本文選題：樹突細胞 + 靶向��； 參考：《揚州大學》2010年博士論文

【摘要】： 世界上三分之一的人感染結(jié)核分枝桿菌(Mycobacterium tuberculosis, MTB),使得結(jié)核病(tuberculosis, TB)成為世界范圍內(nèi)最嚴重的細菌性傳染疾病之一,導致每年160萬人死亡,嚴重威脅人類的健康和公共衛(wèi)生安全。目前人類唯一使用的抗結(jié)核疫苗：卡介苗(Bacillus Calmette-Guerin, BCG),由牛結(jié)核分枝桿菌經(jīng)多次傳代減毒而來。然而BCG對于成人肺結(jié)核的保護效果很不穩(wěn)定,造成BCG保護力有限的可能原因有：(1)BCG的過度減毒,在減毒傳代過程中丟失了編碼保護性抗原的基因序列,例如缺失基因差異區(qū)域；(2)盡管BCG能夠激發(fā)抗MTB免疫應答,然而MTB隱藏于肉芽腫中,特異性效應T細胞很難與其直接發(fā)生作用；(3)BCG在激發(fā)保護性Th1應答的同時也活化了調(diào)節(jié)性T細胞(T regulatory cell, Treg),消減了效應性T細胞有效的保護作用。 BCG保護率的不穩(wěn)定,加上TB與人類免疫缺陷病毒(Human immunodeficiency virus, HIV)的共感染,以及結(jié)核桿菌多重耐藥菌株甚至極端耐藥菌株的出現(xiàn)使得TB呈現(xiàn)全球預警狀態(tài),因此更加理性的設(shè)計新型TB疫苗及尋求更為合理的抗TB免疫策略是亟待解決的研究課題。盡管對于開發(fā)新型TB疫苗已進行了大量的工作,但就目前而言,其中只有少數(shù)能夠與BCG相當或稍優(yōu)于BCG的保護效果。對于樹突細胞(dendritic cell, DC)的分類、功能及其在調(diào)節(jié)免疫應答中重要作用的了解,以及體內(nèi)靶向DC相關(guān)研究工作表現(xiàn)出的較為理想的應用前景,使得體內(nèi)抗原靶向DC成為設(shè)計新型TB疫苗或抗TB免疫策略的新思路。事實上,至目前為止,尚未有關(guān)于體內(nèi)靶向DC,誘導抗TB保護性免疫應答的相關(guān)研究報道。 DC具有相似的細胞形態(tài),大量分布于淋巴組織的T細胞區(qū)域,高表達MHC-Ⅱ類分子,具有突出的持續(xù)攝取環(huán)境中抗原分子并加工遞呈給T細胞的潛能。根據(jù)分化過程、表型、成熟機制及專屬職能等的不同,DC被分為不同的亞群。目前對于小鼠DC的分類已較為成熟,雖然人DC亞群不能完全等同于小鼠DC亞群,但是在小鼠和人中均發(fā)現(xiàn)了漿細胞樣DC,血液來源的淋巴組織駐留型DC,外周遷移型DC以及單核細胞來源的炎性DC等亞群,使得以小鼠為模型的體內(nèi)DC靶向研究具有向人類臨床研究過渡的可能。大量研究數(shù)據(jù)顯示不同DC亞群在支配調(diào)節(jié)如CD4+T細胞分化等獲得性細胞免疫應答中的表現(xiàn)各異,因此利用其特異性表面分子的單克隆抗體進行體內(nèi)抗原靶向的策略來直接操控各DC亞群,能夠調(diào)節(jié)、控制免疫應答的方向,是制備預防性/治療性疫苗的理想方式。 目前抗體介導的體內(nèi)DC抗原靶向研究中,多采用化學耦連或利用基因重組技術(shù)將目的抗原插入針對DC表面受體的抗體分子基因組的方法,實現(xiàn)目的抗原靶向DC。雖然多種DC表面受體分子均能傳遞外源信號,啟動T細胞應答,然而很難預測在應對特定病原體感染時,目的抗原靶向哪一種DC亞群/DC表面受體分子能產(chǎn)生最為理想的抗病原體保護性免疫應答。因此對靶向不同DC亞群產(chǎn)生的免疫應答進行比較,對于發(fā)現(xiàn)最適宜的靶向亞群或靶向分子具有指導意義。本研究開發(fā)出一種簡單方便、可選性高的靶向系統(tǒng)：將MTB免疫優(yōu)勢抗原分子與鏈酶親合素(streptavidin, SA)進行融合表達并四聚體化,在SA與生物素(biotin, biot)間高親和力的作用下,四聚體化的SA融合蛋白可與biot標記的DC表面分子單抗可形成復合物。利用這個靈活的模式,在獲得SA融合蛋白后,利用biot標記的針對不同DC亞群表面受體分子的特異單抗,可快速簡便的將目的抗原靶向小鼠和人相應的DC亞群。本研究中使用的靶向抗原來源于早期分泌抗原靶6(Earlier secreted antigen target 6, ESAT-6)蛋白家族(ESX),這些高度保守、低分子量的MTB保護性抗原由MTBⅦ型分泌系統(tǒng)表達,在小鼠、豚鼠以及不同遺傳背景的人群中表現(xiàn)出較好的免疫原性,在動物模型中表現(xiàn)出保護作用,且這些抗原分子的特異效應性CD4+、CD8+T細胞與MTB感染者體內(nèi)抗MTB保護性應答相關(guān)。本研究中,利用該方便靈活的抗原靶向系統(tǒng),將MTB ESX免疫優(yōu)勢抗原靶向不同的DC表面分子,以期篩選出能產(chǎn)生最為理想的抗結(jié)核免疫應答的DC靶點/DC亞群。 1.抗體介導的結(jié)核分枝桿菌ESX抗原體外靶向DC 首先在體外實驗中對這種抗原靶向方法的功能和特異性進行評價：證明經(jīng)biot標記的DC表面分子單抗(針對：CD11b、CD11c、MHC-Ⅱ、DCIR-2或PDCA-1)的導向作用,ESX-SA融合蛋白可高效的結(jié)合在抗原遞呈細胞表面。靶向漿細胞樣DC表面分子PDCA-1或經(jīng)典DC表面分子CD11b、CD11c、MHC-Ⅱ、DCIR-2的ESX抗原分子可有效的被細胞攝取內(nèi)吞。為評價抗原遞呈細胞對靶向的ESX抗原的加工遞呈能力,利用BCG免疫小鼠制備了ESX抗原(TB10.4, ESX-H)特異性MHC-Ⅱ限制性T細胞雜交瘤。試驗中發(fā)現(xiàn)經(jīng)典DC、漿細胞樣DC或巨噬細胞對靶向的ESX抗原通過MHC-Ⅱ類加工機制進行處理,ESX抗原表位與MHC-Ⅱ分子形成復合物,高效遞呈至ESX特異性T細胞雜交瘤或MTB野生株H37Rv感染的小鼠脾臟T細胞。 2.抗體介導的ESX抗原體內(nèi)靶向不同DC亞群誘導抗結(jié)核免疫應答 對ESX-SA融合抗原靶向DC表面受體分子產(chǎn)生的免疫原性進行比較。這些DC受體分子包括MHC-Ⅱ分子,整合素CD11b、CD11c,漿細胞樣DC抗原-1(plasmocytoid dendritic cell antigen-1, PDCA-1/CD317)和C型凝集素受體(C-type lectin receptors, CLRs):甘露糖受體家族的CD205,唾液酸糖蛋白受體家族的CD207(langerin, Clec4K)、CD209(DC-specific ICAM3-Grabbing non-integrin, DC-SIGN)以及唾液酸糖蛋白受體家族DC免疫受體亞家族的DCIR2(Clec4A)。根據(jù)ESX抗原靶向后：(1)體內(nèi)ESX抗原靶向DC并經(jīng)MHC途徑遞呈的效率；(2)誘導ESX抗原特異性Th1、Th2、Th17和Treg獲得性免疫應答的效果；(3)交叉激活ESX特異性CD8+T細胞的能力；(4)繼BCG初免后DC靶向免疫策略的潛在加強功效,以期篩選出最適宜進行靶向、能夠誘導理想的抗MTB免疫應答的DC亞群或DC表面受體分子。 體內(nèi)研究顯示該靶向系統(tǒng)具有高度特異性,只有經(jīng)DC表面分子抗體靶向的DC亞群表面才能檢測到ESX的結(jié)合,分選出這些DC進行體外培養(yǎng)能夠有效遞呈靶向的ESX抗原至特異性MHC-Ⅱ限制性T細胞雜交瘤。 ESX-SA與biot標記的DC表面分子單抗形成復合物,輔以多聚肌苷酸—胞苷酸(Poly inosinic:Poly cytidylic acid, Poly I:C,聚肌胞)作為DC激活因子免疫小鼠,對產(chǎn)生的抗原特異性T細胞應答進行比較。僅一次注射低至1μg(50pmole)劑量的ESX-SA (ESAT-6-SA),靶向DC表面CD11b、CD11c或CD205可高效誘導ESX特異性Th1、Th17應答,靶向CD207或PDCA-1也能顯著激發(fā)Th1應答,只是程度稍低,而靶向CD209未能誘導抗原特異性的Th1免疫應答。利用biot-SA靶向系統(tǒng),無論靶向何種DC表面受體分子均不能激發(fā)抗原特異性Th2免疫應答。就所有檢測的CLRs而言,靶向CD205能產(chǎn)生最強的Th1應答。對不同劑量靶向抗原誘導免疫應答的水平進行分析比較,即使將0.1μg(5pmole) ESX抗原靶向DC表面CD11b分子仍可檢測到抗原特異性Th1、Th17應答。靶向相同劑量的ESX抗原至DC表面分子,FcyR缺失小鼠與野生型小鼠產(chǎn)生的免疫應答水平相當,且在注射biot-Ctrl Ig-ESX-SA復合物的小鼠體內(nèi)不能檢測到特異性獲得性免疫應答,由此說明,本研究中抗體介導的ESX抗原靶向DC,進而為細胞攝取,加工遞呈及激發(fā)免疫應答的過程中未涉及FcR的作用,具有高度靶向特異性。以減毒活疫苗進行初次免疫,亞單位疫苗進行加強的免疫方法,可能是最為理想有效的抗TB治療性疫苗免疫策略。我們選用ESX家族保護性抗原、亞單位疫苗的熱門候選分子TB10.4來評價繼BCG初免后,TB10.4靶向DC的免疫加強效果。對BCG初免的小鼠,以biot標記單抗將TB10.4-SA靶向DC表面CLRs: CD205、CD207、CD209、DCIR-2或PDCA-1作為加強免疫,可不同程度的增強TB10.4特異性IFN-γ免疫應答；在誘導Th17免疫應答方面,靶向CD205的免疫加強效果最好,其次是CD207和PDCA-1。比較各種免疫條件,發(fā)現(xiàn)只有在BCG初免,TB10.4靶向CD205作為加強免疫的小鼠中檢測到TB10.4特異性的CD8+T細胞的交叉激活。 綜上,本研究開發(fā)出一種新型、可選性高的抗原靶向系統(tǒng),利用DC表面分子特異性單抗的導向作用,將MTB保護性抗原靶向不同DC亞群,可有效開啟或加強抗MTB CD4+和CD8+T細胞免疫反應,至此DC表面CLRs CD205可能是最為理想的靶向候選分子。
[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.
【學位授予單位】：揚州大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392

【引證文獻】

相關(guān)期刊論文 前1條

1 歐珍;陳祥;孟闖;焦新安;;結(jié)核分枝桿菌TB10.4抗原的分子生物學特性研究進展[J];中國人獸共患病學報;2011年11期

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本文編號：2008297