Naturally selected hepatitis C virus polymorphisms confer broad neutralizing antibody resistance

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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Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609780113 ◽

2016 ◽

Vol 113 (45) ◽

pp. 12768-12773 ◽

Cited By ~ 44

Author(s):

Leopold Kong ◽

David E. Lee ◽

Rameshwar U. Kadam ◽

Tong Liu ◽

Erick Giang ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Design ◽

Neutralizing Antibody ◽

Md Simulations ◽

Receptor Binding Site ◽

Structural Investigations ◽

Thermophilic Organisms

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world’s population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen–deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

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Maternal Neutralizing Antibody and Transmission of Hepatitis C Virus to Infants

The Journal of Infectious Diseases ◽

10.1086/593067 ◽

2008 ◽

Vol 198 (11) ◽

pp. 1651-1655 ◽

Cited By ~ 19

Author(s):

Kimberly A. Dowd ◽

Ronald C. Hershow ◽

Sigal Yawetz ◽

Philip LaRussa ◽

Clemente Diaz ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibody

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Hepatitis C Virus Continuously Escapes From Neutralizing Antibody and T-Cell Responses During Chronic Infection in Vivo

Yearbook of Gastroenterology ◽

10.1016/s0739-5930(08)70218-6 ◽

2007 ◽

Vol 2007 ◽

pp. 271-272

Author(s):

K.M. Chang

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Chronic Infection ◽

T Cell Responses ◽

Neutralizing Antibody ◽

Cell Responses

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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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A DNA Vaccine Expressing Fusion Protein E2-NT(gp96) Induces Hepatitis C Virus Cross-Neutralizing Antibody in BALB/c Mice

Hepatitis Monthly ◽

10.5812/hepatmon.96347 ◽

2019 ◽

Vol 19 (9) ◽

Author(s):

Ebrahim Kord ◽

Jean Dubuisson ◽

Thibaut Vausselin ◽

Ali Akbar Amirzargar ◽

Mir Saeed Yekaninejad ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Fusion Protein ◽

Dna Vaccine ◽

Neutralizing Antibody

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A role for B cells to transmit hepatitis C virus infection

10.21203/rs.3.rs-41273/v1 ◽

2020 ◽

Author(s):

Isabelle Desombere ◽

Freya Van Houtte ◽

Ali Farhoudi ◽

Lieven Verhoye ◽

Caroline Buysschaert ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

B Cells ◽

Neutralizing Antibody ◽

Hcv Rna ◽

Serum Samples ◽

Cell Culture Systems ◽

Hcv Transmission

Abstract Hepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge of the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We previously reported that laboratory strains of HCV associated with non-permissive B cells could trans-infect hepatocytes and thereby evade host neutralizing antibody responses, suggesting a role for B cells in HCV transmission. To evaluate this hypothesis, we assessed the ability of B cells and sera from recent (<2 years) or chronic (≥ 2 years) infections to infect humanized liver chimeric mice. HCV was efficiently transmitted by B cells from chronically infected patients whereas the sera were non-infectious. In contrast, we noted that B cells from recently infected patients failed to transmit HCV to the mice, whereas all serum samples were infectious. Only patients with circulating anti-glycoprotein antibodies harbored genomic HCV-RNA in B cells. Taken together, our studies provide direct in vivo evidence for HCV transmission by B cells and these findings may have clinical implications for prophylactic and therapeutic antibody-based vaccine design.

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Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Journal of General Virology ◽

10.1099/jgv.0.001512 ◽

2020 ◽

Author(s):

Mphatso D. Kalemera ◽

Joan Capella-Pujol ◽

Ana Chumbe ◽

Alexander Underwood ◽

Rowena A. Bull ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Natural Infection ◽

Neutralizing Antibody ◽

Complex Type ◽

Functional Studies ◽

Endogenous Expression ◽

F Cells ◽

Large Extracellular Loop ◽

Made In

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

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