【作者】 由庆睿；

【导师】 孔维；

【作者基本信息】 吉林大学 ， 微生物学， 2012， 博士

【摘要】 结核病是由结核分枝杆菌引起的一种高发病率和致死率的传染性疾病。在2010年，世界卫生组织估计约880万的新增感染者，且有150万人死于结核病。自1921年起，世界范围内唯一在人类接种的抗结核疫苗是卡介苗。但是，大量的临床数据表明，BCG的效果存在争议。尽管BCG在控制儿童结核病，如粟粒状结核和脑膜炎，十分有效，但对成人结核的预防效果在不同地区表现出的差异很大（0~80%）。目前认为，BCG的保护性有限，至少部分原因与连续传代造成的某些保护性抗原丢失有关。与很多诱导体液免疫的疫苗不同，由于结核杆菌是细胞内寄生菌，它的清除至少部分程度上要依赖较强的细胞免疫。因此，急需开发新的疫苗或有效的免疫策略，来刺激产生长效的细胞免疫以发挥免疫保护作用，抑制世界范围内结核病的传播。当前，结核杆菌的许多抗原被发现具有保护作用，如Ag85A，Ag85B，TB10.4，ESAT-6，CFP-10，HSP65和Mtb39A。多种抗原联合，可以增加潜在的抗原表位数量以适应更大范围接种人群的需要。Ag85B-ESAT6融合蛋白在小鼠、豚鼠、非人灵长类动物实验中，都表现出很好的保护力，因而被认为是一种非常理想的候选疫苗抗原。由于重复免疫BCG不能够加强原有的保护力，甚至会产生有害的反应，所以异源的初次免疫-加强免疫策略被认为是加强原有BCG免疫反应和保护力的有效方式。迄今，已经有多种疫苗形式被应用于这种策略，如蛋白佐剂疫苗、病毒载体疫苗、DNA疫苗等，并且有一些已取得令人振奋的结果。在这些疫苗类型中，病毒载体疫苗，尤其是腺病毒载体和痘病毒载体疫苗具有强烈的免疫原性，可以同时诱导细胞和体液免疫反应，并具有良好的临床使用安全记录，因而它们成为非常有前景的候选疫苗方式。并且，由于病毒疫苗载体具有潜在的呼吸道黏膜感染能力，使它们可以通过该免疫方式抵御黏膜感染疾病。此外，由于BCG相比使用有限抗原的亚单位疫苗，在理论上具有上千种抗原，因此通过改造BCG，使之获得更好的免疫原性也是改善其效果的一种有效方法。如果改造的BCG配合使用病毒载体疫苗，实施初次免疫-加强免疫的策略，则有可能显著增强其预防结核的效果。本研究旨在开发新型的病毒载体结核疫苗（MVA85B-E6，AD85B-E6）和改善型的菌体疫苗（rBCG），并通过prime-boost免疫策略，联合使用菌体疫苗与病毒载体疫苗，以超越原始BCG所引发的保护力。针对预防性结核疫苗的研究，本文主要分为以下几个部分。第一，本研究制备了Ag85B、ESAT6和Ag85B-ESAT6融合蛋白，并建立了结核疫苗免疫评价的体系。第二，本研究制备了BCG和高效表达Ag85B抗原的rBCG菌体疫苗。鉴定实验和免疫原性实验的数据表明，rBCG能够在无筛选压力的情况下，稳定表达Ag85B蛋白，并且rBCG能够刺激产生增强的、特异的Ag85B免疫反应。不同鼠种对BCG有着明显不同的应答。第三，本研究构建并制备了痘病毒载体疫苗MVA85B-E6，和腺病毒载体疫苗AD85B-E6，它们都能成功的表达Ag85B-ESAT6融合蛋白。AD85B-E6免疫组小鼠产生了强烈的IFN-γ分泌反应，且由CD8T细胞介导，这表明了腺病毒载体刺激CD8T细胞的显著作用。与之相比，MVA85B-E6产生了与BCG相当的免疫保护力，但却只有相对温和的IFN-γ分泌。本研究主要考察了9-2肽特异的CD8T细胞杀伤作用，而这种作用主要是由AD85B-E6免疫引起的。但是，AD85B-E6免疫产生的细胞杀伤作用和IFN-γ反应并不能赋予该免疫组有效的脾、肺保护力。并且，与阴性组小鼠相比，AD85B-E6免疫产生了加重的病理损伤，导致了肺表面结核的散布。第四，在初次免疫-加强免疫的实验中，本研究考察了病毒疫苗对BCG引发的免疫反应的加强作用和对保护力的影响，并且，使用相同的策略考察了病毒疫苗对rBCG的加强作用。数据显示，菌体疫苗被单独的MVA85B-E6或AD85B-E6加强时，未产生保护力的提升。BCG初次免疫后，MVA85B-E6和AD85B-E6联用，能够加强小鼠肺部保护力；而当rBCG初次免疫后，只有先使用AD85B-E6加强免疫，再使用MVA85B-E6才能增强小鼠脾、肺的保护力。实验中，菌体疫苗初次免疫后，先使用AD85B-E6加强免疫，再使用MVA85B-E6的免疫策略始终表现出优于先使用痘病毒再使用腺病毒的加强免疫策略的保护力。并且菌体疫苗初次免疫后，先使用AD85B-E6加强免疫，再使用MVA85B-E6的免疫策略表现出更加强烈的淋巴细胞渗出，它们紧密包裹在局部结核的周围，并且没有大范围扩散到外周的肺组织。结核的持久感染和复发感染，表明单纯的化学疗法不能彻底有效的治愈结核，这促使人们寻找合理的免疫疗法以调节宿主的免疫反应向抗菌免疫的方向发展。近年来，痘病毒载体和腺病毒载体由于其强烈的刺激细胞免疫的能力，已被用于病毒感染的免疫治疗。本研究通过结核暴露后免疫的方式，评价了MVA85B-E6和AD85B-E6对正在发病期结核小鼠的治疗作用。同时，也对另一种有效的预防疫苗Ag85B-ESAT6融合蛋白佐剂的治疗作用加以探索。数据显示，这几种形式的疫苗在均无显著的治疗作用。与之相比，化学治疗药物异烟肼，连续治疗三周，能够发挥明确的治疗作用，改善了病理学表现，但并没有使小鼠达到无菌的状态。这部分结果强调了结核化学治疗中，需要多种抗生素联用及长期用药的合理性。综上所述，本研究成功获得了MVA85B-E6和AD85B-E6病毒载体疫苗，并评价了其单独使用和作为加强疫苗使用的效果，以及它们的治疗作用。实验数据显示，需要寻找更加有效的结核疫苗保护力相关生物学指标，并且配合使用合理的免疫方案对结核疫苗的效果至关重要。更多还原

【Abstract】 Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is aninfectious disease that leads to substantial morbidity and mortality worldwide. In2010, there were an estimated8.8million incident cases of TB and almost1.5million deaths from TB.The only currently licensed anti-TB vaccine, Mycobacterium bovis bacillusCalmette-Gue′rin (BCG), has been used in humans worldwide since1921. However,collective data indicates the efficacy of BCG is somewhat controversial. AlthoughBCG is effective in protecting from severe forms of childhood TB, principallymiliary disease and meningitis, it fails to prevent adult pulmonary TB epidemic witha wide range of efficacy (0to80%). It is considered that the limited protection ofBCG may at least correlate with the lost of some protective antigens after successiveculture. Unlike most antibody-inducing vaccines, the ones against intracellular M. tbneed, at least in part, strong cellular immune responses for protective immunity.Thus, there is an urgent need to develop improved TB vaccines and effectivevaccination strategies, which are capable of promoting long-term cellular immunity.Recently, several candidate antigens, including Ag85A, Ag85B, TB10.4,ESAT-6, CFP-10, HSP65and Mtb39A, were shown to induce protective responses toM. tuberculosis challenge. A combination of some of these antigens would increasethe number of potentially immunogenic epitopes for a given vaccinated population.The well-known Ag85B-ESAT6fusion protein, which has been shown to provideeffective protection against TB in mice, guinea pigs, and non-human primates, maybe an ideal TB vaccine candidate antigen.Since BCG revaccination does not provide enhanced protection, and can beeven deleterious, heterologous prime-boost immunization strategy represents anideal way to extend BCG-initiated immunity and improve the protective efficacy. Todate, several types of TB vaccines such as protein adjuvant formulations, viral-based,and plasmid DNA vaccines have been used as boosters based on this strategy, andsome have achieved encouraging results. Remarkably, recombinant viral vectored,especially adenovirus-and poxvirus-based TB vaccines, which could induce bothcellular and humoral responses with strong immunogenicity and good safety records,would be extremely promising candidates. Furthermore, the potential of recombinant viral carriers for respiratory mucosal vaccination gives them the priority to defensethis mucosal infectious disease. On the other hand, BCG provides thousands ofantigens and stimulates the immune system for prolonged time periods as a liveattenuated vaccine, in contrast to subunit vaccines based on one or limited antigens.Accordingly, attempts have been made to genetically modify BCG to improve itsimmunogenecity. It became more fascinating that a combination vaccine comprises arecombinant BCG vaccine followed by subsequent viral-vectored boosters.This study contains four parts of prophylactic TB vaccines.Firstly, we constructed the proteins of Ag85B, ESAT6and Ag85B-ESAT6fusion protein. We builded up the immunological evaluation system of TB vaccine.Secondly, we generated BCG and rBCG, and evaluated their immunogenecity.We demonstrate that rBCG maintain over expressing Ag85B without selectivepressure and rBCG triggers increased specific cell-mediated immune responses toAg85B. Different strains of mice possess various responses to BCG.Thirdly, we engineered recombinant bivalent poxvirus-based vaccine,MVA85B-E6, and an adenovirus-based vaccine, AD85B-E6, both of which expressthe fusion protein Ag85B-ESAT6. The mice in the AD85B-E6vaccination group hadincreased antigen-specific IFN-γ-producing CD8T cells, thus highlighting the abilityof adenoviral vectors to trigger CD8T cells. In constrast, subcutaneous vaccinationwith MVA85B-E6induced moderate levels of IFN-γ production; however, itgenerated efficient protection in the lungs even beyond that observed with BCGvaccination. In the present study, CTLs were9-2-specific CD8T cells, which weretriggered primarily by vaccination with AD85B-E6. However, the CTL response andIFN-γ production by lymphocytes in the spleens generated by AD85B-E6vaccination did not significantly contribute to protection in the lungs or even in thespleens. Furthermore, aggravating histological damage and more diffuse tubercles inthe lungs were induced by AD85B-E6vaccination compared with the na ve group.Fourthly, in the Prime-boost experiments, we investigated whether theserecombinant viral vaccines could boost conventional BCG-induced immunity, andprovide enhanced protection. Further, we investigated whether they could be givenas booster vaccines on top of recombinant BCG overexpressing Ag85B as a prime.We show that the protective efficacy of mycobacterial vaccine-primed mice is notimproved by either one dose of viral booster; BCG-primed mice receiving two dosesof boosters afford enhanced protection in the lungs; only the rBCG-primed micereceiving AD85B-E6and subsequent MVA85B-E6exhibits enhanced protection against M. tb H37Rv. Bacterial priming plus AD85B-E6boosting followed byMVA85B-E6always affords better protection than that boosted with MVA85B-E6followed by AD85B-E6in the present study. Bacterial priming followed bysequential AD85B-E6and MVA85B-E6boosting, led to compact granulomas withmuch more lymphocytic infiltration than that induced by bacterial vaccine alone, andthe lymphocytes were less diffused to the surrounding parenchyma.The persistent and recurrent infection offers opportunity for immunotherapy,which is a useful complement to chemotherapy with modulating the host immuneresponse in a more anti-pathogen direction to eliminate the bacteria. Recently, MVAvaccinia virus vector and adenoviral vector, which have strong immunogenicity totrigger cell-mediated immune (CMI) response, have been employed inimmunotherapy in viral infection. In the current study, we tested whetherpost-exposure vaccination with the viral vaccines could protect mice against theongoing M. tb infection. In addition, therapeutic effect of Ag85B-ESAT6/DDA/MPLwas also tested, for whose ability to induce protective CMI in prophylactic modelwas reported previously. Disappointingly, they did not exert immunotherapeuticactivity in the absence of chemotherapy. In contrast, single antibiotic treatment withINH for3weeks, generated a significant reduction in bacterial load and improvedpathology. Nevertheless, this short-term chemotherapy did not achieve aculture-negative state, emphasizing that drug combination and duration of antibiotictherapy are important in tuberculosis treatment.In general, we generated MVA85B-E6and AD85B-E6vaccines. We evaluatedthe immune responses and protective efficacy of bacterial vaccines and viralvaccines, when they used alone or in prime-boost strategy. We also investigatedtheir immunotherapeutic effects. Our results suggest that greater efforts must bemade to identify correct biomarkers of vaccine efficacy. Furthermore, appropriateadministration regimens are important for protection against TB.更多还原