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Hepatitis B Virus X Protein Stimulates HBV Replication through Regulating Transcription Factors Associated with Histone Methylation
POSTER PRESENTATIONS FRI-244 HEPATITIS B VIRUS X PROTEIN STIMULATES HBV REPLICATION THROUGH REGULATING TRANSCRIPTION FACTORS ASSOCIATED WITH HISTONE METHYLATION N. Oishi1, M. Honda1, T. Shimakami1, S. Kaneko1. 1Gastroenterology, Kanazawa University Hospital, Kanazawa, Japan E-mail: [email protected] Background and Aims: Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases, from hepatitis to cirrhosis and liver cancer. HBx is a multifunctional protein encoded by the HBV genome that stimulates HBV replication. Previously, we found that HBx has an important role in stimulating HBV transcription and replication and that the transcriptional transactivation function of HBx may be critical for its augmentation effect on HBV replication. However, the molecular mechanism of HBx in transcriptional coactivation remains unclear. In this study, we provide a new insight into the coactivator mechanism and the possibility of a new treatment target for HBV replication and HBV-related hepatocarcinogenesis. Methods: We used a retroviral vector to introduce wild-type HBx (HBxwt) or empty vector (EV) into HepG2 cells. Gene expression profiling was performed using Affymetrix GeneChip Human U133A2.0 ver.2.0 arrays according to the manufacturer’s protocol. Unsupervised hierarchical clustering analysis and class comparison analysis were performed with BRB-Array Tools software version 4.2.2. Transcription factor analysis was performed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems) to identify potential upstream transcription factors (TFs). We used array data from 244 chronic hepatitis B patients for analyzing clinical features. Results: Unsupervised hierarchical clustering analysis of HepG2HBxwt or HepG2-EV cells revealed that the gene expression profiles of these cell lines were significantly different. Class comparison analysis between both cell lines identified 803 HBx-related genes. Six HBx-related activated TFs that affected HBV replication were identified by IPA, small interfering RNA, and inhibitor analyses. Three of these six TFs control HBV replication by modifying DNA or histone methylation. Moreover, these three TFs regulate formation of cccDNA. By analysis of 244 hepatitis B patients, these three TFs were found to be expressed at a significantly highly level in HBeAg(+) HBeAb(−) cases than in HBeAg(−)HBeAb(+) cases. Conclusions: HBx upregulated three TFs associated with DNA or histone methylation. Moreover, HBx stimulates HBV replication and cccDNA formation by modifying DNA or histone methylation. Our study suggests that TFs activated by HBx will be an important therapeutic target against virus replication. FRI-245 FUNCTIONAL VARIANTS AND ABERRANT METHYLATION IN THE SOCS3 PROMOTER REGION INFLUENCE THE PROGRESSION OF HBV-RELATED LIVER DISEASES N.X. Hoan1, H. Van Tong1, D.P. Giang1, N.L. Toan2, L.H. Song3, C.-T. Bock4, P.G. Kremsner1, T.P. Velavan1. 1Institutre for Tropical Medicine, University of Tuebingen, Tuebingen, Germany; 2Depertment of Pathophysiology, Viretnam Military Medical University, Ha Dong, Vietnam; 3Department of Clinical infectious diseases, Tran Hung Dao Hospital, Hanoi; 4Robert Kock Institute, Berlin, Germany E-mail: [email protected] Background and Aims: The clinical manifestations of HBV infection include chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The suppressor of cytokine signaling-3 (SOCS3), a member of SOCS family is a vital regulator of many diseases including HBV-related liver diseases. This study aimed to investigate possible association of SOCS3 promoter variants and SOCS3 methylation with the progression of HBV-related liver diseases. Methods: The SOCS3 promoter region were screened for polymorphisms in a total of 878 HBV-infected patients, defined in subgroups including CHB, LC, HCC and in 272 healthy individuals by S520
direct sequencing. Bisulfite sequencing and Methylation-specific PCR were employed to determine the methylation status in the promoter region and was correlated with SOCS3 expression in 37 tumor and adjacent non-tumor liver specimens. Results: The minor allele rs12953258A was associated with increased susceptibility to HBV infection (HBV vs. Controls: OR = 1.3, 95% CI = 1.1–1.6, adjusted p = 0.03). The minor allele rs111033850C and rs12953258A weree more frequent in HCC patients and in LC patients compared to CHB patients (OR = 1.7, 95%CI = 1.1–2.9, adjusted p = 0.046 and OR = 1.4, 95%CI = 1.1–1.9, adjusted p = 0.017, respectively). HBV viral loads were significantly lower in patients with rs111033850CC compared to those with rs111033850TT and rs111033850TC ( p = 0.034), but significantly higher in patients with rs12953258AA compared to those with rs12953258CC and rs12953258AC. Tumor tissues showed high levels of methylation compared to adjacent non-tumor tissues (OR = 3.0; 95%CI: 1.0–9.8; p = 0.049). Increased SOCS3 expression was observed in HBV-related HCC patients than in non-HBV-related HCC patients ( p = 0.028). Conclusions: The SOCS3 promoter region variants likely regulate the HBV infection and the progression of HBV-related liver diseases. The SOCS3 methylation is a key factor in tumor development in HBV patients. FRI-246 THE BREADTH OF CD8+ T CELL RESPONSES IN CHRONIC AND RESOLVED HEPATITIS B VIRUS INFECTION P. Ehrenmann1, M. Kiraithe1, J. Lang1, F. Jacobi1, R. Thimme1, C. Neumann-Haefelin1. 1Department for Internal Medicine 2, University Hospital Freiburg, Freiburg, Germany E-mail: [email protected] Background and Aims: Hepatits B virus (HBV) infection is one of the main global health problems affecting 350 million people worldwide. Since HBV-specific CD8+ T cells are crucial for viral clearance, T cell based immunotherapies are a promising strategy for clearance of chronic HBV infection. However, only few HBV-specific CD8+ T cell epitopes are known. Therefore, we aim to identify novel HBV-specific CD8+ T cell epitopes and to compare the HBV-specific CD8+ T cell repertoire in chronically HBV infected patients compared to patients who spontaneously eliminated the virus. Methods: PBMC from 69 patients chronically infected with HBV genotype D and 16 patients with resolved HBV infection were screened in an ELISpot-Assay using overlapping peptides (18mers) covering the full HBV genotype D proteome. Positive ELISpot responses were confirmed by intracellular interferon-ɣ staining. Afterwards, epitopes were finemapped and HLA-restriction was determined using EBV-immortalized B cells. Additionally, viral sequences were determined in chronically infected patients and HLA-associated sequence polymorphisms (“footprints”) were identified as indicators for escape mutations. Results: Both, the immunological and the virological analysis revealed new HBV-specific CD8+ T cell epitopes. 43% patients with chronic HBV had responses targeting at least one CD8+ T cell epitope. Interestingly, patients with chronic HBV displayed CD8+ T cell responses targeting epitopes located in the polymerase, core and xproteins but not the HBsAg, while those with resolved HBV had responses to all viral proteins including HBsAg. In total, 70 HBVspecific CD8+ T cell epitopes could be detected. Furthermore, sequencing analysis revealed HLA-footprints which were mostly located in in the polymerase and core, but only few and relatively weak footprints were found in HBsAg. This goes along with the lack of CD8+ T cell responses to HBsAg, indicating that in patients with chronic HBV infection, CD8+ T cell responses do not target HBsAg and thus do not drive viral escape in this viral region. Conclusions: Through the comprehensive analysis of HBV-specific CD8+ T cell responses as well as the analysis of HLA class I footprints, we were able to identify novel HBV-specific CD8+ T cell epitopes and to demonstrate viral escape in some of these epitopes. Additionally,
Journal of Hepatology 2016 vol. 64 | S425–S630
direct sequencing. Bisulfite sequencing and Methylation-specific PCR were employed to determine the methylation status in the promoter region and was correlated with SOCS3 expression in 37 tumor and adjacent non-tumor liver specimens. Results: The minor allele rs12953258A was associated with increased susceptibility to HBV infection (HBV vs. Controls: OR = 1.3, 95% CI = 1.1–1.6, adjusted p = 0.03). The minor allele rs111033850C and rs12953258A weree more frequent in HCC patients and in LC patients compared to CHB patients (OR = 1.7, 95%CI = 1.1–2.9, adjusted p = 0.046 and OR = 1.4, 95%CI = 1.1–1.9, adjusted p = 0.017, respectively). HBV viral loads were significantly lower in patients with rs111033850CC compared to those with rs111033850TT and rs111033850TC ( p = 0.034), but significantly higher in patients with rs12953258AA compared to those with rs12953258CC and rs12953258AC. Tumor tissues showed high levels of methylation compared to adjacent non-tumor tissues (OR = 3.0; 95%CI: 1.0–9.8; p = 0.049). Increased SOCS3 expression was observed in HBV-related HCC patients than in non-HBV-related HCC patients ( p = 0.028). Conclusions: The SOCS3 promoter region variants likely regulate the HBV infection and the progression of HBV-related liver diseases. The SOCS3 methylation is a key factor in tumor development in HBV patients. FRI-246 THE BREADTH OF CD8+ T CELL RESPONSES IN CHRONIC AND RESOLVED HEPATITIS B VIRUS INFECTION P. Ehrenmann1, M. Kiraithe1, J. Lang1, F. Jacobi1, R. Thimme1, C. Neumann-Haefelin1. 1Department for Internal Medicine 2, University Hospital Freiburg, Freiburg, Germany E-mail: [email protected] Background and Aims: Hepatits B virus (HBV) infection is one of the main global health problems affecting 350 million people worldwide. Since HBV-specific CD8+ T cells are crucial for viral clearance, T cell based immunotherapies are a promising strategy for clearance of chronic HBV infection. However, only few HBV-specific CD8+ T cell epitopes are known. Therefore, we aim to identify novel HBV-specific CD8+ T cell epitopes and to compare the HBV-specific CD8+ T cell repertoire in chronically HBV infected patients compared to patients who spontaneously eliminated the virus. Methods: PBMC from 69 patients chronically infected with HBV genotype D and 16 patients with resolved HBV infection were screened in an ELISpot-Assay using overlapping peptides (18mers) covering the full HBV genotype D proteome. Positive ELISpot responses were confirmed by intracellular interferon-ɣ staining. Afterwards, epitopes were finemapped and HLA-restriction was determined using EBV-immortalized B cells. Additionally, viral sequences were determined in chronically infected patients and HLA-associated sequence polymorphisms (“footprints”) were identified as indicators for escape mutations. Results: Both, the immunological and the virological analysis revealed new HBV-specific CD8+ T cell epitopes. 43% patients with chronic HBV had responses targeting at least one CD8+ T cell epitope. Interestingly, patients with chronic HBV displayed CD8+ T cell responses targeting epitopes located in the polymerase, core and xproteins but not the HBsAg, while those with resolved HBV had responses to all viral proteins including HBsAg. In total, 70 HBVspecific CD8+ T cell epitopes could be detected. Furthermore, sequencing analysis revealed HLA-footprints which were mostly located in in the polymerase and core, but only few and relatively weak footprints were found in HBsAg. This goes along with the lack of CD8+ T cell responses to HBsAg, indicating that in patients with chronic HBV infection, CD8+ T cell responses do not target HBsAg and thus do not drive viral escape in this viral region. Conclusions: Through the comprehensive analysis of HBV-specific CD8+ T cell responses as well as the analysis of HLA class I footprints, we were able to identify novel HBV-specific CD8+ T cell epitopes and to demonstrate viral escape in some of these epitopes. Additionally,
Journal of Hepatology 2016 vol. 64 | S425–S630