丙型肝炎病毒多表位融合蛋白的表达及免疫效果的评价

【作者】 邱丰；

【导师】 毕胜利；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2010， 博士

【摘要】 丙型肝炎是一种严重威胁人类生命健康和生活质量的传染性疾病,急性丙型肝炎患者的临床表现包括：乏力,食欲不振,肝脏肿大和叩击痛,部分病人可出现黄疸。这一疾病的致病原为丙型肝炎(丙肝)病毒(hepatitis C virus, HCV),属于黄病毒科丙型肝炎病毒属,它的基因组由一条单链正义RNA构成,全长约9600nt,只含有单一的开放读码框架,编码3011个氨基酸组成的聚合蛋白,该聚合蛋白在翻译的过程中或者翻译后被宿主和病毒蛋白酶切割成至少10种成熟的结构蛋白以及非结构蛋白,包括：核心蛋白C, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A和NS5B。据WHO的统计资料表明,目前全世界约有1.7亿HCV感染者,并且每年新增感染者达300万～400万,所以,研制和开发HCV疫苗来预防HCV感染是当前全世界所共同面临的一个重大而且紧迫的难题。然而迄今为止尚无高效完善的HCV组织培养系统,致使传统减毒疫苗或者灭活疫苗的研制策略困难重重,因此关于HCV疫苗的研究主要集中在HCV重组亚单位蛋白的免疫特性上。此外,疫苗的免疫剂量,免疫方式和免疫程序都是值得深入探讨的问题。一个成功的HCV疫苗必须能够激发出较强的体液免疫反应和细胞免疫反应,体液免疫反应主要指诱导机体产生针对HCV包膜糖蛋白1和2(gpE1和gpE2)的病毒中和抗体,细胞免疫反应包括特异性的受MHC-Ⅱ类分子限制的CD4+辅助性T细胞以及受MHC-Ⅰ类分子限制的CD8+杀伤性T细胞的生成,上述两个类型的T细胞均能够分泌诸如干扰素γ(IFN-γ)之类的抗病毒细胞因子,其中CD8+杀伤性T细胞还具有清除被感染细胞的作用。目前HCV疫苗研究领域中已经诞生了不少候选疫苗,这些研究一般都使用重组HCV包膜糖蛋白gpE1和gpE2作为疫苗中的抗原,但是无论如何在疫苗的前期研究工作中都需要候选分子的高效表达,以便在体外评价其免疫效果,针对HCV的高度变异性以及其他研究中表达的亚单位多肽诱发的体液免疫反应较弱等问题,我们选取了HCV基因组中相对保守并且也是T细胞表位非常集中的NS3区的一段780bp的DNA序列,另外为了提高对B细胞的激活能力,诱生出中和抗体,我们查阅文献后选取了三个保守的广谱中和抗原位点,将这些抗原表位融合表达,可望生成的融合蛋白具备较强的免疫原性并且能够对不同型,不同株的HCV提供保护作用,从而克服阻止HCV的感染,为丙型肝炎疫苗的研制作一些铺垫。本实验中通过微量中和试验以及CTL杀伤实验评价了HCV多表位融合蛋白的体液免疫原性和细胞免疫原性,为HCV疫苗的研制提供了一定的实验基础。另外,该融合蛋白也可作为临床诊断的候选抗原,在提升HCV诊断试剂的灵敏度和特异性方面具有一定的参考价值。一、丙型肝炎病毒多表位融合蛋白在原核系统中的表达根据文献报道：我们挑选出位于HCV包膜蛋白区的三个B细胞表位(aa192～aa202:YEVRNVSGVYH:aa313～aa327:ITGHRMAWDMMMNWS: aa702～aa710:PALSTGLIH)与NS3区一段T细胞表位非常集中的区域融合,利用原核表达系统,以pET-11d作为载体,在大肠埃希菌BL21(DE3)中经IPTG诱导表达获得融合蛋白,之后采用DEAE阴离子交换和镍离子柱亲和层析进行纯化后获得了HCV多表位融合蛋白。通过Western Blot验证融合蛋白的抗原活性。二、丙型肝炎病毒感染模型的建立长期以来,由于缺乏有效的HCV体外细胞培养体系,严重影响了对HCV生命周期的探讨及抗HCV药物的研发。本研究中,我们通过体外转录带有JFH1基因组全长的质粒从而获得了具有感染性的HCV JFH1毒株基因组全长RNA,之后进一步转染Huh7.5细胞建立了HCV的细胞培养体系,该体系能够产生有体外感染活性的HCVcc颗粒,我们使用反转录PCR以及间接免疫荧光两种技术检测到了转染后的细胞培养上清中HCVcc颗粒的存在,并且通过对Huh7.5细胞的再次感染证明了获得的HCVcc颗粒的感染性。三、评价丙型肝炎病毒多表位融合蛋白的细胞免疫原性和体液免疫原性我们通过流式细胞仪分析了免疫后的小鼠脾脏细胞以及外周血淋巴细胞经该多表位蛋白刺激后的增殖情况,显示出了比较好的细胞免疫效果,在小鼠体内产生了能够识别HCV相应抗原位点的典型特异CTL效应,说明免疫应答过程中的T淋巴细胞系统经过此抗原的刺激而得到了有效的激活,并在再次遇到相同的抗原肽时发挥其细胞毒性杀伤细胞效应。另外,该蛋白在家兔体内所诱导的抗体水平迅速升高并且能够维持相当长的时间,当注射到家兔体内的抗原剂量为200ug/只时,HCV中和抗体的检测结果也为阳性,提示HCV多表位融合蛋白可能具有中和HCV野生毒株的能力,可以应用到HCV预防性疫苗的开发上。更多还原

【Abstract】 Hepatitis C is a kind of infectious disease which seriously threatened public health and decreased the quality of everyone’s life. The clinical symptoms of an acute infection with hepatitis C virus include fatigue, myalgia, nausea and vomiting. Other features are jaundice and the yellowish tone of the skin. Hepatitis C virus is the cause of this disease. It belongs to the Hepacivirus genus, Flaviviridae virus family and its genome consists of a single stranded positive sense RNA of approximately 9600 nucleotides, containing a single open reading frame. This open reading frame encodes a precursor poly-protein of 3011 amino acids followed by the cleavage performed by viral proteases or host cell peptidases in the subsequent co-and post-translationally procession to yield at least 10 matured structural and non-structural proteins including core protein C, envelope proteins E1 and E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.The hepatitis C virus is transmitted mainly through blood-to-blood. According to WHO’s statistics, there are more than 170 million of the world population are chronic HCV carriers and 3～4 million HCV new cases were reported every year. It is estimated that there are approximate 40 million HCV carriers in Chinese population and the HCV prevalence is up to 3.2% in our country. Moreover, a large portion of those acute HCV infected individuals will become chronically and about 20% of the chronic infected will develop cirrhosis within a 10～30 year period, and these patients have an elevated risk of developing hepatocellular carcinoma. Histological analysis of liver biopsies is an important tool to determine and follow the chronic liver disease and the progression of fibrosis and cirrhosis. There are many reasons account for the viral immune escape and persistent infection. One of the main factor is that viral genome shows a considerable genetic heterogenecity because of the high mutation rate of 1.9×10-3 nucleotide conversion per gene site every year. Under the host immune pressure and the drug selective pressure, viral mutation occurs and the new kind of HCV quasispecies appears. That will lead to the host immune systems of some infected individuals can not spontaneously clear the viruses efficiently. Therefore, developing an ideal and effective HCV vaccine to prevent HCV infection is a very urgent problem all over the world. However, until very recently, HCV has not been propagated efficiently in cell culture. The major obstacle means that the use of inactivated or live, attenuated viral vaccines has not yet been available. Vaccine approaches have therefore included the use of adjuvanted recombinant polypeptide subunits of the virus in attempts to prime viral neutralizing antibodies to the envelope glycoproteins 1 and 2 (gpEl and gpE2), as well as priming MHC class-Ⅱ-restricted CD4+ T helper (Th) and MHC class-Ⅰ-restricted CD8+ cytotoxic lymphocyte (CTL) responses to these and other viral proteins. Both types of T cell can secrete antiviral cytokines such as interferon-γ(IFN-γ), and CD8+ CTLs have the potential to kill infected cells.HCV vaccine will play a key role in the prevention of HCV infection. Recent studies have made a great progress in developing many HCV vaccine candidates. These studies involved the use of the recombinant HCV envelope glycoproteins gpEl and gpE2 as vaccine antigens. The high yield of the vaccine antigen is very important to the evaluation of immune response in vitro. However, the hypervariability of HCV genome and the weakness of the humoral immune response elicited by HCV subunit peptides have hindered the preparation of HCV vaccines. In order to overcome these obstacles, we selected a conserved HCV NS3 fragment of 780bp and it contains series of T-cell epitopes. Beside of this, our fusion protein also included three broad neutralized B-cell epitopes according to many publications of HCV study to improve the activities of HCV specific B cells and to elicit HCV neutralization antibodies. We expected it had strong immunogenicity and it can neutralize many kinds of HCV strains of different genotypes. Moreover, the micro neutralization test and CTL cytotoxicity assay used in our studies also provided an effective way to measure the humoral immune response and cellular immune response caused by HCV multi-epitope fusion protein. In addition, the fusion protein as a valuable candidate antigen can also be used in clinical diagnosis to improve sensitivity and specificity of present diagnosis reagent. 1. HCV multi-epitope fusion protein expressed in prokaryotic systemAccording to references, three conserved B-cell epitopes (aa192～aa202: YEVRNVSGVYH; aa313～aa327:ITGHRMAWDMMMNWS; aa702～aa710: PALSTGLIH) located within HCV envelop protein were chosen and fused with part of HCV NS3 protein containing series of T-cell epitopes. With the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21(DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein.2. Establish of HCV infectious modelEver since a long time ago, researches about HCV life cycle and developments of anti-HCV drug have been hampered because the severe lack of effective cell culture system in vitro. In this study, we obtain HCV JFH-1 strain full length RNA transcribed from the plasmid template followed by transfection with Huh7.5 cells. RT-PCR and immunofluorescence assay were used in the detection of prepared HCVcc particles and the infectivity was measured by Huh7.5 cell infection.3. Cellular immune response and humoral immune response induced by HCV multi-epitope fusion proteinThe proliferation of PBMC and splenocytes of mice after antigen induction was analyzed by FACS. The results indicated that specific CD8+ cytotoxic lymphocytes could be detected in both PBMC and splenocytes and they could recognize specific antigen sites. We concluded that fusion protein was capable of elicit strong cellular immune response especially in the mice immunized with 100ug of fusion protein.Furthermore, we investigated HCV specific neutralizing antibodies against the virus by micro-neutralization assay and we found that fusion protein was able to elicit neutralizing antibodies which was critical in viral clearance. Taken together the results of humoral immunity, viral neutralization and specific cellular responses suggest that HCV multi-epitope fusion protein is a potential candidate for designing a prophylactic and therapeutic vaccine against HCV.更多还原