Immune modulation by the hepatitis C virus core protein

Hepatitis C virus (HCV) infection is currently the most important cause of chronic viral hepatitis in the world and one of the most frequent indications for liver transplantation. HCV uses different strategies to evade the innate and adaptive immune response, and this evasion plays a key role in determining viral persistence. Several HCV viral proteins have been described as immune modulators. In this review, we will focus on the effect of HCV nucleocapsid core protein in the function of immune cells and its correlation with the findings observed in HCV chronically infected patients. Effects on immune cell function related to both extracellular and intracellular HCV core localization will be considered. This review provides an updated perspective on the mechanisms involved in HCV evasion related to one single HCV protein, which could become a key tool in the development of new antiviral strategies able to control and/or eradicate HCV infection.     

Immunohistochemical detection of hepatitis C virus-infected hepatocytes in chronic liver disease with monoclonal antibodies to core, envelope and NS3 regions of the hepatitis C virus genome.

The localization of hepatitis C virus-infected hepatocytes in the human liver remains unclear despite the development of a serological assay for the antibody to hepatitis C virus. We studied their localization immunohistochemically with monoclonal antibodies to core, envelope and NS3 antigens of hepatitis C virus. We examined 48 liver biopsy samples from C100-3 antibody-positive patients with chronic liver disease (chronic persistent hepatitis, 5 cases; chronic active hepatitis, 41 cases; cirrhosis, 2 cases) and 12 liver biopsy samples from C100-3 antibody-negative patients with chronic liver disease (type B chronic hepatitis, 8 cases; alcoholic liver disease, 4 cases). In the C100-3 antibody-positive group, positive immunostaining for core antigen, envelope antigen and NS3 antigen was found in 23% (11 of 48), 24% (11 of 45) and 24% (11 of 46), respectively. Negative results were obtained in the C100-3 antibody-negative group. Hepatocytes with positive staining were scattered in the lobules, and they were found in the same regions irrespective of whether the antibody to core antigen, to envelope antigen or to NS3 antigen was used. Each positive cell was strongly stained in the cytoplasm; these decorations disappeared after absorption of the primary antibody with purified antigen. mean ALT levels in the patients with positive immunostaining for core, envelope or NS3 antigen (174.8 +/- 105.7 U/L) tended to be higher than in those with negative immunostaining (142.0 +/- 93.8 U/L). On histological evaluation of liver specimens with a scoring system of the histological activity index, intralobular inflammation and fibrosis had higher scores for samples with positive rather than negative immunostaining (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

丙型肝炎病毒核心基因免疫诱生细胞免疫应答研究

目的 研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫在诱生特异性细胞免疫应答中的作用。方法 将包含ＨＣＶ Ｃ基因片段的重组真核表达质粒ｐｃＣＮＡ ＨＣＶ Ｃ,在主宰其可以在小鼠骨髓瘤ＳＰ２／０（Ｈ－２^d）中表达之后,注射ＢＡＬＢ／ｃ小鼠股四头肌。ＥＬＩＳＡ法检测血清中抗体水平;^3Ｈ－ＴｄＲ掺入法测定免疫小鼠脾细胞特异性增殖能力;^51Ｃｒ释放法检测免疫小鼠细胞毒性Ｔ淋巴细胞（ＣＴＬｓ）体外杀伤功能。结     

Immune responses in hepatitis C virus infection and mechanisms of hepatitis C virus persistence

Immune responses against hepatitis C virus (HCV) play a crucial role in the pathogenesis of chronic hepatitis C. HCV infection often persists and leads to chronic hepatitis and eventually cirrhosis. Accumulated data suggest that HCV proteins suppress host immune responses through the suppression of functions of immune cells, such as cytotoxic T lymphocytes, natural killer cells, and dendritic cells. They also suppress the type 1 interferon signaling system. The resulting insufficient immune responses against HCV lead to the sustained infection. The appropriate control of immune responses would contribute to the eradication of HCV and the improvement of hepatitis, but there are still many issues to be clarified. This review describes the scientific evidence to support these emerging concepts, and will touch on the implications for improving antiviral therapy.     

丙型肝炎病毒核心蛋白作用研究进展

丙型肝炎病毒(hepatitis C virus,HCV)是一种主要经过血液传播的肝炎病毒,是造成慢性肝炎、肝硬化甚至肝癌的主要原因之一.目前人们对HCV的致病机制的认识仍不很清楚,也缺乏针对HCV有效的治疗方法及疫苗预防.近年研究发现HCV核心(Core,C)蛋白除作为核壳体蛋白具有病毒颗粒组装功能外,还参与调节细胞凋亡、脂代谢、转录以及抗原呈递等作用,与干扰素抵抗也有密切关系.HCV C蛋白具有广泛的反式激活作用,与宿主细胞蛋白相互作用,是导致病毒持续性感染以及肝细胞癌发生的重要原因.深入认知和分析HCV C蛋白分子生物学特性,对阐明HCV持续性感染与致癌机制以及HCV对肝细胞脂肪变与干扰素疗效的影响等诸多问题具有重要意义.     

Full—length core sequence dependent complex—type glycosylation of hepatitis C virus E2 glycoprotein

AIM：To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV.The purpose of this study is to investigate the affection of context sequences on hepatitis C virus(HCV) E2 processing.METHODS:HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum ME2116 raised against E.coli-derived E2 peptide,respectively Deglycosylation analysis and GNA(Galanthus nivalus)lectin binding assay were performed to study the posttranslational processing of the expressed products.RESULTS:E2 glycoproteins with different molecular weights (-75kDa and -60kDa)were detected using S94 and ME2116,respectively.Deglycosylation analysis showed that this difference was mainly due to different glycosylation.Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form(75kDa)of E2 was complex-type glycosylated,which was readily recognized by homologous patient serum S94.Expression of complex-type glycosylated E2 could not be detected in all of the coretruncated constructs tested,but readily detected in constructs encoding full-length core sequences.CONCLUSION:The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly reprsented more mature forms of E2.As complex-type N-glycans indicated modification by Golgi enzymes,the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER.Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.     

Precore sequence variation in Chinese isolates of hepatitis B virus.

Direct sequencing of polymerase chain reaction-amplified serum hepatitis B virus (HBV) DNA was used to characterize the precore region of HBV from Chinese patients with chronic hepatitis. Two types of mutually exclusive variants were found in hepatitis B e antigen (HBeAg)-negative patients. The first (M1) contains a substitution from proline to serine at codon 15. A second group were infected with a previously described mutant (M2) containing a translational stop codon. HBeAg-positive patients were infected with the wild-type virus or the M1-containing strain. M2 emerged in patients with wild-type infection after seroconversion to anti-HBe, whereas M1 was present during the HBeAg-positive phase. In those with fluctuating HBe status, there was no correlation between prevailing HBe serology and sequence. There was an association between infection with variants and severe chronic hepatitis. Patients infected with strains containing M1 while HBeAg positive had a worse prognosis after seroconversion to anti-HBe.     

Mapping and characterization of B cell linear epitopes in the conservative regions of hepatitis C virus envelope glycoproteins

Forty-eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV-infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera obtained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immunodominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP.     

合并乙型肝炎病毒感染对丙型肝炎病毒核心抗原检测的影响

目的 探讨合并HBV感染对慢性HCV感染者血清丙型肝炎病毒核心抗原(HCVcAg)检出情况的影响. 方法 收集2005年12月-2009年10月慢性丙型肝炎患者和HBV/HCV合并感染者资料,检测血清HCVcAg和HCV RNA,对后者血清进行HBV DNA、HBeAg检测,分析HCVcAg检出率与HBeAg、HBV DNA定量检测的关系.用独立两组多分类的X2检验方法进行统计学分析. 结果 共收集88例慢性丙型肝炎患者和62例HBV/HCV合并感染者资料,血清HCVcAg的检出率分别为72.7％(64/88)和38.7％ (24/62),两者比较,x2= 17.358,P＜0.01,差异有统计学意义.HCV RNA检出率分别为81.8％ (72/88)和53.2％ (33/62),两者比较,x2=20.110,P＜0.01,差异有统计学意义.62例HBV/HCV合并感染者血清中,HBeAg阳性和HBeAg阴性感染者HCVcAg检出率分别为28.6％ (12/42)和60.0％ (12/20),两者比较,x2=5.641,P=0.011,差异有统计学意义.HCV RNA阳性率分别为42.9％ (18/42)和80.0％ (16/20),两者比较,X2=7.547,P＜ 0.01,差异有统计学意义.HBV DNA阳性和阴性时HCVcAg检出率分别为39.1％ (18/46)和37.5％ (6/16),两者比较,P＞0.05,差异无统计学意义.与单纯HCV感染者血清HCVcAg检出率72.7％ (64/88)比较,HBeAg阴性合并感染者为60.0％ (12/20),x2=1.266,P=0.261,差异无统计学意义;HBV DNA阴性合并感染者为37.5％ (6/16),x2=7.635,P＜0.01,差异有统计学意义.结论 HBV/HCV合并感染时HCVcAg检出率较低,可能是由于HBeAg抑制HCV的复制,从而减少HCVcAg的表达所致.     

IL-28B基因变异与丙型肝炎的易感性及其自然清除的关系

目的探讨IL-28B基因变异与丙型肝炎的易感性和丙型肝炎病毒（HCV）自然清除之间的关系。方法利用焦磷酸测序的方法对411例HCV感染者（男性225人,女性186人）及100例健康对照人群进行了IL-28B基因变异位点rs12979860的多态性检测;对HCV感染者进行病毒定量的检测,比较HCV感染人群和健康对照人群,慢性丙型肝炎（CHC）人群和丙型肝炎自愈人群IL-28B基因变异位点的差异以及不同基因型的携带者病毒定量的差异。结果中国健康人群IL-28B基因变异位点rs12979860的最小等位基因频率为6%,与日本人相近,与欧洲人群相差较大。SNP（rs12979860）位点的等位基因频率和基因型频率在HCV感染人群和健康对照人群之间差异无统计学意义（P=0.066;P=0.072）,C/C基因型CHC人群病毒定量水平高于C/T基因型,二者之间差异有统计学意义（P=0.02）;丙型肝炎自愈人群中C/C基因型和C/T基因型差异无统计学意义（P=0.64）。结论 CHC人群rs12979860 SNP基因型与健康人群表达无明显差别,该基因型（rs12979860）不是HCV的易感基因,其CC基因型并不能预测丙型肝炎发生自愈的病程。     

Immune regulation in chronic hepatitis C virus infection

The immunological result of infection with Hepatitis C virus (HCV) depends on the delicate balance between a vigorous immune response that may clear the infection, but with a risk of unspecific inflammation and, or a less inflammatory response that leads to chronic infection. In general, exhaustion and impairment of cytotoxic function of HCV-specific T cells and NK cells are found in patients with chronic HCV infection. In contrast, an increase in immune regulatory functions is found primarily in form of increased IL-10 production possibly due to increased level and function of anti-inflammatory Tregs. Thus, the major immune players during chronic HCV infection are characterized by a decrease of cytotoxic function and increase of inhibitory functions. This may be an approach to diminish intrahepatic and systemic inflammation. Finally, there has been increasing awareness of regulatory functions of epigenetic changes in chronic HCV infection. A vast amount of studies have revealed the complexity of immune regulation in chronic HCV infection, but the interplay between immune regulation in virus and host remains incompletely understood. This review provides an overview of regulatory functions of HCV-specific T cells, NK cells, Tregs, IL-10, and TGF-β, as well as epigenetic changes in the setting of chronic HCV infection.     

Clinical impact of hepatitis B and C virus envelope glycoproteins

Chronic infection by either hepatitis B virus(HBV)or hepatitis C virus(HCV)share epidemiological characteristics with risks for development of severe complications such as liver cirrhosis and hepatocellular carcinoma.HBV and HCV also share a high genetic variability. Among highly variable regions,viral genes encoding surface proteins(hepatitis B surface antigen,E1/E2 HCV glycoproteins)play key roles in the stimulation of the host-related immune response and viral entry into hepatocytes.Specific segments of HBV envelope proteins(preS1,"a"determinant)are crucial in the entry process into permissive cells.HCV entry is a complex multistep process involving multiple cell cofactors (glycosaminoglycans,low density lipoprotein receptor, SR-B1,CD81,claudin-1,occludin,EGFR,EphA2)in the interaction with HCV E1/E2 envelope glycoproteins.In vitro both viruses can be controlled by antibody-me-diated neutralization targeting viral envelope,also essential in preventing HBV infection in vivo as observed through successful vaccination using HBs antigen.But preventive vaccination and/or therapeutic pressure can influence HBV and HCV variability.For HBV,the patterns of antiviral drug resistance in chronic hepatitis are complex and the original pol/S gene overlap has to be taken into account.Treatment-induced HBV mutations in pol could indeed generate S mutants with subsequent modified antigenicity or increased cancer induction.Variability of HBV and HCV envelope proteins combining high exposure to selective pressures and crucial functional roles require investigation in the context of diagnostic,vaccination and treatment tools.In this editorial a synthesis is performed of HBV and HCV envelope properties at the entry step and as antigenic proteins,and the subsequent clinical impact.     

核心蛋白和非结构蛋白5B 直接测序法对丙型肝炎病毒基因分型的影响

目的：分析 HCV 核心蛋白（Core）和非结构蛋白（NS）5B 区直接测序的敏感性和准确性。方法收集慢性丙型肝炎患者血清51份,采用反转录 PCR 对 Core 和 NS5B 区分别扩增,产物直接测序,构建进化树进行基因分型。结果51份样本中,Core 区扩增阳性49份,占96．1％。共检出5种基因亚型：1b 为61．2％（30／49）,2a 为20．4％（10／49）,2b 为2．0％（1／49）,3a 为4．1％（2／49）,6a 为12．2％（6／49）。NS5B 区扩增阳性45份,占88．2％。1b、2a、2b、3a、6a 分别占62．2％（28／45）、20．0％（9／45）、2．2％（1／45）、4．4％（2／45）和11．1％（5／45）。结论与 NS5B 区相比,Core 区引物设计容易,扩增效率和分型成功率较高,可作为 HCV 基因型流行病学调查和临床研究的优先选择。     

Association between hepatitis C virus core protein and carotid atherosclerosis.

A link between certain infectious microorganisms and an increased risk of atherosclerotic disease has been suggested. By analyzing the data of subjects who had undergone general health-screening tests, a possible association between carotid atherosclerosis and seropositivity of antibody against hepatitis C virus (HCV) has been previously reported. In the present study, a possible link between carotid atherosclerosis and HCV core protein positivity was assessed, because it is postulated to be a better marker of viremia and thus persistent infection. Of the 1992 enrolled subjects, 496 (25%) had carotid artery plaque, and 25 (1.3%) were positive for HCV core protein. Carotid artery plaque was positive in 480/1967 (24%) and 16/25 (64%) of the core protein-negative and core protein-positive subjects, respectively (p<0.0001 by chi(2) test). Serum concentrations of transaminases were higher in core protein-positive subjects, but albumin concentrations were not significantly different between the 2 groups. Multivariate logistic regression analysis showed that HCV core protein positivity is an independent predictor of carotid plaque with an odds ratio of 5.61 (95% confidence interval 2.06-15.26, p<0.001). These data further support the possible link between persistent HCV infection and carotid atherosclerosis in the subjects without severe liver dysfunction.     

Quantification of serum hepatitis C virus core protein level in patients chronically infected with different hepatitis C virus genotypes.

BACKGROUND/AIM: A novel fluorescent enzyme immunoassay (FEIA) for the detection and quantification of serum hepatitis C virus (HCV) core protein was developed. The aim of this study was to evaluate the relation among serum HCV core protein level, HCV RNA level, and HCV genotype in patients with chronic HCV infection. PATIENTS AND METHODS: Serum HCV core protein, HCV RNA, HCV genotype were determined in 175 patients using the FEIA, branched DNA assay (Quantiplex HCV RNA ver 1.0), and serologically defined genotyping assay, respectively. For the specificity, all 13 patients seronegative for anti-HCV were negative for serum core antigen and HCV RNA by FEIA and bDNA, respectively. RESULTS: FEIA assay seems to be more sensitive than bDNA for patients with HCV type 2 infection (detection: 83.4% v 63.4%, p < 0.01). There was a good overall correlation between the FEIA and bDNA results. However, when the patients were stratified into their HCV types, a correlation was observed in HCV type 1 but not in type 2 infection. Patients with HCV type 2 infection had a lower serum HCV core protein level (median 56 RFI) compared with type 1 infection (median 149 RFI, p < 0.01). Thirty seven patients subsequently received interferon alpha therapy, patients who showed a complete and sustained response had a lower pretreatment serum HCV core protein level compared with patients who had a relapse and nonresponders (36 v 338 RFI, p < 0.01). CONCLUSIONS: This study showed that FEIA (1) is a good assay for the detection and quantification of serum HCV core protein level, (2) is also very sensitive in detecting HCV core protein in patients with HCV type 2 infection, and (3) may have a role as a predictor of subsequent response to interferon therapy.     

Intra-familial transmission of hepatitis B virus in Italy: phylogenetic sequence analysis and amino-acid variation of the core gene

BACKGROUND/AIMS: Transmission of hepatitis B virus (HBV) in countries of intermediate endemicity, such as Italy, is thought to be primarily horizontal and, to a lesser extent, vertical. Most chronic carriers therefore become infected in infancy or at a very young age. The index cases in such events have been assumed to come from within the family unit or from sources outside the immediate family, with whom the affected person is in close contact. METHODS: We studied a number of Italian families with multiple members chronically infected with HBV. The precore/core region of the virus was amplified from serum derived HBV-DNA, and the sequences subjected to phylogenetic tree analysis. In addition, the extent of amino-acid variation within the core region was correlated to HLA type, determined by allele-specific PCR. RESULTS: The phylogenetic tree analysis provided strong evidence of intra-familial transmission of the virus. Analysis of amino-acid substitutions in the core region in relation to HLA class II alleles from members of the same family showed that these substitutions were restricted in siblings with concordant, and more diverse in those with discordant HLA haplotypes. CONCLUSIONS: This is consistent with major histocompatibility complex class II mediated selection pressure on the virus.     

Immune and non-immune responses to hepatitis C virus infection

The host innate and adaptive immune systems are involved in nearly every step of hepatitis C virus(HCV) infection. In patients,the outcome is determined by a series of complex host-virus interactions,whether it is a natural infection or results from clinical intervention. Strong and persistent CD8+ and CD4+ T-cell responses are critical in HCV clearance,as well as cytokineinduced factors that can directly inhibit virus replication. Newly available direct-acting antivirals(DAAs) are very effective in viral clearance in patients. DAA treatment may further result in the down-regulation of programmed death-1,leading to rapid restoration of HCV-specific CD8+ T cell functions. In this review,we focus on recent studies that address the host responses critical for viral clearance and disease resolution. Additional discussion is devoted to the prophylactic vaccine development as well as to current efforts aimed at understanding the host innate responses against HCV infection. Current theories on how the ubiquitin system and interferon-stimulated genes may affect HCV replication are also discussed.