抗原靶向不同树突细胞亚群诱导抗结核分枝杆菌免疫应答的研究

发布时间：2018-06-12 04:15

本文选题：树突细胞 + 靶向　；参考：《扬州大学》2010年博士论文

【摘要】： 世界上三分之一的人感染结核分枝杆菌(Mycobacterium tuberculosis, MTB),使得结核病(tuberculosis, TB)成为世界范围内最严重的细菌性传染疾病之一,导致每年160万人死亡,严重威胁人类的健康和公共卫生安全。目前人类唯一使用的抗结核疫苗：卡介苗(Bacillus Calmette-Guerin, BCG),由牛结核分枝杆菌经多次传代减毒而来。然而BCG对于成人肺结核的保护效果很不稳定,造成BCG保护力有限的可能原因有：(1)BCG的过度减毒,在减毒传代过程中丢失了编码保护性抗原的基因序列,例如缺失基因差异区域；(2)尽管BCG能够激发抗MTB免疫应答,然而MTB隐藏于肉芽肿中,特异性效应T细胞很难与其直接发生作用；(3)BCG在激发保护性Th1应答的同时也活化了调节性T细胞(T regulatory cell, Treg),消减了效应性T细胞有效的保护作用。 BCG保护率的不稳定,加上TB与人类免疫缺陷病毒(Human immunodeficiency virus, HIV)的共感染,以及结核杆菌多重耐药菌株甚至极端耐药菌株的出现使得TB呈现全球预警状态,因此更加理性的设计新型TB疫苗及寻求更为合理的抗TB免疫策略是亟待解决的研究课题。尽管对于开发新型TB疫苗已进行了大量的工作,但就目前而言,其中只有少数能够与BCG相当或稍优于BCG的保护效果。对于树突细胞(dendritic cell, DC)的分类、功能及其在调节免疫应答中重要作用的了解,以及体内靶向DC相关研究工作表现出的较为理想的应用前景,使得体内抗原靶向DC成为设计新型TB疫苗或抗TB免疫策略的新思路。事实上,至目前为止,尚未有关于体内靶向DC,诱导抗TB保护性免疫应答的相关研究报道。 DC具有相似的细胞形态,大量分布于淋巴组织的T细胞区域,高表达MHC-Ⅱ类分子,具有突出的持续摄取环境中抗原分子并加工递呈给T细胞的潜能。根据分化过程、表型、成熟机制及专属职能等的不同,DC被分为不同的亚群。目前对于小鼠DC的分类已较为成熟,虽然人DC亚群不能完全等同于小鼠DC亚群,但是在小鼠和人中均发现了浆细胞样DC,血液来源的淋巴组织驻留型DC,外周迁移型DC以及单核细胞来源的炎性DC等亚群,使得以小鼠为模型的体内DC靶向研究具有向人类临床研究过渡的可能。大量研究数据显示不同DC亚群在支配调节如CD4+T细胞分化等获得性细胞免疫应答中的表现各异,因此利用其特异性表面分子的单克隆抗体进行体内抗原靶向的策略来直接操控各DC亚群,能够调节、控制免疫应答的方向,是制备预防性/治疗性疫苗的理想方式。目前抗体介导的体内DC抗原靶向研究中,多采用化学耦连或利用基因重组技术将目的抗原插入针对DC表面受体的抗体分子基因组的方法,实现目的抗原靶向DC。虽然多种DC表面受体分子均能传递外源信号,启动T细胞应答,然而很难预测在应对特定病原体感染时,目的抗原靶向哪一种DC亚群/DC表面受体分子能产生最为理想的抗病原体保护性免疫应答。因此对靶向不同DC亚群产生的免疫应答进行比较,对于发现最适宜的靶向亚群或靶向分子具有指导意义。本研究开发出一种简单方便、可选性高的靶向系统：将MTB免疫优势抗原分子与链酶亲合素(streptavidin, SA)进行融合表达并四聚体化,在SA与生物素(biotin, biot)间高亲和力的作用下,四聚体化的SA融合蛋白可与biot标记的DC表面分子单抗可形成复合物。利用这个灵活的模式,在获得SA融合蛋白后,利用biot标记的针对不同DC亚群表面受体分子的特异单抗,可快速简便的将目的抗原靶向小鼠和人相应的DC亚群。本研究中使用的靶向抗原来源于早期分泌抗原靶6(Earlier secreted antigen target 6, ESAT-6)蛋白家族(ESX),这些高度保守、低分子量的MTB保护性抗原由MTBⅦ型分泌系统表达,在小鼠、豚鼠以及不同遗传背景的人群中表现出较好的免疫原性,在动物模型中表现出保护作用,且这些抗原分子的特异效应性CD4+、CD8+T细胞与MTB感染者体内抗MTB保护性应答相关。本研究中,利用该方便灵活的抗原靶向系统,将MTB ESX免疫优势抗原靶向不同的DC表面分子,以期筛选出能产生最为理想的抗结核免疫应答的DC靶点/DC亚群。 1.抗体介导的结核分枝杆菌ESX抗原体外靶向DC 首先在体外实验中对这种抗原靶向方法的功能和特异性进行评价：证明经biot标记的DC表面分子单抗(针对：CD11b、CD11c、MHC-Ⅱ、DCIR-2或PDCA-1)的导向作用,ESX-SA融合蛋白可高效的结合在抗原递呈细胞表面。靶向浆细胞样DC表面分子PDCA-1或经典DC表面分子CD11b、CD11c、MHC-Ⅱ、DCIR-2的ESX抗原分子可有效的被细胞摄取内吞。为评价抗原递呈细胞对靶向的ESX抗原的加工递呈能力,利用BCG免疫小鼠制备了ESX抗原(TB10.4, ESX-H)特异性MHC-Ⅱ限制性T细胞杂交瘤。试验中发现经典DC、浆细胞样DC或巨噬细胞对靶向的ESX抗原通过MHC-Ⅱ类加工机制进行处理,ESX抗原表位与MHC-Ⅱ分子形成复合物,高效递呈至ESX特异性T细胞杂交瘤或MTB野生株H37Rv感染的小鼠脾脏T细胞。 2.抗体介导的ESX抗原体内靶向不同DC亚群诱导抗结核免疫应答对ESX-SA融合抗原靶向DC表面受体分子产生的免疫原性进行比较。这些DC受体分子包括MHC-Ⅱ分子,整合素CD11b、CD11c,浆细胞样DC抗原-1(plasmocytoid dendritic cell antigen-1, PDCA-1/CD317)和C型凝集素受体(C-type lectin receptors, CLRs):甘露糖受体家族的CD205,唾液酸糖蛋白受体家族的CD207(langerin, Clec4K)、CD209(DC-specific ICAM3-Grabbing non-integrin, DC-SIGN)以及唾液酸糖蛋白受体家族DC免疫受体亚家族的DCIR2(Clec4A)。根据ESX抗原靶向后：(1)体内ESX抗原靶向DC并经MHC途径递呈的效率；(2)诱导ESX抗原特异性Th1、Th2、Th17和Treg获得性免疫应答的效果；(3)交叉激活ESX特异性CD8+T细胞的能力；(4)继BCG初免后DC靶向免疫策略的潜在加强功效,以期筛选出最适宜进行靶向、能够诱导理想的抗MTB免疫应答的DC亚群或DC表面受体分子。体内研究显示该靶向系统具有高度特异性,只有经DC表面分子抗体靶向的DC亚群表面才能检测到ESX的结合,分选出这些DC进行体外培养能够有效递呈靶向的ESX抗原至特异性MHC-Ⅱ限制性T细胞杂交瘤。 ESX-SA与biot标记的DC表面分子单抗形成复合物,辅以多聚肌苷酸—胞苷酸(Poly inosinic:Poly cytidylic acid, Poly I:C,聚肌胞)作为DC激活因子免疫小鼠,对产生的抗原特异性T细胞应答进行比较。仅一次注射低至1μg(50pmole)剂量的ESX-SA (ESAT-6-SA),靶向DC表面CD11b、CD11c或CD205可高效诱导ESX特异性Th1、Th17应答,靶向CD207或PDCA-1也能显著激发Th1应答,只是程度稍低,而靶向CD209未能诱导抗原特异性的Th1免疫应答。利用biot-SA靶向系统,无论靶向何种DC表面受体分子均不能激发抗原特异性Th2免疫应答。就所有检测的CLRs而言,靶向CD205能产生最强的Th1应答。对不同剂量靶向抗原诱导免疫应答的水平进行分析比较,即使将0.1μg(5pmole) ESX抗原靶向DC表面CD11b分子仍可检测到抗原特异性Th1、Th17应答。靶向相同剂量的ESX抗原至DC表面分子,FcyR缺失小鼠与野生型小鼠产生的免疫应答水平相当,且在注射biot-Ctrl Ig-ESX-SA复合物的小鼠体内不能检测到特异性获得性免疫应答,由此说明,本研究中抗体介导的ESX抗原靶向DC,进而为细胞摄取,加工递呈及激发免疫应答的过程中未涉及FcR的作用,具有高度靶向特异性。以减毒活疫苗进行初次免疫,亚单位疫苗进行加强的免疫方法,可能是最为理想有效的抗TB治疗性疫苗免疫策略。我们选用ESX家族保护性抗原、亚单位疫苗的热门候选分子TB10.4来评价继BCG初免后,TB10.4靶向DC的免疫加强效果。对BCG初免的小鼠,以biot标记单抗将TB10.4-SA靶向DC表面CLRs: CD205、CD207、CD209、DCIR-2或PDCA-1作为加强免疫,可不同程度的增强TB10.4特异性IFN-γ免疫应答；在诱导Th17免疫应答方面,靶向CD205的免疫加强效果最好,其次是CD207和PDCA-1。比较各种免疫条件,发现只有在BCG初免,TB10.4靶向CD205作为加强免疫的小鼠中检测到TB10.4特异性的CD8+T细胞的交叉激活。综上,本研究开发出一种新型、可选性高的抗原靶向系统,利用DC表面分子特异性单抗的导向作用,将MTB保护性抗原靶向不同DC亚群,可有效开启或加强抗MTB CD4+和CD8+T细胞免疫反应,至此DC表面CLRs CD205可能是最为理想的靶向候选分子。
[Abstract]:1/3 of the people in the world are infected with Mycobacterium tuberculosis (MTB), which make tuberculosis (TB) one of the most serious bacterial infections in the world, causing 1 million 600 thousand deaths a year and a serious threat to human health and public health. At present, the only anti tuberculosis vaccine is used by human beings. Bacillus Calmette-Guerin (BCG), derived from bovine Mycobacterium tuberculosis by multiple passages. However, the protective effect of BCG on adult pulmonary tuberculosis is very unstable. The possible causes of the limited protection of BCG are: (1) the overtoxicity of BCG, the loss of the gene sequence of the protective antigen in the detoxification passage, such as (2) although BCG can stimulate the anti MTB immune response, however, MTB is hidden in granuloma, and the specific effect of T cells is difficult to directly affect it. (3) BCG activates regulatory T cells (T regulatory cell, Treg) while stimulating the protective Th1 response, reducing the effective protective effect of the effector T cells.
The instability of BCG protection rate, combined with the co infection of TB and human immunodeficiency virus (Human immunodeficiency virus, HIV), and the emergence of Mycobacterium tuberculosis multidrug-resistant and even extreme resistant strains make TB present a global warning state, so it is urgent to design a new TB vaccine more rationally and seek more reasonable anti TB immunization strategy. Although a lot of work has been done to develop new TB vaccines, there are only a few of them that can be equivalent to or slightly better than BCG for the protection of BCG. The classification of dendritic cells (dendritic cell, DC), the function and the understanding of the important role in regulating the immune response, and the target DC in the body. The relevant research work has shown an ideal prospect of application, making the antigen targeting DC in the body become a new idea for the design of a new TB vaccine or anti TB immunization strategy. In fact, up to now, there has not been a related report on the target DC in vivo, inducing anti TB protective immune response.
DC has a similar cell morphology, widely distributed in the T cell region of lymphoid tissue, high expression of MHC- class II molecules, and has the potential to continue to ingest the antigen molecules in the environment and to process the potential of T cells. According to the differentiation process, phenotype, maturation mechanism and exclusive function, DC is divided into different subgroups. At present, DC in mice is divided. The classification is more mature, although the human DC subgroup can not be completely equal to the DC subgroup of mice, the plasma cell like DC, the blood source of lymphoid tissue residing DC, the peripheral migratory DC and the inflammatory DC subgroup of monocyte origin are found in mice and people, which makes the study of DC targeting in mice as the model in human clinical research. A large number of research data show that different DC subgroups have different manifestations in the acquired cellular immune response, such as CD4+T cell differentiation, so the strategy of using the monoclonal antibodies of specific surface molecules to directly manipulate the DC subgroups can regulate and control the direction of the immune response. An ideal way to prepare a prophylactic / therapeutic vaccine.
At present, in the study of antibody mediated DC antigen targeting in vivo, the target antigen is inserted into the antibody molecular genome of DC surface receptor by chemical coupling or gene recombination technology. The target antigen target to DC., although various DC surface receptor molecules can transmit the exogenous signal and start the T cell response, however, it is difficult to predict it In response to a specific pathogen infection, the target antigen targeted to the DC subgroup /DC surface receptor molecules can produce the most ideal protective immune response to the disease. Therefore, the comparison of the immune responses to different target DC subsets is of guiding significance for the discovery of the most suitable target subgroup or target molecule. A simple, convenient and highly selective target system: fusion expression of MTB immune dominant antigen molecules and streptavidin (SA) and four polycondensation. Under the high affinity between SA and biotin (biotin, Biot), the four polycondensation of SA fusion protein can form a complex with the DC surface molecular monoclonal antibody labeled with Biot. This flexible model, after obtaining the SA fusion protein, uses Biot labeled specific monoclonal antibodies against different DC subgroup surface receptor molecules, and can quickly and easily target the target antigen to the mouse and the corresponding DC subgroup. The target antigen used in this study is derived from the early secretion of anti target 6 (Earlier secreted antigen target 6, ESAT-6). The protein family (ESX), these highly conserved, low molecular weight MTB protective antigens expressed by the MTB VII secretory system, showed good immunogenicity in mice, guinea pigs and people of different genetic backgrounds, showed protective effects in animal models, and the specific effect CD4+ of these antigen molecules, CD8+T cells and MTB infected bodies. In this study, the MTB ESX immune dominant antigen was targeted to different DC surface molecules by using the convenient and flexible antigen targeting system in this study in order to screen out the DC target /DC subgroup that could produce the most ideal anti tuberculosis immune response.
1. antibody mediated ESX targeting in vitro of Mycobacterium tuberculosis DC
First, the function and specificity of this antigen targeting method are evaluated in vitro: it is proved that the Biot labeled DC surface molecular monoclonal antibody (for CD11b, CD11c, MHC- II, DCIR-2 or PDCA-1) can efficiently combine the ESX-SA fusion protein on the surface of the antigen presenting cell. DC surface molecules CD11b, CD11c, MHC- II, and DCIR-2 ESX antigen molecules can be effectively absorbed by cell uptake. In order to evaluate the ability of the antigen presenting cells to process the targeted ESX antigen, the ESX antigen (TB10.4, ESX-H) specific MHC- II restrictive cell hybridoma was prepared by BCG immunization mice. Or the targeted ESX antigen is treated by the MHC- II processing mechanism, and the ESX epitopes and MHC- II molecules form complex, which can be efficiently presented to ESX specific T cell hybridoma or MTB wild strain H37Rv infected mice spleen T cells.
2. antibody mediated ESX antigen targets different DC subsets in vivo to induce antituberculosis immune response.
The immunogenicity of the ESX-SA fusion antigen targeted to the DC surface receptor molecules is compared. These DC receptor molecules include MHC- II molecules, integrin CD11b, CD11c, plasma cell like DC antigen -1 (plasmocytoid dendritic cell antigen-1,) and the mannose receptor family 5, the CD207 (langerin, Clec4K) of the sialic glycoprotein receptor family, CD209 (DC-specific ICAM3-Grabbing non-integrin, DC-SIGN) and DCIR2 (Clec4A) of the DC immune receptor subfamily of the sialic glycoprotein receptor family. The effect of heterosexual Th1, Th2, Th17 and Treg on the immune response; (3) the ability to cross activate ESX specific CD8+T cells; (4) the potential strengthening effect of DC targeting immunization strategy after BCG early immune response, in order to screen out the most suitable targeting and to induce the ideal anti MTB immune response to the DC subgroup or DC surface receptor molecule.
In vivo studies have shown that the target system is highly specific. Only the DC subgroup surface of the DC surface molecular antibody target can detect the binding of ESX, and the DC can be successfully cultured in vitro to present the targeted ESX antigen to the specific MHC- II restrictive T cell hybridoma.
ESX-SA and Biot labeled DC surface molecular mAb formed complex, supplemented with polyinosinic acid - cytidine acid (Poly inosinic:Poly cytidylic acid, Poly I:C, polymyocytes) as DC activator to immunize mice, and compared the produced antigen specific T cell responses. The CD11b, CD11c or CD205 on the DC surface can efficiently induce ESX specific Th1, Th17 response, and the target CD207 or PDCA-1 can also significantly stimulate the Th1 response, but the target CD209 does not induce the antigen specific Th1 immune response. The target CD205 can produce the strongest Th1 response to all the detected CLRs. Compare the level of the immune response induced by different doses of the target antigen. Even if the 0.1 g (5pmole) ESX antigen is targeted to the DC surface CD11b molecules, the antigen specific Th1, Th17 response, and the ESX antigen of the same dose to the DC surface can be detected. The immune response level produced by the FcyR deficient mice and the wild type mice was similar, and the specific acquired immune response could not be detected in the mice injected with the biot-Ctrl Ig-ESX-SA complex. Thus, the antibody mediated ESX antigen was targeted to DC in this study, and then in the process of cell uptake, processing and stimulating the immune response. It is not involved in the role of FcR, with a highly targeted specificity. The initial immunization of the live attenuated vaccine and the strengthening of subunit vaccines may be the most ideal and effective anti TB therapeutic vaccine immunization strategy. We choose ESX family protective antigen and the hot candidate molecule TB10.4 of subunit vaccine to evaluate TB after BCG first immunity. 10.4 the immunization effect of the target to DC. For the BCG immunized mice, the Biot labeled monoclonal antibody was used to target TB10.4-SA to the DC surface CLRs: CD205, CD207, CD209, DCIR-2 or PDCA-1 as strengthening immunity, and the TB10.4 specific immune response could be enhanced to a different degree. CD207 and PDCA-1. compared the various immune conditions, and found that only in the early BCG immunization, the TB10.4 targeted CD205 as a strengthened immune mouse detected the cross activation of TB10.4 specific CD8+T cells.
To sum up, an antigenic targeting system with high selectivity is developed in this study. Using the guidance of DC surface molecular specific monoclonal antibody, the MTB protective antigen is targeted to different DC subgroups, which can effectively open or strengthen the immune response to MTB CD4+ and CD8+T cells. So DC surface CLRs CD205 may be the most ideal target candidate.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【引证文献】