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Zhang J, Wang Q, Yuan W, Li J, Yuan Q, Zhang J, Xia N, Wang Y, Li J, Tong S. Both middle and large envelope proteins can mediate neutralization of hepatitis B virus infectivity by anti-preS2 antibodies: escape by naturally occurring preS2 deletions. J Virol 2024; 98:e0192923. [PMID: 39078152 PMCID: PMC11334434 DOI: 10.1128/jvi.01929-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2023] [Accepted: 07/02/2024] [Indexed: 07/31/2024] Open
Abstract
Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1/preS2/S, preS2/S, and S domain alone, respectively. S and preS1 domains mediate sequential virion attachment to heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP), respectively, which can be blocked by anti-S and anti-preS1 antibodies. How anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. The late stage of chronic HBV infection often selects for mutated preS2 translation initiation codon to prevent M protein expression, or in-frame preS2 deletions to shorten both L and M proteins. When introduced to infectious clone of genotype C or D, both M-minus mutations and most 5' preS2 deletions sustained virion production. Such mutant progeny viral particles were infectious in NTCP-reconstituted HepG2 cells. Neutralization experiments were performed on the genotype D clone. Although remaining susceptible to anti-preS1 and anti-S neutralizing antibodies, M-minus mutants were only partially neutralized by two anti-preS2 antibodies tested while preS2 deletion mutants were resistant. By infection experiments using viral particles with lost versus increased M protein expression, or a neutralization escaping preS2 deletion only present on L or M protein, we found that both full-length L and M proteins contributed to virus neutralization by the two anti-preS2 antibodies. Thus, immune escape could be a driving force for the selection of M-minus mutations, and especially preS2 deletions. The fact that both L and M proteins could mediate neutralization by anti-preS2 antibodies may shed light on the underlying molecular mechanism.IMPORTANCEThe large (L), middle (M), and small (S) envelope proteins of hepatitis B virus (HBV) contain preS1/preS2/S, preS2/S, and S domain alone, respectively. The discovery of heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP) as the low- and high-affinity HBV receptors could explain neutralizing potential of anti-S and anti-preS1 antibodies, respectively, but how anti-preS2 neutralizing antibodies work remains enigmatic. In this study, we found two M-minus mutants in the context of genotype D partially escaped two anti-preS2 neutralizing antibodies in NTCP-reconstituted HepG2 cells, while several naturally occurring preS2 deletion mutants escaped both antibodies. By point mutations to eliminate or enhance M protein expression, and by introducing preS2 deletion selectively to L or M protein, we found binding of anti-preS2 antibodies to both L and M proteins contributed to neutralization of wild-type HBV infectivity. Our finding may shed light on the possible mechanism(s) whereby anti-preS2 antibodies neutralize HBV infectivity.
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Affiliation(s)
- Jing Zhang
- Department of Pathobiology, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Qianru Wang
- Department of Pathobiology, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Wenqing Yuan
- Liver Research Center, Rhode Island Hospital, The Warren Alpert School of Medicine, Brown University, Providence, Rhode Island, USA
| | - Jing Li
- Liver Research Center, Rhode Island Hospital, The Warren Alpert School of Medicine, Brown University, Providence, Rhode Island, USA
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Quan Yuan
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, China
| | - Jiming Zhang
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Ningshao Xia
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, China
| | - Yongxiang Wang
- Department of Pathobiology, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Jisu Li
- Liver Research Center, Rhode Island Hospital, The Warren Alpert School of Medicine, Brown University, Providence, Rhode Island, USA
| | - Shuping Tong
- Department of Pathobiology, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China
- Liver Research Center, Rhode Island Hospital, The Warren Alpert School of Medicine, Brown University, Providence, Rhode Island, USA
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2
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Gehring AJ, Salimzadeh L. Current and future use of antibody-based passive immunity to prevent or control HBV/HDV infections. Antiviral Res 2024; 226:105893. [PMID: 38679166 DOI: 10.1016/j.antiviral.2024.105893] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2024] [Revised: 04/19/2024] [Accepted: 04/22/2024] [Indexed: 05/01/2024]
Abstract
With the increasing momentum and success of monoclonal antibody therapy in conventional medical practices, there is a revived emphasis on the development of monoclonal antibodies targeting the hepatitis B surface antigen (anti-HBs) for the treatment of chronic hepatitis B (HBV) and hepatitis D (HDV). Combination therapies of anti-HBs monoclonal antibodies, and novel anti-HBV compounds and immunomodulatory drugs presenting a promising avenue to enhanced therapeutic outcomes in HBV/HDV cure regimens. In this review, we will cover the role of antibodies in the protection and clearance of HBV infection, the association of anti-HBV surface antigen antibodies (anti-HBs) in protection against HBV and how antibody effector functions, beyond neutralization, are likely necessary. Lastly, we will review clinical data from previous and ongoing clinical trials of passive antibody therapy to provide a state-of-the-are perspective on passive antibody therapies in combinations with additional novel agents.
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Affiliation(s)
- Adam J Gehring
- Schwartz-Reisman Liver Research Centre, University Health Network, Toronto, ON, Canada; Department of Immunology, University of Toronto, Toronto, ON, Canada.
| | - Loghman Salimzadeh
- Schwartz-Reisman Liver Research Centre, University Health Network, Toronto, ON, Canada; Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada
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Dishlers A, Petrovskis I, Skrastina D, Zarina I, Lieknina I, Jansons J, Akopjana I, Zakova J, Ose V, Sominskaya I. PreS1 Containing HBc VLPs for the Development of a Combined Therapeutic/Prophylactic Hepatitis B Vaccine. Microorganisms 2023; 11:microorganisms11040972. [PMID: 37110395 PMCID: PMC10142831 DOI: 10.3390/microorganisms11040972] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2022] [Revised: 03/31/2023] [Accepted: 04/05/2023] [Indexed: 04/29/2023] Open
Abstract
The available HBV vaccines based on the HBV surface protein are manufactured in yeasts and demonstrate excellent prophylactic but no therapeutic activity and are thus ineffective against chronic HBV infection. Five different HBV core proteins (HBc)-full length and C-terminally truncated-were used for the insertion of the short, preS1,aa 20-47 and long, preS1phil, aa 12-60 + 89-119 fragments. Modified virus-like particles (VLPs) were compared for their biotechnological and immunological properties. The expression level of HBc-preS1 proteins was high for all investigated proteins, allowing us to obtain 10-20 mg of purified VLPs from a gram of biomass with the combination of gel filtration and ion-exchange chromatography to reach approximately 90% purity of target proteins. The immunogenicity of chimeric VLPs was tested in BALB/c mice, showing a high anti-preS1 response and substantial T-cell proliferation after stimulation with HBc protein. Targeted incorporation of oligonucleotide ODN 1668 in modified HBc-preS1 VLPs was demonstrated.
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Affiliation(s)
- Andris Dishlers
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Ivars Petrovskis
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Dace Skrastina
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Ieva Zarina
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Ilva Lieknina
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Juris Jansons
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Inara Akopjana
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Jelena Zakova
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Velta Ose
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
| | - Irina Sominskaya
- Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, 1067 Riga, Latvia
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Beretta M, Mouquet H. Advances in human monoclonal antibody therapy for HBV infection. Curr Opin Virol 2022; 53:101205. [PMID: 35123237 DOI: 10.1016/j.coviro.2022.101205] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2021] [Revised: 12/10/2021] [Accepted: 01/15/2022] [Indexed: 12/17/2022]
Abstract
HBV neutralizing antibodies target the viral envelope antigens (HBsAg) and confer long-term immune protection in vaccinees and infected humans who seroconvert. They recognize various HBsAg epitopes, and can be armed with Fc-dependent effector functions essential for eliminating infected cells and stimulating adaptive immunity. Hundreds of HBsAg-specific monoclonal antibodies (mAbs) were produced from the early 80's, but it is only recently that bona fide human anti-HBV mAbs were generated from vaccinees and seroconverters. Neutralizing HBV mAbs have in vivo prophylactic and therapeutic efficacy in animal models, and the capacity to decrease antigenemia and viremia in infected humans. Thus, polyfunctional, potent and broad human HBV neutralizing mAbs offer novel opportunities to develop effective interventions to prevent and treat HBV infection. Here, we summarize recent findings on the humoral immune response to HBV, and explore the potential of human HBV neutralizing mAbs as immunotherapeutics to help achieving a functional cure for HBV.
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Affiliation(s)
- Maxime Beretta
- Laboratory of Humoral Immunology, Department of Immunology, Institut Pasteur, Paris, 75015, France; INSERM U1222, Paris, 75015, France
| | - Hugo Mouquet
- Laboratory of Humoral Immunology, Department of Immunology, Institut Pasteur, Paris, 75015, France; INSERM U1222, Paris, 75015, France.
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Cross-Protection of Hepatitis B Vaccination among Different Genotypes. Vaccines (Basel) 2020; 8:vaccines8030456. [PMID: 32824318 PMCID: PMC7563454 DOI: 10.3390/vaccines8030456] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2020] [Revised: 08/09/2020] [Accepted: 08/12/2020] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B (HB) vaccination is the most effective method for preventing HB virus (HBV) infection. Universal HB vaccination containing recombinant HB surface antigens (HBsAg) is recommended. Our data revealed that human monoclonal HB surface antibody (anti-HBs) from individuals inoculated with genotype C-based HB vaccine induced cross-protection against HBV genotype A infection. An in vitro infection model demonstrated anti-HBs-positive sera from individuals inoculated with genotype A- or C-based HB vaccine harbored polyclonal anti-HBs that could bind to non-vaccinated genotype HBV. However, because there were low titers of anti-HBs specific for HBsAg of non-vaccinated genotype, high anti-HBs titers would be required to prevent non-vaccinated genotype HBV infection. Clinically, the 2015 Centers for Disease Control and Prevention guidelines state that periodic monitoring of anti-HBs levels after routine HB vaccination is not needed and that booster doses of HB vaccine are not recommended. However, the American Red Cross suggests that HB-vaccine-induced immune memory might be limited; although HB vaccination can prevent clinical liver injury (hepatitis), subclinical HBV infections of non-vaccinated genotypes resulting in detectable HB core antibody could not be completely prevented. Therefore, monitoring anti-HBs levels after routine vaccination might be necessary for certain subjects in high-risk groups.
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Valéa I, Adjei S, Usuf E, Traore O, Ansong D, Tinto H, Owusu Boateng H, Some AM, Buabeng P, Vekemans J, Kotey A, Vandoolaeghe P, Cullinane M, Traskine M, Ouedraogo F, Sambian D, Lievens M, Tahita MC, Jongert E, Lompo P, Idriss A, Borys D, Ouedraogo S, Prempeh F, Schuerman L, Sorgho H, Agbenyega T. Long-term immunogenicity and immune memory response to the hepatitis B antigen in the RTS,S/AS01 E malaria vaccine in African children: a randomized trial. Hum Vaccin Immunother 2020; 16:1464-1470. [PMID: 31951771 PMCID: PMC7482624 DOI: 10.1080/21645515.2019.1695457] [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: 02/04/2023] Open
Abstract
RTS,S/AS01E malaria vaccine contains the hepatitis B virus surface antigen and may thus serve as a potential hepatitis B vaccine. To evaluate the impact of RTS,S/AS01E when implemented in the Expanded Program of Immunization, infants 8-12 weeks old were randomized to receive either RTS,S/AS01E or a licensed hepatitis B control vaccine (HepB), both co-administered with various combinations of the following childhood vaccines: diphtheria-tetanus-acellular pertussis-Haemophilus influenzae type b, trivalent oral poliovirus, pneumococcal non-typeable Haemophilus influenzae protein D conjugate and human rotavirus vaccine. Long-term persistence of antibodies against the circumsporozoite (CS) protein and hepatitis B surface antigen (HBsAg) were assessed, together with the immune memory response to the HB antigen following a booster dose of HepB vaccine. Subgroups receiving RTS,S or the HepB control vaccine were pooled into RTS,S groups and HepB groups, respectively. One month post-HepB booster vaccination, 100% of participants in the RTS,S groups and 98.3% in the control groups had anti-HBs antibody concentrations ≥10 mIU/mL with the geometric mean concentrations (GMCs) at 46634.7 mIU/mL (95% CI: 40561.3; 53617.6) and 9258.2 mIU/mL (95% CI: 6925.3; 12377.0), respectively. Forty-eight months post-primary vaccination anti-CS antibody GMCs ranged from 2.3 EU/mL to 2.7 EU/mL in the RTS,S groups compared to 1.1 EU/mL in the control groups. Hepatitis B priming with the RTS,S/AS01E vaccine was effective and resulted in a memory response to HBsAg as shown by the robust booster response following an additional dose of HepB vaccine. RTS,S/AS01E when co-administered with PHiD-CV, HRV and other childhood vaccines, had an acceptable safety profile.
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Affiliation(s)
- Innocent Valéa
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso.,IRSS, Centre Muraz , Bobo-Dioulasso, Burkina Faso
| | - Samuel Adjei
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | | | - Ousmane Traore
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso
| | - Daniel Ansong
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | - Halidou Tinto
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso
| | - Harry Owusu Boateng
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | | | - Patrick Buabeng
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | | | - Amos Kotey
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | | | | | | | - Florence Ouedraogo
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso
| | - David Sambian
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | | | - Marc Christian Tahita
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso
| | | | - Palpouguini Lompo
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso
| | - Ali Idriss
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | | | - Sayouba Ouedraogo
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso
| | - Frank Prempeh
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
| | | | - Hermann Sorgho
- Unité de Recherche Clinique de Nanoro, Institut de Recherche en Sciences de la Santé , Nanoro, Burkina Faso
| | - Tsiri Agbenyega
- School of Medical Sciences, KNUST (Agogo Presbyterian Hospital) , Kumasi, Ghana
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7
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Corti D, Benigni F, Shouval D. Viral envelope-specific antibodies in chronic hepatitis B virus infection. Curr Opin Virol 2018; 30:48-57. [PMID: 29738926 DOI: 10.1016/j.coviro.2018.04.002] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2018] [Revised: 03/26/2018] [Accepted: 04/02/2018] [Indexed: 12/20/2022]
Abstract
While the cellular immune response associated with acute and chronic HBV infection has been thoroughly studied, the B cell response in chronic hepatitis B and the role of antibodies raised against the HBV envelope antigens in controlling and prevention of infection requires further investigation. The detection of anti-HBs antibodies is considered as one of the biomarkers for functional cure of chronic hepatitis B virus infection, as well as for protective immunity. Indeed, vaccine-induced neutralizing anti-HBs antibodies have been shown to protect against HBV challenge. Yet, the therapeutic potential of viral envelope-specific antibodies and the mechanism involved in protection and prevention of cell-to-cell transmission warrants additional investigative efforts. In this review, we will provide a critical overview of the available preclinical and clinical literature supporting the putative role of active and passive vaccination and neutralizing envelope-specific antibodies for therapeutic intervention in combination regimens intended to cure persistent HBV infection.
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Affiliation(s)
- Davide Corti
- Humabs BioMed SA, A Subsidiary of Vir Biotechnology, 6500 Bellinzona, Switzerland.
| | - Fabio Benigni
- Humabs BioMed SA, A Subsidiary of Vir Biotechnology, 6500 Bellinzona, Switzerland
| | - Daniel Shouval
- Liver Unit, Institute for Gastroenterology and Hepatology, Hadassah-Hebrew University Hospital, P.O. Box 12000, 91120 Jerusalem, Israel.
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8
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Antiviral effects of anti-HBs immunoglobulin and vaccine on HBs antigen seroclearance for chronic hepatitis B infection. J Gastroenterol 2016; 51:1073-1080. [PMID: 26943168 DOI: 10.1007/s00535-016-1189-x] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2016] [Accepted: 02/16/2016] [Indexed: 02/07/2023]
Abstract
BACKGROUND AND AIMS Interferon and nucleotide/nucleoside analogues are the main treatments for chronic hepatitis B. These drugs effectively reduce serum hepatitis B virus (HBV) DNA titers but fail to sufficiently reduce hepatitis B surface antigen (HBsAg) levels. Following the recent identification of sodium taurocholate cotransporting polypeptide as a receptor for HBV entry, inhibition of HBV entry has become an attractive therapeutic target for chronic hepatitis B treatment. We therefore evaluated the antiviral effects of antibody to HBsAg (anti-HBs) immunoglobulin (HBIG), which can inhibit HBV entry, by in an vivo study and a clinical trial. METHODS In the in vivo study, HBV-infected mice were generated from human hepatocyte chimeric mice and treated with HBIG. A clinical trial evaluating HBIG therapy in patients was also performed. RESULTS In the mouse study, HBV DNA titers were reduced and serum HBsAg titers decreased to undetectable levels following high-dose HBIG injection. On the basis of this result, eight chronic hepatitis B patients, who had received long-term nucleotide analogue treatment, were treated with monthly HBIG injections as an additional treatment. After 1 year of treatment, an HBsAg level reduction of more than 1 log IU/mL was observed in four patients, and three patients became anti-HBs positive. No adverse events occurred during HBIG therapy. CONCLUSION These results suggest that monthly HBIG injection might benefit patients with chronic hepatitis B whose HBsAg titer becomes lower following long-term nucleotide/nucleoside analogue treatment.
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Zhang X, Xin L, Li S, Fang M, Zhang J, Xia N, Zhao Q. Lessons learned from successful human vaccines: Delineating key epitopes by dissecting the capsid proteins. Hum Vaccin Immunother 2016; 11:1277-92. [PMID: 25751641 DOI: 10.1080/21645515.2015.1016675] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Recombinant VLP-based vaccines have been successfully used against 3 diseases caused by viral infections: Hepatitis B, cervical cancer and hepatitis E. The VLP approach is attracting increasing attention in vaccine design and development for human and veterinary use. This review summarizes the clinically relevant epitopes on the VLP antigens in successful human vaccines. These virion-like epitopes, which can be delineated with molecular biology, cryo-electron microscopy and x-ray crystallographic methods, are the prerequisites for these efficacious vaccines to elicit functional antibodies. The critical epitopes and key factors influencing these epitopes are discussed for the HEV, HPV and HBV vaccines. A pentamer (for HPV) or a dimer (for HEV and HBV), rather than a monomer, is the basic building block harboring critical epitopes for the assembly of VLP antigen. The processing and formulation of VLP-based vaccines need to be developed to promote the formation and stabilization of these epitopes in the recombinant antigens. Delineating the critical epitopes is essential for antigen design in the early phase of vaccine development and for critical quality attribute analysis in the commercial phase of vaccine manufacturing.
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Affiliation(s)
- Xiao Zhang
- a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics; National Institute of Diagnostics and Vaccine Development in Infectious Diseases; Xiamen University ; Xiamen , Fujian , PR China
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10
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Rapid identification of multi-strain HBV infection in patient by high-throughput DNA sequencing. QUANTITATIVE BIOLOGY 2015. [DOI: 10.1007/s40484-015-0046-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
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11
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Hamada-Tsutsumi S, Iio E, Watanabe T, Murakami S, Isogawa M, Iijima S, Inoue T, Matsunami K, Tajiri K, Ozawa T, Kishi H, Muraguchi A, Joh T, Tanaka Y. Validation of cross-genotype neutralization by hepatitis B virus-specific monoclonal antibodies by in vitro and in vivo infection. PLoS One 2015; 10:e0118062. [PMID: 25693196 PMCID: PMC4333126 DOI: 10.1371/journal.pone.0118062] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2014] [Accepted: 01/05/2015] [Indexed: 02/05/2023] Open
Abstract
Vaccines based on hepatitis B virus (HBV) genotype A have been used worldwide for immunoprophylaxis and are thought to prevent infections by non-A HBV strains effectively, whereas, vaccines generated from genotype C have been used in several Asian countries, including Japan and Korea, where HBV genotype C is prevalent. However, acute hepatitis B caused by HBV genotype A infection has been increasing in Japan and little is known about the efficacy of immunization with genotype C-based vaccines against non-C infection. We have isolated human monoclonal antibodies (mAbs) from individuals who were immunized with the genotype C-based vaccine. In this study, the efficacies of these two mAbs, HB0116 and HB0478, were analyzed using in vivo and in vitro models of HBV infection. Intravenous inoculation of HBV genotype C into chimeric mice with human hepatocytes resulted in the establishment of HBV infection after five weeks, whereas preincubation of the inocula with HB0116 or HB0478 protected chimeric mice from genotype C infection completely. Interestingly, both HB0116 and HB0478 were found to block completely genotype A infection. Moreover, infection by a genotype C strain with an immune escape substitution of amino acid 145 in the hepatitis B surface protein was also completely inhibited by incubation with HB0478. Finally, in vitro analysis of dose dependency revealed that the amounts of HB0478 required for complete protection against genotype C and genotype A infection were 5.5 mIU and 55 mIU, respectively. These results suggested that genotype C-based vaccines have ability to induce cross-genotype immunity against HBV infection.
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Affiliation(s)
- Susumu Hamada-Tsutsumi
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Etsuko Iio
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
- Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Tsunamasa Watanabe
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Shuko Murakami
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Masanori Isogawa
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Sayuki Iijima
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Takako Inoue
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Kayoko Matsunami
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
- Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Kazuto Tajiri
- The Third Department of Internal Medicine, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
- The Department of Immunology, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
| | - Tatsuhiko Ozawa
- The Department of Immunology, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
| | - Hiroyuki Kishi
- The Department of Immunology, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
| | - Atsushi Muraguchi
- The Department of Immunology, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
| | - Takashi Joh
- Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
- * E-mail:
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12
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Watashi K, Urban S, Li W, Wakita T. NTCP and beyond: opening the door to unveil hepatitis B virus entry. Int J Mol Sci 2014; 15:2892-905. [PMID: 24557582 PMCID: PMC3958888 DOI: 10.3390/ijms15022892] [Citation(s) in RCA: 102] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2014] [Revised: 02/13/2014] [Accepted: 02/14/2014] [Indexed: 12/19/2022] Open
Abstract
Chronic hepatitis B virus (HBV) infection, affecting approximately 240 million people worldwide, is a major public health problem that elevates the risk of developing liver cirrhosis and hepatocellular carcinoma. Given that current anti-HBV drugs are limited to interferon-based regimens and nucleos(t)ide analogs, the development of new anti-HBV agents is urgently needed. The viral entry process is generally an attractive target implicated in antiviral strategies. Using primary cells from humans and Tupaia belangeri, as well as HepaRG cells, important determinants of viral entry have been achieved. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as an HBV entry receptor and enabled the establishment of a susceptible cell line that can efficiently support HBV infection. This finding will allow a deeper understanding of the requirements for efficient HBV infection, including the elucidation of the molecular entry mechanism. In addition, pharmacological studies suggest that NTCP is able to serve as a therapeutic target. This article summarizes our current knowledge on the mechanisms of HBV entry and the role of NTCP in this process.
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Affiliation(s)
- Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, 162-8640 Tokyo, Japan.
| | - Stephan Urban
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany.
| | - Wenhui Li
- National Institute of Biological Sciences, No.7 Science Park Road, ZGC Life Science Park, Changping, 102206 Beijing, China.
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, 162-8640 Tokyo, Japan.
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Greiner VJ, Manin C, Larquet E, Ikhelef N, Gréco F, Naville S, Milhiet PE, Ronzon F, Klymchenko A, Mély Y. Characterization of the structural modifications accompanying the loss of HBsAg particle immunogenicity. Vaccine 2014; 32:1049-54. [DOI: 10.1016/j.vaccine.2014.01.012] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2013] [Revised: 12/20/2013] [Accepted: 01/02/2014] [Indexed: 10/25/2022]
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14
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Hu Z, Li M, Huang B, Liu J, Yu L, Chen G. Detection of hepatitis B virus PreS1 antigen using a time-resolved fluoroimmunoassay. J Immunoassay Immunochem 2012; 33:156-65. [PMID: 22471606 DOI: 10.1080/15321819.2011.609576] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The hepatitis B virus (HBV) PreS1 antigen is expressed at the distal most region of the envelope protein and contains the hepatocyte receptor-binding site. The presence of the HBV PreS1 antigen in serum and liver of HBsAg-positive patients is a new marker used for diagnosing HBV infection, and is indicative of viral replication. Our objective is to establish a method of time-resolved fluoroimmunoassay (TRFIA) with higher sensitivity and broader detection range for detecting serum HBV PreS1 antigen. Eu(3+) labeling of antibodies was performed with respective labeling kits, and Eu(3+) fluorescence intensity was measured with an auto DELFIA1235 TRFIA analyzer. The established method was evaluated for its performance. Serum specimens (574 in total) from Wuxi People's Hospital were analyzed for PreS1 antigen using the TRFIA and ELISA. The precision, specificity, and sensitivity of the TRFIA were clearly better than ELISA. The detection limit was 0.01 ng/mL. The average recovery rate for PreS1 antigens was 103.3%. There was significant correlation between the PreS1 antigen results obtained by TRFIA and ELISA in 374 serum samples with HBV >10(3) IU/mL (χ(2) = 25.04, p < 0.01) and 183 HbeAg-positive serum samples (χ(2) = 12.07, p < 0.01). Normal reference ranges were established at 0-0.32 ng/mL based on the values obtained from 100 healthy controls. TRFIA is a significantly effective method for clinical detection of serum HBV PreS1 antigens.
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15
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The structure of HBsAg particles is not modified upon their adsorption on aluminium hydroxide gel. Vaccine 2012; 30:5240-5. [PMID: 22705175 DOI: 10.1016/j.vaccine.2012.05.082] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2012] [Revised: 05/25/2012] [Accepted: 05/30/2012] [Indexed: 11/20/2022]
Abstract
Current Hepatitis B vaccines are based on recombinant Hepatitis B surface antigen (HBsAg) virus-like particles adsorbed on aluminium (Al) gel. These particles exhibit a lipoprotein-like structure with about 70 protein S molecules in association with various types of lipids. To determine whether the adsorption on Al gel affects HBsAg structure, we investigated the effect of adsorption and mild desorption processes on the protein and lipid parts of the particles, using various techniques. Electron microscopy showed that the size and morphology of native and desorbed HBsAg particles were comparable. Moreover, infrared and Raman spectroscopy revealed that the secondary structure of the S proteins was not affected by the adsorption/desorption process. Affinity measurements with Surface Plasmon Resonance showed no difference between native and desorbed HBsAg for HBsAg-specific RF-1 monoclonal antibody. Steady-state and time-resolved fluorescence data of the intrinsic fluorescence of the S proteins further indicated that the adsorption/desorption of HBsAg particles on Al gel did not modify the environment of the most emitting Trp residues, confirming that the conformation of the S proteins remains intact. Moreover, using environment-sensitive 3-hydroxyflavone probes, no significant changes of the lipid core and lipid membrane surface of the HBsAg particles were observed during the adsorption/desorption process. Finally, the ratio between lipids and proteins in the particles was found to be similar before and after the adsorption/desorption process. Taken together, our data show that adsorption on Al gel does not affect the structure of the HBsAg particles.
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16
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Cassidy A, Mossman S, Olivieri A, De Ridder M, Leroux-Roels G. Hepatitis B vaccine effectiveness in the face of global HBV genotype diversity. Expert Rev Vaccines 2012; 10:1709-15. [PMID: 22085174 DOI: 10.1586/erv.11.151] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Recombinant hepatitis B vaccines are of the A2 genotype; one of ten known genotypes whose distribution varies globally. Reports of rare HBV infections in blood donors with an imbalance of non-A2 genotype HBV in vaccinated subjects have raised questions about the cross-protection afforded by HBV-A2 vaccines. Infections in HBV vaccinees were asymptomatic and transient, indicating that vaccination prevented clinical disease. Preclinical data demonstrate cross-reactivity and cross-protection by A2 vaccines against non-A2 HBV genotypes. Substantial improvements in HBV control have been demonstrated in countries with diverse genotype distribution that have introduced universal childhood HBV vaccination programs. Available data show that current HBV-A2 vaccines are highly effective in preventing infections and clinical disease caused by all known HBV genotypes.
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17
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Freed DC, Towne VM, Casimiro DR, Zhao Q, Fu TM. Evaluating functional antibodies in rhesus monkeys immunized with hepatitis B virus surface antigen vaccine with novel adjuvant formulations. Vaccine 2011; 29:9385-90. [PMID: 22001120 DOI: 10.1016/j.vaccine.2011.09.122] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2011] [Revised: 09/06/2011] [Accepted: 09/29/2011] [Indexed: 12/12/2022]
Abstract
Effective and safe novel adjuvants are of great interest to the vaccine research community. In this study, we describe our evaluation of adjuvant formulations containing a TLR9 agonist adjuvant (ISS1018) or ISCOMATRIX™ adjuvant for a two-dose regimen of hepatitis B virus surface antigen virus-like particle vaccine in mice and rhesus macaques. Our results show a 10-20 fold improvement in Ab binding titers determined in an antigen-sandwich assay for adjuvant formulations with ISCOMATRIX™ adjuvant, in comparison to routine aluminum formulation. Furthermore, we optimized a competition assay to evaluate a functional component of immune sera, using a conformation-dependent and protective mAb, RFHBs1, as the probe. Although good correlation was observed between Ab binding titers from the antigen-sandwich assay and functional titers from the in-solution competition against RFHBs1, the latter assessment provided a much more stringent ranking of adjuvant formulations than the former. These results indicate the importance of evaluating functional Abs when assessing and comparing novel adjuvant formulations, as it provides another angle to investigate the effects of change in adjuvant composition on antigenic integrity of the testing vaccines.
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Affiliation(s)
- Daniel C Freed
- Department of Vaccine Basic Research, Department of Vaccine Processing Research and Development, Merck Research Laboratories, West Point, PA, USA
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18
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Asante KP, Abdulla S, Agnandji S, Lyimo J, Vekemans J, Soulanoudjingar S, Owusu R, Shomari M, Leach A, Jongert E, Salim N, Fernandes JF, Dosoo D, Chikawe M, Issifou S, Osei-Kwakye K, Lievens M, Paricek M, Möller T, Apanga S, Mwangoka G, Dubois MC, Madi T, Kwara E, Minja R, Hounkpatin AB, Boahen O, Kayan K, Adjei G, Chandramohan D, Carter T, Vansadia P, Sillman M, Savarese B, Loucq C, Lapierre D, Greenwood B, Cohen J, Kremsner P, Owusu-Agyei S, Tanner M, Lell B. Safety and efficacy of the RTS,S/AS01 E candidate malaria vaccine given with expanded-programme-on-immunisation vaccines: 19 month follow-up of a randomised, open-label, phase 2 trial. THE LANCET. INFECTIOUS DISEASES 2011; 11:741-9. [DOI: 10.1016/s1473-3099(11)70100-1] [Citation(s) in RCA: 84] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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19
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Descamps J, Giffroy D, Remy E, Mortiaux F, Mareschal JC, Ponsar C, Duchene M. A case study of development, validation, and acceptance of a non-animal method for assessing human vaccine potency. ACTA ACUST UNITED AC 2011. [DOI: 10.1016/j.provac.2011.10.018] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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20
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Deresinski SC. Hyperimmune products in the prevention and therapy of infectious disease: a report of a hyperimmune products expert advisory panel. BioDrugs 2009; 14:147-58. [PMID: 18034567 DOI: 10.2165/00063030-200014030-00002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
This paper reviews a meeting at which basic pathophysiology of infections, mechanisms of action of hyperimmune products and pharmacokinetic and pharmacodynamic parameters, as well as currently available hyperimmunes and their potential new targets and uses, were discussed. A hyperimmune product was defined as either a monoclonal antibody or a polyclonal preparation enriched with antibody directed against one or more particular targets. A number of issues were emphasised, including: resistant bacterial pathogens, such as Staphylococcus aureus and Streptococcus pyogenes; the role of hyperimmune intravenous globulins in the prevention of sepsis in low birthweight infants; hepatitis B virus infection associated with liver transplantation; combination therapy; the potential role of hyperimmunes in the prevention and treatment of hepatitis C virus; and the use of immunoglobulins for the prophylaxis of Epstein-Barr virus-related lymphoproliferative disease. Routes of administration were also discussed. It was concluded that the development of hyperimmunes faces numerous obstacles. It was agreed that the use of hyperimmunes in clinical trials must be standardised; clinical trials must be large enough to have sufficient power to demonstrate efficacy with clear-cut end-points, and means need to be developed, in conjunction with regulatory agencies, for the feasible evaluation of combination products. However, progress in all these aspects will provide a wide range of hyperimmunes for future use.
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21
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MISHRA A, DURGAPAL H, MANIVEL V, ACHARYA SK, Rao KVS, Panda SK. Immune response to hepatitis B virus surface antigen peptides during HBV infection. Clin Exp Immunol 2008. [DOI: 10.1111/j.1365-2249.1992.tb07927.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022] Open
Abstract
SUMMARY
Antibody responses of patients with acute (n = 73), fulminant (n = 30) and chronic (n = 51) hepatitis B virus (HBV) infection as well as recovered individuals (n = 7) were studied against three synthetic peptides, Pre-S1 amino acids (aa. 12–32), Pre-S2 amino acids (aa. 120–145), and S amino acids (aa. 124–147) of the envelope region (HBsAg). T cell blastogenic response was investigated in a proportion of the patients (27 acute, nine fulminant, 13 chronic hepatitis and seven recovered individuals) along with seven HBV vaccinated and three normal individuals. The presence of T cell response against S peptide was observed in all the cases (9/9, 100%) during early acute hepatitis. This was suppressed during late stages (8/18, 44%) followed by partial reversal during recovery (5/7, 71 %). T cell response and antibodies to Pre-S1 and Pre-S2 peptides were present only in one-third of the patients throughout these periods. The T cell blastogenic response as well as antibody reactivity against these peptides were absent and minimal in chronic hepatitis. Immune response against envelope protein appears to play a major role in acute hepatic injury due to HBV infection and help in virus clearance.
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Affiliation(s)
| | | | - V MANIVEL
- Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi
| | - S K ACHARYA
- Virology Group, International Centre for Genetic Engineering and Biotechnology, Shaheed Jeet Singh Marg (NII Campus), New Delhi, India
| | - K V S Rao
- Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi
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22
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Kim SH, Shin YW, Hong KW, Chang KH, Ryoo KH, Paik SH, Kim JM, Brotman B, Pfahler W, Prince AM. Neutralization of hepatitis B virus (HBV) by human monoclonal antibody against HBV surface antigen (HBsAg) in chimpanzees. Antiviral Res 2008; 79:188-91. [PMID: 18479762 DOI: 10.1016/j.antiviral.2008.03.006] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2008] [Revised: 03/20/2008] [Accepted: 03/28/2008] [Indexed: 12/13/2022]
Abstract
The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-naïve chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.
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Affiliation(s)
- Se-Ho Kim
- Antibody Engineering Laboratory, Research Center, Green Cross Corp., 341, Bojeong-Dong, Giheung-Gu, Yongin City, Gyunggi-Do, 446-799, Republic of Korea.
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Abstract
Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus (HBV) constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses (e.g. HBV) and avihepadnaviruses (e.g. duck hepatitis B virus [DHBV]). Two important discoveries have been made recently regarding HBV: (1) primary hepatocytes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and (2) a human hepatoma cell line (HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D (CPD) has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes (PDH), investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to: (1) functionally dissect the hepadnaviral entry pathways, (2) perform reverse genetics (e.g. test the fitness of escape mutants), (3) titrate and map neutralizing antibodies, (4) improve current vaccines to combat acute and chronic infections of hepatitis B, and (5) develop entry inhibitors for future clinical applications.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus-Liebig University of Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany.
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24
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Jaoudé GA, Sureau C. Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus. J Virol 2005; 79:10460-6. [PMID: 16051838 PMCID: PMC1182656 DOI: 10.1128/jvi.79.16.10460-10466.2005] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in infectivity of the envelope protein antigenic loop, also exposed to the virion surface and accessible to neutralizing antibodies, remains to be established. In the present study, mutations were created in the antigenic loop of the three envelope proteins, and the resulting mutants were evaluated for their capacity to assist in the maturation and infectivity of HDV. We observed that short internal combined deletions and insertions, affecting residues 109 to 133 in the antigenic loop, were tolerated for secretion of both subviral HBV particles and HDV virions. However, when assayed for infectivity on primary cultures of human hepatocytes or on the recently described HepaRG cell line, virions carrying deletions between residues 118 and 129 were defective. Single amino acid substitutions in this region revealed that Gly-119, Pro-120, Cys-121, Arg-122, and Cys-124 were instrumental in viral entry. These results demonstrate that in addition to a receptor-binding site previously identified in the pre-S1 domain of the L protein, a determinant of infectivity resides in the antigenic loop of HBV envelope proteins.
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Affiliation(s)
- Georges Abou Jaoudé
- Laboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, 6 Rue Alexandre-Cabanel, 75739 Paris, France
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25
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Nainan OV, Khristova ML, Byun K, Xia G, Taylor PE, Stevens CE, Margolis HS. Genetic variation of hepatitis B surface antigen coding region among infants with chronic hepatitis B virus infection. J Med Virol 2002; 68:319-27. [PMID: 12226817 DOI: 10.1002/jmv.10206] [Citation(s) in RCA: 76] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Variants in the amino acid composition of the primary antibody-binding site of hepatitis B surface antigen (HBsAg) have been identified in a number of populations with chronic hepatitis B virus (HBV) infection. Direct sequencing of amplified or cloned PCR products, solid phase detection of sequence-specific PCR products (SP-PCR), and limiting dilution cloning PCR (LDC-PCR) were compared to determine their sensitivity in detecting differing concentrations of HBsAg variants. LDC-PCR had the greatest sensitivity and could detect HBsAg variants at a concentration of 0.1% of the total viral population. HBsAg variants were detected in 51% of infants with chronic HBV infection acquired after postexposure prophylaxis, and more than half of the variants were detected only by the most sensitive methods.
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Affiliation(s)
- Omana V Nainan
- Division of Viral Hepatitis (World Health Organization Collaborating Center for Research and Reference in Viral Hepatitis), National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
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26
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Lewin S, Walters T, Locarnini S. Hepatitis B treatment: rational combination chemotherapy based on viral kinetic and animal model studies. Antiviral Res 2002; 55:381-96. [PMID: 12206877 DOI: 10.1016/s0166-3542(02)00071-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Hepatitis B virus (HBV) causes a generally non-cytopathic infection in the liver. Even though HBV is a DNA virus, it replicates via reverse transcription which is coordinated within the viral nucleocapsid by the virus-specific polymerase. The major transcriptional template is the viral mimichromosome from which the viral DNA exists as a covalently closed circular (ccc) molcule. The virus infects hepatocytes but can also be found in non-hepatocyte reservoirs such as bile-duct epithelium, mesangial cells of the kidney, pancreatic islet cells and lymphoid cells. When patients infected with HBV are treated with either interferon alpha or lamivudine, responses are variable and unpredictable. Sophisticated mathematical models analysing the dynamics of viral clearance during antiviral therapy have recently been applied to chronic hepatitis B. Typically complex profiles, rather than the usual biphasic responses seen with other diseases have been observed, indicating that antiviral efficacy requires substantila improvement. This may be achieved with combination chemotherapy. However, chronic hepatitis B is a complex and heterogeneous disease entity, and the challenge for the future is to define measurable end-points of treatment and address key virological issues such as the role of cccDNA and extra-hepatocyte replication in treatment failure. Clearly, new therapies and effective combination therapy protocols are urgently required in order to improve the present poor response rates in patients undergoing treatment.
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Affiliation(s)
- Sharon Lewin
- Victorian Infectious Diseases Service, Royal Melbourne Hospital, Department of Microbiology and Immunology, The University of Melbourne, Parkville, Australia
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27
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Abraham B, Baine Y, De-Clercq N, Tordeur E, Gerard PP, Manouvriez PL, Parenti DL. Magnitude and quality of antibody response to a combination hepatitis A and hepatitis B vaccine. Antiviral Res 2002; 53:63-73. [PMID: 11684316 DOI: 10.1016/s0166-3542(01)00194-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Interference between antibodies generated by a combination hepatitis A and B vaccine was investigated by evaluating the quantity and quality of anti-hepatitis A virus (HAV) and anti-hepatitis B surface antigen (HBs) antibodies generated by Twinrix (Hepatitis A Inactivated and Hepatitis B (Recombinant) Vaccine). The magnitude of the immune response was determined by a retrospective analysis of eight clinical trials, completed during stepwise development of Twinrix. The functionality of anti-HAV was evaluated by comparison of routine ELISA results with neutralization assays and was further characterized by defining the epitope-specificity of binding. Functionality of the anti-HBs response was not tested because a validated assay was not developed at the time this study was conducted. Results of all analyses demonstrated that the combination vaccine induced high antibody titers against hepatitis A and B and a functional anti-HAV response, with no evidence of immune interference to either viral antigen.
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Affiliation(s)
- Betsy Abraham
- GlaxoSmithKline Pharmaceuticals, Collegeville, PA, USA.
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28
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Thornton SM, Walker S, Zuckerman JN. Management of hepatitis B virus infections in two gibbons and a western lowland gorilla in a zoological collection. Vet Rec 2001; 149:113-5. [PMID: 11504202 DOI: 10.1136/vr.149.4.113] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Since 1996, three primates newly arrived at London Zoo have been found to be infected with hepatitis B virus. The species involved were white-cheeked gibbons (Hylobates leucogenys leucogenys and Hylobates leucogenys siki) and a western lowland gorilla (Gorilla gorilla gorilla). The protocols for the practical management of these cases, including the immunisation of susceptible non-human primates and the staff with recombinant hepatitis B vaccine are described, and the origin and evolution of hepatitis B infection in primates are discussed.
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Affiliation(s)
- S M Thornton
- Veterinary Science Unit, Institute of Zoology, Zoological Society of London
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29
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Heijtink RA, van Bergen P, van Roosmalen MH, Sünnen CM, Paulij WP, Schalm SW, Osterhaus AD. Anti-HBs after hepatitis B immunization with plasma-derived and recombinant DNA-derived vaccines: binding to mutant HBsAg. Vaccine 2001; 19:3671-80. [PMID: 11395201 DOI: 10.1016/s0264-410x(01)00082-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The G145R mutant of the small S-protein is a major escape mutant of hepatitis B virus observed in natural infection, after immunization and HBIG therapy. In a previous study we found that plasma-derived and recombinant DNA-derived vaccine HBsAg reacted differently with monoclonal antibodies sensitive for the G145R change. In the present study we investigated the binding of polyclonal anti-HBs obtained after immunization with plasma vaccine and recombinant DNA vaccine to synthetic peptides (adw(2), adr) and rHBsAg (HepG2) (ayw(3); wild type and a 145R mutant). Anti-HBs binding to synthetic peptids (25-mers, 7aa overlap) from the "a"-loop was significantly reduced by the G145R substitution and by changing the amino acid sequence from adw(2) into adr. With mutant G145R rHBsAg the inhibitory activity of vaccine anti-HBs was decreased compared to rHBsAg wild type. In general only minor differences were observed between plasma vaccine and recombinant DNA vaccine related antibody responses. However, the individual heterogeneity in epitope specific reactivity with its possible consequences for protection (against escape mutants) is not reflected in an anti-HBs titer by standard anti-HBs assays. The presented differentiation in anti-HBs response after immunization may deliver new tools for evaluation of future vaccines.
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Affiliation(s)
- R A Heijtink
- Department of Virology, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, Netherlands.
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30
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Cooreman MP, Leroux-Roels G, Paulij WP. Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen. J Biomed Sci 2001; 8:237-47. [PMID: 11385295 DOI: 10.1007/bf02256597] [Citation(s) in RCA: 134] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.
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Affiliation(s)
- M P Cooreman
- Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
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31
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Maeng CY, Oh MS, Park IH, Hong HJ. Purification and structural analysis of the hepatitis B virus preS1 expressed from Escherichia coli. Biochem Biophys Res Commun 2001; 282:787-92. [PMID: 11401532 DOI: 10.1006/bbrc.2001.4641] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
The preS1 of hepatitis B virus (HBV) is located at the outermost part of the envelope protein and possesses several functionally important regions such as hepatocyte receptor-binding site and virus-neutralizing epitopes. As the first step to understand the structure-function relationship for the preS1 antigen, we have purified the preS1 and performed its structural characterization by circular dichroism (CD) spectroscopy. The preS1 was purified to near homogeneity from bacterially expressed glutathione S-transferase (GST)-preS1 fusion protein by two-step purification, affinity chromatography on glutathione-agarose column, and cation-exchange chromatography on Mono S column. The CD analysis showed that the purified preS1, which was largely unstructured in aqueous solution, acquired a significant (16%) alpha-helical structure when analyzed in 50% trifluoroethanol or 20 mM SDS. The results suggest that the preS1 assumes a mainly unstructured conformation and may form induced secondary structures upon binding to target proteins or under hydrophobic environment.
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Affiliation(s)
- C Y Maeng
- Antibody Engineering Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon, 305-600
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32
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Riddell MA, Li F, Anderson DA. Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies. J Virol 2000; 74:8011-7. [PMID: 10933710 PMCID: PMC112333 DOI: 10.1128/jvi.74.17.8011-8017.2000] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.
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Affiliation(s)
- M A Riddell
- Hepatitis Research Unit and Australian Centre for Hepatitis Virology, Macfarlane Burnet Centre for Medical Research, Fairfield 3078, Victoria, Australia
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33
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Maeng CY, Ryu CJ, Gripon P, Guguen-Guillouzo C, Hong HJ. Fine mapping of virus-neutralizing epitopes on hepatitis B virus PreS1. Virology 2000; 270:9-16. [PMID: 10772975 DOI: 10.1006/viro.2000.0250] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
We identified the epitopes on the preS1 which induce antibodies that neutralize both ad and ay subtypes of hepatitis B virus (HBV). Previously we generated murine monoclonal antibodies KR359 and KR127 that bind specifically to the preS1 of HBV. In this study we have performed fine mappings of the epitopes of the antibodies by examining their reactivity with GST fusion proteins, which contain a series of deletion mutants of the preS1. KR359 and KR127 specifically recognize aa 19-26 and 37-45 of the preS1, respectively. The antibodies neutralized both adr and ayw subtypes of the virus in an in vitro neutralization assay using in vitro infection of adult human hepatocyte primary culture by HBV. The epitopes showed little sequence divergence and the antibodies bound to the preS1 of all the HBV subtypes and variants tested.
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Affiliation(s)
- C Y Maeng
- Antibody Engineering Research Unit, Korea Research Institute of Bioscience and Biotechnology, Taejon, Yuseong, 305-600, Korea
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34
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Seddigh-Tonekaboni S, Waters JA, Jeffers S, Gehrke R, Ofenloch B, Horsch A, Hess G, Thomas HC, Karayiannis P. Effect of variation in the common "a" determinant on the antigenicity of hepatitis B surface antigen. J Med Virol 2000; 60:113-21. [PMID: 10596008 DOI: 10.1002/(sici)1096-9071(200002)60:2<113::aid-jmv2>3.0.co;2-0] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Antibody to the common "a" determinant of hepatitis B surface antigen (HBsAg) protects against infection with hepatitis B virus. A number of variant surface antigens with amino acid substitutions within the "a" determinant have been described in patients around the world. Both wild type and variant HBsAgs were expressed in the yeast Pichia pastoris and the antigens were semi-purified and quantitated. The effect on antigenicity of these changes was investigated in a quantitative fashion using four monoclonal antibodies known to bind to different epitopes within the common "a" determinant. The results suggest that amino acid substitution of T131I, K141E and G145R and insertion of 3 amino acids between residues 123 and 124 markedly affect the antigenic structure of HBsAg. These substitutions and insertions in the viral envelope may lead to evasion of the virus neutralizing antibody response and also to reduce efficiency of detection by immunoassays used for diagnosis and blood-bank screening.
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Affiliation(s)
- S Seddigh-Tonekaboni
- Hepatology Section, Department of Medicine A, Imperial College School of Medicine, London, England
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35
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Cooreman MP, van Roosmalen MH, te Morsche R, Sünnen CM, de Ven EM, Jansen JB, Tytgat GN, de Wit PL, Paulij WP. Characterization of the reactivity pattern of murine monoclonal antibodies against wild-type hepatitis B surface antigen to G145R and other naturally occurring "a" loop escape mutations. Hepatology 1999; 30:1287-92. [PMID: 10534351 DOI: 10.1002/hep.510300508] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The hepatitis B surface antigen (HBsAg) "a" domain harbors major B-cell epitopes. Viruses with mutations in this region emerge after vaccination or during hepatitis B immune globulin (HBIg) prophylaxis. A strain with G145R replacement has been almost invariably isolated as a major escape mutant. We investigated mutant antigen-antibody interactions with direct binding assays. G145R and 16 other naturally occurring recombinant HBsAg mutants were expressed in mammalian Cos-1 cells. The reactivity of a panel of 28 murine anti-hepatitis B surface antigen (anti-HBs) monoclonal antibodies to mutant antigens was measured with enzyme immunoassay and expressed as percentage compared with the wild-type (wt) HBsAg signal for each antibody. All point-mutated proteins displayed diffuse intracellular immunofluorescent labeling corresponding to a secretory pathway. Monoclonal antibodies (mAbs) were classified according to different binding patterns. The effect of mutations on antibody binding differs depending on the amino acid involved and on the location within the "a" loop. As expected, most antibodies had absent or negligible binding (<40%), notably with residue 145 replacements. However, we identified antibodies that reacted with conformational epitopes but nevertheless had adequate reactivity (>40%) with all mutant antigens, including G145R. The effect of G145R was more pronounced than that of G145A. A subgroup of antibodies had substantially increased recognition (>120%) of antigens with mutations in the first loop. We demonstrated that antibodies can be selected or combined that react with all mutants investigated, including G145R. These data offer perspectives for improving anti-HBs-based protection against hepatitis B.
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Affiliation(s)
- M P Cooreman
- Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
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36
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Schinazi RF, Ilan E, Black PL, Yao X, Dagan S. Cell-based and animal models for hepatitis B and C viruses. Antivir Chem Chemother 1999; 10:99-114. [PMID: 10431609 DOI: 10.1177/095632029901000301] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Reliable cell-based assays and animal models have been developed for evaluating agents against hepatitis B virus. Although much progress has been made, in vitro and in vivo assays for hepatitis C virus are still on the horizon. Advances towards establishing inexpensive and reliable experimental models have accelerated the development of therapeutic modalities for these life-threatening viral infections. The characterization of well-defined viral targets coupled with improved molecular diagnostic technologies have illuminated this field.
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Affiliation(s)
- R F Schinazi
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA.
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37
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Davis HL. DNA-based immunization against hepatitis B: experience with animal models. Curr Top Microbiol Immunol 1998; 226:57-68. [PMID: 9479835 DOI: 10.1007/978-3-642-80475-5_4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- H L Davis
- Loeb Research Institute, Ottawa Civic Hospital, Canada
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38
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Waters JA, Bailey C, Love C, Thomas HC. A study of the antigenicity and immunogenicity of a new hepatitis B vaccine using a panel of monoclonal antibodies. J Med Virol 1998. [DOI: 10.1002/(sici)1096-9071(199801)54:1<1::aid-jmv1>3.0.co;2-b] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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39
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Triyatni M, Jilbert AR, Qiao M, Miller DS, Burrell CJ. Protective efficacy of DNA vaccines against duck hepatitis B virus infection. J Virol 1998; 72:84-94. [PMID: 9420203 PMCID: PMC109352 DOI: 10.1128/jvi.72.1.84-94.1998] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The efficacy of DNA vaccines encoding the duck hepatitis B virus (DHBV) pre-S/S and S proteins were tested in Pekin ducks. Plasmid pcDNA I/Amp DNA containing the DHBV pre-S/S or S genes was injected intramuscularly three times, at 3-week intervals. All pre-S/S and S-vaccinated ducks developed total anti-DHBs and specific anti-S antibodies with similar titers reaching 1/10,000 to 1/50,000 and 1/2,500 to 1/4,000, respectively, after the third vaccination. However, following virus challenge, significant differences in the rate of virus removal from the bloodstream and the presence of virus replication in the liver were found between the groups. In three of four S-vaccinated ducks, 90% of the inoculum was removed between <5 and 15 min postchallenge (p.c.) and no virus replication was detected in the liver at 4 days p.c. In contrast, in all four pre-S/S-vaccinated ducks, 90% of the inoculum was removed between 60 and 90 min p.c. and DHBsAg was detected in 10 to 40% of hepatocytes. Anti-S serum abolished virus infectivity when preincubated with DHBV before inoculation into 1-day-old ducklings and primary duck hepatocyte cultures, while anti-pre-S/S serum showed very limited capacity to neutralize virus infectivity in these two systems. Thus, although both DNA vaccines induced high titers of anti-DHBs antibodies, anti-S antibodies induced by the S-DNA construct were highly effective in neutralizing virus infectivity while similar levels of anti-S induced by the pre-S/S-DNA construct conferred only very limited protection. This phenomenon requires further clarification, particularly in light of the development of newer HBV vaccines containing pre-S proteins and a possible discrepancy between anti-HBs titers and protective efficacy.
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Affiliation(s)
- M Triyatni
- Department of Microbiology and Immunology, University of Adelaide, South Australia.
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40
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Ryu CJ, Gripon P, Park HR, Park SS, Kim YK, Guguen-Guillouzo C, Yoo OJ, Hong HJ. In vitro neutralization of hepatitis B virus by monoclonal antibodies against the viral surface antigen. J Med Virol 1997; 52:226-33. [PMID: 9179773 DOI: 10.1002/(sici)1096-9071(199706)52:2<226::aid-jmv18>3.0.co;2-i] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
In vitro HBV infection and neutralization were assayed using an anti-preS1 murine monoclonal antibody (1B3) and anti-preS2 (H69K) and anti-S (CS131A) murine-human chimeric antibodies. The 1B3 (IgG1) and H69K (IgG1) was constructed previously and the CS131A was constructed for this study by expressing stably the chimeric heavy and light chains in Chinese hamster ovary cells and purifying from the culture supernatant. Previous study showed that the H69K and CS131A recognize known virus-neutralizing epitopes, while the 1B3 does not. For the assays, adult human hepatocyte primary culture was infected with the adr or ayw subtype of HBV, and the infectivity and subsequent replication was confirmed both by measuring the kinetics of HB-sAg secretion by the infected cells and detecting the intermediate replicative form of HBV DNA in the cells. Next, the hepatocytes were infected with the adr or ayw subtype of the virus that had been preincubated with various concentrations of each of the antibodies and the neutralization of HBV was analyzed. The results showed that the anti-preS2 and anti-S chimeric antibodies exhibited neutralizing activity against both the adr and ayw subtypes of the virus, with approximately 1,000 and 2,000 times higher specific activity than polyclonal hepatitis B immune globulin, respectively, but the anti-preS1 antibody scarcely neutralized the infection. The neutralizing activities of the antibodies were consistent with their epitope specificity and antigenbinding affinity, suggesting that this neutralization assay is specific. The in vitro neutralization assay will be useful for evaluating the neutralizing activity of anti-HBV antibodies before in vivo testing in chimpanzees.
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Affiliation(s)
- C J Ryu
- Antibody Engineering Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Taejon, Korea
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41
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Kim HS, Kim YK, Ryu SE, Hong HJ. Production of hepatitis B virus preS polypeptide in Escherichia coli by mutation of the 5'-end coding sequence and its purification and characterization. Gene 1996; 177:173-7. [PMID: 8921864 DOI: 10.1016/0378-1119(96)00296-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The preS1 and preS2 antigens (preS Ag) of hepatitis B virus (HBV) elicit virus-neutralizing and protective antibodies which can overcome nonresponsiveness to the currently available vaccine for HBV and also carry the attachment site to HBV hepatocyte receptor. Therefore, in order to study the development of more effective vaccine and the receptor-ligand interaction, it will be helpful to obtain high-level production of the preS Ag from bacteria. We have found that the native preS region gene was not expressed under the control of commonly used promoters in Escherichia coli. By site-directed mutagenesis of some nucleotides at the 5'-end of the preS1 region gene, we have generated a mutant gene which is highly expressed in soluble form in E. coli. The produced polypeptide could be efficiently purified by 20% ammonium sulfate precipitation and a gel permeation chromatography and the purified polypeptide was demonstrated to exhibit the antigenicity and the immunogenicity of the preS1 and preS2 Ag, suggesting that it is functional.
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Affiliation(s)
- H S Kim
- Protein Engineering Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST, Yuseong, Taejon, South Korea
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42
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Kim HS, Hong HJ. Efficient expression, purification and characterization of hepatitis B virus preS1 protein from Escherichia coli. Biotechnol Lett 1995. [DOI: 10.1007/bf00129021] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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43
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Kniskern PJ, Hagopian A, Burke P, Schultz LD, Montgomery DL, Hurni WM, Ip CY, Schulman CA, Maigetter RZ, Wampler DE. Characterization and evaluation of a recombinant hepatitis B vaccine expressed in yeast defective for N-linked hyperglycosylation. Vaccine 1994; 12:1021-5. [PMID: 7975842 DOI: 10.1016/0264-410x(94)90339-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The hepatitis B (HB) virus preS2 + 2 polypeptide (the M or middle envelope polypeptide) is N-glycosylated at the N4 residue of the preS2 domain when expressed in recombinant yeast. Hyperglycosylation at this amino acid residue (the addition of a large number of mannose residues to the core oligosaccharide), which occurs in common yeast strains, results in an HB vaccine with diminished immunogenicity. Hyperglycosylation can be prevented by expressing the preS2 + S polypeptide in mutant yeast strains (e.g. mnn9) which limit N-linked glycosylation to the addition of only core saccharide residues. An HB vaccine prepared from recombinant yeast expressing the non-hyperglycosylated preS2 + 2 polypeptide was of similar immunogenicity in mice to a licensed HB vaccine and was much more immunogenic in humans than the hyperglycosylated preS2 + 2 vaccine.
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Affiliation(s)
- P J Kniskern
- Department of Virus and Cell Biology, Merck Research Laboratories, West Point, PA
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44
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Hertogs K, Depla E, Crabbé T, De Bruin W, Leenders W, Moshage H, Yap SH. Spontaneous development of anti-hepatitis B virus envelope (anti-idiotypic) antibodies in animals immunized with human liver endonexin II or with the F(ab')2 fragment of anti-human liver endonexin II immunoglobulin G: evidence for a receptor-ligand-like relationship between small hepatitis B surface antigen and endonexin II. J Virol 1994; 68:1516-21. [PMID: 8107214 PMCID: PMC236608 DOI: 10.1128/jvi.68.3.1516-1521.1994] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
In a previous study, we have identified endonexin II (E-II) on human liver plasma membranes as a specific, Ca(2+)-dependent, small hepatitis B surface antigen (HBsAg)-binding protein. In this article, we describe the spontaneous development of anti-HBs antibodies in rabbits immunized with native or recombinant human liver E-II and in chickens immunized with the F(ab')2 fragment of rabbit anti-human liver E-II immunoglobulin G. Anti-HBs activity was not observed in rabbits immunized with rat liver E-II. Cross-reactivity of anti-E-II antibodies to HBsAg epitopes was excluded, since anti-HBs and anti-E-II activities can be separated by E-II affinity chromatography. The existence of an anti-idiotypic antibody is further demonstrated by competitive binding of human liver E-II and this antibody (Ab2) to small HBsAg, suggesting that Ab2 mimics a specific E-II epitope that interacts with small HBsAg. In addition, it was demonstrated that anti-HBs antibodies developed in rabbits after immunization with intact human liver E-II or in chickens after immunization with F(ab')2 fragments of rabbit anti-human liver E-II immunoglobulin G recognize the same epitopes on small HBsAg. These findings strongly indicate that human liver E-II is a very specific small HBsAg-binding protein and support the assumption that human liver E-II is the hepatitis B virus receptor protein.
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Affiliation(s)
- K Hertogs
- Division of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Leuven, Belgium
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45
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Abstract
A number of recent technological developments have greatly facilitated the genetic engineering of immunoglobulins. The use of PCR has permitted the variable regions to be rapidly cloned either from a specific hybridoma source or as a gene library from non-immunised cells. The conversion of the rodent antibody into a humanized version is now well established. To develop these antibodies for clinical use has required the development of high level expression systems. For the expression of large multimeric glycoproteins, mammalian cell systems generally provide the highest levels of secreted product and therefore are the methods of choice for producing whole recombinant antibodies. Novel antigen-binding units have been developed by joining the two variable domains of an antibody into single-chain polypeptides. Such fragments can be produced in high yield by secretion from E. coli raising the prospect of bulk preparation of these antibody fragments for the development of low-cost immunopurification and assay reagents. Finally, the ability to screen for antigen binding by displaying immunoglobulin variable regions on the surface of filamentous bacteriaphages has opened up the possibility of bypassing the immune system to generate novel antibody specificities in vitro.
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46
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Ogata N, Ostberg L, Ehrlich PH, Wong DC, Miller RH, Purcell RH. Markedly prolonged incubation period of hepatitis B in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis B surface antigen. Proc Natl Acad Sci U S A 1993; 90:3014-8. [PMID: 8464917 PMCID: PMC46227 DOI: 10.1073/pnas.90.7.3014] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
The protective efficacy of a human monoclonal antibody directed against the a determinant of hepatitis B virus (HBV) surface antigen was studied in a chimpanzee. A single high dose of 5 mg/kg (body weight) of monoclonal antibody SDZ OST 577 was intravenously administered to a chimpanzee, followed by intravenous challenge with 10(3.5) chimpanzee infectious doses of a wild-type HBV, the MS-2 strain (ayw subtype). The passively acquired antibody to HBV surface antigen could be detected for 40 weeks. Serum HBV DNA tested by a "nested" polymerase chain reaction assay was negative through the 36th week after virus challenge but became positive by the 38th week. The chimpanzee subsequently developed acute hepatitis B approximately 1 year after challenge. The nucleotide sequence of the a determinant of the surface gene of the replicated virus was identical with that of the inoculated wild-type virus. Thus, a human monoclonal antibody directed against the a determinant of HBV surface antigen delayed but did not prevent experimental infection of HBV and hepatitis in the chimpanzee. Our results indicate an incomplete ability of this antibody to protect against HBV infection in vivo after a single infusion.
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Affiliation(s)
- N Ogata
- Hepatitis Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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47
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Bouffard P, Mamish D, Baginski I, Mimms A, Lambert V, Trépo C, Zeldis JB. Infection of a leukemic cell line (K562) by hepatitis B virus induces cell growth inhibition. J Hepatol 1993; 17:384-9. [PMID: 7686195 DOI: 10.1016/s0168-8278(05)80222-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Hepatitis B virus inhibits the in vitro growth of the human leukemic cell line K562; however, the mechanism of this growth inhibition is not understood. One to 12 days after exposure, viral DNA and RNA were detected in K562 cells by Southern blot and reverse-transcriptase polymerase chain reaction analyses. Virus-containing serum that was heat-inactivated failed to inhibit cell growth and no viral DNA or RNA was detected in these cells. In addition, murine monoclonal antibodies directed to hepatitis B virus surface epitopes neutralized the virus-induced growth inhibition whereas antibodies to hepatitis B virus core epitopes failed to suppress the inhibition. These results indicate that in vitro infection of K562 cells by hepatitis B virus causes inhibition of hematopoietic cell line growth.
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48
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Waters JA, Brown SE, Steward MW, Howard CR, Thomas HC. Analysis of the antigenic epitopes of hepatitis B surface antigen involved in the induction of a protective antibody response. Virus Res 1992; 22:1-12. [PMID: 1371369 DOI: 10.1016/0168-1702(92)90085-n] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Vaccination with hepatitis B surface antigen (HBsAg) has shown that antibody directed against the common 'a' determinant of this antigen is protective against infection with hepatitis B virus (HBV). In this study the antigenic epitopes of the 'a' determinant have been analysed by competitive inhibition assays and by binding studies to synthetic peptides using a panel of monoclonal antibodies prepared against HBsAg, all of which are shown to recognise the common group determinant. One murine monoclonal antibody used in this study, RFHBs1, has been shown previously to block infectivity of HBV in susceptible chimpanzees ((1983) J. Med. Virol. 16, 89-95). This antibody bound to a cyclical synthetic peptide analogue of amino acids 124 to 137 of the major HBsAg polypeptide.
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Affiliation(s)
- J A Waters
- Department of Medicine, St. Mary's Hospital Medical School, Imperial College, London, U.K
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49
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Petit MA, Capel F, Dubanchet S, Mabit H. PreS1-specific binding proteins as potential receptors for hepatitis B virus in human hepatocytes. Virology 1992; 187:211-22. [PMID: 1736525 DOI: 10.1016/0042-6822(92)90309-d] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Cellular receptors play an important role in viral pathogenesis. Until now, there has been no reliable information on the receptor(s) for hepatitis B virus (HBV). Therefore, we attempted to identify specific receptors in human hepatocytes using an immunological approach. Anti-idiotypic (Ab2) antibodies were raised in rabbits against our monoclonal antibody (MAb1) F35.25. MAb1 F35.25 (i) recognized the hepatocyte receptor binding site on HBV (located between amino acid residues 21 and 47 of the preS1 sequence) and (ii) blocked the attachment of preS1-positive HBV particles to human hepatocytes. The presence of Ab2 antibodies in rabbit sera was determined by the ability of antisera to inhibit Id (Ab1)/antigen (HBV) recognition. Affinity-purified Ab2 IgGs to F35.25 represented an internal image for the preS1 domain 12-53. Our present studies indicate that Ab2 IgGs to F35.25 (i) recognized the membrane-associated structure of the preS1-specific HBV receptor in a HepG2 cell binding assay, as visualized by immunoenzymatic staining; (ii) strongly bound to a major 35-kDa component and to three other related proteins of 50, 43, and 40 kDa in extracts of HepG2 cells; and (iii) reacted with several soluble and membrane-associated proteins in normal human liver cells. The binding was insensitive to reduction. All preS1 binding proteins were V8 protease sensitive and endoglycosidase H resistant. The 35-kDa species was trypsin resistant and generated a band of 32 kDa by endoglycosidase F treatment. Together, our results suggest that the identified preS1-specific binding proteins may be involved in the putative complex structure of the hepatocyte receptor for HBV.
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Affiliation(s)
- W F Carman
- Institute of Virology, University of Glasgow, Scotland
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