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农业生物技术学报  2019, Vol. 27 Issue (3): 488-494    DOI: 10.3969/j.issn.1674-7968.2019.03.013
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
单次重组腺病毒加强BCG初免小鼠后诱导的特异性T细胞免疫应答
李武, 邓光存, 刘晓明, 王玉炯*
宁夏大学 西部特色生物资源保护与利用教育部重点实验室,银川 750021
A Single Dose of Recombinant Adenovirus Boosts BCG-primed Specific T Cell Immune Response in Mice (Mus musculus)
LI Wu, DENG Guang-Cun, LIU Xiao-Ming, WANG Yu-Jiong*
Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western, Ningxia University, Yinchuan 750021, China
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摘要 由结核分枝杆菌(Mycobacterium tuberculosis, Mtb)感染引起的结核病是引起人类死亡的重要传染病之一。到目前为止,卡介苗(Bacillus Calmette-Guérin, BCG)仍然是在全球范围内唯一可用的抗结核疫苗,但其免疫保护效果,尤其是针对成人的保护效果不佳。因此,研制新的、更有效的疫苗来替代BCG或加强BCG的免疫效果势在必行。本研究基于课题组前期关于重组腺病毒载体Ad5-CEAB能够诱导小鼠(Mus musculus)产生抗原特异性免疫应答的研究结果,利用抗原特异性淋巴细胞增殖实验、γ-干扰素酶联免疫斑点实验(interferon-γ enzyme-linked immunospot assay, INF-γ ELISPOT)、CD4+和CD8+ T淋巴细胞流式细胞术分析以及细胞因子酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)等实验和技术对BCG初免-单次重组腺病毒Ad5-CEAB加强后诱导小鼠产生的特异性T细胞免疫应答进行评价。实验以美国费城癌症研究所(Institute of Cancer Research, ICR)选育的6~8周龄雌性ICR小鼠为动物模型,随机分为4组,对照组只接种磷酸缓冲盐溶液(phosphate buffer saline, PBS)或1×106 CFU的BCG,实验组接种BCG后(初免),又经呼吸道接种1×109 PFU 的Ad5-CEAB进行加强。6周后处死小鼠并测定相关免疫学指标,结果显示,与PBS对照组相比,BCG组和BCG初免联合Ad5-CEAB加强组表现出明显的促进T细胞增殖的效应,BCG/Ad5-2组、BCG/Ad5-1组和BCG组刺激指数(stimulate index, SI)均显著高于PBS组(P<0.05),Ad5-CEAB加强2次的效果好于加强1次,但Ad5-CEAB加强1次也能取得显著的促进T细胞增殖的效果;Ad5-CEAB加强组INF-γ分泌细胞的数量显著高于对照组(P<0.05),ELISA测定结果表明,淋巴细胞培养上清液中的白细胞介素-2(interleukin-2, IL-2)和α-肿瘤坏死因子(tumor necrosis factor, TNF-α)的含量均显著高于BCG对照组(P<0.05),且BCG/Ad5-2组和BCG/Ad5-1组间细胞因子的含量并没有显著的差异(P>0.05);流式细胞分析结果表明,Ad5-CEAB组中CD4+和CD8+T细胞比值显著高于对照组(P<0.05),BCG/Ad5-2组和BCG/Ad5-1组间没有显著差异(P>0.05)。本研究结果表明,BCG初免联合1次重组腺病毒加强免疫可以诱导小鼠产生较强的抗原特异性T细胞免疫应答。本研究为开发新的基于重组腺病毒的抗结核疫苗的设计和基于粘膜免疫的异源初免-加强的免疫策略的研究提供了依据。
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李武
邓光存
刘晓明
王玉炯
关键词 结核病结核分枝杆菌初免-加强重组腺病毒T细胞免疫    
Abstract:Tuberculosis (TB) is a fatal disease that is caused by Mycobacterium tuberculosis (Mtb). Mycobacterium bovis Bacillus Calmette Guerin (BCG) has been widely used as the unique vaccine for TB prevention for many years, though its protective efficacy against Mtb infection is limited. The development of better vaccines and vaccination strategies to prevent the global spread of Mtb infection is therefore urgently needed. Previous studies had demonstrated that a recombinant adenoviral vector Ad5-CEAB was able to induce robust antigen-specific immune responses in mice (Mus musculus). In this study, antigen-specific lymphocyte proliferation test, interferon-γ enzyme-linked immunospot assay (INF-γ ELISPOT), flow cytometry analysis of splenocytes as well as cytokines enzyme linked immunosorbent assay (ELISA) were used to examine antigen-specific cellular immunological effects of Ad5-CEAB in the mice primed with BCG and boosted with a single dose of the recombinant adenovirus Ad5-CEAB. Female mice of ICR between 6 and 8 weeks of age were randomly divided into 4 groups (8 mice per group), mice in the control groups were injected subcutaneously with phosphate buffer saline (PBS) or with 1×106 CFU of BCG, while mice in the experimental groups were primed subcutaneously with 1×106 CFU of BCG and then intranasally boosted with 1×109 PFU of Ad5-CEAB once or twice. The mice were euthanized 2 weeks after the final immunization for analysis of T cell immune responses.The results displayed significantly elevated splenic T cell proliferation in mice of BCG/Ad5-2 group, BCG/Ad5-1 group and BCG group as compared to the PBS control group (P<0.05). In addition, the results of IFN-γ-ELISPOT also showed a dramatically increased frequency of Mtb antigen-specific IFN-γ-secreting splenic T cells in mice immunized with BCG or BCG/Ad5-CEAB as compared to the PBS-treated mice (P<0.05). The frequencies of CD4+ and CD8+ T cell populations were higher in these immunized mice relative to the PBS-treated group (P<0.05). Noticeably, the above examined indexes of immune responses in mice immunized with one dose of BCG priming and one dose of Ad5-CEAB boosting (BCG/Ad5-1 group) were statistically greater than that in the BCG group and the PBS group (P<0.05). Furthermore, the results of cytokines ELISA showed that significantly elevated antigen-induced cytokines interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) were found in the BCG/Ad5-1 and BCG/Ad5-2 groups as compared to the BCG and PBS groups (P<0.05). Noticeably, there was no difference in the tested cytokines between the BCG prime-recombinant adenovirus boost groups (P>0.05), an indication that a single dose of recombinant adenovirus boost can effectively stimulate antigen-specific cytokines responses in mice as compared to the BCG alone.These data clearly demonstrate that the single dose of mucosal Ad5-CEAB boost is efficient in stimulating a stronger antigen-specific T cell response in BCG-primed mice as compared to the BCG group as well as PBS control group. Collectively, the results in this study showed that the heterologous prime-boost strategy that subcutaneously primed with BCG and intranasally boosted with a single dose of Ad5-CEAB could elicit robust antigen-specific cellular immune responses in mice. These results provide an additional insight into developing novel Ad-basis TB vaccines and novel mucosal-targeted prime-boost anti-TB vaccination strategies.
Key wordsTuberculosis    Mycobacterium tuberculosis    Prime-boost    Recombinant adenovirus    T cell immunity
收稿日期: 2018-10-08     
ZTFLH:  S859.79  
基金资助:宁夏自然科学基金项目(No. NZ15018)
通讯作者: *wyj@nxu.edu.cn   
引用本文:   
李武, 邓光存, 刘晓明, 王玉炯. 单次重组腺病毒加强BCG初免小鼠后诱导的特异性T细胞免疫应答[J]. 农业生物技术学报, 2019, 27(3): 488-494.
LI Wu, DENG Guang-Cun, LIU Xiao-Ming, WANG Yu-Jiong. A Single Dose of Recombinant Adenovirus Boosts BCG-primed Specific T Cell Immune Response in Mice (Mus musculus). 农业生物技术学报, 2019, 27(3): 488-494.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.03.013     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I3/488
 
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