Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein

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 g/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.Hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, is the major cause of non-A, non-B viral hepatitis. HCV has infected approximately 200 million people worldwide and current estimates suggest that as many as 3 million individuals are newly infected each year (4). Approximately 80% of those infected fail to clear the virus; a chronic infection ensues, frequently leading to severe chronic liver disease, cirrhosis, and hepatocellular carcinoma (2, 41). Current treatments for chronic infection are ineffective for approximately 50% of patients, and there is a pressing need to develop preventative and therapeutic vaccines.Due to the error-prone nature of the RNA-dependent RNA polymerase and the high replicative rate in vivo (30, 46), HCV exhibits a high degree of genetic variability. Crucially, this propensity for genetic change allows the virus to respond to and overcome a variety of selective pressures, including host immunity and antiviral therapy (18,26,37,44,53). HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nucleotide level, respectively (59, 61). A significant challenge for the development of vaccines will lie in identifying protective epitopes that are conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope proteins, the natural targets for the neutralizing response, are two of the most variable proteins (10).The envelope proteins E1 and E2 are responsible for cell binding and entry (5,8,16,51,57). They are N-linked glycosylated (23,31,43,62) transmembrane proteins with a Nterminal ectodomain and a C-terminal hydrophobic membrane anchor (12,21,22). In vitro ex...

Identification of Conserved Residues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding

“…Therefore, we isolated and tested a large number of E1E2 clones for their ability to confer infectivity to HCVpp. A total of 37 full-length E1E2 clones representative of genotypes 1 through to 6 were shown to be functional in the HCVpp entry assay (not shown), some of which have been reported previously (29,39). Amino acid sequence alignments, corresponding to positions 412 to 424, 474 to 495, and 520 to 550 were generated for these E1E2 clones, together with E1E2 sequences of known infectious reference clones available through the Los Alamos HCV Sequence Database (http://hcv.lanl.gov/content/hcv-db/index).…”

“…(12). Similarly, W420A mutation abolished both MAb AP33 (itself a broadly neutralizing antibody [39]) and CD81 binding, indicating overlap between the CD81 binding site and the AP33 epitope. Finally, mutations at L413, N415, and G418 also reduced the binding of MAb AP33, and we have recently shown that these, together with W420, are probable contact residues (47).…”

“…The plasmids expressing the HCV genotype 1a strain H77c-derived full-length E1E2, murine leukemia virus (MLV) Gag-Pol, and the MLV transfer vector carrying the green fluorescent protein (GFP) coding sequence under the control of human cytomegalovirus promoter were described previously (5,39). cDNA sequences encoding full-length E1E2 (representing amino acid residues 170 to 746, encoded by the HCV open reading frame referenced to strain H77c [53]) were generated and cloned into the expression vector pCR3.1 (Invitrogen) and their nucleotide sequences determined as described previously (29).…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…First of all, they can be used in S2 containment facilities, an undeniable advantage in particular for antiviral screens [156]. Secondly, they are flexible tools able to incorporate a collection of patient-derived glycoproteins [157,158] (a detailed protocol for the generation of such HCVpp can be found in reference [159]), a feature particularly useful to characterize cross-neutralizing antibodies [158,160]. Thirdly, HCVpp proved to be a relevant model to study the mechanisms and steps of virus entry, with for instance the description of HCV internalization [146,147], of the cellular signalling cascades activated upon virus entry [141], or to reveal new receptors for the virus [116,119].…”

Cre-estrogen receptor-mediated hepatitis C virus structural protein expression in mice

“…Proteins were electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and probed with a mouse monoclonal antibody (mAb) to HCV core (Anogen, Mississauga, Canada); E2 (clone Ap33) (Owsianka et al, 2005); or rabbit polyclonal antibodies to caspase-3 (Cell Signaling Technology, Beverly, MA), GRP78 or CHOP/ GADD153 (Santa Cruz Biotechnology, San Diego, CA), followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, AL), and ECL detection reagents (Amersham Pharmacia, Buckinghamshire, England). Expression levels were evaluated by quantification of the relative density of each band normalized to β-actin.…”