CN103282375B - 肽支架设计 - Google Patents
肽支架设计 Download PDFInfo
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- CN103282375B CN103282375B CN201180058235.0A CN201180058235A CN103282375B CN 103282375 B CN103282375 B CN 103282375B CN 201180058235 A CN201180058235 A CN 201180058235A CN 103282375 B CN103282375 B CN 103282375B
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Abstract
本发明涉及用于病毒感染的治疗、诊断和预后的新的肽和方法,所述病毒感染包括HCV、HIV、CMV感染和流感。本发明还涉及鉴定并提供用于治疗和诊断的肽的方法。X1‑X2‑X3‑X4‑X5 (I)。
Description
发明领域
本发明涉及用于病毒感染的治疗、诊断和预后的新的肽和方法,所述病毒感染包括HCV、HIV、CMV感染和流感。本发明还涉及鉴定并提供用于治疗和诊断的肽的方法。
发明背景
常规的疫苗开发途径已经使用了已经减毒的完整的有复制能力的病毒(例如萨宾脊髓灰质炎疫苗(Sabin polio vaccine),麻疹,流行性腮腺炎,风疹(MMR))或没有复制能力的灭活的病毒体。偶尔地,灭活的病毒疫苗可以包括裂解疫苗,其中病毒粒子已被破坏。分子技术也已经用来开发亚单位疫苗(例如乙型肝炎疫苗),其仅由乙型肝炎病毒的表面糖蛋白组成。灭活病毒疫苗趋向于诱发针对讨论的病毒的原发性抗体应答,而活的减毒疫苗由于所述疫苗诱发瞬时感染而诱发细胞介导的免疫和抗体应答。
利用成功的接种计划已经消除的唯一的疾病是天花。目前的计划是继续消除脊髓灰质炎。可以通过接种消除的病毒感染的特征为由具有稳定的病毒抗原的病毒(例如,非常低的突变频率、较少的亚型)引起的感染,所述病毒在其他动物物种中缺少储备池(reservoir),由感染结束后不在体内持续的病毒引起的感染(其中接种引起长久持续的免疫)。诸如脊髓灰质炎和麻疹的病毒满足这些标准,而诸如流感病毒(Flu)、HCV和HIV的其蛋白序列变化的病毒不满足这些标准。出于这种原因,需要开发用于这些疾病的疫苗的新的替代的途径。
接种的目的是在感染之前刺激针对特定病原体的免疫应答。当个体暴露于所述病原体时,引发记忆应答,其防止感染的建立。因此,疫苗刺激适应性免疫应答,其与先天免疫不同,是活得长久并且具有记忆。对于适应性免疫系统具有两种主要的方式。体液免疫,其包括发展能够结合病毒粒子的抗体和能够中和感染的某些抗体。细胞介导的免疫,其导致发展细胞毒性T细胞,在I类人白细胞抗原(HLA)的情形中,其杀死暴露病毒表位的感染细胞,以这种方式清除感染的细胞。
提供适于刺激适应性免疫系统的疫苗的挑战在于肽表位需要被抗原呈递细胞摄取。
已经证明一些肽通过似乎是能量不依赖性途径穿过真核细胞的质膜易位。这些肽被定义为细胞穿透肽(CPPs)。与常规技术相比,使用这些细胞穿透肽进行细胞递送提供一些优势。其是非侵入性的、能量不依赖性的,有效用于宽泛范围的细胞类型,并且可以一齐(en masse)用于细胞。
肝炎意指肝脏炎症,可由多种因素引起,包括由毒素、某些药物、一些疾病、酗酒和细菌感染与病毒感染引起。肝炎还是影响肝脏的病毒感染家族的名称;发达国家中最常见的类型是甲型肝炎、乙型肝炎和丙型肝炎。
丙型肝炎是由丙型肝炎病毒(HCV)感染导致的肝病。其严重性范围可以从持续几周的轻微疾病到严重的、终生的疾病。丙型肝炎通过血液传播;最常见的传播形式是通过共用针具或其他用于注射药物的器具。感染可以是“急性的”或“慢性的”。急性HCV感染是在某人暴露于丙型肝炎病毒后前6个月内发生的无临床症状的、短期的疾病。对于大部分人,急性感染引起慢性感染,其可导致长期的并发症,甚至死亡。
HCV是包膜正链核糖核酸(RNA)病毒,直径约50nm,属于黄病毒科(Flaviviridae)肝炎病毒属(Hepacivirus),其在感染的细胞的胞质中复制。唯一已知的HCV储蓄池是人,尽管该病毒在实验中已经传播给黑猩猩。HCV的天然靶标是肝细胞,并且可能是B淋巴细胞。到2008年,已知该病毒六种不同的基因型和超过100种的亚型。复制通过缺乏校正功能的RNA-依赖型RNA聚合酶发生,这导致非常高的突变率。在HCV基因组编码包膜蛋白的高变区中的快速突变允许该病毒逃避宿主的免疫监督。结果,大部分感染HCV的人发展成了慢性感染。
估计全世界有一亿七千万人感染了HCV,等于全球人口的大约3%。每年还大约有3-4百万人感染;估计这些新感染的患者中有80%发展成慢性感染。
HCV的6种基因型具有不同的地理分布。该疾病在早期阶段一般是无临床症状的;大部分患有慢性感染的患者最终发展成并发症,诸如肝纤维化和肝硬化,并且在1-5%的病例中,发展成肝细胞癌。
HCV是世界上非甲型、非乙型肝炎的主要病因。急性HCV感染通常引起慢性肝炎和末期肝硬化。估计多至20%的HCV慢性携带者可在大约20年的时间期间内发展成肝硬化,并且患有肝硬化的那些中有1-4%有发展肝癌的危险。
约9.6kb的HCV病毒单链RNA基因组包含5′-和3′-非编码区(NCRs),并且,在这些NCRs之间,是编码约3000个氨基酸的HCV聚蛋白的约9kb的单一长开放阅读框。
HCV多肽由所述开放阅读框的翻译和共翻译蛋白酶解加工产生。结构蛋白来源于氨基端四分之一的编码区,并且包括衣壳或核心蛋白(约21kDa),E1包膜糖蛋白(约35kDa)和E2包膜糖蛋白(约70kDa,之前称为NS1)和p7(约7kDa)。E2蛋白可能以在C端融合或不融合p7蛋白而存在(Shimotohno等,1995)。已经发现了在核心区中的可变开放阅读框(alternative reading frame),其编码并且表达称为F(Frameshift)蛋白的约17kDa的蛋白(Xu等,2001;Ou&Xu,美国专利申请公开号US2002/0076415)。在该区域,发现了关于其他14-17kDa的ARFPs(可变阅读框蛋白(Alternative Reading Frame Proteins))(即,A1至A4)的ORFs,并且在慢性感染的患者的血清中检测到针对至少A1、A2和A3的抗体(Walewski等,2001)。从HCV编码区的其余部分,产生非结构性HCV蛋白,其包括NS2(约23kDa)、NS3(约70kDa)、NS4A(约8kDa)、NS4B(约27kDa)、NS5A(约58kDa)和NS5B(约68kDa)(Grakoui等,1993)。
流感持续为世界上死亡率和发病率的主要原因。世界卫生组织(WHO)估计季节性流行病每年影响3-5百万患有严重疾病的人,并且导致250,000-500,000例死亡。流感是由正粘病毒科(Orthomyxoviridae)的病毒引起的,其为负链RNA病毒。流感病毒以三种类型存在:甲、乙和丙型,其中只有甲型与流行性疾病相关。发现甲型病毒存在于人和动物中,特别是鸟类中,而且还存在其他哺乳动物如猪中。甲型病毒依据病毒表面蛋白的不同种类和组合进一步分型为多个亚型。在多个亚型中,在2009年,甲型流感(H1N1)亚型和甲型流感(H3N2)亚型在人中传播。甲和乙型流感包括在季节性疫苗中,而丙型流感很少发生,因此不包括在季节性疫苗中。乙型病毒是人特异性的,丙型病毒引起非常轻微的疾病。正粘病毒科的基因组是分区段的。甲和乙型流感病毒具有8个区段,而丙型具有七个区段。流行病可能是由于当两个不同的甲型病毒感染同一细胞时基因区段的重分类而引起。在人群中没有针对这种新型的重分类病毒的免疫。在二十世纪发生了三次流行病:1918年的“西班牙流感”,1957年的“亚洲流感”和1968年的“香港流感”。1918年的流行病在世界范围内致使估计四千万-五千万人死亡。后续的流行病要轻得多,1957年估计有两百万例死亡,1968年有一百万例死亡。2009年6月,WHO宣布由流感病毒H1N1(猪流感)引起的流行病,在2010年8月宣布其结束。
人乳头状瘤病毒由乳头状瘤病毒科(Papillomaviridae)的一组DNA病毒构成,其感染皮肤和黏膜。来源于100种不同的已鉴定的亚型的两组是主要的临床关注病因:引起疣的那些(良性的和生殖器疣),和一组可以导致宫颈癌的12种“高危险性”亚型。后一组已经归因为发生几乎所有类型的宫颈癌的作用因素。在世界范围内,宫颈癌保持为妇女的第二大常见恶性病,并且是发展中国家中女性癌症相关的死亡的主要原因。HPV16和18已经主要与宫颈癌相关,然而,该病毒也是男人和妇女中喉癌的病因。HPV通过接触传播,并且通过擦伤(abraisions)进入皮肤。仅有早期蛋白表达的无效感染(abortive infection)与癌症发生相关。
发明目的
本发明实施方案的目的是提供可以用作免疫原在受试者中刺激适应性免疫应答的肽。
具体地,本发明实施方案的目的是提供这样的肽:所述肽可以被抗原呈递细胞(巨噬细胞和树突细胞)摄取,以使所述肽内的表位被正确地处理并呈递到T淋巴细胞,从而刺激有效的免疫应答。
此外,本发明实施方案的目的是提供可以用作抗原的肽,提供用于在受试者中诱导针对抗原的免疫应答的免疫原性组合物和方法。
发明概述
本发明涉及促进抗原呈递细胞(巨噬细胞和树突细胞)摄取肽表位以使在I和II类HLA的情形中所述表位被正确处理和呈递从而刺激CD4+和CD8+T淋巴细胞两者的肽设计。具有细胞毒性能力的CD8+T淋巴细胞将杀死携带目的表位的已感染细胞。CD4+T淋巴细胞提供‘帮助’以保持有效的CD8+T淋巴细胞应答。
本发明人已经发现,肽构建体-具有特定的模式或支架设计的氨基酸序列-具有有效穿透细胞膜的能力。因此,本发明所述的肽构建体可以用于以能够被巨噬细胞和树突细胞呈递的免疫原性有效量的肽或该肽的片段负载细胞。因此,这些肽构建体可以激发细胞毒性T淋巴细胞免疫(CTL)应答和/或体液免疫应答。
因此,在第一方面,本发明涉及具有下述结构的不超过60个氨基酸的分离的细胞穿透肽:
X1-X2-X3-X4-X5(式I),
其中X1和X3独立地定义任意1,2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的线性序列;X2定义来源于抗原的8-30个氨基酸的线性序列;X4定义来源于所述抗原的8-30个氨基酸的线性序列,所述序列X4不同于X2;并且其中X5是选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的任意一个任选的氨基酸。
应该理解,式I的氨基酸序列是指以标准的N-至C-端方向存在的肽序列,其中提及的第一个氨基酸是可具有氨基(-NH2)基团或备选地-NH3 +基团的N-端氨基酸。提及的最后一个氨基酸是可具有游离的羧基(-COOH)或羧基化基团的C端氨基酸。在一些实施方案中,N-和/或C-端氨基酸被修饰,诸如通过N端乙酰化或C-端酰胺化修饰。式I中所用的符号“-”是指标准的肽键,诸如以“X1-X2”表示X1和X2之间的标准肽键。
还应该理解,本发明所述的肽主要意欲用于合成肽的合成。然而,如果通过通过重组方式产生所述肽,则所述肽可长于60个氨基酸。因此,本发明备选的第一方面涉及包含具有下述结构的肽序列的分离的细胞穿透肽:
X1-X2-X3-X4-X5(式I),
其中X1和X3独立地定义任意1,2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的线性序列;X2定义来源于抗原的8-30个氨基酸的线性序列;X4定义来源于所述抗原的8-30个氨基酸的线性序列,所述序列X4不同于X2;并且其中X5是选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的任意一个任选的氨基酸。
在第二方面,本发明涉及不多于60个氨基酸的分离的肽,所述肽包含在表1或表2中任一个中独立定义的X2和/或X4序列。
在第三方面,本发明涉及包含在表1或表2中任一个中独立定义的X2或X4序列的肽用于在受试者中诱导免疫应答的应用。
在第四方面,本发明涉及由表1或表2中任一个定义的X1-X5组成分离的肽。
在另一个方面,本发明涉及包含两个肽单体的二聚体肽,其中每个肽单体如本发明所述。
在另一个方面,本发明涉及包含两个以上本发明所述的肽的肽组合。
在另一个方面,本发明涉及编码本发明所述的肽的分离的核酸或多核苷酸。
在另一个方面,本发明涉及包含编码本发明所述的肽的核酸或多核苷酸的载体。
在另一个方面,本发明涉及包含含有编码本发明所述的肽的核酸或多核苷酸的载体的宿主细胞。
在另一个方面,本发明涉及包含与药用稀释剂或赋形剂和任选地免疫学佐剂组合的下述各项的免疫原性组合物:至少一个本发明所述的肽,包含两个肽单体的二聚体肽,其中每个肽单体如本发明所述,包含两个以上本发明所述的肽的肽组合,编码本发明所述的肽的核酸或多核苷酸,或包含编码本发明所述的肽的核酸或多核苷酸的载体。
在另一个方面,本发明涉及用于在受试者中诱导针对抗原的免疫应答的方法,所述方法包括施用下述:至少一种本发明所述的肽;包含两个肽单体的二聚体肽,其中每个肽单体如本发明所述;包含两种以上本发明所述的肽的肽组合;编码本发明所述的肽的核酸或多核苷酸;包含编码本发明所述的肽的核酸或多核苷酸的载体;或本发明所述的组合物。
在另一个方面,本发明涉及用于在感染病毒的受试者中减轻和/或延缓所述病毒的病理作用的方法,所述方法包括施用下述:至少一种本发明所述的肽;包含两个肽单体的二聚体肽,其中每个肽单体如本发明所述;包含两种以上本发明所述的肽的肽组合;编码本发明所述的肽的核酸或多核苷酸;包含编码本发明所述的肽的核酸或多核苷酸的载体;或本发明所述的组合物。
在另一个方面,本发明涉及本发明所述的肽用作药物。
在另一个方面,本发明涉及本发明所述的肽用于治疗感染病毒的受试者中所述病毒的病理作用。
发明详述
定义
除非另外说明,当在本公开内容中使用术语诸如“一种(one)”、“一个(a)”或“一个(an)”时,它们是指“至少一个”,或者“一个或多个”。另外,术语“包含(comprising)”意欲指“包括(including)”并且因此除明确说明的那些外还允许出现其他组分、特征、条件或步骤。
“HIV”通常指示人免疫缺陷病毒I。
“HIV疾病”由若个阶段组成,包括本身通常表现为流感样感染的急性HIV感染,以及具有若个非特征症状的早期和中期阶段症状疾病,所述非特征症状诸如皮疹、疲劳、盗汗、轻微体重减轻、口腔溃疡、和由真菌引起的皮肤和指甲感染。大多数HIV感染者在发展成更严重的疾病前将经历诸如这些的轻微的症状。通常认为,最早的轻微症状需要五至七年来显现。随着HIV疾病的进展,一些个体会变得相当不健康,即使他们还未被诊断出患有AIDS(见下),HIV疾病的晚期。典型的问题包括慢性口腔或阴道腐烂(vaginal thrush)(真菌引起的疹或斑),嘴上的复发性疱疹疱(herpes blisters)(感冒疮(cold sores))或生殖器上的复发性疱疹疱,持续的发烧,持久的腹泻,以及显著的体重减轻。“AIDS”是晚期HIV疾病,并且是一种这样的病症,其逐渐地减弱免疫系统的有效性并且使个体易遭受机会性感染和肿瘤。
术语“细胞穿透肽”用在本文中是指具有似乎是能量不依赖性地穿过质膜易位至真核细胞和/或原核细胞的细胞质和/或核隔室(诸如细胞质、核、溶酶体、内质网、高尔基体、线粒体和/或叶绿体)中的能力的任意肽。本发明所述的“细胞穿透肽”的这种穿过质膜易位的能力可以是非侵入性的、能量不依赖性的、不饱和性的、和/或受体不依赖性的。在一个实施方案中,术语“细胞穿透肽”是指一种肽,证明其如通过实施例1的测定检测的那样穿过质膜易位。应该理解,本发明所述的细胞穿透肽可以序列完整无损地穿过膜易位,或者备选地被部分降解,但是以该肽中包含的抗原能够在细胞内呈递从而刺激免疫应答的形式易位。因此,本发明所述的细胞穿透肽是可以证明如通过实施例1的测定检测的那样穿过质膜易位并且可以证明刺激有效的免疫应答的肽。
如在本文中所用,术语“来源于抗原”在指肽来源的资源(诸如病毒等)时,意欲是指已经从该资源获得(例如,分离、纯化等)的肽。优选地,肽可以被遗传改造和/或化学合成以与所述资源的天然肽基本相同。该术语包括已知的天然肽序列的变体的应用,诸如这样的肽序列,其中天然肽序列的1,2,3,4,5,6,7,8,9或10个氨基酸已被任意其他的氨基酸置换,诸如保守置换。备选地,1,2,3,4,5,6,7,8,9或10个氨基酸已被去除或添加到天然肽序列上。因此,在一些实施方案中,本发明所述的肽包含序列X2和/或X4,其定义为来源于抗原的8-30个氨基酸、如8-20个氨基酸的序列,其中抗原的肽序列相对于所述抗原包含1,2,3,4,5,6,7,8,9或10个置换、添加或缺失,诸如在X2和/或X4氨基酸序列的N-或C-端添加精氨酸。用于本发明所述的氨基酸序列的氨基酸可以是L-和/或D-形式。应该理解,L-和D-形式可以用于同一肽序列中的不同氨基酸。在一些实施方案中,肽序列内的氨基酸是L-形式,诸如天然氨基酸。应该理解,任意已知的抗原可以用在本发明所述的构建体中。
在一些具体的实施方案中,本发明所述的氨基酸序列的N端的前1、2或3个氨基酸是D-形式的。认为通过在这些细胞穿透肽的N端具有D-形式的氨基酸在某种程度上延迟了所述肽的N-端修剪以及由此引起的降解。备选地,在一些实施方案中,在本发明所述的氨基酸序列的N端的前1、2或3个氨基酸是β或γ形式的氨基酸。β氨基酸的氨基连接在β碳上而不是如20种标准的天然氨基酸那样连接在α碳上。
备选地,本发明所述的氨基酸序列的N端的前1、2或3个氨基酸可以通过结合氟而被修饰,或者备选地,可以使用环形氨基酸或其他适当的非天然氨基酸。
肽的“变体”或“类似物”是指具有与参比肽(典型地天然的或“亲本”多肽)基本相同的氨基酸序列的肽。肽变体在天然氨基酸序列内的特定位置可以具有一个或多个氨基酸置换、缺失和/或插入。
“保守”氨基酸置换是其中氨基酸残基被带有具有相似理化性质的侧链的氨基酸残基置换的那些置换。具有相似侧链的氨基酸残基家族在本领域中是已知的,并且包括带有碱性侧链的氨基酸(例如,赖氨酸、精氨酸、组氨酸),带有酸性侧链的氨基酸(例如,天冬氨酸、谷氨酸),带有不带电荷的极性侧链的氨基酸(例如,甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸),带有非极性侧链的氨基酸(例如,丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸),带有β分支侧链的氨基酸(例如,苏氨酸、缬氨酸、异亮氨酸)和带有芳香族侧链的氨基酸(例如,酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守氨基酸置换的特定形式包括用不在由遗传密码编码的正常的20个氨基酸中的氨基酸置换的那些。因为本发明优选的实施方案需要使用合成肽,所以在本文公开的肽中设置这种“非天然存在的”氨基酸残基是没有问题的,并且因此可将氨基酸残基侧链中的天然饱和碳链交换为更短或更长的饱和碳链——例如,赖氨酸可以用具有侧链-(CH2)nNH3的氨基酸置换,其中n不是4,而精氨酸可以用具有侧链-(CH2)nNHC(=NH2)NH2的氨基酸置换,其中n不是3,等等。相似地,酸性氨基酸天冬氨酸和谷氨酸可以用具有侧链-(CH2)nCOOH的氨基酸残基置换,其中n>2。
在两个氨基酸序列的情形中,术语“基本相同”是指当被最优化比对时,诸如通过程序GAP或BESTFIT使用默认缺口权重(gap weight)比对时,所述序列共有至少约50%、至少约60%、至少约70%、至少约80%、至少约90%、至少约95%、至少约98%或至少约99%的序列同一性。在一个实施方案中,不相同的残基位置差别在于保守氨基酸置换。典型地使用序列分析软件测定序列同一性。蛋白质分析软件使用分配给不同置换、缺失和其他修饰(包括保守氨基酸置换)的相似性测量来匹配相似的序列。例如,公共可用的GCG软件包含诸如“Gap”和“BestFit”的程序,可以以默认参数使用所述软件来确定紧密相关的多肽(诸如来自不同生物物种的同源多肽)之间或野生型蛋白质与其突变蛋白质之间的序列同源性或序列同一性。例如,参见GCG版本6.1。也可以使用FASTA或ClustalW,使用默认或推荐参数比较多肽序列。GCG版本6.1.中的程序,FASTA(例如,FASTA2和FASTA3)给出查询和检索序列之间的最佳重叠区域的比对和百分比序列同一性(Pearson,Methods Enzymol.(酶学方法)1990;183:63-98;Pearson,Methods Mol.Biol.(分子生物学方法)2000;132:185-219)。当将序列与包含大量来自不同生物体的序列的数据库比较时,或者当推断时,另一种优选的算法是使用默认参数的计算机程序BLAST,尤其是blastp。例如,参见Altschul等,J.Mol.Biol.(分子生物学杂志)1990;215:403-410;Altschul等,Nucleic Acids Res.(核酸研究)1997;25:3389-402(1997);所述文献各自通过引用结合于本文。在两个基本相同的氨基酸序列中,“相对应的”氨基酸位置是通过本文中提及的蛋白质分析软件中的任一个(典型地使用默认参数)比对的那些。
“分离的”分子是这样的分子,所述分子在其中它被发现的组合物中相对于它所属的分子种类是占优势的种类(即,它在组合物中组成所述类型分子的至少约50%,并且在组合物中典型地将组成所述种类分子(例如肽)的至少约70%、至少约80%、至少约85%、至少约90%、至少约95%或更多)。一般,肽分子的组合物在所述组合物中所有存在的肽种类的背景下,或至少在被提议的用途的背景下相对基本上有活性的肽种类,将显示98%-99%的肽分子同质性。
术语“线性序列”用在本文中是指以标准的N-至C-端方向通过标准肽键连接的特定的氨基酸序列。所述肽可以进包含肽键。然而,该术语不排除序列内的氨基酸,诸如在X3内的氨基酸,可以诸如通过侧链与在肽序列内的远端位置的另一个氨基酸(诸如在X3内的远端位置)连接。
在本发明的情形中,除非与上下文矛盾,“治疗(“treatment”或“treating”)”是指预防、缓和、处理、治愈或减轻疾病或病症的一种或多种症状或临床相关的表现。例如,对其中未鉴定出疾病或病症的症状或临床相关表现的患者的“治疗”是预防的或预防性的疗法,而对其中鉴定出疾病或病症的症状或临床相关表现的患者的“治疗”通常不构成预防的或预防性的疗法。
术语“抗原”指示这样的物质,其被免疫系统的特异性识别成分(抗体、T细胞)识别。
在本文的情形中,术语“免疫原”意欲指示这样的物质,所述物质能够诱导个体中的适应性免疫应答,其中所述适应性免疫应答靶向所述免疫原。涉及本发明,免疫原将诱导体液免疫应答和/或细胞介导的免疫应答。换言之,免疫原是能够诱导免疫的抗原。
术语“表位”、“抗原决定簇”和“抗原位点”在本文中被可互换地使用并且指示抗原或免疫原中这样的区域,所述区域被抗体识别(在抗体结合表位的情况中,也称为“B细胞表位”)或者当表位复合到主要组织相容性复合体(MHC)分子时被T细胞受体识别(在T细胞受体结合表位(即“T细胞表位”)的情况中)。
术语“免疫原性有效量”具有其在本领域中的通常含义,即能够诱导免疫应答的免疫原的量,所述免疫应答显著地攻击与所述免疫原享有免疫学特性的病原体。
术语“疫苗”用于这样的组合物,其包含免疫原并且能够诱导能够减小发展病理学病况的风险的免疫应答,或者能够诱导可以帮助治愈病理学病况(或至少减轻病理学病况的症状)的治疗有效的免疫应答。
术语“药用”具有其在本领域中的通常含义,即它用于这样的物质,当治疗所讨论的疾病时,其可以作为人用药物的一部分被接受,并且因此该术语有效地排除了使用将恶化而不是改善受治疗的受试者病况的高毒性物质。
“T辅助淋巴细胞表位”(TH表位)是这样的肽,其结合II类MHC分子并且可以在与II类MHC分子结合的抗原呈递细胞(APC)的表面上呈递。“免疫学载体”通常是这样的物质,其包含一个或许多个TH表位,并且通过保证T辅助淋巴细胞被活化并且增殖来增加针对与其偶联的抗原的免疫应答。已知的免疫学载体的实例是破伤风和白喉类毒素以及匙孔血蓝蛋白(keyhole limpet hemocyanin,KLH)。
在本发明所述的支架设计中,X2和X4定义来源于抗原的8-30个氨基酸如8-20个氨基酸的序列。来源于抗原的这一氨基酸序列在本文中可以称为表位。
优选地,用于本发明所述的支架中的表位是CTL表位。“CTL诱导肽”是能够诱导CTL应答的I类HLA结合肽。在其他实施方案中,用于本发明所述的支架设计中的表位是HTL诱导肽。“HTL诱导肽”是能够诱导HTL应答的II类HLA结合肽。
术语“碱性氨基酸”用在本文中是指按照Kice&Marvell“Modern Principles oforganic Chemsitry(现代有机化学原理)”(Macmillan,1974)或Matthews和van Holde“Biochemistry(生物化学)”Cummings Publishing Company(Cummings出版公司),1996检测具有高于6.3(如高于7.4)的等电点的任意氨基酸,包括天然氨基酸和非天然氨基酸。这一定义内包含精氨酸、赖氨酸、高精氨酸(Har)和组氨酸以及它们的衍生物。适当的非天然碱性氨基酸例如在US6,858,396中所述。适当的带正电荷的氨基酸包括可获自Bachem AG的非天然α氨基酸,并且包括α-氨基-甘氨酸,α,γ-二氨基-丁酸,鸟氨酸,α,β-二氨基-丙酸,α-二氟甲基-鸟氨酸,4-氨基-哌啶-4-羧酸,2,6-二氨基-4-己炔酸,β-(1-哌嗪基)-丙氨酸,4,5-脱氢-赖氨酸,δ-羟基-赖氨酸,ω-羟基-正精氨酸,高精氨酸,ω-氨基-精氨酸,ω-甲基-精氨酸,α-甲基-组氨酸,2,5-二碘-组氨酸,1-甲基-组氨酸,3-甲基-组氨酸,β-(2-吡啶基)-丙氨酸,β-(3-吡啶基)-丙氨酸,β-(2-喹啉基)-丙氨酸,3-氨基-酪氨酸,4-氨基-苯丙氨酸,和菠菜素。此外,任意的单或二羧基氨基酸是合适的带正电荷的氨基酸。
术语“中性氨基酸”用在本文中是指按照Kice&Marvell“Modern Principles oforganic Chemsitry(现代有机化学原理)”(Macmillan,1974)测量具有4.8-6.3的等电点的氨基酸。术语“酸性氨基酸”用在本文中是指按照Kice&Marvell“Modern Principles oforganic Chemsitry(现代有机化学原理)”(Macmillan,1974)测量具有低于4.8的等电点的氨基酸。
除非另外指明,氨基酸以本领域技术人员已知的标准命名缩写和提及,诸如参考国际纯粹与应用化学联合会(international union of pure and applied chemistry,IUPAC)(www.iupac.org)的“nomenclature and symbolism for amino acids andpeptides(氨基酸和肽的命名和符号体系)”。
瓜氨酸(在本文件中以单字母符号“B”表示)和/或高瓜氨酸是公知的非天然氨基酸,在一些情形中,其可以用在X1、X3或X5定义的序列中。
在其他备选的实施方案中,色氨酸或色氨酸衍生物用在X1、X3或X5定义的序列中。可以使用任意适当的色氨酸衍生物。用在本文中时,“色氨酸衍生物”意指非天然的修饰的色氨酸氨基酸残基,包括US7,232,803中公开的那些,诸如三叔丁基色氨酸,二叔丁基色氨酸,7-苯甲氧基色氨酸,高色氨酸,5′-氨基乙基色氨酸(获自RSPAmino Acids AnaloguesInc,Boston,Mass.,USA的侧链Boc和N-αFMOC衍生物),N-乙酰基高色氨酸(TorontoResearch),7-苯甲氧基色氨酸(Toronto Research),高色氨酸(Toronto Research),和已经在吲哚环的1-,2-,5-和/或7-位置优选1-或2-位置被取代的色氨酸残基,例如5′羟基色氨酸。
术语“抗体应答”是指结合目的抗原的抗体(例如,IgM,IgA,IgG)的产生,该应答例如通过抗原ELISA测定血清而测量。
术语“佐剂”用在本文中是指当与抗原一起或同时递送时非特异性增强针对所述抗原的免疫应答的任意化合物。示例性的佐剂包括但不限于,水包油和油包水佐剂,基于铝的佐剂(例如,AIOH,AIPO4等)和Montanide ISA720。
术语“患者”和“受试者”是指可以使用本发明的方法治疗的哺乳动物。
当用于本文时,术语“免疫应答”是指生物体的免疫系统响应抗原的反应性。在脊椎动物中,这可包括抗体产生、细胞介导的免疫的诱导和/或补体激活(例如,与脊椎动物免疫系统防止和消退微生物感染相关的现象)。在优选的实施方案中,术语免疫应答包括但不限于,“淋巴细胞增殖性应答”、“细胞因子应答”和“抗体应答”中的一种或多种。
涉及肽序列的术语“净电荷”用在本文中是指肽序列的总电荷,表示为该肽序列中每个个体氨基酸的电荷总和,其中每个碱性氨基酸给定为+1的电荷,每个酸性氨基酸给定为-1的电荷,每个中性氨基酸给定为0的电荷。因此,净电荷将取决于带电荷的氨基酸的数目和性质。
表1-本发明所述的特定肽
当用于本文时,单字母代码‘Z’是指非天然氨基酸正亮氨酸。
表2-本发明所述的特定肽
抗原 | X1 | X2 | X3 | X4 | X5 |
HCV | R | GYIPLVGAPLG | RRR | VARALAHGVRV | R |
HCV | R | GYLPAVGAPIG | RRR | VIRVIAHGLRL | R |
HCV | RR | GYIPLVGAPLG | RR | VARALAHGVRV | |
HCV | RR | GYIPLVGAPLG | RRR | VARALAHGVRV | |
HCV | RR | SRNLGKVIDTLTC | RR | LMGYIPLVGA | |
HCV | RR | GGGQIVGGVYLLP | RR | GPRLGVRATR | |
HCV | W | GYIPLVGAPLG | RR | VARALAHGVRV | |
HCV | RR | IRNLGRVIETLTLZGYIPLIGA | RR | IRNLGRVIETLTLZGYIPLIGA | R |
Flu | BR | TAYERZCNIL | BRGR | FQTVVQBA | |
cmv | R | NLVPMVATV | BRR | NLVPMVATV | B |
当用于本文时,单字母代码‘Z’是指非天然氨基酸正亮氨酸。
抗原
用于本发明的病毒抗原的实例包括,但不限于,例如,HIV,HCV,CMV,HPV,流感病毒,腺病毒,反转录病毒,小RNA病毒等。反转录病毒抗原的非限制性的实例诸如来自人免疫缺陷病毒(HIV)抗原的反转录病毒抗原,诸如gag,pol和env基因的基因产物,Nef蛋白,反转录酶,和其他HIV成分;肝炎病毒抗原,诸如乙型肝炎病毒的S,M和L蛋白,乙型肝炎病毒的pre-S抗原,和其他肝炎,例如甲型、乙型和丙型肝炎,病毒成分如丙型肝炎病毒RNA;流感病毒抗原,如血凝素和神经氨酸酶和其他流感病毒成分;麻疹病毒抗原,诸如麻疹病毒融合蛋白和其他麻疹病毒成分;风疹病毒抗原,如蛋白E1和E2和其他风疹病毒成分;轮状病毒抗原诸如VP7sc和其他轮状病毒成分;巨细胞病毒抗原,诸如包膜糖蛋白B和其他巨细胞病毒抗原成分;呼吸道合胞病毒抗原,诸如RSV融合蛋白,M2蛋白和其他呼吸道合胞病毒抗原成分;单纯疱疹病毒抗原,诸如即时早期蛋白,糖蛋白D和其他单纯疱疹病毒抗原成分;水痘带状疱疹病毒抗原诸如gpl,gpll和其他水痘带状疱疹病毒抗原成分;日本脑炎病毒抗原,诸如蛋白E,M-E,M-E-NS1,NS1,NS1-NS2A,80%E,和其他日本脑炎病毒抗原成分;狂犬病病毒抗原,诸如狂犬病糖蛋白,狂犬病核蛋白和其他狂犬病病毒抗原成分。对于病毒抗原的另外的实例参见Fundamental Virology(基础病毒学),第二版,Fields,B.N.和Knipe,D.M.编(Raven Press,New York,1991)。
结合在本发明所述的支架设计中的表位可以来源于腺病毒,反转录病毒,小RNA病毒,疱疹病毒,轮状病毒,汉坦病毒,冠状病毒,披膜病毒,黄病毒,棒状病毒,副黏病毒,正黏病毒,布尼亚病毒,沙粒病毒,呼肠病毒,乳头瘤病毒,细小病毒,痘病毒,嗜肝DNA病毒,或海绵样病毒(spongiform virus)。在某些具体的非限制性实例中,病毒抗原是获自HIV,CMV,甲型、乙型和丙型肝炎病毒,流感病毒,麻疹病毒,脊髓灰质炎病毒,天花病毒,风疹病毒;呼吸道合胞病毒,单纯疱疹病毒,水痘带状疱疹病毒,埃巴病毒,日本脑炎病毒,狂犬病病毒,流感病毒和/或感冒病毒。
HCV:
本发明所述的肽可以包含已知抗原。对于来源于HCV的抗原,这些抗原可以来源于丙型肝炎病毒(HCV)的核心蛋白,E1,E2,P7,NS2,NS3,NS4(NS4A与NS4B)和NS5(NS5A与NS5B)蛋白。表位是在免疫的宿主中激发I类和/或II类HLA限制性T淋巴细胞应答的那些表位。更具体地,本发明的I类HLA限制性肽可以结合下述I类HLA组中的至少一种HLA分子:HLA-A*01,HLA-A*02,HLA-A*03,HLA-A*11,HLA-A*24,HLA-B*07,HLA-B*08,HLA-B*35,HLA-B*40,HLA-B*44,HLA-Cw3,HLA-Cw4,HLA-Cw6或HLA-Cw7。本发明的II类HLA限制性肽可以结合下述II类HLA组中的至少一种HLA分子:HLA-DRB1,-DRB2,-DRB3,-DRB4,-DRB5,-DRB6,-DRB7,-DRB8或-DRB9。
例如,按照本发明可以用作表位的结合MHC的HCV肽公开在WO02/34770(ImperialCollege Innovations Ltd),WO01/21189和WO02/20035(Epimmune),WO04/024182(Intercell),WO95/25122(The Scripps Research Institute),WO95/27733(美国政府,Department of Health and Human Services),EP0935662(Chiron),WO02/26785(Immusystems GmbH),WO95/12677(Innogenetics N.V),WO97/34621(Cytel Corp),和EP1652858(Innogenetics N.V.)中。
在其他实施方案中,本发明所述的支架设计包括PADRE肽,诸如WO95/07707(Epimmune)(其内容通过引用包含在本文中)中公开的称为PADRE的通用的T细胞表位。“PanDR结合肽或PADRE肽”是结合多于一种II类HLADR分子的分子家族的一员。PADRE结合大部分的HLA-DR分子,并且在体外和体内刺激人辅助T淋巴细胞(HTL)应答。备选地,可以使用来自通用疫苗如破伤风类毒素(tetanos toxoid)的T辅助表位。
在另一个实施方案中,在组合物或多表位肽中的肽特征在于其来源于HCV蛋白,或者更具体的来源于选自由下列各项组成的组的下述HCV区域中的至少一种:核心蛋白,E1,E2/NS1,NS2,NS3,NS4A,NS4B,NS5A和NS5B。甚至更优选的是,肽特征在于其存在于基因型1a,1b和/或3a的HCV共有序列中。
按照本发明可以使用的其他I类和II类HLA结合肽可以通过如WO03/105058所述的方法-Algonomics、通过Epimmune在WO01/21189中所述的方法和/或通过三个公共数据库预测服务商(分别为Syfpeithi,BIMAS和nHLAPred)鉴定。每种肽可以与作为多个重复的相同肽或与任意其他的肽或表位组合用在本发明的支架设计中,这也是本发明的一个方面。
CMV:
要结合在本发明所述的支架设计中的表位可以来源于巨细胞病毒(CMV),包括CMV糖蛋白gB和gH。
流感:
要结合到本发明所述的支架设计中的表位可以来源于每个亚群(诸如H1N1,H2N2或H3N2)的流感血凝素(HA)或流感神经氨酸酶(NA)、核蛋白(NP),M1,M2,NS1,NEP,PA,PB1,PB1-F2,PB2的片段或部分。
合适的表位可以来源于一种亚型或多于一种亚型(包括H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15或H16)的HA蛋白,或其片段或部分。包含所述HA蛋白的亚型的实例包括A/New Caledonia/20/99(H1N1)A/Indonesia/5/2006(H5N1),A/chicken/NewYork/1995,A/herring gull/DE/677/88(H2N8),A/Texas/32/2003,A/mallard/MN/33/00,A/duck/Shanghai/1/2000,A/northern pintail/TX/828189/02,A/Turkey/Ontario/6118/68(H8N4),A/shoveler/Iran/G54/03,A/chicken/Germany/N/1949(H10N7),A/duck/England/56(H11N6),A/duck/Alberta/60/76(H12N5),A/Gull/Maryland/704/77(H13N6),A/Mallard/Gurjev/263/82,A/duck/Australia/341/83(H15N8),A/black-headed gull/Sweden/5/99(H16N3),B/Lee/40,C/Johannesburg/66,A/PuertoRico/8/34(H1N1),A/Brisbane/59/2007(H1N1),A/Solomon Islands3/2006(H1N1),A/Brisbane10/2007(H3N2),A/Wisconsin/67/2005(H3N2),B/Malaysia/2506/2004,B/Florida/4/2006,A/Singapore/1/57(H2N2),A/Anhui/1/2005(H5N1),A/Vietnam/1194/2004(H5N1),A/Teal/HongKong/W312/97(H6N1),A/Equine/Prague/56(H7N7),A/HongKong/1073/99(H9N2))。
在本发明的一些实施方案中,HA蛋白可以为H1,H2,H3,H5,H6,H7或H9亚型。在另一些实施方案中,H1蛋白可以来自A/New Caledonia/20/99(H1N1),A/PuertoRico/8/34(H1N1),A/Brisbane/59/2007(H1N1)或A/Solomon Islands3/2006(H1N1)毒株。H3蛋白也可以来自A/Brisbane10/2007(H3N2)或A/Wisconsin/67/2005(H3N2)毒株。在另一些实施方案中,H2蛋白可以来自A/Singapore/1/57(H2N2)毒株。H5蛋白可以来自A/Anhui/1/2005(H5N1),A/Vietnam/1194/2004(H5N1)或A/Indonesia/5/2005毒株。在另一些实施方案中,H6蛋白可以来自A/Teal/HongKong/W312/97(H6N1)毒株。H7蛋白可以来自A/Equine/Prague/56(H7N7)毒株。在另一些实施方案中,H9蛋白可以来自A/HongKong/1073/99(H9N2)毒株。在另一些实施方案中,HA蛋白可以来自流感病毒,可以是乙型病毒,包括B/Malaysia/2506/2004或B/Florida/4/2006。流感病毒HA蛋白可以是H5Indonesia。
人免疫缺陷病毒(HIV):
对于HIV,要结合到本发明所述的支架中的表位可以来源于由来源于HIV(包括不同基因亚型)的成员的gp120,gp160,gp41,p24gag或p55gag组成的组。
人乳头瘤病毒(HPV):
对于HPV,要结合到本发明所述的支架中的表位可以来源于由E1,E2,E3,E4,E6和E7,L1和L2蛋白组成的组。表位可以来自任意类型,包括8,11,16,18,31,33,35,39,45,51,52,56,58和59型。
载体,佐剂和赋形剂-递送
本发明所述的分离的细胞穿透肽可以通过多种方式和在多种组合物(在本文中称为“组合物”、“疫苗组合物”或“药物组合物”)中递送。本发明的肽和本发明的药物组合物和疫苗组合物用于施用给哺乳动物,特别是人,来治疗和/或预防病毒感染。将包含本发明的肽的疫苗组合物施用给已感染讨论的病毒的患者或者施用给易受病毒感染危险或否则有病毒感染危险的个体,以激发针对所述具体的抗原的免疫应答,并且因此增强患者自身的免疫应答能力。
多种本领域已知的递送系统可以用来将肽递送到适当的细胞中。肽可以在药用载体中或作为胶状混悬液、或作为粉剂、用或不用稀释剂递送。其可以是“裸露的”或与递送赋形剂缔合,并且使用本领域已知的递送系统递送。
“药用载体”或“药用佐剂”是任意适当的赋形剂、稀释剂、载体和/或佐剂,其本身不诱导对接受所述组合物的个体有害的抗体产生,其也不激发保护作用。优选地,药用载体或佐剂增强由抗原激发的免疫应答。适当的载体或佐剂典型地包括下述非穷举性列举中包括的一种或多种化合物:大的缓慢代谢的大分子,诸如蛋白,多糖,聚乳酸,聚乙醇酸,多聚体氨基酸,氨基酸共聚物和无活性的病毒颗粒;氢氧化铝,磷酸铝(参见国际专利申请公布号WO93/24148),明矾(KAl(SO4)2.12H2O),或这些中的一种与3-0-脱酰基单磷酰脂A组合(参见国际专利申请公布号WO93/19780);N-乙酰基-胞壁酰基-L-苏氨酰基-D-异谷氨酰胺(参见美国专利号4,606,918),N-乙酰基-正胞壁酰基-L-丙氨酰基-D-异谷氨酰胺,N-乙酰基胞壁酰基-L-丙氨酰基-D-异谷氨酰基-L-丙氨酸2-(1′,2′-二棕榈酰基-sn-甘油-3-羟基磷酰氧基)乙胺;RIBI(ImmunoChem Research Inc.,Hamilton,MT,USA),其在2%角鲨烯/吐温80乳液中包含单磷酰脂A(即,去毒的内毒素),海藻糖-6,6-二霉菌酸酯,和细胞壁支架(MPL+TDM+CWS)。三种成分MPL、TDM或CWS中的任一种也可以单独使用或者两两组合使用;佐剂诸如Stimulon(Cambridge Bioscience,Worcester,MA,USA),SAF-1(Syntex);佐剂诸如QS21与3-脱-O-乙酰基单磷酰脂A(3-de-O-acetylated monophosphoryl lipid A)之间的组合(参见国际申请号WO94/00153),其可以进一步补充水包油乳液(例如,参见国际申请号WO95/17210,WO97/01640和WO9856414),其中所述水包油乳液包含可代谢的油和皂苷,或可代谢的油,皂苷和固醇,或者其可以进一步补充细胞因子(参见国际申请号WO98/57659);佐剂诸如MF-59(Chiron)或基于聚[二(羧基苯氧基)磷腈(poly[di(carboxylatophenoxy)phosphazene])的佐剂(Virus Research Institute);基于嵌段共聚物的佐剂,诸如Optivax(Vaxcel,Cytrx)或基于菊糖的佐剂,诸如Algammulin和Gammalnulin(Anutech);完全或不完全弗氏佐剂(分别为CFA或IFA)或Gerbu制剂(Gerbu Biotechnik);皂苷,诸如QuilA,纯化的皂苷,如QS21,QS7或QS17,-七叶素或毛地黄皂苷;免疫刺激性寡核苷酸,包括未甲基化的CpG二核苷酸,诸如[嘌呤-嘌呤-CG-嘧啶-嘧啶]寡核苷酸。这些免疫刺激性寡核苷酸包括A、B和C类CpG分子(Coley Pharmaceuticals),ISS(Dynavax),Immunomers(Hybridon)。免疫刺激性寡核苷酸还可以与下述组合:阳离子肽,例如,如Riedl等(2002)所述;包含皂苷例如Quil A的免疫刺激复合物(ISCOMS);赋形剂和稀释剂,其是固有无毒性和非治疗性的,诸如水、盐水、甘油、乙醇、异丙醇、DMSO、湿润剂或乳化剂、pH缓冲物质、防腐剂等;生物降解和/或生物相容性油,诸如角鲨烷、角鲨烯、二十烷、Tetratetracontane、甘油、花生油、植物油,浓度为例如1-10%或2,5-5%;维生素,诸如维生素C(抗坏血酸或其盐或酯)、维生素E(生育酚)或维生素A;类胡萝卜素,或天然或合成的黄酮类化合物(flavanoids);痕量元素,如硒;任意Toll-样受体配体,如在Barton和Medzhitov(2002)中综述的。
在任意前述包含3-脱-O-乙酰基单磷酰脂A的佐剂中,所述3-脱-O-乙酰基单磷酰脂A可以形成小颗粒(参见国际申请号WO94/21292)。
在任意前述佐剂中,MPL或3-脱-O-乙酰基单磷酰脂A可以被称为RC-529的合成的类似物或被任意其他氨基-烷基氨基葡糖苷4-磷酸酯替换(Johnson等1999,Persing等2002)。备选地,其可以被其他脂质A类似物如OM-197替换(Byl等2003)。
“药用赋形剂”包括下述赋形剂,诸如水、盐水、生理盐水溶液、甘油、乙醇等。所述赋形剂中可以包含辅助物质,诸如湿润剂或乳化剂、pH缓冲物质、防腐剂。例如,本领域已知的递送系统为例如脂肽,包封在聚-DL-丙交酯-共-乙交酯(″PLG″)中的肽组合物,微球体,包含在免疫刺激复合物(ISCOMS)中的肽组合物,多抗原肽系统(MAPs),病毒递送载体,病毒或合成来源的颗粒,佐剂,脂质体,脂质,微粒或微胶囊剂,金粒,纳米颗粒,聚合物,凝聚剂(condensing agents),多糖,聚氨基酸,树状聚体,皂苷,QS21,吸附增强物质,脂肪酸,或裸露的cDNA或颗粒吸附的cDNA。
典型地,疫苗或疫苗组合物制备为可注射的,作为液体溶液或混悬液注射。注射可以是皮下、肌内、静脉内、腹膜内、鞘内、真皮内或表皮内注射。其他类型的施用包括电穿孔、植入、栓剂、口服摄入、肠应用、吸附、烟雾化或鼻喷雾或滴剂。还可以制备适于在注射前溶解在或悬浮在液体赋形剂中的固体形式。制剂还可以被乳化或包封在脂质体中以增强辅助作用。
液体制剂可以包含油、聚合物、维生素、碳水化合物、氨基酸、盐、缓冲剂、白蛋白、表面活性剂或填充剂。优选的碳水化合物包括糖或糖醇,如单糖、二糖、三糖、寡糖或多糖,或水溶性葡聚糖。糖或葡聚糖可以包括果糖、右旋糖、乳糖、葡萄糖、甘露糖、山梨糖、木糖、麦芽糖、蔗糖、葡聚糖、支链淀粉、糊精、α和β环糊精、可溶淀粉、羟乙基淀粉和羧甲基纤维素、或它们的混合物。蔗糖是最优选的。“糖醇”定义为具有-OH基团的C4至C8烃,并且包括半乳糖醇,肌醇,甘露醇,木糖醇,山梨醇,甘油和阿糖醇。甘露醇是最优选的。上文提及的这些糖或糖醇可以单独或组合使用。对所用的量没有固定的限制,只要所述糖或糖醇在水性制剂中可溶即可。优选地,糖或糖醇的浓度为1,0%(w/v)-7,0%(w/v),更优选2,0-6,0%(w/v)。优选的氨基酸包括左旋(L)形式的肉碱、精氨酸和甜菜碱;然而,可以添加其他氨基酸。优选的聚合物包括平均分子量为2,000-3,000的聚乙烯吡咯烷酮(PVP),或平均分子量为3,000-5,000的聚乙二醇(PEG)。还优选在组合物中使用缓冲剂,以使冻干之前或重构之后溶液中的pH变化最小化。可以使用任意的生理缓冲液,但是优选柠檬酸盐、磷酸盐、琥珀酸盐和谷氨酸盐缓冲液或它们的混合物。最优选的是柠檬酸盐缓冲液。优选地,浓度为0,01至0,3摩尔。可以添加到制剂中的表面活性剂显示在欧洲专利申请号EP0 270 799和EP0 268110中。
另外,多肽可以进行化学修饰,例如通过共价缀合到聚合物上,从而增加其循环半衰期。优选的聚合物,以及将其连接到肽上的方法显示在美国专利号4,766,106;4,179,337;4,495,285;和4,609,546中。优选的聚合物是聚氧乙基化多元醇和聚乙二醇(PEG)。PEG在室温可溶于水,并且具有通式:
R(O-CH2-CH2)nO-R
其中R可以是氢,或保护基,诸如烷基或链烷醇基。优选地,保护基具有1-8个碳,更优选地,其为甲基。符号n是正整数,优选1-1000,更优选2-500。PEG具有1000-40,000、更优选2000-20,000、最优选3,000-12,000的平均分子量。优选地,PEG具有至少一个羟基,更优选其为末端羟基。正是该羟基优选被活化。然而,应该理解,反应基团的类型和量可以变化,以获得共价缀合的PEG/本发明的多肽。
水溶性聚氧乙基化多元醇也可以用于本发明。其包括聚氧乙基化山梨醇、聚氧乙基化葡萄糖、聚氧乙基化甘油(POG)等。优选POG。一个原因是由于聚氧乙基化甘油的甘油骨架与例如在动物和人中的甘油一酯、甘油二酯、甘油三酯中天然存在的骨架相同。因此,这种分枝(branching)在体内将不必被视为外源试剂。POG具有与PEG相同范围的优选分子量。POG的结构显示在Knauf等,1988中,并且POG/IL-2缀合物的讨论可见于美国专利号4,766,106。
另一种用于增加循环半衰期的药物递送系统是脂质体。本发明的肽和核酸还可以通过脂质体施用,脂质体作用为靶向特定的组织,诸如淋巴组织,或选择性靶向感染的细胞,以及增加肽和核酸组合物的半衰期。脂质体包括乳剂、泡沫剂、微胶粒、不溶性单层、液晶、磷脂分散液、薄片层等。在这些制剂中,要被递送的肽或核酸结合作为脂质体的一部分或者单独或与结合淋巴样细胞中普遍存在的受体结合的分子(如结合CD45抗原的单克隆抗体)、或与其他治疗性或免疫原性组合物联合包埋。因此,填充有或布置有本发明的需要的肽或核酸或脂质体可以被导向淋巴样细胞位点,然后在那里脂质体递送所述肽和核酸组合物。按照本发明适用的脂质体由标准囊泡形成脂质形成,其通常包括中性和带负电荷的磷脂和固醇,诸如胆固醇。脂质的选择通常考虑例如脂质体尺寸、脂质体在血流中的酸不稳定性和稳定性而指导。多种方法可以用来制备脂质体,如在例如Szoka等,1980和美国专利号4,235,871,4,501,728,4,837,028和5,019,369中所述。
为了靶向免疫系统的细胞,要结合到脂质体中的配体可以包括,例如,对所需要的免疫系统细胞的细胞表面决定簇特异性的抗体或其片段。包含肽的脂质体混悬液可以静脉内、局部(locally)、表面(topically)等施用,施用的剂量特别依据施用方式、被递送的肽和被治疗的疾病的阶段而不同。例如,已知携带免疫原性多肽的脂质体在体内激发CTL应答(Reddy等,1992;Collins等,1992;Fries等,1992;Nabel等,1992)。
在制备脂质药物组合物后,优选将其冻干,以防止降解和无菌保存。用于冻干液体组合物的方法对于本领域技术人员是已知的。在使用之前,可以用无菌稀释剂(例如,Ringer′s溶液、蒸馏水、或无菌盐水)重构该组合物。重构后,所述组合物优选使用本领域技术人员已知的那些方法施用个受试者。
肽用于评估免疫应答的应用
本发明所述的肽可以用作诊断剂。例如,本发明的肽可以用来确定特定个体对使用所述肽或相关肽的治疗方案的敏感性,并且因此可有助于修改现行的治疗方案或者确定感染个体的预后。另外,所述肽还可以用来预测那些个体将有发展慢性病毒感染的实质性危险。
因此,本发明涉及确定暴露于病毒的受试者的结果的方法,所述方法包括确定所述受试者是否具有针对一种或多种本发明所述的肽的免疫应答的步骤。
在本发明的优选实施方案中,本文所述的肽可以用作评价免疫应答的试剂。要被评估的免疫应答可以通过使用可导致产生抗原特异性CTLs或HTLs(其识别或结合将被用作所述试剂的肽)的任意药剂作为免疫原而诱导。所述肽试剂不必用作免疫原。可以用于所述分析的测定系统包括相对最近的技术进展,诸如四聚体、细胞内淋巴因子染色和干扰素释放测定、或ELISPOT测定。
例如,本发明的肽可以用于四聚体染色测定来评估外周血单核细胞在暴露于抗原或免疫原后抗原特异性CTLs的存在。HLA-四聚体复合物用来直接显现抗原特异性CTLS(例如,参见Ogg等,1998;和Altman等,1996),并且确定外周血单核细胞样品中抗原特异性CTL群体的频率。使用本发明的肽的四聚体试剂可以按下述产生:在相应的HLA重链和β2微球蛋白的存在下重折叠结合HLA分子的肽,以产生三分子复合物。所述复合物在重链的羧基端在之前被加工到蛋白中的位点被生物素化。然后,通过加入链霉抗生物素蛋白而诱导四聚体形成。通过荧光标记的链霉抗生物素,所述四聚体可以用来染色抗原特异性细胞。然后可以例如通过流式细胞术鉴定所述细胞。这样的分析可以用于诊断或预后目的。通过该步骤鉴定的细胞还可以用于治疗目的。作为四聚体的备选方案,还可以使用五聚体或二聚体(Current Protocols in Immunology(现代免疫学流程)(2000)单元17.2增补35)。
本发明的肽还可以用作评价免疫回忆应答的试剂(例如,参见Bertoni等,1997和Perma等,1991)。例如,可以使用特定的肽分析来自HCV感染的个体的患者PBMC样品抗原特异性CTLs或HTLs的存在。可以通过培育PBMCs并且用本发明的肽刺激所述细胞而评估包含单核细胞的血液样品。在适当的培育期间后,可以分析扩增的细胞群体,例如,分析细胞毒性活性(CTL)或分析HTL活性。
所述肽还可以用作评价疫苗功效的试剂。
例如,可以使用上述任一种方法分析由接种免疫原的患者获得的PBMCs。所述患者是HL甲型的,并且选择识别所述患者中存在的等位基因特异性分子的肽表位试剂进行分析。疫苗的免疫原性通过在PBMC样品中存在表位特异性CTLs和/或HTLs而显示。
本发明的肽还可以用于制备抗体,使用本领域公知的技术制备(例如,参见CURRENT PROTOCOLS IN IMMUNOLOGY(现代免疫学流程),Wiley/Greene,NY;和AntibodiesA Laboratory Manual(抗体:实验室手册),Harlow和Lane,Cold Spring HarborLaboratory Press,1989)。所述抗体包括在HLA分子的情形中识别肽的那些,即,结合肽-MHC复合物的抗体。
本发明的具体实施方案
本发明涉及一种肽,即,包含下述结构的分离的细胞穿透肽:
X1-X2-X3-X4-X5(式I),
其中X1和X3独立地定义任意1,2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的线性序列;X2定义来源于抗原的8-30个氨基酸的线性序列;X4定义来源于所述抗原的8-30个氨基酸的线性序列,所述序列X4不同于X2;并且其中X5是选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的任意一个任选的氨基酸。
在一些具体的实施方案中,X4的序列与X2的序列相同。
在一些实施方案中,所述分离的细胞穿透肽总共具有不超过60个氨基酸。
在一些实施方案中,X1定义任意1,2,3或4个独立地选自任意碱性氨基酸、色氨酸或其衍生物的氨基酸的线性序列。
在一些实施方案中,X1定义一个瓜氨酸和任意1,2或3个独立地选自任意碱性氨基酸、色氨酸或其衍生物的氨基酸以任意顺序的线性序列。
在一些实施方案中,X3定义任意1,2,3或4个独立地选自任意碱性氨基酸的氨基酸的线性序列。
在一些实施方案中,X1定义任意1,2,3或4个独立地选自任意碱性氨基酸的氨基酸的线性序列。
在一些实施方案中,X3定义一个瓜氨酸和任意1,2或3个独立地选自任意碱性氨基酸的氨基酸以任意顺序的线性序列。
在一些实施方案中,权利要求1定义的式I的X2-X3-X4定义的氨基酸序列不存在于所述抗原的天然序列中。
在一些实施方案中,权利要求1定义的式I的X1-X2-X3-X4定义的氨基酸序列不存在于所述抗原的天然序列中。
在一些实施方案中,权利要求1定义的式I的X1-X2-X3-X4-X5定义的氨基酸序列不存在于所述抗原的天然序列中。
在一些实施方案中,所述肽在实施例1所述的基于肽生物素化的测定中证明穿过质膜易位。
在一些实施方案中,所述独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的1,2,3或4个氨基酸是碱性氨基酸。
在一些实施方案中,所述独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的1,2,3或4个氨基酸是色氨酸或其衍生物。
在一些实施方案中,,所述独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的1,2,3或4个氨基酸是瓜氨酸或其衍生物。
在一些实施方案中,所述选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的一个任选的氨基酸是选自Arg,Lys和His。B).
在一些实施方案中,所述选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的一个任选的氨基酸是色氨酸或其衍生物。
在一些实施方案中,所述选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的一个任选的氨基酸是瓜氨酸或其衍生物。
在一些实施方案中,X2和/或X4定义与所述抗原的天然序列相同的序列。
在一些实施方案中,所述肽能够诱导T淋巴细胞应答。
在一些实施方案中,所述肽能够诱导CD4+和/或CD8+T淋巴细胞应答。
在一些实施方案中,所述抗原是病毒蛋白,诸如衣壳蛋白。
在一些实施方案中,所述病毒蛋白选自丙型肝炎病毒的蛋白,诸如核心蛋白;流感病毒的蛋白,诸如M2蛋白。
在一些实施方案中,所述丙型肝炎病毒的病毒蛋白选自HCV基因型1如亚型1a和1b、基因型2如2a和2b以及基因型3如3a的共有序列。
在一些实施方案中,所述细胞穿透肽为19-60个氨基酸,如20-60个氨基酸,如21-60个氨基酸,如22-60个氨基酸,如23-60个氨基酸,如24-60个氨基酸,如25-60个氨基酸,如26-60个氨基酸,如27-60个氨基酸,如28-60个氨基酸,如29-60个氨基酸,如30-60个氨基酸,如31-60个氨基酸,如32-60个氨基酸,如33-60个氨基酸,如34-60个氨基酸,如35-60个氨基酸。
在一些实施方案中,所述细胞穿透肽为18-60个氨基酸,诸如18-59个氨基酸,诸如18-58个氨基酸,诸如18-57个氨基酸,诸如18-56个氨基酸,诸如18-55个氨基酸,诸如18-54个氨基酸,诸如18-53个氨基酸,诸如18-52个氨基酸,诸如18-51个氨基酸,诸如18-50个氨基酸,诸如18-49个氨基酸,诸如18-48个氨基酸,诸如18-47个氨基酸,诸如18-46个氨基酸,诸如18-45个氨基酸,诸如18-44个氨基酸,诸如18-43个氨基酸,诸如18-42个氨基酸,诸如18-41个氨基酸,诸如18-40个氨基酸,诸如18-39个氨基酸,诸如18-38个氨基酸,诸如18-37个氨基酸,诸如18-35个氨基酸,诸如18-34个氨基酸,诸如18-33个氨基酸,诸如18-32个氨基酸,诸如18-31个氨基酸,诸如18-30个氨基酸,诸如18-29个氨基酸,诸如18-28个氨基酸,诸如18-27个氨基酸,诸如18-26个氨基酸,诸如18-25个氨基酸,诸如18-24个氨基酸,诸如18-23个氨基酸,诸如18-22个氨基酸,诸如18-21个氨基酸,诸如18-20个氨基酸,诸如18-19个氨基酸。
在一些实施方案中,X2的净电荷低于或等于0。
在一些实施方案中,X2的净电荷低于或等于0;并且X1和X3定义独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的2,3或4个氨基酸的序列。
在一些实施方案中,X2的净电荷低于或等于0;并且X4的净电荷高于或等于1。
在一些实施方案中,X2的净电荷低于或等于0;X4的净电荷高于或等于1;并且X1和X3定义独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的2,3或4个氨基酸的序列。
在一些实施方案中,X2和X4的净电荷低于或等于0。
在一些实施方案中,X2和X4的净电荷低于或等于0;并且X1和X3定义独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的2,3或4个氨基酸的序列。
在一些实施方案中,X2和X4的净电荷高于或等于1。
在一些实施方案中,X2和X4的净电荷高于或等于1;并且X1和X3定义独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的1或2个氨基酸的序列。
在一些实施方案中,X2的净电荷高于或等于1;并且X4的净电荷低于或等于0。
在一些实施方案中,X2的净电荷高于或等于1;X4的净电荷低于或等于0;X1定义1或2个具有正电荷的氨基酸的序列;并且X3定义独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的2,3或4个氨基酸的序列。
在一些实施方案中,所述碱性氨基酸独立地选自Arg,Lys和His。
在一些实施方案中,X2和/或X4包含由SEQ ID NO:3的位置135-157定义的氨基酸的序列,或其片段或变体。
在一些实施方案中,X2和/或X4由选自下列的序列组成:GYIPLVGAPLG,GYLPAVGAPIG,GYLPAVGAPI,NYVTGNIPG,NYATGNLPG,NYATGNLPG,VTGNIPGSTYS,IRNLGRVIETLTG,SRNLGKVIDTLTC,IRNLGRVIETLT,GGGQIIGGNYLIP,GGGQIVGGVYLLP,LIFLARSALIV,LIFLARSALIL,LIFLARSALIL,SAYERMCNIL,SAYERZVNIL,TAYERZCNIL,IAYERMCNIL,IAYERMCNIL,LFFKCIYRLFKHGL,LFFKTITRLFBHGL,GLEPLVIAGILA,GSDPLVVAASIV,NLVPMVATV,NLVPMVATV,NIVPZVVTA,PEVIPMFSALS,FIIPXFTALSG,ALGPAATL,GPVVHLTL,LECVYCKQQLL,GVYDFAFRDLC,GVFDYAFRDIN,GATPVDLLGA,GVTPAGLIGV,VARALAHGVRV,VIRVIAHGLRL,GITFSIFLIVS,CSFSIFLLAL,GCSFSIFLLAL,GITFSIYLIVS,LZGYIPLIGA,LMGYIPLVGA,LZGYIPLIGA,PBIGVRATB,GPRLGVRATR,GPRLGVRAT,RGSVAHKS,SALILRGSVAHK,FQTAAQRAMM,FQTAAQRAVZ,FQTVVQBA,FQTAAQRA,GPSTEGVPESM,LLSTEGVPNSZ,GSLVGLLHIVL,ASIVGILHLIL,NLVPMVATV,NIVPZVVTA,TPQDLNTMLN,ALLYGATPYAIG,MMTACQGVG,GQAGDDFS,EVYDFAFRDLC,GFAFRDLCIVY,GFAYRDINLAY,GALNLCLPM,和GALQIBLPL,IRNLGRVIETLTLZGYIPLIGA,或其片段或变体。
在一些实施方案中,X2和/或X4由来源于选自下列的氨基酸序列的序列组成:SEQID NO:1,SEQ ID NO:2,SEQ ID NO:3,SEQ ID NO:4,SEQ IDNO:5,SEQ ID NO:6,SEQ ID NO:7,SEQ ID NO:8,SEQ ID NO:9,SEQ IDNO:10,SEQ ID NO:11,SEQ ID NO:12,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:18,SEQ IDNO:19,SEQ ID NO:20,SEQ ID NO:21,SEQ ID NO:22,SEQ ID NO:23,SEQ ID NO:24,SEQ ID NO:25,SEQ ID NO:26,SEQ ID NO:27,SEQ IDNO:28,SEQ ID NO:29,SEQ ID NO:30,SEQ ID NO:31,SEQ ID NO:32,SEQ ID NO:33,SEQ ID NO:34,SEQ ID NO:35,SEQ ID NO:36,SEQ IDNO:37,SEQ ID NO:38,SEQ ID NO:39,SEQ ID NO:40,SEQ ID NO:41,SEQ ID NO:42,SEQ IDNO:43,SEQ ID NO:44,SEQ ID NO:45,SEQ IDNO:46,SEQ ID NO:126,和SEQ ID NO:198,或其片段或变体。
在一些实施方案中,本发明所述的肽选自:
RRGYIPLVGAPLGBGRVARALAHGVRV,RGYIPLVGAPLGRRVARALAHGVRV,
RGYIPLVGAPLGRRRVARALAHGVRVR,RRGYIPLVGAPLGRRVARALAHGVRV,
RRGYIPLVGAPLGRRRVARALAHGVRV,BRGYIPLVGAPLGRRVARALAHGVRV,
RRRGYIPLVGAPLGBRVARALAHGVRV,RGYIPLVGAPLGKKKVARALAHGVRV,
RGYIPLVGAPLGRRRVARALAHGVRV,KKGYIPLVGAPLGKKVARALAHGVRV,
WGYIPLVGAPLGRRVARALAHGVRV,WWGYIPLVGAPLGRRVARALAHGVRV,
EEGYIPLVGAPLGEEVARALAHGVRV,GGGYIPLVGAPLGGGVARALAHGVRV,
EEGYIPLVGAPLGRRVARALAHGVRV,RRGYIPLVGAPLGLRRVARALAHGVRV,
WWGYIPLVGAPLGRRVARALAHGVRV,WWGYIPLVGAPLGRRRVARALAHGVRV,
WWGYIPLVGAPLGRVARALAHGVRV,RGYIPLVGAPLGRRVARALAHGVRV,
RRGYLPAVGAPIGBRVIRVIAHGLRL,RRGYIPLVGAPLGBRVARALAHGVRV,
GYIPLVGAPLGGVARALAHGVRV,WWGYLPAVGAPIRRVIRVIAHGLRL,
GYIPLVGAPLGGVARALAHGVRV,RRGYIPLVGAPLGBGRVARALAHGVRV,
RGYIPLVGAPLGRRVARALAHGVRV,RGYIPLVGAPLGRRRVARALAHGVRV,
RRGYIPLVGAPLGRRVARALAHGVRV,RRGYIPLVGAPLGRRRVARALAHGVRV,
BRGYIPLVGAPLGRRVARALAHGVRV,RRRGYIPLVGAPLGBRVARALAHGVRV,
RGYIPLVGAPLGKKKVARALAHGVRV,RGYIPLVGAPLGRRRVARALAHGVRV,
KKGYIPLVGAPLGKKVARALAHGVRV,WGYIPLVGAPLGRRVARALAHGVRV,
WWGYIPLVGAPLGRRVARALAHGVRV,RRGYIPLVGAPLGLRRVARALAHGVRV,
RRNYVTGNIPGBRGITFSIFLIVS,WWNYATGNLPGRRCSFSIFLLAL,
WWNYVTGNIPGBRGITFSIFLIVS,WWNYVTGNIPGRRGITFSIFLIVS,
RRNYATGNLPGRRGCSFSIFLLAL,RRVTGNIPGSTYSGBRGITFSIYLIVS,
RRIRNLGRVIETLTGBRLZGYIPLIGA,RRSRNLGKVIDTLTCBRLMGYIPLVGA,
SRNLGKVIDTLTCGFADLMGYIPLVGA,WWIRNLGRVIETLTRRLZGYIPLIGA,
WWSRNLGKVIDTLTCRRLMGYIPLVGA,RRGGGQIIGGNYLIPRBPBIGVRATB,
GGGQIVGGVYLLPRRGPRLGVRATR,RRGGGQIVGGVYLLPRRGPRLGVRATR,
WWGGGQIVGGVYLLPRRGPRLGVRAT,,BRLIFLARSALIVRGSVAHKS,
EDLIFLARSALILRGSVAHKS,BRLIFLARSALILBGRSALILRGSVAHK,
SAYERMCNILKGKFQTAAQRAMM,SAYERZVNILKGKFQTAAQRAVZ,
BRTAYERZCNILBRGRFQTVVQBA,BRIAYERMCNILLBRGKFQTAAQRA,
IAYERMCNILKGKFQTAAQRA,LFFKCIYRLFKHGLKRGPSTEGVPESM,
BRRLFFKTITRLFBHGLRRLLSTEGVPNSZ,BRGLEPLVIAGILARRGSLVGLLHIVL,
BRGSDPLVVAASIVRRASIVGILHLIL,,RNLVPMVATVRRNLVPMVATVB,
RNLVPMVATVBRRNLVPMVATVB,RNIVPZVVTARRNIVPZVVTAB,,
PEVIPMFSALSEGATPQDLNTMLN,RFIIPXFTALSGGRRALLYGATPYAIG,
KALGPAATLEEMMTACQGVG,,RRGPVVHLTLRRRGQAGDDFS,RRGPVVHLTLRRRGQAGDDFS,RRGPVVHLTLRGRRGQAGDDFS,RRLECVYCKQQLLRREVYDFAFRDLC,
RRGVYDFAFRDLCRRGFAFRDLCIVYR,RRGVFDYAFRDINRRGFAYRDINLAYR,
RRGATPVDLLGARRGALNLCLPMR,RRGVTPAGLIGVRRGALQIBLPLR,
RGYLPAVGAPIGRRRVIRVIAHGLRLR,RRSRNLGKVIDTLTCRRLMGYIPLVGA,
RRIRNLGRVIETLTLZGYIPLIGARRIRNLGRVIETLTLZGYIPLIGAR,或其片段或变体。
在一些实施方案中,所述肽由选自下列的序列组成:X1-NYVTGNIPG-X3-GITFSIYLIVS;X1-IRNLGRVIETLT-X3-LZGYIPLIGA;X1-GYLPAVGAPI-X3-VIRVIAHGLRL;X1-GGGQIIGGNYLIP-X3-PBIGVRATB;X1-NYATGNLPG-X3-GCSFSIFLLAL;X1-SRNLGKVIDTLTC-X3-LMGYIPLVGA;X1-GYIPLVGAPL-X3-VARALAHGVRV;X1-GGGQIVGGVYLLP-X3-PRLGVRATR;X1-LTFLVRSVLLI-X3-GSVLIVRGSLVH;X1-TAYERZCNIL-X3-GRFQTVVQBA;X1-SDPLVVAASIV-X3-ASIVGILHLIL;X1-LIFLARSALIL-X3-SALILRGSVAH;X1-IAYERMCNIL-X3-GKFQTAAQRA;and X1-LEPLVIAGILA-X3-GSLVGLLHIVL;X1-NLVPMVATV-X3-NLVPMATV;X1-GYLPAVGAPIG-X3-VIRVIAHGLRL;X1-IRNLGRVIETLTG-X3-LZGYIPLIGA;X1-GVYDFAFRDLC-X3-GFAFRDLCIVYR,X1-GVFDYAFRDIN-X3-GFAYRDINLAYR,X1-GATPVDLLGA-X3-GALNLCLPMR,X1-GVTPAGLIGV-X3-GALQIBLPLR,和X1-IRNLGRVIETLTLZGYIPLIGA-X3-IRNLGRVIETLTLZGYIPLIGA;任选地在所述肽的C端具有X5,其中X1,X3和X5是指式I的X1,X3和X5。
在一些实施方案中,所述肽包含一个或多个半胱氨酸。
在一些实施方案中,所述肽包含分子内键,诸如在两个cys残基之间的分子内二硫键(S-S)。
在另一些实施方案中,所述肽包括分子内键,诸如以缩羰基酯结构部分(COO-CH2-OOC,COO-CHR-OOC或COO-CR2-OOC)的形式。
在一些实施方案中,X2中的N-和/或C-端氨基酸是亲水性或极性氨基酸。
在一些实施方案中,X4中的N-端氨基酸是亲水性或极性氨基酸。
在一些实施方案中,本发明所述的肽不超过58个氨基酸,诸如不超过56,54,52,50,48,46,44,42,40,38,36,34,32,30,28,26,24,22,20,18个氨基酸残基。
在一些实施方案中,X1由2或3个氨基酸组成。
在一些实施方案中,X1由WW,BR或RR组成。
在一些实施方案中,X1由R或W组成。
在一些实施方案中,X3由2或3个氨基酸组成。
在一些实施方案中,X3由WW,BR或RR组成。
在一些实施方案中,X3由RRR,BRR或BRGR组成。
在一些实施方案中,本发明所述的分离的肽由表1或表2中定义的X2或X4的序列组成。
在一些实施方案中,本发明所述的分离的肽包括表1或表2中定义的X2和/或X4的序列或其片段。
在一些实施方案中,X2定义8-25个氨基酸的序列,诸如8-20个氨基酸,诸如8-15个氨基酸,诸如8-11个氨基酸的序列。
在一些实施方案中,X2定义少于25个,诸如少于24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7或6个氨基酸的序列。
在一些实施方案中,X2定义多于8个,诸如多于9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29个氨基酸的序列。
在一些实施方案中,X4定义8-25个氨基酸的序列,诸如8-20个氨基酸,诸如8-15个氨基酸,诸如8-11个氨基酸的序列。
在一些实施方案中,X4定义少于25个,诸如少于24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7或6个氨基酸的序列。
在一些实施方案中,X4定义多于8个,诸如多于9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29个氨基酸的序列。
在一些实施方案中,本发明所述的二聚体肽由两个相同的肽单体组成。
在一些实施方案中,本发明所述的免疫原性组合物采取疫苗组合物的形式。
在一些实施方案中,本发明的细胞穿透肽包含至多60个、至多59个、至多58个、至多57个、至多56个、至多55个、至多54个、至多53个、至多52个、至多51个、至多50个、至多49个、至多48个、至多47个、至多46个、至多45个、至多44个、至多43个、至多42个、至多41个、至多40个、至多39个、至多38个、至多37个、至多36个、至多35个、至多34个、至多33个、至多32个、至多31个、至多30个、至多29个、至多28个、至多27个、至多26个、至多25个、至多24个、至多23个、至多22个、至多21个、至多20个、至多19个、至多18个氨基酸。
在一些实施方案中,本发明的细胞穿透肽包含至少18个、至少19个、至少20个、至少21个、至少22个、至少23个、至少24个、至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少31个、至少32个、至少33个、至少34个、至少35个、至少36个、至少37个、至少38个、至少39个、至少40个、至少41个、至少42个、至少43个、至少44个、至少45个、至少46个、至少47个、至少48个、至少49个、至少50个、至少51个、至少52个、至少53个、至少54个、至少55个、至少56个、至少57个、至少58个、至少59个、至少60个氨基酸残基。
在一些实施方案中,本发明的细胞穿透肽由18个氨基酸残基或19个氨基酸残基或20个氨基酸残基或21个氨基酸残基或22个氨基酸残基或23个氨基酸残基或24个氨基酸残基或25个氨基酸残基或26个氨基酸残基或27个氨基酸残基或28个氨基酸残基或29个氨基酸残基或30个氨基酸残基或31个氨基酸残基或32个氨基酸残基或33个氨基酸残基或34个氨基酸残基或35个氨基酸残基或36个氨基酸残基或37个氨基酸残基或38个氨基酸残基或39个氨基酸残基或40个氨基酸残基或41个氨基酸残基或42个氨基酸残基或43个氨基酸残基或44个氨基酸残基或45个氨基酸残基或46个氨基酸残基或47个氨基酸残基或48个氨基酸残基或49个氨基酸残基或50个氨基酸残基或51个氨基酸残基或52个氨基酸残基或53个氨基酸残基或54个氨基酸残基或55个氨基酸残基或56个氨基酸残基或57个氨基酸残基或58个氨基酸残基或59个氨基酸残基或60个氨基酸残基组成。
在一些实施方案中,本发明的细胞穿透肽不由下述序列组成:RFIIP[Nle]FTALSGGRRALLYGATPYAIG,其中Nle表示正亮氨酸。
在一些实施方案中,X2和/或X4不是来源于HIV。
在一些实施方案中,X4是少于12个氨基酸的线性序列。
在一些实施方案中,X2是少于12个氨基酸的线性序列。
在一些实施方案中,X2和/或X4不包含正亮氨酸。
在一些实施方案中,X2不包含正亮氨酸。
在一些实施方案中,X2和/或X4仅包含天然氨基酸。
在一些实施方案中,X2仅包含天然氨基酸。
在一些实施方案中,X2在来源于HIV时仅包含天然氨基酸。
在一些实施方案中,X2和/或X4来源于HCV,CMV,HPV,流感病毒,腺病毒,或小RNA病毒。
本发明所述的编号的实施方案:
1.一种分离的细胞穿透肽,其包括下述结构:
X1-X2-X3-X4-X5(式I),
其中X1和X3独立地定义任意1,2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的线性序列;X2定义来源于抗原的8-30个氨基酸的线性序列;X4定义来源于所述抗原的8-30个氨基酸的线性序列,所述序列X4不同于X2;并且其中X5是选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的任意一个任选的氨基酸。
2.根据实施方案1所述的分离的肽,其中X1定义任意1,2,3或4个独立地选自任意碱性氨基酸、色氨酸或其衍生物的氨基酸的线性序列。
3.根据实施方案1所述的分离的肽,其中X1定义一个瓜氨酸和任意1,2或3个独立地选自任意碱性氨基酸、色氨酸或其衍生物的氨基酸的任意顺序的线性序列。
4.根据实施方案1-3中任一项所述的分离的肽,其中X3定义任意1,2,3或4个独立地选自任意碱性氨基酸的氨基酸的线性序列。
5.根据实施方案1-3中任一项所述的分离的肽,其中X3定义一个瓜氨酸和任意1,2或3个独立地选自任意碱性氨基酸的氨基酸的任意顺序的线性序列。
6.根据实施方案1-5中任一项所述的分离的肽,其中实施方案1定义的式I的X2-X3-X4定义的氨基酸序列不存在于所述抗原的天然序列中。
7.根据实施方案1-6中任一项所述的分离的肽,其中所述肽在实施例1所述的基于肽生物素化的测定中证明穿过质膜易位。
8.根据实施方案1-7中任一项所述的分离的肽,其中所述选自碱性氨基酸、瓜氨酸、色氨酸或其衍生物的一个任选的氨基酸是选自Arg,Lys和His。
9.根据实施方案1-8中任一项所述的分离的肽,其中X2和/或X4定义与所述抗原的天然序列相同的序列。
10.根据实施方案1-9中任一项所述的分离的肽,其中所述肽能够诱导T淋巴细胞应答。
11.根据实施方案1-10中任一项所述的分离的肽,其中所述抗原是病毒蛋白,诸如衣壳蛋白。
12.根据实施方案11所述的分离的肽,其中所述病毒蛋白选自丙型肝炎病毒的蛋白,诸如核心蛋白;流感病毒的蛋白,诸如M2蛋白。
13.根据实施方案11所述的分离的肽,其中所述丙型肝炎病毒的病毒蛋白选自HCV基因型1如亚型1a和1b、基因型2如2a和2b以及基因型3如3a的共有序列。
14.根据实施方案1-13中任一项所述的分离的肽,其中细胞穿透肽为19-60个氨基酸,如20-60个氨基酸,如21-60个氨基酸,如22-60个氨基酸,如23-60个氨基酸,如24-60个氨基酸,如25-60个氨基酸,如26-60个氨基酸,如27-60个氨基酸,如28-60个氨基酸,如29-60个氨基酸,如30-60个氨基酸,如31-60个氨基酸,如32-60个氨基酸,如33-60个氨基酸,如34-60个氨基酸,如35-60个氨基酸。
15.根据实施方案1-14中任一项所述的分离的肽,所述细胞穿透肽为18-60个,如18-59个,如18-58个,如18-57个,如18-56个,如18-55个,如18-54个,如18-53个,如18-52个,如18-51个,如18-50个,如18-49个,如18-48个,如18-47个,如18-46个,如18-45个,如18-44个,如18-43个,如18-42个,如18-41个,如18-40个,如18-39个,如18-38个,如18-37个,如18-35个氨基酸,如18-34个氨基酸,如18-33个氨基酸,如18-32个氨基酸,如18-31个氨基酸,如18-30个氨基酸,如18-29个氨基酸,如18-28个氨基酸,如18-27个氨基酸,如18-26个氨基酸,如18-25个氨基酸,如18-24个氨基酸,如18-23个氨基酸,如18-22个氨基酸,如18-21个氨基酸,如18-20个氨基酸,如18-19个氨基酸。
16.根据实施方案1-15中任一项所述的分离的肽,其中X2的净电荷低于或等于0;并且其中X1和X3定义2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的序列。
17.根据实施方案1-16中任一项所述的分离的肽,其中X2的净电荷低于或等于0;其中X4的净电荷高于或等于1;并且其中X1和X3定义2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的序列。
18.根据实施方案1-17中任一项所述的分离的肽,其中X2和X4的净电荷低于或等于0,并且其中X1和X3定义2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的序列。
19.根据实施方案1-18中任一项所述的分离的肽,其中X2和X4的净电荷高于或等于1,并且其中X1和X3定义1或2个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的序列。
20.根据实施方案1-19中任一项所述的分离的肽,其中X2的净电荷高于或等于1;其中X4的净电荷低于或等于0;其中X1定义1或2个具有正电荷的氨基酸的序列;并且其中X3定义2,3或4个独立地选自任意碱性氨基酸、瓜氨酸、色氨酸或其衍生物的氨基酸的序列。
21.根据实施方案1-20中任一项所述的分离的肽,其中所述碱性氨基酸独立地选自Arg,Lys和His。
22.根据实施方案1-21中任一项所述的分离的肽,其中X2和/或X4包含由SEQ IDNO:3的位置135-157定义的氨基酸的序列,或其片段或变体。
23.根据实施方案1-22中任一项所述的分离的肽,其中X2和/或X4由选自下列的序列组成:GYIPLVGAPLG,GYLPAVGAPIG,GYLPAVGAPI,NYVTGNIPG,NYATGNLPG,NYATGNLPG,VTGNIPGSTYS,IRNLGRVIETLTG,SRNLGKVIDTLTC,IRNLGRVIETLT,GGGQIIGGNYLIP,GGGQIVGGVYLLP,LIFLARSALIV,LIFLARSALIL,LIFLARSALIL,SAYERMCNIL,SAYERZVNIL,TAYERZCNIL,IAYERMCNIL,IAYERMCNIL,LFFKCIYRLFKHGL,LFFKTITRLFBHGL,GLEPLVIAGILA,GSDPLVVAASIV,NLVPMVATV,NLVPMVATV,NIVPZVVTA,PEVIPMFSALS,FIIPXFTALSG,ALGPAATL,GPVVHLTL,LECVYCKQQLL,GVYDFAFRDLC,GVFDYAFRDIN,GATPVDLLGA,GVTPAGLIGV,VARALAHGVRV,VIRVIAHGLRL,GITFSIFLIVS,CSFSIFLLAL,GCSFSIFLLAL,GITFSIYLIVS,LZGYIPLIGA,LMGYIPLVGA,LZGYIPLIGA,PBIGVRATB,GPRLGVRATR,GPRLGVRAT,RGSVAHKS,SALILRGSVAHK,FQTAAQRAMM,FQTAAQRAVZ,FQTVVQBA,FQTAAQRA,GPSTEGVPESM,LLSTEGVPNSZ,GSLVGLLHIVL,ASIVGILHLIL,NLVPMVATV,NIVPZVVTA,TPQDLNTMLN,ALLYGATPYAIG,MMTACQGVG,GQAGDDFS,EVYDFAFRDLC,GFAFRDLCIVY,GFAYRDINLAY,GALNLCLPM,GALQIBLPL,和IRNLGRVIETLTLZGYIPLIGA,或其片段或变体。
24.根据实施方案1-23中任一项所述的分离的肽,其中X2和/或X4由来源于选自下列的氨基酸序列的序列组成:SEQ ID NO:1,SEQ ID NO:2,SEQ ID NO:3,SEQ ID NO:4,SEQID NO:5,SEQ ID NO:6,SEQ ID NO:7,SEQ ID NO:8,SEQ ID NO:9,SEQ ID NO:10,SEQ IDNO:11,SEQ IDNO:12,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15,SEQ ID NO:16,SEQ IDNO:17,SEQ ID NO:18,SEQ ID NO:19,SEQ ID NO:20,SEQ IDNO:21,SEQ ID NO:22,SEQ IDNO:23,SEQ ID NO:24,SEQ ID NO:25,SEQ ID NO:26,SEQ ID NO:27,SEQ ID NO:28,SEQ IDNO:29,SEQ IDNO:30,SEQ ID NO:31,SEQ ID NO:32,SEQ ID NO:33,SEQ ID NO:34,SEQ IDNO:35,SEQ ID NO:36,SEQ ID NO:37,SEQ ID NO:38,SEQ IDNO:39,SEQ ID NO:40,SEQ IDNO:41,SEQ ID NO:42,SEQ ID NO:43,SEQ ID NO:44,SEQ ID NO:45,SEQ ID NO:46,SEQ IDNO:126,和SEQID NO:198,或其片段或变体。
25.根据实施方案1-24中任一项所述的分离的肽,其中所述肽选自:RRGYIPLVGAPLGBGRVARALAHGVRV(SEQ ID NO:47),
RGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:48),RGYIPLVGAPLGRRRVARALAHGVRVR(SEQID NO:49),RRGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:50),
RRGYIPLVGAPLGRRRVARALAHGVRV(SEQ ID NO:51),BRGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:52),RRRGYIPLVGAPLGBRVARALAHGVRV(SEQ ID NO:53),
RGYIPLVGAPLGKKKVARALAHGVRV(SEQ ID NO:54),RGYIPLVGAPLGRRRVARALAHGVRV(SEQID NO:55),KKGYIPLVGAPLGKKVARALAHGVRV(SEQ ID NO:56),
WGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:57),WWGYIPLVGAPLGRRVARALAHGVRV(SEQID NO:58),EEGYIPLVGAPLGEEVARALAHGVRV(SEQ ID NO:59),
GGGYIPLVGAPLGGGVARALAHGVRV(SEQ ID NO:60),EEGYIPLVGAPLGRRVARALAHGVRV(SEQID NO:61),RRGYIPLVGAPLGLRRVARALAHGVRV(SEQ ID NO:62),
WWGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:63),WWGYIPLVGAPLGRRRVARALAHGVRV(SEQ ID NO:64),WWGYIPLVGAPLGRVARALAHGVRV(SEQID NO:65),
RGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:66),RRGYLPAVGAPIGBRVIRVIAHGLRL(SEQ IDNO:67),RRGYIPLVGAPLGBRVARALAHGVRV(SEQ ID NO:68),GYIPLVGAPLGGVARALAHGVRV(SEQ ID NO:69),WWGYLPAVGAPIRRVIRVIAHGLRL(SEQ ID NO:70),
GYIPLVGAPLGGVARALAHGVRV(SEQ ID NO:71),RRGYIPLVGAPLGBGRVARALAHGVRV(SEQIDNO:72),RGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:73),
RGYIPLVGAPLGRRRVARALAHGVRV(SEQ ID NO:74),RRGYIPLVGAPLGRRVARALAHGVRV(SEQID NO:75),RRGYIPLVGAPLGRRRVARALAHGVRV(SEQ ID NO:76),
BRGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:77),RRRGYIPLVGAPLGBRVARALAHGVRV(SEQ ID NO:78),RGYIPLVGAPLGKKKVARALAHGVRV(SEQ ID NO:79),
RGYIPLVGAPLGRRRVARALAHGVRV(SEQ ID NO:80),KKGYIPLVGAPLGKKVARALAHGVRV(SEQID NO:81),WGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:82),
WWGYIPLVGAPLGRRVARALAHGVRV(SEQ ID NO:83),RRGYIPLVGAPLGLRRVARALAHGVRV(SEQ ID NO:84),RRNYVTGNIPGBRGITFSIFLIVS(SEQ ID NO:85),
WWNYATGNLPGRRCSFSIFLLAL(SEQ ID NO:86),WWNYVTGNIPGBRGITFSIFLIVS(SEQIDNO:87),WWNYVTGNIPGRRGITFSIFLIVS(SEQ ID NO:88),RRNYATGNLPGRRGCSFSIFLLAL(SEQID NO:89),RRVTGNIPGSTYSGBRGITFSIYLIVS(SEQ ID NO:90),
RRIRNLGRVIETLTGBRLZGYIPLIGA(SEQ ID NO:91),RRSRNLGKVIDTLTCBRLMGYIPLVGA(SEQID NO:92),SRNLGKVIDTLTCGFADLMGYIPLVGA(SEQ ID NO:93),
WWIRNLGRVIETLTRRLZGYIPLIGA(SEQ ID NO:94),WWSRNLGKVIDTLTCRRLMGYIPLVGA(SEQID NO:95),RRGGGQIIGGNYLIPRBPBIGVRATB(SEQ ID NO:96),
GGGQIVGGVYLLPRRGPRLGVRATR(SEQ ID NO:97),RRGGGQIVGGVYLLPRRGPRLGVRATR(SEQID NO:98),WWGGGQIVGGVYLLPRRGPRLGVRAT(SEQ ID NO:99),BRLIFLARSALIVRGSVAHKS(SEQ ID NO:100),EDLIFLARSALILRGSVAHKS(SEQ ID NO:101),
BRLIFLARSALILBGRSALILRGSVAHK(SEQ ID NO:102),SAYERMCNILKGKFQTAAQRAMM(SEQID NO:103),SAYERZVNILKGKFQTAAQRAVZ(SEQ ID NO:104),
BRTAYERZCNILBRGRFQTVVQBA(SEQ ID NO:105),BRIAYERMCNILLBRGKFQTAAQRA(SEQIDNO:106),IAYERMCNILKGKFQTAAQRA(SEQ ID NO:107),LFFKCIYRLFKHGLKRGPSTEGVPESM(SEQ ID NO:108),BRRLFFKTITRLFBHGLRRLLSTEGVPNSZ(SEQ ID NO:109),
BRGLEPLVIAGILARRGSLVGLLHIVL(SEQ ID NO:110),BRGSDPLVVAASIVRRASIVGILHLIL(SEQID NO:111),RNLVPMVATVRRNLVPMVATVB(SEQ ID NO:112),RNLVPMVATVBRRNLVPMVATVB(SEQ ID NO:113),RNIVPZVVTARRNIVPZVVTAB(SEQID NO:114),
PEVIPMFSALSEGATPQDLNTMLN(SEQ ID NO:115),RFIIPXFTALSGGRRALLYGATPYAIG(SEQ IDNO:116),KALGPAATLEEMMTACQGVG(SEQ ID NO:117),RRGPVVHLTLRRRGQAGDDFS(SEQID NO:118),RRGPVVHLTLRRRGQAGDDFS(SEQ ID NO:119),RRGPVVHLTLRGRRGQAGDDFS(SEQ ID NO:120),RRLECVYCKQQLLRREVYDFAFRDLC(SEQ ID NO:121),
RRGVYDFAFRDLCRRGFAFRDLCIVYR(SEQ ID NO:122),RRGVFDYAFRDINRRGFAYRDINLAYR(SEQ ID NO:123),RRGATPVDLLGARRGALNLCLPMR(SEQ IDNO:124),
RRGVTPAGLIGVRRGALQIBLPLR(SEQ ID NO:125),RGYLPAVGAPIGRRRVIRVIAHGLRLR(SEQID NO:196),RRSRNLGKVIDTLTCRRLMGYIPLVGA(SEQ ID NO:197),和
RRIRNLGRVIETLTLZGYIPLIGARRIRNLGRVIETLTLZGYIPLIGAR(SEQ ID NO:199),或其片段或变体。
26.根据实施方案1-25中任一项所述的分离的肽,其中所述肽由选自下列的序列组成:X1-NYVTGNIPG-X3-GITFSIYLIVS;X1-IRNLGRVIETLT-X3-LZGYIPLIGA;X1-GYLPAVGAPI-X3-VIRVIAHGLRL;X1-GGGQIIGGNYLIP-X3-PBIGVRATB;X1-NYATGNLPG-X3-GCSFSIFLLAL;X1-SRNLGKVIDTLTC-X3-LMGYIPLVGA;X1-GYIPLVGAPL-X3-VARALAHGVRV;X1-GGGQIVGGVYLLP-X3-PRLGVRATR;X1-LTFLVRSVLLI-X3-GSVLIVRGSLVH;X1-TAYERZCNIL-X3-GRFQTVVQBA;X1-SDPLVVAASIV-X3-ASIVGILHLIL;X1-LIFLARSALIL-X3-SALILRGSVAH;X1-IAYERMCNIL-X3-GKFQTAAQRA;and X1-LEPLVIAGILA-X3-GSLVGLLHIVL;X1-NLVPMVATV-X3-NLVPMATV;X1-GYLPAVGAPIG-X3-VIRVIAHGLRL;X1-IRNLGRVIETLTG-X3-LZGYIPLIGA;X1-GVYDFAFRDLC-X3-GFAFRDLCIVYR,X1-GVFDYAFRDIN-X3-GFAYRDINLAYR,X1-GATPVDLLGA-X3-GALNLCLPMR,X1-GVTPAGLIGV-X3-GALQIBLPLR,和X1-IRNLGRVIETLTLZGYIPLIGA-X3-IRNLGRVIETLTLZGYIPLIGA;任选地在所述肽的C端具有X5,其中X1,X3和X5是指式I的X1,X3和X5。
27.根据实施方案1-26中任一项所述的分离的肽,其中所述肽包含一个或多个半胱氨酸。
28.根据实施方案1-27中任一项所述的分离的肽,其中所述肽包括分子内键,诸如在两个cys残基之间的分子内二硫键(S-S)或缩羰基酯。
29.根据实施方案1-28中任一项所述的分离的肽,其中X2中的N-和/或C-端氨基酸是亲水性或极性氨基酸。
30.根据实施方案1-29中任一项所述的分离的肽,其中X4中的N-端氨基酸是亲水性或极性氨基酸。
31.根据实施方案1-30中任一项所述的分离的肽,其不超过58个氨基酸,诸如不超过56,54,52,50,48,46,44,42,40,38,36,34,32,30,28,26,24,22,20,18个氨基酸残基。
32.根据实施方案1-31中任一项所述的分离的肽,其中X1由2或3个氨基酸组成。
33.根据实施方案32所述的分离的肽,其中X1由WW,BR或RR组成。
34.根据实施方案1-33中任一项所述的分离的肽,其中X3由2或3个氨基酸组成。
35.根据实施方案34所述的分离的肽,其中X3由WW,BR或RR组成。
36.不超过60个氨基酸的分离的肽,其包含独立地在表1或表2中任一项定义的X2和/或X4的序列。
37.根据实施方案36所述的分离的肽,所述肽由独立地在表1或表2中任一项定义的X2或X4的序列组成。
38.根据实施方案1-37中任一项所述的分离的肽,其中X2定义8-25个氨基酸的序列,诸如8-20个氨基酸,诸如8-15个氨基酸的序列。
39.根据实施方案1-38中任一项所述的分离的肽,其中X4定义8-25个氨基酸的序列,诸如8-20个氨基酸,诸如8-15个氨基酸的序列。
40.根据实施方案1-39中任一项所述的分离的肽,其中X2定义少于25个,诸如少于24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7或6个氨基酸的序列。
41.根据实施方案1-40中任一项所述的分离的肽,其中X2定义多于8个,诸如多于9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29个氨基酸的序列。
42.根据实施方案1-41中任一项所述的分离的肽,其中X4定义少于25个,诸如少于24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7或6个氨基酸的序列。
43.根据实施方案1-42中任一项所述的分离的肽,其中X4定义多于8个,诸如多于9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29个氨基酸的序列。
44.根据实施方案1-43中任一项所述的分离的肽,其中本发明的细胞穿透肽不由下述序列组成:RFIIP[Nle]FTALSGGRRALLYGATPYAIG,其中Nle表示正亮氨酸。
45.根据实施方案1-44中任一项所述的分离的肽,其中X2和/或X4不是来源于HIV。
46.根据实施方案1-45中任一项所述的分离的肽,其中X4是少于12个氨基酸的线性序列。
47.根据实施方案1-46中任一项所述的分离的肽,其中X2是少于12个氨基酸的线性序列。
48.根据实施方案1-47中任一项所述的分离的肽,其中X2和/或X4不包含正亮氨酸。
49.根据实施方案1-48中任一项所述的分离的肽,其中X2不包含正亮氨酸。
50.根据实施方案1-49中任一项所述的分离的肽,其中X2和/或X4仅包含天然氨基酸。
51.根据实施方案1-50中任一项所述的分离的肽,其中X2仅包含天然氨基酸。
52.根据实施方案1-51中任一项所述的分离的肽,其中X2在来源于HIV时仅包含天然氨基酸。
53.根据实施方案1-52中任一项所述的分离的肽,其中X2和/或X4来源于HCV、CMV、HPV、流感病毒、腺病毒或小RNA病毒。
54.包含独立地在表1或表2中任一项所定义的X2或X4序列的肽用于在受试者中诱导免疫应答的用途。
55.包含两个肽单体的二聚体肽,其中每个肽单体如实施方案1-53中任一项所定义。
56.根据实施方案55所述的二聚体肽,其中所述两个肽单体是相同的。
57.包含两个以上根据实施方案1-53中任一项所述的肽的肽组合。
58.编码根据实施方案1-53中任一项所述的肽的分离的核酸或多核苷酸。
59.包含根据实施方案58所述的核酸或多核苷酸的载体。
60.包含根据实施方案59所述的载体的宿主细胞。
61.一种免疫原性组合物,所述组合物包含与药用稀释剂或赋形剂以及任选地与免疫学佐剂组合的下列各项:至少一种根据实施方案1-53中任一项所述的肽,根据实施方案55-56中任一项所述的二聚体肽,根据实施方案57所述的肽组合,根据实施方案58所述的核酸或多核苷酸,或根据实施方案59所述的载体。
62.根据实施方案61所述的免疫原性组合物,所述组合物采取疫苗组合物的形式。
63.一种用于在受试者中诱导针对抗原的免疫应答的方法,所述方法包括施用至少一种根据实施方案1-53中任一项所述的肽,根据实施方案55-56中任一项所述的二聚体肽,根据实施方案57所述的肽组合,根据实施方案58所述的核酸或多核苷酸,或根据实施方案59所述的载体;或根据实施方案61-62中任一项所述的组合物。
64.一种用于在感染病毒的受试者中减轻和/或延缓所述病毒的病理作用的方法,所述方法包括施用有效量的至少一种根据实施方案1-53中任一项所述的肽,根据实施方案55-56中任一项所述的二聚体肽,根据实施方案57所述的肽组合,根据实施方案58所述的核酸或多核苷酸,或根据实施方案59所述的载体;或根据实施方案61-62中任一项所述的组合物。
65.根据实施方案1-53中任一项所述的肽,所述肽用作药物。
66.根据实施方案1-53中任一项所述的肽,所述肽用于治疗感染病毒的受试者中所述病毒的病理作用。
序列表(粗体显示的氨基酸表示可以用作本发明式I中定义的任意X2和/或X4的适当的抗原序列)
SEQ ID NO:1:登记号AF009606;丙型肝炎病毒亚型1a聚蛋白基因,完整cds。
MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRL
GVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRG
SRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGAED
GVNYATGNLPGCSFSIFLLALLSCLTVPASAYQVRNSSGLYHVTNDCPNSSIVYEAAD
AILHTPGCVPCVREGNASRCWVAVTPTVATRDGKLPTTQLRRHIDLLVGSATLCSALY
VGDLCGSVFLVGQLFTFSPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPTAALVV
AQLLRIPQAIMDMIAGAHWGVLAGIAYFSMVGNWAKVLVVLLLFAGVDAETHVTGGSA
GRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFNSS
GCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVYC
FTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGA
PPCVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINY
TIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTL
PALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEYVVLLFLLLADARVCSCLWMMLL
ISQAEAALENLVILNAASLAGTHGLVSFLVFFCFAWYLKGRWVPGAVYAFYGMWPLLL
LLLALPQRAYALDTEVAASCGGVVLVGLMALTLSPYYKRYISWCMWWLQYFLTRVEAQ
LHVWVPPLNVRGGRDAVILLMCVVHPTLVFDITKLLLAIFGPLWILQASLLKVPYFVR
VQGLLRICALARKIAGGHYVQMAIIKLGALTGTYVYNHLTPLRDWAHNGLRDLAVAVE
PVVFSRMETKLITWGADTAACGDIINGLPVSARRGQEILLGPADGMVSKGWRLLAPIT
AYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTR
TIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRG
DSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETT
MRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATL
GFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHS
TDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGK
AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVS
TDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRG
KPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQ
DHLEFWEGVFTGLTHIDAHFLSQTKQSGENFPYLVAYQATVCARAQAPPPSWDQMWKC
LIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLA
ALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYQEFDEMEECSQHLPYIEQGMMLAE
QFKQKALGLLQTASRQAEVITPAVQTNWQKLEVFWAKHMWNFISGIQYLAGLSTLPGN
PAIASLMAFTAAVTSPLTTGQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSV
GLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAA
ILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLR
RLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYR
GVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLP
APNYKFALWRVSAEEYVEIRRVGDFHYVSGMTTDNLKCPCQIPSPEFFTELDGVRLHR
FAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRL
ARGSPPSMASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESEN
KVVILDSFDPLVAEEDEREVSVPAEILRKSRRFARALPVWARPDYNPPLVETWKKPDY
EPPVVHGCPLPPPRSPPVPPPRKKRTVVLTESTLSTALAELATKSFGSSSTSGITGDN
TTTSSEPAPSGCPPDSDVESYSSMPPLEGEPGDPDLSDGSWSTVSSGADTEDVVCCSM
SYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVL
DSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVAH
INSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALY
DVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIR
TEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNT
LTCYIKARAACRAAGLQDCTMLVCGDDLVVICESAGVQEDAASLRAFTEAMTRYSAPP
GDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSW
LGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALNCEIYGACYSIEPLDLPPIIQRL
HGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKY
LFNWAVRTKLKLTPIAAAGRLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAG
VGIYLLPNR
SEQ ID NO:2:HCV核心蛋白,H77,登记号AF009606
Genbank编号:2316097
>gi|2316098|gb|AAB66324.1|聚蛋白[丙型肝炎病毒亚型1a]
MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARR
PEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAAR
ALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA
SEQ IDNO:3:
丙型肝炎病毒mRNA,完整cds;登记号M96362M72423,丙型肝炎病毒亚型1b
MSTNPKPQRKTKRNTNRRPQDIKFPGGGQIVGGVYLLPRRGPRL
GVRATRKTSERSQPRGRRQPIPKARRPEGRAWAQPGYPWPLYGNEGLGWAGWLLSPRG
SRPSWGPTDPRRKCGFADLED
GVLSCLTTPVSAYEVRNASGMYHVTNDCSNS SIVYEAAD
MIMHTPGCVPCVREDNSSRCWVALTPTLAARNASVPTTTLRRHVDLLVGVAAFCSAMY
VGDLCGSVFLVSQLFTFSPRRHETVQDCNCSIYPGRVSGHRMAWDMMMNWSPTTALVV
SQLLRIPQAVVDMVTGSHWGILAGLAYYSMVGNWAKVLIAMLLFAGVDGTTHVTGGAQ
GRAASSLTSLFSPGPVQHLQLINTNGSWHINRTALSCNDSLNTGFVAALFYKYRFNAS
GCPERLATCRPIDTFAQGWGPITYTEPHDLDQRPYCWHYAPQPCGIVPTLQVCGPVYC
FTPSPVAVGTTDRFGAPTYRWGANETDVLLLNNAGPPQGNWFGCTWMNGTGFTKTCGG
PPCNIGGVGNNTLTCPTDCFRKHPGATYTKCGSGPWLTPRCLVDYPYRLWHYPCTVNF
TIFKVRMYVGGAEHRLDAACNWTRGERCDLEDRDRSELSPLLLSTTEWQVLPCSFTTL
PALSTGLIHLHQNIVDIQYLYGIGSAVVSFAIKWEYIVLLFLLLADARVCACLWMMLL
VAQAEAALENLVVLNAASVAGAHGILS FIVFFCAAWYIKGRLVPGAAYALYGVWPLLL
LLLALPPRAYAMDREMAASCGGAVFVGLVLLTLSPHYKVFLARFIWWLQYLITRTEAH
LQVWVPPLNVRGGRDAIILLTCVVHPELIFDITKYLLAIFGPLMVLQAGITRVPYFVR
AQGLIRACMLARKVVGGHYVQMVFMKLAALAGTYVYDHLTPLRDWAHTGLRDLAVAVE
PVVFSDMETKVITWGADTAACGDIILALPASARRGKEILLGPADSLEGQGWRLLAPIT
AYSQQTRGLLGCIITSLTGRDKNQVEGEVQVVSTATQSFLATCINGVCWTVFHGAGSK
TLAGPKGPITQMYTNVDQDLVGWPAPPGARSLTPCTCGSSDLYLVTRHADVIPVRRRG
DGRGSLLPPRPVSYLKGSSGGPLLCPSGHAVGILPAAVCTRGVAMAVEFIPVESMETT
MRSPVFTDNPSPPAVPQTFQVAHLHAPTGSGKSTRVPAAYAAQGYKVLVLNPSVAATL
GFGAYMSKAHGIDPNLRTGVRTITTGAPITYSTYGKFLADGGGSGGAYDIIMCDECHS
TDSTTIYGIGTVLDQAETAGARLVVLSTATPPGSVTVPHLNIEEVALSNTGEIPFYGK
AIPIEAIKGGRHLIFCHSKKKCDELAAKLSGLGLNAVAYYRGLDVSVIPTSGDVVVVA
TDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTVPQDAVSRSQRRGRTGRG
RAGIYRFVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETSVRLRAYLNTPGLPVCQ
DHLEFSEGVFTGLTHIDAHFLSQTKQAGENFPYLVAYQATVCARAQAPPPSWDEMWRC
LIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKFIMTCMSADLEVVTSTWVLVGGVLA
ALAAYCLTTGSVVIVGRIILSGKPAIIPDREVLYQEFDEMEECASHLPYFEQGMQLAE
QFKQKALGLLQTATKQAEAAAPVVESKWRALETFWAKHMWNFISGIQYLAGLSTLPGN
PAIRSPMAFTASITSPLTTQHTLLFNILGGWVAAQLAPPSAASAFVGAGIAGAAVGTI
GLGKVLVDILAGYGAGVAGALVAFKIMSGEMPSAEDMVNLLPAILSPGALVVGIVCAA
ILRRHVGPGEGAVQWMNRLIAFASRGNHVSPRHYVPESEPAARVTQILSSLTITQLLK
RLHQWINEDCSTPCSSSWLREIWDWICTVLTDFKTWLQSKLLPRLPGVPFFSCQRGYK
GVWRGDGIMHTTCPCGAQITGHVKNGSMRIVGPKTCSNTWYGTFPINAYTTGPCTPSP
APNYSKALWRVAAEEYVEVTRVGDFHYVTGMTTDNVKCPCQVPAPEFFTEVDGVRLHR
YAPACRPLLREEVVFQVGLHQYLVGSQLPCEPEPDVAVLTSMLTDPSHITAETAKRRL
ARGSPPSLASSSASQLSAPSLKATCTTHHDSPDADLIEANLLWRQEMGGNITRVESEN
KVVILDSFDPLRAEDDEGEISVPAEILRKSRKFPPALPIWAPPDYNPPLLESWKDPDY
VPPVVHGCPLPPTKAPPIPPPRRKRTVVLTESTVSSALAELATKTFGSSGSSAIDSGT
ATAPPDQASGDGDRESDVESFSSMPPLEGEPGDPDLSDGSWSTVSEEASEDVVCCSMS
YTWTGALITPCAAEESKLPINPLSNSLLRHHNMVYATTSRSAGLRQKKVTFDRLQVLD
DHYRDVLKEMKAKASTVKAKLLSVEEACKLTPPHSAKSKFGYGAKDVRSLSSRAVTHI
RSVWKDLLEDTETPISTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYD
VVSTLPQAVMGSSYGFQYSPKQRVEFLVNTWKSKKCPMGFSYDTRCFDSTVTENDIRV
EESIYQCCDLAPEAKLAIKSLTERLYIGGPLTNSKGQNCGYRRCRASGVLTTSCGNTL
TCYLKATAACRAAKLRDCTMLVNGDDLVVICESAGTQEDAASLRVFTEAMTRYSAPPG
DPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETARHTPVNSWL
GNIIMYAPTLWARMILMTHFFSILLAQEQLEKTLDCQIYGACYSIEPLDLPQIIERLH
GLSAFSLHSYSPGEINRVASCLRKLGVPPLRAWRHRARSVRAKLLSQGGRAATCGKYL
FNWAVRTKLKLTPIPAASRLDLSGWFVAGYSGGDIYHSLSRARPRWFMLCLLLLSVGV
GIYLLPNR
SEQ ID NO:4,甲型流感病毒的核壳蛋白
1 MASQGTKRSY EQMETSGERQ NATEIRASVG RMVGGIGRFYIQMCTELKLS DHEGRLIQNS
61 ITIERMVLSA FDERRNKYLE EHPSAGKDPK KTGGPIYRRR DGKWMRELILYDKEEIRRIW
121 RQANNGEDAT AGLTHMMIWH SNLNDATYQR TRALVRTGMD PRMCSLMQGSTLPRRSGAAG
181 AAVKGVGTMV MELIRMIKRGINDRNFWRGE NGRRTRIAYE RMCNILKGKF QTAAQRAMMD
241 QVRESRNPGN AEIEDLIFLA RSALILRGSV AHKSCLPACV YGLAVASGYDFEREGYSLVG
301 IDPFRLLQNS QVFSLIRPNE NPAHKSQLVM MACHSAAFED LRVSSFIRGTRVVPRGQLST
361 RGVQIASNEN METMDSSTLE LRSRYWAIRT RSGGNTNQQR ASAGQISVQPTFSVQRNLPF
421 ERATIMAAFT GNTEGRTSDM RTEIIRMMEN ARPEDVSFQG RGVFELSDEKATNPIVPSFD
481 MSNEGS
SEQ ID NO:5
>gi|73919153|ref|YP_308840.1|基质蛋白2[甲型流感病毒(A/New York/392/2004(H3N2))]
MSLLTEVETPIRNEWGCRCNDSWILDRKR70
REEYRKEQQNAVDADDSHFVSIELE
SEQ ID NO:6
>gi|73919147|ref|YP_308843.1|核壳蛋白[甲型流感病毒(A/New York/392/2004(H3N2))]
MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDHEGRLIQNSLTIEKMVLSA 70
FDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELVLYDKEEIRRIWRQANNGEDATAGLTHIMIWH 140
SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMVKRGINDRNFWRGE 210
NGRKTFKGKDQVRESRNPGNAEIEDSCLPACA280
YGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGT 350
KVSPRGKLSTRGVQIASNENMDNMGSSTLELRSGYWAIRTRSGGNTNQQRASAGQTSVQPTFSVQRNLPF 420
EKSTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSYFFG 490
DNAEEYDN
--
SEQ IDNO:7
>gi|56583270|ref|NP_040979.2|基质蛋白2[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MSLLTEVETPIRNEWGCRCNGSWILDRKG
REEYRKEQQSAVDADDGHFVSIELE
SEQ ID NO:8
>gi|8486130|ref|NP_040982.1|核壳蛋白[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
PA135407C
MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA
FDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWH
SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGE
NGRKTRKGKDQVRESRDPGNAEFEDCLPACV
YGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT
KVVPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPF
DRTTVMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFG
DNAEEYDN
--
SEQ ID NO:9
>gi|73912687|ref|YP_308853.1|膜蛋白M2[甲型流感病毒(A/Korea/426/68(H2N2))]
MSLLTEVETPIRNEWGCRCNDSWILDRKR
REEYRKEQQSAVDADDSHFVSIELE
SEQ ID NO:10
>gi|73921307|ref|YP_308871.1|核蛋白[甲型流感病毒(A/Korea/426/68(H2N2))]
MASQGTKRSYEQMETDGERQNATEIRASVGKMIDGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA
FDERRNKYLEEHPSAGKDPKKTGGPIYKRVDGKWMRELVLYDKEEIRRIWRQANNGDDATAGLTHMMIWH
SNLNDTTYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGE
NGRKTRKGKDQVRESRNPGNAEIEDCLPACV
YGPAIASGYNFEKEGYSLVGIDPFKLLQNSQVYSLIRPNENPAHKSQLVWMACNSAAFEDLRVLSFIRGT
KVSPRGKLSTRGVQIASNENMDTMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPAFSVQRNLPF
DKPTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEMSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFG
DNAEEYDN
SEQ ID NO:11
>gi|330647|gb|AAA45994.1|pp65[人疱疹病毒5]
MASVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHT 70
YFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIH 140
ASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHVVCAHELVCSMENTRATKMQVIG 210
DQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHI 280
MLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRG 350
PQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGAS 420
TSAGRKRKSASSATACTAGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMV 490
ATVQGQNLKYQEFFWDANDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG 541
SEQ ID NO:12
>gi|33330937|gb|AAQ10712.1|推定的转化蛋白E6[人乳头状瘤病毒16型]
MHQKRTAMFQDPQERPGKLPQLCTELQTTIHDIILECVYCKQQLLRRERDGNPYAVC 70
DKCLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWT 140
GRCMSCCRSSRTRRETQL
SEQ ID NO:13
>gi|56583270|ref|NP_040979.2|基质蛋白2[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MSLLTEVETPIRNEWGCRCNGSSDPLAIAANIIGILHLILWILDRLFFKCIYRRFKYGLKGGPSTEGVPK
SMREEYRKEQQSAVDADDGHFVSIELE
SEQ ID NO:14
>gi|8486139|ref|NP_040987.1|PB2蛋白[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRITEMIPER
NEQGQTLWSKMNDAGSDRVMVSPLAVTWWNRNGPMTNTVHYPKIYKTYFERVERLKHGTFGPVHFRNQVK
IRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELQDCKISPLMVAYMLERE
LVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVKNDDVDQSLIIAARNIVRRAAVSADPLASL
LEMCHSTQIGGIRMVDILKQNPTEEQAVGICKAAMGLRISSSFSFGGFTFKRTSGSSVKREEEVLTGNLQ
TLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNF
VNRANQRLNPMHQLLRHFQKDAKVLFQNWGVEPIDNVMGMIGILPDMTPSIEMSMRGVRISKMGVDEYSS
TERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETV
KIQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQQMRDVLGTFDTAQIIKLLPFAAAPPKQSR
MQFSSFTVNVRGSGMRILVRGNSPVFNYNKATKRLTVLGKDAGTLTEDPDEGTAGVESAVLRGFLILGKE
DRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN
SEQ ID NO:15
>gi|8486137|ref|NP_040986.1|聚合酶PA[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MEDFVRQCFNPMIVELAEKTMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIIVELGDPNA
LLKHRFEIIEGRDRTMAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKS
EKTHIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMASRGLWDSFRQSERGEETIEERFEITGT
MRKLADQSLPPNFSSLENFRAYVDGFEPNGYIEGKLSQMSKEVNARIEPFLKTTPRPLRLPNGPPCSQRS
KFLLMDALKLSIEDPSHEGEGIPLYDAIKCMRTFFGWKEPNVVKPHEKGINPNYLLSWKQVLAELQDIEN
EEKIPKTKNMKKTSQLKWALGENMAPEKVDFDDCKDVGDLKQYDSDEPELRSLASWIQNEFNKACELTDS
SWIELDEIGEDVAPIEHIASMRRNYFTSEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCR
TKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIGQVSRP
MFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIESMIEAESSVKEKDMTKEFFENKSETWPIGESPKGVEE
SSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLIVQALRDNLEPGTFDLGGLYEAIEECLINDPWV
LLNASWFNSFLTHALS
SEQ ID NO:16
>gi|8486133|ref|NP_040984.1|非结构性蛋白NS1[甲型流感病毒(A/PuertoRico/8/34(H1N1))]
MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERILK
EESDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPKQKVAGPLCIRMDQAIMDKNIILKANFSVIFDR
LETLILLRAFTEEGAIVGEISPLPSLPGHTAEDVKNAVGVLIGGLEWNDNTVRVSETLQRFAWRSSNENG
RPPLTPKQKREMAGTIRSEV
SEQ ID NO:17
>gi|8486132|ref|NP_040983.1|非结构性蛋白NS2[甲型流感病毒(A/PuertoRico/8/34(H1N1))]
MDPNTVSSFQDILLRMSKMQLESSSEDLNGMITQFESLKLYRDSLGEAVMRMGDLHSLQNRNEKWREQLG
QKFEEIRWLIEEVRHKLKVTENSFEQITFMQALHLLLEVEQEIRTFSFQLI
SEQ ID NO:18
>gi|8486128|ref|NP_040981.1|神经氨酸酶[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MNPNQKIITIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVIL
TGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDRHSNGTVKDRSPY
RALMSCPVGEAPSPYNSRFESVAWSASACHDGMGWLTIGISGPDNGAVAVLKYNGIITETIKSWRKKILR
TQESECACVNGSCFTIMTDGPSDGLASYKIFKIEKGKVTKSIELNAPNSHYEECSCYPDTGKVMCVCRDN
WHGSNRPWVSFDQNLDYQIGYICSGVFGDNPRPKDGTGSCGPVYVDGANGVKGFSYRYGNGVWIGRTKSH
SSRHGFEMIWDPNGWTETDSKFSVRQDVVAMTDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKEKT
IWTSASSISFCGVNSDTVDWSWPDGAELPFTIDK
SEQ ID NO:19
>gi|8486126|ref|NP_040980.1|血细胞凝集素[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLG
KCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSW
PNHNTTKGVTAACSHAGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPSNSKDQQNIYQ
NENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPRYAFALSRGFGS
GIITSNASMHECNTKCQTPLGAINSSLPFQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAI
AGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNIQFTAVGKEFNKLEKR
MENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDN
ECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSL
QCRICI
SEQ ID NO:20
>gi|8486123|ref|NP_040978.1|基质蛋白1[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MSLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPS
ERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTT
EVAFGLVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQAR
QMVQAMRTIGTHPSSSAGLKNDLLENLQAYQKRMGVQMQRFK
SEQ ID NO:21
>gi|83031685|ref|YP_418248.1|PB1-F2蛋白[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MGQEQDTPWILSTGHISTQKRQDGQQTPKLEHRNSTRLMGHCQKTMNQVVMPKQIVYWKQWLSLRNPILV
FLKTRVLKRWRLFSKHE
SEQ ID NO:22
>gi|8486135|ref|NP_040985.1|聚合酶1PB1[甲型流感病毒(A/Puerto RiCo/8/34(H1N1))]
MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKARWTTNTETGAPQLNPID
GPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCIETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAA
TALANTIEVFRSNGLTANESGRLIDFLKDVMESMKKEEMGITTHFQRKRRVRDNMTKKMITQRTIGKRKQ
RLNKRSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKA
KLANVVRKMMTNSQDTELSLTITGDNTKWNENQNPRMFLAMITYMTRNQPEWFRNVLSIAPIMFSNKMAR
LGKGYMFESKSMKLRTQIPAEMLASIDLKYFNDSTRKKIEKIRPLLIEGTASLSPGMMMGMFNMLSTVLG
VSILNLGQKRYTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVDRFYRTCKLHGINMSKKKSYINRTGTF
EFTSFFYRYGFVANFSMELPSFGVSGSNESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR
CHRGDTQIQTRRSFEIKKLWEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNP
FVSHKEIESMNNAVMMPAHGPAKNMEYDAVATTHSWIPKRNRSILNTSQRGVLEDEQMYQRCCNLFEKFF
PSSSYRRPVGISSMVEAMVSRARIDARIDFESGRIKKEEFTEIMKICSTIEELRRQK
SEQ ID NO:23
>gi|8486130|ref|NP_040982.1|核壳蛋白[甲型流感病毒(A/Puerto Rico/8/34(H1N1))]
MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA
FDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWH
SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGE
NGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRDPGNAEFEDLTFLARSALILRGSVAHKSCLPACV
YGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT
KVVPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPF
DRTTVMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFG
DNAEEYDN
SEQ ID NO:24
>gi|73918826|ref|YP_308855.1|聚合酶2[甲型流感病毒(A/Korea/426/1968(H2N2))]
MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKRITEMVPER
NEQGQTLWSKMSDAGSDRVMVSPLAVTWWNRNGPMTSTVHYPKIYKTYFEKVERLKHGTFGPVHFRNQVK
IRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELQDCKISPLMVAYMLERE
LVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSLIIAARNIVRRAAVSADPLASL
LEMCHSTQIGGTRMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSIKREEEVLTGNLQ
TLKIRVHEGYEEFTMVGKRATAILRKATRRLVQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNF
VNRANQRLNPMHQLLRHFQKDAKVLFQNWGIEHIDNVMGMIGVLPDMTPSTEMSMRGIRVSKMGVDEYSS
TERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETV
KIQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQQMRDVLGTFDTTQIIKLLPFAAAPPKQSR
MQFSSLTVNVRGSGMRILVRGNSPVFNYNKTTKRLTILGKDAGTLTEDPDEGTSGVESAVLRGFLILGKE
DRRYGPALSINELSTLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN
SEQ ID NO:25
>gi|73919145|ref|YP_308850.1|血凝素[甲型流感病毒(A/Korea/426/68(H2N2))]
MAIIYLILLFTAVRGDQICIGYHANNSTEKVDTILERNVTVTHAKDILEKTHNGKLCKLNGIPPLELGDC
SIAGWLLGNPECDRLLSVPEWSYIMEKENPRYSLCYPGSFNDYEELKHLLSSVKHFEKVKILPKDRWTQH
TTTGGSWACAVSGKPSFFRNMVWLTRKGSNYPVAKGSYNNTSGEQMLIIWGVHHPNDEAEQRALYQNVGT
YVSVATSTLYKRSIPEIAARPKVNGLGRRMEFSWTLLDMWDTINFESTGNLVAPEYGFKISKRGSSGIMK
TEGTLENCETKCQTPLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNVPQIESRGLFGAIAGFI
EGGWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFNGITNKVNSVIEKMNTQFEAVGKEFSNLEKRLENL
NKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRMQLRDNVKELGNGCFEFYHKCDNECMD
SVKNGTYDYPKYEEESKLNRNEIKGVKLSSMGVYQILAIYATVAGSLSLAIMMAGISFWMCSNGSLQCRI
CI
SEQ ID NO:26
>gi|73912688|ref|YP_308854.1|膜蛋白M1[甲型流感病毒(A/Korea/426/68(H2N2))]
MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPS
ERGLQRRRFVQNALNGNGDPNNMDRAVKLYRKLKREITFHGAKEVALSYSAGALASCMGLIYNRMGAVTT
EVAFAVVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQAR
QMVQAMRAIGTPPSSSAGLKDDLLENLQAYQKRMGVQMQRFK
SEQ ID NO:27
>gi|73912687|ref|YP_308853.1|膜蛋白M2[甲型流感病毒(A/Korea/426/68(H2N2))]
MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIIGILHFILWILDRLFFKCIYRFFKHGLKRGPSTEGVPE
SMREEYRKEQQSAVDADDSHFVSIELE
SEQ ID NO:28
>gi|73912685|ref|YP_308852.1|聚合酶PA[甲型流感病毒(A/Korea/426/68(H2N2))]
MEDFVRQCFNPMIVELAEKAMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIMVELDDPNA
LLKHRFEIIEGRDRTMAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKS
ENTHIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMANRGLWDSFRQSERGEETIEERFEITGT
MRRLADQSLPPNFSCLENFRAYVDGFEPNGYIEGKLSQMSKEVNAKIEPFLKTTPRPIRLPDGPPCFQRS
KFLLMDALKLSIEDPSHEGEGIPLYDAIKCMRTFFGWKEPYIVKPHEKGINPNYLLSWKQVLAELQDIEN
EEKIPRTKNMKKTSQLKWALGENMAPEKVDFDNCRDISDLKQYDSDEPELRSLSSWIQNEFNKACELTDS
IWIELDEIGEDVAPIEHIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCR
TKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIGQMSRP
MFLYVRTNGTSKIKMKWGMEMRPCLLQSLQQIESMVEAESSVKEKDMTKEFFENKSETWPIGESPKGVEE
GSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLVVQALRDNLEPGTFDLGGLYEAIEECLINDPWV
LLNASWFNSFLTHALR
SEQ ID NO:29
>gi|73921833|ref|YP_308877.1|PB1-F2蛋白[甲型流感病毒(A/Korea/426/68(H2N2))]
MGQEQDTPWTQSTEHINIQKRGSGQQTRKLERPNLTQLMDHYLRTMNQVDMHKQTASWKQWLSLRNHTQE
SLKIRVLKRWKLFNKQEWTN
SEQ ID NO:30
>gi|73912683|ref|YP_308851.1|PB1聚合酶亚基[甲型流感病毒(A/Korea/426/68(H2N2))]
MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPID
GPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCLETMEVIQQTRVDKLTQGRQTYDWTLNRNQPAA
TALANTIEVFRSNGLTANESGRLIDFLKDVIESMDKEEMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQ
RLNKRSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVHFVETLARNICEKLEQSGLPVGGNEKKA
KLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRVFLAMITYITRNQPEWFRNVLSIAPIMFSNKMAR
LGKGYMFESKSMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLIDGTVSLSPGMMMGMFNMLSTVLG
VSILNLGQKKYTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVNRFYRTCKLVGINMSKKKSYINRTGTF
EFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR
CHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGSNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNP
FVSHKEIESVNNAVVMPAHGPAKSMEYDAVATTHSWTPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFF
PSSSYRRPVGISSMVEAMVSRARIDARIDFESGRIKKEEFAEIMKICSTIEELRRQK
SEQ ID NO:31
>gi|73921567|ref|YP_308869.1|非结构性蛋白NS2[甲型流感病毒(A/Korea/426/68(H2N2))]
MDSNTVSSFQDILLRMSKMQLGSSSEDLNGMITQFESLKLYRDSLGEAVMRMGDLHSLQNRNGKWREQLG
QKFEEIRWLIEEVRHRLKITENSFEQITFMQALQLLFEVEQEIRTFSFQLI
SEQ ID NO:32
>gi|73921566|ref|YP_308870.1|非结构性蛋白NS1[甲型流感病毒(A/Korea/426/68(H2N2))]
MDSNTVSSFQVDCFLWHVRKQVVDQELGDAPFLDRLRRDQKSLRGRGSTLDLDIEAATRVGKQIVERILK
EESDEALKMTMASAPASRYLTDMTIEELSRDWFMLMPKQKVEGPLCIRIDQAIMDKNIMLKANFSVIFDR
LETLILLRAFTEEGAIVGEISPLPSLPGHTIEDVKNAIGVLIGGLEWNDNTVRVSKTLQRFAWRSSNENG
RPPLTPKQKRKMARTIRSKVRRDKMAD
SEQ ID NO:33
>gi|73921307|ref|YP_308871.1|核蛋白[甲型流感病毒(A/Korea/426/68(H2N2))]
MASQGTKRSYEQMETDGERQNATEIRASVGKMIDGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA
FDERRNKYLEEHPSAGKDPKKTGGPIYKRVDGKWMRELVLYDKEEIRRIWRQANNGDDATAGLTHMMIWH
SNLNDTTYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGE
NGRKTRSAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACV
YGPAIASGYNFEKEGYSLVGIDPFKLLQNSQVYSLIRPNENPAHKSQLVWMACNSAAFEDLRVLSFIRGT
KVSPRGKLSTRGVQIASNENMDTMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPAFSVQRNLPF
DKPTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEMSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFG
DNAEEYDN
SEQ ID NO:34
>gi|73921304|ref|YP_308872.1|神经氨酸酶[甲型流感病毒(A/Korea/426/68(H2N2))]
MNPNQKIITIGSVSLTIATVCFLMQIAILVTTVTLHFKQHECDSPASNQVMPCEPIIIERNITEIVYLNN
TTIEKEICPEVVEYRNWSKPQCQITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPGKCYQFALGQGTTL
DNKHSNDTIHDRIPHRTLLMNELGVPFHLGTRQVCVAWSSSSCHDGKAWLHVCVTGDDKNATASFIYDGR
LMDSIGSWSQNILRTQESECVCINGTCTVVMTDGSASGRADTRILFIEEGKIVHISPLSGSAQHVEECSC
YPRYPDVRCICRDNWKGSNRPVIDINMEDYSIDSSYVCSGLVGDTPRNDDRSSNSNCRNPNNERGNPGVK
GWAFDNGDDVWMGRTISKDLRSGYETFKVIGGWSTPNSKSQINRQVIVDSNNWSGYSGIFSVEGKRCINR
CFYVELIRGRQQETRVWWTSNSIVVFCGTSGTYGTGSWPDGANINFMPI
SEQ ID NO:35
>gi|73919213|ref|YP_308844.1|非结构性蛋白2[甲型流感病毒(A/New York/392/2004(H3N2))]
MDSNTVSSFQDILLRMSKMQLGSSSEDLNGMITQFESLKIYRDSLGEAVMRMGDLHLLQNRNGKWREQLG
QKFEEIRWLIEEVRHRLKTTENSFEQITFMQALQLLFEVEQEIRTFSFQLI
SEQ ID NO:36
>gi|73919212|ref|YP_308845.1|非结构性蛋白1[甲型流感病毒(A/New York/392/2004(H3N2))]
MDSNTVSSFQVDCFLWHIRKQVVDQELSDAPFLDRLRRDQRSLRGRGNTLGLDIKAATHVGKQIVEKILK
EESDEALKMTMVSTPASRYITDMTIEELSRNWFMLMPKQKVEGPLCIRMDQAIMEKNIMLKANFSVIFDR
LETIVLLRAFTEEGAIVGEISPLPSFPGHTIEDVKNAIGVLIGGLEWNDNTVRVSKNLQRFAWRSSNENG
GPPLTPKQKRKMARTARSKV
SEQ ID NO:37
>gi|73919207|ref|YP_308839.1|血凝素[甲型流感病毒(A/New York/392/2004(H3N2))]
MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDS
PHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNES
FNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQISL
YAQASGRITVSTKRSQQTVIPSIGSRPRIRDVPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGK
SSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGA
IAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEG
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCD
NACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNI
RCNICI
SEQ ID NO:38
>gi|73919153|ref|YP_308840.1|基质蛋白2[甲型流感病毒(A/New York/392/2004(H3N2))]
MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIIGILHLILWILDRLFFKCVYRLFKHGLKRGPSTEGVPE
SMREEYRKEQQNAVDADDSHFVSIELE
SEQ ID NO:39
>gi|73919152|ref|YP_308841.1|基质蛋白1[甲型流感病毒(A/New York/392/2004(H3N2))]
MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPS
ERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEIALSYSAGALASCMGLIYNRMGAVTT
EVAFGLVCATCEQIADSQHRSHRQMVATTNPLIKHENRMVLASTTAKAMEQMAGSSEQAAEAMEIASQAR
QMVQAMRAVGTHPSSSTGLRDDLLENLQTYQKRMGVQMQRFK
SEQ ID NO:40
>gi|73919150|ref|YP_308848.1|PB1-F2蛋白[甲型流感病毒(A/New York/392/2004(H3N2))]
MEQEQDTPWTQSTEHTNIQRRGSGRQIQKLGHPNSTQLMDHYLRIMSQVDMHKQTVSWRLWPSLKNPTQV
SLRTHALKQWKSFNKQGWTN
SEQ ID NO:41
>gi|73919149|ref|YP_308847.1|聚合酶PB1[甲型流感病毒(A/New York/392/2004(H3N2))]
MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPID
GPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCLETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAA
TALANTIEVFRSNGLTANESGRLIDFLKDVMESMDKEEMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQ
RVNKRGYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKA
KLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLAMITYITKNQPEWFRNILSIAPIMFSNKMAR
LGKGYMFESKRMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLIDGTASLSPGMMMGMFNMLSTVLG
VSVLNLGQKKYTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINKTGTF
EFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR
CHRGDTQIQTRRSFELKKLWDQTQSRAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDENYRGRLCNPLNP
FVSHKEIESVNNAVVMPAHGPAKSMEYDAVATTHSWNPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFF
PSSSYRRPIGISSMVEAMVSRARIDARIDFESGRIKKEEFSEIMKICSTIEELRRQK
SEQ ID NO:42
>gi|73919147|ref|YP_308843.1|核壳蛋白[甲型流感病毒(A/New York/392/2004(H3N2))]
MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDHEGRLIQNSLTIEKMVLSA
FDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELVLYDKEEIRRIWRQANNGEDATAGLTHIMIWH
SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMVKRGINDRNFWRGE
NGRKTRSAYERMCNILKGKFQTAAQRAMVDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACA
YGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGT
KVSPRGKLSTRGVQIASNENMDNMGSSTLELRSGYWAIRTRSGGNTNQQRASAGQTSVQPTFSVQRNLPF
EKSTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSYFFG
DNAEEYDN
SEQ ID NO:43
>gi|73919136|ref|YP_308842.1|神经氨酸酶[甲型流感病毒(A/New York/392/2004(H3N2))]
MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTN
TTIEKEMCPKLAEYRNWSKPQCDITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTL
NNVHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCVTGDDKNATASFIYNGR
LVDSIVSWSKKILRTQESECVCINGTCTVVMTDGSASGKADTKILFIEEGKIIHTSTLSGSAQHVEECSC
YPRYPGVRCVCRDNWKGSNRPIVDINIKDYSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVK
GWAFDDGNDVWMGRTISEKLRSGYETFKVIEGWSKPNSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINR
CFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI
SEQ ID NO:44
>gi|73919134|ref|YP_308846.1|聚合酶PA[甲型流感病毒(A/New York/392/2004(H3N2))]
MEDFVRQCFNPMIVELAEKAMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIVVELDDPNA
LLKHRFEIIEGRDRTMAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKS
ENTHIHIFSFTGEEIATKADYTLDEESRARIKTRLFTIRQEMANRGLWDSFRQSERGEETIEEKFEISGT
MRRLADQSLPPKFSCLENFRAYVDGFEPNGCIEGKLSQMSKEVNAKIEPFLKTTPRPIKLPNGPPCYQRS
KFLLMDALKLSIEDPSHEGEGIPLYDAIKCIKTFFGWKEPYIVKPHEKGINSNYLLSWKQVLSELQDIEN
EEKIPRTKNMKKTSQLKWALGENMAPEKVDFDNCRDISDLKQYDSDEPELRSLSSWIQNEFNKACELTDS
IWIELDEIGEDVAPIEYIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCR
TKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIGQISRP
MFLYVRTNGTSKVKMKWGMEMRRCLLQSLQQIESMIEAESSIKEKDMTKEFFENKSEAWPIGESPKGVEE
GSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLVVQALRDNLEPGTFDLGGLYEAIEECLINDPWV
LLNASWFNSFLTHALK
SEQ ID NO:45
>gi|73919060|ref|YP_308849.1|聚合酶PB2[甲型流感病毒(A/New York/392/2004(H3N2))]
MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKRITEMVPER
NEQGQTLWSKMSDAGSDRVMVSPLAVTWWNRNGPVASTVHYPKVYKTYFDKVERLKHGTFGPVHFRNQVK
IRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELRDCKISPLMVAYMLERE
LVRKTRFLPVAGGTSSIYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSLIIAARNIVRRAAVSADPLASL
LEMCHSTQIGGTRMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSVKKEEEVLTGNLQ
TLKIRVHEGYEEFTMVGKRATAILRKATRRLVQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNF
VNRANQRLNPMHQLLRHFQKDAKVLFQNWGIEHIDSVMGMVGVLPDMTPSTEMSMRGIRVSKMGVDEYSS
TERVWSIDRFLRVRDQRGNVLLSPEEVSETQGTERLTITYSSSMMWEINGPESVLVNTYQWIIRNWEAV
KIQWSQNPAMLYNKMEFEPFQSLVPKAIRSQYSGFVRTLFQQMRDVLGTFDTTQIIKLLPFAAAPPKQSR
MQFSSLTVNVRGSGMRILVRGNSPVFNYNKTTKRLTILGKDAGTLIEDPDESTSGVESAVLRGFLIIGKE
DRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN
SEQ ID NO:46:CMV蛋白IE122:
>gi|39841910|gb|AAR31478.1|UL122[人疱疹病毒5]
MESSAKRKMDPDNPDEGPSSKVPRPETPVTKATTFLQTMLRKEVNSQLSLGDPLFPELAEESLKTFEQVT
EDCNENPEKDVLAELGDILAQAVNHAGIDSSSTGHTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASAT
PELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQG
EEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLARPDEDSSSSSSSSCSSASDSESESEEMK
CSSGGGASVTSSHHGRGGFGSAASSSLLSCGHQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRG
RVKIDEVSRMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLA
LSTPFLMEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHAQKF
PKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ
SEQ ID NO:126:
>gi|4927721|gb|AAD33253.1|AF125673_2E7[人乳头状瘤病毒16型]
MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQ
STHMCSQKP
实施例1
下述实施例中所用的本发明的肽利用Sheppard,(1978)J.Chem.Soc.,Chem.Commun.(化学学会和化学通信杂志),539的Fmoc-策略通过Schafer-N作为C端酰胺而合成。
细胞穿透测定
将一组肽在N端生物素化,并且如下述图表所示,将长度和类型不同的氨基酸组合添加到肽中序列盒X1,X3和X4。在来自一名个体献血者的生长的细胞上检测所述肽。
本发明所述的肽的氨基酸序列的示意图(每个X定义一段氨基酸序列):
X1 | X2 | X3 | X4 | X5 |
SP_2至SP_17是来自HCV核心蛋白上特定天然结构域的51n_生物素的变体。
关于生物素化肽的细胞内染色
使用96孔U型底聚苯乙烯平板(NUNC,货号:163320)进行人PBMCs染色。简言之,将8ul表1或表2所述的N-或C-端生物素化的肽(即,对于每种肽检测5mM,2.5mM和1.25mM)与40ul来自献血者的PBMC(12.5x106个细胞/ml)一起在37℃温育2小时。然后,细胞用150ulCellwash(BD,货号:349524)洗涤3次,接着将每种细胞沉淀用100ul胰蛋白酶-EDTA(Sigma,货号:T4424)重悬,然后在37℃温育5分钟。然后,将胰蛋白酶化的细胞用150ulCellwash(BD,货号:349524)洗涤3次,接着用BD Cytofix/CytopermTM plus(BD,货号:554715)重悬,然后按照供应商说明在4℃温育20分钟。然后,将细胞用150ul PermWash(BD,货号:554715)洗涤2次。然后,将细胞用链霉抗生物素蛋白-APC(BD,货号:554067)和抗-hCD11c(eBioscience,货号:12-0116)按照供应商说明在4℃染色30分钟,目的是分别显现生物素化的肽和树突细胞。然后,将细胞用150ul PermWash洗涤3次,接着重悬在染色缓冲液(BD,货号:554656)中,之后进行流式细胞术检测。树突细胞门控(gated)为在淋巴细胞区外的CD11c+事件(即,比淋巴细胞高的FSC&SSC信号)。在具有HTS上样器(loader)的FACSCanto II流式细胞仪上共捕获200000个细胞,并且对总细胞和树突细胞绘制关于肽-荧光(即GeoMean)的直方图。
关于生物素化肽的细胞外染色
使用96孔U型底聚苯乙烯平板(NUNC,货号:163320)进行人PBMCs染色。简言之,将8ul表1或表2所述的N-或C-端生物素化的肽(即,对于每种肽检测5mM,2.5mM和1.25mM;所有的肽由Schafer制备)与40ul来自献血者的PBMC(12.5x106个细胞/ml)一起在37℃温育2小时。然后,细胞用150ul Cellwash(BD,货号:349524)洗涤3次,然后将细胞用链霉抗生物素蛋白-APC(BD,货号:554067)和抗-hCD11c(eBioscience,货号:12-0116)按照供应商说明在4℃染色30分钟,目的是分别显现生物素化的肽和树突细胞。然后,将细胞用150ulCellWash(BD,货号:349524)洗涤3次,接着重悬在染色缓冲液(BD,货号:554656)中,之后进行流式细胞术检测。树突细胞门控为在淋巴细胞区外的CD11c+事件(即,比淋巴细胞高的FSC&SSC信号)。在具有HTS上样器(loader)的FACSCantoII流式细胞仪上共捕获200000个细胞,并且对总细胞和树突细胞绘制关于肽-荧光(即GeoMean)的直方图。
可以清楚看出在开头、中间和末端添加精氨酸提高进入细胞的能力。观察到精氨酸的变化决定进入细胞的能力,使用在开头两个和在中间两个精氨酸获得最好的结果-但是不同量的精氨酸的变化都显示了良好的结果。
下表所示的数据是每种检测的肽的几何平均值,其通过FACS Duva软件计算。通过胰蛋白酶化(trypsinating)/Cytofix/Cytoperm的几何平均值:
表3
如表4所示,本发明所述的肽具有通过细胞膜分配的能力。
表4:在DC细胞上使用低浓度肽(208uM)的结果。结果以(关于BC16)降序分类。
肽
实施例2
阳性CTL应答可以备选地通过ELISPOT测定检测。
通过ELISPOT测定检测人IFN-γ细胞毒性T-细胞(CTL)应答
简言之,在第1天,将来自HCV患者的PBMC样品在烧瓶(430000PBMCs/cm2)中在37℃,5%CO2以覆盖量的培养基(RPMI1640Fisher Scientific;货号:PAAE15-039,补充了L-谷氨酰胺(MedProbe货号.13E17-605E)、10%胎牛血清(FBS)(Fisher Scientific货号A15-101)和青霉素/链霉素(Fisher Acientific货号P11-010))温育2小时,以允许单核细胞贴壁。分离非贴壁细胞,洗涤,并且在进一步使用之前冷冻在FBS中的10%V/V DMSO中。将贴壁细胞小心用培养基洗涤,然后在37℃在含有2μg/ml终浓度的hrGM-CSF(Xiamen amoytopbiotech co,货号:3004.9090.90)和1μg/ml hrIL-4(Invitrogen,货号:PHC0043)的培养基中温育至第3天,然后在第6天重复该步骤。在第7天,向用0.5μg/孔抗人γ干扰素包被的ELISPOT(Millipore multiscreen HTS)平板中添加培养的树突细胞(每孔5000-10000个),以及解冻的自体非贴壁细胞(每孔200000个),抗原样品(对于肽抗原终浓度为1-8ug/ml;对于伴刀豆球蛋白A(Sigma,货号:C7275)或PHA(Sigma,货号:L2769)终浓度为5ug/ml)和抗-无反应性抗体(anti-Anergy antibodies,对于抗-PD-1(eBioscience,货号:16-9989-82)和抗-PD-L1(eBioscience,货号:16-5983-82)终浓度均为0.03-0.05ug/ml)。将平板温育过夜,并且按照供应商说明显示斑点。在ELISPOT读数仪(S5UV分析仪)上读取斑点。
表5:核心蛋白的ELISPOT
表5-在具有来源于HCV蛋白的结构域的支架肽上运行的IFN-g ELISPOT的结果。
与无肽和不相关对照肽相比,所述肽具有整体上更高量的T细胞斑点。这清楚地表明所述肽在HCV患者中具有积极的免疫学效应。
实施例3
详细的REVEAL&
快速表位发现系统
对于下述HLA-种类检测表6列出的九聚体的HLA结合性质:
HLA-A1,HLA-A2,HLA-A3,HLA-A11,HLA-A24,HLA-A29,HLA-B7,HLA-B8,HLA-B14,HLA-B15,HLA-B27,HLA-B35,HLA-B40。
合成所述肽作为Prospector定制肽文库。长度为8-15个氨基酸的肽以高平均纯度的0.5-2mg的量合成。在100%的样品上进行通过MALDI-TOF质谱的质量控制。
REVEALTM结合测定检测每种候选肽结合一种或多种I类MHC等位基因和稳定MHC-肽复合物的能力。通过比较与高亲和力和中等亲和力T细胞表位的结合,可以鉴定蛋白序列中最可能的免疫原性肽。检测是基于存在或不存在MHC-肽复合物的天然构象。
相对于阳性对照肽(其为已知的T细胞表位),对每种肽给出评分。测试肽的评分定量报告为所述阳性对照肽产生的信号的百分数,并且该肽表示为具有推定的合格(pass)或不合格(fail)结果。通过包含中间对照肽确认测定的性能,已知所述中间对照肽以较弱的亲和力结合所研究的等位基因。
实施例4
细胞内染色:
制备具有来源于HCV、流感病毒或CMV的X2和X4的本发明所述的肽,并在上述“细胞穿透测定”中所述的实验中检测细胞内染色。
对于相同的肽1-15的细胞外染色:
肽编号 | 抗原 | 中值 | n |
1 | 阴性对照 | 1.00 | 35 |
2 | 支架中的阴性对照 | 1.77 | 20 |
3 | 阳性对照 | 9.15 | 19 |
5 | HCV天然序列 | 2.08 | 21 |
6 | HCV | 2.20 | 29 |
7 | HCV | 29.42 | 12 |
8 | HCV | 3.06 | 28 |
9 | HCV | 2.06 | 23 |
10 | HCV(非支架) | 1.95 | 27 |
11 | HCV(非支架) | 2.62 | 23 |
12 | HCV(天然序列) | 1.76 | 1J |
13 | HCV | 16.80 | 7 |
14 | HCV | 17.00 | 16 |
15 | HCV(非支架) | 2.38 | 12 |
19 | HCV | 1.37 | 19 |
20 | HCV | 126.5 | 7 |
结果:可以看出,与天然肽或在X1,X3和X5中具有其他氨基酸的肽相比,支架形式上的肽具有提高的摄入。
Claims (16)
1.一种分离的细胞穿透肽,其由下述结构组成:
X1-X2-X3-X4-X5 (式I),
其中X1和X3独立地定义任意1,2或3个精氨酸的线性序列;X2和X4独立地定义来自HCV,CMV,HPV或流感病毒的相同抗原或其相对于所述抗原包含1-10个置换,添加或缺失并与天然序列具有至少50%序列同一性的变体序列的8-15个氨基酸的线性序列,所述序列X4不同于X2;并且其中X5是任选的精氨酸。
2.根据权利要求1所述的分离的肽,其中所述肽包含一个或多个半胱氨酸。
3.根据权利要求1所述的分离的肽,其中X2中的N-和/或C-端氨基酸是亲水性或极性氨基酸。
4.根据权利要求1所述的分离的肽,其中X4中的N-端氨基酸是亲水性或极性氨基酸。
5.根据权利要求1所述的分离的肽,其中X1由RR组成,X3由RR组成,和X1和X3每个由RR组成。
6.根据权利要求1所述的分离的肽,其中X2和/或X4是少于12个氨基酸的线性序列。
7.包含两个肽单体的二聚体肽,其中每个肽单体如权利要求1-6中任一项所定义。
8.包含两个以上根据权利要求1-6中任一项所述的肽的肽组合。
9.编码根据权利要求1-6中任一项所述的肽的分离的核酸或多核苷酸。
10.包含根据权利要求9所述的核酸或多核苷酸的载体,或包含所述载体的宿主细胞。
11.一种免疫原性组合物,所述组合物包含与药用稀释剂或赋形剂以及任选地与免疫学佐剂组合的:至少一种根据权利要求1-6中任一项所述的肽,根据权利要求7所述的二聚体肽,根据权利要求8所述的肽组合,根据权利要求9所述的核酸或多核苷酸,或根据权利要求10所述的载体。
12.根据权利要求11所述的免疫原性组合物,所述组合物为疫苗组合物的形式。
13.至少一种根据权利要求1-6中任一项所述的肽,根据权利要求7所述的二聚体肽,根据权利要求8所述的肽组合,根据权利要求9所述的核酸或多核苷酸,或根据权利要求10所述的载体;或根据权利要求11-12中任一项所述的组合物在制备用于在受试者中诱导针对抗原的免疫应答的药物中的应用。
14.有效量的至少一种根据权利要求1-6中任一项所述的肽,根据权利要求7所述的二聚体肽,根据权利要求8所述的肽组合,根据权利要求9所述的核酸或多核苷酸,或根据权利要求10所述的载体;或根据权利要求11-12中任一项所述的组合物在制备用于在感染病毒的受试者中减轻和/或延缓所述病毒的病理作用的药物中的应用。
15.根据权利要求1-6中任一项所述的肽,所述肽用作药物。
16.根据权利要求1-6中任一项所述的肽,所述肽用于治疗感染病毒的受试者中所述病毒的病理作用。
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EP2646459A1 (en) | 2013-10-09 |
CA2819416C (en) | 2019-04-30 |
AU2011335551A1 (en) | 2013-06-20 |
US9550811B2 (en) | 2017-01-24 |
EA201390806A1 (ru) | 2014-04-30 |
US20130337002A1 (en) | 2013-12-19 |
CA2819416A1 (en) | 2012-06-07 |
EP2646459B1 (en) | 2020-01-08 |
EP2646459A4 (en) | 2014-12-03 |
CN103282375A (zh) | 2013-09-04 |
WO2012072088A1 (en) | 2012-06-07 |
AU2011335551B2 (en) | 2016-10-06 |
JP6069212B2 (ja) | 2017-02-01 |
EA025152B1 (ru) | 2016-11-30 |
US20170112917A1 (en) | 2017-04-27 |
ZA201304017B (en) | 2015-02-25 |
JP2014502156A (ja) | 2014-01-30 |
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