A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice

It has been shown previously that measles virus (MV) can be successfully used to express foreign proteins (M. Singh and M. A. Billeter, J. Gen. Virol. 80:101-106, 1998). To develop an inexpensive MV-based vaccine, we generated recombinant MVs that produce structural proteins of hepatitis B virus (HBV). A recombinant virus that expressed the HBV small surface antigen (HBsAg) was analyzed in terms of its replication characteristics, its genetic stability in cell culture, and its immunogenic potential in genetically modified mice. Although this virus showed a progression of replication slightly slower than that of the parental MV, it appeared to stably maintain the added genetic information; it uniformly expressed the appropriately glycosylated HBsAg after 10 serial passages. Genetically modified mice inoculated with this recombinant MV produced humoral immune responses against both HBsAg and MV proteins.     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.     

Hepatitis B virus x protein induces perinuclear mitochondrial clustering in microtubule- and Dynein-dependent manners

The hepatitis B virus (HBV) X protein (HBx) is thought to play a key role in HBV replication and the development of liver cancer. It became apparent that HBx induces mitochondrial clustering at the nuclear periphery, but the molecular basis for mitochondrial clustering is not understood. Since mitochondria move along the cytoskeleton as a cargo of motor proteins, we hypothesized that mitochondrial clustering induced by HBx occurs by an altered intracellular motility. Here, we demonstrated that the treatment of HBx-expressing cells with a microtubule-disrupting drug (nocodazole) abrogated mitochondrial clustering, while the removal of nocodazole restored clustering within 30 to 60 min, indicating that mitochondrial transport is occurring in a microtubule-dependent manner. The addition of a cytochalasin D-disrupting actin filament, however, did not measurably affect mitochondrial clustering. Mitochondrial clustering was further studied by observations of HBV-related hepatoma cells and HBV-replicating cells. Importantly, the abrogation of the dynein activity in HBx-expressing cells by microinjection of a neutralizing anti-dynein intermediate-chain antibody, dynamitin overexpression, or the addition of a dynein ATPase inhibitor significantly suppressed the mitochondrial clustering. In addition, HBx induced the activation of the p38 mitogen-activated protein kinase (MAPK) and inhibition of the p38 kinase activity by SB203580-attenuated HBx-induced mitochondrial clustering. Taken together, HBx activation of the p38 MAPK contributed to the increase in the microtubule-dependent dynein activity. The data suggest that HBx plays a novel regulatory role in subcellular transport systems, perhaps facilitating the process of maturation and/or assembly of progeny particles during HBV replication. Furthermore, mitochondrion aggregation induced by HBx may represent a cellular process that underlies disease progression during chronic viral infection.     

Identification of HLA-A*0201-restricted cytotoxic T-cell epitopes of Trypanosoma cruzi TcP2beta protein in HLA-transgenic mice and patients

Trypanosoma cruzi-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of parasite growth and will play an important part in therapeutic and prophylactic T. cruzi vaccines. The identification of parasite-specific epitopes that are efficiently recognized by CTLs is the first step in the development of future vaccines. HLA-A2 transgenic mice (HHD) were shown to provide a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, since these mice are endowed with a CTL repertoire representative of HLA-A2.1 individuals. Here, we describe the immunological characterization of T-cell epitopes of the T. cruzi ribosomal P2 protein (TcP2beta) that are recognized by HLA-A*0201-restricted CTLs in HLA-transgenic mice and humans. Epitopes identified in the present study do not share sequence homology with the homologous human or murine counterparts and so they should not induce any autoreactive response. Moreover, HHD mice vaccinated with these peptide epitopes have reduced parasitemia after challenge with a lethal T. cruzi infection. Hence, these epitopes represent potential subunit components of multi-protein vaccines to prevent Chagas' disease.     

Hepatitis B virus X protein induces size-selective membrane permeabilization through interaction with cardiolipin

Hepatitis B virus X protein (HBx) functions in a variety of cellular events during the HBV life cycle. In a previous study, we reported that the HBx protein is sufficient to induce mitochondrial membrane permeabilization; however, the exact mechanism of HBx-induced mitochondrial membrane permeabilization has been not proposed. In this study, we report that HBx specifically targets cardiolipin (CL) and induces membrane permeabilization depending on CL concentration in mitochondrial outer membrane–mimic artificial liposomes. Interestingly, HBx-induced membrane permeabilization was enhanced by liposomes containing phosphatidylethanolamine, which plays a crucial role in forming a negative curvature on the membrane. We also show that the 68-117 region of HBx, which interacts with mitochondria, is necessary for membrane permeabilization. We examined the size of the pores formed by HBx and found that HBx permeates fluorescent dyes depending on the hydrodynamic diameter with a pore size of approximately 10 nm. The results of this study suggest that CL is necessary for HBx-induced membrane permeabilization and provide important information that suggests a new strategy for anti-HBV therapy.     

Hepatitis B virus polypeptide X: expression in Escherichia coli and identification of specific antibodies in sera from hepatitis B virus-infected humans.

Sequence analysis of the hepatitis B virus (HBV) genome revealed the presence of an open reading frame (ORF X) which has the potential to encode a 154-amino acid polypeptide. A fusion protein containing 145 of the amino acids encoded by ORF X and 8 amino acids of beta-galactosidase was expressed and characterized in bacterial extracts. Immunoprecipitations with the ORF X fusion protein as a radioactively labeled antigen were performed to screen sera of humans infected with HBV for the presence of antibodies against ORF X-encoded determinants (anti-X). Such antibodies were identified in 9 samples from a set of 26 sera characterized as positive for HBV surface antigen but were not found in 16 normal human sera. The data reported here demonstrate that sera from some patients with markers of HBV infection contain antibodies directed against the polypeptide encoded by ORF X. As such, these findings represent evidence that ORF X constitutes a gene, or a portion of a gene, which is expressed during HBV infection. Although there does not appear to be a direct relationship between anti-X and any individual markers of HBV infection, our data suggest that anti-X is more prevalent in HBV-positive sera containing antibodies to HBe3 antigen (anti-HBe3).     

乙型肝炎病毒x蛋白通过靶向miR-200c诱导DNA异常甲基化

乙型肝炎病毒x (hepatitis B virus x,HBx)蛋白是导致肝癌(hepatocellular Carcinoma,HCC)的重要因素.但HBX在HCC形成过程中表观遗传机制尚有待阐明.本研究发现microRNA-200c (miR-200c)在过表达乙型肝炎病毒的HCC中下调,并且其直接靶向DNA甲基转移酶3A (DNA methyltransferase 3A,DNMT3A).此外,miR-200c和DNMT3A在HB诱发的肝癌组织中呈现负相关.乙型肝炎病毒诱导miR-200c下调,进而引起DNMT3A表达上调,导致细胞中肿瘤相关基因的启动子超甲基化.我们对乙型肝炎病毒诱导的肝癌表观遗传学改变进行了进一步研究,并提出一种基于miRNA的靶向治疗乙型肝炎病毒相关肝癌的潜在方法.     

Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria

Hepatitis B virus X protein (HBx) is essential for viral replication and plays an important role in viral pathogenesis. HBx transactivates many viral and cellular genes and participates in cellular signal transduction pathways, proliferation, and apoptosis. In the present study, we report that HBx induces apoptosis by enhancing the translocation of Bax to mitochondria, followed by inducing the loss of mitochondrial membrane potential and release of cytochrome C. In addition, Bcl-2, inhibitor of Bax, rescues the disruption of mitochondrial membrane potential and DNA fragmentation induced by serum starvation in HepG2-X cells expressing HBx. We also found that HBx binds directly to Bax and interferes with the interaction between Bax and 14-3-3epsilon to enhance the translocation of Bax to mitochondria. Taken together, our data suggest that HBx induces apoptosis by interacting with Bax and enhancing its translocation to mitochondria.     

佐剂CpG-ODN与重组HBsAg疫苗联合免疫乙型肝炎病毒转基因小鼠的免疫应答研究

目的 以CpG-ODN为佐剂与重组HBsAg(rHBsAg)疫苗合用,研究其对乙型肝炎病毒转基因(HBV Tg)小鼠模型的免疫应答效果.方法 40只HBV Tg小鼠随机分为4组,每组小鼠分别注射rHBsAg疫苗(单用rHBsAg组)、rHBsAg疫苗+CpG-ODN(试验组)、rIFNα-2b(IFN组)、生理盐水(对照组).经多次免疫HBV转基因小鼠,于免疫前、后不同时间采血,动态观察各组小鼠血清中HBsAg量、抗-HBs阳性率和HBV DNA的变化,检测肝组织中HBsAg的表达.检测免疫小鼠的外周血T淋巴细胞亚群和白细胞介素2(IL-2)、IL-12(p70)以及γ干扰素(IFN-γ)的含量,分别检测免疫小鼠的脾细胞增殖和细胞毒性T淋巴细胞(CTL)杀伤功能并计算各组小鼠肝组织活性指数(HAI).结果 rHBsAg组和rHBsAg+CpG组在免疫小鼠后2周100%诱导抗-HBs;rHBsAg+CpG组能显著降低血清中的HBsAg量或使HBsAg转阴,rHBsAg+CpG组肝组织中HBsAg的表达量与血清中一样降低,并降低血清中HBV DNA的拷贝数.rHBsAg组的CD3+、CD4+、CD8+细胞在T细胞中所占百分比,IL-2、IL-12(p70)和IFN-γ的含量以及淋巴细胞特异性增殖和杀伤效应均明显高于对照组(P＜0.05).rHBsAg+CpG组与rHBsAg组比较,免疫小鼠产生更强的HBV特异性细胞应答(P＜0.05),且以Th1型细胞免疫应答为主.在rHBsAg+CpG组肝组织中出现大量淋巴细胞,肝脏的HAI在4个组中最高.结论 CpG-ODN作为佐剂可以增强重组HBsAg疫苗诱导HBV转基因小鼠产生抗病毒免疫应答,重组HBsAg疫苗辅以CpG ODN可作为免疫治疗慢性HBV感染的可行性途经.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Nondeletional T-cell receptor transgenic mice: model for the CD4(+) T-cell repertoire in chronic hepatitis B virus infection

Chronicity after infection with the hepatitis B virus (HBV) can occur for a variety of reasons. However, once established, chronicity may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of coexisting with the secreted hepatitis B e antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To ensure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR alpha- and beta-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas produced from immunizing HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency ( approximately 67%) of CD4(+) T cells that expressed a Tg TCR specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4(+) T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4(+) T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg, as measured by cytokine production in vitro and T-helper-cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th1 subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4(+) T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo. The 11/4-12 TCR-Tg lineage may serve as a monoclonal model for the HBe/HBcAg-specific CD4(+) T-cell repertoire present in chronically infected HBV patients.     

Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential

The hepatitis B virus X protein (HBx) has been implicated in the carcinogenicity of this virus as a causative factor by means of its transactivation function in development of hepatocellular carcinoma. However, we and others have recently reported that HBx is located in mitochondria and causes subsequent cell death (Takada, S., Shirakata, Y., Kaneniwa, N., and Koike, K. (1999) Oncogene 18, 6965-6973; Rahmani, Z., Huh, K. W., Lasher, R., and Siddiqui, A. (2000) J. Virol. 74, 2840-2846). In this study, we, therefore, examined the mechanism of HBx-related cell death. Using enhanced green fluorescent protein (EGFP) fusion constructs of HBx, the region required for its mitochondrial localization was mapped to amino acids (aa) 68-117, which is essential for cell death but inactive for transactivation function. In vitro binding analysis supported the notion that the recombinant HBx associates with isolated mitochondria through the region of aa 68-117 without causing redistribution of cytochrome c and apoptosis-inducing factor (AIF). A cytochemical analysis revealed that mitochondrial membrane potential was decreased by HBx association with mitochondria, suggesting that HBx induces dysfunction of permeability transition pore (PTP) complex. Furthermore, PTP inhibitors, reactive oxygen species (ROS) scavengers and Bcl-xL, which are known to stabilize mitochondrial membrane potential, prevented HBx-induced cell death. Collectively, the present results suggest that location of HBx in mitochondria of hepatitis B virus-infected cells causes loss of mitochondrial membrane potential and subsequently induces mitochondria-dependent cell death.     

Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.     

Hepatitis B virus core protein interacts with CD59 to promote complement-mediated liver inflammation during chronic hepatitis B virus infection

The inflammatory response mediated by the immune system is the major cause of hepatitis B virus (HBV)-associated liver injury. Here, we identified CD59, as a novel HBc-interacting protein in hepatocytes by tandem affinity purification (TAP) screening. The expression of CD59 was markedly down-regulated in HBc-transfected HepG2 or HepG2.215 cells, which resulted in an upshift of hepatocyte sensitivity to membrane attack complex (MAC)-induced cell lysis. These results were consistent with the accumulation of MACs in the liver of HBV-infected patients. Additional analyses using laser confocal microscopy, quantitative PCR and flow cytometry revealed that CD59 was specifically translocated to the nucleus upon binding to HBc, which induced the down-regulation of CD59 on both the mRNA and protein levels.     

Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.     

IL-2-engineered nano-APC effectively activates viral antigen-mediated T cell responses from chronic hepatitis B virus-infected patients

Impaired function of virus-specific T cells resulting from virus persistence is one of the major mechanisms underlying the development of chronic hepatitis B viral infection. Previously, we found that IL-2 can restore the effector function of T cells rendered tolerant by Ag persistence. However, systemic administration of IL-2 induces organ pathology and expansion of T regulatory cells. In this study, we show that nano-APC with engineered HLA alleles and IL-2 deliver peptide-MHC complexes, costimulatory molecules, and IL-2 to Ag-responding T cells, resulting in enhanced expression of CD25 and activation of TCR signaling pathways, while suppressing PD-1 expression on viral-responding CD8 T cells from chronic hepatitis B virus patients. The enhanced activation of CD4 and CD8 T cells induced by IL-2-nano-APC was Ag dependent and IL-2-nano-APC did not affect T regulatory cells. At a size of 500 nm, the nano-APC effectively induce immune synapse formation on Ag-specific T cells and accumulate as free particles in the lymphoid organs. These attributes of IL-2-nano-APC or other bioadjuvant-engineered nano-APC have profound implications for their use as a therapeutic strategy in the treatment of chronic hepatitis B virus infection or other chronic viral diseases.     

Variability and conservation in hepatitis B virus core protein

Background  

Hepatitis B core protein (HBVc) has been extensively studied from both a structural and immunological point of view, but the evolutionary forces driving sequence variation within core are incompletely understood.     

Hepatitis B genotype G and high frequency of lamivudine-resistance mutations among human immunodeficiency virus/hepatitis B virus co-infected patients in Brazil

In this study, we evaluated the hepatitis B virus (HBV) genotype distribution and HBV genomic mutations among a group of human immunodeficiency virus-HBV co-infected patients from an AIDS outpatient clinic in S?o Paulo. HBV serological markers were detected by commercially available enzyme immunoassay kits. HBV DNA was detected using in-house nested polymerase chain reaction and quantified by Cobas Amplicor. HBV genotypes and mutations in the basal core promoter (BCP)/pre-core/core regions and surface/polymerase genes were determined by sequencing. Among the 59 patients included in this study, 55 reported prior use of lamivudine (LAM) or tenofovir. HBV DNA was detected in 16/22 patients, with a genotype distribution of A (n = 12,75%), G (n = 2,13%), D (n = 1,6%) and F (n = 1,6%). The sequence data of the two patients infected with genotype G strongly suggested co-infection with genotype A. In 10 patients with viremia, LAM-resistance mutations in the polymerase gene (rtL180M + rtM204V and rtV173L + rtL180M + rtM204V) were found, accompanied by changes in the envelope gene (sI195M, sW196L and sI195M/sE164D). Mutations in the BCP and pre-core regions were identified in four patients. In conclusion, genotype G, which is rarely seen in Brazil, was observed in the group of patients included in our study. A high prevalence of mutations associated with LAM-resistance and mutations associated with anti-HBs resistance were also found among these patients.