Human Monoclonal Antibodies That Inhibit Binding of Hepatitis C Virus E2 Protein to CD81 and Recognize Conserved Conformational Epitopes

Abstract

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 ...

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Despite these difficulties, related viruses, easier to cultivate, for instance bovine viral diarrhoea virus, have been used as surrogate models [38]. Secondly, recombinant forms of the envelope glycoproteins were used to search for viral receptors [33,39] but also as probes for antibody screening or neutralization of binding assays [40,41], to study HCV-induced receptor signalling during entry [42], to delineate E2-receptor interacting surfaces [43,44], or to draw a theoretical model for E2 structure [45]. Most frequently a soluble form of E2 produced in mammalian or insect cells and capable of inhibiting HCV entry [46] is used.…”

Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles

“…CBH-5 (Hadlock et al, 2000), HC84.22, HC84.26 (Keck et al, 2012) andHC33.4 (Keck et al, 2013) were a gift of Steven Foung (Stanford University School of Medicine, Stanford, CA, USA). AR3A, AR3C (Law et al, 2008), AR4A and AR5A (Giang et al, 2012) were a gift from Mansun Law (The Scripps Research Institute, La Jolla, CA, USA).…”

Broadly neutralizing antibodies with few somatic mutations and hepatitis C virus clearance

“…Broadly neutralizing human mAbs (bNAbs) capable of neutralizing diverse HCV strains have been isolated from HCV-infected individuals, proving that antibodies can target relatively conserved regions of the two HCV envelope glycoproteins (E1 and E2), despite the enormous genetic diversity of HCV (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Infusion of bNAbs is protective against infection in animal models of HCV (17,18), and a recent study also showed that bNAbs could abrogate established HCV infection in a humanized transgenic mouse model (19).…”