Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

Cardoso, M. da Silva; Siemoneit, K.; Sturm, Daniela; Krone, Christoph; Moradpour, Darius; Kubanek, B

doi:10.1002/(sici)1096-9071(199805)55:1<28::aid-jmv6>3.0.co;2-q

Cited by 18 publications

(7 citation statements)

References 22 publications

(17 reference statements)

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“…The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus, RMPA95, expressing the envelope proteins E1 and E2 of an HCV genotype 1a (H) strain. Positive clones were fused with the heteromyeloma line K6H6/B5 (14).…”

Section: Methodsmentioning

confidence: 99%

Preclinical Evaluation of Two Neutralizing Human Monoclonal Antibodies against Hepatitis C Virus (HCV): a Potential Treatment To Prevent HCV Reinfection in Liver Transplant Patients

Eren

Landstein

Terkieltaub

et al. 2006

J Virol

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Section: Methodsmentioning

confidence: 99%

Preclinical Evaluation of Two Neutralizing Human Monoclonal Antibodies against Hepatitis C Virus (HCV): a Potential Treatment To Prevent HCV Reinfection in Liver Transplant Patients

Eren

Landstein

Terkieltaub

et al. 2006

J Virol

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“…Intriguingly, intracellular forms of truncated E2, enriched for the presence of monomeric, nonaggregated E2, were found to bind CD81 with greater affinity than the secreted forms (21,28), suggesting that structural differences exist between the intracellular and secreted forms of the E2 glycoprotein. Indeed, several monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes within E2 have provided insight into the conformational state of the HCV envelope glycoproteins (5,9,12,15,16,26,27,38,43). Studies with these MAbs have demonstrated structural differences between truncated forms of E2 and full-length E1E2 complexes.…”

mentioning

confidence: 99%

CD81-Dependent Binding of Hepatitis C Virus E1E2 Heterodimers

Cocquerel

Kuo

Dubuisson

et al. 2003

J Virol

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Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.Hepatitis C virus (HCV) infection is a major health problem affecting an estimated 160 million people worldwide (36). It is a major cause of chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (53), and mixed cryoglobulinemia, a B-lymphocyte proliferative disorder (reviewed in references 6 and 49). HCV is a small enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (33). Its genome encodes a single ϳ3,000-amino-acid polyprotein that is co-and posttranslationally processed by viral and cellular proteases to yield the mature structural and nonstructural proteins (33, 37). The structural proteins-the core protein and envelope glycoproteins E1 and E2-are believed to be the major constituents of HCV particles. The E1 and E2 envelope proteins are N glycosylated in their large N-terminal ectodomains and are anchored into membranes by their hydrophobic C-terminal transmembrane domains (TMDs) (39). These domains have been shown to be endoplasmic reticulum (ER) retention signals (10,12,20,23). E1 and E2 associate to form two types of complexes: properly folded E1E2 heterodimers stabilized by noncovalent interactions and misfolded disulfide-linked aggregates (for a review, see reference 39).The E1E2 noncovalent heterodimer, comprising the viral envelope (reviewed in reference 39), is involved in viral entry (3, 30); however, the mechanism of HCV cell entry is not clear. Several putative cell surface receptors of HCV or recombinant E2 proteins have been identified (1,25,34,45,46,50,51). Among these receptors, human CD81 has been repeatedly shown to interact with recombinant soluble E2, the E1E2 comple...

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“…Abs that arise in HCV-infected individuals in response to viral infection are anticipated to react with the truly native conformation of the viral envelope structure. Recently, several HMAbs have been identified that react with conformational epitopes within E2 (1,11,23,24). Moreover, some of these HMAbs have been shown to have neutralization-of-binding (NOB) activity (1, 23, 24) defined by their ability to neutralize binding of recombinant, truncated HCV-E2 to human cells (37).…”

mentioning

confidence: 99%

Recognition of Native Hepatitis C Virus E1E2 Heterodimers by a Human Monoclonal Antibody

Cocquerel¹,

Quinn²,

Flint³

et al. 2003

J Virol

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The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.Hepatitis C virus (HCV) is the causal agent of hepatitis C, which is a major health problem worldwide (28). HCV is a positive-strand RNA virus (2) that belongs to the Flaviviridae family. Its genome encodes two membrane-associated envelope glycoproteins, E1 and E2, which are N glycosylated in their large N-terminal ectodomains and are anchored into membranes by their C-terminal transmembrane domains (31). These latter domains have been shown to be endoplasmic reticulum (ER) retention signals (5,7,17,20). When expressed in cell culture, the E1 and E2 glycoproteins assemble into noncovalently linked E1E2 heterodimers. These noncovalent E1E2 complexes have been proposed as functional subunits of the HCV particle. In addition, a significant amount of E1 and E2 is also present in high-molecular-weight, disulfide-linked aggregates, thought to result from a nonproductive folding pathway leading to misfolded protein complexes (for review see reference 31).Because of the lack of a suitable cell culture system for in vitro propagation of HCV and the unavailability of virions in sufficient quantities, truncated, secreted versions of E2 have been used as soluble surrogates for native virus particles. Indeed, the identification of CD81 as the putative cellular receptor for HCV is based on its binding to a truncated form of E2 (36). Intriguingly, intracellular forms of truncated E2, enriched for the presence of monomeric, nonaggregated E2, were found to bind CD81 with greater affinity than did the secreted forms (18, 26), suggesting that antigenic or structural differences exist between intracellular and secreted forms of the E2 glycoprotein. Several murine monoclonal antibodies (MAbs) have been shown to recognize conformation-dependent epitopes within E2. Studies using these antibodies (Abs) (including MAb H53) have provided additional insight into the conformational state of the envelope glycoproteins during intracellular processing and folding and have helped to define a native, prebudding form of t...

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Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

Cited by 18 publications

References 22 publications

Preclinical Evaluation of Two Neutralizing Human Monoclonal Antibodies against Hepatitis C Virus (HCV): a Potential Treatment To Prevent HCV Reinfection in Liver Transplant Patients

Preclinical Evaluation of Two Neutralizing Human Monoclonal Antibodies against Hepatitis C Virus (HCV): a Potential Treatment To Prevent HCV Reinfection in Liver Transplant Patients

CD81-Dependent Binding of Hepatitis C Virus E1E2 Heterodimers

Recognition of Native Hepatitis C Virus E1E2 Heterodimers by a Human Monoclonal Antibody

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