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1
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Zankharia US, Kudchodkar S, Khoshnejad M, Perales-Puchalt A, Choi H, Ho M, Zaidi F, Ugen KE, Kim JJ, Weiner DB, Muthumani K. Neutralization of hepatitis B virus by a novel DNA-encoded monoclonal antibody. Hum Vaccin Immunother 2020; 16:2156-2164. [PMID: 32463327 PMCID: PMC7553714 DOI: 10.1080/21645515.2020.1763686] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Hepatitis B virus (HBV) causes a potentially life-threatening liver infection that frequently results in life-long chronic infection. HBV is responsible for 887,000 deaths each year, most resulting from chronic liver diseases and hepatocellular carcinoma. Presently, there are 250 million chronic HBV carriers worldwide who are at a high risk for developing cirrhosis and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer with a strong association with HBV infection. HBV transmission through blood transfusions and perinatal transfer from infected mother to child have been common routes of infection. In the present study, we describe the development of a synthetic DNA plasmid encoding an anti-HBV human monoclonal antibody specific for the common “a determinant region” of HBsAg of hepatitis B virus and demonstrate the ability of this platform at directing in vivo antibody expression. In vivo delivery of this DNA encoded monoclonal antibody (DMAb) plasmid in mice resulted in expression of human IgG over a period of one month following a single injection. Serum antibody was found to recognize the relevant conformational epitope from plasma purified native HBsAg as well as bound HBV in HepG2.2.15 cells. The serum DMAb efficiently neutralized HBV and prevented infection of HepaRG cells in vitro. Additional study of these HBV-DMAb as a possible therapy or immunoprophylaxis for HBV infection is warranted.
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Affiliation(s)
- Urvi S Zankharia
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
| | - Sagar Kudchodkar
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
| | - Makan Khoshnejad
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
| | | | - Hyeree Choi
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
| | - Michelle Ho
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
| | - Faraz Zaidi
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
| | - Kenneth E Ugen
- Department of Molecular Medicine, University of South Florida Morsani College of Medicine , Tampa, FL, USA
| | - Joseph J Kim
- Inovio Pharmaceuticals, Plymouth Meeting , PA, USA
| | - David B Weiner
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
| | - Kar Muthumani
- Vaccine & Immunotherapy Center, The Wistar Institute , Philadelphia, PA, USA
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2
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Wang W, Zhou X, Bian Y, Wang S, Chai Q, Guo Z, Wang Z, Zhu P, Peng H, Yan X, Li W, Fu YX, Zhu M. Dual-targeting nanoparticle vaccine elicits a therapeutic antibody response against chronic hepatitis B. NATURE NANOTECHNOLOGY 2020; 15:406-416. [PMID: 32123380 PMCID: PMC7223715 DOI: 10.1038/s41565-020-0648-y] [Citation(s) in RCA: 151] [Impact Index Per Article: 30.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Accepted: 01/24/2020] [Indexed: 05/09/2023]
Abstract
Chronic hepatitis B is caused by prolonged infection with the hepatitis B virus (HBV), which can substantially increase the risk of developing liver disease. Despite the development of preventive vaccines against HBV, a therapeutic vaccine inducing an effective antibody response still remains elusive. The preS1 domain of the large HBV surface protein is the major viral attachment site on hepatocytes and thus offers a therapeutic target; however, its poor immunogenicity limits clinical translation. Here, we design a ferritin nanoparticle vaccine that can deliver preS1 to specific myeloid cells, including SIGNR1+ dendritic cells (which activate T follicular helper cells) and lymphatic sinus-associated SIGNR1+ macrophages (which can activate B cells). This nanoparticle vaccine induces a high-level and persistent anti-preS1 response that results in efficient viral clearance and partial serological conversion in a chronic HBV mouse model, offering a promising translatable vaccination strategy for the functional cure of chronic hepatitis B.
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Affiliation(s)
- Wenjun Wang
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, China
| | - Xiaoxiao Zhou
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, China
| | - Yingjie Bian
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Shan Wang
- Department of Pediatric Surgical Oncology, Children's Hospital, Chongqing Medical University, Chongqing, China
| | - Qian Chai
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Zhenqian Guo
- Department of Pediatric Surgical Oncology, Children's Hospital, Chongqing Medical University, Chongqing, China
| | - Zhenni Wang
- Department of Pediatric Surgical Oncology, Children's Hospital, Chongqing Medical University, Chongqing, China
| | - Ping Zhu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Hua Peng
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Xiyun Yan
- Key Laboratory of Protein and Peptide Pharmaceutical, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Wenhui Li
- National Institute of Biological Sciences, Beijing, China
| | - Yang-Xin Fu
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Mingzhao Zhu
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
- College of Life Sciences, University of the Chinese Academy of Sciences, Beijing, China.
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3
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Nomura M, Tsuge M, Uchida T, Hiraga N, Kurihara M, Tsushima K, Fujino H, Nakahara T, Murakami E, Abe-Chayama H, Kawaoka T, Miki D, Hiramatsu A, Imamura M, Kawakami Y, Aikata H, Ochi H, Zhang Y, Makokha GN, Hayes CN, Tanaka S, Chayama K. CTL-associated and NK cell-associated immune responses induce different HBV DNA reduction patterns in chronic hepatitis B patients. J Viral Hepat 2018; 25:1555-1564. [PMID: 29998562 DOI: 10.1111/jvh.12970] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2018] [Accepted: 06/07/2018] [Indexed: 12/23/2022]
Abstract
The activation of hepatitis B virus (HBV)-related hepatitis is associated with both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). We analyzed the association between the immune response and changes in the proportion of Pre-S deletion variants. We quantified Pre-S deleted HBV (HBV-del) and wild-type HBV (HBV-wt) DNA levels in sera obtained from HBV-infected mice and chronic hepatitis B patients. In chronic hepatitis B patients, the HBV-del proportion usually increased during or after ALT elevation but did not occur during all ALT elevations. To clarify this difference in the immunological responses, we performed in vivo analyses using HBV-infected human hepatocyte chimeric mice. Although HBV-del proportions did not change in mice with NK cell-associated hepatitis or in mice treated with entecavir, the proportions sharply increased in mice with CTL-associated hepatitis. Furthermore, the number of patients in which HBV-del proportions were greater than 5% was significantly higher in chronic hepatitis B patients than in asymptomatic carriers (P = 0.023). We identified associations between virological response in chronic hepatitis B patients and two different immune responses. The proportion of HBV-del variants could be a useful biomarker for distinguishing between chronic hepatitis and asymptomatic carriers.
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Affiliation(s)
- Motonobu Nomura
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Masataka Tsuge
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan.,Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan
| | - Takuro Uchida
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Nobuhiko Hiraga
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Mio Kurihara
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Ken Tsushima
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Hatsue Fujino
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Takashi Nakahara
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Eisuke Murakami
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Hiromi Abe-Chayama
- Liver Research Project Center, Hiroshima University, Hiroshima, Japan.,Center for Medical Specialist Graduate Education and Research, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Tomokazu Kawaoka
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Daiki Miki
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan.,Laboratory for Digestive Diseases, RIKEN Center for Integrative Medical Sciences, Hiroshima, Japan
| | - Akira Hiramatsu
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Yoshiiku Kawakami
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Hiroshi Aikata
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Hidenori Ochi
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan.,Laboratory for Digestive Diseases, RIKEN Center for Integrative Medical Sciences, Hiroshima, Japan
| | - Yizhou Zhang
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Grace Naswa Makokha
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Clair Nelson Hayes
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan
| | - Shinji Tanaka
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Department of Endoscopy, Hiroshima University Hospital, Hiroshima, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Division of Frontier Medical Science, Programs for Biomedical Research Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.,Liver Research Project Center, Hiroshima University, Hiroshima, Japan.,Laboratory for Digestive Diseases, RIKEN Center for Integrative Medical Sciences, Hiroshima, Japan
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Wu IC, Liu WC, Chang TT. Applications of next-generation sequencing analysis for the detection of hepatocellular carcinoma-associated hepatitis B virus mutations. J Biomed Sci 2018; 25:51. [PMID: 29859540 PMCID: PMC5984823 DOI: 10.1186/s12929-018-0442-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2018] [Accepted: 04/30/2018] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Next-generation sequencing (NGS) is a powerful and high-throughput method for the detection of viral mutations. This article provides a brief overview about optimization of NGS analysis for hepatocellular carcinoma (HCC)-associated hepatitis B virus (HBV) mutations, and hepatocarcinogenesis of relevant mutations. MAIN BODY For the application of NGS analysis in the genome of HBV, four noteworthy steps were discovered in testing. First, a sample-specific reference sequence was the most effective mapping reference for NGS. Second, elongating the end of reference sequence improved mapping performance at the end of the genome. Third, resetting the origin of mapping reference sequence could probed deletion mutations and variants at a certain location with common mutations. Fourth, using a platform-specific cut-off value to distinguish authentic minority variants from technical artifacts was found to be highly effective. One hundred and sixty-seven HBV single nucleotide variants (SNVs) were found to be studied previously through a systematic literature review, and 12 SNVs were determined to be associated with HCC by meta-analysis. From comprehensive research using a HBV genome-wide NGS analysis, 60 NGS-defined HCC-associated SNVs with their pathogenic frequencies were identified, with 19 reported previously. All the 12 HCC-associated SNVs proved by meta-analysis were confirmed by NGS analysis, except for C1766T and T1768A which were mainly expressed in genotypes A and D, but including the subgroup analysis of A1762T. In the 41 novel NGS-defined HCC-associated SNVs, 31.7% (13/41) had cut-off values of SNV frequency lower than 20%. This showed that NGS could be used to detect HCC-associated SNVs with low SNV frequency. Most SNV II (the minor strains in the majority of non-HCC patients) had either low (< 20%) or high (> 80%) SNV frequencies in HCC patients, a characteristic U-shaped distribution pattern. The cut-off values of SNV frequency for HCC-associated SNVs represent their pathogenic frequencies. The pathogenic frequencies of HCC-associated SNV II also showed a U-shaped distribution. Hepatocarcinogenesis induced by HBV mutated proteins through cellular pathways was reviewed. CONCLUSION NGS analysis is useful to discover novel HCC-associated HBV SNVs, especially those with low SNV frequency. The hepatocarcinogenetic mechanisms of novel HCC-associated HBV SNVs defined by NGS analysis deserve further investigation.
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Affiliation(s)
- I-Chin Wu
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan, Republic of China.,Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan, Republic of China
| | - Wen-Chun Liu
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan, Republic of China.,Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan, Republic of China
| | - Ting-Tsung Chang
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan, Republic of China.
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5
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Chen BF. Different pre-S deletion patterns and their association with hepatitis B virus genotypes. World J Gastroenterol 2017. [PMID: 27672298 DOI: 10.3748/wjg.v22.i35.8041.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 09/29/2022] Open
Abstract
AIM To investigate the associations of different types of pre-S deletions with hepatitis B virus (HBV) genotypes. METHODS The sequences of the pre-S region, basal core promoter (BCP) mutation, and precore (PC) mutation were examined through direct DNA sequencing or clonal analysis and sequencing in 273 HBV carriers, namely 55 asymptomatic carriers, 55 carriers with chronic hepatitis (CH), 55 with liver cirrhosis (LC), 53 with liver cirrhotic hepatocellular carcinoma (LC-HCC), and 55 with noncirrhotic HCC. A total of 126 HBV carriers (46.2%) harbored pre-S deletions. The DNA sequences of pre-S deletion mutants from 43 age-matched genotype B (HBV/B)-infected carriers and 43 age-matched genotype C (HBV/C)-infected carriers were further examined, aligned, and compared. RESULTS No significant difference was observed in the mean age distribution (P = 0.464), male sex (P = 0.805), viral load (P = 0.635), or BCP mutation (P = 0.117) between the HBV/B and HBV/C groups. However, the rate of PC mutation was significantly higher in the HBV/B-infected carriers than in the HBV/C-infected carriers (P = 0.003). Both genotypes exhibited a high rate of deletion in the C-terminal half of the pre-S1 region and N-terminus of the pre-S2 region (86.0% and 79.1% in the HBV/B group; 69.8% and 72.1% in the HBV/C group, respectively). Epitope mapping showed that deletion in several epitope sites was frequent in both genotypes, particularly pS1-BT and pS2-B2. Conversely, the rate of pS2-B1 deletion was significantly higher in the HBV/B group (72.1% vs 37.2%, P = 0.002), and the rate of pS2-T deletion was significantly higher in the HBV/C group (48.8% vs 25.6%, P = 0.044). Functional mapping showed that the rate of deletion in three functional sites (the nucleocapsid binding site, start codon of M, and site for viral secretion) located in the N-terminus of the pre-S2 region was significantly higher in the HBV/B group (P < 0.05). One type of N-terminus pre-S1 deletion mutant with deletion of the start codon of the L protein was frequently observed in the HBV/C group (20.9% vs 9.3%, P = 0.228), particularly in the LC patients (42.9% vs 12.5%). Different patterns of pre-S deletions were also found between the HBV/B and HBV/C groups according to different clinical outcomes. In CH patients, deletion in the site for polymerized human serum albumin was more frequent in the HBV/B group (88.9% vs 36.4%, P = 0.028). In the LC-HCC patients, the rate of deletion in the pre-S2 region was significantly higher in the HBV/B group than in the HBV/C group (P < 0.05). CONCLUSION HBV/B- and HBV/C-infected carriers exhibit different patterns of pre-S deletion, which may be associated with the progression of liver diseases.
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Affiliation(s)
- Bing-Fang Chen
- Bing-Fang Chen, School of Medicine, Fu Jen Catholic University, New Taipei City 24205, Taiwan
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6
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Chen BF. Different pre-S deletion patterns and their association with hepatitis B virus genotypes. World J Gastroenterol 2016; 22:8041-8049. [PMID: 27672298 PMCID: PMC5028817 DOI: 10.3748/wjg.v22.i35.8041] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/09/2016] [Revised: 07/24/2016] [Accepted: 08/08/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the associations of different types of pre-S deletions with hepatitis B virus (HBV) genotypes.
METHODS The sequences of the pre-S region, basal core promoter (BCP) mutation, and precore (PC) mutation were examined through direct DNA sequencing or clonal analysis and sequencing in 273 HBV carriers, namely 55 asymptomatic carriers, 55 carriers with chronic hepatitis (CH), 55 with liver cirrhosis (LC), 53 with liver cirrhotic hepatocellular carcinoma (LC-HCC), and 55 with noncirrhotic HCC. A total of 126 HBV carriers (46.2%) harbored pre-S deletions. The DNA sequences of pre-S deletion mutants from 43 age-matched genotype B (HBV/B)-infected carriers and 43 age-matched genotype C (HBV/C)-infected carriers were further examined, aligned, and compared.
RESULTS No significant difference was observed in the mean age distribution (P = 0.464), male sex (P = 0.805), viral load (P = 0.635), or BCP mutation (P = 0.117) between the HBV/B and HBV/C groups. However, the rate of PC mutation was significantly higher in the HBV/B-infected carriers than in the HBV/C-infected carriers (P = 0.003). Both genotypes exhibited a high rate of deletion in the C-terminal half of the pre-S1 region and N-terminus of the pre-S2 region (86.0% and 79.1% in the HBV/B group; 69.8% and 72.1% in the HBV/C group, respectively). Epitope mapping showed that deletion in several epitope sites was frequent in both genotypes, particularly pS1-BT and pS2-B2. Conversely, the rate of pS2-B1 deletion was significantly higher in the HBV/B group (72.1% vs 37.2%, P = 0.002), and the rate of pS2-T deletion was significantly higher in the HBV/C group (48.8% vs 25.6%, P = 0.044). Functional mapping showed that the rate of deletion in three functional sites (the nucleocapsid binding site, start codon of M, and site for viral secretion) located in the N-terminus of the pre-S2 region was significantly higher in the HBV/B group (P < 0.05). One type of N-terminus pre-S1 deletion mutant with deletion of the start codon of the L protein was frequently observed in the HBV/C group (20.9% vs 9.3%, P = 0.228), particularly in the LC patients (42.9% vs 12.5%). Different patterns of pre-S deletions were also found between the HBV/B and HBV/C groups according to different clinical outcomes. In CH patients, deletion in the site for polymerized human serum albumin was more frequent in the HBV/B group (88.9% vs 36.4%, P = 0.028). In the LC-HCC patients, the rate of deletion in the pre-S2 region was significantly higher in the HBV/B group than in the HBV/C group (P < 0.05).
CONCLUSION HBV/B- and HBV/C-infected carriers exhibit different patterns of pre-S deletion, which may be associated with the progression of liver diseases.
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7
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Gao S, Duan ZP, Coffin CS. Clinical relevance of hepatitis B virus variants. World J Hepatol 2015; 7:1086-1096. [PMID: 26052397 PMCID: PMC4450185 DOI: 10.4254/wjh.v7.i8.1086] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Revised: 01/28/2015] [Accepted: 02/12/2015] [Indexed: 02/06/2023] Open
Abstract
The hepatitis B virus (HBV) is a global public health problem with more than 240 million people chronically infected worldwide, who are at risk for end-stage liver disease and hepatocellular carcinoma. There are an estimated 600000 deaths annually from complications of HBV-related liver disease. Antiviral therapy with nucleos/tide analogs (NA) targeting the HBV polymerase (P) can inhibit disease progression by long-term suppression of HBV replication. However, treatment may fail with first generation NA therapy due to the emergence of drug-resistant mutants, as well as incomplete medication adherence. The HBV replicates via an error-prone reverse transcriptase leading to quasispecies. Due to overlapping open reading frames mutations within the HBV P can cause concomitant changes in the HBV surface gene (S) and vice versa. HBV quasispecies diversity is associated with response to antiviral therapy, disease severity and long-term clinical outcomes. Specific mutants have been associated with antiviral drug resistance, immune escape, liver fibrosis development and tumorgenesis. An understanding of HBV variants and their clinical relevance may be important for monitoring chronic hepatitis B disease progression and treatment response. In this review, we will discuss HBV molecular virology, mechanism of variant development, and their potential clinical impact.
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8
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Gong JY, Liu X, Dong Y, Zhou TH, Li JW. Construction and expression of a recombinant eukaryotic expression plasmid containing the preS1-preS2-S genes of hepatitis B virus and the granulocyte-macrophage colony stimulating factor gene: A study of its immunomodulatory effects. Biomed Rep 2012; 1:251-256. [PMID: 24648930 DOI: 10.3892/br.2012.47] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2012] [Accepted: 11/06/2012] [Indexed: 11/06/2022] Open
Abstract
A total of 10-20% of the population remains unresponsive or weakly responsive to hepatitis B vaccine, which is composed of hepatitis B surface antigen HBsAg (S protein). Therefore, it is necessary to develop a hepatitis B vaccine with a better penetrating and responsive rate. In the present study, a plasmid pVAX1-L-GM was constructed and its immunomodulatory effect of as hepatitis B virus (HBV) DNA vaccine was analyzed through the immunization of BALB/c mice. Immune responses were measured after immunization by anti-HBsAg, proliferation of splenocytes, the number of CD4+ and CD8+ molecules, CTL cytotoxicity, cytokines of IFN-γ and IL-2 secretion assays. Following the immunization, mice in the pVAX1-L-GM group produced antibody 2 weeks earlier compared to the control plasmid pVAX1 and pVAX1HBsAg groups and antibody levels showed significant differences. Enhanced HBsAg-specific splenocyte proliferation as well as specific cytotoxic activities of splenic CTLs were also detected. Furthermore, pVAX1-L-GM plasmid increased the number of CD4+ and CD8+ molecules on the surface of the spleen T cell and the level of IFN-γ, IL-2 secretion. pVAX1-L-GM induced a specific immune response in mice and enhanced the immune effect. Thus, a foundation was laid for developing immunogenicity of a better prevention and treatment of HBV via a hepatitis B vaccine.
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Affiliation(s)
- Jun-Yuan Gong
- Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, P.R. China
| | - Xin Liu
- Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, P.R. China
| | - Yan Dong
- Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, P.R. China
| | - Tian-Hong Zhou
- Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, P.R. China
| | - Jun-Wu Li
- Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, P.R. China
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9
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Kao JH, Liu CJ, Jow GM, Chen PJ, Chen DS, Chen BF. Fine mapping of hepatitis B virus pre-S deletion and its association with hepatocellular carcinoma. Liver Int 2012; 32:1373-81. [PMID: 22676233 DOI: 10.1111/j.1478-3231.2012.02826.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/20/2012] [Accepted: 05/01/2012] [Indexed: 01/16/2023]
Abstract
BACKGROUND Naturally occurring pre-S deletion mutants have been identified in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). AIMS This study investigated whether specific deletions within the pre-S region were associated with HCC development. METHODS The virologic characteristics of 56 HBV chronic carriers and 112 age-matched patients with HBV-related HCC were examined. RESULTS The HCC patients had a significantly higher frequency of high viral load, basal core promoter mutation and pre-S deletion than chronic carriers. Sequencing analysis showed that the deleted regions were clustered mainly in the C terminus of pre-S1 (70.5%) and the N terminus of pre-S2 (72.7%) in HCC patients. Immuno-epitope mapping of these pre-S deletion sequences showed that all the deletion regions encompassed T- and B- cell epitopes and the B-cell epitope at amino acid 1-6 of pre-S2 was significantly deleted in HCC patients (60.0% vs. 0.0%; P = 0.036). Functional mapping of these deletion mutants showed that most of HCC patients lost one or more functional sites and the deletion of site for viral secretion (aa 1-5 of pre-S2 domain) was significantly detected in HCC patients than chronic carriers (62.5% vs. 0.0%; P = 0.029). Computational protein function prediction indicated that these mutants may have different molecular functions and participate in other biological processes compared with wild-type pre-S. CONCLUSIONS Deletion of B-cell epitope at amino acid 1-6 of pre-S2 region and the site for virion secretion are significantly associated with the development of HCC in HBV carriers.
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Affiliation(s)
- Jia-Horng Kao
- Division of Gastroenterology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
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Ryu HJ, Kim DY, Park JY, Chang HY, Lee MH, Han KH, Chon CY, Ahn SH. Clinical features and prognosis of hepatocellular carcinoma with respect to pre-S deletion and basal core promoter mutations of hepatitis B virus Genotype C2. J Med Virol 2011; 83:2088-95. [DOI: 10.1002/jmv.22238] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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11
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Tang YZ, Liu L, Pan MM, Wang YM, Deng GH. Evolutionary pattern of full hepatitis B virus genome during sequential nucleos(t)ide analog therapy. Antiviral Res 2011; 90:116-25. [PMID: 21440005 DOI: 10.1016/j.antiviral.2011.03.183] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2010] [Revised: 03/13/2011] [Accepted: 03/21/2011] [Indexed: 12/23/2022]
Abstract
The evolutionary and mutational pattern of full hepatitis B virus (HBV) quasispecies during sequential nucleos(t)ide analog (NUC) therapy remains unclear. In this study, full-length HBV clones were generated from serial serum samples of five chronic hepatitis B patients who received sequential NUC therapies (treated patients) and two untreated patients with acute flares. The evolutionary and mutational patterns of full HBV quasispecies were studied. In the three treated patients who received lamivudine as initial antiviral therapy, nucleotide polymorphism and nonsynonymous divergence all decreased at lamivudine breakthrough but increased after rescue therapies. Conversely, two other treated patients showed a distinct change in divergence during adefovir-telbivudine sequential therapies. Untreated subjects exhibited increased polymorphism and divergence in the preC/C region at ALT flare. Four of the treated patients presented amino acid changes in the "a" determinant during NUC therapy. All of the treated subjects showed amino acid changes within the known T-cell or B-cell epitopes in the surface or core antigen, most of which were accompanied by mutations in reverse transcriptase (RT) region. Co-variations in the core promoter, the preC region and in the known epitopes of the preS gene accompanied by RT mutations, were common. In untreated patients, most of these co-variations located in the preC/C gene. In conclusion, the distribution of genetic variability of HBV shows remarkably different patterns between the treated and untreated subjects and the quasispecies divergence of different regions of HBV may vary remarkably even within a single host.
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Affiliation(s)
- Ying-Zi Tang
- Institute for Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China
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12
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Ahn SH, Yuen L, Han KH, Littlejohn M, Chang HY, Damerow H, Ayres A, Heo J, Locarnini S, Revill PA. Molecular and clinical characteristics of hepatitis B virus in Korea. J Med Virol 2010; 82:1126-34. [PMID: 20513074 DOI: 10.1002/jmv.21844] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Korea is an endemic area of hepatitis B virus (HBV) infection but very little is known about the molecular characteristics of HBV isolates from Korean patients or the association with disease progression. The complete HBV genome sequences from 53 Korean patients with chronic hepatitis B, advanced cirrhosis, or hepatocellular carcinoma (HCC) were analyzed to identify (i) subgenotype distribution and genetic diversity and (ii) signature mutations associated with liver disease progression. With the exception of 1 patient infected with HBV/B, all 52 patients (98.1%) were infected with HBV/C, subgenotype C2. These strains were 98.4% identical and the frequency of amino acid substitutions occurring within key immunological epitopes increased with disease severity. A number of amino acid/nucleotide substitutions were associated with HCC, namely sR24K (HBsAg), SI126T (HBsAg), and pcA1846T (precore gene) mutations (P = 0.029, 0.001, and 0.008, respectively). HBV harboring deletions in the pre-S region were also associated with increased liver disease severity (chronic hepatitis B vs. cirrhosis, P = 0.040; chronic hepatitis B vs. HCC, P = 0.040). Despite the high degree of sequence conservation, several key HBV mutations were associated with disease progression. Prospective studies with larger cohorts of patients are required to evaluate further the clinical manifestation of HBV/C2 in Korea.
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Affiliation(s)
- Sang Hoon Ahn
- Department of Internal Medicine, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Republic of Korea
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13
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Huang X, Qin Y, Zhang P, Tang G, Shi Q, Xu J, Qi F, Shen Q. PreS deletion mutations of hepatitis B virus in chronically infected patients with simultaneous seropositivity for hepatitis-B surface antigen and anti-HBS antibodies. J Med Virol 2010; 82:23-31. [PMID: 19950231 DOI: 10.1002/jmv.21669] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Hepatitis B surface antigen (HBsAg) and anti-HBs antibodies (anti-HBs) may coexist in certain chronic hepatitis B (CHB) patients. This study was designed to further explore the relationship between this coexistence and hepatitis B Virus (HBV) preS deletions. Sera of 28 patients carrying both HBsAg and anti-HBs (Group I) and those of another 28 HBsAg positive but anti-HBs negative patients (Group II) were collected from CHB patients. Direct sequencing of polymerase chain reaction products or sequencing of clones was applied to both groups to determine sequences of HBV preS and S genes. Genotyping of the S gene indicated that all sampled HBVs were either Genosubtype Ba or Genosubtype Ce. Seven samples in Group I harbored HBV preS deletion mutations. Three of the seven samples showed large deletion mutations in 3' terminus of preS1 and co-existence of the mutant type and the full-length wild type, and the remaining four samples showed deletion mutations in 5' terminus of preS2. All mutant strains were found to be genosubtype Ce. Only two samples in Group I showed G145R/A mutation. Only one sample in Group II contained preS deletion mutation. It is therefore concluded that HBV preS deletion mutations are likely to be related to the coexistence of HBsAg and anti-HBs in CHB patients (P-value = 0.024). Some immune reactions may select for the preS deletion in CHB patients with anti-HBs, the possible marker for immune selection.
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Affiliation(s)
- Xiangyan Huang
- Department of Laboratory Diagnostics, Changhai Hospital Affiliate to the Second Military Medical University, Shanghai, China
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Sylvan SPE, Madalinski K, Hellström UB. Anti-preS responses influence the anti-HBs response in newborns after vaccination with the third generation Sci-B-Vac vaccine. Vaccine 2009; 28:446-51. [PMID: 19874926 DOI: 10.1016/j.vaccine.2009.10.023] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2009] [Revised: 09/28/2009] [Accepted: 10/07/2009] [Indexed: 02/06/2023]
Abstract
We analysed the specificity and significance of the antibody response towards the linear preS1 sequence that has been shown to represent the "hepatocyte binding site" comprising amino acids preS1 (21-47) or the specific preS2 (131-140) antibody response to the "polymerised albumin receptor" in relation to the antibody response to hepatitis B surface antigen during immunisation of healthy children with the preS-containing Sci-B-Vac vaccine. Twenty-eight healthy newborns received three doses of the Sci-B-Vac vaccine according to a 0-, 1-, and 6-month scheme. Seventeen (61%) of the 28 newborns had detectable levels of anti-preS1 (21-47) antibodies and 14 (50%) were anti-preS2 (131-140) reactive at 6 and/or 9 months after initiation of the vaccination. The mean levels of anti-HBs were significantly higher in the anti-preS2 (131-140) non-reactive (24580+/-7815IU/l, mean+SEM) compared with the reactive sera (7287+/-2317IU/l, p<0.025). The highest anti-HBs levels were found in newborns who exhibited reactivity towards the aa 21-47 of the preS1 but lacked anti-preS2 (131-140) reactivity.
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Affiliation(s)
- Staffan P E Sylvan
- Department of Communicable Disease Control and Prevention, Uppsala County Council, Sweden.
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15
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Hellström UB, Madalinski K, Sylvan SP. PreS1 epitope recognition in newborns after vaccination with the third-generation Sci-B-Vac vaccine and their relation to the antibody response to hepatitis B surface antigen. Virol J 2009; 6:7. [PMID: 19154574 PMCID: PMC2635352 DOI: 10.1186/1743-422x-6-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2008] [Accepted: 01/20/2009] [Indexed: 12/12/2022] Open
Abstract
Background Sci-B-Vac™ is a recombinant, hepatitis B vaccine derived from a mammalian cell line and containing hepatitis B surface antigen (HBsAg) as well as preS1 and preS2 antigens. Few studies have been performed on the antibody responses to preS1 in relation to the antibody to hepatitis B surface antigen (anti-HBs) response during immunisation of healthy children with preS-containing vaccines. Results In this study 28 healthy newborns were randomly selected to receive either 2.5 ug or 5.0 ug of the Sci-B-Vac vaccine. Children received three doses of vaccine according to a 0-, 1-, 6-month scheme. Antibodies against the S-protein and three synthetic peptides mimicking three B-cell preS1 epitopes, (21–32 amino acid epitope), (32–47 amino acid epitope) and the C-terminal (amino acid epitope 94–117) were determined at 6 and 9 months. Fourteen (50%) of the 28 newborns had detectable levels of anti-preS1 (21–32) antibodies; 15 (54%) were anti-preS1 (32–47) reactive and 12 (43%) were anti-preS1 (94–117) reactive at 6 or 9 months after initiation of the vaccination. Significantly higher levels of anti-HBs were observed in the sera of patients with detectable anti-preS1 (32–47) reactivity (24 550 ± 7375 IU/L, mean ± SEM) as compared with the non-reactive sera (5991 ± 1530 IU/L, p < 0.05). The anti-HBs levels were significantly lower if none (p < 0.05) or one (p < 0.025) of the preS1 (21–32, 32–47, 94–117) peptides were recognised compared with the anti-HBs levels if two or three peptides were recognised. Conclusion Recognition of several preS1 epitopes, and in particular, the epitope contained within the second half of the hepatocyte binding site localised in the hepatitis B surface protein of the third-generation hepatitis B vaccine is accompanied by a more pronounced antibody response to the S-gene-derived protein in healthy newborns.
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Affiliation(s)
- Ulla B Hellström
- Department of Communicable Disease Control and Prevention, Uppsala County Council, Sweden.
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16
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Hellström U, Lindh M, Krogsgaard K, Sylvan S. Demonstration of an association between detection of IgG antibody reactivity towards the C-terminal region of the preS1 protein of hepatitis B virus and the capacity to respond to interferon therapy in chronic hepatitis B. J Gastroenterol Hepatol 2008; 23:804-10. [PMID: 17931371 DOI: 10.1111/j.1440-1746.2007.05174.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
BACKGROUND AND AIM The treatment of hepatitis B virus (HBV) remains complex, with somewhat unpredictable responses. The aim of this study was to determine the predictive value of the pretreatment presence of circulatory antibodies towards a synthetic peptide mimicking the amino acids 94-117 of the preS1 protein of HBV and the capacity to respond to alpha-inteferon (IFN-alpha) treatment. METHODS The anti preS1(94-117) antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA) and the response to INF-alpha therapy was judged by the effect on the viral kinetics as measured by an assay based on quantitative polymerase chain reaction during the treatment and follow up. RESULTS We found a significant (P < 0.001) correlation between the pretreatment presence of anti preS1(94-117) antibodies and a decrease in viral levels on follow up after the end of IFN-alpha therapy. The combined response of HBV DNA suppression (P < 0.001), hepatitis B e antigen (HBeAg) loss (P < 0.0001), anti-HBe seroconversion (P < 0.005) and AST aminotransferase normalization (P < 0.01) was also highly associated with the pretreatment presence of anti preS1(94-117) antibodies. CONCLUSION The positive predictive value (PPV) of anti preS1(94-117) in determining a virological response was 83% and the negative predictive value (NPV) was 100%, indicating that in the absence of pretreatment anti preS1 reactivity virtually no patient has the capacity to respond to IFN-alpha therapy. Our findings may help to improve the efficacy of INF-alpha therapy for chronic hepatitis B (CHB) by guiding the selection of patients for treatment and optimizing the clinical management of the individual patient.
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Affiliation(s)
- Ulla Hellström
- Department of Communicable Disease Control and Prevention, Karolinska Hospital, Stockholm, Sweden
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Ito K, Tanaka Y, Kato M, Fujiwara K, Sugauchi F, Sakamoto T, Shinkai N, Orito E, Mizokami M. Comparison of complete sequences of hepatitis B virus genotype C between inactive carriers and hepatocellular carcinoma patients before and after seroconversion. J Gastroenterol 2007; 42:837-844. [PMID: 17940837 DOI: 10.1007/s00535-007-2100-6] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2007] [Accepted: 07/31/2007] [Indexed: 02/04/2023]
Abstract
BACKGROUND Most patients who acquire chronic hepatitis B virus (HBV) infection by perinatal transmission become inactive carriers (IC) after hepatitis B e (HBe) antigen seroconversion, whereas some patients have persistent abnormal serum transaminase levels and develop hepatocellular carcinoma (HCC) in the anti-HBe-positive phase. The aim of this study was to investigate the HCC-related mutations of HBV. METHODS Complete sequences of HBV were examined among eight IC and eight HCC patients infected with HBV genotype C before and after seroconversion. RESULTS The frequency of the T1653 mutation tended to be higher among HCC patients after seroconversion (16.7% vs. 62.5%; P = 0.086). The prevalence of a basal core promoter double mutation (T1762/A1764) was high among both IC and HCC patients after seroconversion (83.3% vs. 87.5%; P = 0.825). Among the HCC patients, a pre-S deletion mutant was detected in 62.5% patients before seroconversion, and in 37.5% patients after seroconversion. The core deletion mutant was also detected in 50% of HCC patients only before seroconversion. Deletion mutants of the pre-S or core region before seroconversion were significantly associated with HCC patients (0% vs. 62.5%; P = 0.007, 0% vs. 50%; P = 0.021, respectively). CONCLUSIONS Our data showed a significant association of pre-S and core deletion mutants before seroconversion with HCC development. The T1653 mutation after seroconversion was frequently found in HCC patients infected with HBV genotype C. These results suggest that mutations may be predictive factor for development of HCC.
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Affiliation(s)
- Kiyoaki Ito
- Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, 1-Kawasumi, Mizuho, Nagoya 467-8601, Japan
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Chen BF, Liu CJ, Jow GM, Chen PJ, Kao JH, Chen DS. High prevalence and mapping of pre-S deletion in hepatitis B virus carriers with progressive liver diseases. Gastroenterology 2006; 130:1153-68. [PMID: 16618410 DOI: 10.1053/j.gastro.2006.01.011] [Citation(s) in RCA: 232] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/01/2005] [Accepted: 12/21/2005] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS The interactions among pre-S deletion, precore (PC) mutation, and basal core promoter (BCP) mutation in various stages of chronic hepatitis B virus (HBV) infection remain unclear and were thus investigated in this study. METHODS The sequences of the pre-S region and the BCP (A1762T, G1764A) and PC (G1896A) mutations were determined in 46 HBV chronic carriers (CC) and 106 age-matched carriers with different stages of liver diseases, including 38 chronic hepatitis (CH), 18 cirrhosis (LC), and 50 hepatocellular carcinoma (HCC). RESULTS A higher prevalence of pre-S deletion and BCP and PC mutations was found in carriers with progressive liver diseases compared with the CC group. By logistic regression analysis, patients with pre-S deletion and BCP mutation were significantly associated with the development of progressive liver diseases than those without. Combination of mutations rather than single mutation was associated with the development of progressive liver diseases, especially in combination with pre-S deletion. Sequencing analysis showed that the deleted regions were more often in the 3' terminus of pre-S1 and the 5' terminus of pre-S2. Further mapping of these pre-S deletion sequences found that all the deletion regions encompassed T- and B-cell epitopes, and most of them lost 1 or more functional sites. CONCLUSIONS Our data indicate that patients with progressive liver diseases have a higher frequency of pre-S deletion.
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Affiliation(s)
- Bing-Fang Chen
- School of Medicine, Fu Jen Catholic University, Taipei, Taiwan
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Hu WG, Wei J, Xia HC, Yang XX, Li F, Li GD, Wang Y, Zhang ZC. Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins. World J Gastroenterol 2005; 11:2088-94. [PMID: 15810073 PMCID: PMC4305776 DOI: 10.3748/wjg.v11.i14.2088] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitope-based hepatitis B vaccine design.
METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA.
RESULTS: The results in mice showed that the immu-nogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity. More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing.
CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic dom-ains in preS1 (21-119) region. The preS1 (34-59) fragm-ents were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitope-based HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.
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Affiliation(s)
- Wei-Guo Hu
- Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China
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Lu YY, Liu Y, Cheng J, Ling YD, Chen TY, Shao Q, Wang L, Zhang LX. Genes trans-regulated by a novel hepatitis B virus preS2 antigen binding protein S2-29 by cDNA microarray. Shijie Huaren Xiaohua Zazhi 2004; 12:58-61. [DOI: 10.11569/wcjd.v12.i1.58] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the biological functions of a novel hepatitis B virus preS2 antigen binding protein S2-29, and to analyze the gene expression profiles of HepG2 cell transfected with S2-29 gene.
METHODS: S2-29 gene was screened and identified by using yeast two-hybrid system 3 and coimmunoprecipita-tion technique. Full-length encoding frame S2-29 and its amino acid sequences were identified by using bioinformatics method and the recombined eukaryotic expression plasmid pcDNA3.1(-)-S2-29 was constructed and transfected into HepG2 cells. Total mRNA was isolated from the HepG2 cells transfected with pcDNA3.1(-) and pcDNA3.1(-)-S2-29, respectively. cDNA microarray was employed for detecting and analysing of mRNA from the HepG2 cells.
RESULTS: S2-29 cDNA sequence was obtained and identified by yeast two-hybrid screening and the bioinformatics analysis. Among 1 152 genes, there were 10 differences, of which 9 genes were upregulated and 1 gene were downregulated in HepG2 cells transfected with S2-29 protein expression plasmid. These genes differentially down-regulated by S2-29 protein included eukaryotic translation elongation factor 2, MAP-kinase activating death domain, glutathione peroxidase 5, gelsolin-like capping protein (actin filament), NDRG family member 2, prosaposin, SUMO-1 activating enzyme subunit 1, insulin receptor and a novel protein.
CONCLUSION: Microarray technique is successfully used to screen the genes trans-regulated by S2-29, which brings some new clues for studying the trans-regulation and biological function of S2-29.
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Lu YY, Cheng TY, Cheng J, Liang YD, Wang L, Liu Y, Li K, Zhang J, Shao Q, Zhang LX. Screening and identification of a novel gene coding for hepatitis B virus pre-S2 antigen interacting protein S2-29. Shijie Huaren Xiaohua Zazhi 2003; 11:1114-1117. [DOI: 10.11569/wcjd.v11.i8.1114] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM The Pre-S2 region of hepatitis B virus (HBV) has been reported to have complex biological functions. It has human polymerized albumin receptor (PAR) activity, which correlates with viral replication, and it can induce neutralization antibody. As an important part of truncated middle surface proteins (MHBs), the Pre-S2 domain binds PKC alpha/beta and triggers a PKC-dependent activation of the c-Raf-1/MAP2-kinase signal transduction cascade, resulting in activation of transcription factors such as AP-1 and NF-kB. To investigate the biological function of hepatitis B virus (HBV) Pre-S2 protein, we used yeast two-hybrid technique to screen proteins interacting with HBV Pre-S2 antigen in hepatocytes.
METHODS The HBV Pre-S2 gene was amplified by polymerase chain reaction (PCR) and cloned into yeast expression vector PGBKT7 to construct HBV Pre-S2 bait plasmid. The bait plasmid was transformed into yeast AH109 and mated with yeast Y187 containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After being extracted and sequenced, genes were analyzed by bioinformatics. The complete sequence of new gene S2-29 was amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7, then translated by using reticulocyte lysate and analysed by immunoprecipitation technique in vitro.
RESULTS Twenty-six colonies were obtained, among them two colonies were new genes with unknown function and no homeobox genes were found in Genbank by blast. The complete sequence of new gene S2-29 could be amplified from the mRNA of HepG2 cell and the interaction between HBV Pre-S2 antigen and S2-29 was further confirmed by coimmunoprecipitation technique.
CONCLUSION Genes of HBV Pre-S2 interacting proteins were successfully screened. A novel gene S2-29 was cloned and could express in HepG2 cell. The HBV Pre-S2 antigen could interact with S2-29, which brings new clues for studying the biological functions of HBV Pre-S2 and the pathogenesis of HBV infection.
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Affiliation(s)
- Yin-Ying Lu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Tian-Yan Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jun Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yao-Dong Liang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Lin Wang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yan Liu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ke Li
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jian Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Qing Shao
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ling-Xia Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
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Park JH, Lee MK, Kim HS, Kim KL, Cho EW. Targeted destruction of the polymerized human serum albumin binding site within the preS2 region of the HBV surface antigen while retaining full immunogenicity for this epitope. J Viral Hepat 2003; 10:70-9. [PMID: 12558915 DOI: 10.1046/j.1365-2893.2003.00397.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The 55-amino acid (a.a.) preS2 region of the hepatitis B virus (HBV) envelope protein is highly immunogenic, and antibodies against this epitope confer seroprotection against HBV infections. Accordingly, various experimental and clinical studies for developing and evaluating HBV vaccines that include this particular epitope have been reported. However, a pitfall in using preS2 epitopes as part of a vaccinating antigen is that polymerized human serum albumin (pHSA), which is a normal constituent of the human serum, binds to and makes complexes with this particular region. Consequently, it is most likely that the antigen epitope is masked by serum pHSA and subsequently not detected by the immune system. To overcome these limitations, a novel single a.a substitute of the preS2 region was designed that corresponds to a tyrosine to serine exchange at position 140 of preS2. Competitive enzyme-linked immunosorbent assay showed that this substitution completely abolishes pHSA-binding activities in the mutated preS2 peptide, and CD spectra analysis revealed that this property might have been induced by slight conformational changes in its secondary structure. Nevertheless, the original B-cell epitope was still preserved in the mutated preS2 as determined by experimental immunization in mice. In this regard, the preS2(120-145/Y140S) sequence may be an HBV vaccine where epitopes, with intrinsic properties have been deleted without affecting the immunogenicity of the epitope itself. It is expected that the inclusion of this point mutated preS2 epitope will improve the efficacy of conventional preS2-containing HBV vaccines.
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Affiliation(s)
- J-H Park
- Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
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Emir S, Büyükpamuk M, Akyüz C, Kutluk T, Güler E, Cağlar K. The comparison of antibody response to different hepatitis b vaccines with and without pre-S2 antigen in children with cancer. Pediatr Hematol Oncol 2002; 19:227-33. [PMID: 12051588 DOI: 10.1080/08880010252899389] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Children with cancer are at an increased risk of hepatitis B infection and chronic liver disease. Since hepatitis B vaccines containing pre-S2 antigen has been recently reported as being more efficient in providing immunization in healthy individuals, the authors compared antibody response to pre-S2-containing vaccine with no-pre-S2-containing hepatitis B vaccine, when given in double doses to 100 children receiving chemotherapy. Patients, aged 1 to 16 years with negative HBV serology, were vaccinated with 2 different types of HBV vaccines between 1997 and 1999. Group 1 received Gen Hevac B containing pre-S2 (n = 41) in a dose of 20 microg for patients younger than 10 years old and 40 microg for older patients. Group 2 was vaccinated at the same dose with hepatitis B vaccines not containing pre-S2 antigen. All vaccinations were repeated at 0, 1, and 6 months. Serum samples were drawn for determination of anti-HBs titers at 1, 3, 6, and 8 months. After the third dose of vaccine, the seroconversion rate was 72% in group 1 and 62% in group 2. The anti-HBs levels were higher in the group receiving pre-S2-containing hepatitis B vaccine. However, the difference between groups was not statistically significant (p > .05). The administration of pre-S2-containing hepatitis B vaccines may give a better seroconversion and higher antibody response to vaccination in children with cancer. But a further large-scale study is needed to confirm this finding.
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Affiliation(s)
- Suna Emir
- Department of Pediatric Oncology, Hacettepe University Faculty of Medicine, Ankara, Turkey.
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Wei J, Wang YQ, Lu ZM, Li GD, Wang Y, Zhang ZC. Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay. World J Gastroenterol 2002; 8:276-81. [PMID: 11925607 PMCID: PMC4658366 DOI: 10.3748/wjg.v8.i2.276] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119 aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B.
METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119 aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni2+-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed.
RESULTS: preS1(21-119 aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E. coli (30 mg•L⁻¹), and easily purified to high purity over 90% by one step of Ni2+-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119 aa) protein was determined by 150 g•L⁻¹ SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119 aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 mo, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent.
CONCLUSION: The high-purity preS1(21-119 aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.
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Affiliation(s)
- Jun Wei
- Institute of Biochemsitry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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