A systematic analysis of a broadly neutralizing antibody AR3C epitopes on Hepatitis C virus E2 envelope glycoprotein and their cross-reactivity

ABSTRACT Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein. Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 μg/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.

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Cross-genotype characterization of genetic diversity and molecular adaptation in hepatitis C virus envelope glycoprotein genes

Journal of General Virology ◽

10.1099/vir.0.82357-0 ◽

2007 ◽

Vol 88 (2) ◽

pp. 458-469 ◽

Cited By ~ 22

Author(s):

Richard J. P. Brown ◽

Alexander W. Tarr ◽

C. Patrick McClure ◽

Vicky S. Juttla ◽

Nader Tagiuri ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Positive Selection ◽

Envelope Glycoprotein ◽

Neutralizing Antibody ◽

Full Length ◽

Envelope Gene ◽

Adaptive Mutations ◽

Codon Substitution ◽

Sequence Databases

Investigation of the mechanisms underlying hepatitis C virus (HCV) envelope glycoprotein gene evolution will greatly assist rational development of broadly neutralizing antibody-based vaccines or vaccine components. Previously, comprehensive cross-genotype evolutionary studies of E1E2 have not been possible due to the paucity of full-length envelope gene sequences representative of all major HCV genotypes (1–6) deposited in international sequence databases. To address this shortfall, a full-length E1E2 clone panel, corresponding to genotypes of HCV that were previously under-represented, was generated. This panel, coupled with divergent isolates available via international sequence databases, was subjected to high-resolution methods for determining codon-substitution patterns, enabling a fine-scale dissection of the selective pressures operating on HCV E1E2. Whilst no evidence for positive selection was observed in E1, the E2 protein contained a number of sites under strong positive selection. A high proportion of these sites were located within the first hypervariable region (HVR1), and statistical analysis revealed that cross-genotype adaptive mutations were restricted to a subset of homologous positions within this region. Importantly, downstream of HVR1, a differential genotype-specific distribution of adaptive mutations was observed, suggesting that subtly different evolutionary pressures shape present-day genotype diversity in E2 outside HVR1. Despite these observations, it is demonstrated that purifying selection due to functional constraint is the major evolutionary force acting on HCV E1E2. These findings are important in the context of neutralizing-antibody vaccine targeting, as well as in contributing to our understanding of E1E2 function.

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Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.01138-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12473-12482 ◽

Cited By ~ 114

Author(s):

Teresa J. Broering ◽

Kerry A. Garrity ◽

Naomi K. Boatright ◽

Susan E. Sloan ◽

Frantisek Sandor ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Glycoprotein ◽

Cross Reactivity ◽

Human Antibody ◽

Human Monoclonal Antibodies ◽

Genotype 1A ◽

Neutralizing Monoclonal Antibody

ABSTRACT Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.

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Broad Anti-Hepatitis C Virus (HCV) Antibody Responses Are Associated with Improved Clinical Disease Parameters in Chronic HCV Infection

Journal of Virology ◽

10.1128/jvi.02669-15 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4530-4543 ◽

Cited By ~ 17

Author(s):

Rachael E. Swann ◽

Vanessa M. Cowton ◽

Mark W. Robinson ◽

Sarah J. Cole ◽

Stephen T. Barclay ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Chronic Infection ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Hcv Infection ◽

Neutralization Activity ◽

Broadly Neutralizing Antibody ◽

Chronic Hcv Infection ◽

Chronic Hcv

ABSTRACTDuring hepatitis C virus (HCV) infection, broadly neutralizing antibody (bNAb) responses targeting E1E2 envelope glycoproteins are generated in many individuals. It is unclear if these antibodies play a protective or a pathogenic role during chronic infection. In this study, we investigated whether bNAb responses in individuals with chronic infection were associated with differences in clinical presentation. Patient-derived purified serum IgG was used to assess the breadth of HCV E1E2 binding and the neutralization activity of HCV pseudoparticles. The binding and neutralization activity results for two panels bearing viral envelope proteins representing either an intergenotype or an intragenotype 1 group were compared. We found that the HCV load was negatively associated with strong cross-genotypic E1E2 binding (P= 0.03). Overall, we observed only a modest correlation between total E1E2 binding and neutralization ability. The breadth of intergenotype neutralization did not correlate with any clinical parameters; however, analysis of individuals with genotype 1 (gt1) HCV infection (n= 20), using an intragenotype pseudoparticle panel, found a strong association between neutralization breadth and reduced liver fibrosis (P= 0.006). A broad bNAb response in our cohort with chronic infection was associated with a single nucleotide polymorphism (SNP) in theHLA-DQB1gene (P= 0.038), as previously reported in a cohort with acute disease. Furthermore, the bNAbs in these individuals targeted more than one region of E2-neutralizing epitopes, as assessed through cross-competition of patient bNAbs with well-characterized E2 antibodies. We conclude that the bNAb responses in patients with chronic gt1 infection are associated with lower rates of fibrosis and host genetics may play a role in the ability to raise such responses.IMPORTANCEGlobally, there are 130 million to 150 million people with chronic HCV infection. Typically, the disease is progressive and is a major cause of severe liver cirrhosis and hepatocellular carcinoma. While it is known that neutralizing antibodies have a role in spontaneous clearance during acute infection, little is known about their role in chronic infection. In the present work, we investigated the antibody response in a cohort of chronically infected individuals and found that a broadly neutralizing antibody response is protective and is associated with reduced levels of liver fibrosis and cirrhosis. We also found an association between SNPs in class II HLA genes and the presence of a broadly neutralizing response, indicating that antigen presentation may be important for the production of HCV-neutralizing antibodies.

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Toward a Hepatitis C Virus Vaccine: the Structural Basis of Hepatitis C Virus Neutralization by AP33, a Broadly Neutralizing Antibody

Journal of Virology ◽

10.1128/jvi.02052-12 ◽

2012 ◽

Vol 86 (23) ◽

pp. 12923-12932 ◽

Cited By ~ 67

Author(s):

J. A. Potter ◽

A. M. Owsianka ◽

N. Jeffery ◽

D. J. Matthews ◽

Z.-Y. Keck ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Vaccine ◽

Neutralizing Antibody ◽

Virus Neutralization ◽

Structural Basis ◽

Broadly Neutralizing Antibody

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Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1202924109 ◽

2012 ◽

Vol 109 (24) ◽

pp. 9499-9504 ◽

Cited By ~ 103

Author(s):

L. Kong ◽

E. Giang ◽

J. B. Robbins ◽

R. L. Stanfield ◽

D. R. Burton ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibody ◽

Virus Neutralization ◽

Structural Basis ◽

Broadly Neutralizing Antibody

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Escape of Hepatitis C Virus from Epitope I Neutralization Increases Sensitivity of Other Neutralization Epitopes

Journal of Virology ◽

10.1128/jvi.02066-17 ◽

2018 ◽

Vol 92 (9) ◽

Cited By ~ 11

Author(s):

Jun Gu ◽

Joshua Hardy ◽

Irene Boo ◽

Patricia Vietheer ◽

Kathleen McCaffrey ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Vaccine Development ◽

Acquired Resistance ◽

Neutralizing Antibody ◽

Cell Surface Receptor ◽

Antigenic Determinants ◽

Surface Receptor ◽

Broadly Neutralizing Antibody ◽

First Time

ABSTRACTThe 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.IMPORTANCEUnderstanding 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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Cross-reactivity and clinical impact of the antibody response to hepatitis C virus second envelope glycoprotein (E2)

Journal of Medical Virology ◽

10.1002/jmv.1096 ◽

2001 ◽

Vol 65 (1) ◽

pp. 23-29 ◽

Cited By ~ 7

Author(s):

Kenneth G. Hadlock ◽

Robert Gish ◽

Judy Rowe ◽

Sonal S. Rajyaguru ◽

Margaret Newsom ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antibody Response ◽

Envelope Glycoprotein ◽

Cross Reactivity ◽

Clinical Impact ◽

Glycoprotein E2

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Broadly Neutralizing Antibody Mediated Clearance of Human Hepatitis C Virus Infection

Cell Host & Microbe ◽

10.1016/j.chom.2018.10.012 ◽

2018 ◽

Vol 24 (5) ◽

pp. 717-730.e5 ◽

Cited By ~ 35

Author(s):

Valerie J. Kinchen ◽

Muhammad N. Zahid ◽

Andrew I. Flyak ◽

Mary G. Soliman ◽

Gerald H. Learn ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Neutralizing Antibody ◽

Hepatitis C Virus Infection ◽

Human Hepatitis ◽

Broadly Neutralizing Antibody

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Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: Implications for vaccine design

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614942113 ◽

2016 ◽

Vol 113 (45) ◽

pp. E6946-E6954 ◽

Cited By ~ 35

Author(s):

Brian G. Pierce ◽

Zhen-Yong Keck ◽

Patrick Lau ◽

Catherine Fauvelle ◽

Ragul Gowthaman ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Glycoprotein ◽

Rational Design ◽

Neutralizing Antibody ◽

Antibody Binding ◽

Viral Envelope ◽

Viral Escape ◽

E2 Protein ◽

Antibody Recognition

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.

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