沙眼衣原体感染体液免疫应答谱的建立及Tarp蛋白生物学特性的研究
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摘要
沙眼衣原体(Chlamydia trachomatis,简称Ct)是一类专性细胞内寄生的原核微生物,为临床上导致失明的主要病因之一,亦是性传播性疾病(sex transmitted disease, STD)最常见的病原体,Ct感染生殖道后亦可增加HIV传播的危险性及宫颈鳞状细胞癌的发病率。虽然沙眼衣原体感染可以用抗生素治疗,但由于感染后症状常不明显,甚至没有自觉症状,因而很容易被忽视,其诊断和治疗往往被延误,造成Ct在宿主体内持续存在引起盆腔炎和不孕等多种并发症。接种疫苗将是预防和控制衣原体感染性疾病最为有效的方式。由于灭活疫苗不能给沙眼患者提供保护性作用,近十年来,大家主要致力于研究衣原体亚单位疫苗,但由于对衣原体保护性和致病性因子知识的缺乏,至今仍未有成熟的衣原体疫苗问世。从全基因组范围分析衣原体性蛋白所诱导的免疫应答,寻找Ct免疫优势抗原,将有助于筛选衣原体毒力相关因子和保护性蛋白,阐明Ct的致病机制,促进衣原体诊断试剂盒及疫苗的研制。研究目的
1.克隆表达D型Ct全基因组ORFs编码蛋白的GST融合蛋白,为Ct蛋白质组学的研究奠定基础。
2.建立Ct感染人体后体液免疫应答谱,筛选衣原体免疫优势抗原；比较活沙眼衣原体感染及死衣原体接种后抗血清的抗原识别谱,筛选衣原体感染依赖性抗原,为进一步阐明Ct感染的致病机制,Ct血清诊断试剂盒的开发及疫苗的研制提供实验依据。
研究方法
1.依据文献和互联网http://stdgen.northwestern.edu/对D型Ct全基因组ORFs进行分析,给预测的918个衣原体基因或基因片段分别设计特异性引物,采用PCR方法从D型Ct基因组中扩增获取目的基因。构建pGEX-6p-CTs原核表达重组质粒,转化大肠杆菌XL-1blue, IPTG诱导表达重组GST-CTs融合蛋白,建立D型Ct蛋白质组。
2.将衣原体蛋白质组的全部融合蛋白按照ORFs顺序分装到10块96孔微量凹孔板中,建立衣原体全基因组蛋白质阵列。收集Ct生殖道感染女性患者血清,应用ELISA法检测这些抗血清与衣原体全基因组蛋白质阵列的免疫反应性,建立沙眼衣原体感染人体后的体液免疫应答谱,并筛选出衣原体的免疫优势抗原。
3.为鉴定衣原体性蛋白与抗血清反应的特异性：①应用衣原体感染的HeLa细胞或未感染的HeLa细胞裂解物吸附抗血清,同时收集健康人血清,ELISA法分别检测这些血清与衣原体全基因组蛋白质阵列的免疫反应性；②Western blot法检测ELISA法中反应阳性的抗原与该抗血清的免疫反应性；③免疫共沉淀结合Western blot法检测抗血清能否从衣原体感染的HeLa细胞裂解物中沉淀出相应衣原体性蛋白。
4.分别应用死衣原体皮下接种新西兰大白兔,腹腔注射接种Balb/c鼠；活衣原体滴鼻感染Balb/c鼠,阴道感染Balb/c鼠及C57BL/6鼠。收集血清,ELISA法分别检测这些抗血清与衣原体全基因组蛋白质阵列的免疫反应性。①通过比较人类抗血清与兔、小鼠抗血清所识别的衣原体抗原谱,分析兔和小鼠是否适合用作制备抗衣原体性蛋白的抗体和筛选衣原体疫苗效果的动物模型；②通过比较活衣原体感染人类及死衣原体接种的动物抗血清所识别的衣原体抗原谱,鉴定衣原体感染依赖性抗原和感染非依赖性抗原。
5. Western blot法分析衣原体感染依赖性抗原和感染非依赖性抗原在衣原体感染细胞裂解物和纯化的衣原体EB、RB上的表达情况。
研究结果
1.分析得出目前全世界所预测的D型Ct基因组有918个ORFs,包括910个基因组编码基因和8个质粒编码基因。本实验成功克隆表达了908个基因的全长或片段,其中,788个ORFs克隆表达了全长基因,120个ORFs克隆表达了基因片段,共获得933个pGEX-6p-CTs原核表达质粒,并均在E.coli XL1-Blue中表达出相应的重组融合蛋白。尚有10个ORFs未能成功诱导表达出高质量的融合蛋白。它们分别是CT081, CT219, CT267, CT786, CT039.1, CT221.1, CT480.1,CT814.1,DEG02和DEG06。
2.将衣原体蛋白质组的全部GST融合蛋白按照顺序分装到10块96孔微量凹孔板中,每一块板中均有一CPAF阳性对照孔和一GST-alone阴性对照孔,建立了衣原体全基因组蛋白质阵列。
3.收集了99份Ct生殖道感染女性患者血清,应用ELISA法检测这些抗血清与衣原体全基因组蛋白质阵列的免疫反应性,建立了沙眼衣原体感染人体后的体液免疫应答谱,并筛选出27个衣原体免疫优势抗原。其中,719个衣原体抗原被99份抗血清中的一份或一份以上抗血清识别,124个抗原能被10%或10%以上抗血清识别,75个抗原能被20%或20%以上抗血清识别,50个抗原能被30%或30%以上抗血清识别,38个抗原能被40%或40%以上抗血清识别。27个抗原能被50%或50%以上抗血清识别,我们把它定义为衣原体的免疫优势抗原。这27个衣原体免疫优势抗原包括了衣原体膜蛋白：CT681,CT443,CT812; ABC转运子蛋白CT067和CT381；包涵体膜蛋白CT119,CT147,CT442,CT529和CT813；分泌到宿主细胞浆的分泌型蛋白CT858和pCT03 (Pgp3);III型分泌系统相关蛋白CT089和CT456；与代谢有关的酶及蛋白酶CT240,CT798,CT806, CT828和CT841；一些衣原体hypothetical蛋白CT143,CT101, CT694, CT142, CT695, CT875, CT795, CT022。其中11个抗原已经被报道为免疫优势抗原,尚有16个衣原体免疫优势抗原为全球首次报道,分别是：CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT456, CT695, CT798, CT806, CT828, CT841和CT875。
4.应用衣原体感染的HeLa细胞或未感染的HeLa细胞裂解物吸附抗血清,ELISA法分别检测这些血清与衣原体全基因组蛋白质阵列的免疫反应性,发现应用衣原体感染的HeLa细胞裂解物吸附免疫血清后,该血清不再识别Ct全基因组蛋白质阵列,而未感染的HeLa细胞裂解物吸附抗血清后其与Ct全基因组蛋白质阵列的反应性与吸附之前相似。健康人血清亦不与Ct全基因组蛋白质阵列发生反应。结果提示：衣原体感染患者抗血清与衣原体全基因组蛋白质阵列的反应是抗原抗体特异性反应。
5. Western blot法检测ELISA法中反应阳性的抗原与抗血清的免疫反应性,发现大多数抗原Western blot法检测仍然为反应阳性,少数抗原虽在ELISA法检测时反应阳性,但在Western blot法检测时却为反应阴性。免疫共沉淀实验结果显示该抗血清能将ELISA法检测阳性而Western blot法检测阴性的蛋白从感染的HeLa细胞裂解物中沉淀下来,从而证实该抗原是构像依赖性抗原。该结果表明本实验中用到的Ct全基因组蛋白质阵列结合ELISA法检测衣原体抗原组既能检测线性抗原,还能鉴定构像依赖性抗原,因此该方法优于Western blot法。27个衣原体免疫优势抗原中共有10个为构像依赖性抗原,分别是：GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828和GST-pCT03 (Pgp3)。
6.①通过比较人、兔和小鼠三个种属所识别的衣原体抗原,发现许多人类优势识别的抗原亦能被2种啮齿目动物强烈识别,表明啮齿目动物可以被用于研究衣原体引起的免疫反应和评估衣原体疫苗候选抗原的保护作用。②比较活衣原体感染的Balb/c鼠和C57BL/6鼠识别衣原体抗原的数目,发现滴鼻感染和阴道感染的Balb/c鼠均识别54个衣原体抗原,而阴道感染的C57鼠仅能识别27个抗原,死衣原体接种的Balb/c鼠识别29个抗原,提示宿主识别的抗原数与感染的严重程度正相关。③活衣原体感染个体所识别的抗原与灭活衣原体接种个体抗血清所识别的抗原差别尤为显著。人类抗血清识别的719个抗原中,563个抗原不能被死衣原体接种的兔或鼠抗血清识别,为感染依赖性抗原；156个抗原能被死衣原体接种的兔或鼠血清识别,为感染非依赖性抗原。
7. Western blot法分析衣原体感染依赖性抗原和感染非依赖性抗原在衣原体感染细胞裂解物和纯化的衣原体EB、RB上的表达情况。发现大多数感染依赖性抗原主要表达在感染细胞裂解物中,而在EB上表达很少,提示这些蛋白可能为分泌性蛋白,RB合成该蛋白后很快将其分泌至包涵体腔或宿主细胞浆。
结论
1.D型Ct全基因组共预测有918个ORFs。
2.成功地将788个Ct基因的全长和120个Ct基因的片段克隆到pGEX-6p载体中,构建了933个pGEX-6p-CTs原核表达质粒。
3.成功表达了908个衣原体基因全长或片段,制备了933个重组GST-CTs融合蛋白,建立了D型Ct蛋白质组。并建立了D型Ct全基因组蛋白质阵列。
4.建立了Ct生殖道感染女性患者中衣原体B细胞抗原组,成功建立了沙眼衣原体感染后体液免疫应答谱,鉴定出Ct27个免疫优势抗原,全球首次报道16个新的衣原体免疫优势抗原,分别是：CT022,CT067,CT101,CT142,CT143,CT240,CT381,CT442,CT443,CT456,CT695,CT798,CT806,CT828,CT841和CT875。
5.Ct全基因组蛋白质阵列结合ELISA法检测衣原体抗原组既能检测线性抗原,还能鉴定构像依赖性抗原。27个衣原体免疫优势抗原中有10个为构像依赖性抗原,分别是：GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCTO3 (Pgp3)。
6.首次系统的鉴定出563个Ct感染依赖性抗原。在38个能被40%或40%以上患者血清识别的衣原体抗原中,共有14个衣原体感染依赖性抗原,分别是：CT089, CT116, CT142, CT153, CT228, CT442, CT529, CT694, CT798, CT806, CT813, CT828, CT858, CT866。
研究目标
1.进一步分析衣原体Ⅲ型分泌系统相关蛋白与沙眼衣原体生殖道感染患者(sexually transmitted infection,简称STI)患者和沙眼患者抗血清的免疫反应性。
2.制备抗Ⅲ型分泌系统效应蛋白Tarp (CT456)的单克隆抗体。
3.研究Tarp蛋白在衣原体感染HeLa细胞中的表达特征。
4.评估Tarp蛋白重组疫苗的免疫保护效果。
5.鉴定Tarp蛋白的免疫优势区。
研究方法
1.通过互联网搜索目前已报道的CtⅢ型分泌系统相关蛋白。
2.收集8份沙眼和24份Ct生殖道感染患者血清,ELISA法进一步分析CtⅢ型分泌系统相关蛋白的免疫反应性。应用衣原体感染的HeLa细胞或未感染的HeLa细胞裂解物吸附抗血清,同时收集健康人血清,ELISA法分别检测这些血清与衣原体III型分泌系统相关蛋白的免疫反应性以鉴定衣原体性蛋白与抗血清反应的特异性。
3.采用杂交瘤技术制备抗Tarp蛋白的单克隆抗体,间接免疫荧光法筛选阳性克隆,ELISA法鉴定单克隆抗体的特异性,间接免疫荧光法鉴定单克隆抗体的亚类和衣原体种属特异性。
4.间接免疫荧光法检测Tarp蛋白在衣原体感染HeLa细胞中的表达特征。Western blot法检测Tarp蛋白在纯化的EB、RB、D型Ct感染的HeLa细胞裂解液中的表达。
5. Tarp蛋白重组疫苗免疫保护效果的评估：
①克隆表达Ct鼠肺炎型(MoPn) Tarp同源体(TC0741) GST融合蛋白,用PreScissionTM蛋白酶酶切去除GST而获得纯的TC0741重组蛋白；
②建立Balb/c小鼠MoPn生殖道感染模型,分别收集感染小鼠血清和脾细胞,ELISA法检测内源性Tarp蛋白诱导的特异性抗体和T细胞应答。
③TC0741重组蛋白免疫小鼠,分别收集小鼠血清和脾细胞,ELISA法检测外源性Tarp蛋白诱导的特异性抗体的滴度及抗体亚类,间接免疫荧光法检测抗体的特异性；ELISA法检测MoPn刺激脾细胞后培养上清中IFN-γ、IL-4和IL-5的水平。
④以TC0741重组蛋白为免疫原,结合CPG和不完全弗氏佐剂(IFA),采用肌肉注射方式接种Balb/c小鼠,同时设MoPn+CPG+IFA阳性对照组和单纯的CPG+IFA阴性对照组,再以MoPn生殖道攻击小鼠,间接免疫荧光法检测小鼠阴道分泌物中MoPn的IFU值以判断小鼠清除MoPn感染的速度；解剖分离小鼠生殖道组织,H&E(苏木精和曙红)染色法检测小鼠上生殖道组织病理学变化。
6.克隆表达D型Ct Tarp蛋白11个片段的GST融合蛋白,ELISA法检测这11个片段与Ct生殖道感染患者、沙眼患者、兔免疫血清,以及抗Tarp单克隆抗体的免疫反应性,进一步鉴定Tarp蛋白的免疫优势区,为进一步探讨Tarp的生物学功能和保护性作用提供实验依据。
研究结果
1.通过互联网搜索,目前已报道的衣原体III型分泌系统相关蛋白主要有Inc家族蛋白及另外约49个衣原体蛋白。应用ELISA法检测CtIII型分泌系统相关蛋白的免疫反应性,结果显示STI患者和沙眼患者血清均能优势识别D型Ct CT456 (Tarp)、CT089和CT858重组蛋白,其中Tarp蛋白在沙眼患者血清中识别频率更高达100%,其识别频率和平均OD均高于其他蛋白,甚至高于CPAF。尽管D型Ct Inc家族免疫优势蛋白CT119,CT529,CT813和主要外膜蛋白MOMP能被STI患者血清优势识别,但在沙眼患者血清中的识别频率却显著低于STI患者血清。
2.采用杂交瘤技术获得9株稳定分泌抗Tarp蛋白单克隆抗体的杂交瘤细胞株,分别命名为：R4D5,N5B11,R5B6.2,R2H1.2,R12B12, R2H7, R5G8.1, R8G7.2和M4F4。9个mAbs全部为IgG, R5B6.2和R8G7.2为IgG2a, R4D5, R2H1.2, R12B12, R2H7, N5B11, M4F4和R5G8.1均为IgG1。间接免疫荧光法染色结果显示9个mAbs均能特异性的着色Ct血清型A、D、L2,而不着色MoPn、6BC和AR39。
3.间接免疫荧光法检测Tarp蛋白在衣原体感染HeLa细胞上的表达特征,结果显示：在衣原体感染后8h内可见Tarp表达,在13h时Tarp表达消失。24h衣原体包涵体己经非常明显,但仍不能检测到Tarp的表达。直到28h时重新检测到少量Tarp,而且Tarp的表达量随感染时间的进一步延长而逐渐增加。Western blot法检测Tarp蛋白在纯化的EB、RB、HeLa-D裂解液中的表达,发现Tarp主要在纯化的EB和HeLa-D裂解液中表达,而在RB上则不表达。结果提示Tarp蛋白是一个衣原体EB相关蛋白。
4.以MoPn基因组为模板,PCR扩增得到了TC0741基因全长片段,构建了pGEX-6p-TC0741重组质粒；成功表达了GST-TC0741融合蛋白；用PreScissionTM蛋白酶酶切去除GST而获得了纯的TC0741重组蛋白。
5. MoPn感染小鼠血清中产生了高滴度的抗MoPn和抗Tarp的特异性抗体,该血清能识别MoPn抗原和GST-Tarp融合蛋白,但不能识别单纯的GST蛋白。MoPn感染小鼠的脾细胞经Tarp蛋白和MoPn刺激后诱导产生了高浓度的IFN-γ,且IFN-γ的浓度随着Tarp蛋白剂量加大而增高,而GST蛋白却不能刺激感染小鼠的脾细胞产生IFN-γ。各组脾细胞培养上清中基本检测不到IL-4和IL-5的产生。结果提示MoPn感染小鼠后内源性Tarp诱导产生了Tarp特异性体液和细胞免疫应答。
6.给小鼠接种Tarp蛋白+佐剂以评估外源性Tarp蛋白诱导的免疫应答。间接免疫荧光实验检测发现接种MoPn+佐剂和Tarp蛋白+佐剂的小鼠抗血清能使MoPn包涵体染色,而仅接种佐剂的小鼠抗血清却不能使MoPn包涵体染色。MoPn感染HeLa细胞的裂解物能中和MoPn+佐剂组和Tarp蛋白+佐剂组小鼠免疫血清中抗MoPn抗体,经预吸附后,两组血清均不再使MoPn包涵体染色。GST-Tarp蛋白预吸附Tarp蛋白+佐剂组血清后,该血清不再染色MoPn包涵体,但GST-Tarp蛋白预吸附MoPn+佐剂组血清后,该血清仍继续染色MoPn包涵体。ELISA法检测小鼠免疫血清中抗MoPn特异性抗体的滴度和亚类。MoPn+佐剂组和Tarp蛋白+佐剂组小鼠抗血清中产生了高滴度的抗MoPn抗体,而单纯佐剂组血清中则没有抗MoPn抗体产生。MoPn+佐剂组和Tarp蛋白+佐剂组小鼠抗血清中IgG2a的滴度均高于IgG1,即IgG2a/IgGl>1。MoPn+佐剂和Tarp蛋白+佐剂组脾细胞培养上清中IFN-γ的浓度显著高于单纯佐剂组。各组脾细胞培养上清中基本检测不到IL-4和IL-5的产生。结果提示Tarp重组疫苗能够诱导小鼠产生MoPn特异性细胞免疫应答和体液免疫应答,以Thl细胞应答为主。
7.间接免疫荧光法检测小鼠阴道分泌物中MoPn的IFU值以判断小鼠清除MoPn感染的速度,发现单纯接种佐剂组中,在感染后27天阴道分泌物中仍可检测到衣原体。MoPn+佐剂组在感染后第6天,小鼠阴道分泌物中MoPn的IFU值显著低于单纯接种佐剂组和Tarp+佐剂组(P<0.05),第15天已基本检测不到MoPn。Tarp+佐剂组在感染后第21天,小鼠阴道分泌物中MoPn的数量显著低于单纯接种佐剂组(P<0.05),第24天已完全检测不到MoPn,提示此时Tarp+佐剂组小鼠生殖道中MoPn已被完全清除。结果表明：Tarp重组疫苗诱导产生的免疫反应有利于小鼠生殖道内衣原体感染的清除。
8.小鼠生殖道组织大体观察发现Tarp+佐剂组(P<0.05)和MoPn+佐剂组(P<0.01)输卵管双侧积水的发生率均显著小于单纯佐剂组。小鼠生殖道组织切片H&E染色评估炎症的严重程度和生殖道管腔扩张程度结果显示：三组中小鼠子宫角组织的炎症积分和管腔扩张积分情况没有显著差异(P>0.05),而在输卵管部位,Tarp+佐剂组和MoPn+佐剂组的炎症积分和管腔扩张积分均显著低于单纯佐剂接种组(P<0.01)。结果提示Tarp重组疫苗能防止小鼠上生殖道的炎性病理改变,具有显著的保护作用,因此,Tarp是一个有前景的衣原体疫苗候选抗原。
9.成功克隆表达了11个Tarp片段的GST融合蛋白。ELISA法检测这11个片段与Ct生殖道感染患者、沙眼患者、兔免疫血清,以及抗Tarp单克隆抗体的免疫反应性,发现所有Tarp反应阳性抗血清均能识别氨基酸从152～302的区域；STI抗血清和兔抗血清能同时识别氨基酸1-156、310-431和582～682区域；只能被STI抗血清识别的是氨基酸从425-581区域；只能被兔抗血清识别的是氨基酸683-847区域。6个抗Tarp单克隆抗体识别氨基酸152～302的区域,另3个单克隆抗体分别识别氨基酸1～119、120-151,和310～431的区域。
结论
1.Tarp蛋白在STI患者和沙眼患者中均为免疫优势抗原。
2.成功建立了9株稳定分泌抗Tarp蛋白的单克隆抗体杂交瘤细胞株。
3.Tarp是一个衣原体EB相关蛋白。
4.接种Tarp诱导产生了以Thl为主的免疫应答。Tarp重组疫苗与CPG+不完全弗氏佐剂联合免疫在实验小鼠内发挥一定的保护作用,有利于小鼠生殖道内衣原体的清除,显著降低小鼠上生殖道组织的炎性病理改变。Tarp可作为一种新的潜在靶抗原用于衣原体疫苗的研制。
5.氨基酸从152到302的区域,是Tarp蛋白最强的免疫优势区。
Abstract
Chlamydia trachomatis is an obligate intracellular prokaryotic parasite which is the leading cause of preventable blindness and the most prevalent bacterial pathogen causing sexually transmitted disease in clinic. Although chlamydial infection is susceptible to antibiotics treatment, many urogenitally infected individuals don't seek treatment due to lack of obvious clinic symptoms, thus, becoming vulnerable to developing persistent infection and long-term sequelae including ectopic pregnancy and infertility. Vaccination is considered the most effective means for preventing chlamydial infection and diseases. However, the inactivated whole organism-based vaccines failed in human trachoma vaccine trials. Thus, extensive efforts were made to develop effective and safe subunit vaccines in past decades. However, there is still no licensed C. trachomatis vaccine probably due to insufficient knowledge on protective and pathogenic determinants of chlamydial organisms. A genome-wide profiling of the immune response to Chlamydia trachomatis infection and screening immundominant antigens will contribute to identify protective and pathogenic determinants of chlamydial organisms, to clarify chlamydial pathogenesis, and to develop a serum diagnosis kit or a chlamydial vaccine. PartⅠA Genome-Wide Profiling of the Humoral Immune Response to Chlamydia trachomatis Infection in Humans
Objectives
1. Cloning and expression of GST fusion proteins encoded by Chlamydia trachomatis serovar D genome-wide ORFs, to lay the foundation for study on Chlamydial proteomics.
2. To establish the humoral immune response profile to Chlamydia trachomatis infection in humans and screen immundominant antigens; to identify infection-dependent antigens by comparing antigen profiles recognized by live chlamydial organism-infected versus dead organism-immunized hosts. To provide the experimental evidence for clarifying chlamydial pathogenesis, and developing a serum diagnosis kit or a chlamydial vaccine.
Methods
1. The informations of all Chlamydia trachomatis serovar D ORFs were analyzed according to the publised papers and sequences on website http://stdgen.northwestern.edu/. Specific primers of 918 chlamydial genes full length or fragments were synthesized according to the gene sequences. PCR was used to amply the target genes from Chlamydia trachomatis serovar D genomic DNA. The pGEX-6p-CTs expression plasmids were constructed and been transformated into E.coli XL-1 blue. The expression of recombinant Ct proteins were induced by IPTG. The proteome of Chlamydia trachomatis serovar D was established.
2. All the bacterial lysates containing the chlamydial fusion proteins were added to the 96 well microplates in ORF order for setting up the whole genome scale protein array. Serum samples were collected from patients which were urogenitally infected with Chlamydia. The ELISA was carried out to detect the reactivity of STI antisera with the whole genome scale protein array. The humoral immune response profile to Chlamydia trachomatis infection in humans was established and the chlamydial immunodominant antigens were identified.
3. To identify the specificity of the reactivity of the antisera with chlamydial proteins:①Sera from healthy female individuals (without C. trachomatis infection) were harvest and used as negative controls and the reactivities of these sera with the whole genome scale protein array by ELISA. All positively detected antigens were re-measured with the same antibody samples with or without further absorption with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells;②The reactivity of all positively detected antigens were re-measured by Western blot;③The immunoprecipitation and Western blot assays were carried out to detect whether the same antisera can precipitate endogenous chlamydial antigens from chlamydia-infected HeLa cell lysates under a non-denaturing condition.
4. To produce antibodies to the whole chlamydial organisms, rabbits or mice were immunized with UV-inactivated C. trachomatis serovar D organisms; Rabbits were injected intramuscularly while Balb/c mice intraperitoneally. Balb/c mice were infected with live chlamydia EB intranasally. Balb/c and C57BL mice were infected with live chlamydia EB intravaginally. All the antisera were harvested and the reactivity with the whole genome scale protein array by ELISA.①We compared the human-recognized C. trachomatis antigens with those detected by antisera from rabbits and different strains of mice to identify whether these hosts are appropriate for using to produce antibodies to C. trachomatis antigens and/or identify/evaluate chlamydial vaccine candidate antigens;②We compared the antigen profiles recognized by live organism-infected humans versus dead organism-immunized animals to identify infection-dependent antigens and infection-independent antigens.
5. The expressiong of infection-dependent antigens and infection-independent antigens on whole cell lysates samples with or without human antibody precipitation along with the purified chlamydial RB and EB organisms were detected by an Western blot.
Results
1. We found that there were 918 predicted ORFs encoded by C. trachomatis serovar D genome and plasmid.In the current study,908 genes were cloned,788 ORFs were expressed in full length and 120 in one or more fragments. Totally,933 pGEX-6p-CTs were constructed and all of them were expressed as GST fusion proteins in E.coli XL1-Blue. The remaining 10 ORFs were not expressed good quality fusion proteins despite our best efforts. These difficult ORFs include CT081, CT219, CT267, CT786, CT039.1, CT221.1, CT480.1, CT814.1, DEG02 and DEG16.
2. All the Chlamydia GST fusion proteins were arrayed onto ten 96 well microplates in ORF order. To minimize the plate to plate variations, each plate was included with a GST-alone negative control well and CPAF positive control well. Thus,the chlamydial whole genome scale protein array was set up.
3. Antisera from 99 women urogenitally infected with C. trachomatis were collected. The reactivity of these antisera with the chlamydial whole genome scale protein array was detected by ELISA. The humoral immune response profile to Chlamydia trachomatis infection in humans was set up and 27 chlamydial immunodominant antigens were identified. 719 out of the 908 ORF-encoded chlamydial proteins were recognized by at least one of the 99 human antisera. Among the 719 antigens,124 were recognized by 10%,75 by 20%,50 by 30% and 38 by 40% or more antisera respectively. The 27 proteins recognized by 50% or more antisera were designated as immunodominant antigens. These 27 immunodominant antigens cover a wide range of proteins including those localized in the organism membrane such as CT681, CT443, CT812 and the ABC transporters CT067 and CT381, proteins in the inclusion membrane such as CT119, CT147, CT442, CT529 and CT813, and proteins secreted into host cell cytosol such as CT858 and pCT03. In addition, some typeⅢsecretion system- related proteins such as CT089 and CT456, metabolic enzymes and proteases such as CT240, CT798, CT806, CT828 and CT841as well as various hypothetical proteins are also among the immunodominant antigens. Although 11 of the 27 antigens were previously reported to be immunodominant, the remaining 16 proteins represent the newly discovered immundominant antigens in the current study, including CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT456, CT695, CT798, CT806, CT828, CT841 and CT875.
4. The reactivity of positively reacted antigens were re-measured with the same antibody samples with or without further absorption with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells. The results showed that the absorption with chlamydia-infected HeLa lysates blocked antibody binding to all fusion proteins while a similar absorption with HeLa alone lysates failed to do so. No significant reactivity of the serum samples from healthy individuals was found. These observations have demonstrated that human antibody binding to the whole genome scale protein array is specific to chlamydial antigens.
5. The reactivity of all positively detected antigens were re-measured by Western blot and the results showed that Most of the chlamydial GST fusion proteins identified by this antiserum in the proteome array ELISA were also detected by the antiserum in Western blot. However, some antigens were not detected by the serum sample in the Western blot or only minimally detected and the intensity of their reactivity with the human antiserum on the Western blot was incompatible with the OD values. The immunoprecipitation and Western blot assays showed that the antigens which positively reacted in ELISA but not in Western blot were successfully precipitated by the same human antiserum and the amounts of antigens precipitated relative to the amounts of antigens available in the infected cell lysates were largely consistent with the corresponding OD values detected in the proteome array ELISA. Thus, these chlamydial proteins were conformation-dependent antigens. The results demonstrated that our fusion protein-based whole genome scale proteome array ELISA approach could detected both linear and conformation-dependent antigens, so that our method was advantage over the Western blot approaches. Among the 27 immunodominant antigens identified using our proteome array with human sera,10 were not or only minimally detected in Western blot,they were GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCT03 (Pgp3).
6.①As for the types of antigens recognized by the 3 host species, they were human,rabbit and mouth,we found that many antigens dominantly recognized by humans were also strongly recognized by the two rodents species,suggesting that rodents can be used to study chlamydial Immunology and evaluate the protection efficacy of chlamydial vaccine candidate antigens.②A more careful analysis of antigens recognized by the live Chlamydia infected Balb/c and C57BL mice revealed that Intranasal (in) or intravaginal (iv) infection of Balb/c mice both induced antibodies to 54 chlamydial antigens respectively while intravaginal infection of C57BL mice induced antibodies to 27 antigens only, suggestion that a correlation between the number of antigens recognized by the host and the severity of infection.③There were especially significant differences in the types of antigens recognized by antisera from live organism infection and dead organism immunization individuals. When the 719 antigens recognized by human antibodies were resorted based on their recognition by the immunized rabbits and mice, we found that 563 of the 719 human-recognized antigens were not detected by either rabbits or mice, thus classified as infection-dependent antigens, while the remaining 156 as infection-independent antigens since they were recognized by the dead organism-immunized animals.
7. The expressiong of infection-dependent antigens and infection-independent antigens on whole cell lysates samples with or without human antibody precipitation along with the purified chlamydial RB and EB organisms were detected by an Western blot. The results showed that many infection-dependent antigens were detected abundantly in infected whole cell lysates but only minimally in purified EBs, suggesting that all infection-dependent antigens are produced during live infection and many may not be incorporated into the infectious particles.
Conclusions
1. There were 918 predicted ORFs of all Chlamydia trachomatis serovar D genome.
2.788 Chlamydia trachomatis gene full length and 120 gene fragments were successfully cloned into pGEX-6p vector,933 pGEX-6p-CTs plasmids were constructed.
3.908 chlamydial ORFs full length or fragments were successfully expressed and 933 recombinant GST-CTs fusion proteins,Thus Chlamydia trachomatis serovar D proteome. A whole genome scale proteome array of Chlamydia trachomatis serovar D was set up.
4. The B cell ANTIGENome of women urogenitally infected with C. trachomatis were mapped and the humoral immune response profile to Chlamydia trachomatis infection in humans was established.27 chlamydial immunodominant antigens were identified, including 16 proteins were the newly discovered immundominant antigens in the current study, including CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT695, CT798, CT806, CT828, CT841 and CT875.
5. Our fusion protein-based whole genome scale proteome array ELISA approach could detected both linear and conformation-dependent antigens. Among the 27 immunodominant antigens identified using our proteome array with human sera,10 were conformation-dependent antigens, they were GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCT03 (Pgp3).
6. Newly systematically identified 563 Chlamydia trachomatis infection-dependent antigens.14 of the 38 antigens which were recognized by(?)40% patients sera were infection- dependent antigens,they are CT089, CT116, CT142, CT153, CT228, CT442, CT529, CT694, CT798, CT806, CT813, CT828, CT858, CT866.
Objectives
1. The reactivity of TypeⅢSecretion System associated proteins with tients rogenitally infected with C. trachomatis (sexually transmitted infection, TI) and trachoma patients were further detected.
2. The mAbs against TypeⅢSecretion System associated protein Tarp (CT456),which was an immnuodominant antigen,were prepared.
3. The expression of Tarp protein in Chlamydia infected HeLa cells were detected.
4. Protection efficacy of Tarp protein recombinant vaccine was estimated.
5. The immunodominant regions of Tarp protein were identified.
Methods
1. Searching for the reported Chlamydia trachomatis typeⅢsecretion system associated proteins in the internet.
2. Antisera from 24 women urogenitally infected with C. trachomatis and 8 trachoma patients were collected. The reactivity of these antisera with the chlamydial typeⅢsecretion system associated proteins was further detected by ELISA. To identify the specificity of the reactivity of the antisera with chlamydial proteins, sera from healthy female individuals (without C. trachomatis infection) were harvest and the patient sera were preabsorpted with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells, the reactivities of these sera with the t3ssassociated proteins by ELISA.
3. Hybridoma technique was carried out to prepare mAbs against Tarp protein. The specificities to these monoclonal antibodies were determined by ELISA. The isotype and chlamydial species specificity of these monoclonal antibodies were determined by an indirect immunofluores-cence assay.
4. The expression of Tarp protein in Chlamydia infected HeLa cells were detected by an indirect immunofluorescence assay. The expression of Tarp protein in purified EB, RB and Chlamydia infected HeLa cells lysate were detected by Western blot.
5. Evaluation of the immune protection efficacy of Tarp protein recombinant vaccme:
①GST fusion protein of Chlamydia muridarum Tarp(ORF TC0471)was cloned and expressed.The GST-TC0741 protein were cleavaged with a precision protease and freed TC0741 recombinant protein was made.
②Mopn urogenitally infected Balb/c mouse model was set up. The endogenous Tarp-specific antibody and T cell response were measured by ELISA after the antisera and spleen cells of infected mice were collected respectively.
③Mice were immunized with recombinant TC0741 protein,mouse antisera and spleen cells were collected respectively. The exogenous Tarp-specific antibody production and isotypes were detected by ELISA. The specific of anti-MoPn antibody were detected by an indirect immunofluorescence assay. The levels of IFN-γ、IL-4 and IL-5 were detected from the spleen cell culture supernants after the mouse spleen cells were re-stimulated with MoPn antigens by ELISA.
④Balb/c mice were immunized with recombinant TC0741 protein emulsified in the CpF-IFA agjuvant via intramuscular injection, The intact MoPn organisms similarly emulsified in the same adjuvant were used as a positive control, adjuvant alone as a negative control. Thirty days after the immunization, the mice were intravaginally challenged with MoPn. To test the clearance of MoPn infection, the IFU values of MoPn organism shedding were monitored by an indirect immunofluorescence assay.The mouse urogenital tract tissues were isolated and the Histopathological changes were assessed by an H&E(hematoxylin and eosin) dyeing method.
6. To map the immunodominant regions of Tarp protein and provide important information for further understanding the biological function and the protective immunity of Tarp,11 fragments of Tarp were cloned and expressed as GST fusion proteins and the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms and with mAbs against Tarp protein were detected by ELISA.
Results
1. We found that chlamydial typeⅢsecretion system associated proteins was predicted to consist of Inc proteins and other 49 chlamydial proteins or so by searching in the internet.The reactivities were detected by ELISA and the results showed that CT456 (Tarp)、CT089 and CT858 recombinant proteins were immunodominantly recognized by both STI and trachoma antisera,the reactivity ot Tarp even stronger than that of CPAF with 100% recognition frequency and the highest average OD value. The trachoma antisera can't predominantly recognize the three Inc proteins CT119,529 and 813despite the fact that they are immunodominant during human urogenital infection with C. trachomatis. MOMP from C. trachomatis serovar D was lack of recognition by trachoma antisera.
2. Nine hybridoma cell lines stable in secreting specific against Tarp protein monoclonal antibodies were successfully obtained, they were R4D5, N5B11, R5B6.2, R2H1.2, R12B12, R2H7, R5G8.1, R8G7.2 and M4F4. All the 9 mAbs were IgG,2 mAbs (R5B6.2 and R8G7.2) were belong to IgG2a isotype and the other 7 mAbs (R4D5, R2H1.2, R12B12, R2H7, N5B11, M4F4 and R5G8.1) were belong to IgGl isotype. Results of indirect immunofluorescence assay showed that all the 9 mAbs can react strongly with chlamydia serovar A,D,and L2, but not MoPn,6BC,and AR39.
3. The expression of Tarp protein in Chlamydia infected HeLa cells were detected by an indirect immunofluorescence assay. The results showed that:Tarp was detected in the first 8 hours and no longer detectable by 13 hours after infection. Although chlamydial inclusions became very obvious at 24 hours after infection, Tarp remained undetectable. However, by 28 hours after infection, Tarp started to reappear and progressively increased as the incubation continued. Tarp protein was only detected in Chlamydia trachomatis serovar D infected HeLa cell lysates and purified EBs but not RBs on a Western blot. Our results suggesting that Tarp protein was an EB associated protein.
4. TC0471 full length sequence was amplified by PCR from Chlamydia muridarum genomic DNA and pGEX-6p-TC0741 plasmid was constructed; GST-TC0741 fusion protein was successfully expressed; freed TC0741 recombinant protein was made by cleavaged the GST-TC0741 protein with a precision protease.
5. The MoPn infected mice developed high titers of antibodies that recognized both MoPn-derived antigens and Tarp fusion proteins but not the fusion tag GST alone. High levels of IFN-y were induced after the spleen cells from MoPn infected mice were re-srimulated with either Tarp protein or MoPn. The levels of IFN-ywere increased with the amount of Tarp protein. No IL-4 and only minimum levels of IL-5 were detected from the spleen cell culture supernatants of the MoPn infected mice. These observations together have demonstrated that mice infected with MoPn developed Tarp-specific humoral and cellular immune responses.
6. The immune response induced by exogenous Tarp protein was evaluated after mice were immunized with recombinant TC0741 protein emulsified with adjuvant. The antisera induced by MoPn or Tarp but not adjuvant alone visualized antigens in MoPn inclusions in animmunofluorescence assay. the anti-Tarp antibody labeling was blocked by either the immunogen GST-Tarp or MoPn-infected cell lysates while the anti-MoPn antibody labeling was effectively blocked by MoPn-infected cell lysates. We further quantitated the titers of the MoPn antigen-reactive antibodies and isotyped the mouse IgG antibodies in an ELISA Immunization with either MoPn or Tarp with adjuvant but not adjuvant alone induced high titers of antibodies that specifically recognized MoPn endogenous antigens. Immunization with either Tarp or MoPn induced more IgG2a than IgG1,that is IgG2a/IgG1>1. The spleen cells from mice immunized with either MoPn or Tarp produced significantly higher levels of IFN than those from adjuvant alone immunization. No IL-4 and only minimum levels of IL-5 were detected from the spleen cell culture supernatants of the immunized mice. These results further strengthened the conclusion that immunization with Tarp induced a MoPn-specific cellular and humoral immune responses, it was a Thl-dominant immune response.
7. To test the clearance of MoPn infection, the IFU values of MoPn organism shedding were monitored by an indirect immunofluorescence assay. Live MoPn organisms were detected in the vaginal swabs collected from the adjuvant alone group of mice up to 27 days after infection. Immunization with the intact MoPn organisms shortened the infection time course to 15 days with a significant reduction in the infectious titers from the vaginal swabs within 6 days after infection. Importantly, immunization with Tarp resulted in a significant reduction in the vaginal shedding of live organisms on day 21 and most of the Tarp-immunized mice cleared infection by day 24 post infection. Thus, Tarp-induced immunity enhanced the resolution of MoPn infection from the mouse lower genital tracts although not as potently as the intact organism-induced immunity.
8. Gross appearance assessment showed that the incidence of bilateral hydrosalpinx in Tarp-immunized (33%) or MoPn-immunized (10%) mice is significantly lower than that in the adjuvant alone-immunized mice(70%). After, we further evaluated the severity of inflammation microscopically using histology sections. Both the inflammatory cell infiltration and luminal dilatation were blindly semi-quantitated. There was no significant difference in either inflammation or lumenal dilatation scores in the uterine horn tissues between the three groups of mice. However, mice immunized with either Tarp or MoPn displayed significantly lower scores in both inflammatory cell infiltration and lumenal dilatation than the control group of mice, suggesting that the immunity induced by either Tarp or MoPn can reduce the inflammatory damages in the mouse oviducts.
9.11 fragments of Tarp were successfully expressed as GST fusion proteins and the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms and with mAbs against Tarp protein were detected by ELISA. A major immunodominant region was strongly recognized by all antibodies. This region covers amino acids 152 to 302, which consist of three repeats (152-201,202-251 and 252-302). Several other minor immunodominant regions were also identified, including 1-156,310-431 and 582-682, recognized by antisera from both human and rabbit; 425-581, only recognized by human antisera; and 683-847, preferentially recognized by rabbit antisera. This immunodominance was also confirmed by the observation that six out of the nine mAbs bound to the major immunodominant region and that the other three each bound to one of the minor fragments,1-119,120-151 and 310-431.
Conclusions
1. Tarp protein was an immunodominant antigen not only in STI patient but also in trachoma patient.
2. Nine hybridoma cell lines stable in secreting specific against Tarp protein monoclonal antibodies were successfully set up.
3. Tarp was a chlamydial EB associated protein.
4. Immunization of mice with Tarp induced Thl-dominant immunity that significantly reduced the shedding of live organisms from the lower genitaltract and attenuated inflammatory pathologies in the fallopian tube tissues. These observations have demonstrated that Tarp can induce protective immunity against chlamydial infection and pathology in mice,it cab be used as a novel promising chlamydia vaccine candidate antigen.
5. The region covers amino acids 152 to 302 was the most immunodominant region of Tarp protein.
1.克隆表达D型Ct全基因组ORFs编码蛋白的GST融合蛋白,为Ct蛋白质组学的研究奠定基础。
2.建立Ct感染人体后体液免疫应答谱,筛选衣原体免疫优势抗原；比较活沙眼衣原体感染及死衣原体接种后抗血清的抗原识别谱,筛选衣原体感染依赖性抗原,为进一步阐明Ct感染的致病机制,Ct血清诊断试剂盒的开发及疫苗的研制提供实验依据。
研究方法
1.依据文献和互联网http://stdgen.northwestern.edu/对D型Ct全基因组ORFs进行分析,给预测的918个衣原体基因或基因片段分别设计特异性引物,采用PCR方法从D型Ct基因组中扩增获取目的基因。构建pGEX-6p-CTs原核表达重组质粒,转化大肠杆菌XL-1blue, IPTG诱导表达重组GST-CTs融合蛋白,建立D型Ct蛋白质组。
2.将衣原体蛋白质组的全部融合蛋白按照ORFs顺序分装到10块96孔微量凹孔板中,建立衣原体全基因组蛋白质阵列。收集Ct生殖道感染女性患者血清,应用ELISA法检测这些抗血清与衣原体全基因组蛋白质阵列的免疫反应性,建立沙眼衣原体感染人体后的体液免疫应答谱,并筛选出衣原体的免疫优势抗原。
3.为鉴定衣原体性蛋白与抗血清反应的特异性：①应用衣原体感染的HeLa细胞或未感染的HeLa细胞裂解物吸附抗血清,同时收集健康人血清,ELISA法分别检测这些血清与衣原体全基因组蛋白质阵列的免疫反应性；②Western blot法检测ELISA法中反应阳性的抗原与该抗血清的免疫反应性；③免疫共沉淀结合Western blot法检测抗血清能否从衣原体感染的HeLa细胞裂解物中沉淀出相应衣原体性蛋白。
4.分别应用死衣原体皮下接种新西兰大白兔,腹腔注射接种Balb/c鼠；活衣原体滴鼻感染Balb/c鼠,阴道感染Balb/c鼠及C57BL/6鼠。收集血清,ELISA法分别检测这些抗血清与衣原体全基因组蛋白质阵列的免疫反应性。①通过比较人类抗血清与兔、小鼠抗血清所识别的衣原体抗原谱,分析兔和小鼠是否适合用作制备抗衣原体性蛋白的抗体和筛选衣原体疫苗效果的动物模型；②通过比较活衣原体感染人类及死衣原体接种的动物抗血清所识别的衣原体抗原谱,鉴定衣原体感染依赖性抗原和感染非依赖性抗原。
5. Western blot法分析衣原体感染依赖性抗原和感染非依赖性抗原在衣原体感染细胞裂解物和纯化的衣原体EB、RB上的表达情况。
研究结果
1.分析得出目前全世界所预测的D型Ct基因组有918个ORFs,包括910个基因组编码基因和8个质粒编码基因。本实验成功克隆表达了908个基因的全长或片段,其中,788个ORFs克隆表达了全长基因,120个ORFs克隆表达了基因片段,共获得933个pGEX-6p-CTs原核表达质粒,并均在E.coli XL1-Blue中表达出相应的重组融合蛋白。尚有10个ORFs未能成功诱导表达出高质量的融合蛋白。它们分别是CT081, CT219, CT267, CT786, CT039.1, CT221.1, CT480.1,CT814.1,DEG02和DEG06。
2.将衣原体蛋白质组的全部GST融合蛋白按照顺序分装到10块96孔微量凹孔板中,每一块板中均有一CPAF阳性对照孔和一GST-alone阴性对照孔,建立了衣原体全基因组蛋白质阵列。
3.收集了99份Ct生殖道感染女性患者血清,应用ELISA法检测这些抗血清与衣原体全基因组蛋白质阵列的免疫反应性,建立了沙眼衣原体感染人体后的体液免疫应答谱,并筛选出27个衣原体免疫优势抗原。其中,719个衣原体抗原被99份抗血清中的一份或一份以上抗血清识别,124个抗原能被10%或10%以上抗血清识别,75个抗原能被20%或20%以上抗血清识别,50个抗原能被30%或30%以上抗血清识别,38个抗原能被40%或40%以上抗血清识别。27个抗原能被50%或50%以上抗血清识别,我们把它定义为衣原体的免疫优势抗原。这27个衣原体免疫优势抗原包括了衣原体膜蛋白：CT681,CT443,CT812; ABC转运子蛋白CT067和CT381；包涵体膜蛋白CT119,CT147,CT442,CT529和CT813；分泌到宿主细胞浆的分泌型蛋白CT858和pCT03 (Pgp3);III型分泌系统相关蛋白CT089和CT456；与代谢有关的酶及蛋白酶CT240,CT798,CT806, CT828和CT841；一些衣原体hypothetical蛋白CT143,CT101, CT694, CT142, CT695, CT875, CT795, CT022。其中11个抗原已经被报道为免疫优势抗原,尚有16个衣原体免疫优势抗原为全球首次报道,分别是：CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT456, CT695, CT798, CT806, CT828, CT841和CT875。
4.应用衣原体感染的HeLa细胞或未感染的HeLa细胞裂解物吸附抗血清,ELISA法分别检测这些血清与衣原体全基因组蛋白质阵列的免疫反应性,发现应用衣原体感染的HeLa细胞裂解物吸附免疫血清后,该血清不再识别Ct全基因组蛋白质阵列,而未感染的HeLa细胞裂解物吸附抗血清后其与Ct全基因组蛋白质阵列的反应性与吸附之前相似。健康人血清亦不与Ct全基因组蛋白质阵列发生反应。结果提示：衣原体感染患者抗血清与衣原体全基因组蛋白质阵列的反应是抗原抗体特异性反应。
5. Western blot法检测ELISA法中反应阳性的抗原与抗血清的免疫反应性,发现大多数抗原Western blot法检测仍然为反应阳性,少数抗原虽在ELISA法检测时反应阳性,但在Western blot法检测时却为反应阴性。免疫共沉淀实验结果显示该抗血清能将ELISA法检测阳性而Western blot法检测阴性的蛋白从感染的HeLa细胞裂解物中沉淀下来,从而证实该抗原是构像依赖性抗原。该结果表明本实验中用到的Ct全基因组蛋白质阵列结合ELISA法检测衣原体抗原组既能检测线性抗原,还能鉴定构像依赖性抗原,因此该方法优于Western blot法。27个衣原体免疫优势抗原中共有10个为构像依赖性抗原,分别是：GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828和GST-pCT03 (Pgp3)。
6.①通过比较人、兔和小鼠三个种属所识别的衣原体抗原,发现许多人类优势识别的抗原亦能被2种啮齿目动物强烈识别,表明啮齿目动物可以被用于研究衣原体引起的免疫反应和评估衣原体疫苗候选抗原的保护作用。②比较活衣原体感染的Balb/c鼠和C57BL/6鼠识别衣原体抗原的数目,发现滴鼻感染和阴道感染的Balb/c鼠均识别54个衣原体抗原,而阴道感染的C57鼠仅能识别27个抗原,死衣原体接种的Balb/c鼠识别29个抗原,提示宿主识别的抗原数与感染的严重程度正相关。③活衣原体感染个体所识别的抗原与灭活衣原体接种个体抗血清所识别的抗原差别尤为显著。人类抗血清识别的719个抗原中,563个抗原不能被死衣原体接种的兔或鼠抗血清识别,为感染依赖性抗原；156个抗原能被死衣原体接种的兔或鼠血清识别,为感染非依赖性抗原。
7. Western blot法分析衣原体感染依赖性抗原和感染非依赖性抗原在衣原体感染细胞裂解物和纯化的衣原体EB、RB上的表达情况。发现大多数感染依赖性抗原主要表达在感染细胞裂解物中,而在EB上表达很少,提示这些蛋白可能为分泌性蛋白,RB合成该蛋白后很快将其分泌至包涵体腔或宿主细胞浆。
结论
1.D型Ct全基因组共预测有918个ORFs。
2.成功地将788个Ct基因的全长和120个Ct基因的片段克隆到pGEX-6p载体中,构建了933个pGEX-6p-CTs原核表达质粒。
3.成功表达了908个衣原体基因全长或片段,制备了933个重组GST-CTs融合蛋白,建立了D型Ct蛋白质组。并建立了D型Ct全基因组蛋白质阵列。
4.建立了Ct生殖道感染女性患者中衣原体B细胞抗原组,成功建立了沙眼衣原体感染后体液免疫应答谱,鉴定出Ct27个免疫优势抗原,全球首次报道16个新的衣原体免疫优势抗原,分别是：CT022,CT067,CT101,CT142,CT143,CT240,CT381,CT442,CT443,CT456,CT695,CT798,CT806,CT828,CT841和CT875。
5.Ct全基因组蛋白质阵列结合ELISA法检测衣原体抗原组既能检测线性抗原,还能鉴定构像依赖性抗原。27个衣原体免疫优势抗原中有10个为构像依赖性抗原,分别是：GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCTO3 (Pgp3)。
6.首次系统的鉴定出563个Ct感染依赖性抗原。在38个能被40%或40%以上患者血清识别的衣原体抗原中,共有14个衣原体感染依赖性抗原,分别是：CT089, CT116, CT142, CT153, CT228, CT442, CT529, CT694, CT798, CT806, CT813, CT828, CT858, CT866。
研究目标
1.进一步分析衣原体Ⅲ型分泌系统相关蛋白与沙眼衣原体生殖道感染患者(sexually transmitted infection,简称STI)患者和沙眼患者抗血清的免疫反应性。
2.制备抗Ⅲ型分泌系统效应蛋白Tarp (CT456)的单克隆抗体。
3.研究Tarp蛋白在衣原体感染HeLa细胞中的表达特征。
4.评估Tarp蛋白重组疫苗的免疫保护效果。
5.鉴定Tarp蛋白的免疫优势区。
研究方法
1.通过互联网搜索目前已报道的CtⅢ型分泌系统相关蛋白。
2.收集8份沙眼和24份Ct生殖道感染患者血清,ELISA法进一步分析CtⅢ型分泌系统相关蛋白的免疫反应性。应用衣原体感染的HeLa细胞或未感染的HeLa细胞裂解物吸附抗血清,同时收集健康人血清,ELISA法分别检测这些血清与衣原体III型分泌系统相关蛋白的免疫反应性以鉴定衣原体性蛋白与抗血清反应的特异性。
3.采用杂交瘤技术制备抗Tarp蛋白的单克隆抗体,间接免疫荧光法筛选阳性克隆,ELISA法鉴定单克隆抗体的特异性,间接免疫荧光法鉴定单克隆抗体的亚类和衣原体种属特异性。
4.间接免疫荧光法检测Tarp蛋白在衣原体感染HeLa细胞中的表达特征。Western blot法检测Tarp蛋白在纯化的EB、RB、D型Ct感染的HeLa细胞裂解液中的表达。
5. Tarp蛋白重组疫苗免疫保护效果的评估：
①克隆表达Ct鼠肺炎型(MoPn) Tarp同源体(TC0741) GST融合蛋白,用PreScissionTM蛋白酶酶切去除GST而获得纯的TC0741重组蛋白；
②建立Balb/c小鼠MoPn生殖道感染模型,分别收集感染小鼠血清和脾细胞,ELISA法检测内源性Tarp蛋白诱导的特异性抗体和T细胞应答。
③TC0741重组蛋白免疫小鼠,分别收集小鼠血清和脾细胞,ELISA法检测外源性Tarp蛋白诱导的特异性抗体的滴度及抗体亚类,间接免疫荧光法检测抗体的特异性；ELISA法检测MoPn刺激脾细胞后培养上清中IFN-γ、IL-4和IL-5的水平。
④以TC0741重组蛋白为免疫原,结合CPG和不完全弗氏佐剂(IFA),采用肌肉注射方式接种Balb/c小鼠,同时设MoPn+CPG+IFA阳性对照组和单纯的CPG+IFA阴性对照组,再以MoPn生殖道攻击小鼠,间接免疫荧光法检测小鼠阴道分泌物中MoPn的IFU值以判断小鼠清除MoPn感染的速度；解剖分离小鼠生殖道组织,H&E(苏木精和曙红)染色法检测小鼠上生殖道组织病理学变化。
6.克隆表达D型Ct Tarp蛋白11个片段的GST融合蛋白,ELISA法检测这11个片段与Ct生殖道感染患者、沙眼患者、兔免疫血清,以及抗Tarp单克隆抗体的免疫反应性,进一步鉴定Tarp蛋白的免疫优势区,为进一步探讨Tarp的生物学功能和保护性作用提供实验依据。
研究结果
1.通过互联网搜索,目前已报道的衣原体III型分泌系统相关蛋白主要有Inc家族蛋白及另外约49个衣原体蛋白。应用ELISA法检测CtIII型分泌系统相关蛋白的免疫反应性,结果显示STI患者和沙眼患者血清均能优势识别D型Ct CT456 (Tarp)、CT089和CT858重组蛋白,其中Tarp蛋白在沙眼患者血清中识别频率更高达100%,其识别频率和平均OD均高于其他蛋白,甚至高于CPAF。尽管D型Ct Inc家族免疫优势蛋白CT119,CT529,CT813和主要外膜蛋白MOMP能被STI患者血清优势识别,但在沙眼患者血清中的识别频率却显著低于STI患者血清。
2.采用杂交瘤技术获得9株稳定分泌抗Tarp蛋白单克隆抗体的杂交瘤细胞株,分别命名为：R4D5,N5B11,R5B6.2,R2H1.2,R12B12, R2H7, R5G8.1, R8G7.2和M4F4。9个mAbs全部为IgG, R5B6.2和R8G7.2为IgG2a, R4D5, R2H1.2, R12B12, R2H7, N5B11, M4F4和R5G8.1均为IgG1。间接免疫荧光法染色结果显示9个mAbs均能特异性的着色Ct血清型A、D、L2,而不着色MoPn、6BC和AR39。
3.间接免疫荧光法检测Tarp蛋白在衣原体感染HeLa细胞上的表达特征,结果显示：在衣原体感染后8h内可见Tarp表达,在13h时Tarp表达消失。24h衣原体包涵体己经非常明显,但仍不能检测到Tarp的表达。直到28h时重新检测到少量Tarp,而且Tarp的表达量随感染时间的进一步延长而逐渐增加。Western blot法检测Tarp蛋白在纯化的EB、RB、HeLa-D裂解液中的表达,发现Tarp主要在纯化的EB和HeLa-D裂解液中表达,而在RB上则不表达。结果提示Tarp蛋白是一个衣原体EB相关蛋白。
4.以MoPn基因组为模板,PCR扩增得到了TC0741基因全长片段,构建了pGEX-6p-TC0741重组质粒；成功表达了GST-TC0741融合蛋白；用PreScissionTM蛋白酶酶切去除GST而获得了纯的TC0741重组蛋白。
5. MoPn感染小鼠血清中产生了高滴度的抗MoPn和抗Tarp的特异性抗体,该血清能识别MoPn抗原和GST-Tarp融合蛋白,但不能识别单纯的GST蛋白。MoPn感染小鼠的脾细胞经Tarp蛋白和MoPn刺激后诱导产生了高浓度的IFN-γ,且IFN-γ的浓度随着Tarp蛋白剂量加大而增高,而GST蛋白却不能刺激感染小鼠的脾细胞产生IFN-γ。各组脾细胞培养上清中基本检测不到IL-4和IL-5的产生。结果提示MoPn感染小鼠后内源性Tarp诱导产生了Tarp特异性体液和细胞免疫应答。
6.给小鼠接种Tarp蛋白+佐剂以评估外源性Tarp蛋白诱导的免疫应答。间接免疫荧光实验检测发现接种MoPn+佐剂和Tarp蛋白+佐剂的小鼠抗血清能使MoPn包涵体染色,而仅接种佐剂的小鼠抗血清却不能使MoPn包涵体染色。MoPn感染HeLa细胞的裂解物能中和MoPn+佐剂组和Tarp蛋白+佐剂组小鼠免疫血清中抗MoPn抗体,经预吸附后,两组血清均不再使MoPn包涵体染色。GST-Tarp蛋白预吸附Tarp蛋白+佐剂组血清后,该血清不再染色MoPn包涵体,但GST-Tarp蛋白预吸附MoPn+佐剂组血清后,该血清仍继续染色MoPn包涵体。ELISA法检测小鼠免疫血清中抗MoPn特异性抗体的滴度和亚类。MoPn+佐剂组和Tarp蛋白+佐剂组小鼠抗血清中产生了高滴度的抗MoPn抗体,而单纯佐剂组血清中则没有抗MoPn抗体产生。MoPn+佐剂组和Tarp蛋白+佐剂组小鼠抗血清中IgG2a的滴度均高于IgG1,即IgG2a/IgGl>1。MoPn+佐剂和Tarp蛋白+佐剂组脾细胞培养上清中IFN-γ的浓度显著高于单纯佐剂组。各组脾细胞培养上清中基本检测不到IL-4和IL-5的产生。结果提示Tarp重组疫苗能够诱导小鼠产生MoPn特异性细胞免疫应答和体液免疫应答,以Thl细胞应答为主。
7.间接免疫荧光法检测小鼠阴道分泌物中MoPn的IFU值以判断小鼠清除MoPn感染的速度,发现单纯接种佐剂组中,在感染后27天阴道分泌物中仍可检测到衣原体。MoPn+佐剂组在感染后第6天,小鼠阴道分泌物中MoPn的IFU值显著低于单纯接种佐剂组和Tarp+佐剂组(P<0.05),第15天已基本检测不到MoPn。Tarp+佐剂组在感染后第21天,小鼠阴道分泌物中MoPn的数量显著低于单纯接种佐剂组(P<0.05),第24天已完全检测不到MoPn,提示此时Tarp+佐剂组小鼠生殖道中MoPn已被完全清除。结果表明：Tarp重组疫苗诱导产生的免疫反应有利于小鼠生殖道内衣原体感染的清除。
8.小鼠生殖道组织大体观察发现Tarp+佐剂组(P<0.05)和MoPn+佐剂组(P<0.01)输卵管双侧积水的发生率均显著小于单纯佐剂组。小鼠生殖道组织切片H&E染色评估炎症的严重程度和生殖道管腔扩张程度结果显示：三组中小鼠子宫角组织的炎症积分和管腔扩张积分情况没有显著差异(P>0.05),而在输卵管部位,Tarp+佐剂组和MoPn+佐剂组的炎症积分和管腔扩张积分均显著低于单纯佐剂接种组(P<0.01)。结果提示Tarp重组疫苗能防止小鼠上生殖道的炎性病理改变,具有显著的保护作用,因此,Tarp是一个有前景的衣原体疫苗候选抗原。
9.成功克隆表达了11个Tarp片段的GST融合蛋白。ELISA法检测这11个片段与Ct生殖道感染患者、沙眼患者、兔免疫血清,以及抗Tarp单克隆抗体的免疫反应性,发现所有Tarp反应阳性抗血清均能识别氨基酸从152～302的区域；STI抗血清和兔抗血清能同时识别氨基酸1-156、310-431和582～682区域；只能被STI抗血清识别的是氨基酸从425-581区域；只能被兔抗血清识别的是氨基酸683-847区域。6个抗Tarp单克隆抗体识别氨基酸152～302的区域,另3个单克隆抗体分别识别氨基酸1～119、120-151,和310～431的区域。
结论
1.Tarp蛋白在STI患者和沙眼患者中均为免疫优势抗原。
2.成功建立了9株稳定分泌抗Tarp蛋白的单克隆抗体杂交瘤细胞株。
3.Tarp是一个衣原体EB相关蛋白。
4.接种Tarp诱导产生了以Thl为主的免疫应答。Tarp重组疫苗与CPG+不完全弗氏佐剂联合免疫在实验小鼠内发挥一定的保护作用,有利于小鼠生殖道内衣原体的清除,显著降低小鼠上生殖道组织的炎性病理改变。Tarp可作为一种新的潜在靶抗原用于衣原体疫苗的研制。
5.氨基酸从152到302的区域,是Tarp蛋白最强的免疫优势区。
Abstract
Chlamydia trachomatis is an obligate intracellular prokaryotic parasite which is the leading cause of preventable blindness and the most prevalent bacterial pathogen causing sexually transmitted disease in clinic. Although chlamydial infection is susceptible to antibiotics treatment, many urogenitally infected individuals don't seek treatment due to lack of obvious clinic symptoms, thus, becoming vulnerable to developing persistent infection and long-term sequelae including ectopic pregnancy and infertility. Vaccination is considered the most effective means for preventing chlamydial infection and diseases. However, the inactivated whole organism-based vaccines failed in human trachoma vaccine trials. Thus, extensive efforts were made to develop effective and safe subunit vaccines in past decades. However, there is still no licensed C. trachomatis vaccine probably due to insufficient knowledge on protective and pathogenic determinants of chlamydial organisms. A genome-wide profiling of the immune response to Chlamydia trachomatis infection and screening immundominant antigens will contribute to identify protective and pathogenic determinants of chlamydial organisms, to clarify chlamydial pathogenesis, and to develop a serum diagnosis kit or a chlamydial vaccine. PartⅠA Genome-Wide Profiling of the Humoral Immune Response to Chlamydia trachomatis Infection in Humans
Objectives
1. Cloning and expression of GST fusion proteins encoded by Chlamydia trachomatis serovar D genome-wide ORFs, to lay the foundation for study on Chlamydial proteomics.
2. To establish the humoral immune response profile to Chlamydia trachomatis infection in humans and screen immundominant antigens; to identify infection-dependent antigens by comparing antigen profiles recognized by live chlamydial organism-infected versus dead organism-immunized hosts. To provide the experimental evidence for clarifying chlamydial pathogenesis, and developing a serum diagnosis kit or a chlamydial vaccine.
Methods
1. The informations of all Chlamydia trachomatis serovar D ORFs were analyzed according to the publised papers and sequences on website http://stdgen.northwestern.edu/. Specific primers of 918 chlamydial genes full length or fragments were synthesized according to the gene sequences. PCR was used to amply the target genes from Chlamydia trachomatis serovar D genomic DNA. The pGEX-6p-CTs expression plasmids were constructed and been transformated into E.coli XL-1 blue. The expression of recombinant Ct proteins were induced by IPTG. The proteome of Chlamydia trachomatis serovar D was established.
2. All the bacterial lysates containing the chlamydial fusion proteins were added to the 96 well microplates in ORF order for setting up the whole genome scale protein array. Serum samples were collected from patients which were urogenitally infected with Chlamydia. The ELISA was carried out to detect the reactivity of STI antisera with the whole genome scale protein array. The humoral immune response profile to Chlamydia trachomatis infection in humans was established and the chlamydial immunodominant antigens were identified.
3. To identify the specificity of the reactivity of the antisera with chlamydial proteins:①Sera from healthy female individuals (without C. trachomatis infection) were harvest and used as negative controls and the reactivities of these sera with the whole genome scale protein array by ELISA. All positively detected antigens were re-measured with the same antibody samples with or without further absorption with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells;②The reactivity of all positively detected antigens were re-measured by Western blot;③The immunoprecipitation and Western blot assays were carried out to detect whether the same antisera can precipitate endogenous chlamydial antigens from chlamydia-infected HeLa cell lysates under a non-denaturing condition.
4. To produce antibodies to the whole chlamydial organisms, rabbits or mice were immunized with UV-inactivated C. trachomatis serovar D organisms; Rabbits were injected intramuscularly while Balb/c mice intraperitoneally. Balb/c mice were infected with live chlamydia EB intranasally. Balb/c and C57BL mice were infected with live chlamydia EB intravaginally. All the antisera were harvested and the reactivity with the whole genome scale protein array by ELISA.①We compared the human-recognized C. trachomatis antigens with those detected by antisera from rabbits and different strains of mice to identify whether these hosts are appropriate for using to produce antibodies to C. trachomatis antigens and/or identify/evaluate chlamydial vaccine candidate antigens;②We compared the antigen profiles recognized by live organism-infected humans versus dead organism-immunized animals to identify infection-dependent antigens and infection-independent antigens.
5. The expressiong of infection-dependent antigens and infection-independent antigens on whole cell lysates samples with or without human antibody precipitation along with the purified chlamydial RB and EB organisms were detected by an Western blot.
Results
1. We found that there were 918 predicted ORFs encoded by C. trachomatis serovar D genome and plasmid.In the current study,908 genes were cloned,788 ORFs were expressed in full length and 120 in one or more fragments. Totally,933 pGEX-6p-CTs were constructed and all of them were expressed as GST fusion proteins in E.coli XL1-Blue. The remaining 10 ORFs were not expressed good quality fusion proteins despite our best efforts. These difficult ORFs include CT081, CT219, CT267, CT786, CT039.1, CT221.1, CT480.1, CT814.1, DEG02 and DEG16.
2. All the Chlamydia GST fusion proteins were arrayed onto ten 96 well microplates in ORF order. To minimize the plate to plate variations, each plate was included with a GST-alone negative control well and CPAF positive control well. Thus,the chlamydial whole genome scale protein array was set up.
3. Antisera from 99 women urogenitally infected with C. trachomatis were collected. The reactivity of these antisera with the chlamydial whole genome scale protein array was detected by ELISA. The humoral immune response profile to Chlamydia trachomatis infection in humans was set up and 27 chlamydial immunodominant antigens were identified. 719 out of the 908 ORF-encoded chlamydial proteins were recognized by at least one of the 99 human antisera. Among the 719 antigens,124 were recognized by 10%,75 by 20%,50 by 30% and 38 by 40% or more antisera respectively. The 27 proteins recognized by 50% or more antisera were designated as immunodominant antigens. These 27 immunodominant antigens cover a wide range of proteins including those localized in the organism membrane such as CT681, CT443, CT812 and the ABC transporters CT067 and CT381, proteins in the inclusion membrane such as CT119, CT147, CT442, CT529 and CT813, and proteins secreted into host cell cytosol such as CT858 and pCT03. In addition, some typeⅢsecretion system- related proteins such as CT089 and CT456, metabolic enzymes and proteases such as CT240, CT798, CT806, CT828 and CT841as well as various hypothetical proteins are also among the immunodominant antigens. Although 11 of the 27 antigens were previously reported to be immunodominant, the remaining 16 proteins represent the newly discovered immundominant antigens in the current study, including CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT456, CT695, CT798, CT806, CT828, CT841 and CT875.
4. The reactivity of positively reacted antigens were re-measured with the same antibody samples with or without further absorption with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells. The results showed that the absorption with chlamydia-infected HeLa lysates blocked antibody binding to all fusion proteins while a similar absorption with HeLa alone lysates failed to do so. No significant reactivity of the serum samples from healthy individuals was found. These observations have demonstrated that human antibody binding to the whole genome scale protein array is specific to chlamydial antigens.
5. The reactivity of all positively detected antigens were re-measured by Western blot and the results showed that Most of the chlamydial GST fusion proteins identified by this antiserum in the proteome array ELISA were also detected by the antiserum in Western blot. However, some antigens were not detected by the serum sample in the Western blot or only minimally detected and the intensity of their reactivity with the human antiserum on the Western blot was incompatible with the OD values. The immunoprecipitation and Western blot assays showed that the antigens which positively reacted in ELISA but not in Western blot were successfully precipitated by the same human antiserum and the amounts of antigens precipitated relative to the amounts of antigens available in the infected cell lysates were largely consistent with the corresponding OD values detected in the proteome array ELISA. Thus, these chlamydial proteins were conformation-dependent antigens. The results demonstrated that our fusion protein-based whole genome scale proteome array ELISA approach could detected both linear and conformation-dependent antigens, so that our method was advantage over the Western blot approaches. Among the 27 immunodominant antigens identified using our proteome array with human sera,10 were not or only minimally detected in Western blot,they were GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCT03 (Pgp3).
6.①As for the types of antigens recognized by the 3 host species, they were human,rabbit and mouth,we found that many antigens dominantly recognized by humans were also strongly recognized by the two rodents species,suggesting that rodents can be used to study chlamydial Immunology and evaluate the protection efficacy of chlamydial vaccine candidate antigens.②A more careful analysis of antigens recognized by the live Chlamydia infected Balb/c and C57BL mice revealed that Intranasal (in) or intravaginal (iv) infection of Balb/c mice both induced antibodies to 54 chlamydial antigens respectively while intravaginal infection of C57BL mice induced antibodies to 27 antigens only, suggestion that a correlation between the number of antigens recognized by the host and the severity of infection.③There were especially significant differences in the types of antigens recognized by antisera from live organism infection and dead organism immunization individuals. When the 719 antigens recognized by human antibodies were resorted based on their recognition by the immunized rabbits and mice, we found that 563 of the 719 human-recognized antigens were not detected by either rabbits or mice, thus classified as infection-dependent antigens, while the remaining 156 as infection-independent antigens since they were recognized by the dead organism-immunized animals.
7. The expressiong of infection-dependent antigens and infection-independent antigens on whole cell lysates samples with or without human antibody precipitation along with the purified chlamydial RB and EB organisms were detected by an Western blot. The results showed that many infection-dependent antigens were detected abundantly in infected whole cell lysates but only minimally in purified EBs, suggesting that all infection-dependent antigens are produced during live infection and many may not be incorporated into the infectious particles.
Conclusions
1. There were 918 predicted ORFs of all Chlamydia trachomatis serovar D genome.
2.788 Chlamydia trachomatis gene full length and 120 gene fragments were successfully cloned into pGEX-6p vector,933 pGEX-6p-CTs plasmids were constructed.
3.908 chlamydial ORFs full length or fragments were successfully expressed and 933 recombinant GST-CTs fusion proteins,Thus Chlamydia trachomatis serovar D proteome. A whole genome scale proteome array of Chlamydia trachomatis serovar D was set up.
4. The B cell ANTIGENome of women urogenitally infected with C. trachomatis were mapped and the humoral immune response profile to Chlamydia trachomatis infection in humans was established.27 chlamydial immunodominant antigens were identified, including 16 proteins were the newly discovered immundominant antigens in the current study, including CT022, CT067, CT101, CT142, CT143, CT240, CT381, CT442, CT443, CT695, CT798, CT806, CT828, CT841 and CT875.
5. Our fusion protein-based whole genome scale proteome array ELISA approach could detected both linear and conformation-dependent antigens. Among the 27 immunodominant antigens identified using our proteome array with human sera,10 were conformation-dependent antigens, they were GST-CT022, GST-CT067, GST-CT101, GST-CT142, GST-CT240, GST-CT695C, GST-CT798C, GST-CT806, GST-CT828 and GST-pCT03 (Pgp3).
6. Newly systematically identified 563 Chlamydia trachomatis infection-dependent antigens.14 of the 38 antigens which were recognized by(?)40% patients sera were infection- dependent antigens,they are CT089, CT116, CT142, CT153, CT228, CT442, CT529, CT694, CT798, CT806, CT813, CT828, CT858, CT866.
Objectives
1. The reactivity of TypeⅢSecretion System associated proteins with tients rogenitally infected with C. trachomatis (sexually transmitted infection, TI) and trachoma patients were further detected.
2. The mAbs against TypeⅢSecretion System associated protein Tarp (CT456),which was an immnuodominant antigen,were prepared.
3. The expression of Tarp protein in Chlamydia infected HeLa cells were detected.
4. Protection efficacy of Tarp protein recombinant vaccine was estimated.
5. The immunodominant regions of Tarp protein were identified.
Methods
1. Searching for the reported Chlamydia trachomatis typeⅢsecretion system associated proteins in the internet.
2. Antisera from 24 women urogenitally infected with C. trachomatis and 8 trachoma patients were collected. The reactivity of these antisera with the chlamydial typeⅢsecretion system associated proteins was further detected by ELISA. To identify the specificity of the reactivity of the antisera with chlamydial proteins, sera from healthy female individuals (without C. trachomatis infection) were harvest and the patient sera were preabsorpted with lysates made from either HeLa cells alone or C. trachomatis serovar D-infected HeLa cells, the reactivities of these sera with the t3ssassociated proteins by ELISA.
3. Hybridoma technique was carried out to prepare mAbs against Tarp protein. The specificities to these monoclonal antibodies were determined by ELISA. The isotype and chlamydial species specificity of these monoclonal antibodies were determined by an indirect immunofluores-cence assay.
4. The expression of Tarp protein in Chlamydia infected HeLa cells were detected by an indirect immunofluorescence assay. The expression of Tarp protein in purified EB, RB and Chlamydia infected HeLa cells lysate were detected by Western blot.
5. Evaluation of the immune protection efficacy of Tarp protein recombinant vaccme:
①GST fusion protein of Chlamydia muridarum Tarp(ORF TC0471)was cloned and expressed.The GST-TC0741 protein were cleavaged with a precision protease and freed TC0741 recombinant protein was made.
②Mopn urogenitally infected Balb/c mouse model was set up. The endogenous Tarp-specific antibody and T cell response were measured by ELISA after the antisera and spleen cells of infected mice were collected respectively.
③Mice were immunized with recombinant TC0741 protein,mouse antisera and spleen cells were collected respectively. The exogenous Tarp-specific antibody production and isotypes were detected by ELISA. The specific of anti-MoPn antibody were detected by an indirect immunofluorescence assay. The levels of IFN-γ、IL-4 and IL-5 were detected from the spleen cell culture supernants after the mouse spleen cells were re-stimulated with MoPn antigens by ELISA.
④Balb/c mice were immunized with recombinant TC0741 protein emulsified in the CpF-IFA agjuvant via intramuscular injection, The intact MoPn organisms similarly emulsified in the same adjuvant were used as a positive control, adjuvant alone as a negative control. Thirty days after the immunization, the mice were intravaginally challenged with MoPn. To test the clearance of MoPn infection, the IFU values of MoPn organism shedding were monitored by an indirect immunofluorescence assay.The mouse urogenital tract tissues were isolated and the Histopathological changes were assessed by an H&E(hematoxylin and eosin) dyeing method.
6. To map the immunodominant regions of Tarp protein and provide important information for further understanding the biological function and the protective immunity of Tarp,11 fragments of Tarp were cloned and expressed as GST fusion proteins and the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms and with mAbs against Tarp protein were detected by ELISA.
Results
1. We found that chlamydial typeⅢsecretion system associated proteins was predicted to consist of Inc proteins and other 49 chlamydial proteins or so by searching in the internet.The reactivities were detected by ELISA and the results showed that CT456 (Tarp)、CT089 and CT858 recombinant proteins were immunodominantly recognized by both STI and trachoma antisera,the reactivity ot Tarp even stronger than that of CPAF with 100% recognition frequency and the highest average OD value. The trachoma antisera can't predominantly recognize the three Inc proteins CT119,529 and 813despite the fact that they are immunodominant during human urogenital infection with C. trachomatis. MOMP from C. trachomatis serovar D was lack of recognition by trachoma antisera.
2. Nine hybridoma cell lines stable in secreting specific against Tarp protein monoclonal antibodies were successfully obtained, they were R4D5, N5B11, R5B6.2, R2H1.2, R12B12, R2H7, R5G8.1, R8G7.2 and M4F4. All the 9 mAbs were IgG,2 mAbs (R5B6.2 and R8G7.2) were belong to IgG2a isotype and the other 7 mAbs (R4D5, R2H1.2, R12B12, R2H7, N5B11, M4F4 and R5G8.1) were belong to IgGl isotype. Results of indirect immunofluorescence assay showed that all the 9 mAbs can react strongly with chlamydia serovar A,D,and L2, but not MoPn,6BC,and AR39.
3. The expression of Tarp protein in Chlamydia infected HeLa cells were detected by an indirect immunofluorescence assay. The results showed that:Tarp was detected in the first 8 hours and no longer detectable by 13 hours after infection. Although chlamydial inclusions became very obvious at 24 hours after infection, Tarp remained undetectable. However, by 28 hours after infection, Tarp started to reappear and progressively increased as the incubation continued. Tarp protein was only detected in Chlamydia trachomatis serovar D infected HeLa cell lysates and purified EBs but not RBs on a Western blot. Our results suggesting that Tarp protein was an EB associated protein.
4. TC0471 full length sequence was amplified by PCR from Chlamydia muridarum genomic DNA and pGEX-6p-TC0741 plasmid was constructed; GST-TC0741 fusion protein was successfully expressed; freed TC0741 recombinant protein was made by cleavaged the GST-TC0741 protein with a precision protease.
5. The MoPn infected mice developed high titers of antibodies that recognized both MoPn-derived antigens and Tarp fusion proteins but not the fusion tag GST alone. High levels of IFN-y were induced after the spleen cells from MoPn infected mice were re-srimulated with either Tarp protein or MoPn. The levels of IFN-ywere increased with the amount of Tarp protein. No IL-4 and only minimum levels of IL-5 were detected from the spleen cell culture supernatants of the MoPn infected mice. These observations together have demonstrated that mice infected with MoPn developed Tarp-specific humoral and cellular immune responses.
6. The immune response induced by exogenous Tarp protein was evaluated after mice were immunized with recombinant TC0741 protein emulsified with adjuvant. The antisera induced by MoPn or Tarp but not adjuvant alone visualized antigens in MoPn inclusions in animmunofluorescence assay. the anti-Tarp antibody labeling was blocked by either the immunogen GST-Tarp or MoPn-infected cell lysates while the anti-MoPn antibody labeling was effectively blocked by MoPn-infected cell lysates. We further quantitated the titers of the MoPn antigen-reactive antibodies and isotyped the mouse IgG antibodies in an ELISA Immunization with either MoPn or Tarp with adjuvant but not adjuvant alone induced high titers of antibodies that specifically recognized MoPn endogenous antigens. Immunization with either Tarp or MoPn induced more IgG2a than IgG1,that is IgG2a/IgG1>1. The spleen cells from mice immunized with either MoPn or Tarp produced significantly higher levels of IFN than those from adjuvant alone immunization. No IL-4 and only minimum levels of IL-5 were detected from the spleen cell culture supernatants of the immunized mice. These results further strengthened the conclusion that immunization with Tarp induced a MoPn-specific cellular and humoral immune responses, it was a Thl-dominant immune response.
7. To test the clearance of MoPn infection, the IFU values of MoPn organism shedding were monitored by an indirect immunofluorescence assay. Live MoPn organisms were detected in the vaginal swabs collected from the adjuvant alone group of mice up to 27 days after infection. Immunization with the intact MoPn organisms shortened the infection time course to 15 days with a significant reduction in the infectious titers from the vaginal swabs within 6 days after infection. Importantly, immunization with Tarp resulted in a significant reduction in the vaginal shedding of live organisms on day 21 and most of the Tarp-immunized mice cleared infection by day 24 post infection. Thus, Tarp-induced immunity enhanced the resolution of MoPn infection from the mouse lower genital tracts although not as potently as the intact organism-induced immunity.
8. Gross appearance assessment showed that the incidence of bilateral hydrosalpinx in Tarp-immunized (33%) or MoPn-immunized (10%) mice is significantly lower than that in the adjuvant alone-immunized mice(70%). After, we further evaluated the severity of inflammation microscopically using histology sections. Both the inflammatory cell infiltration and luminal dilatation were blindly semi-quantitated. There was no significant difference in either inflammation or lumenal dilatation scores in the uterine horn tissues between the three groups of mice. However, mice immunized with either Tarp or MoPn displayed significantly lower scores in both inflammatory cell infiltration and lumenal dilatation than the control group of mice, suggesting that the immunity induced by either Tarp or MoPn can reduce the inflammatory damages in the mouse oviducts.
9.11 fragments of Tarp were successfully expressed as GST fusion proteins and the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms and with mAbs against Tarp protein were detected by ELISA. A major immunodominant region was strongly recognized by all antibodies. This region covers amino acids 152 to 302, which consist of three repeats (152-201,202-251 and 252-302). Several other minor immunodominant regions were also identified, including 1-156,310-431 and 582-682, recognized by antisera from both human and rabbit; 425-581, only recognized by human antisera; and 683-847, preferentially recognized by rabbit antisera. This immunodominance was also confirmed by the observation that six out of the nine mAbs bound to the major immunodominant region and that the other three each bound to one of the minor fragments,1-119,120-151 and 310-431.
Conclusions
1. Tarp protein was an immunodominant antigen not only in STI patient but also in trachoma patient.
2. Nine hybridoma cell lines stable in secreting specific against Tarp protein monoclonal antibodies were successfully set up.
3. Tarp was a chlamydial EB associated protein.
4. Immunization of mice with Tarp induced Thl-dominant immunity that significantly reduced the shedding of live organisms from the lower genitaltract and attenuated inflammatory pathologies in the fallopian tube tissues. These observations have demonstrated that Tarp can induce protective immunity against chlamydial infection and pathology in mice,it cab be used as a novel promising chlamydia vaccine candidate antigen.
5. The region covers amino acids 152 to 302 was the most immunodominant region of Tarp protein.
引文
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