Publications by authors named "Mansun Law"

72 Publications

Inactivated whole hepatitis C virus vaccine employing a licensed adjuvant elicits cross-genotype neutralizing antibodies in mice.

J Hepatol 2022 May 4;76(5):1051-1061. Epub 2022 Jan 4.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital - Amager and Hvidovre, and Department of Immunology and Microbiolgy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address:

Background & Aims: A prophylactic vaccine is required to eliminate HCV as a global public health threat. We developed whole virus inactivated HCV vaccine candidates employing a licensed adjuvant. Further, we investigated the effects of HCV envelope protein modifications (to increase neutralization epitope exposure) on immunogenicity.

Methods: Whole virus vaccine antigen was produced in Huh7.5 hepatoma cells, processed using a multistep protocol and formulated with adjuvant (MF-59 analogue AddaVax or aluminium hydroxide). We investigated the capacity of IgG purified from the serum of immunized BALB/c mice to neutralize genotype 1-6 HCV (by virus neutralization assays) and to bind homologous envelope proteins (by ELISA). Viruses used for immunizations were (i) HCV5aHi with strain SA13 envelope proteins and modification of an O-linked glycosylation site in E2 (T385P), (ii) HCV5aHi(T385) with reversion of T385P to T385, featuring the original E2 sequence determined in vivo and (iii) HCV5aHi(ΔHVR1) with deletion of HVR1. For these viruses, epitope exposure was investigated using human monoclonal (AR3A and AR4A) and polyclonal (C211 and H06) antibodies in neutralization assays.

Results: Processed HCV5aHi formulated with AddaVax induced antibodies that efficiently bound homologous envelope proteins and broadly neutralized cultured genotype 1-6 HCV, with half maximal inhibitory concentrations of between 14 and 192 μg/ml (mean of 36 μg/ml against the homologous virus). Vaccination with aluminium hydroxide was less immunogenic. Compared to HCV5aHi(T385) with the original E2 sequence, HCV5aHi with a modified glycosylation site and HCV5aHi(ΔHVR1) without HVR1 showed increased neutralization epitope exposure but similar immunogenicity.

Conclusion: Using an adjuvant suitable for human use, we developed inactivated whole HCV vaccine candidates that induced broadly neutralizing antibodies, which warrant investigation in further pre-clinical studies.

Lay Summary: A vaccine against hepatitis C virus (HCV) is needed to prevent the estimated 2 million new infections and 400,000 deaths caused by this virus each year. We developed inactivated whole HCV vaccine candidates using adjuvants licensed for human use, which, following immunization of mice, induced antibodies that efficiently neutralized all HCV genotypes with recognized epidemiological importance. HCV variants with modified envelope proteins exhibited similar immunogenicity as the virus with the original envelope proteins.
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http://dx.doi.org/10.1016/j.jhep.2021.12.026DOI Listing
May 2022

A structured RNA motif locks Argonaute2:miR-122 onto the 5' end of the HCV genome.

Nat Commun 2021 11 25;12(1):6836. Epub 2021 Nov 25.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

microRNAs (miRNAs) form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host miRNAs. For hepatitis C virus (HCV), binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation. Here, we investigate the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins. The crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5' end from enzymatic attack. The trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control. Combined results reveal a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex.
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http://dx.doi.org/10.1038/s41467-021-27177-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8616905PMC
November 2021

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

Gastroenterology 2022 02 13;162(2):562-574. Epub 2021 Oct 13.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

Background & Aims: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays.

Methods: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps.

Results: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones.

Conclusions: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.
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http://dx.doi.org/10.1053/j.gastro.2021.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8792218PMC
February 2022

From Structural Studies to HCV Vaccine Design.

Viruses 2021 05 4;13(5). Epub 2021 May 4.

Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.

Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments of antiviral therapeutics. To achieve global elimination of HCV, an effective cross-genotype vaccine is needed. The failure of previous vaccination trials to elicit an effective cross-reactive immune response demands better vaccine antigens to induce a potent cross-neutralizing response to improve vaccine efficacy. HCV E1 and E2 envelope (Env) glycoproteins are the main targets for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. Therefore, a molecular-level understanding of the nAb responses against HCV is imperative for the rational design of cross-genotype vaccine antigens. Here we summarize the recent advances in structural studies of HCV Env and Env-nAb complexes and how they improve our understanding of immune recognition of HCV. We review the structural data defining HCV neutralization epitopes and conformational plasticity of the Env proteins, and the knowledge applicable to rational vaccine design.
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http://dx.doi.org/10.3390/v13050833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147963PMC
May 2021

Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins.

Immunity 2021 04 5;54(4):781-796.e4. Epub 2021 Mar 5.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.
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http://dx.doi.org/10.1016/j.immuni.2021.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046733PMC
April 2021

Virus reactivation in a non-cirrhotic HBV patient requiring liver transplantation after cessation of nucleoside analogue therapy.

Antivir Ther 2021 Jan-Feb;26(1-2):3-8. Epub 2021 Sep 26.

Division of Gastroenterology/Hepatology, 2697Scripps Clinic, La Jolla, CA, USA.

Nucleos(t)ide analogues (NAs) are a mainstay of therapy for chronic hepatitis B (CHB) infections and have a profound effect on hepatitis B virus (HBV) suppression. We report a rare case of HBV reactivation in a CHB patient without cirrhosis following cessation of NA therapy that resulted in acute liver failure requiring liver transplantation. Investigation of the viral genetics and host immune responses suggest that viral mutations known to promote virus replication are associated with reactivation, whereas adaptive immunity to HBV remained defective in this patient. Viral sequencing may be useful for identifying mutations that are unfavorable for therapy withdrawal.
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http://dx.doi.org/10.1177/13596535211042205DOI Listing
May 2022

Evaluation of a Series of Lipidated Tucaresol Adjuvants in a Hepatitis C Virus Vaccine Model.

ACS Med Chem Lett 2020 Dec 29;11(12):2428-2432. Epub 2020 Oct 29.

Department of Chemistry, Department of Immunology and Microbial Science, The Skaggs Institute for Chemical Biology, The Worm Institute of Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Hepatitis C virus (HCV) infections represent a global health challenge; however, developing a vaccine for treatment of HCV infection has remained difficult as heterogeneous HCV contains distinct genotypes, and each genotype contains various subtypes and different envelope glycoproteins. Currently, there is no effective preventive vaccine for achieving global control over HCV. In our efforts to improve upon current HCV vaccines we designed a synthetically accessible adjuvant platform, wherein we synthesized 11 novel lipidated tucaresol analogues to assess their immunological potential. Using a tucaresol-based adjuvant approach, truncated lipid-variants together with an engineered E1E2 antigen construct, namely E2ΔTM3, elicited antibody (Ab) responses that were significantly higher than tucaresol. In sum, antibody end-point titer values largely corroborated HCV neutralization data with a simplified lipidated tucaresol variant affording the highest end point titer and % neutralization. This study lays the groundwork for additional permutations in tucaresol adjuvant design, including the examination of other proteins in vaccine development.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734796PMC
December 2020

An alternate conformation of HCV E2 neutralizing face as an additional vaccine target.

Sci Adv 2020 07 24;6(30):eabb5642. Epub 2020 Jul 24.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.

To achieve global elimination of hepatitis C virus (HCV), an effective cross-genotype vaccine is needed. The HCV envelope glycoprotein E2 is the main target for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. E2 is structurally flexible and functions in engaging host receptors. Many nAbs bind to the "neutralizing face" on E2, including several broadly nAbs encoded by the germline gene family that bind to a similar conformation (A) of this face. Here, a previously unknown conformation (B) of the neutralizing face is revealed in crystal structures of two of four additional E2-V1-69 nAb complexes. In this conformation, the E2 front-layer region is displaced upon antibody binding, exposing residues in the back layer for direct antibody interaction. This E2 B structure may represent another conformational state in the viral entry process that is susceptible to antibody neutralization and thus provide a new target for rational vaccine development.
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http://dx.doi.org/10.1126/sciadv.abb5642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380959PMC
July 2020

Into the Unknown: A Chemical Biology Approach Provides Mechanistic Insight into HCV Entry.

Cell Chem Biol 2020 07;27(7):767-769

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

In this issue of Cell Chemical Biology, Hu et al. (2020) demonstrate that chlorcyclizine blocks HCV fusion by targeting the putative fusion peptide on the viral envelope glycoprotein E1. The study provides new insights into the viral fusion machinery, presenting an opportunity to study novel antivirals against HCV.
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http://dx.doi.org/10.1016/j.chembiol.2020.06.018DOI Listing
July 2020

Proof of concept for rational design of hepatitis C virus E2 core nanoparticle vaccines.

Sci Adv 2020 04 15;6(16):eaaz6225. Epub 2020 Apr 15.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are responsible for cell entry, with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell-based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved substantial yield and purity, as well as enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B cell patterns associated with nanoparticle-induced antibody responses, which target the conserved neutralizing epitopes on E2 and cross-neutralize HCV genotypes.
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http://dx.doi.org/10.1126/sciadv.aaz6225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159917PMC
April 2020

Antibody Responses in Hepatitis C Infection.

Authors:
Mansun Law

Cold Spring Harb Perspect Med 2021 03 1;11(3). Epub 2021 Mar 1.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California 92109, USA.

Antibody responses in hepatitis C virus (HCV) have been a rather mysterious research topic for many investigators working in the field. Chronic HCV infection is often associated with dysregulation of immune functions particularly in B cells, leading to abnormal lymphoproliferation or the production of autoantibodies that exacerbate inflammation and extrahepatic diseases. When considering the antiviral function of antibody, it was difficult to endorse its role in HCV protection, whereas T-cell response has been shown unequivocally critical for natural recovery. Recent breakthroughs in the study of HCV and antigen-specific antibody responses provide important insights into viral vulnerability to antibodies and the immunogenetic and structural properties of the neutralizing antibodies. The new knowledge reinvigorates HCV vaccine research by illuminating a new path for the rational design of vaccine antigens to elicit broadly neutralizing antibodies for protection.
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http://dx.doi.org/10.1101/cshperspect.a036962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919400PMC
March 2021

Antibody Responses to Immunization With HCV Envelope Glycoproteins as a Baseline for B-Cell-Based Vaccine Development.

Gastroenterology 2020 03 4;158(4):1058-1071.e6. Epub 2019 Dec 4.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California. Electronic address:

Background & Aims: We investigated antibody responses to hepatitis C virus (HCV) antigens E1 and E2 and the relevance of animal models for vaccine development. We compared antibody responses to vaccination with recombinant E1E2 complex in healthy volunteers, non-human primates (NHPs), and mice.

Methods: We analyzed 519 serum samples from participants in a phase 1 vaccine trial (ClinicalTrials.gov identifier NCT00500747) and compared them with serum or plasma samples from C57BL/6J mice (n = 28) and rhesus macaques (n = 4) immunized with the same HCV E1E2 antigen. Blood samples were collected at different time points and analyzed for antibody binding, neutralizing activity, and epitope specificity. Monoclonal antibodies from the immunized NHPs were isolated from single plasmablasts and memory B cells, and their immunogenetic properties were characterized.

Results: Antibody responses of the volunteers, NHPs, and mice to the non-neutralizing epitopes on the E1 N-terminus and E2 hypervariable region 1 did not differ significantly. Antibodies from volunteers and NHPs that neutralized heterologous strains of HCV primarily interacted with epitopes in the antigen region 3. However, the neutralizing antibodies were not produced in sufficient levels for broad neutralization of diverse HCV isolates. Broadly neutralizing antibodies similar to the human V1-69 class antibody specific for antigen region 3 were produced in the immunized NHPs.

Conclusions: In an analysis of vaccinated volunteers, NHPs, and mice, we found that recombinant E1E2 vaccine antigen induces high-antibody titers that are insufficient to neutralize diverse HCV isolates. Antibodies from volunteers and NHPs bind to the same neutralizing epitopes for virus neutralization. NHPs can therefore be used as a preclinical model to develop HCV vaccines. These findings also provide useful baseline values for development of vaccines designed to induce production of neutralizing antibodies.
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http://dx.doi.org/10.1053/j.gastro.2019.11.282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371413PMC
March 2020

Author Correction: Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

Nat Biomed Eng 2019 Oct;3(10):842

Department of Bioengineering, University of California San Diego, San Diego, CA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41551-019-0456-6DOI Listing
October 2019

Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

Nat Biomed Eng 2019 10 22;3(10):806-816. Epub 2019 Jul 22.

Department of Bioengineering, University of California San Diego, San Diego, CA, USA.

Protein-based therapeutics can activate the adaptive immune system, leading to the production of neutralizing antibodies and the clearance of the treated cells mediated by cytotoxic T cells. Here, we show that the sequential use of immune-orthogonal orthologues of CRISPR-associated protein 9 (Cas9) and adeno-associated viruses (AAVs) evades adaptive immune responses and enables effective gene editing using repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major histocompatibility complex (MHC) proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors and 167 AAV VP1-capsid-protein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologues-which we validated for three Cas9 orthologues in mice-yet we anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo gene editing is uncompromised when using multiple dosing with orthologues of AAVs and Cas9 in mice that were previously immunized against the AAV vector and the Cas9 cargo. Multiple dosing with protein orthologues may allow for sequential regimens of protein therapeutics that circumvent pre-existing immunity or induced immunity.
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http://dx.doi.org/10.1038/s41551-019-0431-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783354PMC
October 2019

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 05 30;116(20):10039-10047. Epub 2019 Apr 30.

Copenhagen Hepatitis C Program, Department of Infectious Diseases, Copenhagen University Hospital, DK-2650 Hvidovre, Denmark;

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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http://dx.doi.org/10.1073/pnas.1822002116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525505PMC
May 2019

V1-69 antiviral broadly neutralizing antibodies: genetics, structures, and relevance to rational vaccine design.

Curr Opin Virol 2019 02 16;34:149-159. Epub 2019 Mar 16.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Broadly neutralizing antibodies (bnAbs) are potential therapeutic molecules and valuable tools for studying conserved viral targets for vaccine and drug design. Interestingly, antibody responses to conserved epitopes can be highly convergent at the molecular level. Human antibodies targeting a number of viral antigens have often been found to utilize a restricted set of immunoglobulin germline genes in different individuals. Here we review recent knowledge on V1-69-encoded antibodies in antiviral responses to influenza virus, HCV, and HIV-1. These antibodies share common genetic and structural features, and often develop neutralizing activity against a broad spectrum of viral strains. Understanding the genetic and structural characteristics of such antibodies and the target epitopes should help advance novel strategies to elicit bnAbs through vaccination.
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http://dx.doi.org/10.1016/j.coviro.2019.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266006PMC
February 2019

Genetic and structural insights into broad neutralization of hepatitis C virus by human V1-69 antibodies.

Sci Adv 2019 01 2;5(1):eaav1882. Epub 2019 Jan 2.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.

An effective vaccine to the antigenically diverse hepatitis C virus (HCV) must target conserved immune epitopes. Here, we investigate cross-neutralization of HCV genotypes by broadly neutralizing antibodies (bNAbs) encoded by the relatively abundant human gene family . We have deciphered the molecular requirements for cross-neutralization by this unique class of human antibodies from crystal structures of HCV E2 in complex with bNAbs. An unusually high binding affinity is found for germ line-reverted versions of V1-69 precursor antibodies, and neutralization breadth is acquired during affinity maturation. Deep sequencing analysis of an HCV-immune B cell repertoire further demonstrates the importance of the gene family in the generation of HCV bNAbs. This study therefore provides critical insights into immune recognition of HCV with important implications for rational vaccine design.
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http://dx.doi.org/10.1126/sciadv.aav1882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314831PMC
January 2019

Standardized Method for the Study of Antibody Neutralization of HCV Pseudoparticles (HCVpp).

Methods Mol Biol 2019 ;1911:441-450

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Hepatitis C virus (HCV) pseudoparticles (HCVpp) are generated by cotransfection of HCV envelope (E1 and E2) genes along with a retroviral packaging/reporter construct into HEK293T cells. Enveloped particles bearing HCV E1E2 proteins on their surface are released through a retroviral budding process into the supernatant. Viral E1E2 glycoproteins facilitate a single round of receptor-mediated entry of HCVpp into hepatoma cells, which can be quantified by reporter gene expression. These HCVpp have been employed to study mechanisms of HCV entry into hepatoma cells, as well as HCV neutralization by immune sera or HCV-specific monoclonal antibodies.
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http://dx.doi.org/10.1007/978-1-4939-8976-8_30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398441PMC
June 2019

Detection of Antibodies to HCV E1E2 by Lectin-Capture ELISA.

Methods Mol Biol 2019 ;1911:421-432

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.

Enzyme-linked immunosorbent assays (ELISAs) enable rapid detection and quantitation of antibodies in samples. Such assays can be highly sensitive and can be performed in most laboratories with basic equipment. Although detecting binding antibodies to the surface proteins of most pathogens by ELISA is not always indicative of antibody function, i.e., neutralizing activity of antibodies, the results can be used as a first step toward more in-depth analysis of antibody responses. Here we describe a method that can be used to standardize ELISAs for the detection of HCV envelope antibodies across laboratories and provide adaptations of the method to further characterize antibody responses in serum samples.
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http://dx.doi.org/10.1007/978-1-4939-8976-8_28DOI Listing
June 2019

Probing the Antigenicity of HCV Envelope Glycoproteins by Phage Display Antibody Technology.

Methods Mol Biol 2019 ;1911:381-393

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.

The envelope glycoproteins E1 and E2 of hepatitis C virus form a heterodimeric complex on the viral surface. They are the targets of neutralizing antibodies and are being investigated as potential vaccine antigens. Because of the high level of cysteine residues and N-glycosylation sites in the polypeptide sequences, it is technically challenging to produce pure, folded recombinant E1, E2, and E1E2 complex for downstream analysis. In this chapter, the methods we used to isolate a panel of human antibodies specific to diverse antigenic regions on the glycoproteins are discussed. The antibodies have been found to be valuable reagents for the study of HCV envelope glycoproteins, including the determination of the first E2 core domain structure.
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http://dx.doi.org/10.1007/978-1-4939-8976-8_26DOI Listing
June 2019

Simultaneous Quantification of Hepatitis C Virus Envelope Glycoproteins E1 and E2 by Dual-Color Fluorescence Immunoblot Analysis.

Methods Mol Biol 2019 ;1911:295-304

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.

The hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are crucial for HCV assembly and entry, and are promising vaccine antigens. However, they are challenging to study because of technical difficulties in protein production and in quality control for protein folding and glycosylation. To study E1 and E2 in different experimental systems, e.g. infected cells, virus culture, virus-like particles, and clinical samples, a standardized method to accurately quantify the glycoproteins will be essential for most research projects. Here we outline a sensitive assay based on dual-color fluorescence immunoblot and the Odyssey imaging system to detect and quantify HCV E1 and E2 glycoproteins either using a purified E1E2 complex, or an engineered protein standard containing E1 and E2 at equal molar ratio. The method is capable of simultaneously detecting and quantifying as little as 7 ng of E1 and 5 ng of E2 in HCV pseudoparticles, and will be useful to quantify E1 and E2 from a wide variety of samples.
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http://dx.doi.org/10.1007/978-1-4939-8976-8_20DOI Listing
June 2019

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms.

J Virol 2019 02 5;93(4). Epub 2019 Feb 5.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Hvidovre, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Yearly, ∼2 million people become hepatitis C virus (HCV) infected, resulting in an elevated lifetime risk for severe liver-related chronic illnesses. Characterizing epitopes of broadly neutralizing antibodies (NAbs), such as AR3A, is critical to guide vaccine development. Previously identified alanine substitutions that can reduce AR3A binding to expressed H77 envelope were introduced into chimeric cell culture-infectious HCV recombinants (HCVcc) H77(core-NS2)/JFH1. Substitutions G523A, G530A, and D535A greatly reduced fitness, and S424A, P525A, and N540A, although viable, conferred only low-level AR3A resistance. Using highly NAb-sensitive hypervariable region 1 (HVR1)-deleted HCVcc, H77/JFH1 and J6(core-NS2)/JFH1, we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1, or J6/JFH1 with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1, and D431G in J6/JFH1 M345T increased infectivity and conferred low-level AR3A resistance to H77/JFH1 but not H77/JFH1 L438S and F442Y conferred high-level AR3A resistance to H77/JFH1 but abrogated the infectivity of H77/JFH1. D431G conferred AR3A resistance to J6/JFH1 but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1 but not J6/JFH1 while decreasing scavenger receptor class B type I coreceptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other genotype 2a recombinants [JFH1 or T9(core-NS2)/JFH1]. Although the data indicate that AR3A has a high barrier to resistance, our approach permitted identification of low-level resistance substitutions. Also, the HVR1-dependent effects on AR3A resistance substitutions suggest a complex role of HVR1 in virus escape and receptor usage, with important implications for HCV vaccine development. Hepatitis C virus (HCV) is a leading cause of liver-related mortality, and limited treatment accessibility makes vaccine development a high priority. The vaccine-relevant cross-genotype-reactive antibody AR3A has shown high potency, but the ability of the virus to rapidly escape by mutating the AR3A epitope (barrier to resistance) remains unexplored. Here, we succeeded in inducing only low-level AR3A resistance, indicating a higher barrier to resistance than what we have previously reported for AR5A. Furthermore, we identify AR3A resistance substitutions that have hypervariable region 1 (HVR1)-dependent effects on HCV viability and on broad neutralization sensitivity. One of these substitutions increased envelope breathing and decreased scavenger receptor class B type I HCV coreceptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and the role of HVR1 in protecting HCV from AR3-targeting antibodies. These viral escape mechanisms should be taken into consideration in future HCV vaccine development.
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http://dx.doi.org/10.1128/JVI.01909-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364003PMC
February 2019

Interrogation of Antigen Display on Individual Vaccine Nanoparticles for Achieving Neutralizing Antibody Responses against Hepatitis C Virus.

Nano Lett 2018 12 21;18(12):7832-7838. Epub 2018 Nov 21.

Department of Pharmaceutical Sciences , University of Michigan , Ann Arbor , Michigan 48109 , United States.

Elicitation of neutralizing antibody responses against hepatitis C virus (HCV) has been a challenging goal. While the E2 subunit of the HCV envelope glycoprotein complex is a promising target for generating cross-genotype neutralizing antibodies, vaccinations with soluble E2 immunogens generally induce weak neutralizing antibody responses. Here, E2 immunogens (i.e., E2.661 and E2c.661) were loaded into lipid-based nanovaccines and examined for induction of neutralizing antibody responses. Compared with soluble E2 immunogens, E2 nanoparticles elicited 6- to 20-fold higher E2-specific serum IgG titers in mice. Importantly, E2 vaccine nanoparticles analyzed at a single particle level with a flow cytometry-based method revealed interesting dynamics between epitope display on the surfaces of nanoparticles in vitro and induction of neutralizing antibody responses in vivo. E2c.661 nanoparticles that are preferentially bound by a broadly neutralizing antibody, HCV1, in vitro elicit neutralizing antibody responses against both autologous and heterologous HCV virions in vivo. In stark contrast, E2.661 nanoparticles with reduced HCV1-antibody binding in vitro mainly induce autologous neutralizing antibody responses in vivo. These results show that rationale antigen design coupled with interrogation of epitope display on vaccine nanoparticles at a single particle level may aid in vaccine development toward achieving neutralizing antibody responses in vivo.
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http://dx.doi.org/10.1021/acs.nanolett.8b03601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465111PMC
December 2018

Hepatitis C Virus-Escape Studies for Human Monoclonal Antibody AR4A Reveal Isolate-Specific Resistance and a High Barrier to Resistance.

J Infect Dis 2019 01;219(1):68-79

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital.

Global control of hepatitis C virus (HCV) depends on development of a prophylactic vaccine. We studied escape for cross-genotype-reactive neutralizing antibody AR4A, providing valuable information for HCV vaccine design. We cultured HCV core-NS2 recombinants H77 (genotype 1a)/JFH1 or the highly antibody-susceptible hypervariable region 1 (HVR1)-deleted variants H77/JFH1∆HVR1 and J6(genotype 2a)/JFH1∆HVR1 in Huh7.5 cells with AR4A. Long-term AR4A exposure of H77/JFH1 and H77/JFH1∆HVR1 did not yield resistance. However, J6/JFH1∆HVR1 developed the envelope-E2 substitutions I696T or I696N, which reduced AR4A binding (I696N > I696T). I696N conferred greater AR4A resistance than I696T in J6/JFH1∆HVR1, whereas the reverse was observed in J6/JFH1. This was because I696N but not I696T conferred broadly increased antibody neutralization susceptibility to J6/JFH1. I696N and I696T abrogated infectivity of H77/JFH1 and broadly increased neutralization susceptibility of S52 (genotype 3a)/JFH1. In conclusion, I696 is in the AR4A epitope, which has a high barrier to resistance, thus strengthening the rationale for its inclusion in rational HCV vaccine designs.
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http://dx.doi.org/10.1093/infdis/jiy481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455953PMC
January 2019

Immunogenetic and structural analysis of a class of HCV broadly neutralizing antibodies and their precursors.

Proc Natl Acad Sci U S A 2018 07 28;115(29):7569-7574. Epub 2018 Jun 28.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037;

Elicitation of broadly neutralizing antibodies (bnAbs) is a leading strategy in rational vaccine design against antigenically diverse pathogens. Here, we studied a panel of monoclonal antibodies (mAbs) from mice immunized with the hepatitis C virus (HCV) envelope glycoproteins E1E2. Six of the mAbs recognize the conserved E2 antigenic site 412-423 (AS412) and cross-neutralize diverse HCV genotypes. Immunogenetic and structural analysis revealed that the antibodies originated from two different germline (GL) precursors and bind AS412 in a β-hairpin conformation. Intriguingly, the anti-HCV activity of one antibody lineage is associated with maturation of the light chain (LC), whereas the other lineage is dependent on heavy-chain (HC) maturation. Crystal structures of GL precursors of the LC-dependent lineage in complex with AS412 offer critical insights into the maturation process of bnAbs to HCV, providing a scientific foundation for utilizing the mouse model to study AS412-targeting vaccine candidates.
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http://dx.doi.org/10.1073/pnas.1802378115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055136PMC
July 2018

The Neutralizing Face of Hepatitis C Virus E2 Envelope Glycoprotein.

Front Immunol 2018 11;9:1315. Epub 2018 Jun 11.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, United States.

The high genetic variability of hepatitis C virus, together with the high level of glycosylation on the viral envelope proteins shielding potential neutralizing epitopes, pose a difficult challenge for vaccine development. An effective hepatitis C virus (HCV) vaccine must target conserved epitopes and the HCV E2 glycoprotein is the main target for such neutralizing antibodies (NAbs). Recent structural investigations highlight the presence of a highly conserved and accessible surface on E2 that is devoid of N-linked glycans and known as the E2 neutralizing face. This face is defined as a hydrophobic surface comprising the front layer (FL) and the CD81 binding loop (CD81bl) that overlap with the CD81 receptor binding site on E2. The neutralizing face consists of highly conserved residues for recognition by cross-NAbs, yet it appears to be high conformationally flexible, thereby presenting a moving target for NAbs. Three main overlapping neutralizing sites have been identified in the neutralizing face: antigenic site 412 (AS412), antigenic site 434 (AS434), and antigenic region 3 (AR3). Here, we review the structural analyses of these neutralizing sites, either as recombinant E2 or epitope-derived linear peptides in complex with bNAbs, to understand the functional and preferred conformations for neutralization, and for viral escape. Collectively, these studies provide a foundation and molecular templates to facilitate structure-based approaches for HCV vaccine development.
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http://dx.doi.org/10.3389/fimmu.2018.01315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008530PMC
June 2018

Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis.

PLoS Pathog 2017 12 18;13(12):e1006735. Epub 2017 Dec 18.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, United States of America.

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.
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http://dx.doi.org/10.1371/journal.ppat.1006735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749897PMC
December 2017

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

mBio 2017 02 28;8(1). Epub 2017 Feb 28.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA

Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches. Both epitope-focused and trimer-based strategies are currently being explored in HIV-1 vaccine development, which aims to elicit broadly neutralizing antibodies (bNAbs) targeting conserved epitopes on the viral envelope (Env). However, little is known about the differences in antibody response to these bNAb targets presented by foreign scaffolds and native Env. In this study, a systematic effort was undertaken to design multivalent epitope scaffolds and soluble gp140.681 trimers with a complete antigenic surface, and to comparatively analyze the antibody responses elicited by these antigens to the N332 supersite and MPER in a mouse model. This study will inform both epitope-focused and trimer-based vaccine design and will facilitate integration of the two vaccine strategies.
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http://dx.doi.org/10.1128/mBio.00036-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347340PMC
February 2017

Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A.

PLoS Pathog 2017 02 23;13(2):e1006214. Epub 2017 Feb 23.

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.

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3-4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2-6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1-deleted viruses to rapidly generate escape viruses aiding our understanding of the divergent escape pathways used by HCV to evade AR5A.
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http://dx.doi.org/10.1371/journal.ppat.1006214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358973PMC
February 2017

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

J Virol 2017 Jan 16;91(1). Epub 2016 Dec 16.

Li Ka Shing Institute of Virology, Department of Medical Microbiology & Immunology, University of Alberta, Edmonton, Canada

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.

Importance: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.
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http://dx.doi.org/10.1128/JVI.01552-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165201PMC
January 2017
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