融合基因ag85B-mpt64_190-198-mtb8.4在抗结核新型重组卡介苗以及亚单位疫苗研究中的应用

【作者】 郄亚卿；

【导师】 王洪海；

【作者基本信息】 复旦大学 ， 微生物学， 2008， 博士

【摘要】 结核病（Tuberculosis,TB）是一种古老而漫长的感染性疾病,是长期危害人类健康的严重疾病之一。近年来,由于HIV和结核分枝杆菌双重感染,以及耐药型结核分枝杆菌的流行,导致结核病的发病率和死亡率大幅度上升。目前全球大约有20亿人感染上结核病,感染人数接近全世界人口的三分之一,其中每年有85%的死于结核病的患者,以及95%的疑似新发结核病例,都发生在发展中国家。结核病严重威胁着人类健康,联合国已将结核病列为21世纪重点控制的三大疾病之一。目前研制成功的唯一的能够预防结核病的疫苗——卡介苗,虽然已在全球范围内广泛使用,但在近期研究中发现,其许多临床试验结果并不是十分有效,免疫预防效果不稳定,在全球范围的免疫保护力检测,由0-80%不等,并且对成人几乎无效,对已感染患者更是没有治疗作用,可以说卡介苗并不是预防肺结核的理想疫苗,研究和开发新的结核疫苗势在必行!现阶段研究结核病疫苗主要集中在以下几个领域:1.以BCG为载体,重组进新基因的重组BCG使其比亲代卡介苗具有更强的抗原性和免疫性,以代替现在使用的卡介苗。2.结核蛋白疫苗,用于BCG免疫后的加强免疫。3.营养缺陷型结核减毒活疫苗4.结核DNA疫苗本研究就是针对结核疫苗研究的两个方面,重组BCG研究和亚单位疫苗的研究来开展工作的。一.重组BCG疫苗的研究由于BCG预防免疫效果不稳定,我们需要研究免疫效应强于BCG的疫苗才能有效提高对结核病的防护作用。对BCG加以改良的方式也有多种:将BCG缺失的重要的保护性抗原重组入BCG,或是在BCG中过量表达主要的保护性抗原,促进抗原的释放和呈递,增强机体免疫反应。而由于重组多价抗原疫苗,往往能够比单一抗原疫苗激发更强的免疫反应,因此在近些年研究中,也越来越受到重视。本研究工作主要是选择结核分枝杆菌最重要的保护性抗原Ag85B,ESAT6,以及Mtb8.4,与具CD8⁺T细胞表位的一段Mpt64抗原的肽段和结核杆菌休眠期表达的抗原HSPX,以不同的组合方式,构建了三株重组BCG疫苗,其中以大肠杆菌-结核分枝杆菌穿梭质粒pMV261为载体的有rBCG-Ag85B-mpt64_{（190-198）}-Mtb8.4和rBCG-Ag85B-ESAT6-Rv2031c（HSPX）,另一个是以大肠杆菌-结核分枝杆菌穿梭质粒pMV361为载体构建的rBCG-Ag85B-ESAT6。均为国内外首次报道。将上述三株重组BCG和BCG分别免疫小鼠,通过分析IFN-γ的分泌,IgG水平、以及IgG2a/IgG1,比较了它们的免疫原性。研究结果表明,三株重组BCG针对特异抗原均可以引起特异性的较BCG强的细胞免疫反应。其中P261-rBCG-Ag85B-Mpt64_{（190-198）}-Mtb8.4的免疫原性较高,而且还能诱导比其它重组BCG更强的Th1型免疫反应,这正是成功抗击结核病的关键因素。因此我们选择P261-rBCG-Ag85B-Mpt64_{（190-198）}-Mtb8.4（P261-rBCG-AMM）作为候选疫苗,进一步做了以下研究工作:在免疫原性水平上,P261-rBCG-AMM不仅能够诱导产生特异性较BCG强的IFN-γ水平,并且还能激起体内特异的CD4⁺和CD8⁺活性T细胞,能够诱导人单核细胞产生与BCG水平相类似的TNF-α,能够长期有效的诱导比BCG高的Th1型免疫反应;在毒力攻击保护实验中,研究结果显示,攻毒后6周P261-rBCG-AMM免疫的小鼠肺部菌落数较少,净体重增加。肺部组织的病理改变要轻于BCG免疫组,抗酸染色结果显示,随着免疫时间的延长,重组BCG免疫的小鼠肺部组织的结核杆菌数最少。因此P261-rBCG-AMM能够产生与BCG相当甚至更好的保护作用。另外,我们通过比较重组BCG和BCG的生长曲线,以及免疫小鼠后,脏器内菌落数的变化,发现重组BCG并没有因为加入新的重组基因而导致生长状态发生改变,其生长曲线仍和BCG的类似,而且脏器内菌落数随着时间变化的趋势也与BCG大体吻合。这就证明重组BCG能够激起较强的免疫保护反应,并不是因为重组BCG在动物体内数量的增加,而是因为重组BCG表达有重组基因的结果。关于P261-rBCG-AMM的免疫原性和免疫保护性评价也属首次报道。二.融合基因在蛋白疫苗研究中的应用将Ag85B、MPT64_190-198和Mtb8.4三种重要的保护性抗原物质融合表达,构建了融合多肽Ag85B-Mpt64_{（190-198）}-Mtb8.4（AMM）。表达纯化AMM蛋白,将其和Ag85B蛋白分别与IFA混合免疫小鼠。研究表明它可以激活比单个Ag85B蛋白更有效的细胞免疫反应,以及更强的Th1型免疫反应。AMM的两个组分Ag85B和MPT64_190-198均可刺激免疫小鼠的脾淋巴细胞表达分泌IFN-γ。另外将AMM蛋白与不同佐剂（包括FIA,MPL,壳聚糖微球体）混合,免疫小鼠后,观察免疫指标,结果发现,FIA的辅助免疫功能最强,壳聚糖微球体虽然辅助功能稍逊于FIA,但是由于其使用安全,经过必要的改造,有可能发展为一个良好的佐剂。重组蛋白疫苗AMM被证实也是一种潜在的新型抗结核疫苗。可以进行免疫保护性等相关评价。更多还原

【Abstract】 Tuberculosis remains a serious world health problem.Infection with the causative agent,Mycobacterium tuberculosis,is a major clinical burden with 8 million new cases and approximately 2 million deaths per year.Ninety percent of the estimated deaths from TB and 95%of the estimated eight million new cases of TB each year occur in developing countries,which comprise 85%of the world’s population. Incidence is increasing fastest in African countries that are affected by HIV,followed by Eastern Europe and the former Soviet Union,both of which are plagued by multi-drug resistant strains of TB that present an ominous global threat.Mycobacterium boris bacille Calmette G’eurin（BCG） vaccine,an attenuated strain of M.bovis,is the only vaccine currently available against Mycobacterium tuberculosis and the most widely used vaccine,with over three billion administered doses.BCG protects children efficiently against miliary and meningeal TB,but the protective efficiency against adult pulmonary TB ranges from 0%to 80%based on large,well-controlled field trials.Therefore,it is urgently needed to develop more effective and satisfactory BCG vaccines against TB.This has prompted the search for new,improved TB vaccines.A heap of promising new approaches has been developed during the last two decades.Dozens of vaccine candidates have been tested in recent years in animal models,including subunit protein/peptide vaccines in adjuvants,DNA vaccines,rationally attenuated strains of M.tb,recombinant mycobacterias and live vectors expressing genes coding for immuno-dominant mycobacterial antigens or mycobacterial lipids.And our research is focus on two kinds of tuberculosis vaccines:recombinant BCG and subunit protein vaccine.1.Recombinant BCG:As BCG vaccination is widely accepted within current immunization programmes, is associated with minimal side effects and the vaccine is amenable to genetic manipulation the opportunity exists to modify BCG to render it a more effective vaccine.There are a number of key observations,which provide the basis for a rationale approach to improving BCG.First,BCG lacks a number of genes that are present in parental M.bovis and M. tuberculosis which can insertion in it.Second,genes encoding immunodominant antigens or immunostimulatory cytokines can be over-expression in BCG.Third,the multigenic vaccine approach is attractive because broad immune responses can be generated by simultaneously targeting several antigens.We constructed three recombinant BCG strains expressing Ag85B,mpt64,mtb8.4 ESAT-6 and HSPX:rBCG-Ag85B-Mpt64_{（190-198）}-Mtb8.4 and rBCG-Ag85B-ESAT6-Rv2031c（HSPX） are both in vector pMV261,while rBCG-Ag85B-ESAT6 is in vector pMV361.First,we compared immune responses between three recombinant BCG vaccines and BCG.After C57BL/6 mice immunized with BCG and rBCG vaccines,we studied the level of antibody response,IgG2a/IgG1 and the amount of IFN-γsecreted,and found that P261-rBCG-Ag85B-Mpt64_{（190-198）}-Mtb8.4 could induce stronger cellular immunity than the others and enhance Th1 response.Then,we chose P261-rBCG-Ag85B-Mpt64_{（190-198）}-Mtb8.4 as our candidate vaccine and did some more researches on it.In cellular immune response, P261-rBCG-Ag85B-Mpt64_{（190-198）}-Mtb8.4 could not only elicited higher IFN-γthan BCG,but also generated more antigen-specific CD4 T cells and CD8 T cells than those vaccinated with BCG.Moreover,P261-rBCG-Ag85B-Mpt64_{（190-198）}-Mtb8.4 induced TNF-αsecretion in THP-1 cells in vitro and the level was similar with BCG did.The protective efficacy of rBCG was also determined.The results demonstrated that the recombinant BCG strain could confer similar or even better protective efficacy than others.The protective effect of rBCG could relate to a difference in the persistence of the recombinant strain within the host.To investigate this,we determined if expression of the recombinant genes ag85B-mpt64_{（190-198）}-mtb8.4 influenced the growth of BCG in vivo and in vitro.Mice were infected intravenous with rBCG and BCG and the growth in the lungs,liver and spleen was monitored.In all cases,the bacterial load did not differ significantly between rBCG and BCG, irrespective of the time after infection.Then we compared the growth characteristics of rBCG and BCG:there was no any significant difference in proliferation characteristics between rBCG and BCG.These results suggest that the improved protective efficacy was not due to increased persistence of rBCG in vivo,but was related directly to expression in BCG of the Ag85B-Mpt64_{（190-198）}-Mtb8.4 fusion protein2.Ag85B-Mpt64_{（190-198）}-Mtb8.4 in the subunit vaccine of TBSince Ag85B,MPT64_190-198,and Mtb8.4 were confirmed to be the effective protective antigens（or antigen epitopes）,they were fused together to construct a new subunit vaccine（AMM）.To compare antigen-specific immune response primed by single and fusion protein immunization,humoral immune responses were examined in mice immunized with Ag85B and AMM formulated in Freund’s Incomplete Adjuvant （FIA）.The results indicate that the humoral immune response against Ag85B,the cellular immune responses elicited by Ag85B and Mpt64_190-198 induced by the fusion protein AMM was stronger than that induced by single Ag85B.The function of adjuvants in subunit vaccine was also determined.AMM fusion with different adjuvants（FIA,chitosan and MPL） vaccinated in mice.And the results showed that chitosan and FIA could help AMM to elicit strong cellular and humoral immune responses.Having these essential elements of a successful vaccine,AMM could be a strong candidate subunit vaccine for further study.更多还原