结核杆菌抗原CFP21的CTL表位筛选与改造
|
摘要
结核病(Tuberculosis, TB)是由结核杆菌(Mycobacterium tuberculosis,Mtb)引起的一种慢性传染病,目前仍然是导致传染病发病率和死亡率较高的疾病之一。尤其是近年来,由于人体免疫缺陷病毒双重感染的出现而造成个体机会性感染愈发严重。目前唯一可用的用于结核病预防的疫苗是卡介苗(Bacillus Calmette-Guerin, BCG),但其在预防成年人患肺结核的有效性方面受到了限制,并且伴随着结核杆菌多重耐药菌株的出现,人们迫切需要研制新型的抗结核疫苗。
由CD4+辅助性T(Th1)细胞和CD8+毒性T淋巴细胞(CTL)所介导的细胞免疫反应在抗结核感染保护性免疫中发挥着主导作用。结核新型疫苗的研究依赖于能被CD8+T细胞识别从而产生IFN-γ而发挥杀伤效应的结核抗原及其相应表位的鉴定。分泌蛋白以及细胞壁蛋白是结核杆菌主要的免疫保护抗原,并且位于Mtb基因组和BCG差别区(RD区)内的分泌蛋白由于其在BCG中的缺失,逐渐成为疫苗候选抗原的热点。优势抗原表位的鉴定,对发展更有效的结核病治疗性多表位肽疫苗具有重要的意义,并且能够为基础研究提供理论基础。
结核杆菌分泌蛋白CFP21 (culture filtrate proteins 21,培养滤液蛋白21)位于RD2区,具有高度的免疫原性。CFP21能够诱导结核感染结核患者产生T细胞增殖反应以及释放高水平IFN-γ,发挥细胞杀伤作用,是抗结核疫苗设计的理想候选抗原。
CFP21 HLA-A*0201/03限制性表位的鉴定与改造主要通过以下工作来完成：我们首先运用在线数据库,以SYFPEITHI初步预测,结合BIMAS和NetCTL 1.2在线分析,初步筛选出一系列的天然表位肽。已有相关文献报道,通过对抗原表位锚定位点的氨基酸替换,能够增强表位与HLA-Ⅰ分子的结合力。因此,我们通过将天然表位,按1Y、2L和/或9L进行氨基酸置换,获得改造肽,利用数据库再次分析,以期寻求结合力更高的表位。综合分析预测结果,选取在至少两种数据库中评分结果位于前10的表位肽,分别是4条母体肽p5 (SLVRIVGVV)、p13 (VVATTLALV)、p134 (AVADHVAAV)、p189 (NIMAHVSYV)以及3条改造肽p189-1Y2L9L(YLMAHVSYL)、p134-1Y2L (YLADHVAAV)、p134-1Y2L9L (YLADHVAAL)。对已经筛选出来的表位,我们采用标准Fmoc方案合成多肽,产物经反向高效液相色谱(RP-HPLC)分析、纯化,获得了纯度高于95%的九肽产物。经质谱测定,分子量所得测定值与理论值相符合。
通过内源性抗原提呈途径中所必需的抗原多肽转运蛋白(TAP)缺陷的T2细胞结合力以及肽/HLA稳定性实验,对初步筛选的表位进行验证。结果显示,相对于母体肽p134,改造肽p134-1Y2L、p134-1Y2L9L与HLA分子具有更高的结合力与稳定性。通过结合力及稳定性的初步筛选,在7条候选肽中,只有母体肽p134以及相应的两条改造肽,用于后续ELISPOT以及细胞毒杀伤作用的T细胞免疫活性检测。通过HLA-A*0201+PPD+/HLA-A*03+PPD+健康供者外周血单核细胞(PBMCs)诱导出能够分泌IFN-γ的细胞数的分析以及特异性CTL的LDH杀伤活性检测显示,母体肽p134以及两条改造肽p134-1Y2L和p134-1Y2L9L都能够诱导T细胞反应,但只有p134诱导出的CTL对靶细胞具有明显的杀伤效应。通过上述的工作,我们证明,CFP21134-142 (AVADHVAAV),是CFP21潜在的免疫优势HLA-A*0201/03限制性表位。
本课题鉴定出一个源于结核杆菌分泌蛋白CFP21的HLA-A*0201/03限制性表位—p134 (AVADHVAAV)。该表位能有效激发HLA-A*0201和HLA-A*03限制性CTL的免疫应答,且为一种多等位基因广谱CTL表位,能够为结核病的疫苗设计及免疫治疗奠定基础。
Tuberculosis, caused by the bacterium, Mycobacterium tuberculosis (Mtb), remains a leading cause of infectious disease morbidity and mortality, which has emerged as a major opportunistic infection in individuals with HIV/AIDS. The only tuberculosis (TB) vaccine currently available is the attenuated Mycobacterium bovis strain Bacillus Calmette-Guerin (BCG), which is ineffective on preventing pulmonary TB in adults. The uncertain efficacy of the BCG vaccine for TB in adults, and the emergence of extensively drug resistant Mtb strains have further enhanced the urgency of the development of novel TB vaccines.
The cellular arm of the immune response mediated by CD4+ type 1 helper T (Th1) and CD8+ cytotoxic T lymphocytes (CTL) has been determined to be a pivotal component of protective immunity against Mtb. In order to develop new vaccines against tuberculosis, attention has been directed towards the identification of antigens/epitopes recognized by CD8+ T cells secreting interferon-γ(IFN-γ). Secreted and surface-exposed cell wall proteins seem to play a pivotal role in the induction of protective cellular immunity against TB. Recently, secreted proteins encoded by different regions of difference (RDs) of the Mtb genome have been considered as attractive candidates owing to their absence in most BCG strains. Detailed knowledge of the epitopes recognized by immune responses can aid in peptide-based anti-TB vaccines development, and provide important tools for basic research.
A major secreted protein, CFP21, locating in RD2 was demonstrated to be an immunodominant antigen by elevating T cell proliferate response and production of cytokines such as IFN-y. CFP21 could also induce an optimum level of cytotoxic T cell activity. These findings suggested that CFP21 could be used as an attractive candidate antigen.
In the present study, to identify novel CFP21-derived HLA-A*0201/03 restricted epitopes, a series of native peptides were predicted with various prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Recent studies suggested that modification of amino acids at the anchor position of epitopes could result in enhancing the binding affinity to HLA-I molecules. Here, we designed analogues of the native peptides by altering the native peptides with tyrosine at position 1 (1Y), leucine at position 2 (2L) and/or position 9 (9L). Based on the three prediction programs, four native peptides, p5 (SLVRIVGVV), p13 (VVATTLALV), p134 (AVADHVAAV), p189 (NIMAHVSYV) and three of their analogues, p189-1Y2L9L (YLMAHVSYL), p134-1Y2L (YLADHVAAV), p134-1Y2L9L (YLADHVAAL) with prediction scores in the top 10 detected by at least two of the programs were selected and synthesized for further study. Peptides were synthesized by standard solid phase Fmoc strategy and were purified to more than 95% purity by reverse phase high performance liquid chromatography (RP-HPLC). Their molecular weights were confirmed by electro-spray ionization mass spectrometry (ESI-MS).
To evaluate the binding affinity of these peptides to HLA-A*0201/03 molecules and the stability of the peptide/HLA-A complexes in vitro, TAP-deficient T2 cells were used. In concordance with the binding affinities, these two analogues p134-1Y2L and p134-1Y2L9L showed more potent binding stabilities than the native peptide, p134. Based on the results of the T2 binding assay and peptide/HLA stability assay, among the seven peptides studied, only p134 and its two analogues were selected to investigate their ability to induce T cell response by using ELISPOT and cytotoxicity assay. Subsequent IFN-γrelease and LDH release assays by using PBMCs from HLA-A*0201+/03+ PPD+ healthy donors showed that the native peptide p134 and its two analogues p134-1Y2L and p134-1Y2L9L could induce potent T cell response, but only p134-induced CTLs could potently lyse the peptide-loaded target cells. All these results demonstrated that the amino acid sequence of 134-142 (AVADHVAAV) in CFP21 could be an immunogenic CTL epitope which could be both processed and presented endogenously via HLA-A*0201/03.
In the present study, a novel HLA-A*0201/03 restricted T cell epitope, p134 (AVADHVAAV), was identified in a secreted protein of Mtb, CFP21. The potent T cell response activity and the potential to be a broad-spectrum epitope made p134 a promising epitope for vaccination and immunotherapy against TB.
由CD4+辅助性T(Th1)细胞和CD8+毒性T淋巴细胞(CTL)所介导的细胞免疫反应在抗结核感染保护性免疫中发挥着主导作用。结核新型疫苗的研究依赖于能被CD8+T细胞识别从而产生IFN-γ而发挥杀伤效应的结核抗原及其相应表位的鉴定。分泌蛋白以及细胞壁蛋白是结核杆菌主要的免疫保护抗原,并且位于Mtb基因组和BCG差别区(RD区)内的分泌蛋白由于其在BCG中的缺失,逐渐成为疫苗候选抗原的热点。优势抗原表位的鉴定,对发展更有效的结核病治疗性多表位肽疫苗具有重要的意义,并且能够为基础研究提供理论基础。
结核杆菌分泌蛋白CFP21 (culture filtrate proteins 21,培养滤液蛋白21)位于RD2区,具有高度的免疫原性。CFP21能够诱导结核感染结核患者产生T细胞增殖反应以及释放高水平IFN-γ,发挥细胞杀伤作用,是抗结核疫苗设计的理想候选抗原。
CFP21 HLA-A*0201/03限制性表位的鉴定与改造主要通过以下工作来完成：我们首先运用在线数据库,以SYFPEITHI初步预测,结合BIMAS和NetCTL 1.2在线分析,初步筛选出一系列的天然表位肽。已有相关文献报道,通过对抗原表位锚定位点的氨基酸替换,能够增强表位与HLA-Ⅰ分子的结合力。因此,我们通过将天然表位,按1Y、2L和/或9L进行氨基酸置换,获得改造肽,利用数据库再次分析,以期寻求结合力更高的表位。综合分析预测结果,选取在至少两种数据库中评分结果位于前10的表位肽,分别是4条母体肽p5 (SLVRIVGVV)、p13 (VVATTLALV)、p134 (AVADHVAAV)、p189 (NIMAHVSYV)以及3条改造肽p189-1Y2L9L(YLMAHVSYL)、p134-1Y2L (YLADHVAAV)、p134-1Y2L9L (YLADHVAAL)。对已经筛选出来的表位,我们采用标准Fmoc方案合成多肽,产物经反向高效液相色谱(RP-HPLC)分析、纯化,获得了纯度高于95%的九肽产物。经质谱测定,分子量所得测定值与理论值相符合。
通过内源性抗原提呈途径中所必需的抗原多肽转运蛋白(TAP)缺陷的T2细胞结合力以及肽/HLA稳定性实验,对初步筛选的表位进行验证。结果显示,相对于母体肽p134,改造肽p134-1Y2L、p134-1Y2L9L与HLA分子具有更高的结合力与稳定性。通过结合力及稳定性的初步筛选,在7条候选肽中,只有母体肽p134以及相应的两条改造肽,用于后续ELISPOT以及细胞毒杀伤作用的T细胞免疫活性检测。通过HLA-A*0201+PPD+/HLA-A*03+PPD+健康供者外周血单核细胞(PBMCs)诱导出能够分泌IFN-γ的细胞数的分析以及特异性CTL的LDH杀伤活性检测显示,母体肽p134以及两条改造肽p134-1Y2L和p134-1Y2L9L都能够诱导T细胞反应,但只有p134诱导出的CTL对靶细胞具有明显的杀伤效应。通过上述的工作,我们证明,CFP21134-142 (AVADHVAAV),是CFP21潜在的免疫优势HLA-A*0201/03限制性表位。
本课题鉴定出一个源于结核杆菌分泌蛋白CFP21的HLA-A*0201/03限制性表位—p134 (AVADHVAAV)。该表位能有效激发HLA-A*0201和HLA-A*03限制性CTL的免疫应答,且为一种多等位基因广谱CTL表位,能够为结核病的疫苗设计及免疫治疗奠定基础。
Tuberculosis, caused by the bacterium, Mycobacterium tuberculosis (Mtb), remains a leading cause of infectious disease morbidity and mortality, which has emerged as a major opportunistic infection in individuals with HIV/AIDS. The only tuberculosis (TB) vaccine currently available is the attenuated Mycobacterium bovis strain Bacillus Calmette-Guerin (BCG), which is ineffective on preventing pulmonary TB in adults. The uncertain efficacy of the BCG vaccine for TB in adults, and the emergence of extensively drug resistant Mtb strains have further enhanced the urgency of the development of novel TB vaccines.
The cellular arm of the immune response mediated by CD4+ type 1 helper T (Th1) and CD8+ cytotoxic T lymphocytes (CTL) has been determined to be a pivotal component of protective immunity against Mtb. In order to develop new vaccines against tuberculosis, attention has been directed towards the identification of antigens/epitopes recognized by CD8+ T cells secreting interferon-γ(IFN-γ). Secreted and surface-exposed cell wall proteins seem to play a pivotal role in the induction of protective cellular immunity against TB. Recently, secreted proteins encoded by different regions of difference (RDs) of the Mtb genome have been considered as attractive candidates owing to their absence in most BCG strains. Detailed knowledge of the epitopes recognized by immune responses can aid in peptide-based anti-TB vaccines development, and provide important tools for basic research.
A major secreted protein, CFP21, locating in RD2 was demonstrated to be an immunodominant antigen by elevating T cell proliferate response and production of cytokines such as IFN-y. CFP21 could also induce an optimum level of cytotoxic T cell activity. These findings suggested that CFP21 could be used as an attractive candidate antigen.
In the present study, to identify novel CFP21-derived HLA-A*0201/03 restricted epitopes, a series of native peptides were predicted with various prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Recent studies suggested that modification of amino acids at the anchor position of epitopes could result in enhancing the binding affinity to HLA-I molecules. Here, we designed analogues of the native peptides by altering the native peptides with tyrosine at position 1 (1Y), leucine at position 2 (2L) and/or position 9 (9L). Based on the three prediction programs, four native peptides, p5 (SLVRIVGVV), p13 (VVATTLALV), p134 (AVADHVAAV), p189 (NIMAHVSYV) and three of their analogues, p189-1Y2L9L (YLMAHVSYL), p134-1Y2L (YLADHVAAV), p134-1Y2L9L (YLADHVAAL) with prediction scores in the top 10 detected by at least two of the programs were selected and synthesized for further study. Peptides were synthesized by standard solid phase Fmoc strategy and were purified to more than 95% purity by reverse phase high performance liquid chromatography (RP-HPLC). Their molecular weights were confirmed by electro-spray ionization mass spectrometry (ESI-MS).
To evaluate the binding affinity of these peptides to HLA-A*0201/03 molecules and the stability of the peptide/HLA-A complexes in vitro, TAP-deficient T2 cells were used. In concordance with the binding affinities, these two analogues p134-1Y2L and p134-1Y2L9L showed more potent binding stabilities than the native peptide, p134. Based on the results of the T2 binding assay and peptide/HLA stability assay, among the seven peptides studied, only p134 and its two analogues were selected to investigate their ability to induce T cell response by using ELISPOT and cytotoxicity assay. Subsequent IFN-γrelease and LDH release assays by using PBMCs from HLA-A*0201+/03+ PPD+ healthy donors showed that the native peptide p134 and its two analogues p134-1Y2L and p134-1Y2L9L could induce potent T cell response, but only p134-induced CTLs could potently lyse the peptide-loaded target cells. All these results demonstrated that the amino acid sequence of 134-142 (AVADHVAAV) in CFP21 could be an immunogenic CTL epitope which could be both processed and presented endogenously via HLA-A*0201/03.
In the present study, a novel HLA-A*0201/03 restricted T cell epitope, p134 (AVADHVAAV), was identified in a secreted protein of Mtb, CFP21. The potent T cell response activity and the potential to be a broad-spectrum epitope made p134 a promising epitope for vaccination and immunotherapy against TB.
引文
[1]唐神结,肖和平.结核病免疫研究进展.国外医学内科学分册2002；29：369-372.
[2]吴建林,曹慧.耐药结核病流行现状与控制策略.寄生虫病与感染性疾病2009；7：51-4.
[3]Rajasekaran S, Chandrasekar C, Mahilmaran A, et al. HIV coinfection among multidrug resistant and extensively drug resistant tuberculosis patients--a trend. J Indian Med Assoc 2009; 107:281-2,284-6.
[4]Roy E, Lowrie DB, Jolles SR. Current strategies in TB immunotherapy. Curr Mol Med 2007; 7：373-86.
[5]Hernandez-Pando R, Castanon M, Espitia C, et al. Recombinant BCG vaccine candidates. Curr Mol Med 2007; 7:365-72.
[6]Romano M, Huygen K. DNA vaccines against mycobacterial diseases. Expert Rev Vaccines 2009; 8:1237-50.
[7]Sable SB, Kalra M, Verma I, et al. Tuberculosis subunit vaccine design:The conflict of antigenicity and immunogenicity. Clin Immunol 2007; 122:239-51.
[8]Doherty TM, Andersen P. Vaccines for tuberculosis:novel concepts and recent progress. Clin Microbiol Rev 2005; 18:687-702.
[9]Nicod LP. Immunology of tuberculosis. Swiss Med Wkly 2007; 137:357-62.
[10]van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002; 15:294-309.
[11]Barker LF, Brennan MJ, Rosenstein PK, et al. Tuberculosis vaccine research:the impact of immunology. Curr Opin Immunol 2009; 21:331-8.
[12]Cooper AM, Khader SA. The role of cytokines in the initiation, expansion, and control of cellular immunity to tuberculosis. Immunol Rev 2008; 226:191-204.
[13]Belardelli F. Role of interferons and other cytokines in the regulation of the immune response. APMIS 1995; 103:161-79.
[14]冯经华,尹凤鸣,严振球.目前结核分枝杆菌感染中重要免疫细胞和细胞因子作用的研究进展.临床军医杂志2007；35：291-95.
[15]Cutler A, Brombacher F. Cytokine therapy. Ann N Y Acad Sci 2005; 1056:16-29.
[16]Carlisle J, Evans W, Hajizadeh R, et al. Multiple mycobacterium antigens induce interferon-γ production from sarcoidosis peripheral blood mononuclear cells. Clin Exp Immunol 2007; 150:460-8.
[17]Russell MS, Dudani R, Krishnan L, et al. IFN-gamma expressed by T cells regulates the persistence of antigen presentation by limiting the survival of dendritic cells. J Immunol 2009; 183:7710-8.
[18]Kaufmann SH. The contribution of immunology to the rational design of novel antibacterial vaccines. Nat Rev Microbiol 2007; 5:491-503.
[19]Sud D, Bigbee C, Flynn JL, et al. Contribution of CD8+ T cells to control of Mycobacterium tuberculosis infection. J Immunol 2006; 176:4296-314.
[20]Kamath A, Woodworth JSM, Behar SM. Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection. J Immunol 2006; 177:6361-69.
[21]Woodworth JS, Behar SM. Mycobacterium tuberculosis-specific CD8+ T cells and their role in immunity. Crit Rev Immunol 2006; 26:317-52.
[22]Nemeth J, Winkler HM, Karlhofer F, et al. T cells co-producing Mycobacterium tuberculosis-specific type 1 cytokines for the diagnosis of latent tuberculosis. Eur Cytokine Netw 2010; 21:34-9.
[23]Woodworth JS, Wu Y, Behar SM. Mycobacterium tuberculosis-specific CD8+ T cells require perforin to kill target cells and provide protection in vivo. J Immunol 2008; 181: 8595-603.
[24]Canaday DH, Wilkinson RJ, Li Q, et al. CD4(+) and CD8(+) T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism. J Immunol 2001; 167:2734-42.
[25]Sonnenberg MG, Belisle JT. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gelelectrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Infect Immun 1997; 65:4515-4524.
[26]刘忠华,万康林,陈创夫.结核分枝杆菌主要分泌蛋白的研究进展.中国人兽共患病学报2007；23：722-24.
[27]Trajkovic V, Natarajan K, Sharma P. Immunomodulatory action of mycobacterial secretory proteins. Microbes Infect 2004; 6:513-519.
[28]Nagai S, Wiker HG, Harboe M, et al. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun 1991; 59:372-82.
[29]Lim JH, Kim HJ, Lee KS, et al. Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate. FEMS Microbiol Lett 2004; 232:51-59.
[30]Geluk A, van Meijgaarden KE, Franken KLMC, et al. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans. J Immunol 2000; 165:6463-71.
[31]Palma C, Iona E, Giannoni F, et al. The Ag85B protein of Mycobacterium tuberculosis may turn a protective immune response induced by Ag85B-DNA vaccine into a potent but non-protective Thl immune response in mice. Cell Microbiol 2007; 9:1455-65.
[32]Teixeira FM, Teixeira HC, Ferreira AP, et al. DNA vaccine using Mycobacterium bovis Ag85B antigen induces partial protection against experimental infection in BALB/c mice. Clin Vaccine Immunol 2006; 13:930-5
[33]Takamura S, Matsuo K, Takebe Y, et al. Ag85B of mycobacteria elicits effective CTL responses through activation of robust Thl immunity as a novel adjuvant in DNA vaccine. J Immunol 2005; 175:2541-7.
[34]Okkels LM, Brock I, Follmann F, et al. PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis:strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family. Infect Immun 2003; 71:6116-6123.
[35]Mustafa AS, Al-attiyah R, Hanif SN, et al. Efficient testing of pools of large numbers of peptides covering 12 open reading frames of Mycobacterium tuberculosis RD1 and identification of major antigens and immunodominant peptides recognized by human Thl cells. Clin Vaccine Immunol 2008; 15:916-24.
[36]张海,伍静,徐志凯.结核分枝杆菌RD1区研究进展.生物技术通报2007；18：267-69.
[37]Dietrich J, Aagaard C, Leah R, et al. Exchanging ESAT6 with TB 10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine:efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J Immunol 2005; 174:6332-9.
[38]Wang QM, Kang L, Wang XH. Improved cellular immune response elicited by a ubiquitin-fused ESAT-6 DNA vaccine against Mycobacterium tuberculosis. Microbiol Immunol 2009; 53:384-90.
[39]Aagaard CS, Hoang TT, Vingsbo-Lundberg C, et al. Quality and vaccine efficacy of CD4+ T cell responses directed to dominant and subdominant epitopes in ESAT-6 from Mycobacterium tuberculosis. J Immunol 2009; 183:2659-68.
[40]Grover A, Ahmed MF, Verma I, et al. Expression and purification of the Mycobacterium tuberculosis complex-restricted antigen CFP21 to study its immunoprophylactic potential in mouse model. Protein Expr Purif 2006; 48:274-80.
[41]Hovav AH, Fishman Y, Bercovier H. Gamma interferon and monophosphory lipid A-trehalose dicorynomycolate are efficient adjuvants for Mycobacterium tuberculosis multivalent acellular vaccine. Infect Immun 2005; 73:250-7.
[42]Sablel SB. Kalra M, Verma I, et al. Tuberculosis subunit vaccine design:the conflict of antigenicity and immunogenicity. Clin Immunol 2007; 122:239-51.
[43]Kalra M, Grover A, Mehta N, et al. Supplementation with RD antigens enhances the protective efficacy of BCG in tuberculous mice. Clin Immunol 2007; 125:173-83.
[44]Wei ML, Cresswell P. HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 1992; 356:443-6.
[45]Falk K, Rotzschke O, Stevanovic S, et al. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 1991; 351:290-6.
[46]Wang R, Doolan DL, Le TP, et al. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 1998; 282:476-80.
[47]Mustafa AS, Shaban FA. ProPred analysis and experimental evaluation of promiscuous T-cell epitopes of three major secreted antigens of Mycobacterium tuberculosis. Tuberculosis 2006; 86:115-124.
[48]Mustafa AS, Shaban FA, Al-Attiyah R, et al. Human Thl cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class Ⅱ molecules. Scand J Immunol 2003; 57:125-34.
[49]Shams H, Klucar P, Weis SE, et al. Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+T cells in the context of multiple HLA alleles. J Immunol 2004; 173:1966-77.
[50]Geluk A, Taneja V, van Meijgaarden KE, et al. Identification of HLA class Ⅱ-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class Ⅱ-deficient mice. Proc Natl Acad Sci USA 1998; 95:10797-802.
[51]Molder T, Adojaan M, Kaldma K, et al. Elicitation of broad CTL response against HIV-1 by the DNA vaccine encoding artificial multi-component fusion protein MultiHIV--study in domestic pigs. Vaccine 2009; 28:293-8.
[52]Lamberth K, Rφder G, Harndahl M, et al. The peptide-binding specificity of HLA-A*3001 demonstrates membership of the HLA-A3 supertype. Immunogenetics 2008; 60:633-43.
[53]阮志华,吴玉章,支轶等.肿瘤血管内皮抗原TEM8的HLA-2.1限制性低亲和性CTL表位预测及改造分析.第三军医大学学报2005；27：1106-08.
[54]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[55]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.
[56]Lazoura E, Apostolopoulos V. Rational peptide-based vaccine design for cancer immunotherapeutic applications. Curr Med Chem 2005; 12:629-39.
[57]Lazoura E, Lodding J, Farrugia W, et al. Enhanced major histocompatibility complex class Ⅰ binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide. Immunology 2006; 119:306-16.
[58]Persson YA, Blomgran-Julinder R, Rahman S, et al. Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria. Microbes Infect 2008; 10: 233-40.
[59]Quintana FJ, Carmi P, Mor F, et al. DNA fragments of the human 60-kDa heat shock protein (HSP60) vaccinate against adjuvant arthritis:identification of a regulatory HSP60 peptide. J Immunol 2003; 171:3533-41.
[60]印春生.合成肽疫苗研究进展.中国兽药杂志2005；39：29-33.
[61]祝秉东,王洪海.结核疫苗研究的历史与现状.中华结核和呼吸杂志2007；30：387-82.
[62]Ding FX, Wang F, Lu YM, et al. Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009; 49: 1492-502.
[63]Fonseca DP, Benaissa-Trouw B, van EM, et al. Induction of cell-mediated immunity against Mycobacterium tuberculosis using DNA vaccines encoding cytotoxic and helper T-cell epitopes of the 38-kilodalton protein. Infect Immun 2001; 69:4839-4845.
[64]Wang QM, Sun SH, Hu ZL, et al. Epitope DNA vaccines against tuberculosis:spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine. Scand J Immunol 2004; 60:219-225.
[65]De Groot AS, McMurry J, Marcon L, et al. Developing an epitope-driven tuberculosis (TB) vaccine. Vaccine 2005; 23:2121-31.
[1]Wang B, Yao K, Liu GY, et al. Computational prediction and identification of Epstein-Barr virus latent membrane protein 2A antigen-specific CD8+ T-cell epitopes. Cell Mol Immunol 2009; 6:97-103.
[2]Mommaas B, KampJ, Drijfhout JW, et al. Identification of a novel HLA-B60-restricted T cell epitope of the minor histocompatibility antigen HA-1 locus. J Immunol 2002; 169 3131-6.
[3]Kesmir C, Nussbaum AK, Schild AK, et al. Prediction of proteasome cleavage motifs by neural networks. Protein Eng 2002; 15:287-96.
[4]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[5]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.
[6]Lazoura E, Apostolopoulos V. Rational peptide-based vaccine design for cancer immunotherapeutic applications. Curr Med Chem 2005; 12:629-39.
[7]唱凯,李贤龙,贾延辉等.T细胞抗原表位预测的研究方法进展.生物学杂志2009；26：65-68.
[8]高艳锋,翟明霞,祁峰等.肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位的预测.现代肿瘤学2008；16：1850-52.
[9]Lazoura E, Lodding J, Farrugia W, et al. Enhanced major histocompatibility complex class Ⅰ binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide. Immunology 2006; 119:306-16.
[10]Mays LE, Wilson JM. Identification of the murine AAVrh32.33 capsid-specific CD8+ T cell epitopes. J Gene Med 2009; 11:1095-102.
[11]Lim JB, Kim HO, Jeong SH, et al. Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1) epitopes as targets for CD8+ HCMV-specific cytotoxic T lymphocytes. J Transl Med 2009; 7:72-83.
[12]Gao YF, Sun ZQ, Qi F, et al. Identification of a new broad-spectrum CD8+ T cell epitope from over-expressed antigen COX-2 in esophageal carcinoma. Cancer Lett 2009; 284: 55-61.
[13]Semeniuk CA, Capina RE, Mendoza MG, et al. Identification and characterization of HLA-A*0301 epitopes in HIV-1 gag proteins using a novel approach. J Immunol Methods 2010; 352:118-25.
[14]Shingler WH, Chikoti P, Kingsman SM, et al. Identification and functional validation of MHC class Ⅰ epitopes in the tumor-associated antigen 5T4. Int Immunol 2008; 20:1057-66.
[15]陈燕,娄加陶,吴传勇等.KLIANNTRV是结核抗原Ag85A的HLA-A*0201限制性CTL表位.现代检验医学杂志2007；22：1-3.
[16]吴传勇,娄加陶,蒋廷旺等.结核杆菌抗原Ag85C的HLA-A*0201限制性CD8+CTL表位的预测及鉴定.第二军医大学学报2007；28：349-54.
[17]Mina Suzuki, Taiki Aoshi, Toshi Nagata, et al. Identification of Murine H2-Dd-and H2-Ab-Restricted T-Cell Epitopes on a Novel Protective Antigen, MPT51, of Mycobacterium tuberculosis. Infect Immun 2004; 72:3829-3837.
[18]Aoshi T, Nagata T, Suzuki M, et al. Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening. Infect Immun 2008; 76:1565-71.
[19]Adams HP, Koziol JA. Prediction of binding to MHC class Ⅰ molecules. J Immunol Methods 1995; 185:181-90.
[20]Parker KC, Shields M, DiBrino M, et al. Peptide binding to MHC class Ⅰ molecules: implications for antigenic peptide prediction. Immunol Res 1995; 14:34-57.
[21]Yu K, Petrovsky N, Schonbach C, et al. Methods for prediction of peptide binding to MHC molecules:a comparative study. Mol Med 2002; 8:137-48.
[22]唐艳,吴玉章,林治华等.酪氨酸激酶相关蛋白22(180-188)表位的修饰和初步鉴定.第三军医大学学报2005；27：309-12.
[23]阮志华,吴玉章,支轶等.肿瘤血管内皮抗原TEM8的HLA-2.1限制性低亲和性CTL表位预测及改造分析.第三军医大学学报2005；27：1106-08.
[24]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[25]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.
[1]Merrifield RB. Solid phase peptide synthesis I:the synthesis of a tetrapeptide. J Am Chem Soc 1963; 85:2149-2154.
[2]王德心.固相有机合成—原理及应用指南.北京：化学工业出版社,2004,1-138.
[3]Swinnen D, Hilvert D. Facile, fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters. Org Lett 2000; 2:2439-2442.
[4]郑彦慧,张艳平,张浩等.Fmoc保护氨基酸与Wang树脂的缩合反应.高等学校化学学报2008；29：1769-72.
[5]景文鹏,吴蕾.Fmoc固相合成神经降压素.中国现代医生2008；46：39-40.
[6]李从岩,徐琪,颜炜群.固相合成法制备环肽Hymen istatin-1及其含statine的类似物.中国抗生素杂志2008；33：678-81.
[7]王卫国,杨明,李佩华等.多肽合成中氨基酸活化酯的制备研究.化学试剂2007；29：391-3.
[8]王丙莲,孟庆军,张利群等.HPLC-MS/MS在活性多肽检测中的应用.食品研究与开发2007；28：159-61.
[9]齐崴,贾辰熙,何志敏.反相高效液相色谱与质谱联用分析合成七肽的消旋产物.分析化学研究报告2006；34：1244-48.
[10]Fanciulli G, Azara E, Wood TD, et al. Quantification of Gluten Exorphin A5 in cerebrospinal fluid by liquid chromatography-mass spectrometry. J Chroma B 2006; 833: 204-209.
[1]Tang Y, Lin ZH, Ni B, et al. An altered peptide ligand for naive cytotoxic T lymphocyte epitope of TRP-2(180-188) enhanced immunogenicity. Cancer Immunol Immunother 2007; 56:319-29.
[2]Li F, Yang D, Wang YQ, et al. Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen. Cancer Immunol Immunother 2009; 58: 2039-49.
[3]He YD, Mao LW, Lin ZH, et al. Identification of a common HLA-A*0201-restricted epitope among SSX family members by mimicking altered peptide ligands strategy. Mol Immunol 2008; 25:2455-64.
[4]Boss CN, Griinebach F, Brauer K, et al. Identification and characterization of T-cell epitopes deduced from RGS5, a novel broadly expressed tumor antigen. Clin Cancer Res 2007; 13: 3347-55.
[5]Tanaka F, Fujie T, Go H, et al. Efficient induction of antitumor cytoxic T lymphocytes from a healthy donor using HLA-A2-restricted MAGE-3 peptide in vitro. Cancer Immunol Immunother 1997; 44:21-6.
[6]Vertuani S, Sette A, Sidney J, et al. Improved immunogenicity of an immunodominant epitope of the Her-2/neu protooncogene by alterations of MHC contact residues. J Immunol 2004; 172:3501-8.
[7]Ding FX, Wang F, Lu YM, et al. Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009; 49: 1492-502.
[8]Wang B, Yao K, Liu GY, et al. Computational prediction and identification of Epstein-Barr virus latent membrane protein 2A antigen-specific CD8+ T-cell epitopes. Cell Mol Immunol 2009; 6:97-103.
[9]祁峰.肿瘤抗原环氧合酶-2的HLA限制性广谱CTL表位筛选与改造.硕士学位论文.郑州：郑州大学,2007.
[10]阮志华,吴玉章,支轶等.肿瘤血管内皮抗原TEM8的HLA-2.1限制性低亲和性CTL表位预测及改造分析.第三军医大学学报2005；27：1106-08.
[11]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[12]孙伟红.人磷脂酰乙醇胺结合蛋白4(hPEBP4)的HLA-A*0201限制性CD8+ CTL表位的鉴定及功能研究.博士学位论文.上海：第二军医大学,2006.
[13]Mashishi T, Gray CM. The ELISPOT assay:an easily transferable method for measuring cellular responses and identifying T cell epitopes. Clin Chem Lab Med 2002; 40:903-10.
[14]JR, Kuta EG, Turk E, et al. A panel of MHC class Ⅰ restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J Immunol Methods 2002; 260:157-72.
[15]Schmittel A, Keilholz U, Thiel E, et al. Quantification of tumor-specific T lymphocytes with the ELISPOT assay. J Immunother 2000; 23:289-95.
[16]Kim GG, Donnenberg VS, Donnenberg AD, et al. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4h 51Cr-release assay. J Immunol Methods.2007; 325:51-66.
[17]Savage P, Millrain M, Dimakou S, et al. Expansion of CD8+ cytotoxic T cells in vitro and in vivo using MHC class I tetramers. Tumour Biol 2007; 28:70-6.
[1]吴建林,曹慧.耐药结核病流行现状与控制策略.寄生虫病与感染性疾病2009；7：51-4.
[2]Roy E, Lowrie DB, Jolles SR. Current strategies in TB immunotherapy. Curr Mol Med 2007; 7:373-86.
[3]Cooper AM. Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009; 27:393-422.
[4]唐神结,肖和平.结核病免疫研究进展.国外医学内科学分册2002；29：369-372.
[5]van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002; 15:294-309.
[6]Grotzke JE, Harriff MJ, Siler AC, et al. The Mycobacterium tuberculosis phagosome is a HLA-I processing competent organelle. PLoS Pathog 2009; 5:e1000374.
[7]Zinkernagel RM. Doherty PC. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 1974; 248:701-702.
[8]Townsend AR, McMichael AJ, Carter NP, et al. Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell 1984; 39: 13-25.
[9]Townsend AR, Rothbard J, Gotch FM, et al. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986; 44:959-68.
[10]Falk K, Rotzschke O, Rammensee HG. Cellular peptide composition governed by major histocompatibility complex class I molecules. Nature 1990; 348:248-51.
[11]Rotzschke O, Falk K, Deres K, et al. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature 1990; 348:252-4.
[12]Rotzschke O, Falk K, Wallny HJ, et al. Characterization of naturally occurring minor histocompatibility peptides including H-4 and H-Y. Science 1990; 249:283-7.
[13]Ding FX, Wang F, Lu YM, et al. Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009; 49: 1492-502.
[14]De Groot AS, McMurry J, Marcon L, et al. Developing an epitope-driven tuberculosis (TB) vaccine. Vaccine 2005; 23:2121-31.
[15]陈燕,娄加陶,吴传勇等.KLIANNTRV是结核抗原Ag85A的HLA-A*0201限制性CTL表位.现代检验医学杂志2007；22：1-3.
[16]吴传勇,娄加陶,蒋廷旺等.结核杆菌抗原Ag85C的HLA-A*0201限制性CD8+CTL表位的预测及鉴定.第二军医大学学报2007；28：349-54.
[17]Geluk A, van Meijgaarden KE, Franken KLMC, et al. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans. J Immunol 2000; 165:6463-71.
[18]Aoshi T, Nagata T, Suzuki M, et al. Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening. Infect Immun 2008; 76:1565-71.
[19]Frieder M, Lewinsohn DM. T-cell epitope mapping in Mycobacterium tuberculosis using pepmixes created by micro-scale SPOT-synthesis. Methods Mol Biol 2009; 524:369-82.
[20]He YD, Mao LW, Lin ZH, et al. Identification of a common HLA-A*0201-restricted epitope among SSX family members by mimicking altered peptide ligands strategy. Mol Immunol 2008; 25:2455-64.
[21]Boss CN, Grunebach F, Brauer K, et al. Identification and characterization of T-cell epitopes deduced from RGS5, a novel broadly expressed tumor antigen. Clin Cancer Res 2007; 13:3347-55.
[22]Mays LE, Wilson JM. Identification of the murine AAVrh32.33 capsid-specific CD8+ T cell epitopes. J Gene Med 2009; 11:1095-102.
[23]Lazoura E, Lodding J, Farrugia W, et al. Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide. Immunology 2006; 119:306-16.
[24]Lazoura E, Apostolopoulos V. Rational peptide-based vaccine design for cancer immunotherapeutic applications. Curr Med Chem 2005; 12:629-39.
[25]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.
[2]吴建林,曹慧.耐药结核病流行现状与控制策略.寄生虫病与感染性疾病2009；7：51-4.
[3]Rajasekaran S, Chandrasekar C, Mahilmaran A, et al. HIV coinfection among multidrug resistant and extensively drug resistant tuberculosis patients--a trend. J Indian Med Assoc 2009; 107:281-2,284-6.
[4]Roy E, Lowrie DB, Jolles SR. Current strategies in TB immunotherapy. Curr Mol Med 2007; 7：373-86.
[5]Hernandez-Pando R, Castanon M, Espitia C, et al. Recombinant BCG vaccine candidates. Curr Mol Med 2007; 7:365-72.
[6]Romano M, Huygen K. DNA vaccines against mycobacterial diseases. Expert Rev Vaccines 2009; 8:1237-50.
[7]Sable SB, Kalra M, Verma I, et al. Tuberculosis subunit vaccine design:The conflict of antigenicity and immunogenicity. Clin Immunol 2007; 122:239-51.
[8]Doherty TM, Andersen P. Vaccines for tuberculosis:novel concepts and recent progress. Clin Microbiol Rev 2005; 18:687-702.
[9]Nicod LP. Immunology of tuberculosis. Swiss Med Wkly 2007; 137:357-62.
[10]van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002; 15:294-309.
[11]Barker LF, Brennan MJ, Rosenstein PK, et al. Tuberculosis vaccine research:the impact of immunology. Curr Opin Immunol 2009; 21:331-8.
[12]Cooper AM, Khader SA. The role of cytokines in the initiation, expansion, and control of cellular immunity to tuberculosis. Immunol Rev 2008; 226:191-204.
[13]Belardelli F. Role of interferons and other cytokines in the regulation of the immune response. APMIS 1995; 103:161-79.
[14]冯经华,尹凤鸣,严振球.目前结核分枝杆菌感染中重要免疫细胞和细胞因子作用的研究进展.临床军医杂志2007；35：291-95.
[15]Cutler A, Brombacher F. Cytokine therapy. Ann N Y Acad Sci 2005; 1056:16-29.
[16]Carlisle J, Evans W, Hajizadeh R, et al. Multiple mycobacterium antigens induce interferon-γ production from sarcoidosis peripheral blood mononuclear cells. Clin Exp Immunol 2007; 150:460-8.
[17]Russell MS, Dudani R, Krishnan L, et al. IFN-gamma expressed by T cells regulates the persistence of antigen presentation by limiting the survival of dendritic cells. J Immunol 2009; 183:7710-8.
[18]Kaufmann SH. The contribution of immunology to the rational design of novel antibacterial vaccines. Nat Rev Microbiol 2007; 5:491-503.
[19]Sud D, Bigbee C, Flynn JL, et al. Contribution of CD8+ T cells to control of Mycobacterium tuberculosis infection. J Immunol 2006; 176:4296-314.
[20]Kamath A, Woodworth JSM, Behar SM. Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection. J Immunol 2006; 177:6361-69.
[21]Woodworth JS, Behar SM. Mycobacterium tuberculosis-specific CD8+ T cells and their role in immunity. Crit Rev Immunol 2006; 26:317-52.
[22]Nemeth J, Winkler HM, Karlhofer F, et al. T cells co-producing Mycobacterium tuberculosis-specific type 1 cytokines for the diagnosis of latent tuberculosis. Eur Cytokine Netw 2010; 21:34-9.
[23]Woodworth JS, Wu Y, Behar SM. Mycobacterium tuberculosis-specific CD8+ T cells require perforin to kill target cells and provide protection in vivo. J Immunol 2008; 181: 8595-603.
[24]Canaday DH, Wilkinson RJ, Li Q, et al. CD4(+) and CD8(+) T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism. J Immunol 2001; 167:2734-42.
[25]Sonnenberg MG, Belisle JT. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gelelectrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Infect Immun 1997; 65:4515-4524.
[26]刘忠华,万康林,陈创夫.结核分枝杆菌主要分泌蛋白的研究进展.中国人兽共患病学报2007；23：722-24.
[27]Trajkovic V, Natarajan K, Sharma P. Immunomodulatory action of mycobacterial secretory proteins. Microbes Infect 2004; 6:513-519.
[28]Nagai S, Wiker HG, Harboe M, et al. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun 1991; 59:372-82.
[29]Lim JH, Kim HJ, Lee KS, et al. Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate. FEMS Microbiol Lett 2004; 232:51-59.
[30]Geluk A, van Meijgaarden KE, Franken KLMC, et al. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans. J Immunol 2000; 165:6463-71.
[31]Palma C, Iona E, Giannoni F, et al. The Ag85B protein of Mycobacterium tuberculosis may turn a protective immune response induced by Ag85B-DNA vaccine into a potent but non-protective Thl immune response in mice. Cell Microbiol 2007; 9:1455-65.
[32]Teixeira FM, Teixeira HC, Ferreira AP, et al. DNA vaccine using Mycobacterium bovis Ag85B antigen induces partial protection against experimental infection in BALB/c mice. Clin Vaccine Immunol 2006; 13:930-5
[33]Takamura S, Matsuo K, Takebe Y, et al. Ag85B of mycobacteria elicits effective CTL responses through activation of robust Thl immunity as a novel adjuvant in DNA vaccine. J Immunol 2005; 175:2541-7.
[34]Okkels LM, Brock I, Follmann F, et al. PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis:strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family. Infect Immun 2003; 71:6116-6123.
[35]Mustafa AS, Al-attiyah R, Hanif SN, et al. Efficient testing of pools of large numbers of peptides covering 12 open reading frames of Mycobacterium tuberculosis RD1 and identification of major antigens and immunodominant peptides recognized by human Thl cells. Clin Vaccine Immunol 2008; 15:916-24.
[36]张海,伍静,徐志凯.结核分枝杆菌RD1区研究进展.生物技术通报2007；18：267-69.
[37]Dietrich J, Aagaard C, Leah R, et al. Exchanging ESAT6 with TB 10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine:efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J Immunol 2005; 174:6332-9.
[38]Wang QM, Kang L, Wang XH. Improved cellular immune response elicited by a ubiquitin-fused ESAT-6 DNA vaccine against Mycobacterium tuberculosis. Microbiol Immunol 2009; 53:384-90.
[39]Aagaard CS, Hoang TT, Vingsbo-Lundberg C, et al. Quality and vaccine efficacy of CD4+ T cell responses directed to dominant and subdominant epitopes in ESAT-6 from Mycobacterium tuberculosis. J Immunol 2009; 183:2659-68.
[40]Grover A, Ahmed MF, Verma I, et al. Expression and purification of the Mycobacterium tuberculosis complex-restricted antigen CFP21 to study its immunoprophylactic potential in mouse model. Protein Expr Purif 2006; 48:274-80.
[41]Hovav AH, Fishman Y, Bercovier H. Gamma interferon and monophosphory lipid A-trehalose dicorynomycolate are efficient adjuvants for Mycobacterium tuberculosis multivalent acellular vaccine. Infect Immun 2005; 73:250-7.
[42]Sablel SB. Kalra M, Verma I, et al. Tuberculosis subunit vaccine design:the conflict of antigenicity and immunogenicity. Clin Immunol 2007; 122:239-51.
[43]Kalra M, Grover A, Mehta N, et al. Supplementation with RD antigens enhances the protective efficacy of BCG in tuberculous mice. Clin Immunol 2007; 125:173-83.
[44]Wei ML, Cresswell P. HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 1992; 356:443-6.
[45]Falk K, Rotzschke O, Stevanovic S, et al. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 1991; 351:290-6.
[46]Wang R, Doolan DL, Le TP, et al. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 1998; 282:476-80.
[47]Mustafa AS, Shaban FA. ProPred analysis and experimental evaluation of promiscuous T-cell epitopes of three major secreted antigens of Mycobacterium tuberculosis. Tuberculosis 2006; 86:115-124.
[48]Mustafa AS, Shaban FA, Al-Attiyah R, et al. Human Thl cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class Ⅱ molecules. Scand J Immunol 2003; 57:125-34.
[49]Shams H, Klucar P, Weis SE, et al. Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+T cells in the context of multiple HLA alleles. J Immunol 2004; 173:1966-77.
[50]Geluk A, Taneja V, van Meijgaarden KE, et al. Identification of HLA class Ⅱ-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class Ⅱ-deficient mice. Proc Natl Acad Sci USA 1998; 95:10797-802.
[51]Molder T, Adojaan M, Kaldma K, et al. Elicitation of broad CTL response against HIV-1 by the DNA vaccine encoding artificial multi-component fusion protein MultiHIV--study in domestic pigs. Vaccine 2009; 28:293-8.
[52]Lamberth K, Rφder G, Harndahl M, et al. The peptide-binding specificity of HLA-A*3001 demonstrates membership of the HLA-A3 supertype. Immunogenetics 2008; 60:633-43.
[53]阮志华,吴玉章,支轶等.肿瘤血管内皮抗原TEM8的HLA-2.1限制性低亲和性CTL表位预测及改造分析.第三军医大学学报2005；27：1106-08.
[54]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[55]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.
[56]Lazoura E, Apostolopoulos V. Rational peptide-based vaccine design for cancer immunotherapeutic applications. Curr Med Chem 2005; 12:629-39.
[57]Lazoura E, Lodding J, Farrugia W, et al. Enhanced major histocompatibility complex class Ⅰ binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide. Immunology 2006; 119:306-16.
[58]Persson YA, Blomgran-Julinder R, Rahman S, et al. Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria. Microbes Infect 2008; 10: 233-40.
[59]Quintana FJ, Carmi P, Mor F, et al. DNA fragments of the human 60-kDa heat shock protein (HSP60) vaccinate against adjuvant arthritis:identification of a regulatory HSP60 peptide. J Immunol 2003; 171:3533-41.
[60]印春生.合成肽疫苗研究进展.中国兽药杂志2005；39：29-33.
[61]祝秉东,王洪海.结核疫苗研究的历史与现状.中华结核和呼吸杂志2007；30：387-82.
[62]Ding FX, Wang F, Lu YM, et al. Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009; 49: 1492-502.
[63]Fonseca DP, Benaissa-Trouw B, van EM, et al. Induction of cell-mediated immunity against Mycobacterium tuberculosis using DNA vaccines encoding cytotoxic and helper T-cell epitopes of the 38-kilodalton protein. Infect Immun 2001; 69:4839-4845.
[64]Wang QM, Sun SH, Hu ZL, et al. Epitope DNA vaccines against tuberculosis:spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine. Scand J Immunol 2004; 60:219-225.
[65]De Groot AS, McMurry J, Marcon L, et al. Developing an epitope-driven tuberculosis (TB) vaccine. Vaccine 2005; 23:2121-31.
[1]Wang B, Yao K, Liu GY, et al. Computational prediction and identification of Epstein-Barr virus latent membrane protein 2A antigen-specific CD8+ T-cell epitopes. Cell Mol Immunol 2009; 6:97-103.
[2]Mommaas B, KampJ, Drijfhout JW, et al. Identification of a novel HLA-B60-restricted T cell epitope of the minor histocompatibility antigen HA-1 locus. J Immunol 2002; 169 3131-6.
[3]Kesmir C, Nussbaum AK, Schild AK, et al. Prediction of proteasome cleavage motifs by neural networks. Protein Eng 2002; 15:287-96.
[4]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[5]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.
[6]Lazoura E, Apostolopoulos V. Rational peptide-based vaccine design for cancer immunotherapeutic applications. Curr Med Chem 2005; 12:629-39.
[7]唱凯,李贤龙,贾延辉等.T细胞抗原表位预测的研究方法进展.生物学杂志2009；26：65-68.
[8]高艳锋,翟明霞,祁峰等.肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位的预测.现代肿瘤学2008；16：1850-52.
[9]Lazoura E, Lodding J, Farrugia W, et al. Enhanced major histocompatibility complex class Ⅰ binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide. Immunology 2006; 119:306-16.
[10]Mays LE, Wilson JM. Identification of the murine AAVrh32.33 capsid-specific CD8+ T cell epitopes. J Gene Med 2009; 11:1095-102.
[11]Lim JB, Kim HO, Jeong SH, et al. Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1) epitopes as targets for CD8+ HCMV-specific cytotoxic T lymphocytes. J Transl Med 2009; 7:72-83.
[12]Gao YF, Sun ZQ, Qi F, et al. Identification of a new broad-spectrum CD8+ T cell epitope from over-expressed antigen COX-2 in esophageal carcinoma. Cancer Lett 2009; 284: 55-61.
[13]Semeniuk CA, Capina RE, Mendoza MG, et al. Identification and characterization of HLA-A*0301 epitopes in HIV-1 gag proteins using a novel approach. J Immunol Methods 2010; 352:118-25.
[14]Shingler WH, Chikoti P, Kingsman SM, et al. Identification and functional validation of MHC class Ⅰ epitopes in the tumor-associated antigen 5T4. Int Immunol 2008; 20:1057-66.
[15]陈燕,娄加陶,吴传勇等.KLIANNTRV是结核抗原Ag85A的HLA-A*0201限制性CTL表位.现代检验医学杂志2007；22：1-3.
[16]吴传勇,娄加陶,蒋廷旺等.结核杆菌抗原Ag85C的HLA-A*0201限制性CD8+CTL表位的预测及鉴定.第二军医大学学报2007；28：349-54.
[17]Mina Suzuki, Taiki Aoshi, Toshi Nagata, et al. Identification of Murine H2-Dd-and H2-Ab-Restricted T-Cell Epitopes on a Novel Protective Antigen, MPT51, of Mycobacterium tuberculosis. Infect Immun 2004; 72:3829-3837.
[18]Aoshi T, Nagata T, Suzuki M, et al. Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening. Infect Immun 2008; 76:1565-71.
[19]Adams HP, Koziol JA. Prediction of binding to MHC class Ⅰ molecules. J Immunol Methods 1995; 185:181-90.
[20]Parker KC, Shields M, DiBrino M, et al. Peptide binding to MHC class Ⅰ molecules: implications for antigenic peptide prediction. Immunol Res 1995; 14:34-57.
[21]Yu K, Petrovsky N, Schonbach C, et al. Methods for prediction of peptide binding to MHC molecules:a comparative study. Mol Med 2002; 8:137-48.
[22]唐艳,吴玉章,林治华等.酪氨酸激酶相关蛋白22(180-188)表位的修饰和初步鉴定.第三军医大学学报2005；27：309-12.
[23]阮志华,吴玉章,支轶等.肿瘤血管内皮抗原TEM8的HLA-2.1限制性低亲和性CTL表位预测及改造分析.第三军医大学学报2005；27：1106-08.
[24]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[25]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.
[1]Merrifield RB. Solid phase peptide synthesis I:the synthesis of a tetrapeptide. J Am Chem Soc 1963; 85:2149-2154.
[2]王德心.固相有机合成—原理及应用指南.北京：化学工业出版社,2004,1-138.
[3]Swinnen D, Hilvert D. Facile, fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters. Org Lett 2000; 2:2439-2442.
[4]郑彦慧,张艳平,张浩等.Fmoc保护氨基酸与Wang树脂的缩合反应.高等学校化学学报2008；29：1769-72.
[5]景文鹏,吴蕾.Fmoc固相合成神经降压素.中国现代医生2008；46：39-40.
[6]李从岩,徐琪,颜炜群.固相合成法制备环肽Hymen istatin-1及其含statine的类似物.中国抗生素杂志2008；33：678-81.
[7]王卫国,杨明,李佩华等.多肽合成中氨基酸活化酯的制备研究.化学试剂2007；29：391-3.
[8]王丙莲,孟庆军,张利群等.HPLC-MS/MS在活性多肽检测中的应用.食品研究与开发2007；28：159-61.
[9]齐崴,贾辰熙,何志敏.反相高效液相色谱与质谱联用分析合成七肽的消旋产物.分析化学研究报告2006；34：1244-48.
[10]Fanciulli G, Azara E, Wood TD, et al. Quantification of Gluten Exorphin A5 in cerebrospinal fluid by liquid chromatography-mass spectrometry. J Chroma B 2006; 833: 204-209.
[1]Tang Y, Lin ZH, Ni B, et al. An altered peptide ligand for naive cytotoxic T lymphocyte epitope of TRP-2(180-188) enhanced immunogenicity. Cancer Immunol Immunother 2007; 56:319-29.
[2]Li F, Yang D, Wang YQ, et al. Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen. Cancer Immunol Immunother 2009; 58: 2039-49.
[3]He YD, Mao LW, Lin ZH, et al. Identification of a common HLA-A*0201-restricted epitope among SSX family members by mimicking altered peptide ligands strategy. Mol Immunol 2008; 25:2455-64.
[4]Boss CN, Griinebach F, Brauer K, et al. Identification and characterization of T-cell epitopes deduced from RGS5, a novel broadly expressed tumor antigen. Clin Cancer Res 2007; 13: 3347-55.
[5]Tanaka F, Fujie T, Go H, et al. Efficient induction of antitumor cytoxic T lymphocytes from a healthy donor using HLA-A2-restricted MAGE-3 peptide in vitro. Cancer Immunol Immunother 1997; 44:21-6.
[6]Vertuani S, Sette A, Sidney J, et al. Improved immunogenicity of an immunodominant epitope of the Her-2/neu protooncogene by alterations of MHC contact residues. J Immunol 2004; 172:3501-8.
[7]Ding FX, Wang F, Lu YM, et al. Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009; 49: 1492-502.
[8]Wang B, Yao K, Liu GY, et al. Computational prediction and identification of Epstein-Barr virus latent membrane protein 2A antigen-specific CD8+ T-cell epitopes. Cell Mol Immunol 2009; 6:97-103.
[9]祁峰.肿瘤抗原环氧合酶-2的HLA限制性广谱CTL表位筛选与改造.硕士学位论文.郑州：郑州大学,2007.
[10]阮志华,吴玉章,支轶等.肿瘤血管内皮抗原TEM8的HLA-2.1限制性低亲和性CTL表位预测及改造分析.第三军医大学学报2005；27：1106-08.
[11]Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides:implication in the identification of cryptic tumor epitopes. Eur J Immunol 2000; 30:3411-21.
[12]孙伟红.人磷脂酰乙醇胺结合蛋白4(hPEBP4)的HLA-A*0201限制性CD8+ CTL表位的鉴定及功能研究.博士学位论文.上海：第二军医大学,2006.
[13]Mashishi T, Gray CM. The ELISPOT assay:an easily transferable method for measuring cellular responses and identifying T cell epitopes. Clin Chem Lab Med 2002; 40:903-10.
[14]JR, Kuta EG, Turk E, et al. A panel of MHC class Ⅰ restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J Immunol Methods 2002; 260:157-72.
[15]Schmittel A, Keilholz U, Thiel E, et al. Quantification of tumor-specific T lymphocytes with the ELISPOT assay. J Immunother 2000; 23:289-95.
[16]Kim GG, Donnenberg VS, Donnenberg AD, et al. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4h 51Cr-release assay. J Immunol Methods.2007; 325:51-66.
[17]Savage P, Millrain M, Dimakou S, et al. Expansion of CD8+ cytotoxic T cells in vitro and in vivo using MHC class I tetramers. Tumour Biol 2007; 28:70-6.
[1]吴建林,曹慧.耐药结核病流行现状与控制策略.寄生虫病与感染性疾病2009；7：51-4.
[2]Roy E, Lowrie DB, Jolles SR. Current strategies in TB immunotherapy. Curr Mol Med 2007; 7:373-86.
[3]Cooper AM. Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009; 27:393-422.
[4]唐神结,肖和平.结核病免疫研究进展.国外医学内科学分册2002；29：369-372.
[5]van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002; 15:294-309.
[6]Grotzke JE, Harriff MJ, Siler AC, et al. The Mycobacterium tuberculosis phagosome is a HLA-I processing competent organelle. PLoS Pathog 2009; 5:e1000374.
[7]Zinkernagel RM. Doherty PC. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 1974; 248:701-702.
[8]Townsend AR, McMichael AJ, Carter NP, et al. Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell 1984; 39: 13-25.
[9]Townsend AR, Rothbard J, Gotch FM, et al. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986; 44:959-68.
[10]Falk K, Rotzschke O, Rammensee HG. Cellular peptide composition governed by major histocompatibility complex class I molecules. Nature 1990; 348:248-51.
[11]Rotzschke O, Falk K, Deres K, et al. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature 1990; 348:252-4.
[12]Rotzschke O, Falk K, Wallny HJ, et al. Characterization of naturally occurring minor histocompatibility peptides including H-4 and H-Y. Science 1990; 249:283-7.
[13]Ding FX, Wang F, Lu YM, et al. Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma. Hepatology 2009; 49: 1492-502.
[14]De Groot AS, McMurry J, Marcon L, et al. Developing an epitope-driven tuberculosis (TB) vaccine. Vaccine 2005; 23:2121-31.
[15]陈燕,娄加陶,吴传勇等.KLIANNTRV是结核抗原Ag85A的HLA-A*0201限制性CTL表位.现代检验医学杂志2007；22：1-3.
[16]吴传勇,娄加陶,蒋廷旺等.结核杆菌抗原Ag85C的HLA-A*0201限制性CD8+CTL表位的预测及鉴定.第二军医大学学报2007；28：349-54.
[17]Geluk A, van Meijgaarden KE, Franken KLMC, et al. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans. J Immunol 2000; 165:6463-71.
[18]Aoshi T, Nagata T, Suzuki M, et al. Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening. Infect Immun 2008; 76:1565-71.
[19]Frieder M, Lewinsohn DM. T-cell epitope mapping in Mycobacterium tuberculosis using pepmixes created by micro-scale SPOT-synthesis. Methods Mol Biol 2009; 524:369-82.
[20]He YD, Mao LW, Lin ZH, et al. Identification of a common HLA-A*0201-restricted epitope among SSX family members by mimicking altered peptide ligands strategy. Mol Immunol 2008; 25:2455-64.
[21]Boss CN, Grunebach F, Brauer K, et al. Identification and characterization of T-cell epitopes deduced from RGS5, a novel broadly expressed tumor antigen. Clin Cancer Res 2007; 13:3347-55.
[22]Mays LE, Wilson JM. Identification of the murine AAVrh32.33 capsid-specific CD8+ T cell epitopes. J Gene Med 2009; 11:1095-102.
[23]Lazoura E, Lodding J, Farrugia W, et al. Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide. Immunology 2006; 119:306-16.
[24]Lazoura E, Apostolopoulos V. Rational peptide-based vaccine design for cancer immunotherapeutic applications. Curr Med Chem 2005; 12:629-39.
[25]Ruppert J, Sidney J, Celis E, et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 1993; 74:929-37.