高变区1抑制丙型肝炎病毒包膜蛋白E2诱生交叉中和抗体

发布时间：2018-01-08 15:11

本文关键词：高变区1抑制丙型肝炎病毒包膜蛋白E2诱生交叉中和抗体　出处：《第二军医大学》2011年博士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒(hepatitis C virus,HCV)属于黄病毒科,主要经血液传播,是引起人类丙型肝炎的病原体。全球约有1.7亿人感染HCV,每年新增感染者达300万-400万。我国HCV感染者估计近4000万。HCV感染易慢性化,其慢性化率可高达85%,部分慢性丙型肝炎患者可发展为肝硬化,甚至肝细胞癌。目前主要采用聚乙二醇干扰素α联合利巴韦林治疗丙型肝炎,其效果明显受HCV基因型影响。2、3型HCV感染对该疗法的持续病毒学应答率(SVR)可达到75%,但1型HCV感染的SVR不到50%。目前全球范围内经血液以及血制品途径感染的丙型肝炎病例与上世纪九十年代以前相比有大幅下降,但在发展中国家血液和血制品仍然是传播HCV的重要途径。此外,每年散发性以及传播途径不明的丙型肝炎新发病例仍高居不下。控制HCV感染的关键措施还是依赖于开发出有效的疫苗,从1989年HCV基因组被克隆以来,丙型肝炎疫苗的研究在欧美和日本等发达国家一直是研究热点,但迄今未有有效的疫苗问世。 HCV包膜蛋白E2位于HCV蛋白前体第384-746位氨基酸残基,由一个大的N端膜外区和一个C端疏水锚定区组成,与HCV另一包膜蛋白E1通过非共价键形成异源性二聚体。E2蛋白是介导病毒吸附和进入宿主细胞的关键蛋白,是诱导宿主产生HCV中和抗体的关键抗原,因此是疫苗研究的最主要靶标。然而,包膜蛋白高度变异,尤其E2蛋白氨基末端的含有27个氨基酸残基的高变区1(hypervariable region 1,HVR1)是HCV蛋白中变异频率最高的区域,也是公认的HCV中和抗体表位所在区域。HVR1是介导HCV与受体SR-BI结合的关键区域,HVR1与SR-BI的作用通过几种不同的机制促进HCV细胞侵入。HVR1抗体和SR-BI抗体均能阻断HCV感染。HVR1的高度变异曾被认为是遏制丙肝疫苗发展的瓶颈问题,近几年来基于HCV假病毒(HCVpp)以及细胞培养产生的HCV(HCVcc)的中和试验发现,HVR1并不是唯一的中和抗体表位所在区域,在HCV包膜E2蛋白中还存在一系列构象依赖的中和表位和线性中和表位,其中一些表位在不同基因型、亚型间高度保守。本实验室前期分别用分泌表达E2的表达质粒以及删除了HVR1的E2表达质粒免疫小鼠,然后检测、分析小鼠血清中的抗体以及病毒中和活性,结果显示:E2质粒免疫的小鼠血清中,HVR1抗体在总E2抗体中占很大比重,针对HVR1以外区域的抗体在中E2抗体中只占较小比重;删除HVR1可显著增强E2质粒免疫诱导的针对HVR1以外区域的抗体,小鼠免疫血清与其他基因型E2蛋白的交叉反应以及对其他基因型HCVpp的交叉中和活性也随之显著增强。这和急性HCV感染者血清中可检测到HVR1抗体而难以检测到E2抗体相似。结果提示:HVR1有可能抑制E2蛋白中保守表位的免疫原性,从而从另一个方面导致HCV免疫逃避。目前基于HCV包膜蛋白为主要靶抗原的疫苗均含有HVR1,这类疫苗免疫黑猩猩只能抵抗同株病毒攻击,但不能抵抗异株的攻击。HVR1的存在可能是这类疫苗免疫保护效果不理想的重要原因,删除HVR1也许能增强疫苗免疫对异株病毒攻击的保护效果。核酸疫苗在大动物的免疫效果远远不及在小动物的免疫效果,目前还没有核酸疫苗用于人类。为了进一步探讨用删除了HVR1的包膜E2蛋白作为HCV疫苗的发展前景,本研究用CHO细胞表达了E2以及删除了HVR1的E2蛋白,用亲和层析纯化出了两种E2蛋白,分析两种E2蛋白的构象、受体结合功能和抗原性,通过小动物免疫分析HVR1对E2蛋白免疫原性的影响,与我们用核酸免疫获得的结果进行对比和验证,探讨缺失HVR1的E2蛋白在丙肝疫苗中的应用前景。一、稳定表达HCV包膜E2-人IgG Fc融合蛋白的CHO细胞株的建立我们首先分别拼接HCV包膜蛋白E2-Fc和E2Δ-Fc融合基因(E2Δ指缺失HVR1),使E2与人IgG Fc段作为融合蛋白表达,方便目的蛋白的亲和层析纯化。将融合基因插入具有自主知识产权的CHO细胞表达质粒载体pCIDA-GS-neo(专利号:ZL 200610024202.5,发明名称:一种用哺乳动物细胞高效分泌表达丙型肝炎病毒包膜蛋白E2的方法),然后分别将构建的表达质粒转染HEK 293T细胞,用ELISA及Western blotting方法检测培养上清中E2-Fc以及E2Δ-Fc融合蛋白,证实两者均可有效表达且表达水平相当。尔后,我们分别将这两种E2-Fc融合蛋白表达质粒及EGFP表达质粒(GS-EGFP)分别转染CHO细胞,以蛋氨酸亚氨基代砜(methionine sulphoximine,MSX)在不含谷氨酰胺的培养基内筛选表达目的蛋白的细胞克隆,用有限稀释法对粗筛出的细胞克隆进行进一步筛选,从而建立和获得了稳定分泌表达E2-Fc和E2Δ-Fc融合蛋白的细胞株。紧接着,我们对筛选出的贴壁CHO细胞株进行了无血清无蛋白培养的驯化,通过逐步下降培养基内血清的浓度,使细胞由贴壁状态转为悬浮生长,随后对悬浮细胞株的培养体系进行了放大,从75T方瓶培养经由250 ml三角烧瓶的小量放大,最后实现在Wave Bioreactor EH2/10细胞培养系统中2L细胞培养袋的灌流培养。细胞密度由最初的2×105cells/ml,达到最后的2×107cells/ml,细胞活率始终保持在80%以上,目的蛋白的分泌量也在灌流培养开始后稳定在3-4 mg/L左右。二、E2-Fc、E2Δ-Fc融合蛋白纯化及功能鉴定以上述的细胞培养上清为原料,离心去除细胞,继以GE公司超滤系统(QuixStand? Benchtop System)对上清液进行浓缩和超滤,将其浓缩约10倍,并去除其中分子量低于30 KD的蛋白质。随后,以ProteinA亲和层析柱HiTrap? Protein A HP Columns从超滤液中纯化融合蛋白,通过对柱压、洗脱条件、收集方式等参数的优化,从每升培养上清中可纯化出3-4 mg目的蛋白。然后用截留分子量为30 KD的离心型蛋白超滤柱以低温低速离心的方式将纯化出蛋白样品的缓冲液置换为pH为7.0的磷酸盐缓冲液。用构象依赖性单抗对最终纯化产物进行的Western blotting分析表明E2蛋白保持了天然空间构象。以糖苷酶EndoH、PNGase F分别对两种蛋白进行去糖基化处理,然后观察其分子量的变化,结果提示这两种蛋白的糖基化程度和形式相似。ELISA及Pull down实验显示E2-Fc,E2Δ-Fc均能与人CD81大胞外环(hCD81-LEL)结合,E2Δ-Fc结合活性较E2-Fc为高。检测两种蛋白与细胞膜表面表达的人CD81和SR-BI分子的结合活性,结果显示缺失HVR1后,E2蛋白与SR-BI的结合能力明显降低,与CD81的结合略有增强。流式细胞术检测发现两种蛋白均能与HCV易感的Huh7.5细胞结合且效率相当。将与E2蛋白结合后的Huh7.5细胞裂解,通过免疫共沉淀的方法检测介导E2蛋白与细胞结合的分子,结果显示,E2-Fc能将CD81及SR-BI共沉淀下来,而HVR1缺失的E2Δ-Fc共沉淀较多的CD81及少量SR-BI,提示HVR1是介导E2蛋白与靶细胞表面SR-BI结合的重要区域。两种蛋白均能抑制HCVpp和HCVcc的感染性,抑制率最高可达90%以上,且株特异性不强。三、删除HVR1增强E2-Fc融合蛋白的免疫原性分别将上述两种融合蛋白联合弗氏佐剂免疫小鼠,于0、3和8周每只小鼠皮下注射10μg佐剂乳化的融合蛋白,初免第2、7和10周采血检测小鼠血清中的E2ΔHVR1抗体及HVR1抗体,发现E2-Fc免疫组10只小鼠中,有5只E2ΔHVR1及HVR1抗体均为阳性,4只E2ΔHVR1抗体单阳性,1只两种抗体一直是阴性。E2ΔHVR1及HVR1抗体滴度随免疫次数增多逐步升高,E2Δ-Fc蛋白的确能诱导更高的E2ΔHVR1抗体水平,其滴度约为E2-Fc免疫组的5-10倍。证实HVR1的存在抑制了E2ΔHVR1抗体的产生。尔后,为进一步检测小鼠血清抗体中和活性,我们将各组小鼠血清混合后纯化出IgG,行病毒感染中和实验,结果显示,两组IgG对于与蛋白来源同株的H77株HCVpp感染中和效率没有显著性差异,中和率在80%以上。但对于异株HCV,E2-Fc组中和活性显著低于E2Δ-Fc组。含有HVR1的E2-Fc融合蛋白诱导的抗体中,HVR1抗体阳性则其交叉中和活性明显低于HVR1抗体阴性者,也说明HVR1抑制交叉中和抗体的产生。这些结果与我们DNA免疫的研究一致,高度提示缺失HVR1可有力促进HCV包膜E2蛋白诱导交叉中和抗体。此外,考虑到Fc段本身有特殊的免疫学功能,可能有助于免疫应答,尤其是细胞免疫应答的诱导。本研究中,采用融合蛋白免疫小鼠,该融合蛋白是否能在动物体内诱生针对E2蛋白特异且高效的细胞免疫反应,是我们需要进一步验证的内容。小结本研究着眼于丙型肝炎病毒蛋白疫苗的开发思路,在前期DNA免疫的基础上,构建了稳定表达E2-Fc/E2Δ-Fc的无蛋白无血清悬浮CHO细胞培养体系并成功纯化出相应的目的蛋白。功能试验结果显示,HVR1缺失并未明显影响E2蛋白的天然构象,HVR1删除后,E2蛋白与其受体CD81的结合增强,与SR-BI的结合能力减弱。动物免疫结果显示,HVR1的缺失显著增强了E2蛋白诱导的交叉中和抗体。综上所述,该研究首次从蛋白层面揭示了HVR1对E2蛋白结构、功能及免疫原性的影响,为HCV蛋白疫苗的开发提供了新思路。
[Abstract]:Hepatitis C virus (hepatitis C, virus, HCV) belongs to the family Flaviviridae, mainly through blood transmission, is caused by human hepatitis C virus pathogens. Approximately 170 million people worldwide are infected with HCV, new infections each year up to 3 million -400 million. China's HCV infected.HCV estimates that nearly 40 million of the infection is likely to slow, high rate of chronicity 85%, part of patients with chronic hepatitis C cirrhosis, and hepatocellular carcinoma. At present the main pegylated interferon alpha combined with Leigh Bhave Lin in the treatment of hepatitis C, the effect was significantly affected by the influence of HCV genotypes on the sustained virologic response to therapy of HCV infection rate of.2,3 (SVR) can reach 75%, but the 1 type of HCV infection SVR to 50%. worldwide through infected blood and blood products of hepatitis C patients and the last century before 90s compared to a sharp decline, but in the blood and blood products in developing countries is still. An important way to broadcast HCV. In addition, each of the sporadic and the transmission of unknown new cases of HCV infection still remains high. Key measures to control HCV infection is dependent on the development of an effective vaccine, since the 1989 HCV genome was cloned on hepatitis C vaccine in Europe and Japan and other developed countries have been studied hot, but so far no effective vaccine available.
HCV envelope protein E2 in HCV protein precursor in 384-746 amino acid residues, by a large N ectodominant terminal and a C terminal hydrophobic anchor region, and another HCV envelope protein E1 by non covalent bond to form heterologous dimers with two.E2 is the key protein mediated virus adsorption and entry the host cell is the key antigen induce neutralizing antibody to HCV, it is the main target of vaccine research. However, the envelope protein is highly variable and hypervariable region of 27 amino acid residues containing especially E2 protein amino terminal 1 (hypervariable region 1, HVR1) is a regional variation in the highest frequency of HCV protein HCV, is recognized as a neutralizing epitope region of.HVR1 is the key area of HCV mediated by receptor binding of SR-BI, HVR1 and SR-BI promote HCV cell invasion of.HVR1 antibody and SR-BI antibody can block the infection of HCV.HVR through several different mechanisms The height variation of 1 was considered to be a bottleneck in HCV vaccine development in recent years, based on the HCV virus (HCVpp) and cell culture produced HCV (HCVcc) found the neutralization test, HVR1 is not the only neutralizing epitope region, also depends on a series of conformational neutralization epitopes and linear neutralizing epitopes in HCV envelope protein E2, some epitopes in different genotypes and subtypes are highly conserved.
Ourprevious respectively with secretory expression plasmid E2 and the deletion of HVR1 expression plasmid of E2 immunized mice, then detection, analysis of antibody and virus neutralizing activity in serum of mice showed that the serum E2 of mice immunized, HVR1 antibody accounted for a large proportion in the total E2 antibody, antibody to outside the HVR1 area only a small proportion in E2 antibody; deletion of HVR1 significantly enhanced the antibody to HVR1 outside the E2 plasmid induced immune cross reaction, immune serum of mice with other genotypes of E2 protein and HCVpp of other genotypes cross neutralizing activity also increased significantly. And the acute HCV infection detected HVR1 antibody but it is difficult to detect the serum E2 antibody. The results suggest that HVR1 may inhibit the conservative form immunogenicity of the E2 protein, which can lead to HCV immune escape from another.. The HCV envelope protein based on the main target antigen vaccine containing HVR1, this kind of immune vaccine can resist chimpanzee monoecious virus attack, may exist but they cannot resist the different strains of the.HVR1 attack is an important reason for this type of vaccine immune protection effect is not ideal, perhaps can enhance the protective effect of deletion of HVR1 vaccine on different strains of virus attack.
The nucleic acid vaccine on the immune effect of large animal is far less than in the immune effect of small animal, there is no vaccine for human use. In order to further investigate the deletion of HVR1 envelope protein E2 as the development prospects of HCV vaccine, the expression of E2 and deletion of HVR1 E2 protein in CHO cells, purified by affinity chromatography two kinds of E2 protein, conformation analysis of two E2 protein, receptor binding function and antigenicity, through small animal immune analysis on the effect of HVR1 on E2 protein immunogenicity, were compared and validated with the results obtained by our DNA immunization, and to discuss the application of HVR1 deletion of E2 protein in hepatitis C vaccine.
Establishment of a CHO cell line stably expressing the HCV envelope E2- human IgG Fc fusion protein
We are first splicing HCV envelope protein E2-Fc and E2 -Fc fusion gene (E2 Delta HVR1, which refers to the lack of E2 and IgG) Fc as a fusion protein, convenient protein affinity chromatography. The fusion gene was inserted with independent intellectual property rights CHO expression plasmid vector pCIDA-GS-neo (Patent No.: ZL 200610024202.5. The invention: a method for mammalian cells secreting expression of hepatitis C virus envelope protein E2), then the plasmid was transfected into HEK cells by 293T, ELISA and Western blotting method to detect the culture supernatant of E2-Fc and E2 -Fc fusion protein, both of which can be confirmed and the expression level is quite effective. Then, we are the two kinds of E2-Fc fusion protein expression plasmid and expression plasmid EGFP (GS-EGFP) CHO cells were transfected with methionine imino sulfone (methionine sulphoximine, MSX generation) Clones were screened for the expression of target protein in the culture medium without glutamine, the cell clones were further screened by the screening method of limited dilution to establish and obtain a stable expression and secretion of E2-Fc and E2 a -Fc fusion protein in cell line. Then, we carried out domestication and serum-free culture of selected protein adherent CHO cells, through the medium of serum concentrations gradually decreased, the cell growth by adherence to the suspension, then the training system of suspension cell lines were amplified, amplified by 250 ml three 75T from the small angle flask flask culture, and finally realize the cultivation of 2L cell culture bag perfusion the culture system of Wave Bioreactor in EH2/10 cells. The cell density from 2 * 105cells/ml, reaching 2 * 107cells/ml, cell survival rate has remained above 80%, the secretion of protein in irrigation The flow culture began to stabilize at about 3-4 mg/L.
Purification and functional identification of two, E2-Fc, E2 Delta -Fc fusion protein
In the cell culture supernatant as raw materials and centrifuged to remove cells, followed by GE (QuixStand Benchtop System ultrafiltration system?) concentration and ultrafiltration of supernatant, which is concentrated about 10 times, and the removal of the molecular weight of less than 30 KD protein. Then, using ProteinA affinity chromatography column HiTrap Protein A HP Columns? The fusion protein was purified by ultrafiltration, the column pressure, elution conditions, optimization parameter collection methods, from per liter of culture supernatant can be purified 3-4 mg protein. Then with MWCO of 30 KD type centrifugal ultrafiltration column protein by low temperature and low speed centrifugal method and purified protein buffer replacement the samples of pH 7 phosphate buffer. With conformation dependent monoclonal antibody analysis of the purified products were Western blotting showed that E2 protein conformation. In order to maintain the natural glycosidase enzymes EndoH, PNGase and F respectively to two Protein deglycosylation treatment, then observe the changes of molecular weight, the results showed that two kinds of protein glycosylation and similar forms.ELISA and Pull down showed that E2-Fc, E2 -Fc and CD81 a large extracellular loop (hCD81-LEL) combined with E2, a -Fc binding activity was higher than E2-Fc binding activity. Detection of two kinds of protein expression and cell surface CD81 and SR-BI molecules, the results showed that HVR1 deletion, binding ability of E2 protein and SR-BI were significantly decreased, and the combination of CD81 is slightly enhanced. Flow cytometry assay showed that two proteins were HCV and susceptible Huh7.5 cell binding and efficiency Huh7.5. After cell lysis combined with E2 protein, molecular methods, by immunoprecipitation detection mediated by E2 protein binding to the cells showed that E2-Fc CD81 and SR-BI can be co precipitated, and deletion of the HVR1 E2 -Fc CD81 and a little more precipitation SR-BI indicates that HVR1 is an important region that mediates the binding of E2 protein to SR-BI on target cell surface. All two proteins can inhibit the infectivity of HCVpp and HCVcc, the inhibition rate is above 90%, and the plant specificity is not strong.
Three, deleting the immunogenicity of HVR1 enhanced E2-Fc fusion protein
The above two kinds of immunized mice with Freund's adjuvant in 0,3 and 8 weeks each mice were injected subcutaneously with 10 g adjuvant fusion protein, immunization of 2,7 and 10 weeks of blood testing E2 HVR1 antibody and HVR1 antibody in sera of mice immunized with E2-Fc, found in a group of 10 mice. 5 E2 HVR1 and HVR1 antibodies were positive, 4 E2 HVR1 1 single antibody positive, only two kinds of antibodies has been negative for.E2 and HVR1 HVR1 antibody titer with immunization times increased gradually increased, E2 Delta -Fc protein can indeed induce higher E2 HVR1 antibody level, its titer was about 5-10 times E2-Fc immune group. To confirm the presence of HVR1 inhibited E2 HVR1 antibodies. Then, for the further detection of serum antibody neutralizing activity, we will in serum were mixed and purified for IgG, virus neutralization test, results showed that the two groups for IgG and protein from strain H77 Strain HCVpp infection efficiency and no significant difference, and the rate is more than 80%. But for different strains of HCV, E2-Fc group of neutralizing activity was significantly lower than that in E2 group. A -Fc HVR1 containing E2-Fc fusion protein induced by HVR1 antibody, the antibody positive cross neutralizing activity was significantly lower than that of HVR1 antibody negative, indicating that HVR1 suppress the cross neutralizing antibodies. These results are consistent with our DNA immunity, highly suggestive of deletion of HVR1 can effectively promote the HCV envelope protein E2 induced cross neutralizing antibodies. In addition, taking into account the Fc itself has special immunological function, may contribute to the immune response, especially the induction of cellular immune response. In this study, the mice were immunized with fusion protein, whether the fusion protein in animal in vivo induced cellular immune responses against E2 protein specifically and efficiently, we need to further verify the contents.
Summary
This study focuses on the development of hepatitis C virus protein vaccine, based on the previous DNA immunity, constructed the stable expression of E2-Fc/E2 Delta -Fc protein free serum-free CHO cell culture system and purified protein. The corresponding objective function test results show that HVR1 deficiency did not affect E2 protein natural conformation HVR1 after deletion, enhanced binding protein E2 and its receptor CD81, and weaken the binding ability of SR-BI. Animal immunization results showed that loss of HVR1 significantly enhanced the cross neutralizing antibodies induced by E2 protein. In summary, this study first from the protein level revealed that HVR1 of E2 protein structure, function and influence of immunogenicity. Provides a new idea for the development of HCV protein vaccine.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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