Neutralizing Monoclonal Antibodies against Hepatitis C Virus E2 Protein Bind Discontinuous Epitopes and Inhibit Infection at a Postattachment Step

Abstract

The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77. Hepatitis C virus (HCV) is a blood-borne hepatotropic virus that infects ϳ170 million people worldwide. Approximately 70% of infected individuals progress to chronic liver disease, which carries an increased risk of cirrhosis and hepatocellular carcinoma (7). In general, treatment of chronic HCV infection is complicated by resistance due to extensive genetic diversity. HCV has been classified into seven major genotypes, which differ by ϳ30% at the nucleotide level (4), and this positivesense, single-stranded RNA virus has a capacity for rapid evolution of variant viruses during persistent infection. The current treatment, pegylated ␣ 2a interferon (IFN-␣ 2a ) and ribavirin, has variable side effects and response rates depending on the virus and host genotype (16). No vaccine is currently available, and preclinical development has been hampered by a lack of understanding of which conserved epitopes on the HCV structural proteins should be targeted.HCV contains an ϳ9.6-kb RNA genome that is translated as a single polyprotein and then cleaved by viral and host proteases into structural proteins (core, E1, and E2), p7, and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (39). Viral attachment and entry are mediated by the envelope glycoproteins, E1 and E2. Four attachment or entry receptors that are required for infection of hepatocytes have been identified, including CD81 (53), scavenger receptor B1 (SR-B1) (56), and the tight-junction proteins claudin 1 (CLDN1) (14) and occludin (OCLN) (54). The importance of E2 binding to the large extracellular loop of CD81 has been established in vitro (13,18,28,50,53), and interactions between E2 hypervariable region 1 (HVR1) and SR-B1

Structural evidence for a bifurcated mode of action in the antibody-mediated neutralization of hepatitis C virus

“…Several segments of the E2 protein have been identified as key components of conformational or linear epitopes that are critical to antibody-mediated neutralization of HCV in vitro (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Interestingly, naturally evoked antibodies and those produced in vitro that are specifically directed against a short peptide located in the E2 protein between residues 427-446, also known as epitope II, displayed one of three activities: virus neutralization, E2 binding but no neutralization, or interference with virus neutralization (15,16).…”

“…We thus hypothesized that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interactions between an antibody and a specific set of amino acid residues. A significant amount of information on several candidate HCV E2-binding sites has been generated in recent years by epitope-mapping techniques (7)(8)(9)(10)(11)(12)(13)(14)(15)(16); however, the underlying mechanism at the atomic level is still poorly understood. Here, we present the crystal structure of the epitope II peptide complexed with a neutralizing monoclonal antibody, mAb#8.…”

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

“…This finding may relate to differences in immunogenicity of HCV glycoprotein antigens in mice compared to other hosts. As mentioned above, murine MAbs isolated through immunization with E2 proteins have tended to target linear epitopes (53,54), and reduced efficacy toward heterologous genotypes has been reported (55). Another plausible explanation for the lower neutralization of HCVpp SA13 (5a) from vaccinated mice is the immunization schedule.…”

Characterization of a Novel Specific Mouse Monoclonal Antibody Targeted to Envelope Protein E1/E2 of Hepatitis C Virus

“…The high sensitivity was detected till 2.00 µg/ml of the coated antigen. Several mouse monoclonal antibodies targeting HCV core and envelope regions are already known and these antibodies are mainly used for antigen detection (Cagnon et al, 2004, Tabll et al, 2008and Shi et al, 2014.The monoclonal antibody (7G9) is targeted to the envelope glycoproteins E1 and E2.These envelope glycoproteins E1 and E2 are essential in the attachment and entry of the virus ( Sabo et al ,2011).E2 causes not only viral attachment to the cell membrane at its entrance but enhances also the neutralizing antibodies in the host. The envelope proteins play a major role in attachment and entry of virions into the body (Mazumdar et al, 2011) and (Park et al, 2013).…”