Ag85B-ESAT6重组结核疫苗研究
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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。
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.
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。
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.
引文
[1] World Health Organization. Global tuberculosis control. WHO Report,2011.
[2] World Health Organization. Global tuberculosis control. WHO Report,2010.
[3] COLE S T, BROSCH R, PARKHILL J, et al. Deciphering the biology of Mycobacteriumtuberculosis from the complete genome sequence [J]. Nature,1998,393:537-544.
[4] KAUFMANN S H, HUSSEY G, LAMBERT P H. New vaccines for tuberculosis [J].Lancet,2010,375:2110-2119.
[5] GLATMAN-FREEDMAN A. The role of antibody-mediated immunity in defense againstMycobacterium tuberculosis: advances toward a novel vaccine strategy [J]. Tuberculosis(Edinburgh),2006,86:191-197.
[6] HAMASUR B, HAILE M, PAWLOWSKI A, et al. A mycobacterial lipoarabinomannan specificmonoclonal antibody and its F(ab') fragment prolong survival of mice infected withMycobacterium tuberculosis [J]. Clinical and Experimental Immunology,2004,138:30-38.
[7] KAUFMANN S H. How can immunology contribute to the control of tuberculosis?[J]. NatureReviews. Immunology,2001,1:20-30.
[8] ARMSTRONG J A, HART P D. Phagosome-lysosome interactions in cultured macrophagesinfected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations onbacterial survival [J]. Journal of Experimental Medicine,1975,142:1-16.
[9] STURGILL-KOSZYCKI S, SCHLESINGER P H, CHAKRABORTY P, et al. Lack ofacidification in Mycobacterium phagosomes produced by exclusion of the vesicularproton-ATPase [J]. Science,1994,263:678-681.
[10] BRIGHTBILL H D, LIBRATY D H, KRUTZIK S R, et al. Host defense mechanisms triggeredby microbial lipoproteins through toll-like receptors [J]. Science,1999,285:732-736.
[11] VAN CREVEL R, OTTENHOFF T H, VAN DER MEER J W. Innate immunity toMycobacterium tuberculosis [J]. Clinical Microbiology Reviews,2002,15:294-309.
[12] KAUFMANN S H. Novel tuberculosis vaccination strategies based on understanding the immuneresponse [J]. Journal of Internal Medicine,2010,267:337-353.
[13] BALAJI K N, BOOM W H. Processing of Mycobacterium tuberculosis bacilli by humanmonocytes for CD4+alphabeta and gammadelta T cells: role of particulate antigen [J]. Infectionand Immunity,1998,66:98-106.
[14] PFEIFER J D, WICK M J, ROBERTS R L, et al. Phagocytic processing of bacterial antigens forclass I MHC presentation to T cells [J]. Nature,1993,361:359-362.
[15] KOVACSOVICS-BANKOWSKI M, ROCK K L. A phagosome-to-cytosol pathway forexogenous antigens presented on MHC class I molecules [J]. Science,1995,267:243-246.
[16] CARUSO A M, SERBINA N, KLEIN E, et al. Mice deficient in CD4T cells have onlytransiently diminished levels of IFN-gamma, yet succumb to tuberculosis [J]. Journal ofImmunology,1999,162:5407-5416.
[17] COWLEY S C, ELKINS K L. CD4+T cells mediate IFN-gamma-independent control ofMycobacterium tuberculosis infection both in vitro and in vivo [J]. Journal ofImmunology,2003,171:4689-4699.
[18] SCANGA C A, MOHAN V P, YU K, et al. Depletion of CD4(+) T cells causes reactivation ofmurine persistent tuberculosis despite continued expression of interferon gamma and nitric oxidesynthase2[J]. Journal of Experimental Medicine,2000,192:347-358.
[19] SERBINA N V, LAZAREVIC V, FLYNN J L. CD4(+) T cells are required for the development ofcytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection [J]. Journal ofImmunology,2001,167:6991-7000.
[20] FENG C G, BRITTON W J. CD4+and CD8+T cells mediate adoptive immunity to aerosolinfection of Mycobacterium bovis bacillus Calmette-Guerin [J]. Journal of InfectiousDiseases,2000,181:1846-1849.
[21] LADEL C H, DAUGELAT S, KAUFMANN S H. Immune response to Mycobacterium bovisbacille Calmette Guerin infection in major histocompatibility complex class I-and II-deficientknock-out mice: contribution of CD4and CD8T cells to acquired resistance [J]. EuropeanJournal of Immunology,1995,25:377-384.
[22] SILVA C L, LOWRIE D B. Identification and characterization of murine cytotoxic T cells thatkill Mycobacterium tuberculosis [J]. Infection and Immunity,2000,68:3269-3274.
[23] FAZAL N, LAMMAS D A, RAHELU M, et al. Lysis of human macrophages by cytolytic CD4+T cells fails to affect survival of intracellular Mycobacterium bovis-bacille Calmette-Guerin(BCG)[J]. Clinical and Experimental Immunology,1995,99:82-89.
[24] PITHIE A D, LAMMAS D A, FAZAL N, et al. CD4+cytolytic T cells can destroy autologousand MHC-matched macrophages but fail to kill intracellular Mycobacterium bovis-BCG [J].FEMS Immunology and Medical Microbiology,1995,11:145-154.
[25] LALVANI A, HILL A V. Cytotoxic T-lymphocytes against malaria and tuberculosis: from naturalimmunity to vaccine design [J]. Clinical Science (London),1998,95:531-538.
[26] FLYNN J L, GOLDSTEIN M M, TRIEBOLD K J, et al. Major histocompatibility complex classI-restricted T cells are required for resistance to Mycobacterium tuberculosis infection [J].Proceedings of the National Academy of Sciences of the United States ofAmerica,1992,89:12013-12017.
[27] WINAU F, WEBER S, SAD S, et al. Apoptotic vesicles crossprime CD8T cells and protectagainst tuberculosis [J]. Immunity,2006,24:105-117.
[28] BROOKES R H, PATHAN A A, MCSHANE H, et al. CD8+T cell-mediated suppression ofintracellular Mycobacterium tuberculosis growth in activated human macrophages [J]. EuropeanJournal of Immunology,2003,33:3293-3302.
[29] TURNER J, D'SOUZA C D, PEARL J E, et al. CD8-and CD95/95L-dependent mechanisms ofresistance in mice with chronic pulmonary tuberculosis [J]. American Journal of Respiratory Celland Molecular Biology,2001,24:203-209.
[30] SUD D, BIGBEE C, FLYNN J L, et al. Contribution of CD8+T cells to control ofMycobacterium tuberculosis infection [J]. Journal of Immunology,2006,176:4296-4314.
[31] VAN PINXTEREN L A, CASSIDY J P, SMEDEGAARD B H, et al. Control of latentMycobacterium tuberculosis infection is dependent on CD8T cells [J]. European Journal ofImmunology,2000,30:3689-3698.
[32] FLYNN J L, ERNST J D. Immune responses in tuberculosis [J]. Current Opinion inImmunology,2000,12:432-436.
[33] COOPER A M. Cell-mediated immune responses in tuberculosis [J]. Annual Review ofImmunology,2009,27:393-422.
[34] STENGER S, ROSAT J P, BLOOM B R, et al. Granulysin: a lethal weapon of cytolytic T cells [J].Immunology Today,1999,20:390-394.
[35] BASTIAN M, BRAUN T, BRUNS H, et al. Mycobacterial lipopeptides elicit CD4+CTLs inMycobacterium tuberculosis-infected humans [J]. Journal of Immunology,2008,180:3436-3446.
[36] JELLISON E R, KIM S K, WELSH R M. Cutting edge: MHC class II-restricted killing in vivoduring viral infection [J]. Journal of Immunology,2005,174:614-618.
[37] STENGER S, MAZZACCARO R J, UYEMURA K, et al. Differential effects of cytolytic T cellsubsets on intracellular infection [J]. Science,1997,276:1684-1687.
[38] SERBINA N V, LIU C C, SCANGA C A, et al. CD8+CTL from lungs of Mycobacteriumtuberculosis-infected mice express perforin in vivo and lyse infected macrophages [J]. Journal ofImmunology,2000,165:353-363.
[39] STENGER S, HANSON D A, TEITELBAUM R, et al. An antimicrobial activity of cytolytic Tcells mediated by granulysin [J]. Science,1998,282:121-125.
[40] LAZAREVIC V, FLYNN J. CD8+T cells in tuberculosis [J]. American Journal of Respiratoryand Critical Care Medicine,2002,166:1116-1121.
[41] PATHAN A A, WILKINSON K A, WILKINSON R J, et al. High frequencies of circulatingIFN-gamma-secreting CD8cytotoxic T cells specific for a novel MHC class I-restrictedMycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease [J].European Journal of Immunology,2000,30:2713-2721.
[42] BELKAID Y, ROUSE B T. Natural regulatory T cells in infectious disease [J]. NatureImmunology,2005,6:353-360.
[43] JIANG H, CHESS L. Regulation of immune responses by T cells [J]. New England Journal ofMedicine,2006,354:1166-1176.
[44] HORWITZ D A, ZHENG S G, GRAY J D. Natural and TGF-beta-induced Foxp3(+)CD4(+)CD25(+) regulatory T cells are not mirror images of each other [J]. Trends inImmunology,2008,29:429-435.
[45] GOLDSACK L, KIRMAN J R. Half-truths and selective memory: Interferon gamma, CD4(+) Tcells and protective memory against tuberculosis [J]. Tuberculosis (Edinburgh),2007,87:465-473.
[46] NGAI P, MCCORMICK S, SMALL C, et al. Gamma interferon responses of CD4and CD8T-cellsubsets are quantitatively different and independent of each other during pulmonaryMycobacterium bovis BCG infection [J]. Infection and Immunity,2007,75:2244-2252.
[47] TASCON R E, STAVROPOULOS E, LUKACS K V, et al. Protection against Mycobacteriumtuberculosis infection by CD8+T cells requires the production of gamma interferon [J]. Infectionand Immunity,1998,66:830-834.
[48] XING Z, ZGANIACZ A, WANG J, et al. Enhanced protection against fatal mycobacterialinfection in SCID beige mice by reshaping innate immunity with IFN-gamma transgene [J].Journal of Immunology,2001,167:375-383.
[49] FEUERER M, EULENBURG K, LODDENKEMPER C, et al. Self-limitation of Th1-mediatedinflammation by IFN-gamma [J]. Journal of Immunology,2006,176:2857-2863.
[50] XING Z, ZGANIACZ A, SANTOSUOSSO M. Role of IL-12in macrophage activation duringintracellular infection: IL-12and mycobacteria synergistically release TNF-alpha and nitric oxidefrom macrophages via IFN-gamma induction [J]. Journal of Leukocyte Biology,2000,68:897-902.
[51] OKADA M, KITA Y, NAKAJIMA T, et al. Evaluation of a novel vaccine (HVJ-liposome/HSP65DNA+IL-12DNA) against tuberculosis using the cynomolgus monkey model of TB [J].Vaccine,2007,25:2990-2993.
[52] MACLEOD M K, CLAMBEY E T, KAPPLER J W, et al. CD4memory T cells: what are theyand what can they do?[J]. Seminars in Immunology,2009,21:53-61.
[53] ANDERSEN P, SMEDEGAARD B. CD4(+) T-cell subsets that mediate immunological memoryto Mycobacterium tuberculosis infection in mice [J]. Infection and Immunity,2000,68:621-629.
[54] KAMATH A, WOODWORTH J S, BEHAR S M. Antigen-specific CD8+T cells and thedevelopment of central memory during Mycobacterium tuberculosis infection [J]. Journal ofImmunology,2006,177:6361-6369.
[55] MASOPUST D, VEZYS V, USHERWOOD E J, et al. Activated primary and memory CD8Tcells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin [J]. Journalof Immunology,2004,172:4875-4882.
[56] SERBINA N V, FLYNN J L. CD8(+) T cells participate in the memory immune response toMycobacterium tuberculosis [J]. Infection and Immunity,2001,69:4320-4328.
[57] WILLIAMS M A, BEVAN M J. T cell memory: fixed or flexible?[J]. NatureImmunology,2005,6:752-754.
[58] DUTTON R W, BRADLEY L M, SWAIN S L. T cell memory [J]. Annual Review ofImmunology,1998,16:201-223.
[59] KALIA V, SARKAR S, GOURLEY T S, et al. Differentiation of memory B and T cells [J].Current Opinion in Immunology,2006,18:255-264.
[60] SALLUSTO F, GEGINAT J, LANZAVECCHIA A. Central memory and effector memory T cellsubsets: function, generation, and maintenance [J]. Annual Review ofImmunology,2004,22:745-763.
[61] MORA J R, VON ANDRIAN U H. T-cell homing specificity and plasticity: new concepts andfuture challenges [J]. Trends in Immunology,2006,27:235-243.
[62] DAVIS S S. Nasal vaccines [J]. Adv Drug Deliv Rev,2001,51:21-42.
[63] ILLUM L, DAVIS S S. Nasal vaccination: a non-invasive vaccine delivery method that holdsgreat promise for the future [J]. Adv Drug Deliv Rev,2001,51:1-3.
[64] BOYAKA P N, MCGHEE J R. Cytokines as adjuvants for the induction of mucosal immunity [J].Adv Drug Deliv Rev,2001,51:71-79.
[65] FREYTAG L C, CLEMENTS J D. Mucosal adjuvants [J]. Vaccine,2005,23:1804-1813.
[66] TOKA F N, PACK C D, ROUSE B T. Molecular adjuvants for mucosal immunity [J].Immunological Reviews,2004,199:100-112.
[67] CHEN L, WANG J, ZGANIACZ A, et al. Single intranasal mucosal Mycobacterium bovis BCGvaccination confers improved protection compared to subcutaneous vaccination againstpulmonary tuberculosis [J]. Infection and Immunity,2004,72:238-246.
[68] SANTOSUOSSO M, MCCORMICK S, ZHANG X, et al. Intranasal boosting with anadenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovisBCG immunization against pulmonary tuberculosis [J]. Infection andImmunity,2006,74:4634-4643.
[69] SANTOSUOSSO M, ZHANG X, MCCORMICK S, et al. Mechanisms of mucosal and parenteraltuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustainedaccumulation of immune protective CD4and CD8T cells within the airway lumen [J]. Journal ofImmunology,2005,174:7986-7994.
[70] DIETRICH J, ANDERSEN C, RAPPUOLI R, et al. Mucosal administration of Ag85B-ESAT-6protects against infection with Mycobacterium tuberculosis and boosts prior bacillusCalmette-Guerin immunity [J]. Journal of Immunology,2006,177:6353-6360.
[71] ORME I M. Beyond BCG: the potential for a more effective TB vaccine [J]. Molecular MedicineToday,1999,5:487-492.
[72] ORME I M, MCMURRAY D N, BELISLE J T. Tuberculosis vaccine development: recentprogress [J]. Trends in Microbiology,2001,9:115-118.
[73] LY L H, MCMURRAY D N. Tuberculosis: vaccines in the pipeline [J]. Expert Review ofVaccines,2008,7:635-650.
[74] WANG J, XING Z. Tuberculosis vaccines: the past, present and future [J]. Expert Review ofVaccines,2002,1:341-354.
[75] LY L H, MCMURRAY D N. Tuberculosis: vaccines in the pipeline [J]. Expert Review ofVaccines,2008,7:635-650.
[76] NASSER EDDINE A, BAUMANN S, KAUFMANN S H E. New tuberculosis vaccinesapproaching clinical trial-An overview [J]. Drug Discovery Today: TherapeuticStrategies,2006,3:113-119.
[77] RAMSHAW I A, RAMSAY A J. The prime-boost strategy: exciting prospects for improvedvaccination [J]. Immunology Today,2000,21:163-165.
[78] PARK S H, YANG S H, LEE C G, et al. Efficient induction of T helper1CD4+T-cell responsesto hepatitis C virus core and E2by a DNA prime-adenovirus boost [J].Vaccine,2003,21:4555-4564.
[79] REYES-SANDOVAL A, BERTHOUD T, ALDER N, et al. Prime-boost immunization withadenoviral and modified vaccinia virus Ankara vectors enhances the durability andpolyfunctionality of protective malaria CD8+T-cell responses [J]. Infection andImmunity,2010,78:145-153.
[80] TATSIS N, LIN S W, HARRIS-MCCOY K, et al. Multiple immunizations with adenovirus andMVA vectors improve CD8+T cell functionality and mucosal homing [J].Virology,2007,367:156-167.
[81] BERESFORD B, SADOFF J C. Update on research and development pipeline: tuberculosisvaccines [J]. Clinical Infectious Diseases,2010,50Suppl3:S178-183.
[82] STANFORD J, STANFORD C, GRANGE J. Immunotherapy with Mycobacterium vaccae in thetreatment of tuberculosis [J]. Frontiers in Bioscience,2004,9:1701-1719.
[83] STANFORD J L, STANFORD C A. Immunotherapy with Mycobacterium vaccae and thetreatment of tuberculosis [J]. Society for Applied Bacteriology SymposiumSeries,1996,25:81S-86S.
[84] CARDONA P J. RUTI: a new chance to shorten the treatment of latent tuberculosis infection [J].Tuberculosis (Edinburgh),2006,86:273-289.
[85] CARDONA P J, AMAT I, GORDILLO S, et al. Immunotherapy with fragmented Mycobacteriumtuberculosis cells increases the effectiveness of chemotherapy against a chronical infection in amurine model of tuberculosis [J]. Vaccine,2005,23:1393-1398.
[86] DLUGOVITZKY D, FIORENZA G, FARRONI M, et al. Immunological consequences of threedoses of heat-killed Mycobacterium vaccae in the immunotherapy of tuberculosis [J]. RespiratoryMedicine,2006,100:1079-1087.
[87] DE BRUYN G, GARNER P. Mycobacterium vaccae immunotherapy for treating tuberculosis [J].Cochrane Database of Systematic Reviews,2003,CD001166.
[88] SKINNER M A, YUAN S, PRESTIDGE R, et al. Immunization with heat-killed Mycobacteriumvaccae stimulates CD8+cytotoxic T cells specific for macrophages infected with Mycobacteriumtuberculosis [J]. Infection and Immunity,1997,65:4525-4530.
[89] LOWRIE D B, TASCON R E, BONATO V L, et al. Therapy of tuberculosis in mice by DNAvaccination [J]. Nature,1999,400:269-271.
[90] ZHU D, JIANG S, LUO X. Therapeutic effects of Ag85B and MPT64DNA vaccines in a murinemodel of Mycobacterium tuberculosis infection [J]. Vaccine,2005,23:4619-4624.
[91] CHANGHONG S, HAI Z, LIMEI W, et al. Therapeutic efficacy of a tuberculosis DNA vaccineencoding heat shock protein65of Mycobacterium tuberculosis and the human interleukin2fusion gene [J]. Tuberculosis (Edinburgh),2009,89:54-61.
[92] OKADA M, KITA Y, NAKAJIMA T, et al. Novel prophylactic and therapeutic vaccine againsttuberculosis [J]. Vaccine,2009,27:3267-3270.
[93] REPIQUE C J, LI A, COLLINS F M, et al. DNA immunization in a mouse model of latenttuberculosis: effect of DNA vaccination on reactivation of disease and on reinfection with asecondary challenge [J]. Infection and Immunity,2002,70:3318-3323.
[94] TURNER J, RHOADES E R, KEEN M, et al. Effective preexposure tuberculosis vaccines fail toprotect when they are given in an immunotherapeutic mode [J]. Infection andImmunity,2000,68:1706-1709.
[95] HU X D, CHEN S T, YU D H, et al. Immunotherapy with combined DNA vaccines is an effectivetreatment for M. bovis infection in cattle [J]. Vaccine,2009,27:1317-1322.
[96] WU J, MA H, QU Q, et al. Incorporation of immunostimulatory motifs in the transcribed regionof a plasmid DNA vaccine enhances Th1immune responses and therapeutic effect againstMycobacterium tuberculosis in mice [J]. Vaccine,2011,29:7624-7630.
[97] HA S J, JEON B Y, YOUN J I, et al. Protective effect of DNA vaccine during chemotherapy onreactivation and reinfection of Mycobacterium tuberculosis [J]. Gene Therapy,2005,12:634-638.
[98] HA S J, JEON B Y, KIM S C, et al. Therapeutic effect of DNA vaccines combined withchemotherapy in a latent infection model after aerosol infection of mice with Mycobacteriumtuberculosis [J]. Gene Therapy,2003,10:1592-1599.
[99] YU D H, HU X D, CAI H. Efficient tuberculosis treatment in mice using chemotherapy andimmunotherapy with the combined DNA vaccine encoding Ag85B, MPT-64and MPT-83[J].Gene Therapy,2008,15:652-659.
[100]HABERSETZER F, HONNET G, BAIN C, et al. A poxvirus vaccine is safe, induces T-cellresponses, and decreases viral load in patients with chronic hepatitis C [J].Gastroenterology,2011,141:890-899.
[101]SOLOFF A C, LIU X, GAO W, et al. Adenovirus5-and35-based immunotherapy enhances thestrength but not breadth or quality of immunity during chronic SIV infection [J]. EuropeanJournal of Immunology,2009,39:2437-2449.
[102]HUNG C F, MA B, MONIE A, et al. Therapeutic human papillomavirus vaccines: current clinicaltrials and future directions [J]. Expert Opinion on Biological Therapy,2008,8:421-439.
[103]BRODIN P, ROSENKRANDS I, ANDERSEN P, et al. ESAT-6proteins: protective antigens andvirulence factors?[J]. Trends in Microbiology,2004,12:500-508.
[104]RENSHAW P S, LIGHTBODY K L, VEVERKA V, et al. Structure and function of the complexformed by the tuberculosis virulence factors CFP-10and ESAT-6[J]. EMBOJournal,2005,24:2491-2498.
[105]ANTOINE G, SCHEIFLINGER F, DORNER F, et al. The complete genomic sequence of themodified vaccinia Ankara strain: comparison with other orthopoxviruses [J].Virology,1998,244:365-396.
[106]MCSHANE H, BROOKES R, GILBERT S C, et al. Enhanced immunogenicity of CD4(+) t-cellresponses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boostvaccination regimen for murine tuberculosis [J]. Infection and Immunity,2001,69:681-686.
[107]PERERA P Y, DERRICK S C, KOLIBAB K, et al. A multi-valent vaccinia virus-basedtuberculosis vaccine molecularly adjuvanted with interleukin-15induces robust immuneresponses in mice [J]. Vaccine,2009,27:2121-2127.
[108]MCSHANE H, BEHBOUDI S, GOONETILLEKE N, et al. Protective immunity againstMycobacterium tuberculosis induced by dendritic cells pulsed with both CD8(+)-andCD4(+)-T-cell epitopes from antigen85A [J]. Infection and Immunity,2002,70:1623-1626.
[109]IMLER J L. Adenovirus vectors as recombinant viral vaccines [J]. Vaccine,1995,13:1143-1151.
[110] RADOSEVIC K, WIELAND C W, RODRIGUEZ A, et al. Protective immune responses to arecombinant adenovirus type35tuberculosis vaccine in two mouse strains: CD4and CD8T-cellepitope mapping and role of gamma interferon [J]. Infection and Immunity,2007,75:4105-4115.
[111] WANG J, THORSON L, STOKES R W, et al. Single mucosal, but not parenteral, immunizationwith recombinant adenoviral-based vaccine provides potent protection from pulmonarytuberculosis [J]. Journal of Immunology,2004,173:6357-6365.
[112] DERRICK S C, YANG A L, MORRIS S L. A polyvalent DNA vaccine expressing anESAT6-Ag85B fusion protein protects mice against a primary infection with Mycobacteriumtuberculosis and boosts BCG-induced protective immunity [J]. Vaccine,2004,23:780-788.
[113] WEINRICH OLSEN A, VAN PINXTEREN L A, MENG OKKELS L, et al. Protection of micewith a tuberculosis subunit vaccine based on a fusion protein of antigen85b and esat-6[J].Infection and Immunity,2001,69:2773-2778.
[114] OLSEN A W, WILLIAMS A, OKKELS L M, et al. Protective effect of a tuberculosis subunitvaccine based on a fusion of antigen85B and ESAT-6in the aerosol guinea pig model [J].Infection and Immunity,2004,72:6148-6150.
[115] LANGERMANS J A, DOHERTY T M, VERVENNE R A, et al. Protection of macaques againstMycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen85B and ESAT-6[J]. Vaccine,2005,23:2740-2750.
[2] World Health Organization. Global tuberculosis control. WHO Report,2010.
[3] COLE S T, BROSCH R, PARKHILL J, et al. Deciphering the biology of Mycobacteriumtuberculosis from the complete genome sequence [J]. Nature,1998,393:537-544.
[4] KAUFMANN S H, HUSSEY G, LAMBERT P H. New vaccines for tuberculosis [J].Lancet,2010,375:2110-2119.
[5] GLATMAN-FREEDMAN A. The role of antibody-mediated immunity in defense againstMycobacterium tuberculosis: advances toward a novel vaccine strategy [J]. Tuberculosis(Edinburgh),2006,86:191-197.
[6] HAMASUR B, HAILE M, PAWLOWSKI A, et al. A mycobacterial lipoarabinomannan specificmonoclonal antibody and its F(ab') fragment prolong survival of mice infected withMycobacterium tuberculosis [J]. Clinical and Experimental Immunology,2004,138:30-38.
[7] KAUFMANN S H. How can immunology contribute to the control of tuberculosis?[J]. NatureReviews. Immunology,2001,1:20-30.
[8] ARMSTRONG J A, HART P D. Phagosome-lysosome interactions in cultured macrophagesinfected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations onbacterial survival [J]. Journal of Experimental Medicine,1975,142:1-16.
[9] STURGILL-KOSZYCKI S, SCHLESINGER P H, CHAKRABORTY P, et al. Lack ofacidification in Mycobacterium phagosomes produced by exclusion of the vesicularproton-ATPase [J]. Science,1994,263:678-681.
[10] BRIGHTBILL H D, LIBRATY D H, KRUTZIK S R, et al. Host defense mechanisms triggeredby microbial lipoproteins through toll-like receptors [J]. Science,1999,285:732-736.
[11] VAN CREVEL R, OTTENHOFF T H, VAN DER MEER J W. Innate immunity toMycobacterium tuberculosis [J]. Clinical Microbiology Reviews,2002,15:294-309.
[12] KAUFMANN S H. Novel tuberculosis vaccination strategies based on understanding the immuneresponse [J]. Journal of Internal Medicine,2010,267:337-353.
[13] BALAJI K N, BOOM W H. Processing of Mycobacterium tuberculosis bacilli by humanmonocytes for CD4+alphabeta and gammadelta T cells: role of particulate antigen [J]. Infectionand Immunity,1998,66:98-106.
[14] PFEIFER J D, WICK M J, ROBERTS R L, et al. Phagocytic processing of bacterial antigens forclass I MHC presentation to T cells [J]. Nature,1993,361:359-362.
[15] KOVACSOVICS-BANKOWSKI M, ROCK K L. A phagosome-to-cytosol pathway forexogenous antigens presented on MHC class I molecules [J]. Science,1995,267:243-246.
[16] CARUSO A M, SERBINA N, KLEIN E, et al. Mice deficient in CD4T cells have onlytransiently diminished levels of IFN-gamma, yet succumb to tuberculosis [J]. Journal ofImmunology,1999,162:5407-5416.
[17] COWLEY S C, ELKINS K L. CD4+T cells mediate IFN-gamma-independent control ofMycobacterium tuberculosis infection both in vitro and in vivo [J]. Journal ofImmunology,2003,171:4689-4699.
[18] SCANGA C A, MOHAN V P, YU K, et al. Depletion of CD4(+) T cells causes reactivation ofmurine persistent tuberculosis despite continued expression of interferon gamma and nitric oxidesynthase2[J]. Journal of Experimental Medicine,2000,192:347-358.
[19] SERBINA N V, LAZAREVIC V, FLYNN J L. CD4(+) T cells are required for the development ofcytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection [J]. Journal ofImmunology,2001,167:6991-7000.
[20] FENG C G, BRITTON W J. CD4+and CD8+T cells mediate adoptive immunity to aerosolinfection of Mycobacterium bovis bacillus Calmette-Guerin [J]. Journal of InfectiousDiseases,2000,181:1846-1849.
[21] LADEL C H, DAUGELAT S, KAUFMANN S H. Immune response to Mycobacterium bovisbacille Calmette Guerin infection in major histocompatibility complex class I-and II-deficientknock-out mice: contribution of CD4and CD8T cells to acquired resistance [J]. EuropeanJournal of Immunology,1995,25:377-384.
[22] SILVA C L, LOWRIE D B. Identification and characterization of murine cytotoxic T cells thatkill Mycobacterium tuberculosis [J]. Infection and Immunity,2000,68:3269-3274.
[23] FAZAL N, LAMMAS D A, RAHELU M, et al. Lysis of human macrophages by cytolytic CD4+T cells fails to affect survival of intracellular Mycobacterium bovis-bacille Calmette-Guerin(BCG)[J]. Clinical and Experimental Immunology,1995,99:82-89.
[24] PITHIE A D, LAMMAS D A, FAZAL N, et al. CD4+cytolytic T cells can destroy autologousand MHC-matched macrophages but fail to kill intracellular Mycobacterium bovis-BCG [J].FEMS Immunology and Medical Microbiology,1995,11:145-154.
[25] LALVANI A, HILL A V. Cytotoxic T-lymphocytes against malaria and tuberculosis: from naturalimmunity to vaccine design [J]. Clinical Science (London),1998,95:531-538.
[26] FLYNN J L, GOLDSTEIN M M, TRIEBOLD K J, et al. Major histocompatibility complex classI-restricted T cells are required for resistance to Mycobacterium tuberculosis infection [J].Proceedings of the National Academy of Sciences of the United States ofAmerica,1992,89:12013-12017.
[27] WINAU F, WEBER S, SAD S, et al. Apoptotic vesicles crossprime CD8T cells and protectagainst tuberculosis [J]. Immunity,2006,24:105-117.
[28] BROOKES R H, PATHAN A A, MCSHANE H, et al. CD8+T cell-mediated suppression ofintracellular Mycobacterium tuberculosis growth in activated human macrophages [J]. EuropeanJournal of Immunology,2003,33:3293-3302.
[29] TURNER J, D'SOUZA C D, PEARL J E, et al. CD8-and CD95/95L-dependent mechanisms ofresistance in mice with chronic pulmonary tuberculosis [J]. American Journal of Respiratory Celland Molecular Biology,2001,24:203-209.
[30] SUD D, BIGBEE C, FLYNN J L, et al. Contribution of CD8+T cells to control ofMycobacterium tuberculosis infection [J]. Journal of Immunology,2006,176:4296-4314.
[31] VAN PINXTEREN L A, CASSIDY J P, SMEDEGAARD B H, et al. Control of latentMycobacterium tuberculosis infection is dependent on CD8T cells [J]. European Journal ofImmunology,2000,30:3689-3698.
[32] FLYNN J L, ERNST J D. Immune responses in tuberculosis [J]. Current Opinion inImmunology,2000,12:432-436.
[33] COOPER A M. Cell-mediated immune responses in tuberculosis [J]. Annual Review ofImmunology,2009,27:393-422.
[34] STENGER S, ROSAT J P, BLOOM B R, et al. Granulysin: a lethal weapon of cytolytic T cells [J].Immunology Today,1999,20:390-394.
[35] BASTIAN M, BRAUN T, BRUNS H, et al. Mycobacterial lipopeptides elicit CD4+CTLs inMycobacterium tuberculosis-infected humans [J]. Journal of Immunology,2008,180:3436-3446.
[36] JELLISON E R, KIM S K, WELSH R M. Cutting edge: MHC class II-restricted killing in vivoduring viral infection [J]. Journal of Immunology,2005,174:614-618.
[37] STENGER S, MAZZACCARO R J, UYEMURA K, et al. Differential effects of cytolytic T cellsubsets on intracellular infection [J]. Science,1997,276:1684-1687.
[38] SERBINA N V, LIU C C, SCANGA C A, et al. CD8+CTL from lungs of Mycobacteriumtuberculosis-infected mice express perforin in vivo and lyse infected macrophages [J]. Journal ofImmunology,2000,165:353-363.
[39] STENGER S, HANSON D A, TEITELBAUM R, et al. An antimicrobial activity of cytolytic Tcells mediated by granulysin [J]. Science,1998,282:121-125.
[40] LAZAREVIC V, FLYNN J. CD8+T cells in tuberculosis [J]. American Journal of Respiratoryand Critical Care Medicine,2002,166:1116-1121.
[41] PATHAN A A, WILKINSON K A, WILKINSON R J, et al. High frequencies of circulatingIFN-gamma-secreting CD8cytotoxic T cells specific for a novel MHC class I-restrictedMycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease [J].European Journal of Immunology,2000,30:2713-2721.
[42] BELKAID Y, ROUSE B T. Natural regulatory T cells in infectious disease [J]. NatureImmunology,2005,6:353-360.
[43] JIANG H, CHESS L. Regulation of immune responses by T cells [J]. New England Journal ofMedicine,2006,354:1166-1176.
[44] HORWITZ D A, ZHENG S G, GRAY J D. Natural and TGF-beta-induced Foxp3(+)CD4(+)CD25(+) regulatory T cells are not mirror images of each other [J]. Trends inImmunology,2008,29:429-435.
[45] GOLDSACK L, KIRMAN J R. Half-truths and selective memory: Interferon gamma, CD4(+) Tcells and protective memory against tuberculosis [J]. Tuberculosis (Edinburgh),2007,87:465-473.
[46] NGAI P, MCCORMICK S, SMALL C, et al. Gamma interferon responses of CD4and CD8T-cellsubsets are quantitatively different and independent of each other during pulmonaryMycobacterium bovis BCG infection [J]. Infection and Immunity,2007,75:2244-2252.
[47] TASCON R E, STAVROPOULOS E, LUKACS K V, et al. Protection against Mycobacteriumtuberculosis infection by CD8+T cells requires the production of gamma interferon [J]. Infectionand Immunity,1998,66:830-834.
[48] XING Z, ZGANIACZ A, WANG J, et al. Enhanced protection against fatal mycobacterialinfection in SCID beige mice by reshaping innate immunity with IFN-gamma transgene [J].Journal of Immunology,2001,167:375-383.
[49] FEUERER M, EULENBURG K, LODDENKEMPER C, et al. Self-limitation of Th1-mediatedinflammation by IFN-gamma [J]. Journal of Immunology,2006,176:2857-2863.
[50] XING Z, ZGANIACZ A, SANTOSUOSSO M. Role of IL-12in macrophage activation duringintracellular infection: IL-12and mycobacteria synergistically release TNF-alpha and nitric oxidefrom macrophages via IFN-gamma induction [J]. Journal of Leukocyte Biology,2000,68:897-902.
[51] OKADA M, KITA Y, NAKAJIMA T, et al. Evaluation of a novel vaccine (HVJ-liposome/HSP65DNA+IL-12DNA) against tuberculosis using the cynomolgus monkey model of TB [J].Vaccine,2007,25:2990-2993.
[52] MACLEOD M K, CLAMBEY E T, KAPPLER J W, et al. CD4memory T cells: what are theyand what can they do?[J]. Seminars in Immunology,2009,21:53-61.
[53] ANDERSEN P, SMEDEGAARD B. CD4(+) T-cell subsets that mediate immunological memoryto Mycobacterium tuberculosis infection in mice [J]. Infection and Immunity,2000,68:621-629.
[54] KAMATH A, WOODWORTH J S, BEHAR S M. Antigen-specific CD8+T cells and thedevelopment of central memory during Mycobacterium tuberculosis infection [J]. Journal ofImmunology,2006,177:6361-6369.
[55] MASOPUST D, VEZYS V, USHERWOOD E J, et al. Activated primary and memory CD8Tcells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin [J]. Journalof Immunology,2004,172:4875-4882.
[56] SERBINA N V, FLYNN J L. CD8(+) T cells participate in the memory immune response toMycobacterium tuberculosis [J]. Infection and Immunity,2001,69:4320-4328.
[57] WILLIAMS M A, BEVAN M J. T cell memory: fixed or flexible?[J]. NatureImmunology,2005,6:752-754.
[58] DUTTON R W, BRADLEY L M, SWAIN S L. T cell memory [J]. Annual Review ofImmunology,1998,16:201-223.
[59] KALIA V, SARKAR S, GOURLEY T S, et al. Differentiation of memory B and T cells [J].Current Opinion in Immunology,2006,18:255-264.
[60] SALLUSTO F, GEGINAT J, LANZAVECCHIA A. Central memory and effector memory T cellsubsets: function, generation, and maintenance [J]. Annual Review ofImmunology,2004,22:745-763.
[61] MORA J R, VON ANDRIAN U H. T-cell homing specificity and plasticity: new concepts andfuture challenges [J]. Trends in Immunology,2006,27:235-243.
[62] DAVIS S S. Nasal vaccines [J]. Adv Drug Deliv Rev,2001,51:21-42.
[63] ILLUM L, DAVIS S S. Nasal vaccination: a non-invasive vaccine delivery method that holdsgreat promise for the future [J]. Adv Drug Deliv Rev,2001,51:1-3.
[64] BOYAKA P N, MCGHEE J R. Cytokines as adjuvants for the induction of mucosal immunity [J].Adv Drug Deliv Rev,2001,51:71-79.
[65] FREYTAG L C, CLEMENTS J D. Mucosal adjuvants [J]. Vaccine,2005,23:1804-1813.
[66] TOKA F N, PACK C D, ROUSE B T. Molecular adjuvants for mucosal immunity [J].Immunological Reviews,2004,199:100-112.
[67] CHEN L, WANG J, ZGANIACZ A, et al. Single intranasal mucosal Mycobacterium bovis BCGvaccination confers improved protection compared to subcutaneous vaccination againstpulmonary tuberculosis [J]. Infection and Immunity,2004,72:238-246.
[68] SANTOSUOSSO M, MCCORMICK S, ZHANG X, et al. Intranasal boosting with anadenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovisBCG immunization against pulmonary tuberculosis [J]. Infection andImmunity,2006,74:4634-4643.
[69] SANTOSUOSSO M, ZHANG X, MCCORMICK S, et al. Mechanisms of mucosal and parenteraltuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustainedaccumulation of immune protective CD4and CD8T cells within the airway lumen [J]. Journal ofImmunology,2005,174:7986-7994.
[70] DIETRICH J, ANDERSEN C, RAPPUOLI R, et al. Mucosal administration of Ag85B-ESAT-6protects against infection with Mycobacterium tuberculosis and boosts prior bacillusCalmette-Guerin immunity [J]. Journal of Immunology,2006,177:6353-6360.
[71] ORME I M. Beyond BCG: the potential for a more effective TB vaccine [J]. Molecular MedicineToday,1999,5:487-492.
[72] ORME I M, MCMURRAY D N, BELISLE J T. Tuberculosis vaccine development: recentprogress [J]. Trends in Microbiology,2001,9:115-118.
[73] LY L H, MCMURRAY D N. Tuberculosis: vaccines in the pipeline [J]. Expert Review ofVaccines,2008,7:635-650.
[74] WANG J, XING Z. Tuberculosis vaccines: the past, present and future [J]. Expert Review ofVaccines,2002,1:341-354.
[75] LY L H, MCMURRAY D N. Tuberculosis: vaccines in the pipeline [J]. Expert Review ofVaccines,2008,7:635-650.
[76] NASSER EDDINE A, BAUMANN S, KAUFMANN S H E. New tuberculosis vaccinesapproaching clinical trial-An overview [J]. Drug Discovery Today: TherapeuticStrategies,2006,3:113-119.
[77] RAMSHAW I A, RAMSAY A J. The prime-boost strategy: exciting prospects for improvedvaccination [J]. Immunology Today,2000,21:163-165.
[78] PARK S H, YANG S H, LEE C G, et al. Efficient induction of T helper1CD4+T-cell responsesto hepatitis C virus core and E2by a DNA prime-adenovirus boost [J].Vaccine,2003,21:4555-4564.
[79] REYES-SANDOVAL A, BERTHOUD T, ALDER N, et al. Prime-boost immunization withadenoviral and modified vaccinia virus Ankara vectors enhances the durability andpolyfunctionality of protective malaria CD8+T-cell responses [J]. Infection andImmunity,2010,78:145-153.
[80] TATSIS N, LIN S W, HARRIS-MCCOY K, et al. Multiple immunizations with adenovirus andMVA vectors improve CD8+T cell functionality and mucosal homing [J].Virology,2007,367:156-167.
[81] BERESFORD B, SADOFF J C. Update on research and development pipeline: tuberculosisvaccines [J]. Clinical Infectious Diseases,2010,50Suppl3:S178-183.
[82] STANFORD J, STANFORD C, GRANGE J. Immunotherapy with Mycobacterium vaccae in thetreatment of tuberculosis [J]. Frontiers in Bioscience,2004,9:1701-1719.
[83] STANFORD J L, STANFORD C A. Immunotherapy with Mycobacterium vaccae and thetreatment of tuberculosis [J]. Society for Applied Bacteriology SymposiumSeries,1996,25:81S-86S.
[84] CARDONA P J. RUTI: a new chance to shorten the treatment of latent tuberculosis infection [J].Tuberculosis (Edinburgh),2006,86:273-289.
[85] CARDONA P J, AMAT I, GORDILLO S, et al. Immunotherapy with fragmented Mycobacteriumtuberculosis cells increases the effectiveness of chemotherapy against a chronical infection in amurine model of tuberculosis [J]. Vaccine,2005,23:1393-1398.
[86] DLUGOVITZKY D, FIORENZA G, FARRONI M, et al. Immunological consequences of threedoses of heat-killed Mycobacterium vaccae in the immunotherapy of tuberculosis [J]. RespiratoryMedicine,2006,100:1079-1087.
[87] DE BRUYN G, GARNER P. Mycobacterium vaccae immunotherapy for treating tuberculosis [J].Cochrane Database of Systematic Reviews,2003,CD001166.
[88] SKINNER M A, YUAN S, PRESTIDGE R, et al. Immunization with heat-killed Mycobacteriumvaccae stimulates CD8+cytotoxic T cells specific for macrophages infected with Mycobacteriumtuberculosis [J]. Infection and Immunity,1997,65:4525-4530.
[89] LOWRIE D B, TASCON R E, BONATO V L, et al. Therapy of tuberculosis in mice by DNAvaccination [J]. Nature,1999,400:269-271.
[90] ZHU D, JIANG S, LUO X. Therapeutic effects of Ag85B and MPT64DNA vaccines in a murinemodel of Mycobacterium tuberculosis infection [J]. Vaccine,2005,23:4619-4624.
[91] CHANGHONG S, HAI Z, LIMEI W, et al. Therapeutic efficacy of a tuberculosis DNA vaccineencoding heat shock protein65of Mycobacterium tuberculosis and the human interleukin2fusion gene [J]. Tuberculosis (Edinburgh),2009,89:54-61.
[92] OKADA M, KITA Y, NAKAJIMA T, et al. Novel prophylactic and therapeutic vaccine againsttuberculosis [J]. Vaccine,2009,27:3267-3270.
[93] REPIQUE C J, LI A, COLLINS F M, et al. DNA immunization in a mouse model of latenttuberculosis: effect of DNA vaccination on reactivation of disease and on reinfection with asecondary challenge [J]. Infection and Immunity,2002,70:3318-3323.
[94] TURNER J, RHOADES E R, KEEN M, et al. Effective preexposure tuberculosis vaccines fail toprotect when they are given in an immunotherapeutic mode [J]. Infection andImmunity,2000,68:1706-1709.
[95] HU X D, CHEN S T, YU D H, et al. Immunotherapy with combined DNA vaccines is an effectivetreatment for M. bovis infection in cattle [J]. Vaccine,2009,27:1317-1322.
[96] WU J, MA H, QU Q, et al. Incorporation of immunostimulatory motifs in the transcribed regionof a plasmid DNA vaccine enhances Th1immune responses and therapeutic effect againstMycobacterium tuberculosis in mice [J]. Vaccine,2011,29:7624-7630.
[97] HA S J, JEON B Y, YOUN J I, et al. Protective effect of DNA vaccine during chemotherapy onreactivation and reinfection of Mycobacterium tuberculosis [J]. Gene Therapy,2005,12:634-638.
[98] HA S J, JEON B Y, KIM S C, et al. Therapeutic effect of DNA vaccines combined withchemotherapy in a latent infection model after aerosol infection of mice with Mycobacteriumtuberculosis [J]. Gene Therapy,2003,10:1592-1599.
[99] YU D H, HU X D, CAI H. Efficient tuberculosis treatment in mice using chemotherapy andimmunotherapy with the combined DNA vaccine encoding Ag85B, MPT-64and MPT-83[J].Gene Therapy,2008,15:652-659.
[100]HABERSETZER F, HONNET G, BAIN C, et al. A poxvirus vaccine is safe, induces T-cellresponses, and decreases viral load in patients with chronic hepatitis C [J].Gastroenterology,2011,141:890-899.
[101]SOLOFF A C, LIU X, GAO W, et al. Adenovirus5-and35-based immunotherapy enhances thestrength but not breadth or quality of immunity during chronic SIV infection [J]. EuropeanJournal of Immunology,2009,39:2437-2449.
[102]HUNG C F, MA B, MONIE A, et al. Therapeutic human papillomavirus vaccines: current clinicaltrials and future directions [J]. Expert Opinion on Biological Therapy,2008,8:421-439.
[103]BRODIN P, ROSENKRANDS I, ANDERSEN P, et al. ESAT-6proteins: protective antigens andvirulence factors?[J]. Trends in Microbiology,2004,12:500-508.
[104]RENSHAW P S, LIGHTBODY K L, VEVERKA V, et al. Structure and function of the complexformed by the tuberculosis virulence factors CFP-10and ESAT-6[J]. EMBOJournal,2005,24:2491-2498.
[105]ANTOINE G, SCHEIFLINGER F, DORNER F, et al. The complete genomic sequence of themodified vaccinia Ankara strain: comparison with other orthopoxviruses [J].Virology,1998,244:365-396.
[106]MCSHANE H, BROOKES R, GILBERT S C, et al. Enhanced immunogenicity of CD4(+) t-cellresponses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boostvaccination regimen for murine tuberculosis [J]. Infection and Immunity,2001,69:681-686.
[107]PERERA P Y, DERRICK S C, KOLIBAB K, et al. A multi-valent vaccinia virus-basedtuberculosis vaccine molecularly adjuvanted with interleukin-15induces robust immuneresponses in mice [J]. Vaccine,2009,27:2121-2127.
[108]MCSHANE H, BEHBOUDI S, GOONETILLEKE N, et al. Protective immunity againstMycobacterium tuberculosis induced by dendritic cells pulsed with both CD8(+)-andCD4(+)-T-cell epitopes from antigen85A [J]. Infection and Immunity,2002,70:1623-1626.
[109]IMLER J L. Adenovirus vectors as recombinant viral vaccines [J]. Vaccine,1995,13:1143-1151.
[110] RADOSEVIC K, WIELAND C W, RODRIGUEZ A, et al. Protective immune responses to arecombinant adenovirus type35tuberculosis vaccine in two mouse strains: CD4and CD8T-cellepitope mapping and role of gamma interferon [J]. Infection and Immunity,2007,75:4105-4115.
[111] WANG J, THORSON L, STOKES R W, et al. Single mucosal, but not parenteral, immunizationwith recombinant adenoviral-based vaccine provides potent protection from pulmonarytuberculosis [J]. Journal of Immunology,2004,173:6357-6365.
[112] DERRICK S C, YANG A L, MORRIS S L. A polyvalent DNA vaccine expressing anESAT6-Ag85B fusion protein protects mice against a primary infection with Mycobacteriumtuberculosis and boosts BCG-induced protective immunity [J]. Vaccine,2004,23:780-788.
[113] WEINRICH OLSEN A, VAN PINXTEREN L A, MENG OKKELS L, et al. Protection of micewith a tuberculosis subunit vaccine based on a fusion protein of antigen85b and esat-6[J].Infection and Immunity,2001,69:2773-2778.
[114] OLSEN A W, WILLIAMS A, OKKELS L M, et al. Protective effect of a tuberculosis subunitvaccine based on a fusion of antigen85B and ESAT-6in the aerosol guinea pig model [J].Infection and Immunity,2004,72:6148-6150.
[115] LANGERMANS J A, DOHERTY T M, VERVENNE R A, et al. Protection of macaques againstMycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen85B and ESAT-6[J]. Vaccine,2005,23:2740-2750.