HCV感染者体内病毒NS3区部分区段的演变观测

目的　观察２ 例慢性 Ｈ Ｃ Ｖ 携带者及１ 例 Ｈ Ｃ Ｖ Ｒ Ｎ Ａ 转阴者体内包含 Ｃ Ｔ Ｌ 抗原表位的 Ｈ Ｃ Ｖ Ｎ Ｓ３ 区部分区段的长期演变。方法　通过反转录 Ｐ Ｃ Ｒ 扩增, Ｍ１３ 亚克隆,对３ 例 Ｈ Ｃ Ｖ 感染者的 Ｈ Ｃ Ｖ Ｎ Ｓ３ 区部分区段的一级结构进行测定。对感染者的 Ｈ Ｌ Ａ 进行分型。根据基序（ ｍｏｔｉｆ） 及 Ｈ Ｌ Ａ 分型资料预测该区段中的 Ｃ Ｔ Ｌ 抗原表位。结果　文献报道的 Ｈ Ｌ Ａ Ａ２ 限制的抗原表位在无 Ｈ Ｌ Ａ Ａ２ 的感染者 Ｃ 中,１９９１ 年至１９９６ 年氨基酸序列无变异。具有 Ｈ Ｌ Ａ Ａ２ 的感染者 Ｗ, Ｚ 部分氨基酸序列中该表位的起始密码子发生无义突变,测序资料中包含稳定的变异位点的肽段,符合 Ｍ Ｈ Ｃ结合肽的基序（ ｍｏｔｉｆ） 。结论　无义突变可能与 Ｃ Ｔ Ｌ 的免疫压力有关。稳定变异位点产生的原因可能是免疫逃避。     

丙型肝炎病毒（HCV）E2／NS1相对保守区多肽抗原在检？…

目的 研究（ＨＣＶ）Ｅ２／ＮＳ１相对保守区多肽抗原在检测抗－ＨＣＶ中的意义。方法 利用ＨＣＶＥ２／ＮＳ１基因编码的膜区糖蛋白合成Ｅ２／ＮＳ１相对保守区多肽抗原,建立酶免疫试验（ＥＩＡ）,对９６例ＨＣＶ感染者及４０例正常献血员进行ＨＣＶＥ２／ＮＳ１抗体的检测,同时平行检测ＨＣＶＲＮＡ肝炎为１３．５５％,慢性丙型肝炎为２５．０４％,无症状感染者为２．０８％;正常献血员中发现３例抗ＨＣＶＥ２／ＮＳ１抗体     

中国株HGV NS3 区基因的原核表达及其产物的抗原性分析

目的研究ＨＧＶＮＳ３区基因产物的抗原性,并探讨ＮＳ３蛋白在血清学检测中的应用。方法将中国株ＨＧＶＮＥ３区的３个基因片段分别克隆到ｐＲＳＥＴＢ和ｐＲＳＥＴＣ质粒载体中,构建成原核表达载体。ＩＰＴＧ诱导下,在大肠杆菌ＢＬ２１中高效表达,获得３个重组蛋白,用Ｗｅｓｔｅｒｎｂｌｏｔ和ＥＬＩＳＡ法分别对表达产物进行分析。结果所构建的表达载体均得到高效表达,得到的重组蛋白ＰＡ、Ｐ３和Ｐ４的分子量分别为４２０００,３００００和２４０００,在Ｗｅｓｔｅｒｎｂｌｏｔ和ＥＬＩＳＡ反应中均可被ＨＧＶ阳性血清识别,其中ＨＧＶＮＳ３区Ｎ端的基因产物抗原性较强。结论中国株ＨＧＶＮＳ３区的Ｎ端存在优势的抗原决定簇,其基因产物有较好的抗原性。     

初次及再次感染HCV后不同功能区抗体的研究

１４例初次ＨＣＶ感染者及１１例再次ＨＣＶ感染者系列血清系在无法检测ＨＣＶ时留存的２８９位手术受血者系列血清中筛选获得。系列血清包括受血前、受血后不同时期收集的血清。回顾性地研究其不同功能区抗体出现的动态变化，抗Ｃ与抗ＮＳ３抗体有早期诊断价值。抗ＮＳ３、抗Ｃ、抗ＮＳ５及抗ＮＳ４抗体在ＨＣＶ感染后系列血清中检出率分别为８４．７６％、７９．２７％、７２．５４％和６８．３９％。感染过程中各区抗体可以全部出现、部分出现或单独出现抗Ｃ、抗ＮＳ３及抗ＮＳ５抗体，未发现单独含抗Ｅ、抗ＮＳ１及抗ＮＳ４区抗体的血清。抗Ｅ、抗ＮＳ１及抗ＮＳ４抗体消失较早。研究表明：以ＨＣＶＣ区、ＮＳ３区及ＮＳ５区编码的优势抗原包被的ＥＬＩＳＡ抗体诊断试剂盒将提高ＨＣＶ的诊断。     

人原发性肝内胆管细胞癌中HCVRNA及其NS3区抗原的分布及意义

人原发性肝内胆管细胞癌中ＨＣＶＲＮＡ及其ＮＳ３区抗原的分布及意义西安第四军医大学病理学教研室应用原位分子杂交及免疫组化方法，使用地高辛标记ＨＣＶ５＇非编码区探针及抗ＨＣＶＮＳ３区Ｃ３３ｃ单克隆抗体，对３５例人原发性肝内胆管细胞癌（ＰＩＣ）和癌旁肝组织...     

从HCV蛋白一级结构预测其C,E,NS5区的CD_4~ T细胞抗原位点

我们根据ＣＤ４＋Ｔ细胞识别抗原位点的物理化学和生物学特征，设计了一个具有查找两亲性螺旋状结构（ａｍｐｈｉｐａｔｈｉｃｈｅｌｉｘｓｔｒｕｃｔｕｒｅ）肽段功能的计算机程序。用该程序对ＨＣＶ－１型病毒Ｃ、Ｅ（Ｅ１、Ｅ２／ＮＳ１）、ＮＳ５蛋白一级结构进行分析，发现这些蛋白区存在ＣＤ４＋Ｔ细胞识别位点。此结果支持了ＣＤ４＋Ｔ细胞对ＨＣＶＣ，Ｅ，ＮＳ５区可发生增殖反应的结论。提示该程序可作为一种预测ＣＤ４＋Ｔ细胞识别ＨＣＶ抗原位点的方法。     

ANALYSIS OF MOTIF IN TCR Vβ HV4 SEQUENCES BINDING SUPERANTIGEN TSST-1

首先对４１种人和小鼠的Ｔ细胞受体β链可变基因编码肽段（Ｖβ）的氨基酸序列进行多序列对准，就Ｖβ之第四高变区（ＨＶ４）片段进行比较，分析与超抗原毒素休克综合征毒素－１（ＴＳＳＴ－１）结合的四种Ｖβ（小鼠Ｖβ３、Ｖβ１５、Ｖβ１７和人Ｖβ２）之ＨＶ４序列内是否存在特定的氨基酸残基排列模式。结果发现：小鼠Ｖβ３和Ｖβ１７的ＨＶ４具有特异的ＲＦＳＡＸＣＸＳＮＳ模式，而小鼠Ｖβ１５和人Ｖβ２的ＨＶ４则含独特的ＫＦＸＩＸＨ模式。提示：与ＴＳＳＴ－１结合的四种Ｖβ所对应的Ｔ细胞识别表位可能不止一个。     

尼莫地平对大鼠脑血管痉挛缺血性脑损害的防治作用

目的：探讨尼莫地平（ＮＤ）对蛛网膜下腔出血（ＳＡＨ）后脑血管痉挛（ＣＶＳ）缺血性脑损害的保护作用。方法：应用非开颅大鼠模型,观察ＳＡＨ组和ＮＤ处理组２４ｈ内微区脑血流量（ＣＢＦ）和海马组织Ｃａ含量动态变化及３天后海马ＣＡ１区形态学改变。结果：在ＳＡＨ后２４ｈ内,ＳＡＨ组ＣＢＦ明显而持续降低,海马组织Ｃａ含量逐渐增加,３天后海马ＣＡ１区神经元明显受损。ＮＤ使上述改变均减轻。结论：ＮＤ通过对微循环的改善增加ＳＡＨ后ＣＶＳ时ＣＢＦ,通过阻断脑缺血时的有害代谢环节而减轻ＣＶＳ缺血性神经元损伤。     

20例中国人HCVⅡ/1b型高变区1序列变异的动态观察

目的 动态研究中国人群ＨＣＶⅡ／１ｂ型包膜蛋白Ｅ２／ＮＳ１高变区１（ＨＶＲ１）序列变异规律、意义及影响因素。方法 应用逆转录巢式ＰＣＲ技术从２０例ＨＣＶⅡ／１ｂ型感染的中国病人血清中扩增了ＨＣＶ部分包膜区基因片段（ｎｔ１４４９～１５８６，ＨＣＶ－Ｊ），纯化后直接采用双脱氧链末端终止法进行序列分析。结果 中国人群ＨＣＶ ＨＶＲ１位于氨基酸（ＡＡ）３８４～４１０位，有６个较保守的ＡＡ位点；３８５位Ｔｈ     

中国株HGV NS3区基因的原核表达及其产物的抗原性分析

目的 研究ＨＧＶ ＮＳ３区基因产物的抗原性,并探讨ＮＳ３蛋白在血清学检测中的应用。方法 将中国株ＨＧＶ ＮＥ３区的３个基因片段分别克隆到ｐＲＳＥＴ Ｂ和ｐＲＳＥＴ Ｃ质粒载体中,构建成原核表达载体。ＩＰＴＧ诱导下,在大肠杆菌ＢＬ２１中高效表达,获得３个重组蛋白,用Ｗｅｓｔｅｒｎ ｂｌｏｔ和ＥＬＩＳＡ法分别对表达产物进行分析。结果 所构建的表达载体均得到高效表达,得到的重组蛋白ＰＡ,Ｐ３和Ｐ４的分子     

Genetic variability of hepatitis C virus non-structural protein 3 and virus-specific CD8+ response in patients with chronic hepatitis C

Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus.     

The hepatitis C virus NS5A protein and response to interferon α: mutational analyses in patients with chronic HCV genotype 3a infection from India

The hepatitis C virus (HCV) non-structural (NS)5A protein is linked to interferon α resistance in vitro and higher numbers of NS5A amino acid (aa) variations in HCV 1a/b isolates are associated with virologic response to interferon α-based therapy in vivo. Here, we aimed to study NS5A aa variations in Indian patients undergoing interferon α/ribavirin treatment infected with HCV 3a. The NS5A region [aa 2194–2401, comprising interferon sensitivity determining region, protein kinase resource (PKR) binding domain, V3 region] was sequenced from pre-treatment sera of 24 patients with HCV 3a infection. Mean number and physicochemical properties of aa variations (conserved vs. non-conserved) were assessed. Additionally, published NS5A sequences [NS5A region (n = 61), PKR binding domain (n = 111)] of characterized HCV 3a isolates were analyzed. The mean number of NS5A aa variations was not correlated with treatment response in our cohort. When all available NS5A sequences were included, a higher number of non-conserved aa variations within PKR binding domain and an extended V3 region of NS5A was associated with virologic response (P = 0.004 and 0.05, respectively). Mutational analyses of a large number of NS5A sequences suggest, that a higher number of non-conserved aa variations within the PKR binding domain and the extended V3 region is correlated with virologic response in HCV 3a infected patients. Ankur Goyal and Wolf P. Hofmann contributed equally to this work.     

Hypervariable 5′-terminus of hepatitis C virus E2/NS1 encodes antigenically distinct variants

Synthetic peptides representing sequences encoded at the 5′-terminus of E2/NS1 in hepatitis C virus (HCV) were constructed. Peptides synthesized based on the sequences of four distinct HCV isolates were used to develop enzyme immunoassays (EIAs) for detection of antibodies in chronic HCV patients and in HCV-infected plasma donors. HCV sequence-specific antibodies were detected among patients with chronic HCV from the United States and Italy at frequencies of 22.2% and 55.8%, respectively. Similarly, sequence-specific antibodies were detected in 54.6% of U.S. and 55.6% of Japanese commercial plasma donors who had previous evidence of HCV exposure. Our data support earlier findings of geographic variability among HCV variants. The region encoded by amino acids (aa) 380–436 was shown to contain at least one variant-specific and one conserved epitope. The data further indicate that a majority of patients chronically infected with HCV (58.1% U.S., 68.8% Italy) have antibodies directed to the 5′-terminus of the E2/NS1 gene product. We conclude that genotypic variability within the E2/NS1 gene of HCV results in antigenically distinct variants. © 1993 Wiley-Liss, Inc.     

Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.     

HCV螺旋酶N端HLA-A2限制性CTL表位的变异和CTL免疫应答

目的 :研究丙型肝炎病毒 (HCV)螺旋酶N端HLA A2限制性细胞毒性T细胞 (CTL)表位的变异及其增殖反应。方法 :对两例慢性HCV感染者随访 7年 ,用第 1年和第 5年的外周血提取病毒RNA ,再以RT PCR扩增HCV螺旋酶N端基因 ,并亚克隆及测序。根据测序结果 ,合成CTL抗原表位肽段。用血清学方法进行HLA分型。从感染者第 7年外周血中分离淋巴细胞 ,检测CTL对抗原肽的增殖反应。结果 :在具有HLA A2表型的感染者中 ,第 1年病毒抗原表位的起始氨基酸均存在琥珀突变 ,5年后该突变消失。在无HLA A2表型的感染者中 ,其感染后 5年病毒抗原表位肽段既无共有序列的变异 ,也无琥珀突变。HCV感染后 7年 ,两例感染者的淋巴细胞对该抗原表位肽段均无增殖反应。结论 :螺旋酶N端抗原表位出现的无义突变 ,可能与病毒的免疫逃避有关。在慢性感染的后期 ,病毒感染者对螺旋酶N端的CTL表位肽不出现免疫应答。     

IL28B polymorphism is not associated with HCV protease diversity in patients co-infected with HIV and HCV treated with pegylated interferon and ribavirin

Recent studies have demonstrated that IL28B polymorphisms predict therapeutic responses in chronic hepatitis C virus (HCV)‐treated patients; however, the effect on HCV viral diversity, particularly on the HCV protease gene, is not clear. This study sought to evaluate the effect of IL28B polymorphisms on HCV diversity at NS3/4 protease region, which may influence therapeutic response to an HCV protease inhibitor based regimen. Twenty‐two patients co‐infected with HIV and HCV genotype 1, treatment‐naïve on stable HIV antiretroviral therapy initiating interferon‐based treatment were evaluated. Plasma HCV NS3 gene diversity was analyzed by clonal analysis at baseline and end of treatment. IL28B (rs12979860) genotypes were tested for associations with virologic outcomes and diversity parameters. There was similar baseline NS3 diversity in patients with CC (favorable) genotype compared to those with CT/TT (unfavorable) genotypes. There was no significant association between IL28B genotype and baseline NS3 nucleotide p‐distance, dS‐dN, amino acid p‐distance, or nucleotide changes. Among patients without a sustained virologic response, between baseline and follow‐up there was a significant trend towards decreased diversity after treatment among patients with favorable genotype, which was not observed in unfavorable genotypes. In patients treated with peginterferon/ribavirin therapy, IL28B polymorphism was not associated with enhanced NS3 diversity at baseline. Among non‐SVR patients with the less favorable genotype, there was no change in diversity after treatment. This suggests that IL28B genotype is unlikely to have a negative impact on subsequent HCV PI efficacy in patients co‐infected with HIV and HCV patients who have previously failed HCV therapy. J. Med. Virol. 84:1522–1527, 2012. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Mapping of serotype-specific, immunodominant epitopes in the NS-4 region of hepatitis C virus (HCV): use of type-specific peptides to serologically differentiate infections with HCV types 1, 2, and 3.

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenicity of the NS-4 protein was investigated by epitope mapping and by enzyme-linked immunosorbent assay with branched oligopeptides. Epitope mapping of the region between amino acid residues 1679 and 1768 in the HCV polyprotein revealed two major antigenic regions (1961 to 1708 and 1710 to 1728) that were recognized by antibody elicited upon natural infection of HCV. The antigenic regions were highly variable between variants of HCV, with only 50 to 60% amino acid sequence similarity between types 1, 2, and 3. Although limited serological cross-reactivity between HCV types was detected between peptides, particularly in the first antigenic region of NS-4, type-specific reactivity formed the principal component of the natural humoral immune response to NS-4. Type-specific antibody to particular HCV types was detected in 89% of the samples from anti-HCV-positive blood donors and correlated almost exactly with genotypic analysis of HCV sequences amplified from the samples by polymerase chain reaction. Whereas almost all blood donors appeared to be infected with a single virus type (97%), a higher proportion of samples (40%) from hemophiliacs infected from transfusion of non-heat-inactivated clotting factor contained antibody to two or even all three HCV types, providing evidence that long-term exposure may lead to multiple infection with different variants of HCV.     

Modulation of the peripheral T-Cell response by CD4 mutants of hepatitis C virus: transition from a Th1 to a Th2 response

A disturbing feature of hepatitis C virus (HCV) is its long-term persistence in roughly 85% of those infected. Escape mutants may play a major role in HCV persistence. Our previous studies have identified a human leukocyte antigen DRB1*15 (HLA-DRB1*15) restricted Th1 epitope in the HCV NS3 protein, NS3(358-375), and escape variants of this epitope that may emerge under immune selection. Such variants attenuate or fail to stimulate T-cell proliferation. Here we provide data from peripheral blood mononuclear cells derived from four HLA-DRB1*15 patients chronically infected with HCV, and report that naturally occurring single amino acid substitutions in the Th1 epitope NS3(358-375) fail to stimulate proliferation, which is accompanied by a shift in cytokine secretion patterns from one characteristic of a Th1 antiviral responses to a Th2 form. Further, in one patient, we demonstrate that HCV variant peptides can effectively inhibit host polyclonal peripheral T-cell proliferation. We speculate that this phenomenon may be a factor in chronic HCV infection.     

Usefulness of the phage display technology for the identification of a hepatitis C virus NS4A epitope recognized early in the course of the disease

A dodecapeptide phage-displayed library was screened with the mouse monoclonal antibody (mAb) 2E3C2 which competed with human antibodies for the binding to the HCV c100 recombinant protein. Four mimotopes shared a consensus motif with the HCV 1701-1707 sequence corresponding to the carboxyl-terminal domain of the non-structural protein NS4A. However, these mimotopes reacted with 2E3C2 only, whereas the corresponding NS4 epitope defined at the sequence 1698-1709 and displayed on phage was recognized by both 2E3C2 and sera from HCV infected patients. Using the Spot method of multiple peptide synthesis and alanine replacement analysis, the respective reactivities of mAb 2E3C2 and anti-NS4A human antibodies against NS4 were shown to be directed against two slightly different overlapping minimal linear sequences and to involve different critical residues. The phage clone displaying the NS4 epitope was used to study the specific recognition of this epitope by different individual HCV positive sera as well as by two seroconversion panels of sera from HCV infected patients. Compared with the detection by RIBA of the different HCV antigens and c100 particularly, these results indicated that the antibodies directed against the NS4 (1698-1709) epitope were produced early during the course of the disease and decreased later.     

Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.