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1
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Paul SS, Patwa SM, Tan YJ. Development of monoclonal antibodies to target the large surface protein of hepatitis B virus and their use in therapeutic and diagnostic applications. J Viral Hepat 2023; 30:870-878. [PMID: 37525419 DOI: 10.1111/jvh.13880] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/07/2023] [Revised: 07/11/2023] [Accepted: 07/23/2023] [Indexed: 08/02/2023]
Abstract
Over 250 million people are living with chronic infection caused by the hepatitis B virus (HBV). HBV has three surface proteins, namely small (SHBs), medium (MHBs) and large (LHBs), and they play different roles in the virus life cycle. The approved hepatitis B vaccine only contains the SHBs protein and many studies have focused on characterising the functional domains in SHBs. Although the LHBs protein is less studied, recent studies have shown that it plays important roles in mediating viral entry, replication and assembly. Over the years, there have been major advancements in monoclonal antibody (mAb) discovery tools and multiple mAbs have been developed to specifically target the preS1 domain in LHBs. We summarise the HBV infection systems and antibody discovery strategies that have been utilised by various research groups to assess the potential use of anti-preS1 mAbs as therapeutic antibodies against HBV or in the development of new diagnostic assays.
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Affiliation(s)
| | - Som Mohanlal Patwa
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore City, Singapore
- Infectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore
| | - Yee-Joo Tan
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore City, Singapore
- Infectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore
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2
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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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3
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Seitz S, Habjanič J, Schütz AK, Bartenschlager R. The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle. Annu Rev Virol 2020; 7:263-288. [PMID: 32600157 DOI: 10.1146/annurev-virology-092818-015508] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.
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Affiliation(s)
- Stefan Seitz
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany;
| | - Jelena Habjanič
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Anne K Schütz
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany; .,Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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Jiang B, Wu Q, Kuhnhenn L, Akhras S, Spengler C, Boller K, Peiffer KH, Hildt E. Formation of semi-enveloped particles as a unique feature of a hepatitis B virus PreS1 deletion mutant. Aliment Pharmacol Ther 2019; 50:940-954. [PMID: 31240738 DOI: 10.1111/apt.15381] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2019] [Revised: 04/29/2019] [Accepted: 05/29/2019] [Indexed: 12/12/2022]
Abstract
BACKGROUND Naturally occurring variants with deletions or mutations in the C-terminal PreS1 domain from hepatitis B virus (HBV) chronically infected patients have been shown to promote HBsAg retention, inhibit HBsAg secretion and change the extracellular appearance of PreS1-containing HBV particles (filaments and virions). AIMS To study the impact of N-terminal deletion in preS1 domain on viral secretion and morphogenesis. METHODS An HBV mutant with 15 amino acids (aa 25-39) deletion in N-terminal preS1 was isolated. Intracellular and extracellular HBsAg were quantified by Western blot. Subcellular HBsAg distribution was analysed by confocal laser scanning microscopy. The viral morphology was characterised by sucrose density gradient ultracentrifugation, Western blot, electron microscopy, HBV mixed ELISA and HBV particle gel essay. RESULTS Expression of this mutant genome released higher amounts of HBsAg in the form of shorter filaments. A significant fraction of semi-enveloped virions was observed in the supernatant that has been unprecedented so far. Stepwise insertion of aa 25-31, aa 32-39 and aa 25-39 increased the length of filaments. The rescue of aa 25-31 and aa 25-39 drastically reduced the amounts of extracellular HBsAg and semi-enveloped virions, while such effects could not be observed after insertion of aa 32-39, arguing against a simple spacer function of this region. The deletion and rescued mutants do not differ in subcellular HBsAg distribution and colocalisation with ER, Golgi and multivesicular bodies markers arguing against differences in release pathways. CONCLUSION N-terminal PreS1-domain (aa 25-31) determines HBsAg secretion and triggers proper assembly of PreS1-containing particles.
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Affiliation(s)
- Bingfu Jiang
- Division of Virology, Paul-Ehrlich-Institut, Langen, Germany
| | - Qingyan Wu
- Division of Virology, Paul-Ehrlich-Institut, Langen, Germany
| | - Lisa Kuhnhenn
- Division of Virology, Paul-Ehrlich-Institut, Langen, Germany.,Department of Gastroenterology and Hepatology, J. W. Goethe University, Frankfurt, Germany
| | - Sami Akhras
- Division of Virology, Paul-Ehrlich-Institut, Langen, Germany
| | | | - Klaus Boller
- Department of Immunology, Paul-Ehrlich-Institut, Langen, Germany
| | - Kai-Henrik Peiffer
- Division of Virology, Paul-Ehrlich-Institut, Langen, Germany.,Department of Gastroenterology and Hepatology, J. W. Goethe University, Frankfurt, Germany
| | - Eberhard Hildt
- Division of Virology, Paul-Ehrlich-Institut, Langen, Germany.,German Center for Infection Research (DZIF), Germany
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5
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Peiffer KH, Kuhnhenn L, Jiang B, Mondorf A, Vermehren J, Knop V, Susser S, Walter D, Dietz J, Carra G, Finkelmeier F, Zeuzem S, Sarrazin C, Hildt E. Divergent preS Sequences in Virion-Associated Hepatitis B Virus Genomes and Subviral HBV Surface Antigen Particles From HBV e Antigen-Negative Patients. J Infect Dis 2019. [PMID: 29528436 DOI: 10.1093/infdis/jiy119] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Background Hepatitis B virus (HBV) surface proteins (HBsAg) coat the viral particle and form subviral particles (SVPs). Loss of HBsAg represents a functional cure and is an important treatment goal. Methods We analyzed the impact of the HBV genotypes A-E and pre-S mutations on SVP expression in hepatitis B virus e antigen (HBeAg)-negative chronic HBV-infected patients. A HBV genome harboring a preS1-deletion was analyzed in hepatoma cells. Results We observed a genotype-specific ratio of the 3 surface proteins (SHBs/MHBs/LHBs), reflecting differences in the morphology and composition of SVPs. Deletions/mutations in the preS1/preS2 domain, detected in released viral genomes, did not affect the molecular weight of MHBs and LHBs in these patients. In contrast, LHB molecular weight was altered in vitro using an HBV genome harboring a preS1-deletion derived from one of these patients. Conclusion Differences in composition of SVPs may result in genotype-specific immunogenicity and pathogenesis. In the patients with preS-mutations, secreted HBsAg and released viral genomes cannot be derived from the same genetic source. As viral genomes are derived from covalently closed circular DNA (cccDNA), HBsAg is presumably derived from integrated DNA. This important HBsAg source should be considered for novel antiviral strategies in HBeAg-negative chronic HBV-infected patients.
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Affiliation(s)
- Kai-Henrik Peiffer
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany.,Paul Ehrlich Institute, Division of Virology, Langen, Germany
| | - Lisa Kuhnhenn
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany.,Paul Ehrlich Institute, Division of Virology, Langen, Germany
| | - Bingfu Jiang
- Paul Ehrlich Institute, Division of Virology, Langen, Germany
| | - Antonia Mondorf
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Johannes Vermehren
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Viola Knop
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Simone Susser
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Dirk Walter
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Julia Dietz
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Gert Carra
- Paul Ehrlich Institute, Division of Virology, Langen, Germany
| | - Fabian Finkelmeier
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Stefan Zeuzem
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany
| | - Christoph Sarrazin
- University Hospital Frankfurt, Department of Gastroenterology and Hepatology, Langen, Germany.,St. Josefs Hospital, Department of Gastroenterology, Wiesbaden, Germany
| | - Eberhard Hildt
- Paul Ehrlich Institute, Division of Virology, Langen, Germany.,German Center for Infection Research, Germany
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Pumpens P, Renhofa R, Dishlers A, Kozlovska T, Ose V, Pushko P, Tars K, Grens E, Bachmann MF. The True Story and Advantages of RNA Phage Capsids as Nanotools. Intervirology 2016; 59:74-110. [DOI: 10.1159/000449503] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2016] [Accepted: 08/30/2016] [Indexed: 11/19/2022] Open
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The Hepatitis B Virus Core Variants that Expose Foreign C-Terminal Insertions on the Outer Surface of Virus-Like Particles. Mol Biotechnol 2016; 57:1038-49. [PMID: 26446016 PMCID: PMC4619458 DOI: 10.1007/s12033-015-9895-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The major immunodominant region (MIR) and N-terminus of the hepatitis B virus (HBV) core (HBc) protein were used to expose foreign insertions on the outer surface of HBc virus-like particles (VLPs). The additions to the HBc positively charged arginine-rich C-terminal (CT) domain are usually not exposed on the VLP surface. Here, we constructed a set of recombinant HBcG vectors in which CT arginine stretches were substituted by glycine residues. In contrast to natural HBc VLPs and recombinant HBc VLP variants carrying native CT domain, the HBcG VLPs demonstrated a lowered capability to pack bacterial RNA during expression in Escherichia coli cells. The C-terminal addition of a model foreign epitope from the HBV preS1 sequence to the HBcG vectors resulted in the exposure of the inserted epitope on the VLP surface, whereas the same preS1 sequences added to the native CT of the natural HBc protein remained buried within the HBc VLPs. Based on the immunisation of mice, the preS1 epitope added to the HBcG vectors as a part of preS1(20-47) and preS1phil sequences demonstrated remarkable immunogenicity. The same epitope added to the original C-terminus of the HBc protein did not induce a notable level of anti-preS1 antibodies. HBcG vectors may contribute to the further development of versatile HBc VLP-based vaccine and gene therapy applications.
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8
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Pumpens P, Grens E. The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016. Mol Biol 2016; 50:489-509. [DOI: 10.1134/s0026893316040099] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Accepted: 01/14/2016] [Indexed: 01/02/2025]
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9
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Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes. Viruses 2015; 7:4204-29. [PMID: 26230706 PMCID: PMC4576179 DOI: 10.3390/v7082818] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2015] [Revised: 07/20/2015] [Accepted: 07/27/2015] [Indexed: 12/21/2022] Open
Abstract
Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.
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10
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Li X, Pushko P, Tretyakova I. Recombinant Hemagglutinin and Virus-Like Particle Vaccines for H7N9 Influenza Virus. ACTA ACUST UNITED AC 2015; 6. [PMID: 26523241 DOI: 10.4172/2157-7560.1000287] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Cases of H7N9 human infection were caused by a novel, avian-origin H7N9 influenza A virus that emerged in eastern China in 2013. Clusters of human disease were identified in many cities in China, with mortality rates approaching 30%. Pandemic concerns were raised, as historically, influenza pandemics were caused by introduction of novel influenza A viruses into immunologically naïve human population. Currently, there are no approved human vaccines for H7N9 viruses. Recombinant protein vaccine approaches have advantages in safety and manufacturing. In this review, we focused on evaluation of the expression of recombinant hemagglutinin (rHA) proteins as candidate vaccines for H7N9 influenza, with the emphasis on the role of oligomeric and particulate structures in immunogenicity and protection. Challenges in preparation of broadly protective influenza vaccines are discussed, and examples of broadly protective vaccines are presented including rHA stem epitope vaccines, as well as recently introduced experimental multi-HA VLP vaccines.
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Affiliation(s)
- Xiaohui Li
- Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, PR China 200240 ; Genor Biopharma Co., Ltd. 1690 Zhangheng Road, Shanghai, PR China 201203
| | - Peter Pushko
- Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD, U.S.A
| | - Irina Tretyakova
- Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD, U.S.A
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11
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Lee G, Liu S. Monoclonal antibodies against hepatitis B viral surface antigens and epitope grouping. Monoclon Antib Immunodiagn Immunother 2015; 34:90-5. [PMID: 25897606 DOI: 10.1089/mab.2014.0079] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Monoclonal antibodies (MAbs) were generated against subtypes (ad/ad/rw) of the human hepatitis B viral surface antigen (HBsAg). Among dozens of antibodies that were generated, the majority was shown to commonly react with various ad/ay subtypes of the S protein. Epitope(s) of these antibodies were grouped by various immunoassay methods, and at least four distinct epitope regions were identified. Some of these antibodies were selected to formulate sandwich enzyme immunoassays for quantitative determinations of HBsAg in reconstituted specimens. Epitope-defined monoclonal antibodies with high affinity and specificity might be suitable for formulations as vaccines (containing a mixture of humanized monoclonal antibodies) for passive immunization in humans for immunoprophylaxis of HBV infection.
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Affiliation(s)
- Gregory Lee
- UBC Center for Reproductive Health , Vancouver, British Columbia, Canada
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12
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Wang X, Li Q, Davies M. Development of antibody arrays for monoclonal antibody Higher Order Structure analysis. Front Pharmacol 2013; 4:103. [PMID: 23970865 PMCID: PMC3748713 DOI: 10.3389/fphar.2013.00103] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2013] [Accepted: 07/31/2013] [Indexed: 11/17/2022] Open
Abstract
Antibody arrays were developed to probe a monoclonal antibody's three-dimensional structure (3-D structure). Peptides with overlapping regions were designed to cover the whole mAb light chain and heavy chain, respectively, and used to generate polyclonal antibodies after the conjugation of the peptides to a carrier protein, KLH. It was shown that good peptide specificity was achieved from the antibodies generated. Using more than 30 different polyclonal antibodies to measure the surface epitope distribution, it was shown that the mAb antibody array can detect epitope exposure as low as 0.1% of defined mAb populations. This ELISA-based analysis of mAb epitope exposure can be considered as a measurement of “conformational impurity” in biologics development, similar to the analysis of other product-related impurities such as different forms of glycosylation, deamidation, and oxidation. This analysis of “conformational impurity” could provide valuable information on the mAb conformational comparability for biosimilar mAbs as well as novel mAbs, especially in the area of protein immunogenicity. Furthermore, stability studies indicated that there are several conformational “hot spots” in many mAbs tested, especially in the hinge region. This antibody array technology can be used for novel mAb Higher Order Structure (HOS) analysis during process and formulation development. Another important area of application is for biosimilar mAb development where the innovator molecule and biosimilar molecule could be compared based on their systemic “fingerprint” from the 30 plus antibodies.
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Affiliation(s)
- Xing Wang
- Array Bridge Inc. St. Louis, MO, USA
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13
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Niedre-Otomere B, Bogdanova A, Skrastina D, Zajakina A, Bruvere R, Ose V, Gerlich WH, Garoff H, Pumpens P, Glebe D, Kozlovska T. Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies. J Viral Hepat 2012; 19:664-73. [PMID: 22863271 DOI: 10.1111/j.1365-2893.2012.01594.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.
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Affiliation(s)
- B Niedre-Otomere
- Biomedical Research and Study Centre, University of Latvia, Riga, Latvia, Germany
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14
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Roseman AM, Borschukova O, Berriman JA, Wynne SA, Pumpens P, Crowther RA. Structures of hepatitis B virus cores presenting a model epitope and their complexes with antibodies. J Mol Biol 2012; 423:63-78. [PMID: 22750730 PMCID: PMC3465560 DOI: 10.1016/j.jmb.2012.06.032] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2012] [Revised: 05/16/2012] [Accepted: 06/20/2012] [Indexed: 12/22/2022]
Abstract
The core shell of hepatitis B virus is a potent immune stimulator, giving a strong neutralizing immune response to foreign epitopes inserted at the immunodominant region, located at the tips of spikes on the exterior of the shell. Here, we analyze structures of core shells with a model epitope inserted at two alternative positions in the immunodominant region. Recombinantly expressed core protein assembles into T = 3 and T = 4 icosahedral shells, and atomic coordinates are available for the T = 4 shell. Since the modified protein assembles predominantly into T = 3 shells, a quasi-atomic model of the native T = 3 shell was made. The spikes in this T = 3 structure resemble those in T = 4 shells crystallized from expressed protein. However, the spikes in the modified shells exhibit an altered conformation, similar to the DNA containing shells in virions. Both constructs allow full access of antibodies to the foreign epitope, DPAFR from the preS1 region of hepatitis B virus surface antigen. However, one induces a 10-fold weaker immune response when injected into mice. In this construct, the epitope is less constrained by the flanking linker regions and is positioned so that the symmetry of the shell causes pairs of epitopes to come close enough to interfere with one another. In the other construct, the epitope mimics the native epitope conformation and position. The interaction of native core shells with an antibody specific to the immunodominant epitope is compared to the constructs with an antibody against the foreign epitope. Our findings have implications for the design of vaccines based on virus-like particles.
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Affiliation(s)
- A M Roseman
- MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
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15
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N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus. J Hepatol 2011; 55:29-37. [PMID: 21145866 DOI: 10.1016/j.jhep.2010.10.019] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Revised: 08/29/2010] [Accepted: 10/30/2010] [Indexed: 12/20/2022]
Abstract
BACKGROUNDS & AIMS The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized. METHODS We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro. RESULTS The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies binding to the C-terminal part of preS1 did not neutralize HBV infectivity for susceptible hepatocyte cultures. In contrast, antibodies elicited by the complete N-terminal attachment site of preS1(2-48) strongly neutralized with an IC50<3μg/ml of total immunoglobulin. Interestingly, antibodies against the very N-terminal part of preS1(1-21) could not neutralize infectivity although this sequence contains the most conserved and essential part of the attachment site. These antibodies reacted well with non-myristoylated preS1 peptides but only weakly with myristoylated preS1 peptides, natural HBsAg or HBV. CONCLUSIONS N-terminal myristic acid obviously favors a topology of LHBs that makes the most essential part of the preS1 attachment site inaccessible for neutralizing antibodies, whereas antibodies to neighbouring sequences neutralized very well. Thus, addition of the preS1(2-48) peptide in a highly immunogenic form to the current hepatitis B vaccine may improve protective immunity and reduce selection of escape mutations.
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16
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Bremer CM, Saniewski M, Wend UC, Torres P, Lelie N, Gerlich WH, Glebe D. Transient occult hepatitis B virus infection in a blood donor with high viremia. Transfusion 2011; 49:1621-9. [PMID: 19413737 DOI: 10.1111/j.1537-2995.2009.02188.x] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Screening of blood donors for viral nucleic acids has recently been introduced in several countries. With the use of transcription-mediated amplification, a blood donor was detected who had 90,000 copies of hepatitis B virus (HBV) DNA/mL but no hepatitis B surface antigen (HBsAg) or antibody to hepatitis B core antigen (anti-HBc). One month later, anti-HBc and hepatitis B surface antibody (anti-HBs) appeared; HBV DNA disappeared after 2 months. This study asked why HBsAg was undetectable in this rare case of transient occult HBV infection. STUDY DESIGN AND METHODS The HBV DNA in the first sample was cloned and sequenced to identify mutations. The physical nature of the virus was examined by polyethylene glycol precipitation, DNase digestion, density gradient centrifugation, and immunoprecipitation. RESULTS Several mutations were found all over the genome, but the HBs antigen loop was unchanged. A stop mutation in the precore region led to loss of hepatitis B e antigen (HBeAg) expression. No HBV DNA–containing immune complexes were present. The plasma did not contain nonencapsidated HBV DNA that could explain the absence of HBsAg. The virus was immune precipitated by antibodies against HBsAg or preS1 antigen. The ratio of HBV to HBsAg subviral particles was estimated to be 1 in less than 20 whereas in overt cases the ratio is 1 in more than 1000. CONCLUSION The acute resolving occult HBV infection was caused by an HBeAg-negative variant, which otherwise was almost normal. The negative HBsAg result was probably due to an unusually low production of surplus HBsAg. The absence of the viral immunomodulator HBeAg and the early appearance of anti-HBs suggested a rapid noncytolytic HBsAg-specific T-cell response leading to low expression of HBsAg.
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Affiliation(s)
- Corinna M Bremer
- Institute of Medical Virology, Justus Liebig University, Giessen, Germany
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17
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Construction and immunological evaluation of multivalent hepatitis B virus (HBV) core virus-like particles carrying HBV and HCV epitopes. CLINICAL AND VACCINE IMMUNOLOGY : CVI 2010; 17:1027-33. [PMID: 20410327 DOI: 10.1128/cvi.00468-09] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcDelta protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-gamma) production profile.
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Côté I, Poulos BT, Redman RM, Lightner DV. Development and characterization of a monoclonal antibody against Taura syndrome virus. JOURNAL OF FISH DISEASES 2009; 32:989-996. [PMID: 19602090 DOI: 10.1111/j.1365-2761.2009.01082.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
We produced a panel of monoclonal antibodies (MAbs) from the fusion of Taura syndrome virus variants from Belize (TSV-BZ) immunized BALB/cJ mouse spleen cells and non-immunoglobulin secreting SP2/0 mouse myeloma cells. One antibody, 2C4, showed strong specificity and sensitivity for TSV in dot-blot immunoassay and immunohistochemistry (IHC) analysis. The MAb reacted against native TSV-BZ, TSV variants from Sinaloa, Mexico (TSV-SI) and TSV variants from Hawaii (TSV-HI) in dot-blot immunoassay. By IHC, the antibody identified the virus in a pattern similar to the digoxigenin-labelled TSV-cDNA probe for the TSV-BZ, TSV-HI and TSV-SI variants, but not for the TSV variants from Venezuela (TSV-VE) and the TSV variants from Thailand (TSV-TH). MAb 2C4 did not react against other shrimp pathogens or with normal shrimp tissue. Western blot analysis showed a strong reaction against CP2, a region of high antigenic variability amongst TSV variants. This antibody has potential diagnostic application in detection and differentiation of certain TSV biotypes.
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Affiliation(s)
- I Côté
- Department of Veterinary Sciences and Microbiology, The University of Arizona, Tucson, AZ 85721, USA.
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19
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Chi SW, Kim DH, Lee SH, Chang I, Han KH. Pre-structured motifs in the natively unstructured preS1 surface antigen of hepatitis B virus. Protein Sci 2007; 16:2108-17. [PMID: 17766372 PMCID: PMC2204132 DOI: 10.1110/ps.072983507] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The preS1 surface antigen of hepatitis B virus (HBV) is known to play an important role in the initial attachment of HBV to hepatocytes. We have characterized structural features of the full-length preS1 using heteronuclear NMR methods and discovered that this 119-residue protein is inherently unstructured without a unique tertiary structure under a nondenaturing condition. Yet, combination of various NMR parameters shows that the preS1 contains "pre-structured" domains broadly covering its functional domains. The most prominent domain is formed by residues 27-45 and overlaps with the putative hepatocyte-binding domain (HBD) encompassing residues 21-47, within which two well-defined pre-structured motifs, formed by Pro(32)-Ala(36) and Pro(41)-Phe(45) are found. Additional, somewhat less prominent, pre-structured motifs are also formed by residues 11-18, 22-25, 37-40, and 46-50. Overall results suggest that the preS1 is a natively unstructured protein (NUP) whose N-terminal 50 residues, populated with multiple pre-structured motifs, contribute critically to hepatocyte binding.
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Affiliation(s)
- Seung-Wook Chi
- Molecular Cancer Research Center, Division of Molecular Therapeutics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
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20
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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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21
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Alves Vianna CO, da Silva E Mouta Júnior S, da Glória Teixeira Martins M, Batoreu NM, Queiroz JL, Gomes SA, Magalhães de Andrade Góes AC, Garcia Armoa GR, Marques CH, Baroni de Moraes MT. Evaluation of murine monoclonal antibodies targeting different epitopes of the hepatitis B virus surface antigen by using immunological as well as molecular biology and biochemical approaches. J Immunol Methods 2006; 313:38-47. [PMID: 16753174 DOI: 10.1016/j.jim.2006.03.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2005] [Revised: 03/06/2006] [Accepted: 03/08/2006] [Indexed: 01/22/2023]
Abstract
The hepatitis B virus surface protein (HBsAg) displays the major B cells antigenic determinants that can induce protective immunity and prevent the hepatitis B virus (HBV) infection, a major health problem. A panel of murine monoclonal antibodies against the HBsAg (MAb anti-HBs), raised after mice immunization with a pool of plasma of hepatitis chronic carriers, has been established. Mainly using simple immunological tools such as enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, we could trace the location of the epitopes on the HBsAg determinants. We also report the use of two specific methodology approaches based on molecular biology and biochemical techniques such as, respectively, cloning and expression of preS1 major neutralizing epitope of the HBsAg in Escherichia coli and ELISA accomplished to chemical reduction with dithiothreitol (DTT), which were able to complete the MAb anti-HBs characterization. Our results showed that the majority of the MAbs anti-HBs were directed to the HBV common determinant a. One MAb recognizes a discontinuous epitope present in all forms of the HBsAg when evaluated by Western blot.
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Affiliation(s)
- Carlos Otávio Alves Vianna
- Laboratory of Monoclonal Antibodies Technology, Bio-Manguinhos, Oswaldo Cruz Foundation, FIOCRUZ, P.O. Box 926, Rio de Janeiro, Brazil
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22
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Glebe D, Urban S, Knoop EV, Cag N, Krass P, Grün S, Bulavaite A, Sasnauskas K, Gerlich WH. Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes. Gastroenterology 2005; 129:234-45. [PMID: 16012950 DOI: 10.1053/j.gastro.2005.03.090] [Citation(s) in RCA: 210] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Studies on the early steps in the life cycle of hepatitis B virus have been hampered by the lack of readily available target cells. In this study, we mapped a defined virus attachment site to primary hepatocytes that is essential for infection. METHODS We used purified virus particles from human carrier plasma as an inoculum and primary cultures of tupaia hepatocytes as susceptible target cells and studied the inhibitory effect of amino-terminally acylated preS1-derived lipopeptides on infection interference. RESULTS Infectivity of virus could be blocked efficiently in this system by amino-terminally acylated peptides containing amino acids 2-18 from the preS1 domain. The addition of amino acids 28-48 enhanced the inhibitory capacity, whereas amino acids 49-78 did not contribute to inhibition. Myristoylated preS1 peptides 2-48 bound strongly to tupaia hepatocytes but not to nonhepatic cells or rodent hepatocytes and thereby inhibited infection even at concentrations of 1 nmol/L completely. Particles consisting only of the small hepatitis B surface protein-the active component of current hepatitis B vaccines-did not bind at all to tupaia hepatocytes, but the addition of the preS1 domain to the particles allowed binding. CONCLUSIONS The preS1 sequence 2-48 mediates attachment of the virus to its target cells, whereas the small surface protein seems to be involved in other steps. These findings indicate that the current subunit hepatitis B vaccines may be improved by the addition of distinct preS1 epitopes. Moreover, preS1 lipopeptides are promising candidates for specific antiviral therapy against hepatitis B infections.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus-Liebig University Giessen, Germany.
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23
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Xin ZT, Liu C, Dong B, Gao YP, Shao NS, Liu W, Zhang J, Dong J, Ling SG, Xue YN. A subtractive fluorescence-activated cell-sorting strategy to identify mimotopes of HBV–preS protein from bacterially displayed peptide library. J Immunol Methods 2004; 293:13-21. [PMID: 15541273 DOI: 10.1016/j.jim.2004.06.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2004] [Revised: 05/28/2004] [Accepted: 06/03/2004] [Indexed: 11/28/2022]
Abstract
A novel subtractive fluorescence-activated cell-sorting (FACS) strategy using a model system is described here to identify disease-specific (DS) epitopes from a bacterially displayed random peptide library. In this process, preimmune serum was used as "Driver " to block any common binding sites on the bacterial surface and the labeled anti-preS IgG polyclonal antibodies from immunized serum were used as "Tester" to enrich preS-specific mimotopes. Bacterial clones were identified out of this pool through an "antigen-independent" procedure only using both different sera samples. After four rounds of sub-FACS screening, 41 out of 50 bacterial clones were identified as reacting with the immunized serum but not reacting with the pre-immune one. Two motif sequences HQLD and DPAF were obtained from 13 clones. Immunization of mice with two representative bacterial clones elicited a strong specific response against native preS antigen in comparison with the control. This technique may provide a useful technology platform for high-throughput screening of disease-related epitope which is of importance to develop vaccine against some infectious diseases whose pathogen or immunodominant antigen is still unknown.
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Affiliation(s)
- Zhong-Tao Xin
- Department of Biochemistry, Beijing Institute of Basic Medical Sciences, P.O. Box 130 (3), Beijing, 100850, People's Republic of China
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24
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Kazaks A, Borisova G, Cvetkova S, Kovalevska L, Ose V, Sominskaya I, Pumpens P, Skrastina D, Dislers A. Mosaic hepatitis B virus core particles presenting the complete preS sequence of the viral envelope on their surface. J Gen Virol 2004; 85:2665-2670. [PMID: 15302960 DOI: 10.1099/vir.0.79810-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc–preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium- and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76–85 in the MIR, the incorporation level of HBc–preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.
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Affiliation(s)
- Andris Kazaks
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Galina Borisova
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Svetlana Cvetkova
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Larisa Kovalevska
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Velta Ose
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Irina Sominskaya
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Paul Pumpens
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Dace Skrastina
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
| | - Andris Dislers
- Biomedical Research and Study Centre, University of Latvia, 1 Ratsupites Street, LV-1067 Riga, Latvia
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25
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Glebe D, Aliakbari M, Krass P, Knoop EV, Valerius KP, Gerlich WH. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus. J Virol 2003; 77:9511-21. [PMID: 12915565 PMCID: PMC187384 DOI: 10.1128/jvi.77.17.9511-9521.2003] [Citation(s) in RCA: 146] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2003] [Accepted: 06/03/2003] [Indexed: 12/17/2022] Open
Abstract
The susceptibility of the tree shrew Tupaia belangeri to human hepatitis B virus (HBV) has been demonstrated both in vivo and in vitro. In this study, we show that purified HBV infects primary T. belangeri hepatocyte cultures in a very specific manner, as detected by HBV covalently closed circular DNA, mRNA, HBV e antigen, and HBsAg production. A monoclonal antibody (MAb), MA18/7, directed against the pre-S1 domain of the large HBs protein, which has been shown to neutralize infectivity of HBV for primary human hepatocytes, also blocked infection of primary Tupaia hepatocytes. MAbs against the pre-S2 domain of HBs inhibited infection only partially, whereas an S MAb and polyvalent anti-HBs antibodies neutralized infection completely. Thus, both pre-S1 and S antigens are necessary for infection in the tupaia. Using subviral particles, >70% of primary Tupaia hepatocytes are capable of specific binding of pre-S1-rich HBsAg, showing localization in distinct membrane areas. The data show that the early steps of HBV infection in Tupaia hepatocyte cultures are comparable to those in the human system.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology. Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, 35392 Giessen, Germany.
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26
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Voronkova T, Grosch A, Kazaks A, Ose V, Skrastina D, Sasnauskas K, Jandrig B, Arnold W, Scherneck S, Pumpens P, Ulrich R. Chimeric bacteriophage fr virus-like particles harboring the immunodominant C-terminal region of hamster polyomavirus VP1 induce a strong VP1-specific antibody response in rabbits and mice. Viral Immunol 2003; 15:627-43. [PMID: 12513932 DOI: 10.1089/088282402320914557] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1.
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Lundkvist A, Meisel H, Koletzki D, Lankinen H, Cifire F, Geldmacher A, Sibold C, Gött P, Vaheri A, Krüger DH, Ulrich R. Mapping of B-cell epitopes in the nucleocapsid protein of Puumala hantavirus. Viral Immunol 2002; 15:177-92. [PMID: 11952140 DOI: 10.1089/088282402317340323] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Hantavirus nucleocapsid protein (N) has been proven to induce highly protective immune responses in animal models. The knowledge on the mechanisms behind N-induced protection is still limited, although recent data suggest that both cellular and humoral immune responses are of importance. For a detailed B-cell epitope mapping of Puumala hantavirus (PUUV) N, we used recombinant N derivatives of the Russian strain CG18-20 and the Swedish strain Vranica/Hällnäs, as well as overlapping synthetic peptides corresponding to the Finnish prototype strain Sotkamo. The majority of a panel of monoclonal antibodies (mAbs) reacted with proteins derived from all included PUUV strains demonstrating the antigenic similarity of these proteins. In line with previous results, the epitopes of most mAbs were mapped within the 80 N-terminal amino acids of N. The present study further revealed that the epitopes of four mAbs raised against native viral N were located within amino acids 14-45, whereas one mAb raised against recombinant N was mapped to amino acids 14-39. Differences between the reactivity of the PUUV strains Vranica/Hällnäs and CG18-20 N suggested the importance of amino acid position 35 for the integrity of the epitopes. In line with the patterns obtained by the truncated recombinant proteins, mapping by overlapping peptides (PEPSCAN) confirmed a complex recognition pattern for most analyzed mAbs. Together, the results revealed the existence of several, partially overlapping, and discontinuous B-cell epitopes. In addition, based on differences within the same competition group, novel epitopes were defined.
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Affiliation(s)
- A Lundkvist
- Swedish Institute for Infectious Disease Control, and Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm.
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28
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Sominskaya I, Paulij W, Jansons J, Sobotta D, Dreilina D, Sunnen C, Meisel H, Gerlich WH, Pumpens P. Fine-mapping of the B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen. J Immunol Methods 2002; 260:251-61. [PMID: 11792393 DOI: 10.1016/s0022-1759(01)00551-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
In this study, we report the exact localization and substitutional characterization of a B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen. A set of deletion variants containing preS2 sequences of different length was generated on the basis of frCP as a carrier. It was found after Western blot analysis that three monoclonal antibodies (MAbs) (2-11B1, 3-11C2, HB.OT10) recognized the linear preS2 sequence within the amino acid (aa) stretch 3-WNSTTFHQTLQDP-13. The importance of each aa residue of the epitope was proved by comparison of antibody binding to alanine-substituted peptides in both free-peptide and Pepscan variants.
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Affiliation(s)
- Irina Sominskaya
- Biomedical Research and Study Centre, University of Latvia, Ratsupites Str. 1, LV-1067, Riga, Latvia.
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Paran N, Geiger B, Shaul Y. HBV infection of cell culture: evidence for multivalent and cooperative attachment. EMBO J 2001; 20:4443-53. [PMID: 11500372 PMCID: PMC125578 DOI: 10.1093/emboj/20.16.4443] [Citation(s) in RCA: 68] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Hepadnaviruses do not infect cultured cells, therefore our knowledge of the mechanism of the early stages of virus-cell interaction is rather poor. In this study, we show that dimethylsulfoxide (DMSO)-treated HepG2 hepatoblastoma cells are infected efficiently by serum-derived hepatitis B virus (HBV) as monitored by viral gene expression and replication markers. To measure virus attachment, a variety of HBV surface proteins (HBsAgs) were conjugated to polystyrene beads and their capacity to attach cells was visualized and quantified by light microscopy at a single-cell resolution. Remarkably, DMSO increases the attachment efficiency by >200-fold. We further identify the QLDPAF sequence within preS1 as the receptor-binding viral domain epitope. Interestingly, a similar sequence is shared by several cellular, bacterial and viral proteins involved in cell adhesion, attachment and fusion. We also found that the small HBsAg contains a secondary attachment site that recognizes a distinct receptor on the cell membrane. Furthermore, we provide evidence in support of multivalent HBV attachment with synergistic interplay. Our data depict a mechanistic view of virus attachment and ingestion.
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Affiliation(s)
- Nir Paran
- Departments of
Molecular Genetics and Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author e-mail:
| | - Benjamin Geiger
- Departments of
Molecular Genetics and Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author e-mail:
| | - Yosef Shaul
- Departments of
Molecular Genetics and Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author e-mail:
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Ren S, Nassal M. Hepatitis B virus (HBV) virion and covalently closed circular DNA formation in primary tupaia hepatocytes and human hepatoma cell lines upon HBV genome transduction with replication-defective adenovirus vectors. J Virol 2001; 75:1104-16. [PMID: 11152483 PMCID: PMC114016 DOI: 10.1128/jvi.75.3.1104-1116.2001] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is a hepatotropic DNA-containing virus that replicates via reverse transcription. Because of its narrow host range, there is as yet no practical small-animal system for HBV infection. The hosts of the few related animal viruses, including woodchuck hepatitis B virus and duck hepatitis B virus, are either difficult to keep or only distantly related to humans. Some evidence suggests that tree shrews (tupaias) may be susceptible to infection with human HBV, albeit with low efficiency. Infection efficiency depends on interactions of the virus with factors on the surface and inside the host cell. To bypass restrictions during the initial entry phase, we used recombinant replication-defective adenovirus vectors, either with or without a green fluorescent protein marker gene, to deliver complete HBV genomes into primary tupaia hepatocytes. Here we show that these cells, like the human hepatoma cell lines HepG2 and Huh7, are efficiently transduced by the vectors and produce all HBV gene products required to generate the secretory antigens HBsAg and HBeAg, replication-competent nucleocapsids, and enveloped virions. We further demonstrate that covalently closed circular HBV DNA is formed. Therefore, primary tupaia hepatocytes support all steps of HBV replication following deposition of the genome in the nucleus, including the intracellular amplification cycle. These data provide a rational basis for in vivo experiments aimed at developing tupaias into a useful experimental animal system for HBV infection.
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Affiliation(s)
- S Ren
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, D-79106 Freiburg, Germany
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31
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D'Mello F, Howard CR. An improved selection procedure for the screening of phage display peptide libraries. J Immunol Methods 2001; 247:191-203. [PMID: 11150550 DOI: 10.1016/s0022-1759(00)00318-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Peptide sequences that bind to a wide range of ligands such as monoclonal antibodies, receptors and carbohydrates have been successfully identified after screening phage display peptide libraries. However, these procedures tend to select mainly medium to low affinity-binding clones. A modified screening procedure has been developed in order to improve the efficiency of this process such that high avidity/affinity binding clones are preferentially selected. Three different solid phase binding surfaces were evaluated for the attachment of antibody during the screening procedure and a stepwise decrease in the pH of the elution buffer introduced during the final round of biopanning. The monoclonal antibody MA 18/7 was used to screen a 15-mer peptide library. This antibody is well-characterised and its binding site has been mapped to residues 28-37 of the pre-S1 protein of the hepatitis B virus. The antibody was either biotinylated and attached to polystyrene plates via a streptavidin-biotin 'bridge', or bound directly to 1/4 in. polystyrene beads, or to 11 microm latex beads. A significant enrichment of binding clones was observed when the monoclonal antibody was attached directly to polystyrene or latex beads as compared to the biotinylated antibody. All mimotopes identified after biopanning with the antibody attached to the polystyrene beads possessed a central core motif, identical or similar to the sequence DPAF contained within the epitope binding site of MA 18/7 on the native pre-S molecule. However, this motif was only observed in 30% of clones isolated after biopanning using the 11 microm latex beads and in 2% of clones isolated after biopanning on the streptavidin-coated plates. Immunoblotting with the monoclonal antibody MA 18/7 confirmed binding to clones containing the DPAF sequence or a similar motif. A stepwise reduction in the pH of the elution buffer in the final round of biopanning resulted in the removal of clones that possessed low affinity binding motifs, thereby increasing the percentage of clones containing high affinity binding motifs in the final elution step at pH 2.0. Thus, the combined use of polystyrene beads and a stepwise decrease in the pH of the elution buffer in the final round of biopanning resulted in the elimination of non-binding clones and an increase in the efficiency in isolating high affinity binding clones.
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Affiliation(s)
- F D'Mello
- Royal Veterinary College, Department of Pathology and Infectious Diseases, Royal College Street, London NW1 OTU, UK
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32
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Gedvilaite A, Frömmel C, Sasnauskas K, Micheel B, Ozel M, Behrsing O, Staniulis J, Jandrig B, Scherneck S, Ulrich R. Formation of immunogenic virus-like particles by inserting epitopes into surface-exposed regions of hamster polyomavirus major capsid protein. Virology 2000; 273:21-35. [PMID: 10891404 DOI: 10.1006/viro.2000.0392] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We generated highly immunogenic virus-like particles that are based on the capsid protein VP1 of the hamster polyomavirus (HaPV-VP1) and harbor inserted foreign epitopes. The HaPV-VP1 regions spanning amino acids 81-88 (position 1), 222/223 (2), 244-246 (3), and 289-294 (4) were predicted to be surface exposed. An epitope of the pre-S1 region of the hepatitis B virus (designated S1; amino acid sequence DPAFR) was introduced into the predicted positions of VP1. All VP1/S1 fusion proteins were expressed in yeast and generated virus-like particles. Immunoassays using the S1-specific monoclonal antibody MA18/7 and immunization of C57Bl6 mice with different VP1/S1 constructs showed a pronounced reactivity and a strong S1-specific antibody response for particles carrying the insert in position 1, 2, 1+2, and 1+3. Our results suggest that HaPV-VP1 represents a highly flexible carrier moiety for the insertion of foreign sequences offering a broad range of potential uses, especially in vaccine development.
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MESH Headings
- Amino Acid Sequence
- Animals
- Antibodies, Monoclonal/immunology
- Antibodies, Viral/immunology
- Antigens, Viral/chemistry
- Antigens, Viral/genetics
- Antigens, Viral/immunology
- Antigens, Viral/metabolism
- Capsid/chemistry
- Capsid/genetics
- Capsid/immunology
- Capsid/metabolism
- Capsid Proteins
- Cricetinae
- Enzyme Multiplied Immunoassay Technique
- Epitopes/chemistry
- Epitopes/genetics
- Epitopes/immunology
- Epitopes/metabolism
- Genetic Vectors/genetics
- Genetic Vectors/immunology
- Hepatitis B Surface Antigens/chemistry
- Hepatitis B Surface Antigens/genetics
- Hepatitis B Surface Antigens/immunology
- Hepatitis B Surface Antigens/metabolism
- Mice
- Mice, Inbred C57BL
- Microscopy, Immunoelectron
- Models, Molecular
- Molecular Sequence Data
- Mutagenesis, Insertional/genetics
- Peptide Fragments/chemistry
- Peptide Fragments/genetics
- Peptide Fragments/immunology
- Peptide Fragments/metabolism
- Polyomavirus/chemistry
- Polyomavirus/genetics
- Polyomavirus/immunology
- Polyomavirus/metabolism
- Protein Conformation
- Protein Precursors/chemistry
- Protein Precursors/genetics
- Protein Precursors/immunology
- Protein Precursors/metabolism
- Recombinant Fusion Proteins/chemistry
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/immunology
- Recombinant Fusion Proteins/metabolism
- Saccharomyces cerevisiae/genetics
- Sequence Alignment
- Vaccines, Synthetic/chemistry
- Vaccines, Synthetic/genetics
- Vaccines, Synthetic/immunology
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Affiliation(s)
- A Gedvilaite
- Institute of Biotechnology, Vilnius, LT-2028, Lithuania
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33
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Küttner G, Kramer A, Schmidtke G, Giessmann E, Dong L, Roggenbuck D, Scholz C, Seifert M, Stigler RD, Schneider-Mergener J, Porstmann T, Höhne W. Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments. Mol Immunol 1999; 36:669-83. [PMID: 10509818 DOI: 10.1016/s0161-5890(99)00074-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.
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Affiliation(s)
- G Küttner
- Institut für Biochemie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Germany.
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34
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Sasnauskas K, Buzaite O, Vogel F, Jandrig B, Razanskas R, Staniulis J, Scherneck S, Krüger DH, Ulrich R. Yeast cells allow high-level expression and formation of polyomavirus-like particles. Biol Chem 1999; 380:381-6. [PMID: 10223341 DOI: 10.1515/bc.1999.050] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Polyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated in E. coli or insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeast Saccharomyces cerevisiae for expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels in Saccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.
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35
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Borisova G, Borschukova O, Skrastina D, Dislers A, Ose V, Pumpens P, Grens E. Behavior of a short preS1 epitope on the surface of hepatitis B core particles. Biol Chem 1999; 380:315-24. [PMID: 10223334 DOI: 10.1515/bc.1999.043] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79-81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody MA18/7 and specific immunogenicity of the preS1 epitope depended on the location and length of the deletion. While chimeras with deletions within the stretch 79-88 presented the preS1 epitope on their surface and demonstrated remarkable preS1 immunogenicity, the corresponding chimeras without any deletion or with a more prolonged deletion (79-93) were unable to provide such presentation and possessed a lower specific preS1 immunogenicity. Deletion of the stretch 79-81 was sufficient to avoid the intrinsic HBc immunogenicity of the core particles, although chimeras with deleted major immunodominant region retained their property to be recognized by human polyclonal or hyperimmune anti-HBc antibodies.
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Affiliation(s)
- G Borisova
- Biomedical Research and Study Centre, University of Latvia, Riga
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36
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Vasiljeva I, Kozlovska T, Cielens I, Strelnikova A, Kazaks A, Ose V, Pumpens P. Mosaic Qbeta coats as a new presentation model. FEBS Lett 1998; 431:7-11. [PMID: 9684855 DOI: 10.1016/s0014-5793(98)00716-9] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The new protein carrier was developed on the basis of recombinant RNA phage Qbeta capsid. C-terminal UGA extension of the short form of Qbeta coat, so-called A1 extension, served as a target for presentation of foreign peptides on the outer surface of mosaic Qbeta particles. In conditions of enhanced UGA suppression, the proportion of A1-extended to short coats in mosaic particles dropped from 48% to 14%, with an increase of the length of A1 extension. A model insertion, short preS1 epitope 31-DPAFR-35 of hepatitis B surface antigen, demonstrated superficial location on the mosaic Qbeta particles and ensured specific antigenicity and immunogenicity.
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Affiliation(s)
- I Vasiljeva
- Biomedical Research and Study Centre, University of Latvia, Riga
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37
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Ulrich R, Nassal M, Meisel H, Krüger DH. Core particles of hepatitis B virus as carrier for foreign epitopes. Adv Virus Res 1998; 50:141-82. [PMID: 9520999 DOI: 10.1016/s0065-3527(08)60808-8] [Citation(s) in RCA: 115] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
To be effective as vaccines, most monomeric proteins and peptides either require chemical coupling to high molecular weight carriers or application together with adjuvants. More recently, recombinant DNA techniques have been used to insert foreign epitopes into proteins with inherent multimerization capacity, such as particle-forming viral capsid or envelope proteins. The core protein of hepatitis B virus (HBcAg), because of its unique structural and immunological properties, has gained widespread interest as a potential antigen carrier. Foreign sequences of up to approximately 40 amino acid residues at the N terminus, 50 or 100 amino acids in the central immunodominant c/e 1 epitope region of HBcAg, and up to 100 or even more residues at the C terminus, did not interfere with particle formation. The humoral immunogenicity of inserted epitopes is determined by the immunogenicity of the peptide itself and its surface exposure, and is influenced by the route of application. The probably flexible and surface-exposed c/e1 region emerged as the most promising insertion site. When applied together with adjuvants approved for human and veterinary use, or even without adjuvants, such chimeric particles induced B and T cell immune responses against the inserted epitopes. In some cases neutralizing antibodies, cytotoxic T cells and protection against challenge with the intact pathogen were demonstrated. Major factors for the potentiated immune response against the foreign epitopes are the multimeric structure of chimeric HBcAg that results in a high epitope density per particle, and the provision of T cell help by the carrier moiety. Beyond its use as subunit vaccine, chimeric HBcAg produced in attenuated Salmonella strains may be applicable as live vaccine.
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Affiliation(s)
- R Ulrich
- Charité Medical School, Humboldt University, Berlin, Germany
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38
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D'Mello F, Partidos CD, Steward MW, Howard CR. Definition of the primary structure of hepatitis B virus (HBV) pre-S hepatocyte binding domain using random peptide libraries. Virology 1997; 237:319-26. [PMID: 9356343 DOI: 10.1006/viro.1997.8774] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The pre-S-specific monoclonal antibody MA 18/7 has been shown to inhibit the binding of HBV to HepG2 cells and liver membranes. This antibody can thus be used to identify the critical residues of the pre-S region involved in the hepatocyte-binding domain. Using overlapping 7-mer peptides representing the pre-S region of HBV, the epitope recognized by MA 18/7 was shown to contain sequences from both the pre-S1 and pre-S2 regions, thus indicating that the hepatocyte-binding domain is conformationally dependent. To further characterize the primary structure of the hepatocyte-binding domain on the pre-S protein, a phage-displayed 15-mer peptide library and a 8-mer solid phase peptide library were used to analyze the fine specificity of the monoclonal antibody MA 18/7. Several mimotopes were identified with the phage-displayed peptide library, the majority of which possess a central motif with at least three identical residues present within the native pre-S1 sequence. No significant consensus sequences were found when these mimotopes were compared to the pre-S2 sequence. Mimotopes identified using the solid-phase peptide library also contained a similar motif. All phage mimotopes and a single mimotope from the solid-phase peptide library competed with recombinant HBsAg particles containing the pre-S1 region for binding to MA 18/7. Mouse antisera raised against four mimotopes from the phage display library reacted with HBsAg particles containing pre-S sequences. The data show that the structure of the pre-S molecule around the conserved DPAF motif in the pre-S region may have a functional role in binding HBV to cellular receptors, and that the central motif identified in mimotopes of this region may offer a novel strategy target for the improvement of existing hepatitis B vaccines which, at present, are mostly devoid of pre-S specificities.
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Affiliation(s)
- F D'Mello
- Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College Street, London, NW1 OTU, United Kingdom
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39
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Budkowska A, Maillard P, Theret N, Groh F, Possehl C, Topilko A, Crainic R. Activation of the envelope proteins by a metalloproteinase enables attachment and entry of the hepatitis B virus into T-lymphocyte. Virology 1997; 237:10-22. [PMID: 9344903 DOI: 10.1006/viro.1997.8758] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Previously, we identified an HBV binding factor (HBV-BF), a 50-kDa serum glycoprotein which interacts with HBV envelope proteins and which is also located in the membrane of normal human hepatocyte (A. Budkowska et al. (1993) J. Virol. 67, 4316). Here we show that HBV-BF is a neutral metalloproteinase which shares substrate specificity and properties with a newly described family of membrane type matrix metalloproteinases. HBV-BF treatment of the HBV resulted in the cleavage of the N-terminal part of the middle HBV envelope protein at the pre-S2(136-141) amino acid sequence VRGLYF/L (containing a single arginine cleavage site). HBV-BF affected the reactivity of the large HBV protein with pre-S1-specific MAbs, probably inducing the conformational change of the pre-S1 domain. The HBV-BF-digested virus remained morphologically intact with unchanged S antigenic determinants. The structural modifications of the viral envelope proteins induced by HBV-BF enabled cell membrane attachment and viral entry into the T-lymphocyte. Both processes were blocked by the metalloproteinase inhibitor 1,10 phenanthroline. Thus, the host-dependent proteolytic activation of the envelope proteins seems to be essential for the HBV entry into the cell. HBV-BF under a membrane bound or a secreted form could be (one of) the molecule(s) responsible for the HBV proteolytic activation.
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Affiliation(s)
- A Budkowska
- Epidémiologie Moléculaire des Entérovirus, Chimie Organique, Station Centrale de Microscopie Electronique, Institut Pasteur, 25 Rue du Dr. Roux, Paris, 75724, USA.
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40
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Germaschewski V, Murray K. Screening a monoclonal antibody with a fusion-phage display library shows a discontinuity in a linear epitope within PreS1 of hepatitis B virus. J Med Virol 1995; 45:300-5. [PMID: 7539834 DOI: 10.1002/jmv.1890450311] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The epitope recognized by the monoclonal antibody MA18/7, specific for the PreS1-domain of the hepatitis B virus surface antigen, has been defined precisely by means of a library of fusion-phage carrying random hexapeptides on the tip of filamentous phage fd particles. Phage, isolated after only one round of affinity selection, displayed hexapeptides showing strong conservation of the PreS1 primary sequence in the region 19-23 with three noncontiguous residues, DP (20 and 21) and F (23) appearing in phage that bound the antibody. The importance of these core residues was supported by comparing the antibody binding of individual phage in solution, which provided relative dissociation constants for these interactions. Replacement of F (23) by Y was the only substitution observed in the three core residues, and resulted in somewhat weaker binding. Synthetic tetra- and hexapeptides containing these key residues inhibited the reaction between the phage and the antibody.
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Affiliation(s)
- V Germaschewski
- Institute of Cell and Molecular Biology, University of Edinburgh, Scotland, UK
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41
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Hofmann C, Sandig V, Kirillova I, Jennings G, Rudolph M, Schlag P, Strauss M. Hepatocyte-specific binding of L/S-HBV particles expressed in insect cells. BIOLOGICAL CHEMISTRY HOPPE-SEYLER 1995; 376:173-8. [PMID: 7612194 DOI: 10.1515/bchm3.1995.376.3.173] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The genome of hepatitis B virus (HBV) codes for three surface antigen proteins. Two of them are essential components of infectious viral particles. Whereas expression of the small (S) antigen led to formation of virus-like particles in different systems so far, secretion of neither the large antigen nor budding of virus-like particles containing both antigens could be observed. Using modified large antigen genes in dual expression vectors we were able to demonstrate secretion of virus-like particles in the baculovirus insect cell system. N-terminal fusion of an insect protein (melittin) derived signal sequence and destruction of the myristylation site resulted in secretion of the large antigen. Particles consisting of about 95% small and 5% large antigen bind specifically to hepatocytes. These pseudovirions could serve as a HBV vaccine and as a useful component of future hepatocyte-specific gene transfer vehicles.
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Affiliation(s)
- C Hofmann
- Max-Planck-Gesellschaft and Humboldt Universität, Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany
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42
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Bruss V, Lu X, Thomssen R, Gerlich WH. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. EMBO J 1994; 13:2273-9. [PMID: 8194518 PMCID: PMC395089 DOI: 10.1002/j.1460-2075.1994.tb06509.x] [Citation(s) in RCA: 141] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The preS domain at the N-terminus of the large envelope protein (LHBs) of the hepatitis B virus is involved in (i) envelopment of viral nucleocapsids and (ii) binding to the host cell. While the first function suggests a cytosolic location of the preS domain during virion assembly, the function as an attachment site requires its translocation across the lipid bilayer and final exposure on the virion surface. We compared the transmembrane topology of newly synthesized LHBs in the endoplasmic reticulum (ER) membrane with its topology in the envelope of secreted virions. Protease sensitivity and the absence of glycosylation suggest that the entire preS domain of newly synthesized LHBs remains at the cytosolic side of ER vesicles. However, virions secreted from transfected cell cultures or isolated from the blood of persistent virus carriers expose antibody binding sites and proteolytic cleavage sites of the preS domain at their surface in approximately half of the LHBs molecules. Thus, preS domains appear to be transported across the viral lipid barrier by a novel post-translational translocation mechanism to fulfil a dual function in virion assembly and attachment to the host cell.
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Affiliation(s)
- V Bruss
- Department of Medical Microbiology, University of Göttingen, Germany
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43
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Seifer M, Standring DN. Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid or the viral replication origin, DR1. J Virol 1993; 67:4513-20. [PMID: 7687299 PMCID: PMC237835 DOI: 10.1128/jvi.67.8.4513-4520.1993] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The double-stranded DNA genome of hepatitis B virus (HBV) is reverse transcribed from the viral pregenome RNA template by a virally encoded reverse transcriptase enzyme (RT) that possesses both priming and elongation activities. Prior efforts have failed to express an active form of HBV RT outside the nucleocapsid in animal cells or to release it from viral nucleocapsids, thus restricting the characterization of this important enzyme. Here, we have engineered epitope-tagged HBV RT proteins and expressed them in Xenopus oocytes via a synthetic RT mRNA which does not include the viral capsid protein or the known initiation site for viral DNA synthesis, DR1. We demonstrate the production of an immunoprecipitable 96-kDa HBV RT protein and show, using a simple in vitro RT assay, that oocyte lysates containing this protein possess an activity that (i) catalyzes an RNA-dependent deoxynucleotide triphosphate polymerization reaction by using an as-yet-unidentified RNA template and (ii) is sensitive to the RT inhibitors actinomycin D and phosphonoformate. Experiments with the chain terminator ddATP suggest that a significant amount of chain elongation occurs in our in vitro reaction. Electrophoretic analysis reveals a heterogeneous array of RT reaction products with sizes ranging from about 100 bases to far larger than that of the input RT mRNA. These products appear to contain covalently bound protein, consistent with the notion that the RT protein may have primed their synthesis. We conclude that HBV RT activity can be uncoupled from both the nucleocapsid and the replication origin, DR1. Our results raise the possibility that unless HBV employs novel mechanisms to regulate its constitutively active RT, cellular RNAs may be reverse transcribed during HBV infection, with potential implications for the development of HBV-related liver cancer. The use of the oocyte system should facilitate studies of HBV RT, including the development of HBV RT inhibitors for antiviral therapy.
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Affiliation(s)
- M Seifer
- Hormone Research Institute, University of California, San Francisco, California 94143-0534
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44
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Abstract
There are two identified liver-specific attachment sites in the preS2 domain and one in the preS1 domain. Which mechanism leads to attachment in vivo is not known. The subsequent penetration seems to require proteolysis which does not occur spontaneously in HepG2 cells, but presumably in vivo. The role of the small HBs protein for attachment remains enigmatic so far, but it must have a function because an escape mutant exists against a monoclonal antibody which binds to an epitope of the small protein. The occurrence of this escape mutant in vaccinated persons proves that the standard hepatitis B vaccine does induce neutralizing antibodies, but it also suggests very strongly that the neutralizing preS epitopes be included in future hepatitis B vaccines.
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Affiliation(s)
- W H Gerlich
- Institute of Medical Virology, University of Giessen, Germany
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