白的解旋酶结构域中定位的cd8+t细胞表位的9-mer肽刺激t细胞，通过ifn-γelispot和ifn-γ细胞内染色来分析cmi。表5中提供的数据总结了不存在(模拟的(mock))或存在ns39-mer肽的孵化之后每百万个脾细胞的斑点形成细胞的数目。数据表明，表达hcv-ns的cv32和cv33载体都刺激强烈的t细胞反应。根据通过注射109vp/剂量获得的对cv32载体的第一个阳性结果的观察，cv32de1e3nsmut载体的免疫效力好像比人类c亚群ad6de1e3nsmut载体低大约100倍。用mrkad6nsmut的平行试验表明，107vp/动物的剂量足够刺激细胞介导的免疫。因此，这些结果证实了，在用表达gag的载体的实验中也观察到的(参见表3)、相对于人类c亚群载体(例如had5和had6)，cv32衍生的载体的更低的免疫效力。表5.在用mrkad6nsmut、cv32nsmut或cv33nsmut免疫的小鼠中的hcvns特异性t细胞反应实施例8：抗ad5的先存免疫性不取消chad3gag引发的抗gagcmi为了评估针对高血清型流行度的ad5的先存免疫性对chad3免疫的影响，在0和2周在四头肌中两次注射1010vp的ad5wt来预先免疫各5只balbc小鼠的4个群组。作为对照，各5只小鼠的两个群组在相同的时间点仅用缓冲液注射。然后用106和107vp/小鼠的ad5gag或chad3gag载体免疫ad5预免疫的小鼠的群组。用106和107vp/小鼠的ad5gag或chad3gag载体免疫对照(幼稚)小鼠的群组。在第4周通过如上所述的中和分析使用ad5和chad3seap载体评估抗ad5和chad3中和免疫性。对用含有在balbc小鼠中定位的gag表位的gag9-mer肽刺激的纯化脾细胞进行elispot分析来评估抗gag免疫性。在附图36中报告的结果证明，抗ad5免疫性不取消chad3gag引发的抗gagcmi，而如所预料的，抗ad5免疫性完全地阻断了ad5gag免疫。实施例9：chad3hcea免疫在表达人类cea的转基因小鼠中引发强烈的cea特异性免疫反应还在表达人类cea的转基因小鼠(clarke，petal.cancerres.(1998)58(7)：1469-77.)中评估了在此公开和要求权利的chad载体引发针对自体抗原的免疫反应从而破坏耐受性的能力。用已经描述的1010vpchad3hcea或ad5hcea在四头肌中注射各8只小鼠的群组。在用包含从aa497到末端(aa703)的人类cea氨基酸序列的15-mer肽的库刺激的pbmc上，每周监视针对cea的免疫反应，直到第75天。通过ics确定响应于cea肽库孵化的分泌干扰素-γ的cd4-cd8+t细胞，来评估抗cea免疫性。在附图37中报告的结果表明，与表达相同转基因的ad5相比，chad3hcea载体免疫刺激了针对人类cea的更加持久的cd8+t细胞反应。灵长类免疫研究方法和材料：免疫方案还评估了在此公开和要求权利的chad载体在恒河猴(在此称为猴)中引发cmi的能力。麻痹恒河猴(开他敏/甲苯噻嗪)，使用结核菌素注射器(becton-dickinson)以0.5ml等分量向两个三角肌中肌肉内递送疫苗。在所有情况中，恒河猴体重在3-10公斤之间，在1ml缓冲液中施用每个疫苗的总剂量。从在免疫期间的几个时间点收集的血液样品制备血清和外周血单核细胞(pbmc)。所有动物照料和处理都根据动物照料和使用委员会机构(institutionalanimalcareandusecommittee)提供的标准，根据guideforcareanduseoflaboratoryanimals，instituteoflaboratoryanimalresources，nationalresearchcouncil中阐述的原则进行。elispot分析根据早先描述的方案(allenetal.，2001j.virol.75(2)：738-749)进行对恒河猴的ifn-γelispot分析，有某些修改。对于gag特异性刺激，从涵盖完整hiv-1gag序列具有10-aa重叠的20-aa的肽制备肽库(synpepcorp.，dublin，ca)。对于hcvns特异性刺激，从涵盖ns3到ns5b的完整hcv-ns序列的具有10-aa重叠的15-aa肽制备6个肽库。用涵盖完整蛋白序列的具有10-aa重叠的15-aa肽进行her2/neu和cea特异性刺激。向每个加样孔添加50μl2-4×105个外周血单核细胞(pbmc)；使用beckmancoulterz2粒子分析仪以设置在80fl的较低的尺寸截止(cut-off)对细胞进行计数。将50μl培养基或每种肽的浓度为8μg/ml的gag肽库添加到pbmc。样品在37℃，5％co2孵化20-24小时。相应地发展出斑点，用根据imagepro平台(silverspring，md)定制的成像仪和自动计数子程序处理平板；将计数值标准化为106个细胞输入。细胞内细胞因子染色(ics)向1ml的2×106pbmc/ml完全rpmi培养基(17×100mm圆底聚丙烯试管(sarstedt，newton，nc))中，添加抗hcd28(克隆l293，becton-dickinson)和抗hcd49d(克隆l25，becton-dickinson)单克隆抗体到1μg/ml的终浓度。对于gag特异性刺激，添加10μl的肽库(每种肽0.4mg/ml)。对hcvns特异性刺激使用类似的条件。在37℃孵化试管1小时，之后添加20μl的5mg/ml布雷菲德菌素a(brefeldina)(sigma)。在37℃、5％co2、90％湿度孵化细胞16小时。向每个试管添加4mlpbs/2％fbs，在1200rmp10分钟将细胞团粒化。将细胞重悬浮在pbs/2％fbs中，使用几种荧光标记的mabs对表面标记进行染色(30分钟，4℃)：每试管20μl抗hcd3-apc，克隆fn-18(biosource)；20μl抗hcd8-percp，克隆ski(bectondickinson，franklinlakes，nj)；和20μl抗hcd4-pe，克隆sk3(bectondickinson)。这个阶段的样品处理在黑暗中进行。洗涤细胞，在750μl1×facsperm缓冲液(bectondickinson)中在室温下孵化10分钟。将细胞团粒化，重悬浮在pbs/2％fbs中，添加0.1μgfitc-抗hifn-γ，克隆md-1(biosource)。孵化30分钟后，洗涤细胞并重悬浮在pbs中。使用bectondickinsonfacscalibur仪器的所有四个彩色通道对样品进行分析。为了分析数据，首先除去(gated)低的侧向和前向分散的淋巴细胞群体；将用于细胞因子阳性事件的常用的荧光截止用于cd4+和cd8+群体，并用于样品的模拟和gag肽反应试管。实施例10：在猴中使用chadδe1-gag载体的同源激发一强化方式引发gag特异性t细胞在第0周和第4周给予各3只动物的群组肌肉注射任何一种以下构建体：1010vp的cv-32δe1-gag；或1010vpcv33δe1-gag；或1010vp和108vp的mrkad5δe1gag。以规律的4周间隔收集的pbmc在elispot分析中进行分析。结果在表6中提供，其表明不存在(模拟)或存在gag肽库的情况下孵化之后每百万个pbmc中斑点形成细胞的数目，确定了cv32δe1gag和cv33δe1gag能够在非人灵长类中诱导显著水平的gag特异性t细胞。有趣的是注意到，在单次剂量之后(第4周)，cv32δe1-gag反应与mrkad5δe1-gag108vp剂量相当，比mrkad5-gag1010vp/剂的更低。cv33δe1-gag1010vp/剂诱导了与mrkad5-gag1010vp/剂的反应相当的反应。在第8周第二剂之后证实了这个结果。表6.用mrkad5δe1-gag、cv32δe1-gag、cv33δe1-gag免疫的猴中的gag特异性t细胞反应实施例11：在异源的激发一强化方案中chad载体引发hcvns特异性t细胞反应在独立的实验中，在第0、4周以108或1010vp每只动物向两只和三只猴的组给予mrkad6nsoptmut载体的免疫接种。在第24周以相同的剂量用相同的病毒对动物进行强化，在第104周以1010vp每只动物用cv33-nsmut再次强化。结果在表7和8中呈现，其总结了不存在(模拟的)或存在hcvns肽库的孵化之后每百万个pbmc的斑点形成细胞的数目。在第一剂之后第4周在注射了1010vp的动物中(表8)，和在第8周(第2剂以后)在注射了108的动物中(表7)，通过ifn-γelispot评定的t细胞免疫性显示了峰值反应。第24周的注射没有使反应强化(“同源强化”)而在注射cv33-nsmut之后观察到强烈的强化效果(“异源强化”)。表7.用108vp/动物的mrkad6nsoptmut免疫和用cv33-nsmut强化的猴中的hcvns特异性t细胞反应表8.用1010vp/动物的mrkad6nsoptmut免疫和用cv33-nsmut强化的猴中的hcvns特异性t细胞反应在第二实验中评估用黑猩猩ad载体异源强化的效率。在第0周和第4周用mrkad5gag(1010vp/动物)、mrkad6nsmut(1010vp/动物)或用两种载体的组合(每个载体1010vp/动物)免疫各三只猴的群组，然后在第24周用相同的免疫原强化(同源强化)。用相同的免疫原进行同源强化；用cv33gag、cv32nsmut或用组合的这两种载体进行异源强化。表9中提供的结果总结了不存在(模拟的)或存在hcvns肽库的孵化之后每百万个pbmc的斑点形成细胞的数目。在第51周用cv33gag(1010vp/动物)、cv32nsmut(1010vp/动物)和用两种载体的组合(每种载体1010vp/动物)再次强化相同的群组。在表9中提供的结果进一步表明，由于反应低于注射第一剂疫苗后第4周观察到的峰值，同源强化不是很有效。在用异源黑猩猩载体免疫接种后在第54周通过ifn-γelispot测量到强烈的强化效果。表9.在用mrkad5gag或mrkad6nsoptmut以1010vp/动物免疫的猴中，黑猩猩ad载体的免疫接种有效地强化gag和hcvns特异性t细胞反应实施例12：用包含taa的chad载体进行疫苗接种在猴中破坏耐受性并引发taa特异性t细胞反应在各四只猴的群组中进行实验，所述实验被设计来确定黑猩猩腺病毒载体是否有足够的免疫原性来破坏对自体抗原的耐受性，并记录黑猩猩载体用于强化人类腺病毒载体激发的免疫反应的实用性。在第0、2和4周用包含肿瘤相关抗原cea的ad5de1rhcea(1011vp)三次注射免疫动物，随后在第16、18和20周用cv33de1rhcea(1011vp)接种。用恒河猴cea肽通过ifn-γelispot测量t细胞反应。结果在附图34中报告，其提供了在不存在(dmso)或存在恒河猴ceac和d肽库的孵化之后每百万pbmc中斑点形成细胞的数目，确定了基于用两种不同的ad血清型的接种的免疫方案在非人灵长类中引起针对cea的持续的t细胞反应。虽然已经参考本发明的某些优选的实施方式详细描述了本发明，要理解的是，修改和变化都处在所描述的和要求权利的精神和范围之内。序列表<110>merck&co.,inc.institutodiricerchedibiologiamolecolarep.angelettis.p.a.&lt;120&gt;黑猩猩腺病毒疫苗载运体&lt;130&gt;itr0048ypct&lt;150&gt;60/538,799&lt;151&gt;2004-01-23&lt;160&gt;125&lt;170&gt;fastseqforwindowsversion4.0&lt;210&gt;1&lt;211&gt;37741&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad3基因组&lt;400&gt;1catcatcaataatataccttattttggattgaagccaatatgataatgagatgggcggcg60cgaggcggggcgcggggcgggaggcgggtttgggggcgggccggcgggcggggcggtgtg120gcggaagtggactttgtaagtgtggcggatgtgacttgctagtgc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atgggatagtactaagtacacaggtgaaacacttgctacaagctccttcaccttctcc1320tacattgcccaagaatga1338&lt;210&gt;10&lt;211&gt;1278&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad9纤维&lt;400&gt;10atgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgca60gacaacgcaccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaa120gagaagcccctgggggtgttgtccctgcgactggccgaccccgtcaccaccaagaacggg180gaaatcaccctcaagctgggagagggggtggacctcgactcctcgggaaaactcatctcc240aacacggccaccaaggccgctgcccctctcagtttttccaacaacaccatttcccttaac300atggatcaccccttttacactaaagatggaaaattagccttacaagtttctccaccatta360aatatactgagaacaagcattctaaacacactagctttaggttttggatcaggtttagga420ctccgtggctctgccttggcagtacagttagtctctccacttacatttgatactgatgga480aacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagc540aacataagctgggctaaaggtttaaaatttgaagatggagccatagcaaccaacattgga600aatgggttagagtttggaagcagtagtacagaaacaggtgtcgatgatgcttacccaatc660caagttaaacttggatctggccttagctttgacagtacaggagccataatggctggtaac720aaagaagacgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaata780ctcgcagaaaatgatgcaaaactaacactttgcttgactaaatgtggtagtcaaatactg840gccactgtgtcagtcttagttgtaggaagtggagacctaaaccccattactggcaccgta900agcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattct960acactaaaaaaatactgggggtataggcagggagatagcatagatggcactccatatgcc1020aatgctgtaggattcatgcccaatttaaaagcttatccaaagtcacaaagttctactact1080aaaaataatatagtagggcaagtatacatgaatggagatgtttcaaaacctatgcttctc1140actataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatac1200acctggactaatggaagctatgttggagcaacatttggagctaactcttataccttctcc1260tacatcgcccaagaatga1278&lt;210&gt;11&lt;211&gt;1278&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad10纤维&lt;400&gt;11atgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgca60gacaacgcaccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaa120gagaagcccctgggggtgctgtccctgcgactggccgaccccgtcaccaccaagaacggg180gaaatcaccctcaagctgggagagggggtggacctcgactcctcgggaaaactcatctcc240aacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaac300atggatcaccccttttacactaaagatggaaaattatccttacaagtttctccaccatta360aatatactgagaacaagcattctaaacacactagctttaggttttggatcaggtttagga420ctccgtggctctgccttggcagtacagttagtctctccacttacatttgatactgatgga480aacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagc540aacataagctgggctaaaggtttaaaatttgaagatggagccatagcaaccaacattgga600aatgggttagagtttggaagcagtagtacagaaacaggtgttgatgatgcttacccaatc660caagttaaacttggatctggccttagctttgacagtacaggagccataatggctggtaac720aaagaagacgataaactcactttgtggacaacacctgatccatcgccaaactgtcaaata780ctcgcagaaaatgatgcaaaactaacactttgcttgactaaatgtggtagtcaaatactg840gccactgtgtcagtcttagttgtaggaagtggaaacctaaaccccattactggcaccgta900agcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattct960acactaaaaaaatactgggggtataggcagggagatagcatagatggcactccatatacc1020aatgctgtaggattcatgcccaatttaaaagcttatccaaagtcacaaagttctactact1080aaaaataatatagtagggcaagtatacatgaatggagatgtttcaaaacctatgcttctc1140actataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatac1200acctggactaatggaagctatgttggagcaacatttggggctaactcttataccttctca1260tacatcgcccaagaatga1278&lt;210&gt;12&lt;211&gt;1737&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad11纤维&lt;400&gt;12atgaagcgcaccaaaacgtctgacgagagcttcaaccccgtgtacccctatgacacggaa60aacggtcctccctccgtccctttcctcacccctcccttcgtgtctcccgatggattccaa120gagagcccccccggggtcctgtctctgaacctggccgagcccctggtcacttcccacggc180atgctcgccctgaaaatgggaagtggcctctccctggacgacgccggcaacctcacctct240caagatgtcaccaccactacccctcccctgaaaaaaaccaagaccaacctcagcctagaa300acctcagcccccctgactgtgagcacctcaggcgccctcaccctagcagccgccgttccc360ctggcggtggccggcacctccctcaccatgcaatcagaggcccccctgacagtccaagat420gcaaaactcaccctggccaccaagggccccctgaccgtgtctgaaggcaaactagccttg480cagacctcggccccgctgacggccgctgacagcagcaccctcacaatcagcgccacaccg540ccccttagcacaagcaatggcagcttgggtattgacatgcaagcccccatttacactact600aacggaaaactgggacttaactttggtgctcccctgcatgtggtagacagcctaaatgca660ctgactgtagtgactggccaaggtcttacgataaacggtacagccctacaaactagagtc720tcaggtgccctcaactatgactcatcaggaaacctagaattgagagctgcagggggtatg780cgagttgatgcaaatggcaaacttatccttgacgtagcttacccatttgatgctcaaaac840aacctcagccttagacttggacagggacccctgtttgttaactctgcccacaacttggat900gttaactacaacagaggcctctacctgttcacatctggaaataccaaaaagctagaagtt960aatatcaaaacagccaaaggcctcatttatgatgacactgctatagcaatcaatccaggc1020gatgggctagagtttggctcaggctcagatacaaatccattaaaaactaaacttggattg1080ggactagagtatgactccagcagagccataattgctaagctgggaaccggcctaagcttt1140gacaacacaggtgccatcacagtgggcaacaaaaatgatgacaagcttaccttgtggacc1200acaccagacccctctcccaactgtagaatttattcagaaaaagatgctaaatttacacta1260gttttaactaaatgcggcagtcaggtgttggccagcgtttctgttttatctgtaaaaggc1320agccttgcgcccatcagtggcacagtaactagcgctcagattattctcagatttgatgaa1380aatggagttctactaagcaattcttctcttgacccccaatactggaactacagaaaaggt1440gaccttacagagggcactgcatataccaacgcagtgggatttatgcccaacctcacagca1500tacccaaaaacacagagtcaaactgctaaaagcaacattgtaagccaggtttacttgaat1560ggggacaaatccaaacccatgatcctcaccattaccctcaatggaactaatgaaacaggg1620gatgctacagttagcacttactccatgtcattctcatggaattggaatggaagtaattac1680attaatgaaacgttccaaaccaactctttcaccttctcctacatcgcccaagaataa1737&lt;210&gt;13&lt;211&gt;1632&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad16纤维&lt;400&gt;13atgaagcgcaccaaaacgtctgacaagagcttcaaccccgtgtacccctatgacacggaa60aacggtcctccctccgtccctttcctcacccctcccttcgtgtctcccgatggattccaa120gagagcccccccggggtcctgtctctgaacctggccgagcccctggtcacttcccacggc180atgctcgccctgaaaatgggaagtggcctctccctggacgacgccggcaacctcacctct240caagatgtcaccaccactacccctcccctgaaaaaaaccaagaccaacctcagcctagaa300acctcagcccccctgactgtgagcacctcaggcgccctcaccctagcagccgccgccccc360ctggcggtggccggcacctccctcaccatgcaatcagaggcccccctgacagtccaagat420gcaaaactcaccctggccaccaagggccccctgaccgtgtctgaaggcaaactggccttg480cagacctcggccccgctgacggccgctgacagcagcaccctcaccgttagcgccacacca540cccatcagtgtaagcagtggaagtttgggcttagacatggaagacccaatgtatactcat600gatggaaaactgggaataagaattgggggcccactgagagtagtagacagcctgcacaca660ctgactgtagttaccggaaatggaatagctgtagataacaatgccctccaaactagagtt720acgggcgccctgggttatgacacatcaggaaacctacaactgagagccgcggggggtatg780cgaattgatgcaaatggccaacttatccttgatgtggcatacccatttgatgctcaaaac840aatctcagccttagacttggtcagggacccctgtatgtaaacacagaccacaacctagat900ttgaattgcaacagaggtctgaccacaactaccaccaacaacacaaaaaaacttgaaact960aaaattggctcaggcttagactatgataccaatggtgctgttattattaaacttggcact1020ggtgtcagctttgacagcacaggtgccctaagtgtgggaaacactggcgatgataaactg1080actctgtggacaaccccagacccatctccaaattgcagaattcactcagacaaagactgc1140aagtttactctagtcctaactaagtgtggaagtcaaatcctggcttctgtcgccgcccta1200gcggtgtcaggaaatctggcttcaataacaggcaccgtttccagcgttaccatctttctc1260agatttgatcagaatggagtgcttatggaaaactcctcgctagacaagcagtactggaac1320ttcagaaatggtaattcaaccaatgccaccccctacaccaatgcagttgggtttatgcca1380aacctcgcagcataccccaagacacagagccagactgcaaaaaacaacattgtaagtcag1440gtttacttgaatggggacaaatccaaacccatgacccttaccattaccctcaatggaact1500aatgaatccagtgaaactagccaggtgagtcactactccatgtcatttacgtgggcttgg1560gagagtgggcaatatgccaccgaaacctttgccaccaattcctttaccttctcttacatt1620gctgaacaataa1632&lt;210&gt;14&lt;211&gt;1632&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad17纤维&lt;400&gt;14atgaagcgcaccaaaacgtctgacgagagcttcaaccccgtgtacccctatgacacggaa60agcggccctccctccgtccctttcctcacccctcccttcgtgtctcccgatggattccaa120gaaagcccccccggggtcctgtctctgaacctggccgagcccctggtcacttcccacggc180atgcttgccctgaaaatgggaagtggcctctccctggacgacgctggcaaccttacctct240caagatattacctccactacccctcccctcaaaaaaaccaagaccaacctcagcctagaa300acctcatcccccctaactgtaagcacctcaggcgccctcaccgtagcagccgccgctccc360ctggcggtggccggcacctccctcaccatgcaatcagaggcccccctggcagtacaggat420gcaaaactcaccctggccaccaaaggccccctgaccgtgtctgaaggcaaactggccttg480caaacatcggccccgctgacggccgctgacagcagcaccctcaccgttagctccactcca540ccaattagtgtaagcagtggaagtttgggcttggacatggaagaccccatgtatactcac600gatggaaaactgggaataagaattgggggtccactaagagtagtagacagcttgcacaca660ctcactgtagttaccggaaatggactaactgtagataacaatgccctccaaactagagtt720acgggcgccctaggttatgacacatcaggaaatctacaactgagagccgcagggggtatg780cgaattgatgcaaatggccaacttatccttgatgtggcatacccatttgatgctcaaaac840aatctcagccttagacttggtcagggacccctgtatgtaaatacagaccacaacctggat900ttaaattgcaacagaggtctaaccacaactaccaccaacaacacaaaaaaacttgagact960aaaattagctcaggcttagactatgacaccaatggtgctgtcattattaaacttggcact1020ggtctaagcttcgacaacacaggcgccctaactgtgggaaacactggtgatgataaactg1080actctgtggacgaccccagacccatctccaaattgcagaattcactcagacaaagactgc1140aagtttactctcgtcctaactaagtgtggaagccaaatcctggcctctgtcgccgcccta1200gcggtatcaggaaatctggcttcgataacaggcaccgttgccagcgttaccatctttctt1260agatttgatcagaatggagtgcttatggaaaactcctcactagacaagcagtactggaac1320ttcagaaatggcaattcaactaatgctgccccctacaccaacgcagttgggttcatgcca1380aacctcgcagcgtaccccaaaacgcaaagccagactgctaaaaacaacattgtaagtcag1440gtttacttgaatggagacaaatccaaacccatgacccttaccatcaccctcaatggaact1500aatgaatccagtgaaactagtcaggtgagtcactactccatgtcatttacatgggcttgg1560gaaagcgggcaatatgccactgaaacctttgccaccaactccttcaccttttcttacatt1620gctgaacaataa1632&lt;210&gt;15&lt;211&gt;1632&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad19纤维&lt;400&gt;15atgaagcgcaccaaaacgtctgacaagagcttcaaccccgtgtacccctatgacacggaa60aacggtcctccctccgtccctttcctcacccctcccttcgtgtctcccgatggattccaa120gagagcccccccggggtcctgtctctgaacctggccgagcccctggtcacttcccacggc180atgctcgccctgaaaatgggaagtggcctctccctggacgacgccggcaacctcacctct240caagatgtcaccaccactacccctcccctgaaaaaaaccaagaccaacctcagcctagaa300acctcagcccccctgactgtgagcacctcaggcgccctcaccctagcagccgccgccccc360ctggcggtggccggcacctccctcaccatgcaatcagaggcccccctgacagtccaagat420gcaaaactcaccctggccaccaagggccccctgaccgtgtctgaaggcaaactggccttg480cagacctcggccccgctgacggccgctgacagcagcaccctcaccgttagcgccacacca540cccatcagtgtaagcagtggaagtttgggcttagacatggaagacccaatgtatactcat600gatggaaaactgggaataagaattgggggcccactgagagtagtagacagcctgcacaca660ctgactgtagttaccggaaatggaatagctgtagataacaatgccctccaaactagagtt720acgggcgccctgggttatgacacatcaggaaacctacaactgagagccgcggggggtatg780cgaattgatgcaaatggccaacttatccttgatgtggcatacccatttgatgctcaaaac840aatctcagccttagacttggtcagggacccctgtatgtaaacacagaccacaacctagat900ttgaattgcaacagaggtctgaccacaactaccaccaacaacacaaaaaaacttgaaact960aaaattggctcaggcttagactatgataccaatggtgctgttattattaaacttggcact1020ggtgtcagctttgacagcacaggtgccctaagtgtgggaaacactggcgatgataaactg1080actctgtggacaaccccagacccatctccaaattgcagaattcactcagacaaagactgc1140aagtttactctagtcctaactaagtgtggaagtcaaatcctggcttctgtcgccgcccta1200gcggtgtcaggaaatctggcttcaataacaggcaccgtttccagcgttaccatctttctc1260agatttgatcagaatggagtgcttatggaaaactcctcgctagacaagcagtactggaac1320ttcagaaatggtaattcaaccaatgccaccccctacaccaatgcagttgggt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tc1440accctgcccaccaacaccaacacctacgattacatgaacgggagagtggtgcctccctcg1500ctggtggacgcctacatcaacatcggggcgcgctggtcgctggaccccatggacaacgtg1560aatcccttcaaccaccaccgcaacgcgggcctgcgctaccgctccatgctcctgggcaac1620gggcgctacgtgcccttccacatccaggtgccccagaaatttttcgccatcaagagcctc1680ctgctcctgcccgggtcctacacctacgagtggaacttccgcaaggacgtcaacatgatc1740ctgcagagctccctcggcaacgacctgcgcacggacggggcctccatctccttcaccagc1800atcaacctctacgccaccttcttccccatggcgcacaacacggcctccacgctcgaggcc1860atgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggcggccaacatg1920ctctaccccatcccggccaacgccaccaacgtgcccatctccatcccctcgcgcaactgg1980gccgccttccgcggctggtccttcacgcgcctcaagacccgcgagacgccctcgctgggc2040tccgggttcgacccctacttcgtctactcgggctccatcccctacctcgacggcaccttc2100tacctcaaccacaccttcaagaaggtctccatcaccttcgactcctccgtcagctggccc2160ggcaacgaccgcctcctgacgcccaacgagttcgaaatcaagcgcaccgtcgacggagag2220ggatacaacgtggcccagtgcaacatgaccaaggactggttcctggtccagatgctggcc2280cactacaacatcggctaccagggcttctacgtgcccgagggctacaaggaccgcatgtac2340tccttcttccgcaacttccagcccatgagccgccaggtggtggacgaggtcaactacaag2400gactaccaggccgtcaccctggcctaccagcacaacaactcgggcttcgtcggctacctc2460gcgcccaccatgcgccagggccagccctaccccgccaactacccgtacccgctcatcgga2520aagagcgccgtcaccagcgtcacccagaaaaagttcctctgcgacagggtcatgtggcgc2580atccccttctccagcaacttcatgtccatgggcgcgctcaccgacctcggccagaacatg2640ctctatgccaactccgcccacgcgctagacatgaatttcgaagtcgaccccatggatgag2700tccacccttctctatgttgtcttcgaagtcttcgacgtcgtccgagtgcaccagccccac2760cgcggcgtcatcgaggccgtctacctgcgcacccccttctcggccggtaacgccaccacc2820taa2823&lt;210&gt;18&lt;211&gt;2823&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad5六邻体&lt;400&gt;18atggccaccccatcgatgctgccccagtgggcgtacatgcacatcgccggacaggacgct60tcggagtacctgagtccgggtctggtgcagttcgcccgcgccacagacacctacttcagt120ctggggaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgc180agccagcggctgacgctgcgcttcgtgcccgtggaccgcgaggacaacacctactcgtac240aaagtgcgctacacgctggccgtgggcgacaaccgcgtgctggacatggccagcacctac300tttgacatccgcggcgtgctggatcggggccctagcttcaaaccctactccggcaccgcc360tacaacagcctggctcccaagggagcgcccaattccagccagtgggagcaaaaaaagact420ggcaataatgccaatggagatacggagaatgtcacttatggtgtagctgccatgggagga480attgacatcgataaaaatggccttcaaattggaaccgatgacaccaaagatgacgataat540gaaatttatgcagacaaaacatatcagcctgagccgcaaataggagaggaaaactggcaa600gaaacatattcctactatggaggtagagctcttaaaaaagataccaaaatgaagccatgc660tatggctcatttgccagacctaccaatgtgaaaggaggacaggcaaaaataaaaacagat720ggagatgttaagtcatttgacatagacctagccttctttgatattcccaattctggcgcg780ggaaatggcacaaatgttaacgatgatccagatatggttatgtatacagaaaatgtaaat840ctggaaaccccagatactcatattgtgtacaaaccaggaacttcagatgacagctcaaag900gtcaacttgtgtcagcaatccatgcctaacagacccaattatattggcttcagagacaat960tttattgggcttatgtactacaacagcactggcaatatgggtgtgctggctggtcaggcc1020tctcaactgaatgccgtggtggacttgcaagacagaaacacagagctgtcctaccagctc1080ttgcttgactctctgggtgacagaaccaggtatttcagtatgtggaatcaggcggtggac1140agttatgatcctgatgtgcgcattattgaaaaccatggtgtggaggatgaattgccaaac1200tattgcttccccttggatggagcaggcaccaattcggtttaccaaggtgttaaaccaaaa1260actgacaatggcaacgatcagtgggaaacagattccacagtttcaagtcacaatcagata1320tgcaaaggcaatatctatgccatggagatcaacctccaggccaacctgtggagaagtttt1380ctctactcgaacgtggccctgtacctgcccgattcttacaagtacacgccggccaacatc1440accctgcccaccaacaccaacacctacgattacatgaacgggagagtggtgcctccctcg1500ctggtggacgcctacatcaacatcggggcgcgctggtcgctggaccccatggacaacgtg1560aatcccttcaaccaccaccgcaacgcgggcctgcgctaccgctccatgctcctgggcaac1620gggcgctacgtgcccttccacatccaggtgccccagaaattttttgccatcaagagcctc1680ctgctcctgcccgggtcctacacctacgagtggaacttccgcaaggacgtcaacatgatc1740ctgcagagctccctcggcaacgacctgcgcacggacggggcctccatctccttcaccagc1800atcaacctctacgccaccttcttccccatggcgcacaacacggcctccacgctcgaggcc1860atgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggcggccaacatg1920ctctaccccatcccggccaacgccaccaacgtgcccatctccatcccctcgcgcaactgg1980gccgccttccgcggctggtccttcacgcgcctcaagacccgcgagacgccctcgctgggc2040tccgggttcgacccctacttcgtctactcgggctccatcccctacctcgacggcaccttc2100tacctcaaccacaccttcaagaaggtctccatcaccttcgactcctccgtcagctggccc2160ggcaacgaccgcctcctgacgcccaacgagttcgaaatcaagcgcaccgtcgacggagag2220ggatacaacgtggcccagtgcaacatgaccaaggactggttcctggtccagatgctggcc2280cactacaacatcggctaccagggcttctacgtgcccgagggctacaaggaccgcatgtac2340tccttcttccgcaacttccagcccatgagccgccaggtcgtggacgaggtcaactacaag2400gactaccaggccgtcaccctggcctaccagcacaacaactcgggcttcgtcggctacctc2460gcgcccaccatgcgccagggccagccctaccccgccaactacccctacccgctcatcggc2520aagagcgccgtcgccagcgtcacccagaaaaagttcctctgcgaccgggtcatgtggcgc2580atccccttctccagcaacttcatgtccatgggcgcgctcaccgacctcggccagaacatg2640ctctacgccaactccgcccacgcgctagacatgaatttcgaagtcgaccccatggatgag2700tccacccttctctatgttgtcttcgaagtcttcgacgtcgtccgagtgcaccagccccac2760cgcggcgtcatcgaggccgtctacctgcgcacccccttctcggccggtaaagccaccacc2820taa2823&lt;210&gt;19&lt;211&gt;2823&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad7六邻体&lt;400&gt;19atggccaccccatcgatgctgccccagtgggcgtacatgcacatcgccggacaggacgct60tcggagtacctgagtccgggtctggtgcagttcgcccgcgccacagacacctacttcagt120ctggggaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgc180agccagcggctgacgctgcgcttcgtgcccgtggaccgcgaggacaacacctactcgtac240aaagtgcgctacacgctggccgtgggcgacaaccgcgtgctggacatggccagcacctac300tttgacatccgcggcgtgctggaccggggccctagcttcaaaccttactccggcaccgct360tacaacagcctggcccccaagggagcacccaattccagccagtgggagcaaaaaaagact420ggcaaaaatgccaatggagatacggagaatgtcacttatggtgtagctgccatgggagga480attgacatcgataaaaatggccttcaaattggaaccgatgacaccaaagatggcgataat540gaaatttatgcagacaaaacatatcagcctgagccgcaaataggagaggaaaactggcaa600gaaacatattcctactatggaggtagagctcttaaaaaagataccaaaatgaagccatgc660tatggctcatttgctagacctaccaatgtgaaaggaggacaggcaaaaataaaaacagat720ggagatgttaagtcatttgacatagacctagccttctttgatattccaaattctggcgcg780ggaaatggcacaaatgttaacgatgatccagatatggttatgtatacagaaaatgtaaat840ctggaaaccccagatactcatattgtgtacaaaccaggaacttcagatgacagctccgag900gtcaacttgtgtcagcaatccatgcctaacagacccaattatattggcttcagagacaat960tttattgggcttatgtactacaacagcactggcaatatgggtgtgctggctggtcaggcc1020tctcaactgaatgccgtggtggacttgcaagacagaaacacagagctgtcctaccagctc1080ttgcttgactctctgggtgacagaaccaggtatttcagtatgtggaatcaggcggtggac1140agttatgatcctgatgtgcgcattattgaaaaccatggtgtggaggatgaattgccaaac1200tattgcttccccttggatggagcaggcaccaattcggtttaccaaggtgttaaaccaaaa1260actgacaatggcaacgatcagtgggaaacagattccacagtttcaagtcacaatcagata1320tgcaaaggcaatatctatgccatggagatcaatctccaggccaacctgtggagaagtttc1380ctctactcgaacgtggccctgtacctgcccgattcttacaagtacacgccggccaacatc1440accctgcccaccaacaccaacacctacgattacatgaacgggagagtggtgcctccctcg1500ctggtggatgcctacatcaacatcggagcgcgctggtcgctggaccccatggacaacgtc1560aatcccttcaaccaccaccgcaatgcggggctgcgctaccgctccatgctcctgggcaac1620gggcgctacgtgcccttccacatccaggtgccccagaaatttttcgccatcaagagcctt1680ctgctcctgcccgggtcctacacctacgagtggaacttccgcaaggacgtcaacatgatc1740ctgcagagctccctcggcaacgacctgcgcacggacggggcctccatctccttcaccagc1800atcaacctctacgccaccttcttccccatggcgcacaacacggcctccacgctcgaggcc1860atgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggcggccaacatg1920ctctaccccatcccggccaacgccaccaacgtgcccatctccatcccctcgcgcaactgg1980gccgccttccgcggctggtccttcacgcgcctcaagaccaaggagacgccctcgctgggc2040tccgggttcgacccatacttcgtctactcgggctccatcccctacctcgacggcaccttc2100tacctcaaccacaccttcaagaaggtctccatcaccttcgattcctccgtcagctggccc2160ggcaacgaccggctcctgacgcccaacgagttcgaaatcaagcgcaccgtcgacggcgag2220ggatacaacgtggcccagtgcaacatgaccaaggactggttcctggtccagatgctggcc2280cactacaacatcggctaccagggcttctacgtgcccgagggctacaaggaccgcatgtac2340tccttcttccgcaacttccagcccatgagccgccaggtggtggacgaggtcaactacaag2400gactaccaggccgtcaccctggcctaccagcacaacaactcgggcttcgtcggctacctc2460gcgcccaccatgcgccagggccagccctaccccgccaactacccgtacccgctcatcggc2520aagagcgccgtcaccagcgtcacccagaaaaagttcctctgcgacagggtcatgtggcgc2580atccccttctccagcaacttcatgtccatgggcgcgctcaccgacctcgggcagaacatg2640ctctatgccaactccgcccacgcgctagacatgaatttcgaagtcgaccccatggatgag2700tccacccttctctatgttgtcttcgaagtcttcgacgtcgtccgagtgcaccagccccac2760cgcggcgtcatcgaggccgtctacctgcgcacccccttctcggccggtaacgccaccacc2820taa2823&lt;210&gt;20&lt;211&gt;2793&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad9六邻体&lt;400&gt;20atggccaccccatcgatgctgccccagtgggcgtacatgcacatcgccggacaggacgct60tcggagtacctgagtccgggtctggtgcagttcgcccgcgccacagacacctacttcagt120ctggggaacaagtttaggaaccccacggtggcacccacgcacgatgtgaccaccgaccgc180agccagcggctgacgctgcgcttcgtgcccgtggaccgcgaggacaacacctactcgtac240aaagtgcgctacacgctggccgtgggcgacaaccgcgtgctggacatggccagcacctac300tttgacatccgcggcgtgctggatcggggccctagcttcaaaccctactccggcaccgct360tacaacagcctggctcccaagggagcgcccaacacttgccagtggacatatactgataac420caaactgagaaaacagccacatatggaaatgcacccgtagagggcattaacattacaaaa480gatggcattcaacttggaactgacagcgatggtcaggcaatctatgcagacgaaacttat540cagcccgaacctcaggtgggagatcctgaatggcatgataccacaggtacagaagaaaaa600tatggaggcagagcgcttaaacctgccaccgacatgaaaccttgctatggctcttttgcc660aagccaactaatgttaagggaggtcaggccaaaagcagaacaaaaactgatggaacaact720gagcctgatattgacatggccttttttgatggcagaaatgcaacaacagctggtttgact780ccagaaattgttttgtatactgaaaatgtggatctggaaactccagatacccatattgta840tacaaggcaggcacagatgacagcagctcttctatcaatttgggtcagcagtccatgccc900aacagacccaactacattggcttcagagacaactttatcgggctcatgtactacaacagc960actggcaatatgggtgtactggctggacaggcctcccagctgaatgctgtggtggacttg1020caggacagaaacactgaactgtcctaccagctcttgcttgactctctgggtgacagaacc1080aggtatttcagtatgtggaatcaggcggtggacagttatgaccccgatgtgcgcattatt1140gaaaatcacggtgtggaggatgaactccccaactattgcttccccctgaatgctgtgggt1200agaacaaatagttatcagggaattaaacccaatggaggcgatccagctacatgggccaaa1260gatgaaagcgtcaatgattctaatgaattgggcaagggcaatcctttcgccatggagatc1320aacatccaggccaacctgtggcggaacttcctctacgcgaacgtggcgctgtacctgccc1380gactcctacaagtacacgccggccaacatcacgctgcccgccaacaccaacacctacgat1440tacatgaacggccgcgtggtggcgccctcgctggtggacgcctacatcaacatcggggcg1500cgctggtcgctggaccccatggacaacgtcaaccccttcaaccaccaccgcaacgcgggc1560ctgcgctaccgctccatgctcctgggcaacgggcgctacgtgcccttccacatccaggtg1620ccccaaaagtttttcgccatcaagagcctcctgctcctgcccgggtcctacacctacgag1680tggaacttccgcaaggacgtcaacatgatcctgcagagctccctcggcaacgacctgcgc1740acggacggggcctccatcgccttcaccagcatcaacctctacgccaccttcttccccatg1800gcgcacaacaccgcctccacgctcgaggccatgctgcgcaacgacaccaacgaccagtcc1860ttcaacgactacctctcggcggccaacatgctctaccccatcccggccaacgccaccaac1920gtgcccatctccatcccctcgcgcaactgggccgccttccgcggatggtccttcacgcgc1980ctcaagacccgcgagacgccctcgctaggctccgggttcgacccctacttcgtctactcg2040ggctccatcccctacctcgacggcaccttctacctcaaccacaccttcaagaaggtctcc2100atcaccttcgactcctccgtcagctggcccggcaacgaccgcctcctgacgcccaacgag2160ttcgaaatcaagcgcaccgtcgacggagagggatacaacgtggcccagtgcaacatgacc2220aaggactggttcctggtccagatgctggcccactacaacatcggctaccagggcttctac2280gtgcccgagggctacaaggaccgcatgtactccttcttccgcaacttccagcccatgagc2340cgccaggtcgtggacgaggtcaactacaaggactaccaggccgtcaccctggcctaccag2400cacaacaactcgggcttcgtcggctacctcgcgcccaccatgcgccagggccagccctac2460cccgccaactacccctacccgctcatcggcaagagcgccgtcgccagcgtcacccagaaa2520aagttcctctgcgaccgggtcatgtggcgcatccccttctccagcaacttcatgtccatg2580ggcgcgctcaccgacctcggccagaacatgctctacgccaactccgcccacgcgctagac2640atgaatttcgaagtcgaccccatggatgagtccacccttctctatgttgtcttcgaagtc2700ttcgacgtcgtccgagtgcaccagccccaccgcggcgtcatcgaggccgtctacctgcgc2760acgcccttctcggccggcaacgccaccacctaa2793&lt;210&gt;21&lt;211&gt;2793&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad10六邻体&lt;400&gt;21atggccaccccatcgatgctgccccagtgggcgtacatgcacatcgccggacaggacgct60tcggagtacctgagtccgggtctggtgcagttcgcccgcgccacagacacctacttcagt120ctggggaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgc180agccagcggctgacgctgcgcttcgtgcccgtggaccgcgaggacaacacctactcgtac240aaagtgcgctacacgctggccgtgggcgacaaccgcgtgctggacatggccagcacctac300tttgacatccgcggcgtgctggatcggggccctagcttcaaaccctactccggcaccgcc360tacaacagcctggctcccaagggagcgcccaacacttgccagtggacatatactgataac420caaactgagaaaacagccacatatggaaatgcgcctgtgcaaggcattagtattacaaaa480gatggtattcaacttggaactgacactgatgatcagcccatttatgcagataaaacttat540caaccagagcctcaagtgggtgatgctgaatggcatgacatcactggtactgatgaaaaa600tatggaggcagagctctcaagcctgacaccaaaatgaagccctgctatggttcttttgcc660aagcctaccaataaagaaggaggtcaggcaaatgtgaaaaccgaaacaggcggtaccaaa720gaatatgacattgacatggcattcttcgataatcgaagtgcagctgcggctggcctggcc780ccagaaattgttttgtatactgagaatgtggatctggaaactccagatactcatattgta840tacaaggcaggcacagatgacagcagctcttctatcaatttgggtcagcagtccatgccc900aacagacccaactacattggcttcagagacaactttatcggtctcatgtactacaacagc960actggcaatatgggtgtactggctggtcaggcctcccagctgaatgctgtggtggacttg1020caggacagaaacactgaactgtcctaccagctcttgcttgactctctgggtgacagaacc1080aggtattttagtatgtggaatcaggcggtggacagttatgaccccgatgtgcgcattatt1140gaaaatcacggtgtggaggatgaactccctaattattgcttcccccttaatgctgtgggt1200agaactgatacttaccagggaattaaggccaatggtgctgatcaaaccacatggaccaaa1260gatgatactgttaatgatgctaatgaattgggcaagggcaatcctttcgccatggagatc1320aacatccaggccaacctgtggcggaacttcctctacgcgaacgtggccctgtacctgccc1380gactcctacaagtacacgccggccaacatcacgctgcccaccaacaccaacacctacgat1440tacatgaacggccgcgtggtggcgccctcgctggtggacgcctacatcaacatcggggcg1500cgctggtcgctggaccccatggacaacgtcaaccccttcaaccaccaccgcaacgcgggc1560ctgcgctaccgctccatgctcctgggcaacgggcgctacgtgcccttccacatccaggtg1620ccccaaaagttcttcgccatcaagagcctcctgctcctgcccgggtcctacacctacgag1680tggaacttccgcaaggacgtcaacatgatcctgcagagctccctcggcaacgacctgcgc1740acggacggggcctccatcgccttcaccagcatcaacctctacgccaccttcttccccatg1800gcgcacaacaccgcctccacgctcgaggccatgctgcgcaacgacaccaacgaccagtcc1860ttcaacgactacctctcggcggccaacatgctctaccccatcccggccaatgccaccaac1920gtgcccatctccatcccctcgcgcaactgggccgccttccgcggatggtccttcacgcgc1980ctcaagacccgcgagacgccctcgctaggctccgggttcgacccctacttcgtctactcg2040ggctccatcccctacctcgacggcaccttctacctcaaccacaccttcaagaaggtctcc2100atcaccttcgactcctccgtcagctggcccggcaacgaccgcctcctgacgcccaacgag2160ttcgaaatcaagcgcaccgtcgacggagaggggtacaacgtggcccagtgcaacatgacc2220aaggactggttcctggtccagatgctggcccactacaacatcggctaccagggcttctac2280gtgcccgagggctacaaggaccgcatgtactccttcttccgcaacttccagcccatgagc2340cgccaggtcgtggacgaggtcaactacaaggactaccaggccgtcaccctggcctaccag2400cacaacaactcgggcttcgtcggctacctcgcgcccaccatgcgccagggccagccctac2460cccgccaactacccctacccgctcatcggcaagagcgccgtcgccagcgtcacccagaaa2520aagttcctctgcgaccgggtcatgtggcgcatccccttctccagcaacttcatgtccatg2580ggcgcgctcaccgacctcggccagaacatgctctacgccaactccgcccacgcgctagac2640atgaatttcgaagtcgaccccatggatgagtccacccttctctatgttgtcttcgaagtc2700ttcgacgtcgtccgagtgcaccagccccaccgcggcgtcatcgaggccgtctacctgcgc2760acgcccttctcggccggcaacgccaccacctaa2793&lt;210&gt;22&lt;211&gt;2883&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad11六邻体&lt;400&gt;22atggcgaccccatcgatgatgccgcagtggtcgtacatgcacatctcgggccaggacgcc60tcggagtacctgagtcccgggctggtgcagttcgctcgcgccaccgagagctacttcagt120ctgagtaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgg180tcccagcgcctgacgctgcggttcatccccgtggaccgcgaggacaccgcgtactcgtac240aaggcgcggttcaccctggccgtgggcgacaaccgcgtgctggacatggcctccacctac300tttgacatccgcggcgtgctggaccgcggccccaccttcaagccctactccggcaccgcy360tacaactccctggcccccaagggcgctcccaactcctgcgagtgggagcaagaggaaact420caggcagttgaagaagcagcagaagaggaggaagaagatgctgacggtcaagctgaggaa480gagcaagcagctaccaaaaagactcatgtatatgctcaggctcccctttccggcgaaaaa540attagcaaagacggtctgcagataggaacggacgctacagcaaccgaacaaaaacctatt600tatgcagaccctacattccagcccgaaccccaaatcggggagtcccagtggaatgaggca660gatgctacagtcgctggtggtagagtgctcaagaaaaccactcccatgaaaccatgctat720ggttcctatgcaagacccacgaatgctaatggaggtcagggtgtactagcggcaaatgcc780caaggacagctagaatctcaggttgaaatgcaattcttttcaacttctgaaaacgcccgt840aacgaggctaacaacattcagcccaaattggtgctgtatagcgaggatgtgcacatggag900accccggatacacacctctcttacaagcccacaaaaagcgatgacaattctaaagttatg960ctgggccaacaggccatgcccaacaggcctaattacattggcttcagagacaactttatc1020ggtctcatgtactacaacagcactggcaacatgggagtgcttgcaggtcaggcctctcag1080ttgaatgcagtggtggacttgcaagacagaaacacagaactgtcctaccagctcttgctt1140gattccatgggtgacagaaccagatatttctccatgtggaatcaggcagtggacagttat1200gacccagatgtcagaattattgaaaatcatggaactgaagacgagctccccaactattgt1260ttccctctgggcggcataggggtaactgacacttaccaggctgttaagaccaacaatggc1320aataatgggggtcaggtgacttggacaaaagatgaaacttttgcagagcgcaatgagata1380ggggtgggaaacaatttcgccatggagatcaacctcaatgccaacctgtggaggaacttc1440ctgtactccaacgtggccctgtacctgccagacaagcttaagtacaacccctccaacgtg1500gacatctctgacaaccccaacacctacgattacatgaacaagcgagtggtggccccgggg1560ctggtggactgctacatcaacctgggcgcgcgctggtcgctggactacatggacaacgtc1620aaccctttcaaccaccaccgcaacgcgggcctgcgctaccgctccatgctcctgggcaac1680gggcgctacgtgcccttccacatccaggtgccccagaagttctttgccatcaagaacctc1740ctcctcctgccgggctcctacacctacgagtggaacttcaggaaggatgtcaacatggtc1800ctccagagctctctgggcaacgatctcagggtggacggggccagcatcaagttcgagagc1860atctgcctctacgccaccttcttccccatggcccacaacaccgcctccacgctcgaggcc1920atgctcaggaacgacaccaacgaccagtccttcaatgactacctctccgccgccaacatg1980ctctaccccatccccgccaacgccaccaacgtccccatctccatcccctcgcgcaactgg2040gcggccttccgcggctgggccttcacccgcctcaagaccaaggagaccccctccctgggc2100tcgggattcgacccctactacacctactcgggatccattccctacctggacggcaccttc2160tacctcaaccacactttcaagaaggtctcggtcaccttcgactcctcggtcagctggccg2220ggcaacgaccgcctgctcacccccaacgagttcgagatcaagcgctcggtcgacggggag2280ggctacaacgtggcccagtgcaacatgaccaaggactggttcctggtccagatgctggcc2340aactacaacatcggctaccagggcttctacatcccagagagctacaaggacaggatgtac2400tccttcttcaggaacttccagcccatgagccggcaggtggtggaccagaccaagtacaag2460gactaccaggaggtgggcatcatccaccagcacaacaactcgggcttcgtgggctacctc2520gcccccaccatgcgcgagggacaggcctaccccgccaacttcccctacccgctcataggc2580aagaccgcggtcgacagcatcacccagaaaaagttcctctgcgaccgcaccctctggcgc2640atccccttctccagcaacttcatgtccatgggtgcgctcacggacctgggccagaacctg2700ctctatgccaactccgcccacgcgctcgacatgaccttcgaggtcgaccccatggacgag2760cccacccttctctatgttctgttcgaagtctttgacgtggtccgggtccaccagccgcac2820cgcggcgtcatcgagaccgtgtacctgcgcacgcccttctcggccggcaacgccaccacc2880taa2883&lt;210&gt;23&lt;211&gt;2835&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad16六邻体&lt;400&gt;23atggccaccccatcgatgctgccccagtgggcgtacatgcacatcgccggacaggacgct60tcggagtacctgagtccgggtctggtgcagttcgcccgcgccacagacacctacttcagt120ctggggaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgc180agccagcggctgacgctgcgcttcgtgcccgtggaccgcgaggacaacacctactcgtac240aaagtgcgctacacgctggccgtgggcgacaaccgcgtgctggacatggccagcacctac300tttgacatccgcggcgtgctggaccggggccctagcttcaaaccctactccggcaccgcc360tacaacagcctggcccccaagggagctcccaattccagtcagtgggagcagacggagaac420gggggcggacaggctacgactaaaacacacacctatggagttgccccaatgggtggaact480aatattacagtcgacggactacaaattggaactgacgctacagctgatacggaaaaacca540atttatgctgataaaacattccaacctgagcctcagataggagaggaaaactggcaagaa600actgaaagcttttatggcggtagggctcttaagaaagacacaaacatgaagccttgttat660ggctcatttgccagacctaccaatgaaaagggaggtcaagctaaacttaaagttggagct720gatgggctgccgaccaaagaatttgacatagacctagcattctttgatactcctggtggc780actgtgaccggaggtacagaggagtataaagcagatattgttatgtataccgaaaacacg840tatctggaaactccagacacacatgtggtgtataaaccaggcaaggataacacaagttct900aaaattaacctggtccagcagtctatgcccaacaggcccaactacattgggtttagggac960aactttattgggctcatgtattacaacagcactggcaatatgggtgtgctggccggtcag1020gcttctcagttgaatgctgtggttgacttgcaagacagaaacactgaactgtcttaccag1080ctcttgcttgactctttgggtgacagaaccaggtatttcagtatgtggaatcaggcggtg1140gacagttatgatcctgatgtgcgcattattgaaaaccatggtgtggaagatgaacttccc1200aactattgcttccccctggatgggtctggcactaacgccgcttaccaaggtgtgaaagta1260aaaaatggtcaagatggtgatgttgagagcgaatgggaaaaagatgatactgtcgcagct1320cgaaatcaattatgcaagggcaacatttttgccatggagatcaatctccaggccaacctg1380tggagaagttttctctactcgaacgtggccctgtacctgcccgattcttacaagtacacg1440ccggccaacatcaccctgcccaccaacaccaacacctacgattacatgaacgggagagtg1500gtgcctccctcgctggtggacgcctacatcaacatcggggcgcgctggtcgctggacccc1560atggacaacgtcaatcccttcaaccaccatcgcaacgcggggctgcgctaccgctccatg1620ctcctgggcaacgggcgctacgtgcccttccacatccaggtgccccagaaatttttcgcc1680attaagagcctcctgctcctgcccgggtcctacacctacgagtggaacttccgcaaggac1740gtcaacatgatcctgcagagctccctcggcaacgacctgcgcacggacggggcctccatc1800tccttcaccagcatcaacctctacgccaccttcttccccatggcgcacaacaccgcctcc1860acgctcgaggccatgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcg1920gcggccaacatgctctaccccatcccggccaacgccaccaacgtgcccatctccatcccc1980tcgcgcaactgggccgccttccgcggctggtccttcacgcgcctcaagaccaaggagacg2040ccctcgctgggctccgggttcgacccctacttcgtctactcgggctccatcccctacctc2100gacggcaccttctacctcaaccacaccttcaagaaggtctccatcaccttcgactcctcc2160gtcagctggcccggcaacgaccggctcctgacgcccaacgagttcgaaatcaagcgcacc2220gtcgacggcgagggctacaacgtggcccagtgcaacatgaccaaggactggttcctggtc2280cagatgctggcccactacaacatcggctaccagggcttctacgtgcccgagggctacaag2340gaccgcatgtactccttcttccgcaacttccagcccatgagccgccaggtcgtggacgag2400gtcaactacaaggactaccaggccgtcaccctggcctaccagcacaacaactcgggcttc2460gtcggctacctcgcgcccaccatgcgccagggccagccctaccccgccaactacccctac2520ccgctcatcggcaagagcgccgtcgccagcgtcacccagaaaaagttcctctgcgaccgg2580gtcatgtggcgcatccccttctccagcaacttcatgtccatgggcgcgctcaccgacctc2640ggccagaacatgctctacgccaactccgcccacgcgctagacatgaatttcgaagtcgac2700cccatggatgagtccacccttctctatgttgtcttcgaagtcttcgacgtcgtccgagtg2760caccagccccaccgcggcgtcatcgaggccgtctacctgcgcacccccttctcggccggt2820aacgccaccacctaa2835&lt;210&gt;24&lt;211&gt;2883&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad17六邻体&lt;400&gt;24atggcgaccccatcgatgatgccgcagtggtcgtacatgcacatctcgggccaggacgcc60tcngagtacctgagccccgggctggtgcagttcgcccgcgccaccgagagctacttcagc120ctgagtaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgg180tctcagcgcctgacgctgcggttcattcccgtggaccgcgaggacaccgcgtactcgtac240aaggcgcggttcaccctggccgtgggcgacaaccgcgtgctggacatggcctccacctac300tttgacatccgcggggtgctggaccggggtcccactttcaagccctactctggcaccgcc360tacaactccctggcccccaagggcgctcccaactcctgcgagtgggagcaagaggaaact420caggcagttgaagaagcagcagaagaggaagaagaagatgctgacggtcaagctgaggaa480gagcaagcagctaccaaaaagactcatgtatatgctcaggctcccctttctggcgaaaaa540attagtaaagatggtctgcaaataggaacggacgctacagctacagaacaaaaacctatt600tatgcagaccctacattccagcccgaaccccaaatcggggagtcacagtggaatgaggca660gatgctacagtcgccggcggtagagtgctaaagaaatctactcccatgaaaccatgctat720ggttcctatgcaagacccacaaatgctaatggaggtcagggtgtactaacggcaaatgcc780cagggacagctagaatctcaggttgaaatgcaattcttttcaacttctgaaaacgcccgt840aacgagactaacaacattcagcccaaattggtgctgtatagtgaggatgtgcacatggag900accccggatacgcacctttcttacaagcccgcaaaaagcgatgacaattcaaaaatcatg960ctgggtcagcagtccatgcccaacagacctaattacatcggcttcagagataactttatc1020ggcctcatgtattacaatagcactggcaacatgggagtgcttgcaggtcaggcctctcag1080ttgaatgcagtggtggacttgcaagacagaaacacagaactgtcctaccagctcttgctt1140gattccatgggtgacagaaccagatacttttccatgtggaatcaggcagtggacagttat1200gacccagatgttagaattattgaaaatcatggaactgaagacgagctccccaactattgt1260ttccctctgggtggcataggggtaactgacacttaccaggctgttaaaaccaacaatggc1320aataacgggggccaggtgacttggacaaaagatgaaacttttgcagatcgcaatgaaata1380ggggtgggaaacaatttcgctatggagataaacctcagtgccaacctgtggagaaacttc1440ctgtactccaacgtggcgctgtacctaccagacaagcttaagtacaacccctccaatgtg1500gacatctctgacaaccccaacacctacgattacatgaacaagcgagtggtggccccgggg1560ctggtggactgctacatcaacctgggcgcgcgctggtcgctggactacatggacaacgtc1620aaccccttcaaccaccaccgcaatgcgggcctgcgctaccgctccatgctcctgggcaac1680gggcgctacgtgcccttccacatccaggtgccccagaagttctttgccatcaagaacctc1740ctcctcctgccgggctcctacacctacgagtggaacttcaggaaggatgtcaacatggtc1800ctccagagctctctgggtaacgatctcagggtggacggggccagcatcaagttcgagagc1860atctgcctctacgccaccttcttccccatggcccacaacacggcctccacgctcgaggcc1920atgctcaggaacgacaccaacgaccagtccttcaatgactacctctccgccgccaacatg1980ctctaccccatacccgccaacgccaccaacgtccccatctccatcccctcgcgcaactgg2040gcggccttccgcggctgggccttcacccgcctcaagaccaaggagaccccctccctgggc2100tcgggattcgacccctactacacctactcgggctccattccctacctggacggcaccttc2160tacctcaaccacactttcaagaaggtctcggtcaccttcgactcctcggtcagctggccg2220ggcaacgaccgtctgctcacccccaacgagttcgagatcaagcgctcggtcgacggggag2280ggctacaacgtggcccagtgcaacatgaccaaggactggttcctggtccagatgctggcc2340aactacaacatcggctaccagggcttctacatcccagagagctacaaggacaggatgtac2400tccttcttcaggaacttccagcccatgagccggcaggtggtggaccagaccaagtacaag2460gactaccaggaggtgggcatcatccaccagcacaacaactcgggcttcgtgggctacctc2520gcccccaccatgcgcgagggacaggcctaccccgccaacttcccctatccgctcataggc2580aagaccgcggtcgacagcatcacccagaaaaagttcctctgcgaccgcaccctctggcgc2640atccccttctccagcaacttcatgtccatgggtgcgctctcggacctgggccagaacttg2700ctctacgccaactccgcccacgccctcgacatgaccttcgaggtcgaccccatggacgag2760cccacccttctctatgttctgttcgaagtctttgacgtggtccgggtccaccagccgcac2820cgcggcgtcatcgagaccgtgtacctgcgtacgcccttctcggccggcaacgccaccacc2880taa2883&lt;210&gt;25&lt;211&gt;2877&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad19六邻体&lt;400&gt;25atggcgaccccatcgatgatgccgcagtggtcgtacatgcacatctcgggccaggacgcc60tcggagtacctgagccccgggctggtgcagttcgcccgcgccaccgagagctacttcagt120ctgagtaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgg180tcccagcgcctgacgctgcggttcatccccgtggaccgcgaggacaccgcgtactcgtac240aaggcgcggttcaccctggccgtgggcgacaaccgcgtgctggacatggcctccacctac300tttgacatccgcggcgtgctggaccgcggccccaccttcaagccctactccggcaccgcc360tacaactccctggcccccaagggcgctcccaactcttgtgagtgggagcaattagaagaa420gcccaggccgctttggaagacgaagaattagaagatgaagacgaggaaccacaggatgag480gcgcctgtgaaaaagacccatgtatacgctcaggctcccctttctggagaagaaattact540aaagacggtttgcaaatagggtcagataacacagaagctcagtctaagcctatatatgca600gaccctacattccagcccgaaccccaaatcggggagtcccagtggaacgaggcagatgct660acagtcgctggtggtagagtgctcaagaaaaccactcccatgaaaccatgctatggttcc720tatgcaagacccacgaatgctaatggaggtcagggtgtgctggtggctgatgataagggg780gtccttcaatctaaagttgaattgcaatttttttcaaatactactactcttaatcagcgg840gagggtaatgatacaaaaccaaaagtagtgctgtatagcgaggatgtgcacatggaaaca900ccagacacccacatttcttacaagcccacaaaaagcgatgacaattctaaagttatgctg960ggccaacagtccatgcccaacaggcctaattacatcggcttcagagacaactttatcggt1020ctcatgtactacaacagcactggcaacatgggagtgcttgcaggtcaggcctctcagttg1080aatgcagtggtggacttgcaagacagaaacacagaactgtcctaccagctcttgcttgat1140tccatgggtgacagaaccagatatttctccatgtggaatcaggcagtggacagttatgac1200ccggatgtcagaattattgaaaatcatggaaccgaagacgagctccccaactattgtttt1260cctctgggtggcataggggtaactgacacttaccaggtcattaaaactaatggcaatggt1320caagcagacccaacctgggaaaaagatacagagtttgcagaccgcaatgaaataggggtg1380ggaaacaatttcgccatggagatcaacctcaatgccaacctgtggaggaacttcctgtac1440tccaacgtggccctgtacctgccagacaagcttaagtacaacccctccaacgtggacatc1500tctgacaaccccaacacctacgattacatgaacaagcgagtggtggccccggggctggtg1560gactgctacatcaacctgggcgcgcgctggtcgctggactacatggacaacgtcaacccc1620ttcaaccaccaccgcaacgcgggcctgcgctaccgctccatgctcctgggcaacgggcgc1680tacgtgcccttccacatccaggtgccccagaagttctttgccatcaagaacctcctcctc1740ctgccgggctcctacacctacgagtggaacttcaggaaggatgtcaacatggtcctccag1800agctctttgggcaacgatctcagggtggacggggccagcatcaagttcgagagcatctgc1860ctctacgccaccttcttccccatggcccacaacaccgcctccacgctcgaggccatgctc1920aggaacgacaccaacgaccagtccttcaatgactacctctccgccgccaacatgctctac1980cccatccccgccaacgccaccaacgtccctatctccatcccctcgcgcaactgggcggcc2040ttccgcggctgggccttcacccgcctcaagaccaaggagacaccctccctgggctcggga2100ttcgacccctactacacctactcgggatccattccctacctggacggcaccttctacctc2160aaccacactttcaagaaggtctcggtcaccttcgactcctcggtcagctggccgggcaac2220gaccgcctgctcacccccaacgagttcgagatcaagcgctcggtcgacggggagggctac2280aacgtggcccagtgcaacatgaccaaggactggttcctggtccagatgctggccaactac2340aacatcggctaccagggcttctacatcccagagagctacaaggacaggatgtactccttc2400ttcaggaacttccagcccatgagccggcaggtggtggaccaaaccaagtacaaggactac2460caggaggtgggcatcatccaccagcacaacaactcgggcttcgtgggctacctcgccccc2520accatgcgcgagggacaggcctaccccgccaacttcccctacccgctcataggcaagacc2580gcggtcgacagcatcacccagaaaaagttcctctgcgaccgcaccctctggcgcatcccc2640ttctccagcaacttcatgtccatgggtgcgctcacggacctgggccagaacctgctctat2700gccaactccgcccacgcgctcgacatgaccttcgaggtcgaccccatggacgagcccacc2760cttctctatgttctgttcgaagtctttgacgtggtccgggtccaccagccgcaccgcggc2820gtcatcgagaccgtgtacctgcgcacgcccttctcggccggcaacgccaccacctaa2877&lt;210&gt;26&lt;211&gt;49&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;pola&lt;400&gt;26aataaaagatctttattttcattagatctgtgtgttggttttttgtgtg49&lt;210&gt;27&lt;211&gt;37&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;27atggaattcgtttaaaccatcatcaataatatacctc37&lt;210&gt;28&lt;211&gt;23&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;28cgctggcactcaagagtggcctc23&lt;210&gt;29&lt;211&gt;37&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;29atgaagcttgtttaaacccatcatcaataatatacct37&lt;210&gt;30&lt;211&gt;26&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;30atctagacagcgtccatagcttaccg26&lt;210&gt;31&lt;211&gt;44&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;31atgctacgtagcgatcgcgtgagtagtgtttgggggtgggtggg44&lt;210&gt;32&lt;211&gt;33&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;32taggcgcgccgcttctcctcgttcaggctggcg33&lt;210&gt;33&lt;211&gt;38&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;33gatctagttagtttaaacgaattcggatctgcgacgcg38&lt;210&gt;34&lt;211&gt;37&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;34ttcgatcatgtttaaacgaaattaagaattcggatcc37&lt;210&gt;35&lt;211&gt;33&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;35tattctgcgatcgctgaggtgggtgagtgggcg33&lt;210&gt;36&lt;211&gt;31&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;36taggcgcgcccttaaacggcatttgtgggag31&lt;210&gt;37&lt;211&gt;27&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;37cgtctagaagacccgagtcttaccagt27&lt;210&gt;38&lt;211&gt;39&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;38cgggatccgtttaaaccatcatcaataatataccttatt39&lt;210&gt;39&lt;211&gt;37&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;39atggaattcgtttaaaccatcatcaataatatacctt37&lt;210&gt;40&lt;211&gt;45&lt;212&gt;dna&lt;213&gt;人工序列&lt;220&gt;&lt;223&gt;寡聚物&lt;400&gt;40atgacgcgatcgctgatatcctataataataaaacgcagactttg45&lt;210&gt;41&lt;211&gt;2880&lt;212&gt;dna&lt;213&gt;黑猩猩腺病毒-chad3六邻体&lt;400&gt;41atggcgaccccatcgatgatgccgcagtggtcgtacatgcacatctcgggccaggacgcc60tcggagtacctgagccccgggctggtgcagttcgcccgcgccaccgagagctacttcagc120ctgagtaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgg180tctcagcgcctgacgctgcggttcattcccgtggaccgcgaggacaccgcgtactcgtac240aaggcgcggttcaccctggccgtgggcgacaaccgcgtgctggacatggcctccacctac300tttgacatccgcggggtgctggaccggggtcccactttcaagccctactctggcaccgcc360tacaactccctggcccccaagggcgctcccaactcctgcgagtgggagcaagaggaaact420caggcagttgaagaagcagcagaagaggaagaagaagatgctgacggtcaagctgaggaa480gagcaagcagctaccaaaaagactcatgtatatgctcaggctcccctttctggcgaaaaa540attagtaaagatggtctgcaaataggaacggacgctacagctacagaacaaaaacctatt600tatgcagaccctacattccagcccgaaccccaaatcggggagtcccagtggaatgaggca660gatgctacagtcgccggcggtagagtgctaaagaaatctactcccatgaaaccatgctat720ggttcctatgcaagacccacaaatgctaatggaggtcagggtgtactaacggcaaatgcc780cagggacagctagaatctcaggttgaaatgcaattcttttcaacttctgaaaacgcccgt840aacgaggctaacaacattcagcccaaattggtgctgtatagtgaggatgtgcacatggag900accccggatacgcacctttcttacaagcccgcaaaaagcgatgacaattcaaaaatcatg960ctgggtcagcagtccatgcccaacagacctaattacatcggcttcagagacaactttatc1020ggcctcatgtattacaatagcactggcaacatgggagtgcttgcaggtcaggcctctcag1080ttgaatgcagtggtggacttgcaagacagaaacacagaactgtcctaccagctcttgctt1140gattccatgggtgacagaaccagatacttttccatgtggaatcaggcagtggacagttat1200gacccagatgttagaattattgaaaatcatggaactgaagacgagctccccaactattgt1260ttccctctgggtggcataggggtaactgacacttaccaggctgttaaaaccaacaatggc1320aataacgggggccaggtgacttggacaaaagatgaaacttttgcagatcgcaatgaaata1380ggggtgggaaacaatttcgctatggagatcaacctcagtgccaacctgtggagaaacttc1440ctgtactccaacgtggcgctgtacctaccagacaagcttaagtacaacccctccaatgtg1500gacatctctgacaaccccaacacctacgattacatgaacaagcgagtggtggccccgggg1560ctggtggactgctacatcaacctgggcgcgcgctggtcgctggactacatggacaacgtc16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