鼻咽癌(Nasopharyngeal carcinoma,NPC)与EB病毒(Epstein-Barr virus,EBV)感染密切相关。鼻咽癌细胞中表达的EB病毒潜伏膜蛋白2(Latent membrane protein 2,LMP2)是免疫疗法的理想靶抗原。靶向EBV-LMP2疫苗已被证明是安全有效的,可在NP...鼻咽癌(Nasopharyngeal carcinoma,NPC)与EB病毒(Epstein-Barr virus,EBV)感染密切相关。鼻咽癌细胞中表达的EB病毒潜伏膜蛋白2(Latent membrane protein 2,LMP2)是免疫疗法的理想靶抗原。靶向EBV-LMP2疫苗已被证明是安全有效的,可在NPC患者体内增强特异性细胞毒性T淋巴细胞(Cytotoxic T lymphocyte,CTL)应答。目前面临的挑战是如何提高NPC患者的临床反应率,其关键在于阐明LMP2特异性CTL的细胞毒性。为研究rAd-LMP2疫苗诱导的LMP2A特异性CTL对肿瘤细胞的杀伤效果,本研究利用rMVA-LMP2-GFP感染P815细胞(H-2d)构建表达EBV-LMP2A和GFP蛋白的P815-LMP2-GFP靶细胞,用rAd-LMP2疫苗免疫BALB/c小鼠(H-2d)来诱生LMP2A特异性的CTL细胞作为效应细胞。然后,应用活细胞成像技术观察LMP2A特异性CTL对P815-LMP2-GFP靶细胞的杀伤过程。结果显示成功构建了P815-LMP2-GFP靶细胞和LMP2A特异性CTL效应细胞,并以成像的方式实时动态记录了LMP2A特异性CTL识别并杀死靶细胞的过程。P815-LMP2-GFP靶细胞可在2h内被LMP2A特异性CTL识别并杀伤。本研究建立了一种直接监测LMP2A特异性CTL识别并杀伤靶细胞过程的动态可视化方法。因此,本研究为肿瘤疫苗的体外抗肿瘤免疫效果的研究提供了新手段和新思路。展开更多
Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persist...Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.展开更多
Epstein-Barr virus （EBV） is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 （LMP2） of EBV plays a crucial role in the pathogenesis of EBV-assoc...Epstein-Barr virus （EBV） is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 （LMP2） of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte （CTL）, helper T cell （Th） and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195-232 and LMP2aa419-436. The EBV-LMP2m protein was expressed in E. coil BL21 （DE3） after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma （NPC） patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA （60.53% sensitivity and 76.86% specificity）. The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients.展开更多
文摘鼻咽癌(Nasopharyngeal carcinoma,NPC)与EB病毒(Epstein-Barr virus,EBV)感染密切相关。鼻咽癌细胞中表达的EB病毒潜伏膜蛋白2(Latent membrane protein 2,LMP2)是免疫疗法的理想靶抗原。靶向EBV-LMP2疫苗已被证明是安全有效的,可在NPC患者体内增强特异性细胞毒性T淋巴细胞(Cytotoxic T lymphocyte,CTL)应答。目前面临的挑战是如何提高NPC患者的临床反应率,其关键在于阐明LMP2特异性CTL的细胞毒性。为研究rAd-LMP2疫苗诱导的LMP2A特异性CTL对肿瘤细胞的杀伤效果,本研究利用rMVA-LMP2-GFP感染P815细胞(H-2d)构建表达EBV-LMP2A和GFP蛋白的P815-LMP2-GFP靶细胞,用rAd-LMP2疫苗免疫BALB/c小鼠(H-2d)来诱生LMP2A特异性的CTL细胞作为效应细胞。然后,应用活细胞成像技术观察LMP2A特异性CTL对P815-LMP2-GFP靶细胞的杀伤过程。结果显示成功构建了P815-LMP2-GFP靶细胞和LMP2A特异性CTL效应细胞,并以成像的方式实时动态记录了LMP2A特异性CTL识别并杀死靶细胞的过程。P815-LMP2-GFP靶细胞可在2h内被LMP2A特异性CTL识别并杀伤。本研究建立了一种直接监测LMP2A特异性CTL识别并杀伤靶细胞过程的动态可视化方法。因此,本研究为肿瘤疫苗的体外抗肿瘤免疫效果的研究提供了新手段和新思路。
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02A229)
文摘Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.
基金This work was supported by a grant from the National Natural Science Foundation of China （No： 81372447）.
文摘Epstein-Barr virus （EBV） is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 （LMP2） of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte （CTL）, helper T cell （Th） and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195-232 and LMP2aa419-436. The EBV-LMP2m protein was expressed in E. coil BL21 （DE3） after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma （NPC） patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA （60.53% sensitivity and 76.86% specificity）. The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients.
