约氏疟原虫基因减毒子孢子疫苗保护性抗原的研究

发布时间：2018-04-13 19:50

本文选题：疟疾 + 疫苗　；参考：《第三军医大学》2014年博士论文

【摘要】：疟疾目前仍然是最主要的全球健康威胁之，每年因疟疾感染而死亡的人数约62.7万人，至今临床上尚无高效可行的疟疾疫苗问世。红前期阶段为疟原虫入侵人体的首个时期，阻断疟原虫子孢子在肝脏中发育和繁殖，可防止肝期裂殖子入血感染红细胞，从而出现临床症状，针对红前期设计的疫苗可从源头上控制疟疾感染。目前认为，最为有效的红前期疟疾疫苗是减毒子孢子疫苗，减毒子孢子诱导的CD8+T细胞免疫反应是其最主要的保护性免疫机制，也成为衡量疟疾疫苗有效性的金标准。然而，减毒子孢子的大量获得及运输、保存方面存在的困难，使其大规模的生产及现场应用受到限制，亚单位疫苗将会成为新代疫苗的主要研发方向，但至今仅鉴定出少数的红前期疫苗候选抗原，因此，迫切需要发展新的高效红前期保护性抗原筛选方法，筛选出更多介导CD8+T细胞免疫反应的靶抗原，进步认识清除感染细胞的免疫机制，为研制出高效的红前期疟疾疫苗奠定坚实基础。本研究首先通过DNA疫苗评价了约氏疟原虫（Plasmodium yoelii）候选抗原PyTmp21在小鼠体内诱导的红前期保护作用。其次，，利用肝脏晚期发育停滞P.yoeliifabb/f-基因减毒子孢子疫苗小鼠保护性模型，采用水动力法尾静脉注射（Hydrodynamic tail vein injection, HTVI）表达特异性抗原的荧光素酶报告融合质粒DNA入小鼠肝细胞内，结合活体成像系统（In Vivo Imaging System, IVIS）实时监测荧光素酶报告基因在免疫小鼠肝细胞内表达量，从而鉴定减毒子孢子疫苗诱导特异性CD8+T细胞免疫应答杀伤肝细胞的靶抗原，为进步筛选Pyfabb/f-基因减毒子孢子诱导保护性免疫相关的红前期抗原建立技术平台。最后，利用该技术平台探讨Pyfabb/f-基因减毒子孢子能否诱导产生针对新候选抗原PyTmp21的特异性细胞免疫反应并清除表达该抗原的肝细胞，从而评价PyTmp21是否为潜在的减毒子孢子疫苗诱导的保护性相关抗原。主要研究结果如下：一、PyTmp21DNA疫苗免疫后可抑制小鼠红前期疟原虫感染的肝脏虫荷量及诱导消除性保护作用为了鉴定新候选抗原PyTmp21能否在体内诱导保护性免疫，我们利用DNA疫苗免疫小鼠后发现，PyTmp21免疫可显著减少子孢子攻击感染小鼠肝脏内的疟原虫虫荷量及诱导60%消除性保护效率。二、建立HTVI/IVIS筛选Pyfabb/f-基因减毒子孢子诱导的特异性CD8+T细胞免疫反应相关保护性抗原的研究平台 1、通过HTVI法将PyCSP-Luc重组质粒注射入正常小鼠体内，IVIS系统观察证实质粒DNA在小鼠肝脏内持续、稳定高表达，并可实时监测与荧光素酶融合的目的蛋白在肝脏的表达量。通过流式细胞术证实HTVI注射后质粒DNA主要表达在肝细胞内，为目的蛋白的表达定位提供依据。 2、利用Pyfabb/f-基因减毒子孢子免疫小鼠诱导产生完全保护性免疫反应，HTVI将PyCSP-Luc质粒DNA攻击免疫小鼠，IVIS观察证实免疫小鼠肝脏内荧光素酶表达量减低。进步抗体耗竭实验证实CD8+T细胞介导了清除表达靶抗原的肝细胞。再者，对质粒攻击后的免疫小鼠肝脏淋巴细胞进行流式细胞内染色发现，CSP特异性CD8+T细胞数量明显增加且IFN-γ分泌水平增加。本实验表明Py fabb/f-基因减毒子孢子免疫小鼠可诱导产生CSP特异性CD8+T细胞免疫反应，并介导清除表达该抗原表位的肝细胞，HTVI与IVIS相结合技术可作为有效的工具用于筛选基因减毒子孢子诱导CD8+T细胞清除肝细胞的靶抗原。三、利用HTVI/IVIS研究平台鉴定Py fabb/f-基因减毒子孢子诱导保护性免疫相关的红前期抗原为了鉴定基因减毒子孢子疫苗保护模型诱导的保护性免疫相关红前期抗原，我们利用HTVI/IVIS研究平台检测Pyfabb/f-基因减毒子孢子能否诱导产生针对新候选抗原PyTmp21的特异性T细胞免疫反应并杀伤表达该抗原的肝细胞。我们发现2次Pyfabb/f-子孢子同源免疫后，HTVI法将PyTmp21-Luc质粒DNA攻击免疫小鼠，未能观察到肝脏内荧光素酶表达量减低现象。然而，利用DNA+子孢子或子孢子+DNA异源性免疫-加强免疫策略发现，PyTmp21-Luc质粒DNA攻击免疫小鼠后其在肝脏的表达量明显减低，提示Pyfabb/f-减毒子孢子可诱导针对PyTmp21抗原的特异性免疫反应并杀伤表达该抗原的肝细胞。本实验表明，同源性减毒子孢子免疫小鼠后可能主要诱导产生针对优势抗原CSP的有效免疫反应，而不足以诱导产生针对非CSP抗原的免疫反应，因此，我们通过异源性免疫-加强免疫策略证实Pyfabb/f-基因减毒子孢子可诱导产生有效的PyTmp21抗原特异性T细胞免疫反应，PyTmp21抗原为潜在的减毒子孢子疫苗诱导的保护性相关抗原。我们的研究显示HTVI/IVIS方法可用于检测基因减毒子孢子疫苗诱导的CD8+T细胞特异性杀伤肝细胞的靶抗原。采用异源性免疫-加强免疫策略证实Pyfabb/f-基因减毒子孢子疫苗诱导了针对PyTmp21抗原的特异性免疫反应，并清除了表达该抗原的肝细胞，PyTmp21作为新的红前期抗原参与减毒子孢子疫苗诱导的保护性免疫反应。异源性免疫-加强免疫策略结合HTVI/IVIS技术可作为有效的工具用于筛选减毒子孢子全虫疫苗诱导的新红前期非CSP保护性抗原，为进步认识减毒全虫疫苗的保护性免疫机制、筛选出更多的亚单位疫苗候选抗原并设计出高效的疟疾疫苗奠定基础。
[Abstract]:Malaria remains a major threat to global health, the number of infections and deaths due to malaria each year about 627 thousand people, so far there are no clinically effective malaria vaccine. The advent of preerythrocytic stage for the first time the parasite invade the body, blocking the sporozoite development and reproduction in the liver, which can prevent the liver stage merozoites infect red blood cells into the blood, and clinical symptoms, according to the design of the vaccine preerythrocytic malaria infection from the source control. At present, the most effective preerythrocytic malaria vaccine is attenuated sporozoite vaccine, attenuated sub spore CD8+T cells induced immune response is the most important protective immune mechanism also, become the gold standard to measure the effectiveness of malaria vaccine. However, a large number of sporozoites obtained reduced toxicity and transportation, preservation of the difficulties, the large-scale production and application is limited, sub unit A vaccine will become the main development direction of the new generation of vaccines, but has only identified preerythrocytic vaccine candidate antigen, few therefore, there is an urgent need to develop protective antigens of early high Xiaohong new screening method, screened more target antigen to CD8+T cells mediated by the immune response, clear progress in understanding the immune mechanism of infected cells, for developed to lay a solid foundation for effective preerythrocytic malaria vaccine.
Plasmodium yoelii evaluation based on the DNA (Plasmodium yoelii) vaccine candidate antigen PyTmp21 in preerythrocytic protective effect of mice. Secondly, the use of advanced liver stagnation of growth of P.yoeliifabb/f- gene attenuated sporozoite vaccine induced protection of mice model, using the method of hydrodynamic tail vein injection (Hydrodynamic tail vein injection, HTVI) expression. The specific antigen of luciferase reporter fusion plasmid DNA into mouse liver cells, combined with in vivo imaging system (In Vivo Imaging System, IVIS) expression of luciferase reporter gene in real-time monitoring of liver cell immunity in mice, thereby reducing identification of target antigen attenuated sporozoite vaccine induced specific CD8+T cell immune response to kill liver cells, for screening progress Pyfabb/f- gene attenuated sporozoites induce protective immune related antigen preerythrocytic technology platform. Finally, using the technology The platform of Pyfabb/f- gene attenuated sporozoites could induce specific cellular immune responses to the new candidate antigen PyTmp21 and clear expression of the antigen in liver cells, so as to evaluate whether PyTmp21 is a potential attenuated sporozoite vaccine induced protective antigen. The main research results are as follows:
1. Immunization of PyTmp21DNA vaccine inhibits the amount of liver worms and induced protective effects of the mice infected with Plasmodium red Plasmodium
In order to identify new candidate antigen PyTmp21 can induce protective immunity in vivo, we use the DNA vaccine in mice and found that PyTmp21 immunization can significantly reduce the sporozoite infection against malaria burden in mouse liver and induced by 60% to eliminate protection efficiency.
Two, establishment of a HTVI/IVIS screening platform for screening specific CD8+T cell immune response related protective antigens induced by Pyfabb/f- gene attenuated sporozoites
1, PyCSP-Luc recombinant plasmid was injected into normal mice by HTVI method, IVIS system observation confirmed that the plasmid DNA sustained in mouse liver, high expression, and real-time monitoring and fusion protein luciferase expression in liver volume. The main expression in liver cells confirmed by HTVI after injection of plasmid DNA by flow cytometry and to provide the basis for the expression and localization of the protein.
2, attenuated sporozoite immunized mice induced by fully protective immune response by Pyfabb/f- gene, HTVI PyCSP-Luc plasmid DNA mice attacked, IVIS observation confirmed the expression decreased luciferase immune mice liver. The progress of antibody depletion experiments confirmed that CD8+T cells mediated by the expression of clear target antigen of liver cells. Furthermore, after the attack on plasmid immunization of mice liver lymphocytes by flow cytometry were found, the number of CSP specific CD8+T cells were significantly increased and IFN- secretion levels increased. The experimental results show that the Py fabb/f- gene attenuated sporozoite immunized mice can induce CSP specific CD8+T cell immune response, and mediated expression of the epitope of clearing liver cells HTVI, combined with IVIS Technology can be used as an effective tool for screening the target antigen gene attenuated sporozoite induced CD8+T cell depletion of liver cells.
Three, using the HTVI/IVIS research platform to identify the protective immune related prophase antigen induced by the sporozoites of the Py fabb/f- gene attenuated
In order to reduce the immunity related gene identification of preerythrocytic antigen protection induced by attenuated sporozoite vaccine protection model, we use the HTVI/IVIS research platform for detection of Pyfabb/f- gene attenuated sporozoites could induce specific T cell immune response to new candidate antigen PyTmp21 and antigen expression of the destruction of liver cells. We found that 2 Pyfabb/f- homologous sporozoite after immunization, HTVI PyTmp21-Luc plasmid DNA attack mice were not observed the expression of luciferase in the liver to reduce phenomenon. However, the use of DNA+ +DNA sporozoites or sporozoites of heterologous immune booster immunization strategy, PyTmp21-Luc plasmid DNA mice attacked the expression in liver was significantly decreased, suggesting that the specific immune response Pyfabb/f- attenuated sporozoites can induce the PyTmp21 antigen expression of the antigen and kill liver cells. The experimental results show that the homology of attenuated Sporozoite immunized mice may induce effective immune response against dominant antigen CSP, but not enough to induce immune responses to non CSP antigens in us by heterologous immunity boost immunization strategy confirmed that the Pyfabb/f- gene attenuated sporozoites could induce PyTmp21 antigen specific T cell immune response effectively, PyTmp21 as a potential antigen attenuated sporozoite vaccine induced protective antigen.
Our study shows that the HTVI/IVIS method can be used to detect target gene by antigen-specific CD8+T cell attenuated sporozoite vaccine induced killer liver cells. Using heterologous immune booster immunization strategy confirmed that the Pyfabb/f- gene attenuated sporozoite vaccine induced specific immune response to PyTmp21 antigens, and clear expression of the antigen in liver cells PyTmp21, as a new preerythrocytic antigen in reducing spore vaccine induced protective immune reaction. Heterologous immune toxin boost immunization strategy combined with HTVI/IVIS technology can be used as an effective tool for the screening of attenuated elicitor spore parasite vaccine new preerythrocytic non CSP protective antigen, understanding the mechanism of protective immunity of attenuated parasite the vaccine for progress, screened more subunit vaccine candidate antigens and designed to lay the foundation for effective malaria vaccine.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R531.3

【共引文献】