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Publication numberCN1832956 B
Publication typeGrant
Application numberCN 200480022891
PCT numberPCT/US2004/018962
Publication date20 Nov 2013
Filing date14 Jun 2004
Priority date13 Jun 2003
Also published asCA2529145A1, CA2529145C, CN1832956A, EP1638987A2, EP1638987A4, EP1638987B1, EP2292634A2, EP2292634A3, US8173786, US20070041941, US20120276142, WO2005000235A2, WO2005000235A3
Publication number200480022891.5, CN 1832956 B, CN 1832956B, CN 200480022891, CN-B-1832956, CN1832956 B, CN1832956B, 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 B
Abstract  translated from Chinese
公开了融合蛋白和编码融合蛋白的核酸分子。 It discloses a fusion protein and a nucleic acid molecule encoding the fusion protein. 公开了包含与IL-15蛋白序列连接的非IL-15信号肽的融合蛋白和包含与非IgE蛋白序列连接的IgE信号肽的融合蛋白。 It discloses compositions comprising a non-IL-15 and IL-15 signal peptide sequence linked protein and a fusion protein comprising the signal peptide and non-IgE IgE protein sequence of the fusion protein is connected. 公开了包含所述核酸分子的载体以及包含所述载体的宿主细胞;还公开了编码融合蛋白的重组疫苗和减毒活病原体及其使用方法。 Discloses a vector comprising the nucleic acid molecule and a host cell comprising said vector; also discloses recombinant vaccines encoding fusion proteins and live attenuated pathogens, and methods of use. 公开了在给予IL-15和CD40L之后,在有或没有免疫原的情况下的免疫调节效应,也公开了用于传递所述蛋白的各种核酸分子及其组合物与所述组合物的使用方法。 After administration of the disclosed IL-15 and CD40L, with or without conditions immunogen immunomodulatory effect, also discloses the use of a variety of proteins for delivering the nucleic acid molecules and combinations thereof and the composition Methods.
Claims(9)  translated from Chinese
1.一种编码融合蛋白的分离的核酸分子,所述融合蛋白由IgE信号肽与非IgE蛋白序列连接组成,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,而且其中所述非IgE蛋白为IL-15。 A fusion protein encoding isolated nucleic acid molecule, said fusion protein signal peptide and non-IgE-IgE protein sequence linked composition, wherein said non-IgE IgE signal peptide and the protein sequence derived from the same animal species, and wherein said non-IgE protein is IL-15.
2.权利要求1的分离的核酸分子,其中所述分离的核酸分子是质粒。 Isolated nucleic acid molecule of claim 1, wherein said isolated nucleic acid molecule is a plasmid.
3.权利要求1或2的核酸分子,所述核酸分子掺入到病毒载体中。 1 or a nucleic acid molecule of claim 2, wherein the nucleic acid molecule incorporated into a viral vector.
4.一种注射用药物组合物,所述组合物包含权利要求1-3任一项的核酸分子。 An injectable pharmaceutical composition, said composition comprising a nucleic acid molecule of claim any one of 1-3.
5.一种重组疫苗,所述疫苗包含权利要求1-3任一项的核酸分子。 A recombinant vaccine, said vaccine comprising a nucleic acid molecule of claim any one of claims 1-3.
6.—种减毒活病原体,其中包含权利要求1-3任一项的核酸分子。 6.- kinds of live attenuated pathogens, which comprises a nucleic acid molecule of claim any one of claims 1-3.
7.—种由IgE信号肽与非IgE蛋白连接组成的融合蛋白,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,而且其中所述非IgE蛋白为IL-15。 7.- kinds of IgE-IgE signal peptide and non-connected to form a protein fusion protein, wherein said non-IgE IgE signal peptide and the protein derived from the same animal species, and wherein said non-IgE protein is IL-15.
8.—种体外细胞培养物,所述细胞培养物包括含权利要求1-3任一项的核酸分子的细胞,其中所述核酸序列与在所述细胞中表达所需的调节元件操作性连接。 8.- species in vitro cell culture, said cell culture comprising a nucleic acid molecule comprising a cell according to claim any one of claims 1-3, wherein said nucleic acid sequence with the desired expression regulatory elements operatively connected to said cell .
9.一种制备融合蛋白的方法,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所述融合蛋白的核酸分子的细胞,其中所述核酸分子包含在所述细胞中表达所述融合蛋白所需的调节元件,所述培养时间足以让所述细胞表达所述融合蛋白;并且所述融合蛋白是权利要求7的融合蛋白。 9. A method for preparing a fusion protein, said method comprising the cells were cultured under conditions sufficient time required for fusion protein, said cell comprising encoding said fusion protein comprising a nucleic acid molecule of the cell, wherein said nucleic acid molecule comprising expressing said fusion protein regulatory element required, the incubation time sufficient to allow the cells express the fusion protein in the cell; and the fusion protein is a fusion protein of claim 7.
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's

发明领域 Field of the Invention

[0001] 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。 [0001] The present invention relates to improved vaccines, immunogens for prophylactic and / or improved methods for treating immune individuals, and to improved immunotherapeutic compositions and improved immunotherapy methods.

[0002] 发明背景 [0002] Background of the Invention

[0003] 免疫治疗涉及调节个体免疫应答,以达到所需的治疗效果。 [0003] Immunotherapy involves adjusting the 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。 Immunotherapy drugs relates to such compositions: When administered to an individual, the individual can regulate the immune system and ultimately reduce unwanted immune response-related symptoms, or alleviate the symptoms of the final immune response by increasing needs. 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 an integral part of the vaccination program, in which the vaccine administered to an individual, so that the individual is exposed to the immunogen, the individual will produce anti-immune response to the immunogen. 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., cellular immunity or humoral immunity), which is the treatment or prevention of a particular disorder, infection or disease required.

[0004] 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。 [0004] The vaccine is used to generate an individual immunized against the target antigen, 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 include cancer-associated tumor antigens and antigens involved in autoimmune diseases, cell-associated antigens.

[0005] 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。 [0005] In designing such vaccines, it has been recognized that, to produce the target antigen in the vaccine can effectively induce cellular immunity in the immune system of vaccinated individual cells. 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 vaccine, use nontoxic recombinant vector vaccines and DNA vaccine causes the generation of each antigen in the vaccinated individual cells, the result of the immune system to induce cell-mediated immunity. 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 humoral response.

[0006]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。 [0006] The cellular immune response, to provide protection against a pathogen infection and provide treatment pathogen infection, cancer or an effective immune-mediated therapy of autoimmune diseases, it is often 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 the vaccine vaccinated individual cells (e.g., live attenuated vaccines, recombinant vaccines using non-toxic carrier and DNA vaccine) are generally preferred.

[0007] 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。 [0007] While such individual to make effective vaccines are usually produced against pathogen infection or a prophylactic or therapeutic immune disease in humans, but the need for improved vaccines. 需要产生增强免疫应答的组合物和方法。 We need to produce compositions and methods to enhance the immune response.

[0008] 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。 [0008] 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.

[0009] 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。 [0009] Gene therapy involves the gene delivery to needy individuals or from a gene encoding a protein 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。 Various strategies have been developed to deliver proteins, because individuals do not produce enough of the corresponding genes and / or complete functional proteins. 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。 Thus, within the gene therapy can compensate the missing functionality intact enough 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 a therapeutically effective amount designed to produce the protein construct, provided to the patient 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 offers an alternative method for delivering therapeutic protein drugs. 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.

[0010] 除了直接将核酸分子给予个体之外,经常还可给予蛋白。 [0010] In addition to direct nucleic acid molecule administered to an individual 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。 By the method of producing such recombinant 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 a need for improved compositions and methods for producing proteins.

[0011] 发明概述 [0011] Summary of the Invention

[0012] 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。 [0012] The present invention relates to a vaccine containing a recombinant nucleic acid molecule and so generating anti-immunogen individual method, the nucleic acid molecule comprises a nucleic acid sequence encoding an immunogen and the nucleic acid sequences encoding the fusion protein and optionally a nucleic acid sequence encoding CD40L, said fusion protein comprising a non-IL-15 and IL-15 signal sequence protein sequence connected; said method comprising administering to the subject a recombinant vaccine.

[0013] 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。 [0013] The present invention relates to a live attenuated pathogen comprising a nucleic acid molecule and to produce anti-pathogen immunity so that the individual method, the nucleic acid molecule comprises a nucleic acid sequence encoding nucleic acid sequence and optionally encodes CD40L fusion protein, said fusion protein comprises Non-IL-15 IL-15 signal sequence protein sequence connected; said method comprising administering to the subject a live attenuated pathogen.

[0014] 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。 [0014] The present invention relates to an isolated nucleic acid molecule, said nucleic acid molecule comprises a nucleic acid sequence and nucleic acid sequences encoding CD40L protein encoding IL-15 protein and optionally a nucleic acid sequence encoding an immunogen.

[0015] 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。 [0015] 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 one or two or more nucleic acid nucleic acid sequence encoding an immunogen containing molecules.

[0016] 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⑶40L的核酸序列。 [0016] The present invention relates to a method for regulating an immune response in an individual, said method comprising administering to a subject a composition comprising one or more nucleic acid molecules comprising a nucleic acid sequence encoding a nucleic acid sequence encoding ⑶40L 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。 Nucleic acid sequences encoding the various different proteins can be on the same nucleic acid molecules and / or different nucleic acid molecules, or both.

[0017] 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。 [0017] The present invention relates to a method of inducing the individual to produce an immune response in the original, said method comprising administering to a subject an anti immunity comprising one or more nucleic acid molecules of the composition, the nucleic acid molecule comprises a nucleic acid sequence encoding IL-15 protein, nucleic acid sequences and nucleic acid sequences encoding CD40L encoding immunogens. 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。 Nucleic acid sequences encoding the various proteins may be the same of different nucleic acid molecules and / or different nucleic acid molecules or a combination thereof on.

[0018] 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。 [0018] The present invention relates to a recombinant vaccine containing a nucleic acid molecule and makes the individual to produce anti-immunogen method, the nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen, a nucleic acid sequence encoding a nucleic acid sequence encoding CD40L of IL-15 protein; said method comprising administering to the subject the recombinant vaccine.

[0019] 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。 [0019] The present invention relates to a nucleic acid Zi live attenuated pathogens and enable individuals to produce anti-pathogen immunity, said nucleic acid molecule comprising a nucleic acid sequence of a nucleic acid sequence encoding CD40L encoding IL-15 protein; said method comprising give individuals the live attenuated pathogens.

[0020] 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。 [0020] The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, said fusion protein from the non-IgE and IgE signal peptide sequence linked protein composition, wherein the IgE signal peptide and the protein sequence derived from non-IgE the same animal species.

[0021] 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。 [0021] The present invention relates to a host cell in vitro culture, the culture expression vector comprising in operable in the host cell, said expression vector comprising a fusion protein encoded by the IgE and non-IgE signal peptide sequence linked protein consisting of nucleic acid sequence; relates to the nucleic acid molecule; and relates to a host cell comprising said vector.

[0022] 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。 [0022] 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 sequences, as well as the desired expression and regulatory element operably linked to a nucleic acid sequence encoding an immunogen; the said fusion protein comprising IgE and non-IgE protein signal peptide sequence of the connection.

[0023] 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IgE蛋白序列连接的IgE信号肽。 [0023] 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 which encodes a fusion protein comprising a nucleic acid sequence nucleic acid molecule with a nucleic acid molecule encoding an immunogen comprising a nucleic acid sequence is completely different, the fusion protein comprises a signal peptide and non-IgE IgE protein sequence connection.

[0024] 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。 [0024] The present invention relates to an isolated fusion protein, said fusion protein comprising the signal peptide and non-IgE IgE protein sequence connection. [0025] 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。 [0025] The present invention relates to a method for regulating an immune response in an individual, said method comprising administering to the subject a composition, said composition comprising a nucleic acid molecule: a nucleic acid molecule encoding a nucleic acid sequence contained in a fusion protein, said fusion protein comprises Immune regulate IgE signal peptide-protein linked.

[0026] 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。 [0026] The present invention relates to a method of inducing the individual to produce an immune response in the original, said method comprising administering to the subject anti-immune nucleic acid molecule, the nucleic acid molecule encoding a fusion protein comprising a nucleic acid sequence and a nucleic acid sequence encoding the original immunization; the fusion IgE immunomodulatory protein contains a 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 molecules and / or different nucleic acid molecules.

[0027] 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。 [0027] The present invention relates to a recombinant vaccine containing a nucleic acid molecule and makes the individual to produce anti-immunogen method, the nucleic acid molecule comprising a nucleic acid sequence encoding immunogens and nucleic acid sequences encoding the fusion protein, the fusion protein comprises immunomodulatory IgE signal sequence of the protein connected; said method comprising administering to the subject a recombinant vaccine.

[0028] 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。 [0028] The present invention relates to a live attenuated pathogen comprising a nucleic acid molecule and to produce anti-pathogen immunity so that the individual method, the nucleic acid molecule comprises a nucleic acid sequence encoding a fusion protein, IgE signal sequence immunomodulatory protein comprises connecting said fusion protein ; said method comprising administering to the subject a live attenuated pathogen.

[0029] 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。 [0029] The present invention relates to nucleic acid molecules, vectors and host cells, the nucleic acid molecule comprises a nucleic acid sequence encoding a fusion protein, said fusion protein comprising the signal peptide and the IL -15 IgE protein sequence connected; said vector comprising said nucleic acid molecule; the host cell comprising said vector.

[0030] 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。 [0030] The present invention relates to a fusion protein, said fusion protein comprising the signal peptide of IgE and IL-15 protein sequence connection.

[0031] 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。 [0031] 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 comprising IL-15 IgE protein sequence of the signal peptide of the connection. 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 a fusion protein containing the coding and / or nucleic acid molecule immunogen or nucleic acid sequence is present on each of the nucleic acid molecule.

[0032] 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⑶40L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。 [0032] The present invention relates to a method for regulating an immune response in an individual, said method comprising administering to a subject a nucleic acid molecule comprising one or more compositions comprising a nucleic acid sequence encoding the fusion protein, and optionally comprises a nucleic acid sequence encoding ⑶40L, said fusion protein comprising the signal peptide IgE and IL-15 protein connection. 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。 Nucleic acid sequences encoding the various different proteins can be on the same nucleic acid molecules and / or different nucleic acid molecules, or both.

[0033] 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。 [0033] The present invention relates to a method of inducing the individual to produce an immune response in the original, said method comprising administering to a subject an anti immunity comprising one or more nucleic acid molecules of the composition, the nucleic acid molecule encoding a fusion protein comprising a nucleic acid sequence encoding immune original nucleic acid sequence and optionally a nucleic acid sequence encoding CD40L; said fusion protein comprises a signal peptide IgE and IL-15 protein sequence connection. 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。 Nucleic acid sequences encoding the various proteins may be the same of different nucleic acid molecules and / or different nucleic acid molecules or a combination thereof on.

[0034] 附图简述 [0034] Brief Description

[0035] 图1描绘来自实施例1的数据,显示用IL-15和抗⑶3单克隆抗体刺激人PBMC后,产生IFN-Y。 [0035] Figure 1 depicts the data from the embodiment of Example 1, after the display, and anti-IL-15 monoclonal antibody ⑶3 stimulate human PBMC, to produce IFN-Y. PBMC得自接受三联疗法(HAART)的HIV-1慢性感染者。 PBMC from receiving triple therapy (HAART) in HIV-1 chronic infection. 所有供体的病毒量都低于500拷贝/ml,他们的⑶4计数高于500细胞/ml。 All donor virus amount is lower than 500 copies / ml, they ⑶4 count above 500 cells / ml. 为了确定IL-15增加IFN-Y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。 To determine the increase in IL-15 produced IFN-Y can be used as indicators of whether effector function, cells IL-15 and anti-CD3 stimulation, and then analyzed by standard ELISPOT assay (ELIspot assay).

[0036] 图2描绘来自实施例1的数据,显示用IL-15和抗⑶3单克隆抗体刺激人PBMC后,IFN-Y的产生主要是由⑶8介导的。 [0036] Figure 2 depicts data from Example 1, display ⑶3 and anti-IL-15 monoclonal antibody after stimulating human PBMC, IFN-Y are generated mainly by ⑶8 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⑶4或⑶8 T细胞,然后用IL-15和抗⑶3进行刺激,再用标准酶联免疫斑点测定法进行分析。 1, PBMC from receiving triple therapy (HAART) in HIV-1 infected individuals have chronic depleted ⑶4 or ⑶8 T cells, and then stimulated with IL-15 and anti-⑶3, then the standard ELISPOT assays were analyzed.

[0037] 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- Y。 [0037] FIG. 3A, 3B, 3C and 3D drawing data from Example 1, the display HIV-1 peptide and IL-15 stimulation of human PBMC after antigen-specific IFN- Y. 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-Y的能力。 PBMC from acceptance of triple therapy (HAART) in HIV-1 chronically infected, and analyze their response to 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-Y. ⑶8已耗尽,用HIV-1肽和IL-5刺激后,评价IFN- Y的产生(图3D)。 ⑶8 depleted, with HIV-1 peptide and IL-5 stimulation, evaluation of the generation of IFN- Y (FIG. 3D).

[0038] 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。 [0038] FIGS. 4A, 4B and 4C depict data from Example 1 showing with HIV-1DNA vaccine and IL-15 after immunization, HIV-1 antigen-specific cellular immune response. 在第O周和第2周,给Balb/c小鼠共注射50 μ gpCenv或pCgag以及SOygpIL-M和IL-15表达质粒。 At O weeks and two weeks, to Balb / c mice were co-injected 50 μ gpCenv or pCgag and SOygpIL-M and IL-15 expression plasmid. 最后免疫后2周,收获脾细胞。 Two weeks after the last immunization, spleen cells were 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 Fig. 4A, by 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 Fig. 4B, for HIV-1 antigen-specific chemokine secretion levels 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-1 β的分泌。 In the first three days, supernatants were harvested, MIP-1 β assay of secreted. 在图4C中,对抗原特异性IFN-Y分泌水平进行评价。 In Figure 4C, antigen-specific IFN-Y secretion levels were evaluated. 将脾细胞重悬浮,浓度为5X IO6细胞/ml。 The spleen cells were resuspended, at a concentration of 5X IO6 cells / ml. 将100 μ I等分试样加入到96孔平底微量滴定板的各孔中。 The 100 μ I aliquot was added to each well of a 96-well flat-bottomed microtiter plate. 将重组ρ24蛋白加入到各孔中,一式三份,得到终浓度5 μ g/ml和I μ g/ml。 Ρ24 recombinant protein was added to each well, in triplicate, to give a final concentration of 5 μ g / ml and I μ g / ml. 将细胞于37C /5% CO2孵育3天,收获上清液。 The cells at 37 C / 5% CO2 and incubated three days supernatants were 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 kit for measuring secreted cytokine levels.

[0039] 图5A和图5B描绘来自实施例1的数据,显示Thl细胞因子的胞内染色。 [0039] Figures 5A and 5B depict data from Example 1, showing intracellular staining of Thl cytokines. 小鼠接受两次注射:单独的pCgag或者pCgag加上pIL-lOTNA质粒。 Mice received two injections: Separate pCgag or pCgag plus pIL-lOTNA 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。 A week later, spleen cells harvested, P55 peptide pools containing solution (containing 127 kinds of 15-mers across 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 staining extracellular and intracellular mice with anti-staining. 显示CD3+/CD8+淋巴细胞的应答。 Show CD3 + / CD8 + lymphocyte responses. 图5A显示IFN-Y的数据。 5A shows IFN-Y data. 图5B显示肿瘤坏死因子-α的数据。 5B shows tumor necrosis factor -α data. 斑点印迹显示⑶3+/⑶8+淋巴细胞的应答。 Dot blot show ⑶3 + / ⑶8 + lymphocyte responses.

[0040] 图6描绘来自实施例1鼠T辅助细胞增殖测定的数据。 [0040] Figure 6 depicts Example 1 from mouse T helper cell proliferation assay data implemented. 在第O周和第2周,给Balb/c小鼠共注射50 μ g pCgag或pCenv和50 μ g质粒,所述质粒表达IL-2R依赖性Thl细胞因子IL-2或IL-15的cDNA。 At O weeks and two weeks, to Balb / c mice were co-injected 50 μ g pCgag or pCenv and 50 μ g plasmid, the plasmid cDNA IL-2R-dependent Thl cytokines IL-2 or IL-15 in . 将含5 X IO5细胞的100 μ I等分试样立即加入到96孔平底微量滴定板的各孔中。 Cells containing 5 X IO5 aliquot of 100 μ I immediately added to each well flat-bottom 96-well microtiter plate. 将重组Ρ24蛋白加入到各孔中,一式三份,得到终浓度5 μ g/ml和I μ g/ml0测定刺激指数。 Ρ24 recombinant protein was added to each well, in triplicate, to give a final concentration of 5 μ g / ml and I μ g / ml0 stimulation index was measured. 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, unrelated proteins play a role in the control. 同样,pCgag或对照通常针对无关gp 120蛋白来说,SI为I。 Similarly, pCgag or control for unrelated gp 120 protein generally speaking, SI was I. 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, using PHA or Con A (Sigma) as a positive control polyclonal stimulus.

[0041] 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。 [0041] FIG. 7 depicts the data from the embodiment of Example 1, i.e. after immunization with DNA vaccine pCgag Balb / c mice, the Gag epitope mapping. 在第O周和第2周,给Balb/c小鼠共注射50 μ g pCgag和50 μ g pIL_15质粒或表达基因IL-15的载体骨架或载体骨架。 At O weeks and two weeks, to Balb / c mice were co-injected 50 μ g pCgag and 50 μ g pIL_15 plasmid or expression vector backbone or 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 splenocytes with 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- Y的能力。 Peptide into a series of 22 kinds of mixed and combined liquid matrix form, the ability to activate cells to produce IFN- Y measurement.

[0042] 图8A、图8B和图8C描绘来自实施例1的数据,显示在对来自⑶4敲除小鼠的脾细胞进行刺激后,产生IFN-Y。 [0042] FIGS. 8A, 8B and 8C depict data from Example 1, showing the pair from ⑶4 knockout mice were stimulated spleen cells to produce IFN-Y. 图8A,在第O周和第2周,给Balb/c小鼠共注射50 μ g pCgag和50 μ g pIL-15即IL-15表达质粒。 FIGS. 8A, in the first week and the first two weeks O, to Balb / c mice were injected a total of 50 μ g pCgag and 50 μ g pIL-15 that is 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- Y。 Two weeks after the last immunization, spleen cells were harvested by enzyme-linked immunosorbent spot assay, HIV-1-specific determination produced IFN- Y. 图8B,Cd4tmlKmv小鼠用pCgag、用和不用pIL_15进行免疫。 Figure 8B, Cd4tmlKmv mice were pCgag, with and without pIL_15 immunization. 图8C,Cd4tmlKmv小鼠用pCgag以及pIL-15、p⑶40L或这两者进行免疫。 Figure 8C, Cd4tmlKmv mice treated with pCgag and pIL-15, p⑶40L or both immunization. 在最后一次免疫后两周,收获脾细胞,在用HIV-1 Gag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-1 Gag特异性IFN- Y。 Two weeks after the last immunization, spleen cells harvested after in vitro stimulation with HIV-1 Gag peptide, enzyme-linked immunosorbent spot assay, HIV-1 Gag-specific determination produced IFN- Y.

[0043] 图9描绘来自实施例2的数据,显示IL-15和⑶40L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。 [0043] Figure 9 depicts data from Example 2 showing IL-15 and the vaccine site ⑶40L locally generated, can replace the need for T cell help for CD8 effector T cells for amplification.

[0044] 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。 [0044] FIGS. 10, 11, 12A-C, FIG. 13A-B, Figures 14 and 15 relates to the content described in Example 3.

[0045] 图16涉及实施例4中记载的内容。 [0045] Example 16 relates to the content of the fourth embodiment described.

[0046] 优选实施方案的详述 [0046] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] 定义 [0047] defines

[0048] 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。 [0048] As used herein, the term "target protein" refers to a gene construct of the present invention, the 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, desirable 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, prevention and / or treatment of individual-specific infection or disease associated with the target protein.

[0049] 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。 [0049] As used herein, the term "genetic construct" refers to a DNA or RNA molecule, which comprises a coding 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 comprises a start signal and stop signal adjustment element operatively connected to said adjustment element comprises accepting said nucleic acid molecule capable of directing the expression of individual cells in a promoter and polyadenylation signal.

[0050] 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。 [0050] As used herein, the term "expressible form" refers to a coding sequence containing the necessary encoding the target protein or immunomodulating protein operably linked elements regulating gene construct, such that when present in individual cells, expression of the coding can be sequence. ` `

[0051] 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。 [0051] As used herein, the term "sharing an epitope" refers to a protein with another protein comprising at least an epitope identical or substantially similar to an epitope.

[0052] 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。 [0052] As used herein, the term "substantially similar epitope" means a protein having an epitope different structures, but still induce cellular or humoral immune response epitopes which cross-reacted with the protein.

[0053] 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。 [0053] As used herein, the term "intracellular pathogen" refers to a virus or pathogenic organism: copy, or at least a part of the life cycle in a host cell, thereby producing pathogen proteins or protein causes the generation of pathogen.

[0054] 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。 [0054] As used herein, the term "hyperproliferative disease" means characterized by excessive cell proliferation diseases and disorders.

[0055] 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。 [0055] As used herein, the term "hyperproliferative-associated protein" refers to a hyperproliferative disease-associated proteins.

[0056] 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。 [0056] As used herein the term "immunomodulating protein" refers to an individual regulation of the immune system protein, 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、0x40、0x40 LIGAND、NKG2D、F461811 或MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、CD30、CD153(CD30L)、Fos、c_jun、Sp-1、Apl、Ap_2、p38、p65Rel、MyD88、IRAK、TRAF6、IkB、NIK、SAP K、SAPl、JNK2、JNK1B2、JNKlBl、JNK2B2、JNK2B1、JNK1A2、JNK2A1、JNK3A1、JNK3A2、NF-κ -B2、p49 剪接形式、NF-κ -B2、plOO 剪接形式、NF- κ -B2、pl05 剪接形式、NF- κ -B 50Κ 链前体、NF κ B ρ50、人IL-1 α、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TNE-β、人白介素12,MadCAM-KNGF IL-7、VEGF、TNF-R、Fas、CD40L、IL-4, CSF、G-CSF, GM-CSF, M-CSF, LFA-3、I CAM-3,ICAM-2, ICAM-1、PECAM、P150.95、Mac_l、LFA-1、CD34、RANTES、IL-8、ΜΙΡ-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, 0x40,0x40 LIGAND, NKG2D, F461811 or MICA, MICB, NKG2A, NKG2B, NKG2C , NKG2E, NKG2F, CD30, CD153 (CD30L), Fos, c_jun, Sp-1, Apl, Ap_2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, NIK, SAP K, SAPl, JNK2, JNK1B2, JNKlBl, JNK2B2 , JNK2B1, JNK1A2, JNK2A1, JNK3A1, JNK3A2, NF-κ -B2, p49 splice form, NF-κ -B2, plOO splice form, NF- κ -B2, pl05 splice form, before NF- κ -B 50Κ chain body , NF κ B ρ50, human IL-1 α, human IL-2, human IL-4, murine IL-4, human IL-5, human IL-10, human IL-15, human IL-18, human TNF- α, people TNE-β, human interleukin 12, MadCAM-KNGF IL-7, VEGF, TNF-R, Fas, CD40L, IL-4, CSF, G-CSF, GM-CSF, M-CSF, LFA-3, I CAM-3, ICAM-2, ICAM-1, PECAM, P150.95, Mac_l, LFA-1, CD34, RANTES, IL-8, ΜΙΡ-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).

[0057] 概述 [0057] Overview

[0058] 本发明来自以下发现。 [0058] The present invention is derived from the discovery. I)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。 I) when IL-15 signal peptide is not present, the higher the level of IL-15 expression, regardless of the expressed IL-15 protein is a "truncated" of IL-15 protein, or IL-15 containing a non-signal peptide ( particularly IgE signal peptide) fusion proteins of IL-15 connector 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 IL-15 protein or fusion protein containing IL-15 and non-signal peptide (particularly IgE signal peptide) connected to IL-15 protein sequence, in for delivering IL-15 protein as an immunomodulator vaccine proteins and constructs are particularly useful. 2)涉及IL-15和⑶40L传递的疫苗和免疫调节组合物特别有用。 2) relates to IL-15, and vaccines and immunomodulatory compositions ⑶40L pass 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) a fusion protein comprising the signal peptide helps to enhance IgE expression, produce proteins in vaccines and gene therapy drugs are particularly useful, e.g., for protein delivery (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-15 Kozak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 a nucleic acid molecule comprising a nucleic acid molecule comprising a nucleotide sequence encoding the following proteins: the lack of IL-15 comprises a signal peptide (preferably lack of IL-15 Kozak region and untranslated regions) of human IL-15 protein coding sequence; or to provide a non-human IL-15 with 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-15 Kozak区和非翻译区。 IL-15 coding sequence is preferably lacking IL-15 signal sequence, and preferably the lack of IL-15 Kozak region and untranslated regions. 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⑶40L的核苷酸序列。 In some preferred embodiments, the present invention provides vectors, vaccines and immunomodulatory compositions and methods, wherein a nucleic acid molecule comprising a nucleic acid molecule comprising a nucleotide sequence encoding a protein of the following: 1) IL-15 proteins, e.g., lack of 2) a nucleotide sequence encoding human ⑶40L; and IL-15 fusion proteins IL-15 protein signal peptide, or comprise a non-IL-15 signal peptide (e.g., IgE signal peptide) connected to IL-15 protein sequence. 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 preferably the lack of IL-15Kozak region and untranslated regions. 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 a 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.

[0059] 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 Gene [0059] contains IgE and non-IgE protein signal sequences connected signal sequence fusion proteins and IgE and non-IgE protein coding construct connection

[0060] 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。 [0060] Thus, one aspect of the present invention generally relates to fusion proteins comprising the gene encoding the signal sequence and IgE and non-IgE protein connected to the non-connected IgE IgE protein signal sequence construct, and the construct in an expression vector , vaccines and immunomodulatory compositions purposes. 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.

[0061] 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。 [0061] According to some embodiments, there is provided a composition comprising an isolated nucleic acid molecule, the isolated nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein and non-IgE IgE protein sequence operably linked signal sequence.

[0062] 非IgE蛋白的特性取决于构建体的预定用途。 [0062] characteristics of non-IgE protein depends on the intended use of body build. 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, gene therapy embodiments, the protein sequence is such that the desired protein, e.g., the patient lacks a sufficient amount of 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 proteins include enzymes required, for example, DNA enzymes, growth factors (e.g., growth hormone (human, bovine, porcine), coagulation factors, insulin, dystrophin protein desired, etc. Such proteins may also be: When When the expression of the patient, can provide a therapeutic benefit proteins, such as erythropoietin, IL-2, GM-CSF, TPA, etc. In some embodiments, the non-IgE protein sequence is an immunogen. The vaccine can be used to construct , which can provide the original expression immunization, as a target of an 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 The expression, as the target of an immune response, but the immunomodulating effect of the composition is desired in accordance with specific aspects of the condition of the patient being treated up or down the patient's immune system or immune system. upregulation immunomodulatory agent of the immune system can be used such as immunosuppressive therapy or patients suffering from infectious diseases, and reduction of the immune system for the treatment of immune modulators such as autoimmune diseases, organ transplant, tissue transplant patients or patients receiving cell therapy, for them, immunity suppression is necessary. In some embodiments, IgE and non-IgE signal sequence protein sequence connection for use in a system needs to generate the IgE protein. In a preferred embodiment, IgE signal peptide sequence linked to its protein on is derived from the same animal species. In a preferred method, the receiving animals and animal sources constructed IgE protein sequence of the signal peptide and the body are the same species. The fusion protein will be considered non-immunogenic.

[0063] 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。 [0063] In some embodiments, a composition comprising the construct of the coding sequence and the non-IgE protein sequence as immunomodulatory protein IgE connected signal, it may also be included on the same nucleic acid molecule or on different nucleic acid molecules that encode nucleic acid sequence immunogen. 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, the following immunogens 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.

[0064] 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。 [0064] As described above, the protein sequence is preferably non-IgE IL-15 protein, and more preferably the lack of IL-15 IL-15 protein signal sequence, still more preferably the lack of IL-15 signal sequence, region, and lack of IL-15Kozak untranslated lack of IL-15 IL-15 protein sequences. 在一些优选的实施方案中,所述组合物还包括编码⑶40L的核苷酸序列。 In some preferred embodiments, the composition further comprises a nucleotide sequence encoding ⑶40L. 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 a 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 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 compositions.

[0065] 在一些优选的实施方案中,核酸构建体是质粒。 [0065] 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.

[0066] 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。 [0066] contains the gene as a non-IgE protein sequence of connected immunomodulatory protein IgE signal sequence construct of the present invention can be incorporated directly into certain aspects of 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 these pathogens 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I L-15 Kozak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, IL-15 and more preferably the lack of IL-15 protein signal sequence, and still more preferably the lack of IL-15 signal sequence, the lack of I L-15 Kozak region and lack of IL- untranslated sequence of 15 IL-15 protein. 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 provided for carrying nucleotide sequences encoding CD40L attenuated pathogens.

[0067] 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。 One aspect of the [0067] contains IgE and non-IgE signal sequence operably linked to the protein sequence of the fusion proteins of the present invention also. 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 protein sequence portion is an enzyme. 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 of non-IgE protein sequence portion is an 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 a non-IgE protein sequence portion 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.

[0068] 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 Genes [0068] IL-15 comprising a non-IL-15 signal sequence protein connected non-fusion proteins and IL-15 signal sequence encoding IL-15 protein constructs connection

[0069] 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。 [0069] Another general aspect of the present invention relates to a non-IL-15 gene comprises a signal sequence encoding a fusion protein and a non-IL-15 signal sequence and the IL-15 protein with the IL-15 connector connection protein constructs, as well as vaccines and immunomodulatory compositions use body the building. 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 the 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 construct from dogs, cats, horses, cattle, sheep, swine or other species IL-15.

[0070] 本发明的该方面来自这样的观察结果:天然IL-15 mRNA所表达的蛋白序列中含有抑制表达的信号或元件。 [0070] This aspect of the invention comes from the observation that: protein sequence of native IL-15 mRNA expression contained in the signal or component to inhibit expression. 通过去除这些抑制元件,得到更好的表达。 By removing these inhibitory components to give 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 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-15 Kozak区和非翻译区并去除抑制元件。 In addition, removal of IL-15 Kozak region and untranslated region and the removal of 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, it is the lack of IL-15 IL-15 sequence encoding the signal peptide of the amino acid sequence of mature IL-15 protein.

[0071] 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。 [0071] According to some embodiments, there is provided a composition comprising an isolated nucleic acid molecule, the isolated nucleic acid molecule comprising a nucleic acid sequence of a fusion protein non-IL-15 signal sequence encoding IL-15 protein connection. 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 connected components. 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 for 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。 So comprising human IL-15 fusion protein non-immunogenic in humans is non-immunogenic.

[0072] 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。 [0072] According to some embodiments, there is provided a composition comprising construct the body, the fusion protein coding sequence comprises a non-IL-15 and IL-15 signal sequence protein connected to the construction, the sequence may also comprise a nucleic acid molecule in the same upper or different nucleic acid molecule that encodes the immunogen nucleic acid sequences. 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, the following immunogens 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.

[0073] 在优选的实施方案中,组合物还包含编码⑶40L的核苷酸序列。 [0073] In a preferred embodiment, the composition further comprises a nucleotide sequence encoding ⑶40L. 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 included 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 compositions.

[0074] 在一些优选的实施方案中,核酸构建体是质粒。 [0074] 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.

[0075] 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 [0075] IL-15 contains a non-signal sequence and IL-15 fusion protein-protein linked 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 these pathogens 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 (IL-15 preferably 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 pathogens is also provided having a nucleotide sequence encoding a CD40L.

[0076] 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。 [0076] contains a non-IL-15 signal sequence IL-15 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 connected components. 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.

[0077] 包含编码IL-15和⑶40L的基因构建体的组合物及其使用方法 [0077] The composition comprising the gene encoding IL-15 and ⑶40L the constructs and methods of use

[0078] 本发明的另一个总的方面涉及包含编码IL-15和⑶40L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。 [0078] Another aspect of the present invention generally relates to compositions comprising encoding IL-15 gene construct and ⑶40L body composition and the construct use in vaccine and immunomodulatory compositions. 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 the 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 construct from dogs, cats, horses, cattle, sheep, swine 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 in natural form, i.e. with 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11-15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-11-15), most preferably lacking 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.

[0079] 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⑶40L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⑶40L的第二核酸序列。 [0079] According to some embodiments, there is provided a composition comprising an isolated nucleic acid molecule or composition of two different isolated nucleic acid molecules, the former comprising a nucleic acid sequence encoding IL-15 and ⑶40L, the latter comprising encoding IL- comprising a first nucleic acid sequence and a second nucleic acid sequence encoding ⑶40L 15. 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.

[0080] 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⑶40L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。 [0080] According to some embodiments, there is provided a composition comprising body construct, said construct comprising a coding sequence of IL-15 and ⑶40L, the sequence may also be included on the same nucleic acid molecule or on different nucleic acid molecules that encode the nucleic acid molecule immunogen. 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, the following immunogens 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.

[0081] 包含免疫原编码序列的组合物可用作疫苗。 [0081] The composition comprising an immunogen coding sequence can be used as a vaccine. 不包含免疫原编码序列的组合物可用作免疫调节组合物。 The composition does not contain a coding sequence for an immunogen compositions are useful as immunomodulators. 在一些实施方案中,也提供蛋白免疫原,作为IL-15和⑶40L联用而增强的免疫应答的目标。 In some embodiments, also provide protein immunogens, as IL-15 and ⑶40L goals combined with enhanced immune response.

[0082] 在一些优选的实施方案中,核酸构建体是质粒。 [0082] 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, adenovirus, adeno-associated virus, retrovirus, or any other acceptable viral vector, as a vaccine or gene therapy vectors.

[0083] 包含编码IL-15和⑶40L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。 [0083] contains a gene encoding IL-15 and ⑶40L 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 these pathogens 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 (IL-15 preferably lack a signal sequence) is connected to human IgE signal sequence.

[0084] 疫苗和免疫调节组合物 [0084] The composition of vaccines and immunomodulators

[0085] 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。 [0085] In accordance with some embodiments of the invention, the composition of the present invention comprising 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 immunomodulatory protein coding nucleic acid molecule is taken into individual cells, a nucleotide sequence that encodes an immunomodulatory protein expressed in the cell, so the protein available to individuals. 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 for administering a composition protein coding sequence is located on a single nucleic acid molecule, said one or more different combinations of encoding a transcription factor or intermediate factors * (intermediate factor) different nucleic acid molecules comprises, as part of recombinant vaccines and as attenuated vaccine component.

[0086] 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。 [0086] In accordance with some aspects of the present invention, compositions and methods for the prevention of pathogens, 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, such as live attenuated vaccines, cell vaccines, recombinant vaccine or nucleic acid vaccine or DNA vaccine.

[0087] 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。 [0087] The present invention relates to compositions and methods of use deliver immunomodulatory protein.

[0088] 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。 [0088] You can use any well-known techniques, including DNA injection (also known as DNA vaccination), a recombinant vector (eg adenovirus, recombinant adeno-associated virus and recombinant vaccinia virus), to deliver nucleic acid molecules.

[0089] 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。 [0089] 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, priority application number 676,594 and cited, said of each patent literature 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 programs described in the application, an alternative method to pass DNA are described in U.S. Patent Nos. 4,945,050 and No. 5,036,006, both of which documents are incorporated by reference herein.

[0090] 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。 [0090] routes of administration include but are not limited to intramuscular, intranasal (intransally), intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular (intraoccularly) and oral and topical, transdermal, inhalation or suppository or mucosal tissue, such as irrigation through vaginal, rectal, urethral, buccal and sublingual tissue. 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 constructs can include, but are not limited to, the following ways to give: the traditional syringe, needle-free injection device, or "microprojectile bombardment gene gun (microprojectilebombardment gene guns)".

[0091] 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。 [0091] When the cellular uptake of genetic constructs may be retained in the cell as a functional extrachromosomal molecule and / or integrated into the 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, linear DNA will be integrated 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 the DNA introduced into cells, it can be added to promote DNA integrated into the chromosome of the reagents. 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, the RNA 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。 It is also expected to provide the genetic construct as a linear minichromosome including a centromere, 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 in the cell chromosome or integrated 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, a 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 enhancers. 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 to elements, receiving their individual is operable, it is necessary.

[0092] 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。 [0092] start codon and stop codon are generally considered to be part of a nucleotide sequence encoding a 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 an individual, which 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。 Initiation codon and termination codon within the coding sequence must reading frame.

[0093] 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。 [0093] As used promoter and polyadenylation signal in individual cells must work.

[0094] 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。 [0094] The present invention practice, especially examples of promoters who use gene vaccine used in the production include, but are not limited to promoters from the following sources: simian virus 40 (SV40), murine mammary tumor virus (MMTV) promoter, human immunodeficiency virus (MV) such as BIV long terminal repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine promoter (muscle creatine) and human metallothionein (metalothionein) et al gene.

[0095] 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 invention [0095] This, in particular, with examples of human gene vaccine used in the production of 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 SV40 polyadenylation signal.

[0096] 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。 [0096] 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 enhanced promoter from CMV, RSV and EBV's.

[0097] 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。 [0097] 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pVAXl、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。 Plasmid pVAXl from Invitrogen (San Diego, CA) is, pCEP4 and pREP4 containing Epstein Barr virus origin of replication and core antigen EBNA-1 coding region which produces high copy copy episomal not integrated.

[0098] 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。 [0098] In some preferred embodiments related to immunization applications, the administration of a nucleic acid molecule comprising the nucleic acid molecule encoding a target protein, immunoregulatory proteins and polynucleotides can further enhance the immune response against said target protein of a gene sequence. 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、Y-干扰素、血小板衍生生长因子(I3DGF)、TNF、GM-CSF、表皮生长因子(EGF)、IL-U 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 following code: e.g. α- interferon, Y- interferon, platelet derived growth factor (I3DGF), TNF, GM-CSF, epidermal growth factor (EGF), IL-U 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 the signal sequence from IgE IL-15.

[0099] 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。 [0099] You can add additional elements, the elements play a role in target cell destruction, if for any reason you need to eliminate cells receiving the genetic construct 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。 The drug may be administered to a subject ganciclovir, the drug will cause any cells of the selective killing tk, thus, providing a method to selectively destroy cells having the gene constructs.

[0100] 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。 [0100] In order to produce the maximum quantity protein, regulatory sequences may choose to accept the construct of cells in gene expression. 此外,可以选择在细胞中最有效转录的密码子。 Additionally, you can choose the most efficient transcription in cells codons. 本领域普通技术人员可制备在细胞中起作用的DNA构建体。 DNA functioning in a cell to those of ordinary skill in the construct may be prepared.

[0101] 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。 [0101] One method of the present invention comprises the steps of administering nucleic acid molecules according to the following route: the intramuscular, intranasal, intraperitoneal, subcutaneous, intradermal or topical, or by lavage to mucosal tissue, mucosal tissue selected from the inhalation, vaginal, rectal, urethral, buccal and sublingual tissue.

[0102] 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。 [0102] In some embodiments, the nucleic acid molecule and a polynucleotide function enhancer or a genetic vaccine facilitator agent is administered together cells. 多核苷酸功能增强子描述于1994年I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。 US and International Application No. order 5,593,972,5,962,428 No. PCT polynucleotide sequence function enhancer described in 26 January 1994, I filed / US94 / 00899, each application is incorporated herein by reference in. 基因疫苗易化剂描述于1994年4月I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 the April 1994 I filed the order of US No. 021,579, the documents 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 nucleic acid molecule may be administered in combination as a mixture with the nucleic acid molecule administered, the nucleic acid molecule or administered simultaneously, separately administered before or after. 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、Y-干扰素、GM-CSF、血小板衍生生长因子(H)GF)、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, it can play the role of transfer agent and / or replication and / or inflammatory agents and other agents can be administered in combination with a GVF include growth factors, cytokines and lymphokines such as α - interferon, Y- interference Su, GM-CSF, platelet derived growth factor (H) GF), TNF, epidermal growth factor (EGF), ILA, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15 and fibroblast growth factor, surface active agents such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant is similar, LPS analogs (including monophosphoryl lipid A (WL), muramyl peptides), benzoquinone matter and vesicles such as squalene and squalene, hyaluronic acid may also be administered in combination with the gene construct. 在一些实施方案中,免疫调节蛋白可用作GVF。 In some embodiments, an immunomodulatory protein can be used as GVF. 在一些实施方案中,核酸分子与PLG —起提供,以增强传递/摄取。 In some embodiments, the nucleic acid molecule PLG - since provided to enhance delivery / uptake.

[0103] 本发明的药物组合物包含约I纳克至约2000微克DNA。 [0103] The pharmaceutical compositions of the present invention comprise about I 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 present invention comprise from 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 DNA. 在一些优选的实施方案中,药物组合物含有约 In some preferred embodiments, the pharmaceutical compositions contain about

0.1至约500微克DNA。 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I至约350微克DNA。 In some preferred embodiments, the pharmaceutical composition contains from about I 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.

[0104] 本发明的药物组合物可按照所用的给药方式来配制。 The pharmaceutical composition of [0104] the present invention may be administered according to the formulation 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 case, they sterile, non-pyrogenic and non-particles. 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, additives may isotonicity 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.

[0105] 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。 [0105] According to some embodiments of the present invention, there is provided an immunogen to induce an immune response anti-method, or composition of the present invention will be given by individuals. 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。 The vaccine may be live attenuated vaccines, cell vaccine, a recombinant vaccine or nucleic acid vaccine or DNA vaccine.

[0106] 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。 [0106] 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, 223,424,10 5,225,336、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 And live attenuated vaccine using recombinant vectors to deliver foreign antigens in the vaccine instance described in U.S. Patent No. 4,722,848,5,017,487,5, 077,044,5, 110,587,5, 112, 749,5, 174,993,5, 223,424,10 5,225,336,5,240,703,5, 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, above Each literature

通过引用结合到本文中。 Incorporated by reference herein. 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 gene construct, comprising a regulatory sequence operatively connected to and immunomodulatory protein encoding nucleotide sequence, said regulatory sequences affect the expression can play a role in a vaccine. 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 present invention.

[0107] 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。 [0107] The present invention provides an improved method of immunization of an individual, comprising the steps of: administering a gene construct individual cells, 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 comprising a regulatory sequence operably linked to and immunomodulatory protein encoding nucleotide sequence, said regulatory sequences affect the expression can play a role in a vaccine. 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 Immunogen

[0108] 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。 [0108] The present invention is for inducing immune enhancing response against a 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 generate the individual causes of child disease and pathogen immunity, so 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 can be used to control hyperproliferative diseases and disorders (such as cancer), namely by inducing an immune response against the target protein, the target protein is associated with excessive proliferation of 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, namely by inducing an immune response against the target protein, the target protein is involved in autoimmune diseases associated with cell-specific protein.

[0109] 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。 [0109] 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 proteins. 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 the 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 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.

[0110] 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。 [0110] In some embodiments, forms of expression and discovery can expressed in the form of two immunomodulatory protein coding sequence may encode a target protein sequence are given on the same nucleic acid molecule in the individual.

[0111] 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。 [0111] In some embodiments, can be expressed in the form of a sequence encoding the target protein is present on each of the nucleic acid molecule, not containing encoding one or more of the immunomodulatory protein can be expressed in the form of a sequence of nucleic acid molecules. 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, it can 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 encoding without containing one or more immunomodulatory proteins sequence 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 plurality 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, can be expressed in the form of a sequence encoding the target protein is present on the respective nucleic acid molecules encoding both without containing immunomodulatory protein sequences in one or more nucleic acid molecules can be expressed in the form of the while the two immunomodulatory proteins present on the respective nucleic acid molecules containing the coding without 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 elements are passed to the individual.

[0112] 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。 [0112] provides a nucleic acid molecule as plasmid DNA, recombinant vector or nucleic acid molecule as part of an 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 passing encoding the target protein and / or one or two immunomodulatory protein nucleic acid molecule can also be the target protein and / or one or two immunomodulatory proteins as protein to pass, or use * to replace a nucleic acid molecule encoding them.

[0113] 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。 [0113] Gene constructs may include gene expression regulatory element operably linked to the desired coding and the 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 the nucleotide sequence encoding the target protein and the other in the form of an expressible gene construct encoding an immunomodulatory protein comprises a 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 molecules containing a combination of gene constructs incorporated into 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。 Resulting in an enhanced immune response against the target protein. [0114] 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。 [0114] The present invention may be used to generate anti-self immunity 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(listeria)和志贺氏菌(shigella)等原核生物。 The present invention is particularly useful to make the following individuals to produce anti-pathogen immunity, the pathogen-infected cells and no encapsulation membrane (encapsulated), such as viruses and gonorrhea (gonorrhea), Listeria (listeria) 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 can also be used to generate anti-self immune protozoan pathogen, the pathogen intracellular pathogens, including pathogens in the life cycle of a certain stage. 表I列举了可用于制备本发明疫苗的一些病毒科属。 Table I lists some viral families and genera can be used in vaccine preparation of the present invention. 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 a peptide DNA constructs can be used in a vaccine, the peptide comprises at least an epitope: listed in the table shows the antigens and other pathogens antigen epitope identical or substantially similar epitope. 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 can also 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.

[0115]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。 [0115] 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 target of the coding sequence, making it possible to establish a protective immune response against the immunogenic protein response. 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) or extracellular infection is intracellular, all pathogen antigens induce 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 a variety of pathogens antigens 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。 Gene 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.

[0116] 表I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。 [0116] Table I and Table 2 lists the number of 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 methods to produce anti-pathogen immunity against HIV, HSV, HCV, WNV or HBV.

[0117] 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。 [0117] Another aspect of the present invention provides a method for imparting a protective immune response anti-hyperproliferative cells (which is characteristic of hyperproliferative diseases), and provides 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 excessive proliferative diseases include all forms of cancer and psoriasis.

[0118] 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。 [0118] It has been found, comprising encoding immunogenic "hyperproliferative cells" nucleotide sequence-related protein construct into individual cells, resulting in the generation of 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 diseases, it will contain the nucleotide sequence of the gene encoding the excessive proliferation of disease-related protein constructs administered to an individual.

[0119] 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。 [0119] In order for the hyperproliferative-associated protein to be an effective immunogenic target, compared to normal cells, hyperproliferative cells must be specific or to higher levels of protein production. 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。 Including the target antigen protein, and peptide fragments thereof; which comprises 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, a hyperproliferative-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 nearly identical proteins, which produce the protein does not normally exist 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 oncogene protein encoded by the 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 outside, anti-cancer therapeutic target protein and protective program also includes a variable zone B-cell lymphoma antigen produced by T-cell lymphomas and T cell receptor variable region, and 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17-1A所识别的蛋白和叶酸结合蛋白或PSA。 Other tumor-associated proteins can also be used as a target protein, e.g., higher levels of the protein was found in tumor cells, comprising the monoclonal antibody recognized protein 17-1A and folate binding proteins or PSA.

[0120] 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。 [0120] Although the present invention may be used to generate an individual anti several types of cancer in one or more of immunity, but the present invention is particularly useful for preventive immunization individual, the individual susceptible to developing certain cancers, or the individual 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, make it possible to produce an individual and the risk of cancer probability was estimated 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 be predicted probability of any one particular ontogeny.

[0121] 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。 [0121] Similarly, individuals who have cancer and those treated to eliminate the cancer or to ease the individual, but also particularly easy 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。 Therefore, once an individual suffering from certain types of cancer and that has the risk of recurrence, they may be immune to the immune system ready to cope with any future emergence of cancer.

[0122] 本发明提供治疗罹患过度增殖性疾病的个体的方法。 [0122] 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 drugs, guidance and start the individual's immune system to produce the target protein against hyperproliferative cells.

[0123] 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。 [0123] The present invention provides methods of treating suffering from autoimmune diseases and disorders of the individual methods through given broad-spectrum protective immune response, the immune response is against including cell receptors and produce "self" directed antibody-producing cells, including autoimmune-related targets.

[0124] 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)和溃瘍性结肠炎。 [0124] T cell mediated autoimmune diseases include rheumatoid arthritis guide (RA), multiple sclerosis (MS), Sjogren's syndrome (Sjogren's syndrome), sarcoidosis, insulin dependent diabetes mellitus (IDDM ), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis (Wegener's granulomatosis), Crohn's disease (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 endogenous antigen binding and cause autoimmune diseases inflammatory cascade of T-cell receptor. 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。 Anti-T cell vaccination variable region, will induce an immune response including CTL, including, to eliminate these T cells.

[0125] 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。 [0125] In RA, several specific variable regions of T cell receptors involved in the disease (TCR) have been characterized. 这些TCR包括@-3、@-14、20 νβ-17和Va-17。 The TCR include @ -3, @ -14,20 νβ-17 and Va-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 said protein constructs vaccination, will induce targeted T cells involved in RA immune response. 参见Howell,MD等,1991 Proc.Nat.Acad.Sc1.USA 88:10921-10925 ;Piliard, X.等,1991 Science 253:325-329 ;ffilliams,WV等,1992 J Clin.1nvest.90:326-333 ;以上每篇文献都通过引用结合到本文中。 See Howell, MD, etc., 1991 Proc.Nat.Acad.Sc1.USA 88: 10921-10925; Piliard, X. et, 1991 Science 253: 325-329; ffilliams, WV, etc., 1992 J Clin.1nvest.90: 326 -333; above each article are incorporated by reference herein. 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-1O。 These include VfP and TCR Va-1O. 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 `body protein constructs vaccination, it will induce targeted T cells involved in MS immune response. 参见Wucherpfennig, KW等,1990 Science 248:1016-1019 ;0ksenberg, JR等,1990 Nature 345:344-346 ;以上每篇文献都通过引用结合到本文中。 See Wucherpfennig, KW et, 1990 Science 248: 1016-1019; 0ksenberg, JR et, 1990 Nature 345: 344-346; above each article are incorporated by reference herein.

[0126] 在硬皮病中,涉及该病的TCR的若干特异性可变区已经表征。 [0126] In scleroderma, several specific variable regions of TCR relates to the disease have been characterized. 这些TCR包括V β _6、Vβ _8、Vβ —14 和Va —16、Va _3C、Va _7、Va —14、Va —15、Va —16、Va —28 和Va -12。 These include TCR V β _6, Vβ _8, Vβ -14 and Va -16, Va _3C, Va _7, Va -14, Va -15, Va -16, Va -28 and Va -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 said protein constructs vaccination, will induce targeted T cells involved in scleroderma immune response.

[0127] 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。 [0127] 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 can be removed in the presence of the sample, using standard techniques to identify TCR variable region. 用这些信息,可以制备基因疫苗。 With these information, we can be prepared genetic vaccine.

[0128] 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。 [0128] B-cell mediated autoimmune diseases, including lupus (SLE), Graves disease (Grave 's disease), myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cold 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 endogenous antigen binding and cause autoimmune diseases inflammatory cascade 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 region, will induce an immune response including CTL, including, to eliminate these antibody-producing B cells.

[0129] 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。 [0129] In order to treat patients suffering from B-cell mediated autoimmune disease patient, must be involved in autoimmune activity of 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。 You can use standard technology, the variable region of these antibodies were identified. 用这些信息,可以制备基因疫苗。 With these information, we can be prepared genetic vaccine.

[0130] 在SLE的情况下,认为一种抗原是DNA。 [0130] In the case of SLE, considered one antigen is 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 a DNA construct, said DNA construct encodes present in the serum anti-DNA antibody variable region.

[0131] TCR可变区和抗体可变区的共同结构特征是众所周知的。 [0131] Common structural features 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 ofHealth 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, e.g., the method described in the following literature methods: Kabat et, 1987 Sequence ofProteins of Immunological Interest USDepartment ofHealth and HumanServices, Bethesda MD, the literature by hereby 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 Sc1.USA87:1066,所述文献通过引用结合到本文中。 In addition, the general method of cloning functional variable regions of the antibody can be found in Chaudhary, VK, etc., 1990Proc.Natl.Acad Sc1.USA87: 1066, the documents incorporated by reference herein.

[0132] 重组蛋白的制备 [0132] Preparation of Recombinant Protein

[0133] 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。 [0133] The present invention relates to a host cell culture in vitro, the host cell comprising operable in the host cell expression vector, said expression vector comprising a signal peptide encoded by the IgE and non-IgE protein sequence of the fusion protein consisting connection nucleic acid sequence; relates to the nucleic acid molecule; and relates to a host cell 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 connection.

[0134] 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。 [0134] by conventional methods using readily available starting materials, prepared as described above fusion protein. 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 sequence encoding the desired protein preparation, the protein can be prepared using recombinant techniques known in the art.

[0135] 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。 [0135] Those skilled in the art can use well-known techniques, DNA encoding the fusion protein is inserted into a commercially available expression vector for the well-known expression system. 市售质粒pYES2 (Invitrogen, San Diego,Calif.)可用于在酿酒酵母(Sc erevisiae)中进行制备。 Commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) Can be used for the preparation of Saccharomyces cerevisiae (Sc erevisiae) in. 市售MaxBac™(Invitrogen, SanDiego, Calif.)完整杆状病毒表达载体可用于在昆虫细胞中进行制备。 Commercial MaxBac ™ (Invitrogen, SanDiego, Calif.) Complete baculovirus expression vector can be prepared for in insect cells. 市售质粒pcDNAI (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 pcDNAI (Invitrogen, San Diego, Calif.) Can be used for the preparation of Chinese hamster ovary cells in 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。 Those skilled 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.

[0136] 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。 [0136] Those skilled in the art may use other commercially available expression vectors and systems, using well-known methods and readily available materials 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 system for the control sequence contained (such as promoters and polyadenylation signals, preferably enhancer) is readily available and known in the art can be used in a variety of hosts. 参见例如Sambrook等,MolecularCloning aLaboratory Manual,第二版,Cold Spring Harbor Press (1989)。 See e.g., Sambrook et al, MolecularCloning 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 to give a series of processing in the form of protein.

[0137]目前,各种各样的真核宿主也可用于制备重组外源蛋白。 [0137] 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 (directly IgE signal peptide) transformed eukaryotic host.

[0138] 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。 [0138] 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。 It can be used when a suitable promoter and terminal sequences and enhancers, for these types of hosts are compatible and operational, such as 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.

[0139] 适于所需宿主的表达系统的构建细节是本领域已知的。 [0139] adapted to the desired expression system of the host Construction details 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 the selected expression vector and used to transform the compatible host which is then cultured and maintained under the following conditions: wherein 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 (from the culture medium or lysing the cells or preferably by) recovering the thus produced protein of the invention.

[0140] 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。 [0140] 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.

[0141] 实施例 [0141] Example

[0142] 实施例1 [0142] Example 1

[0143] 引言 [0143] Introduction

[0144] 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。 [0144] 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 combination therapy program established virus rarely affect 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。 To date, combination therapy has not yet led to viral clearance, and the current treatment options have considerable side effects, it will ultimately affect patient compliance and affect 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,⑶8+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, ⑶8 + T cell response to the control HIV-1 infection and relieve 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 has not been fully elucidated, 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,在网比亚,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)。 In addition, net Shakespeare, a group of high-exposure, but HIV-negative individuals lack antibody response, but it shows that the anti-HIV-1 CD8 + T cell immune response (following references 8 and 9). 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 load. 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 persistent contradiction between 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 not clear the virus, may be partly because of CTL escape mutants (following references 10-14), which is immune pathogenesis of virus may, for example, affect Nef dependency class I MHC downward or Vpr or Env host immunity (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 (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 HIV-1 immune pathogenesis limit produce an effective CD8 response, then provide HIV-1 antigen in antiretroviral therapy, it is able in a limited way to strengthen the CD8 memory 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, the CD8 + T cell expansion is an important help. 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, CD8 + memory T cell survival, sustained antigen presentation, it was not accidental, (hereinafter Ref. 22), but may depend on the surrounding environment to produce specific cytokines.

[0145] 显著影响⑶8+T细胞的一个所述细胞因子是白介素-15(IL_15)。 [0145] a significant impact ⑶8 + T cells, the cytokine interleukin -15 (IL_15). Waldmann及其同事首次报道,IL-15是一种15 kDa蛋白,使用IL-2受体复合物的Y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 15 kDa protein, the use of chain and β chain Y IL-2 receptor complex, the complex IL-2 is a T cell receptor complex signal, 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 seems to stimulate memory CD8 + T cells play a role in the phenotype. 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 IL-15 in HIV-1 infection of research. 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)。 It has been shown, IL-15 to reduce apoptosis isolated from HIV-1 infected lymphocytes (hereinafter Ref. 24) and increased natural killer cell activity and proliferation (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 (following reference 30) 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-Y (IFN-Y)的产生具有直接作用(以下参考文献31和32)。 It is important, IL-15 seems to HIV-1 effector T cell proliferation and IFN -Y (IFN-Y) produces a direct effect (following 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-Y。 However, IL-15 in a number of tests for the HIV-1-positive serum did not stimulate IFN-Y. 因此,研究了IL-15对抗原特异性⑶8+Τ细胞免疫应答的作用。 Therefore, the study of the IL-15 antigen-specific cellular immune responses ⑶8 + Τ role. [0146] 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。 [0146] The role of IL-15 isolated HIV-1 seropositive patients with chronic infection from the T cells were studied. 发现,rhIL-15增强了⑶8 T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⑶8+IFN-Y的产生。 Discovery, rhIL-15 enhances ⑶8 T cell proliferation, and it is important, IL-15 in all patients have increased the effect of antigen-specific ⑶8 + IFN-Y production. 在免疫模型中,IL-15增强了⑶8+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 ⑶8 + effector cell function, which is the study of the immune system, the 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 levels of lysis can express target HIV-1 antigen, while providing IL-15 时. 这一作用发生在⑶4+T细胞剧烈增殖不存在的情况下。 This effect occurs in the case of ⑶4 + T cell proliferation severe does not exist. 然而,在⑶4敲除(KO)小鼠中,在产生⑶8效应细胞应答时,IL-15不能完全忽略⑶4辅助的需要。 However, ⑶4 knockout (KO) mice produce ⑶8 effector cells in response time, IL-15 can not be completely ignored ⑶4 auxiliary needs. 这些结果表明,IL-15在⑶8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 proliferation ⑶8 memory cells are highly effective, but alone it began to produce IL-15 is not enough.

[0147] 材料与方法 [0147] Materials and Methods

[0148] 人PBMC的酶联免疫斑点测定 ELISPOT assay [0148] human PBMC

[0149] 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。 [0149] by standard ELISPOT assay, for basic ficoll-hypaque technique by HIV-1-positive volunteers from isolated PBMC, evaluate effector function. 将PBMC重悬于含10% FCS(RlO)的RPMI中,浓度为IXlO6 细胞/ml。 The resuspended PBMC containing 10% FCS (RlO) in RPMI at a concentration of IXlO6 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15 μ g/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 15 μ g / ml, was used to coat 96-well nitrocellulose plate (Millipore, Bedford, MA). 膜板在4C孵育过夜。 Membrane plate and incubated overnight at 4 C. 用200 μ I PBS洗涤膜板6次。 It was washed with 200 μ I PBS six times the diaphragm. 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50 μ g/ μ I的DMS0。 The mixture was sterilized 122 kinds of peptides (mixture) preparation of a mixture (cocktail), concentration (for each peptide) 50 μ g / μ I of DMS0 is. 所述肽是一系列重叠肽,长度为15个氨基酸,包括所有HIV-1 Gag (AIDS Reagent和ReferenceRepository, ARRR)。 The peptide is a series of overlapping peptides, 15 amino acids in length, including all HIV-1 Gag (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(100 μ 101.0 X IO6细胞/ml),再加入100 μ I肽混合物(稀释I: 200 (溶于R10),有或没有50ng/ml IL-15,终浓度25ng/ml)。 100,000 were added to the PBMC antibody-coated nitrocellulose sheet each well (100 μ 101.0 X IO6 cells / ml), was added 100 μ I peptide mixture (diluted I: 200 (dissolved R10), with or No 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5μ g/ml PHA作为阳性对照。 With 5μ g / ml PHA as a positive control. 将各板在37C孵育约24小时。 The plates were incubated at 37 C for about 24 hours. 然后用200 μ I PBS洗涤各板6次。 Then 200 μ I with PBS the plates were washed 6 times. 将100 μ I抗体7-Β6-1-生物素(Mabtech)加入到每孔中,浓度为I μ g/ml的PBS。 The 100 μ I 7-Β6-1- biotin antibody (Mabtech) was added to each well at a concentration of I μ g / 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200 μ IPBS洗涤各板6次。 Then washed with 200 μ IPBS plates 6 times. 将100 μ I链霉抗生物素-ALP (Mabtech)加入到每孔中,浓度为I μ g/ml的PBS。 The 100 μ I streptavidin-biotin -ALP (Mabtech) was added to each well at a concentration of I μ g / 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200 μ I PBS洗涤各板6次。 200 μ I with PBS the plates were washed 6 times. 将100μ I底物溶液(BCIP/NBT,Sigma)加入到每孔中。 The 100μ I substrate solution (BCI P / 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 (DynalBiotech, Lake success, NY ;0slo, NO)来耗尽CD8 和CD4 群体。 Or in combination with CD8-specific monoclonal antibody conjugates or with CD4-specific monoclonal antibody-conjugated Dynabeads (DynalBiotech, Lake success, NY; 0slo, NO) to deplete CD8 and CD4 populations.

[0150] 用抗⑶3单克隆抗体对PBMC的共刺激 [0150] The monoclonal antibody anti ⑶3 PBMC costimulation

[0151]在有或没有 IL-15 (50ng/ml)的情况下,用结合Dynabeads (DynalBiotech)的对⑶3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- Y。 [0151] in the presence or absence of IL-15 (50ng / ml) of the case, with binding Dynabeads (DynalBiotech) of a monoclonal antibody specific for ⑶3 stimulate an isolated HIV-1 seropositive patients from PBMC, and then ELISPOT assay as described above, the analysis produced IFN- Y. 使用或者与CD8特异性单克隆抗体偶联或者与0)4特异性单克隆抗体偶联的Dynabeads (Dynal Biotech)来耗尽0)8和0)4群体。 Or in combination with CD8-specific monoclonal antibody conjugates or with 0) 4 specific monoclonal antibody-conjugated Dynabeads (Dynal Biotech) to drain 0) 8 and 0) 4 groups.

[0152] 用CD40L对PBMC的共刺激 [0152] The costimulatory CD40L on PBMC

[0153] 测定⑶40L蛋白与IL-15和肽混合物的联用,其浓度为250 μ g/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-Y。 [0153] Determination of ⑶40L protein and IL-15 and peptide mixture in combination with a concentration of 250 μ g / ml, as described above, then by ELISPOT assay analysis produced IFN-Y.

[0154] 在小鼠中进行质粒免疫 [0154] plasmid immunization in mice

[0155] 在第O周和第2周,雌性Balb/c小鼠用50 μ g pCgag或pCenv和50 μ g质粒一起进行接种,所述质粒如前所述表达IL-2R依赖性Thl细胞因子IL-15的基因(以下参考文献33)。 [0155] In the first week and the first two weeks O, female Balb / c mice were treated with 50 μ g pCgag or pCenv and 50 μ g plasmid were seeded, the IL-2R expression plasmid as described previously dependent Thl cytokine IL-15 gene (hereinafter, Ref. 33). 也采用Cd4tmlKmv定向突变的纯合型小鼠。 Also used Cd4tmlKmv directed homozygous mutant mice. 这些小鼠因⑶4基因突变而在⑶4+T细胞发育中被完全阻断;其90%的循环T-细胞是CD8+。 These mice because ⑶4 gene mutation is completely blocked in ⑶4 + 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O周和第2周,B6.129S6-Cd4tmlKmv用50 μ g pCgag和50 μ g质粒一起进行接种,所述质粒表达⑶40L、IL-15或两者的组合。 In the first week and the first two weeks O, B6.129S6-Cd4tmlKmv with 50 μ g pCgag and 50 μ g plasmid were inoculated together, the plasmid ⑶40L, IL-15 or a combination of 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。 Qiagen column was prepared for all 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.

[0156] 鼠细胞毒性T淋巴细胞测定 [0156] Determination of murine cytotoxic T lymphocyte

[0157] 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。 [0157] In five hours 51Cr release CTL assay using recombinant vaccinia virus-infected cells as targets, evaluate CTL responses. 在接种和体外刺激后一周,分离脾细胞。 In the week after vaccination and in vitro stimulation, splenocytes isolated. 用相关痘苗病毒感染的细胞刺激效应子。 With related vaccinia virus-infected cells stimulate effector. P815 用vDKl (gag/pol) (ARRR)或vMN462 (ARRR) (env)感染。 P815 infection with vDKl (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-SHM,不含Con A (Becton Dickinson Labware, Bedford, MA)。 CTL medium consisted of 1: Iscove's Modified 1 Dulbecco 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-SHM, without Con A (Becton Dickinson Labware, Bedford, MA). 通过在37C感染3 X 106P815 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 C infections 3 X 106P815 cells (MOI MOI 10) for 12 hours to prepare vaccinia infection targets. 进行标准铬释放测定,其中靶细胞用20 μ Ci/ml Na251CrO4标记120分钟,然后与受刺激的效应脾细胞一起在37C孵育6小时。 Standard chromium release assay, wherein the target cells with 20 μ Ci / ml Na251CrO4 mark 120 minutes, and then stimulated with effect splenocytes were incubated at 37 C 6 hours. 在效应子:靶标(E: T)比例为50: I至12.5: I的范围内,测定CTL裂解。 In the effector: target (E: T) ratio of 50: I 12.5: within the range I was determined CTL lysis. 收获上清液,在LKB CliniGamma Y-计数器上计数。 Supernatants were harvested, counted on a LKB CliniGamma Y- counter. 按照下式求出特异性裂解的百分数: According to the following equation percentage specific lysis:

[0158] [0158]

Figure CN1832956BD00201

[0159] 通过使靶细胞在含1% Triton X-100培养基中裂解,求出最大释放。 [0159] 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" count value exceeds "maximum release" of 20%, I think that the determination is invalid.

[0160] ⑶8+T细胞的完全裂解 [0160] ⑶8 + complete lysis of T cells

[0161] 通过用抗⑶8单克隆抗体(Pharmingen, San Diego, CA)进行处理,然后通过在37C与兔补体(Sigma) —起保温45分钟,从脾细胞中去除⑶8+T细胞(以下参考文献33)。 [0161] by treatment with anti-⑶8 monoclonal antibody (Pharmingen, San Diego, CA), and then at 37 C with rabbit complement (Sigma) - 45 minutes from the insulation removed ⑶8 + T cells from the spleen cells (hereinafter Ref. 33).

[0162] 鼠T辅助细胞增殖测定 [0162] murine T helper cell proliferation assay

[0163] 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。 [0163] The lymphocyte proliferation assay, total lymphocyte immune activity evaluation and detection of dividing cells have specific antigens. 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, from spleen cells harvested, removing red cells, then washed several times with fresh medium, to prepare lymphocytes. 分离的细胞以5X IO6细胞/ml的浓度重悬浮。 Isolated cells at a concentration of 5X IO6 cells / ml of resuspended. 将含5X IO5细胞的100 μ I等分试样立即加入到96孔平底微量滴定板的每孔中。 5X IO5 cells containing 100 μ I aliquot was added immediately to each well of a 96 well flat bottom microtiter plate. 将重组ρ24蛋白加入到各孔中,一式三份,得到终浓度5 μ g/ml和I μ g/ml。 Ρ24 recombinant protein was added to each well, in triplicate, to give a final concentration of 5 μ g / ml and I μ g / ml. 将细胞于37C /5% CO2孵育3天。 The cells at 37 C / 5% CO2 and incubated for 3 days. 将I μ Ci的氚化胸苷加入到各孔中,然后将细胞在37C孵育12-18小时。 The I μ Ci of tritiated thymidine was added to each well, and the cells were incubated at 37 C 12-18 hours. 收获各板中的细胞,在Beta读板仪(Wallac, Turku, Finland)上测定所掺入的氣化胸苷的数量。 Harvesting the cells in each plate, the number of gasification of the thymidine incorporation assay in Beta plate reader (Wallac, Turku, Finland) on. 按下式求出刺激指数: Stimulation index calculated by the following formula:

[0164] 刺激指数(SI)=(实验计数/自发计数) [0164] The stimulation index (SI) = (experimental count / spontaneous count)

[0165]自发计数孔中包含10%胎牛血清,起到无关蛋白对照作用。 [0165] Spontaneous count wells containing 10% fetal bovine serum, unrelated proteins play a role in the 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I。 Similarly, spleen cells from immunized mice or control pCgag generally unrelated protein for its target for SI as I. 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, using PHA or Con A (Sigma) as a positive control polyclonal stimulus.

[0166] 受刺激鼠细胞的细胞因子和趋化因子分析[0167] 从脾脏收获淋巴细胞,将分离的细胞重悬浮,浓度为5X IO6细胞/ml。 [0166] murine cells stimulated cytokine and chemokine analysis [0167] harvested from spleen lymphocytes isolated cells were resuspended, at a concentration of 5X IO6 cells / ml. 将含5X IO5细胞的100 μ I等分试样加入到96孔平底微量滴定板的各孔中。 5X IO5 cells containing 100 μ I aliquot was added to each well of a 96-well flat-bottomed microtiter plate. 将重组Ρ24蛋白或包膜蛋白加入到各孔中,一式三份,得到终浓度5 μ g/ml和I μ g/ml。 Ρ24 recombinant protein or an envelope protein was added to each well, in triplicate, to give a final concentration of 5 μ g / ml and I μ g / ml. 将细胞于37C /5% CO2孵育3天,收获上清液。 The cells at 37 C / 5% CO2 and incubated three days supernatants were harvested. 用市售ELISA试剂盒测定细胞因子和趋化因子。 Determination of cytokines using commercially available ELISA kits and chemokines.

[0168] 受刺激鼠细胞的干扰素-Y的胞内染色 [0168] in the murine cells stimulated intracellular staining of IFN -Y

[0169] 小鼠接受两次注射:pCgag DNA或者pCgag DNA质粒加上pIL-15。 [0169] Mice received two injections: 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。 A week later, spleen cells harvested, P55 peptide pools containing solution (containing 122 kinds of 15-mers across 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-Y进行胞内染色。 After stimulation, cells were treated with anti-mouse CD3 antibody and anti-mouse CD8 antibody extracellular staining, followed by anti-mouse IFN-Y intracellular staining performed. 描点曲线显示CD3+/⑶8+淋巴细胞的应答。 Plot the curve shows CD3 + / ⑶8 + lymphocyte responses.

[0170] 表位作图 [0170] Epitope mapping

[0171] 将脾细胞重悬于含10% FCS(RlO)的RPMI中,浓度为IX IO6细胞/ml。 [0171] The spleen cells were resuspended in RPMI containing 10% FCS (RlO) in a concentration of IX IO6 cells / ml. 将得自AIDS Reference和Reagent Repository的系列122种妝,混合成为每种合并液中含10种肽的混合物,终浓度为20 μ g/ml/肽。 Obtained from the AIDS Reference and Reagent Repository of series 122 kinds of makeup, blended into each combined liquid mixture containing 10 kinds of peptides, a final concentration of 20 μ g / 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 combined solution, a total of 22 kinds of 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 form of each pool was used to stimulate spleen cells. IFN-Y的产生通过酶联免疫斑点测定法(R和D系统)评价。 IFN-Y generated by ELISpot assay (R and D Systems) Evaluation. 将各板于37C孵育约24小时。 The plates at 37 C for about 24 hours. 每个样品按一式三份进行测定。 Each sample was measured in triplicate.

[0172] 结果 [0172] Results

[0173] 用⑶3和IL-15对淋巴细胞进行刺激 [0173] The ⑶3 and IL-15 stimulated lymphocytes

[0174] 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。 [0174] Evaluation of IL-15 in a synergistic manner, together with the T cell receptor stimulation, empower effector T cell activation. PBMC是从HIV-1感染者中分离的。 PBMC is 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 —起孵育过夜。 Binding antibody PBMC stimulated with anti-CD3 surface, then with the IL-15 - were incubated overnight. 不出所料,⑶3 单独刺激PBMC,能诱导IFN- Y的产生,而单独补充IL-15却只能诱导低应答至无应答。 Unsurprisingly, ⑶3 alone stimulate PBMC, induced IFN- Y production, which alone can only add IL-15-induced low response to no response. 然而,当淋巴细胞用⑶3和IL-15 —起刺激时,观察到细胞分泌IFN- Y的数量增加好几倍(图1)。 However, when using ⑶3 lymphocytes and IL-15 - from the stimulus when the observed number of cells to secrete IFN- Y is increased several times (Figure 1). 受刺激群体耗尽⑶4+或⑶8+T细胞,然后补充IL-15,再次检测活性。 Stimulated population depletion ⑶4 + or ⑶8 + T cells, and then add IL-15, to detect the activity again. CD8细胞的损失再次耗尽活化信号。 Loss of CD8 cells depleted activation signals again. 数据表明,来自HIV-1慢性感染者的⑶8+效应T细胞,可以因IL-15/⑶3刺激而扩增(图2)。 Data show that, ⑶8 + effector T cells from chronically infected with HIV-1 can be due to IL-15 / ⑶3 amplified stimulation (Figure 2).

[0175] HIV-1阳性样品经IL-15刺激后产生抗原特异性IFN- Y [0175] HIV-1 antigen-specific IFN- Y-positive samples were stimulated with IL-15

[0176] 对IL-15增强HIV-1抗原特异性⑶8+应答的能力,进行体外评价。 [0176] The IL-15 to enhance HIV-1 antigen-specific responses ⑶8 + 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 combination antiretroviral therapy (HAART) in HIV-1 chronically infected, 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- Y的能力。 After using HIV-1-specific peptide stimulation, IL-15 in the case of the presence or absence of evaluation capacity of PBMC secrete IFN- Y. PBMC用包含HIV-1 gag蛋白完整可读框的重叠HIV-1 15个氨基酸肽进行刺激。 Overlapping HIV-1 PBMC with HIV-1 gag protein contains a complete open reading frame of 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-Y产生增加(图3A和图3B),IFN-y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 receiving peptide stimulation, when after treatment with IL-15, showed increased production of IFN-Y (FIGS. 3A and 3B), IFN-y produced in the presence or absence of IL-15 has 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 partial 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 CD8 cell population is depleted, CD8-mediated IFN-γ in the generation of reactive been lost (Fig. 3D).

[0177] IL-15在小鼠体内增加CD8+CTL应答 [0177] IL-15 increase in CD8 + CTL responses in mice

[0178] 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-Y的产生。 [0178] over the response to HIV-1 and IL-15 in the study to determine IL-15 in the initial stimulation of T cell populations can increase IFN-Y production. 然而,尚不清楚,IL-15对⑶8+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 ⑶8 + 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 the delivery and studied HIV-1 antigen 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 with or IL-15 expression plasmid or control plasmid injected together, and then compare 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: I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: I effector: target, compared to the By using envelope plasmid and control vector are observed in 11% lysis (Figure 4A). 这些结果是⑶8 T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 ⑶8 T cell-dependent, suggesting that IL-15 significantly affect effector T cell response.

[0179] 小鼠在抗原刺激后IL-15诱导MIP-1 β和IFN_g的分泌 Secretion [0179] mice after antigen stimulation induced by IL-15 and MIP-1 β IFN_g of

[0180] 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-1 β作为免疫活化标记的表达分布型。 [0180] vaccine induced immune response can be further amplified through checking β - chemokines MIP-1 β as an expression profile of 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-1 β,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-1 β, play a key role in the proliferation of immune cells (hereinafter, Reference 24). 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 chemokines stimulate T cells generated by the quantity and quality can provide additional knowledge of 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-1 β释放进行分析和检测。 The supernatant (such as the Materials and Methods section) stimulated T cells of MIP-1 β release for analysis and testing. 用IL-15共同免疫,结果导致高水平分泌MIP-1 β (图4Β)。 IL-15 together with immunization, resulting in high levels of secreted MIP-1 β (Figure 4Β).

[0181] 也对上清液中产生的Thl细胞因子IFN-Y进行评价。 [0181] also produced in the supernatant Thl cytokine IFN-Y 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 cell for CTL assay, after recombinant vaccinia virus expressing HIV-1 envelope effector cells infected 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-Y (120pg/ml)。 Figure 4C shows that compared with single injection of plasmid vaccine or control mice were injected spleen cells from mice IL-15 induced higher levels of IFN-Y (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, these studies did not observe any significant cultures produced IL-4 (data not shown).

[0182] IFN- Y和TNF- α的胞内染色 [0182] IFN- and TNF- α Y intracellular staining

[0183] 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。 [0183] In order to quantitatively study of HIV-1 vaccine T cell responses were intracellular cytokine staining assay. 处死免疫动物,收获脾细胞,在含Ρ55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 medium containing Ρ55 mixture and brefeldin A five hours in vitro. 通过流式细胞术,测定⑶8+⑶3+Τ细胞`的IFN-Y或TNF-α的产生(图5A和图5B)。 By flow cytometry to measure ⑶8 + ⑶3 + `generate Τ cells of IFN-Y or TNF-α (Fig. 5A and 5B). 共同接种IL-15的动物表现出高⑶8效应T细胞应答,2.6%的⑶8+T细胞产生IFN- Y,3.7%的产生TNF- α。 IL-15 co-vaccinated animals exhibit high ⑶8 effector T cell response, 2.6% of ⑶8 + T cells to produce IFN- Y, 3.7% of the produce TNF- α. 这些数据表明,IL-15对功能性⑶8+T细胞应答显示出巨大影响。 These data indicate, IL-15 in functional ⑶8 + T cell responses showed tremendous impact.

[0184] 用IL-15和HIV-1疫苗共同免疫的鼠脾细胞中淋巴细胞增殖 [0184] The IL-15 and HIV-1 vaccine co-immunized mouse spleen cells, lymphocyte proliferation

[0185] 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。 [0185] T helper cell activation and proliferation of humoral and cellular immune amplification, it 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, splenocytes from immunized evaluated the proliferation in mice 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,清楚地看至Li,在共注射IL-2质粒的小鼠中,对于gpl20 env蛋白的脾细胞增殖显著增加。 However, IL-2 used as a control to clearly see Li, co-injection of IL-2 in mice plasmid for splenocyte proliferation gpl20 env protein significantly increased. 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 control, pCgag alone or pCEnv + IL-15 combined with immunized mice who were injected mouse spleen cells of IL-2, leading to at least three times higher stimulation index (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⑶8 T细胞功能。 The data further demonstrated that in the absence of T cell help dramatic amplification, IL-15 seems to increase ⑶8 T cell function. 这也证明,IL-15所引起的扩增并不依赖于IL-2。 This also proves that IL-15 induced amplification is not dependent on IL-2. 这表明,在这种情况下,⑶4及⑶8效应子功能的扩大。 This suggests that, in this case, the expansion of ⑶4 and ⑶8 effector function.

[0186] 表位作图 [0186] Epitope mapping

[0187] 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? [0187] IL-15 treatment to enhance the role of CD8 + T cell responses, whether it is the table because of the increased number of bits in response to (ie: epitope extension), or because of the same epitope-specific CD8 + T cells The overall increase in the number? 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, using ELISpot assay and a series of peptides (mixed matrix combined liquid form) 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)。 The epitope mapping to Gag 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AMQMLKET1-SEQIDN0:2(以下参考文献35)为优势CD8表位。 After Paterson, who previously with recombinant Listeria monocytogenes bacteria (L.monocytogenes) HIV-1 vaccine immunization has been determined AMQMLKET1-SEQIDN0: 2 (The following references 35) were the dominant CD8 epitope. 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 namely 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 the only group Gag immune, it was observed that the response does not increase the number of epitopes. 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 increased 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 in animals immunized subdominant epitope is clear evidence. IL-15影响CD8效应细胞的扩增。 IL-15 effect amplification of CD8 effector cells.

[0188] ⑶4敲除小鼠 [0188] ⑶4 knockout mice

[0189] 我们观察到,在来自HIV-1感染者的PBMC中,IL-15使抗原特异性⑶8 T细胞扩增。 [0189] We observed in PBMC from HIV-1 infection in, IL-15 antigen-specific ⑶8 T 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,⑶4辅助T细胞对IL-15免疫扩增的贡献引起疑问。 Therefore, ⑶4 helper T cells to IL-15 contributions to immune amplification 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 CD4 cell populations to induce CD8 effect ability, 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。 Cd4tmlKmv orientation of homozygous mutant mice (following reference 36) 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 the CD4 + T cell development, and therefore most of the 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- Y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)。 Using plasmid co-immune model (which in normal mice per million splenocytes an average of about 200 cells are induced to produce IFN- Y), at absolutely no circumstances CD4 cells, IL-15 induced not save 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,CD4 T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 involve amplification of CD4 (Fig. 6), so that the defect is a matter of course because of the lack of another function provided by T helper cells, CD4 T helper cells also provide help to CD8 amplification, i.e., by antigen presenting cells (APC) activation. 在这一APC活化模型中,APC上的⑶40与T细胞⑶40配体的连接,上调B7的表达,使T细胞活化。 APC activation in this model, ⑶40 connection on the T cell APC ⑶40 ligand B7 regulated expression of T cell activation. 在I类MHC肽呈递的情况下,B7分子对⑶8 T细胞扩增提供共刺激。 In the case of class I MHC presented peptides, B7 molecules ⑶8 T cell co-stimulation provided amplification. 而且,Bourgeois等(以下参考文献37)证明,⑶40L能直接影响CD8记忆细胞的发育。 Moreover, Bourgeois (hereinafter Ref. 37) prove, ⑶40L can directly affect memory CD8 cell development.

[0190] 在⑶4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。 [0190] Defects in ⑶4 auxiliaries and show a lack of co-stimulatory level, it is next to be considered and studied. 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 treated with IL-15 and CD40L containing pCgag plasmids and co-immunoprecipitation. 使用锚定的⑶40L分子。 Use anchored ⑶40L molecules. 锚定的⑶40L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 )。 Anchored ⑶40L is expressed locally and in the transport of immune cells, but not secretion, which makes the test more complicated (the following references 38). 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 (following references 38) in plasmid model. 的确,当研究pCgag与P⑶40L联合使用时,在⑶40 KO小鼠中,诱导Gag特异性⑶8免疫应答(图8)。 Indeed, when used in combination study pCgag and P⑶40L in ⑶40 KO mice induced ⑶8 Gag-specific immune responses (Figure 8). 该数据进一步表明,IL-15直接影响记忆⑶8淋巴细胞。 The data further indicate, IL-15 directly affects memory ⑶8 lymphocytes. 当⑶4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 ⑶4 cells do not exist, IL-15 from the juvenile cells failed to induce antigen-specific CD8 cell responses.

[0191] 讨论 [0191] Discussion

[0192] HIV-1特异性⑶8免疫应答的维持和增强一直是许多研究的来源。 [0192] maintaining HIV-1-specific immune responses and enhance ⑶8 been the 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)。 Was observed in the mouse model, IL-15 exists, cell division can lead to memory (hereinafter Reference 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 functional analysis and research by the lack of IL12, IL-15 or its specific receptor transgenic mice genetically carried out, it 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、⑶44hi⑶8+T细胞提供有效而有区别的刺激。 Indeed, Zhang and his collaborators (hereinafter ref. 39) demonstrated in vivo mouse models, IL-15 on memory phenotype, ⑶44hi⑶8 + T cells provide effective 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 (hereinafter Ref. 40) reported that memory CD8 + T cell division by IL-15 stimulation. However, IL-2 inhibition. 也发现,IL-2抑制⑶8+记忆T细胞的增殖。 Also found that, IL-2 proliferation inhibition ⑶8 + memory T cells.

[0193] 本文所公开的研究工作证明,IL-15对体外和体内诱导⑶8+效应T细胞也特别有效。 [0193] The studies disclosed herein demonstrate, IL-15 in vitro and in vivo induction ⑶8 + effector T cells are particularly effective. 当与肽和IL-15 —起孵育时,从HIV-1感染者分离的⑶8+T细胞能以抗原特异性方式分泌IFN-Y。 When the peptides and IL-15 - from the incubation time from HIV-1 infected patients isolated ⑶8 + T cells can secrete antigen-specific manner IFN-Y. IL-15与TCR协同作用,刺激淋巴细胞产生IFN-Y并确保效应子表型。 IL-15 and TCR synergies, stimulate lymphocytes to produce IFN-Y and make sure 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 resulted in 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 suggests that HIV infection, certain cells are activated portion and the active portion of such 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 in all patients, when using the IL-15 antigen and HIV-1 co-stimulation PBMC, a significant increase in effector function.

[0194] 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。 [0194] Currently, von Adrian and his colleagues (following reference 41) suggested that lymphocyte IL-15 stimulation leads to CD8 + T cells are directly processed into the juvenile cell memory 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 in this paper show that the combination TCR of CD8 + T cells may lead to more complete activation, suggesting that IL-15 alone the role of 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 it shows, IL-15 leads to non-functional memory cells (Ref. 41 hereinafter). 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⑶8+T细胞。 The data demonstrate that the human and mouse studies, IL-15 can result in a complete functional amplification ⑶8 + T cells. 在小鼠中,IL-15显著增加⑶8+T细胞应答以及增加趋化因子和IFN-Y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 ⑶8 + T cell responses, and increased chemokine and IFN-Y responses, which clearly indicates that antigen-specific amplification and build on previous work (following references 33 and 42). 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CD4 T细胞在发生CD8应答方面具有重要作用。 However, CD4 T 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⑶40L,可以遏止对⑶4 T细胞的需要。 In CD4 knockout mice by using ⑶40L, we can curb the need for ⑶4 T cells. 这一发现对病毒感染的免疫治疗极其重要。 This discovery is extremely important for viral infection immunotherapy.

[0195] 许多免疫治疗策略都集中于扩增⑶8+Τ细胞应答。 [0195] Many immunotherapeutic strategies have focused on the amplification ⑶8 + Τ cell response. 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。 In turn, said that the absence of such assistance, resulting in invalid CD8 + T cell response. 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。 Generally require chronic infection in CD4 + helper, in order to maintain control of viral replication, examples of which may be HIV-1 infection. Serbina等(以下参考文献43)证明,⑶8+细胞毒性T细胞的发展依赖于⑶4+Τ细胞。 Serbina (hereinafter reference 43) demonstrated that, ⑶8 + cytotoxic T cell development depends on ⑶4 + Τ cells. 他们还观察到,在⑶4 T细胞敲除小鼠中具有降低的IL-15产生。 They also observed that, ⑶4 T cell knockout mice have reduced in IL-15 produced. 然而,IL-15并不由⑶4+Τ细胞产生。 However, IL-15 does not generate the ⑶4 + Τ 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⑶4+Τ细胞增加IL-15产生的某些反馈机制,而在降低⑶4辅助的情况下,最终CD8+T细胞功能也下降。 There may be some increase in cells ⑶4 + Τ feedback mechanisms produced IL-15, and in the case of reducing the auxiliary ⑶4 final CD8 + T cell function is also decreased. 该反馈机制可解释,为什么6个患者中有3个IFN-Y产生是在单独添加IL-15之后。 The feedback mechanism may explain why the six patients had three IFN-Y generation is added separately after IL-15. 因此,在⑶4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⑶8+Τ淋巴细胞。 Thus, in the absence of ⑶4 and IL-15 in the case of a low level, in HIV-1 seropositive patients, the virus can only be part of the residual ⑶8 + Τ lymphocyte activation. 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, you can add IL-15, to change the defects caused by the immune suppression caused by the virus. 应该在免疫治疗HIV-1的领域中,考虑该假说的意义。 It should be in the field of immunotherapy of HIV-1, considering the significance of the Hypothesis.

[0196] 总的来说,IL-15在小鼠中增强⑶8+Τ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。 [0196] In general, IL-15 enhanced ⑶8 + Τ cell effector function in mice and enhance the function of T cells isolated positive patients infected with HIV-1 from 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。 It should be considered, as a supplement to the IL-15 active immunotherapy, as adjuvant therapy for HAART.

[0197] 参考文献(其各自通过引用结合到本文中) [0197] Reference (which each herein incorporated by reference)

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APlenotropic Cytokine with Diverse Receptor / SignalingPathways Whose Expressionis Controlled at Multiple Pathways (Plenotropic cytokine having different receptor / signaling pathway, which is expressed in many ways controlled) .1mmunity 4: 329- 336. [0221] 24.Chang, KH, Kim, JM, Kim HY, Sorig, YG, Choi, YH, Park, YS, Cho, JH , Hong, SK., 2000, Spontaneous Programmed Cell Death ofPeripheral Blood Mononuclear Cellsfrom HIV-1nfected Persons isDecreased with IL-15 (HIV infected peripheral blood mononuclear cell spontaneous programmed cell death, with the IL-15 and lower) .Yonsei Medical Journal, 41:. 112-118 [0222] 25.Naora H ., Gougeon, ML, 1999, Enhanced Survival and PotentExpansion ofthe Natural Killer Population of HIV Infected Individuals byExogenous IL_15 (Xi Bu source IL-15 increased survival of HIV-infected persons and efficient amplification of natural killer group) .1mmunology Letters, 68: 359-367. 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Kacani, L., Sprizl GM., Erdei, A., Dierich, MP, 1999, Interleukin-15Enhances HIV-1 Driven Polyclonal B-Cell Response inVitro (interleukin -15 in vitro to enhance HIV-1 driven polyclonal B cell response ) .Expand Clin Immun, 16:. 167-179 [0226] 29.Kacani, L., Stoiber H., Dierich, MP, 1997, Role of IL-15 in HIV-1Associated Hypergammaglobulinanemia (IL-15 in the HIV-1 Related high hypogammaglobulinemia Role) .Clin Exp Imm 108:. 14-18 [0227] 30.Agostini, C., Zambello, R., Perrin A., Piazzo, F., Siviero, M., Basso, U., Borfolin, M., Trentin L., Semenatao, G., 1999, CD8 T-CellInfiltration inExtravascular Tissues of Patients with HumanImunodeeiciency Virus Infection.1nterleukin 15 Upmodulates Co-Stimulatory Pathways Involved in the AntigenPresenting Cells of T-celllnteraction (people immunodeficiency virus infection extravascular tissue CD8 T cell infiltration. 白介素15上调涉及T细胞相互作用抗原呈递细胞的共同剌激途径).Blood,93:1277-1286.[0228] 31.Patki,AH.,Quinone S.,Mateo, ME.,Dorazio, D.,Yen-Lieberman Β.,BoomWH, Thomas, EK., Lederm`ann MM,1996, Activation of antigen Inducaed LymphocuteProliferation by Interleukin-15 Without the Mitogenic Effect og Interleukin-2That May InduceHuman Immunodefeciency Virus-1 Expression (由白介素-15 激活抗原诱导的淋巴细胞增殖,没有对可诱导人免疫缺陷病毒-1表达的白介素_2的促有丝分裂原效应).JCI,98:616-621.[0229] 32.Lucey DR.,Pinto LA,Bethke,FR.,Rusnak J.,Mekher GP, Hashemi,FN,Landay, AL,Kessler,HA, f Paxton, RJ.,Grabstein,K.,Shearer GM.,In VitroImmunologic and Virologic Effects of Interleukin-15 on Peripheral BloodMononuclear Cells from Normal Donors andHuman Immunodeficiency Virus Type-1Infected Patients (白介素-15对来自正常供体和人免疫缺陷病毒I型感染者的外周血单核细胞的体外免疫学和病毒学效应).Clin Diagn Lab Imm,4:43-48.[0230] 33.Kim, JJ.Trivedi, NN.,Nottingham,LK.,Morrison, L,Tsai, A.,Hu, Y.,Mahalingarn, S.,Dang, K.,Aim,L,Doyle, NK.,Wilson, D.,Chattergoon,MA.,Chalian,AA.,Boyer, JD.,Agadjanyan, M.,Weiner, DB.,1998, Modulation of amplitutude anddirection of in vivo immuneresponses by co-administration of cassettes withDNA immunogens (共同给予DNA免疫原盒,对体内免疫应答的幅度和方向的调节),Eur.JImmunol.28:1089-1103.[0231] 34.Kim JJ.Tsai A.Nottingham LK.Morrison L.Cunning DM.0h J.Lee DJ.DangK.Dentchev T.Chalian AA.Agadjanyan MG.Weiner DB,1999,Intracellular adhesionmolecule-1 modulates beta—chemokines anddirectly costimulates T cells invivo (胞内粘附分子-1在体内调节β -趋化因子并直接共同刺激T细胞),Journal ofClinical Investigation.103:869-77.[0232] 35.Mata, M.,Travers, PJ.Liu, Q.,Frankel,FR.,Paterson, Y.,1998,The MHCClass I Restricted Immune Response to HIV-1 gag in Balb/cMice Selects a SingleEpitope That Does not Have a Predictable MHCBinding-Motif and Binds to KdThrough Interactions Between aGlutamine at PS and Pocket D (在Balb/c 小鼠中,针对HIV-1 gag的MHCI类限制性免疫应答选择单个表位,所述表位没有可预测的MHC结合基序,并通过在PS的谷氨酰胺与口袋之间的相互作用而结合Kd),Jo of Immun., 161:2985-2993.[0233] 36.McCarrick III JW, Parties JR, Seong RH,Solter D,Knowles BB.1993.Positive-negative selection gene targeting with the diphtheria toxinA-chaingene in mouse embryonic stem cells (在小鼠胚胎干细胞中,用白喉毒素A链基因进行正-负选择基因打革E ).Transgenic Res 2:183-190.[0234] 37.Bourgeois,C.,Rocha,B.,Tanchot,C.,A Role for CD40Expression on CD8+TCells in the Generation of CD8+T Cell Memory (在CD8+T 细胞记忆产生过程中,CD40表达对CD8+T 细胞的作用),2002,Science 297:2060-2063.[0235] 38.Sin J1.Kim JJ.Zhang D.Weiner DB.Modulation of cellularresponses byplasmid CD40L:CD40L plas mid vectors enhance antigen-specific helper T cell typeI CD4+T cell-mediated protective immunityagainst herpes simplex virus type 2in vivo (质粒CD40L对细胞应答的调节:CD40L质粒载体在体内增强抗原特异性辅助T细胞I型⑶4+T细胞介导的针对单纯疱疹病毒2型的保护性免疫).Human GeneTherapy.12:1091-102,2001.[0236] 39.Zhang, X.,Sun,S.,Hwang, 1.,Tough,DF.Sprent, 1998,J.,Immunity,8:591-599.[0237] 40.Ku,CC.,Murakami, M.,Sakamoto,A.,KappIer,J.,Marrack.P.,2000,Controlof Homerostasis of CD8 Memory T Cells by OpposingCytokines (相反细胞因子对CD8记忆T细胞稳态的控制),Science,288:675-678.[0238] 41.Manjunath, N.,Shankar, P.,Wan, J.,Weninger, W.,Crowley, MA.Hieshmina,K., Springer, TA., Fan, X., Shen, H., Lieberman, J., vonAndrian, UH.,2001, Effectordifferentiation is not prerequisite forgeneration of memory cytotoxic Tlymphocytes (效应细胞分化对于记忆细胞毒性T淋巴细胞的产生来说并非必要),Journalof Clinicallnvestigation, 108:871-878.[0239] 42.Moore AC.Kong WP.Chakrabarti BK.Nabel GJ.Effects ofantigen andgenetic adjuvants on immune responses to humanimmunodeficiency virus DNAvaccines in mice (在小鼠中,抗原和遗传佐剂对针对人免疫缺陷病毒DNA疫苗的免疫应答的作用).Journal ofVirology.76(1):243-50,2002 年I 月.[0240] 43.Serbina NV.Lazarevic V.Flynn JL 2001,CD4(+)T cells arerequired forthe development of cytotoxic CD8 (+)T cells duringMycobacterium tuberculosisinfection (在结核分枝杆菌感染中,CD4 (+) T细胞对细胞毒CD8 (+) T细胞发育来说是必需的).Journal ofImmunology.167:6991-7000.[0241] 实施例2 Interleukin-15 regulation involving T cell co-stimulation pathway interaction of antigen-presenting cells) .Blood, 93:. 1277-1286 [0228] 31.Patki, AH, Quinone S., Mateo, ME, Dorazio, D.,.. Yen-Lieberman Β., BoomWH, Thomas, EK., Lederm`ann MM, 1996, Activation of antigen Inducaed LymphocuteProliferation by Interleukin-15 Without the Mitogenic Effect og Interleukin-2That May InduceHuman Immunodefeciency Virus-1 Expression (activated by the interleukin -15 antigen-induced lymphocyte proliferation, no to human immunodeficiency virus-1-induced expression of interleukin-_2 promoting mitogen effect) .JCI, 98: 616-621 [0229] 32.Lucey DR, Pinto LA, Bethke.. , FR., Rusnak J., Mekher GP, Hashemi, FN, Landay, AL, Kessler, HA, f Paxton, RJ., Grabstein, K., Shearer GM., In VitroImmunologic and Virologic Effects of Interleukin-15 on Peripheral BloodMononuclear Cells from Normal Donors andHuman Immunodeficiency Virus Type-1Infected Patients (in vitro immunologic and virologic effects of interleukin-15 on from a normal donor and the human immunodeficiency virus type I infection of peripheral blood mononuclear cells) .Clin Diagn Lab Imm, 4:... 43-48 [0230] 33.Kim, JJ.Trivedi, NN, Nottingham, LK, Morrison, L, Tsai, A., Hu, Y., Mahalingarn, S., Dang, K., Aim , L, Doyle, NK., Wilson, D., Chattergoon, MA., Chalian, AA., Boyer, JD., Agadjanyan, M., Weiner, DB., 1998, Modulation of amplitutude anddirection of in vivo immuneresponses by co -administration of cassettes withDNA immunogens (co-administered DNA immunogen box, the amplitude and direction of immune response regulation), Eur.JImmunol.28: 1089-1103 [0231] 34.Kim JJ.Tsai A.Nottingham LK.. Morrison L.Cunning DM.0h J.Lee DJ.DangK.Dentchev T.Chalian AA.Agadjanyan MG.Weiner DB, 1999, Intracellular adhesionmolecule-1 modulates beta-chemokines anddirectly costimulates T cells invivo (intracellular adhesion molecule-1 in body regulate β - chemokine and co-stimulation of T cells directly), Journal ofClinical Investigation.103: 869-77 [0232] 35.Mata, M., Travers, PJ.Liu, Q., Frankel, FR, Paterson.. , Y., 1998, The MHCClass I Restricted Immune Response to HIV-1 gag in Balb / cMice Selects a SingleEpitope That Does not Have a Predictable MHCBinding-Motif and Binds to KdThrough Interactions Between aGlutamine at PS and Pocket D (in Balb / c mice against HIV-1 gag in MHCI-restricted immune responses to select a single epitope, the epitope is no predictable MHC binding motifs, and by between glutamine and PS interaction pocket combined with Kd), Jo of Immun, 161:.. 2985-2993 [0233] 36.McCarrick III JW, Parties JR, Seong RH, Solter D, Knowles BB.1993.Positive-negative selection gene targeting with the diphtheria toxinA-chaingene in mouse embryonic stem cells (mouse embryonic stem cells, with the diphtheria toxin A chain gene were positive - negative selection gene dozen leather E) .Transgenic Res 2: 183-190 [0234] 37.Bourgeois, C., Rocha,. B., Tanchot, C., A Role for CD40Expression on CD8 + TCells in the Generation of CD8 + T Cell Memory (CD8 + T cell memory in the selection process, CD40 expression of CD8 + T cells role), 2002, Science 297: 2060-2063 [0235] 38.Sin J1.Kim JJ.Zhang D.Weiner DB.Modulation of cellularresponses byplasmid CD40L:. CD40L plas mid vectors enhance antigen-specific helper T cell typeI CD4 + T cell-mediated protective immunityagainst herpes simplex virus type 2in vivo (plasmid CD40L regulation of cellular responses: CD40L plasmid vector in vivo to enhance antigen-specific T helper cell type I ⑶4 + for simple T cell-mediated herpes simplex virus type 2 protective immunity) .Human GeneTherapy. 12:. 1091-102,2001 [0236] 39.Zhang, X., Sun, S., Hwang, 1., Tough, DF.Sprent, 1998, J, Immunity, 8:. 591-599 [0237]. 40.Ku, CC., Murakami, M., Sakamoto, A., KappIer, J., Marrack.P., 2000, Controlof Homerostasis of CD8 Memory T Cells by OpposingCytokines (opposite cytokines on CD8 memory T cell homeostasis control), Science, 288:. 675-678 [0238] 41.Manjunath, N., Shankar, P., Wan, J., Weninger, W., Crowley, MA.Hieshmina, K, Springer, TA,.. Fan, X., Shen, H., Lieberman, J., vonAndrian, UH., 2001, Effectordifferentiation is not prerequisite forgeneration of memory cytotoxic Tlymphocytes (for differentiated effector cell memory generation of cytotoxic T lymphocytes is not necessary), Journalof Clinicallnvestigation, 108:. 871-878 [0239] 42.Moore AC.Kong WP.Chakrabarti BK.Nabel GJ.Effects ofantigen andgenetic adjuvants on immune responses to humanimmunodeficiency virus DNAvaccines in mice (in mice, antigens and genetic adjuvant against human immunodeficiency virus DNA vaccine immune response action) on .Journal ofVirology.76 (1):. 243-50,2002 on I January [0240] 43.Serbina NV.Lazarevic V.Flynn JL 2001, CD4 (+ ) T cells arerequired forthe development of cytotoxic CD8 (+) T cells duringMycobacterium tuberculosisinfection (Mycobacterium tuberculosis infection, CD4 (+) T cells to cytotoxic CD8 (+) T cell development is required) .Journal ofImmunology .167: 6991-7000 [0241] Example 2

[0242] 需要⑶4(+) Th细胞并产生IFN-Y,以在免疫抑制个体体内以及抗肿瘤免疫中控制病毒复制。 [0242] need ⑶4 (+) Th cells and produce IFN-Y, in immunosuppressed individuals with anti-tumor immunity in vivo and in 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⑶40L来替代。 Data from experiments demonstrated that the T cell help needed to amplify CD8 effector T cells, the vaccine can be locally produced IL-15 parts and ⑶40L instead. 用通过基因敲除II类MHC的β -链(MHC IIΚ0)而消除⑶4 (+) T细胞的小鼠进行实验,揭示出用抗原gag+IL-15+⑶40L初次免疫所述动物,导致CD8 T细胞的活化。 By using the MHC class II knockout of β - chain (MHC IIΚ0) eliminated ⑶4 (+) T cells in mouse experiments, revealed by an antigen gag + IL-15 + ⑶40L primary immunization of the animal, resulting in CD8 T cell activation. 用IFN-Y的产生(以斑点表示)测定活化。 With IFN-Y production (indicated by dots) measured activation. 在该测定中多于50点的为阳性。 More than 50 points in this assay was positive. 这些数据见图9,说明了用于测定不依赖⑶4(+)T细胞辅助的效应⑶8T细胞的活化的简单方法。 The data shown in Figure 9 shows a simple method for the determination does not depend ⑶4 (+) T cell help effect ⑶8T cell activation. 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 a great significance.

[0243] 实施例3 [0243] Example 3

[0244] 人、小鼠和猿猴IL-15 cDNA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。 [0244] human, mouse and simian IL-15 cDNA encoding 162 amino acids are precursor protein (aa) residues, leader sequence containing 48 aa residues after removal will give its 114 aa residues 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 has activity on mouse cells. 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 the forecast is similar to IL-2 and its four other members of the helical bundle cytokine family. (Grabstein, K.等(1994) Science 264:965, Anderson, D.Μ.等(1995) Genomics25:701 ;和Bamford, RN等(1995) Cytokine 7:595, Brandhuber, BJ等(1987) Science238:1707,所述文献都通过引用结合到本文中。) (Grabstein, K. et (1994) Science 264: 965, Anderson, D.Μ. etc. (1995) Genomics25: 701; and Bamford, RN, etc. (1995) Cytokine 7: 595, Brandhuber, BJ et (1987) Science238: 1707, the documents are incorporated by reference herein.)

[0245] IL-15 mRNA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。 [0245] IL-15 mRNA in heart, lung, liver, placenta, skeletal muscle, adherent peripheral blood mononuclear cells, APC (dendritic cells) and epithelial cells and fibroblast cell lines can be detected. 然而,IL-15 mRNA在含有高水平IL-2mRNA的活化外周血T细胞中没能检出。 However, IL-15 mRNA 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.1mmunol.154:483 ;P.Wilkinson (Armitage, RJ et (1995) J.1mmunol.154: 483; P.Wilkinson

F.Liew (1995) J.Exp.Med.181: 1255 ;Grabstein, K.等(1994) Science 264:965 ;Giri,JG等(1994) EMBO J.13:2822 #PGiri,JG等(1995) EMBO J.15:3654,每篇文献都通过引用结合到本文中)。 F.Liew (1995) J.Exp.Med.181: 1255; Grabstein, K. et (1994) Science 264: 965; Giri, JG et (1994) EMBO J.13: 2822 # PGiri, JG et al. (1995) EMBO J.15: 3654, each document are incorporated by reference herein).

[0246] 因为IL-15是原型Thl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。 [0246] Because IL-15 is a prototype Thl cytokine, but also because as T cells, NK cells, LAK cells, and TIL stimulating actives, IL-15 is an exciting candidate, as a molecular adjuvant may and DNA vaccines (e.g., an HIV vaccine) in combination with, 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.

[0247] RNA印迹分析表明IL-15的广泛组成型表达。 [0247] RNA blot analysis indicated that extensive constitutive expression 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。 Control transcriptional expression occurs in translation and transport (intracellular transport) level. IL-15 mRNA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-15 mRNA contains many prevent its translation into protein component comprising 1) 5 IL-15 Translation 'AUG with upstream AUG, which is effective interference (five mice, who have 12); 2) IL -15 initiation codon of the coding sequence with weak KOZAK area (GTAATGA); 3) C- terminal mature IL-15 protein coding sequence negative elements. (Grabstein 等(1994)Science 264:965-968,Bamford 等(1996)PNAS 93:2897-2902 ;Bamford 等(1998)J.1mmunol 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 et (1994) Science 264: 965-968, Bamford et (1996) PNAS 93: 2897-2902; Bamford et (1998) J.1mmunol 160: 4418-4426; and Kozak et (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 the expression.

[0248] 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。 [0248] Natural IL-15 isoform contains two leader peptide: 21 amino acid signal peptide (SSP), or 48 amino acid signal peptide (LSP) (Waldmann et, Ann.Rev> Immunol (1999) 17.: 19-49, which is incorporated herein by reference.

[0249] 进行下述策略:通过优化用于免疫的IL-15 DNA构建体,来增加IL-15的表达。 [0249] subjected to the following strategy: optimized for IL-15 DNA immunization constructs, 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 the signal peptide starting IL-15, thus inhibiting upstream AUG is not present in the final messenger of IL-15. 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 element PCR antisense primer designed to remove. 所述引物见图10。 The primer shown in Figure 10.

[0250] 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。 [0250] subjected to the following strategy: by substitution with IgE leader sequence of 48 amino acid IL-15 signal peptide (LSP), 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。 Sense primer designed from the 48 amino acids of LSP beginning, and 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 a strong KOZAK zone (GCCGCCACC-SEQ ID NO: 4). 有义引物设计成含有IgEill导序列加上ATG起始位点的序列。 Sense primer designed to IgEill leader sequence plus the ATG start site containing sequences. 所述引物见图11。 The primer shown in Figure 11.

[0251] 制备不同的构建体,用于转染RD细胞。 Different constructs [0251] Preparation of the body, for the transfection of 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-15 SSP-左)的编码序列和人48个氨基酸信号肽(IL-15 LSP-右)的编码序列的人构建体的表达比较。 13A shows, Bag 21 contains amino acid signal peptide and IL-15 connection (IL-15 SSP- left) coding sequence of human 48 amino acid signal peptide (IL-15 LSP- right) Construction of the coding sequence of human Comparison of expression of the body. 图12B显示,包含48个氨基酸信号肽(人IL-15 LSP-左)的编码序列和IgE信号肽(人IL-15-1gE-右)的编码序列的人构建体的表达比较。 12B shows, the signal peptide contains 48 amino acids (human IL-15 LSP- left) and IgE signal peptide coding sequence (human IL-15-1gE- right) coding sequence comparison of expression of the human construct. 图12C显示,包含48个氨基酸信号肽(恒河猴IL-15 LSP-左)的编码序列和IgE信号肽(恒河猴IL-15-1gE-右)的编码序列的恒河猴构建体的表达比较。 12C shows, contains 48 amino acid signal peptide (rhesus IL-15 LSP- left) of the rhesus monkey coding sequence and IgE signal peptide coding sequence (rhesus IL-15-1gE- right) construct Comparison of expression.

[0252] 对不同构建体所产生的IL-15蛋白,测定IL-15的生物活性。 [0252] The different constructs produced protein IL-15, IL-15 was measured biological activity. 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-15-1gE-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-15-1gE- right) construct coding sequence 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-15 LSP-左)的编码序列和IgE信号肽(恒河猴IL-15-1gE-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, the signal peptide contains 48 amino acids (rhesus IL-15 LSP- left) and rhesus monkeys IgE signal peptide coding sequence (rhesus IL-15-1gE- right) of the coding sequence of the construct Comparison of the biological activity of IL-15 between.

[0253] 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。 [0253] and insertion sequences having IgE signal peptide coding sequence of the IL-15 coding sequence of an expression vector pVAX, prepared constructs. 也得到编码HIV-1 Gag的构建体。 Also encodes HIV-1 Gag construct. 进行免疫学实验,用IL-15工程质粒以及HIV-1 Gag,比较对免疫应答的效应。 Immunological experiments with plasmid IL-15 engineering and HIV-1 Gag, 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 programs in accordance with FIG. 14, for Balb / c mice were vaccinated.

[0254] 在第三次免疫后5周,通过比较对抗原特异性IFN- Y产生的再次刺激,研究免疫应答。 [0254] In the five weeks after the third immunization, by comparison with the antigen-specific IFN- Y generated again stimulated 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-1 Gag的构建体的小鼠,接种编码HIV-1 Gag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-1 Gag以及IgE信号肽的构建体的小鼠。 Vaccine group comprising naive mice, mice inoculated carrier PCDN3 inoculated encoding HIV-1 Gag construct mice inoculated encoding HIV-1 Gag body and IL-15 (48 amino acid signal peptide is connected) of construct mice inoculated encoding HIV-1 Gag and IgE signal peptide of mouse body construct.

[0255] 实施例4 [0255] Example 4

[0256] 构建工程化IL-15质粒疫苗,即通过除去天然IL-15 Kozak区、AUG和UTR。 [0256] Construction of engineered IL-15 plasmid vaccine, namely by removing natural IL-15 Kozak region, AUG and UTR. 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 levels higher than the relevant wild-type plasmid 30-50 times. 在用工程化IgE信号-1L-15和HIV-1 gag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-1 gag构建体免疫的小鼠高几倍。 With engineered IgE signal -1L-15 and HIV-1 gag construct common immunized mice, the immune responses observed significant than single HIV-1 gag constructs mice immunized several times higher. 数据见图16。 Data shown in Figure 16.

[0257] 实施例5 [0257] Example 5

[0258] 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。 [0258] encoding immunomodulatory protein isolated cDNA construct can be used as raw material to 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., constructs described above.

[0259] 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。 [0259] Using standard techniques and readily available raw materials, can be prepared by a nucleic acid molecule encoding immunomodulatory proteins, and incorporated into the construct as described above, vectors, vaccines and the like.

[0260] Genbank检索号AF031167是指人IL-15 mRNA的完整编码序列。 [0260] Genbank accession numbers AF031167 refers to human IL-15 mRNA of the complete coding sequence. Genbank检索号Y09908、X91233、X94223和Χ94222也是指人IL-15序列。 Genbank accession numbers Y09908, X91233, X94223 and Χ94222 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.

[0261] Genbank检索号L07414是指人⑶40-配体mRNA的完整编码序列。 [0261] Genbank accession number L07414 refers to the complete coding sequence of human ⑶40- ligand mRNA. 其通过引用结合到本文中。 Which is incorporated herein by reference.

[0262] 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。 [0262] Bax of nucleotide and amino acid sequence of GENBANK Accession No. L22473, which is incorporated by reference herein.

[0263] 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。 [0263] TRAIL nucleotide sequence and amino acid sequence of GENBANK Accession No. U37518 or AF023849, which is incorporated by reference herein.

[0264] 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 [0264] TRAILrecDRC5 of U90875 or AF016266, which is incorporated by reference herein. 通过引用结合到本文中的还有TRAIL-R2 AFO16849 ;TRAIL-R3 AF014794;和TRAIL-R4 AF021232。 Incorporated herein by reference, there TRAIL-R2 AFO16849; TRAIL-R3 AF014794; and TRAIL-R4 AF021232.

[0265] 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。 [0265] RANK nucleotide sequence and amino acid sequence of GENBANK Accession No. AF018253, which is incorporated by reference herein.

[0266] RANK配体的核苷酸序列和氨基酸序列的GENBANK检索号为AFO19047或AF333234,其通过引用结合到本文中。 [0266] RANK ligand GENBANK accession numbers nucleotide and amino acid sequence of AFO19047 or AF333234, which is incorporated by reference herein.

[0267] 0x40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。 GENBANK Accession No. nucleotide and amino acid sequences [0267] 0x40 for X75962, which is incorporated by reference herein.

[0268] 0x40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。 [0268] 0x40 nucleotide and amino acid sequence of GENBANK accession number X79929 or ligand of AB007839, which is incorporated by reference herein.

[0269] 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。 [0269] NKG2D the nucleotide and amino acid sequence of GENBANK accession number is AF461811 or X54870, which is incorporated by reference herein.

[0270] MICA的核苷酸序列和氨基酸序列的GENBANK检索号为X92841,其通过弓丨用结合到本文中。 [0270] MICA of nucleotide and amino acid sequence of GENBANK Accession No. X92841, its use by bow Shu incorporated herein.

[0271] 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。 [0271] The nucleotide and amino acid sequences of the MICB, GENBANK accession number is U65416, which is incorporated by reference herein.

[0272] 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。 GENBANK Accession No. nucleotide and amino acid sequences [0272] NKG2A is X54867, which is incorporated by reference herein.

[0273] 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。 GENBANK Accession No. nucleotide and amino acid sequences [0273] NKG2B is X54868, which is incorporated by reference herein.

[0274] 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。 [0274] NKG2C of nucleotide and amino acid sequence of GENBANK Accession No. X54869 or Aj0016984, which is incorporated by reference herein.

[0275] 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。 GENBANK Accession No. nucleotide and amino acid sequences [0275] NKG2E is L14542, which is incorporated by reference herein.

[0276] 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。 [0276] NKG2F of nucleotide and amino acid sequence of GENBANK accession numbers for the AH006173, U96845 or U96846, which is incorporated by reference herein.

[0277] CD30的核苷酸序列和氨基酸序列的GENBANK检索号为M83554,(Durkop,H等,Cell68 (3),421-427 (1992)),其通过引用结合到本文中。 GENBANK Accession No. nucleotide and amino acid sequences [0277] CD30 is M83554, (Durkop, H, etc., Cell68 (3), 421-427 (1992)), which is incorporated by reference herein.

[0278] ⑶153 (⑶30L)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。 [0278] ⑶153 (⑶30L) nucleotide and amino acid sequence of GENBANK Accession No. L09753, (Smith, CA, etc., Cell 73 (7), 1349-1360 (1993)), which is incorporated by reference herein.

[0279] 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。 [0279] Fos the nucleotide sequence of GENBANK Accession No. K00650 or V01512, which is incorporated by reference herein.

[0280] 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。 [0280] GENBANK Accession No. c-jun nucleotide sequence is J04111 or M29039, which is incorporated by reference herein.

[0281] 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。 [0281] GENBANK Accession No. Sp-1 nucleotide sequence is BC021101, BC005250, BC002878, M31126, J02893 or X15102, which is incorporated by reference herein.

[0282] Apl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 [0282] Apl 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, 1988Cell 54: 541_552, which by reference

用结合到本文中。 With incorporated herein.

[0283] 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。 The nucleotide sequence of [0283] Ap-2 of GENBANK accession numbers for the M36711, which is incorporated by reference herein.

[0284] 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。 GENBANK Accession No. nucleotide sequence [0284] p38 is U66243, which is incorporated by reference herein.

[0285] 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。 [0285] GENBANK Accession No. p65Rel nucleotide sequence is L19067, which is incorporated by reference herein.

[0286] 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。 GENBANK Accession No. nucleotide sequence [0286] MyD88 is U70451, which is incorporated by reference herein.

[0287] 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。 [0287] IRAK GENBANK nucleotide sequence accession numbers for the NM001569, which is incorporated by reference herein.

[0288] 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。 GENBANK Accession No. nucleotide sequence [0288] TRAF6 is U78798, which is incorporated by reference herein.

[0289] IkB的核苷酸序列可在以下文献中找到:Gilmore等,Trends Genetl993年12月;9(12):427-33,其通过引用结合到本文中。 [0289] IkB nucleotide sequence can be found in the following documents: Gilmore, etc., Trends Genetl993年 Dec; 9 (12): 427-33, which is incorporated by reference herein.

[0290] 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。 [0290] NIK nucleotide sequence of GENBANK Accession No. Y10256, which is incorporated by reference herein.

[0291] 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。 [0291] The nucleotide sequence of SAP K can be found in the following documents: Franklin, etc., Oncogene 1995 年 12 月 7 days; 11 (11): 2365-74, which is incorporated by reference herein.

[0292] SAPl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。 [0292] SAPl nucleotide sequence of GENBANK Accession No. M85164 or M85165, which is incorporated by reference herein.

[0293] 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。 GENBANK Accession No. nucleotide sequence [0293] JNK2 is L31951, which is incorporated by reference herein.

[0294] 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。 GENBANK Accession No. nucleotide sequence [0294] JNK1B2 is U35005; which is incorporated by reference herein.

[0295] JNKlBl的核苷酸序列的GENBANK检索号为U35004 ;其通过引用结合到本文中。 [0295] JNKlBl GENBANK nucleotide sequence accession number is U35004; which is incorporated by reference herein.

[0296] 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。 GENBANK Accession No. nucleotide sequence [0296] JNK2B2 is U35003; which is incorporated by reference herein. [0297] 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。 GENBANK Accession No. nucleotide sequence [0297] JNK2B1 is U35002; which is incorporated by reference herein.

[0298] JNKIA2的核苷酸序列的GENBANK检索号为U34822 ;其通过引用结合到本文中。 GENBANK Accession No. nucleotide sequence [0298] JNKIA2 is U34822; which is incorporated by reference herein.

[0299] 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。 GENBANK Accession No. nucleotide sequence [0299] JNK2A1 is U34821; which is incorporated by reference herein.

[0300] 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。 GENBANK Accession No. nucleotide sequence [0300] JNK3A1 is U34820; which is incorporated by reference herein.

[0301] 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。 GENBANK Accession No. nucleotide sequence [0301] JNK3A2 is U34819, which is incorporated by reference herein.

[0302] 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。 [0302] NF- κ -B2 namely p49 splice form of the nucleotide sequence of GENBANK Accession No. A57034, which is incorporated by reference herein.

[0303] NF- κ -B2即plOO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。 [0303] NF- κ -B2 That plOO splice form of the nucleotide sequence of GENBANK Accession No. A42024, which is incorporated by reference herein.

[0304] NF- κ -B2即pl05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。 [0304] NF- κ -B2 That pl05 splice form of the nucleotide sequence of GENBANK Accession No. S17233, which is incorporated by reference herein.

[0305] 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。 [0305] NF- κ nucleotide sequence of GENBANK Accession No. -B 50K chain precursor of A37867, which is incorporated by reference herein.

[0306] NF κ B p50 的核苷酸序列描述于:Meyer R.等(1991) Proc.Natl.Acad.Sc1.USA88(3),966 970,其通过引用结合到本文中。 [0306] NF κ B p50 nucleotide sequences are described: Meyer R. et (1991) Proc.Natl.Acad.Sc1.USA88 (3), 966 970, which is incorporated by reference herein.

[0307] 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 ^ (1985)Nature 315:641-647和检索号Swissprot P01583,其各自通过引用结合到本文中。 [0307] human IL-1 nucleotide and amino acid sequences are well known, as shown in the following documents: Telford et (1986) Nucl.Acids Res.14: 9955-9963, Furutani et (1985) Nucl.Acids Res. 14: 3167-3179, March ^ (1985) Nature 315: 641-647 and Accession No. Swissprot P01583, each of which is incorporated herein by reference.

``[0308] 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=HolbiOok等(1984) Proc.Natl.Acad.Sc1.USA 81:1634-1638, Fujita 等(1983) Proc.Natl.Acad.Sc1.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 P01585,其各自通过引用结合到本文中。 `` [0308] nucleotide and amino acid sequence of human IL-2 is well known, as shown in the following references = HolbiOok etc. (1984) Proc.Natl.Acad.Sc1.USA 81: 1634-1638, Fujita et al (1983 ) Proc.Natl.Acad.Sc1.USA 80: 7437-7441, Fuse, etc. (1984) Nucl.Acids Res.12: 9323-9331, Taniguchi et (1983) nature 302: 305-310, Meada etc. (1983) Biochem .Biophys.Res.Comm.115: 1040-1047, Devos et (1983) Nucl.Acids Res.11: 4307_4323, retrieve number Swissprot P01585, which each incorporated by reference herein.

[0309] 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.1mmunol.142:274-282 Otsuka 等(1987)Nucl.AcidsRes.15:333_344,Yokota 等(1986)Proc.Natl.Acad.Sci USA 83:5894_5898,Noma 等(I 984)Nature 319:640_646,Lee 等 [0309] The nucleotide and amino acid sequences of human IL-4 is well known, as shown in the following documents: Arai et (1989) J.1mmunol.142: 274-282 Otsuka et (1987) Nucl.AcidsRes.15: 333_344 , Yokota et (1986) Proc.Natl.Acad.Sci USA 83: 5894_5898, Noma, etc. (I 984) Nature 319: 640_646, Lee, etc.

(1986) Proc.Natl.Acad.Sc1.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。 (1986) Proc.Natl.Acad.Sc1.USA 83: 2061_2063, and Accession No. Swissprot 05112 (Accession No. murine IL-4 is Swissprot 07750), which are each incorporated by reference herein.

[0310] 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 [0310] The nucleotide and amino acid sequences of human IL-5 is well known, as shown in the following documents: Campbell et

(1987)Proc.Natl.Acad.Sc1.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.Sc1.USA 84:7388_7392,和检索号Swissprot P05113,其各自通过引用结合到本文中。 (1987) Proc.Natl.Acad.Sc1.USA 84: 6629-6633, Tanabe et (1987) J.Biol.Chem.262: 16580-16584, Campbell et (1988) Eur.J.Biochem.174: 345_352, Azuma et (1986) Nucl.Acids Res.14: 9149-9158, Yokota et (1986) Proc.Natl.Acad.Sc1.USA 84: 7388_7392, and Accession No. Swissprot P05113, which is incorporated herein by their reference.

[0311] 人IL-1O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.Sc1.USA 88:1172_1176,和检索号Swissprot P22301。 [0311] Nucleotide sequences and amino acid sequences of IL-1O is well known, as shown in the following documents: Viera et (1991) Proc.Natl.Acad.Sc1.USA 88: 1172_1176, and Accession No. Swissprot P22301.

[0312] 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SwissprotU03099,其各自通过引用结合到本文中。 [0312] human IL-15 nucleotide and amino acid sequences are well known, as shown in the following references = Grabstein etc. (1994) Science 264: 965_968, and Accession No. SwissprotU03099, their respective incorporated by reference herein.

[0313] 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.1mmunol.156:4274_4279,检索号为D49950,其各自通过引用结合到本文中。 [0313] The nucleotide and amino acid sequences of human IL-18 are well known, as shown in the following references = Ushio, etc. (1996) J.1mmunol.156: 4274_4279, retrieve number D49950, their respective incorporated by reference herein .

[0314] 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SwissprotP01375,其各自通过引用结合到本文中。 [0314] The nucleotide and amino acid sequences human TNF-α is well known, as shown in the following documents: Pennica, (1984) Nature 312: 724_729, retrieve number SwissprotP01375, their respective incorporated by reference herein.

[0315] 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 P01374,其各自通过引用结合到本文中。 Nucleotide and amino acid sequence [0315] human TNF-β are well known, as shown in the following documents: Gray, (1984) Nature 312: 721_724, and Accession No. Swessprot P01374, which each herein incorporated by reference. 人IL-1O氨基酸序列是众所周知的,示于以下文献:Viera等(1991)Proc.Natl.Acad.Sc1.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-1O amino acid sequence is known, as shown in the following documents: Viera et (1991) Proc.Natl.Acad.Sc1.USA 88: 1172-1176, and Accession No. Swissprot P22301, which each herein incorporated by reference.

[0316] 人白介素12 mRNA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。 [0316] human interleukin-12 mRNA complete coding sequence shown in Genbank accession numbers AF180563 (P40 mRNA) and AF180562 (P35 mRNA) and US Patent No. 5,840,530, each of which is incorporated herein by reference.

[0317] MadCAM-1 的序列信息参见Genbank 检索号U80016 (Leung, E.等,Immunogenetics46(2),111-119(1997)),其各自通过引用结合到本文中。 Sequence information, see Genbank [0317] MadCAM-1 search Number U80016 (Leung, E. et, Immunogenetics46 (2), 111-119 (1997)), which are each incorporated herein by reference.

[0318] MadCAM-1 的序列信息参见Genbank 检索号U43628 (Shyjan, A.Μ.等,J.1mmunol.156 (8),2851-2857 (1996)),其各自通过引用结合到本文中。 Sequence information, see Genbank [0318] MadCAM-1 search Number U43628 (Shyjan, A.Μ. etc., J.1mmunol.156 (8), 2851-2857 (1996)), which are each incorporated herein by reference.

[0319] NGF 的序列信息参见Genbank 检索号M57399 (Kretschmer, PJ等,GrowthFactors 5,99-114 (1991)),其各自通过引用结合到本文中。 [0319] NGF sequence information see Genbank accession number M57399 (Kretschmer, PJ etc., GrowthFactors 5,99-114 (1991)), which are each incorporated by reference herein.

[0320] IL-7 的序列信息参见Genbank检索号J04156 (Goodwin, RG等,Proc.Natl.Acad.Sc1.USA86(1), 302-306 (1989)),其各自通过弓I用结合到本文中。 Sequence information [0320] IL-7 See Genbank accession number J04156 (Goodwin, RG, etc., Proc.Natl.Acad.Sc1.USA86 (1), 302-306 (1989)), by their respective bow I used herein incorporated in.

[0321] 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。 [0321] VEGF sequence information parameters See Genbank accession number M32977 (Leung, DW, etc., Science 246,1306-1309 (1989)), which are each incorporated by reference herein.

[0322] TNF-R 的序列信息参见Genbank 检索号M60275 (Gray, PW等,Proc.Natl.Acad.Sc1.USA87,7380-7384 (1990)),其各自通过弓I用结合到本文中。 [0322] TNF-R sequence information see Genbank accession number M60275 (Gray, PW et, Proc.Natl.Acad.Sc1.USA87,7380-7384 (1990)), I bow with by their respective incorporated herein.

[0323] TNF-R的序列信息参见Genbank检索号M63121 (Himmler,A.等,DNA Cell Biol.9,705-715 (1990)),其各自通过弓I用结合到本文中。 [0323] TNF-R sequence information see Genbank accession number M63121 (Himmler, A., Etc., DNA Cell Biol.9,705-715 (1990)), I bow with by their respective incorporated herein.

[0324] 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。 [0324] Fas sequence information see Genbank Accession No. M67454 (Itoh, N. et, Cell 66 (2), 233-243 (1991)), which are each incorporated by reference herein.

[0325] 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。 [0325] CD40L sequence information see Genbank Accession No. L07414 (Gauchat, JFM, etc., FEBS Lett, 315,259-266 (1992), which are each incorporated by reference herein.

[0326] IL-4 的序列信息参见Genbank 检索号M23442 (Arai, N.等,J.1mmunol.142(1),274-282 (1989)),其各自通过弓I用结合到本文中。 [0326] IL-4 sequence information, see Genbank accession numbers M23442 (Arai, N. et, J.1mmunol.142 (1), 274-282 (1989)), I bow with by their respective incorporated herein.

[0327] IL-4 的序列信息参见Genbank 检索号M13982 (Yokota, T.等,Proc.Natl.Acad.Sc1.USA83 (16),5894-5898 (1986)),其各自通过弓I用结合到本文中。 [0327] IL-4 sequence information, see Genbank accession numbers M13982 (Yokota, T. et, Proc.Natl.Acad.Sc1.USA83 (16), 5894-5898 (1986)), I bow with by their respective binding to herein.

[0328] 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。 [0328] CSF sequence information see Genbank accession number M37435 (Wong, GG, etc., Science 235 (4795), 1504-1508 (1987)), which are each incorporated herein by reference.

[0329] G-CSF 的序列信息参见Genbank 检索号X03656 (Nagata, S.等,EMBO J.5 (3),575-581 (1986)),其各自通过弓I用结合到本文中。 [0329] G-CSF sequence information see Genbank Accession No. X03656 (Nagata, S. et, EMBO J.5 (3), 575-581 (1986)), through their respective I bow with incorporated herein.

[0330] 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。 [0330] G-CSF sequence information see Genbank Accession No. X03655 (Nagata, S. et, EMBO J.5 (3), 575-581 (1986)), which is incorporated herein by reference. [0331] GM-CSF 的序列信息参见Genbank检索号Ml 1220 (Lee, F.等,Proc.Ntl.Acad.Sc1.USA(13) ,4360-4364(1985)),其通过引用结合到本文中。 [0331] GM-CSF sequence information see Genbank Accession No. Ml 1220 (Lee, F. et, Proc.Ntl.Acad.Sc1.USA (13), 4360-4364 (1985)), which is incorporated herein by reference .

[0332] GM-CSF 的序列信息参见Genbank 检索号M10663(ffong, GG等,Science228 (4701),810-815 (1985)),其通过引用结合到本文中。 [0332] GM-CSF sequence information see Genbank accession number M10663 (ffong, GG, etc., Science228 (4701), 810-815 (1985)), which is incorporated herein by reference.

[0333] 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。 [0333] M-CSF sequence information see Genbank Accession No. M27087 (Takahashi, M. et, Biochem.Biophys.Res.Commun.161 (2), 892-901 (1989)), which is incorporated herein by reference.

[0334] M-CSF 的序列信息参见Genbank 检索号M37435(Wong GG等,Science235(4795),1504-1508 (1987)),其通过引用结合到本文中。 [0334] M-CSF sequence information see Genbank accession number M37435 (Wong GG et, Science235 (4795), 1504-1508 (1987)), which is incorporated herein by reference.

[0335] 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。 Sequence information [0335] LFA-3, see Genbank accession numbers Y00636 (Wallner, BP, etc., J.Exp.Med.166 (4), 923-932 (1987)), which is incorporated by reference herein.

[0336] 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。 Sequence information [0336] ICAM-3, see Genbank accession number X69819, which is incorporated by reference herein.

[0337] ICAM-2 的序列信息参见Genbank 检索号X15606 (Staunton, DE等,Nature339 (6219),61-64 (1989)),其通过引用结合到本文中。 Sequence information [0337] ICAM-2 See Genbank accession number X15606 (Staunton, DE, etc., Nature339 (6219), 61-64 (1989)), which is incorporated herein by reference.

[0338] 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。 [0338] ICAM-1 sequence information see Genbank accession number J03132 (Staunton, DE, etc., Cell 52 (6), 925-933 (1988)), which is incorporated by reference herein.

[0339] 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。 Sequence information [0339] PECAM See Genbank Accession No. M28526 (Newman, PJ et, Science 247,1219-1222 (1990), which is incorporated herein by reference.

[0340] 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。 [0340] P150.95 sequence information see Genbank accession numbers Y00093 (Corbi, AL, etc., EMBO J.6 (13), 4023-4028 (1987)), which is incorporated by reference herein.

[0341] 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。 Sequence information, see Genbank [0341] Mac-1 search Number J03925 (Corbi, AL, etc., J.Biol.Chem.263 (25), 12403-12411 (1988)), which is incorporated herein by reference.

[0342] LFA-1 的序列信息参见Genbank 检索号Y00796 (Larson.R.等,J.CellBiol.108 (2),703-712 (1989)),其通过引用结合到本文中。 [0342] LFA-1 sequence information, see Genbank Accession No. Y00796 (Larson.R. Et, J.CellBiol.108 (2), 703-712 (1989)), which is incorporated herein by reference.

[0343] CD34 的序列信息参见Genbank 检索号M81104 (Simmons, DL等,J.1mmunol.148,267-271 (1992)),其通过引用结合到本文中。 [0343] CD34 sequence information see Genbank accession number M81104 (Simmons, DL, etc., J.1mmunol.148,267-271 (1992)), which is incorporated by reference herein.

[0344] RANTES 的序列信息参见Genbank 检索号M21121 (Schall,TJ等,J.1mmunol.141,1018-1025 (1988)),其通过引用结合到本文中。 [0344] RANTES sequence information see Genbank Accession No. M21121 (Schall, TJ et, J.1mmunol.141,1018-1025 (1988)), which is incorporated herein by reference.

[0345] IL-8 的序列信息参见Genbank 检索号M28130 (Mukaida, N.等,J.1mmunol.143(4),1366-1371 (1989)),其通过引用结合到本文中。 [0345] IL-8 sequence information, see Genbank Accession No. M28130 (Mukaida, N. et, J.1mmunol.143 (4), 1366-1371 (1989)), which is incorporated herein by reference.

[0346] MIP-1 α 的序列信息参见Genbank 检索号U72395 (Fridell,R.Α.等,J.Cell.ScillO(ptll),1325-1331 (1997)),其通过引用结合到本文中。 [0346] MIP-1 α sequence information, see Genbank Accession No. U72395 (Fridell, R.Α. Et, J.Cell.ScillO (ptll), 1325-1331 (1997)), which is incorporated herein by reference.

[0347] 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。 [0347] E-selecton sequence information see Genbank accession number M24736 (Bevilacqua, MP, etc., Science 243 (4895), 1160-1165 (1989)), which is incorporated by reference herein.

[0348] CD2 的序列信息参见Genbank 检索号M14362 (Sewell, ff.A.等,Proc.Natl.Acad.Sc1.U.SA83,8718-8722 (1986) ;Proc.Natl.Acad.Sc1.USA84,7256-7256 (1987)),其通过引用结合到本文中。 [0348] CD2 sequence information see Genbank accession number M14362 (Sewell, ff.A. etc., Proc.Natl.Acad.Sc1.U.SA83,8718-8722 (1986); Proc.Natl.Acad.Sc1.USA84, 7256-7256 (1987)), which is incorporated herein by reference.

[0349] MCP-1 的序列信息参见Genbank 检索号S69738(Li,YS等,Mol.Cell.Biochem.126 (I),61-68 (1993)),其通过引用结合到本文中。 Sequence information, see Genbank [0349] MCP-1 in Accession No. S69738 (Li, YS, etc, Mol.Cell.Biochem.126 (I), 61-68 (1993)), which is incorporated herein by reference.

[0350] 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。 [0350] 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.

[0351] P-selection 的序列信息参见Genbank 检索号M25322 (Johnston, G.1.等,Cell56,1033-1044(1989),其通过引用结合到本文中。 [0351] P-selection sequence information see Genbank Accession No. M25322 (Johnston, G.1. Et, Cell56,1033-1044 (1989), which is incorporated herein by reference.

[0352] FLT 的序列信息参见Genbank 检索号X94263 (Mandriota, SJ等,J.Biol.Chem.271 (19),11500-11505 (1996)),其通过引用结合到本文中。 [0352] FLT sequence information see Genbank Accession No. X94263 (Mandriota, SJ et, J.Biol.Chem.271 (19), 11500-11505 (1996)), which is incorporated herein by reference.

[0353] FLT 的序列信息参见Genbank 检索号X51602 (Shibuya, M.等,Oncogene 5(4),519-524 (1990) Hah, HJ等Hum.Mol.Genet.2 (12),2204 (1993)),其通过引用结合到本文中。 [0353] FLT sequence information see Genbank Accession No. X51602 (Shibuya, M. et, Oncogene 5 (4), 519-524 (1990) Hah, HJ et Hum.Mol.Genet.2 (12), 2204 (1993) ), which is incorporated herein by reference.

[0354] 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。 [0354] Apo-1 sequence information, see Genbank Accession No. X63717 (Oehm et, J.Biol.Chem., (1992), 267 (15), 10709-15), which is incorporated by reference herein.

[0355] 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。 [0355] Fas sequence information see Genbank Accession No. M67454 (Itoh et, Cell, (1991), 66 (2), 233-43), which is incorporated herein by reference.

[0356] TNFR-1 的序列信息参见Genbank检索号M67454(Nophar等,EMBO J.,1990,9(10),3269-78),其通过弓I用结合到本文中。 [0356] TNFR-1 sequence information see Genbank accession numbers M67454 (Nophar etc., EMBO J., 1990,9 (10), 3269-78), which I used by bow incorporated herein.

[0357] 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。 See Genbank accession number sequence information [0357] p55 of M58286 (Loetscher et, Cell, 1990,61,351-359), which is incorporated herein by reference.

[0358] WSL-1 的序列信息参见Genbank 检索号Y09392 (Kitson 等,Nature, 1996,384 (6607),372-5),其通过弓I用结合到本文中。 [0358] WSL-1 sequence information, see Genbank Accession No. Y09392 (Kitson et, Nature, 1996,384 (6607), 372-5), by which the bow I with incorporated herein.

[0359] DR3 的序列信息参见Genbank 检索号U72763 (Chinnaiyan 等,Science, 1996,274 (5829),990-2),其通过弓I用结合到本文中。 Sequence information [0359] DR3 See Genbank accession number U72763 (Chinnaiyan, etc., Science, 1996,274 (5829), 990-2), which I used by bow incorporated herein.

[0360] TRAMP 的序列信息参见Genbank 检索号U75381 (Bodmer 等,Immunity, 1997,6 (I),79-88),其通过引用结合到本文中。 [0360] TRAMP sequence information see Genbank Accession No. U75381 (Bodmer et, Immunity, 1997,6 (I), 79-88), which is incorporated herein by reference.

[0361] 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。 [0361] Apo-3 sequence information, see Genbank Accession No. U74611 (Marsters et, Curr.Biol., 1996,6 (12), 1669-76), which is incorporated by reference herein.

[0362] AIR的序列信息参见Genbank检索号U78029,其通过引用结合到本文中。 [0362] AIR sequence information see Genbank accession number U78029, which is incorporated by reference herein.

[0363] LARD 的序列信息参见Genbank检索号U94512 (Screaton 等,Proc.Natl.Acad.Sc1.USA, 1997,94(9),4615-19),其通过引用结合到本文中。 [0363] LARD sequence information see Genbank Accession No. U94512 (Screaton et, Proc.Natl.Acad.Sc1.USA, 1997,94 (9), 4615-19), which is incorporated herein by reference.

[0364] NGRF 的序列信息参见Genbank 检索号M14764 (Johnson 等,Cell, 1986,47 (4),545-554),其通过弓I用结合到本文中。 [0364] NGRF sequence information see Genbank Accession No. M14764 (Johnson et, Cell, 1986,47 (4), 545-554), by which the bow I with incorporated herein.

[0365] 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。 [0365] DR4 (TRAIL) sequence information see Genbank Accession No. U90875 (Pan et, Science, 1997,276 (5309), 111-113), which is incorporated herein by reference.

[0366] 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。 Sequence information [0366] DR5 See Genbank Accession No. AF012535 (Sheridan et, Science, 1997,1227 (5327), 818-821), which is incorporated herein by reference.

[0367] 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。 Sequence information [0367] KILLER See Genbank Accession No. AF022386 (Wu et, Nat.Genet.17 (2), 141-143 (1997)), which is incorporated herein by reference.

[0368] TRA1L-R2的序列信息参见Genbank检索号AF020501,其通过引用结合到本文中。 [0368] TRA1L-R2 sequence information, see Genbank accession number AF020501, which is incorporated herein by reference.

[0369] TRICK2的序列信息参见Genbank检索号AF018657。 [0369] TRICK2 sequence information see Genbank accession numbers AF018657.

[0370] DR6的序列信息参见Genbank检索号AF068868,其通过引用结合到本文中。 Sequence information [0370] DR6 See Genbank accession number AF068868, which is incorporated herein by reference.

[0371] 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。 [0371] ICE sequence information see Genbank accession number U13697, U13698 and U13699 (Alnemri, ES, etc., J.Biol.Chem.270 (9), 4312-4317 (1995)), which is incorporated herein by reference.

[0372] 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。 [0372] VLA-1 sequence information, see Genbank Accession No. X17033 (Takada. Et, J.Biol.Chem.109 (1), 397-407 (1989)), which is incorporated herein by reference.

[0373] 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。 [0373] CD86 (B7.2) sequence information see Genbank Accession No. U04343 (Azuma et, Nature.366 (6450), 76 (1993)), which is incorporated herein by reference.

[0374]表 I [0374] Table I

[0375]小 RNA 病毒科(Picornavirus Family) [0375] small RNA virus family (Picornavirus Family)

[0376] 属:鼻病毒属(Rhinovirus):(医学)是约50%感冒的病因 [0376] the case: rhinoviruses genus (Rhinovirus) :( Medicine) is the cause of about 50% cold

[0377] 肠道病毒属(Etherovirus):(医学)包括脊髓灰质炎 [0377] enterovirus (Etherovirus) :( Medicine) including polio

[0378] 病毒(poliovirus)、柯萨奇病毒(coxsackievirus)、埃 [0378] virus (poliovirus), Coxsackie virus (coxsackievirus), Egypt

[0379] 可病毒(echovirus)和人肠道病毒(human enterovirus) [0379] to the virus (echovirus) and human enterovirus (human enterovirus)

[0380] 例如甲型肝炎病毒(hepatitis A virus) [0380] such as hepatitis A virus (hepatitis A virus)

[0381] 口疮病毒属(Apthoviruse):(兽医)是口蹄疫病毒 [0381] mouth sores virus genus (Apthoviruse) :( veterinarian) foot and mouth disease virus

[0382] 靶抗原:VP1、VP2、VP3、VP4、VPG [0382] target antigens: VP1, VP2, VP3, VP4, VPG

[0383]杯状病毒科(Calcivirus Family) [0383] Caliciviridae (Calcivirus Family)

[0384] 属:诺沃克病毒组(Norwalk group of Virus):(医学)这 [0384] Genus: Norovirus group (Norwalk group of Virus) :( Medicine) This

[0385] 些病毒是流行性胃肠炎的重要致病因子 [0385] These viruses are an important causative agent of epidemic gastroenteritis

[0386]披膜病毒科(Togavirus Family) [0386] Togaviridae (Togavirus Family)

[0387] 属:甲病毒属(Alphavirus):(医学和兽医)实例包括 [0387] genera: alphavirus (Alphavirus) :( medical and veterinary) Examples include

[0388] Senilis virus、罗斯河病毒(Ross River virus)、东方 [0388] Senilis virus, Ross River virus (Ross River virus), East

[0389] 马脑炎病毒和西方马脑炎病毒(Eastern & Western [0389] equine encephalitis virus and Western equine encephalitis virus (Eastern & Western

[0390] Equine encephalitis virus) [0390] Equine encephalitis virus)

[0391] 呼肠孤病毒属(Reovirus):(医学)风疫病毒(Rubella [0391] reovirus genus (Reovirus) :( Medicine) wind disease virus (Rubella

[0392] virus) [0392] virus)

[0393]黄病毒科(Flariviridue Family) [0393] Flaviviridae (Flariviridue Family)

[0394] 实例包括:(医学)登革病毒(dengue virus)、黄热病 [0394] Examples include :( Medical) dengue virus (dengue virus), yellow fever

[0395] 毒(yellow fever virus)、日本脑炎病毒(Japanese [0395] Toxicity (yellow fever virus), Japanese encephalitis virus (Japanese

[0396] encephalitis virus)、圣路易斯脑炎病毒(St.Louis [0396] encephalitis virus), St. Louis encephalitis virus (St.Louis

[0397] encephalitis virus)和蝶传脑炎病毒(tick borne [0397] encephalitis virus) and butterfly-borne encephalitis virus (tick borne

[0398] encephalitis virus)。 [0398] encephalitis virus). 西尼罗病毒(West Nile virus) West Nile virus (West Nile virus)

[0399] (Genbank NC001563, AF533540, AF404757, [0399] (Genbank NC001563, AF533540, AF404757,

[0400] AF404756, AF404755, AF404754, AF404753, [0400] AF404756, AF404755, AF404754, AF404753,

[0401] AF481864, M12294, AF317203, AF196835, [0401] AF481864, M12294, AF317203, AF196835,

[0402] AF260969, AF260968, AF260967, AF206518 和 [0402] AF260969, AF260968, AF260967, AF206518 and

[0403] AF202541) [0403] AF202541)

[0404] 代表性靶抗原: [0404] Representative target antigens:

[0405] E [0405] E

[0406] NS5 [0406] NS5

[0407] C[0408] 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 [0407] C [0408] Hepatitis C virus :( internal medicine) of this virus is not a science, but is believed to still be togavirus

[0409] 病毒(togavirus)或黄病毒(fIavivirus)。 [0409] virus (togavirus) or flavivirus (fIavivirus). 与披膜病毒科最相似。 The most similar Togaviridae.

[0410] 冠状病毒科(Coronavirus Family):(医学和兽医) [0410] coronavirus family (Coronavirus Family) :( medical and veterinary)

[0411] 传染性支气管炎病毒(Infectious bronchitis virus)(家 [0411] IBV (Infectious bronchitis virus) (Home

[0412] 禽) [0412] birds)

[0413] 猪传染性胃肠病毒(Porcine transmissible [0413] Porcine contagious gastrointestinal virus (Porcine transmissible

[0414] gastroenteric virus)(猪) [0414] gastroenteric virus) (pig)

[0415] 猪血凝脑脊髓炎病毒(Porcine hemaglutinating [0415] swine hemagglutination encephalomyelitis virus (Porcine hemaglutinating

[0416] encephalomyelitis virus)(猪) [0416] encephalomyelitis virus) (pig)

[0417] 猫传染性腹膜炎病毒(Feline infectious peritonitis [0417] Feline infectious peritonitis virus (Feline infectious peritonitis

[0418] virus)(猫) [0418] virus) (cat)

[0419] 猫肠道冠状病毒(Feline enteric coronavirus)(猫) [0419] cat intestinal coronavirus (Feline enteric coronavirus) (cat)

[0420] 犬冠状病毒(Canine coronavirus)(狗) [0421] SARS 相关性冠状病毒(SARS associated [0420] canine coronavirus (Canine coronavirus) (dog) [0421] SARS-related coronavirus (SARS associated

[0422] coronavirus) [0422] coronavirus)

[0423] 人呼吸道冠状病毒(Human respiratory coronavirus) [0423] Human respiratory coronavirus (Human respiratory coronavirus)

[0424] 引起约40种感冒 [0424] cause about 40 kinds of cold

[0425] EX.224E,0C43 [0425] EX.224E, 0C43

[0426] 注释-冠状病毒可引起非甲、非乙或非丙型肝炎 [0426] Notes - coronavirus can cause non-A, non-B or hepatitis C

[0427] 靶抗原: [0427] target antigens:

[0428] El-也称为M或基质蛋白 [0428] El- also called M or matrix protein

[0429] E2-也称为S或剌突(Spike)蛋白 [0429] E2- also called S or assassination sudden (Spike) protein

[0430] E3-也称为BE 或hemagglutin-elterose 糖蛋白(不 [0430] E3- also known BE or hemagglutin-elterose glycoprotein (not

[0431] 存在于所有冠状病毒中) [0431] present in all coronaviruses)

[0432] N-核壳 [0432] N- nucleocapsid

[0433]弹状病毒科(Rhabdovirus Family) [0433] Rhabdoviridae family (Rhabdovirus Family)

[0434] 属:水疱病毒属(Vesiliovirus) [0434] Genus: blisters virus genus (Vesiliovirus)

[0435] 狂犬病毒属(Lyssavirus):(医学和兽医)狂犬病 [0435] lyssavirus (Lyssavirus) :( medical and veterinary) rabies

[0436] 靶抗原:G蛋白 [0436] Target antigen: G protein

[0437] N 蛋白 [0437] N protein

[0438]丝状病毒科(Filoviridue Family):(医学) [0438] filamentous virus family (Filoviridue Family) :( Medicine)

[0439] 出血热病毒(Hemorrhagic fever virus),例如马尔堡 [0439] hemorrhagic fever virus (Hemorrhagic fever virus), such as Marburg

[0440] 病毒(Marburg virus)和埃博拉病毒(Ebola virus) [0440] virus (Marburg virus) and Ebola virus (Ebola virus)

[0441]副粘病毒科(Paramyxovirus Family): [0441] paramyxovirus (Paramyxovirus Family):

[0442] 属:副粘病毒属(Paramyxovirus):(医学和兽医) [0442] genera: paramyxovirus genus (Paramyxovirus) :( medical and veterinary)

[0443] 目思腺炎病毒(Mumps virus)、新城疫病毒(New Castle [0443] CONTENTS think pancreatitis virus (Mumps virus), Newcastle disease virus (New Castle

[0444] disease virus)(鸡的重要病原体) [0444] disease virus) (important pathogen of chickens)

[0445] 麻疹病毒属(Mortilliviiais):(医学和兽医)麻疹、犬 [0445] Morbillivirus (Mortilliviiais) :( medical and veterinary) Measles, canine

[0446] 瘟热[0447] 肺病毒属(Pneuminvirus):(医学和兽医) [0446] distemper [0447] lung virus genus (Pneuminvirus) :( medical and veterinary)

[0448] 呼吸道合胞病毒(Respiratory syncytial virus) [0448] Respiratory syncytial virus (Respiratory syncytial virus)

[0449]正粘病毒科(Orthomyxovirus Family)(医学) [0449] Orthomyxoviridae (Orthomyxovirus Family) (Medicine)

[0450] 流感病毒(Influenza virus) [0450] Influenza virus (Influenza virus)

[0451]布尼亚病毒科(Bungavirus Family) [0451] Bunyaviridae (Bungavirus Family)

[0452] 属:布尼亚病毒属(Bungavirus):(医学)加州脑炎病毒 [0452] Genus: Bunia virus genus (Bungavirus) :( Medical) California encephalitis virus

[0453] (California encephalitis virus LA Crosse) [0453] (California encephalitis virus LA Crosse)

[0454] 白蛉热病毒属(Phlebovirus):(医学)立夫特山谷热 [0454] sandfly fever virus genus (Phlebovirus) :( Medical) Rift Valley fever

[0455] 病毒(Rift Valley Fever virus) [0455] virus (Rift Valley Fever virus)

[0456] 汉坦病毒属(Hantavirus):Puremala是一种出血热 [0456] hantavirus (Hantavirus): Puremala is a hemorrhagic fever

[0457] 病毒 [0457] virus

[0458] 纳伊罗病毒(Nairvirus)(兽医)内罗毕绵羊病 [0458] Na Yiluo virus (Nairvirus) (Veterinary) Nairobi sheep disease

[0459] 还有许多未命名的布尼亚病毒 [0459] There are many unnamed Bunia virus

[0460]沙粒病毒科(Arenavirus Family)(医学) [0461] LCM,拉沙热病毒(Lassi fever virus) [0460] Arenaviridae (Arenavirus Family) (Medicine) [0461] LCM, Lassa fever virus (Lassi fever virus)

[0462]呼肠孤病毒科(Reovirus Family) [0462] reoviridae (Reovirus Family)

[0463] 属:呼肠孤病毒属(Reovirus):可能的人病原体 [0463] Genus: reovirus genus (Reovirus): Possible human pathogen

[0464] 轮状病毒属(Rotavirus):小儿急性胃肠炎 [0464] rotavirus (Rotavirus): children with acute gastroenteritis

[0465] 环状病毒属(Orbivirus):(医学和兽医) [0465] circovirus genus (Orbivirus) :( medical and veterinary)

[0466] 科罗拉多蜱热、Lebombo (人)马器质性脑病、蓝舌 [0466] Colorado tick fever, Lebombo (person) horse organic encephalopathy, bluetongue

[0467]逆转病毒科(Retrovirus Family) [0467] retroviral Branch (Retrovirus Family)

[0468] 亚科: [0468] subfamilies:

[0469] 致癌RNA病毒亚科(Oncorivirinal):(兽医)(医学) [0469] oncogenic RNA virus subfamily (Oncorivirinal) :( Veterinary) (Medical)

[0470] 猫白血病病毒(feline leukemia virus)、HTLVI 和 [0470] feline leukemia virus (feline leukemia virus), HTLVI and

[0471] HTLVII [0471] HTLVII

[0472] 慢病毒亚科(Lentivirinal):(医学和兽医)HIVjg [0472] lentivirus subfamily (Lentivirinal) :( medical and veterinary) HIVjg

[0473] 免疫缺陷病毒(feline immunodeficiency virus)、马 [0473] immunodeficiency virus (feline immunodeficiency virus), MA

[0474] 传染性贫血病毒(equine infections, anemia virus) [0474] infectious anemia virus (equine infections, anemia virus)

[0475] 泡沫病毒亚科(Spumavirinal) [0475] foamy virus subfamily (Spumavirinal)

[0476]乳多空病毒科(Papovavirus Family) [0476] Papovaviridae (Papovavirus Family)

[0477] 亚科: [0477] subfamilies:

[0478] 多瘤病毒属(Polyomavirus):(医学)BKU 和JQJ [0478] polyomavirus genus (Polyomavirus) :( Medical) BKU and JQJ

[0479] 病毒 [0479] virus

[0480] 亚科: [0480] subfamilies:

[0481] 乳头瘤病毒属(Papillomavirus):(医学)癌症或恶 [0481] Papillomavirus genus (Papillomavirus) :( Medicine) for cancer or evil

[0482] 性乳头瘤相关的许多病毒类型 [0482] Many of the papilloma virus type-related

[0483]腺病毒(Adenovirus)(医学) [0483] adenovirus (Adenovirus) (Medicine)

[0484] EX AD7,ARD.,0.B.-引起呼吸道疾病_某些腺 [0484] EX AD7, ARD., 0.B.- cause respiratory _ some glands

[0485] 病毒例如275引起肠炎[0486]细小 DNA 病毒科(Parvovirus Family)(兽医) [0485] enteritis caused by viruses such as 275 [0486] small DNA virus family (Parvovirus Family) (veterinarian)

[0487] 猫细小DNA病毒(Feline parvovirus):引起猫肠炎 [0487] cat small DNA viruses (Feline parvovirus): Cat cause enteritis

[0488] 猫肠炎病毒(Feline panleucopeniavirus) [0488] cat enteritis virus (Feline panleucopeniavirus)

[0489] 犬细小DNA 病毒(Canine parvovirus) [0489] canine parvovirus DNA virus (Canine parvovirus)

[0490] 猪细小DNA 病毒(Porcine parvovirus) [0490] Porcine DNA virus (Porcine parvovirus)

[0491]痕疫病毒科(Herpesvirus Family) [0491] marks the plague virus family (Herpesvirus Family)

[0492] 亚科:甲型痕疫病毒亚科(Alphaherpesviridue) [0492] subfamilies: the epidemic of influenza virus subfamily marks (Alphaherpesviridue)

[0493] 属:单纯疱疫病毒属(Simplexvirus)(医学) [0493] Genus: herpes simplex virus genus Phytophthora (Simplexvirus) (Medicine)

[0494] HSVI (Genbank X14112,NC001806),HSVII [0494] HSVI (Genbank X14112, NC001806), HSVII

[0495] (NC001798) [0495] (NC001798)

[0496] 水痘病毒属(Varicellovirus):(医学兽医)伪狂犬病- [0496] varicella virus genus (Varicellovirus) :( veterinary medicine) Pseudorabies -

[0497] 水痘-带状疱疹 [0497] varicella - zoster

[0498] 亚科:乙型痕疫病毒亚科(Betaherpesviridue) [0499] 属:巨细胞病毒属(Cytomegalovirus)(医学) [0498] subfamilies: subfamily B mark disease virus (Betaherpesviridue) [0499] Genus: Cytomegalovirus (Cytomegalovirus) (Medicine)

[0500] HCMV [0500] HCMV

[0501 ] 鼠巨细胞病毒属(Muromegalovirus) [0501] murine cytomegalovirus (Muromegalovirus)

[0502] 亚科:丙型痕疫病毒亚科(Gammaherpesviridue) [0502] subfamilies: Hepatitis mark disease virus subfamily (Gammaherpesviridue)

[0503] 属:淋巴隐伏病毒属(Lymphocryptovirus)(医学) [0503] Genus: Concealed lymph virus genus (Lymphocryptovirus) (Medicine)

[0504] EBV_(伯基特淋巴瘤(Burkitts Iympho)) [0504] EBV_ (Burkitt's lymphoma (Burkitts Iympho))

[0505] 蛛猴疱疫病毒属(Rhadinovirus) [0505] spider monkey herpes virus genus Phytophthora (Rhadinovirus)

[0506]痕病毒科(Poxvirus Family) [0506] marks the virus family (Poxvirus Family)

[0507] 亚科:脊椎动物痘病毒亚科(Chordopoxviridue)(医学-兽 [0507] subfamilies: subfamily vertebrate poxviruses (Chordopoxviridue) (medicine - Beast

[0508] 医) [0508] Medical)

[0509] 属:天花病毒属(Variola)(天花) [0509] the case: the case of smallpox virus (Variola) (smallpox)

[0510] 痘苗病毒属(Vaccinia)(牛痘) [0510] Vaccinia virus genus (Vaccinia) (vaccinia)

[0511] 副痘病毒属(Parapoxivirus)-兽医 [0511] Parapoxvirus genus (Parapoxivirus) - Veterinary

[0512] 禽痘病毒属(Auipoxvirus)-兽医 [0512] fowl pox virus genus (Auipoxvirus) - Veterinary

[0513] 山羊痘病毒属(Capripoxvirus) [0513] goat pox virus genus (Capripoxvirus)

[0514] 野兔痘病毒属(Leporipoxvirus) [0514] hare poxvirus genus (Leporipoxvirus)

[0515] 猪痘病毒属(Suipoxvirus) [0515] swine pox virus genus (Suipoxvirus)

[0516] 亚科:昆虫痘病毒亚科(Entemopoxviridue) [0516] subfamilies: entomopoxviruses subfamily (Entemopoxviridue)

[0517]嗜肝 DNA 病毒科(Hepadnavirus Family) [0517] hepatotropic DNA virus family (Hepadnavirus Family)

[0518] 乙型肝炎病毒(hepatitis B virus) [0518] Hepatitis B virus (hepatitis B virus)

[0519]未分类 丁型肝炎病毒(hepatitis delta virus) [0519] Unclassified hepatitis D virus (hepatitis delta virus)

[0520]表 2 [0520] Table 2

[0521] 细菌病原体 [0521] bacterial pathogens

[0522] 致病性革兰氏阳性球菌包括:肺炎球菌(pneumococcal);葡 [0522] pathogenic Gram-positive cocci include: pneumococcal (pneumococcal); Portugal

[0523] 萄球菌(staphylococcal);和链球菌(streptococcal)。 [0523] aureus (staphylococcal); and streptococci (streptococcal). 致病性革 Pathogenicity leather

[0524] 兰氏阴性球菌包括:脑膜炎球菌(menignococcal);和淋病球[0525] 菌(gonococcal)。 [0524] Gram-negative bacteria include: meningococcal (menignococcal); and gonorrhea ball [0525] bacteria (gonococcal).

[0526] 致病性革兰氏阴性肠杆菌包括:肠杆菌科 [0526] Gram-negative pathogenic E. coli include: Enterobacteriaceae

[0527] (enterobacteriaceae);假单胞菌属(pseudomonas)、不动杆菌 [0527] (enterobacteriaceae); Pseudomonas (pseudomonas), Acinetobacter

[0528] 属(acinetobacteria)和艾肯氏菌属(eikenella)、melioidosis ;沙 [0528] genus (acinetobacteria) and Aiken Shi genus (eikenella), melioidosis; sand

[0529] 门氏菌属(sahnonella);志贺氏菌属(shigellosis);嗜血杆菌 [0529] Salmonella spp (sahnonella); Shigella (shigellosis); Haemophilus

[0530] 属(hemophilus);软下推菌属(chancroid);布鲁氏菌属 [0530] genus (hemophilus); soft push down the genus (chancroid); Brucella

[0531] (brucellosis) ;土拉热菌属(tularemia);耶尔森氏菌属(yersinia [0531] (brucellosis); Tula hot genus (tularemia); Yersinia (yersinia

[0532] (巴斯德氏菌属(pasteurella));念球状链杆菌(streptobacillus [0532] (Pasteurella (pasteurella)); read the spherical streptobacillus (streptobacillus

[0533] mortiliformis)和螺菌属(spirillum);单核细胞增生李斯特氏 [0533] mortiliformis) and snail species (spirillum); Listeria monocytogenes

[0534] 菌(listeria monocytogenes);猪红斑丹毒丝菌(erysipelothrix [0534] bacteria (listeria monocytogenes); Erysipelothrix rhusiopathiae bacteria (erysipelothrix

[0535] rhusiopathiae);白喉(diphtheria)、霍乱(cholera)、炭疽 [0535] rhusiopathiae); diphtheria (diphtheria), cholera (cholera), anthrax

[0536] (anthrax);性病肉芽肿(donovanosis (腹股沟肉芽肿(granuloma [0536] (anthrax); venereal granuloma (donovanosis (groin granuloma (granuloma

[0537] inguinale));和巴尔通体病(bartonellosis)。 [0537] inguinale)); and Bartonella disease (bartonellosis).

[0538] 致病性厌氧菌包括:破伤风(tetanus));肉毒中毒(botulism); [0538] pathogenic anaerobic bacteria include: tetanus (tetanus)); botulism (botulism);

[0539] 其它梭状芽孢杆菌;结核(tuberculosis);麻风(leprosy);和 [0539] Other Clostridium; tuberculosis (tuberculosis); leprosy (leprosy); and

[0540] 其它分枝杆菌。 [0540] Other mycobacteria. 致病性螺旋体病包括:梅毒(syphilis);-密 Pathogenic leptospirosis include: syphilis (syphilis); - close

[0541] 螺旋体病(treponematoses):雅司病(yaws)、品他病(pinta)和 [0541] leptospirosis (treponematoses): yaws (yaws), pinta (pinta) and

[0542] 地方性梅毒;和钩端螺旋体病(leptospirosis)。 [0542] endemic syphilis; and leptospirosis (leptospirosis).

[0543] 由高等病原菌和致病性真菌引起的其它感染包括:放线菌 [0543] Other infections caused by higher pathogen and pathogenic fungi include: actinomycosis

[0544] 病(actinomycosis);诺卡氏菌病(nocardiosis);隐球菌病 [0544] disease (actinomycosis); Nocardiosis (nocardiosis); cryptococcosis

[0545] (cryptococcosis)、芽生菌病(blastomycosis)、组织胞衆菌病 [0545] (cryptococcosis), blastomycosis (blastomycosis), all the bacteria cell tissue disease

[0546] (histoplasmosis)和球抱子菌病(coccidioidomycosis);念珠菌 [0546] (histoplasmosis) and ball spores salmonellosis (coccidioidomycosis); candidiasis

[0547] 病(candidiasis)、曲霉病(aspergillosis)和毛霉病 [0547] disease (candidiasis), aspergillosis (aspergillosis) and mucormycosis

[0548] (mucormycosis);抱子丝菌病(sporotrichosis);类球抱子菌病 [0548] (mucormycosis); silk bacteria spores disease (sporotrichosis); quasi-spherical spores fungal disease

[0549] (paracoccidiodomycosis)、petriellidiosis、球拟酵母菌病 [0549] (paracoccidiodomycosis), petriellidiosis, Torulopsis yeast disease

[0550] (torulopsosis)、足分支菌病(mycetoma)和着色真菌病 [0550] (torulopsosis), foot branch salmonellosis (mycetoma) and colored fungal disease

[0551] (chromomycosis);和皮肤真菌病(dermatophytosis) ◦ [0551] (chromomycosis); and fungal skin disease (dermatophytosis) ◦

[0552] 立克次氏体感染包括立克次氏体病(rickettsial和 [0552] rickettsial infections include rickettsial disease (rickettsial and

[0553] rickettsioses)。 [0553] rickettsioses).

[0554] 支原体(mycoplasma)和衣原体(chlamydial)感染包括: [0554] mycoplasma (mycoplasma) and chlamydia (chlamydial) infections include:

[0555] 肺炎支原体(mycoplasma pneumoniae);性病性淋巴肉 [0555] Mycoplasma pneumoniae (mycoplasma pneumoniae); lymphogranuloma venereum meat

[0556] 芽肿瘤(lymphogranuloma venereum);婴鸟鶴热 [0556] bud tumor (lymphogranuloma venereum); infant Bird Crane heat

[0557] (psittacosis);和围产期衣原体感染。 [0557] (psittacosis); and perinatal chlamydial infections.

[0558] 致病性真核生物 [0558] pathogenic eukaryotes

[0559] 致病性原生动物和寄生虫及其感染包括:阿米巴病 [0559] pathogenic protozoa and parasites and infections include: amebiasis

[0560] (amebiasis);症疾(malaria);利什曼病(leishmaniasis);维虫 [0560] (amebiasis); disease disease (malaria); leishmaniasis (leishmaniasis); dimensional worm

[0561] 病(trypanosomiasis);弓形体病(toxoplasmosis);卡氏肺囊虫 [0561] sickness (trypanosomiasis); toxoplasmosis (toxoplasmosis); Pneumocystis carinii

[0562] (pneurnocystis carinii);巴贝虫病(babesiosis);贾第鞭毛虫 [0562] (pneurnocystis carinii); babesiosis (babesiosis); Giardia lamblia

[0563] 病(giardiasis);旋毛虫病(trichinosis);丝虫病(filariasis);血[0564] 吸虫病(schistosomiasis));线虫(nematode);吸虫(trematode [0563] disease (giardiasis); trichinosis (trichinosis); filariasis (filariasis); blood [0564] trematodes (schistosomiasis)); nematodes (nematode); flukes (trematode

[0565] 或fluke);和绦虫(cestode (tapeworm))感染。 [0565] or fluke); and tapeworm (cestode (tapeworm)) infection.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
WO2002029088A Title not available
Classifications
International ClassificationC07K14/705, A61P37/00, A61K, C07K14/54, A61P35/00, C07K14/00, C07K14/52, C07H21/04, A61K48/00
Cooperative ClassificationC07K14/5443, A61K2039/55527, C07K14/70578, C07K2319/02, A61K2039/53
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