结核分枝杆菌H37Rv感染期表面蛋白筛选及宿主相关免疫应答研究
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摘要
目前，结核病仍是致死率最高的疾病之一，全球几乎1/3人口为潜在结核患者，该人群中10%有发展为活动性结核的可能。MTB致病机理复杂，疫苗研制及诊断研究已成为研究的热点。筛选候选疫苗及检测抗原，有助于深入了解其致病机制。表面蛋白（分泌蛋白、跨膜蛋白等)介导细菌与宿主的相互作用，与宿主最初接触过程中能破坏宿主组织，引起一系列级联反应，在MTB致病过程中起重要作用，并影响宿主免疫防御。MTB表面蛋白具有靶向免疫细胞并在其内部潜伏增殖能力，了解MTB表面蛋白和宿主细胞的相互作用有利于了解MTB的致病机制。
本文构建MTB H37Rv全基因组表面蛋白噬菌体展示文库，直接完成了表面蛋白筛选、鉴别、表达纯化及功能分析过程，MTB H37Rv全基因组噬菌体展示文库库容量为105，经过表面蛋白筛选后共得到840个阳性克隆。随机挑选其中200个克隆测序分析，共得到47个包含信号肽序列的表面蛋白，因此预知840个阳性克隆将有188个表面蛋白，与目前软件预测MTB所含表面蛋白总数（179个）相近，显示出该展示系统的高效性。
利用MTB表面蛋白噬菌体展示文库进行差减筛选，得到6个能与结核患者血清特异性结合的表面蛋白（Ag85B, ESAT-6, MPT-64, LpqA, cpsA和PapA2)。这些抗原在MTB毒力菌株中具有高表达性，从而导致其对于结核患者血清和BCG免疫血清显示出不同的反应性。在活动性结核和治疗后痰培养转阴性结核患者之间cpsA和PapA2噬菌体融合蛋白的血清学反应敏感性分别从80.1%降到13.3%和从79.3%降到14.0%。揭示在抗结核治疗后抗原特异性血清学反应的动态变化，为筛选感染早期和潜伏期重要抗原提供了有效方法。
利用生物信息软件将筛选的6个MTB表面蛋白进行B细胞表位预测，并合成17个B细胞表位多肽，ELISA方法检测17个表位多肽与结核患者血清以及BCG免疫血清免疫学反应。结果显示8条多肽对结核患者血清显示特异性反应。同时表位多肽A1、C3和P3对痰培养阳性血清反应敏感性明显高于对治疗后痰培养阴性血清。利用ELISPOT和流式细胞检测方法来评价在活动性结核患者以及治疗后痰培养转阴性结核患者之间cpsA, PapA2和PaPA2噬菌体融合蛋白和表位多肽A1、C3、P3抗原特异性IFN-γ反应差别。结果显示，接受治疗后cpsA和PapA2噬菌体融合蛋白和表位多肽C3和A1抗原特异性IFN-γ反应性明显降低。验证了从感染期到恢复期抗原特异性血清学和T细胞免疫反应动态变化过程。证明PapA2、CspA表面蛋白是结核感染期的毒力因子，为进一步筛选结核疫苗候选抗原提供理论基础。
将cpsA, PapA2噬菌体融合蛋白联合Ag85B+ESAT-6噬菌体融合蛋白构建多价疫苗，并对其免疫原性进行分析。应用多色流式细胞分析方法将不同免疫组的抗原特异性多功能T细胞亚群进行评价归类，从而评价出抗结核免疫有效的宿T细胞亚群组成。结果显示，新联合蛋白组诱导高质量体液免疫和细胞免疫反应，明显优于单抗原免疫，而表位多肽激发的免疫反应相对较弱。cpsA和PapA2噬菌体融合蛋白联合免疫组能激发Ag85B抗原特异性白细胞介素-2（IL-2）,肿瘤坏死因子-α（TNF-α）和γ-干扰素（IFN-γ）三种细胞因子、IL-2和TNF-α两种细胞因子以及TNF-α单细胞因子的高表达，从而证明了cpsA和PapA2噬菌体融合蛋白可引起记忆性免疫应答。
本研究成功构建MTB表面蛋白噬菌体展示文库，并利用该文库进行差减筛选，从而筛选到6个能与结核患者血清特异性结合的表面蛋白，对筛选表面蛋白进行B细胞表位预测并合成相应表位多肽。利用ELISA和流式细胞检测方法来评价在活动性结核患者以及治疗后痰培养转阴性结核患者之间噬菌体融合蛋白和表位多肽抗原特异性血清学反应及IFN-γ反应差别，揭示抗结核治疗后抗原特异性血清学及IFN-γ反应性变化趋势，为筛选结核感染期相关毒力因子提供平台，亦为筛选疫苗候选基因提供理论基础。同时将cpsA, PapA2噬菌体融合蛋白联合Ag85B+ESAT-6噬菌体融合蛋白构建多价疫苗，从体液免疫反应水平、细胞因子表达水平、多功能细胞因子表达数量、T细胞亚群比例等多个指标对各组合疫苗所诱导免疫反应进行综合评价，亦对抗结核免疫有效的宿主T细胞亚群组成进行系统诠释，为结核疫苗提供新的评测标准。
To this day, TB remains one of the leading causes of mortality. Additionally, about one thirdof the world population has latent TB and10%of them develop the disease during their lifetime.Owing to the complex pathogenic mechanism of MTB, vaccine development and diagnosticstudies have become a hot topic. Surface proteins consisting of secreted and transmembraneproteins play a central role in MTB pathogenesis, as they are used by the bacteria for nutrientuptake, adherence to host proteins and modulation of the host immune response. Surface proteinsalso allow MTB to target and proliferate inside immune cells. Understanding the interactionbetween surface proteins of MTB and the host cell is therefore likely to help us better understandthe pathogenesis of MTB.
In this study, we constructed a MTB surface proteins phage display library with840positiveclones, which can be used for direct selection, identification, expression, purification andfunctional research on surface proteins of MTB. After sequence analysis of a randomly selected200clones, there were140clones with a signal sequence, and47distinct ORFs predicted toencode secretome proteins. When extrapolated to the840clones, there are about188surfaceproteins, which is similar to the number of secreted proteins with signal peptide predicted bysoftware（179)，which showed the efficiency of our display system.
We also made an innovative iterative screening with the phage display MTB surface proteinlibrary to identify antigens with specific reactivity against antibodies in the sera of TB patients. Asa result, we identified six antigens（MPT64, Ag85B, ESAT-6, PapA2, LpqA and CpsA)，and theAg85B, PapA2, LpqA and CspA proteins sharing>99%identity with BCG are still to be identified.These antigens may be expressed differently in virulent stains of MTB and the BCG strains, whichmay contribute to higher specific reactivity with sera from active TB compared to the reactivitywith the serum from BCG vaccinations.
The sensitivity of cpsA and PapA2phage display proteins in active TB patients and sputum-negative TB patients after anti-TB treatment ranged from80.1%to13.3%and79.3%to14.0%respectively. Our research revealed the dynamic changes of the antigen-specific serologicalreaction after anti-TB treatment, and provided an effective method for the screening of importantantigens on the earlier infection and incubation stage.
The B-cell epitope of these six MTB surface proteins were predicted with the biologicalinformation software, then synthesised17B-cell epitope peptides. The serum reaction of these17epitope peptides between the TB patient serum and BCG immune serum were detected usingELISA method. As a result,6peptides showed a specific reaction to the sera of TB patients. Onthe other hand, the reaction sensitivity of epitope peptide A1, C3and P3to the sputum culturepositive serum were significantly higher than that of sputum culture-negative serum after anti-TBtreatment.
The differences of antigen-specific IFN-γ responses of the cpsA, LpqA, PaPA2phage fusionproteins and the epitope peptides A1, C3and P3between active TB patients and sputumsmear-negative TB after anti-TB treatment were determined using flow cytometry and ELISPOT.As a result, after the treatment, the antigen specific IFN-γ response of cpsA and PapA2and theepitope peptide C3and A1decreased greatly. Our research have verified a antigen specificserological and IFN-γ response dynamic changes between the active stage and the infectionconvalescent stage, the cpsA and PaPA2should be the virulent factors of the MTB exhibits greatersecretory activity in the active stage, which would have great impact on diagnosis, monitoring oftreatment and vaccination strategies.
New subunit vaccines of MTB were obtained by combining Ag85B＋ESAT-6with cpsA andPapA2phage display protein, the antigen-specific multifunctional T cell proliferation in thedifferent vaccination groups were analysised using multicolor flow cytometry, then made ajudgment on the valuable CD4+T cell marker for vaccine-take in TB vaccine trials. Aftervaccination, the combination vaccination groups induced qualitatively and quantitatively differenthumoral and cellular immune responses compared to and single component. The cpsA and PapA2phage display proteins can stimulate highly expression of IFN-γ, interleukin-12（IL-2), and tumornecrosis factor-α （TNF-α）three cytokines, IL-2and TNF-α two cytokines, and TNF-α CD4+Tcell populations, which proved that these two phage display proteins can stimulate a memoryimmune response.
In this study, we successfully constructed a MTB surface proteins phage display library, which can be used for direct selection, identification, expression, purification and functionalresearch on surface proteins of MTB, and made a new iterative screening with this library toidentify antigens with specific reactivity against antibodies in the sera of TB patients. As a resultwe identified six antigens, the B-cell epitope of these six MTB surface proteins were predictedwith the biological information software, and synthesised17B-cell epitope peptides. Thedifferences of antigen-specific serological reaction and IFN-γ responses of these six phage fuseproteins and the B cell epitope peptides between active TB patients and sputum smear-negativeTB after anti-TB treatment were determined using flow cytometry and ELISPOT, hoping to revealthe change trend of the antigen-specific serological and IFN-γ reaction after anti-TB treatment,which provided a platform for the screening of the virulent factor in the TB active stage, andprovide theoretical basis for the selecting of TB vaccine relative antigens, At last, we constructedsubunit vaccine by combining Ag85B＋ESAT-6with cpsA and PapA2phage display proteins.Then a comprehensive analysis of the immune response from each combination group were madefrom the humoral immune response, cytokine expression levels, the number of multifunctionalcytokine expression and the proportion of multiple T cell subsets, which helped to make asystematic interpretation of the useful anti-TB host T cell subsets and also helped to provide aneffective platform and evaluation standards for the TB vaccine development.
本文构建MTB H37Rv全基因组表面蛋白噬菌体展示文库，直接完成了表面蛋白筛选、鉴别、表达纯化及功能分析过程，MTB H37Rv全基因组噬菌体展示文库库容量为105，经过表面蛋白筛选后共得到840个阳性克隆。随机挑选其中200个克隆测序分析，共得到47个包含信号肽序列的表面蛋白，因此预知840个阳性克隆将有188个表面蛋白，与目前软件预测MTB所含表面蛋白总数（179个）相近，显示出该展示系统的高效性。
利用MTB表面蛋白噬菌体展示文库进行差减筛选，得到6个能与结核患者血清特异性结合的表面蛋白（Ag85B, ESAT-6, MPT-64, LpqA, cpsA和PapA2)。这些抗原在MTB毒力菌株中具有高表达性，从而导致其对于结核患者血清和BCG免疫血清显示出不同的反应性。在活动性结核和治疗后痰培养转阴性结核患者之间cpsA和PapA2噬菌体融合蛋白的血清学反应敏感性分别从80.1%降到13.3%和从79.3%降到14.0%。揭示在抗结核治疗后抗原特异性血清学反应的动态变化，为筛选感染早期和潜伏期重要抗原提供了有效方法。
利用生物信息软件将筛选的6个MTB表面蛋白进行B细胞表位预测，并合成17个B细胞表位多肽，ELISA方法检测17个表位多肽与结核患者血清以及BCG免疫血清免疫学反应。结果显示8条多肽对结核患者血清显示特异性反应。同时表位多肽A1、C3和P3对痰培养阳性血清反应敏感性明显高于对治疗后痰培养阴性血清。利用ELISPOT和流式细胞检测方法来评价在活动性结核患者以及治疗后痰培养转阴性结核患者之间cpsA, PapA2和PaPA2噬菌体融合蛋白和表位多肽A1、C3、P3抗原特异性IFN-γ反应差别。结果显示，接受治疗后cpsA和PapA2噬菌体融合蛋白和表位多肽C3和A1抗原特异性IFN-γ反应性明显降低。验证了从感染期到恢复期抗原特异性血清学和T细胞免疫反应动态变化过程。证明PapA2、CspA表面蛋白是结核感染期的毒力因子，为进一步筛选结核疫苗候选抗原提供理论基础。
将cpsA, PapA2噬菌体融合蛋白联合Ag85B+ESAT-6噬菌体融合蛋白构建多价疫苗，并对其免疫原性进行分析。应用多色流式细胞分析方法将不同免疫组的抗原特异性多功能T细胞亚群进行评价归类，从而评价出抗结核免疫有效的宿T细胞亚群组成。结果显示，新联合蛋白组诱导高质量体液免疫和细胞免疫反应，明显优于单抗原免疫，而表位多肽激发的免疫反应相对较弱。cpsA和PapA2噬菌体融合蛋白联合免疫组能激发Ag85B抗原特异性白细胞介素-2（IL-2）,肿瘤坏死因子-α（TNF-α）和γ-干扰素（IFN-γ）三种细胞因子、IL-2和TNF-α两种细胞因子以及TNF-α单细胞因子的高表达，从而证明了cpsA和PapA2噬菌体融合蛋白可引起记忆性免疫应答。
本研究成功构建MTB表面蛋白噬菌体展示文库，并利用该文库进行差减筛选，从而筛选到6个能与结核患者血清特异性结合的表面蛋白，对筛选表面蛋白进行B细胞表位预测并合成相应表位多肽。利用ELISA和流式细胞检测方法来评价在活动性结核患者以及治疗后痰培养转阴性结核患者之间噬菌体融合蛋白和表位多肽抗原特异性血清学反应及IFN-γ反应差别，揭示抗结核治疗后抗原特异性血清学及IFN-γ反应性变化趋势，为筛选结核感染期相关毒力因子提供平台，亦为筛选疫苗候选基因提供理论基础。同时将cpsA, PapA2噬菌体融合蛋白联合Ag85B+ESAT-6噬菌体融合蛋白构建多价疫苗，从体液免疫反应水平、细胞因子表达水平、多功能细胞因子表达数量、T细胞亚群比例等多个指标对各组合疫苗所诱导免疫反应进行综合评价，亦对抗结核免疫有效的宿主T细胞亚群组成进行系统诠释，为结核疫苗提供新的评测标准。
To this day, TB remains one of the leading causes of mortality. Additionally, about one thirdof the world population has latent TB and10%of them develop the disease during their lifetime.Owing to the complex pathogenic mechanism of MTB, vaccine development and diagnosticstudies have become a hot topic. Surface proteins consisting of secreted and transmembraneproteins play a central role in MTB pathogenesis, as they are used by the bacteria for nutrientuptake, adherence to host proteins and modulation of the host immune response. Surface proteinsalso allow MTB to target and proliferate inside immune cells. Understanding the interactionbetween surface proteins of MTB and the host cell is therefore likely to help us better understandthe pathogenesis of MTB.
In this study, we constructed a MTB surface proteins phage display library with840positiveclones, which can be used for direct selection, identification, expression, purification andfunctional research on surface proteins of MTB. After sequence analysis of a randomly selected200clones, there were140clones with a signal sequence, and47distinct ORFs predicted toencode secretome proteins. When extrapolated to the840clones, there are about188surfaceproteins, which is similar to the number of secreted proteins with signal peptide predicted bysoftware（179)，which showed the efficiency of our display system.
We also made an innovative iterative screening with the phage display MTB surface proteinlibrary to identify antigens with specific reactivity against antibodies in the sera of TB patients. Asa result, we identified six antigens（MPT64, Ag85B, ESAT-6, PapA2, LpqA and CpsA)，and theAg85B, PapA2, LpqA and CspA proteins sharing>99%identity with BCG are still to be identified.These antigens may be expressed differently in virulent stains of MTB and the BCG strains, whichmay contribute to higher specific reactivity with sera from active TB compared to the reactivitywith the serum from BCG vaccinations.
The sensitivity of cpsA and PapA2phage display proteins in active TB patients and sputum-negative TB patients after anti-TB treatment ranged from80.1%to13.3%and79.3%to14.0%respectively. Our research revealed the dynamic changes of the antigen-specific serologicalreaction after anti-TB treatment, and provided an effective method for the screening of importantantigens on the earlier infection and incubation stage.
The B-cell epitope of these six MTB surface proteins were predicted with the biologicalinformation software, then synthesised17B-cell epitope peptides. The serum reaction of these17epitope peptides between the TB patient serum and BCG immune serum were detected usingELISA method. As a result,6peptides showed a specific reaction to the sera of TB patients. Onthe other hand, the reaction sensitivity of epitope peptide A1, C3and P3to the sputum culturepositive serum were significantly higher than that of sputum culture-negative serum after anti-TBtreatment.
The differences of antigen-specific IFN-γ responses of the cpsA, LpqA, PaPA2phage fusionproteins and the epitope peptides A1, C3and P3between active TB patients and sputumsmear-negative TB after anti-TB treatment were determined using flow cytometry and ELISPOT.As a result, after the treatment, the antigen specific IFN-γ response of cpsA and PapA2and theepitope peptide C3and A1decreased greatly. Our research have verified a antigen specificserological and IFN-γ response dynamic changes between the active stage and the infectionconvalescent stage, the cpsA and PaPA2should be the virulent factors of the MTB exhibits greatersecretory activity in the active stage, which would have great impact on diagnosis, monitoring oftreatment and vaccination strategies.
New subunit vaccines of MTB were obtained by combining Ag85B＋ESAT-6with cpsA andPapA2phage display protein, the antigen-specific multifunctional T cell proliferation in thedifferent vaccination groups were analysised using multicolor flow cytometry, then made ajudgment on the valuable CD4+T cell marker for vaccine-take in TB vaccine trials. Aftervaccination, the combination vaccination groups induced qualitatively and quantitatively differenthumoral and cellular immune responses compared to and single component. The cpsA and PapA2phage display proteins can stimulate highly expression of IFN-γ, interleukin-12（IL-2), and tumornecrosis factor-α （TNF-α）three cytokines, IL-2and TNF-α two cytokines, and TNF-α CD4+Tcell populations, which proved that these two phage display proteins can stimulate a memoryimmune response.
In this study, we successfully constructed a MTB surface proteins phage display library, which can be used for direct selection, identification, expression, purification and functionalresearch on surface proteins of MTB, and made a new iterative screening with this library toidentify antigens with specific reactivity against antibodies in the sera of TB patients. As a resultwe identified six antigens, the B-cell epitope of these six MTB surface proteins were predictedwith the biological information software, and synthesised17B-cell epitope peptides. Thedifferences of antigen-specific serological reaction and IFN-γ responses of these six phage fuseproteins and the B cell epitope peptides between active TB patients and sputum smear-negativeTB after anti-TB treatment were determined using flow cytometry and ELISPOT, hoping to revealthe change trend of the antigen-specific serological and IFN-γ reaction after anti-TB treatment,which provided a platform for the screening of the virulent factor in the TB active stage, andprovide theoretical basis for the selecting of TB vaccine relative antigens, At last, we constructedsubunit vaccine by combining Ag85B＋ESAT-6with cpsA and PapA2phage display proteins.Then a comprehensive analysis of the immune response from each combination group were madefrom the humoral immune response, cytokine expression levels, the number of multifunctionalcytokine expression and the proportion of multiple T cell subsets, which helped to make asystematic interpretation of the useful anti-TB host T cell subsets and also helped to provide aneffective platform and evaluation standards for the TB vaccine development.
引文
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