The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.Hepatitis C virus (HCV), a member of the family Flaviviridae, expresses its proteins from a 9.5-kb positive-sense RNA genome (18). The virus is highly variable, with more than nine distinct genotypes (1, 18). Most patients progress from acute to chronic disease in spite of a robust immune response. Nonetheless, evidence for a humoral immune response providing at least partial protection in clinical and animal model studies is accumulating (6, 9-11, 29, 37) and suggests that neutralizing antibodies have a role in the containment of HCV infection. For a protective immune response, the important viral gene products are the envelope proteins, designated E1 and E2. Both sequence analyses of different isolates and sequential studies of virus isolates in infected patients suggest that the HCV E2 protein is under immune selection leading to selection of variants in the amino-terminal domain of HCV E2, designated hypervariable region 1 (HVR-1) (1, 9, 16-18, 20, 37, 39, 40). Antibodies to HVR-1 appear to mediate virus neutralization in cell culture and chimpanzee protection studies (10, 37). Unfortunately, antibodies to HVR-1 tend to be isolate specific and over time drive the selection of new viral variants that the existing immune response does not recognize (9,20,37,40). Although there has been progress at inducing a broader immune response to HVR-1-related sequences (31), the high mutability of HVR-1 sequences in vivo may allow for the selection of immune escape mutants even against antibodies that recognize the majority of HVR-1 isolates.Studies using HCV ...

. Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.Hepatitis C virus (HCV) infects over 170 million individuals worldwide. Although acute infection is usually silent, most HCV infections progress to chronicity that is not cleared by an apparently robust immune response (3, 24). The virus is a member of the family Flaviviridae (37), with a 9.5-kb positivestrand RNA genome that encodes three structural proteins, the capsid and viral envelope proteins E1 and E2, and at least six nonstructural proteins, NS2 to NS5b (29). The envelope proteins are thought to be the primary mediators of virion attachment and cell entry (13). HCV E2 is a ϳ70-kDa glycoprotein that shows large variations among HCV genotypes and contains a 27-amino-acid (aa) sequence at its amino terminus that is highly variable and is designated the hypervariable region 1, or HVR1 (reviewed in references 3 and 6). This linear region on E2 is likely to be involved in virus infection, since neutralizing antisera to HVR1 have been reported in in vitro and in vivo models, although other studies showed that HCV with HVR1 deleted remains infectious (15,19,34,41,47). Unfortunately, a leading contributor to disease progression is the emergence of new viral mutants or "quasispecies" in HVR1 induced by immune selection. Increased diversity or mutations in HVR1 correlate with progressive disease, and decreased diversity correlates with resolving disease (14). HCV E2 is thought to mediate attachment to target cells and binds to human CD81, a member of the tetraspannin family of proteins (28). Interaction of E2 with CD81 on B or T cells has been reported to result in B-cell aggregation and a lowering of the threshold for T-and B-cell activation (17, 43). Other alternative receptors that have been proposed include the lowdensity lipoprotein receptor (1, 44), two receptors on HepG2 cells, the s...

Hepatitis C virus (HCV)-associated B cell lymphomas were previously shown to express a restricted repertoire of immunoglobulin V(H) and V(L) genes, V(H)1-69 and VkappaA27, respectively. Although this suggests a role for antigen selection in the pathogenesis of these lymphomas, the driving antigen involved in the clonal expansion has not been identified. B cell response to a viral antigen, the HCV envelope glycoprotein 2 (E2), was analyzed in an asymptomatic HCV-infected patient. Single B cells, immortalized as hybridomas and selected for binding E2, were analyzed for their V gene usage. Sequences of these V region genes demonstrated that each hybridoma expressed unique V(H) and V(L) genes. Remarkably, these anti-E2 hybridomas preferentially used the V(H)1-69 gene. Analysis of replacement to silent mutation ratios indicated that the genes underwent somatic mutation and antigenic selection. In a separate report, human anti-E2 antibodies were also shown to express the same V(H) gene. These data strengthen the hypothesis that the HCV-associated lymphomas are derived from clonally expanded B cells stimulated by HCV.

Hepatitis C virus (HCV) infection is associated with extrahepatic B-cell lymphoproliferative disorders. To determine whether a viral antigen drives this B-cell expansion, the B-cell receptors were cloned from HCV-associated lymphomas and were expressed as soluble immunoglobulins. The rescued immunoglobulins were then tested for their ability to bind the HCV-E2 envelope glycoprotein, an antigen that was previously implicated in the pathogenesis of HCV-associated B-cell diseases. One of 2 lymphoma immunoglobulin test cases bound the E2 protein in a manner identical to a bona fide human anti-E2 antibody. Moreover, it bound E2 from multiple viral genotypes, suggesting reactivity with a conserved E2 epitope. IntroductionMore than 100 million people worldwide are infected by hepatitis C virus (HCV). 1 Infection causes prolonged and persistent disease in the majority of carriers, often leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (reviewed in Houghton 2 and Liang et al 3 ). HCV is also the leading cause of mixed cryoglobulinemia (MC), a systemic immune complex-mediated disorder characterized by polyclonal or monoclonal B-cell proliferation. 4,5 MC can evolve into overt B-cell non-Hodgkin lymphoma (NHL), 6,7 although the incidence of malignant transformation appears to be restricted to certain areas of the world.In type II MC, immunoglobulin immune complexes contain both monoclonal immunoglobulin M (IgM) and polyclonal IgG. 8 The monoclonal IgM has rheumatoid factor (RF) activity and is encoded mostly by a restricted set of variable (V) region genes, specifically V H 1-69 (also known as 51p1) and V3-A27 (also known as v325). 9-12 The same set of V region genes is expressed by the majority of HCV-associated NHL, suggesting a malignant progression from type II MC. 13 Immunoglobulin V region genes expressed by these lymphomas exhibit an ongoing process of somatic mutations, indicative of antigen selection. 13 Moreover, the histologic presentation of many HCV-associated NHLs is typical of germinal center (GC) and post-GC B cells. 14 Therefore, it is likely that lymphomagenesis occurs when B cells proliferate in response to antigen. To date, the antigen driving these extrahepatic lymphoproliferative diseases is unknown.Nevertheless, immune responses occurring in HCV patients against one HCV antigen, the E2 envelope glycoprotein (E2), suggest that the restricted V gene usage seen in the lymphoproliferative disorders may be linked to this antigen. V region genes from human anti-E2 antibodies, derived from B cells of HCV-infected individuals, show similar V gene bias to that observed in HCV-associated MC and NHL. 15,16 These studies implicate the specific immune response to the E2 antigen in the pathogenesis of B-cell lymphoproliferative diseases and potentially in HCV-associated lymphomas.A possibility exists that cognate B cells, which bind E2 via their specific B-cell receptor (BCR) could engage 2 signaling complexes simultaneously-the BCR and the CD19/CD21/CD81 complex (CD81 is the putative E...