【作者】 董慧；

【导师】 焦新安； Claude Leclerc； Laleh Majlessi；

【作者基本信息】 扬州大学 ， 预防兽医学， 2010， 博士

【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。更多还原

【Abstract】 One-third of the Earth’s population is infected with Mycobacterium tuberculosis, making the pulmonary tuberculosis the most widely spread infectious disease, leading to 1.6 million deaths annually. The only vaccine in use against infection with M. tuberculosis, the live attenuated M. bovis BCG (Bacillus Calmette-Guerin), is not able to protect efficiently against the adult pulmonary tuberculosis in endemic zones. The limited efficiency of BCG in the control of M. tuberculosis infection can be explained by the following hypothesis:(ⅰ) BCG could be attenuated, due to the deletion of genes coding for protective immunogens like the deletion of the "Regions of Differences" (RD). (ⅱ) Despite the strong anti-mycobacterial Thl responses induced by BCG, intracellular M. tuberculosis sequestered inside the granuloma, may be inaccessible to these effector T cells. (ⅲ) Following BCG immunization, induction of regulatory T cells (Treg), in parallel to the induction of anti-mycobacterial Thl responses, could reduce the efficiency of the protective effect against M. tuberculosis infection.With the resurgence of tuberculosis in immuno-compromised individuals and the rapid expansion of multi-drug resistant and extensively drug-resistant tuberculosis, the need of a better rational design of new strategies of anti-tuberculosis vaccines is reinforced. Despite intense research on live attenuated and/or sub-unit anti-tuberculosis vaccines, a very few vaccine candidates display improved protective effect and their efficiency remains limited. Here, we drived the extensive knowledge available on the properties of dendritic cells (DC) and antigen targeting to DC subsets, towards the practical in vivo antigen delivery to DC in anti-tuberculosis vaccination. Indeed, so far, addressing M. tuberculosis-derived protein antigens to DC subset(s) and/or DC surface receptor(s), for the induction of protective anti-mycobacterial immunity, has not been investigated.Despite their shared morphology, abundance in T-cell areas of lymphoid tissues, high MHC-Ⅱexpression and outstanding potential to continuously probe the environment, process and present antigens to T cells, DC are divided into different subsets, according to their ontogenic origin, phenotype, maturation programs and specialized functions. Although the well-established classification of the mouse DC subsets can’t be transposed to the human DC populations, plasmacytoid DC, blood-derived lymphoid tissue resident DC, peripheral migratory DC and monocyte-derived inflammatory DC have been distinguished in both mice and humans. Numerous evidences argue that the magnitude of adaptive immune responses, as well as differentiation and specialization of CD4+ T cells into Th1, Th2 or Th17, are dictated by different DC subsets with specialized activities. The mobilization of different DC subsets by their direct in vivo targeting through specific surface markers represents a promising pathway to design well-controlled immunization strategies for the development of preventive and/or therapeutic vaccines.So far, the antigen targeting strategy is limited by the requirement of individual chemical coupling or genetic insertion of each immunogen of interest to mAbs specific to each of the numerous DC surface receptors, candidate for antigen addressing. It is largely admitted that DC translate information from different surface receptors into an activation program that orients the Th cell differentiation. However since all the rules governing functions of DC subsets are not yet understood, it remains difficult to predict which DC subsets/DC surface receptors are the most appropriate to be targeted in order to optimize the protective immunity against a given pathogen. Therefore, comparison of the properties and impacts of various DC subsets on the generation of pathogen-specific adaptive responses may help identify the most adapted DC subset(s), able to tailor the most adapted and protective adaptive immunity. To this end, we have designed a versatile antigen targeting approach, prominent mycobacterial immunogens are genetically fused to streptavidin (SA), the resulted fusion proteins are tetramerized to optimize their high affinity interaction with biotin (biot). Such SA fusion tetramers are then complexed to biot-conjugated mAbs, specific to diverse DC surface receptors. Therefore, in this flexible model, once such antigen-SA fusion proteins are produced, they can be readily carried and delivered to different mouse and human DC subsets by simple use of individual biot-mAbs of a large panel of specificities against DC surface receptors, with expression profiles restricted to given DC subsets. Potent mycobacterial antigens included in this study were selected among highly-conserved, low-molecular weight immunogens belonging to the Early Secreted Antigenic Target,6 kDa (ESAT-6) protein family (ESX) of M. tuberculosis. These proteins, actively secreted by the typeⅦsecretion system of mycobacteria, are known for their marked immunogenicity in mice, guinea pig and in ethnically different human populations, and for their protective potential in animal tuberculosis models. Moreover, the presence of CD4+ and CD8+ effector T cells specific to such proteins is directly correlated to the natural anti-mycobacterial protection in M. tuberculosis-infected humans. Based on this verstaile antigen targeting approach, we aim to identify the most appropriate DC receptor(s) to which the targeting of M. tuberculosis-derived ESX immunogens can induce optimized immune responses with anti-tuberculosis protective potential.PartⅠ. In vitro mAb-mediated M. tuberculosis-derived ESX antigen targeting to DCWe first showed in vitro that this Ab-based antigen targeting system allows delivery of ESX immunogens to various antigen-presenting cell surface receptors, either integrins, C-type lectins DCIR-2, MHC-Ⅱmolecule or CD317 (plasmacytoid DC Ag-1, PDCA-1). Importantly, ESX antigens bound to CDllb, CDllc, DCIR-2, MHC-II on conventional DC or to PDCA-1 on plasmacytoid DC were efficiently endocytosed, most probably due to the cross-linking of the targeted surface receptors by the biot-mAbs, leading to their capping together with the bound ESX-SA cargo. To evaluate the presentation of ESX antigen by targeted APC, we developed MHC-II-restricted T cell hybridomas specific to ESX antigen (TB10.4, ESX-H) from BCG immunized mice. By use of such T-cell hybridomas in in vitro presentation assays that we set up, we demonstrated that ESX antigens delivered to conventional or plasmacytoid dendritic cells or to macropahges, were highly efficiently addressed to MHC-II presentation machinery and processed. The ESX-derived epitopes were then loaded on MHC-II molecules and presented, in a highly sensitive manner, to ESX-specific, MHC-Ⅱ-restricted T-cell hybridoma or the polyclonal splenocytes from M. tuberculosis infected mice.PartⅡ. mAb-based ESX antigen targeting to different DC subsets to induce anti-mycobacterial immunityWe characterized the immunogenicity of several ESX proteins fused to SA (ESX-SA), targeted to different DC surface receptors by complexing them to biot-mAbs specific to:MHC-Ⅱmolecules, CD11b or CD11cβ2 integrins, CD317 (plasmacytoid DC Ag-1, PDCA-1) or C-type lectin receptors of:(ⅰ) mannose receptor family, i.e., CD205 (DEC205), (ⅱ) asialoglycoprotein receptor family, i.e., CD207 (Langerin, Clec4K), or CD209 (DC-specific ICAM3-grabbing non-integrin, DC-SIGN), or (ⅲ) DC immunoreceptor (DCIR) subfamily of asialoglycoprotein receptor, i.e., DCIR-2 (Clec4A). We explored this model to select the most appropriate DC subsets or DC surface receptors to target in anti-tuberculosis vaccination on the basis of:(ⅰ) in vivo capture/processing and presentation of ESX antigens by MHC molecules, (ⅱ) in vivo outcome of the ESX-specific Th1, Th2, Th17 or Treg adaptive responses, (ⅲ) in vivo cross presentation of ESX antigens and cross priming of specific CD8+ T cells, (ⅳ) boost effect of such immunization subsequent to BCG priming.This antigen delivery system was highly specific in vivo, as only the DC subsets, targeted with ESX-SA complexed to selected biot-mAbs displayed ESX antigens at their surface, and when positively sorted ex vivo, were able to present ESX antigens to the ESX-specific, MHC-II-restricted T-cell hybridomas.By use of different biot-mAbs specific toβ2 integrins, diverse C-type lectins or PDCA-1, in the presence of the DC maturation signal, Poly inosinic:Poly cytidylic acid (PolyⅠ:C), we directly compared the in vivo efficiency of ESX targeting to different DC subsets/DC surface receptors in the induction of T-cell responses. Remarkably, a single injection of only 1μg (=50 pmoles)/mouse of ESX-SA complexed to biot-mAbs specific to CDllb, CDllc or CD205, induced specific, intense and highly sensitive lympho-proliferative, Thl and Th17-but not Th2-responses. ESX-SA complexed to biot-mAbs specific to CD207 or PDCA-1 induced less intense and less sensitive, yet still marked Thl responses, while ESX targeting to CD209 failed to induce such responses. Among the C-type lectins evaluated in the present study, CD205 was the most efficient at inducing Thl cells in primary responses to ESX antigens.ESX targeting to DC surface receptors allowed substantial reduction of the effective dose of antigen for immunization without impairment of T-cell immunity, as exemplified by the low dose of 5 pmoles (=0.1μg)/mouse of ESX-SA, complexed to biot-anti-CDllb mAbs which induced highly significant ESX-specific Thl and Th17 responses. Importantly, the facts that:(ⅰ) FcγR -/- and WT mice mounted comparable adaptive immune responses to mAb-mediated ESX targeting to DC and (ⅱ) ESX-SA fusion proteins complexed to biot-control Ig did not induce detectable adaptive immune responses, show that the mechanism responsible of targeting, endocytosis and further antigen presentation does not involve FcR.As priming with live attenuated mycobacteria followed by boosting with subunit vaccines, is of the most promising prophylactic anti-tuberculosis vaccination strategies, we focused particular interest in the boosting potential of ESX antigen targeting to DC subsets. We selected for this study the TB10.4 (Rv0288, ESX-H) antigen, a promising protective ESX antigen with highest interest in development of innovative sub-unit vaccine candidate. In mice primed with BCG and then boosted with TB10.4-SA targeted to CD205, CD207, CD209 or DCIR-2, a comparable boost effect of IFN-γresponses was obtained. The best boost effect at the level of Th17 response was obtained with TB10.4 targeting to CD205, followed by CD207 and PDCA-1. Using the strategy of TB10.4 antigen targeting to DC subsets, among all the conditions evaluated and the DC surface receptors targeted, we only detected efficient TB10.4-specific CD8+ T-cell cross priming in mice which were primed with BCG and then boosted with TB10.4 targeted to CD205.Thus, we have developed a new and versatile approach to target promising mycobacterial immunogens to different DC subsets in order to efficiently initiate or boost anti-mycobacterial CD4+ and CD8+ T-cell immunity. So far, CD205 C-type lectin seems to be the best DC surface marker to induce such T-cell responses.更多还原