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Thorselius CE, Wolfisberg R, Fahnøe U, Scheel TKH, Holmbeck K, Bukh J. Norway rat hepacivirus resembles hepatitis C virus in terms of intra-host evolution and escape from neutralizing antibodies. J Hepatol 2025:S0168-8278(25)00163-1. [PMID: 40096950 DOI: 10.1016/j.jhep.2025.02.044] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2024] [Revised: 02/05/2025] [Accepted: 02/24/2025] [Indexed: 03/19/2025]
Abstract
BACKGROUND & AIMS Norway rat hepacivirus 1 (NrHV) is an attractive surrogate model for evaluating vaccine strategies against hepatitis C virus (HCV). Yet the immune response in NrHV infections remains poorly understood, particularly the role of neutralizing antibodies (nAbs). Here, we explore nAb development and viral evolution during chronic NrHV infection of inbred rats to understand neutralization and viral escape dynamics. METHODS Lewis rats inoculated with the NrHV RHV-rn1 strain were monitored for >52 weeks. Viremia was quantified by reverse-transcription quantitative PCR, and NrHV nAbs were characterized by infectious cell culture-based neutralization assays and challenge experiments. Viral evolution was followed over time by whole open reading frame deep sequencing. RESULTS In most animals, high levels of nAbs appeared after 20 to 45 weeks of infection, coinciding with the emergence of numerous mutations in the envelope proteins. Incorporation of these E1/E2 mutations into cell culture-adapted RHV-rn1 reduced sensitivity to neutralization by autologous contemporary serum. Five key recurrent E1/E2 substitutions (E209K, R224Q, V275I, T500K, and L569P) were identified, collectively impairing neutralization by serum, with E209K in E1 alone proving sufficient for escape from neutralization. In contrast, NrHV-infected rats devoid of nAbs displayed fewer envelope mutations. Finally, pretreatment of cells with rat serum with high-titer nAbs led to partial control of NrHV infection, and passive immunization with such sera protected SCID mice from subsequent challenge. CONCLUSIONS This study demonstrates the correlation between nAbs and viral evolution during long-term NrHV infection. The observed humoral immunity for NrHV infection closely resembles that of chronic HCV infection, where late-emerging high-level nAbs fail to clear evolving viral populations, thereby contributing to evasion from adaptive immune responses. Preexisting antibodies do, however, protect from infection. IMPACT AND IMPLICATIONS The findings from this study provide compelling scientific justification for using Norway rat hepacivirus (NrHV) as a model to investigate immune responses and associated vaccine strategies against hepatitis C virus (HCV). By demonstrating that delayed neutralizing antibody development and viral escape through envelope mutations mirror patterns observed in HCV infections, this research offers valuable insights into the adaptive immune dynamics underlying chronic hepacivirus infections. These results are particularly important for researchers and vaccine developers aiming to better understand immune evasion mechanisms and refine HCV vaccine candidates. Practical applications include utilizing NrHV as a preclinical platform to test and optimize vaccine formulations and evaluate passive immunization strategies aimed at controlling HCV-related disease burdens while considering limitations related to host and viral variability.
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Affiliation(s)
- Caroline E Thorselius
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Raphael Wolfisberg
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Ulrik Fahnøe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Troels K H Scheel
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Kenn Holmbeck
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
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2
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Olesen CH, Augestad EH, Troise F, Bukh J, Prentoe J. In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus. PLoS Pathog 2021; 17:e1009720. [PMID: 34280245 PMCID: PMC8321405 DOI: 10.1371/journal.ppat.1009720] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 07/29/2021] [Accepted: 06/15/2021] [Indexed: 12/18/2022] Open
Abstract
Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.
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Affiliation(s)
- Christina Holmboe Olesen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Elias H. Augestad
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Fulvia Troise
- Ceinge Biotecnologie Avanzate Via Gaetano Salvatore, Napoli, Italy
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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3
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Mekonnen ZA, Masavuli MG, Yu W, Gummow J, Whelan DM, Al-Delfi Z, Torresi J, Gowans EJ, Grubor-Bauk B. Enhanced T Cell Responses Induced by a Necrotic Dendritic Cell Vaccine, Expressing HCV NS3. Front Microbiol 2020; 11:559105. [PMID: 33343515 PMCID: PMC7739890 DOI: 10.3389/fmicb.2020.559105] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Accepted: 10/28/2020] [Indexed: 12/21/2022] Open
Abstract
A vaccine that induces potent, broad and sustained cell-mediated immunity, resulting in effective memory has the potential to restrict hepatitis C (HCV) virus infection. Early, multi-functional CD4+ and CD8+ T cell responses against non-structural protein 3 (NS3) have been associated with HCV clearance. Necrotic cells generate strong immune responses and represent a major antigenic source used by dendritic cells (DC) for processing and presentation, but there is conflicting evidence as to their immunogenicity in vaccination. Immunization with DC loaded with viral antigens has been done in the past, but to date the immunogenicity of live vs. necrotic DC vaccines has not been investigated. We developed a DC2.4 cell line stably expressing HCV NS3, and compared the NS3-specific responses of live vs. necrotic NS3 DC. Vaccination of mice with necrotic NS3 DC increased the breadth of T-cell responses and enhanced the production of IL-2, TNF-α, and IFN-γ by effector memory CD4+ and CD8+T cells, compared to mice vaccinated with live NS3 DC. A single dose of necrotic NS3 DC vaccine induced a greater influx and activation of cross-presenting CD11c+ CD8α+ DC and necrosis-sensing Clec9A+ DC in the draining lymph nodes. Furthermore, using a hydrodynamic challenge model necrotic NS3 DC vaccination resulted in enhanced clearance of NS3-positive hepatocytes from the livers of vaccinated mice. Taken together, the data demonstrate that necrotic DC represent a novel and exciting vaccination strategy capable of inducing broad and multifunctional T cell memory.
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Affiliation(s)
- Zelalem A Mekonnen
- Viral Immunology Group, Discipline of Surgery, Basil Hetzel Institute for Translational Medicine, University of Adelaide, Adelaide, SA, Australia
| | - Makutiro G Masavuli
- Viral Immunology Group, Discipline of Surgery, Basil Hetzel Institute for Translational Medicine, University of Adelaide, Adelaide, SA, Australia
| | - Wenbo Yu
- Viral Immunology Group, Discipline of Surgery, Basil Hetzel Institute for Translational Medicine, University of Adelaide, Adelaide, SA, Australia.,Centre for Cancer Biology, University of South Australia, Adelaide, SA, Australia
| | - Jason Gummow
- Gene Silencing and Expression Laboratory, Robinson Research Institute, The University of Adelaide, Adelaide, SA, Australia
| | - Dawn M Whelan
- Viral Immunology Group, Discipline of Surgery, Basil Hetzel Institute for Translational Medicine, University of Adelaide, Adelaide, SA, Australia
| | - Zahraa Al-Delfi
- Viral Immunology Group, Discipline of Surgery, Basil Hetzel Institute for Translational Medicine, University of Adelaide, Adelaide, SA, Australia
| | - Joseph Torresi
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia
| | - Eric J Gowans
- Viral Immunology Group, Discipline of Surgery, Basil Hetzel Institute for Translational Medicine, University of Adelaide, Adelaide, SA, Australia
| | - Branka Grubor-Bauk
- Viral Immunology Group, Discipline of Surgery, Basil Hetzel Institute for Translational Medicine, University of Adelaide, Adelaide, SA, Australia
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V H1-69 antiviral broadly neutralizing antibodies: genetics, structures, and relevance to rational vaccine design. Curr Opin Virol 2019; 34:149-159. [PMID: 30884330 DOI: 10.1016/j.coviro.2019.02.004] [Citation(s) in RCA: 95] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2018] [Accepted: 02/07/2019] [Indexed: 12/15/2022]
Abstract
Broadly neutralizing antibodies (bnAbs) are potential therapeutic molecules and valuable tools for studying conserved viral targets for vaccine and drug design. Interestingly, antibody responses to conserved epitopes can be highly convergent at the molecular level. Human antibodies targeting a number of viral antigens have often been found to utilize a restricted set of immunoglobulin germline genes in different individuals. Here we review recent knowledge on VH1-69-encoded antibodies in antiviral responses to influenza virus, HCV, and HIV-1. These antibodies share common genetic and structural features, and often develop neutralizing activity against a broad spectrum of viral strains. Understanding the genetic and structural characteristics of such antibodies and the target epitopes should help advance novel strategies to elicit bnAbs through vaccination.
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Human Norovirus Epitope D Plasticity Allows Escape from Antibody Immunity without Loss of Capacity for Binding Cellular Ligands. J Virol 2019; 93:JVI.01813-18. [PMID: 30355694 PMCID: PMC6321922 DOI: 10.1128/jvi.01813-18] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Accepted: 10/12/2018] [Indexed: 02/02/2023] Open
Abstract
Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two cocirculating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands.IMPORTANCE Human norovirus causes ∼20% of all acute gastroenteritis and ∼200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30 years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.
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Guo X, Zhong JY, Li JW. Hepatitis C Virus Infection and Vaccine Development. J Clin Exp Hepatol 2018; 8:195-204. [PMID: 29892184 PMCID: PMC5992307 DOI: 10.1016/j.jceh.2018.02.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2017] [Accepted: 02/08/2018] [Indexed: 12/12/2022] Open
Abstract
In the twenty-seven years since the discovery of hepatitis C virus (HCV) the majority of individuals exposed to HCV establish a persistent infection, which is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. In developed nations, the cure rates of HCV infection could be over 90% with direct-acting antiviral (DAA) regimens, which has made the great progress in global eradication. However, the cost of these treatments is so expensive that the patients in developing nations, where the disease burden is the most severe, could not afford it, which highly restricted its access. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in risk groups due to limited surveillance. Consequently a protective vaccine and likely emergence of drug-resistant viral variants call for further studies of HCV biology. In the current review, the development and the progress of preventive and therapeutic vaccines against the HCV have been reviewed in the context of peptide vaccines, recombinant protein vaccines, HCV-like particle, DNA vaccines and viral vectors expressing HCV genes.
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Affiliation(s)
- Xuan Guo
- Research Institute of Chemical Defense, No.1 Huaiyin Road, Beijing 102205, China
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, No.1 Dali Road, Tianjin 300050, China
| | - Jin-Yi Zhong
- Research Institute of Chemical Defense, No.1 Huaiyin Road, Beijing 102205, China
| | - Jun-Wen Li
- Department of Environment and Health, Tianjin Institute of Health and Environmental Medicine, No.1 Dali Road, Tianjin 300050, China
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7
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Balasco N, Barone D, Iaccarino E, Sandomenico A, De Simone A, Ruvo M, Vitagliano L. Intrinsic structural versatility of the highly conserved 412-423 epitope of the Hepatitis C Virus E2 protein. Int J Biol Macromol 2018; 116:620-632. [PMID: 29758309 DOI: 10.1016/j.ijbiomac.2018.05.055] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Revised: 05/09/2018] [Accepted: 05/10/2018] [Indexed: 12/14/2022]
Abstract
HCV infection is a major threaten for human health as it affects hundreds of million people worldwide. Here we investigated the conformational properties of the 412-423 fragment of the envelope E2 protein, one of the most immunogenic regions of the virus proteome whose characterization may provide interesting insights for anti-HCV vaccine development. The spectroscopic characterization of the polypeptide unravels its unexpected tendency to form amyloid-like aggregates. When kept in monomeric state, it shows a limited tendency to adopt regular secondary structure. Enhanced molecular dynamics simulations, starting from four distinct conformational states, highlight its structural versatility. Interestingly, all multiform conformational states of the polypeptide detected in crystallographic complexes with antibodies are present in the structural ensemble of all simulations. This observation corroborates the idea that known antibodies recognize this region through a conformational selection mechanism. Accordingly, the design of effective anti-HCV vaccines should consider the intrinsic flexibility of this region. The structural versatility of the 412-423 region is particularly puzzling if its remarkable sequence conservation is considered. It is likely that flexibility and sequence conservation are important features that endow this epitope with the ability to accomplish distinct functions such as immunity escape and interaction with host receptors.
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Affiliation(s)
- Nicole Balasco
- Institute of Biostructures and Bioimaging, CNR, Naples I-80134, Italy.
| | - Daniela Barone
- Institute of Biostructures and Bioimaging, CNR, Naples I-80134, Italy; Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Università della Campania "Luigi Vanvitelli", Caserta 81100, Italy
| | - Emanuela Iaccarino
- Institute of Biostructures and Bioimaging, CNR, Naples I-80134, Italy; Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Università della Campania "Luigi Vanvitelli", Caserta 81100, Italy
| | | | - Alfonso De Simone
- Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, UK
| | - Menotti Ruvo
- Institute of Biostructures and Bioimaging, CNR, Naples I-80134, Italy
| | - Luigi Vitagliano
- Institute of Biostructures and Bioimaging, CNR, Naples I-80134, Italy.
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Guo Y, Yu H, Zhong Y, He Y, Qin X, Qin Y, Zhou Y, Zhang P, Zhang Y, Li Z, Jia Z. Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation. Medicine (Baltimore) 2018; 97:e0208. [PMID: 29642144 PMCID: PMC5908620 DOI: 10.1097/md.0000000000010208] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
We used lectin microarray and mass spectrometric analysis to identify the N-linked glycosylation patterns of hepatitis C virus (HCV) particles. HCV J6/JFH-1 chimeric cell culture (HCVcc) in the culture supernatant was concentrated and purified by ultrafiltration and sucrose gradient ultracentrifugation. Twelve fractions were collected from the top and analyzed for viral infectivity and HCV RNA content after sucrose gradient separation. HCV RNA and proteins were separated by ultracentrifugation in a continuous 10% to 60% sucrose gradient to purify viral particles based on their sedimentation velocities. HCVcc particles were found mainly in fractions 6 to 8, as determined by quantitative polymerase chain reaction (qPCR) analysis for HCV RNA and ELISA of the HCV core protein. The N-glycans on HCV proteins were analyzed by lectin microarray and mass spectrometry. We identified that 32 of 37 lectins displayed the positive binding signals and 16 types of N-glycoforms of which the major HCV glycoforms were high mannose-type N-linked oligosaccharides, hybrid N-glycans, and fucosylated N-glycans. Our study provided new detailed information regarding the majority of the glycan-protein profile, complementing to previous findings of glycan-HCV protein interactions.
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Affiliation(s)
- Yonghong Guo
- Department of Infectious Diseases, The Second Affiliated Hospital, Xi’an Jiaotong University
| | - Hanjie Yu
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Yaogang Zhong
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Yu He
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Xinmin Qin
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Yuan Qin
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Yun Zhou
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Peixin Zhang
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Ying Zhang
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
| | - Zheng Li
- Laboratory for Functional Glycomics, College of Life Science, Northwest University
| | - Zhansheng Jia
- Department of Infectious Diseases and Center of Liver Diseases, Tangdu Hospital, The Fourth Military Medical University, Baqiao District, Xi’an, Shaanxi, China
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A Prominent Site of Antibody Vulnerability on HIV Envelope Incorporates a Motif Associated with CCR5 Binding and Its Camouflaging Glycans. Immunity 2017; 45:31-45. [PMID: 27438765 DOI: 10.1016/j.immuni.2016.06.026] [Citation(s) in RCA: 118] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2016] [Revised: 03/24/2016] [Accepted: 04/28/2016] [Indexed: 01/12/2023]
Abstract
The dense patch of high-mannose-type glycans surrounding the N332 glycan on the HIV envelope glycoprotein (Env) is targeted by multiple broadly neutralizing antibodies (bnAbs). This region is relatively conserved, implying functional importance, the origins of which are not well understood. Here we describe the isolation of new bnAbs targeting this region. Examination of these and previously described antibodies to Env revealed that four different bnAb families targeted the (324)GDIR(327) peptide stretch at the base of the gp120 V3 loop and its nearby glycans. We found that this peptide stretch constitutes part of the CCR5 co-receptor binding site, with the high-mannose patch glycans serving to camouflage it from most antibodies. GDIR-glycan bnAbs, in contrast, bound both (324)GDIR(327) peptide residues and high-mannose patch glycans, which enabled broad reactivity against diverse HIV isolates. Thus, as for the CD4 binding site, bnAb effectiveness relies on circumventing the defenses of a critical functional region on Env.
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Abdelhafez TH, Bader El Din NG, Tabll AA, Mashaly MM, Dawood RM, Yassin NA, El-Awady MK. Mice Antibody Response to Conserved Nonadjuvanted Multiple Antigenic Peptides Derived from E1/E2 Regions of Hepatitis C Virus. Viral Immunol 2017; 30:359-365. [PMID: 28402196 DOI: 10.1089/vim.2016.0123] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Synthetic peptides are one of the hepatitis C virus (HCV)-specific small molecules that have antiviral activity and represent a target for HCV vaccine. This study aims to determine the lowest concentration of adjuvanted and non-adjuvanted (multiple antigenic peptide [MAP]) form of three conserved HCV envelope peptides that can induce murine immunogenic responses and evaluate the neutralization capacities of the generated antibodies (Abs) against HCV in cultured Huh7.5 cells. In this study, three HCV synthetic peptides, E1 peptide (a.a 315-323) and E2 peptides (a.a 412-419 and a.a 516-531) were synthesized. Female Balb/c mice were immunized with different concentration of either adjuvanted linear peptides or nonadjuvanted MAP peptides to determine the lowest dose that generates Ab responses enough to confer viral neutralization in vitro. The humoral responses targeting these peptides in immunized mice sera were measured by enzyme-linked immunosorbent assay (ELISA). Viral neutralization capacities of the generated mice Abs were assessed using Huh7.5 cells infected with the HCVcc infectious system (J6/JFH-1). The results of this study showed that the MAPs induce higher Ab titers than adjuvanted linear peptides after 4 weeks of immunization (p = 0.003). The viral neutralization experiments showed that the immunized mice sera contain anti E1/E2 Abs that blocked HCVcc (J6/JFH-1) entry into Huh7.5 cells. In conclusion, the three HCV envelope MAP peptides are more immunogenic and produce higher neutralizing Abs than linear peptides; therefore, they can be essential components for HCV vaccine.
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Affiliation(s)
- Tawfeek H Abdelhafez
- 1 Department of Microbial Biotechnology, National Research Center , Dokki, Giza, Egypt
| | - Noha G Bader El Din
- 1 Department of Microbial Biotechnology, National Research Center , Dokki, Giza, Egypt
| | - Ashraf A Tabll
- 1 Department of Microbial Biotechnology, National Research Center , Dokki, Giza, Egypt
| | - Mohammad M Mashaly
- 2 Department of Chemistry, Faculty of Science, Damietta University , Damietta, Egypt
| | - Reham M Dawood
- 1 Department of Microbial Biotechnology, National Research Center , Dokki, Giza, Egypt
| | - Nemat A Yassin
- 3 Department of Pharmacology, National Research Center , Dokki, Giza, Egypt
| | - Mostafa K El-Awady
- 1 Department of Microbial Biotechnology, National Research Center , Dokki, Giza, Egypt
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11
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Das S, Mullick R, Kumar A, Tandon H, Bose M, Gouthamchandra K, Chandra M, Ravishankar B, Khaja MN, Srinivasan N, Das S, Melkote Subbarao S, Karande AA. Identification of a novel epitope in the C terminus of hepatitis C virus-E2 protein that induces potent and cross-reactive neutralizing antibodies. J Gen Virol 2017; 98:962-976. [PMID: 28221101 DOI: 10.1099/jgv.0.000735] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis, but an effective vaccine is still not available to prevent infection. Use of neutralizing antibodies could be a potential therapeutic option. In this study, the presence of anti-HCV antibodies in HCV-infected patients was assessed from 50 patients and the presence of neutralizing antibodies was examined using 'hepatitis C virus-like particles'. Antibodies from two samples exhibited significant inhibitory activity, suggesting that these may neutralize viral infection. Antigenic determinants generating the neutralizing antibodies from these two samples were delineated by epitope mapping using the core, E1 and E2 regions and a stretch of 45 amino acid peptide (E2C45) derived from the C-terminal region of HCV-E2 protein (aa 634-679) was designed. Results suggest that this hitherto uncharacterized region has the potential to generate neutralizing antibodies against HCV and thus be effective in preventing virus entry into liver cells. Computational analysis of the structure of the modelled peptide (E2C45) suggested high conformational entropy for this region. Furthermore, E2C45 peptide-generated antibodies could block virus entry and monoclonal antibodies generated against this peptide could also significantly reduce virus replication in a cell culture system. It is possible that the inhibition could be partly due to a conformational alteration of the CD81-binding region, preventing virus attachment to liver cells. In conclusion, this work focused on the discovery of a novel epitope at the C terminus of E2 that induces potent neutralizing antibodies in HCV-infected patients.
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Affiliation(s)
- Soma Das
- Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
| | - Ranajoy Mullick
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | - Anuj Kumar
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | - Himani Tandon
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India
| | - Mihika Bose
- Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
| | - K Gouthamchandra
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | - Madhavi Chandra
- Bioviz Technologies Pvt Ltd, Sagar Society, Road No. 2, Banjara Hills, Hyderabad 500 034, India
| | | | - M N Khaja
- Bioviz Technologies Pvt Ltd, Sagar Society, Road No. 2, Banjara Hills, Hyderabad 500 034, India
| | | | - Saumitra Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | - Shaila Melkote Subbarao
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
| | - Anjali Anoop Karande
- Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
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12
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Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice. J Virol 2016; 90:10486-10498. [PMID: 27630242 DOI: 10.1128/jvi.01462-16] [Citation(s) in RCA: 54] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2016] [Accepted: 09/06/2016] [Indexed: 12/18/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines. IMPORTANCE A prophylactic vaccine with high efficacy and low cost is urgently needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Here, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus, our work identified a promising broadly protective HCV vaccine candidate that should be considered for further preclinical and clinical development.
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13
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Beaumont E, Roch E, Chopin L, Roingeard P. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties. PLoS One 2016; 11:e0151626. [PMID: 26966906 PMCID: PMC4788456 DOI: 10.1371/journal.pone.0151626] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2016] [Accepted: 03/01/2016] [Indexed: 02/07/2023] Open
Abstract
Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.
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Affiliation(s)
- Elodie Beaumont
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| | - Emmanuelle Roch
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| | - Lucie Chopin
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
| | - Philippe Roingeard
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
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14
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O’Shea D, Law J, Egli A, Douglas D, Lund G, Forester S, Lambert J, Law M, Burton D, Tyrrell D, Houghton M, Humar A, Kneteman N. Prevention of hepatitis C virus infection using a broad cross-neutralizing monoclonal antibody (AR4A) and epigallocatechin gallate. Liver Transpl 2016; 22:324-32. [PMID: 26389583 PMCID: PMC4769112 DOI: 10.1002/lt.24344] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/03/2015] [Accepted: 09/01/2015] [Indexed: 01/13/2023]
Abstract
The anti-hepatitis C virus (HCV) activity of a novel monoclonal antibody (mAb; AR4A) and epigallocatechin gallate (EGCG) were studied in vitro using a HCV cell culture system and in vivo using a humanized liver mouse model capable of supporting HCV replication. Alone, both exhibit reliable cross-genotype HCV inhibition in vitro, and combination therapy completely prevented HCV infection. In vitro AR4A mAb (alone and combined with EGCG) robustly protects against the establishment of HCV genotype 1a infection. EGCG alone fails to reliably protect against an HCV challenge. In conclusion, AR4A mAb represents a safe and efficacious broadly neutralizing antibody against HCV applicable to strategies to safely prevent HCV reinfection following liver transplantation, and it lends further support to the concept of HCV vaccine development. The poor bioavailability of EGCG limits HCV antiviral activity in vitro.
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Affiliation(s)
- D. O’Shea
- Transplant Infectious Diseases, Alberta Transplant Institute, Department of Medicine, University of Alberta,Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada,To whom correspondence should be addressed: Daire O’Shea, Consultant in Infectious Diseases, Regional Infectious Diseases Unit, Crewe Road, Edinburgh EH4 2XU, Scotland, UK, Tel: +441315372862, Fax: +441315372878,
| | - J Law
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada
| | - A Egli
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada
| | - D Douglas
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada
| | - G Lund
- KMT Hepatech, University of Alberta, Edmonton, Alberta, Canada
| | - S Forester
- Dept. of Food Science, Pennsylvania State University, PA
| | - J Lambert
- Dept. of Food Science, Pennsylvania State University, PA
| | - M Law
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla CA 92037, USA
| | - D.R. Burton
- Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla CA 92037, USA,Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA
| | - D.L.J. Tyrrell
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada
| | - M. Houghton
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada
| | - A. Humar
- Toronto General Hospital, University Health Network, Multi-Organ Transplant Program Toronto, Ontario, Canada
| | - N Kneteman
- Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada,Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
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15
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Kumar A, Das S, Mullick R, Lahiri P, Tatineni R, Goswami D, Bhat P, Torresi J, Gowans EJ, Karande AA, Das S. Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy. Vaccine 2015; 34:1115-25. [PMID: 26700891 DOI: 10.1016/j.vaccine.2015.11.061] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Revised: 10/15/2015] [Accepted: 11/19/2015] [Indexed: 12/17/2022]
Abstract
Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy.
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Affiliation(s)
- Anuj Kumar
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
| | - Soma Das
- Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
| | - Ranajoy Mullick
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
| | - Priyanka Lahiri
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
| | - Ranjitha Tatineni
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
| | - Debashree Goswami
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
| | - Prasanna Bhat
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
| | - Joseph Torresi
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3000, Australia
| | - Eric James Gowans
- Virology Laboratory, Discipline of Surgery, University of Adelaide, Basil Hetzel Institute for Translational Medicine, Adelaide, SA 5011, Australia
| | - Anjali Anoop Karande
- Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
| | - Saumitra Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
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16
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Kachko A, Frey SE, Sirota L, Ray R, Wells F, Zubkova I, Zhang P, Major ME. Antibodies to an interfering epitope in hepatitis C virus E2 can mask vaccine-induced neutralizing activity. Hepatology 2015; 62:1670-82. [PMID: 26251214 PMCID: PMC4681649 DOI: 10.1002/hep.28108] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2014] [Accepted: 08/03/2015] [Indexed: 12/13/2022]
Abstract
UNLABELLED Hepatitis C virus (HCV) neutralization occurring at the E2 region 412-426 (EP-I) could be enhanced when antibodies directed specifically to the E2 region 434-446 (EP-II) were removed from serum samples of persistently infected patients and vaccinated chimpanzees, a phenomenon of so-called antibody interference. Here, we show that this type of interference can be observed in individuals after immunization with recombinant E1E2 proteins. One hundred twelve blinded serum samples from a phase I, placebo-controlled, dose escalation trial using recombinant HCV E1E2 with MF59C.1 adjuvant in healthy HCV-negative adults were tested in enzyme-linked immunosorbent assay for binding reactivity to peptides representing the E2 regions 412-426 (EP-I) and 434-446 (EP-II). All samples were subsequently tested for neutralizing activity using cell-culture HCV 1a(H77)/2a chimera, HCV pseudotype particles (HCVpp) H77, and HCVpp HCV-1 after treatment to remove EP-II-specific antibodies or mock treatment with a control peptide. Among the 112 serum samples, we found 22 double positive (EP-I and EP-II), 6 EP-II positive only, 14 EP-I positive only, and 70 double negative. Depleting EP-II antibodies from double-positive serum samples increased 50% inhibitory dose (ID50) neutralizing antibody titers (up to 4.9-fold) in up to 72% of samples (P ≤ 0.0005), contrasting with ID50 neutralization titer increases in 2 of 70 double-negative samples (2.9%; P > 0.5). In addition, EP-I-specific antibody levels in serum samples showed a significant correlation with ID50 neutralization titers when EP-II antibodies were removed (P < 0.0003). CONCLUSION These data show that antibodies to the region 434-446 are induced during immunization of individuals with recombinant E1E2 proteins, and that these antibodies can mask effective neutralizing activity from EP-I-specific antibodies. Elicitation of EP-II-specific antibodies with interfering capacity should be avoided in producing an effective cross-neutralizing vaccine aimed at the HCV envelope proteins.
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Affiliation(s)
- Alla Kachko
- Division of Viral ProductsCBER/FDASilver SpringMD
| | - Sharon E. Frey
- Division of Infectious Diseases, Allergy and ImmunologySaint Louis University School of MedicineSt LouisMO
| | - Lev Sirota
- Division of Biostatistics, Office of Biostatistics and EpidemiologyCBER/FDASilver SpringMD
| | - Ranjit Ray
- Division of Infectious Diseases, Allergy and ImmunologySaint Louis University School of MedicineSt LouisMO
| | | | | | - Pei Zhang
- Division of HematologyCBER/FDASilver SpringMD
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17
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Deng K, Liu R, Rao H, Jiang D, Wang J, Xie X, Wei L. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection. PLoS One 2015; 10:e0138756. [PMID: 26406225 PMCID: PMC4583415 DOI: 10.1371/journal.pone.0138756] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2015] [Accepted: 09/03/2015] [Indexed: 01/07/2023] Open
Abstract
Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection.
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Affiliation(s)
- Kai Deng
- Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, China
| | - Ruyu Liu
- Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Huiying Rao
- Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, China
| | - Dong Jiang
- Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing Key Laboratory of Emerging infectious Diseases, Beijing, China
| | - Jianghua Wang
- Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, China
| | - Xingwang Xie
- Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, China
| | - Lai Wei
- Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, China
- * E-mail:
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18
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Tarr AW, Khera T, Hueging K, Sheldon J, Steinmann E, Pietschmann T, Brown RJP. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design. Viruses 2015; 7:3995-4046. [PMID: 26193307 PMCID: PMC4517138 DOI: 10.3390/v7072809] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2015] [Revised: 06/19/2015] [Accepted: 07/08/2015] [Indexed: 12/13/2022] Open
Abstract
In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design.
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Affiliation(s)
- Alexander W Tarr
- School of Life Sciences, Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham NG7 2RD, UK.
| | - 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 Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Kathrin Hueging
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - Julie Sheldon
- Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - 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 Centrefor Infection Research (HZI), Hannover D-30625, Germany.
| | - 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 Centrefor Infection Research (HZI), Hannover D-30625, Germany.
- German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Braunschweig 38124, 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 Centrefor Infection Research (HZI), Hannover D-30625, Germany.
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19
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Low cross-neutralization of hepatitis C correlates with liver disease in immunocompromized patients. AIDS 2015; 29:1025-33. [PMID: 26125137 DOI: 10.1097/qad.0000000000000651] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
BACKGROUND AND AIMS Chronic hepatitis C virus (HCV) infection causes severe liver disease in HIV-infected patients and liver transplant recipients. The impact of serum and immunoglobulin on viral entry was analysed in these patients. METHOD Sera from 60 anti-HCV positive patients, including 30 who were also anti-HIV positive, were tested with HCVpp from different genotypes (1a, 1b, 3 and 4) and with HCVcc (H77/JFH1). Seventeen HIV-seropositive and 13 HIV-seronegative patients with decompensated liver disease were studied before and after liver transplant. RESULTS Serum neutralization was markedly lower after liver transplant and in HIV patients than in mono-infected immune-competent individuals. This effect was due to low antibody-mediated neutralization. In HIV patients, low neutralization was correlated with low lymphocyte T CD4 cell counts and the severity of liver disease. To characterize neutralization, we tested HCVpp lacking hypervariable region (HVR1) and SR-BI receptor cholesterol transfer inhibition by BLT-4. These experiments showed that neutralization was strongly dependent on the HVR1 and the SR-BI receptor. HVR1 sequences showed that selective pressures were low in immune-compromised patients and highly correlated to HCV neutralization after liver transplant. Neutralization experiments were reproduced with HCV strain JFH1. CONCLUSION Serum neutralization in HIV-coinfected patients and HCV-infected liver transplant recipients is poor enhancing HCV entry through HVR1/SR-BI interplay. This may contribute to the severity of hepatitis C in these settings.
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20
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Alhammad YMO, Maharajh S, Butcher R, Eden JS, White PA, Poumbourios P, Drummer HE. Longitudinal Sequence and Functional Evolution within Glycoprotein E2 in Hepatitis C Virus Genotype 3a Infection. PLoS One 2015; 10:e0126397. [PMID: 25970466 PMCID: PMC4430534 DOI: 10.1371/journal.pone.0126397] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2015] [Accepted: 04/01/2015] [Indexed: 01/16/2023] Open
Abstract
The E2 glycoprotein of Hepatitis C virus (HCV) is a major target of the neutralizing antibody (NAb) response with the majority of epitopes located within its receptor binding domain (RBD; 384–661). Within E2 are three variable regions located at the N-terminus (HVR1; 384–411), and internally at 460–480 (HVR2) and 570–580 [intergenotypic variable region (igVR)], all of which lie outside a conserved core domain that contains the CD81 binding site, essential for attachment of virions to host cells and a major target of NAbs. In this study, we examined the evolution of the E1 and E2 region in two patients infected with genotype 3a virus. Whereas one patient was able to clear the acute infection, the other developed a chronic infection. Mutations accumulated at multiple positions within the N-terminal HVR1 as well as within the igVR in both patients over time, whereas mutations in HVR2 were observed only in the chronically infected patient. Mutations within or adjacent to the CD81 contact site were observed in both patients but were less frequent and more conservative in the patient that cleared his/her infection. The evolution of CD81 binding function and antigenicity was examined with longitudinal E2 RBD sequences. The ability of the RBD to bind CD81 was completely lost by week 108 in the patient that developed chronic HCV. In the second patient, the ability of the week 36 RBD, just prior to viral clearance, to bind CD81 was reduced ~50% relative to RBD sequences obtained earlier. The binding of a NAb specific to a conserved epitope located within E2 residues 411–428 was significantly reduced by week 108 despite complete conservation of its epitope suggesting that E2 antigenicity is allosterically modulated. The exposure of non-neutralizing antibody epitopes was similarly explored and we observed that the epitope of 3 out of 4 non-NAbs were significantly more exposed in the RBDs representing the late timepoints in the chronic patient. By contrast, the exposure of non-neutralizing epitopes was reduced in the patient that cleared his/her infection and could in part be attributed to sequence changes in the igVR. These studies reveal that during HCV infection, the exposure of the CD81 binding site on E2 becomes increasingly occluded, and the antigenicity of the E2 RBD towards both neutralizing and non-neutralizing antibodies is modulated via allosteric mechanisms.
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Affiliation(s)
- Yousef M. O. Alhammad
- Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, 3004, Australia
- Department of Microbiology, Monash University, Clayton, Victoria, Australia
| | - Sanvir Maharajh
- Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, 3004, Australia
- Department of Microbiology, Monash University, Clayton, Victoria, Australia
| | - Rebecca Butcher
- Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, 3004, Australia
| | - John-Sebastian Eden
- School of Biological Sciences and Sydney Medical School, Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia
| | - Peter A. White
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia
| | - Pantelis Poumbourios
- Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, 3004, Australia
- Department of Microbiology, Monash University, Clayton, Victoria, Australia
| | - Heidi E Drummer
- Centre for Biomedical Research, Burnet Institute, 85 Commercial Rd, Melbourne, 3004, Australia
- Department of Microbiology, Monash University, Clayton, Victoria, Australia
- Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- * E-mail:
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21
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Bailey JR, Dowd KA, Snider AE, Osburn WO, Mehta SH, Kirk GD, Thomas DL, Ray SC. CD4+ T-Cell-Dependent Reduction in Hepatitis C Virus-Specific Neutralizing Antibody Responses After Coinfection With Human Immunodeficiency Virus. J Infect Dis 2015; 212:914-23. [PMID: 25754978 DOI: 10.1093/infdis/jiv139] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2014] [Accepted: 02/26/2015] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Human immunodeficiency virus (HIV) infection leads to lower rates of hepatitis C virus (HCV) clearance after acute infection, higher HCV viremia, and accelerated progression of HCV-related fibrosis. The mechanisms underlying this acceleration of HCV progression by HIV are poorly understood, but HIV-induced dysfunction in the anti-HCV humoral immune response may play a role. METHODS To define the effect of HIV coinfection on the anti-HCV antibody response, we measured anti-HCV envelope binding antibody titers, neutralizing antibody (nAb) titers, and nAb breadth of serum from HCV-infected subjects isolated longitudinally before and after incident HIV infection. RESULTS A significant reduction in HCV envelope-specific binding antibody and nAb titers was detected in subjects with CD4(+) T-cell counts <350/mm(3) after HIV infection, and subjects with CD4(+) T-cell counts <200/mm(3) also showed a reduction in nAb breadth. Subjects who maintained CD4(+) T-cell counts ≥350/mm(3) displayed little to no decline in antibody levels. CONCLUSIONS Depletion of CD4(+) T cells by HIV infection results in a global decline in the anti-HCV envelope antibody response, including binding antibody titers, nAb titers, and nAb breadth.
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Affiliation(s)
| | - Kimberly A Dowd
- Viral Pathogenesis Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
| | - Anna E Snider
- Division of Infectious Diseases, Department of Medicine
| | | | - Shruti H Mehta
- Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore
| | - Gregory D Kirk
- Division of Infectious Diseases, Department of Medicine Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore
| | | | - Stuart C Ray
- Division of Infectious Diseases, Department of Medicine Department of Oncology, Johns Hopkins University School of Medicine
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Meshram RJ, Gacche RN. Effective epitope identification employing phylogenetic, mutational variability, sequence entropy, and correlated mutation analysis targeting NS5B protein of hepatitis C virus: from bioinformatics to therapeutics. J Mol Recognit 2015; 28:492-505. [PMID: 25727409 DOI: 10.1002/jmr.2466] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2014] [Revised: 11/21/2014] [Accepted: 01/16/2015] [Indexed: 12/13/2022]
Abstract
Hepatitis C virus (HCV) is considered as a foremost cause affecting numerous human liver-related disorders. An effective immuno-prophylactic measure (like stable vaccine) is still unavailable for HCV. We perform an in silico analysis of nonstructural protein 5B (NS5B) based CD4 and CD8 epitopes that might be implicated in improvement of treatment strategies for efficient vaccine development programs against HCV. Here, we report on effective utilization of knowledge obtained from multiple sequence alignment and phylogenetic analysis for investigation and evaluation of candidate epitopes that have enormous potential to be used in formulating proficient vaccine, embracing multiple strains prevalent among major geographical locations. Mutational variability data discussed herein focus on discriminating the region under active evolutionary pressure from those having lower mutational potential in existing experimentally verified epitopes, thus, providing a concrete framework for designing an effective peptide-based vaccine against HCV. Additionally, we measured entropy distribution in NS5B residues and pinpoint the positions in epitopes that are more susceptible to mutations and, thus, account for virus strategy to evade the host immune system. Findings from this study are expected to add more details on the sequence and structural aspects of NS5B protein, ultimately facilitating our understanding about the pathophysiology of HCV and assisting advance studies on the function of NS5B antigen on the epitope level. We also report on the mutational crosstalk between functionally important coevolving residues, using correlated mutation analysis, and identify networks of coupled mutations that represent pathways of allosteric communication inside and among NS5B thumb, finger, and palm domains.
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23
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Immunological mechanisms controlling hepatitis C virus infection. ASIAN PACIFIC JOURNAL OF TROPICAL DISEASE 2015. [DOI: 10.1016/s2222-1808(14)60633-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Luo K, Li S, Jiang L, Zuo T, Qing J, Shi X, Liu Y, Wu H, Chen X, Zhang L. Combinatorial library-based profiling of the antibody response against hepatitis C virus in humans. J Gen Virol 2014; 96:52-63. [PMID: 25274855 DOI: 10.1099/vir.0.069278-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The antibody response plays a crucial role against hepatitis C virus (HCV) infection, and our understanding of this intricate progress in vivo is far from complete. We previously reported a novel and robust technique based on a large combinatorial viral antigen library displayed on the surface of the yeast Saccharomyces cerevisiae, allowing comprehensive profiling of polyclonal antibody responses in vivo in both qualitative and quantitative terms. Here, we report the generation and application of a combinatorial library of HCV strain JFH1 envelope glycoprotein to profile the antibody response in four HCV chronically infected individuals. By systematic analysis of the location and frequency of antigenic fragments along the JFH1 envelope glycoprotein, we showed that the major binding antibody response was targeted to E2 (80.9-99.8 %), whilst that against E1 was relatively small (0.3-19.0 %). A total of five major antigenic domains (D1-D5) were identified: one was within E1 and an additional four within E2, despite substantial variability among the different individuals. However, serum absorption with the yeast clones containing the antigenic domain D1 resulted in more reduction in neutralizing antibody activity against pseudotyped HCV than those in E2, suggesting that E1 contains additional neutralizing epitopes. Our results have provided additional insights into the HCV-specific antibody response in humans and should assist in a better understanding of protective antibody immunity and in guiding the development of effective vaccines and therapeutics against HCV infection.
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Affiliation(s)
- Kan Luo
- Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, PR China
| | - Shu Li
- Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, PR China
| | - Liwei Jiang
- Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, PR China
| | - Teng Zuo
- Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, PR China
| | - Jie Qing
- Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, PR China
| | - Xuanling Shi
- Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, PR China
| | - Yali Liu
- Center for Infectious Diseases, Beijing You'an Hospital, Capital Medical University, Beijing, PR China
| | - Hao Wu
- Center for Infectious Diseases, Beijing You'an Hospital, Capital Medical University, Beijing, PR China
| | - Xinyue Chen
- Center for Infectious Diseases, Beijing You'an Hospital, Capital Medical University, Beijing, PR China
| | - Linqi Zhang
- Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, PR China
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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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26
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Ikram A, Anjum S, Tahir M. In Silico Identification and Conservation Analysis of B-cell and T-Cell Epitopes of Hepatitis C Virus 3a Genotype Enveloped Glycoprotein 2 From Pakistan: A Step Towards Heterologous Vaccine Design. HEPATITIS MONTHLY 2014; 14:e9832. [PMID: 24976845 PMCID: PMC4071360 DOI: 10.5812/hepatmon.9832] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/28/2013] [Revised: 07/22/2013] [Accepted: 10/17/2013] [Indexed: 12/11/2022]
Abstract
BACKGROUND Hepatitis C virus (HCV) is known for the eminent global disease burden responsible for encumbering public health. Development of an effective vaccine is the major need of the day; however, several obstacles loom ahead of this objective. One of the major barriers is that as a RNA virus, it mutates rapidly resulting in high sequence divergence and several viral isolates in the world. Theglycoprotein 2 (gpE2) is the primary component of HCV envelope with direct interaction with the host cell surface receptors; it is an indispensable target of neutralizing antibodies and hence, should be a fundamental component of vaccine design. OBJECTIVES This study focused on B-cells and T-cells epitopes prediction in HCV gpE2, particularly in 3a genotype, in Pakistan and identification of the conserved epitopes among various 3a isolates at global level, principally conserved across HCV major genotypes. MATERIALS AND METHODS Epitope finding was done by using online available bioinformatics tools including Immune Epitope Database (IEDB), ProPred-I, and ProPred. Conservation of these epitopes was found by aligning selected gpE2 sequences using MultAlin online software and conservancy analysis tool available at IEDB. RESULTS Many B-cell and T-cell epitopes predicted in gpE2 were found conserved among HCV 3a genotypes whereas few were conserved in other genotypes anticipating these epitopes as potential candidates of producing strong B-cell and T-cell response against HCV 3a and other genotypes. CONCLUSIONS HCV gpE2 is an ideal target for HCV vaccine. Prediction of epitope immunogenicity and characterization on the basis of peptide sequences will be significantly helpful for development of a heterologous vaccine against HCV variants.
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Affiliation(s)
- Aqsa Ikram
- Atta-Ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan
| | - Sadia Anjum
- Atta-Ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan
- Corresponding Author: Sadia Anjum, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan. Tel: +92-5190856152 Fax+92-5190856102, E-mail:
| | - Muhammad Tahir
- Atta-Ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan
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27
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Osburn WO, Snider AE, Wells BL, Latanich R, Bailey JR, Thomas DL, Cox AL, Ray SC. Clearance of hepatitis C infection is associated with the early appearance of broad neutralizing antibody responses. Hepatology 2014; 59:2140-51. [PMID: 24425349 PMCID: PMC4043926 DOI: 10.1002/hep.27013] [Citation(s) in RCA: 215] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Accepted: 01/10/2014] [Indexed: 12/12/2022]
Abstract
UNLABELLED The contribution of humoral immune responses to spontaneous control of hepatitis C virus (HCV) infection remains unclear. We assessed neutralizing antibody (nAb) responses during acute HCV infection to determine whether infection outcome is associated with the nAb response, specifically, its timing or breadth (neutralization of multiple genotype-matched variants). A representative genotype 1 HCV pseudoparticle (HCVpp) library, consisting of 19 genetically distinct genotype 1 HCVpp that comprise the natural variability of genotype 1 E1E2 sequences, was used to assess anti-genotype 1 nAb responses during acute infection in at-risk persons followed prospectively. Neutralization of individual library HCVpp by the last viremic plasma sample obtained before clearance was compared to either 1-year post-initial viremia or clearance time-matched specimens obtained from subjects developing persistent infection. In persistently infected persons nAb responses were delayed then progressively broadened, whereas in persons who controlled viremia broader responses were detected early and contracted after clearance of viremia. Surprisingly, the breadth of anti-genotype 1 nAb responses was not dependent on subjects' infection genotype. Also, individual library HCVpp neutralization sensitivity was not associated with any known E2 sequence determinants. Interestingly, two single nucleotide polymorphisms in the HLA-DQ locus were associated with nAb breadth. CONCLUSION Control of HCV infection is associated with more rapid development of a broad nAb response, independent of the infection viral genotype, providing further evidence for the role of nAb in controlling HCV infection and the potential benefit of generating broad anti-HCV nAb responses by vaccination.
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Affiliation(s)
- William O. Osburn
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
| | - Anna E. Snider
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
| | - Brittany L. Wells
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
| | - Rachel Latanich
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
| | - Justin R. Bailey
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
| | - David L. Thomas
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
| | - Andrea L. Cox
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
| | - Stuart C. Ray
- Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205
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Hamed MR, Brown RJ, Zothner C, Urbanowicz RA, Mason CP, Krarup A, McClure CP, Irving WL, Ball JK, Harris M, Hickling TP, Tarr AW. Recombinant human L-ficolin directly neutralizes hepatitis C virus entry. J Innate Immun 2014; 6:676-84. [PMID: 24854201 PMCID: PMC6741592 DOI: 10.1159/000362209] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2013] [Revised: 03/16/2014] [Accepted: 03/16/2014] [Indexed: 12/25/2022] Open
Abstract
L-ficolin is a soluble pattern recognition molecule expressed by the liver that contributes to innate immune defense against microorganisms. It is well described that binding of L-ficolin to specific pathogen-associated molecular patterns activates the lectin complement pathway, resulting in opsonization and lysis of pathogens. In this study, we demonstrated that in addition to this indirect effect, L-ficolin has a direct neutralizing effect against hepatitis C virus (HCV) entry. Specific, dose-dependent binding of recombinant L-ficolin to HCV glycoproteins E1 and E2 was observed. This interaction was inhibited by soluble L-ficolin ligands. Interaction of L-ficolin with E1 and E2 potently inhibited entry of retroviral pseudoparticles bearing these glycoproteins. L-ficolin also inhibited entry of cell-cultured HCV in a calcium-dependent manner. Neutralizing concentrations of L-ficolin were found to be circulating in the serum of HCV-infected individuals. This is the first description of direct neutralization of HCV entry by a ficolin and highlights a novel role for L-ficolin as a virus entry inhibitor.
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Affiliation(s)
- Mohamed R. Hamed
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
- Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt
| | - Richard J.P. Brown
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Carsten Zothner
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - Richard A. Urbanowicz
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Christopher P. Mason
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Anders Krarup
- Biochemistry Department, University of Oxford, Oxford, UK
| | - C. Patrick McClure
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - William L. Irving
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Jonathan K. Ball
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
| | - Timothy P. Hickling
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
| | - Alexander W. Tarr
- School of Life Sciences, and Nottingham Digestive Diseases Biomedical Research Unit, University of Nottingham, Nottingham, UK
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29
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Zhu YZ, Qian XJ, Zhao P, Qi ZT. How hepatitis C virus invades hepatocytes: The mystery of viral entry. World J Gastroenterol 2014; 20:3457-3467. [PMID: 24707128 PMCID: PMC3974512 DOI: 10.3748/wjg.v20.i13.3457] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Revised: 12/03/2013] [Accepted: 01/05/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infection is a global health problem, with an estimated 170 million people being chronically infected. HCV cell entry is a complex multi-step process, involving several cellular factors that trigger virus uptake into the hepatocytes. The high- density lipoprotein receptor scavenger receptor class B type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the main receptors that mediate the initial step of HCV infection. In addition, the virus uses cell receptor tyrosine kinases as entry regulators, such as epidermal growth factor receptor and ephrin receptor A2. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced.
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30
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Larrubia JR, Moreno-Cubero E, Lokhande MU, García-Garzón S, Lázaro A, Miquel J, Perna C, Sanz-de-Villalobos E. Adaptive immune response during hepatitis C virus infection. World J Gastroenterol 2014; 20:3418-3430. [PMID: 24707125 PMCID: PMC3974509 DOI: 10.3748/wjg.v20.i13.3418] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2013] [Revised: 09/28/2013] [Accepted: 11/29/2013] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infection affects about 170 million people worldwide and it is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV is a hepatotropic non-cytopathic virus able to persist in a great percentage of infected hosts due to its ability to escape from the immune control. Liver damage and disease progression during HCV infection are driven by both viral and host factors. Specifically, adaptive immune response carries out an essential task in controlling non-cytopathic viruses because of its ability to recognize infected cells and to destroy them by cytopathic mechanisms and to eliminate the virus by non-cytolytic machinery. HCV is able to impair this response by several means such as developing escape mutations in neutralizing antibodies and in T cell receptor viral epitope recognition sites and inducing HCV-specific cytotoxic T cell anergy and deletion. To impair HCV-specific T cell reactivity, HCV affects effector T cell regulation by modulating T helper and Treg response and by impairing the balance between positive and negative co-stimulatory molecules and between pro- and anti-apoptotic proteins. In this review, the role of adaptive immune response in controlling HCV infection and the HCV mechanisms to evade this response are reviewed.
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Aguilar-Noriega D, Alvarez-Lajonchere L, Brown E, Santana FL, Dubuisson J, Wychowski C, Guerra I, Martínez-Donato G, Pérez A, Amador-Cañizares Y, Dueñas-Carrera S. A chimeric protein encompassing hepatitis C virus epitopes is able to elicit both humoral and cell-mediated immune responses in mice. Biotechnol Appl Biochem 2014; 61:627-36. [PMID: 24575938 DOI: 10.1002/bab.1223] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2013] [Accepted: 02/19/2014] [Indexed: 12/14/2022]
Abstract
Hepatitis C virus (HCV) infection is a worldwide health problem. Vaccines against this pathogen are not available and advances in this field are limited because of the high genetic variability of the virus, inaccessibility of animal models, and incomplete definition of immunological correlates of protection. In the present work, a chimeric protein, Eq1, encompassing HCV amino acid regions from structural antigens, was generated. Eq1 was expressed in GC-366 bacterial cells. After cell disruption, Eq1 was purified from the insoluble fraction by sequential steps of differential solubilization and metal chelating affinity chromatography. Eq1 was specifically recognized by anti-HCV positive human sera. Moreover, immunization of BALB/c mice with different doses of Eq1 formulated either in Alum or Freund's incomplete adjuvant elicited both humoral- and cellular-specific immune responses. Doses of 20 µg of Eq1 induced the strongest cell-mediated immune responses and only the formulation of this dose in Alum elicited a neutralizing antibody response against heterologous cell culture HCV. All these data together indicate that Eq1 is immunogenic in mice and might be an interesting component of vaccine candidates against HCV infection.
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32
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Bukong TN, Kodys K, Szabo G. A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry. Int J Pept Res Ther 2014; 20:269-276. [PMID: 25379035 DOI: 10.1007/s10989-013-9390-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin-moesin-radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry.
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Affiliation(s)
- Terence N Bukong
- Department of Medicine, University of Massachusetts Medical School, LRB208, 364 Plantation Street, Worcester, MA 01605, USA
| | - Karen Kodys
- Department of Medicine, University of Massachusetts Medical School, LRB208, 364 Plantation Street, Worcester, MA 01605, USA
| | - Gyongyi Szabo
- Department of Medicine, University of Massachusetts Medical School, LRB208, 364 Plantation Street, Worcester, MA 01605, USA
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33
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Bellier B, Klatzmann D. Virus-like particle-based vaccines against hepatitis C virus infection. Expert Rev Vaccines 2014; 12:143-54. [DOI: 10.1586/erv.13.10] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Anjum S, Wahid A, Afzal MS, Albecka A, Alsaleh K, Ahmad T, Baumert TF, Wychowski C, Qadri I, Penin F, Dubuisson J. Additional glycosylation within a specific hypervariable region of subtype 3a of hepatitis C virus protects against virus neutralization. J Infect Dis 2013; 208:1888-1897. [PMID: 23908491 DOI: 10.1093/infdis/jit376] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND The envelope glycoprotein E2 of hepatitis C virus (HCV) contains several hypervariable regions. Interestingly, 2 regions of intragenotypic hypervariability within E2 have been described as being specific to HCV subtype 3a. Based on their amino acid position in E2, they were named HVR495 and HVR575. Here, we further investigated these regions in order to better understand their role in HCV infection. METHODS Sequences of HCV envelope glycoproteins from Pakistani patients infected with subtype 3a were cloned and compared with other subtype 3a sequences. The entry functions and the sensitivity to antibody neutralization of selected HCV glycoprotein sequences were tested in the HCV pseudotyped particles (HCVpp) system. In addition, the cell-cultured HCV system (HCVcc) was also used to confirm some of the data obtained with the HCVpp system. RESULTS We observed interesting new features within HVR495 and HVR575 for several subtype 3a isolates. Indeed, changes in glycosylation sites were observed with the appearance of a new glycosylation site within HVR495. Importantly, HCVpp and HCVcc that contained this new HVR495 glycosylation site were less sensitive to antibody neutralization. CONCLUSIONS We identified a new glycosylation site within the HVR495 region of HCV subtype 3a that has a protective effect against antibody neutralization.
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Affiliation(s)
- Sadia Anjum
- Institut Pasteur de Lille, Center for Infection and Immunity of Lille
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35
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Sequence and functional analysis of the envelope glycoproteins of hepatitis C virus variants selectively transmitted to a new host. J Virol 2013; 87:13609-18. [PMID: 24109215 DOI: 10.1128/jvi.02119-13] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) remains a challenging public health problem worldwide. The identification of viral variants establishing de novo infections and definition of the phenotypic requirements for transmission would facilitate the design of preventive strategies. We explored the transmission of HCV variants in three cases of acute hepatitis following needlestick accidents. We used single-genome amplification of glycoprotein E1E2 gene sequences to map the genetic bottleneck upon transmission accurately. We found that infection was likely established by a single variant in two cases and six variants in the third case. Studies of donor samples showed that the transmitted variant E1E2 amino acid sequences were identical or closely related to those of variants from the donor virus populations. The transmitted variants harbored a common signature site at position 394, within hypervariable region 1 of E2, together with additional signature amino acids specific to each transmission pair. Surprisingly, these E1E2 variants conferred no greater capacity for entry than the E1E2 derived from nontransmitted variants in lentiviral pseudoparticle assays. Mutants escaping the antibodies of donor sera did not predominate among the transmitted variants either. The fitness parameters affecting the selective outgrowth of HCV variants after transmission in an immunocompetent host may thus be more complex than those suggested by mouse models. Human antibodies directed against HCV envelope effectively cross-neutralized the lentiviral particles bearing E1E2 derived from transmitted variants. These findings provide insight into the molecular mechanisms underlying HCV transmission and suggest that viral entry is a potential target for the prevention of HCV infection.
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36
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Tarr AW, Lafaye P, Meredith L, Damier-Piolle L, Urbanowicz RA, Meola A, Jestin JL, Brown RJP, McKeating JA, Rey FA, Ball JK, Krey T. An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell transmission. Hepatology 2013; 58:932-9. [PMID: 23553604 DOI: 10.1002/hep.26430] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/06/2013] [Accepted: 03/25/2013] [Indexed: 12/26/2022]
Abstract
UNLABELLED Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver-transplanted patients a challenging task. Virus-specific therapeutic antibodies are generally safe and well-tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy-chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage-display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture-derived particles by interfering with the E2-CD81 interaction. In contrast to some of the most broadly neutralizing human anti-E2 monoclonal antibodies, D03 efficiently inhibited HCV cell-to-cell transmission. CONCLUSION This is the first description of a potent and broadly neutralizing HCV-specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell-to-cell transmission.
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Affiliation(s)
- Alexander W Tarr
- School of Molecular Medical Sciences, The University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom
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37
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Lu J, Tao W, Li R, Xiang Y, Zhang N, Xiang X, Xie Q, Zhong J. Construction and characterization of infectious hepatitis C virus chimera containing structural proteins directly from genotype 1b clinical isolates. Virology 2013; 443:80-8. [DOI: 10.1016/j.virol.2013.04.030] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2013] [Revised: 04/10/2013] [Accepted: 04/27/2013] [Indexed: 12/17/2022]
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Esteban-Riesco L, Depaulis F, Moreau A, Bacq Y, Dubois F, Goudeau A, Gaudy-Graffin C. Rapid and sustained autologous neutralizing response leading to early spontaneous recovery after HCV infection. Virology 2013; 444:90-9. [PMID: 23890816 DOI: 10.1016/j.virol.2013.05.037] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2013] [Revised: 03/27/2013] [Accepted: 05/29/2013] [Indexed: 01/12/2023]
Abstract
After HCV infection, the association between the humoral response and viral sequence evolution remains unclear. We investigated the mechanisms leading to early HCV clearance and spontaneous recovery in two patients. The early evolution of the HCV envelope glycoproteins, and the infectivity spectrum of variants were explored using retroviral pseudoparticles bearing HCV envelopes. Ability of the autologous neutralizing response to control these variants was analyzed. For the first case, the maximum neutralizing activity was for serum collected between two and three months post ALT peak, this activity was still detectable after 30 months. For the second case, autologous neutralizing activity against the variant isolated at the ALT peak was detected in every serum collected between 4 days and 13 months after. The neutralizing response was sustained beyond the time at which the virus was cleared. This raise interesting questions about the role of such antibodies in case of re-exposure.
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Structural and antigenic definition of hepatitis C virus E2 glycoprotein epitopes targeted by monoclonal antibodies. Clin Dev Immunol 2013; 2013:450963. [PMID: 23935648 PMCID: PMC3722892 DOI: 10.1155/2013/450963] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2013] [Accepted: 06/10/2013] [Indexed: 12/24/2022]
Abstract
Hepatitis C virus (HCV) is the major cause of chronic liver disease as well as the major indication for liver transplantation worldwide. Current standard of care is not completely effective, not administrable in grafted patients, and burdened by several side effects. This incomplete effectiveness is mainly due to the high propensity of the virus to continually mutate under the selective pressure exerted by the host immune response as well as currently administered antiviral drugs. The E2 envelope surface glycoprotein of HCV (HCV/E2) is the main target of the host humoral immune response and for this reason one of the major variable viral proteins. However, broadly cross-neutralizing monoclonal antibodies (mAbs) directed against HCV/E2 represent a promising tool for the study of virus-host interplay as well as for the development of effective prophylactic and therapeutic approaches. In the last few years many anti-HCV/E2 mAbs have been evaluated in preclinical and clinical trials as possible candidate antivirals, particularly for administration in pre- and post-transplant settings. In this review we summarize the antigenic and structural characteristics of HCV/E2 determined through the use of anti-HCV/E2 mAbs, which, given the absence of a crystal structure of this glycoprotein, represent currently the best tool available.
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40
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Wahid A, Dubuisson J. Virus-neutralizing antibodies to hepatitis C virus. J Viral Hepat 2013; 20:369-76. [PMID: 23647953 DOI: 10.1111/jvh.12094] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/10/2013] [Accepted: 02/26/2013] [Indexed: 02/06/2023]
Abstract
For a long time, the lack of an appropriate cell culture system has hampered the study of neutralizing antibody responses against hepatitis C virus (HCV). However, the last decade has seen the development of several model systems that have significantly advanced our understanding of viral entry and antibody neutralization. Studies of acutely infected patients suggest that a strong and early production of neutralizing antibodies may contribute to control the virus during the acute phase of HCV infection and facilitate viral elimination by cellular immune responses. It also emerges that the early antibody response mainly targets hypervariable region 1 (HVR1) of the envelope glycoprotein E2. This host response can lead to viral escape from neutralization by rapid amino acid changes in this hypervariable region. In contrast, cross-reactive neutralizing antibodies seem to appear later during HCV infection, and several mechanisms contribute to reduce their accessibility to their cognate epitopes. These include the masking of major conserved neutralizing epitopes by HVR1, specific N-linked glycans and the lipid moiety of the viral particle. Other potential mechanisms of evasion from the neutralizing antibody response include a modulation by high-density lipoproteins and interfering antibodies as well as the capacity of the virus to be transferred by cell-to-cell contacts. Finally, the recent identification of several highly conserved neutralizing epitopes provides some opportunities for the design and development of vaccine candidates that elicit a protective humoral immune response.
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Affiliation(s)
- A Wahid
- Center for Infection & Immunity of Lille CIIL, Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Univ Lille Nord de France, Lille, France
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41
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Beaumont E, Patient R, Hourioux C, Dimier-Poisson I, Roingeard P. Chimeric hepatitis B virus/hepatitis C virus envelope proteins elicit broadly neutralizing antibodies and constitute a potential bivalent prophylactic vaccine. Hepatology 2013; 57:1303-13. [PMID: 23150224 DOI: 10.1002/hep.26132] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Revised: 10/17/2012] [Accepted: 10/17/2012] [Indexed: 12/12/2022]
Abstract
UNLABELLED The development of a prophylactic vaccine against hepatitis C virus (HCV) has become an important medical priority, because 3-4 million new HCV infections are thought to occur each year worldwide. Hepatitis B virus (HBV) is another major human pathogen, but infections with this virus can be prevented with a safe, efficient vaccine, based on the remarkable ability of the envelope protein (S) of this virus to self-assemble into highly immunogenic subviral particles. Chimeric HBV-HCV envelope proteins in which the N-terminal transmembrane domain of S was replaced with the transmembrane domain of the HCV envelope proteins (E1 or E2) were efficiently coassembled with the wild-type HBV S protein into subviral particles. These chimeric particles presented the full-length E1 and E2 proteins from a genotype 1a virus in an appropriate conformation for formation of the E1-E2 heterodimer. Produced in stably transduced Chinese hamster ovary cells and used to immunize New Zealand rabbits, these particles induced a strong specific antibody (Ab) response against the HCV and HBV envelope proteins in immunized animals. Sera containing anti-E1 or anti-E2 Abs elicited by these particles neutralized infections with HCV pseudoparticles and cell-cultured viruses derived from different heterologous 1a, 1b, 2a, and 3 strains. Moreover, the anti-hepatitis B surface response induced by these chimeric particles was equivalent to the response induced by a commercial HBV vaccine. CONCLUSIONS Our results provide support for approaches based on the development of bivalent HBV-HCV prophylactic vaccine candidates potentially able to prevent initial infection with either of these two hepatotropic viruses.
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Affiliation(s)
- Elodie Beaumont
- INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France
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42
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Koutsoudakis G, Forns X, Pérez-Del-Pulgar S. [The molecular biology of hepatitis C virus]. GASTROENTEROLOGIA Y HEPATOLOGIA 2013; 36:280-93. [PMID: 23490024 DOI: 10.1016/j.gastrohep.2012.11.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/09/2012] [Accepted: 11/13/2012] [Indexed: 12/12/2022]
Abstract
Since the discovery of the hepatitis C virus (HCV), a plethora of experimental models have evolved, allowing the virus's life cycle and the pathogenesis of associated liver diseases to be investigated. These models range from inoculation of cultured cells with serum from patients with hepatitis C to the use of surrogate models for the study of specific stages of the HCV life cycle: retroviral pseudoparticles for the study of HCV entry, replicons for the study of HCV replication, and the HCV cell culture model, which reproduces the entire life cycle (replication and production of infectious particles). The use of these tools has been and remains crucial to identify potential therapeutic targets in the different stages of the virus's life cycle and to screen new antiviral drugs. A clear example is the recent approval of two viral protease inhibitors (boceprevir and telaprevir) in combination with pegylated interferon and ribavirin for the treatment of chronic hepatitis C. This review analyzes the advances made in the molecular biology of HCV and highlights possible candidates as therapeutic targets for the treatment of HCV infection.
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Affiliation(s)
- George Koutsoudakis
- Servicio de Hepatología, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Barcelona, España
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43
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Control of regulatory T cells is necessary for vaccine-like effects of antiviral immunotherapy by monoclonal antibodies. Blood 2012; 121:1102-11. [PMID: 23264590 DOI: 10.1182/blood-2012-06-432153] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Regulatory T cells (Tregs) down-regulate immunity and are associated with chronic viral infections, suggesting that their inhibition might be used to treat life-threatening diseases. Using the FrCasE mouse retroviral model, we have recently shown that short mAb-based immunotherapies can induce life-long protective immunity. This finding has a potentially important therapeutical impact because mAbs are increasingly used to treat severe viral infections. We now report that poor anti-FrCasE immunity in infected mice is due to Treg expansion in secondary lymphoid organs because depletion of Tregs restored humoral and cytotoxic T lymphocyte (CTL) antiviral responses. Kinetic analyses show that Treg expansion is not a consequence of chronicity, but rather is associated with viral spread. Moreover, Treg adoptive transfers indicate that production of the immunosuppressive cytokine IL-10 is essential for preventing a protective immune response. Finally, treatment of infected mice with a virus-neutralizing IgG2a shortly after infection prevents Treg expansion and limits immunosuppressive activity. This effect is rapid, necessary for the development of protective immunity, and depends on mAb effector functions. Therefore, manipulating Tregs may be necessary to confer robust antiviral immunity in the context of mAb-based therapy. This concept likely applies to cancer treatment because vaccine-like effects of mAbs have also been observed in certain cancer immunotherapies.
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Reyes-del Valle J, de la Fuente C, Turner MA, Springfeld C, Apte-Sengupta S, Frenzke ME, Forest A, Whidby J, Marcotrigiano J, Rice CM, Cattaneo R. Broadly neutralizing immune responses against hepatitis C virus induced by vectored measles viruses and a recombinant envelope protein booster. J Virol 2012; 86:11558-66. [PMID: 22896607 PMCID: PMC3486281 DOI: 10.1128/jvi.01776-12] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2012] [Accepted: 08/06/2012] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.
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Affiliation(s)
- Jorge Reyes-del Valle
- Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
| | - Cynthia de la Fuente
- Laboratory of Virology and Infectious Diseases, Center for the Study of Hepatitis C, Rockefeller University, New York, New York, USA
| | - Mallory A. Turner
- Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
| | - Christoph Springfeld
- Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
| | - Swapna Apte-Sengupta
- Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
| | - Marie E. Frenzke
- Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
| | - Amelie Forest
- Laboratory of Virology and Infectious Diseases, Center for the Study of Hepatitis C, Rockefeller University, New York, New York, USA
| | - Jillian Whidby
- Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USA
| | - Joseph Marcotrigiano
- Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USA
| | - Charles M. Rice
- Laboratory of Virology and Infectious Diseases, Center for the Study of Hepatitis C, Rockefeller University, New York, New York, USA
| | - Roberto Cattaneo
- Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
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Sautto G, Mancini N, Diotti RA, Solforosi L, Clementi M, Burioni R. Anti-hepatitis C virus E2 (HCV/E2) glycoprotein monoclonal antibodies and neutralization interference. Antiviral Res 2012; 96:82-9. [PMID: 22898087 DOI: 10.1016/j.antiviral.2012.07.013] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Revised: 07/27/2012] [Accepted: 07/30/2012] [Indexed: 01/06/2023]
Abstract
The suggested HCV escape mechanism consisting in the elicitation of antibody (Ab) subpopulations interfering with the neutralizing activity of other Abs has recently been questioned. In particular, it was originally reported that Abs directed against the 436-447 region (epitope II) of HCV/E2 glycoprotein may interfere with the neutralizing Abs directed against the 412-423 region (epitope I) involved in the binding to CD81. In this paper, we investigate on the molecular features of this phenomenon describing an anti-HCV/E2 monoclonal Ab (mAb) (e509) endowed with a weak neutralizing activity, and whose epitope is centered on epitope II. Interestingly, e509 influenced the potent neutralizing activity of AP33, one of the best characterized anti-HCV/E2 mAb, whereas it did not show any interfering activity against two other broadly neutralizing mAbs (e20 and e137), whose epitopes partially overlap with that of e509 and which possibly displace it from the antigen. These data may give a possible clue to interpret the conflicting studies published to date on the mechanism of interference, suggesting the existence of at least two groups of broadly neutralizing anti-HCV/E2 Abs: (i) those whose epitope is focused on the 412-423 CD81-binding region and whose activity may be hampered by other Abs directed against the 436-447 region, and (ii) those directed against CD81-binding regions but whose epitope contains also residues within the 436-447 region recognized by interfering mAbs, thus competing with them for binding. The conflicting results of previous studies may therefore depend on the relative amount of each of these two populations in the polyclonal preparations used. Overall, a better comprehension of this phenomenon may be of importance in the set up of novel mAb-based anti-HCV therapeutic strategies.
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Affiliation(s)
- Giuseppe Sautto
- Laboratorio di Microbiologia e Virologia, Università Vita-Salute San Raffaele, Milano, Italy
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A meta-analysis of the existing knowledge of immunoreactivity against hepatitis C virus (HCV). PLoS One 2012; 7:e38028. [PMID: 22675428 PMCID: PMC3364976 DOI: 10.1371/journal.pone.0038028] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2012] [Accepted: 04/29/2012] [Indexed: 02/06/2023] Open
Abstract
Approximately 3% of the world population is infected by HCV, which represents a major global health challenge. Almost 400 different scientific reports present immunological data related to T cell and antibody epitopes derived from HCV literature. Analysis of all HCV-related epitope hosted in the Immune Epitope Database (IEDB), a repository of freely accessible immune epitope data, revealed more than 1500 and 1900 distinct T cell and antibody epitopes, respectively. The inventory of all data revealed specific trends in terms of the host and the HCV genotypes from which sequences were derived. Upon further analysis we found that this large number of epitopes reflects overlapping structures, and homologous sequences derived from different HCV isolates. To access and visualize this information we developed a novel strategy that assembles large sets of epitope data, maps them onto reference genomes and displays the frequency of positive responses. Compilation of the HCV immune reactivity from hundreds of different studies, revealed a complex and thorough picture of HCV immune epitope data to date. The results pinpoint areas of more intense reactivity or research activities at the level of antibody, CD4 and CD8 responses for each of the individual HCV proteins. In general, the areas targeted by the different effector immune functions were distinct and antibody reactivity was positively correlated with hydrophilicity, while T cell reactivity correlated with hydrophobicity. At the sequence level, epitopes frequently recognized by both T cell and B cell correlated with low variability, and our analysis thus highlighted areas of potential interest for practical applications. The human reactivity was further analyzed to pinpoint differential patterns of reactivity associated with acute versus chronic infection, to reveal the apparent impact of glycosylation on T cell, but not antibody responses, and to highlight a paucity of studies involved antibody epitopes associated with virus neutralization.
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Huret C, Desjardins D, Miyalou M, Levacher B, Amadoudji Zin M, Bonduelle O, Combadière B, Dalba C, Klatzmann D, Bellier B. Recombinant retrovirus-derived virus-like particle-based vaccines induce hepatitis C virus-specific cellular and neutralizing immune responses in mice. Vaccine 2012; 31:1540-7. [PMID: 22634300 DOI: 10.1016/j.vaccine.2012.05.025] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2012] [Revised: 05/07/2012] [Accepted: 05/12/2012] [Indexed: 10/28/2022]
Abstract
While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses.
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