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Garbuglia AR, Pauciullo S, Zulian V, Del Porto P. Update on Hepatitis C Vaccine: Results and Challenges. Viruses 2024; 16:1337. [PMID: 39205311 PMCID: PMC11359353 DOI: 10.3390/v16081337] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2024] [Revised: 08/15/2024] [Accepted: 08/20/2024] [Indexed: 09/04/2024] Open
Abstract
Therapy against the Hepatitis C virus (HCV) has significantly improved with the introduction of direct-acting antiviral drugs (DAAs), achieving over 95% sustained virological response (SVR). Despite this, the development of an effective anti-HCV vaccine remains a critical challenge due to the low number of patients treated with DAAs and the occurrence of HCV reinfections in high-risk groups. Current vaccine strategies aim to stimulate either B-cell or T-cell responses. Vaccines based on E1 and E2 proteins can elicit broad cross-neutralizing antibodies against all major HCV genotypes, though with varying efficiencies and without full protection against infection. In humans, the neutralizing antibodies induced by such vaccines mainly target the AR3 region, but their levels are generally insufficient for broad neutralization. Various HCV proteins expressed through different viral vectors have been utilized to elicit T cell immune responses, showing sustained expansion of HCV-specific effector memory T cells and improved proliferation and polyfunctionality of memory T cells over time. However, despite these advancements, the frequency and effectiveness of T-cell responses remain limited.
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Affiliation(s)
- Anna Rosa Garbuglia
- Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani” (IRCCS), 00149 Rome, Italy; (S.P.); (V.Z.)
| | - Silvia Pauciullo
- Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani” (IRCCS), 00149 Rome, Italy; (S.P.); (V.Z.)
| | - Verdiana Zulian
- Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani” (IRCCS), 00149 Rome, Italy; (S.P.); (V.Z.)
| | - Paola Del Porto
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, 00100 Rome, Italy;
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2
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Santisteban Celis IC, Matoba N. Lectibodies as antivirals. Antiviral Res 2024; 227:105901. [PMID: 38734211 DOI: 10.1016/j.antiviral.2024.105901] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Revised: 05/02/2024] [Accepted: 05/05/2024] [Indexed: 05/13/2024]
Abstract
Growing concerns regarding the emergence of highly transmissible viral diseases highlight the urgent need to expand the repertoire of antiviral therapeutics. For this reason, new strategies for neutralizing and inhibiting these viruses are necessary. A promising approach involves targeting the glycans present on the surfaces of enveloped viruses. Lectins, known for their ability to recognize specific carbohydrate molecules, offer the potential for glycan-targeted antiviral strategies. Indeed, numerous studies have reported the antiviral effects of various lectins of both endogenous and exogenous origins. However, many lectins in their natural forms, are not suitable for use as antiviral therapeutics due to toxicity, other unfavorable pharmacological effects, and/or unreliable manufacturing sources. Therefore, improvements are crucial for employing lectins as effective antiviral therapeutics. A novel approach to enhance lectins' suitability as pharmaceuticals could be the generation of recombinant lectin-Fc fusion proteins, termed "lectibodies." In this review, we discuss the scientific rationale behind lectin-based antiviral strategies and explore how lectibodies could facilitate the development of new antiviral therapeutics. We will also share our perspective on the potential of these molecules to transcend their potential use as antiviral agents.
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Affiliation(s)
- Ian Carlosalberto Santisteban Celis
- Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA; Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville School of Medicine, Louisville, KY, USA
| | - Nobuyuki Matoba
- Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA; Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville School of Medicine, Louisville, KY, USA; UofL Health - Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA.
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3
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Chaudhuri D, Datta J, Majumder S, Giri K. Repurposing of drug molecules from FDA database against Hepatitis C virus E2 protein using ensemble docking approach. Mol Divers 2024; 28:1175-1188. [PMID: 37061608 DOI: 10.1007/s11030-023-10646-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Accepted: 03/31/2023] [Indexed: 04/17/2023]
Abstract
Hepatitis C virus, a member of the Flaviviridae family and genus Hepacivirus, is an enveloped, positively single stranded RNA virus. Its surface consists of a heterodimer of E1 and E2 proteins which play a crucial role in receptor binding and membrane fusion. In this study we have used in silico virtual screening by utilizing ensemble docking on the approved drugs. These drugs can bind with high efficiency to the 36 prominent conformations of the CD81 binding site clustered from a total of 3 µs MD simulation data on the E2 protein. We started with 9213 compounds from the FDA list of drugs and progressively came down to 5 compounds which have been seen to bind with very high efficiency to not only all the conformations but also the two predicted druggable pockets that encompass the CD81 binding site. MM/PBSA binding energy calculations also point to the highly stable interaction of the compounds to the E2 protein. This study may in future broaden the arsenal of therapeutics for use against HCV infection and lead to more effective care against the virus.
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Affiliation(s)
- Dwaipayan Chaudhuri
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India
| | - Joyeeta Datta
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India
| | - Satyabrata Majumder
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India
| | - Kalyan Giri
- Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073, India.
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4
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Tam EH, Peng Y, Cheah MXY, Yan C, Xiao T. Neutralizing antibodies to block viral entry and for identification of entry inhibitors. Antiviral Res 2024; 224:105834. [PMID: 38369246 DOI: 10.1016/j.antiviral.2024.105834] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Revised: 02/01/2024] [Accepted: 02/07/2024] [Indexed: 02/20/2024]
Abstract
Neutralizing antibodies (NAbs) are naturally produced by our immune system to combat viral infections. Clinically, neutralizing antibodies with potent efficacy and high specificity have been extensively used to prevent and treat a wide variety of viral infections, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Immunodeficiency Virus (HIV), Dengue Virus (DENV) and Hepatitis B Virus (HBV). An overwhelmingly large subset of clinically effective NAbs operates by targeting viral envelope proteins to inhibit viral entry into the host cell. Binding of viral envelope protein to the host receptor is a critical rate limiting step triggering a cascade of downstream events, including endocytosis, membrane fusion and pore formation to allow viral entry. In recent years, improved structural knowledge on these processes have allowed researchers to also leverage NAbs as an indispensable tool in guiding discovery of novel antiviral entry inhibitors, providing drug candidates with high efficacy and pan-genus specificity. This review will summarize the latest progresses on the applications of NAbs as effective entry inhibitors and as important tools to develop antiviral therapeutics by high-throughput drug screenings, rational design of peptidic entry inhibitor mimicking NAbs and in silico computational modeling approaches.
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Affiliation(s)
- Ee Hong Tam
- School of Biological Sciences, Nanyang Technological University 637551, Singapore; Institute of Structural Biology, Nanyang Technological University 636921, Singapore
| | - Yu Peng
- School of Biological Sciences, Nanyang Technological University 637551, Singapore; Institute of Structural Biology, Nanyang Technological University 636921, Singapore
| | - Megan Xin Yan Cheah
- Institute of Molecular and Cell Biology, A*STAR (Agency of Science, Technology and Research) 138673, Singapore
| | - Chuan Yan
- Institute of Molecular and Cell Biology, A*STAR (Agency of Science, Technology and Research) 138673, Singapore
| | - Tianshu Xiao
- School of Biological Sciences, Nanyang Technological University 637551, Singapore; Institute of Structural Biology, Nanyang Technological University 636921, Singapore.
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5
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Stiving AQ, Foreman DJ, VanAernum ZL, Durr E, Wang S, Vlasak J, Galli J, Kafader JO, Tsukidate T, Li X, Schuessler HA, Richardson DD. Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2024; 35:62-73. [PMID: 38032172 DOI: 10.1021/jasms.3c00309] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/01/2023]
Abstract
Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.
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Affiliation(s)
- Alyssa Q Stiving
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - David J Foreman
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Zachary L VanAernum
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Eberhard Durr
- Infectious Diseases and Vaccines Discovery, Merck & Co., Inc., West Point, Pennsylvania 19486, United States
| | - Shiyi Wang
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Josef Vlasak
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Jennifer Galli
- Infectious Diseases and Vaccines Discovery, Merck & Co., Inc., West Point, Pennsylvania 19486, United States
| | - Jared O Kafader
- Departments of Chemistry and Molecular Biosciences, The Chemistry of Life Processes Institute, The Proteomics Center of Excellence at Northwestern University, Evanston, Illinois 60208, United States
| | - Taku Tsukidate
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Xuanwen Li
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Hillary A Schuessler
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
| | - Douglas D Richardson
- Analytical Research and Development, Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, New Jersey 07065, United States
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6
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Newby ML, Allen JD, Crispin M. Influence of glycosylation on the immunogenicity and antigenicity of viral immunogens. Biotechnol Adv 2024; 70:108283. [PMID: 37972669 PMCID: PMC10867814 DOI: 10.1016/j.biotechadv.2023.108283] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 10/04/2023] [Accepted: 11/09/2023] [Indexed: 11/19/2023]
Abstract
A key aspect of successful viral vaccine design is the elicitation of neutralizing antibodies targeting viral attachment and fusion glycoproteins that embellish viral particles. This observation has catalyzed the development of numerous viral glycoprotein mimetics as vaccines. Glycans can dominate the surface of viral glycoproteins and as such, the viral glycome can influence the antigenicity and immunogenicity of a candidate vaccine. In one extreme, glycans can form an integral part of epitopes targeted by neutralizing antibodies and are therefore considered to be an important feature of key immunogens within an immunization regimen. In the other extreme, the existence of peptide and bacterially expressed protein vaccines shows that viral glycosylation can be dispensable in some cases. However, native-like glycosylation can indicate native-like protein folding and the presence of conformational epitopes. Furthermore, going beyond native glycan mimicry, in either occupancy of glycosylation sites or the glycan processing state, may offer opportunities for enhancing the immunogenicity and associated protection elicited by an immunogen. Here, we review key determinants of viral glycosylation and how recombinant immunogens can recapitulate these signatures across a range of enveloped viruses, including HIV-1, Ebola virus, SARS-CoV-2, Influenza and Lassa virus. The emerging understanding of immunogen glycosylation and its control will help guide the development of future vaccines in both recombinant protein- and nucleic acid-based vaccine technologies.
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Affiliation(s)
- Maddy L Newby
- School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK
| | - Joel D Allen
- School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
| | - Max Crispin
- School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
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7
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Zhang H, Bull RA, Quadeer AA, McKay MR. HCV E1 influences the fitness landscape of E2 and may enhance escape from E2-specific antibodies. Virus Evol 2023; 9:vead068. [PMID: 38107333 PMCID: PMC10722114 DOI: 10.1093/ve/vead068] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2023] [Revised: 09/27/2023] [Accepted: 11/16/2023] [Indexed: 12/19/2023] Open
Abstract
The Hepatitis C virus (HCV) envelope glycoprotein E1 forms a non-covalent heterodimer with E2, the main target of neutralizing antibodies. How E1-E2 interactions influence viral fitness and contribute to resistance to E2-specific antibodies remain largely unknown. We investigate this problem using a combination of fitness landscape and evolutionary modeling. Our analysis indicates that E1 and E2 proteins collectively mediate viral fitness and suggests that fitness-compensating E1 mutations may accelerate escape from E2-targeting antibodies. Our analysis also identifies a set of E2-specific human monoclonal antibodies that are predicted to be especially resilient to escape via genetic variation in both E1 and E2, providing directions for robust HCV vaccine development.
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Affiliation(s)
- Hang Zhang
- Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, SAR, China
| | - Rowena A Bull
- School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia
- The Kirby Institute for Infection and Immunity, Sydney, NSW 2052, Australia
| | - Ahmed Abdul Quadeer
- Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, SAR, China
- Department of Electrical and Electronic Engineering, University of Melbourne, Parkville, VIC 3010, Australia
| | - Matthew R McKay
- Department of Electrical and Electronic Engineering, University of Melbourne, Parkville, VIC 3010, Australia
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia
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8
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Olofsson S, Bally M, Trybala E, Bergström T. Structure and Role of O-Linked Glycans in Viral Envelope Proteins. Annu Rev Virol 2023; 10:283-304. [PMID: 37285578 DOI: 10.1146/annurev-virology-111821-121007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
N- and O-glycans are both important constituents of viral envelope glycoproteins. O-linked glycosylation can be initiated by any of 20 different human polypeptide O-acetylgalactosaminyl transferases, resulting in an important functional O-glycan heterogeneity. O-glycans are organized as solitary glycans or in clusters of multiple glycans forming mucin-like domains. They are functional both in the viral life cycle and in viral colonization of their host. Negatively charged O-glycans are crucial for the interactions between glycosaminoglycan-binding viruses and their host. A novel mechanism, based on controlled electrostatic repulsion, explains how such viruses solve the conflict between optimized viral attachment to target cells and efficient egress of progeny virus. Conserved solitary O-glycans appear important for viral uptake in target cells by contributing to viral envelope fusion. Dual roles of viral O-glycans in the host B cell immune response, either epitope blocking or epitope promoting, may be exploitable for vaccine development. Finally, specific virus-induced O-glycans may be involved in viremic spread.
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Affiliation(s)
- Sigvard Olofsson
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden;
| | - Marta Bally
- Department of Clinical Microbiology, Wallenberg Centre for Molecular Medicine and Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden
| | - Edward Trybala
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden;
| | - Tomas Bergström
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden;
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9
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Nguyen NH, Jarvi NL, Balu-Iyer SV. Immunogenicity of Therapeutic Biological Modalities - Lessons from Hemophilia A Therapies. J Pharm Sci 2023; 112:2347-2370. [PMID: 37220828 DOI: 10.1016/j.xphs.2023.05.014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 05/17/2023] [Accepted: 05/17/2023] [Indexed: 05/25/2023]
Abstract
The introduction and development of biologics such as therapeutic proteins, gene-, and cell-based therapy have revolutionized the scope of treatment for many diseases. However, a significant portion of the patients develop unwanted immune reactions against these novel biological modalities, referred to as immunogenicity, and no longer benefit from the treatments. In the current review, using Hemophilia A (HA) therapy as an example, we will discuss the immunogenicity issue of multiple biological modalities. Currently, the number of therapeutic modalities that are approved or recently explored to treat HA, a hereditary bleeding disorder, is increasing rapidly. These include, but are not limited to, recombinant factor VIII proteins, PEGylated FVIII, FVIII Fc fusion protein, bispecific monoclonal antibodies, gene replacement therapy, gene editing therapy, and cell-based therapy. They offer the patients a broader range of more advanced and effective treatment options, yet immunogenicity remains the most critical complication in the management of this disorder. Recent advances in strategies to manage and mitigate immunogenicity will also be reviewed.
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Affiliation(s)
- Nhan H Nguyen
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY, USA; Currently at Truvai Biosciences, Buffalo, NY, USA
| | - Nicole L Jarvi
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY, USA
| | - Sathy V Balu-Iyer
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY, USA.
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10
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Fukui A, Maruzuru Y, Ohno S, Nobe M, Iwata S, Takeshima K, Koyanagi N, Kato A, Kitazume S, Yamaguchi Y, Kawaguchi Y. Dual impacts of a glycan shield on the envelope glycoprotein B of HSV-1: evasion from human antibodies in vivo and neurovirulence. mBio 2023; 14:e0099223. [PMID: 37366623 PMCID: PMC10470582 DOI: 10.1128/mbio.00992-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Accepted: 05/11/2023] [Indexed: 06/28/2023] Open
Abstract
Identification of the mechanisms of viral evasion from human antibodies is crucial both for understanding viral pathogenesis and for designing effective vaccines. Here we show in cell cultures that an N-glycan shield on the herpes simplex virus 1 (HSV-1) envelope glycoprotein B (gB) mediated evasion from neutralization and antibody-dependent cellular cytotoxicity due to pooled γ-globulins derived from human blood. We also demonstrated that the presence of human γ-globulins in mice and immunity to HSV-1 induced by viral infection in mice significantly reduced replication in their eyes of a mutant virus lacking the glycosylation site but had little effect on the replication of its repaired virus. These results suggest that an N-glycan shield on a specific site of HSV-1 envelope gB mediated evasion from human antibodies in vivo and from HSV-1 immunity induced by viral infection in vivo. Notably, we also found that an N-glycan shield on a specific site of HSV-1 gB was significant for HSV-1 neurovirulence and replication in the central nervous system of naïve mice. Thus, we have identified a critical N-glycan shield on HSV-1 gB that has dual impacts, namely evasion from human antibodies in vivo and viral neurovirulence. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latent and recurrent infections in humans. To produce recurrent infections that contribute to transmission of the virus to new human host(s), the virus must be able to evade the antibodies persisting in latently infected individuals. Here, we show that an N-glycan shield on the specific site of the envelope glycoprotein B (gB) of HSV-1 mediates evasion from pooled γ-globulins derived from human blood both in cell cultures and mice. Notably, the N-glycan shield on the specific site of gB was also significant for HSV-1 neurovirulence in naïve mice. Considering the clinical features of HSV-1 infection, these results suggest that the glycan shield not only facilitates recurrent HSV-1 infections in latently infected humans by evading antibodies but is also important for HSV-1 pathogenesis during the initial infection.
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Affiliation(s)
- Ayano Fukui
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Yuhei Maruzuru
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Research Center for Asian Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Shiho Ohno
- Division of Structural Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Miyagi, Japan
| | - Moeka Nobe
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Shuji Iwata
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Kosuke Takeshima
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Naoto Koyanagi
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Research Center for Asian Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Akihisa Kato
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Research Center for Asian Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Shinobu Kitazume
- Department of Clinical Laboratory Sciences, School of Health Sciences, Fukushima Medical University, Fukushima, Japan
| | - Yoshiki Yamaguchi
- Division of Structural Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Miyagi, Japan
| | - Yasushi Kawaguchi
- Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Research Center for Asian Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- The University of Tokyo, Pandemic Preparedness, Infection and Advanced Research Center, Tokyo, Japan
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11
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Shajahan A, Pepi LE, Kumar B, Murray NB, Azadi P. Site specific N- and O-glycosylation mapping of the spike proteins of SARS-CoV-2 variants of concern. Sci Rep 2023; 13:10053. [PMID: 37344512 PMCID: PMC10284906 DOI: 10.1038/s41598-023-33088-0] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 04/06/2023] [Indexed: 06/23/2023] Open
Abstract
The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N- and O- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.
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Affiliation(s)
- Asif Shajahan
- Vaccine Production Program, Vaccine Research Center, National Institutes of Health, 9 W Watkins Mill Rd, Gaithersburg, MD, 20877, USA.
| | - Lauren E Pepi
- Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, GA, 30602, USA
| | - Bhoj Kumar
- Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, GA, 30602, USA
| | - Nathan B Murray
- Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, GA, 30602, USA
| | - Parastoo Azadi
- Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, GA, 30602, USA.
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12
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Shajahan A, Pepi L, Kumar B, Murray N, Azadi P. Site Specific N- and O-glycosylation mapping of the Spike Proteins of SARS-CoV-2 Variants of Concern. RESEARCH SQUARE 2022:rs.3.rs-2188138. [PMID: 36415454 PMCID: PMC9681045 DOI: 10.21203/rs.3.rs-2188138/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profile of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N-terminal domain (NTD) and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N- and O- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.
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13
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Feng T, Zhang J, Chen Z, Pan W, Chen Z, Yan Y, Dai J. Glycosylation of viral proteins: Implication in virus-host interaction and virulence. Virulence 2022; 13:670-683. [PMID: 35436420 PMCID: PMC9037552 DOI: 10.1080/21505594.2022.2060464] [Citation(s) in RCA: 41] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Glycans are among the most important cell molecular components. However, given their structural diversity, their functions have not been fully explored. Glycosylation is a vital post-translational modification for various proteins. Many bacteria and viruses rely on N-linked and O-linked glycosylation to perform critical biological functions. The diverse functions of glycosylation on viral proteins during viral infections, including Dengue, Zika, influenza, and human immunodeficiency viruses as well as coronaviruses have been reported. N-linked glycosylation is the most common form of protein modification, and it modulates folding, transportation and receptor binding. Compared to N-linked glycosylation, the functions of O-linked viral protein glycosylation have not been comprehensively evaluated. In this review, we summarize findings on viral protein glycosylation, with particular attention to studies on N-linked glycosylation in viral life cycles. This review informs the development of virus-specific vaccines or inhibitors.
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Affiliation(s)
- Tingting Feng
- Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou, China
| | - Jinyu Zhang
- Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou, China
| | - Zhiqian Chen
- Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou, China
| | - Wen Pan
- Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou, China
| | - Zhengrong Chen
- Department of Respiratory Medicine, Children's Hospital of Soochow University, Soochow University, Suzhou, China
| | - Yongdong Yan
- Department of Respiratory Medicine, Children's Hospital of Soochow University, Soochow University, Suzhou, China
| | - Jianfeng Dai
- Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou, China
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14
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Zaki MYW, Fathi AM, Samir S, Eldafashi N, William KY, Nazmy MH, Fathy M, Gill US, Shetty S. Innate and Adaptive Immunopathogeneses in Viral Hepatitis; Crucial Determinants of Hepatocellular Carcinoma. Cancers (Basel) 2022; 14:1255. [PMID: 35267563 PMCID: PMC8909759 DOI: 10.3390/cancers14051255] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Revised: 02/01/2022] [Accepted: 02/04/2022] [Indexed: 02/08/2023] Open
Abstract
Viral hepatitis B (HBV) and hepatitis C (HCV) infections remain the most common risk factors for the development of hepatocellular carcinoma (HCC), and their heterogeneous distribution influences the global prevalence of this common type of liver cancer. Typical hepatitis infection elicits various immune responses within the liver microenvironment, and viral persistence induces chronic liver inflammation and carcinogenesis. HBV is directly mutagenic but can also cause low-grade liver inflammation characterized by episodes of intermittent high-grade liver inflammation, liver fibrosis, and cirrhosis, which can progress to decompensated liver disease and HCC. Equally, the absence of key innate and adaptive immune responses in chronic HCV infection dampens viral eradication and induces an exhausted and immunosuppressive liver niche that favors HCC development and progression. The objectives of this review are to (i) discuss the epidemiological pattern of HBV and HCV infections, (ii) understand the host immune response to acute and chronic viral hepatitis, and (iii) explore the link between this diseased immune environment and the development and progression of HCC in preclinical models and HCC patients.
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Affiliation(s)
- Marco Y. W. Zaki
- Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61732, Egypt; (A.M.F.); (N.E.); (M.H.N.); (M.F.)
- National Institute for Health Research Birmingham Liver Biomedical Research Unit and Centre for Liver and Gastrointestinal Research, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
| | - Ahmed M. Fathi
- Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61732, Egypt; (A.M.F.); (N.E.); (M.H.N.); (M.F.)
| | - Samara Samir
- Department of Biochemistry, Faculty of Pharmacy, Sohag University, Sohag 82524, Egypt;
| | - Nardeen Eldafashi
- Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61732, Egypt; (A.M.F.); (N.E.); (M.H.N.); (M.F.)
| | - Kerolis Y. William
- Department of Internal Medicine, Faculty of Medicine, Cairo University, Cairo 12613, Egypt;
| | - Maiiada Hassan Nazmy
- Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61732, Egypt; (A.M.F.); (N.E.); (M.H.N.); (M.F.)
| | - Moustafa Fathy
- Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61732, Egypt; (A.M.F.); (N.E.); (M.H.N.); (M.F.)
| | - Upkar S. Gill
- Barts Liver Centre, Centre for Immunobiology, Barts & The London School of Medicine & Dentistry, QMUL, London E1 2AT, UK;
| | - Shishir Shetty
- National Institute for Health Research Birmingham Liver Biomedical Research Unit and Centre for Liver and Gastrointestinal Research, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
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15
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Kumbhar PS, Pandya AK, Manjappa AS, Disouza JI, Patravale VB. Carbohydrates-based diagnosis, prophylaxis and treatment of infectious diseases: Special emphasis on COVID-19. CARBOHYDRATE POLYMER TECHNOLOGIES AND APPLICATIONS 2021. [PMCID: PMC7935400 DOI: 10.1016/j.carpta.2021.100052] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
COVID-19 pandemic is taking a dangerous turn due to unavailability of approved and effective vaccines and therapy. Currently available diagnostic techniques are time-consuming, expensive, and maybe impacted by the mutations produced in the virus. Therefore, investigation of novel, rapid, and economic diagnosis techniques, prophylactic vaccines and targeted efficacious drug delivery systems as treatment strategy is imperative. Carbohydrates are essential biomolecules which also act as markers in the realization of immune systems. Moreover, they exhibit antiviral, antimicrobial, and antifungal properties. Carbohydrate-based vaccines and therapeutics including stimuli sensitive systems can be developed successfully and used effectively to fight COVID-19. Thus, carbohydrate-based diagnostic, prophylactic and therapeutic alternatives could be promising to defeat COVID-19 propitiously. Morphology of SARS-CoV-2 and its relevance in devising combat strategies has been discussed. Carbohydrate-based approaches for tackling infectious diseases and their importance in the design of various diagnostic and treatment modalities have been reviewed.
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16
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Dobrica M, van Eerde A, Tucureanu C, Onu A, Paruch L, Caras I, Vlase E, Steen H, Haugslien S, Alonzi D, Zitzmann N, Bock R, Dubuisson J, Popescu C, Stavaru C, Liu Clarke J, Branza‐Nichita N. Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice. PLANT BIOTECHNOLOGY JOURNAL 2021; 19:2027-2039. [PMID: 34002936 PMCID: PMC8486241 DOI: 10.1111/pbi.13631] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 04/27/2021] [Accepted: 05/13/2021] [Indexed: 05/03/2023]
Abstract
Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.
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Affiliation(s)
| | | | - Catalin Tucureanu
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Adrian Onu
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Lisa Paruch
- NIBIO ‐ Norwegian Institute of Bioeconomy ResearchÅsNorway
| | - Iuliana Caras
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Ene Vlase
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Hege Steen
- NIBIO ‐ Norwegian Institute of Bioeconomy ResearchÅsNorway
| | | | - Dominic Alonzi
- Oxford Glycobiology InstituteDepartment of BiochemistryUniversity of OxfordOxfordUK
| | - Nicole Zitzmann
- Oxford Glycobiology InstituteDepartment of BiochemistryUniversity of OxfordOxfordUK
| | - Ralph Bock
- Max Planck Institute of Molecular Plant PhysiologyPotsdam‐GolmGermany
| | - Jean Dubuisson
- Université LilleCNRSINSERMCHU LilleInstitut Pasteur de LilleU1019‐UMR 9017‐CIIL‐Center for Infection and Immunity of LilleLilleFrance
| | | | - Crina Stavaru
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
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17
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Frumento N, Flyak AI, Bailey JR. Mechanisms of HCV resistance to broadly neutralizing antibodies. Curr Opin Virol 2021; 50:23-29. [PMID: 34329953 PMCID: PMC8500940 DOI: 10.1016/j.coviro.2021.07.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 07/06/2021] [Accepted: 07/07/2021] [Indexed: 12/12/2022]
Abstract
Broadly neutralizing antibodies (bNAbs) block infection by genetically diverse hepatitis C virus (HCV) isolates by targeting relatively conserved epitopes on the HCV envelope glycoproteins, E1 and E2. Many amino acid substitutions conferring resistance to these bNAbs have been characterized, identifying multiple mechanisms of bNAb escape. Some resistance substitutions follow the expected mechanism of directly disrupting targeted epitopes. Interestingly, other resistance substitutions fall in E2 domains distant from bNAb-targeted epitopes. These substitutions, which can confer broad resistance to multiple bNAbs, act by less clearly defined mechanisms. Some modulate binding of HCV to cell surface receptors, while others may induce conformational changes in the E2 protein. In this review, we discuss mechanisms of HCV bNAb resistance and implications for HCV vaccine development.
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Affiliation(s)
- Nicole Frumento
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA
| | - Andrew I Flyak
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA
| | - Justin R Bailey
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
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18
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Gruszewska E, Grytczuk A, Chrostek L. Glycosylation in viral hepatitis. Biochim Biophys Acta Gen Subj 2021; 1865:129997. [PMID: 34474116 DOI: 10.1016/j.bbagen.2021.129997] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Revised: 08/26/2021] [Accepted: 08/26/2021] [Indexed: 01/12/2023]
Abstract
BACKGROUND The interaction between hepatitis viruses and host cells is regulated by glycans exposed on the surfaces of human and viruses cells. As the biosynthesis and degradation of human glycoproteins take place at the highest level in the liver, the changes in glycosylation of serum proteins may potentially be useful in the diagnosis of liver pathology. On the other hand, specific alterations in viruses envelope glycans could cause large changes in the entry process of hepatitis viruses into a host cells. SCOPE OF REVIEW Unique alterations in glycosylation of specific proteins can be detected in HBV and HCV infected patients especially with confirmed fibrosis/cirrhosis. On the other hand, viral envelope proteins that bind to host cells are glycosylated. These glycosylated proteins play a key role in recognition, binding and penetration of the host cells. In this review we summarized the knowledge about significance of glycosylation for viral and host factors. MAJOR CONCLUSIONS Glycosylation changes in single serum glycoproteins are noticed in the sera of patients with viral hepatitis. However, a more specific biomarker for the diagnosis of chronic hepatitis than that of a single glycosylated molecule is systemic investigation of complete set of glycan structures (N-glycome). Glycans play important roles in the viral biology cycle especially as a connecting element with host receptors. GENERAL SIGNIFICANCE The interaction between virus glycoproteins and cellular receptors, which are also glycoproteins, determines the possibility of virus penetration into host cells. Therefore these glycans can be the targets for the developing of novel treatment strategies of viral hepatitis.
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Affiliation(s)
- Ewa Gruszewska
- Department of Biochemical Diagnostics, Medical University of Bialystok, Waszyngtona 15A, 15-269 Bialystok, Poland
| | - Agnieszka Grytczuk
- Department of Laboratory Diagnostics, University Clinical Hospital in Bialystok, Bialystok, Poland
| | - Lech Chrostek
- Department of Biochemical Diagnostics, Medical University of Bialystok, Waszyngtona 15A, 15-269 Bialystok, Poland.
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19
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Roberts DS, Mann M, Melby JA, Larson EJ, Zhu Y, Brasier AR, Jin S, Ge Y. Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry. J Am Chem Soc 2021; 143:12014-12024. [PMID: 34328324 PMCID: PMC8353889 DOI: 10.1021/jacs.1c02713] [Citation(s) in RCA: 52] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry, and the S protein glycosylation plays key roles in altering the viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants as well as other O-glycoproteins in general.
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Affiliation(s)
- David S Roberts
- Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
| | - Morgan Mann
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States
| | - Jake A Melby
- Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
| | - Eli J Larson
- Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
| | - Yanlong Zhu
- Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States
- Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States
| | - Allan R Brasier
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States
- Institute for Clinical and Translational Research, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States
| | - Song Jin
- Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
| | - Ying Ge
- Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
- Department of Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States
- Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53705, United States
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20
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Schön K, Lepenies B, Goyette-Desjardins G. Impact of Protein Glycosylation on the Design of Viral Vaccines. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2021; 175:319-354. [PMID: 32935143 DOI: 10.1007/10_2020_132] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Glycans play crucial roles in various biological processes such as cell proliferation, cell-cell interactions, and immune responses. Since viruses co-opt cellular biosynthetic pathways, viral glycosylation mainly depends on the host cell glycosylation machinery. Consequently, several viruses exploit the cellular glycosylation pathway to their advantage. It was shown that viral glycosylation is strongly dependent on the host system selected for virus propagation and/or protein expression. Therefore, the use of different expression systems results in various glycoforms of viral glycoproteins that may differ in functional properties. These differences clearly illustrate that the choice of the expression system can be important, as the resulting glycosylation may influence immunological properties. In this review, we will first detail protein N- and O-glycosylation pathways and the resulting glycosylation patterns; we will then discuss different aspects of viral glycosylation in pathogenesis and in vaccine development; and finally, we will elaborate on how to harness viral glycosylation in order to optimize the design of viral vaccines. To this end, we will highlight specific examples to demonstrate how glycoengineering approaches and exploitation of different expression systems could pave the way towards better self-adjuvanted glycan-based viral vaccines.
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Affiliation(s)
- Kathleen Schön
- Immunology Unit and Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hanover, Germany
- Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Hanover, Germany
| | - Bernd Lepenies
- Immunology Unit and Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hanover, Germany.
| | - Guillaume Goyette-Desjardins
- Immunology Unit and Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hanover, Germany.
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21
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Where to Next? Research Directions after the First Hepatitis C Vaccine Efficacy Trial. Viruses 2021; 13:v13071351. [PMID: 34372558 PMCID: PMC8310243 DOI: 10.3390/v13071351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2021] [Revised: 07/03/2021] [Accepted: 07/08/2021] [Indexed: 11/17/2022] Open
Abstract
Thirty years after its discovery, the hepatitis C virus (HCV) remains a leading cause of liver disease worldwide. Given that many countries continue to experience high rates of transmission despite the availability of potent antiviral therapies, an effective vaccine is seen as critical for the elimination of HCV. The recent failure of the first vaccine efficacy trial for the prevention of chronic HCV confirmed suspicions that this virus will be a challenging vaccine target. Here, we examine the published data from this first efficacy trial along with the earlier clinical and pre-clinical studies of the vaccine candidate and then discuss three key research directions expected to be important in ongoing and future HCV vaccine development. These include the following: 1. design of novel immunogens that generate immune responses to genetically diverse HCV genotypes and subtypes, 2. strategies to elicit broadly neutralizing antibodies against envelope glycoproteins in addition to cytotoxic and helper T cell responses, and 3. consideration of the unique immunological status of individuals most at risk for HCV infection, including those who inject drugs, in vaccine platform development and early immunogenicity trials.
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22
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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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23
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LeBlanc EV, Kim Y, Capicciotti CJ, Colpitts CC. Hepatitis C Virus Glycan-Dependent Interactions and the Potential for Novel Preventative Strategies. Pathogens 2021; 10:pathogens10060685. [PMID: 34205894 PMCID: PMC8230238 DOI: 10.3390/pathogens10060685] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 05/25/2021] [Accepted: 05/28/2021] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infections continue to be a major contributor to liver disease worldwide. HCV treatment has become highly effective, yet there are still no vaccines or prophylactic strategies available to prevent infection and allow effective management of the global HCV burden. Glycan-dependent interactions are crucial to many aspects of the highly complex HCV entry process, and also modulate immune evasion. This review provides an overview of the roles of viral and cellular glycans in HCV infection and highlights glycan-focused advances in the development of entry inhibitors and vaccines to effectively prevent HCV infection.
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Affiliation(s)
- Emmanuelle V. LeBlanc
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
| | - Youjin Kim
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
| | - Chantelle J. Capicciotti
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
- Department of Chemistry, Queen’s University, Kingston, ON K7L 3N6, Canada
- Department of Surgery, Queen’s University, Kingston, ON K7L 3N6, Canada
| | - Che C. Colpitts
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
- Correspondence:
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Structural and Biophysical Characterization of the HCV E1E2 Heterodimer for Vaccine Development. Viruses 2021; 13:v13061027. [PMID: 34072451 PMCID: PMC8227786 DOI: 10.3390/v13061027] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Revised: 05/20/2021] [Accepted: 05/25/2021] [Indexed: 02/07/2023] Open
Abstract
An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.
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25
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Zhao X, Chen H, Wang H. Glycans of SARS-CoV-2 Spike Protein in Virus Infection and Antibody Production. Front Mol Biosci 2021; 8:629873. [PMID: 33928117 PMCID: PMC8076860 DOI: 10.3389/fmolb.2021.629873] [Citation(s) in RCA: 66] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Accepted: 01/22/2021] [Indexed: 12/18/2022] Open
Abstract
Viral protein glycosylation represents a successful strategy employed by the parasite to take advantage of host-cell machinery for modification of its own proteins. The resulting glycans have unneglectable roles in viral infection and immune response. The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which presents on the surface of matured virion and mediates viral entry into the host, also undergoes extensive glycosylation to shield it from the human defense system. It is believed that the ongoing COVID-19 pandemic with more than 90,000,000 infections and 1,900,000 deaths is partly due to its successful glycosylation strategy. On the other hand, while glycan patches on S protein have been reported to shield the host immune response by masking "nonself" viral peptides with "self-glycans," the epitopes are also important in eliciting neutralizing antibodies. In this review, we will summarize the roles of S protein glycans in mediating virus-receptor interactions, and in antibody production, as well as indications for vaccine development.
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Affiliation(s)
| | | | - Hongliang Wang
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
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26
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Roberts DS, Mann MW, Melby JA, Larson EJ, Zhu Y, Brasier AR, Jin S, Ge Y. Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2021. [PMID: 33688648 DOI: 10.1101/2021.02.28.433291] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.
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27
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Seitz C, Casalino L, Konecny R, Huber G, Amaro RE, McCammon JA. Multiscale Simulations Examining Glycan Shield Effects on Drug Binding to Influenza Neuraminidase. Biophys J 2020; 119:2275-2289. [PMID: 33130120 PMCID: PMC7732775 DOI: 10.1016/j.bpj.2020.10.024] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 10/08/2020] [Accepted: 10/21/2020] [Indexed: 12/18/2022] Open
Abstract
Influenza neuraminidase is an important drug target. Glycans are present on neuraminidase and are generally considered to inhibit antibody binding via their glycan shield. In this work, we studied the effect of glycans on the binding kinetics of antiviral drugs to the influenza neuraminidase. We created all-atom in silico systems of influenza neuraminidase with experimentally derived glycoprofiles consisting of four systems with different glycan conformations and one system without glycans. Using Brownian dynamics simulations, we observe a two- to eightfold decrease in the rate of ligand binding to the primary binding site of neuraminidase due to the presence of glycans. These glycans are capable of covering much of the surface area of neuraminidase, and the ligand binding inhibition is derived from glycans sterically occluding the primary binding site on a neighboring monomer. Our work also indicates that drugs preferentially bind to the primary binding site (i.e., the active site) over the secondary binding site, and we propose a binding mechanism illustrating this. These results help illuminate the complex interplay between glycans and ligand binding on the influenza membrane protein neuraminidase.
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Affiliation(s)
- Christian Seitz
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California.
| | - Lorenzo Casalino
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California
| | - Robert Konecny
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California
| | - Gary Huber
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California
| | - Rommie E Amaro
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California.
| | - J Andrew McCammon
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California; Department of Pharmacology, University of California, San Diego, La Jolla, California
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28
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Lin CH, Yang CY, Ou SC, Wang M, Lo CY, Tsai TL, Wu HY. The Impacts of Antivirals on the Coronavirus Genome Structure and Subsequent Pathogenicity, Virus Fitness and Antiviral Design. Biomedicines 2020; 8:E376. [PMID: 32987828 PMCID: PMC7601523 DOI: 10.3390/biomedicines8100376] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Revised: 09/17/2020] [Accepted: 09/23/2020] [Indexed: 01/29/2023] Open
Abstract
With the global threat of SARS-CoV-2, much effort has been focused on treatment and disease control. However, how coronaviruses react to the treatments and whether the surviving viruses have altered their characteristics are also unanswered questions with medical importance. To this end, bovine coronavirus (BCoV), which is in the same genus as SARS-CoV-2, was used as a test model and the findings were as follows. With the treatment of antiviral remdesivir, the selected BCoV variant with an altered genome structure developed resistance, but its pathogenicity was not increased in comparison to that of wild type (wt) BCoV. Under the selection pressure of innate immunity, the genome structure was also altered; however, neither resistance developed nor pathogenicity increased for the selected BCoV variant. Furthermore, both selected BCoV variants showed a better efficiency in adapting to alternative host cells than wt BCoV. In addition, the previously unidentified feature that the spike protein was a common target for mutations under different antiviral treatments might pose a problem for vaccine development because spike protein is a common target for antibody and vaccine designs. The findings derived from this fundamental research may contribute to the disease control and treatments against coronaviruses, including SARS-CoV-2.
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Affiliation(s)
- Ching-Hung Lin
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan; (C.-H.L.); (C.-Y.Y.); (C.-Y.L.); (T.-L.T.)
| | - Cheng-Yao Yang
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan; (C.-H.L.); (C.-Y.Y.); (C.-Y.L.); (T.-L.T.)
| | - Shan-Chia Ou
- Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan;
| | - Meilin Wang
- Department of Microbiology and Immunology, Chung Shan Medical University, Taichung 40201, Taiwan;
| | - Chen-Yu Lo
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan; (C.-H.L.); (C.-Y.Y.); (C.-Y.L.); (T.-L.T.)
| | - Tsung-Lin Tsai
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan; (C.-H.L.); (C.-Y.Y.); (C.-Y.L.); (T.-L.T.)
| | - Hung-Yi Wu
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan; (C.-H.L.); (C.-Y.Y.); (C.-Y.L.); (T.-L.T.)
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29
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Hepatitis C virus vaccine design: focus on the humoral immune response. J Biomed Sci 2020; 27:78. [PMID: 32631318 PMCID: PMC7338099 DOI: 10.1186/s12929-020-00669-4] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Accepted: 06/26/2020] [Indexed: 02/06/2023] Open
Abstract
Despite the recent development of safe and highly effective direct-acting antivirals, hepatitis C virus (HCV) infection remains a significant health problem. In 2016, the World Health Organization set out to reduce the rate of new HCV infections by 90% by 2030. Still, global control of the virus does not seem to be achievable in the absence of an effective vaccine. Current approaches to the development of a vaccine against HCV include the production of recombinant proteins, synthetic peptides, DNA vaccines, virus-like particles, and viral vectors expressing various antigens. In this review, we focus on the development of vaccines targeting the humoral immune response against HCV based on the cumulative evidence supporting the important role of neutralizing antibodies in protection against HCV infection. The main targets of HCV-specific neutralizing antibodies are the glycoproteins E1 and E2. Recent advances in the knowledge of HCV glycoprotein structure and their epitopes, as well as the possibility of getting detailed information on the human antibody repertoire generated by the infection, will allow rational structure-based antigen design to target specific germline antibodies. Although obtaining a vaccine capable of inducing sterilizing immunity will be a difficult task, a vaccine that prevents chronic hepatitis C infections, a more realistic goal in the short term, would have a considerable health impact.
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30
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Center RJ, Boo I, Phu L, McGregor J, Poumbourios P, Drummer HE. Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine. J Biol Chem 2020; 295:7179-7192. [PMID: 32299914 PMCID: PMC7247312 DOI: 10.1074/jbc.ra120.013015] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2020] [Revised: 04/13/2020] [Indexed: 12/13/2022] Open
Abstract
The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture-derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor-binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture-derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.
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Affiliation(s)
- Rob J Center
- Burnet Institute, 85 Commercial Road, Melbourne 3004, Australia; Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne 3000, Australia
| | - Irene Boo
- Burnet Institute, 85 Commercial Road, Melbourne 3004, Australia
| | - Lilian Phu
- Burnet Institute, 85 Commercial Road, Melbourne 3004, Australia; Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne 3000, Australia
| | - Joey McGregor
- Burnet Institute, 85 Commercial Road, Melbourne 3004, Australia; Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne 3000, Australia
| | - Pantelis Poumbourios
- Burnet Institute, 85 Commercial Road, Melbourne 3004, Australia; Department of Microbiology, Monash University, Clayton 3056, Australia
| | - Heidi E Drummer
- Burnet Institute, 85 Commercial Road, Melbourne 3004, Australia; Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne 3000, Australia; Department of Microbiology, Monash University, Clayton 3056, Australia.
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31
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Stejskal L, Lees WD, Moss DS, Palor M, Bingham RJ, Shepherd AJ, Grove J. Flexibility and intrinsic disorder are conserved features of hepatitis C virus E2 glycoprotein. PLoS Comput Biol 2020; 16:e1007710. [PMID: 32109245 PMCID: PMC7065822 DOI: 10.1371/journal.pcbi.1007710] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Revised: 03/11/2020] [Accepted: 02/04/2020] [Indexed: 12/15/2022] Open
Abstract
The glycoproteins of hepatitis C virus, E1E2, are unlike any other viral fusion machinery yet described, and are the current focus of immunogen design in HCV vaccine development; thus, making E1E2 both scientifically and medically important. We used pre-existing, but fragmentary, structures to model a complete ectodomain of the major glycoprotein E2 from three strains of HCV. We then performed molecular dynamic simulations to explore the conformational landscape of E2, revealing a number of important features. Despite high sequence divergence, and subtle differences in the models, E2 from different strains behave similarly, possessing a stable core flanked by highly flexible regions, some of which perform essential functions such as receptor binding. Comparison with sequence data suggest that this consistent behaviour is conferred by a network of conserved residues that act as hinge and anchor points throughout E2. The variable regions (HVR-1, HVR-2 and VR-3) exhibit particularly high flexibility, and bioinformatic analysis suggests that HVR-1 is a putative intrinsically disordered protein region. Dynamic cross-correlation analyses demonstrate intramolecular communication and suggest that specific regions, such as HVR-1, can exert influence throughout E2. To support our computational approach we performed small-angle X-ray scattering with purified E2 ectodomain; this data was consistent with our MD experiments, suggesting a compact globular core with peripheral flexible regions. This work captures the dynamic behaviour of E2 and has direct relevance to the interaction of HCV with cell-surface receptors and neutralising antibodies.
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Affiliation(s)
- Lenka Stejskal
- Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, London, United Kingdom
- Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom
| | - William D. Lees
- Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom
| | - David S. Moss
- Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom
| | - Machaela Palor
- Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, London, United Kingdom
| | - Richard J. Bingham
- Department of Biological Sciences, University of Huddersfield, Huddersfield, United Kingdom
| | - Adrian J. Shepherd
- Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom
| | - Joe Grove
- Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, London, United Kingdom
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32
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Wang D. Coronaviruses' sugar shields as vaccine candidates. CURRENT TRENDS IN IMMUNOLOGY 2020; 21:17-23. [PMID: 32606565 PMCID: PMC7326345] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
A successful global healthcare response relies on versatile vaccines and production of broadly virus-neutralizing antibodies by the immune system to protect us from emerging infectious diseases. The present 2019 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic highlights the urgent need for development of anti-viral biodefense. Due to the genetic and proteomic diversities of viral pathogens, establishing versatile anti-viral vaccines or therapeutic agents is highly challenging. Carbohydrate antigens represent an important class of immunological targets for vaccine development and immunotherapy against microbial infections. In this mini review, some concepts and strategies for exploring the potential of immunogenic sugar moieties as CoV vaccine candidates are presented.
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Affiliation(s)
- Denong Wang
- Tumor Glycomics Laboratory, SRI International Biosciences Division, 333 Ravenswood Avenue, Menlo Park, CA, USA
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33
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Cooperation between somatic mutation and germline-encoded residues enables antibody recognition of HIV-1 envelope glycans. PLoS Pathog 2019; 15:e1008165. [PMID: 31841553 PMCID: PMC6936856 DOI: 10.1371/journal.ppat.1008165] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 12/30/2019] [Accepted: 10/29/2019] [Indexed: 11/19/2022] Open
Abstract
Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline VHDJH rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination. The viral pathogen HIV-1 uses sugar molecules, called glycans, from the host to densely cover its envelope protein. Most broadly neutralizing HIV-1 antibodies interact with glycans on the HIV-1 envelope protein. For this reason, the vaccine induction of anti-HIV-1 glycan antibodies is a principal goal. Since vaccine-induced anti-HIV-1 glycan antibodies are rare, it has not been determined how antibodies develop during vaccination to recognize HIV-1 glycans. Here, we elucidated the amino acids required for a primate antibody induced by HIV-1 vaccination to interact with HIV envelope glycans. Genetic and functional analyses showed the putative antibody germline nucleotide sequence encoded amino acids that were required for glycan reactivity. Somatic mutation also introduced critical amino acids that were required for glycan recognition. Unusually, the somatic mutations were not required in order to form direct contacts with antigen, but instead functioned to improve antibody flexibility and to form its glycan binding site. These results define the molecular development of a vaccine-induced HIV-1 glycan antibody, providing insight into why vaccines rarely elicit antibodies against the glycans on the HIV-1 outer coat protein.
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Identifying immunologically-vulnerable regions of the HCV E2 glycoprotein and broadly neutralizing antibodies that target them. Nat Commun 2019; 10:2073. [PMID: 31061402 PMCID: PMC6502829 DOI: 10.1038/s41467-019-09819-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Accepted: 04/02/2019] [Indexed: 02/06/2023] Open
Abstract
Isolation of broadly neutralizing human monoclonal antibodies (HmAbs) targeting the E2 glycoprotein of Hepatitis C virus (HCV) has sparked hope for effective vaccine development. Nonetheless, escape mutations have been reported. Ideally, a potent vaccine should elicit HmAbs that target regions of E2 that are most difficult to escape. Here, aimed at addressing this challenge, we develop a predictive in-silico evolutionary model for E2 that identifies one such region, a specific antigenic domain, making it an attractive target for a robust antibody response. Specific broadly neutralizing HmAbs that appear difficult to escape from are also identified. By providing a framework for identifying vulnerable regions of E2 and for assessing the potency of specific antibodies, our results can aid the rational design of an effective prophylactic HCV vaccine. A good vaccine should direct the immune response to virus regions that are most difficult to escape. Here, Quadeer et al. develop a predictive in-silico evolutionary model for HCV E2 which identifies one such antigenic region and identifies multiple broadly neutralizing human antibodies that appear difficult to escape from.
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35
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Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations. Proc Natl Acad Sci U S A 2019; 116:10039-10047. [PMID: 31040211 DOI: 10.1073/pnas.1822002116] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
About two million new cases of hepatitis C virus (HCV) infections annually underscore the urgent need for a vaccine. However, this effort has proven challenging because HCV evades neutralizing antibodies (NAbs) through molecular features of viral envelope glycoprotein E2, including hypervariable region 1 (HVR1) and N-linked glycans. Here, we observe large variation in the effects of removing individual E2 glycans across HCV strains H77(genotype 1a), J6(2a), and S52(3a) in Huh7.5 cell infections. Also, glycan-mediated effects on neutralization sensitivity were completely HVR1-dependent, and neutralization data were consistent with indirect protection of epitopes, as opposed to direct steric shielding. Indeed, the effect of removing each glycan was similar both in type (protective or sensitizing) and relative strength across four nonoverlapping neutralization epitopes. Temperature-dependent neutralization (e.g., virus breathing) assays indicated that both HVR1 and protective glycans stabilized a closed, difficult to neutralize, envelope conformation. This stabilizing effect was hierarchical as removal of HVR1 fully destabilized closed conformations, irrespective of glycan status, consistent with increased instability at acidic pH and high temperatures. Finally, we observed a strong correlation between neutralization sensitivity and scavenger receptor BI dependency during viral entry. In conclusion, our study indicates that HVR1 and glycans regulate HCV neutralization by shifting the equilibrium between open and closed envelope conformations. This regulation appears tightly linked with scavenger receptor BI dependency, suggesting a role of this receptor in transitions from closed to open conformations during entry. This importance of structural dynamics of HCV envelope glycoproteins has critical implications for vaccine development and suggests that similar phenomena could contribute to immune evasion of other viruses.
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36
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Urbanowicz RA, Wang R, Schiel JE, Keck ZY, Kerzic MC, Lau P, Rangarajan S, Garagusi KJ, Tan L, Guest JD, Ball JK, Pierce BG, Mariuzza RA, Foung SKH, Fuerst TR. Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells. J Virol 2019; 93:e01403-18. [PMID: 30651366 PMCID: PMC6430559 DOI: 10.1128/jvi.01403-18] [Citation(s) in RCA: 53] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Accepted: 12/19/2018] [Indexed: 02/07/2023] Open
Abstract
The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.
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Affiliation(s)
- Richard A Urbanowicz
- School of Life Sciences, The University of Nottingham, Nottingham, United Kingdom
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and The University of Nottingham, Nottingham, United Kingdom
| | - Ruixue Wang
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
| | - John E Schiel
- University of Maryland Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology, Rockville, Maryland, USA
| | - Zhen-Yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
| | - Melissa C Kerzic
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
| | - Patrick Lau
- Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
| | - Sneha Rangarajan
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
| | - Kyle J Garagusi
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
| | - Lei Tan
- Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
| | - Johnathan D Guest
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
| | - Jonathan K Ball
- School of Life Sciences, The University of Nottingham, Nottingham, United Kingdom
- NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and The University of Nottingham, Nottingham, United Kingdom
| | - Brian G Pierce
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
| | - Roy A Mariuzza
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
| | - Steven K H Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
| | - Thomas R Fuerst
- W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
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Dijkstra M, Bawono P, Abeln S, Feenstra KA, Fokkink W, Heringa J. Motif-Aware PRALINE: Improving the alignment of motif regions. PLoS Comput Biol 2018; 14:e1006547. [PMID: 30383764 PMCID: PMC6233922 DOI: 10.1371/journal.pcbi.1006547] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2018] [Revised: 11/13/2018] [Accepted: 10/05/2018] [Indexed: 11/21/2022] Open
Abstract
Protein or DNA motifs are sequence regions which possess biological importance. These regions are often highly conserved among homologous sequences. The generation of multiple sequence alignments (MSAs) with a correct alignment of the conserved sequence motifs is still difficult to achieve, due to the fact that the contribution of these typically short fragments is overshadowed by the rest of the sequence. Here we extended the PRALINE multiple sequence alignment program with a novel motif-aware MSA algorithm in order to address this shortcoming. This method can incorporate explicit information about the presence of externally provided sequence motifs, which is then used in the dynamic programming step by boosting the amino acid substitution matrix towards the motif. The strength of the boost is controlled by a parameter, α. Using a benchmark set of alignments we confirm that a good compromise can be found that improves the matching of motif regions while not significantly reducing the overall alignment quality. By estimating α on an unrelated set of reference alignments we find there is indeed a strong conservation signal for motifs. A number of typical but difficult MSA use cases are explored to exemplify the problems in correctly aligning functional sequence motifs and how the motif-aware alignment method can be employed to alleviate these problems. The most important functional parts of proteins are often small—but very specific—sequence motifs. Moreover, these motifs tend to be strongly conserved during evolution due to their functional role. Nevertheless, when trying to align protein sequences of the same family, it is often very difficult to align such motifs using standard multiple sequence alignment methods. Aligning functional residues correctly is essential to detect motif conservation, which can be used to filter out spuriously occurring motifs. Additionally, many downstream analyses, such as phylogenetics, are strongly reliant on alignment quality. We have developed a sequence alignment program named Motif-Aware PRALINE (MA-PRALINE) that incorporates information about motifs explicitly. Motifs are provided to MA-PRALINE in the PROSITE pattern syntax; it then scans the input sequences for instances of the pattern and provides a score bonus to matching sequence positions. Our method provides a reproducible alternative to editing alignments by hand in order to account for motif conservation, which is a tedious and error-prone process. We will show that MA-PRALINE allows the alignment of motif-rich regions to be fine-tuned while not degrading the rest of the alignment. MA-PRALINE is available on GitHub as open source software; this allows it to be easily tailored to similar problems. We apply MA-PRALINE on the HIV-1 envelope glycoprotein (gp120) to get an improved alignment of the N-terminal glycosylation motifs. The presence of these motifs is essential for the virus in evading the immune response of the host.
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Affiliation(s)
- Maurits Dijkstra
- Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
- * E-mail:
| | - Punto Bawono
- Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
| | - Sanne Abeln
- Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
| | - K. Anton Feenstra
- Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
| | - Wan Fokkink
- Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
| | - Jaap Heringa
- Department of Computer Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
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Prentoe J, Bukh J. Hypervariable Region 1 in Envelope Protein 2 of Hepatitis C Virus: A Linchpin in Neutralizing Antibody Evasion and Viral Entry. Front Immunol 2018; 9:2146. [PMID: 30319614 PMCID: PMC6170631 DOI: 10.3389/fimmu.2018.02146] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Accepted: 08/30/2018] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infection is the cause of about 400,000 annual liver disease-related deaths. The global spread of this important human pathogen can potentially be prevented through the development of a vaccine, but this challenge has proven difficult, and much remains unknown about the multitude of mechanisms by which this heterogeneous RNA virus evades inactivation by neutralizing antibodies (NAbs). The N-terminal motif of envelope protein 2 (E2), termed hypervariable region 1 (HVR1), changes rapidly in immunoglobulin-competent patients due to antibody-driven antigenic drift. HVR1 contains NAb epitopes and is directly involved in protecting diverse antibody-specific epitopes on E1, E2, and E1/E2 through incompletely understood mechanisms. The ability of HVR1 to protect HCV from NAbs appears linked with modulation of HCV entry co-receptor interactions. Thus, removal of HVR1 increases interaction with CD81, while altering interaction with scavenger receptor class B, type I (SR-BI) in a complex fashion, and decreasing interaction with low-density lipoprotein receptor. Despite intensive efforts this modulation of receptor interactions by HVR1 remains incompletely understood. SR-BI has received the most attention and it appears that HVR1 is involved in a multimodal HCV/SR-BI interaction involving high-density-lipoprotein associated ApoCI, which may prime the virus for later entry events by exposing conserved NAb epitopes, like those in the CD81 binding site. To fully elucidate the multifunctional role of HVR1 in HCV entry and NAb evasion, improved E1/E2 models and comparative studies with other NAb evasion strategies are needed. Derived knowledge may be instrumental in the development of a prophylactic HCV vaccine.
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Affiliation(s)
- Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Copenhagen, Denmark.,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, Hvidovre Hospital, Copenhagen, Denmark.,Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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Bagdonaite I, Vakhrushev SY, Joshi HJ, Wandall HH. Viral glycoproteomes: technologies for characterization and outlook for vaccine design. FEBS Lett 2018; 592:3898-3920. [PMID: 29961944 DOI: 10.1002/1873-3468.13177] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2018] [Revised: 06/13/2018] [Accepted: 06/26/2018] [Indexed: 12/27/2022]
Abstract
It has long been known that surface proteins of most enveloped viruses are covered with glycans. It has furthermore been demonstrated that glycosylation is essential for propagation and immune evasion for many viruses. The recent development of high-resolution mass spectrometry techniques has enabled identification not only of the precise structures but also the positions of such post-translational modifications on viruses, revealing substantial differences in extent of glycosylation and glycan maturation for different classes of viruses. In-depth characterization of glycosylation and other post-translational modifications of viral envelope glycoproteins is essential for rational design of vaccines and antivirals. In this Review, we provide an overview of techniques used to address viral glycosylation and summarize information on glycosylation of enveloped viruses representing ongoing public health challenges. Furthermore, we discuss how knowledge on glycosylation can be translated to means to prevent and combat viral infections.
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Affiliation(s)
- Ieva Bagdonaite
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
| | - Sergey Y Vakhrushev
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
| | - Hiren J Joshi
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
| | - Hans H Wandall
- Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Denmark
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40
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Bagdonaite I, Wandall HH. Global aspects of viral glycosylation. Glycobiology 2018; 28:443-467. [PMID: 29579213 PMCID: PMC7108637 DOI: 10.1093/glycob/cwy021] [Citation(s) in RCA: 175] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2017] [Revised: 02/10/2018] [Accepted: 03/21/2018] [Indexed: 12/15/2022] Open
Abstract
Enveloped viruses encompass some of the most common human pathogens causing infections of different severity, ranging from no or very few symptoms to lethal disease as seen with the viral hemorrhagic fevers. All enveloped viruses possess an envelope membrane derived from the host cell, modified with often heavily glycosylated virally encoded glycoproteins important for infectivity, viral particle formation and immune evasion. While N-linked glycosylation of viral envelope proteins is well characterized with respect to location, structure and site occupancy, information on mucin-type O-glycosylation of these proteins is less comprehensive. Studies on viral glycosylation are often limited to analysis of recombinant proteins that in most cases are produced in cell lines with a glycosylation capacity different from the capacity of the host cells. The glycosylation pattern of the produced recombinant glycoproteins might therefore be different from the pattern on native viral proteins. In this review, we provide a historical perspective on analysis of viral glycosylation, and summarize known roles of glycans in the biology of enveloped human viruses. In addition, we describe how to overcome the analytical limitations by using a global approach based on mass spectrometry to identify viral O-glycosylation in virus-infected cell lysates using the complex enveloped virus herpes simplex virus type 1 as a model. We underscore that glycans often pay important contributions to overall protein structure, function and immune recognition, and that glycans represent a crucial determinant for vaccine design. High throughput analysis of glycosylation on relevant glycoprotein formulations, as well as data compilation and sharing is therefore important to identify consensus glycosylation patterns for translational applications.
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Affiliation(s)
- Ieva Bagdonaite
- Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, Copenhagen N, Denmark
| | - Hans H Wandall
- Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, Copenhagen N, Denmark
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41
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Cowton VM, Singer JB, Gifford RJ, Patel AH. Predicting the Effectiveness of Hepatitis C Virus Neutralizing Antibodies by Bioinformatic Analysis of Conserved Epitope Residues Using Public Sequence Data. Front Immunol 2018; 9:1470. [PMID: 30013555 PMCID: PMC6036255 DOI: 10.3389/fimmu.2018.01470] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2018] [Accepted: 06/13/2018] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) is a global health issue. Although direct-acting antivirals are available to target HCV, there is currently no vaccine. The diversity of the virus is a major obstacle to HCV vaccine development. One approach toward a vaccine is to utilize a strategy to elicit broadly neutralizing antibodies (bNAbs) that target highly-conserved epitopes. The conserved epitopes of bNAbs have been mapped almost exclusively to the E2 glycoprotein. In this study, we have used HCV-GLUE, a bioinformatics resource for HCV sequence data, to investigate the major epitopes targeted by well-characterized bNAbs. Here, we analyze the level of conservation of each epitope by genotype and subtype and consider the most promising bNAbs identified to date for further study as potential vaccine leads. For the most conserved epitopes, we also identify the most prevalent sequence variants in the circulating HCV population. We examine the distribution of E2 sequence data from across the globe and highlight regions with no coverage. Genotype 1 is the most prevalent genotype worldwide, but in many regions, it is not the dominant genotype. We find that the sequence conservation data is very encouraging; several bNAbs have a high level of conservation across all genotypes suggesting that it may be unnecessary to tailor vaccines according to the geographical distribution of genotypes.
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Affiliation(s)
| | | | | | - Arvind H. Patel
- MRC-University of Glasgow Centre for Virus Research, Garscube Campus, Glasgow, Scotland, United Kingdom
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42
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Mazalovska M, Kouokam JC. Lectins as Promising Therapeutics for the Prevention and Treatment of HIV and Other Potential Coinfections. BIOMED RESEARCH INTERNATIONAL 2018; 2018:3750646. [PMID: 29854749 PMCID: PMC5964492 DOI: 10.1155/2018/3750646] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Revised: 03/20/2018] [Accepted: 04/01/2018] [Indexed: 12/20/2022]
Abstract
Human immunodeficiency virus-acquired immunodeficiency syndrome (HIV/AIDS) remains a global health problem. Current therapeutics specifically target the viral pathogen at various stages of its life cycle, although complex interactions between HIV and other pathogenic organisms are evident. Targeting HIV and concomitant infectious pathogens simultaneously, both by therapeutic regimens and in prevention strategies, would help contain the AIDS pandemic. Lectins, a ubiquitous group of proteins that specifically bind glycosylated molecules, are interesting compounds that could be used for this purpose, with demonstrated anti-HIV properties. In addition, potential coinfecting pathogens, including other enveloped viruses, bacteria, yeasts and fungi, and protozoa, display sugar-coated macromolecules on their surfaces, making them potential targets of lectins. This review summarizes the currently available findings suggesting that lectins should be further developed to simultaneously fight the AIDS pandemic and concomitant infections in HIV infected individuals.
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Affiliation(s)
- Milena Mazalovska
- Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA
- Center for Predictive Medicine, University of Louisville, Louisville, KY 40202, USA
| | - J. Calvin Kouokam
- Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA
- Center for Predictive Medicine, University of Louisville, Louisville, KY 40202, USA
- James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202, USA
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Dustin LB. Innate and Adaptive Immune Responses in Chronic HCV Infection. Curr Drug Targets 2018; 18:826-843. [PMID: 26302811 DOI: 10.2174/1389450116666150825110532] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2015] [Revised: 07/25/2015] [Accepted: 07/27/2015] [Indexed: 12/14/2022]
Abstract
Hepatitis C virus (HCV) remains a public health problem of global importance, even in the era of potent directly-acting antiviral drugs. In this chapter, I discuss immune responses to acute and chronic HCV infection. The outcome of HCV infection is influenced by viral strategies that limit or delay the initiation of innate antiviral responses. This delay may enable HCV to establish widespread infection long before the host mounts effective T and B cell responses. HCV's genetic agility, resulting from its high rate of replication and its error prone replication mechanism, enables it to evade immune recognition. Adaptive immune responses fail to keep up with changing viral epitopes. Neutralizing antibody epitopes may be hidden by decoy structures, glycans, and lipoproteins. T cell responses fail due to changing epitope sequences and due to exhaustion, a phenomenon that may have evolved to limit immune-mediated pathology. Despite these difficulties, innate and adaptive immune mechanisms do impact HCV replication. Immune-mediated clearance of infection is possible, occurring in 20-50% of people who contract the disease. New developments raise hopes for effective immunological interventions to prevent or treat HCV infection.
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Affiliation(s)
- Lynn B Dustin
- University of Oxford, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, United Kingdom
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Lavie M, Hanoulle X, Dubuisson J. Glycan Shielding and Modulation of Hepatitis C Virus Neutralizing Antibodies. Front Immunol 2018; 9:910. [PMID: 29755477 PMCID: PMC5934428 DOI: 10.3389/fimmu.2018.00910] [Citation(s) in RCA: 80] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Accepted: 04/12/2018] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) envelope glycoprotein heterodimer, E1E2, plays an essential role in virus entry and assembly. Furthermore, due to their exposure at the surface of the virion, these proteins are the major targets of anti-HCV neutralizing antibodies. Their ectodomain are heavily glycosylated with up to 5 sites on E1 and up to 11 sites on E2 modified by N-linked glycans. Thus, one-third of the molecular mass of E1E2 heterodimer corresponds to glycans. Despite the high sequence variability of E1 and E2, N-glycosylation sites of these proteins are generally conserved among the seven major HCV genotypes. N-glycans have been shown to be involved in E1E2 folding and modulate different functions of the envelope glycoproteins. Indeed, site-directed mutagenesis studies have shown that specific glycans are needed for virion assembly and infectivity. They can notably affect envelope protein entry functions by modulating their affinity for HCV receptors and their fusion activity. Importantly, glycans have also been shown to play a key role in immune evasion by masking antigenic sites targeted by neutralizing antibodies. It is well known that the high mutational rate of HCV polymerase facilitates the appearance of neutralization resistant mutants, and occurrence of mutations leading to glycan shifting is one of the mechanisms used by this virus to escape host humoral immune response. As a consequence of the importance of the glycan shield for HCV immune evasion, the deletion of N-glycans also leads to an increase in E1E2 immunogenicity and can induce a more potent antibody response against HCV.
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Affiliation(s)
- Muriel Lavie
- University of Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection & Immunity of Lille, Lille, France
| | - Xavier Hanoulle
- University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France
| | - Jean Dubuisson
- University of Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection & Immunity of Lille, Lille, France
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Can Broadly Neutralizing Monoclonal Antibodies Lead to a Hepatitis C Virus Vaccine? Trends Microbiol 2018; 26:854-864. [PMID: 29703495 DOI: 10.1016/j.tim.2018.04.002] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Revised: 03/09/2018] [Accepted: 04/02/2018] [Indexed: 12/20/2022]
Abstract
While licensed vaccines elicit protective antibody responses against a variety of viral infections, an effective vaccine for hepatitis C virus (HCV) has remained elusive. The extraordinary genetic diversity of HCV and the ability of the virus to evade the immune response have hindered vaccine development efforts. However, recent studies have greatly expanded the number of well characterized broadly neutralizing human monoclonal antibodies (bNAbs) against HCV. These bNAbs target relatively conserved HCV epitopes, prevent HCV infection in animal models, and are associated with spontaneous clearance of human HCV infection. In this review, recent high-resolution bNAb epitope mapping and structural analysis of bNAb-epitope complexes that may serve as a guide for vaccine development are discussed along with major obstacles.
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Escape of Hepatitis C Virus from Epitope I Neutralization Increases Sensitivity of Other Neutralization Epitopes. J Virol 2018; 92:JVI.02066-17. [PMID: 29467319 PMCID: PMC5899191 DOI: 10.1128/jvi.02066-17] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 02/05/2018] [Indexed: 01/01/2023] Open
Abstract
The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24–peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable. IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins.
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47
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Fuerst TR, Pierce BG, Keck ZY, Foung SKH. Designing a B Cell-Based Vaccine against a Highly Variable Hepatitis C Virus. Front Microbiol 2018; 8:2692. [PMID: 29379486 PMCID: PMC5775222 DOI: 10.3389/fmicb.2017.02692] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Accepted: 12/26/2017] [Indexed: 02/06/2023] Open
Abstract
The ability to use structure-based design and engineering to control the molecular shape and reactivity of an immunogen to induce protective responses shows great promise, along with corresponding advancements in vaccine testing and evaluation systems. We describe in this review new paradigms for the development of a B cell-based HCV vaccine. Advances in test systems to measure in vitro and in vivo antibody-mediated virus neutralization include retroviral pseudotype particles expressing HCV E1E2 glycoproteins (HCVpp), infectious cell culture-derived HCV virions (HCVcc), and surrogate animal models mimicking acute HCV infection. Their applications have established the role of broadly neutralizing antibodies to control HCV infection. However, the virus has immunogenic regions in the viral envelope glycoproteins that are associated with viral escape or non-neutralizing antibodies. These regions serve as immunologic decoys that divert the antibody response from less prominent conserved regions mediating virus neutralization. This review outlines the immunogenic regions on E2, which are roughly segregated into the hypervariable region 1 (HVR1), and five clusters of overlapping epitopes designated as antigenic domains A-E. Understanding the molecular architecture of conserved neutralizing epitopes within these antigenic domains, and how other antigenic regions or decoys deflect the immune response from these conserved regions will provide a roadmap for the rational design of an HCV vaccine.
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Affiliation(s)
- Thomas R Fuerst
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, United States.,Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, United States
| | - Brian G Pierce
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, United States.,Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, United States
| | - Zhen-Yong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, United States
| | - Steven K H Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, United States
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48
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Verma AK, Gupta S, Singh SP, Nagpure NS. An update on mechanism of entry of white spot syndrome virus into shrimps. FISH & SHELLFISH IMMUNOLOGY 2017; 67:141-146. [PMID: 28587833 DOI: 10.1016/j.fsi.2017.06.007] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Revised: 05/29/2017] [Accepted: 06/03/2017] [Indexed: 05/19/2023]
Abstract
Host-parasite relationships can be best understood at the level of protein-protein interaction between host and pathogen. Such interactions are instrumental in understanding the important stages of life cycle of pathogen such as adsorption of the pathogen on host surface followed by effective entry of pathogen into the host body, movement of the pathogen across the host cytoplasm to reach the host nucleus and replication of the pathogen within the host. White Spot Disease (WSD) is a havoc for shrimps and till date no effective treatment is available against the disease. Moreover information regarding the mechanism of entry of White Spot Syndrome Virus (WSSV) into shrimps, as well as knowledge about the protein interactions occurring between WSSV and shrimp during viral entry are still at very meagre stage. A cumulative and critically assessed information on various viral-shrimp interactions occurring during viral entry can help to understand the exact pathway of entry of WSSV into the shrimp which in turn can be used to device drugs that can stop the entry of virus into the host. In this context, we highlight various WSSV and shrimp proteins that play role in the entry mechanism along with the description of the interaction between host and pathogen proteins.
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Affiliation(s)
- Arunima Kumar Verma
- Department of Zoology, Autonomous Government P.G. College, Satna, Madhya Pradesh, India.
| | - Shipra Gupta
- Bioinformatics Centre, Biotech Park, Sector-G, Jankipuram, Lucknow, 226021, Uttar Pradesh, India
| | - Shivesh Pratap Singh
- Department of Zoology, Autonomous Government P.G. College, Satna, Madhya Pradesh, India
| | - Naresh Sahebrao Nagpure
- Fish Genetics and Biotechnology Division, ICAR-Central Institute of Fisheries Education, Panch Marg, Off Yari Road, Mumbai, 40006, India
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49
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Bukh J. The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control. J Hepatol 2016; 65:S2-S21. [PMID: 27641985 DOI: 10.1016/j.jhep.2016.07.035] [Citation(s) in RCA: 161] [Impact Index Per Article: 17.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2016] [Accepted: 07/29/2016] [Indexed: 12/11/2022]
Abstract
The discovery of hepatitis C virus (HCV) in 1989 permitted basic research to unravel critical components of a complex life cycle for this important human pathogen. HCV is a highly divergent group of viruses classified in 7 major genotypes and a great number of subtypes, and circulating in infected individuals as a continuously evolving quasispecies destined to escape host immune responses and applied antivirals. Despite the inability to culture patient viruses directly in the laboratory, efforts to define the infectious genome of HCV resulted in development of experimental recombinant in vivo and in vitro systems, including replicons and infectious cultures in human hepatoma cell lines. And HCV has become a model virus defining new paradigms in virology, immunology and biology. For example, HCV research discovered that a virus could be completely dependent on microRNA for its replication since microRNA-122 is critical for the HCV life cycle. A number of other host molecules critical for HCV entry and replication have been identified. Thus, basic HCV research revealed important molecules for development of host targeting agents (HTA). The identification and characterization of HCV encoded proteins and their functional units contributed to the development of highly effective direct acting antivirals (DAA) against the NS3 protease, NS5A and the NS5B polymerase. In combination, these inhibitors have since 2014 permitted interferon-free therapy with cure rates above 90% among patients with chronic HCV infection; however, viral resistance represents a challenge. Worldwide control of HCV will most likely require the development of a prophylactic vaccine, and numerous candidates have been pursued. Research characterizing features critical for antibody-based virus neutralization and T cell based virus elimination from infected cells is essential for this effort. If the world community promotes an ambitious approach by applying current DAA broadly, continues to develop alternative viral- and host- targeted antivirals to combat resistant variants, and invests in the development of a vaccine, it would be possible to eradicate HCV. This would prevent about 500 thousand deaths annually. However, given the nature of HCV, the millions of new infections annually, a high chronicity rate, and with over 150 million individuals with chronic infection (which are frequently unidentified), this effort remains a major challenge for basic researchers, clinicians and communities.
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Affiliation(s)
- Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
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50
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Wasilewski LN, Ray SC, Bailey JR. Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles. J Gen Virol 2016; 97:2883-2893. [PMID: 27667373 DOI: 10.1099/jgv.0.000608] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
A better understanding of natural variation in neutralization resistance and fitness of diverse hepatitis C virus (HCV) envelope (E1E2) variants will be critical to guide rational development of an HCV vaccine. This work has been hindered by inadequate genetic diversity in viral panels and by a lack of standardization of HCV entry assays. Neutralization assays generally use lentiviral pseudoparticles expressing HCV envelope proteins (HCVpp) or chimeric full-length viruses that are replication competent in cell culture (HCVcc). There have been few systematic comparisons of specific infectivities of E1E2-matched HCVcc and HCVpp, and to our knowledge, neutralization of E1E2-matched HCVpp and HCVcc has never been compared using a diverse panel of human broadly neutralizing monoclonal antibodies (bNAbs) targeting distinct epitopes. Here, we describe an efficient method for introduction of naturally occurring E1E2 genes into a full-length HCV genome, producing replication-competent chimeric HCVcc. We generated diverse panels of E1E2-matched HCVcc and HCVpp and measured the entry-mediating fitness of E1E2 variants using the two systems. We also compared neutralization of E1E2-matched HCVcc and HCVpp by a diverse panel of human bNAbs targeting epitopes across E1E2. We found no correlation between specific infectivities of E1E2-matched HCVcc versus HCVpp, but found a very strong positive correlation between relative neutralization resistance of these same E1E2-matched HCVcc and HCVpp variants. These results suggest that quantitative comparisons of neutralization resistance of E1E2 variants can be made with confidence using either HCVcc or HCVpp, allowing the use of either or both systems to maximize diversity of neutralization panels.
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Affiliation(s)
- Lisa N Wasilewski
- Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Stuart C Ray
- Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.,Department of Oncology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Justin R Bailey
- Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
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