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Publication numberCN1832956 A
Publication typeApplication
Application numberCN 200480022891
PCT numberPCT/US2004/018962
Publication date13 Sep 2006
Filing date14 Jun 2004
Priority date13 Jun 2003
Also published asCA2529145A1, CA2529145C, CN1832956B, EP1638987A2, EP1638987A4, EP1638987B1, EP2292634A2, EP2292634A3, US8173786, US20070041941, US20120276142, WO2005000235A2, WO2005000235A3
Publication number200480022891.5, CN 1832956 A, CN 1832956A, CN 200480022891, CN-A-1832956, CN1832956 A, CN1832956A, CN200480022891, CN200480022891.5, PCT/2004/18962, PCT/US/2004/018962, PCT/US/2004/18962, PCT/US/4/018962, PCT/US/4/18962, PCT/US2004/018962, PCT/US2004/18962, PCT/US2004018962, PCT/US200418962, PCT/US4/018962, PCT/US4/18962, PCT/US4018962, PCT/US418962
InventorsDB维纳, M库茨勒, AK楚, J杨, JD博伊尔
Applicant宾夕法尼亚州大学信托人
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Nucleic acid sequences encoding and compositions comprising ige signal peptide and/or il-15 and methods for using the same
CN 1832956 A
Abstract
Fusion proteins and nucleic acid molecules encoding fusion proteins are disclosed. Fusion proteins comprising non-IL-15 signal peptide linked to IL-15 protein sequences and fusion proteins comprising an IgE signal peptide linked to non-IgE protein sequences are disclosed. Vectors comprising such nucleic acid molecules; and to host cells comprising such vectors are disclosed as well as recombinant vaccines and live attenuated pathogens encoding fusion proteins, and methods of using the same, are disclosed. The immunomodulatory effect following delivery of IL-15 and CD40L, with or without immunogens, is disclosed as are various nucleic acid molecules and compositions thereof used for delivering such proteins and methods of using such compositions.
Claims(53)  translated from Chinese
1.一种分离的核酸分子,所述核酸分子包含编码包含与白介素(IL)-15蛋白或其功能性片段连接的非IL-15信号肽的非免疫原性融合蛋白的核酸序列;或者所述核酸分子包含编码包含与IL-15蛋白或其功能性片段连接的非IL-15信号肽的融合蛋白的核酸序列,其中所述非IL-15信号肽与所述IL-15来自相同物种的基因。 1. An isolated nucleic acid molecule, comprising the nucleic acid molecule comprises a coding -15 protein or a functional fragment was ligated with the interleukin (IL) of the non-immunogenic Non-IL-15 signal peptide fusion protein nucleic acid sequence; or the said nucleic acid molecule comprises a nucleic acid sequence encoding a IL-15 protein or functional fragment thereof connected to the non-IL-15 signal peptide fusion protein, wherein said non-IL-15 signal peptide and the IL-15 from the same species genes.
2.权利要求1的分离的核酸分子,其中所述非IL-15信号肽是IgE信号肽。 Isolated nucleic acid molecule of claim 1, wherein said non-IL-15 signal peptide is a signal peptide IgE.
3.权利要求1的分离的核酸分子,其中所述融合蛋白由与IL-15蛋白或其功能性片段连接的非IL-15信号肽组成。 Isolated nucleic acid molecule of claim 1, wherein the fusion protein consists of a non-IL-15 signal peptide and IL-15 protein or functional fragment thereof connected components.
4.权利要求1-3中任一项的分离的核酸分子,其中所述IL-15蛋白或其功能性片段是缺乏IL-15信号肽的IL-15蛋白或缺乏IL-15信号肽的IL-15蛋白的功能性片段。 1-3 isolated nucleic acid molecule of any one of claim 1, wherein the IL-15 protein or functional fragment thereof is the lack of IL-15 protein IL-15 signal peptide or absence of IL IL-15 signal peptide -15 protein functional fragments.
5.权利要求1-4中任一项的分离的核酸分子,其中编码所述IL-15蛋白或其功能性片段的核酸序列缺乏IL-15Kozak区和/或IL-155′非翻译区和/或IL-153′非翻译区。 1-4 5. The isolated nucleic acid molecule of claim any one of, wherein the nucleic acid sequence encoding the IL-15 protein or a functional fragment lacking IL-15Kozak region and / or IL-155 'untranslated region and / or IL-153 'untranslated region.
6.权利要求1-5中任一项的分离的核酸分子,所述核酸分子还包含编码CD40L或其功能性片段的核苷酸序列。 1-5 isolated nucleic acid molecule of any one of claim 1, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding a CD40L or a functional fragment thereof.
7.一种分离的核酸分子,所述核酸分子包含编码IL-15蛋白或其功能性片段的核酸序列和编码CD40L或其功能性片段的核苷酸序列。 7. An isolated nucleic acid molecule, the nucleic acid molecule comprises a nucleic acid sequence and the nucleotide sequence encoding CD40L or functional fragment thereof encoding IL-15 protein or a functional fragment thereof.
8.权利要求1-7中任一项的分离的核酸分子,所述核酸分子还包含编码免疫原的核酸序列。 1-7 isolated nucleic acid molecule of any one of claim 1, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding an immunogen.
9.权利要求8的分离的核酸分子,其中所述免疫原是病原体抗原、癌相关抗原或涉及自身免疫性疾病的细胞相关抗原。 Isolated nucleic acid molecule of claim 8, wherein the immunogen is a pathogen antigen, a cancer-associated antigen or a cell involved in autoimmune disease-associated antigen.
10.权利要求9的分离的核酸分子,其中所述免疫原是病原体抗原。 Isolated nucleic acid molecule of claim 9, wherein said immunogen is a pathogen antigen.
11.权利要求10的分离的核酸分子,其中所述病原体抗原是从选自以下的病原体来的:人免疫缺陷病毒(HIV)、HSV、HCV和WNV。 Isolated nucleic acid molecule of claim 10, wherein the pathogen antigen is from a pathogen selected from the following to: the human immunodeficiency virus (HIV), HSV, HCV, and WNV.
12.权利要求1-11中任一项的分离的核酸分子,其中IL-15编码序列缺乏IL-15信号肽。 1-11 isolated nucleic acid molecule of any one of claim 1, wherein the IL-15 IL-15 coding sequence lacking the signal peptide.
13.权利要求1-12中任一项的分离的核酸分子,其中IL-15编码序列缺乏IL-15Kozak区和/或IL-15 5'非翻译区和/或IL-15 3'非翻译区。 1-12 isolated nucleic acid molecule of any one of claims 13, wherein the IL-15 coding sequence lacking IL-15Kozak region and / or IL-15 5 'untranslated region and / or IL-15 3' untranslated region .
14.权利要求1-13中任一项的分离的核酸分子,其中所述分离的核酸分子是质粒。 1-13 isolated nucleic acid molecule of any one of claims 14, wherein said isolated nucleic acid molecule is a plasmid.
15.权利要求1-13中任一项的核酸分子,所述核酸分子掺入到病毒载体中。 A nucleic acid molecule of any one of claim 1 to 13, wherein the nucleic acid molecule incorporated into a viral vector.
16.一种组合物,所述组合物包含权利要求1-15中任一项的核酸分子和包含编码免疫原的核酸序列的核酸分子。 16. A composition, said composition comprising a nucleic acid molecule of claim and one of the nucleic acid molecule comprises a nucleic acid sequence encoding an immunogen according to any of 1-15.
17.权利要求16的组合物,其中所述免疫原是病原体抗原、癌相关抗原或涉及自身免疫性疾病的细胞相关抗原。 17. The composition of claim 16, wherein the immunogen is a pathogen antigen, a cancer-associated antigen or a cell involved in autoimmune disease-associated antigen.
18.权利要求17的组合物,其中所述免疫原是病原体抗原。 18. The composition of claim 17, wherein the immunogen is a pathogen antigen.
19.权利要求18的组合物,其中所述病原体抗原是从选自以下的病原体来的:HIV、HSV、HCV和WNV。 19. The composition of claim 18, wherein the pathogen antigen is selected from the pathogen to the: HIV, HSV, HCV, and WNV.
20.一种组合物,所述组合物包含权利要求1-19中任一项的核酸分子并且还包含编码CD40L的核苷酸序列的核酸分子。 20. A composition, said composition comprising a nucleic acid molecule as claimed in any one of claim 1 to 19 and further comprising a nucleic acid molecule encoding a nucleotide sequence of CD40L.
21.一种注射用药物组合物,所述组合物包含权利要求1-15中任一项的核酸分子或权利要求16-20中任一项的组合物。 21. A pharmaceutical composition for injection, said composition comprising a nucleic acid molecule of claim or any one of Claims 1 to 15 the composition of claim any one of 16-20.
22.一种重组疫苗,所述疫苗包含权利要求1-13中任一项的核酸分子。 22. A recombinant vaccine, said vaccine comprising a nucleic acid molecule of any one of claims 1 to 13.
23.权利要求22的重组疫苗,其中所述重组疫苗是重组痘苗病毒疫苗。 23. The recombinant vaccine of claim 22, wherein said recombinant vaccine is a recombinant vaccinia virus vaccine.
24.一种减毒活病原体,其中包含权利要求1-13中任一项的核酸分子。 24. A live attenuated pathogens, which comprises a nucleic acid molecule as claimed in any one of claim 1-13.
25.一种包含与IL-15蛋白序列连接的非IL-15信号序列的非免疫原性融合蛋白或者一种包含与IL-15蛋白序列连接的非IL-15信号序列的融合蛋白,其中所述非IL-15信号序列与所述IL-15蛋白序列来自相同的物种。 25. A non-IL-15 and IL-15 signal sequence protein sequence linked comprising non-immunogenic fusion protein or a fusion protein comprising a non-IL-15 signal sequence and the IL-15 protein sequence connection, wherein said non-IL-15 signal sequence and the IL-15 protein sequences from the same species.
26.权利要求25的融合蛋白,其中所述非IL-15信号序列是IgE信号序列。 26. The fusion protein of claim 25, wherein the non-IL-15 signal sequence is a signal sequence IgE.
27.权利要求25或26的融合蛋白,所述融合蛋白由与IL-15蛋白序列连接的非IL-15信号序列组成。 The fusion protein of claim 26 or 25, wherein said fusion protein by non-IL-15 and IL-15 signal sequence protein sequence linked composition.
28.权利要求25-27中任一项的融合蛋白,其中所述IL-15蛋白序列缺乏IL-15信号序列。 According to any one of claims 25-27 28. A fusion protein, wherein said IL-15 IL-15 protein sequence lacking a signal sequence.
29.一种组合物,所述组合物包含两种核酸分子,第一种核酸分子包含编码IL-15蛋白的核酸序列,第二种核酸分子包含编码CD40L蛋白的核苷酸序列。 29. A composition, the composition comprises two nucleic acid molecules, the first nucleic acid molecule comprising a nucleic acid sequence encoding IL-15 protein, a second nucleic acid molecule comprises a nucleotide sequence encoding a CD40L protein.
30.权利要求29的组合物,所述组合物还包含编码免疫原的核酸序列。 30. The composition of claim 29, wherein the composition further comprises a nucleic acid sequence encoding an immunogen.
31.权利要求29或30的组合物,其中所述免疫原是病原体抗原、癌相关抗原或涉及自身免疫性疾病的细胞相关抗原。 The composition of claim 30 or 29, wherein the immunogen is a pathogen antigen, a cancer-associated antigen or a cell involved in autoimmune disease-associated antigen.
32.权利要求31的组合物,其中所述免疫原是病原体抗原。 32. The composition of claim 31, wherein the immunogen is a pathogen antigen.
33.权利要求32的组合物,其中所述免疫原是从选自以下病原体来的病原体抗原:HIV、HSV、HCV和WNV。 33. The composition of claim 32, wherein the immunogen is selected from the pathogen to pathogen antigens: HIV, HSV, HCV, and WNV.
34.权利要求29-33中任一项的组合物,其中所述分离的核酸分子是质粒。 The composition of any one of claims 29-33 34., wherein said isolated nucleic acid molecule is a plasmid.
35.一种调节个体免疫应答的方法,所述方法包括给予所述个体权利要求58或63的组合物。 35. A method of modulating an immune response in an individual, said method comprising administering to said subject the composition of claim 58 or 63.
36.一种诱导个体产生抗免疫原的免疫应答的方法,所述方法包括给予所述个体权利要求59、62或64-67中任一项的组合物。 36. A method of inducing anti-immunogen individual generating an immune response, said method comprising administering to said subject the composition of claim any one of claims 59, 62 or 64-67.
37.一种分离的核酸分子,所述核酸分子包含编码融合蛋白的核酸序列,所述融合蛋白由与非IgE蛋白序列连接的IgE信号肽组成,其中所述IgE信号肽和所述非IgE蛋白序列来源于相同物种的动物。 37. An isolated nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, said fusion protein consists of a signal peptide and non-IgE IgE protein sequence linked composition, wherein said non-IgE IgE signal peptide and the protein sequences from animals of the same species.
38.一种分离的核酸分子,所述核酸分子包含编码融合蛋白的核酸序列,所述融合蛋白由与非IgE蛋白序列连接的IgE信号肽组成,其中所述非IgE蛋白是酶或其功能性片段。 38. An isolated nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, said fusion protein consists of a signal peptide and non-IgE IgE protein sequence linked composition, wherein said non-IgE protein is an enzyme or a functional fragments.
39.一种分离的核酸分子,所述核酸分子包含编码融合蛋白的核酸序列,所述融合蛋白由与非IgE蛋白序列连接的IgE信号肽组成,其中所述非IgE蛋白是免疫调节蛋白或其功能性片段。 39. An isolated nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, said fusion protein consists of a signal peptide and non-IgE IgE protein sequence linked composition, wherein said non-IgE protein is an immunomodulatory protein or functional fragments.
40.权利要求39的分离的核酸分子,其中所述融合蛋白由与免疫调节蛋白或其功能性片段连接的IgE信号肽组成。 Isolated nucleic acid molecule of claim 39, wherein the fusion protein consists of a signal peptide with immunomodulatory IgE protein or functional fragment thereof connected components.
41.权利要求39-40中任一项的分离的核酸分子,其中所述IgE信号肽和所述非IgE蛋白序列来源于相同物种的动物。 39-40 isolated nucleic acid molecule of any one of claims 41, wherein said non-IgE IgE signal peptide and the protein sequence derived from the same animal species.
42.权利要求39-41中任一项的分离的核酸分子,其中所述分离的核酸分子是质粒。 39-41 isolated nucleic acid molecule of any one of claims 42, wherein said isolated nucleic acid molecule is a plasmid.
43.权利要求39-41中任一项的核酸分子,所述核酸分子掺入到病毒载体中。 The nucleic acid molecule of any one of claims 39-41 43., wherein the nucleic acid molecule incorporated into a viral vector.
44.一种注射用药物组合物,所述组合物包含权利要求39-41中任一项的核酸分子。 44. A pharmaceutical composition for injection, said composition comprising a nucleic acid molecule of claim of any one of 39-41.
45.一种重组疫苗,所述疫苗包含权利要求39-41中任一项的核酸分子。 45. A recombinant vaccine, said vaccine comprising a nucleic acid molecule of any one of claims 39-41.
46.一种减毒活病原体,其中包含权利要求39-41中任一项的核酸分子。 46. A live attenuated pathogens, which comprises a nucleic acid molecule of claim of any one of 39-41.
47.一种融合蛋白,所述蛋白包含与非IgE蛋白连接的IgE信号肽,其中所述IgE信号肽和所述非IgE蛋白来源于相同物种的动物。 47. A fusion protein, said protein comprising the signal peptide and non-IgE-IgE-protein linked, wherein said non-IgE IgE signal peptide and the protein derived from the same animal species.
48.一种融合蛋白,所述蛋白包含与非IgE蛋白连接的IgE信号肽,其中所述非IgE蛋白是酶或其功能性片段。 48. A fusion protein, said protein comprising the signal peptide and non-IgE-IgE-protein linked, wherein said non-IgE protein is an enzyme or functional fragment thereof.
49.权利要求47的融合蛋白,其中所述非IgE蛋白序列是免疫调节蛋白或其功能性片段。 49. The fusion protein of claim 47, wherein said non-IgE protein sequence is immunomodulatory protein or functional fragment thereof.
50.权利要求47的融合蛋白,所述蛋白由与免疫调节蛋白或其功能性片段连接的IgE信号肽组成。 50. The fusion protein of claim 47, said protein consists of a signal peptide with immunomodulatory IgE protein or functional fragment thereof connected components.
51.权利要求49或50的融合蛋白,其中所述IgE信号肽和所述非IgE蛋白来源于相同物种的动物。 49 or 50 of the fusion protein of claim 51., wherein said non-IgE IgE signal peptide and the protein derived from the same animal species.
52.一种体外细胞培养物,所述细胞培养物包括含编码融合蛋白的核酸序列的核酸分子的细胞,所述融合蛋白由与非IgE蛋白序列连接的IgE信号肽组成,其中所述核酸序列在所述细胞中与表达所需的调节元件操作性连接。 52. An in vitro cell culture, said cell culture comprising a nucleic acid sequence encoding a fusion protein comprising the nucleic acid molecule of the cell, the fusion protein consists of a signal peptide and non-IgE IgE protein sequence linked composition, wherein the nucleic acid sequence regulatory elements operable in said cell and expression of the desired connection.
53.一种制备非IgE蛋白的方法,所述方法包括将细胞在融合蛋白表达所需的条件下培养足够时间,所述细胞包括含编码融合蛋白的核酸序列的核酸分子的细胞,所述融合蛋白由与非IgE蛋白序列连接的IgE信号肽组成,其中所述核酸序列在所述细胞中与表达所需的调节元件操作性连接,所述培养时间足以让所述细胞表达所述融合蛋白。 53. A method of preparing a non-IgE protein, said method comprising the cells were cultured under conditions sufficient time required for expression of the fusion protein, said cell comprising a nucleic acid sequence encoding a fusion protein comprising the nucleic acid molecule of the cell, the fusion protein signal peptide and non-IgE-IgE protein sequence linked composition, wherein said nucleic acid sequence in said cell and regulatory elements necessary for expression operably linked, the incubation time is sufficient for the cell expressing the fusion protein.
Description  translated from Chinese
编码IgE信号肽和/或IL-15的核酸序列与包含IgE信号肽和/或IL-15的组合物及其使用方法 Encoding IgE signal peptide and / or nucleic acid sequence of IL-15 with the compositions and methods of use comprising IgE signal peptide and / or IL-15 in

发明领域本发明涉及改良疫苗、用于针对免疫原的预防性和/或治疗性免疫个体的改进方法,并且涉及改进的免疫治疗组合物和改进的免疫治疗方法。 Field of the Invention The present invention relates to improved vaccines, preventive against the immunogen and / or improved methods for treating immune individuals, and to improved immunotherapeutic compositions and improved immunotherapy methods.

发明背景免疫治疗涉及调节个体免疫应答,以达到所需的治疗效果。 BACKGROUND OF THE INVENTION Immunotherapy for modulating an individual's immune response, to achieve the desired therapeutic effect. 免疫治疗药涉及这样的组合物:当给予个体时,能调节个体的免疫系统,最终减少与不想要的免疫应答相关的症状,或者最终通过增加所需免疫应答而缓解症状。 Immunotherapeutic agents relates to such compositions: When administered to an individual, the individual can regulate the immune system, and ultimately reduce unwanted symptoms associated with the immune response, or an immune response and ultimately alleviate symptoms by increasing desired. 在某些情况下,免疫治疗是接种方案的组成部分,其中将疫苗给予个体,使个体暴露给免疫原,个体将产生抗该免疫原的免疫应答。 In some cases, immunotherapy is part of a vaccination protocol in which the vaccine will be administered to a subject, so that the individual exposed to the immunogen, the individual will produce anti-immunogen immune response. 在这样的情况下,免疫治疗药增加免疫应答和/或选择性加强免疫应答部分(例如细胞免疫或体液免疫),这是治疗或预防特定病症、感染或疾病所需要的。 In such a case, immunotherapeutic agents to increase the immune response and / or selectivity of strengthening the immune response portion (e.g., cell-mediated immunity or humoral immunity), which is the treatment or prevention of the particular condition, infection or disease require.

疫苗用于使个体产生抗靶抗原的免疫,所述靶抗原例如变应原、病原体抗原或涉及人类疾病的细胞相关抗原。 For allowing an individual to produce a vaccine against the target antigen immunization, the target antigen, for example allergens, pathogen antigens or cells involved in human disease-associated antigen. 涉及人类疾病的细胞相关抗原包括癌相关肿瘤抗原和涉及自身免疫性疾病的细胞相关抗原。 Relates to cell-associated human diseases including cancer-associated tumor antigens and antigens involved in autoimmune diseases, cell-associated antigens.

在设计这样的疫苗中,已经认识到,在接种个体细胞中产生靶抗原的疫苗能有效诱导免疫系统的细胞免疫。 In designing such vaccines, it has been recognized that, to produce the target antigen in the vaccine vaccinated individual cells can induce cellular immunity in the immune system. 具体地讲,减毒活疫苗、使用无毒载体的重组疫苗和DNA疫苗各自在接种个体细胞中引起抗原的产生,结果诱导免疫系统的细胞免疫。 Specifically, live attenuated vaccines, recombinant vaccines using non-toxic carrier and DNA vaccines each induced an antigen in the vaccinated individual cells, induction of cellular immunity results of the immune system. 另一方面,死疫苗即灭活疫苗以及仅包含蛋白的亚单位疫苗不能诱导良好的细胞免疫应答,尽管它们能诱导体液应答。 On the other hand, the dead and only the inactivated vaccine i.e. a vaccine comprising a protein subunit vaccines can not induce good cellular immune response, even though they can induce a humoral response.

细胞免疫应答,对于提供抗病原体感染的保护性以及提供治疗病原体感染、癌症或自身免疫性疾病的有效的免疫介导疗法来说,通常是必需的。 Cellular immune response, for providing protection against a pathogen infection and to treat a pathogen infection, an effective therapy for cancer or immune-mediated autoimmune disease, it is usually necessary. 因此,在接种个体细胞中产生靶抗原的疫苗(例如减毒活疫苗、使用无毒载体的重组疫苗和DNA疫苗)通常是优选的。 Thus, to produce the target antigen in a vaccine vaccinated individual cells (e.g., live attenuated vaccines, recombinant vaccines using non-toxic carrier and DNA vaccine) are generally preferred.

尽管这类疫苗通常能使个体有效产生针对病原体感染或人类疾病的预防性或治疗性免疫,但是需要改良疫苗。 Although such vaccines are usually effective to produce a make individual against pathogen infection or a prophylactic or therapeutic immune human diseases, but a need for improved vaccines. 需要产生增强免疫应答的组合物和方法。 Need to produce compositions and methods to enhance the immune response.

同样,尽管某些免疫治疗药可用于调节患者的免疫应答,但是仍需要改进的免疫治疗组合物和方法。 Similarly, although certain drugs can be used in immunotherapy modulating the immune response in patients, but is still a need for improved immunotherapeutic compositions and methods.

基因治疗涉及将基因传递给有需要的个体,或者能从所述基因编码的蛋白获益的个体。 Gene therapy involves gene delivery to an individual in need, or from a protein encoded by the gene to benefit individuals. 已经开发出各种策略,以传递蛋白,因为个体没有产生足够和/或完整功能蛋白的相应基因。 Has developed a variety of strategies to deliver proteins, because the individual does not produce enough of the corresponding gene and / or a fully functional protein. 因此,基因治疗能补偿所缺失的足够的完整功能性内源蛋白。 Therefore, within the gene therapy is sufficient to compensate for the lack of a complete functional protein. 在某些基因治疗策略中,使用设计用来产生治疗有效量蛋白的构建体,给患者提供治疗有效的蛋白。 In certain gene therapy strategies using the design to generate a therapeutically effective amount of the protein constructs, providing the patient with a therapeutically effective protein. 基因治疗提供一种用于传递蛋白治疗药的替代方法。 Gene therapy provides an alternative method for delivering a therapeutic agent protein. 仍需要改进的基因治疗组合物和方法。 Gene therapy is still a need for improved compositions and methods.

除了直接将核酸分子给予个体之外,经常还可给予蛋白。 In addition to the nucleic acid molecule administered to a subject directly to outside, often also given protein. 由重组方法产生的这类蛋白通常是制备它们的最有效方式。 Produced by recombinant methods such proteins is that they are usually the most effective manner. 仍然需要改进的生产蛋白的组合物和方法。 Still need to improve a composition and method for producing proteins.

发明概述本发明涉及含核酸分子的重组疫苗和使个体产生抗免疫原免疫的方法,所述核酸分子包含编码免疫原的核酸序列和编码融合蛋白的核酸序列以及任选编码CD40L的核酸序列,所述融合蛋白包含与IL-15蛋白序列连接的非IL-15信号序列;所述方法包括给予个体所述重组疫苗。 Summary of the Invention The present invention relates to a vaccine containing a recombinant nucleic acid molecule and so that the individual to produce anti-immunogen method, the nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen and the nucleic acid sequence encoding a fusion protein and optionally a nucleic acid sequence encoding CD40L, and the said fusion protein comprising a non-IL-15 and IL-15 signal sequence protein sequence connected; said method comprising administering to the individual a recombinant vaccine.

本发明涉及包含核酸分子的减毒活病原体和使个体产生抗病原体免疫的方法,所述核酸分子包含编码融合蛋白的核酸序列以及任选编码CD40L的核酸序列,所述融合蛋白包含与IL-15蛋白序列连接的非IL-15信号序列;所述方法包括给予个体所述减毒活病原体。 The present invention relates to live attenuated pathogens and make the individual to produce anti-pathogen immunity method comprising the nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein and optionally a nucleic acid sequence encoding CD40L, said fusion protein comprising the IL-15 Non-IL-15 signal sequence protein sequence connected; said method comprising administering to the subject a live attenuated pathogen.

本发明涉及分离的核酸分子,所述核酸分子包含编码IL-15蛋白的核酸序列和编码CD40L蛋白的核酸序列以及任选编码免疫原的核酸序列。 The present invention relates to isolated nucleic acid molecule, the nucleic acid molecule comprises a nucleic acid sequence and nucleic acid sequence encoding CD40L protein encoding IL-15 protein and optionally a nucleic acid sequence encoding an immunogen.

本发明涉及包含这样的核酸分子的组合物:含有编码IL-15蛋白的核酸序列的核酸分子和含有编码CD40L蛋白的核酸序列的核酸分子,以及任选在以上一种或两种核酸分子上含有编码免疫原的核酸序列。 The present invention relates to compositions comprising such nucleic acid molecules: nucleic acid molecule and a nucleic acid molecule the nucleic acid sequence encoding IL-15 protein comprising a nucleic acid sequence encoding a protein containing CD40L, and optionally at least one or both of a nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen.

本发明涉及调节个体免疫应答的方法,所述方法包括给予个体包含一种或多种核酸分子的组合物,其包含编码IL-15蛋白的核酸序列和编码CD40L的核酸序列。 The present invention relates to a method for regulating an immune response in an individual, said method comprising administering to the subject one or more compositions comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a nucleic acid sequence encoding CD40L IL-15 protein. 编码各种不同蛋白的不同核酸序列可以在同一核酸分子和/或不同核酸分子上,或者两者皆有。 A variety of different nucleic acid sequences encoding different proteins can be on the same nucleic acid molecule and / or different nucleic acid molecules, or both.

本发明涉及诱导个体产生抗免疫原的免疫应答的方法,所述方法包括给予个体包含一种或多种核酸分子的组合物,所述核酸分子包含编码IL-15蛋白的核酸序列、编码免疫原的核酸序列和编码CD40L的核酸序列。 The present invention relates to a method of inducing anti-immunogen to produce an individual an immune response, said method comprising administering to a subject comprising one or more nucleic acid molecules of the composition, the nucleic acid molecule comprises a nucleic acid sequence encoding IL-15 protein, encoded immunogen The nucleic acid sequence and the nucleic acid sequence encoding CD40L. 编码各种不同蛋白的不同核酸序列可以在同一核酸分子和/或不同核酸分子或者其组合上。 A variety of different nucleic acid sequences encoding different proteins can be the same nucleic acid molecule and / or different nucleic acid molecules or a combination thereof on.

本发明涉及含核酸分子的重组疫苗和使个体产生抗免疫原免疫的方法,所述核酸分子包含编码免疫原的核酸序列、编码IL-15蛋白的核酸序列和编码CD40L的核酸序列;所述方法包括给予个体所述重组疫苗。 The present invention relates to a vaccine containing a recombinant nucleic acid molecule and so that the individual to produce anti-immunogen method, the nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen, IL-15 protein encoding nucleic acid sequence and a nucleic acid sequence encoding CD40L; said method comprising administering to an individual the recombinant vaccine.

本发明涉及包含核酸分子的减毒活病原体和使个体产生抗病原体免疫的方法,所述核酸分子包含编码IL-15蛋白的核酸序列和编码CD40L的核酸序列;所述方法包括给予个体所述减毒活病原体。 The present invention relates to live attenuated pathogens and make the individual to produce anti-pathogen immunity method comprising the nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence encoding a nucleic acid sequence encoding CD40L IL-15 protein; the method comprising administering to a subject the Save poison live pathogens.

本发明涉及核酸分子,其包含编码融合蛋白的核酸序列,所述融合蛋白由与非IgE蛋白序列连接的IgE信号肽组成,其中所述IgE信号肽和所述非IgE蛋白序列来源于相同物种的动物。 The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, said fusion protein consists of a signal peptide and non-IgE IgE protein sequence linked composition, wherein said non-IgE IgE signal peptide and the protein sequences derived from the same species animals.

本发明涉及宿主细胞体外培养物,所述培养物包含在所述宿主细胞中可操作的表达载体,所述表达载体包含编码由与非IgE蛋白序列连接的IgE信号肽组成的融合蛋白的核酸序列;涉及所述核酸分子;和涉及包含所述载体的宿主细胞。 The present invention relates to a host cell culture in vitro, the cultures were included in the expression vector operable in the host cell, the nucleic acid sequence of the fusion protein encoded by a vector comprising a signal peptide and non-IgE IgE protein sequence consisting of the expression of the connection ; relates to the nucleic acid molecule; and relates to a host cell comprising the vector.

本发明涉及核酸分子,所述核酸分子包含与表达所需调节元件操作性连接且编码融合蛋白的核酸序列,以及与表达所需调节元件操作性连接且编码免疫原的核酸序列;所述融合蛋白包含与非IgE蛋白序列连接的IgE信号肽。 The present invention relates to a nucleic acid molecule, the nucleic acid molecule comprises the regulatory elements necessary for expression operably linked to the fusion protein and the encoding nucleic acid sequence, as well as the regulatory elements required for expression of a nucleic acid sequence operatively connected and the original encoding immunity; said fusion protein contains IgE and non-IgE protein signal peptide sequence of the connection.

本发明涉及组合物,所述组合物包含这样的核酸分子:含有编码融合蛋白的核酸序列的核酸分子和含有编码免疫原的核酸序列的核酸分子,其中含有编码融合蛋白的核酸序列的核酸分子与含有编码免疫原的核酸序列的核酸分子完全不同,所述融合蛋白包含与非IEg蛋白序列连接的IgE信号肽。 The present invention relates to a composition, said composition comprising a nucleic acid molecule: a nucleic acid molecule encoding a fusion protein and a nucleic acid sequence comprising a nucleic acid molecule encoding an immunogen comprising a nucleic acid sequence, wherein the nucleic acid molecule encodes a fusion protein comprising a nucleic acid sequence and nucleic acid molecule encoding an immunogen comprising a nucleic acid sequence is completely different, the fusion protein comprises a signal peptide and non-IgE protein sequence IEg connected.

本发明涉及分离的融合蛋白,所述融合蛋白包含与非IgE蛋白序列连接的IgE信号肽。 The present invention relates to an isolated fusion protein, said fusion protein comprising the signal peptide and non-IgE IgE protein sequence attached.

本发明涉及调节个体免疫应答的方法,所述方法包括给予个体组合物,所述组合物包含这样的核酸分子:含有编码融合蛋白的核酸序列的核酸分子,所述融合蛋白包含与免疫调节蛋白连接的IgE信号肽。 The present invention relates to a method for regulating an immune response in an individual, said method comprising administering to a subject composition, said composition comprising a nucleic acid molecule: a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, said fusion protein comprising protein linked with immunoregulatory The IgE signal peptide.

本发明涉及诱导个体产生抗免疫原的免疫应答的方法,所述方法包括给予个体核酸分子,所述核酸分子含有编码融合蛋白的核酸序列和含有编码免疫原的核酸序列;所述融合蛋白包含与免疫调节蛋白连接的IgE信号肽。 The present invention relates to a method of inducing anti-immunogen to produce an individual an immune response, said method comprising administering to the subject a nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein and a nucleic acid sequence encoding the original immunization; said fusion protein comprises immunomodulatory IgE signal peptide-protein linked. 编码不同蛋白的不同核酸序列可以在同一核酸分子和/或不同核酸分子上。 Different nucleic acid sequences encoding different proteins can be the same nucleic acid molecule and / or different nucleic acid molecules.

本发明涉及含核酸分子的重组疫苗和使个体产生抗免疫原免疫的方法,所述核酸分子包含编码免疫原的核酸序列和编码融合蛋白的核酸序列,所述融合蛋白包含与免疫调节蛋白连接的IgE信号序列;所述方法包括给予个体所述重组疫苗。 The present invention relates to a vaccine containing a recombinant nucleic acid molecule and so that the individual to produce anti-immunogen method, the nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen and the nucleic acid sequence encoding a fusion protein, said fusion protein comprising a protein linked with immunoregulatory IgE signal sequence; the method comprising administering to the subject a recombinant vaccine.

本发明涉及包含核酸分子的减毒活病原体和使个体产生抗病原体免疫的方法,所述核酸分子包含编码融合蛋白的核酸序列,所述融合蛋白包含与免疫调节蛋白连接的IgE信号序列;所述方法包括给予个体所述减毒活病原体。 The present invention relates to live attenuated pathogens and make the individual to produce anti-pathogen immunity method comprising the nucleic acid molecule, the nucleic acid molecule comprises a nucleic acid sequence encoding a fusion protein, IgE signal sequence comprises immunomodulatory protein-protein linked the fusion; the said method comprising administering to the subject a live attenuated pathogen.

本发明涉及核酸分子、载体和宿主细胞,所述核酸分子包含编码融合蛋白的核酸序列,所述融合蛋白包含与IL-15蛋白序列连接的IgE信号肽;所述载体包含所述核酸分子;所述宿主细胞包含所述载体。 The present invention relates to nucleic acid molecules, vectors and host cells, the nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, said fusion protein comprising the signal peptide of IgE and IL-15 protein sequence connected; said vector comprising the nucleic acid molecule; the said host cell comprising the vector.

本发明涉及融合蛋白,所述融合蛋白包含与IL-15蛋白序列连接的IgE信号肽。 The present invention relates to a fusion protein, said fusion protein comprising the signal peptide of IgE and IL-15 protein sequence attached.

本发明涉及组合物,所述组合物包含这样的核酸分子:含有编码融合蛋白的核酸序列的核酸分子和含有编码免疫原的核酸序列的核酸分子;所述融合蛋白包含与IL-15蛋白序列连接的IgE信号肽。 The present invention relates to a composition, said composition comprising a nucleic acid molecule: a nucleic acid molecule encoding a fusion protein comprising a nucleic acid sequence and a nucleic acid molecule encoding an immunogen comprising a nucleic acid sequence; said fusion protein comprises a connected sequence of IL-15 protein The IgE signal peptide. 任选编码CD40L的核酸序列可存在于包含编码融合蛋白和/或免疫原的核酸序列的核酸分子上或存在于各自的核酸分子上。 Optionally a nucleic acid sequence encoding CD40L may be present in the fusion protein comprises a coding and / or nucleic acid molecule immunogen or a nucleic acid sequence present on the respective nucleic acid molecules.

本发明涉及调节个体免疫应答的方法,所述方法包括给予个体包含一种或多种核酸分子的组合物,其包含编码融合蛋白的核酸序列,并任选包含编码CD40L的核酸序列,所述融合蛋白包含与IL-15蛋白连接的IgE信号肽。 The present invention relates to a method for regulating an immune response in an individual, said method comprising administering to a subject comprising one or more nucleic acid molecules of the composition, comprising a nucleic acid sequence encoding a fusion protein, and optionally a nucleic acid sequence encoding CD40L, said fusion IgE protein comprising the signal peptide and IL-15 protein attached. 编码各种不同蛋白的不同核酸序列可以在同一核酸分子和/或不同核酸分子上,或者两者皆有。 A variety of different nucleic acid sequences encoding different proteins can be on the same nucleic acid molecule and / or different nucleic acid molecules, or both.

本发明涉及诱导个体产生抗免疫原的免疫应答的方法,所述方法包括给予个体包含一种或多种核酸分子的组合物,所述核酸分子包含编码融合蛋白的核酸序列、编码免疫原的核酸序列和任选编码CD40L的核酸序列;所述融合蛋白包含与IL-15蛋白序列连接的IgE信号肽。 The present invention relates to a method of inducing anti-immunogen to produce an individual an immune response, said method comprising administering to a subject comprising one or more nucleic acid molecules of the composition, the nucleic acid molecule comprises a nucleic acid sequence encoding a fusion protein, a nucleic acid encoding the immunogen sequence and optionally a nucleic acid sequence encoding CD40L; said fusion protein comprising the signal peptide IgE and IL-15 protein sequence attached. 编码各种不同蛋白的不同核酸序列可以在同一核酸分子和/或不同核酸分子或者其组合上。 A variety of different nucleic acid sequences encoding different proteins can be the same nucleic acid molecule and / or different nucleic acid molecules or a combination thereof on.

附图简述图1描绘来自实施例1的数据,显示IL-15和抗CD3单克隆抗体刺激人PBMC后,产生IFN-γ。 1 is a graph depicting data from Example 1 showing IL-15 and anti-CD3 monoclonal antibody after stimulation of human PBMC, to produce IFN-γ. PBMC得自接受三联疗法(HAART)的HIV-1慢性感染者。 PBMC from accepting triple therapy (HAART) in HIV-1 chronically infected. 所有供体的病毒量都低于500拷贝/ml,他们的CD4计数高于500细胞/ml。 All donor virus load are below 500 copies / ml, their CD4 count above 500 cells / ml. 为了确定IL-15增加IFN-γ产生是否可作为效应子功能的指标,将细胞用IL-15和抗CD3刺激,然后用标准酶联免疫斑点测定法(ELIspot assay)进行分析。 In order to determine IL-15 to increase IFN-γ is produced can be used as indicators of effector function, IL-15 cells were treated with anti-CD3 stimulation and then analyzed using a standard ELISPOT assay (ELIspot assay).

图2描绘来自实施例1的数据,显示用IL-15和抗CD3单克隆抗体刺激人PBMC后,IFN-γ的产生主要是由CD8介导的。 Figure 2 depicts data from Example 1, IL-15 and the display after anti-CD3 monoclonal antibody stimulation of human PBMC, IFN-γ is produced primarily by CD8 mediated. 如图1所示,来自接受三联疗法(HAART)的HIV-1慢性感染者的PBMC已耗尽了CD4或CD8T细胞,然后用IL-15和抗CD3进行刺激,再用标准酶联免疫斑点测定法进行分析。 1, HIV-1 infection of PBMC from chronic accept triple therapy (HAART) has been depleted of CD4 or CD8T cells, and then stimulated with IL-15 and anti-CD3, and then measuring the standard ELISPOT Method for analysis.

图3A、3B、3C和3D描绘来自实施例1的数据,显示用HIV-1肽和IL-15刺激人PBMC后,产生抗原特异性IFN-γ。 Figure 3A, 3B, 3C and 3D depict data from Example 1, with HIV-1 peptide display and IL-15 after stimulation of human PBMC, an antigen-specific IFN-γ. 对来自接受三联疗法(HAART)的HIV-1慢性感染者的PBMC,在标准酶联免疫斑点测定法中分析其响应25ng/ml IL-15(图3A和图3C)和响应HIV-1Gag肽与IL-15联用(图3B和图3C)而分泌IFN-γ的能力。 PBMC from the acceptance of triple therapy (HAART) in HIV-1 infected patients with chronic, analyze its response 25ng / ml IL-15 (Fig. 3A and Fig. 3C) and the response in HIV-1Gag peptide standard ELISPOT assay in combination with IL-15 (Fig. 3B and Fig. 3C) and the ability to secrete IFN-γ. CD8已耗尽,用HIV-1肽和IL-5刺激后,评价IFN-γ的产生(图3D)。 CD8 depleted, with HIV-1 peptide and IL-5 stimulation, evaluation of IFN-γ production (Fig. 3D).

图4A、图4B和图4C描绘来自实施例1的数据,显示在用HIV-1DNA疫苗和IL-15免疫后,HIV-1抗原特异性细胞免疫应答。 Figures 4A, 4B and 4C depict data from Example 1, displayed after using HIV-1DNA vaccine and IL-15 immunization, HIV-1 antigen-specific cellular immune responses. 在第0周和第2周,给Balb/c小鼠共注射50μg pCenv或pCgag以及50μgpIL-15和IL-15表达质粒。 At 0 weeks and two weeks, to Balb / c mice were co-injected 50μg pCenv or pCgag and 50μgpIL-15 and IL-15 expression plasmid. 最后免疫后2周,收获脾细胞。 2 weeks after the last immunization, spleen cells are harvested. 在图4A中,通标准铬释放测定法,测定脾细胞的抗HIV-1包膜和重组痘苗病毒感染的P815细胞的CTL活性。 In Figure 4A, through a standard chromium release assay to measure CTL activity of spleen cells against HIV-1 envelope and recombinant vaccinia virus-infected P815 cells. 在图4B中,对HIV-1抗原特异性趋化因子的分泌水平进行分析。 In Figure 4B, for HIV-1 antigen-specific chemokine secretion were analyzed. 脾细胞用HIV-1 env重组痘苗病毒感染的P815细胞进行刺激。 Spleen cells with HIV-1 env recombinant vaccinia virus-infected P815 cells were stimulated. 在第3天,收获上清液,测定MIP-1β的分泌。 On day 3, supernatants were harvested, the MIP-1β secretion was measured. 在图4C中,对抗原特异性IFN-γ分泌水平进行评价。 In Figure 4C, of antigen-specific IFN-γ secretion was evaluated. 将脾细胞重悬浮,浓度为5106细胞/ml。 The spleen cells were resuspended, at a concentration of 5 106 cells / ml. 将100μl等分试样加入到96孔平底微量滴定板的各孔中。 The 100μl aliquots were added to each well of a 96-well flat-bottom microtiter plate. 将重组p24蛋白加入到各孔中,一式三份,得到终浓度5μg/ml和1μg/ml。 The recombinant p24 protein was added to each well, in triplicate, to give a final concentration of 5μg / ml and 1μg / ml. 将细胞于37℃/5%CO2孵育3天,收获上清液。 The cells were at 37 ℃ / 5% CO2 incubation for 3 days the supernatant was harvested. 用市售ELISA试剂盒,测定所分泌的细胞因子水平。 Using a commercially available ELISA kits, measuring secreted cytokine levels.

图5A和图5B描绘来自实施例1的数据,显示Th1细胞因子的胞内染色。 Figures 5A and 5B depict from the data in Example 1, showing intracellular staining of Th1 cytokines. 小鼠接受两次注射:单独的pCgag或者pCgag加上pIL-15DNA质粒。 Mice received two injections: Separate pCgag or pCgag plus pIL-15DNA plasmids. 一周后,收获脾细胞,在含有p55肽合并液(含127种15聚体,其跨越HIV-1 p55,具有11个氨基酸的重叠)和布雷菲德菌素A的培养基中体外培养5小时。 One week later, spleen cells harvested, containing p55 peptide pools (containing 127 kinds of 15-mers, which spans HIV-1 p55, with overlapping 11 amino acids) and brefeldin A medium in vitro 5 hours . 刺激后,细胞用抗小鼠CD3抗体和抗小鼠CD8抗体进行胞外染色,然后用抗小鼠进行胞内染色。 After stimulation, cells were treated with anti-mouse CD3 antibody and anti-mouse CD8 antibody was used for extracellular staining, and then with anti-mouse were intracellular staining. 显示CD3+/CD8+淋巴细胞的应答。 Display CD3 + / CD8 + lymphocyte responses. 图5A显示IFN-γ的数据。 Figure 5A shows the IFN-γ data. 图5B显示肿瘤坏死因子-α的数据。 Figure 5B shows the tumor necrosis factor -α data. 斑点印迹显示CD3+/CD8+淋巴细胞的应答。 Dot blot show CD3 + / CD8 + lymphocyte responses.

图6描绘来自实施例1鼠T辅助细胞增殖测定的数据。 Figure 6 depicts Example 1 from the murine T helper cell proliferation assay data implementation. 在第0周和第2周,给Balb/c小鼠共注射50μg pCgag或pCenv和50μg质粒,所述质粒表达IL-2R依赖性Th1细胞因子IL-2或IL-15的cDNA。 At Week 0 and Week 2, to Balb / c mice co-injected with 50μg 50μg pCgag or pCenv plasmid, the plasmid expressing IL-2R dependent Th1 cytokine cDNA IL-2 or IL-15's. 将含5105细胞的100μl等分试样立即加入到96孔平底微量滴定板的各孔中。 Containing 5 105 cells in 100μl aliquot was added immediately to each well of a 96-well flat-bottom microtiter plate. 将重组p24蛋白加入到各孔中,一式三份,得到终浓度5μg/ml和1μg/ml。 The recombinant p24 protein was added to each well, in triplicate, to give a final concentration of 5μg / ml and 1μg / ml. 测定刺激指数。 Determination of the stimulation index. 自发计数孔中包含10%胎牛血清,起到无关蛋白对照作用。 Spontaneous count wells containing 10% fetal bovine serum, play a role in unrelated protein control. 同样,pCgag或对照通常针对无关gp 120蛋白来说,SI为1。 Similarly, pCgag or control for unrelated gp 120 proteins generally speaking, SI 1. 为了确保细胞健康,采用PHA或Con A(Sigma)作为多克隆刺激物阳性对照。 To ensure the healthy cells, the use of PHA or Con A (Sigma) as a positive control polyclonal stimulus.

图7描绘来自实施例1的数据,即用DNA疫苗pCgag免疫Balb/c小鼠后,Gag的表位作图。 Figure 7 depicts an embodiment of the data from Example 1, i.e., after immunization with DNA vaccine pCgag Balb / c mice, the Gag epitope mapping. 在第0周和第2周,给Balb/c小鼠共注射50μg pCgag和50μg pIL-15质粒或表达基因IL-15的载体骨架或载体骨架。 At Week 0 and Week 2, to Balb / c mice co-injected 50μg pCgag and 50μg pIL-15 or a plasmid or expression vector backbone vector backbone of IL-15 gene. 分离脾细胞,在标准酶联免疫斑点测定法中用一系列肽进行编排。 Isolated spleen cells, using standard ELISPOT assay orchestrate a series of peptides. 肽混合成一系列22种合并液的矩阵形式,测定其激活细胞产生IFN-γ的能力。 Peptide mixed into a series of 22 kinds of matrix form combined liquid and its ability to activate the cells to produce IFN-γ was measured.

图8A、图8B和图8C描绘来自实施例1的数据,显示在对来自CD4敲除小鼠的脾细胞进行刺激后,产生IFN-γ。 Figure 8A, 8B and 8C depict the data from Example 1, showing the pair from CD4 knockout mouse spleen cells were stimulated to produce IFN-γ. 图8A,在第0周和第2周,给Balb/c小鼠共注射50μg pCgag和50μg pIL-15即IL-15表达质粒。 Figure 8A, at Week 0 and Week 2, to Balb / c mice co-injected 50μg pCgag and 50μg pIL-15 i.e., IL-15 expression plasmid. 在最后一次免疫后两周,收获脾细胞,采用酶联免疫斑点测定法,测定产生的HIV-1特异性IFN-γ。 Two weeks after the last immunization, spleen cells were harvested, using the ELISPOT assay to measure produced by HIV-1-specific IFN-γ. 图8B,Cd4tmlKmv小鼠用pCgag、用和不用pIL-15进行免疫。 Figure 8B, Cd4tmlKmv mice with pCgag, with and without pIL-15 immunization. 图8C,Cd4tmlKmv小鼠用pCgag以及pIL-15、pCD40L或这两者进行免疫。 Figure 8C, Cd4tmlKmv mice with pCgag and pIL-15, pCD40L or both immunization. 在最后一次免疫后两周,收获脾细胞,在用HIV-1Gag肽进行体外刺激后,采用酶联免疫斑点测定法,测定产生的HIV-1Gag特异性IFN-γ。 Two weeks after the last immunization, spleen cells harvested, after in vitro stimulation with HIV-1Gag peptides, using ELISPOT assay, determination of HIV-1Gag produce specific IFN-γ.

图9描绘来自实施例2的数据,显示IL-15和CD40L在疫苗部位的局部产生,可以取代对T细胞辅助的需要,供CD8效应T细胞扩增用。 Figure 9 depicts data from Example 2 showing IL-15 and CD40L in the local site generation vaccines, can replace the need for T cell help for CD8 effector T cell amplification.

图10、图11、图12A-C、图13A-B、图14和图15涉及实施例3中记载的内容。 Figure 10, Figure 11, Figure 12A-C, FIG. 13A-B, FIG. 14 and FIG. 15 relates to the content described in Example 3.

图16涉及实施例4中记载的内容。 Figure 16 relates to the content described in Example 4.

优选实施方案的详述定义本文所用的术语“靶蛋白”是指本发明基因构建体所编码的肽和蛋白,其作为免疫应答的靶蛋白。 Detailed Description of the preferred embodiments defined herein, the term "target protein" refers to a gene construct of the present invention, peptides and proteins encoded, as the immune response of the target protein. 术语“靶蛋白”和“免疫原”可互换使用,是指可诱导免疫应答的蛋白。 The term "target protein" and "immunogen" are used interchangeably, refer to a protein can induce an immune response. 靶蛋白是免疫原性蛋白,其与病原体蛋白或不需要的细胞类型(例如癌细胞或涉及自身免疫性疾病的细胞)的蛋白共享至少一个表位,希望产生抗这些蛋白的免疫应答。 Target protein is an immunogenic protein, protein with a pathogen or undesirable cell types (e.g., cancer cells or cells involved in autoimmune diseases) proteins share at least one epitope, desired to produce an immune response against these proteins. 针对靶蛋白的免疫应答将会保护个体,预防和/或治疗个体与所述靶蛋白相关的特异性感染或疾病。 Immune response against the target protein will protect the individual, preventing and / or treating individual-specific infection or disease associated with the target protein.

本文所用的术语“基因构建体”是指DNA或RNA分子,其包含编码靶蛋白或免疫调节蛋白的核苷酸序列。 As used herein, the term "genetic construct" refers to a DNA or RNA molecule, which comprises encoding a target protein or immunomodulating protein nucleotide sequence. 编码序列包括与调节元件操作性连接的起始信号和终止信号,所述调节元件包括在接受所述核酸分子的个体细胞中能指导表达的启动子和聚腺苷酸化信号。 Coding sequence including start and stop signals adjusting element operatively connected to said adjusting element comprises accepting individual cells of the nucleic acid molecule capable of directing the expression of a promoter and a polyadenylation signal.

本文所用的术语“可表达形式”是指含有与编码靶蛋白或免疫调节蛋白的编码序列操作性连接的必要调节元件的基因构建体,使得当存在于个体细胞中时,可以表达编码序列。 As used herein, the term "expressible form" refers to a target protein or encoding immunomodulatory protein coding sequence operably linked elements necessary to modulate gene construct, such that when present in individual cells, expression of the coding sequence can.

本文所用的术语“共享表位”是指,蛋白包含至少一个与另一蛋白表位相同或基本类似的表位。 As used herein, the term "sharing an epitope" refers to proteins comprising at least one protein with another epitope on the same or substantially similar epitopes.

本文所用的术语“基本类似的表位”是指具有与蛋白表位不同的结构、但仍能诱导细胞或体液免疫应答的表位,其与所述蛋白有交叉反应。 As used herein, the term "substantially similar epitope" refers to proteins having an epitope different structure, but still induce cellular or humoral immune response epitope cross-reactive with the protein.

本文所用的术语“胞内病原体”是指这样的病毒或病原生物:其复制或生命周期的至少一部分是在宿主细胞内进行、因而产生病原体蛋白或引起病原体蛋白的产生。 As used herein, the term "intracellular pathogen" refers to a virus or pathogenic organism: copy or at least part of the life cycle is performed in a host cell, thereby producing the protein or pathogens causing pathogen protein produced.

本文所用的术语“过度增殖性疾病”是指特征为细胞过度增殖的疾病和障碍。 As used herein, the term "hyperproliferative diseases" is characterized by excessive cell proliferation refers to diseases and disorders.

本文所用的术语“过度增殖相关蛋白”是指与过度增殖性疾病相关的蛋白。 As used herein, the term "hyperproliferative-associated protein" refers to a hyperproliferative disease-related proteins.

本文所用的术语“免疫调节蛋白”是指调节个体免疫系统的蛋白,所述个体已接受免疫调节蛋白。 As used herein the term "immunomodulating protein" refers to a protein regulating an individual's immune system, the individual has accepted immunomodulatory protein. 免疫调节蛋白的实例包括:IL-15、CD40L、TRAIL;TRAILrecDRC5、TRAIL-R2、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、NKG2D、F461811或MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、CD30、CD153(CD30L)、Fos、c-jun、Sp-1、Ap1、Ap-2、p38、p65Re1、MyD88、IRAK、TRAF6、IkB、NIK、SAP K、SAP1、JNK2、JNK1B2、JNK1B1、JNK2B2、JNK2B1、JNK1A2、JNK2A1、JNK3A1、JNK3A2、NF-κ-B2、p49剪接形式、NF-κ-B2、p100剪接形式、NF-κ-B2、p105剪接形式、NF-κ-B 50K链前体、NFκB p50、人IL-1α、人IL-2、人IL-4、鼠IL-4、人IL-5、人IL-10、人IL-15、人IL-18、人TNF-α、人TNF-β、人白介素12、MadCAM-1、NGF IL-7、VEGF、TNF-R、Fas、CD40L、IL-4、CSF、G-CSF、GM-CSF、M-CSF、LFA-3、ICAM-3、ICAM-2、ICAM-1、PECAM、P150.95、Mac-1、LFA-1、CD34、RANTES、IL-8、MIP-la、E-selecton、CD2、MCP-1、L-selecton、P-selecton、FLT、Apo-1、Fas、TNFR-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、DR4(TRAIL)、DR5、KILLER、TRAIL-R2、TRICK2、DR6、ICE、VLA-1和CD86(B7.2)。 Examples of immunomodulatory proteins include: IL-15, CD40L, TRAIL; TRAILrecDRC5, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, F461811 or MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, CD30, CD153 (CD30L), Fos, c-jun, Sp-1, Ap1, Ap-2, p38, p65Re1, MyD88, IRAK, TRAF6, IkB, NIK, SAP K, SAP1, JNK2, JNK1B2, JNK1B1, JNK2B2, JNK2B1, JNK1A2, JNK2A1, JNK3A1, JNK3A2, NF-κ-B2, p49 splice form, NF-κ-B2, p100 splice form, NF-κ-B2, p105 splice form, NF-κ- B 50K chain precursor, NFκB p50, human IL-1α, human IL-2, human IL-4, murine IL-4, human IL-5, human IL-10, human IL-15, human IL-18, human TNF-α, human TNF-β, human interleukin-12, MadCAM-1, NGF IL-7, VEGF, TNF-R, Fas, CD40L, IL-4, CSF, G-CSF, GM-CSF, M-CSF, LFA-3, ICAM-3, ICAM-2, ICAM-1, PECAM, P150.95, Mac-1, LFA-1, CD34, RANTES, IL-8, MIP-la, E-selecton, CD2, MCP- 1, L-selecton, P-selecton, FLT, Apo-1, Fas, TNFR-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4 (TRAIL), DR5, KILLER , TRAIL-R2, TRICK2, DR6, ICE, VLA-1 and CD86 (B7.2).

概述本发明来自以下发现。 Summary of the Invention from the following findings. 1)当IL-15信号肽不存在时,IL-15蛋白表达水平更高,无论所表达的IL-15蛋白是“截短”的IL-15蛋白,还是含有与非IL-15信号肽(特别是IgE信号肽)连接的IL-15蛋白序列的融合蛋白。 1) When the IL-15 signal peptide does not exist, a higher level of IL-15 protein expression, regardless of the expressed IL-15 protein is a "truncated" of the IL-15 protein, or IL-15 containing a non-signal peptide ( particularly IgE signal peptide) fusion protein attached IL-15 protein sequence. 缺乏IL-15信号肽的IL-15蛋白,无论是“截短”的IL-15蛋白还是含有与非IL-15信号肽(特别是IgE信号肽)连接的IL-15蛋白序列的融合蛋白,在用于传递IL-15蛋白作为免疫调节蛋白的疫苗和构建体中,都特别有用。 Lack of IL-15 IL-15 signal peptide protein, whether it is "truncated" of the IL-15 protein or a fusion protein containing IL-15 and non-signal peptide (particularly IgE signal peptide) attached IL-15 protein sequence, In the IL-15 protein for delivery as an immunomodulator and a vaccine protein constructs, are particularly useful. 2)涉及IL-15和CD40L传递的疫苗和免疫调节组合物特别有用。 2) IL-15 and relates to vaccines and immunomodulatory compositions CD40L delivery particularly useful. 3)包含IgE信号肽的融合蛋白有助于增强表达,在蛋白的产生、疫苗和基因治疗药中是特别有用的,例如用于传递蛋白(例如免疫调节蛋白)。 3) the fusion protein comprises a signal peptide IgE contributes to enhance expression, produce proteins in vaccines and gene therapeutic agent is particularly useful, for example for transfer protein (e.g., immunomodulatory proteins). 在一些优选的实施方案中,本发明提供载体、疫苗和免疫调节组合物和方法,其中包含核酸分子,所述核酸分子包含编码以下蛋白的核苷酸序列:包含缺乏IL-15信号肽(优选缺乏IL-15Kozak区和非翻译区)的人IL-15编码序列的蛋白;或提供具有非IL-15信号肽(优选IgE信号序列)的人IL-15编码序列的融合蛋白。 In some preferred embodiments, the present invention provides vectors, vaccines and immunomodulatory compositions and methods, wherein the nucleic acid molecule comprising a nucleic acid molecule comprising a nucleotide sequence encoding the following proteins: the lack of IL-15 comprising a signal peptide (preferably lack of IL-15Kozak and non-translated regions) of human IL-15 protein coding sequence; or providing a non-human IL-15 having a signal peptide (preferably IgE signal sequence) IL-15 fusion protein coding sequence. IL-15编码序列优选缺乏IL-15信号序列,及优选缺乏IL-15Kozak区和非翻译区。 IL-15 coding sequence is preferably lacking IL-15 signal sequence, and the lack of IL-15Kozak preferred and non-translated region. 在一些优选的实施方案中,本发明提供载体、疫苗和免疫调节组合物和方法,其中包含核酸分子,所述核酸分子包含编码以下蛋白的核苷酸序列:1)IL-15蛋白,例如缺乏IL-15信号肽的IL-15蛋白,或包含与非IL-15信号肽(例如IgE信号肽)连接的IL-15蛋白序列的融合蛋白;2)编码人CD40L的核苷酸序列。 In some preferred embodiments, the present invention provides vectors, vaccines and immunomodulatory compositions and methods, wherein the nucleic acid molecule comprising a nucleic acid molecule comprising a nucleotide sequence encoding the following proteins: 1) IL-15 protein, such as lack of IL-15 IL-15 protein signal peptide, or a fusion protein comprising IL-15 and non-signal peptide (e.g., IgE signal peptide) connected to IL-15 protein sequence; a nucleotide sequence 2) encoding the human CD40L. IL-15编码序列优选缺乏IL-15信号序列,和优选缺乏IL-15Kozak区和非翻译区。 IL-15 coding sequence is preferably lacking IL-15 signal sequence, and the lack of IL-15Kozak preferred and non-translated region. 在一些优选的实施方案中,本发明提供载体、疫苗和免疫调节组合物和方法,其中包含核酸分子,所述核酸分子包含编码IgE信号肽与非IgE蛋白序列(优选人IL-15蛋白序列)连接的融合蛋白的核苷酸序列。 In some preferred embodiments, the present invention provides vectors, vaccines and immunomodulatory compositions and methods, wherein the nucleic acid molecule comprising the nucleic acid molecule comprises a signal peptide encoding IgE and non-IgE protein sequence (preferably human IL-15 protein sequence) connecting the nucleotide sequence of the fusion protein.

包含与非IgE蛋白连接的IgE信号序列的融合蛋白和编码与非IgE蛋白连接的IgE信号序列的基因构建体因此,本发明的一个总的方面涉及包含与非IgE蛋白连接的IgE信号序列的融合蛋白和编码与非IgE蛋白连接的IgE信号序列的基因构建体,以及所述构建体在表达载体、疫苗和免疫调节组合物中的用途。 Gene comprises a signal sequence and a non-IgE IgE-protein linked the fusion protein and IgE signal sequence encoding a non-IgE-protein linked construct Accordingly, a general aspect of the invention relates to a fusion protein linked with a non-IgE-IgE signal sequence gene protein and IgE signal sequence encoding a non-IgE-protein linked construct expression vectors, vaccines and immunomodulatory compositions use thereof, and the construct. 关于这一方面,提供几个不同的实施方案和形式。 On this aspect, several different embodiments and forms.

根据一些实施方案,提供包含分离的核酸分子的组合物,所述分离的核酸分子包含编码与非IgE蛋白序列操作性连接的IgE信号序列的融合蛋白的核酸序列。 According to some embodiments, providing a composition comprising the isolated nucleic acid molecule, the isolated nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein with the non-IgE IgE protein sequence operably linked signal sequence.

非IgE蛋白的特性取决于构建体的预定用途。 Characteristics of non-IgE protein in the body depends on the intended use of the building. 例如,对于基因治疗实施方案来说,蛋白序列将是这样的所需蛋白,例如患者所缺乏的足量功能性或完整功能性蛋白。 For example, for gene therapy embodiments, the protein sequence is such that the desired protein, e.g., a sufficient amount of the patient lacks a functional or complete functional protein. 这类所需蛋白的实例包括酶,例如DNA酶、生长因子(例如生长激素(人、牛、猪)、凝血因子、胰岛素、肌营养不良蛋白等。所需蛋白也可是这样的蛋白:当在患者体内表达时,能提供治疗性益处的蛋白,例如红细胞生成素、IL-2、GM-CSF、TPA等。在一些实施方案中,非IgE蛋白序列是免疫原。所述构建体可用于疫苗,其中可提供免疫原的表达,作为免疫应答的目标。在一些实施方案中,非IgE蛋白序列是免疫调节蛋白。所述构建体可用于疫苗以及免疫调节组合物,所述疫苗可提供免疫原的表达,作为免疫应答的目标,而所述免疫调节组合物所需的效果是按照所治疗患者的病症上调或下调患者的免疫系统或免疫系统的具体方面。上调免疫系统的免疫调节剂可用于治疗罹患例如免疫抑制或传染性疾病的患者,而下调免疫系统的免疫调节剂可用于治疗例如自身免疫性疾病、接受器官移植、组织移植的患者或接受细胞治疗的患者,对于他们来说,免疫抑制是必要的。在一些实施方案中,IgE信号序列与非IgE蛋白序列连接,用于在一个需要产生IgE蛋白的系统中使用。在优选的实施方案中,IgE信号肽与其上连接的蛋白序列是来源于相同物种的动物。在优选的方法中,接受所述构建体的动物与IgE信号肽和蛋白序列的来源动物是相同的物种。所述融合蛋白将被认为是非免疫原性的。 Examples of such desired proteins include enzymes, such as DNA enzymes, growth factors (e.g., growth hormone (human, bovine, porcine), coagulation factors, insulin, dystrophin, etc. may also be desired protein such proteins: When When expression of the patient's body to provide therapeutic benefit protein, e.g., erythropoietin, IL-2, GM-CSF, TPA, etc. In some embodiments, the non-IgE protein sequence is an immunogen. The construct can be used in vaccines , which can provide the original expression immunization, as a target of the immune response. In some embodiments, the non-IgE protein sequence is immunomodulatory protein. The construct can be used in vaccines and immunomodulatory compositions, the vaccine immunogen may be provided expression, as the target of the immune response, and the immunomodulatory effect of the composition is desired in accordance with specific aspects of the condition being treated, the patient up or down by the patient's immune system or immune system. upregulation of the immune system of immune modulators can be used for such as immunosuppressive therapy or patients suffering from infectious diseases, and reduced immune system for the treatment of immune modulators such as autoimmune diseases, organ transplant patients or patients receiving tissue transplants cell therapy, for them, the immune suppression is necessary. In some embodiments, IgE signal sequence connection with non-IgE protein sequence, for use in a system needs to generate IgE proteins In a preferred embodiment, IgE signal peptide sequence linked to its protein on is derived from the same species of animals. In a preferred method, the source of animal acceptance of the animal with the IgE construct the signal peptide and the protein sequence is the same species as the body. The fusion protein will be considered non-immunogenic.

在一些实施方案中,包含与作为免疫调节蛋白的非IgE蛋白序列连接的IgE信号的编码序列的构建体的组合物,也可包含在同一核酸分子上或不同核酸分子上,即编码免疫原的核酸序列上。 In some embodiments, comprises a coding sequence and non-IgE protein sequence as immunomodulatory protein IgE signal connection compositions construct, may be included in or on different nucleic acid molecules on the same nucleic acid molecule, i.e., encoding the immunogen nucleic acid sequence. 通常,下述免疫原可以是任何免疫原性蛋白,包括变应原、病原体抗原、癌相关抗原或涉及自身免疫性疾病的细胞相关抗原。 Typically, following the immunogen may be any immunogenic protein including allergens, pathogen antigens, cancer-associated antigen or a cell involved in autoimmune disease-associated antigen. 在优选的实施方案中,免疫原是病原体抗原,最优选的病原体选自HIV、HSV、HCV和WNV。 In a preferred embodiment, the immunogen is a pathogen antigen, most preferably a pathogen selected from HIV, HSV, HCV, and WNV.

如上所述,非IgE蛋白序列优选为IL-15蛋白,更优选为缺乏IL-15信号序列的IL-15蛋白,还更优选为缺乏IL-15信号序列、缺乏IL-15Kozak区和缺乏IL-15非翻译序列的IL-15蛋白。 As described above, the protein sequence is preferably non-IgE IL-15 protein, and more preferably the lack of IL-15 protein signal sequence of IL-15, still more preferably the lack of IL-15 signal sequence, the lack of IL-15Kozak area and lack of IL- 15 untranslated sequence of IL-15 protein. 在一些优选的实施方案中,所述组合物还包括编码CD40L的核苷酸序列。 In some preferred embodiments, the composition further comprises a nucleotide sequence encoding CD40L. 该核苷酸序列可包含在同一核酸分子上(作为融合蛋白)或不同的分子上。 The nucleotide sequence may comprise the nucleic acid molecule in the same (as a fusion protein) or different molecules. CD40L可包含在含免疫原编码序列的疫苗组合物中,得到改良疫苗。 CD40L may be included in a vaccine composition containing an immunogen coding sequence, to give improved vaccines. 在其它实施方案中,CD40L可包含在不含免疫原编码序列的免疫调节组合物中,得到改良的免疫调节组合物。 In other embodiments, CD40L may be included in the immunomodulatory composition is free immunogen coding sequence, to give improved immunomodulatory composition.

在一些优选的实施方案中,核酸构建体是质粒。 In some preferred embodiments, the nucleic acid construct is a plasmid. 在一些优选的实施方案中,核酸分子掺入到病毒载体中,所述病毒载体例如痘苗病毒、腺病毒、腺病毒相关病毒、逆转录病毒,或其它任何可接受的病毒载体,用作疫苗或基因治疗载体。 In some preferred embodiments, the nucleic acid molecule incorporated into a viral vector, the viral vector such as vaccinia, adenovirus, adeno-associated virus, retrovirus, or any other acceptable viral vector, as a vaccine or Gene therapy vectors.

包含与作为免疫调节蛋白的非IgE蛋白序列连接的IgE信号序列的基因构建体可直接掺入到本发明某些方面的减毒活病原体中。 Gene contains a non-IgE protein sequence as immunomodulatory protein connection IgE signal sequence construct of the present invention can be incorporated directly into certain aspects of live attenuated pathogens. 下面列出这些用作疫苗的病原体的实例。 Listed below are examples of such pathogens useful as vaccines. 在优选的实施方案中,免疫调节蛋白是IL-15,更优选缺乏IL-15信号序列的IL-15蛋白,还更优选缺乏IL-15信号序列、缺乏IL-15Kozak区和缺乏IL-15非翻译序列的IL-15蛋白。 In a preferred embodiment, the immunomodulatory protein is IL-15, and more preferably the lack of IL-15 protein signal sequence of IL-15, still more preferably the lack of IL-15 signal sequence, the lack of IL-15Kozak area and lack of IL-15 non- IL-15 protein translation sequence. 在一些实施方案中,还提供携带编码CD40L的核苷酸序列的减毒病原体。 In some embodiments, also provides nucleotide sequences encoding CD40L carrying the attenuated pathogen.

包含与非IgE蛋白序列操作性连接的IgE信号序列的融合蛋白也是本发明的一个方面。 Contains non-IgE IgE signal sequence of the protein sequence operably linked fusion protein is also an aspect of the present invention. 在一些实施方案中,融合蛋白的非IgE蛋白序列部分是酶。 In some embodiments, the fusion protein of non-IgE portion is an enzyme protein sequences. 在一些实施方案中,融合蛋白的非IgE蛋白序列部分是免疫原。 In some embodiments, the fusion protein is part of the non-IgE protein sequence immunogen. 在一些实施方案中,融合蛋白的非IgE蛋白序列部分是免疫调节蛋白。 In some embodiments, the fusion protein is part of the non-IgE protein sequence immunomodulatory protein. 优选的非IgE蛋白序列是IL-15蛋白,最优选缺乏IL-15信号序列。 Preferred non-IgE IL-15 protein is a protein sequence, and most preferably lacking IL-15 signal sequence.

包含与IL-15蛋白连接的非IL-15信号序列的融合蛋白和编码与IL-15蛋白连接的非IL-15信号序列的基因构建体本发明的另一个总的方面涉及包含与IL-15蛋白连接的非IL-15信号序列的融合蛋白和编码与IL-15蛋白连接的非IL-15信号序列的基因构建体,以及所述构建体在疫苗和免疫调节组合物中的用途。 General aspect of the gene fusion protein and non-IL-15 signal sequence encoding the IL-15 protein comprising a non-attached IL-15 and IL-15 signal sequence protein construct another connection of the present invention relates to compositions comprising and IL-15 Non-IL-15 gene signal sequence and a fusion protein encoding a non-IL-15 signal sequence protein linked with IL-15 protein constructs in vaccines and connected immunomodulatory compositions use thereof, and the construct. 关于这一方面,提供几个不同的实施方案和形式。 On this aspect, several different embodiments and forms. 通常,IL-15是指人IL-15。 Typically, IL-15 refers to a human IL-15. 然而,构建体也可指来自狗、猫、马、牛、猪或羊等其它物种的IL-15。 However, it can also mean constructs from dogs, cats, horses, cattle, sheep, pigs or other species IL-15.

本发明的该方面来自这样的观察结果:天然IL-15mRNA所表达的蛋白序列中含有抑制表达的信号或元件。 This aspect of the invention from such observations: protein sequence of native IL-15mRNA expressed by signals or elements contained inhibit expression. 通过去除这些抑制元件,得到更好的表达。 By removal of these inhibitory components, better expression. 在优选的实施方案中,IL-15编码序列缺乏IL-15信号肽的编码序列,优选在其位置上提供另一种信号蛋白,例如IgE信号蛋白。 In a preferred embodiment, IL-15 coding sequence of IL-15 coding sequence lacking the signal peptide, preferably another signal protein to provide in its position, e.g. IgE signal protein. 此外,去除IL-15Kozak区和非翻译区并去除抑制元件。 In addition, the removal of IL-15Kozak and non-translated region and remove suppression components. 构建体中优选包含的仅IL-15序列,是编码缺乏IL-15信号肽的成熟IL-15蛋白的氨基酸序列的IL-15序列。 Construction of only IL-15 sequence preferably comprises the body, is the lack of IL-15 sequence encoding the signal peptide of the IL-15 amino acid sequence of mature IL-15 protein.

根据一些实施方案,提供包含分离的核酸分子的组合物,所述分离的核酸分子包含编码与IL-15蛋白连接的非IL-15信号序列的融合蛋白的核酸序列。 According to some embodiments, providing a composition comprising the isolated nucleic acid molecule, the isolated nucleic acid molecule comprising a nucleic acid sequence of a fusion protein of non-IL-15 signal sequence encoding the IL-15 protein attached. 在一些优选的实施方案中,融合蛋白由与IL-15蛋白连接的非IL-15信号序列组成。 In some preferred embodiments, the fusion protein consists of a non-IL-15 and IL-15 signal sequence protein linked composition. 在一些优选的实施方案中,IL-15蛋白缺乏IL-15信号序列。 In some preferred embodiments, IL-15 IL-15 protein lacks a signal sequence. 在一些优选的实施方案中,融合蛋白对于IL-15序列所来源的物种来说是非免疫原性的。 In some preferred embodiments, the fusion protein to the IL-15 sequence derived species is non-immunogenic. 因此包含人IL-15的非免疫原性融合蛋白在人体内是非免疫原性的。 Therefore comprise a human IL-15 fusion protein non-immunogenic in humans non-immunogenic.

根据一些实施方案,提供包含构建体的组合物,所述构建体包含与IL-15蛋白连接的非IL-15信号序列的融合蛋白编码序列,所述序列也可包括在同一核酸分子上或不同核酸分子上,即编码免疫原的核酸序列上。 According to some embodiments, to provide compositions containing the construct body, the fusion protein coding sequence comprises a non-IL-15 and IL-15 signal sequence protein connection of the building, the sequence may also comprise a nucleic acid molecule on the same or different nucleic acid molecule, i.e. a nucleic acid encoding the immunogen sequence. 通常,下述免疫原可以是任何免疫原性蛋白,包括变应原、病原体抗原、癌相关抗原或涉及自身免疫性疾病的细胞相关抗原。 Typically, following the immunogen may be any immunogenic protein including allergens, pathogen antigens, cancer-associated antigen or a cell involved in autoimmune disease-associated antigen. 在优选的实施方案中,免疫原是病原体抗原,最优选的病原体选自HIV、HSV、HCV和WNV。 In a preferred embodiment, the immunogen is a pathogen antigen, most preferably a pathogen selected from HIV, HSV, HCV, and WNV.

在优选的实施方案中,组合物还包含编码CD40L的核苷酸序列。 In a preferred embodiment, the composition further comprises a nucleotide sequence encoding CD40L. 该核苷酸序列可包含在同一核酸分子上作为融合蛋白,或不同分子上。 The nucleotide sequence may be contained on the same nucleic acid molecule as a fusion protein, or a different molecule. CD40L也包含在含免疫原编码序列的疫苗组合物中,得到改良疫苗。 CD40L is also included in the vaccine composition containing an immunogen coding sequence, to give improved vaccines. 在其它实施方案中,CD40L可包含在不含免疫原编码序列的免疫调节组合物中,得到改良的免疫调节组合物。 In other embodiments, CD40L may be included in the immunomodulatory composition is free immunogen coding sequence, to give improved immunomodulatory composition.

在一些优选的实施方案中,核酸构建体是质粒。 In some preferred embodiments, the nucleic acid construct is a plasmid. 在一些优选的实施方案中,核酸分子掺入到病毒载体中,所述病毒载体例如痘苗病毒、腺病毒、腺病毒相关病毒、逆转录病毒,或其它任何可接受的病毒载体,用作疫苗或基因治疗载体。 In some preferred embodiments, the nucleic acid molecule incorporated into a viral vector, the viral vector such as vaccinia, adenovirus, adeno-associated virus, retrovirus, or any other acceptable viral vector, as a vaccine or Gene therapy vectors.

包含与IL-15蛋白连接的非IL-15信号序列的融合蛋白的核苷酸序列的基因构建体可直接掺入到本发明相同方面的减毒活病原体中。 Gene contains a non-IL-15 and IL-15 signal sequence protein linked the fusion protein nucleotide sequence construct may be incorporated directly into the same aspect of the present invention, the live attenuated pathogens. 下面列出这些用作疫苗的病原体的实例。 Listed below are examples of such pathogens useful as vaccines. 在优选的实施方案中,人IL-15(优选缺乏IL-15信号序列)与人IgE信号序列连接。 In a preferred embodiment, the human IL-15 (preferably IL-15 lack a signal sequence) is connected to human IgE signal sequence. 在一些实施方案中,还提供具有编码CD40L的核苷酸序列的减毒病原体。 In some embodiments, the attenuated pathogen is also provided having a nucleotide sequence encoding CD40L.

包含与IL-15蛋白序列连接的非IL-15信号序列的融合蛋白是本发明的一方面。 Comprising a non-IL-15 and IL-15 signal sequence protein sequence of the fusion protein is connected to an aspect of the invention. 在一些优选的实施方案中,融合蛋白由与IL-15蛋白连接的非IL-15信号序列组成。 In some preferred embodiments, the fusion protein consists of a non-IL-15 and IL-15 signal sequence protein linked composition. 在一些优选的实施方案中,IL-15蛋白缺乏IL-15信号序列。 In some preferred embodiments, IL-15 IL-15 protein lacks a signal sequence. 在一些优选的实施方案中,信号序列是IgE信号序列。 In some preferred embodiments, the signal sequence is a signal sequence IgE. 序列优选人序列。 Sequence preferably a human sequence. 在一些优选的实施方案中,融合蛋白是非免疫原性的。 In some preferred embodiments, the fusion protein non-immunogenic. 非免疫原性是指这样的蛋白:对于IL-15序列所来源的物种来说,所述蛋白是非免疫原性的。 Refers to a non-immunogenic protein: for IL-15 sequence derived species, said protein non-immunogenic.

包含编码IL-15和CD40L的基因构建体的组合物及其使用方法本发明的另一个总的方面涉及包含编码IL-15和CD40L的基因构建体的组合物以及所述构建体在疫苗和免疫调节组合物中的用途。 Another general aspect comprises encoding IL-15 compositions and methods of use and CD40L gene constructs of the present invention relates to compositions comprising IL-15 and CD40L encoding gene construct and the body composition of the constructs in vaccines and immunization adjusting the composition of use. 关于这一方面,提供几个不同的实施方案和形式。 On this aspect, several different embodiments and forms. 通常,IL-15是指人IL-15。 Typically, IL-15 refers to a human IL-15. 然而,构建体也可指来自狗、猫、马、牛、猪或羊等其它物种的IL-15。 However, it can also mean constructs from dogs, cats, horses, cattle, sheep, pigs or other species IL-15. IL-15可以是天然形式,即带有IL-15信号序列。 IL-15 may be a native form, i.e., IL-15 signal sequence. 优选IL-15是融合蛋白的组成部分(所述融合蛋白包含非I1-15信号序列),最优选还缺乏IL-15信号序列。 IL-15 is preferably an integral part of a fusion protein (fusion protein comprising the signal sequence of non-I1-15), most preferably also a lack of IL-15 signal sequence. 在优选的实施方案中,IL-15与IgE信号序列连接。 In a preferred embodiment, IL-15 is connected to IgE signal sequence.

根据一些实施方案,提供包含一种分离的核酸分子的组合物或两个不同的分离的核酸分子的组合物,前者包含编码IL-15和CD40L的核酸序列,后者包含编码IL-15的第一核酸序列及包含编码CD40L的第二核酸序列。 According to some embodiments, providing a composition comprising an isolated nucleic acid molecule or a composition of two different isolated nucleic acid molecules, the former comprising a nucleic acid sequence encoding IL-15 and CD40L, which includes a first encoding of IL-15 a nucleic acid sequence and comprising a second nucleic acid sequence encoding CD40L. 在一些优选的实施方案中,包含IL-15的蛋白对于IL-15序列所来源的物种来说是非免疫原性的。 In some preferred embodiments, the IL-15 protein comprising the sequence for the species of IL-15 is derived is non-immunogenic.

根据一些实施方案,提供包含构建体的组合物,所述构建体包含IL-15和CD40L的编码序列,所述序列也可包括在同一核酸分子上或在不同核酸分子上,即编码免疫原的核酸分子上。 According to some embodiments, to provide compositions comprising body construct, the construct comprising a coding sequence of IL-15 and CD40L, the sequences may also be included on the same nucleic acid molecule or on different nucleic acid molecules, i.e., encoding the immunogen nucleic acid molecule. 通常,下述免疫原可以是任何免疫原性蛋白,包括变应原、病原体抗原、癌相关抗原或涉及自身免疫性疾病的细胞相关抗原。 Typically, following the immunogen may be any immunogenic protein including allergens, pathogen antigens, cancer-associated antigen or a cell involved in autoimmune disease-associated antigen. 在优选的实施方案中,免疫原是病原体抗原,最优选的病原体选自HIV、HSV、HCV和WNV。 In a preferred embodiment, the immunogen is a pathogen antigen, most preferably a pathogen selected from HIV, HSV, HCV, and WNV.

包含免疫原编码序列的组合物可用作疫苗。 The composition comprises an immunogen coding sequence as vaccines. 不包含免疫原编码序列的组合物可用作免疫调节组合物。 Composition does not contain a coding sequence may be used as immunogen immunomodulatory composition. 在一些实施方案中,也提供蛋白免疫原,作为IL-15和CD40L联用而增强的免疫应答的目标。 In some embodiments, also provide protein immunogens, as IL-15 and CD40L target combined with enhanced immune response.

在一些优选的实施方案中,核酸构建体是质粒。 In some preferred embodiments, the nucleic acid construct is a plasmid. 在一些优选的实施方案中,核酸分子掺入到病毒载体中,例如痘苗病毒、腺病毒、腺病毒相关病毒、逆转录病毒,或其它任何可接受的病毒载体,用作疫苗或基因治疗载体。 In some preferred embodiments, the nucleic acid molecule incorporated into a viral vector, such as vaccinia virus, adenovirus, adeno-associated virus, retrovirus, or any other acceptable viral vector, as a vaccine or gene therapy vectors.

包含编码IL-15和CD40L的核苷酸序列的基因构建体可直接掺入到本发明相同方面的减毒活病原体中。 Comprising a gene encoding IL-15 and CD40L construct nucleotide sequences can be incorporated directly into the same aspect of the present invention, the live attenuated pathogens. 下面列出这些用作疫苗的病原体的实例。 Listed below are examples of such pathogens useful as vaccines. 在优选的实施方案中,人IL-15(优选缺乏IL-15信号序列)与人IgE信号序列连接。 In a preferred embodiment, the human IL-15 (preferably IL-15 lack a signal sequence) is connected to human IgE signal sequence.

疫苗和免疫调节组合物按照本发明的一些实施方案,本发明的组合物包含基因构建体,所述构建体包含免疫原和/或免疫原性蛋白编码序列。 Vaccines and immunomodulatory compositions according to some embodiments of the invention, the composition of the invention comprises a gene construct, said construct comprising an immunogen and / or an immunogenic protein coding sequence. 将所述组合物给予个体,以调节个体免疫系统的活性,因而增强抗所述免疫原的免疫应答。 The composition is administered to an individual to modulate the activity of an individual's immune system, thereby enhancing the anti-immune response of the immunogen. 当编码免疫调节蛋白的核酸分子被个体细胞摄入时,编码免疫调节蛋白的核苷酸序列在细胞中表达,因此将蛋白提供给个体。 When encoding immunoregulatory proteins nucleic acid molecule is taken into individual cells, a nucleotide sequence that encodes an immunomodulatory protein expressed in the cell, and therefore the protein to the individual. 本发明的其它方面提供给予组合物中位于单个核酸分子上的蛋白编码序列的方法,所述组合物包含编码一种或多种不同转录因子或中间因子(intermediate factor)的不同核酸分子,作为重组疫苗的组成部分和作为减毒疫苗的组成部分。 Other aspects of the present invention provides a method of administering the composition is located in protein coding sequence on a single nucleic acid molecule, said one or more different combinations of encoding the transcription factor or intermediate factors (intermediate factor) of different nucleic acid molecule comprises, as a recombinant components of the vaccine and as part of attenuated vaccines.

按照本发明的一些方面,提供针对病原体或异常的疾病相关细胞的预防性和/或治疗性免疫个体的组合物和方法。 In accordance with some aspects of the invention, there is provided compositions and methods for the prevention of a pathogen or disease-related abnormal cells and / or therapeutic immune individuals. 疫苗可以是任何类型的疫苗,例如减毒活疫苗、细胞疫苗、重组疫苗或核酸疫苗或DNA疫苗。 The vaccine may be any type of vaccine, e.g., live attenuated vaccines, cell vaccines, recombinant vaccine or a nucleic acid vaccine or DNA vaccine.

本发明涉及用于传递免疫调节蛋白的组合物及其使用方法。 The present invention relates to compositions and their use for delivering an immune regulatory protein.

可使用任何众所周知的技术,包括DNA注射(亦称DNA疫苗接种)、重组载体(例如重组腺病毒、重组腺病毒相关病毒和重组痘苗病毒),来传递核酸分子。 May use any well known techniques, including DNA injection (also known as DNA vaccination), recombinant vectors (e.g. recombinant adenovirus, recombinant adenovirus associated virus and recombinant vaccinia virus), to deliver a nucleic acid molecule.

DNA疫苗描述于美国专利第5,593,972、5,739,118、5,817,637、5,830,876、5,962,428、5,981,505、5,580,859、5,703,055、5,676,594号及其中引用的优先权申请,所述的每篇专利文献都通过引用结合到本文中。 DNA vaccines are described in U.S. Patent No. 5,593,972,5,739,118,5,817,637,5,830,876,5,962,428,5,981,505,5,580,859,5,703,055,5,676,594 its priority application cited, according to the patent literature of each incorporated by reference herein. 除了这些申请中描述的传递方案以外,传递DNA的替代方法描述于美国专利第4,945,050和5,036,006号,这两篇文献都通过引用结合到本文中。 In addition to these transfer schemes described in the application, alternative transfer DNA are described in U.S. Patent No. 4,945,050 and No. 5,036,006, both of which documents are incorporated by reference herein.

给药途径包括但不限于肌内、鼻内(intransally)、腹膜内、皮内、皮下、静脉内、动脉内、眼内(intraoccularly)和口服以及局部、经皮、吸入或栓剂或粘膜组织,例如通过阴道、直肠、尿道、口腔和舌下组织灌洗。 Routes of administration include but are not limited to intramuscular, intranasal (intransally), intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular (intraoccularly) and oral as well as topical, transdermal, inhalation or suppository or mucosal tissue, e.g., by vaginal, rectal, urethral, buccal and sublingual tissue lavage. 优选的给药途径包括粘膜组织、肌内、腹膜内、皮内和皮下注射。 Preferred routes of administration include mucosal tissue, intramuscular, intraperitoneal, intradermal and subcutaneous injection. 基因构建体可通过包括但不限于以下方式给予:传统的注射器、无针注射装置或“微弹轰击基因枪(microprojectilebombardment gene guns)”。 Gene construct can include but are not limited to, the following manner to give: traditional syringes, needleless injection devices, or "microprojectile bombardment gene guns (microprojectilebombardment gene guns)".

当被细胞摄取时,基因构建体可保留在细胞中,作为功能性染色体外分子和/或整合到细胞染色体DNA上。 When cellular uptake, the gene construct may be retained in the cell, as a functional extrachromosomal molecule and / or integrate into the cell chromosome DNA. 可以将DNA导入细胞中,在此它可作为分离的遗传物质,以质粒形式存在。 DNA can be introduced into the cell, where it can be used as separate genetic material in the form of a plasmid. 或者,可以将能整合到染色体上的线状DNA导入细胞中。 Alternatively, the linear DNA can integrate into the chromosome into a cell. 当DNA导入细胞时,可以添加促进DNA整合到染色体中的试剂。 When introducing DNA into the cell, may be added to promote DNA integration into the chromosome of the reagent. 可用于促进整合的DNA序列也可包括在DNA分子中。 DNA sequences can be used to promote integration may also be included in the DNA molecule. 或者,可将RNA给予细胞。 Alternatively, RNA may be administered cells. 也预期提供基因构建体,作为线状小染色体,包括着丝粒、端粒和复制起点。 Is also expected to provide gene constructs, as small linear chromosomes, including centromeres, telomeres and replication. 基因构建体可在生活在细胞内的减毒活微生物体或重组微生物载体中保留部分遗传物质。 Gene constructs may remain part of the genetic material in the cell in the live attenuated live microorganisms or recombinant microbial vectors. 基因构建体可以是重组病毒疫苗基因组的组成部分,其中遗传物质或者整合在细胞染色体上或者仍存在于染色体外。 Gene constructs may be part of genomes of recombinant viral vaccines where the genetic material, or integrated in the chromosome or extrachromosomal still present. 基因构建体包括核酸分子基因表达所需的调节元件。 Gene construct comprises the regulatory elements necessary for gene expression of a nucleic acid molecule. 这些元件包括:启动子、起始密码子、终止密码子和聚腺苷酸化信号。 These elements include: a promoter, an initiation codon, stop codon and polyadenylation signal. 另外,编码靶蛋白或免疫调节蛋白的序列的基因表达常需要增强子。 In addition, the gene encoding the target protein expression or immunoregulatory protein sequences often require an enhancer. 这些编码所需蛋白的序列和调节元件操作性连接的元件,在接受它们的个体中是可操作的,这是必要的。 These sequences encoding the desired protein and a regulatory element operably linked elements, receiving their individual is operable, it is necessary.

起始密码子和终止密码子通常被认为是编码所需蛋白的核苷酸序列的组成部分。 Start codon and stop codon are generally considered part of the nucleotide sequence encoding the desired protein. 然而,这些元件在接受所述基因构建体的个体中能发挥功能,这是必要的。 However, these elements receiving the genetic construct capable of functioning in individuals, it is necessary. 起始密码子和终止密码子在编码序列内必须符合读框。 Start codon and a stop codon within the coding sequence must be in reading frame.

所用的启动子和聚腺苷酸化信号在个体细胞中必须起作用。 Promoter and polyadenylation signal used in the individual cells must be worked.

本发明实践、尤其是人用基因疫苗生产中所用的启动子的实例包括但不限于来自以下来源的启动子:猿猴病毒40(SV40)、小鼠乳腺瘤病毒(MMTV)启动子、人免疫缺陷病毒(MV)例如BIV长末端重复序列(LTR)启动子、莫洛尼病毒、ALV、巨细胞病毒(CMV)例如CMV立即早期启动子、Epstein Barr病毒(EBV)、劳斯肉瘤病毒(RSV)以及来自人基因的启动子,例如人肌动蛋白、人肌球蛋白、人血红蛋白、人肌酸(muscle creatine)和人金属硫蛋白(metalothionein)等人基因的启动子。 Practice of the invention, especially people with examples of promoter DNA vaccine used in the production include, but are not limited to, a promoter from the following sources: the simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, Human Immunodeficiency virus (MV) such as BIV long terminal repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) immediate-early promoter such as CMV, Epstein Barr virus (EBV), Rous sarcoma virus (RSV) as well as promoters from human genes such as human actin, promoter of the human myosin, human hemoglobin, human muscle creatine (muscle creatine) and the human metallothionein (metalothionein) et al., Gene.

本发明实践、尤其是人用基因疫苗生产中所用的聚腺苷酸化信号的实例包括但不限于SV40聚腺苷酸化信号和LTR聚腺苷酸化信号。 Practice of the present invention, in particular instances gene vaccine production for human use as used polyadenylation signals include, but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. 具体地讲,使用pCEP4质粒(Invitrogen,San Diego CA)中的SV40聚腺苷酸化信号,称为SV40聚腺苷酸化信号。 Specifically, using pCEP4 plasmid (Invitrogen, San Diego CA) of the SV40 polyadenylation signal, called the SV40 polyadenylation signal.

除了DNA表达所需的调节元件之外,DNA分子中也可包含其它元件。 In addition to the regulatory elements required for DNA expression, DNA molecule may also contain other elements. 所述其它元件包括增强子。 The other elements include enhancers. 增强子可选自以下增强子,包括但不限于:人肌动蛋白、人肌球蛋白、人血红蛋白、人肌酸和病毒增强子,例如来自CMV、RSV和EBV的增强子。 Enhancer enhancer selected from the following, including, but not limited to: human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers, such as enhancers from CMV, RSV and EBV's.

基因构建体可提供哺乳动物复制起点,以便在染色体外保留构建体并在细胞中产生多拷贝构建体。 Gene construct mammalian origin of replication may be provided in order to retain the construct extrachromosomally and produce multiple copies of the construct in the cell body. 来自Invitrogen(San Diego,CA)的质粒pVAX1、pCEP4和pREP4含有Epstein Barr病毒复制起点和核心抗原EBNA-1编码区,其产生高拷贝的未整合的附加体的复制。 PVAX1 plasmid from Invitrogen (San Diego, CA) is, pCEP4 and pREP4 contains Epstein Barr virus origin of replication and core antigen EBNA-1 coding region which produces high copy replication of the episome is not integrated.

在免疫应用相关的一些优选实施方案中,给予核酸分子,所述核酸分子包含编码靶蛋白、免疫调节蛋白以及还能进一步增强抗所述靶蛋白免疫应答的蛋白的基因的核苷酸序列。 In some preferred embodiments related to immunization applications, the administration of a nucleic acid molecule, comprising a nucleic acid molecule encoding the target protein, immunoregulatory protein and nucleotide sequence can further enhance the immune response against said target protein protein gene. 所述基因的实例是编码以下的其它细胞因子和淋巴因子的基因:例如α-干扰素、γ-干扰素、血小板衍生生长因子(PDGF)、TNF、GM-CSF、表皮生长因子(EGF)、IL-1、IL-2、IL-4、IL-6、IL-10、IL-12和IL-15,其中IL-15包括缺失信号序列并任选包括来自IgE的信号序列的IL-15。 Examples of such genes are genes of other cytokines and lymphokines encoding the following: e.g., interferon α-, γ- interferon, platelet derived growth factor (PDGF), TNF, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15, IL-15 which comprises a deletion of a signal sequence and optionally including a signal sequence from IgE IL-15.

可以添加额外元件,所述元件起到细胞破坏的靶标作用,如果因任何原因需要消除接受基因构建体的细胞的话。 Additional components may be added, the element play the role of the target cell destruction, if for any reason need to eliminate cells undergo genetic construct the words. 基因构建体中可包含呈可表达形式的疱疹胸苷激酶(tk)基因。 Gene construct expressible form can be included in the form of the herpes thymidine kinase (tk) gene. 可将药物更昔洛韦给予个体,该药物将会引起对产生tk的任何细胞的选择性杀伤,因此,提供选择性破坏具有所述基因构建体的细胞的方法。 Drug ganciclovir may be administered to a subject, the drug will cause the cells of any of the selective killing tk, thus, providing a method to selectively destroy cells having the gene constructs.

为了最大量地产生蛋白,可以选择适合于在接受构建体的细胞中进行基因表达的调节序列。 In order to produce the maximum quantity protein, you can choose to accept construct regulatory sequences for gene expression in cells in. 此外,可以选择在细胞中最有效转录的密码子。 In addition, cells can be selected in the most efficient transcription codon. 本领域普通技术人员可制备在细胞中起作用的DNA构建体。 Cells play a role in DNA to those of ordinary skill in the construct can be prepared.

本发明的一个方法包括按以下途径给予核酸分子的步骤:肌内、鼻内、腹膜内、皮下、皮内或局部,或者通过向粘膜组织灌洗,所述粘膜组织选自吸入、阴道、直肠、尿道、口腔和舌下组织。 A method of the present invention comprises the steps according to the following route of administration of the nucleic acid molecule: intramuscular, intranasal, intraperitoneal, subcutaneous, intradermal or topical, or by lavage to mucosal tissue, the mucosal tissue is selected from inhalation, vaginal, rectal , urethral, buccal and sublingual tissue.

在一些实施方案中,将核酸分子与多核苷酸功能增强子或基因疫苗易化剂一起给予细胞。 In some embodiments, the nucleic acid molecule and a polynucleotide function enhancer or a genetic vaccine facilitator agent is administered together with the cells. 多核苷酸功能增强子描述于1994年1月26日申请的美国顺序号5,593,972、5,962,428和国际申请顺序号PCT/US94/00899,每件申请都通过引用结合到本文中。 Polynucleotide function enhancer described in January 26, 1994 to apply for the United States and the international application number order 5,593,972,5,962,428 sequence number PCT / US94 / 00899, each application are incorporated herein by reference. 基因疫苗易化剂描述于1994年4月1日申请的美国顺序号021,579,所述文献通过引用结合到本文中。 Genetic vaccine facilitator agents are described in April 1, 1994 U.S. application sequence number 021,579, said document incorporated by reference herein. 与核酸分子联合给予的活性助剂可以作为与核酸分子的混合物给予,或者在给予核酸分子同时、之前或之后单独给予。 Coagent administered in conjunction with nucleic acid molecules can be used as a mixture with the nucleic acid molecule administered, the nucleic acid molecule or administered simultaneously, before or after the separate administration. 另外,可起到转染剂和/或复制剂和/或炎性试剂的作用并能与GVF联合给药的其它试剂包括生长因子、细胞因子和淋巴因子,例如α-干扰素、γ-干扰素、GM-CSF、血小板衍生生长因子(PDGF)、TNF、表皮生长因子(EGF)、ILA、IL-2、IL-4、IL-6、IL-10、IL-12和IL-15以及成纤维细胞生长因子、表面活性剂例如免疫刺激复合物(ISCOMS)、弗氏不完全佐剂、LPS类似物(包括单磷脂酰脂质A(WL)、胞壁酰肽)、苯醌类似物和囊泡例如角鲨烯和角鲨烯,透明质酸也可与基因构建体联合给予。 In addition, may function transfecting agents and / or replication and / or inflammatory agents and other agents can be co-administered with a GVF include growth factors, cytokines and lymphokines, such as interferon α-, γ- interference Su, GM-CSF, platelet derived growth factor (PDGF), TNF, epidermal growth factor (EGF), ILA, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15 as well as fibroblast growth factor, surface active agents such as immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs (including single phosphatidyl lipid A (WL), muramyl peptides), quinone analogs and vesicles such as squalene and squalane, hyaluronic acid can also be administered in combination with gene constructs. 在一些实施方案中,免疫调节蛋白可用作GVF。 In some embodiments, the immunomodulatory protein can be used as GVF. 在一些实施方案中,核酸分子与PLG一起提供,以增强传/摄取。 In some embodiments, the nucleic acid molecule is provided with PLG to enhance transmission / uptake.

本发明的药物组合物包含约1纳克至约2000微克DNA。 The pharmaceutical composition of the present invention comprise from about 1 nanogram to about 2000 micrograms of DNA. 在一些优选的实施方案中,本发明的药物组合物包含约5纳克至约1000微克DNA。 In some preferred embodiments, the pharmaceutical compositions of the invention comprise about 5 nanogram to about 1000 micrograms of DNA. 在一些优选的实施方案中,药物组合物含有约10纳克至约800微克DNA。 In some preferred embodiments, the pharmaceutical compositions contain about 10 nanograms to about 800 micrograms of DNA. 在一些优选的实施方案中,药物组合物含有约0.1至约500微克DNA。 In some preferred embodiments, the pharmaceutical composition contains from about 0.1 to about 500 micrograms of DNA. 在一些优选的实施方案中,药物组合物含有约1至约350微克DNA。 In some preferred embodiments, the pharmaceutical composition contains from about 1 to about 350 micrograms of DNA. 在一些优选的实施方案中,药物组合物含有约25至约250微克DNA。 In some preferred embodiments, the pharmaceutical composition contains from about 25 to about 250 micrograms of DNA. 在一些优选的实施方案中,药物组合物含有约10至约200微克DNA。 In some preferred embodiments, the pharmaceutical composition contains from about 10 to about 200 micrograms of DNA.

本发明的药物组合物可按照所用的给药方式来配制。 The pharmaceutical compositions according to the present invention can be administered formulated used. 在药物组合物是注射用药物组合物的情况下,它们无菌、无热源和无颗粒。 In the pharmaceutical composition is a pharmaceutical composition for injection of the case, they are sterile, pyrogen-free and free of particulates. 优选使用等渗制剂。 Isotonic formulation is preferably used. 通常,等渗性添加剂可包括氯化钠、葡萄糖、甘露醇、山梨醇和乳糖。 Typically, isotonicity additive may include sodium chloride, dextrose, mannitol, sorbitol and lactose. 在某些情况下,优选等渗溶液例如磷酸缓冲盐溶液。 In some cases, isotonic solutions such as preferably phosphate buffered saline. 稳定剂包括明胶和白蛋白。 Stabilizers include gelatin and albumin. 在一些实施方案中,向制剂中添加血管收缩药。 In some embodiments, adding a vasoconstrictor to the formulation.

根据本发明的一些实施方案,提供诱导抗免疫原免疫应答的方法,即通过将本发明的组合物给予个体。 According to some embodiments of the invention, there is provided a method immunogens to induce anti-immune response, i.e. the compositions of this invention will be administered to a subject by. 疫苗可以是减毒活疫苗、细胞疫苗、重组疫苗或核酸疫苗或DNA疫苗。 Vaccines may be live attenuated vaccines, cell vaccines, recombinant vaccines or DNA vaccines or DNA vaccines.

除了使用免疫调节蛋白编码序列的可表达形式来改进基因疫苗之外,本发明还涉及改良的减毒活疫苗和改良疫苗,其使用重组载体来传递编码抗原的外源基因。 In addition to using expressible forms of immunomodulating protein coding sequence to improve genetic vaccines addition, the present invention also relates to improved attenuated live vaccines and improved vaccines which use recombinant vectors to deliver foreign genes encoding the antigen. 减毒活疫苗和使用重组载体来传递外源抗原的疫苗的实例描述于美国专利第4,722,848、5,017,487、5,077,044、5,110,587、5,112,749、5,174,993、5,223,424、105,225,336、5,240,703、5,242,829、5,294,441、5,294,548、5,310,668、5,387,744、5,389,368、5,424,065、5,451,499、5,453,364、5,462,734、5,470,734和5,482,713,以上每篇文献都通过引用结合到本文中。 And live attenuated vaccine using recombinant vectors to deliver foreign antigens instance vaccines are described in U.S. Patent Nos 4,722,848,5,017,487,5,077,044,5,110,587,5,112,749,5,174,993,5,223,424,105,225,336,5,240,703,5,242,829,5,294,441,5,294,548,5,310,668,5,387,744, 5,389,368,5,424,065,5,451,499,5,453,364,5,462,734,5,470,734 and 5,482,713, each article above are incorporated herein by reference. 提供基因构建体,其包含与调节序列操作性连接且编码免疫调节蛋白的核苷酸序列,所述调节序列可在疫苗中起到影响表达的作用。 Provides a gene construct comprising a regulatory sequence operatively connected to the immunomodulatory protein and encoding nucleotide sequence, the regulatory sequence can affect the expression in a vaccine effect. 将基因构建体掺入到减毒活疫苗和重组疫苗中,以产生本发明的改良疫苗。 The gene construct is incorporated into attenuated live vaccines and recombinant vaccines to produce improved vaccines of the invention.

本发明提供免疫个体的改进方法,其包括以下步骤:将基因构建体给予个体细胞,作为疫苗组合物的组成部分,所述疫苗组合物包括DNA疫苗、减毒活疫苗和重组疫苗。 The present invention provides an improved method of immunization of an individual, comprising the steps of: a gene construct administered to an individual cell, as part of the vaccine composition, said vaccine composition including DNA vaccines, attenuated live vaccines and recombinant vaccines. 基因构建体包含与调节序列操作性连接且编码免疫调节蛋白的核苷酸序列,所述调节序列可在疫苗中起到影响表达的作用。 Gene construct contains regulatory sequences operably linked to and encoding immunomodulatory protein nucleotide sequence, the regulatory sequence can affect the expression in a vaccine effect. 改良疫苗导致增强的细胞免疫应答。 Improved vaccines result in an enhanced cellular immune response.

免疫原本发明用于诱发增强的抗靶蛋白的免疫应答,所述靶蛋白即与病原体、变应原或个体自身“异常”细胞特异性相关的蛋白。 Originally invention for inducing immune enhanced immune response against the target protein, the target protein i.e. pathogens, allergens or the individual's own "abnormal" cell-specific proteins associated. 本发明可用于使个体产生抗病原因子和病原生物的免疫,使得抗病原体蛋白的免疫应答提供抗病原体的保护性免疫。 The present invention may be used to produce an individual antiviral reasons promoter and pathogen immunity, so that the immune response against a pathogen protein provides protective immunity against a pathogen. 本发明可用于控制过度增殖性疾病和障碍(例如癌症),即通过诱发抗靶蛋白的免疫应答,所述靶蛋白是与过度增殖细胞特异性相关的蛋白。 The present invention may be used to control the hyperproliferative diseases and disorders (e.g. cancer), i.e., by inducing an immune response against the target protein, the target protein is associated with a hyperproliferative cell-specific proteins. 本发明可用于控制自身免疫性疾病和障碍,即通过诱发抗靶蛋白的免疫应答,所述靶蛋白是与涉及自身免疫性疾病的细胞特异性相关的蛋白。 The present invention can be used to control autoimmune diseases and disorders, i.e., by inducing an immune response against the target protein, the target protein is involved in autoimmune diseases associated with cell-specific proteins.

根据本发明的一些方面,将编码靶蛋白和免疫调节蛋白的DNA或RNA导入个体组织细胞中,在此进行表达,因而产生所编码的蛋白。 In accordance with some aspects of the invention, encoding a target protein and immunomodulating proteins is introduced DNA or RNA in individual cells, here expressed, thereby producing the encoded protein. 在个体细胞中,编码靶蛋白和一种或两种免疫调节蛋白的DNA或RNA序列与表达所需的调节元件连接。 The required adjustment in the individual cells, encoding a target protein and one or both immunomodulating proteins and expression of DNA or RNA sequence element is connected. DNA表达的调节元件包括启动子和聚腺苷酸化信号。 DNA expression regulatory elements include a promoter and a polyadenylation signal. 另外,其它元件,例如Kozak区,也可以包含在基因构建体中。 In addition, other elements, e.g., Kozak region, may also be included in the gene construct.

在一些实施方案中,发现编码靶蛋白序列的可表达形式以及编码两种免疫调节蛋白序列的可表达形式都在给予个体的同一核酸分子上。 In some embodiments, and can be found in the form of the expression of two forms of immunomodulating protein expression of the coding sequence may encode a target protein sequence are given on the same nucleic acid molecule in the individual.

在一些实施方案中,编码靶蛋白序列的可表达形式存在于各自的核酸分子上,而不在含有编码一种或多种免疫调节蛋白的序列可表达形式的核酸分子上。 In some embodiments, expressible forms of sequences encoding the target protein is present on the respective nucleic acid molecules, instead of containing one or more encoding the immunomodulatory protein sequence expressible form of a nucleic acid molecule. 在一些实施方案中,编码靶蛋白序列的可表达形式与编码一种或多种免疫调节蛋白的序列的可表达形式存在于一个核酸分子上,而不在含有编码一种或多种免疫调节蛋白的序列可表达形式的核酸分子上。 In some embodiments, may be in the form of expression of the coding sequence of the target protein with one or more immunomodulatory proteins can be in the form of expression of the coding sequence present on a nucleic acid molecule, and not containing encoding one or more immunomodulatory proteins sequences can be expressed in form of a nucleic acid molecule. 可以按照本发明,产生并传递多种不同核酸分子,并且传递给个体。 According to the present invention can produce and deliver a variety of different nucleic acid molecules, and passed to the individual. 例如,在一些实施方案中,编码靶蛋白序列的可表达形式存在于各自的核酸分子上,而不在含有编码这两种免疫调节蛋白中的一种或多种的序列可表达形式的核酸分子上,而所述两种免疫调节蛋白存在于各自的核酸分子上,而不在含有编码一种或多种免疫调节蛋白的序列的可表达形式的核酸分子上。 For example, in some embodiments, expressible forms of sequences encoding the target protein is present on the respective nucleic acid molecules, instead of containing both immunomodulatory protein encoded in one or more sequences may be expressed in the form of a nucleic acid molecule , while the two immunomodulatory proteins present on the respective nucleic acid molecules, rather than containing the encoding one or more immunomodulatory proteins can be expressed in the form of a sequence of a nucleic acid molecule. 在这些情况下,将所有三种分子都传递给个体。 In these cases, all three molecules are delivered to the individual.

可提供核酸分子,作为质粒DNA、重组载体的核酸分子或作为减毒疫苗或细胞疫苗所提供的遗传物质的组成部分。 Nucleic acid molecule may be provided as plasmid DNA, recombinant vector or a nucleic acid molecule as part of attenuated vaccine or cell vaccine provided genetic material. 或者,在一些实施方案中,除了传递编码靶蛋白和/或一种或两种免疫调节蛋白的核酸分子以外,还可以将靶蛋白和/或一种或两种免疫调节蛋白作为蛋白来传递,或者是用于替代编码它们的核酸分子。 Alternatively, in some embodiments, in addition to transmitting encoded target protein and / or one or both immunomodulating proteins nucleic acid molecules, can also be the target protein and / or one or both immunomodulating proteins as proteins to pass, or is used to replace the nucleic acid molecules encoding them.

基因构建体可包括与基因表达所需调节元件操作性连接且编码靶蛋白或免疫调节蛋白的核苷酸序列。 Gene construct the gene may comprise regulatory elements necessary for expression operably linked to and encoding a target protein or immunomodulating protein nucleotide sequence. 根据本发明,提供以下基因构建体的组合:一个基因构建体包含编码靶蛋白的核苷酸序列的可表达形式,而另一个基因构建体包含编码免疫调节蛋白的核苷酸序列的可表达形式。 According to the present invention, there is provided a combination of the following gene construct: a genetic construct comprises an expressible form of a nucleotide sequence encoding the target protein and the other gene construct expressible form comprises an immunomodulatory protein encoding nucleotide sequence . 将包含基因构建体组合的DNA或RNA分子掺入到活细胞中,导致DNA或RNA的表达并产生靶蛋白和一种或多种免疫调节蛋白。 DNA or RNA molecule containing the gene construct will be incorporated into the compositions of the living cells, resulting in the expression of DNA or RNA and produce the target protein and one or more immunomodulatory proteins. 导致增强的抗靶蛋白的免疫应答。 Enhanced immune response against the target protein.

本发明可用于使个体产生抗所有病原体的免疫,所述病原体例如病毒、原核生物和病原性真核生物,例如单细胞病原生物和多细胞寄生虫。 The present invention may be used to generate anti-self immune all pathogens, said pathogens such as viruses, prokaryote and pathogenic eukaryotic organisms such as unicellular pathogenic organisms and multicellular parasites. 本发明特别用于使个体产生抗以下病原体的免疫,所述病原体感染细胞并且没有包囊膜(encapsulated),例如病毒和淋病(gonorrhea)、李斯特氏菌(1isteria)和志贺氏菌(shigella)等原核生物。 The present invention is particularly useful to make an individual to produce anti-pathogen immunity following a pathogen-infected cells and no encapsulation membrane (encapsulated), such as viruses and gonorrhea (gonorrhea), Listeria (1isteria) and Shigella (shigella) and other prokaryotes. 另外,本发明也可用于使个体产生抗原生动物病原体的免疫,所述病原体包括处于胞内病原体的生命周期某一阶段的病原体。 Further, the present invention also may be used to generate anti-protozoan pathogen individual immunized, the pathogen intracellular pathogens, including pathogens in the life cycle of a certain stage. 表1列举了可用于制备本发明疫苗的一些病毒科属。 Table 1 lists some of the virus used in the vaccine can Genus present invention is prepared. 包含编码肽的DNA序列的DNA构建体可用于疫苗,所述肽至少包含这样的表位:与该表所列举的抗原等病原体抗原上展示的表位相同或基本类似的表位。 Comprising a DNA sequence encoding the peptide can be used to construct a DNA vaccine, the peptide comprises at least such an epitope: listed in the table show the antigen and other pathogen antigen epitope identical or substantially similar epitopes. 此外,本发明也可用于使个体产生抗其它病原体的免疫,所述病原体包括原核病原体和真核原生动物病原体,以及多细胞寄生虫,例如表2所列举的那些。 Further, the present invention also may be used to produce an individual immunized against other pathogens, the pathogens, including prokaryotic and eukaryotic protozoan pathogens pathogens, as well as multicellular parasites, such as those listed in Table 2.

为了生产基因疫苗以保护机体免遭病原体感染,基因构建体中必须包含编码免疫原性蛋白的遗传物质,作为靶的编码序列,使得可以建立针对所述免疫原性蛋白的保护性免疫应答。 In order to produce a genetic vaccine to protect the body against pathogen infection, genetic construct must contain the genetic material encoding immunogenic protein, as a coding sequence of the target, making it possible to establish an immune response against the immunogenic protein protective immunity. 对于病原体是在胞内感染(本发明对胞内感染特别有用)还是胞外感染来说,所有病原体抗原都诱导保护性反应,是不太可能的。 For pathogen infection (infection of the present invention is particularly useful for intracellular) infections, in the extracellular or intracellular, all pathogen antigens inducing a protective response, is unlikely. 因为DNA和RNA比较小,可以相对容易地生产,所以本发明提供采用多种病原体抗原接种的额外优点。 Because DNA and RNA is relatively small, relatively easy to produce, so the additional advantage of more pathogen antigens of the present invention provides the use of vaccination. 基因疫苗中所用的基因构建体可包括编码许多病原体抗原的遗传物质,例如,一个构建体中可包含若干病毒基因,由此提供多种靶标。 DNA vaccine used in the gene construct may include genetic material encoding antigens of many pathogens, e.g., a construct may comprise a number of viral genes, thereby providing multiple targets.

表1和表2列举了用于制备基因疫苗的一些病原因子和病原生物,以保护个体免遭其感染。 Table 1 and Table 2 lists some pathogenic factors and pathogenic organisms used to prepare DNA vaccine to protect individuals from infection. 在一些优选的实施方案中,使个体产生抗病原体免疫的方法针对HIV、HSV、HCV、WNV或HBV。 In some preferred embodiments, so that the individual method of producing anti-pathogen immunity against HIV, HSV, HCV, WNV or HBV.

本发明的另一方面提供赋予抗过度增殖细胞(其是过度增殖性疾病的特征)的保护性免疫应答的方法,并提供治疗罹患过度增殖性疾病的个体的方法。 Another aspect of the present invention having anti-hyperproliferative cell (which is characteristic of hyperproliferative diseases) method protective immune response and to provide a method of treating individuals suffering from hyperproliferative diseases. 过度增殖性疾病的实例包括癌症和银屑病的所有形式。 Examples of over-proliferative diseases, including all forms of cancer and psoriasis.

已经发现,将包含编码免疫原性“过度增殖细胞”相关蛋白的核苷酸序列的基因构建体导入个体细胞,导致在接种的个体细胞中产生这些蛋白。 It has been found, comprising encoding immunogenic "hyperproliferative cell" nucleotide sequence of the gene related protein construct into individual cells, resulting in these proteins in the vaccinated cells of an individual. 为了产生抗过度增殖性疾病的免疫,将包含编码过度增殖性疾病相关蛋白的核苷酸序列的基因构建体给予个体。 In order to generate an immune anti-hyperproliferative disease, comprising the nucleotide sequence of the gene encoding the excessive proliferative disease related protein constructs administered to an individual.

为了让过度增殖相关蛋白成为有效的免疫原性靶标,与正常细胞相比,过度增殖细胞中必须专一性或以更高水平产生蛋白。 In order for the hyperproliferative-associated protein to be an effective immunogenic target, compared to normal cells, hyperproliferative cells or be specific to a higher level produce the protein. 靶抗原包括所述蛋白、其片段和肽;其包括存在于所述蛋白上的至少一个表位。 Target antigens include the proteins, fragments thereof and peptides; comprising at least one epitope present in the protein. 在一些实例中,过度增殖相关蛋白是蛋白编码基因的突变产物。 In some instances, excessive proliferation-associated protein is the product of the mutant protein-coding genes. 与正常蛋白相比,突变基因编码除了具有稍微不同的氨基酸序列外几乎相同的蛋白,这产生正常蛋白上不存在的不同表位。 Compared with normal protein, in addition to mutations in the gene encoding the amino acid sequence having a slightly different outer almost the same protein, which produce the protein does not normally exist in different epitopes. 所述靶蛋白包括例如以下癌基因所编码的蛋白:myb、myc、fyn和易位基因bcr/abl、ras、src、P53、neu、trk和EGRF。 The target proteins include, for example proteins encoded by oncogenes following: myb, myc, fyn, and the translocation gene bcr / abl, ras, src, P53, neu, trk and EGRF. 除了癌基因产物作为靶抗原之外,抗癌治疗用靶蛋白和保护性方案还包括B细胞淋巴瘤产生的抗原的可变区和T细胞淋巴瘤的T细胞受体的可变区,在一些实施方案中也可用自身免疫性疾病的靶抗原。 In addition to oncogene products as target antigens addition, anti-cancer therapeutic target protein and further comprising a protective program variable region produced by B-cell lymphoma antigen and T cell lymphoma T cell receptor variable region in some embodiments, the target antigen may also be an autoimmune disease. 其它肿瘤相关蛋白也可用作靶蛋白,例如在肿瘤细胞中发现呈更高水平的蛋白,包括单克隆抗体17-IA所识别的蛋白和叶酸结合蛋白或PSA。 Other tumor-associated proteins can also be used as target proteins, such as higher levels of protein was found in tumor cells, comprising the monoclonal antibody 17-IA and folate binding protein recognized proteins or PSA.

尽管本发明可用于使个体产生抗几种类型癌症中的一种或多种的免疫,但是本发明特别可用于预防性免疫个体,所述个体易于发展特定癌症,或者所述个体已经罹患癌症并易于复发。 While the present invention may be used to generate anti-self several types of cancer with one or more immune, but the present invention is particularly useful for the prophylactic immunization of individuals, the individual susceptible to developing a particular cancer, or the subject has cancer and easy to relapse. 遗传学和技术以及流行病学的发展,使人们能够对个体产生癌症的概率和风险进行测算和评价。 Genetics and epidemiology of development and technology, to enable people to generate the probability of an individual and the risk of cancer were measured and evaluated. 采用遗传筛选和/或家族健康史,可以预测特定个体发生几种类型癌症中任何一种的概率。 Genetic screening and / or family health history, several types of cancer can predict the probability of any one particular individual occurrence.

同样,那些已经罹患癌症的个体和那些经治疗消除癌症或得到缓解的个体,也特别易于复发。 Similarly, individuals who have cancer and those treated to eliminate the cancer or to ease the individual, but also particularly prone to relapse. 作为治疗方案的一部分,可对经诊断确认患有癌症的个体进行抗癌免疫,以便控制癌症复发。 As part of the treatment regimen can confirm diagnosed with cancer anticancer immune individuals to control cancer recurrence. 因此,一旦得知个体患有某类癌症并具有复发危险时,可以对他们进行免疫,使其免疫系统做好准备,以应付未来出现的任何癌症。 So, once that individuals suffering from certain types of cancer and has a risk of recurrence can be immunized against them, so that the immune system is ready to deal with any future cancers arise.

本发明提供治疗罹患过度增殖性疾病的个体的方法。 The present invention provides a method of treating individuals suffering from hyperproliferative diseases. 在该方法种,引入基因构建体,作为免疫治疗药,指导和启动个体免疫系统,以对付产生靶蛋白的过度增殖细胞。 In this kind of method, the introduction of gene constructs, as immunotherapeutic agents, guidance and start the individual's immune system to produce the target protein against hyperproliferative cells.

本发明提供治疗罹患自身免疫性疾病和障碍的个体的方法,即通过给予广谱保护性免疫应答,所述免疫应答是针对包括细胞受体和产生“自身”定向抗体的细胞在内的自身免疫相关靶标的。 The present invention provides therapeutic suffering from autoimmune diseases and disorders of the individual methods, i.e., by administering a broad spectrum of protective immune response, said immune response comprises a cell receptor and against a "self" cell directed antibodies, including autoimmune related targets.

T细胞介导的自身免疫性疾病包括类风湿性关节炎(RA)、多发性硬化(MS)、斯耶格伦综合征(Sjogren's syndrome)、结节病、胰岛素依赖性糖尿病(IDDM)、自身免疫性甲状腺炎、反应性关节炎、关节强硬性脊椎炎、硬皮病、多肌炎、皮肌炎、银屑病、脉管炎、韦格纳肉芽肿病(Wegener's granulomatosis)、节段性回肠炎(Crohn's disease)和溃疡性结肠炎。 Autoimmune diseases mediated by T cells including rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome (Sjogren's syndrome), sarcoidosis, insulin dependent diabetes mellitus (IDDM), itself autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis (Wegener's granulomatosis), segmental ileitis (Crohn's disease) and ulcerative colitis. 这些疾病的特征都是结合内源性抗原并引起自身免疫性疾病相关的炎性级联的T细胞受体。 These diseases are characterized by binding of endogenous antigens and autoimmune diseases caused by the inflammatory cascade associated with T-cell receptors. 抗T细胞可变区的接种,将会诱导包括CTL在内的免疫应答,以消除这些T细胞。 Vaccinated against the variable region of T cells, including CTL will induce an immune response, including, to eliminate these T cells.

在RA中,涉及该病的T细胞受体(TCR)的若干特异性可变区已经表征。 In RA, several specific variable regions of T cell receptors involved in the disease (TCR) have been characterized. 这些TCR包括Vβ-3、Vβ-14、20Vβ-17和Vα-17。 These include TCR Vβ-3, Vβ-14,20Vβ-17 and Vα-17. 因此,用编码至少一种所述蛋白的DNA构建体进行接种,将会诱导靶向涉及RA的T细胞的免疫应答。 Thus, DNA encoding at least one of the protein construct were inoculated, will induce target T cells involved in RA immune response. 参见Howell,MD等,1991Proc.Nat.Acad.Sci.USA 88:10921-10925;Piliard,X.等,1991Science 253:325-329;Williams,WV等,1992 J Clin Invest.90:326-333;以上每篇文献都通过引用结合到本文中。 See Howell, MD, etc., 1991Proc.Nat.Acad.Sci.USA 88: 10921-10925; Piliard, X, etc., 1991Science 253:. 325-329; Williams, WV, etc., 1992 J Clin Invest.90: 326-333; above each article are incorporated herein by reference. 在MS中,涉及该病的TCR的若干特异性可变区已经表征。 In MS, several specific variable regions of the TCR involved disease have been characterized. 这些TCR包括VfP和Va-10。 These include VfP and TCR Va-10. 因此,用编码至少一种所述蛋白的DNA构建体进行接种,将会诱导靶向涉及MS的T细胞的免疫应答。 Thus, DNA encoding at least one of the protein construct were inoculated, will induce target T cells involved in MS immune response. 参见Wucherpfennig,KW等,1990Science 248:1016-1019;Oksenberg,JR等,1990Nature 345:344-346;以上每篇文献都通过引用结合到本文中。 See Wucherpfennig, KW, etc., 1990Science 248: 1016-1019; Oksenberg, JR, etc., 1990Nature 345: 344-346; each reference above are incorporated by reference herein.

在硬皮病中,涉及该病的TCR的若干特异性可变区已经表征。 In scleroderma, several specific variable regions of the TCR involved disease have been characterized. 这些TCR包括Vβ-6、Vβ-8、Vβ-14和Vα-16、Vα-3C、Vα-7、Vα-14、Vα-15、Vα-16、Vα-28和Vα-12。 These include TCR Vβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12. 因此,用编码至少一种所述蛋白的DNA构建体进行接种,将会诱导靶向涉及硬皮病的T细胞的免疫应答。 Thus, DNA encoding at least one of the protein construct were inoculated, will induce target T cells involved in scleroderma immune response.

为了治疗罹患T细胞介导的自身免疫性疾病(特别是TCR可变区已经表征的)的患者,可以进行滑膜活检。 For the treatment of autoimmune disease suffering from T cell-mediated (especially TCR variable region has been characterized) patients, synovial biopsy can be performed. 可以取出所存在的T细胞样品,用标准技术鉴定TCR可变区。 T cells in a sample can be taken out of the present, using standard techniques to identify TCR variable region. 用这些信息,可以制备基因疫苗。 With these information, you can prepare DNA vaccine.

B细胞介导的自身免疫性疾病包括狼疮(SLE)、格雷夫斯病(Grave's disease)、重症肌无力、自身免疫性溶血性贫血、自身免疫性血小板减少症、哮喘、冷球蛋白血症、原发性胆硬化和恶性贫血。 Autoimmune diseases, including B-cell mediated lupus (SLE), Graves' disease (Grave's disease), myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, Primary biliary sclerosis and pernicious anemia. 这些疾病的特征都是结合内源性抗原并引起自身免疫性疾病相关的炎性级联的抗体。 These diseases are characterized by binding of endogenous antigens and autoimmune diseases caused by the inflammatory cascade associated antibodies. 抗抗体可变区的接种,将会诱导包括CTL在内的免疫应答,以消除这些产生抗体的B细胞。 Anti-vaccination antibody variable regions, will induce an immune response including CTL, including, to eliminate these antibody-producing B cells.

为了治疗罹患B细胞介导的自身免疫性疾病的患者,必须对涉及自身免疫活性的抗体可变区进行鉴定。 In order to treat patients suffering from B-cell mediated autoimmune disease in patients, involving the autoimmune activity must be an antibody variable regions were identified. 可以进行活检,可以取出炎症部位所存在的抗体样品。 Biopsy can be performed, the site of inflammation can be taken out antibodies present in the sample. 可以使用标准技术,对这些抗体的可变区进行鉴定。 Using standard techniques, variable regions of these antibodies were identified. 用这些信息,可以制备基因疫苗。 With these information, you can prepare DNA vaccine.

在SLE的情况下,认为一种抗原是DNA。 In the case of SLE, one antigen is believed DNA. 因此,在有待进行抗SLE免疫的患者中,可以对他们的血清筛选抗DNA抗体,可以制备包含DNA构建体的疫苗,所述DNA构建体编码血清中存在的抗DNA抗体可变区。 Thus, in patients to be immunized anti SLE, their sera can be screened for anti-DNA antibodies, vaccines can be prepared comprising the DNA construct, the DNA construct encodes present in the serum anti-DNA antibody variable region.

TCR可变区和抗体可变区的共同结构特征是众所周知的。 Common structural features of TCR variable region and an antibody variable region is well known. 通常可按照以下众所周知的方法,找到编码特定TCR或抗体的DNA序列,所述方法例如描述于以下文献的方法:Kabat等,1987 Sequence ofProteins of Immunological Interest USDepartment of Health and HumanServices,Bethesda MD,所述文献通过引用结合到本文中。 Generally in accordance with the following well-known methods, to find a DNA sequence encoding a particular TCR or antibody, the method such as described in the following literature methods: Kabat, etc., 1987 Sequence ofProteins of Immunological Interest USDepartment of Health and HumanServices, Bethesda MD, the literature incorporated by reference herein. 另外,从抗体克隆功能性可变区的通用方法可参见Chaudhary,VK等,1990Proc.Natl.Acad Sci.USA 87:1066,所述文献通过引用结合到本文中。 Further, from the General Method antibody clone functional variable regions can be found in Chaudhary, VK, etc., 1990Proc.Natl.Acad Sci.USA 87: 1066, said document incorporated by reference herein.

重组蛋白的制备本发明涉及体外宿主细胞培养物,所述宿主细胞包含在所述宿主细胞中可操作的表达载体,所述表达载体包含编码由与非IgE蛋白序列连接的IgE信号肽组成的融合蛋白的核酸序列;涉及所述核酸分子;和涉及包含所述载体的宿主细胞。 Preparation of recombinant proteins in vitro The present invention relates to a host cell culture, the host cell comprising operable in said host cell an expression vector, said expression vector comprising a fusion of a signal peptide encoded by IgE and non-IgE protein sequence consisting connection a nucleic acid sequence of the protein; relates to the nucleic acid molecule; and host cells comprising said vector. 本发明也涉及产生融合蛋白的方法,所述方法包括培养宿主细胞的步骤。 The present invention also relates to a method of producing a fusion protein, said method comprising the steps of culturing a host cell. 本发明涉及分离的融合蛋白,其包含与非IgE蛋白序列连接的IgE信号肽。 The present invention relates to an isolated fusion protein comprising the signal peptide and non-IgE IgE protein sequence attached.

可以通过常规方法,使用易得的原料,如上所述地制备融合蛋白。 By conventional methods, using readily available starting materials, fusion protein was prepared as described above. 制备编码所需蛋白的合适DNA序列,就可以使用本领域已知的重组技术来制备蛋白。 Suitable DNA sequences encoding the desired protein preparation, the protein can be prepared using recombinant techniques known in the art.

本领域普通技术人员可以使用众所周知的技术,将编码融合蛋白的DNA插入到市售表达载体中,用于众所周知的表达系统中。 Those of ordinary skill in the art can use well-known, the DNA encoding the fusion protein is inserted into a commercially available expression vector for the well-known expression systems. 市售质粒pYES2(Invitrogen,San Diego,Calif.)可用于在酿酒酵母(S.cerevisiae)中进行制备。 Commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) Can be used in yeast (S.cerevisiae) are prepared. 市售MaxBacTM(Invitrogen,San Diego,Calif.)完整杆状病毒表达载体可用于在昆虫细胞中进行制备。 Commercially available MaxBacTM (Invitrogen, San Diego, Calif.) Complete baculovirus expression vector can be used for preparing in insect cells. 市售质粒pcDNA I(Invitrogen,San Diego,Calif.)可用于在中国仓鼠卵巢细胞等哺乳动物细胞中进行制备。 Commercially available plasmid pcDNA I (Invitrogen, San Diego, Calif.) Can be used for the preparation of the Chinese hamster ovary cells and other mammalian cells. 本领域普通技术人员可使用这些市售表达载体系统或其它系统,用常规技术和易得原料来制备融合蛋白。 Ordinary skill in the art can use these commercial expression vectors systems or other systems, using conventional techniques and readily available starting materials for preparing the fusion protein.

本领域普通技术人员可使用其它市售表达载体和系统,使用众所周知的方法和易得原料,来制备载体。 Those of ordinary skill may use other commercially available expression vectors and systems, using well-known methods and readily available starting material, to prepare the carrier. 含有所需控制序列(例如启动子和聚腺苷酸化信号,优选增强子)的表达系统是易得的,并且本领域已知可用于各种宿主。 Expression systems containing the desired control sequences (such as promoters and polyadenylation signals, and preferably enhancers) are readily available and known in the art can be used for a variety of hosts. 参见例如Sambrook等,Molecular Cloning aLaboratory Manual,第二版,Cold Spring Harbor Press(1989)。 See, e.g., Sambrook et al., Molecular Cloning aLaboratory Manual, Second Edition, Cold Spring Harbor Press (1989). 因此,所需蛋白既可在原核系统中也可在真核系统中制备,得到一系列加工形式的蛋白。 Therefore, the desired protein can also be produced in eukaryotic systems in prokaryotic systems, get a series of processing in the form of protein.

目前,各种各样的真核宿主也可用于制备重组外源蛋白。 Currently, a wide variety of eukaryotic hosts are also useful for the preparation of recombinant foreign proteins. 可以用产生所需蛋白(直接使用IgE信号肽)的表达载体转化真核宿主。 Expression vectors can be used to produce the desired protein (signal peptide directly using IgE) transformed eukaryotic host.

常用的真核系统包括但不限于酵母细胞、真菌细胞、昆虫细胞、哺乳动物细胞、鸟类细胞和高等植物细胞。 Commonly used eukaryotic systems include, but are not limited to yeast cells, fungal cells, insect cells, mammalian cells, avian cells, and cells of higher plants. 可以使用合适的启动子以及末端序列和增强子,其用于这些宿主类型时是相容的和可操作的,例如杆状病毒多角体启动子。 Can be used when an appropriate promoter and terminal sequences and enhancers, for these types of hosts are compatible and operable, e.g., the baculovirus polyhedrin promoter. 如上所述,启动子可以是组成型或诱导型的。 As described above, the promoter may be constitutive or inducible. 例如,在哺乳动物系统中,小鼠金属硫蛋白启动子可以因添加重金属离子而被诱导。 For example, in mammalian systems, the mouse metallothionein promoter by the addition of heavy metal ions can be induced.

适于所需宿主的表达系统的构建细节是本领域已知的。 Construction details of expression systems suitable for desired host are known in the art. 对于蛋白的重组制备来说,编码它的DNA适宜连接所选的表达载体并用于转化相容性宿主,然后在以下条件下培养和维持它们:其中发生外源基因的表达。 For the preparation of recombinant proteins, the DNA encoding it is suitably connected to an expression vector and used to transform a selected host compatibility, they are then cultured and maintained under the following conditions: wherein the expression of the foreign gene occurs. 从培养物中(或者可以通过裂解细胞或者优选从培养基中)回收由此产生的本发明蛋白。 From the culture medium (or from the culture medium by lysing the cells or preferably) recovering the resulting protein of the invention.

本领域普通技术人员可以使用众所周知的技术,使用所述表达系统,分离所产生的融合受体蛋白或其片段。 Those of ordinary skill in the art can use well-known, the use of the expression system, separating the resulting receptor fusion protein or fragment thereof.

实施例实施例1引言对许多HIV-1阳性个体来说,抗逆转录病毒的联合疗法在感染者中成功地降低了病毒量,导致良好的预后。 EXAMPLES Example 1 Introduction For many HIV-1 positive individuals, the antiretroviral combination therapy in infected persons succeeded in reducing the viral load, resulting in good prognosis. 然而,一些实验室已经报道,联合用药方案很少影响已建立的病毒储存库(以下参考文献1-3)。 However, some laboratories have reported that the combination of established programs rarely affect virus repository (following references 1-3). 迄今为止,联合疗法尚未导致病毒的清除,而且目前的治疗方案具有相当大的副作用,最终会影响患者的依从性并影响病程。 So far, the combination therapy has not yet led to viral clearance, and the current treatment options have considerable side effects, will ultimately affect patient compliance and to influence the course. 因此,非常需要开发替代疗法,包括用于HIV-1的潜在的免疫疗法。 Therefore, a great need to develop alternative therapies, including potential for immunotherapy of HIV-1. 据信,CD8+T细胞应答对于控制HIV-1感染和缓解病程来说,是重要的。 It is believed, CD8 + T cell response to the control HIV-1 infection and mitigation course, is important. 尽管HIV-1特异性CD8+T细胞应答在控制病毒复制中的确切功能尚未完全阐明,但是,已经确定,HIV-1血清阳性个体中长期不发展与特异性CD8+T细胞介导的细胞应答之间的相关性(以下参考文献4-7)。 Although HIV-1-specific CD8 + T cell responses in the control of viral replication in the exact function is not fully understood, however, have been identified, HIV-1-seropositive individuals do not develop long-term and specific CD8 + T cell-mediated cellular responses correlation between the (following references 4-7). 另外,在冈比亚,一组高暴露、但HIV阴性的个体缺乏抗体应答,但却显示出抗HIV-1的CD8+T细胞免疫应答(以下参考文献8和9)。 Further, in the Gambia, a set of high exposure, but HIV-negative individuals lack of antibody response, but it shows that the anti-HIV-1 CD8 + T cell immune response (Ref. 8 and 9 below). 的确,HIV-1感染后,增强的细胞免疫应答被诱导,同时伴随病毒量的下降。 Indeed, after HIV-1 infection, enhanced cellular immune response is induced, accompanied by decreased viral loads. 然而,尽管存在高水平的HIV特异性细胞毒性T淋巴细胞(CTL),但HIV-1感染仍然没有清除。 However, despite the presence of high levels of HIV-specific cytotoxic T lymphocytes (CTL), but HIV-1 infection is still not clear. 高CD8介导的应答与持续的病程之间的矛盾值得深思。 Duration of response and ongoing conflict between the high CD8-mediated worth considering. CTL不能清除病毒,可能部分因为CTL逃逸突变体(以下参考文献10-14),这是病毒可能的免疫致病机制,例如Nef相关性I类MHC的下调或Vpr或Env对宿主免疫力的影响(以下参考文献15-18)。 CTL can clear the virus, may be partly because CTL escape mutants (The following references 10-14), which is immune pathogenesis virus may, for example, affect Nef dependency class I MHC downward or Vpr or Env host immunity (The following references 15-18). 另一个问题是CD8+T淋巴细胞缺乏有效的CD4+T细胞辅助(以下参考文献19和20)。 Another problem is the lack of effective CD8 + T lymphocytes of CD4 + T cell help (The following references 19 and 20). 据观察,循环CD8+细胞可具有受损的功能(以下参考文献21)。 It was observed that circulating CD8 + cells may have impaired function (hereinafter, reference 21). 如果HIV-1免疫发病机理限制产生有效的CD8应答的话,在抗逆转录病毒疗法中提供HIV-1抗原,则能以有限的方式强化CD8记忆细胞和效应细胞。 If the immune pathogenesis of HIV-1 restriction produce effective CD8 response, then provide HIV-1 antigens in antiretroviral therapy, is able in a limited way to strengthen the CD8 memory cells and effector cells. 这些事件能对克服疾病提供潜在影响。 These events offer the potential impact on overcoming the disease. 然而,为CD8+T细胞扩增提供帮助是重要的。 However, CD8 + T cell expansion for help is important. 鉴于此,人们发现,CD8+记忆T细胞的存活,对持续的抗原呈递来说,并非偶然,(以下参考文献22),但是可能依赖于周围环境中特异性细胞因子的产生。 In view of this, it was found that, CD8 + memory T cell survival, sustained antigen presentation, it was not accidental (Reference 22 below), but may depend on the surrounding environment to produce specific cytokines.

显著影响CD8+T细胞的一个所述细胞因子是白介素-15(IL-15)。 A significant effect of the CD8 + T cell cytokine is interleukin -15 (IL-15). Waldmann及其同事首次报道,IL-15是一种15kDa蛋白,使用IL-2受体复合物的γ链和β链,所述复合物是IL-2受体与信号T细胞的复合物,其具有独特α链(以下参考文献23)。 Waldmann and colleagues first reported, IL-15 is a 15kDa protein, using the IL-2 γ-chain and β-chain receptor complex, the complex is IL-2 receptor and signals T cell complex, which has a unique α-chain (hereinafter, Reference 23). IL-15表现出抗凋亡活性,看来在刺激记忆CD8+T细胞表型中起到作用。 IL-15 exhibits anti-apoptotic activity, it appears to stimulate the memory phenotype of CD8 + T cells play a role. 许多研究小组正在对IL-15在HIV-1感染中的作用进行研究。 Many research groups are the role of IL-15 in HIV-1 infection to study. 已经证明,IL-15减少从HIV-1感染者分离的淋巴细胞的细胞凋亡(以下参考文献24)并增加天然杀伤细胞的活性和增殖(以下参考文献25-27)。 Has been shown, IL-15 reduced apoptosis isolated from HIV-1 infected lymphocytes (Ref. 24 below) and increased natural killer cell activity and proliferation (The following references 25-27). 也认为,IL-15与HIV-1感染者的B细胞增殖(以下参考文献28和29)和巨噬细胞活化(以下参考文献30)有关。 Also believes, IL-15 and HIV-1-infected B cell proliferation (following references 28 and 29), and macrophage activation (Ref. 30 below) related. 重要的是,IL-15看来也对HIV-1效应T细胞的增殖和干扰素-γ(IFN-γ)的产生具有直接作用(以下参考文献31和32)。 Importantly, IL-15 also appears on the HIV-1 effector T cell proliferation and interferon -γ (IFN-γ) is produced having a direct effect (hereinafter, references 31 and 32). 然而,IL-15在许多检验为HIV-1血清阳性的患者中却不能刺激IFN-γ。 However, IL-15 in a number of tests for HIV-1-positive serum did not stimulate IFN-γ. 因此,研究了IL-15对抗原特异性CD8+T细胞免疫应答的作用。 Therefore, the study of the IL-15 antigen-specific CD8 + T cell immune response role.

对IL-15对从慢性感染的HIV-1血清阳性患者分离的T细胞的作用进行了研究。 The role of IL-15 isolated HIV-1 seropositive patients with chronic infection from T cells were studied. 发现,rhIL-15增强了CD8T细胞的增殖,而且重要的是,IL-15在所有患者中都增加了的效应抗原特异性CD8+IFN-γ的产生。 Found, rhIL-15 enhances proliferation CD8T cells, and importantly, IL-15 in all patients have increased the effect of antigen-specific CD8 + IFN-γ production. 在免疫模型中,IL-15增强了CD8+效应细胞的功能,这是在免疫模型系统中进行的研究。 Immunization model, IL-15 enhances the function of CD8 + effector cells, which is the study of the immune system model performed. 来自小鼠的CD8+淋巴细胞能够以更高水平裂解表达HIV-1抗原的靶标,当提供IL-15时。 CD8 + lymphocytes from mice with higher expression levels of cleaved target HIV-1 antigen, while providing IL-15 时. 这一作用发生在CD4+T细胞剧烈增殖不存在的情况下。 This effect occurs in the case of CD4 + T cells in the absence of the intense proliferation. 然而,在CD4敲除(KO)小鼠中,在产生CD8效应细胞应答时,IL-15不能完全忽略CD4辅助的需要。 However, in CD4 knockout (KO) mice, in response when generating CD8 effector cells, IL-15 can not be completely ignored CD4 helper needs. 这些结果表明,IL-15在CD8记忆细胞的增殖中是高度有效的,但是单独的IL-15对其开始产生并不足够。 These results indicate that, IL-15 is highly effective in proliferating CD8 memory cells, but IL-15 alone is not sufficient to generate its start.

材料与方法人PBMC的酶联免疫斑点测定通过标准酶联免疫斑点测定,对于通过基本ficoll-hypaque技术从HIV-1阳性志愿者分离的PBMC,评价效应子功能。 Materials and Methods Human PBMC was determined by standard ELISpot ELISpot was measured for by basic ficoll-hypaque technique from HIV-1 positive volunteers separated PBMC, evaluation effector function. 将PBMC重悬于含10%FCS(R10)的RPMI中,浓度为1106细胞/ml。 PBMC were resuspended in 10% FCS (R10) in RPMI at a concentration of 1 106 cells / ml. 将抗体1-DIK(Mabtech,Mariemont OH;Nacka,SE)在0.1M碳酸盐-碳酸氢盐溶液(pH 9.6)中稀释至15μg/ml,用于包被96孔硝酸纤维素膜板(Millipore,Bedford,MA)。 Antibody 1-DIK (Mabtech, Mariemont OH; Nacka, SE) in 0.1M carbonate - bicarbonate solution was diluted (pH 9.6) medium to 15μg / ml, was used to coat 96-well nitrocellulose plate (Millipore , Bedford, MA). 膜板在4℃孵育过夜。 Membrane plates were incubated overnight at 4 ℃. 用200μlPBS洗涤膜板6次。 The membranes were washed with 200μlPBS plate 6 times. 将122种无菌肽的混合物(mixture)配制成混合物(cocktail),浓度(对于每种肽)为50μg/μl的DMSO。 The mixture was 122 kinds sterile peptide (mixture) formulated as a mixture (cocktail), concentration (for each peptide) was 50μg / μl of DMSO. 所述肽是一系列重叠肽,长度为15个氨基酸,包括所有HIV-1Gag(AIDS Reagent和ReferenceRepository,ARRR)。 The peptide is a series of overlapping peptides, 15 amino acids in length, including all HIV-1Gag (AIDS Reagent and ReferenceRepository, ARRR). 将100,000个PBMC加入到硝酸纤维素抗体包被板的每孔中(100μl@1.0106细胞/m1),再加入100μl肽混合物(稀释1∶200(溶于R10),有或没有50ng/ml IL-15,终浓度25ng/ml)。 The PBMC was added to 100,000 antibody-coated nitrocellulose plates each well (100μl@1.0106 cells / m1), then add 100μl peptide mixture (diluted 1:200 (dissolved R10), with or without 50ng / ml IL-15, a final concentration of 25ng / ml). 每个样品按一式三份进行测定。 Each sample was measured in triplicate. 用5μg/ml PHA作为阳性对照。 With 5μg / ml PHA as positive controls. 将各板在37℃孵育约24小时。 The plates were incubated at 37 ℃ for about 24 hours. 然后用200μlPBS洗涤各板6次。 The plates were then washed 6 times 200μlPBS. 将100μl抗体7-B6-1-生物素(Mabtech)加入到每孔中,浓度为1μg/ml的PBS。 The 100μl Antibody 7-B6-1- biotin (Mabtech) was added to each well at a concentration of 1μg / ml in PBS. 各板在室温下孵育2-4小时。 The plates were incubated at room temperature for 2-4 hours. 然后用200μl PBS洗涤各板6次。 Then each plate was washed with 200μl PBS six times. 将100μl链霉抗生物素-ALP(Mabtech)加入到每孔中,浓度为1μg/ml的PBS。 The 100μl streptavidin-biotin -ALP (Mabtech) was added to each well at a concentration of 1μg / ml in PBS. 各板在室温下孵育1-2小时。 The plates were incubated at room temperature for 1-2 hours. 用200μl PBS洗涤各板6次。 Each plate was washed with 200μl PBS six times. 将100μl底物溶液(BCIP/NBT,Sigma)加入到每孔中。 The 100μl substrate solution (BCIP / NBT, Sigma) was added to each well. 用自来水除去显影液。 With tap water to remove the developer. 使用或者与CD8特异性单克隆抗体偶联或者与CD4特异性单克隆抗体偶联的Dynabeads(Dynal Biotech,Lake success,NY;Oslo,NO)来耗尽CD8和CD4群体。 Or in combination with CD8-specific monoclonal antibody conjugated with a CD4-specific monoclonal antibody or conjugated Dynabeads (Dynal Biotech, Lake success, NY; Oslo, NO) to deplete CD4 and CD8 populations.

用抗CD3单克隆抗体对PBMC的共刺激在有或没有IL-15(50ng/ml)的情况下,用结合Dynabeads(DynalBiotech)的对CD3具有特异性的单克隆抗体,刺激从HIV-1血清阳性患者分离的PBMC,然后通过如上所述的酶联免疫斑点测定法,分析产生的IFN-γ。 With anti-CD3 monoclonal antibody stimulation of PBMC in the co-presence or absence of IL-15 (50ng / ml) of the case, with binding Dynabeads (DynalBiotech) of a monoclonal antibody specific for CD3 stimulated serum from HIV-1 positive patients with isolated PBMC, then by ELISPOT assay as described above, the analysis produced IFN-γ. 使用或者与CD8特异性单克隆抗体偶联或者与CD4特异性单克隆抗体偶联的Dynabeads(Dynal Biotech)来耗尽CD8和CD4群体。 Or in combination with CD8-specific monoclonal antibody conjugated with a CD4-specific monoclonal antibody or conjugated Dynabeads (Dynal Biotech) to deplete CD4 and CD8 populations.

用CD40L对PBMC的共刺激测定CD40L蛋白与IL-15和肽混合物的联用,其浓度为250μg/ml,然后通过如上所述的酶联免疫斑点测定法,分析产生的IFN-γ。 Determination of IL-15 and CD40L protein peptide mixture in combination with a concentration of 250μg / ml, as described above, then by ELISPOT assay, analysis of IFN-γ produced by the co-stimulatory CD40L on PBMC.

在小鼠中进行质粒免疫在第0周和第2周,雌性Balb/c小鼠用50μg pCgag或pCenv和50μg质粒一起进行接种,所述质粒如前所述表达IL-2R依赖性Th1细胞因子IL-15的基因(以下参考文献33)。 In plasmid immunized mice at week 0 and 2 weeks, female Balb / c mice were immunized with 50μg pCgag or pCenv and 50μg plasmid were inoculated together, the IL-2R expression plasmid as described previously dependent Th1 cytokine IL-15 gene (following Ref. 33). 也采用Cd4tmlKmv定向突变的纯合型小鼠。 Also used Cd4tmlKmv directed mutation homozygous mice. 这些小鼠因CD4基因突变而在CD4+T细胞发育中被完全阻断;其90%的循环T-细胞是CD8+。 These mice by CD4 gene mutation was completely blocked in the CD4 + T cell development; 90% of the circulating T- cells are CD8 +. 纯合突变型小鼠在辅助T细胞活性和其它T细胞应答上也显示出II类限制性缺陷。 Homozygous mutant mice on the helper T cell activity and other T-cell responses also showed class II restricted defects. 在第0周和第2周,B6.129S6-Cd4tmlKmv用50μg pCgag和50μg质粒一起进行接种,所述质粒表达CD40L、IL-15或两者的组合。 At Week 0 and Week 2, B6.129S6-Cd4tmlKmv with 50μg pCgag and 50μg plasmid were inoculated together, the plasmid expressing the combinatorial CD40L, IL-15 or both. 用Qiagen柱制备所有DNA,最终的制剂是0.25%布比卡因的等渗柠檬酸缓冲液。 All preparative column Qiagen DNA, the final formulation is 0.25% bupivacaine isotonic citrate buffer. 第二次注射后一周,切取脾脏。 Week, spleens were cut after the second injection.

鼠细胞毒性T淋巴细胞测定在5小时51Cr释放CTL测定中,使用重组痘苗病毒感染的细胞作为靶标,评价CTL应答。 Murine cytotoxic T lymphocytes was measured in five hours 51Cr release CTL assay, using recombinant vaccinia virus-infected cells as targets, evaluation of the CTL response. 在接种和体外刺激后一周,分离脾细胞。 In one week after inoculation and in vitro stimulation, splenocytes isolated. 用相关痘苗病毒感染的细胞刺激效应子。 With relevant vaccinia-infected cells stimulate effector. P815用vDK1(gag/pol)(ARRR)或vMN462(ARRR)(env)感染。 P815 infected with vDK1 (gag / pol) (ARRR) or vMN462 (ARRR) (env). 如前所述,刺激物用0.1%戊二醛固定,然后与脾细胞一起(比例为1∶20)在CTL培养基中孵育4-5天。 As mentioned earlier, stimuli with 0.1% glutaraldehyde, and then together with the spleen cells (ratio 1:20) were incubated for 4-5 days in CTL medium. CTL培养基由1∶1的Iscove改良Dulbecco Media(Gibc-BRL,Grand Island,NY)和Hanks平衡盐溶液(Gibco-BRL)组成,后者含有10%胎牛血清1640(Gibco-BRL)和10%RAT-T-STIM,不含Con A(Becton Dickinson Labware,Bedford,MA)。 CTL medium consisted of Iscove's Modified Dulbecco 1:1 Media (Gibc-BRL, Grand Island, NY) and Hanks balanced salt solution (Gibco-BRL) composition, which contains 10% fetal calf serum 1640 (Gibco-BRL) and 10 % RAT-T-STIM, without Con A (Becton Dickinson Labware, Bedford, MA). 通过在37℃感染3106P815细胞(感染复数MOI为10)达12小时,制备痘苗病毒感染的靶标。 By 37 ℃ infection 3 106P815 cells (multiplicity of infection MOI of 10) for 12 hours to prepare a vaccinia infected targets. 进行标准铬释放测定,其中靶细胞用20μCi/ml Na251CrO4标记120分钟,然后与受刺激的效应脾细胞一起在37℃孵育6小时。 Standard chromium release assay, wherein the target cells with 20μCi / ml Na251CrO4 mark 120 minutes, and then incubated with the stimulated effector splenocytes at 37 ℃ 6 hours. 在效应子∶靶标(E∶T)比例为50∶1至12.5∶1的范围内,测定CTL裂解。 In the effector: target (E:T) ratio is within the range of 50:1 to 12.5:1 was measured CTL lysis. 收获上清液,在LKB CliniGammaγ-计数器上计数。 Supernatants were harvested, counted in a LKB CliniGammaγ- counter. 按照下式求出特异性裂解的百分数: According to the following equation percentage specific lysis: 通过使靶细胞在含1%Triton X-100培养基中裂解,求出最大释放。 By containing the target cells in 1% Triton X-100 lysis medium, maximum release is obtained. 如果“自发释放”计数值超过“最大释放”的20%的话,认为测定无效。 If the "spontaneous release" counts more than "maximum release" of 20%, then that determination is invalid.

CD8+T细胞的完全裂解通过用抗CD8单克隆抗体(Pharmingen,San Diego,CA)进行处理,然后通过在37℃与兔补体(Sigma)一起保温45分钟,从脾细胞中去除CD8+T细胞(以下参考文献33)。 Complete lysis of CD8 + T cells by treatment with anti-CD8 monoclonal antibody (Pharmingen, San Diego, CA), and then by 37 ℃ with rabbit complement (Sigma) incubated 45 minutes together, CD8 + T cells were removed from splenocytes (Reference 33 below).

鼠T辅助细胞增殖测定采用淋巴细胞增殖测定,评价淋巴细胞的总免疫活性并检测对分裂细胞具有特异性的抗原。 Murine T helper cell proliferation was measured by lymphocyte proliferation assay, a total evaluation of immunocompetent lymphocytes and detection of mitotic cells having an antigen specificity. 按照以下参考文献34所述,从脾脏收获淋巴细胞,去除红细胞,然后用新鲜培养基洗涤几次,以制备淋巴细胞。 34 according to the following references, harvested from spleen lymphocytes, red blood cell removal, and then washed several times with fresh medium, to prepare lymphocytes. 分离的细胞以5106细胞/ml的浓度重悬浮。 Isolated cells at a concentration of 5 106 cells / ml were resuspended. 将含5105细胞的100μl等分试样立即加入到96孔平底微量滴定板的每孔中。 Containing 5 105 cells in 100μl aliquot was added immediately to each well of a 96-well flat bottom microtiter plate. 将重组p24蛋白加入到各孔中,一式三份,得到终浓度5μg/ml和1μg/ml。 The recombinant p24 protein was added to each well, in triplicate, to give a final concentration of 5μg / ml and 1μg / ml. 将细胞于37℃/5%CO2孵育3天。 The cells were at 37 ℃ / 5% CO2 incubated for 3 days. 将1μCi的氚化胸苷加入到各孔中,然后将细胞在37℃孵育12-18小时。 The 1μCi of tritiated thymidine was added to each well, and the cells were incubated at 37 ℃ 12-18 hours. 收获各板中的细胞,在Beta读板仪(Wallac,Turku,Finland)上测定所掺入的氚化胸苷的数量。 Harvest the cells in each plate, the number of the incorporated tritiated thymidine was measured in Beta plate reader (Wallac, Turku, Finland) on. 按下式求出刺激指数:刺激指数(SI)=(实验计数/自发计数)自发计数孔中包含10%胎牛血清,起到无关蛋白对照作用。 Stimulation index calculated by the following formula: stimulation index (SI) = (experimental counts / spontaneous count) spontaneous counting holes contained 10% fetal bovine serum, played the role of unrelated protein control. 同样,来自pCgag或对照免疫小鼠的脾细胞通常针对其无关蛋白靶标来说SI为1。 Similarly, spleen cells from immunized or control mice pCgag generally unrelated protein target for which it is an SI. 为了确保细胞健康,采用PHA或Con A(Sigma)作为多克隆刺激物阳性对照。 To ensure the healthy cells, the use of PHA or Con A (Sigma) as a positive control polyclonal stimulus.

受刺激鼠细胞的细胞因子和趋化因子分析从脾脏收获淋巴细胞,将分离的细胞重悬浮,浓度为5106细胞/ml。 Stimulation of murine cells by cytokines and chemokines analysis harvested lymphocytes from the spleen, the isolated cells were resuspended, at a concentration of 5 106 cells / ml. 将含5105细胞的100μl等分试样加入到96孔平底微量滴定板的各孔中。 Containing 5 105 cells in 100μl aliquots were added to each well of a 96-well flat-bottom microtiter plate. 将重组p24蛋白或包膜蛋白加入到各孔中,一式三份,得到终浓度5μg/ml和1μg/ml。 The recombinant p24 protein or envelope protein was added to each well, in triplicate, to give a final concentration of 5μg / ml and 1μg / ml. 将细胞于37℃/5%CO2孵育3天,收获上清液。 The cells were at 37 ℃ / 5% CO2 incubation for 3 days the supernatant was harvested. 用市售ELISA试剂盒测定细胞因子和趋化因子。 Cytokines measured using a commercially available ELISA kit and chemokines.

受刺激鼠细胞的干扰素-γ的胞内染色小鼠接受两次注射:pCgag DNA或者pCgag DNA质粒加上pIL-15。 Intracellular staining of mouse interferon-stimulated mouse cells receiving two injections -γ: pCgag DNA or pCgag DNA plasmid plus pIL-15. 一周后,收获脾细胞,在含有p55肽合并液(含122种15聚体,其跨越HIV-1 p55,具有11个氨基酸的重叠)和布雷菲德菌素A的培养基中体外培养5小时。 One week later, spleen cells harvested, containing p55 peptide pools (containing 122 kinds of 15-mers, which spans HIV-1 p55, with overlapping 11 amino acids) and brefeldin A medium in vitro 5 hours . 刺激后,细胞用抗小鼠CD3抗体和抗小鼠CD8抗体进行胞外染色,然后用抗小鼠IFN-γ进行胞内染色。 After stimulation, cells were treated with anti-mouse CD3 antibody and anti-mouse CD8 antibody was used for extracellular staining, and then with anti-mouse IFN-γ intracellular staining performed. 描点曲线显示CD3+/CD8+淋巴细胞的应答。 Plot the curve shows the CD3 + / CD8 + lymphocyte responses.

表位作图将脾细胞重悬于含10%FCS(R10)的RPMI中,浓度为1106细胞/ml。 Epitope mapping spleen cells resuspended in 10% FCS (R10) in RPMI at a concentration of 1 106 cells / ml. 将得自AIDS Reference和Reagent Repository的系列122种肽,混合成为每种合并液中含10种肽的混合物,终浓度为20μg/ml/肽。 Obtained from the AIDS Reference and Reagent Repository series of 122 kinds of peptides, each pool was to provide a mixture containing 10 kinds of peptides, the final concentration of 20μg / ml / peptide. 每种肽都包含在两个不同的合并液中,总共有22种肽合并液。 Each peptide is contained in two different combiner solution, total of 22 peptides were combined. 以矩阵形式排列各合并液,用于脾细胞刺激。 Arranged in a matrix of each pool was used to stimulate spleen cells. IFN-γ的产生通过酶联免疫斑点测定法(R和D系统)评价。 IFN-γ is produced by ELISPOT assay (R and D systems) was evaluated. 将各板于37℃孵育约24小时。 The plates were incubated at 37 ℃ for about 24 hours. 每个样品按一式三份进行测定。 Each sample was measured in triplicate.

结果用CD3和IL-15对淋巴细胞进行刺激评价IL-15以协同方式,与T细胞受体刺激一起,增强T细胞效应子活化的能力。 The results with CD3 and IL-15 stimulation on lymphocyte evaluate IL-15 in a synergistic manner, together with the T cell receptor stimulation, enhance the ability of activated effector T cells. PBMC是从HIV-1感染者中分离的。 PBMC are isolated from HIV-1 infected patients. PBMC用抗CD3表面结合抗体进行刺激,然后与IL-15一起孵育过夜。 PBMC were stimulated with a combination of anti-CD3 antibody surface, and then incubated overnight with IL-15. 不出所料,CD3单独刺激PBMC,能诱导IFN-γ的产生,而单独补充IL-15却只能诱导低应答至无应答。 Unsurprisingly, CD3 stimulation alone PBMC, can induce IFN-γ production, while a separate supplement to induce IL-15, but only a low response to no response. 然而,当淋巴细胞用CD3和IL-15一起刺激时,观察到细胞分泌IFN-γ的数量增加好几倍(图1)。 However, when stimulating lymphocytes together with CD3 and IL-15, IFN-γ secreting cells was observed to increase in the number of several times (Fig. 1). 受刺激群体耗尽CD4+或CD8+T细胞,然后补充IL-15,再次检测活性。 Stimulated CD4 + population depleted or CD8 + T cells, and then added IL-15, detecting the activity again. CD8细胞的损失再次耗尽活化信号。 CD8 cell depleted loss again activating signal. 数据表明,来自HIV-1慢性感染者的CD8+效应T细胞,可以因IL-15/CD3刺激而扩增(图2)。 Data show that, CD8 + effector T cells from HIV-1 chronic infection can be due to IL-15 / CD3 stimulation and amplification (Fig. 2).

HIV-1阳性样品经IL-15刺激后产生抗原特异性IFN-γ对IL-15增强HIV-1抗原特异性CD8+应答的能力,进行体外评价。 HIV-1 antigen-specific IFN-γ-positive samples by IL-15 stimulation of IL-15 to enhance HIV-1 antigen-specific CD8 + response ability, in vitro evaluation. 从接受抗逆转录病毒联合治疗(HAART)的HIV-1慢性感染者中,采集样品。 From receiving antiretroviral combination therapy (HAART) in HIV-1-infected patients with chronic, collect samples. 在用HIV-1特异性肽刺激后,在IL-15存在或不存在的情况下,评价PBMC分泌IFN-γ的能力。 After using the HIV-1 specific peptide stimulation, in the case of IL-15 the presence or absence of, evaluating the ability of PBMC secrete IFN-γ. PBMC用包含HIV-1gag蛋白完整可读框的重叠HIV-1 15个氨基酸肽进行刺激。 PBMC with HIV-1gag protein containing the complete open reading frame of overlapping HIV-1 15 amino acid peptide stimulation. 来自接受肽刺激的患者的PBMC,当用IL-15处理后,表现出IFN-γ产生增加(图3A和图3B),IFN-γ产生在有无IL-15的情况下具有显著性差异(p=009),(图3C)。 PBMC from patients undergoing stimulation of peptide, when treated with IL-15, showed increased production of IFN-γ (Fig. 3A and Fig. 3B), IFN-γ produced in the presence or absence of IL-15 has a significant difference ( p = 009), (Fig. 3C). 一些患者单用IL-15刺激,就会高水平分泌INF-γ(图3A),表明它们具有部分T细胞活化,所述活化被阻断并需要补充细胞因子才有效。 Some patients with IL-15 stimulation alone, will secrete high levels of INF-γ (Fig. 3A), indicating that they have a portion T cell activation, the activation is blocked and need to be supplemented cytokines valid. 这一活性清楚表明,当CD8细胞群体被耗尽时,CD8在介导IFN-γ的产生中的活性已经丧失(图3D)。 This activity clearly shows that when the CD8 cell populations were depleted, CD8-mediated IFN-γ in the generation of the active has been lost (Fig. 3D).

IL-15在小鼠体内增加CD8+CTL应答以上对HIV-1应答和IL-15的研究,确定IL-15在初次刺激的T细胞群体中能增加IFN-γ的产生。 IL-15 increased CD8 + CTL responses than research on HIV-1 and IL-15 response in mice, to determine IL-15 stimulated T cells in the initial population can increase IFN-γ production. 然而,尚不清楚,IL-15对CD8+T细胞的体内功能诱导具有什么样的效应。 However, it is unclear, IL-15 对 CD8 + T cells induced in vivo function of what kind of effect has. 为了解决该问题,使用小鼠模型系统。 To solve this problem, using a mouse model system. 给小鼠接种HIV-1质粒,作为传递HIV-1抗原并研究CD8免疫在体内诱导的措施。 Mice were inoculated with HIV-1 plasmid, as a transfer HIV-1 antigen and studies in vivo induction of CD8 immune measures. HIV-1表达质粒是与或者表达IL-15的质粒、或者对照质粒一起注射的,然后比较所得免疫应答。 HIV-1 expression plasmid is an expression plasmid with or IL-15, or injected with a control plasmid, and then compares the resulting immune response. 在大量CTL测定中,与表达HIV-1包膜和IL-15的质粒一起注射,导致约40%表达HIV-1包膜的靶标裂解,其比例为50∶1效应子∶靶标,相比之下,用包膜质粒和对照载体则观察到11%裂解(图4A)。 In a large number of CTL assay, the HIV-1 envelope expressing plasmid and injection of IL-15 together, resulting in about 40% of HIV-1 envelope expressing target lysis in a ratio of 50:1 effector: target, compared to the By using envelope plasmid and the control vector to 11% lysis was observed (Figure 4A). 这些结果是CD8T细胞依赖性的,表明IL-15对效应T细胞应答的显著影响。 These results are CD8T cell-dependent, suggesting that IL-15 significantly affect the effector T cell response.

小鼠在抗原刺激后IL-15诱导MIP-1β和IFN-g的分泌疫苗诱导的细胞免疫应答可进一步扩增,即通过检查β-趋化因子MIP-1β作为免疫活化标记的表达分布型。 Mice after antigen stimulation IL-15-induced MIP-1β and IFN-g secretion vaccine-induced cellular immune response can be further amplified, i.e., by checking β- chemotactic factor expression profile of MIP-1β as the immune activation markers. 趋化因子是免疫和炎症反应的重要调节剂。 Chemokines are important immune and inflammatory response modifiers. 它们在白细胞从血管进入宿主防御的外围部位的分子调控中尤为重要。 They are particularly important in the molecular regulation of white blood cells from blood vessels into the peripheral part of the host's defense. 此外,先前已经报道过,T细胞产生的趋化因子包括MIP-1β,在细胞免疫增殖中起到关键作用(以下参考文献24)。 In addition, has been previously reported, T cells produce chemokines including MIP-1β, play a key role in the proliferation of immune cells (Reference 24 below). 因此,受刺激T细胞所产生的趋化因子的水平,可提供抗原特异性细胞免疫应答的量和质方面的补充知识。 Thus, by the level of chemokine stimulation of T cells generated, may provide additional knowledge of the quantity and quality of the antigen-specific cellular immune response. 对受刺激T细胞的上清液(如材料与方法小节所述)的MIP-1β释放进行分析和检测。 The supernatant (as described in Section Materials and Methods) stimulated T cells, release of MIP-1β were analyzed and detected. 用IL-15共同免疫,结果导致高水平分泌MIP-1β(图4B)。 Co-immunization with IL-15, resulting in high levels of secretion of MIP-1β (FIG. 4B).

也对上清液中产生的Th1细胞因子IFN-γ进行评价。 Also produced in the supernatant of the Th1 cytokine IFN-γ was evaluated. 在细胞用于CTL测定之前,在用表达HIV-1包膜的重组痘苗病毒所感染的效应细胞进行为期3天的淋巴细胞刺激之后,得到样品。 Before measuring cell for CTL after recombinant vaccinia virus expressing HIV-1 envelope infected effector cells for a period of three days of lymphocyte stimulation, to obtain a sample. 图4C表明,与单独注射质粒疫苗或对照的小鼠相比,共注射IL-15的小鼠的脾细胞诱导产生更高水平的IFN-γ(120pg/ml)。 Figure 4C shows that, compared with the separate injection of a plasmid vaccine or control mice were injected spleen cells of IL-15 in mice induced higher levels of IFN-γ (120pg / ml). 相比之下,在这些研究中没有观察到任何培养物明显产生的IL-4(数据未显示)。 In contrast, in these studies did not observe any significant cultures produced IL-4 (data not shown).

IFN-γ和TNF-α的胞内染色为了定量研究对HIV-1疫苗的T细胞应答,进行胞内细胞因子染色测定。 Intracellular staining of IFN-γ and TNF-α in order to quantitatively study of HIV-1 vaccine T cell responses, were intracellular cytokine staining was measured. 处死免疫动物,收获脾细胞,在含p55混合物和布雷菲德菌素A的培养基中体外培养5小时。 Immunized animals were sacrificed, spleen cells harvested, in a medium containing a mixture of p55 and Brefeldin A in the in vitro cultured for 5 hours. 通过流式细胞术,测定CD8+CD3+T细胞的IFN-γ或TNF-α的产生(图5A和图5B)。 By flow cytometry, measuring CD8 + CD3 + T cells to produce IFN-γ or TNF-α (Fig. 5A and 5B). 共同接种IL-15的动物表现出高CD8效应T细胞应答,2.6%的CD8+T细胞产生IFN-γ,3.7%的产生TNF-α。 IL-15 co-vaccinated animals exhibited high CD8 effector T cell response, 2.6% of CD8 + T cells to produce IFN-γ, 3.7% of the produced TNF-α. 这些数据表明,IL-15对功能性CD8+T细胞应答显示出巨大影响。 These data indicate that, IL-15 in functional CD8 + T cell responses showed great impact.

用IL-15和HIV-1疫苗共同免疫的鼠脾细胞中淋巴细胞增殖T辅助淋巴细胞的活化和增殖对体液免疫和细胞免疫扩增来说,是至关重要的。 With IL-15 and HIV-1 vaccine together immunized mice splenocytes proliferation of T helper lymphocytes lymphocyte activation and proliferation of both humoral and cellular immune amplification, is essential. 在基础淋巴细胞增殖测定中,评价了来自免疫小鼠的脾细胞在响应重组HIV-1抗原刺激时的增殖能力。 On the basis of lymphocyte proliferation assay was evaluated from immunized mice spleen cells proliferation in response to recombinant HIV-1 antigen stimulation. 看来IL-15对增殖应答没有重大影响(图6)。 IL-15 appears no significant effect on the proliferative response (Figure 6). 然而,IL-2用作对照,清楚地看到,在共注射IL-2质粒的小鼠中,对于gp120env蛋白的脾细胞增殖显著增加。 However, IL-2 used as a control, clearly seen, in the IL-2 plasmid was co-injected mice, a significant increase in proliferation of spleen cells for gp120env protein. 比起用对照、pCgag单用或pCEnv+IL-15联用免疫的小鼠来说,共注射IL-2的小鼠脾细胞,导致刺激指数至少高出3倍(图6)。 Compared with the control, pCgag alone or pCEnv + IL-15 combined with immunized mice, the spleen cells of mice co-injected IL-2, leading to the stimulation index of at least 3-fold higher (Fig. 6). 该数据进一步证明,在没有T细胞辅助剧烈扩增时,IL-15看来也能增加CD8T细胞功能。 The data further demonstrated that in the absence of T cell help intense amplification, IL-15 appears to also increase CD8T cell function. 这也证明,IL-15所引起的扩增并不依赖于IL-2。 This also proves the amplification caused by IL-15 does not depend on IL-2. 这表明,在这种情况下,CD4及CD8效应子功能的扩大。 This suggests that, in this case, the expansion of CD4 and CD8 effector function.

表位作图IL-15处理对CD8+T细胞应答的增强作用,是否是因为所响应的表位数量的增加(即:表位延伸),还是因为对同一表位具有特异性的CD8+T细胞数量的总体增加? Epitope mapping of the IL-15 treatment on enhancement of CD8 + T cell responses, whether it is because the table in response to increase in the number of bits (i.e.: epitope extension), or because of having the same epitope specificity of the CD8 + T The overall increase in the number of cells? 为了解决这些问题,使用酶联免疫斑点测定和一系列得自AIDS Reference和Reagent Repository的肽(按矩阵形式混合成合并液),鉴定出两个表位。 To solve these problems, the use of ELISPOT assay and a series of peptides (mixed into a matrix form combined liquid) were obtained from the AIDS Reference and Reagent Repository, identified two epitopes. 将优势表位作图到Gag氨基酸197-211(AMQMLKETMEEAAE-SEQ ID NO:1)(图7)。 Plotting Gag epitope to amino acids 197-211 (AMQMLKETMEEAAE-SEQ ID NO: 1) (Fig. 7). Paterson等人先前在用重组单核细胞增生李斯特氏菌(L.monocytogenes)HIV-1疫苗进行免疫后,已经确定AMQMLKETI-SEQID NO:2(以下参考文献35)为优势CD8表位。 After Paterson, who was previously with recombinant Listeria monocytogenes bacteria (L.monocytogenes) HIV-1 vaccine for immunization, have been identified AMQMLKETI-SEQID NO: 2 (Ref. 35 below) was the dominant CD8 epitopes. 进而确定了亚优势表位即Gag氨基酸293-307(FRDVDRFYKTRAE-SEQ ID NO:3)(图7)。 And to determine the sub-dominant epitopes that Gag amino acids 293-307 (FRDVDRFYKTRAE-SEQ ID NO: 3) (Fig. 7). 在仅用Gag免疫的小组中,观察到所响应表位的数量没有增加。 In Gag immunized only group, the number of responses was observed no increase in the epitope. 然而,IL-15极大地增加了对这些表位应答的数量级。 However, IL-15 significantly increases the response to these epitopes magnitude. 仅在用IL-15共同免疫的动物中是亚优势表位的清楚证据。 Only together with IL-15 immunized animals are subdominant epitope clear evidence. IL-15影响CD8效应细胞的扩增。 Effect of IL-15 was amplified CD8 effector cells.

CD4敲除小鼠我们观察到,在来自HIV-1感染者的PBMC中,IL-15使抗原特异性CD8T细胞扩增。 CD4 knockout mice we observed in PBMC from HIV-1 infected persons in, IL-15 antigen-specific CD8T cell expansion. 我们也观察到,在我们的疫苗模型中,显著的CD8效应细胞诱导不依赖于CD4扩增。 We also observed that, in our model, a vaccine, a significant induction of CD8 cells is not dependent on CD4 amplification effect. 因此,CD4辅助T细胞对IL-15免疫扩增的贡献引起疑问。 Therefore, CD4 helper T cells to IL-15 immune amplification contribution of doubt. 为了解决这一问题,对IL-15在完全没有CD4细胞的情况下诱导CD8效应群体的能力,进行了调查。 To solve this problem, for IL-15 in the case where there is no induction of CD4 CD8 cell populations capacity effect, were investigated. 对Cd4tmlKmv定向突变的纯合型小鼠(以下参考文献36)进行免疫。 Directed mutagenesis of Cd4tmlKmv homozygous mice (Ref. 36 below) were immunized. 这些小鼠在CD4+T细胞发育中被阻断,因此大多数循环淋巴细胞是CD8细胞。 These mice are blocked in CD4 + T cell development, most circulating lymphocytes are CD8 cells. 利用质粒共同免疫模型(其中在正常小鼠中每一百万个脾细胞中平均约有200个IFN-γ产生细胞被诱导),在绝对没有CD4细胞的情况下,IL-15不能拯救诱导的CD8效应子功能(图8B)。 Common immune model using plasmid (which in normal mice per million spleen cells in an average of about 200 IFN-γ-producing cells was induced), in the case where absolutely no CD4 cells, IL-15 does not rescue induced CD8 effector function (Fig. 8B). 因为看来IL-15的作用不涉及CD4扩增(图6),所以该缺陷理所当然是因为缺乏T辅助细胞所提供的另一功能,CD4T辅助细胞也提供对CD8扩增的帮助,即通过抗原呈递细胞(APC)的活化。 Because the role of IL-15 seems not to involve CD4 amplification (Fig. 6), so that the defect is a matter of course because of the lack of another function provided by T helper cells, CD4T helper cells also provide help for CD8 amplification, i.e., by an antigen presenting cells (APC) activation. 在这一APC活化模型中,APC上的CD40与T细胞CD40配体的连接,上调B7的表达,使T细胞活化。 APC activation in this model, CD40 APC on connection with T cell CD40 ligand B7 upregulated expression of T cell activation. 在I类MHC肽呈递的情况下,B7分子对CD8T细胞扩增提供共刺激。 In the case of class I MHC presentation of peptides, B7 molecules provide costimulatory CD8T cell expansion. 而且,Bourgeois等(以下参考文献37)证明,CD40L能直接影响CD8记忆细胞的发育。 Moreover, Bourgeois et al. (Ref. 37 below) prove, CD40L can directly affect the development of CD8 memory cells.

在CD4辅助方面的缺陷表现为缺乏共刺激水平,这一点是下一项要考虑和研究的。 Defects in CD4 helper's performance is the lack of co-stimulatory level, it is next to consider and research. 为了检验该假说,小鼠用含有IL-15和CD40L以及pCgag的质粒进行共免疫。 To test this hypothesis, mice were treated with IL-15 and CD40L containing pCgag plasmids and co-immunoprecipitation. 使用锚定的CD40L分子。 Use anchored CD40L molecule. 锚定的CD40L是局部表达的并在转运的免疫细胞,但是不能分泌,这使实验变得更复杂(以下参考文献38)。 The anchor is a partial expression of CD40L and transport immune cells, but not secreted, which makes more complicated experiments (Reference 38 below). 这样的接种可在质粒模型中提供共刺激(以下参考文献38)。 Such a vaccination can provide costimulatory (Reference 38 below) in plasmid model. 的确,当研究pCgag与pCD40L联合使用时,在CD40KO小鼠中,诱导Gag特异性CD8免疫应答(图8)。 Indeed, when the study pCgag when used in combination with pCD40L, in CD40KO mice, induced Gag-specific CD8 immune response (Fig. 8). 该数据进一步表明,IL-15直接影响记忆CD8淋巴细胞。 The data further indicate, IL-15 directly affects memory CD8 lymphocytes. 当CD4细胞不存在时,IL-15从稚细胞不能诱导抗原特异性CD8细胞应答。 When the absence of CD4 cells, IL-15 from juvenile cells can not induce antigen-specific CD8 cell responses.

讨论HIV-1特异性CD8免疫应答的维持和增强一直是许多研究的来源。 Discuss HIV-1-specific CD8 sustain immune responses and enhancement have been a source of many studies. 近期研究已经报道,IL-15在支持记忆细胞存活中起到重要作用。 Recent studies have reported, IL-15 plays an important role in supporting the memory cell survival. 观察到在小鼠模型中,IL-15的存在,可导致记忆细胞分裂(以下参考文献39)。 Observed in the mouse model, IL-15 exists, can cause memory cell division (hereinafter ref. 39). 离体功能分析以及用遗传上缺乏IL12、IL-15或其特异性受体的转基因小鼠进行的研究,在表征IL-15所起的作用中是重要的。 In vitro studies using functional analysis and the lack of IL12, IL-15, or a specific receptor transgenic mice genetically carried out, is important in the characterization of IL-15 in the role. 的确,Zhang及其合作者(以下参考文献39)证明,在体内小鼠模型中,IL-15对记忆表型、CD44hi CD8+T细胞提供有效而有区别的刺激。 Indeed, Zhang and coworkers (ref. 39 below) demonstrated that in vivo mouse model, IL-15 on memory phenotype, CD44hi CD8 + T cells provide an efficient and differentiated stimulation. 另外,Ku等(以下参考文献40)报道,记忆CD8+T细胞的分裂是由IL-15刺激的,但是被IL-2抑制。 In addition, Ku et al (Reference 40 below) reported that memory CD8 + T cell division is stimulated by IL-15, IL-2 but was suppressed. 也发现,IL-2抑制CD8+记忆T细胞的增殖。 Also found, IL-2 inhibited the proliferation of CD8 + memory T cells.

本文所公开的研究工作证明,IL-15对体外和体内诱导CD8+效应T细胞也特别有效。 Research work disclosed herein proof, IL-15 in vitro and in vivo induction of CD8 + effector T cells are also particularly effective. 当与肽和IL-15一起孵育时,从HIV-1感染者分离的CD8+T细胞能以抗原特异性方式分泌IFN-γ。 When incubated with peptide and IL-15, from HIV-1 infected individuals isolated CD8 + T cells capable of secreting antigen specific manner IFN-γ. IL-15与TCR协同作用,刺激淋巴细胞产生IFN-γ并确保效应子表型。 IL-15 and TCR synergistically stimulate lymphocytes to produce IFN-γ and ensure effector phenotype. 在某些患者中,在抗原不存在的情况下,IL-15导致IFN-γ的产生。 In some patients, in the case of absence of antigen, IL-15 led to IFN-γ production. 这表明,在HIV感染中,某些细胞被部分激活,而这类部分激活状态可被IL-15拯救。 This indicates that, in HIV infection, certain cells are activated part, and such part of active IL-15 can be saved. 然而,重要的是,在所有患者中,当用IL-15和HIV-1抗原共同刺激PBMC时,效应子功能显著增加。 However, it is important that in all patients, IL-15 when using HIV-1 antigen and costimulatory PBMC, a significant increase in effector function.

目前,von Adrian及其同事(以下参考文献41)提出,对淋巴细胞的IL-15刺激能导致CD8+T细胞直接从稚细胞加工成记忆细胞表型。 Currently, von Adrian and colleagues (Ref. 41 hereinafter) made of lymphocyte IL-15 stimulation can lead to CD8 + T cells directly processed into memory cells from juvenile cell phenotype. 然而,本文的数据表明,TCR的结合可导致CD8+T淋巴细胞更完全活化,表明IL-15单用对稚细胞的作用是最小的。 However, the data herein indicate that binding of the TCR CD8 + T lymphocytes can lead to a more fully activated, indicating that the role of IL-15 alone to juvenile cells is minimal. 另外,也表明,IL-15导致非功能性记忆细胞(以下参考文献41)。 Further, also shows, IL-15 leads to non-functional memory cells (Reference 41 below). 该数据证明,在人以及小鼠研究中,IL-15扩增都能导致完全功能性CD8+T细胞。 The data demonstrate that, in the human and mouse studies, IL-15 can result in a complete functional amplify CD8 + T cells. 在小鼠中,IL-15显著增加CD8+T细胞应答以及增加β-趋化因子和IFN-γ应答,这清楚表明抗原特异性扩增并建立在先前的工作上(以下参考文献33和42)。 In mice, IL-15 significantly increased CD8 + T cell responses and increasing β- chemokine and IFN-γ responses, which clearly indicates that antigen-specific amplification and build on previous work (Ref. 33 and 42 below ). 在CD4+T细胞扩增不存在的情况下,观察到CD8+T细胞功能的扩增。 In the case of CD4 + T cell expansion does not exist, CD8 + T cells was observed in the amplification function. 然而,对于CD4T细胞在发生CD8应答方面具有重要作用。 However, CD4T cells play an important role in the occurrence of CD8 response. 在CD4敲除小鼠的研究中,通过使用CD40L,可以遏止对CD4T细胞的需要。 In CD4 knockout mice, by using CD40L, the need to curb CD4T cells. 这一发现对病毒感染的免疫治疗极其重要。 This finding is extremely important to viral infection immunotherapy.

许多免疫治疗策略都集中于扩增CD8+T细胞应答。 Many immunotherapeutic strategies have focused on the amplification of CD8 + T cell responses. HIV-1感染通过病毒诱导的免疫抑制,使免疫治疗变得更为复杂,所述免疫抑制能导致缺乏有效的CD4+T细胞辅助。 HIV-1 infection by virus-induced immunosuppression, immune therapy becomes more complex, the immunosuppression can lead to a lack of effective CD4 + T cell help. 反过来说,认为缺乏这样的辅助,造成无效CD8+T细胞应答。 On the other hand, that the lack of such a secondary, leading to inefficient CD8 + T cell responses. 在通常的慢性感染中需要CD4+的辅助,以维持对病毒复制的控制,这可能是HIV-1感染的实例。 In a typical chronic infections need of CD4 + helper, in order to maintain control of viral replication, which could be an instance HIV-1 infection. Serbina等(以下参考文献43)证明,CD8+细胞毒性T细胞的发展依赖于CD4+T细胞。 Serbina etc. (Reference 43 below) demonstrated that, CD8 + cytotoxic T cell development is dependent on CD4 + T cells. 他们还观察到,在CD4T细胞敲除小鼠中具有降低的IL-15产生。 They also observed that, CD4T cell knockout mice have reduced IL-15 produced in. 然而,IL-15并不由CD4+T细胞产生。 However, IL-15 is not produced by CD4 + T cells. 它主要由基质细胞、单核细胞和巨噬细胞产生。 It mainly consists of stromal cells, monocytes and macrophages. 可能有CD4+T细胞增加IL-15产生的某些反馈机制,而在降低CD4辅助的情况下,最终CD8+T细胞功能也下降。 There may be some increase in CD4 + T cell IL-15 produces a feedback mechanism, and in the case of a reduced CD4 helper, CD8 + T cell function ultimately decreases. 该反馈机制可解释,为什么6个患者中有3个IFN-γ产生是在单独添加IL-15之后。 The feedback mechanism could explain why six patients in the three IFN-γ produced was added separately after IL-15. 因此,在CD4不存在且IL-15处于较低水平的情况下,在HIV-1血清阳性患者中,残留病毒仅可部分激活CD8+T淋巴细胞。 Thus, in the absence of CD4 and IL-15 in the case of low level, in HIV-1 seropositive patients, residual virus is only partially activated CD8 + T lymphocytes. 重要的是,由此看来,可以添加IL-15,以更改由病毒免疫抑制所引起的缺陷。 Importantly, this, can be added IL-15, to change the defect virus by the immune suppression caused. 应该在免疫治疗HIV-1的领域中,考虑该假说的意义。 Should be in the field of immunotherapy of HIV-1 in consideration of the significance of this hypothesis.

总的来说,IL-15在小鼠中增强CD8+T细胞效应子功能并增强从HIV-1感染阳性患者分离的CD8+T细胞的功能。 In general, IL-15 enhanced CD8 + T cell effector function in mice and enhances T cell function isolated positive HIV-1 infected patients from the CD8 +. 应该考虑,将IL-15作为主动免疫疗法的补充,用作HAART的辅助治疗。 Should be considered, as a supplement to the IL-15 active immunotherapy, is used as adjuvant therapy of HAART.

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实施例2需要CD4(+)Th细胞并产生IFN-γ,以在免疫抑制个体体内以及抗肿瘤免疫中控制病毒复制。 Example 2 requires CD4 (+) Th cells and produce IFN-γ, to the immunosuppressed individual and the anti-tumor immunity in vivo in the control of viral replication. 来自实验的数据证明,对于T细胞辅助以扩增CD8效应T细胞的需要,可以被疫苗部位局部产生IL-15和CD40L来替代。 Data from experiments demonstrate for T cell help needed to amplify CD8 effector T cells, the vaccine can be parts of the local generating IL-15 and CD40L instead. 用通过基因敲除II类MHC的β-链(MHC II KO)而消除CD4(+)T细胞的小鼠进行实验,揭示出用抗原gag+IL-15+CD40L初次免疫所述动物,导致CD8T细胞的活化。 By using the knockout of β- chain MHC class II (MHC II KO) eliminate CD4 (+) T cells in mice experiment, revealed with an antigen gag IL-15 + CD40L + primary immunization of the animal, resulting in CD8T cell activation. 用IFN-γ的产生(以斑点表示)测定活化。 With IFN-γ production (indicated by dots) measured activation. 在该测定中多于50点的为阳性。 More than 50 points were positive in this assay. 这些数据见图9,说明了用于测定不依赖CD4(+)T细胞辅助的效应CD8T细胞的活化的简单方法。 The data shown in Figure 9, illustrates a simple method of T cell helper effector CD8T cell activation does not depend for measuring CD4 (+). 这些研究对于无免疫应答个体的治疗来说,具有重要意义。 These studies for the treatment of immunocompromised individuals, it has great significance.

实施例3人、小鼠和猿猴IL-15cDNA均编码162个氨基酸(aa)残基的前体蛋白,含有48个aa残基的前导序列,将其切除后,得到114个aa残基的成熟IL-15。 3 embodiment, mouse and simian IL-15cDNA are encoded 162 amino acid (aa) residue precursor protein, containing 48 aa residues leader sequence, which after removal, to give 114 aa residues of mature IL-15. 人IL-15与猿猴和小鼠IL-15分别共享约97%和73%的序列同一性。 Human and simian IL-15 and IL-15 mice were shared about 97% and 73% sequence identity. 人IL-15和猿猴IL-15对小鼠细胞都有活性。 Simian human IL-15 and IL-15 on mouse cells have activity. 尽管IL-15的结构尚未确定,但是预测其类似于IL-2和四螺旋束细胞因子家族的其它成员。 While the structure of IL-15 has not been determined, but it is similar to the prediction IL-2 and other members of the four-helix bundle cytokine family. (Grabstein,K.等(1994)Science 264:965,Anderson,DM等(1995)Genomics 25:701;和Bamford,RN等(1995)Cytokine 7:595,Brandhuber,BJ等(1987)Science 238:1707,所述文献都通过引用结合到本文中。)IL-15mRNA在心脏、肺、肝脏、胎盘、骨骼肌、粘附外周血单核细胞、APC(树突细胞)和上皮细胞系以及成纤维细胞系中都可检出。 (Grabstein, K, etc. (1994) Science 264: 965, Anderson, DM, etc. (1995) Genomics 25:. 701; and Bamford, RN, etc. (1995) Cytokine 7: 595, Brandhuber, BJ, etc. (1987) Science 238: 1707 , the literature are incorporated by reference herein.) IL-15mRNA in heart, lung, liver, placenta, skeletal muscle, peripheral blood mononuclear cells adhered, APC (dendritic cells) and epithelial and fibroblast cell lines lines can be detected. 然而,IL-15mRNA在含有高水平IL-2mRNA的活化外周血T细胞中没能检出。 However, IL-15mRNA in activated peripheral blood T cells contain high levels of IL-2mRNA could not be detected. IL-15已经显示出刺激天然杀伤细胞、活化外周血T淋巴细胞、肿瘤浸润淋巴细胞(TIL)和B细胞的生长。 IL-15 has been shown to stimulate natural killer cells, activated peripheral blood T lymphocytes, tumor infiltrating lymphocytes (TIL) and B cell growth. 另外,IL-15也已经显示出是人血T淋巴细胞的趋化因子,在NK细胞中能诱导淋巴因子活化杀伤(LAK)活性,并能诱导溶细胞性效应细胞的产生。 In addition, IL-15 has also been shown to be human blood T lymphocytes chemokines, in NK cells induces lymphokine-activated killer (LAK) activity and induce cytolytic effector cells generated. (Armitage,RJ等(1995)J.Immunol.154:483;P.Wilkinson和F.Liew(1995)J.Exp.Med.181:1255;Grabstein,K.等(1994)Science 264:965;Giri,JG等(1994)EMBO J.13:2822;和Giri,JG等(1995)EMBO J.15:3654,每篇文献都通过引用结合到本文中)。 (Armitage, RJ, etc. (1995) J.Immunol.154: 483; P.Wilkinson and F.Liew (1995) J.Exp.Med.181: 1255; Grabstein, K, etc. (1994) Science 264: 965; Giri. , JG, etc. (1994) EMBO J.13: 2822; and Giri, JG, etc. (1995) EMBO J.15: 3654, each reference are incorporated herein by reference).

因为IL-15是原型Th1细胞因子,而且由于其作为T细胞、NK细胞、LAK细胞和TIL刺激物的活性,IL-15是一种令人兴奋的候选物,作为分子佐剂可与DNA疫苗(例如HIV疫苗)联用,以增强细胞免疫应答。 Because IL-15 is a prototype of Th1 cytokines, but also because as T cells, NK cells, LAK cells and TIL stimulating actives, IL-15 is an exciting candidate, with DNA as a molecular adjuvant vaccine (e.g., an HIV vaccine) MS, in order to enhance the cellular immune response. IL-15增加HIV特异性CTL,而IL-15的过量产生与节段性回肠炎等炎性疾病有关。 IL-15 increase in HIV-specific CTL, and excessive production of IL-15 with Crohn Yandeng inflammatory diseases.

RNA印迹分析表明IL-15的广泛组成型表达。 RNA blot analysis indicated that the constitutive expression of a wide of IL-15. 表达的控制发生在转录后的翻译和转运(胞内运输)水平。 Controlling the expression of the transcription occurs after translation and transport (intracellular transport) levels. IL-15mRNA中包含许多妨碍其翻译成蛋白的元件,包括1)5′AUG带有上游AUG,其干扰有效的IL-15翻译(小鼠有5个,人有12个);2)IL-15编码序列的起始密码子具有弱KOZAK区(GTAATGA);3)IL-15成熟蛋白编码序列的C-端存在负元件。 IL-15mRNA contains many prevent its translation into protein components, including 1) 5'AUG IL-15 translation with upstream AUG, which interfere with effective (mice 5, people have 12); 2) IL- initiation codon 15 of the coding sequence with weak KOZAK region (GTAATGA); C- terminal 3) IL-15 mature protein coding sequence of a negative element. (Grabstein等(1994)Science 264:965-968,Bamford等(1996)PNAS 93:2897-2902;Bamford等(1998)J.Immunol 160:4418-4426;和Kozak等(1991)J.Cell Biol.115:887-903,所述文献各自通过引用结合到本文中。这三个控制元件各自可被消除,以改进表达。 (Grabstein, etc. (1994) Science 264: 965-968, Bamford, etc. (1996) PNAS 93: 2897-2902; Bamford, etc. (1998) J.Immunol 160: 4418-4426; and Kozak, etc. (1991) J.Cell Biol. 115: 887-903, each of the documents incorporated by reference herein in each of the three control elements can be eliminated to improve expression.

天然IL-15同种型含有两个前导肽:21个氨基酸的信号肽(SSP)或48个氨基酸的信号肽(LSP)(Waldmann等,Ann.Rev>Immunol.(1999)17:19-49,其通过引用结合到本文中。 Natural IL-15 isoforms contain two leader peptide: 21 amino acid signal peptide (SSP) or a 48 amino acid signal peptide (LSP) (Waldmann, etc., Ann.Rev> Immunol (1999) 17:. 19-49 , which is incorporated herein by reference.

进行下述策略:通过优化用于免疫的IL-15DNA构建体,来增加IL-15的表达。 Subjected to the following strategy: by optimizing the IL-15DNA constructs used for immunization, to increase the expression of IL-15. 引物设计成从信号肽开始扩增IL-15,因此上游抑制性AUG在最终IL-15信使中不存在。 Primers were designed to amplify from the start signal peptide IL-15, thus inhibiting the upstream AUG is not present in the final IL-15 messenger. 引物设计成包括强KOZAK区(GCCGCCACC)。 Primers were designed to include strong KOZAK area (GCCGCCACC). C-端负调节元件用PCR反义引物设计来除去。 C- terminal negative regulatory elements antisense PCR primers were designed to remove. 所述引物见图10。 The primer shown in Figure 10.

进行下述策略:通过用IgE前导序列取代48个氨基酸的IL-15信号肽(LSP),来增加IL-15的表达。 Subjected to the following strategy: 48 by substitution of IL-15 amino acid signal peptide (LSP) with IgE leader sequence to increase the expression of IL-15. 有义引物设计成从48个氨基酸的LSP后开始,而反义引物从终止位点扩增。 The sense primer designed from the 48 amino acids of LSP begun, and the antisense primer from the termination site amplification. 引物设计成包括强KOZAK区(GCCGCCACC-SEQ ID NO:4)。 Primers were designed to include strong KOZAK area (GCCGCCACC-SEQ ID NO: 4). 有义引物设计成含有IgE前导序列加上ATG起始位点的序列。 Sense primer designed to IgE leader sequence plus the ATG start site sequence comprises. 所述引物见图11。 The primers shown in Figure 11.

制备不同的构建体,用于转染RD细胞。 Preparation of different constructs for transfection RD cells. 测定不同构建体的IL-15蛋白的产生。 Determination of IL-15 produced a different protein constructs. 数据见图12A-C。 Data shown in Figure 12A-C. 图13A显示,包含与IL-15连接的21个氨基酸信号肽(IL-15SSP-左)的编码序列和人48个氨基酸信号肽(IL-15LSP-右)的编码序列的人构建体的表达比较。 Figure 13A shows that expression of the coding sequence and the human signal peptide comprising 21 amino acids with IL-15 connection (IL-15SSP- left) of the 48 amino acid signal peptide (IL-15LSP- right) coding sequence were constructed Comparison . 图12B显示,包含48个氨基酸信号肽(人IL-15LSP-左)的编码序列和IgE信号肽(人IL-15-IgE-右)的编码序列的人构建体的表达比较。 Figure 12B shows, contains 48 amino acid signal peptide (human IL-15LSP- left) and IgE signal peptide coding sequence (human IL-15-IgE- right) comparison of coding sequences of the expression construct human body. 图12C显示,包含48个氨基酸信号肽(恒河猴IL-15LSP-左)的编码序列和IgE信号肽(恒河猴IL-15-IgE-右)的编码序列的恒河猴构建体的表达比较。 Figure 12C shows that expression of the rhesus contains 48 amino acid signal peptide (Rhesus IL-15LSP- left) and IgE signal peptide coding sequence (rhesus monkey IL-15-IgE- right) of the coding sequence construct Compare.

对不同构建体所产生的IL-15蛋白,测定IL-15的生物活性。 Different constructs produced IL-15 protein, was measured biological activity of IL-15. 数据见图13A和图13B。 Data shown in Figure 13A and 13B. 图13A显示,在包含48个氨基酸信号肽(人IL-15LSP-左)和IgE信号肽(人IL-15-IgE-右)的编码序列的人构建体之间的IL-15生物活性比较。 13A shows, in the signal peptide contains 48 amino acids (human IL-15LSP- left) and IgE signal peptide (human IL-15-IgE- right) of the coding sequence construct human IL-15 Comparison between biological activity of the body. 图13B显示,包含48个氨基酸信号肽(恒河猴IL-15LSP-左)的编码序列和IgE信号肽(恒河猴IL-15-IgE-右)的编码序列的恒河猴构建体之间的IL-15生物活性比较。 13B shows, rhesus contains 48 amino acids signal peptide (Rhesus IL-15LSP- left) and IgE signal peptide coding sequence (rhesus monkey IL-15-IgE- right) of the coding sequence between the body construct Comparison of IL-15 biological activity.

用具有与IgE信号肽编码序列连接的IL-15编码序列的插入序列的表达载体pVAX,制备构建体。 IL-15 having the insertion sequence IgE signal peptide coding sequence linked coding sequence of an expression vector pVAX, prepared constructs. 也得到编码HIV-1Gag的构建体。 Also encodes HIV-1Gag constructs. 进行免疫学实验,用IL-15工程质粒以及HIV-1Gag,比较对免疫应答的效应。 Immunologically experiments, IL-15 works with the plasmid and HIV-1Gag, comparison of the effects of the immune response. 按照图14的免疫方案,对Balb/c小鼠进行接种。 Immunization program in Figure 14, for Balb / c mice were inoculated.

在第三次免疫后5周,通过比较对抗原特异性IFN-γ产生的再次刺激,研究免疫应答。 In five weeks after the third immunization, restimulated by comparison with the antigen-specific IFN-γ generated immune response. 数据见图15。 Data shown in Figure 15. 疫苗组包括首次用于实验的小鼠,接种载体pCDN3的小鼠,接种编码HIV-1Gag的构建体的小鼠,接种编码HIV-1Gag和IL-15(连接48个氨基酸信号肽)的构建体的小鼠,接种编码HIV-1Gag以及IgE信号肽的构建体的小鼠。 Vaccine group comprising naive mice inoculated carrier pCDN3 mice, the mice were inoculated with constructs encoding HIV-1Gag inoculated encoding HIV-1Gag and IL-15 (48 amino acid signal peptide is connected) construct Construction of mouse body mice inoculated encoding HIV-1Gag and IgE signal peptide.

实施例4构建工程化IL-15质粒疫苗,即通过除去天然IL-15Kozak区、AUG和UTR。 Example 4 Construction of engineered IL-15 plasmid vaccine through natural IL-15Kozak area, AUG and UTR removed. 提供具有IgE信号肽编码序列的工程化IL-15质粒。 Providing a signal peptide coding sequence having IgE engineered IL-15 plasmids. 工程化IL-15的表达水平比相关野生型质粒高30-50倍。 Engineered IL-15 expression level is higher than the relevant wild-type plasmid 30-50 times. 在用工程化IgE信号-IL-15和HIV-1gag构建体共同免疫的小鼠中,所观察到的免疫应答明显比单用HIV-1gag构建体免疫的小鼠高几倍。 With engineered IgE signal -IL-15 and HIV-1gag construct common immunized mice, the immune responses observed significantly build than single HIV-1gag mice immunized several times higher. 数据见图16。 Data shown in Figure 16.

实施例5编码免疫调节蛋白的分离的cDNA可用作构建可产生免疫调节蛋白的构建体的原料。 Example 5 isolated cDNA encoding immune regulatory protein may be used as the implementation of build materials can produce immunomodulatory protein construct. 在一些实施方案中,提供这样的构建体:其中一个以下免疫调节蛋白的编码序列与IgE信号肽连接。 In some embodiments, providing such constructs: One of the following immunomodulating protein coding sequence and IgE signal peptide. 在一些实施方案中,提供所述构建体作为疫苗和免疫调节组合物的组成部分,例如如上所述的构建体。 In some embodiments, the construct is provided as part of vaccines and immunomodulatory compositions, e.g., the construct described above.

用标准技术和易得原料,可以制备编码免疫调节蛋白的核酸分子,并掺入到如上所述的构建体、载体、疫苗等中。 Using standard techniques and readily available starting material, can be prepared by a nucleic acid molecule encoding an immunomodulatory protein, and incorporated into the construct as described above, vector, vaccine and the like.

Genbank检索号AF031167是指人IL-15mRNA的完整编码序列。 Genbank accession numbers AF031167 refers to the complete coding sequence of the human IL-15mRNA of. Genbank检索号Y09908、X91233、X94223和X94222也是指人IL-15序列。 Genbank accession numbers Y09908, X91233, X94223 and X94222 also refers to human IL-15 sequence. 它们都通过引用结合到本文中。 Which are incorporated by reference herein.

Genbank检索号L07414是指人CD40-配体mRNA的完整编码序列。 Genbank accession number L07414 refers to the complete coding sequence of the human CD40- ligand mRNA. 其通过引用结合到本文中。 Which is incorporated herein by reference.

Bax的核苷酸序列和氨基酸序列的GENBANK检索号为L22473,其通过引用结合到本文中。 Bax nucleotide sequence and amino acid sequence of GENBANK Accession No. L22473, which is incorporated by reference herein.

TRAIL的核苷酸序列和氨基酸序列的GENBANK检索号为U37518或AF023849,其通过引用结合到本文中。 TRAIL nucleotide sequence and amino acid sequence of GENBANK Accession No. U37518 or AF023849, which is incorporated by reference herein.

TRAILrecDRC5的核苷酸序列和氨基酸序列的GENBANK检索号为U90875或AF016266,其通过引用结合到本文中。 GENBANK Accession No. nucleotide and amino acid sequences TRAILrecDRC5 is U90875 or AF016266, which is incorporated by reference herein. 通过引用结合到本文中的还有TRAIL-R2 AF016849;TRAIL-R3 AF014794;和TRAIL-R4 AF021232。 Incorporated by reference herein as well as TRAIL-R2 AF016849; TRAIL-R3 AF014794; and TRAIL-R4 AF021232.

RANK的核苷酸序列和氨基酸序列的GENBANK检索号为AF018253,其通过引用结合到本文中。 RANK nucleotide sequence and amino acid sequence of GENBANK Accession No. AF018253, which is incorporated by reference herein.

RANK配体的核苷酸序列和氨基酸序列的GENBANK检索号为AF019047或AF333234,其通过引用结合到本文中。 GENBANK Accession No. nucleotide sequence and amino acid sequence of RANK ligand is AF019047 or AF333234, which is incorporated by reference herein.

Ox40的核苷酸序列和氨基酸序列的GENBANK检索号为X75962,其通过引用结合到本文中。 Ox40 nucleotide sequence and amino acid sequence of GENBANK Accession No. X75962, which is incorporated by reference herein.

Ox40配体的核苷酸序列和氨基酸序列的GENBANK检索号为X79929或AB007839,其通过引用结合到本文中。 GENBANK Accession No. nucleotide and amino acid sequences for Ox40 ligand X79929 or AB007839, which is incorporated by reference herein.

NKG2D的核苷酸序列和氨基酸序列的GENBANK检索号为AF461811或X54870,其通过引用结合到本文中。 NKG2D nucleotide sequence and amino acid sequence of GENBANK Accession No. AF461811 or X54870, which is incorporated by reference herein.

MICA的核苷酸序列和氨基酸序列的GENBANK检索号为X92841,其通过引用结合到本文中。 MICA nucleotide sequence and amino acid sequence of GENBANK Accession No. X92841, which is incorporated by reference herein.

MICB的核苷酸序列和氨基酸序列的GENBANK检索号为U65416,其通过引用结合到本文中。 MICB nucleotide sequence and amino acid sequence of GENBANK Accession No. U65416, which is incorporated by reference herein.

NKG2A的核苷酸序列和氨基酸序列的GENBANK检索号为X54867,其通过引用结合到本文中。 NKG2A nucleotide sequence and amino acid sequence of GENBANK Accession No. X54867, which is incorporated by reference herein.

NKG2B的核苷酸序列和氨基酸序列的GENBANK检索号为X54868,其通过引用结合到本文中。 NKG2B nucleotide sequence and amino acid sequence of GENBANK Accession No. X54868, which is incorporated by reference herein.

NKG2C的核苷酸序列和氨基酸序列的GENBANK检索号为X54869或Aj0016984,其通过引用结合到本文中。 NKG2C nucleotide sequence and amino acid sequence of GENBANK Accession No. X54869 or Aj0016984, which is incorporated by reference herein.

NKG2E的核苷酸序列和氨基酸序列的GENBANK检索号为L14542,其通过引用结合到本文中。 NKG2E nucleotide sequence and amino acid sequence of GENBANK Accession No. L14542, which is incorporated by reference herein.

NKG2F的核苷酸序列和氨基酸序列的GENBANK检索号为AH006173、U96845或U96846,其通过引用结合到本文中。 NKG2F nucleotide sequence and amino acid sequence of GENBANK Accession No. AH006173, U96845 or U96846, which is incorporated by reference herein.

CD30的核苷酸序列和氨基酸序列的GENBANK检索号为M83554,(Durkop,H等,Cell 68(3),421-427(1992)),其通过引用结合到本文中。 CD30 nucleotide sequence and amino acid sequence of GENBANK Accession No. M83554, (Durkop, H, etc., Cell 68 (3), 421-427 (1992)), which is incorporated by reference herein.

CD153(CD30L)的核苷酸序列和氨基酸序列的GENBANK检索号为L09753,(Smith,CA等,Cell 73(7),1349-1360(1993)),其通过引用结合到本文中。 CD153 (CD30L) nucleotide sequence and amino acid sequence of GENBANK Accession No. L09753, (Smith, CA, etc., Cell 73 (7), 1349-1360 (1993)), which is incorporated by reference herein.

Fos的核苷酸序列的GENBANK检索号为K00650或V01512,其通过引用结合到本文中。 Fos GENBANK nucleotide sequence accession number for K00650 or V01512, which is incorporated by reference herein.

c-jun的核苷酸序列的GENBANK检索号为J04111或M29039,其通过引用结合到本文中。 c-jun nucleotide sequence of GENBANK Accession No. J04111 or M29039, which is incorporated by reference herein.

Sp-1的核苷酸序列的GENBANK检索号为BC021101、BC005250、BC002878、M31126、J02893或X15102,其通过引用结合到本文中。 Sp-1 nucleotide sequence of GENBANK accession numbers for BC021101, BC005250, BC002878, M31126, J02893 or X15102, which is incorporated by reference herein.

Ap1的核苷酸序列可在以下文献中找到:Lee等,1987 Cell 49:741-752,Rauscher等,1988 Science 240:1010-1016,和Chiu等,1988Cell 54:541-552,其通过引用结合到本文中。 Ap1 nucleotide sequence can be found in the following documents: Lee, etc., 1987 Cell 49: 741-752, Rauscher, etc., 1988 Science 240: 1010-1016, and Chiu et al, 1988Cell 54: 541-552, which is incorporated by reference herein.

Ap-2的核苷酸序列的GENBANK检索号为M36711,其通过引用结合到本文中。 GENBANK Accession No. nucleotide sequence Ap-2 of M36711, which is incorporated by reference herein.

p38的核苷酸序列的GENBANK检索号为U66243,其通过引用结合到本文中。 p38 nucleotide sequence of GENBANK Accession No. U66243, which is incorporated by reference herein.

p65Rel的核苷酸序列的GENBANK检索号为L19067,其通过引用结合到本文中。 p65Rel nucleotide sequence of GENBANK Accession No. L19067, which is incorporated by reference herein.

MyD88的核苷酸序列的GENBANK检索号为U70451,其通过引用结合到本文中。 MyD88 nucleotide sequence of GENBANK Accession No. U70451, which is incorporated by reference herein.

IRAK的核苷酸序列的GENBANK检索号为NM001569,其通过引用结合到本文中。 IRAK nucleotide sequence of GENBANK Accession No. NM001569, which is incorporated by reference herein.

TRAF6的核苷酸序列的GENBANK检索号为U78798,其通过引用结合到本文中。 TRAF6 nucleotide sequence of GENBANK Accession No. U78798, which is incorporated by reference herein.

IkB的核苷酸序列可在以下文献中找到:Gilmore等,Trends Genet1993年12月;9(12):427-33,其通过引用结合到本文中。 IkB nucleotide sequence can be found in the following documents: Gilmore, etc., Trends Genet1993 Dec; 9 (12): 427-33, which is incorporated by reference herein.

NIK的核苷酸序列的GENBANK检索号为Y10256,其通过引用结合到本文中。 NIK nucleotide sequence of GENBANK Accession No. Y10256, which is incorporated by reference herein.

SAP K的核苷酸序列可在以下文献中找到:Franklin等,Oncogene.1995年12月7日;11(11):2365-74,其通过引用结合到本文中。 The nucleotide sequence of SAP K can be found in the following documents: Franklin, etc., Oncogene.1995 on 7 December; 11 (11): 2365-74, which is incorporated by reference herein.

SAP1的核苷酸序列的GENBANK检索号为M85164或M85165,其通过引用结合到本文中。 SAP1 nucleotide sequence of GENBANK Accession No. M85164 or M85165, which is incorporated by reference herein.

JNK2的核苷酸序列的GENBANK检索号为L31951,其通过引用结合到本文中。 JNK2 nucleotide sequence of GENBANK Accession No. L31951, which is incorporated by reference herein.

JNK1B2的核苷酸序列的GENBANK检索号为U35005;其通过引用结合到本文中。 JNK1B2 nucleotide sequence of GENBANK Accession No. U35005; which is incorporated by reference herein.

JNK1B1的核苷酸序列的GENBANK检索号为U35004;其通过引用结合到本文中。 JNK1B1 nucleotide sequence of GENBANK Accession No. U35004; which is incorporated by reference herein.

JNK2B2的核苷酸序列的GENBANK检索号为U35003;其通过引用结合到本文中。 JNK2B2 nucleotide sequence of GENBANK Accession No. U35003; which is incorporated by reference herein.

JNK2B1的核苷酸序列的GENBANK检索号为U35002;其通过引用结合到本文中。 JNK2B1 nucleotide sequence of GENBANK Accession No. U35002; which is incorporated by reference herein.

JNK1A2的核苷酸序列的GENBANK检索号为U34822;其通过引用结合到本文中。 JNK1A2 nucleotide sequence of GENBANK Accession No. U34822; which is incorporated by reference herein.

JNK2A1的核苷酸序列的GENBANK检索号为U34821;其通过引用结合到本文中。 JNK2A1 nucleotide sequence of GENBANK Accession No. U34821; which is incorporated by reference herein.

JNK3A1的核苷酸序列的GENBANK检索号为U34820;其通过引用结合到本文中。 JNK3A1 nucleotide sequence of GENBANK Accession No. U34820; which is incorporated by reference herein.

JNK3A2的核苷酸序列的GENBANK检索号为U34819,其通过引用结合到本文中。 JNK3A2 nucleotide sequence of GENBANK Accession No. U34819, which is incorporated by reference herein.

NF-κ-B2即p49剪接形式的核苷酸序列的GENBANK检索号为A57034,其通过引用结合到本文中。 NF-κ-B2 i.e. p49 splice form of the nucleotide sequence of GENBANK Accession No. A57034, which is incorporated by reference herein.

NF-κ-B2即p100剪接形式的核苷酸序列的GENBANK检索号为A42024,其通过引用结合到本文中。 NF-κ-B2 i.e. p100 splice form of the nucleotide sequence of GENBANK Accession No. A42024, which is incorporated by reference herein.

NF-κ-B2即p105剪接形式的核苷酸序列的GENBANK检索号为S17233,其通过引用结合到本文中。 NF-κ-B2 i.e. p105 splice form of the nucleotide sequence of GENBANK Accession No. S17233, which is incorporated by reference herein.

NF-κ-B 50K链前体的核苷酸序列的GENBANK检索号为A37867,其通过引用结合到本文中。 The nucleotide sequence of GENBANK Accession No. NF-κ-B 50K chain precursor of A37867, which is incorporated by reference herein.

NFκB p50的核苷酸序列描述于:Meyer R.等(1991)Proc.Natl.Acad.Sci.USA 88(3),966970,其通过引用结合到本文中。 NFκB p50 nucleotide sequences are described in: Meyer R., etc. (1991) Proc.Natl.Acad.Sci.USA 88 (3), 966970, which is incorporated by reference herein.

人IL-1的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Telford等(1986)Nucl.Acids Res.14:9955-9963,Furutani等(1985)Nucl.Acids Res.14:3167-3179,March等(1985)Nature 315:641-647和检索号Swissprot PO1583,其各自通过引用结合到本文中。 Human IL-1 nucleotide and amino acid sequences are well known, are shown in the following documents: Telford, etc. (1986) Nucl.Acids Res.14: 9955-9963, Furutani, etc. (1985) Nucl.Acids Res.14: 3167 -3179, March, etc. (1985) Nature 315: 641-647 and Accession No. Swissprot PO1583, their respective incorporated herein by reference.

人IL-2的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Holbrook等(1984)Proc.Natl.Acad.Sci.USA 81:1634-1638,Fujita等(1983)Proc.Natl.Acad.Sci.USA 80:7437-7441,Fuse等(1984)Nucl.Acids Res.12:9323-9331,Taniguchi等(1983)nature 302:305-310,Meada等(1983)Biochem.Biophys.Res.Comm.115:1040-1047,Devos等(1983)Nucl.Acids Res.11:4307-4323,检索号为Swissprot PO 1585,其各自通过引用结合到本文中。 The nucleotide sequence and amino acid sequence of human IL-2 are well known, are shown in the following documents: Holbrook, etc. (1984) Proc.Natl.Acad.Sci.USA 81: 1634-1638, Fujita, etc. (1983) Proc.Natl. Acad.Sci.USA 80: 7437-7441, Fuse, etc. (1984) Nucl.Acids Res.12: 9323-9331, Taniguchi, etc. (1983) nature 302: 305-310, Meada etc. (1983) Biochem.Biophys.Res. Comm.115: 1040-1047, Devos, etc. (1983) Nucl.Acids Res.11: 4307-4323, Accession No. for Swissprot PO 1585, which are each incorporated by reference herein.

人IL-4的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Arai等(1989)J.Immunol.142:274-282Otsuka等(1987)Nucl.AcidsRes.15:333-344,Yokota等(1986)Proc.Natl.Acad.Sci USA 83:5894-5898,Noma等(1984)Nature 319:640-646,Lee等(1986)Proc.Natl.Acad.Sci.USA 83:2061-2063,和检索号Swissprot 05112(鼠IL-4的检索号为Swissprot 07750),其各自通过引用结合到本文中。 The nucleotide sequence and amino acid sequence of human IL-4 is well known, are shown in the following documents: Arai, etc. (1989) J.Immunol.142: 274-282Otsuka etc. (1987) Nucl.AcidsRes.15: 333-344, Yokota etc. (1986) Proc.Natl.Acad.Sci USA 83: 5894-5898, Noma, etc. (1984) Nature 319: 640-646, Lee, etc. (1986) Proc.Natl.Acad.Sci.USA 83: 2061-2063, and Accession No. Swissprot 05112 (Accession No. murine IL-4 as Swissprot 07750), which are each incorporated herein by reference.

人IL-5的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Campbell等(1987)Proc.Natl.Acad.Sci.USA 84:6629-6633,Tanabe等(1987)J.Biol.Chem.262:16580-16584,Campbell等(1988)Eur.J.Biochem.174:345-352,Azuma等(1986)Nucl.Acids Res.14:9149-9158,Yokota等(1986)Proc.Natl.Acad.Sci.USA 84:7388-7392,和检索号Swissprot PO5113,其各自通过引用结合到本文中。 The nucleotide sequence and amino acid sequence of human IL-5 is well known, are shown in the following literature: Campbell, etc. (1987) Proc.Natl.Acad.Sci.USA 84: 6629-6633, Tanabe, etc. (1987) J.Biol. Chem.262: 16580-16584, Campbell, etc. (1988) Eur.J.Biochem.174: 345-352, Azuma, etc. (1986) Nucl.Acids Res.14: 9149-9158, Yokota, etc. (1986) Proc.Natl. Acad.Sci.USA 84: 7388-7392, and Accession No. Swissprot PO5113, their respective incorporated herein by reference.

人IL-10的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Viera等(1991)Proc.Natl.Acad.Sci.USA 88:1172-1176,和检索号Swissprot P22301。 The nucleotide sequence and amino acid sequence of human IL-10 are well known, are shown in the following documents: Viera, etc. (1991) Proc.Natl.Acad.Sci.USA 88: 1172-1176, and Accession No. Swissprot P22301.

人IL-15的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Grabstein等(1994)Science 264:965-968,和检索号SwissprotUO3099,其各自通过引用结合到本文中。 Human IL-15 nucleotide and amino acid sequences are well known, are shown in the following documents: Grabstein, etc. (1994) Science 264: 965-968, and Accession No. SwissprotUO3099, their respective incorporated by reference herein.

人IL-18的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Ushio等(1996)J.Immunol.156:4274-4279,检索号为D49950,其各自通过引用结合到本文中。 Human IL-18 nucleotide and amino acid sequences are well known, are shown in the following documents: Ushio, etc. (1996) J.Immunol.156: 4274-4279, Accession No. as D49950, which are each incorporated by reference herein.

人TNF-α的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Pennica,(1984)Nature 312:724-729,检索号为SwissprotPO1375,其各自通过引用结合到本文中。 The nucleotide sequence and amino acid sequence of human TNF-α is well known, are shown in the following literature: Pennica, (1984) Nature 312: 724-729, Accession No. for SwissprotPO1375, their respective incorporated by reference herein.

人TNF-β的核苷酸序列和氨基酸序列是众所周知的,示于以下文献:Gray,(1984)Nature 312:721-724,和检索号Swessprot PO1374,其各自通过引用结合到本文中。 The nucleotide sequence and amino acid sequence of human TNF-β are well known, are shown in the following literature: Gray, (1984) Nature 312: 721-724, and Accession No. Swessprot PO1374, their respective incorporated by reference herein. 人IL-10氨基酸序列是众所周知的,示于以下文献:Viera等(1991)Proc.Natl.Acad.Sci.USA 88:1172-1176,和检索号Swissprot P22301,其各自通过引用结合到本文中。 Human IL-10 amino acid sequence are well known, are shown in the following documents: Viera, etc. (1991) Proc.Natl.Acad.Sci.USA 88: 1172-1176, and Accession No. Swissprot P22301, each of which is incorporated by reference herein.

人白介素12mRNA的完整编码序列示于Genbank检索号AF180563(P40 mRNA)和AF180562(P35 mRNA)和美国专利第5,840,530号,其各自通过引用结合到本文中。 Human interleukin 12mRNA the complete coding sequence is shown in Genbank accession number AF180563 (P40 mRNA) and AF180562 (P35 mRNA) and US Patent No. 5,840,530, each of which is incorporated by reference herein.

MadCAM-1的序列信息参见Genbank检索号U80016(Leung,E.等,Immunogenetics 46(2),111-119(1997)),其各自通过引用结合到本文中。 MadCAM-1 sequence information, see Genbank Accession No. U80016 (Leung, E., Etc., Immunogenetics 46 (2), 111-119 (1997)), which are each incorporated by reference herein.

MadCAM-1的序列信息参见Genbank检索号U43628(Shyjan,AM等,J.Immunol.156(8),2851-2857(1996)),其各自通过引用结合到本文中。 MadCAM-1 sequence information, see Genbank Accession No. U43628 (Shyjan, AM, etc., J.Immunol.156 (8), 2851-2857 (1996)), which are each incorporated herein by reference.

NGF的序列信息参见Genbank检索号M57399(Kretschmer,PJ等,Growth Factors 5,99-114(1991)),其各自通过引用结合到本文中。 NGF sequence information see Genbank Accession No. M57399 (Kretschmer, PJ, etc., Growth Factors 5,99-114 (1991)), which are each incorporated herein by reference.

IL-7的序列信息参见Genbank检索号J04156(Goodwin,RG等,Proc.Natl.Acad.Sci.USA86(1),302-306(1989)),其各自通过引用结合到本文中。 IL-7 sequence information, see Genbank Accession No. J04156 (Goodwin, RG, etc., Proc.Natl.Acad.Sci.USA86 (1), 302-306 (1989)), which are each incorporated herein by reference.

VEGF的序列信息参见Genbank检索号M32977(Leung,DW等,Science 246,1306-1309(1989)),其各自通过引用结合到本文中。 VEGF sequence information see Genbank Accession No. M32977 (Leung, DW, etc., Science 246,1306-1309 (1989)), which are each incorporated by reference herein.

TNF-R的序列信息参见Genbank检索号M60275(Gray,PW等,Proc.Natl.Acad.Sci.USA87,7380-7384(1990)),其各自通过引用结合到本文中。 TNF-R sequence information, see Genbank Accession No. M60275 (Gray, PW, etc., Proc.Natl.Acad.Sci.USA87,7380-7384 (1990)), which are each incorporated by reference herein.

TNF-R的序列信息参见Genbank检索号M63121(Himmler,A.等,DNA Cell Biol.9,705-715(1990)),其各自通过引用结合到本文中。 TNF-R sequence information, see Genbank Accession No. M63121 (Himmler, A., Etc., DNA Cell Biol.9,705-715 (1990)), which are each incorporated by reference herein.

Fas的序列信息参见Genbank检索号M67454(Itoh,N.等,Cell 66(2),233-243(1991)),其各自通过引用结合到本文中。 Fas sequence information see Genbank Accession No. M67454 (Itoh, N., Etc., Cell 66 (2), 233-243 (1991)), which are each incorporated by reference herein.

CD40L的序列信息参见Genbank检索号L07414(Gauchat,JFM等,FEBS Lett,315,259-266(1992),其各自通过引用结合到本文中。 CD40L sequence information see Genbank Accession No. L07414 (Gauchat, JFM, etc., FEBS Lett, 315,259-266 (1992), which are each incorporated by reference herein.

IL-4的序列信息参见Genbank检索号M23442(Arai,N.等,J.Immunol.142(1),274-282(1989)),其各自通过引用结合到本文中。 IL-4 sequence information, see Genbank Accession No. M23442 (Arai, N., Etc., J.Immunol.142 (1), 274-282 (1989)), which are each incorporated herein by reference.

IL-4的序列信息参见Genbank检索号M13982(Yokota,T.等,Proc.Natl.Acad.Sci.USA83(16),5894-5898(1986)),其各自通过引用结合到本文中。 IL-4 sequence information, see Genbank Accession No. M13982 (like Yokota, T., Proc.Natl.Acad.Sci.USA83 (16), 5894-5898 (1986)), which are each incorporated herein by reference.

CSF的序列信息参见Genbank检索号M37435(Wong,GG等,Science 235(4795),1504-1508(1987)),其各自通过引用结合到本文中。 CSF sequence information see Genbank Accession No. M37435 (Wong, GG, etc., Science 235 (4795), 1504-1508 (1987)), which are each incorporated herein by reference.

G-CSF的序列信息参见Genbank检索号X03656(Nagata,S.等,EMBO J.5(3),575-581(1986)),其各自通过引用结合到本文中。 G-CSF sequence information see Genbank Accession No. X03656 (Nagata, S., Etc., EMBO J.5 (3), 575-581 (1986)), which are each incorporated by reference herein.

G-CSF的序列信息参见Genbank检索号X03655(Nagata,S.等,EMBO J.5(3),575-581(1986)),其通过引用结合到本文中。 G-CSF sequence information see Genbank Accession No. X03655 (Nagata, S., Etc., EMBO J.5 (3), 575-581 (1986)), which is incorporated herein by reference.

GM-CSF的序列信息参见Genbank检索号M11220(Lee,F.等,Proc.Ntl.Acad.Sci.USA(13),4360-4364(1985)),其通过引用结合到本文中。 GM-CSF sequence information see Genbank Accession No. M11220 (Lee, F., Etc., Proc.Ntl.Acad.Sci.USA (13), 4360-4364 (1985)), which is incorporated herein by reference.

GM-CSF的序列信息参见Genbank检索号M10663(Wong,GG等,Science 228(4701),810-815(1985)),其通过引用结合到本文中。 GM-CSF sequence information see Genbank Accession No. M10663 (Wong, GG, etc., Science 228 (4701), 810-815 (1985)), which is incorporated by reference herein.

M-CSF的序列信息参见Genbank检索号M27087(Takahashi,M.等,Biochem.Biophys.Res.Commun.161(2),892-901(1989)),其通过引用结合到本文中。 M-CSF sequence information see Genbank Accession No. M27087 (like Takahashi, M., Biochem.Biophys.Res.Commun.161 (2), 892-901 (1989)), which is incorporated herein by reference.

M-CSF的序列信息参见Genbank检索号M37435(Wong GG等,Science 235(4795),1504-1508(1987)),其通过引用结合到本文中。 M-CSF sequence information see Genbank Accession No. M37435 (Wong GG, etc., Science 235 (4795), 1504-1508 (1987)), which is incorporated herein by reference.

LFA-3的序列信息参见Genbank检索号Y00636(Wallner,BP等,J.Exp.Med.166(4),923-932(1987)),其通过引用结合到本文中。 LFA-3 sequence information, see Genbank Accession No. Y00636 (Wallner, BP, etc., J.Exp.Med.166 (4), 923-932 (1987)), which is incorporated herein by reference.

ICAM-3的序列信息参见Genbank检索号X69819,其通过引用结合到本文中。 ICAM-3 sequence information, see Genbank Accession No. X69819, which is incorporated by reference herein.

ICAM-2的序列信息参见Genbank检索号X15606(Staunton,DE等,Nature 339(6219),61-64(1989)),其通过引用结合到本文中。 ICAM-2 sequence information, see Genbank Accession No. X15606 (Staunton, DE, etc., Nature 339 (6219), 61-64 (1989)), which is incorporated by reference herein.

ICAM-1的序列信息参见Genbank检索号J03132(Staunton,DE等,Cell 52(6),925-933(1988)),其通过引用结合到本文中。 ICAM-1 sequence information, see Genbank Accession No. J03132 (Staunton, DE, etc., Cell 52 (6), 925-933 (1988)), which is incorporated by reference herein.

PECAM的序列信息参见Genbank检索号M28526(Newman,PJ等,Science 247,1219-1222(1990),其通过引用结合到本文中。 PECAM sequence information see Genbank Accession No. M28526 (Newman, PJ, etc., Science 247,1219-1222 (1990), which is incorporated herein by reference.

P150.95的序列信息参见Genbank检索号Y00093(Corbi,AL等,EMBO J.6(13),4023-4028(1987)),其通过引用结合到本文中。 P150.95 sequence information see Genbank Accession No. Y00093 (Corbi, AL, etc., EMBO J.6 (13), 4023-4028 (1987)), which is incorporated herein by reference.

Mac-1的序列信息参见Genbank检索号J03925(Corbi,AL等,J.Biol.Chem.263(25),12403-12411(1988)),其通过引用结合到本文中。 Mac-1 sequence information, see Genbank Accession No. J03925 (Corbi, AL, etc., J.Biol.Chem.263 (25), 12403-12411 (1988)), which is incorporated herein by reference.

LFA-1的序列信息参见Genbank检索号Y00796(Larson.R.等,J.Cell Biol.108(2),703-712(1989)),其通过引用结合到本文中。 LFA-1 sequence information, see Genbank Accession No. Y00796 (Larson.R. Etc., J.Cell Biol.108 (2), 703-712 (1989)), which is incorporated by reference herein.

CD34的序列信息参见Genbank检索号M81104(Simmons,DL等,J.Immunol.148,267-271(1992)),其通过引用结合到本文中。 CD34 sequence information, see Genbank Accession No. M81104 (Simmons, DL, etc., J.Immunol.148,267-271 (1992)), which is incorporated herein by reference.

RANTES的序列信息参见Genbank检索号M21121(Schall,TJ等,J.Immunol.141,1018-1025(1988)),其通过引用结合到本文中。 RANTES sequence information see Genbank Accession No. M21121 (Schall, TJ, etc., J.Immunol.141,1018-1025 (1988)), which is incorporated herein by reference.

IL-8的序列信息参见Genbank检索号M28130(Mukaida,N.等,J.Immunol.143(4),1366-1371(1989)),其通过引用结合到本文中。 IL-8 sequence information, see Genbank Accession No. M28130 (Mukaida, N., Etc., J.Immunol.143 (4), 1366-1371 (1989)), which is incorporated herein by reference.

MIP-1α的序列信息参见Genbank检索号U72395(Fridell,RA等,J。Cell.Sci 110(pt 11),1325-1331(1997)),其通过引用结合到本文中。 MIP-1α sequence information see Genbank Accession No. U72395 (Fridell, RA, etc, J.Cell.Sci 110 (pt 11), 1325-1331 (1997)), which is incorporated herein by reference.

E-selecton的序列信息参见Genbank检索号M24736(Bevilacqua,MP等,Science 243(4895),1160-1165(1989)),其通过引用结合到本文中。 E-selecton sequence information see Genbank Accession No. M24736 (Bevilacqua, MP, etc., Science 243 (4895), 1160-1165 (1989)), which is incorporated herein by reference.

CD2的序列信息参见Genbank检索号M14362(Sewell,WA等,Proc.Natl.Acad.Sci.USA83,8718-8722(1986);Proc.Natl.Acad.Sci.USA84,7256-7256(1987)),其通过引用结合到本文中。 CD2 sequence information see Genbank Accession No. M14362 (Sewell, WA, etc., Proc.Natl.Acad.Sci.USA83,8718-8722 (1986); Proc.Natl.Acad.Sci.USA84,7256-7256 (1987)), which is incorporated herein by reference.

MCP-1的序列信息参见Genbank检索号S69738(Li,YS等,Mol.Cell.Biochem.126(1),61-68(1993)),其通过引用结合到本文中。 Sequence information, see Genbank MCP-1's Accession No. S69738 (Li, YS, etc., Mol.Cell.Biochem.126 (1), 61-68 (1993)), which is incorporated herein by reference.

L-selection的序列信息参见Genbank检索号X16150(Tedder,TF等,J.Exp.Med.170(1),123-133(1989)),其通过引用结合到本文中。 L-selection sequence information see Genbank Accession No. X16150 (Tedder, TF, etc., J.Exp.Med.170 (1), 123-133 (1989)), which is incorporated herein by reference.

P-selection的序列信息参见Genbank检索号M25322(Johnston,GI等,Cell 56,1033-1044(1989),其通过引用结合到本文中。 P-selection sequence information see Genbank Accession No. M25322 (Johnston, GI, etc., Cell 56,1033-1044 (1989), which is incorporated herein by reference.

FLT的序列信息参见Genbank检索号X94263(Mandrota,SJ等,J.Biol.Chem.271(19),11500-11505(1996)),其通过引用结合到本文中。 FLT sequence information see Genbank Accession No. X94263 (Mandrota, SJ, etc., J.Biol.Chem.271 (19), 11500-11505 (1996)), which is incorporated herein by reference.

FLT的序列信息参见Genbank检索号X51602(Shibuya,M.等,Oncogene 5(4),519-524(1990)Han,HJ等Hum.Mol.Genet.2(12),2204(1993)),其通过引用结合到本文中。 FLT sequence information see Genbank Accession No. X51602 (Shibuya, M., Etc., Oncogene 5 (4), 519-524 (1990) Han, HJ etc. Hum.Mol.Genet.2 (12), 2204 (1993)), which incorporated by reference herein.

Apo-1的序列信息参见Genbank检索号X63717(Oehm等,J.Biol.Chem.,(1992),267(15),10709-15),其通过引用结合到本文中。 Apo-1 sequence information, see Genbank Accession No. X63717 (Oehm, etc., J.Biol.Chem., (1992), 267 (15), 10709-15), which is incorporated by reference herein.

Fas的序列信息参见Genbank检索号M67454(Itoh等,Cell,(1991),66(2),233-43),其通过引用结合到本文中。 Fas sequence information see Genbank Accession No. M67454 (Itoh, etc., Cell, (1991), 66 (2), 233-43), which is incorporated herein by reference.

TNFR-1的序列信息参见Genbank检索号M67454(Nophar等,EMBO J.,1990,9(10),3269-78),其通过引用结合到本文中。 TNFR-1 sequence information, see Genbank Accession No. M67454 (Nophar, etc., EMBO J., 1990,9 (10), 3269-78), which is incorporated by reference herein.

p55的序列信息参见Genbank检索号M58286(Loetscher等,Cell,1990,61,351-359),其通过引用结合到本文中。 p55 sequence information see Genbank Accession No. M58286 (Loetscher, etc., Cell, 1990,61,351-359), which is incorporated herein by reference.

WSL-1的序列信息参见Genbank检索号Y09392(Kitson等,Nature,1996,384(6607),372-5),其通过引用结合到本文中。 WSL-1 sequence information, see Genbank Accession No. Y09392 (Kitson, etc, Nature, 1996,384 (6607), 372-5), which is incorporated herein by reference.

DR3的序列信息参见Genbank检索号U72763(Chinnaiyan等,Science,1996,274(5829),990-2),其通过引用结合到本文中。 DR3 sequence information see Genbank Accession No. U72763 (Chinnaiyan, etc., Science, 1996,274 (5829), 990-2), which is incorporated herein by reference.

TRAMP的序列信息参见Genbank检索号U75381(Bodmer等,Immunity,1997,6(1),79-88),其通过引用结合到本文中。 TRAMP sequence information see Genbank Accession No. U75381 (Bodmer, etc., Immunity, 1997,6 (1), 79-88), which is incorporated herein by reference.

Apo-3的序列信息参见Genbank检索号U74611(Marsters等,Curr.Biol.,1996,6(12),1669-76),其通过引用结合到本文中。 Apo-3 sequence information, see Genbank Accession No. U74611 (Marsters etc., Curr.Biol., 1996,6 (12), 1669-76), which is incorporated by reference herein.

AIR的序列信息参见Genbank检索号U78029,其通过引用结合到本文中。 AIR sequence information see Genbank Accession No. U78029, which is incorporated by reference herein.

LARD的序列信息参见Genbank检索号U94512(Screaton等,Proc.Natl.Acad.Sci.USA,1997,94(9),4615-19),其通过引用结合到本文中。 LARD sequence information see Genbank Accession No. U94512 (Screaton, etc., Proc.Natl.Acad.Sci.USA, 1997,94 (9), 4615-19), which is incorporated herein by reference.

NGRF的序列信息参见Genbank检索号M14764(Johnson等,Cell,1986,47(4),545-554),其通过引用结合到本文中。 NGRF sequence information see Genbank Accession No. M14764 (Johnson, etc., Cell, 1986,47 (4), 545-554), which is incorporated herein by reference.

DR4(TRAIL)的序列信息参见Genbank检索号U90875(Pan等,Science,1997,276(5309),111-113),其通过引用结合到本文中。 DR4 (TRAIL) sequence information, see Genbank Accession No. U90875 (Pan, etc., Science, 1997,276 (5309), 111-113), which is incorporated herein by reference.

DR5的序列信息参见Genbank检索号AF012535(Sheridan等,Science,1997,1227(5327),818-821),其通过引用结合到本文中。 DR5 sequence information see Genbank Accession No. AF012535 (Sheridan, etc., Science, 1997,1227 (5327), 818-821), which is incorporated herein by reference.

KILLER的序列信息参见Genbank检索号AF022386(Wu等,Nat.Genet.17(2),141-143(1997)),其通过引用结合到本文中。 KILLER sequence information see Genbank Accession No. AF022386 (Wu, etc., Nat.Genet.17 (2), 141-143 (1997)), which is incorporated herein by reference.

TRAIL-R2的序列信息参见Genbank检索号AF020501,其通过引用结合到本文中。 TRAIL-R2 sequence information see Genbank accession number AF020501, which is incorporated herein by reference.

TRICK2的序列信息参见Genbank检索号AF018657。 TRICK2 sequence information see Genbank accession number AF018657.

DR6的序列信息参见Genbank检索号AF068868,其通过引用结合到本文中。 DR6 sequence information see Genbank accession number AF068868, which is incorporated herein by reference.

ICE的序列信息参见Genbank检索号U13697、U13698和U13699(Alnemri,ES等,J.Biol.Chem.270(9),4312-4317(1995)),其通过引用结合到本文中。 ICE sequence information see Genbank Accession No. U13697, U13698 and U13699 (Alnemri, ES, etc., J.Biol.Chem.270 (9), 4312-4317 (1995)), which is incorporated herein by reference.

VLA-1的序列信息参见Genbank检索号X17033(Takada.等,J.Biol.Chem.109(1),397-407(1989)),其通过引用结合到本文中。 VLA-1 sequence information, see Genbank Accession No. X17033 (Takada., Etc., J.Biol.Chem.109 (1), 397-407 (1989)), which is incorporated herein by reference.

CD86(B7.2)的序列信息参见Genbank检索号U04343(Azuma等,Nature.366(6450),76(1993)),其通过引用结合到本文中。 CD86 (B7.2) of the sequence information see Genbank Accession No. U04343 (Azuma, etc., Nature.366 (6450), 76 (1993)), which is incorporated herein by reference.

表1小RNA病毒科(Picornavirus Family)属:鼻病毒属(Rhinovirus):(医学)是约50%感冒的病因肠道病毒属(Etherovirus):(医学)包括脊髓灰质炎病毒(poliovirus)、柯萨奇病毒(coxsackievirus)、埃可病毒(echovirus)和人肠道病毒(human enterovirus)例如甲型肝炎病毒(hepatitis A virus)口疮病毒属(Apthoviruse):(兽医)是口蹄疫病毒靶抗原:VP1、VP2、VP3、VP4、VPG杯状病毒科(Calcivirus Family)属:诺沃克病毒组(Norwalk group of Virus):(医学)这些病毒是流行性胃肠炎的重要致病因子披膜病毒科(Togavirus Family)属:甲病毒属(Alphavirus):(医学和兽医)实例包括Senilis virus、罗斯河病毒(Ross River virus)、东方马脑炎病毒和西方马脑炎病毒(Eastern & WesternEquine encephalitis virus)呼肠孤病毒属(Reovirus):(医学)风疹病毒(Rubellavirus)黄病毒科(Flariviridue Family)实例包括:(医学)登革病毒(dengue virus)、黄热病毒(yellow fever virus)、日本脑炎病毒(Japaneseencephalitis virus)、圣路易斯脑炎病毒(St.Louisencephalitis virus)和蜱传脑炎病毒(tick borneencephalitts virus)。 Table 1 small RNA virus family (Picornavirus Family) Genus: rhinovirus genus (Rhinovirus) :( medicine) is the cause of about 50% of colds enterovirus (Etherovirus) :( Medicine) includes poliovirus (poliovirus), KE Saatchi virus (coxsackievirus), ECHO virus (echovirus) and human enterovirus (human enterovirus) such as hepatitis A virus (hepatitis A virus) virus, mouth sores (Apthoviruse) :( veterinarian) is FMDV target antigens: VP1, VP2, VP3, VP4, VPG Caliciviridae (Calcivirus Family) Genus: Norwalk virus group (Norwalk group of Virus) :( Medical) these viruses are an important causative agent of epidemic gastroenteritis Togaviridae (Togavirus Family) genera: alphavirus (Alphavirus) :( medical and veterinary) Examples include Senilis virus, Ross River virus (Ross River virus), Eastern equine encephalitis virus and Western equine encephalitis virus (Eastern & amp; WesternEquine encephalitis virus) call reovirus genus (Reovirus) :( medicine) Rubella virus (Rubellavirus) Flaviviridae (Flariviridue Family) Examples include medical :() Dengue virus (dengue virus), yellow fever virus (yellow fever virus), Japanese encephalitis virus (Japaneseencephalitis virus), St. Louis encephalitis virus (St.Louisencephalitis virus) and tick-borne encephalitis virus (tick borneencephalitts virus). 西尼罗病毒(West Nile virus)(Genbank NC001563,AF533540,AF404757,AF404756,AF404755,AF404754,AF404753,AF481864,M12294,AF317203,AF196835,AF260969,AF260968,AF260967,AF206518和AF202541)代表性靶抗原:ENS5C丙型肝炎病毒:(医学)这类病毒不在一个科内,但是据信仍是披膜病毒(togavirus)或黄病毒(flavivirus)。 WNV (West Nile virus) (Genbank NC001563, AF533540, AF404757, AF404756, AF404755, AF404754, AF404753, AF481864, M12294, AF317203, AF196835, AF260969, AF260968, AF260967, AF206518 and AF202541) representative target antigens: ENS5C propionate :( hepatitis virus within medicine) is not a kind of virus families, but it is believed still togavirus (togavirus) or yellow virus (flavivirus). 与披膜病毒科最相似。 The most similar Togaviridae. 冠状病毒科(Coronavirus Family):(医学和兽医)传染性支气管炎病毒(Infectious bronchitis virus)(家禽)猪传染性胃肠病毒(Porcine transmissiblegastroenteric virus)(猪)猪血凝脑脊髓炎病毒(Porcine hemaglutinatingencephalomyelitis virus)(猪)猫传染性腹膜炎病毒(Feline infectious peritonitisvirus)(猫)猫肠道冠状病毒(Feline enteric coronavirus)(猫)犬冠状病毒(Canine coronavirus)(狗)SARS相关性冠状病毒(SARS associatedcoronavirus)人呼吸道冠状病毒(Human respiratory coronavirus)引起约40种感冒EX.224E,OC43注释-冠状病毒可引起非甲、非乙或非丙型肝炎靶抗原: Coronavirus family (Coronavirus Family) :( medical and veterinary) infectious bronchitis virus (Infectious bronchitis virus) (poultry) gastrointestinal infectious swine virus (Porcine transmissiblegastroenteric virus) (pig) swine encephalomyelitis virus hemagglutinin (Porcine hemaglutinatingencephalomyelitis virus) (pig) feline infectious peritonitis virus (Feline infectious peritonitisvirus) (cat) cat gut coronavirus (Feline enteric coronavirus) (cat) Canine coronavirus (Canine coronavirus) (dog) SARS-related coronavirus (SARS associatedcoronavirus) Human respiratory coronavirus (Human respiratory coronavirus) caused about 40 kinds of cold EX.224E, OC43 comments - coronavirus can cause non-A, non-B hepatitis or target antigens:

E1-也称为M或基质蛋白E2-也称为S或刺突(Spike)蛋白E3-也称为BE或hemagglutin-elterose糖蛋白(不存在于所有冠状病毒中)N-核壳弹状病毒科(Rhabdovirus Family)属:水疱病毒属(vesiliovirus)狂犬病毒属(Lyssavirus):(医学和兽医)狂犬病靶抗原:G蛋白N蛋白丝状病毒科(Filoviridue Family):(医学)出血热病毒(Hemorrhagic fever virus),例如马尔堡病毒(Marburg virus)和埃博拉病毒(Ebola virus)副粘病毒科(Paramyxovirus Family):属:副粘病毒属(Paramyxovirus):(医学和兽医)腮腺炎病毒(Mumps virus)、新城疫病毒(New Castledisease virus)(鸡的重要病原体)麻疹病毒属(Morbillivirus):(医学和兽医)麻疹、犬瘟热肺病毒属(Pneuminvirus):(医学和兽医)呼吸道合胞病毒(Respiratory syncytial virus)正粘病毒科(Orthomyxovirus Family)(医学)流感病毒(Influenza virus)布尼亚病毒科(Bungavirus Family)属:布尼亚病毒属(Bungavirus):(医学)加州脑炎病毒(California encephalitis virus LA Crosse)白蛉热病毒属(Phlebovirus):(医学)立夫特山谷热病毒(Rift Valley Fever virus) E1- also called M or matrix protein E2- also called S or Spike (Spike), also known as protein E3- BE or hemagglutin-elterose glycoprotein (not present in all coronaviruses) N- nucleocapsid Rhabdovirus Branch (Rhabdovirus Family) Genus: blisters virus is (vesiliovirus) lyssavirus (Lyssavirus) :( medical and veterinary) rabies target antigen: G protein N protein of filamentous virus family (Filoviridue Family) :( medicine) hemorrhagic fever virus (Hemorrhagic fever virus), such as the Marburg virus (Marburg virus) and Ebola virus (Ebola virus) Paramyxoviridae (Paramyxovirus Family): Genus: Paramyxovirus (Paramyxovirus) :( medical and veterinary) mumps virus (Mumps virus), Newcastle disease virus (New Castledisease virus) (important pathogen of chickens) Morbillivirus (Morbillivirus) :( medical and veterinary) measles, canine distemper virus, lung (Pneuminvirus) :( medical and veterinary) respiratory syncytial virus (Respiratory syncytial virus) Orthomyxoviridae (Orthomyxovirus Family) (medicine) influenza virus (Influenza virus) Bunyaviridae (Bungavirus Family) Genus: Bunia virus genus (Bungavirus) :( Medical) California encephalitis virus ( California encephalitis virus LA Crosse) sandfly fever virus genus (Phlebovirus) :( Medical) Rift Valley fever virus (Rift Valley Fever virus)

汉坦病毒属(Hantavirus):Puremala是一种出血热病毒纳伊罗病毒(Nairvirus)(兽医)内罗毕绵羊病还有许多未命名的布尼亚病毒沙粒病毒科(Arenavirus Family)(医学)LCM,拉沙热病毒(Lassi fever virus)呼肠孤病毒科(Reovirus Family)属:呼肠孤病毒属(Reovirus):可能的人病原体轮状病毒属(Rotavirus):小儿急性胃肠炎环状病毒属(Orbivirus):(医学和兽医)科罗拉多蜱热、Lebombo(人)马器质性脑病、蓝舌逆转病毒科(Retrovirus Family)亚科:致癌RNA病毒亚科(Oncorivirinal):(兽医)(医学)猫白血病病毒(feline leukemia virus)、HTLVI和HTLVII慢病毒亚科(Lentivirinal):(医学和兽医)HIV、猫免疫缺陷病毒(feline immunodeficiency virus)、马传染性贫血病毒(equine infections,anemia virus)泡沫病毒亚科(Spumavirinal)乳多空病毒科(Papovavirus Family)亚科:多瘤病毒属(Polyomavirus):(医学)BKU和JCU病毒亚科:乳头瘤病毒属(Papillomavirus):(医学)癌症或恶性乳头瘤相关的许多病毒类型腺病毒(Adenovirus)(医学) Hantavirus genus (Hantavirus): Puremala is a viral hemorrhagic fever virus Na Yiluo (Nairvirus) (Veterinary) Nairobi sheep disease and many unnamed Bunia virus Arenaviridae (Arenavirus Family) (medicine) LCM , Lassa fever virus (Lassi fever virus) Reoviridae (Reovirus Family) Genus: reovirus genus (Reovirus): possible human pathogen Rotavirus (Rotavirus): children with acute gastroenteritis circovirus genus (Orbivirus) :( medical and veterinary) Colorado tick fever, Lebombo (people) horse organic encephalopathy, bluetongue virus reverse Branch (Retrovirus Family) subfamily: oncogenic RNA virus subfamily (Oncorivirinal) :( Veterinary) (Medical ) feline leukemia virus (feline leukemia virus), HTLVI and HTLVII lentivirus subfamily (Lentivirinal) :( medical and veterinary) HIV, feline immunodeficiency virus (feline immunodeficiency virus), equine infectious anemia virus (equine infections, anemia virus) foamy virus subfamily (Spumavirinal) Papovaviridae (Papovavirus Family) subfamily: polyomavirus genus (Polyomavirus) :( Medical) BKU and JCU virus subfamily: papillomavirus genera (Papillomavirus) :( medicine) or cancer malignant papilloma virus associated with many types of adenovirus (Adenovirus) (medicine)

EX AD7,ARD.,OB—引起呼吸道疾病—某些腺病毒例如275引起肠炎细小DNA病毒科(Parvovirus Family)(兽医)猫细小DNA病毒(Feline parvovirus):引起猫肠炎猫肠炎病毒(Feline panleucopeniavirus)犬细小DNA病毒(Canine parvovirus)猪细小DNA病毒(Porcine parvovirus)疱疹病毒科(Herpesvirus Family)亚科:甲型疱疹病毒亚科(Alphaherpesviridue)属:单纯疱疹病毒属(Simplexvirus)(医学)HSVI (Genbank X14112,NC001806),HSVII(NC001798)水痘病毒属(Varicellovirus):(医学兽医)伪狂犬病-水痘-带状疱疹亚科:乙型疱疹病毒亚科(Betaherpesviridue)属:巨细胞病毒属(Cytomegalovirus)(医学)HCMV鼠巨细胞病毒属(Muromegalovirus)亚科:丙型疱疹病毒亚科(Gammaherpesviridue)属:淋巴隐伏病毒属(Lymphocryptovirus)(医学)EBV-(伯基特淋巴瘤(Burkitts lympho))蛛猴疱疹病毒属(Rhadinovirus)痘病毒科(Poxvirus Family)亚科:脊椎动物痘病毒亚科(Chordopoxviridue)(医学-兽医)属:天花病毒属(Variola)(天花)痘苗病毒属(Vaccinia)(牛痘)副痘病毒属(Parapoxivirus)-兽医禽痘病毒属(Auipoxvirus)-兽医山羊痘病毒属(Capripoxvirus)野兔痘病毒属(Leporipoxvirus)猪痘病毒属(Suipoxvirus)亚科:昆虫痘病毒亚科(Entemopoxviridue)嗜肝DNA病毒科(Hepadnavirus Family)乙型肝炎病毒(hepatitis B virus)未分类丁型肝炎病毒(hepatitis delta virus)表2细菌病原体致病性革兰氏阳性球菌包括:肺炎球菌(pneurnococcal);葡萄球菌(staphylococcal);和链球菌(streptococcal)。 EX AD7, ARD, OB- cause respiratory disease - some adenoviruses such as small DNA virus enteritis caused 275 families (Parvovirus Family) (veterinarian) cat small DNA viruses (Feline parvovirus):. Enteritis caused by cat cat enteritis virus (Feline panleucopeniavirus) canine parvovirus DNA virus (Canine parvovirus) porcine parvovirus DNA virus (Porcine parvovirus) herpes virus family (Herpesvirus Family) subfamily: alpha herpes virus subfamily (Alphaherpesviridue) Genus: herpes simplex virus, (Simplexvirus) (medicine) HSVI (Genbank X14112, NC001806), HSVII (NC001798) varicella-zoster virus genus (Varicellovirus) :( veterinary medicine) pseudorabies - varicella - zoster subfamilies: Herpes B virus subfamily (Betaherpesviridue) Genus: Cytomegalovirus (Cytomegalovirus) ( Medical) HCMV murine cytomegalovirus (Muromegalovirus) subfamily: hepatitis herpes virus subfamily (Gammaherpesviridue) Genus: lymphatic insidious virus genus (Lymphocryptovirus) (medicine) EBV- (Burkitt's lymphoma (Burkitts lympho)) spider monkey Herpes virus genus (Rhadinovirus) poxviridae (Poxvirus Family) subfamily: vertebrate poxvirus subfamily (Chordopoxviridue) (Medicine - Veterinary) Genus: smallpox virus, (Variola) (smallpox) vaccinia virus genus (Vaccinia) (vaccinia) Parapoxvirus genus (Parapoxivirus) - Veterinary fowl pox virus genus (Auipoxvirus) - Veterinary goat pox virus genus (Capripoxvirus) rabbit pox virus genus (Leporipoxvirus) swine pox virus genus (Suipoxvirus) subfamily: entomopoxviruses subfamily (Entemopoxviridue) hepatotropic DNA virus family (Hepadnavirus Family) HBV (hepatitis B virus) Unclassified hepatitis D virus (hepatitis delta virus) Table 2 bacterial pathogens pathogenic Gram-positive cocci include: pneumococcal (pneurnococcal); grapes aureus (staphylococcal); and streptococci (streptococcal). 致病性革兰氏阴性球菌包括:脑膜炎球菌(menignococcal);和淋病球菌(gonococcal)。 Pathogenic Gram-negative cocci include: meningococcal (menignococcal); and gonorrhea gonorrhoeae (gonococcal).

致病性革兰氏阴性肠杆菌包括:肠杆菌科(enterobacteriaceae);假单胞菌属(pseudomonas)、不动杆菌属(acinetobacteria)和艾肯氏菌属(eikenella)、melioidosis;沙门氏菌属(sahnonella);志贺氏菌属(shigellosis);嗜血杆菌属(hemophilus);软下疳菌属(chancroid);布鲁氏菌属(brucellosis);土拉热菌属(tularemia);耶尔森氏菌属(yersinia(巴斯德氏菌属(pasteurella));念球状链杆菌(streptobacillusmortiliformis)和螺菌属(spirillum);单核细胞增生李斯特氏菌(listeria monocytogenes);猪红斑丹毒丝菌(erysipelothrixrhusiopathiae);白喉(diphtheria)、霍乱(cholera)、炭疽(anthrax);性病肉芽肿(donovanosis(腹股沟肉芽肿(granulomainguinale));和巴尔通体病(bartonellosis)。 Pathogenic Gram-negative Enterobacteriaceae, including: Enterobacteriaceae (enterobacteriaceae); Pseudomonas (pseudomonas), Acinetobacter (acinetobacteria) and Aiken Shi genus (eikenella), melioidosis; Salmonella (sahnonella ); Shigella (shigellosis); Haemophilus (hemophilus); chancroid genus (chancroid); Brucella (brucellosis); Tula hot genus (tularemia); Yersinia genus (yersinia (Pasteurella (pasteurella)); read globular chain bacilli (streptobacillusmortiliformis) and snail species (spirillum); Listeria monocytogenes bacteria (listeria monocytogenes); Erysipelothrix rhusiopathiae bacteria (erysipelothrixrhusiopathiae ); diphtheria (diphtheria), cholera (cholera), anthrax (anthrax); venereal granuloma (donovanosis (groin granuloma (granulomainguinale)); and Bartonella disease (bartonellosis).

致病性厌氧菌包括:破伤风(tetanus));肉毒中毒(botulism);其它梭状芽孢杆菌;结核(tuberculosis);麻风(leprosy);和其它分枝杆菌。 Pathogenic anaerobic bacteria include: tetanus (tetanus)); botulism (botulism); other clostridia; tuberculosis (tuberculosis); leprosy (leprosy); and other mycobacteria. 致病性螺旋体病包括:梅毒(syphilis);—密螺旋体病(treponematoses):雅司病(yaws)、品他病(pinta)和地方性梅毒;和钩端螺旋体病(leptospirosis)。 Pathogenic leptospirosis include: syphilis (syphilis); - Treponema disease (treponematoses): Yaws (yaws), pinta (pinta) and local syphilis; and leptospirosis (leptospirosis).

由高等病原菌和致病性真菌引起的其它感染包括:放线菌病(actinomycosis);诺卡氏菌病(nocardiosis);隐球菌病(cryptococcosis)、芽生菌病(blastomycosis)、组织胞浆菌病(histoplasmosis)和球孢子菌病(coccidioidomycosis);念珠菌病(candidiasis)、曲霉病(aspergillosis)和毛霉病(mucormycosis);孢子丝菌病(sporotrichosis);类球孢子菌病(paracoccidiodomycosis)、petriellidiosis、球拟酵母菌病(torulopsosis)、足分支菌病(mycetoma)和着色真菌病(chromomycosis);和皮肤真菌病(dermatophytosis)。 Other infections caused by higher pathogen and pathogenic fungi include: actinomycosis (actinomycosis); nocardiosis (nocardiosis); cryptococcosis (cryptococcosis), blastomycosis (blastomycosis), histoplasmosis (histoplasmosis), and coccidioidomycosis (coccidioidomycosis); candidiasis (candidiasis), aspergillosis (aspergillosis) and mucormycosis (mucormycosis); sporotrichosis (sporotrichosis); class coccidioidomycosis (paracoccidiodomycosis), petriellidiosis , Torulopsis yeast disease (torulopsosis), foot fungus disease branch (mycetoma) and colored fungal disease (chromomycosis); and fungal skin disease (dermatophytosis).

立克次氏体感染包括立克次氏体病(rckettsial和rickettsioses)。 Rickettsial infections include rickettsial disease (rckettsial and rickettsioses).

支原体(mycoplasma)和衣原体(chlamydial)感染包括:肺炎支原体(mycoplasma pneurnoniae);性病性淋巴肉芽肿瘤(lymphogranuloma venereum);鹦鹉热(psittacosis);和围产期衣原体感染。 Mycoplasma (mycoplasma) and chlamydia (chlamydial) infections include: Mycoplasma pneumoniae (mycoplasma pneurnoniae); lymphoid granuloma venereal tumor (lymphogranuloma venereum); psittaci (psittacosis); and perinatal chlamydial infections.

致病性真核生物致病性原生动物和寄生虫及其感染包括:阿米巴病(amebiasis);疟疾(malaria);利什曼病(leishmaniasis);锥虫病(trypanosomiasis);弓形体病(toxoplasmosis);卡氏肺囊虫(pneumocystis carinii);巴贝虫病(babesiosis);贾第鞭毛虫病(giardiasis);旋毛虫病(trichinosis);丝虫病(filariasis);血吸虫病(schistosomiasis));线虫(nematode);吸虫(trematode或fluke);和绦虫(cestode(tapeworm))感染。 Pathogenic eukaryotes pathogenic protozoa and parasites and infections include: amebiasis (amebiasis); malaria (malaria); leishmaniasis (leishmaniasis); trypanosomiasis (trypanosomiasis); toxoplasmosis (toxoplasmosis); Pneumocystis carinii (pneumocystis carinii); babesiosis (babesiosis); giardiasis (giardiasis); trichinosis (trichinosis); filariasis (filariasis); schistosomiasis (schistosomiasis) ); elegans (nematode); flukes (trematode or fluke); and tapeworm (cestode (tapeworm)) infection.

序列表<110>宾夕法尼亚州大学信托人(The Trustees of the University of Pennsylvania)<120>编码IgE信号肽和/或IL-15的核酸序列与包含IgE信号肽和/或IL-15的组合物及其使用方法<130>UPAP0020-500<150>US 60/478,205<151>2003-06-13<150>US 60/478,210<151>2003-06-13<160>9<170>PatentIn version 3.2<210>1<211>14<212>PRT<213>人工序列<220> 120 & gt;; encoding IgE signal peptide nucleic acid sequence and / or IL-15 in combination containing IgE signal peptide and / or IL-15 and the sequence table & lt; 110 & gt; Pennsylvania State University Trustee (The Trustees of the University of Pennsylvania) & lt thereof and methods of using & lt; 130 & gt; UPAP0020-500 & lt; 150 & gt; US 60 / 478,205 & lt; 151 & gt; 2003-06-13 & lt; 150 & gt; US 60 / 478,210 & lt; 151 & gt; 2003-06-13 & lt; 160 & gt; 9 & lt; 170 & gt ; PatentIn version 3.2 & lt; 210 & gt; 1 & lt; 211 & gt; 14 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>化学合成的肽<400>1Ala Met Gln Met Leu Lys Glu Thr Met Glu Glu Ala Ala Glu1 5 10<210>2<211>9<212>PRT<213>人工序列 & Lt; 223 & gt; chemically synthesized peptide & lt; 400 & gt; 1Ala Met Gln Met Leu Lys Glu Thr Met Glu Glu Ala Ala Glu1 5 10 & lt; 210 & gt; 2 & lt; 211 & gt; 9 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence

<220> & Lt; 220 & gt;

<223>化学合成的肽<400>2Ala Met Gln Met Leu Lys Glu Thr Ile1 5<210>3<211>13<212>PRT<213>人工序列<220> & Lt; 223 & gt; chemically synthesized peptide & lt; 400 & gt; 2Ala Met Gln Met Leu Lys Glu Thr Ile1 5 & lt; 210 & gt; 3 & lt; 211 & gt; 13 & lt; 212 & gt; PRT & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>化学合成的肽<400>3Phe Arg Asp Val Asp Arg Phe Tyr Lys Thr Arg Ala Glu1 5 10<210>4<211>47<212>DNA<213>人工序列<220> & Lt; 223 & gt; chemically synthesized peptide & lt; 400 & gt; 3Phe Arg Asp Val Asp Arg Phe Tyr Lys Thr Arg Ala Glu1 5 10 & lt; 210 & gt; 4 & lt; 211 & gt; 47 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>寡核苷酸引物<400>4gcccccgtcg acgccgccac catgagaatt tcgaaaccac atttgag 47<210>5<211>37<212>DNA<213>人工序列 & Lt; 223 & gt; Oligonucleotide primers & lt; 400 & gt; 4gcccccgtcg acgccgccac catgagaatt tcgaaaccac atttgag 47 & lt; 210 & gt; 5 & lt; 211 & gt; 37 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence

<220> & Lt; 220 & gt;

<223>寡核苷酸引物<400>5atcgggctcg agtcaagaag tgttgatgaa catttgg 37<210>6<211>39<212>DNA<213>人工序列<220> & Lt; 223 & gt; Oligonucleotide primers & lt; 400 & gt; 5atcgggctcg agtcaagaag tgttgatgaa catttgg 37 & lt; 210 & gt; 6 & lt; 211 & gt; 39 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>寡核苷酸引物<400>6gcccccggta ccgccgccac catggtattg ggaaccata 39<210>7<211>33<212>DNA<213>人工序列<220> & Lt; 223 & gt; Oligonucleotide primers & lt; 400 & gt; 6gcccccggta ccgccgccac catggtattg ggaaccata 39 & lt; 210 & gt; 7 & lt; 211 & gt; 33 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>寡核苷酸引物<400>7atcgggggat cctcaagaag tgttgatgaa cat 33<210>8<211>47<212>DNA<213>人工序列<220> & Lt; 223 & gt; Oligonucleotide primers & lt; 400 & gt; 7atcgggggat cctcaagaag tgttgatgaa cat 33 & lt; 210 & gt; 8 & lt; 211 & gt; 47 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>寡核苷酸引物 & Lt; 223 & gt; oligonucleotide primer

<400>8gcccccgaat tcgccgccac catggattgg acttggatct tattttt 47<210>9<211>49<212>DNA<213>人工序列<220> & Lt; 400 & gt; 8gcccccgaat tcgccgccac catggattgg acttggatct tattttt 47 & lt; 210 & gt; 9 & lt; 211 & gt; 49 & lt; 212 & gt; DNA & lt; 213 & gt; artificial sequence & lt; 220 & gt;

<223>寡核苷酸引物<400>9agttgctgct gctactagag ttcattctaa ctgggtgaat gtaataagt 49 & Lt; 223 & gt; Oligonucleotide primers & lt; 400 & gt; 9agttgctgct gctactagag ttcattctaa ctgggtgaat gtaataagt 49

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Classifications
International ClassificationC07K14/705, C07K14/54, C07K14/00, C07K14/52, A61K48/00, A61K, A61P35/00, C07H21/04, A61P37/00
Cooperative ClassificationC07K14/70578, A61K2039/55527, C07K14/5443, C07K2319/02, A61K2039/53
European ClassificationC07K14/54Q, C07K14/705R
Legal Events
DateCodeEventDescription
13 Sep 2006C06Publication
8 Nov 2006C10Request of examination as to substance
17 Jan 2007CORBibliographic change or correction in the description
Free format text: CORRECT: PRIORITY; FROM: MISSING THE SECOND ARTICLE OF PRIORITY TO: 2003.6.13 US 60/478,210
20 Nov 2013C14Granted