Article(id=1203002057281204306, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203002056400396334, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2023.06.0643, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1659888000000, receivedDateStr=2022-08-08, revisedDate=null, revisedDateStr=null, acceptedDate=1670256000000, acceptedDateStr=2022-12-06, onlineDate=1764747641152, onlineDateStr=2025-12-03, pubDate=1687881600000, pubDateStr=2023-06-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764747641152, onlineIssueDateStr=2025-12-03, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764747641152, creator=13701087609, updateTime=1764747641152, updator=13701087609, issue=Issue{id=1203002056400396334, tenantId=1146029695717560320, journalId=1189873630562394117, year='2023', volume='48', issue='6', pageStart='627', pageEnd='748', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1764747640943, creator=13701087609, updateTime=1764747714497, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1203002364979540735, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203002056400396334, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1203002364979540736, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1203002056400396334, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=643, endPage=647, ext={EN=ArticleExt(id=1203002057520279636, articleId=1203002057281204306, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Research progress and challenge of tuberculosis vaccine, columnId=1203002057176342576, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Prevention, Diagnosis and Treatment of Tuberculosis, runingTitle=null, highlight=null, articleAbstract=

Currently, the bacillus Calmette-Guerin (BCG) is the only licensed vaccine against tuberculosis, but its protective effect is highly controversial. Therefore, it is particularly important to develop a new and effective tuberculosis vaccine. Meanwhile, with the in-depth understanding of the pathogenic mechanism of Mycobacterium tuberculosis and the continuous update of vaccine research and development technology, there will be a major breakthrough in tuberculosis vaccine. Considering the safety, economic cost and protective effect, multi-stage antigen subunit vaccine or multi-epitope vaccine has good application prospects. This paper systematically reviews the types and characteristics of current tuberculosis vaccines, and discuss the problems and challenges in four aspects: antigen selection, animal model selection, the evaluation indicators of vaccine efficacy and the population specificity in the research of new tuberculosis vaccines, which lays a theoretical foundation for the research of new tuberculosis vaccine.

, correspAuthors=Hai-Can Liu, authorNote=null, correspAuthorsNote=
* E-mail:
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卡介苗是目前唯一应用于预防人类结核病的疫苗,但其保护效果备受争议,研究新型高效的结核病疫苗尤为重要。从安全性、经济成本及保护效果等方面考虑,多阶段抗原亚单位疫苗或多表位疫苗具有较好的应用前景。随着对结核分枝杆菌致病机制的深入了解以及疫苗研发技术的不断更新,结核病疫苗会有重大突破。本文论述结核病疫苗的类型及其特性,并针对结核病新型疫苗研究中抗原选择、动物模型选择、疫苗效果评价指标及人群特异性存在的问题和挑战进行讨论,旨在为新型结核病疫苗的研究提供参考。

, correspAuthors=刘海灿, authorNote=null, correspAuthorsNote=
刘海灿,E-mail:
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范雪亭,副研究员,主要从事结核病预防与控制的相关研究

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结核病疫苗研究进展及挑战
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范雪亭 , 万康林 , 王瑞白 , 赵秀芹 , 刘海灿 *
解放军医学杂志 | 结核病的预防、诊断与治疗专题 2023,48(6): 643-647
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解放军医学杂志 | 结核病的预防、诊断与治疗专题 2023, 48(6): 643-647
结核病疫苗研究进展及挑战
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范雪亭, 万康林, 王瑞白, 赵秀芹, 刘海灿*
作者信息
  • 中国疾病预防控制中心传染病预防控制所/传染病预防控制国家重点实验室,北京 102206
  • 范雪亭,副研究员,主要从事结核病预防与控制的相关研究

通讯作者:

刘海灿,E-mail:
Research progress and challenge of tuberculosis vaccine
Xue-Ting Fan, Kang-Lin Wan, Rui-Bai Wang, Xiu-Qin Zhao, Hai-Can Liu*
Affiliations
  • State Key Laboratory for Infectious Disease Prevention and Control/National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
出版时间: 2023-06-28 doi: 10.11855/j.issn.0577-7402.2023.06.0643
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卡介苗是目前唯一应用于预防人类结核病的疫苗,但其保护效果备受争议,研究新型高效的结核病疫苗尤为重要。从安全性、经济成本及保护效果等方面考虑,多阶段抗原亚单位疫苗或多表位疫苗具有较好的应用前景。随着对结核分枝杆菌致病机制的深入了解以及疫苗研发技术的不断更新,结核病疫苗会有重大突破。本文论述结核病疫苗的类型及其特性,并针对结核病新型疫苗研究中抗原选择、动物模型选择、疫苗效果评价指标及人群特异性存在的问题和挑战进行讨论,旨在为新型结核病疫苗的研究提供参考。

结核病  /  卡介苗  /  结核病新型疫苗

Currently, the bacillus Calmette-Guerin (BCG) is the only licensed vaccine against tuberculosis, but its protective effect is highly controversial. Therefore, it is particularly important to develop a new and effective tuberculosis vaccine. Meanwhile, with the in-depth understanding of the pathogenic mechanism of Mycobacterium tuberculosis and the continuous update of vaccine research and development technology, there will be a major breakthrough in tuberculosis vaccine. Considering the safety, economic cost and protective effect, multi-stage antigen subunit vaccine or multi-epitope vaccine has good application prospects. This paper systematically reviews the types and characteristics of current tuberculosis vaccines, and discuss the problems and challenges in four aspects: antigen selection, animal model selection, the evaluation indicators of vaccine efficacy and the population specificity in the research of new tuberculosis vaccines, which lays a theoretical foundation for the research of new tuberculosis vaccine.

tuberculosis  /  bacillus Calmette-Guerin  /  new tuberculosis vaccine
范雪亭, 万康林, 王瑞白, 赵秀芹, 刘海灿. 结核病疫苗研究进展及挑战. 解放军医学杂志, 2023 , 48 (6) : 643 -647 . DOI: 10.11855/j.issn.0577-7402.2023.06.0643
Xue-Ting Fan, Kang-Lin Wan, Rui-Bai Wang, Xiu-Qin Zhao, Hai-Can Liu. Research progress and challenge of tuberculosis vaccine[J]. Medical Journal of Chinese People’s Liberation Army, 2023 , 48 (6) : 643 -647 . DOI: 10.11855/j.issn.0577-7402.2023.06.0643
结核病(tuberculosis,TB)是一种由结核分枝杆菌引起的慢性传染性疾病,严重威胁着人类的健康。随着耐药结核及TB合并人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染的出现,TB的防治面临着更大的挑战。卡介苗(bacille Calmette-Guerin,BCG)是目前唯一应用于人的TB疫苗[1],对儿童原发型肺结核、粟粒型肺结核及结核性脑膜炎等严重TB均有较好的免疫效果,但对成人TB的保护效果极不稳定,且不同地域的免疫保护效果(为0~80%)差别较大[2-3]。此外,研究发现BCG只有10~20年的保护效果[4]。因此,研究新型、有效且安全的TB疫苗迫在眉睫,也是实现世界卫生组织(WHO)设定的“2035年将TB发病率降低90%”这一目标的关键。
目前,TB疫苗主要包括以下4类:减毒活疫苗、灭活疫苗、亚单位疫苗、病毒载体疫苗等[5-6]。根据WHO发布的报告,目前已有23种候选结核疫苗处于临床试验阶段,虽然这些疫苗在安全性、免疫原性等方面表现极佳,但是由于缺乏对TB发病机制及宿主免疫保护机制的认识,它们还不足以完全取代BCG。本文就国内外TB疫苗的研究进展进行综述,并围绕现有TB疫苗面临的问题及挑战进行讨论,以期为TB疫苗的深入研究提供参考。
减毒活疫苗是结核分枝杆菌或母牛分枝杆菌中的某些毒力基因缺失而获得的疫苗[7]。减毒活疫苗中存在许多BCG缺失的重要免疫显性抗原区域,同时,毒力基因片段的缺失为疫苗的安全性提供了保证。该类疫苗可以在体内长久存在并有限复制,因此可诱导机体产生更特异且更持久的免疫反应[8]。MTBVAC是唯一进入人体临床试验阶段的减毒活疫苗,该菌株是在临床分离株Mt103基础上删减了两个毒力基因phoPfadD26,但是保留了BCG缺失的RD1区的早期分泌抗原6(early secreted antigen target 6,ESAT-6)及培养滤液蛋白(10 kD-culture filtrate protein,CFP-10),具有更好的免疫原性[9]。该疫苗是作为BCG的替代品及免疫治疗剂而设计的,目前已经进入Ⅲ期临床试验阶段[10]。但是,减毒活疫苗因疫苗菌株可以在体内长时间存活,可能存在引起不良反应的风险,因此对其安全性必须进行长时间的评估。有研究对伴有严重联合免疫缺陷小鼠的安全性进行评估显示,MTBVAC的毒力比BCG更弱,即安全性更高[11]。此外,Ⅰ期临床试验数据也显示,与BCG相比,MTBVAC表现出良好的安全性及耐受性,且在与BCG相同的剂量下,MTBVAC组多功能性CD4+中央记忆型T细胞的出现频率更高[79],这一数据表明减毒活疫苗可能产生比BCG更强的细胞免疫。
为进一步提高BCG的免疫效果,目前研究者主要致力于两个方面的工作:一是提高BCG的自身免疫效果,二是使用其他类型的疫苗进行加强免疫[12]。研究发现,最有效提高BCG免疫效果的策略即重组BCG(rBCG),主要包括过表达结核分枝杆菌中的免疫优势抗原(BCG中已存在或不存在的抗原)的重组BCG、表达细胞因子的重组BCG及营养缺陷型重组BCG。
目前,重组BCG中过表达的免疫优势抗原主要涉及Ag85B、Ag85A、HspX及RD1区抗原。rBCG30是一种将Ag85B过量表达而构建的疫苗,研究证实其Ag85B的表达水平是原始株的5~6倍,该疫苗是首个经临床前研究证明比BCG更有效的TB疫苗,已完成Ⅰ期临床研究,但最终因存在抗生素抗性基因而被终止研究[13]。VPM1002是目前关注度较高的一种rBCG,携带李斯特菌溶素(Hly)而自身缺失脲霉素C基因,可以刺激机体产生较强的Th17及Th1类细胞反应,且在小鼠中表现出比BCG更强的保护效果[14]。此外,VPM1002还可诱导更多的中央记忆型T细胞(Tcm)[15]、更强的自噬及炎症反应[16]。目前该疫苗已经开始开展Ⅲ期临床试验。
上述策略构建的TB疫苗具有较强的免疫原性及保护效果,不仅对婴幼儿具有较好的保护作用,还可为成年人提供保护,同时延长了保护期,具有巨大的发展潜力及应用前景。
灭活疫苗是指利用物理或化学方法将病原微生物杀死后制备的一种疫苗,可以是整个细菌组分或裂解片段即菌体细胞提取物[17]。与减毒活疫苗相比,灭活疫苗的免疫原性相对较弱,因此需要提高免疫剂量及免疫次数以增强免疫效果,其安全性较高,也是新型TB疫苗研究的热点。
TB灭活疫苗的研究可以追溯至19世纪,Opie等[18]通过热灭活方式构建了一种多剂量TB灭活疫苗,在动物模型及人体中均证实其是有效的预防疫苗,但由于BCG的成功上市而没有进一步研发。灭活疫苗既可用作预防性疫苗,也可用作治疗性疫苗。在灭活疫苗中,RUTI是一种包含结核分枝杆菌细胞壁的脂质体,是一种能减少抗生素的使用、改善潜伏感染及结核患者治疗效果的治疗性疫苗。Ⅰ期临床试验数据表明其具有较好的免疫原性及安全性,Ⅱ期临床试验数据显示,RUTI在感染HIV与未感染HIV的结核分枝杆菌潜伏感染者中均显示出较强的免疫原性及较好的安全性[19]。另外,Ⅱa期临床试验数据表明,RUTI对活跃期及潜伏期的结核分枝杆菌中表达的抗原均表现出明显的体液及细胞免疫反应[20]
此外,大量临床研究显示,Vaccae(微卡,热灭活的母牛分枝杆菌)可明显提高耐药结核患者的痰液转阴率,能改善复治性肺结核患者的细胞免疫功能,有助于病灶吸收[21]。目前,我国已经将Vaccae批准为TB患者的辅助性治疗疫苗,该疫苗也在我国开展了Ⅲ期临床试验,以评估其预防潜伏感染转化为活动性结核的有效性。虽然目前几种灭活疫苗在安全性、免疫原性及辅助治疗方面取得了较好的成绩,但能否替代BCG最终进入市场取决于进一步的评估结果。
亚单位疫苗是一种包含病原体中具有保护效果的蛋白或多肽的疫苗,它可以包含一种或多种抗原或者抗原表位,一般联合佐剂使用。结核分枝杆菌约有4000个抗原,目前用于疫苗研究的抗原主要包括3类:(1)早期分泌蛋白,主要涉及ESAT-6、Ag85B、Ag85A及MPT64等抗原;(2)细胞壁蛋白,主要包括热休克蛋白HspX等[22];(3)细胞壁相关的PE/PPE家族蛋白等。结核分枝杆菌生长周期较长,根据代谢状态的不同可分为复苏期、复制期及休眠期等多个阶段[23]。结核分枝杆菌在不同的代谢阶段表达的抗原谱也不尽相同,即不同阶段的优势抗原不同。且结核分枝杆菌感染宿主后所处的阶段并不唯一,因此理想的TB亚单位疫苗最好包含多阶段的优势抗原[24]。目前有两种包含多阶段抗原的TB亚单位疫苗进入了临床研究阶段,即H56(Ag85B、ESAT-6、Rv2660c)及ID93(Rv2608、Rv3619、Rv3620、Rv1813),它们均处于Ⅱa期临床试验,且都显示出较好的临床免疫效果。
随着生物信息技术的不断发展,抗原表位预测技术越来越成熟。表位疫苗是一种通过生物信息学技术预测抗原表位,然后利用基因工程技术体外表达或者人工合成病原微生物抗原表位区而构建的疫苗[25]。当前表位疫苗在抗病毒及抗菌方面都有较广泛的应用。实验研究也表明结核分枝杆菌某些抗原表位区的免疫原性比全抗原效果更好[26],为今后研发新型TB疫苗提供了新的思路。但是,表位疫苗也存在一定的问题,如多肽较小,需要使用免疫佐剂,而目前应用于人类疫苗的佐剂种类有限。
DNA疫苗又称基因疫苗,是指将免疫原性及保护效果较好的抗原编码基因与表达载体相连而构建的疫苗。与传统疫苗相比,DNA疫苗允许导入的基因在宿主体内表达,并向免疫系统展示特定的编码蛋白,随后刺激机体特异性的细胞免疫及体液免疫[27]。随着对结核分枝杆菌感染宿主机制认识的深入以及TB亚单位疫苗研发中积累的大量经验,目前已有多种TB DNA疫苗在临床前动物实验中取得了理想的效果。已报道的效果较好的TB DNA疫苗主要包括CFP10、ESAT-6、Ag85复合物及热休克蛋白等。另外,TB DNA疫苗研发策略中除了包含结核分枝杆菌抗原的编码基因外,还可将一些细胞因子的编码基因插入其中,以增强疫苗的免疫效果。
mRNA疫苗是将各类微生物的mRNA用于预防或治疗的生物制剂。与传统疫苗相比,该类疫苗可同时诱导体液免疫及细胞免疫,且具有研发周期短、成本低以及便于标准化生产等优点,但其稳定性及转染效率较差,限制了该类疫苗的发展。近几年,随着对mRNA疫苗设计的不断优化及递送系统的研发,mRNA疫苗再次成为研究热点,尤其在应对突发重大传染病如新型冠状病毒感染方面发挥了重要作用。此外,mRNA疫苗基于内源性表达系统,可以诱导Th1型细胞免疫应答,对于机体抵抗结核分枝杆菌的感染具有重要作用,因此该类疫苗也将是TB新型疫苗的突破口。但该类结核疫苗的难点依然是抗原的筛选。
随着对结核分枝杆菌感染机制认识的深入以及生物技术的不断发展,TB新型疫苗研发取得了一定的成果。但是,目前新型的TB疫苗均处于临床试验阶段,开发一种普遍有效且可以替代或增强BCG免疫效果的疫苗依然面临着巨大挑战。
抗原的选择对于疫苗的研发具有决定性作用,但是结核分枝杆菌的抗原约有4000种,且目前很多抗原的功能尚不完全清楚,因此筛选具有强免疫原性的抗原是如今TB疫苗研究的重点及难点。如何选择免疫原性强的抗原,以及开发的疫苗是单一抗原还是多种抗原,是目前面临的主要问题。另外,结核分枝杆菌感染宿主后会有不同的代谢状态,选择单一代谢状态的抗原还是多种代谢状态的抗原都需要考虑。抗原的选择必须明确结核分枝杆菌与宿主互相作用的动态机制,以及病原体的致病机制,同时考虑抗原本身的免疫原性及抗原在结核分枝杆菌感染或动态互相作用时发挥的作用。另外,多抗原组合作为疫苗候选时,抗原的排列顺序及连接方式都会影响疫苗的免疫效果。
合适的动物模型是疫苗评价时首要考虑的问题。不同的动物模型对结核分枝杆菌的敏感性不同,产生的免疫特点也不同。目前研究证实,动物模型并不能完全模拟临床感染状态[28],例如,目前用于评价TB疫苗免疫效果的动物模型主要是小鼠和豚鼠,但是这两种动物模型都存在一定的局限性。小鼠是最常用的疫苗评价模型,但是其感染结核分枝杆菌不能形成明显的干酪样坏死病变。豚鼠虽然可以形成明显的病变,但不能诱导明显的细胞免疫。灵长类动物如恒河猴、猕猴等虽然与人类症状相似,但是成本较高,限制了其应用。合适的动物模型可为TB疫苗研究提供有力支撑,也是TB疫苗研究中的重要工具。
TB疫苗的效果评价主要包括免疫原性、动物体内结核分枝杆菌载菌量、感染结核分枝杆菌后存活率及存活时间[29-30]。长期以来,研究人员普遍认为Th1类细胞免疫对于抵抗结核分枝杆菌的感染具有重要意义,因此结核疫苗免疫原性的评价主要围绕Th1类细胞免疫反应,尤其是γ干扰素(IFN-γ)、肿瘤坏死因子(TNF)-α等细胞因子,以及CD4+ T、CD8+ T细胞增殖分化比例。同时,大量研究证实宿主感染结核分枝杆菌后,体内也会大量表达IFN-γ,因此IFN-γ、TNF-α等Th1类细胞因子与疫苗的保护效果是否具有相关性需进一步验证。在南非新生儿群体中开展的一项研究发现,新生儿接种BCG 2年后,结核分枝杆菌特异性CD4+ T细胞及分泌的IFN-γ水平与保护效果无相关性[31]。另外,B细胞在介导TB疫苗效力方面的作用也逐渐凸显,但尚未完全了解其具体功能。在动物模型及人类中开展的相关研究已经提供了明确的证据,即增殖的抗原特异性B细胞定植于保护性肉芽肿内,这些肉芽肿是高度特殊化的空间结构,可以控制结核分枝杆菌的进一步感染[32]。此外,最近研究证实,来自潜伏感染者的抗体能促进噬菌体成熟、炎症小体激活及结核分枝杆菌细胞内巨噬细胞的杀伤作用[33]。因此,除了T细胞外,B细胞也可能参与了疫苗诱导的抗TB免疫反应。因此TB疫苗的免疫学评价指标需要进一步探索,这对于疫苗的研究具有重要的意义。
TB疫苗面临的另一重要问题是人群多样性。与实验动物模型不同,年龄、性别及地理位置是导致人群对疫苗反应不同的重要影响因素。其次,不同人种的基因存在差异,因此对于疫苗的反应也各不相同。因此,想要获得针对不同年龄、地理位置及基因的人群均有保护效果的疫苗存在较大的难度,这也是目前TB疫苗研究面临的问题之一。
尽管广泛推广了TB的标准治疗方案、现代诊断方法及疫苗接种,但全球TB流行并未得到有效控制,TB依然对人类健康造成巨大的威胁。另外,耐药结核及HIV合并感染的出现,导致TB的预防及控制面临着更大的挑战。目前TB预防及控制的首要任务是预防未感染的人感染TB,有效的新型疫苗是WHO终止TB战略的关键。虽然目前TB疫苗研究面临很多难题,但是随着疫苗研发技术的发展以及对TB致病机制的逐步了解,在开发改进TB疫苗方面取得的进展令人鼓舞,希望用于人类预防及治疗的新许可TB疫苗在不久的将来成为现实。同时,根据目前TB疫苗的研究进展,从安全性、可行性以及长期免疫效果方面考虑,TB亚单位疫苗尤其是多阶段抗原亚单位疫苗或多表位疫苗具有较好的应用前景。
  • “十三五”传染病重大专项(2018ZX10731301-002)
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doi: 10.11855/j.issn.0577-7402.2023.06.0643
  • 接收时间:2022-08-08
  • 首发时间:2025-12-03
  • 出版时间:2023-06-28
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  • 收稿日期:2022-08-08
  • 录用日期:2022-12-06
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Major Projects of the 13th Five-Year Plan Special for Infectious Diseases(2018ZX10731301-002)
“十三五”传染病重大专项(2018ZX10731301-002)
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    中国疾病预防控制中心传染病预防控制所/传染病预防控制国家重点实验室,北京 102206

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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