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Keyhole Limpet Hemocyanin-Conjugated Peptides from Hepatitis C Virus Glycoproteins Elicit Neutralizing Antibodies in BALB/c Mice. J Immunol Res 2021; 2021:3108157. [PMID: 33532506 PMCID: PMC7834783 DOI: 10.1155/2021/3108157] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 12/18/2020] [Accepted: 01/05/2021] [Indexed: 12/22/2022] Open
Abstract
Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.
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Identifying immunologically-vulnerable regions of the HCV E2 glycoprotein and broadly neutralizing antibodies that target them. Nat Commun 2019; 10:2073. [PMID: 31061402 PMCID: PMC6502829 DOI: 10.1038/s41467-019-09819-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Accepted: 04/02/2019] [Indexed: 02/06/2023] Open
Abstract
Isolation of broadly neutralizing human monoclonal antibodies (HmAbs) targeting the E2 glycoprotein of Hepatitis C virus (HCV) has sparked hope for effective vaccine development. Nonetheless, escape mutations have been reported. Ideally, a potent vaccine should elicit HmAbs that target regions of E2 that are most difficult to escape. Here, aimed at addressing this challenge, we develop a predictive in-silico evolutionary model for E2 that identifies one such region, a specific antigenic domain, making it an attractive target for a robust antibody response. Specific broadly neutralizing HmAbs that appear difficult to escape from are also identified. By providing a framework for identifying vulnerable regions of E2 and for assessing the potency of specific antibodies, our results can aid the rational design of an effective prophylactic HCV vaccine. A good vaccine should direct the immune response to virus regions that are most difficult to escape. Here, Quadeer et al. develop a predictive in-silico evolutionary model for HCV E2 which identifies one such antigenic region and identifies multiple broadly neutralizing human antibodies that appear difficult to escape from.
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Khera T, Behrendt P, Bankwitz D, Brown RJP, Todt D, Doepke M, Khan AG, Schulze K, Law J, Logan M, Hockman D, Wong JAJX, Dold L, Gonzalez-Motos V, Spengler U, Viejo-Borbolla A, Ströh LJ, Krey T, Tarr AW, Steinmann E, Manns MP, Klein F, Guzman CA, Marcotrigiano J, Houghton M, Pietschmann T. Functional and immunogenic characterization of diverse HCV glycoprotein E2 variants. J Hepatol 2019; 70:593-602. [PMID: 30439392 DOI: 10.1016/j.jhep.2018.11.003] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Revised: 10/04/2018] [Accepted: 11/02/2018] [Indexed: 02/08/2023]
Abstract
BACKGROUND & AIMS Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an hepatitis C virus vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity. METHODS We created hepatitis C virus variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies. RESULTS Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing. CONCLUSIONS Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies. LAY SUMMARY Conserved viral epitopes can be made considerably more accessible for binding of potently neutralizing antibodies by deletion of hypervariable region 1 and selected glycosylation sites. Recombinant E2 proteins carrying these mutations are unable to elicit cross-neutralizing antibodies suggesting that exposure of conserved epitopes is not sufficient to focus antibody responses on production of cross-neutralizing antibodies.
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Affiliation(s)
- Tanvi Khera
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Patrick Behrendt
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
| | - Dorothea Bankwitz
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Richard J P Brown
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Daniel Todt
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Mandy Doepke
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Abdul Ghafoor Khan
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-9806, USA
| | - Kai Schulze
- Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - John Law
- Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada
| | - Michael Logan
- Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada
| | - Darren Hockman
- Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada
| | - Jason Alexander Ji-Xhin Wong
- Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada
| | - Leona Dold
- Institute of Virology, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; German Centre for Infection Research (DZIF), partner site Cologne, Germany
| | | | - Ulrich Spengler
- Department of Internal Medicine 1, Rheinische Friedrich-Wilhelms-University Bonn, Bonn, Germany
| | | | - Luisa J Ströh
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany
| | - Thomas Krey
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
| | - Alexander W Tarr
- NIHR Nottingham Digestive Diseases Biomedical Research Centre and School of Life Sciences, The University of Nottingham, Nottingham, UK
| | - Eike Steinmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany; Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
| | - Michael P Manns
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany
| | - Florian Klein
- Institute of Virology, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; German Centre for Infection Research (DZIF), partner site Cologne, Germany
| | - Carlos A Guzman
- Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Joseph Marcotrigiano
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-9806, USA
| | - Michael Houghton
- Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada
| | - Thomas Pietschmann
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany.
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Chi PB, Chattopadhyay S, Lemey P, Sokurenko EV, Minin VN. Synonymous and nonsynonymous distances help untangle convergent evolution and recombination. Stat Appl Genet Mol Biol 2016; 14:375-89. [PMID: 26061623 DOI: 10.1515/sagmb-2014-0078] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
When estimating a phylogeny from a multiple sequence alignment, researchers often assume the absence of recombination. However, if recombination is present, then tree estimation and all downstream analyses will be impacted, because different segments of the sequence alignment support different phylogenies. Similarly, convergent selective pressures at the molecular level can also lead to phylogenetic tree incongruence across the sequence alignment. Current methods for detection of phylogenetic incongruence are not equipped to distinguish between these two different mechanisms and assume that the incongruence is a result of recombination or other horizontal transfer of genetic information. We propose a new recombination detection method that can make this distinction, based on synonymous codon substitution distances. Although some power is lost by discarding the information contained in the nonsynonymous substitutions, our new method has lower false positive probabilities than the comparable recombination detection method when the phylogenetic incongruence signal is due to convergent evolution. We apply our method to three empirical examples, where we analyze: (1) sequences from a transmission network of the human immunodeficiency virus, (2) tlpB gene sequences from a geographically diverse set of 38 Helicobacter pylori strains, and (3) hepatitis C virus sequences sampled longitudinally from one patient.
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Antibody Response to Hypervariable Region 1 Interferes with Broadly Neutralizing Antibodies to Hepatitis C Virus. J Virol 2016; 90:3112-22. [PMID: 26739044 DOI: 10.1128/jvi.02458-15] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2015] [Accepted: 12/30/2015] [Indexed: 12/21/2022] Open
Abstract
UNLABELLED Hypervariable region 1 (HVR1) (amino acids [aa] 384 to 410) on the E2 glycoprotein of hepatitis C virus contributes to persistent infection by evolving escape mutations that attenuate binding of inhibitory antibodies and by blocking access of broadly neutralizing antibodies to their epitopes. A third proposed mechanism of immune antagonism is that poorly neutralizing antibodies binding to HVR1 interfere with binding of other superior neutralizing antibodies. Epitope mapping of human monoclonal antibodies (HMAbs) that bind to an adjacent, conserved domain on E2 encompassing aa 412 to 423 revealed two subsets, designated HC33 HMAbs. While both subsets have contact residues within aa 412 to 423, alanine-scanning mutagenesis suggested that one subset, which includes HC33.8, has an additional contact residue within HVR1. To test for interference of anti-HVR1 antibodies with binding of antibodies to aa 412 to 423 and other E2 determinants recognized by broadly neutralizing HMAbs, two murine MAbs against HVR1 (H77.16) and aa 412 to 423 (H77.39) were studied. As expected, H77.39 inhibited the binding of all HC33 HMAbs. Unexpectedly, H77.16 also inhibited the binding of both subsets of HC33 HMAbs. This inhibition also was observed against other broadly neutralizing HMAbs to epitopes outside aa 412 to 423. Combination antibody neutralization studies by the median-effect analysis method with H77.16 and broadly reactive HMAbs revealed antagonism between these antibodies. Structural studies demonstrated conformational flexibility in this antigenic region, which supports the possibility of anti-HVR1 antibodies hindering the binding of broadly neutralizing MAbs. These findings support the hypothesis that anti-HVR1 antibodies can interfere with a protective humoral response against HCV infection. IMPORTANCE HVR1 contributes to persistent infection by evolving mutations that escape from neutralizing antibodies to HVR1 and by shielding broadly neutralizing antibodies from their epitopes. This study provides insight into a new immune antagonism mechanism by which the binding of antibodies to HVR1 blocks the binding and activity of broadly neutralizing antibodies to HCV. Immunization strategies that avoid the induction of HVR1 antibodies should increase the inhibitory activity of broadly neutralizing anti-HCV antibodies elicited by candidate vaccines.
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Kolesanova EF, Sobolev BN, Moysa AA, Egorova EA, Archakov AI. [Way to the peptide vaccine against hepatitis C]. BIOMEDIT︠S︡INSKAI︠A︡ KHIMII︠A︡ 2015; 61:254-64. [PMID: 25978391 DOI: 10.18097/pbmc20156102254] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
In order to surpass the problem of genetic variability of hepatitis C virus envelope proteins during vaccine development, we used the so-called "reverse vaccinology"approach--"from genome to vaccine". Database of HCV protein sequences was designed, viral genome analysis was performed, and several highly conserved sites were revealed in HCV envelope proteins in the framework of this approach. These sites demonstrated low antigenic activity in full-size proteins and HCV virions: antibodies against these sites were not found in all hepatitis C patients. However, two sites, which contained a wide set of potential T-helper epitope motifs, were revealed among these highly conserved ones. We constructed and prepared by solid-phase peptide synthesis several artificial peptide constructs composed of two linker-connected highly conserved HCV envelope E2 protein sites; one of these sites contained a set of T-helper epitope motifs. Experiments on laboratory animals demonstrated that the developed peptide constructs manifested immunogenicity compared with one of protein molecules and were able to raise antibodies, which specifically bound HCV envelope proteins. We succeeded in obtaining antibodies reactive with HCV from hepatitis C patient plasma upon the immunization with some constructs. An original preparation of a peptide vaccine against hepatitis C is under development on the basis of these peptide constructs.
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Affiliation(s)
| | - B N Sobolev
- Institute of Biomedical Chemistry, Moscow, Russia
| | - A A Moysa
- Institute of Biomedical Chemistry, Moscow, Russia
| | - E A Egorova
- Institute of Biomedical Chemistry, Moscow, Russia
| | - A I Archakov
- Institute of Biomedical Chemistry, Moscow, Russia
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Yusim K, Richardson R, Tao N, Dalwani A, Agrawal A, Szinger J, Funkhouser R, Korber B, Kuiken C. Los alamos hepatitis C immunology database. ACTA ACUST UNITED AC 2015; 4:217-25. [PMID: 16309340 DOI: 10.2165/00822942-200504040-00002] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The Los Alamos Hepatitis C Virus (HCV) Sequence Database (http://hcv.lanl.gov or http://hcv-db.org) was officially launched in September 2003. The sister HCV Immunology Database was made public in September 2004. The HCV Immunology Database is based on the Human Immunodeficiency Virus (HIV) Immunology Database. The HCV Immunology Database contains a curated inventory of immunological epitopes in HCV and their interaction with the immune system, with associated retrieval and analysis tools. This article describes in detail the types of data and services that the new database offers, the tools provided and the database framework. The data and some of the HCV database tools are available for download for non-commercial use.
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Affiliation(s)
- Karina Yusim
- Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, USA
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Urbaczek AC, Ximenes VF, Afonso A, Generoso WC, Nogueira CT, Tansini A, Cappelini LTD, Malagó Júnior W, da Silva FH, da Fonseca LM, da Costa PI. Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors. Mem Inst Oswaldo Cruz 2015; 110:534-42. [PMID: 26018451 PMCID: PMC4501418 DOI: 10.1590/0074-02760140441] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2014] [Accepted: 03/20/2015] [Indexed: 02/08/2023] Open
Abstract
Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.
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Affiliation(s)
- Ana Carolina Urbaczek
- Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil
| | - Valdecir Farias Ximenes
- Departamento de Química, Faculdade de Ciências, Universidade Estadual Paulista, Bauru, SP, Brasil
| | - Ana Afonso
- Unidade de Parasitologia Médica e Microbiologia,, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal
| | - Wesley Cardoso Generoso
- Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil
| | - Camila Tita Nogueira
- Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil
| | - Aline Tansini
- Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil
| | - Luciana Teresa Dias Cappelini
- Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brasil
| | - Wilson Malagó Júnior
- Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil
| | | | - Luiz Marcos da Fonseca
- Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil
| | - Paulo Inácio da Costa
- Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, Bauru, SP, Brasil
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Lange M, Fiedler M, Bankwitz D, Osburn W, Viazov S, Brovko O, Zekri AR, Khudyakov Y, Nassal M, Pumpens P, Pietschmann T, Timm J, Roggendorf M, Walker A. Hepatitis C virus hypervariable region 1 variants presented on hepatitis B virus capsid-like particles induce cross-neutralizing antibodies. PLoS One 2014; 9:e102235. [PMID: 25014219 PMCID: PMC4094522 DOI: 10.1371/journal.pone.0102235] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2014] [Accepted: 06/16/2014] [Indexed: 01/01/2023] Open
Abstract
Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses.
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Affiliation(s)
- Milena Lange
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Melanie Fiedler
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | | | - William Osburn
- Department of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America
| | - Sergei Viazov
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Olena Brovko
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | | | - Yury Khudyakov
- Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
| | - Michael Nassal
- Department of Internal Medicine II, University Hospital Freiburg, Freiburg, Germany
| | - Paul Pumpens
- Department of Recombinant biotechnology, Latvian Biomedical Research and Study Centre, Riga, Latvia
| | | | - Jörg Timm
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Michael Roggendorf
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Andreas Walker
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
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Drummer HE. Challenges to the development of vaccines to hepatitis C virus that elicit neutralizing antibodies. Front Microbiol 2014; 5:329. [PMID: 25071742 PMCID: PMC4080681 DOI: 10.3389/fmicb.2014.00329] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2014] [Accepted: 06/16/2014] [Indexed: 12/24/2022] Open
Abstract
Despite 20 years of research, a vaccine to prevent hepatitis C virus (HCV) infection has not been developed. A vaccine to prevent HCV will need to induce broadly reactive immunity able to prevent infection by the 7 genetically and antigenically distinct genotypes circulating world-wide. HCV encodes two surface exposed glycoproteins, E1 and E2 that function as a heterodimer to mediate viral entry. Neutralizing antibodies (NAbs) to both E1 and E2 have been described with the major NAb target being E2. The function of E2 is to attach virions to host cells via cell surface receptors that include, but is not limited to, the tetraspanin CD81 and scavenger receptor class B type 1. However, E2 has developed a number of immune evasion strategies to limit the effectiveness of the NAb response and possibly limit the ability of the immune system to generate potent NAbs in natural infection. Hypervariable regions that shield the underlying core domain, subdominant neutralization epitopes and glycan shielding combine to make E2 a difficult target for the immune system. This review summarizes recent information on the role of NAbs to prevent HCV infection, the targets of the NAb response and structural information on glycoprotein E2 in complex with neutralizing antibodies. This new information should provide a framework for the rational design of new vaccine candidates that elicit highly potent broadly reactive NAbs to prevent HCV infection.
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Affiliation(s)
- Heidi E Drummer
- Viral Fusion Laboratory, Centre for Biomedical Research, Burnet Institute Melbourne, VIC, Australia. ; Department of Microbiology, Monash University Clayton, VIC, Australia ; Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne Parkville, VIC, Australia
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11
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Studies on the role of neutralizing antibodies against envelope genes in resolving HCV pseudo-particles infection. Mol Biol Rep 2014; 41:3945-50. [PMID: 24566682 DOI: 10.1007/s11033-014-3262-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2013] [Accepted: 02/11/2014] [Indexed: 10/25/2022]
Abstract
Characterization of antibodies targeting the attachment and entry of the viral particles into host cells is important for studding antibody mediated neutralization. Antibodies against the envelope glycoproteins (EGP) have neutralizing capacity and can prevent HCV infections. System based on HCV pseudo typed-particles (HCVpp) stably expressing EGP can be used for screening of HCV anti envelope neutralizing antibodies in the serum of patients with acute and chronic HCV infections. The aim of the current study was to check HCVpp as a useful tool for the detection of anti-HCV envelope antibodies in the serum of HCV infected patients and to test the binding potential of these antiviral molecules to EGP of HCV 3a. Previously developed HCVpp harboring unmodified glycoproteins from local isolates in 293T cell line were used in this study. HCVpp were pre incubated with different concentrations of anti E1 antibody and different E2 antibodies to check antiviral activity. Further we used serum samples with low/medium (≤800,000 IU/mL), and high (>800,000 IU/mL) viral titer from chronic HCV male and female patients. Infection was done in Huh-7 cells for 1 h at 37 oC. Infectivity was checked through Luciferase assay. Considerable decrease in HCVpp infectivity with anti-envelope antibodies was observed in dose dependent manner. Maximum inhibition was seen when 5 µg/ml of monoclonal anti E1 antibody used. Further increase in concentration exhibited no decrease in infectivity which suggests that other factors are also involved in causing infection. Various well characterized E2-specific monoclonal antibodies (mAbs) have been screened for their capability to reduce infection in Huh-7 cells. Three of the four mAbs specific for the E2 had no effect on the infectivity of HCVpp. Confirmation sensitive antibody H53 showed maximum inhibition of infectivity. HCV ELISA positive samples from both male and female patients were used to neutralize the HCVpp. The neutralizing antibody response was observed in both males and females patients and do not assemble the rapidly evolving HCV envelope glycoproteins. That is why in spite the presence of neutralizing antibodies in the blood they fail to resolve infections. Moreover E1 antibodies insignificantly (>0.05) inhibit HCVpp infectivity while E2 antibodies significantly (<0.05) inhibit HCVpp infection. Based on the results of this study it is concluded that anti-envelope antibodies particularly the anti-E2 could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential for developing passive and active immunization for hepatitis C along with interferon therapy.
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Das S, Shetty RK, Kumar A, Shridharan RN, Tatineni R, Chi G, Mukherjee A, Das S, Subbarao SM, Karande AA. Monoclonal antibodies against Hepatitis C genotype 3a virus like particle inhibit virus entry in cell culture system. PLoS One 2013; 8:e53619. [PMID: 23341957 PMCID: PMC3546081 DOI: 10.1371/journal.pone.0053619] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2012] [Accepted: 11/30/2012] [Indexed: 02/05/2023] Open
Abstract
The envelope protein (E1-E2) of Hepatitis C virus (HCV) is a major component of the viral structure. The glycosylated envelope protein is considered to be important for initiation of infection by binding to cellular receptor(s) and also known as one of the major antigenic targets to host immune response. The present study was aimed at identifying mouse monoclonal antibodies which inhibit binding of virus like particles of HCV to target cells. The first step in this direction was to generate recombinant HCV-like particles (HCV-LPs) specific for genotypes 3a of HCV (prevalent in India) using the genes encoding core, E1 and E2 envelop proteins in a baculovirus expression system. The purified HCV-LPs were characterized by ELISA and electron microscopy and were used to generate monoclonal antibodies (mAbs) in mice. Two monoclonal antibodies (E8G9 and H1H10) specific for the E2 region of envelope protein of HCV genotype 3a, were found to reduce the virus binding to Huh7 cells. However, the mAbs generated against HCV genotype 1b (D2H3, G2C7, E1B11) were not so effective. More importantly, mAb E8G9 showed significant inhibition of the virus entry in HCV JFH1 cell culture system. Finally, the epitopic regions on E2 protein which bind to the mAbs have also been identified. Results suggest a new therapeutic strategy and provide the proof of concept that mAb against HCV-LP could be effective in preventing virus entry into liver cells to block HCV replication.
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Affiliation(s)
- Soma Das
- Department of Biochemistry, Indian Institute of Science, Bangalore, India
| | - Rohini K. Shetty
- Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore, India
| | - Anuj Kumar
- Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore, India
| | | | - Ranjitha Tatineni
- Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore, India
| | - Giriprakash Chi
- Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore, India
| | - Anirban Mukherjee
- Department of Biochemistry, Indian Institute of Science, Bangalore, India
| | - Saumitra Das
- Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore, India
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Wang Y, Keck ZY, Foung SKH. Neutralizing antibody response to hepatitis C virus. Viruses 2011; 3:2127-45. [PMID: 22163337 PMCID: PMC3230844 DOI: 10.3390/v3112127] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2011] [Revised: 10/18/2011] [Accepted: 10/22/2011] [Indexed: 12/14/2022] Open
Abstract
A critical first step in a "rational vaccine design" approach for hepatitis C virus (HCV) is to identify the most relevant mechanisms of immune protection. Emerging evidence provides support for a protective role of virus neutralizing antibodies, and the ability of the B cell response to modify the course of acute HCV infection. This has been made possible by the development of in vitro cell culture models, based on HCV retroviral pseudotype particles expressing E1E2 and infectious cell culture-derived HCV virions, and small animal models that are robust tools in studies of antibody-mediated virus neutralization. This review is focused on the immunogenic determinants on the E2 glycoprotein mediating virus neutralization and the pathways in which the virus is able to escape from immune containment. Encouraging findings from recent studies provide support for the existence of broadly neutralization antibodies that are not associated with virus escape. The identification of conserved epitopes mediating virus neutralization that are not associated with virus escape will facilitate the design of a vaccine immunogen capable of eliciting broadly neutralizing antibodies against this highly diverse virus.
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Affiliation(s)
- Yong Wang
- Department of Pathology, School of Medicine, Stanford University, Stanford, CA 94305, USA.
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Vieyres G, Dubuisson J, Patel AH. Characterization of antibody-mediated neutralization directed against the hypervariable region 1 of hepatitis C virus E2 glycoprotein. J Gen Virol 2011; 92:494-506. [PMID: 21084495 PMCID: PMC3081231 DOI: 10.1099/vir.0.028092-0] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2010] [Accepted: 11/17/2010] [Indexed: 12/15/2022] Open
Abstract
The hypervariable region 1 (HVR1) comprising the first 27 aa of E2 glycoprotein is a target for neutralizing antibodies against hepatitis C virus (HCV), but the mechanisms of this neutralization in the cell-culture-infectious genotype 2a strain JFH1 HCV virus (HCVcc) system are unknown. Two rabbit polyclonal sera, R1020 and R140, recognizing the HVR1 of the genotype 1a isolates H77c and Glasgow (Gla), respectively, and a Gla HVR1-specific mouse mAb AP213 have been described previously. However, attempts to generate of antibodies to the JFH1 HVR1 were unsuccessful. Therefore, this study produced chimeric JFH1 HCVcc viruses harbouring the H77c or Gla HVR1 to assess the reactivity of antibodies to this region and their effects on virus infectivity. The inter-genotypic HVR1 swap did not significantly affect virus infectivity. The genotype 1a HVR1-specific antibodies neutralized chimeric viruses in an isolate-dependent manner, underlining the role of HVR1 in HCV infection. The neutralizing antibodies reacted mainly with the C-terminal portion of HVR1, and detailed mapping identified A17, F20 and Q21 in the Gla HVR1 sequence and T21 (and possibly L20) in the corresponding H77c sequence as key epitope residues for AP213 and R140, and R1020, respectively. Importantly, none of the antibodies inhibited in vitro binding of viral envelope glycoproteins to the best-characterized HCV receptor, CD81, or to the glycosaminoglycan attachment factors. However, the HVR1 antibodies were capable of post-attachment neutralization. Overall, this study emphasizes the role of HVR1 in HCVcc entry and provides new tools to study this region further in the context of complete virions.
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Affiliation(s)
- Gabrielle Vieyres
- MRC – University of Glasgow Centre for Virus Research, University of Glasgow, Church Street, Glasgow G11 5JR, UK
- Institut Pasteur de Lille, Center for Infection & Immunity of Lille (CIIL), F-59019 Lille, France
- Inserm U1019, F-59019 Lille, France
- CNRS UMR8204, F-59021 Lille, France
- Université Lille Nord de France, F-59000 Lille, France
| | - Jean Dubuisson
- Institut Pasteur de Lille, Center for Infection & Immunity of Lille (CIIL), F-59019 Lille, France
- Inserm U1019, F-59019 Lille, France
- CNRS UMR8204, F-59021 Lille, France
- Université Lille Nord de France, F-59000 Lille, France
| | - Arvind H. Patel
- MRC – University of Glasgow Centre for Virus Research, University of Glasgow, Church Street, Glasgow G11 5JR, UK
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Zeisel MB, Fofana I, Fafi-Kremer S, Baumert TF. Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies. J Hepatol 2011; 54:566-76. [PMID: 21146244 DOI: 10.1016/j.jhep.2010.10.014] [Citation(s) in RCA: 138] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/09/2010] [Revised: 10/12/2010] [Accepted: 10/14/2010] [Indexed: 12/18/2022]
Abstract
Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Preventive modalities are absent and the current antiviral treatment is limited by resistance, toxicity, and high costs. Viral entry is required for initiation, spread, and maintenance of infection, and thus is a promising target for antiviral therapy. HCV entry is a highly orchestrated process involving viral and host cell factors. These include the viral envelope glycoproteins E1 and E2, CD81, scavenger receptor BI, and tight junction proteins claudin-1 and occludin. Recent studies in preclinical models and HCV-infected patients have demonstrated that the virus has developed multiple strategies to escape host immune responses during viral entry. These include evasion from neutralizing antibodies and viral spread by cell-cell transmission. These challenges have to be taken into account for the design of efficient antiviral strategies. Thus, a detailed understanding of the mechanisms of viral entry and escape is a prerequisite to define viral and cellular targets and develop novel preventive and therapeutic antivirals. This review summarizes the current knowledge about the molecular mechanisms of HCV entry into hepatocytes, highlights novel targets and reviews the current preclinical and clinical development of compounds targeting entry. Proof-of-concept studies suggest that HCV entry inhibitors are a novel and promising class of antivirals widening the preventive and therapeutic arsenal against HCV infection.
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16
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AbdulQawi K, Youssef A, Metwally MA, Ragih I, AbdulHamid M, Shaheen A. Prospective study of prevalence and risk factors for hepatitis C in pregnant Egyptian women and its transmission to their infants. Croat Med J 2010; 51:219-28. [PMID: 20564765 DOI: 10.3325/cmj.2010.51.219] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
AIM To estimate the hepatitis C virus (HCV) vertical transmission rate, the effect of potential risk factors, and the pattern of HCV antibody response and viremia in HCV-infected infants in Benha, Egypt. METHODS A total of 1224 pregnant women who were treated at Benha University Hospital, Egypt, were included in the study. They completed a questionnaire about risk factors for HCV acquisition and suspected risk factors for mother-to-infant transmission and were tested for HCV antibody using a third-generation ELISA test. Women positive for HCV antibody were tested for HCV RNA by polymerase chain reaction. Peripheral blood of infants of positive HCV-RNA women was tested for HCV antibody and HCV-RNA at 1 and after 6 months of age. RESULTS Out of 1224 pregnant women, 105 (8.6%; 95% confidence interval, 7.05-10.17) were positive for HCV antibody. Only 83 (6.8%; 5.39-7.21) were positive for HCV-RNA. HCV infection was associated with older age (1.16; 1.1-1.2, P=0.001), blood transfusion (2.69; 1.2-6.0, P=0.016), and HCV infection of the husband (5.47; 1.4-21, P=0.014) or other household members (2.29; 1.2-4.6, P=0.019). Out of 53 infants tested at first month, 43 (81%; 71-92%) were positive for HCV antibody, but only 7 (13%; 4.1-22%) were positive for HCV-RNA. After 6 months, only 2 (3.8%; 0-8.95%) remained positive for HCV RNA. CONCLUSIONS The prevalence of HCV in pregnant women in Egypt is lower than previously reported and the potential risk factors associated with HCV infection suggest intra-familial transmission. The frequency of vertical transmission of HCV in Egypt is not substantially different from other countries and does not play a role in the high prevalence of HCV in Egypt.
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Affiliation(s)
- Khaled AbdulQawi
- Department of Pediatrics, Benha University, 2 Saad Zaghloul st, Benha, PO Box:113, Qualyabia governorate, Egypt.
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Xiu BS, Feng XY, He J, Wang GH, Zhang XY, Zhang HQ, Song XG, Chen K, Ling SG, Zhu CX, Wei L, Rao HY. Evaluation of cross-reactive antibody response to HVR1 in chronic hepatitis C. World J Gastroenterol 2010; 16:4460-6. [PMID: 20845515 PMCID: PMC2941071 DOI: 10.3748/wjg.v16.i35.4460] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: To evaluate the presence and cross-reactive antibodies against hypervariable region 1 (HVR1) in hepatitis C virus (HCV) infected patients and its relationship with the progression of the disease.
METHODS: Sixteen representative HVR1 proteins selected from a unique set of 1600 natural sequences were used to semiquantitate the cross-reactivity of HVR1 antibodies in the sera of HCV patients. Fifty-five chronic HCV patients including 23 with asymptomatic mild hepatitis, 18 with chronic hepatitis and 16 with liver cirrhosis patients were studied.
RESULTS: The degree of the cross-reactivity of anti-HVR1 antibodies in 23 patients with mild asymptomatic hepatitis was 3.09 ± 2.68, which was significantly lower than in those with chronic hepatitis (5.44 ± 3.93, P < 0.05) and liver cirrhosis (7.44 ± 3.90, P < 0.01). No correlation was observed between the broadness of the cross-reactivity anti-HVR1 antibodies and patient’s age, infection time, serum alanine aminotransferase activity, or serum HCV-RNA concentration. It was the breath of cross-reactivity rather than the presence of anti-HVR1 antibody in HCV sera that was associated with the progression of liver disease.
CONCLUSION: The broadly cross-reactive HVR1 antibodies generated in natural HCV patients can not neutralize the virus, which results in persistent infection in patients with chronic hepatitis.
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18
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Hughes MG, Rosen HR. Human liver transplantation as a model to study hepatitis C virus pathogenesis. Liver Transpl 2009; 15:1395-411. [PMID: 19877210 PMCID: PMC2954677 DOI: 10.1002/lt.21866] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Hepatitis C is a leading etiology of liver cancer and a leading reason for liver transplantation. Although new therapies have improved the rates of sustained response, a large proportion of patients (approximately 50%) fail to respond to antiviral treatment, thus remaining at risk for disease progression. Although chimpanzees have been used to study hepatitis C virus biology and treatments, their cost is quite high, and their use is strictly regulated; indeed, the National Institutes of Health no longer supports the breeding of chimpanzees for study. The development of hepatitis C virus therapies has been hindered by the relative paucity of small animal models for studying hepatitis C virus pathogenesis. This review presents the strengths of human liver transplantation and highlights the advances derived from this model, including insights into viral kinetics and quasispecies, viral receptor binding and entry, and innate and adaptive immunity. Moreover, consideration is given to current and emerging antiviral therapeutic approaches based on translational research results.
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Affiliation(s)
- Michael G. Hughes
- Department of Surgery, Medical University of South Carolina, Charleston, SC
| | - Hugo R. Rosen
- Department of Medicine, Divisions of Gastroenterology & Hepatology and Liver Transplantation; University of Colorado Health Sciences Center & National Jewish Hospital, and Denver VA
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19
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Shebl FM, El-Kamary SS, Saleh DA, Abdel-Hamid M, Mikhail N, Allam A, El-Arabi H, Elhenawy I, El-Kafrawy S, El-Daly M, Selim S, El-Wahab AA, Mostafa M, Sharaf S, Hashem M, Heyward S, Stine OC, Magder LS, Stoszek S, Strickland GT. Prospective cohort study of mother-to-infant infection and clearance of hepatitis C in rural Egyptian villages. J Med Virol 2009; 81:1024-31. [PMID: 19382251 PMCID: PMC3235472 DOI: 10.1002/jmv.21480] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Although persistent transmission of hepatitis C virus (HCV) from infected mothers to their infants is reported in 4-8%, transient HCV perinatal infection also occurs. This prospective cohort study determined perinatal HCV infection- and early and late clearance-rates in 1,863 mother-infant pairs in rural Egyptian villages. This study found 15.7% and 10.9% of pregnant women had HCV antibodies (anti-HCV) and HCV-RNA, respectively. Among 329 infants born of these mothers, 33 (10.0%) tested positive for both anti-HCV and HCV-RNA 2 months following birth-29 (12.5%) having HCV-RNA positive mothers and 4 (with transient infections) having mothers with only anti-HCV. Fifteen remained HCV-RNA positive at one and/or 2 years (persistent infections), while 18 cleared both virus and antibody by 1 year (transient infections). Among the 15 persistent cases, 7 cleared their infections by 2 or 3 years. At 2- to 6- and at 10- to 12-month maternally acquired anti-HCV was observed in 80% and 5% of infants, respectively. Four perinatally infected and one transiently infected infant were confirmed to be infected by their mothers by the sequence similarity of their viruses. Viremia was 155-fold greater in mothers of infants with persistent than mothers of infants with transient infections. Maternal-infant transmission of HCV is more frequent than generally reported. However, both early and late clearance of infection frequently occurs and only 15 (4.6%) and 8 (2.4%) infants born of HCV-RNA positive mothers had detectable HCV-RNA at one and 2-3 years of age. Investigating how infants clear infection may provide important information about protective immunity to HCV.
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Affiliation(s)
- Fatma M. Shebl
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
| | - Samer S. El-Kamary
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
| | - Doa’a A. Saleh
- Department of Community Medicine, Cairo University Faculty of Medicine, Cairo, Egypt
| | - Mohamed Abdel-Hamid
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
- National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt
| | - Nabiel Mikhail
- National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt
| | - Alif Allam
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
| | - Hanaa El-Arabi
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
| | - Ibrahim Elhenawy
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
| | - Sherif El-Kafrawy
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
- National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt
| | - Mai El-Daly
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
- National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt
| | - Sahar Selim
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
| | | | - Mohamed Mostafa
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
| | - Soraya Sharaf
- National Liver Institute, Menoufia University, Shibin El Kom, Egypt
| | - Mohamed Hashem
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
| | - Scott Heyward
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
| | - O. Colin Stine
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
| | - Laurence S. Magder
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
| | - Sonia Stoszek
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
| | - G. Thomas Strickland
- Department of Epidemiology and Preventive Medicine or Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland
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20
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Gottwein JM, Bukh J. Cutting the gordian knot-development and biological relevance of hepatitis C virus cell culture systems. Adv Virus Res 2008; 71:51-133. [PMID: 18585527 DOI: 10.1016/s0065-3527(08)00002-x] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Worldwide approximately 180 million people are chronically infected with hepatitis C virus (HCV). HCV isolates exhibit extensive genetic heterogeneity and have been grouped in six genotypes and various subtypes. Additionally, several naturally occurring intergenotypic recombinants have been described. Research on the viral life cycle, efficient therapeutics, and a vaccine has been hampered by the absence of suitable cell culture systems. The first system permitting studies of the full viral life cycle was intrahepatic transfection of RNA transcripts of HCV consensus complementary DNA (cDNA) clones into chimpanzees. However, such full-length clones were not infectious in vitro. The development of the replicon system and HCV pseudo-particles allowed in vitro studies of certain aspects of the viral life cycle, RNA replication, and viral entry, respectively. Identification of the genotype 2 isolate JFH1, which for unknown reasons showed an exceptional replication capability and resulted in formation of infectious viral particles in the human hepatoma cell line Huh7, led in 2005 to the development of the first full viral life cycle in vitro systems. JFH1-based systems now enable in vitro studies of the function of viral proteins, their interaction with each other and host proteins, new antivirals, and neutralizing antibodies in the context of the full viral life cycle. However, several challenges remain, including development of cell culture systems for all major HCV genotypes and identification of other susceptible cell lines.
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Affiliation(s)
- Judith M Gottwein
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark
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21
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Li H, McMahon BJ, McArdle S, Bruden D, Sullivan DG, Shelton D, Deubner H, Gretch DR. Hepatitis C virus envelope glycoprotein co-evolutionary dynamics during chronic hepatitis C. Virology 2008; 375:580-91. [PMID: 18343477 DOI: 10.1016/j.virol.2008.02.012] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2007] [Revised: 01/16/2008] [Accepted: 02/12/2008] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) envelope glycoprotein co-evolution was studied in 14 genotype 1-infected and treatment-naive subjects, including 7 with mild and 7 with severe liver disease. Cassettes encoding the envelope 1 gene (E1) and hypervariable region (HVR1) of the envelope 2 gene were isolated at 38 different time points over 81 follow-up years. There were no significant differences in age, gender, alcohol use, or viral load between the mild and severe disease groups. Virus from subjects with severe disease had significantly slower evolution in HVR1, and significant divergent evolution of E1 quasispecies, characterized by a preponderance of synonymous mutations, compared to virus from subjects with mild disease. Phylogenetic comparisons indicated higher similarity between amino acid sequences of the E1 and HVR1 regions with mild disease versus severe disease (r=0.44 versus r=0.17, respectively; P=0.01). In summary, HCV envelope quasispecies co-evolution differs during mild versus severe disease.
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Affiliation(s)
- Hui Li
- Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA, USA
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22
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Keck ZY, Machida K, Lai MMC, Ball JK, Patel AH, Foung SKH. Therapeutic control of hepatitis C virus: the role of neutralizing monoclonal antibodies. Curr Top Microbiol Immunol 2008; 317:1-38. [PMID: 17990788 DOI: 10.1007/978-3-540-72146-8_1] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Liver failure associated with hepatitis C virus (HCV) accounts for a substantial portion of liver transplantation. Although current therapy helps some patients with chronic HCV infection, adverse side effects and a high relapse rate are major problems. These problems are compounded in liver transplant recipients as reinfection occurs shortly after transplantation. One approach to control reinfection is the combined use of specific antivirals together with HCV-specific antibodies. Indeed, a number of human and mouse monoclonal antibodies to conformational and linear epitopes on HCV envelope proteins are potential candidates, since they have high virus neutralization potency and are directed to epitopes conserved across diverse HCV genotypes. However, a greater understanding of the factors contributing to virus escape and the role of lipoproteins in masking virion surface domains involved in virus entry will be required to help define those protective determinants most likely to give broad protection. An approach to immune escape is potentially caused by viral infection of immune cells leading to the induction hypermutation of the immunoglobulin gene in B cells. These effects may contribute to HCV persistence and B cell lymphoproliferative diseases.
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Affiliation(s)
- Z Y Keck
- Department of Pathology, Stanford Medical School Blood Center, Palo Alto, CA 94304, USA
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23
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Kusov Y, Gauss-Müller V, Morace G. Immunogenic epitopes on the surface of the hepatitis A virus capsid: Impact of secondary structure and/or isoelectric point on chimeric virus assembly. Virus Res 2007; 130:296-302. [PMID: 17640757 DOI: 10.1016/j.virusres.2007.06.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2007] [Revised: 06/07/2007] [Accepted: 06/08/2007] [Indexed: 11/17/2022]
Abstract
Hepatitis A virus (HAV) protein 2A has the capacity to harbor and expose a short foreign epitope. The chimeric virus, HAV-gp41, bearing seven amino acids of the 2F5 epitope of the HIV glycoprotein gp41, was shown to replicate in cell culture and laboratory animals and to induce a humoral immune response. As an extension of this work, we now investigated the possibility to insert longer epitopes, their impact on genetic stability, and the production of chimeric HAV. Twenty-seven amino acid residues of either HIV gp41, comprising the 2F5 epitope, or of a mimotope (F78) of the hypervariable region 1 of the hepatitis C virus (HCV) envelope protein E2 were inserted near the C-terminus of HAV 2A and viral capsid formation and replication were studied. The genome of the chimeric virus (HAV-F78) had reduced replication ability, yet the sedimentation profile of the chimeric particles was unchanged and the HCV sequence was maintained over serial viral passages. In contrast, no capsids were formed when an extended HIV epitope of 27 residues was inserted, precluding the rescue of infectious chimeric virus. Based on structural analyses, the data suggest that the isoelectric point (pI) and/or the secondary structure of the chimeric proteins are essential determinants that affect HAV particle formation for which protein 2A serves as an assembly signal.
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Affiliation(s)
- Yuri Kusov
- Institute of Medical Molecular Biology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany
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Falkowska E, Kajumo F, Garcia E, Reinus J, Dragic T. Hepatitis C virus envelope glycoprotein E2 glycans modulate entry, CD81 binding, and neutralization. J Virol 2007; 81:8072-9. [PMID: 17507469 PMCID: PMC1951298 DOI: 10.1128/jvi.00459-07] [Citation(s) in RCA: 127] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Hepatitis C virus (HCV) is a major human pathogen that causes serious liver disease, including cirrhosis and hepatocellular carcinoma. The primary target cells of HCV are hepatocytes, and entry is restricted by interactions of the envelope glycoproteins, E1 and E2, with cellular receptors. E1 and E2 form noncovalently linked heterodimers and are heavily glycosylated. Glycans contribute to protein folding and transport as well as protein function. In addition, glycans associated with viral envelopes mask important functional domains from the immune system and attenuate viral immunogenicity. Here, we explored the role of N- and O-linked glycans on E2, which is the receptor binding subunit of the HCV envelope. We identified a number of glycans that are critical for viral entry. Importantly, we showed that the removal of several glycans significantly increased the inhibition of entry by sera from HCV-positive individuals. Only some of the glycans that affected entry and neutralization were also important for CD81 binding. Our results show that HCV envelope-associated glycans play a crucial role in masking functionally important regions of E2 and suggest a new strategy for eliciting highly neutralizing antibodies against this virus.
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Affiliation(s)
- Emilia Falkowska
- Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA
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25
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Haqshenas G, Dong X, Netter H, Torresi J, Gowans EJ. A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo. J Gen Virol 2007; 88:895-902. [PMID: 17325362 DOI: 10.1099/vir.0.82467-0] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.
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Affiliation(s)
- G Haqshenas
- The Macfarlane Burnet Institute, GPO Box 2284, Melbourne, VIC 3001, Australia
| | - X Dong
- Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
- The Macfarlane Burnet Institute, GPO Box 2284, Melbourne, VIC 3001, Australia
| | - H Netter
- Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
| | - J Torresi
- Department of Medicine (RMH/WH), University of Melbourne, Centre for Clinical Research Excellence, Royal Melbourne Hospital, Parkville, VIC, Australia
| | - E J Gowans
- Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
- The Macfarlane Burnet Institute, GPO Box 2284, Melbourne, VIC 3001, Australia
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Abstract
Hepatitis C virus (HCV) exists in different forms in the circulation of infected people: lipoprotein bound and lipoprotein free, enveloped and nonenveloped. Viral particles with the highest infectivity are associated with lipoproteins, whereas lipoprotein-free virions are poorly infectious. The detection of HCV's envelope proteins E1 and E2 in lipoprotein-associated virions has been challenging. Because lipoproteins are readily endocytosed, some forms of HCV might utilize their association with lipoproteins rather than E1 and E2 for cell attachment and internalization. However, vaccination of chimpanzees with recombinant envelope proteins protected the animals from hepatitis C infection, suggesting an important role for E1 and E2 in cell entry. It seems possible that different forms of HCV use different receptors to attach to and enter cells. The putative receptors and the assays used for their validation are discussed in this review.
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Mancini N, Carletti S, Perotti M, Romanò L, Craxì RDS, Craxì A, Zanetti AR, Clementi M, Burioni R. Modulation of epitope-specific anti-hepatitis C virus E2 (anti-HCV/E2) antibodies by anti-viral treatment. J Med Virol 2006; 78:1304-11. [PMID: 16927283 DOI: 10.1002/jmv.20704] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
The dynamic features of three specific anti-hepatitis C virus (HCV) antibody subpopulations directed against different conformational epitopes of the viral E2 protein (HCV/E2) have been evaluated in patients with primary and persistent HCV infection; the three subpopulations are present in patients infected with different HCV genotypes and have shown a different activity using a pseudovirus neutralization assay (antibodies e301 and e137 exhibiting high neutralizing activity, while antibody e509 enhancement of HCV infectivity). In sequential samples from five patients with primary HCV infection and different virological outcome, all samples tested negative with the single exception of the e509 antibody in a patient not clearing the virus. In sequential samples from 28 patients with persistent infection under treatment with pegylated interferon-alpha plus ribavirin (14 sustained virological responders and 14 non-responders), the therapy did not selectively influence titers of the two neutralizing antibody subpopulations; otherwise, a net increase of the e509 antibody subpopulation related to enhancement of HCV infectivity was observed in non-responders, but not in sustained virological responders (P = 0.0156). This increase was not related to the trend of total anti-HCV/E2 response. The data indicate that a specific antibody response against these epitopes is elicited only late during the infection, thus not influencing virus clearance during primary infection, and that a selective increase of the antibody subpopulation enhancing virus infectivity is observed only in the cohort of patients not responding to antiviral therapy.
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Affiliation(s)
- Nicasio Mancini
- Laboratorio di Microbiologia, Università Vita-Salute San Raffaele, Istituto Scientifico San Raffaele, Milano, Italy
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28
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El-Awady MK, Tabll AA, Atef K, Yousef SS, Omran MH, El-Abd Y, Bader-Eldin NG, Salem AM, Zohny SF, El-Garf WT. Antibody to E1 peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro. World J Gastroenterol 2006. [PMID: 16688798 DOI: 10.3748/wjg.v12.i16.2530.] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 09/29/2022] Open
Abstract
AIM To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus (HCV). Specific polyclonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS Hyper-immune HCV E1 antibodies were incubated over night at 4 degree Celsius with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/ mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RT-PCR and flow cytometry. RESULTS Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera (72%) showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.
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Affiliation(s)
- Mostafa K El-Awady
- Department of Biomedical Technology, National Research Center, Dokki, Cairo, Egypt.
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29
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El-Awady MK, Tabll AA, Atef K, Yousef SS, Omran MH, El-Abd Y, Bader-Eldin NG, Salem AM, Zohny SF, El-Garf WT. Antibody to E1 peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro. World J Gastroenterol 2006; 12:2530-2535. [PMID: 16688798 PMCID: PMC4087985 DOI: 10.3748/wjg.v12.i16.2530] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/02/2005] [Revised: 12/26/2005] [Accepted: 01/14/2006] [Indexed: 02/06/2023] Open
Abstract
AIM To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus (HCV). Specific polyclonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS Hyper-immune HCV E1 antibodies were incubated over night at 4 degree Celsius with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/ mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RT-PCR and flow cytometry. RESULTS Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera (72%) showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.
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Affiliation(s)
- Mostafa K El-Awady
- Department of Biomedical Technology, National Research Center, Dokki, Cairo, Egypt.
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30
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Valli MB, Serafino A, Crema A, Bertolini L, Manzin A, Lanzilli G, Bosman C, Iacovacci S, Giunta S, Ponzetto A, Clementi M, Carloni G. Transmission in vitro of hepatitis C virus from persistently infected human B-cells to hepatoma cells by cell-to-cell contact. J Med Virol 2006; 78:192-201. [PMID: 16372297 DOI: 10.1002/jmv.20527] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Virus cell-to-cell spread has been reported for many different viruses and may contribute to pathogenesis of viral disease. The role played by cell-to-cell contact in hepatitis C virus (HCV) transmission was studied in vitro by cell co-cultivation experiments. A human lymphoblastoid B-cell line, infected persistently with HCV in vitro (TO.FE(HCV)), was used as HCV donor [Serafino et al., 2003]; recipient cells were the human hepatoma HepG2 cell line. Both cell types were co-cultured for 48 hr to allow the cell-to-cell contacts. The hepatoma HepG2 cells are not permissive to free-virus infection, but they were infected successfully using TO.FE(HCV) cells as source of virus. The kinetics of viral RNA synthesis and the percentage of infected cells were compared in cell-mediated-and cell-free-viral infection. After co-cultivation, a consistent proportion of hepatoma cells replicated HCV and stably expressed viral antigens. Virus produced was infectious as demonstrated by the ability to reinfect fresh B-cells. This cell model shows that permissiveness to HCV infection can be achieved in vitro in non-permissive hepatoma cells by direct cell-to-cell contacts with infected human B-cells. This mechanism of virus spread may also play a pathogenic role in vivo.
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Affiliation(s)
- Maria Beatrice Valli
- Institute of Neurobiology and Molecular Medicine, National Research Council (CNR), Area di Ricerca CNR TorVergata, Roma, Italy
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31
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McHutchison JG, Bartenschlager R, Patel K, Pawlotsky JM. The face of future hepatitis C antiviral drug development: recent biological and virologic advances and their translation to drug development and clinical practice. J Hepatol 2006; 44:411-21. [PMID: 16364491 DOI: 10.1016/j.jhep.2005.12.001] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Affiliation(s)
- John G McHutchison
- Division of Gastroenterology, Duke Clinical Research Institute, Duke University Medical Centre, 2400 Pratt Street, Room 0311, Terrace Level, Durham, NC 27707, USA.
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32
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Hughes MG, Chong TW, Smith RL, Evans HL, Iezzoni JC, Sawyer RG, Rudy CK, Pruett TL. HCV infection of the transplanted liver: changing CD81 and HVR1 variants immediately after liver transplantation. Am J Transplant 2005; 5:2504-13. [PMID: 16162201 DOI: 10.1111/j.1600-6143.2005.01060.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The second envelope protein at hypervariable region 1 (HVR1) has been implicated in contributing to hepatitis C virus (HCV)-host cell interactions and CD81 (a multifunctional protein) has been demonstrated to act as a cell surface receptor for HCV and may interact directly with HVR1. The purpose of the current study was to determine if certain HVR1 quasispecies variants more effectively associate with and infect allografts after liver transplantation than other HVR1 variants and whether CD81 receptor expression changes after transplantation. Blood and allograft samples were obtained from the peritransplant period in seven patients. Clones of RT-PCR product were directly sequenced to identify HVR1 quasispecies variants. Explanted liver and serial allograft biopsies in recipients with HCV were examined by immunohistochemistry (IHC) for CD81 expression. Examination of HVR1 sequences demonstrated that only a fraction of the quasispecies variants recovered from each patient's blood sampled immediately prior to transplantation associated with and infected the allografts. Genetic diversity at HVR1 decreased with reperfusion but did not significantly decrease with infection. Expression of CD81 varied during the immediate post-transplant period. In conclusion, HVR1 quasispecies variants differentially associate with, and infect allografts, after liver transplantation. Additionally, allografts express variable amounts of CD81 after transplantation.
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Affiliation(s)
- Michael G Hughes
- Department of Surgery, Surgical Infectious Disease Laboratory, University of Virginia, Charlottesville, Virginia, USA.
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33
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Pollack H, Hou Z, Hughes AL, Borkowsky W. Perinatal transmission and viral evolution of hepatitis C virus quasispecies in infants coinfected with HIV. J Acquir Immune Defic Syndr 2005; 36:890-9. [PMID: 15220695 DOI: 10.1097/00126334-200408010-00002] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
OBJECTIVES Three HIV/hepatitis C virus (HCV)-coinfected children and the mothers of 2 were studied to examine the nature of perinatal HCV infection in HIV-coinfected infants and to assess the evolution of viral quasispecies thereafter. Sequences of the hypervariable region in the N terminus of the E2/NS1 region (HVR-1) of the children and their mothers were compared. HCV quasispecies changes in the infants were tracked over several years. METHODS Sequence similarity comparisons and phylogenetic trees were derived from cDNA of plasma isolates. Quantitation of plasma HCV and HIV was performed in the children, as well as CD4 T-cell percentage and liver transaminases. RESULTS Phylogenetic analysis of the mother-child pairs suggested that transmission of multiple dominant and nondominant variants identified in the mother were seen. HCV diversification in the children was seen as early as 2 months of life. The child with the best immune status and HIV control demonstrated the most diversification throughout. CONCLUSION Multiples HCV variants transmitted from mother to child and their early changes in the child may be related to maternal antibody. Variation after the 1st year of life may reflect immunologic pressure from the child. There was no trend suggesting that the presence or absence of selective immunologic pressure affected HCV load or liver transaminase values.
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Affiliation(s)
- Henry Pollack
- Department of Pediatrics, The Saul Krugman Division of Infectious Diseases and Immunology, New York University School of Medicine, NY 10016, USA
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34
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Zehender G, De Maddalena C, Bernini F, Ebranati E, Monti G, Pioltelli P, Galli M. Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome. J Virol 2005; 79:9145-56. [PMID: 15994809 PMCID: PMC1168762 DOI: 10.1128/jvi.79.14.9145-9156.2005] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.
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Affiliation(s)
- Gianguglielmo Zehender
- Istituto di Malattie Infettive e Tropicali, Università di Milano c/o Ospedale L. Sacco, Via G.B. Grassi 74, 20157 Milan, Italy.
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35
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Li C, Allain JP. Chimeric monoclonal antibodies to hypervariable region 1 of hepatitis C virus. J Gen Virol 2005; 86:1709-1716. [PMID: 15914849 DOI: 10.1099/vir.0.80912-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Two chimeric monoclonal antibodies (cAbs), 2P24 and 15H4, to hypervariable region 1 (HVR1) of hepatitis C virus (HCV) were constructed by grafting the variable regions of murine monoclonal antibodies (mAbs) 2P24 and 15H4 to a human IgG1 kappa constant region. Two cAb-producing cell lines were adapted to serum-free media. Both cAb 2P24 and cAb 15H4 cell lines produced 3-5 microg antibodies ml(-1) after 3-5 days culture. cAbs retained binding characteristics similar to those observed in the original mAbs. There was no clear difference in affinity between binding of cAbs and mAbs to seven HVR1 peptides. Mixtures of biotinylated cAbs or mAbs reacted with 32 (86 %) and 31 (84 %) of 37 HVR1 peptides, respectively, but not with non-HVR1 control peptides. HCV from 16 out of 18 (89 %) random HCV-containing plasmas was captured by the mixture of biotinylated cAbs. The capture from IgG-depleted plasmas suggested that cAbs captured mainly free rather than complexed HCV, irrespective of genotype. A mixture of the two cAbs inhibited HCV binding to Molt-4 cells in a dose-dependent manner. These cAbs may be useful for prevention of nosocomial HCV infection and passive immunization to prevent HCV reinfection after liver transplantation.
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Affiliation(s)
- Chengyao Li
- National Blood Service, Division of Transfusion Medicine, East Anglia Blood Centre, Long Road, Cambridge CB2 2PT, UK
| | - Jean-Pierre Allain
- Department of Haematology, Division of Transfusion Medicine, East Anglia Blood Centre, Long Road, Cambridge CB2 2PT, UK
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Polyak SJ, Sullivan DG, Austin MA, Dai JY, Shuhart MC, Lindsay KL, Bonkovsky HL, Di Bisceglie AM, Lee WM, Morishima C, Gretch DR. Comparison of amplification enzymes for Hepatitis C Virus quasispecies analysis. Virol J 2005; 2:41. [PMID: 15847697 PMCID: PMC1090623 DOI: 10.1186/1743-422x-2-41] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2005] [Accepted: 04/22/2005] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Hepatitis C virus (HCV) circulates as quasispecies (QS), whose evolution is associated with pathogenesis. Previous studies have suggested that the use of thermostable polymerases without proofreading function may contribute to inaccurate assessment of HCV QS. In this report, we compared non-proofreading (Taq) with proofreading (Advantage High Fidelity-2; HF-2) polymerases in the sensitivity, robustness, and HCV QS diversity and complexity in the second envelope glycoprotein gene hypervariable region 1 (E2-HVR1) on baseline specimens from 20 patients in the HALT-C trial and in a small cohort of 12 HCV/HIV co-infected patients. QS diversity and complexity were quantified using heteroduplex mobility assays (HMA). RESULTS The sensitivities of both enzymes were comparable at 50 IU/ml, although HF-2 was more robust and slightly more sensitive than Taq. Both enzymes generated QS diversity and complexity scores that were correlated (r = 0.68; p < 0.0001, and r = 0.47; p < 0.01; Spearman's rank correlation). QS diversity was similar for both Taq and HF-2 enzymes, although there was a trend for higher diversity in samples amplified by Taq (p = 0.126). Taq amplified samples yielded complexity scores that were significantly higher than HF-2 samples (p = 0.033). HALT-C patients who were HCV positive or negative following 20 weeks of pegylated IFN plus ribavirin therapy had similar QS diversity scores for Taq and HF-2 samples, and there was a trend for higher complexity scores from Taq as compared with HF-2 samples. Among patients with HCV and HIV co-infection, HAART increased HCV QS diversity and complexity as compared with patients not receiving therapy, suggesting that immune reconstitution drives HCV QS evolution. However, diversity and complexity scores were similar for both HF-2 and Taq amplified specimens. CONCLUSION The data suggest that while Taq may overestimate HCV QS complexity, its use does not significantly affect results in cohort-based studies of HCV QS analyzed by HMA. However, the use of proofreading enzymes such as HF-2 is recommended for more accurate characterization of HCV QS in vivo.
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Affiliation(s)
- Stephen J Polyak
- Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
- Department of Microbiology, University of Washington, Seattle, WA, USA
- Department of Pathobiology, University of Washington, Seattle, WA, USA
| | - Daniel G Sullivan
- Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
| | - Michael A Austin
- Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
| | - James Y Dai
- Department of Biostatistics, University of Washington, Seattle, WA, USA
| | | | - Karen L Lindsay
- Division of Gastrointestinal and Liver Diseases, University of Southern California, Los Angeles, CA, USA
| | - Herbert L Bonkovsky
- Liver-Biliary-Pancreatic Center and the General Clinical Research Center, University of Connecticut Health Center, Farmington, CT, USA
| | - Adrian M Di Bisceglie
- Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine, St. Louis, MO, USA
| | - William M Lee
- Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Chihiro Morishima
- Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
- Department of Pediatrics, University of Washington, Seattle, WA, USA
| | - David R Gretch
- Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
- Department of Medicine, University of Washington, Seattle, WA, USA
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37
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Meunier JC, Engle RE, Faulk K, Zhao M, Bartosch B, Alter H, Emerson SU, Cosset FL, Purcell RH, Bukh J. Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1. Proc Natl Acad Sci U S A 2005; 102:4560-5. [PMID: 15767578 PMCID: PMC555507 DOI: 10.1073/pnas.0501275102] [Citation(s) in RCA: 207] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The lack of a cell culture system to support hepatitis C virus (HCV) replication has hampered studies of this frequent cause of chronic liver disease. However, pseudotyped retroviral particles (pp) bearing the HCV envelope glycoproteins have provided a different approach to HCV studies. We used genotype 1a pp to detect neutralizing antibodies (NtAb) in eight chimpanzees and four humans infected with 1a strains, and developed pp of genotypes 2a, 3a, 4a, 5a, and 6a to study crossreactivity. NtAb was detected in one of four chimpanzees and none of three humans with acute resolving infection, suggesting that NtAb is not required for HCV clearance. NtAb were detected at high titer in two of four chimpanzees and, in Patient H, all with persistent infection; responses paralleled humoral responses to envelope 1 and 2 proteins and, in some cases, correlate also with antibodies to the hypervariable region 1, previously thought to be the primary site of neutralization. NtAb raised during 1a infections could neutralize HCVpp of genotypes 4a, 5a, and 6a but had only limited reactivity against 2a and 3a. The detection of high-titer NtAb with cross-genotype reactivity has important implications for the development of active and passive immune-prophylaxis strategies against HCV. Finally, we found that HCVpp infectivity was enhanced by human or chimpanzee sera; apolipoprotein C1 alone or as a component of high-density lipoproteins caused this enhancement. Future studies of the in vivo role of apolipoprotein C1 might provide additional insights into the infection process of HCV.
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Affiliation(s)
- Jean-Christophe Meunier
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1. Proc Natl Acad Sci U S A 2005. [PMID: 15767578 DOI: 10.1073/pnas.0501275102.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The lack of a cell culture system to support hepatitis C virus (HCV) replication has hampered studies of this frequent cause of chronic liver disease. However, pseudotyped retroviral particles (pp) bearing the HCV envelope glycoproteins have provided a different approach to HCV studies. We used genotype 1a pp to detect neutralizing antibodies (NtAb) in eight chimpanzees and four humans infected with 1a strains, and developed pp of genotypes 2a, 3a, 4a, 5a, and 6a to study crossreactivity. NtAb was detected in one of four chimpanzees and none of three humans with acute resolving infection, suggesting that NtAb is not required for HCV clearance. NtAb were detected at high titer in two of four chimpanzees and, in Patient H, all with persistent infection; responses paralleled humoral responses to envelope 1 and 2 proteins and, in some cases, correlate also with antibodies to the hypervariable region 1, previously thought to be the primary site of neutralization. NtAb raised during 1a infections could neutralize HCVpp of genotypes 4a, 5a, and 6a but had only limited reactivity against 2a and 3a. The detection of high-titer NtAb with cross-genotype reactivity has important implications for the development of active and passive immune-prophylaxis strategies against HCV. Finally, we found that HCVpp infectivity was enhanced by human or chimpanzee sera; apolipoprotein C1 alone or as a component of high-density lipoproteins caused this enhancement. Future studies of the in vivo role of apolipoprotein C1 might provide additional insights into the infection process of HCV.
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Kurihara C, Tsuzuki Y, Hokari R, Nakashima H, Mataki N, Kuroki M, Kawaguchi A, Nagao S, Kondo T, Miura S. A highly hydrophobic domain within hypervariable region 1 may be related to the entry of hepatitis C virus into cultured human hepatoma cells. J Med Virol 2004; 74:546-55. [PMID: 15484277 DOI: 10.1002/jmv.20213] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Interaction of the envelope glycoprotein of hepatitis C virus (HCV) with a cellular receptor(s) is thought to be essential for the initial steps of HCV infection. However, the mechanisms of HCV infection remain unclear. The aim of the present study was to determine the features of HCV that enable efficient entry of the virus into human hepatocytes. Variations of hypervariable region 1 (HVR1) sequences in HCV inocula and in infected human hepatoblastoma HepG2 cells were examined. Immunofluorescence of inoculated HepG2 cells with anti-HCV core antibodies demonstrated that HCV structural proteins were expressed in the cytoplasm, and their entry into HepG2 cells was confirmed. When the HVR1 amino acid sequences were compared, HVR1 quasispecies in the inoculated cells showed more uniformity than those of the inocula. Although there were no statistically significant differences between the two groups, hydrophobic residues were observed more frequently in the HVR1 amino acids from inoculated cells than in the HVR1 amino acids from the inocula. Results of hydropathy analysis revealed that highly hydrophobic domains exist in the middle of HVR1 in the inoculated cells in 7 of 10 patients. The results suggest that limited HCV populations are able to enter HepG2 cells and that the highly hydrophobic domain existing within the HVR1 may play an important role in the entry of HCV into cells.
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Affiliation(s)
- Chie Kurihara
- Second Department of Internal Medicine, National Defense Medical College, Saitama, Japan
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Burioni R, Mancini N, Carletti S, Perotti M, Grieco A, Canducci F, Varaldo PE, Clementi M. Cross-reactive pseudovirus-neutralizing anti-envelope antibodies coexist with antibodies devoid of such activity in persistent hepatitis C virus infection. Virology 2004; 327:242-8. [PMID: 15351212 DOI: 10.1016/j.virol.2004.06.042] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2004] [Revised: 05/17/2004] [Accepted: 06/18/2004] [Indexed: 12/09/2022]
Abstract
Most RNA viruses have evolved mechanisms to avoid neutralizing antibody responses, and it is generally believed that variability of envelope-encoding regions is the major molecular basis of this phenomenon. However, it has been hypothesized that other mechanisms can be involved. Recent experimental data indicate that in hepatitis C virus (HCV) infection, the anti-envelope humoral response includes cross-reactive antibody clones able to neutralize vesicular stomatitis virus (VSV) pseudotypes containing HCV E1 and E2 glycoproteins (HCV/VSV pseudotype) as well as other clones devoid of such activity. In this work, we demonstrate that natural infection with a large variety of HCV isolates belonging to different genotypes elicits HCV/VSV pseudotype-neutralizing cross-reactive anti-envelope antibodies together with clones unable to neutralize this pseudovirus. This was shown by designing a novel strategy for quantitation of serum antibodies binding selectively to single viral cross-reactive conformational epitopes. These data can be useful not only for a better understanding of the virus-host interplay in important viral diseases, but also for the development of an effective anti-HCV vaccine.
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Affiliation(s)
- Roberto Burioni
- Istituto di Microbiologia, Facoltà di Medicina e Chirurgia, Università di Ancona, 60020 Ancona, Italy.
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Keck ZY, Op De Beeck A, Hadlock KG, Xia J, Li TK, Dubuisson J, Foung SKH. Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions. J Virol 2004; 78:9224-32. [PMID: 15308717 PMCID: PMC506923 DOI: 10.1128/jvi.78.17.9224-9232.2004] [Citation(s) in RCA: 119] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Mechanisms of virion attachment, interaction with its receptor, and cell entry are poorly understood for hepatitis C virus (HCV) because of a lack of an efficient and reliable in vitro system for virus propagation. Infectious HCV retroviral pseudotype particles (HCVpp) were recently shown to express native E1E2 glycoproteins, as defined in part by HCV human monoclonal antibodies (HMAbs) to conformational epitopes on E2, and some of these antibodies block HCVpp infection (A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung, F. L. Cosset, and J. Dubuisson, J. Virol. 78:2994-3002, 2004). Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.
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Affiliation(s)
- Zhen-Yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
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Zhu LX, Liu J, Ye Y, Xie YH, Kong YY, Li GD, Wang Y. A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses. World J Gastroenterol 2004; 10:2488-92. [PMID: 15300890 PMCID: PMC4572147 DOI: 10.3748/wjg.v10.i17.2488] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine.
METHODS: Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes. These cells were subjected to HCV antigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals.
RESULTS: Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-γ secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-γ but did not change the profile of IL-4 secretion.
CONCLUSION: Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.
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Affiliation(s)
- Li-Xin Zhu
- State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Yue-Yang Road 320, Shanghai 200031, China
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Heo TH, Chang JH, Lee JW, Foung SKH, Dubuisson J, Kang CY. Incomplete Humoral Immunity against Hepatitis C Virus Is Linked with Distinct Recognition of Putative Multiple Receptors by E2 Envelope Glycoprotein. THE JOURNAL OF IMMUNOLOGY 2004; 173:446-55. [PMID: 15210804 DOI: 10.4049/jimmunol.173.1.446] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Little is known about the role of the humoral immune response to hepatitis C virus (HCV). This study provides molecular evidence for the mechanism by which neutralizing Abs from the sera of chronic HCV patients have lower inhibitory activities against the binding of HCV E2 envelope protein to human hepatoma cell lines than to a lymphoma cell line. E2 binds to several putative receptors, specifically human CD81; human scavenger receptor, class B, type 1; and heparan sulfate. We have shown that E2 binds to target cells via these receptors in a noncompetitive manner. Thus, incomplete inhibition of one of the receptors leads to only a partial E2 blockade and, possibly, evasion of the host immune response. We demonstrated that the difference in and reduction of inhibition was closely related to impaired blockade of E2 binding to scavenger receptor, class B, type 1, and heparan sulfate. We have also shown that soluble E2 protein binds to multiple soluble receptors via separate binding domains on E2, providing further evidence for the distinct recognition of multiple cellular receptors by E2. This report suggests a novel finding that biased humoral immune responses to HCV E2 might provide an alternative mechanism for viral escape without the involvement of mutation. Additionally, our data give crucial consideration to the development of HCV vaccines that stimulate protective humoral immune responses.
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Affiliation(s)
- Tae-Hwe Heo
- Laboratory of Immunology, Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Korea
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Hughes MG, Rudy CK, Chong TW, Smith RL, Evans HL, Iezzoni JC, Sawyer RG, Pruett TL. E2 quasispecies specificity of hepatitis C virus association with allografts immediately after liver transplantation. Liver Transpl 2004; 10:208-16. [PMID: 14762858 DOI: 10.1002/lt.20060] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus-liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end-stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase-polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants.
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Affiliation(s)
- Michael G Hughes
- University of Virginia, Department of Surgery, Surgical Infectious Disease Laboratory, Charlottesville, VA, USA.
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Zhang XX, Deng Q, Zhang SY, Liu J, Cai Q, Lu ZM, Wang Y. Broadly cross-reactive mimotope of hypervariable region 1 of hepatitis C virus derived from DNA shuffling and screened by phage display library. J Med Virol 2004; 71:511-7. [PMID: 14556263 DOI: 10.1002/jmv.10521] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The hypervariable region 1 (HVR1) is the target of neutralizing antibodies but with isolate specificity. The aim of this study was to obtain immunogenic mimotopes of HVR1, which can react broadly with different HVR1 antibodies and could be one of the candidate immunogens in an effective vaccine against HCV. Thirty-one HVR1 cDNA fragments were digested by DNase I into a pool of random fragments and reassembled by repeated cycles of annealing in the presence of DNA polymerase to their original size. The shuffled HVR1 was then inserted into the gene III phagemid vector pCANTAB-5E and displayed on the surface of the phage. Eight individual phages were selected after four rounds of biopanning against anti-HVR1. ELISA was carried out on immobilized purified phages, respectively, to detect their reactivity with a panel of sera. DNA sequences of the inserts were analyzed and compared with the consensus sequences defined by Puntoriero et al. [(1998) EMBO J 17:3521-3533]. The reactivity of the eight selected clones to the 30 sera were from 53.3 to 80%. Among these, phage 13 (ETYVSGGSAARNAYGLTSLFTVGPAQK, aa 384-410) reacted most broadly. None of the selected sequences encoded for peptides corresponded to known HVR1 from original viral isolates. The two high reactive phages had the similar amino acid sequences with the consensus, which might play a particular role in determining the frequency of reactivity. In conclusion, this study has used effectively DNA shuffling combined with phage display technology to identify broadly cross-reactive mimotopes recognized by human polyclonal antibodies. Mimotope 13 and 23 appeared to be most reactive immunologically and could be candidate immunogens. Efforts are now underway to identify their neutralizing antibodies by immunization of animals.
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Affiliation(s)
- Xin-Xin Zhang
- State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China
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Bartosch B, Bukh J, Meunier JC, Granier C, Engle RE, Blackwelder WC, Emerson SU, Cosset FL, Purcell RH. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proc Natl Acad Sci U S A 2003; 100:14199-204. [PMID: 14617769 PMCID: PMC283569 DOI: 10.1073/pnas.2335981100] [Citation(s) in RCA: 242] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.
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Affiliation(s)
- Birke Bartosch
- Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, Institut National de la Santé et de la Recherche Médicale U412, IFR 128, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France
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Frasca L, Scottà C, Del Porto P, Nicosia A, Pasquazzi C, Versace I, Masci AM, Racioppi L, Piccolella E. Antibody-selected mimics of hepatitis C virus hypervariable region 1 activate both primary and memory Th lymphocytes. Hepatology 2003; 38:653-63. [PMID: 12939592 DOI: 10.1053/jhep.2003.50387] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
An ideal strategy that leads to a vaccine aimed at controlling viral escape may be that of preventing the replication of escape mutants by eliciting a T- and B-cell repertoire directed against many viral variants. The hypervariable region 1 (HVR1) of the putative envelope 2 protein that presents B and T epitopes shown to induce protective immunity against hepatitis C virus (HCV), might be suitable for this purpose if its immunogenicity can be improved by generating mimics that induce broad, highly cross-reactive, anti-HVR1 responses. Recently we described a successful approach to select HVR1 mimics (mimotopes) incorporating the variability found in a great number of viral variants. In this report we explore whether these mimotopes, designed to mimic B-cell epitopes, also mimic helper T-cell epitopes. The first interesting observation is that mimotopes selected for their reactivity to HVR1-specific antibodies of infected patients also do express HVR1 T-cell epitopes, suggesting that similar constraints govern HVR1-specific humoral and cellular immune responses. Moreover, some HVR1 mimotopes stimulate a multispecific CD4(+) T-cell repertoire that effectively cross-reacts with HVR1 native sequences. This may significantly limit effects as a T-cell receptor (TCR) antagonist frequently exerted by natural HVR1-variants on HVR1-specific T-cell responses. In conclusion, these data lend strong support to using HVR1 mimotopes in vaccines designed to prevent replication of escape mutants.
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Affiliation(s)
- Loredana Frasca
- Department of Cellular and Developmental Biology, La Sapienza University, Rome, Italy
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Kaplan M, Gawrieh S, Cotler SJ, Jensen DM. Neutralizing antibodies in hepatitis C virus infection: a review of immunological and clinical characteristics. Gastroenterology 2003; 125:597-604. [PMID: 12891562 DOI: 10.1016/s0016-5085(03)00882-5] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
- Mitchell Kaplan
- Department of Internal Medicine, Rush Presbyterian St. Luke's Medical Center, Rush University, Chicago, IL 60612, USA
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Xiu BS, Ling SG, Song XG, Zhang HQ, Chen K, Zhu CX. Cross-reactivity of hypervariable region 1 chimera of hepatitis C virus. World J Gastroenterol 2003; 9:1256-60. [PMID: 12800235 PMCID: PMC4611795 DOI: 10.3748/wjg.v9.i6.1256] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/04/2003] [Revised: 02/04/2003] [Accepted: 02/16/2003] [Indexed: 02/06/2023] Open
Abstract
AIM To analyze the amino acid sequences of hypervariable region 1 (HVR1) of HCV isolates in China and to construct a combinatorial chimeric HVR1 protein having a very broad high cross-reactivity. METHODS All of the published HVR1 sequences from China were collected and processed with a computer program. Several representative HVR1's sequences were formulated based on a consensus profile and homology within certain subdivision. A few reported HVR1 mimotope sequences were also included for a broader representation. All of them were cloned and expressed in E.coli. The cross-reactivity of the purified recombinant HVR1 antigens was tested by ELISA with a panel of sera from HCV infected patients in China. Some of them were further ligated together to form a combinatorial HVR1 chimera. RESULTS Altogether 12 HVR1(s) were selected and expressed in E.coli and purified to homogeneity. All of these purified antigens showed some cross-reactivity with sera in a 27 HCV positive panel. Recombinant HVR1s of No. 1, 2, 4, and 8# showing broad cross-reactivities and complementarity with each other, were selected for the ligation elements. The chimera containing these 4 HVR1s was highly expressed in E.coli. The purified chimeric antigen could react not only with all the HCV antibody positive sera in the panel but also with 90/91 sera of HCV -infected patients. CONCLUSION The chimeric antigen was shown to have a broad cross-reactivity. It may be helpful for solving the problem caused by high variability of HCV, and in the efforts for a novel vaccine against the virus.
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Affiliation(s)
- Bing-Shui Xiu
- Laboratory of Molecular Virology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, P.R.China
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