CN104884082A - 用作诱导抗原特异性t细胞反应的免疫原性增强剂的融合蛋白 - Google Patents

用作诱导抗原特异性t细胞反应的免疫原性增强剂的融合蛋白 Download PDF

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CN104884082A
CN104884082A CN201380063867.5A CN201380063867A CN104884082A CN 104884082 A CN104884082 A CN 104884082A CN 201380063867 A CN201380063867 A CN 201380063867A CN 104884082 A CN104884082 A CN 104884082A
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seq
fusion rotein
antigen
aminoacid sequence
binding domain
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CN104884082B (zh
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周维宜
吴嘉茂
吴俊明
章修纲
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Biocybernetic Gene Vaccine Co Ltd
TheVax Genetics Vaccine Co Ltd
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Biocybernetic Gene Vaccine Co Ltd
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Abstract

本发明公开了一种疫苗组合物，其包含用于诱导增强的病原体抗原特异性T细胞反应的融合蛋白。所述融合蛋白包含：(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于融合蛋白的N端；(b)长度为34-112个氨基酸残基的转位肽，其包含与SEQ ID NO:4、SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:6至少90％相同的氨基酸序列，其位于APC结合域或CD91受体结合域的C端；(c)病原体的抗原，其位于转位肽的C端；(d)核输出信号，其包含SEQ ID NO:13的氨基酸序列；及(e)内质网滞留序列，其位于融合蛋白的C端。

Description

用作诱导抗原特异性T细胞反应的免疫原性增强剂的融合蛋白
技术领域
本发明主要涉及融合蛋白及免疫学。
背景技术
分子生物学已使亚单位疫苗的制造成为可能，其中，免疫原为母蛋白或母复合物的片段或亚单位。所需要的是可诱发T细胞致敏反应又能足够灵活的并入来自许多感染源的毒株的序列的稳定疫苗的开发。
发明内容
一方面，本发明涉及一种融合蛋白，其包含：
(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端；
(b)长度为34-112个氨基酸残基的转位肽，其包含与SEQ ID NO:4、SEQID NO:2、SEQ ID NO:3或SEQ ID NO:6至少90％相同的氨基酸序列，其位于所述APC结合域或CD91受体结合域的C端；
(c)病原体的抗原，其位于所述转位肽的C端；
(d)核输出信号，其包含SEQ ID NO:13的氨基酸序列；及
(e)内质网(ER)滞留序列，其位于所述融合蛋白的C端。
在本发明的一个实施方式中，所述APC结合域或所述CD91受体结合域为多肽，其包含与选自SEQ ID NO:1及SEQ ID NO:8-SEQ ID NO:11的序列至少90％相同的氨基酸序列。
在本发明的另一个实施方式中，所述核输出信号包含SEQ ID NO:14的氨基酸序列。
在本发明的另一个实施方式中，所述内质网滞留序列包含SEQ ID NO:15的氨基酸序列。
在本发明的另一个实施方式中，所述核输出信号位于所述转位肽与所述抗原之间。
在本发明的另一个实施方式中，所述核输出信号位于所述抗原与所述内质网滞留序列之间。
在本发明的另一个实施方式中，所述核输出信号及所述ER滞留序列形成包含与SEQ ID NO:12至少90％相同的氨基酸序列的融合肽。
在本发明的另一个实施方式中，所述转位肽的长度为34-61个氨基酸残基。
在本发明的另一个实施方式中，所述转位肽的长度为34-46个氨基酸残基。
在本发明的另一个实施方式中，所述APC结合域或所述CD91受体结合域无假单胞菌外毒素A(PE)结合域I的氨基酸序列。
在本发明的另一个实施方式中，所述APC结合域或所述CD91受体结合域包含SEQ ID NO:8的氨基酸序列。
在本发明的另一个实施方式中，所述APC结合域或所述CD91受体结合域的氨基酸序列为SEQ ID NO:1。
在本发明的另一个实施方式中，所述抗原为病原体的两种或更多种抗原肽的融合抗原。
在本发明的另一个实施方式中，所述ER滞留序列的长度为多于4个氨基酸残基。
在本发明的另一个实施方式中，所述转位肽包含与SEQ ID NO:4、SEQID NO:2、SEQ ID NO:3或SEQ ID NO:6至少95％相同的氨基酸序列。
在本发明的另一个实施方式中，所述APC结合域或所述CD91受体结合域显示出识别并结合于选自树突细胞、单核细胞、B细胞及淋巴细胞的抗原呈递细胞(APC)上的受体的特征。
在本发明的另一个实施方式中，所述病原体选自PRRSV、PCV、FMDV、CSFV、NDV、传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、流感病毒、假狂犬病病毒、细小病毒、假狂犬病病毒、猪水疱病毒(SVDV)、痘病毒、轮状病毒、支原体肺炎、疱疹病毒、传染性支气管炎及传染性粘液囊症病毒。
另一方面，本发明实质上由下列各项组成，或由下列各项组成：
(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端；
(b)长度为34-112个氨基酸残基转位肽，其包含与SEQ ID NO:4、SEQ IDNO:2、SEQ ID NO:3或SEQ ID NO:6至少90％相同的氨基酸序列，其位于所述APC结合域或CD91受体结合域的C端；
(c)病原体的抗原，其位于所述转位肽的C端；
(d)核输出信号，其包含SEQ ID NO:13的氨基酸序列；及
(e)内质网滞留序列，其位于所述融合蛋白的C端。
又一方面，本发明涉及一种包含前述融合蛋白及佐剂的疫苗组合物。
再一方面，本发明涉及一种用于诱导增强的病原体抗原特异性T细胞反应的方法，其包含：向有此需要的受试者施用包含治疗有效量的前述融合蛋白的疫苗组合物；并由此诱导增强的病原体抗原特异性T细胞反应。
本发明还涉及将前述融合蛋白或疫苗组合物用于诱导增强的病原体抗原特异性T细胞反应，或将前述融合蛋白用于制造诱导增强的病原体抗原特异性T细胞反应的药物。
附图说明
图1A为显示全长铜绿假单胞菌外毒素A(PE)及PE的部分片段的示意图。
图1B-C显示载体图谱。
图2-5分别为显示根据本发明的融合蛋白诱发增强的CD8+/IFN-γ+T细胞(图2A-5A)及CD4+/IFN-γ+T细胞(图2B-5B)介导的免疫原性的图。
图6显示动物组别、用于免疫所述动物的疫苗与剂量，以及免疫接种程序表。
图7-8分别为显示接种不同融合蛋白或安慰剂的动物组别的肿瘤大小曲线及无肿瘤小鼠百分比。
具体实施方式
由于对于本领域的技术人员来说在此的各种修改和变化将明显可见，所以在以下仅意欲用作说明的实施例中更具体地描述本发明。现在，将详述本发明的各种实施方式。参考附图，图中相同标号均代表相同部分。如在本说明书及随后的整个权利要求中使用的，除非上下文另外载明，否则“一”、“一个”和“该”的意思包括复数。并且，如在本说明书及随后的整个权利要求中使用的，除非上下文另外载明，否则“在…中”的意思包括“位于其中”及“位于其上”。此外，为读者的便利标题及小标题可以用于说明书中，其不影响本发明的范围。因此，以下将具体定义用于本说明书的若干术语。
定义
本说明书所使用的术语通常在本发明上下文内及使用各术语的特定上下文中具有它们在本领域中的常规涵义。用以描述本发明的特定用语将在下文中或在本说明书的别处加以说明，以向从业者提供有关本发明的描述的额外指导。为便利，某些术语可以例如使用斜体和/或引号突出显示，突出显示的使用不影响术语的范围与意义；无论是否突出显示，同一术语在相同上下文中的范围与意义皆相同。将意识到的是同一件事的表达方式不止一种。因此，替代用语及同义词可以用作任一一种或多种在此讨论的术语，也无任何特殊意义来归因于是否在此详细说明或讨论术语。本文提供某些术语之同义词。但使用某一或多个同义词并不表示排除其他同义词。本说明书的任何地方的实施例(包括在此处所讨论的术语的实施例)的使用仅为用作说明的，而非用于局限本发明或任何示例性术语的范围与意义。同样地，本发明并不局限于本说明书给出的各种实施方式。
除非另有定义，否则在此使用的所有技术与科学术语具有与本发明相关领域的普通技术者之一通常所了解的相同。在有相互冲突的情况下，则以本说明书及其所提供的定义为解释依据。
术语“抗原呈递细胞(APC)或辅助细胞”指呈现与其表面上的主要组织相容性复合体(MHC)复合的外来抗原的细胞。T细胞可以使用它们的T细胞受体(TCR)来识别这些复合物。这些细胞加工抗原，并向T细胞呈递它们。专业抗原呈递细胞的主要种类为树突细胞(DC)、巨噬细胞(其亦为CD4+，因此易受HIV感染)、单核细胞及若干B细胞。
术语“抗原呈递细胞(APC)结合域”指可结合至抗原呈递细胞(APC)的区域(其为多肽)。所述APC结合域可为包含与选自SEQ ID NO:1及SEQID NO:8-SEQ ID NO:11的序列至少90％相同的氨基酸序列的多肽。APC结合域为可识别并结合于APC上的受体的配体。
分化簇91(CD91)为在细胞膜中形成受体且参与受体介导的内吞作用的蛋白。
术语“PEt”指长度为34-112个氨基酸残基的转位肽(或转位域)。PEt可包含与SEQ ID NO:2-SEQ ID NO:4及SEQ ID NO:6至少90％相同的氨基酸序列。例如，PEt的氨基酸序列可为PE的a.a.280-a.a.313片段(SEQ IDNO:4)、a.a.268-a.a.313片段(SEQ ID NO:3)、a.a.253-a.a.313片段(SEQID NO:2)或a.a.253-a.a.364片段(SEQ ID NO:6)。换言之，PEt的氨基酸序列可含有PE区域II(a.a.253至a.a.364；SEQ ID NO:6)的任一区，只要其包含a.a.280-a.a.313(SEQ ID NO:4)必需序列(即，必需片段)即可。
PE407(SEQ ID NO:7)在先前专利(美国专利第7,335,361B2号)中描述为PE(ΔIII)。
术语“最小转位肽”指PE253-313(SEQ ID NO:2)，其可将抗原转位至靶细胞的细胞质内。
术语“内质网(ER)滞留序列”指作用为帮助抗原从细胞质至ER的转位，并将所述抗原留在ER的内腔中的肽。ER滞留序列包含序列Lys Asp GluLeu(KDEL；SEQ ID NO:15)或RDEL。ER滞留序列可包含序列KDEL、RDEL、KDELKDELKDEL(K3；SEQ ID NO:16)、KKDLRDELKDEL(K3；SEQ ID NO:17)、KKDELRDELKDEL(K3；SEQ ID NO:18)或KKDELRVELKDEL(K3；SEQ ID NO:19)。
核输出信号(NES)指蛋白中具有4个疏水性残基的短氨基酸序列，其为利用核传输，通过核孔复合体从细胞核输出至细胞质而靶向蛋白。输出蛋白(exportin)可识别并结合NES。疏水性残基最常见的间隔为LxxKLxxLxLx(SEQID NO:13)，其中，“L”为亮氨酸，“K”为赖氨酸，且“x”为任一种天然氨基酸。例如，人工NES可包含序列Leu Gln Lys Lys Leu Glu Glu Leu Glu LeuAla(LQKKLEELELA；SEQ ID NO:14)。
术语“NESK”指NES与ER滞留信号的融合肽(即，融合至ER滞留信号的NES)。NESK为具有核输出信号(NES)及ER滞留序列的功能的人工肽。因此，其可通过所述核孔复合体将抗原从细胞核输出至细胞质，并帮助抗原从细胞质转位至ER，而且将抗原留在ER的内腔中。例如，NESK的氨基酸序列可为LQKKLEELELAKDEL(SEQ ID NO:12)。
抗原可为病原体蛋白、多肽或肽，其为造成由所述病原体引起的疾病的原因，或可于所述病原体感染的宿主体内诱导免疫反应，或为多肽在肿瘤细胞内特异表达的肿瘤相关抗原(TAA)。所述抗原可选自病原体或癌症细胞，其包括但不限于人乳头瘤病毒(HPV)、猪繁殖及呼吸综合症病毒(PRRSV)、人类免疫缺陷病毒-1(HIV-1)、登革热病毒、丙型肝炎病毒(HCV)、乙型肝炎病毒(HBV)、猪环状病毒2(PCV2)、典型猪瘟病毒(CSFV)、口蹄疫病毒(FMDV)、新城病病毒(NDV)、传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、流感病毒、假狂犬病病毒、细小病毒、假狂犬病病毒、猪水疱病毒(SVDV)、痘病毒、轮状病毒、支原体肺炎、疱疹病毒、传染性支气管炎、或传染性粘液囊症病毒、非小细胞肺癌、乳癌、黑色素瘤、淋巴瘤、结肠癌、肝细胞癌，及它们的组合。例如，HPV E7蛋白(E7)、HCV核心蛋白(HCV核心)、HBV X蛋白(HBx)选做开发疫苗的抗原。所述抗原可为选自一种或多种病原体蛋白的两种或更多种抗原)融合而成的融合抗原。例如，PRRSV ORF6与ORF5片段的融合抗原，或PRRSV与PCV2病原体的抗原蛋白的融合。
术语“治疗”或“疗法”指对已患癌症、或已遭受感染、或已出现倾向于该疾病的症状或易染病体质的有此需要的受试者施用有效量的融合蛋白，藉以治愈、减轻、解除、治疗、改善或预防疾病、其症状或倾向于它的易染病体质。基于任何适当诊断方法的结果能够识别出这样的受试者。
术语“有效量”指给予治疗的受试者产生治疗效果所需的活性化合物的量。如本领域中的技术人员所公认的，有效量将会根据施用途径、赋形剂的使用以及与其他治疗法共用的可能性而有所变化。
本发明涉及一种增强抗原递送并调节细胞介导的免疫反应的融合蛋白。所述融合蛋白包含：(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端；(b)长度为34-112个氨基酸残基的转位肽，其包含与SEQ ID NO:2至SEQ ID NO:4及SEQ ID NO:6至少90％相同的氨基酸序列，其位于所述APC结合域或CD91受体结合域的C端；(c)病原体的抗原，其位于所述转位肽的C端；(d)核输出信号(NES)；及(e)内质网(ER)滞留序列，所述ER滞留序列位于所述融合蛋白的C端，其中，NES包含SEQID NO:13的氨基酸序列。
以融合蛋白PE313-ORF2-NESK为例，策略在于本发明的融合蛋白刺激可识别第2型猪环状病毒(PCV2)壳体蛋白ORF2(抗原)的T细胞的制造与活化。融合蛋白自N端至C端依序包含PE区域I(APC结合域)，长度为34-112个氨基酸残基的转位肽(例如，PE区域II的a.a.253-313)，截短的PCV2ORF2蛋白(移除N端核定位信号)，NES信号，及ER滞留序列(KDEL)。以PE313-ORF2-NESK诱发增强的ORF2特异性T细胞免疫反应的基础机理包含下列步骤：a)结合至树突细胞(或抗原呈递细胞)表面受体(CD91)；b)由内吞作用内在化；c)传输至ER，并于转位肽前方以弗林蛋白酶进行蛋白质水解；d)加工，并通过MHC I复合体呈递；及e)活化抗原特异性CD4+及CD8+T细胞。CD4+Th1细胞可有效率地刺激并增强细胞毒性CD8+T细胞免疫反应。同时，适应性免疫系统的以上两种技术具有杀死PCV2及PCV2感染细胞的特异性和效力。
这里所述融合蛋白PE313-ORF2-NESK在许多方面均与Lai于美国专利第7,335,361号中公开的融合蛋白疫苗PE407-Ag-K3不同。首先，PE313(SEQ IDNO:5)的长度为94个氨基酸残基，比PE407(SEQ ID NO:7)短，其优点在于可将由PE的额外片段所诱发的不需要的体液反应降至最低。其次，缩短所述ER滞留序列。仅需要一个KDER或RDER而不需使用K3(即，3个KDER)。再者，仅细胞溶质抗原可被加工并经由MHC第I型通道呈递，因此，因为提高抗原移位进入细胞溶质的机会，将NES信号加入融合蛋白中有利于增强病原体抗原特异性T细胞反应。病原体的抗原可输入至细胞核内。通过结合NES信号，输入细胞核内的抗原便可通过融合蛋白的NES信号而输出至细胞质。
实施例
以下给出不意欲限制本发明的范围的根据本发明的实施方式的示例性仪器、设备、方法及其相关的结果。请注意，为读者的便利各标题或子标题用于实施例，其不应限制本发明的范围。此外，在此提出或公开某些理论；但是，只要本发明为据其本身实施而不考虑任何特定学说或实行方案，它们无论正确或错误，都不应限制本发明的范围。
实施例1
表达载体的构建
图1A显示PE含有3个区域(I、II及III)。PE407为PE从a.a.1至a.a 407的区域。PE407不含细胞毒素区域III，并且因此含有区域I及II。PE313为PE从a.a.1至a.a.313的区域，因此，PE313仅含有PE的区域Ia及区域II的部分N端区域。
图1B-C显示表达载体的结构，在有(下图)或无(上图)核输出信号(NES)的情况下，各结构均包含抗原呈递细胞(APC)结合域、转位肽、抗原；及内质网(ER)滞留序列(上图为K3或下图为K)，所述ER滞留序列位于融合蛋白的C端。如下产生质粒pTac-2-PE313-NESK、pTac-2-PE407-K3、pTac-2-RAP1-PE268-313-NESK及pTac-2-RAP1-PE268-313-K3：NdeIPE313-(EcoRI, XhoI)-NESKXhoI、NdeIPE407-(EcoRI,XhoI)-K3XhoI、NdeIRAP1-(EcoRI)-PE268-313-(EcoRI, XhoI)-NESKXhoI及NdeIRAP1-(EcoRI)-PE268-313-(EcoRI,XhoI)-K3XhoI片段以聚合酶连锁反应法(PCR)合成，然后利用卡那霉素抗药基因将其连接入pUC18骨干中以形成各质粒。
之后再将编码目的病原体的抗原或融合抗原的目标DNA插入前述质粒中以产生表达融合蛋白的表达载体。例如，合成编码第2型猪环状病毒(PCV2)ORF2(SEQ ID NO:20)、典型猪瘟病毒(CSFV)E2(SEQ ID NO:21)、口蹄疫病毒(FMDV)VP1-3A(SEQ ID NO:24)及新城病病毒(NDV)FHN(SEQ ID NO:27)的抗原的DNA片段并分别插入质粒pTac-2-PE313-NESK与pTac-2-PE407-K3中以产生下列表达载体：(1)PE313-ORF2-NESK；(2)PE407-ORF2-K3；(3)PE313-E2-NESK；(4)PE407-E2-K3；(5)PE313-VP1-3A-NESK；(6)PE407-VP1-3A-K3；(7)PE313-FHN-NESK及(8)PE407-FHN-K3。合成编码第16型人类乳头瘤病毒E7(SEQ ID NO:28)的抗原的DNA片段并分别插入质粒pTac-2-PE407-K3、pTac-2-RAP1-PE268-313-NESK及pTac-2-RAP1-PE268-313-K3中以产生下列表达载体：(9)PE407-E7-K3；(10)RAP1-PE268-313-E7-NESK及(11)RAP1-PE268-313-E7-K3。
实施例2
蛋白表达
在37℃下在含有25ppm卡那霉素的Luria Bertani培养基中分别培养含有用于融合蛋白(1)PE313-ORF2-NESK；(2)PE407-ORF2-K3；(3)PE313-E2-NESK；(4)PE407-E2-K3；(5)PE313-VP1-3A-NESK；(6)PE407-VP1-3A-K3；(7)PE313-FHN-NESK；(8)PE407-FHN-K3；(9)PE407-E7-K3；(10)RAP1-PE268-313-E7-NESK及(11)RAP1-PE268-313-E7-K3的表达的质粒的大肠杆菌(E.coli)BL21细胞。当培养基到达早期对数生长期(A600＝0.1至0.4)时，加入异丙基-1-硫代-β-D-半乳糖苷(IPTG)，使其最终浓度为0.5至2mM以达诱导的目的。诱导4小时后收集细胞，并且随即在-70℃下储存。所述融合蛋白以如前述的(Liao等人，1995，Appl.Microbiol.Biotechnol.43:498-507)尿素萃取法纯化，之后在4℃下，以50倍体积的TNE缓冲液(50mMTris、50mM NaCl及1mM EDTA)进行隔夜透析，来再折叠。再折叠后的蛋白经过SDS-PAGE分析，并使用Bradford蛋白检测试剂盒(Pierce)进行定量分析。分析结果显示，大部分的再折叠蛋白在非还原条件下为单体，显示所述融合蛋白可轻易再折叠且未聚集。
实施例3
PCV2亚单位疫苗免疫原性分析
小鼠以每周一次、连续3周，经皮下注射接种0.1ml PCV2亚单位疫苗，疫苗含有40μg PE313-ORF2-NESK或PE407-ORF2-K3，以及磷酸铝(缓慢释放融合蛋白的蛋白质吸收剂；10％v/v)与10μg皂角苷(萃取自皂皮树(Quillajasaponaria)的佐剂)。对照组(安慰剂)仅注射佐剂而不注射融合蛋白。所有小鼠均在最后一次免疫后14天处死，并收集其脾脏。脾细胞经分离后，在37℃下及1μg/ml GolgiPlug(BD Pharmingen,San Diego,CA)存在的条件下，在含有或不含有重组ORF2蛋白的6孔培养板(108个细胞/2ml/孔)中培养16小时。刺激过后的脾细胞以FACScan缓冲液清洗，并以藻红蛋白共轭单克隆大鼠抗小鼠CD8a抗体及AF700共轭单克隆大鼠抗小鼠CD4抗体染色。使用根据制造商(BD Pharmingen)的说明的Cytofix/Cytoperm试剂盒进行细胞内细胞因子染色细胞。以AF488共轭大鼠抗小鼠IFN-γ染色细胞内的IFN-γ，以便量测免疫反应及细胞因子水平。使用Gallios流式细胞计数及Kaluza分析软件(Beckman Coulter)进行流式细胞计数分析。
图2A-B分别显示接种安慰剂(仅含佐剂而不含融合蛋白)或融合蛋白的小鼠的脾细胞中CD8+及CD4+IFN-γT细胞的数量。用ORF2刺激的脾细胞中由CD4+及CD8+T细胞所产生的IFN-γ通过经流式细胞计数的细胞内染色来检测。柱状图显示来自有(灰色柱)或没有(黑色柱)ORF2肽刺激的各组的ORF2特异性IFN-γ+CD4+T细胞的数量(图2B)及IFN-γ+CD8+T细胞的数量(图2A)。结果显示，已经接种PE313-ORF2-NESK的小鼠由ORF2肽刺激而产生的ORF2特异性CD4+IFN-γ+及CD8+IFN-γ+T细胞高于已经接种PE407-ORF2-K3的小鼠组。
实施例4
CSFV亚单位疫苗免疫原性分析
除了加入重组E2蛋白来刺激培养中的脾细胞之外，使用相同的免疫接种程序表及剂量为小鼠接种CSFV亚单位疫苗，其中亚单位疫苗含有PE313-E2-NESK或PE407-E2-K3，并且，分离、培养脾细胞，并以上述流式细胞计数法分析。
图3A-B分别显示接种安慰剂(仅含佐剂而不含融合蛋白)或融合蛋白的小鼠的脾细胞中CD8+及CD4+IFN-γT细胞的数量。用E2刺激的脾细胞中由CD4+及CD8+T细胞所产生的IFN-γ通过经流式细胞计数的细胞内染色来检测。柱状图显示来自有(灰色柱)或没有(黑色柱)E2肽刺激的各组的E2特异性IFN-γ+CD4+T细胞的数量(图3B)及IFN-γ+CD8+T细胞的数量(图3A)。结果显示，已经接种PE313-E2-NESK的小鼠由E2肽刺激而产生的E2特异性CD4+IFN-γ+及CD8+IFN-γ+T细胞高于已经接种PE407-E2-K3的小鼠组。
实施例5
FMDV亚单位疫苗免疫原性分析
除了加入重组VP1-3A蛋白来刺激培养中的脾细胞之外，使用相同的免疫接种程序表及剂量为小鼠接种FMDV亚单位疫苗，其中亚单位疫苗含有PE313-VP1-3A-NESK或PE407-VP1-3A-K3，并且，分离、培养脾细胞，并以上述流式细胞计数法分析。
图4A-B显示接种安慰剂或融合蛋白的小鼠的脾细胞中CD8+及CD4+IFN-γT细胞的数量。用VP1-3A刺激的脾细胞中由CD4+及CD8+T细胞所产生的IFN-γ通过经流式细胞计数的细胞内染色来检测。柱状图显示来自有(灰色柱)或没有(黑色柱)VP1-3A肽刺激的各组的VP1-3A特异性IFN-γ+CD4+T细胞的数量(图4B)及IFN-γ+CD8+T细胞的数量(图4A)。结果显示，已经接种PE313-VP1-3A-NESK的小鼠由VP1-3A肽刺激而产生的VP1-3A特异性CD4+IFN-γ+及CD8+IFN-γ+T细胞高于已经接种PE407-VP1-3A-K3的小鼠组。
实施例6
NDV亚单位疫苗免疫原性分析
除了加入重组FHN蛋白来刺激培养中的脾细胞之外，使用相同的免疫接种程序表及剂量为小鼠接种FMDV亚单位疫苗，其中亚单位疫苗含有PE313-FHN-NESK或PE407-FHN-K3，并且，分离、培养脾细胞，并以流式细胞计数法分析。
图5A-B显示接种安慰剂或融合蛋白的小鼠的脾细胞中CD8+及CD4+IFN-γT细胞的数量。用FHN刺激的脾细胞中由CD4+及CD8+T细胞所产生的IFN-γ通过经流式细胞计数的细胞内染色来检测。柱状图显示来自有(灰色柱)或没有(黑色柱)FHN肽刺激的各组的FHN特异性的IFN-γ+CD4+T细胞的数量(图5B)及IFN-γ+CD8+T细胞的数量(图5A)。结果显示，已经接种PE313-FHN-NESK的小鼠其经由FHN肽刺激而产生的FHN特异性CD4+IFN-γ+及CD8+IFN-γ+T细胞高于已经接种PE407-FHN-K3的小鼠组。
实施例7
由第16型人乳头瘤病毒E7蛋白诱导的肿瘤生长的增强的抑制
使用上述相似方法表达和再重叠所述融合蛋白PE407-E7-K3、RAP1-PE268-313-E7-K3及RAP1-PE268-313-E7-NESK。小鼠经皮下注射之用2x103个TC-01细胞(含有第16型HPV E7基因的小鼠肺部上皮细胞)激发，以诱导第16型HPV上皮癌。TC-01细胞激发后十二天，小鼠以每周一次、连续3周经皮下注射接种安慰剂(PBS)、PE407-E7-K3(100mg/剂)、RAP1-PE268-313-E7-K3(100μg/剂)或RAP1-PE268-313-E7-NESK(100μg/剂)(均以AS04C(GlaxoSmithKline)为佐剂)(图6)。AS04C为细胞毒性T淋巴细胞增强佐剂，包含MPL(单磷酯脂质A，一种免疫增效剂)及磷酸铝(一种用于抗原递送的蛋白质吸收剂)。术语“K3”指包含KDEL的ER滞留序列。例如，K3可为氨基酸序列KDELKDELKDEL(SEQ ID NO:16)。术语“NESK”指包含核输出信号及ER滞留序列的融合肽。在本发明的一个实施方式中，所述NESK为氨基酸序列LQKKLEELELAKDEL(SEQ ID NO:12)。记录各组的肿瘤大小及无肿瘤动物数(图7及8)。肿瘤生长由以AS04C为佐剂的疫苗PE407-E7-K3、RAP1-PE268-313-E7-K3及RAP1-PE268-313-E7-NESK显著抑制。但是，在抑制肿瘤生长及提高无肿瘤动物的百分比方面，疫苗RAP1-PE268-313-E7-NESK优于PE407-E7-K3，并且比RAP1-PE268-313-E7-K3更好。
实施例8
产生下列融合蛋白：PE313-NES-抗原-K、PE1-252-PE268-313-NES-抗原-K、PE1-252-PE280-313-NES-抗原-K。此外，融合蛋白PE313-抗原-NESK的PE区域Ia的片段(PE1-252)由RAP1区域3(SEQ ID NO:8)、A2M最小(SEQ ID NO:9)、HIV-Tat最小(SEQ ID NO:10)或HSPs最小(SEQ ID NO:11)代替以分别产生融合蛋白RAP1区域3-PE253-313-抗原-NESK、A2M-PE253-313-抗原-NESK、Tat-PE253-313-抗原-NESK及HSP-PE253-313-抗原-NESK、RAP1区域3-PE268-313-抗原-NESK、A2M-PE268-313-抗原-NESK、Tat-PE268-313-抗原-NESK及HSP-PE268-313-抗原-NESK疫苗、RAP1区域3-PE280-313-抗原-NESK、A2M-PE280-313-抗原-NESK、Tat-PE280-313-抗原-NESK及HSP-PE280-313-抗原-NESK。RAP1区域3-PE253-313-NES-抗原-K、A2M-PE253-313-NES-抗原-K、Tat-PE253-313-NES-抗原-K及HSP-PE253-313-NES-抗原-K、RAP1区域3-PE268-313-NES-抗原-K、A2M-PE268-313-NES-抗原-K、Tat-PE268-313-NES-抗原-K及HSP-PE268-313-NES-抗原-K疫苗、RAP1区域3-PE280-313-NES-抗原-K、A2M-PE280-313-NES-抗原-K、Tat-PE280-313-NES-抗原-K及HSP-PE280-313-NES-抗原-K。使用上述相似方法检测由这些疫苗增强的细胞介导的免疫反应。
表1显示制造各种融合蛋白所用肽的序列编号。
表1
*：粗体字母代表人工核输出信号的氨基酸序列；画线的字母代表内质网滞留信号的氨基酸序列。
**：VP1-3A肽为一种由VP1的a.a.127-a.a.176及3A的a.a.21-a.a.35组成的融合抗原；即，FMDV VP1肽(SEQ ID NO:22)及FMDV 3A肽(SEQ ID NO:23)的融合。
***：FHN肽为一种由融合蛋白的a.a.65-a.a.82及血凝素-神经氨酸酶的a.a.101-a.a.111组成的融合抗原；即，NDV F肽(SEQ ID NO:25)及NDV HN肽(SEQ ID NO:26)的融合。
总之，分析结果已证明含有位于N末端的APC结合域、转位域，并接续病原体的抗原，以及位于羧基末端的NESK融合肽的融合蛋白在增强细胞介导的免疫反应、抑制肿瘤生长和/或提高无肿瘤动物百分比方面为超过在羧基末端无NESK融合肽的PE-融合蛋白的改良的设计。
当已说明并描述本发明的实施方式时，本领域的技术人员可以进行各种修改和改善。本发明并不局限于说明的特定形式，并且所有未偏离本发明的实质与范围的修改均在所附权利要求定义的范围中。
选择并描述实施方式与实施例，以便解释本发明的原理及它们的实际应用，使本领域中的其他技术人员利用本发明及各种实施方式，和具有如适合实际预期使用的各种修改。有关本发明而不偏离其实质和范围的可选择的实施方式对于本领域的技术人员来说将明显可见。因此，本发明的范围由所附权利要求，而非由以上叙述及其中所描述的示例性实施方式定义。
在本发明的说明书中引用并讨论可能包括专利、专利申请案及各种出版物的参考文献。提供该参考文献的引用和/或讨论仅用来阐明本发明的说明，而非承认任一该参考文献为在此描述的本发明的“现有技术”。本说明书中所引用与讨论的所有参考文献，在此通过引用将其全部内容并入本文，且以如各参考文献分别通过引用并入的相同程度并入本文。

Claims (20)

1.一种融合蛋白，其包含：
(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端；
(b)长度为34-112个氨基酸残基的转位肽，其包含与SEQ ID NO:4、SEQID NO:2、SEQ ID NO:3或SEQ ID NO:6至少90％相同的氨基酸序列，其位于所述APC结合域或CD91受体结合域的C端；
(c)病原体的抗原，其位于所述转位肽的C端；
(d)核输出信号，其包含SEQ ID NO:13的氨基酸序列；及
(e)内质网(ER)滞留序列，其位于所述融合蛋白的C端。
2.如权利要求1所述的融合蛋白，其中，所述APC结合域或所述CD91受体结合域为包含与选自SEQ ID NO:1及SEQ ID NO:8至SEQ ID NO:11的序列至少90％相同的氨基酸序列的多肽。
3.如权利要求1所述的融合蛋白，其中，所述核输出信号包含SEQ ID NO:14的氨基酸序列。
4.如权利要求1所述的融合蛋白，其中，所述内质网滞留序列包含SEQID NO:15的氨基酸序列。
5.如权利要求1所述的融合蛋白，其中，所述核输出信号位于所述转位肽与所述抗原之间。
6.如权利要求1所述的融合蛋白，其中，所述核输出信号位于所述抗原与所述内质网滞留序列之间。
7.如权利要求1所述的融合蛋白，其中，所述核输出信号及所述ER滞留序列形成包含与SEQ ID NO:12至少90％相同的氨基酸序列的融合肽。
8.如权利要求1所述的融合蛋白，其中，所述转位肽的长度为34-61个氨基酸残基。
9.如权利要求1所述的融合蛋白，其中，所述APC结合域或所述CD91受体结合域无假单胞菌外毒素A(PE)结合域I的氨基酸序列。
10.如权利要求9所述的融合蛋白，其中，所述APC结合域或所述CD91受体结合域包含SEQ ID NO:8的氨基酸序列。
11.如权利要求1所述的融合蛋白，其中，所述APC结合域或所述CD91受体结合域的氨基酸序列为SEQ ID NO:1。
12.如权利要求11所述的融合蛋白，其中，所述转位肽的长度为34-61个氨基酸残基。
13.如权利要求12所述的融合蛋白，其中，所述核输出信号及所述ER滞留序列形成具有与SEQ ID NO:12至少90％相同的氨基酸序列的融合肽。
14.如权利要求11所述的融合蛋白，其中，所述转位肽的长度为34-46个氨基酸残基。
15.如权利要求10所述的融合蛋白，其中，所述转位肽的长度为34-61个氨基酸残基。
16.如权利要求15所述的融合蛋白，其中，所述核输出信号及所述ER滞留序列形成具有与SEQ ID NO:12至少90％相同的氨基酸序列的融合肽。
17.如权利要求1所述的融合蛋白，其中，所述抗原为病原体两种或更多抗原肽的融合抗原。
18.如权利要求1所述的融合蛋白，其实质上由下列各项组成：
(a)抗原呈递细胞(APC)结合域或CD91受体结合域，其位于所述融合蛋白的N端；
(b)长度为34-112个氨基酸残基的转位肽，其包含与SEQ ID NO:4、SEQID NO:2、SEQ ID NO:3或SEQ ID NO:6至少90％相同的氨基酸序列，其位于所述APC结合域或CD91受体结合域的C端；
(c)病原体的抗原，其位于所述转位肽的C端；
(d)核输出信号，其包含SEQ ID NO:13的氨基酸序列；及
(e)内质网(ER)滞留序列，其位于所述融合蛋白的C端。
19.一种包含如权利要求1所述的融合蛋白及佐剂的疫苗组合物。
20.如上述权利要求中的任意一项所述的融合蛋白或疫苗组合物，其用于诱导增强的病原体抗原特异性T细胞反应。
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