从HCV蛋白一级结构预测其C,E,NS5区的CD4^＋T细胞抗…

我们根据ＣＤ４＾＋Ｔ细胞识别抗原位占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结构（ａｍｐｈｉｐａｔｈｉｃ ｈｅｌｉｘ ｓｔｒｕｃｔｕｒ）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）、Ｎ５蛋白一级结构进行分析，发现这些蛋白区存在的ＣＤ４＾＋Ｔ细胞识别位点。此结果支持了ＣＤ４＾＋Ｔ细胞对ＨＣＶ Ｃ，Ｅ，ＮＳ５区可发生增殖反应的结论。提示该程度可作为一种预测Ｃ     

丙型肝炎病毒结构蛋白在痘苗病毒中的表达

为研究中国丙型肝炎病毒（ＨＣＶ）的抗原性及在细胞内的加工，将丙型肝炎病毒（ＨＣＶ）５’非编码区（ＮＴＲ）和结构基因（Ｃｏｒｅ＋Ｅ１＋Ｅ２／ＮＳ１）插入痘苗病毒表达载体ｐＪＳＡ１１７５中，转染ＴＫ－１４３细胞，经纯化得到丙型肝炎（ＨＣＶ）重组痘苗病毒ｖＪＳＡ１１７５ＣＥ株。Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交表明，ＨＣＶ结构基因存在于痘苗病毒之中。Ｗｅｓｔｅｒｎｂｌｏｔ分析发现，ｖＪＳＡ１１７５ＣＥ表达蛋白带位于９０ｋＤａ，为一多聚蛋白；此蛋白为分泌型，分泌量与细胞裂解物内量大致相同     

鼠抗丙型肝炎病毒（HCV）NS3区单克隆抗体的研制及特性分析

用淋巴细胞杂交瘤技术，制备出５株抗ＨＣＶ－ＮＳ３区单克隆抗体（ＭｃＡｂ）分别命名为Ｃ７－１，３，６，８，９。单抗特性测定结果显示５株单抗均为ＨＣＶ－ＮＳ３区特异性，与ＨＣＶ其它区域的抗原及宿主菌成份均无交叉反应，此５株单抗识别ＮＳ３区上两个不完全相关的抗原位点，其中Ｃ７－１，３，６株识别同一抗原决定簇，Ｓ７－９的识别位点与此３株有一定的相关，Ｃ７－８则识别与此４株不完全相关的位点。ＨＣＶ－ＮＳ３抗     

MTEC 1分泌的趋化因子引起特定亚群胸腺细胞的定向迁移

分析胸腺髓质上皮样细胞系ＭＴＥＣ１分泌的化学趋化因子对胸腺细胞亚群的趋化作用。方法以抗体加补体杀伤结合免疫磁珠及ｐａｎｎｉｎｇ法，将小鼠胸腺细胞分离纯化，获得ＣＤ４＋ＣＤ８＋（ＤＰ），ＣＤ４－ＣＤ８－（ＤＮ），ＣＤ４＋ＣＤ８－（ＣＤ４ＳＰ）及ＣＤ４－ＣＤ８＋（ＣＤ８ＳＰ）四亚群细胞，用Ｂｏｙｄｅｎ小室分析ＭＴＥＣ１┐ＳＮ对四群胸腺细胞的趋化作用。结果ＭＴＥＣ１┐ＳＮ对ＤＰ及ＣＤ４ＳＰ胸腺细胞有趋化活性（ＣＩ＝６．６±１．０及６．１±１．８）；对ＣＤ８ＳＰ细胞有中度趋化活性（ＣＩ＝３．２±１．０）；对ＤＮ趋化活性微弱（ＣＩ＝１．３±０．６）。化学趋化因子ＭＣＰ┐１纯品对ＣＤ４ＳＰ胸腺细胞显示强趋化活性（ＣＩ＝５．６），对ＤＮ胸腺细胞则无可测出趋化活性。结论ＭＴＥＣ１分泌的化学趋化因子对ＤＰ，ＣＤ４ＳＰ及ＣＤ８ＳＰ胸腺细胞有显著趋化作用，对ＤＮ胸腺细胞几乎无趋化作用。提示此类化学趋化因子有趋使胸腺发育中后期阶段的细胞向胸腺髓质区迁移和定位的作用。     

STUDY ON PERIPHERAL BLOOD MONONUCLEAR CELL(PBMC) PROLIFERATIVE RESPONSES TO DIFFERENT HCV ANTIGENS IN PATIENTS WITH HEPATITIS C

为探讨丙型肝炎（ＨＣ）病人细胞免疫功能和丙型肝炎病毒（ＨＣＶ）的致病机制及机体对其免疫保护作用,收集２４例ＨＣ病人（急性３例,慢性２１例）,用３Ｈ－ＴｄＲ掺入法研究病人外周血单个核细胞（ＰＢＭＣ）对不同ＨＣＶ抗原增殖反应,并用流式细胞仪（ＦＡＣＳ）检测了ＰＢＭＣ中ＣＤ４＋、ＣＤ８＋淋巴细胞亚群在ＨＣＶ抗原刺激后的变化。结果：ＨＣ病人ＰＢＭＣ对ＨＣＶ合成肽ＣＰ９,ＮＳ４和基因重组抗原Ｃ,Ｅ１,Ｅ２,ＮＳ３刺激后出现不同程度增殖反应,刺激指数（ＳＩ）分别为１．６９±０．５１,１．６１±０．５４,１．６８±０．５８,１．４９±０．４４,１．４４±０．４４和１．３３±０．３３。３例急性ＨＣ中２例病人的ＰＢＭＣ对ＨＣＶ抗原呈有效增殖反应（ＳＩ≥２．１）,且血清ＨＣＶＲＮＡ阴转伴ＡＬＴ正常。细胞表型分析显示：增殖的细胞表型是ＣＤ４＋淋巴细胞,而ＣＤ８＋淋巴细胞增殖反应较弱。结论：ＨＣ病人ＰＢＭＣ确实存在对ＨＣＶ抗原的增殖反应;ＣＤ４＋淋巴细胞比ＣＤ８＋淋巴细胞增殖反应要强,急性ＨＣ病人ＰＢＭＣ对ＨＣＶ抗原有效的增殖反应预示可能有良好的临床愈合     

五种丙型肝炎病毒(HCV)基因重组抗原斑点酶免疫反应检测血清抗-HCV

五种丙型肝炎病毒（ＨＣＶ）基因重组抗原斑点酶免疫反应检测血清抗－ＨＣＶ谭德明刘双虎胡国龄刘安国刘国珍应用五种丙型肝炎病毒（ＨＣＶ）基因重组抗原，包括ＨＣＶ核心蛋白（Ｃｏｒｅ），包膜蛋白１（Ｅ１）、包膜蛋白２（Ｅ２／ＮＳ１）、ＮＳ３和ＮＳ５抗原〔１，２...     

丙型肝炎病毒NS3蛋白单克隆杂交瘤细胞株的建立及初步鉴定

应用国产基因工程表达的丙型肝炎病毒（ＨＣＶ）ＮＳ３区抗原免疫小鼠，然后取其脾细胞与小鼠骨髓瘤细胞系ＳＰ２／０融合，筛选出４株稳定分泌抗ＨＣＶＮＳ３区蛋白单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株，分别命名为２Ｂ６，２Ｆ３，３Ｄ８，３Ｄ９，经初步研究表明这４株单抗与ＮＳ３抗原具有良好的反应性，与ＨＣＶ核心区多肽及乙型肝炎病毒表面抗原和ｅ抗原均无反应。抑制实验表明这４株抗体分别针对ＮＳ３抗原分子上的２个不同的抗原决定簇。     

小鼠胸腺基质细胞系（MTEC1）对裸鼠骨髓细胞的诱…

在体外建立的小鼠胸腺基质细胞系（ＭＴＥＣ１）及其培养上清（ＭＴＥＣ１－ＳＮ）与裸鼠骨髓（ＢＭ）细胞共同培养，通过直接荧光素标记抗体，ＦＡＣＳ分析其细胞表面标志表明，ＭＴＥＣ１及ＭＴＥＣ１－ＳＮ均有诱导促进Ｔｈｙ·１＾－ＣＤ＾－４ＣＤ＾－８的ＢＭ细胞表达Ｔｈｙ·１，ＣＤ４及ＣＤ８分子的作用。在培养或共育三天时，ＭＴＥＣ１－ＳＮ可促进ＢＭ细胞分化形成ＣＤ＾＋４Ｔｈｙ·１＾－，ＣＤ＾－４Ｔｈｙ·１＾＋细     

McAb共激活T细胞介导肝癌细胞前后的膜分子与TCRVβ基因的表达

Ｔ淋巴细胞活化是一个涉及多种膜表面分子和受体以及一系列相关多肽的复杂过程，为了使Ｔ细胞发挥更好的识别和杀伤癌细胞的功能，采用抗ＣＤ３、ＣＤ２８、ＣＤ８０（Ｂ７１）、ＣＤ２、ＣＤ５８ＭｃＡｂ分别刺激健康人ＰＢＬｓ后作用肝癌细胞，对作用前后ＰＢＬｓ用ＦＡＣＳ进行表型分析，结果发现：作用后ＣＤ３和ＣＤ８分子表达比作用前明显增高，而ＣＤ４分子无显著变化，同时基因家族采用ＲＴＰＣＲＳｏｕｔｈｅｒｎ印迹分析ＴＣＲＶβ基因１～２０亚家族表达水平与特征，健康人ＰＢＬｓ分别加入ＩＬ２、ＰＨＡ、抗ＣＤ３和ＣＤ３＋ＣＤ２８、ＣＤ２８＋ＣＤ８０、ＣＤ２＋ＣＤ５８作用肝癌细胞（ＢＥＬ７４０２）前表达水平平均约为５％，作用ＢＥＬ７４０２后表达水平约为１３％～２５％，其特征为Ｖβ７增高，这提示在癌抗原的参与下ＭｃＡｂ共刺激的Ｔ细胞活化，ＴＣＲ接受ＡＰＣ相应抗原的刺激，具有该ＴＣＲ的淋巴细胞迅速增殖而成为针对抗原的Ｔ细胞克隆，发挥其识别和杀伤癌细胞的作用，这将为肿瘤生物治疗的研究提供分子免疫学依据。     

PEA单抗相对亲和力及抗原结合位点的测定

用绿脓杆菌外毒素Ａ（ＥＰＡ）免疫后的ＢＡＬＢ／Ｃ小鼠脾细胞，经融合后制备出４株抗ＰＥＡ的单克隆抗体。从５０％最大结合的单克隆抗体浓度分析可知，４株单克隆抗体的相对亲和力为４Ｄ１－４Ｄ３〉１Ｄ１－５Ｆ１１〉１Ｄ７－４Ｃ１０〉４Ｈ５－３Ｅ９。通过ＥＬＩＳＡ相加实验证实：４株单克隆抗体均识别相同的ＰＥＡ抗原位点。     

HCV感染外周血单个核细胞及其对T淋巴细胞亚群的影响

目的：研究丙型肝炎病毒（ＨＣＶ）感染外周血单个核细胞（ＰＢＭＣ）的情况及其对Ｔ淋巴细胞亚群的影响。方法：运用非同位素原位杂交（ＮＩＳＨ）法和链酶亲和素－生物素（ＳＡＢＣ）法分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ－ＲＮＡ和非结构（Ｎｏｎｓｔｒｕｃｔｕｒａｌ,ＮＳ）蛋白ＮＳ５抗原,同时用ＳＡＢＣ法检测其Ｔ淋巴细胞亚群。结果：８例（４０．０％）患者的ＰＢＭＣ中ＨＣＶ－ＲＮＡ呈构（Ｎｏｎｓｔｒｕ     

Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection

The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, E2/NS1, NS2, NS3, NS4, and NS5 regions. The U. S. Food and Drug Administration-licensed 2.0G immunoassay for the detection of anti-HCV uses proteins from the core, NS3, and NS4 regions (McHutchinson et al., Hepatology 15:19-25, 1992). The 3.0G enzyme-linked immunosorbent assay includes the protein from the NS5 region (Uyttendaele et al., Vox Sang. 66:122-129, 1994). The necessity of detecting antibodies to viral envelope proteins (E1 and E2) and to different genotype samples has been demonstrated previously (Chien et al., Lancet 342:933, 1993; Lok et al., Hepatology 18:497-502, 1993). In this study we have attempted to improve the sensitivity of the anti-HCV assay by developing a single multiple-epitope fusion antigen (MEFA; MEFA-6) which incorporates all of the major immunodominant epitopes from the seven functional regions of the HCV genome. A nucleic acid sequence consisting of proteins from the viral core, E1, E2, NS3, NS4, and NS5 regions and different subtype-specific regions of the NS4 region was constructed, cloned, and expressed in yeast. The epitopes present on this antigen can be detected by epitope-specific monoclonal and polyclonal antibodies. In a competition assay, the MEFA-6 protein competed with 83 to 96% of genotype-specific antibodies from HCV genotype-specific peptides. This recombinant antigen was subsequently used to design an anti-HCV chemiluminescent immunoassay. We designed our assay using a monoclonal anti-human immunoglobulin G antibody bound to the solid phase. Because MEFA-6 is fused with human superoxide dismutase (h-SOD), we used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection. Our results indicate that MEFA-6 exposes all of the major immunogenic epitopes. Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.     

Characterization of tick-borne encephalitis virus-specific human T lymphocyte responses by stimulation with structural TBEV proteins expressed in a recombinant baculovirus

Very little information is available on human T cell responses following exposure to tick-borne encephalitis virus (TBEV) proteins, largely because the virus is a dangerous pathogen and relatively large amounts of purified antigen would be required for the functional characterization of cellular immune responses. We have produced recombinant TBEV proteins using the baculovirus expression system and tested them for their capacity to stimulate T cells in vitro. T lymphocytes from TBEV vaccinated individuals were characterized. The recombinant E and C proteins triggered CD4+ but not CD8+ cells to proliferate and to produce IFN-gamma and IL-5. T cell responses against recombinant NS3 protein were not detected. T cell lines with specificity for the E protein were also established. These lines were CD4+ and had a TH0 cytokine production pattern. Our results demonstrate the utility of recombinant viral proteins to study the generation and characterization of TBEV specific T cell responses.     

Priming and stimulation of hepatitis C virus-specific CD4+ and CD8+ T cells against HCV antigens NS4, NS5a or NS5b from HCV-naive individuals: implications for prophylactic vaccine

Hepatitis C virus (HCV) is a devastating human pathogen, yet there is no vaccine available for this virus. From studies with acute or chronic HCV-infected humans and chimpanzees, T-cell responses against HCV-derived conserved non-structural antigens have been correlated with viral clearance. In this study, recombinant adenoviral vectors containing HCV-derived NS4, NS5a or NS5b genes were employed to endogenously express the HCV antigens in human dendritic cells (DCs). The DCs expressing these HCV antigens exhibited normal phenotype and function. Intriguingly, we found that the DCs expressing HCV NS4, NS5a or NS5b antigens were able to significantly stimulate autologous T cells obtained from uninfected healthy individuals. These T cells produced various cytokines and proliferated in an HCV antigen-dependent manner. Evidence of both CD4(+) and CD8(+) T-cell responses generated in vitro against HCV NS4, NS5a or NS5b were obtained. HCV NS4 was much less stimulatory for CD4(+) and CD8(+) T cells than NS5. Further, in secondary assays, the CD4(+) T cells primed in vitro exhibited HCV antigen-specific proliferative responses against recombinant protein antigens. In summary, we provide conclusive evidence of in vitro stimulation of CD4(+) and CD8(+) T cells from HCV-naive individuals against HCV antigens NS4, NS5a and NS5b. The studies with naive T cells represent early events in the induction of cellular immune responses, which most likely govern the outcome of HCV infection. These studies have significant implications in designing vaccines for HCV infection in both prophylactic and therapeutic settings.     

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。     

Molecular epidemiology of hepatitis C virus among different groups of people in the province of Fujian, China

Genotyping of hepatitis C virus (HCV) can provide valuable information for prognosis and treatment duration prediction. To explore the genetic diversity of HCV in Fujian Province, China, 112, 104 and 48 anti-HCV-positive serum samples were collected from volunteer blood donors, IDUs and patients, respectively, from Jan 2008 to Dec 2008 and were genotyped through sequence analysis, followed by phylogenetic analysis in the C/E1 and NS5B regions. Genotypes could be determined for 85.61 and 84.85 % of samples in the C/E1 and NS5B region, respectively. 6a was the most prevalent subtype, which accounted for 42.04 and 43.75 % in the C/E1 and NS5B region, respectively. Mixed infection and potential recombination were detected in this study. Kappa tests indicated that similar results were obtained by two genotyping methods targeting the C/E1 and NS5B regions. The differences in the main prevalent subtype between the three target groups suggest diversity of HCV prevalence in different populations.     

Diagnostic possibilities of an immunoenzyme test-system for determining the spectrum of antibodies to structural and nonstructural hepatitis C virus antigens

A new enzyme immunoassay EIA-HCV-Spectr test system constructed on the base of recombinant proteins and synthetic peptides allows separate detection of antibodies to E1/E2, core, HS3, NS4, and NS5 antigens of hepatitis C virus (HCV). The system is highly specific and more sensitive than the test systems used in screening studies, which allows its use as a final test for antiHCV antibodies. Antibodies to various HCV antigens were analyzed using this test system in patients with acute and chronic hepatitis C and asymptomatic donors with antiHCV. In acute hepatitis C during the first-second week after clinical manifestation, antibodies to nonstructural virus proteins are detected 3-4 times less often than in chronic hepatitis C. Acute hepatitis C is characterized by the presence of antibodies only to core antigen (66%). In chronic condition combinations of antibodies to structural and nonstructural HCV antigens predominate: core + NS4, core + NS3 + NS4, core + NS3 + NS5, core + NS4 + NS5, and core + NS3 + NS4 + NS5. In asymptomatic donors with antiHCV and in patients with chronic hepatitis C the spectra of antibodies were similar in 45.7% cases.     

Design of novel conformational and genotype-specific antigens for improving sensitivity of immunoassays for hepatitis C virus-specific antibodies

The current commercially licensed enzyme-linked immunosorbent assays (ELISAs) for hepatitis C virus (HCV) mainly use recombinant proteins containing linear epitopes. There is evidence, however, that conformational epitopes of HCV are more immunoreactive. Thus, we have designed an HCV antibody assay that employs a conformational protein, NS3NS4a PI (with functional protease and helicase activities), and a linear fusion protein, multiple-epitope fusion antigen 7.1 (MEFA 7.1) or MEFA 7.2. We have shown that NS3NS4a PI detects early-seroconversion conformation-sensitive antibodies better than c33c antigen. The correct conformation of NS3NS4a PI also cross-reacts with different genotype samples better than the c33c antigen. MEFA 7.1 and MEFA 7.2 incorporate all the major immunodominant and genotype-specific epitopes of HCV core, E1, E2 hypervariable region 1 (HVR1), E2 HVR1-plus-HVR2 consensus, NS3, NS4, and NS5. Since MEFA 7.1 is degraded by the active NS3NS4a PI protease, we designed a second MEFA 7.2 construct in which the six protease cleavage sites found in MEFA 7.1 were eliminated by amino acid mutation. We demonstrate here that MEFA 7.2 remains intact in the presence of NS3NS4a PI and preserves the epitopes present in MEFA 7.1. Compared to currently licensed assays, an ELISA incorporating a combination of the two antigens NS3NS4a PI and MEFA 7.1 or 7.2 demonstrates better serotype sensitivity and detects seroconversion earlier in many commercially available panels. We believe that an assay using NS3NS4a PI and MEFA 7.1 or 7.2 may have the potential to replace current HCV immunoassays for better sensitivity.     

Hepatitis C virus-related resistance mechanisms to interferon alpha-based antiviral therapy.

Only 50-60% of the patients chronically infected with the hepatitis C virus (HCV) achieve a sustained virologic response to the current standard antiviral therapy consisting of pegylated interferon alpha in combination with ribavirin. The definite reasons for virologic response or non-response to interferon alpha-based therapy are unknown. Besides host and treatment efficacy factors, it is presumable that HCV is able to antagonize the antiviral activity of interferon alpha. So far, among the different HCV proteins, the envelope (E)2 protein, the non-structural (NS)3/4A protein, and the NS5A protein have been associated with interferon alpha resistance mechanisms in vitro. The clinical significance of amino acid mutations within these HCV proteins in HCV isolates from patients who did or did not respond to interferon alpha-based therapy was investigated in multiple studies. Within the E2 (HVR2, CD81 binding sites, PePHD) and the NS3/4A proteins no specific mutations in correlation with virologic response to interferon alpha-based therapy were observed. For the NS5A protein, mutations within the interferon sensitivity determining region (ISDR) and the complete NS5A protein may be of importance for response to interferon alpha-based treatment in patients infected with HCV subtype 1a/b.     

Trans-complementation of HCV replication by non-structural protein 5A

Although establishment of the subgenomic replicon system has considerably facilitated genetic analysis of HCV replication, many details remain largely unknown. To initially test whether HCV replication could be affected in trans, complementation studies were conducted in which defective replicon RNAs carrying a luciferase reporter were introduced into stable cells bearing functional replicons. The NS3 protease and the NS5B viral polymerase genes on the transfected replicons were rendered null by active site mutations and shown not to be complemented in trans by functional proteins expressed from the endogenous replicons. A new strategy was also developed to examine whether adapted copies of NS4B and NS5A could enhance the replication of transfected replicons carrying non-adapted genes. The replication efficiency of a replicon carrying two adaptive mutations in NS3 (E1202G and T1280I) had previously been shown to be greatly enhanced by the presence of a third mutation in either NS4B (K1846T) or NS5A (S2197P). A partially adapted luciferase replicon carrying only the two NS3 mutations was used to transfect cells containing replicons bearing the adapted NS4B or NS5A. Using this approach, NS5A, but not NS4B, was found to trans-complement. In a final confirmatory study, ectopically expressed NS5A also complemented the HCV replicon genome bearing the non-adapted NS5A. These studies strongly suggest that HCV non-structural proteins, with the exception of NS5A, can only act in cis on the RNA from which they were translated.