Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33

Abstract

The mouse monoclonal antibody (MAb) AP33, recognizing a 12 amino acid linear epitope in the hepatitis C virus (HCV) E2 glycoprotein, potently neutralizes retroviral pseudoparticles (HCVpp) carrying genetically diverse HCV envelope glycoproteins. Consequently, this antibody and its epitope are highly relevant to vaccine design and immunotherapeutic development. The rational design of immunogens capable of inducing antibodies that target the AP33 epitope will benefit from a better understanding of this region. We have used complementary approaches, which include random peptide phage display mapping and alanine scanning mutagenesis, to identify residues in the HCV E2 protein critical for MAb AP33 binding. Four residues crucial for MAb binding were identified, which are highly conserved in HCV E2 sequences. Three residues within E2 were shown to be critical for binding to the rat MAb 3/11, which previously was shown to recognize the same 12 amino acid E2 epitope as MAb AP33 antibody, although only two of these were shared with MAb AP33. MAb AP33 bound to a panel of functional E2 proteins representative of genotypes 1-6 with higher affinity than MAb 3/11. Similarly, MAb AP33 was consistently more efficient at neutralizing infectivity by diverse HCVpp than MAb 3/11. Importantly, MAb AP33 was also able to neutralize the cell culture infectious HCV clone JFH-1. In conclusion, these data identify important protective determinants and will greatly assist the development of vaccine candidates based on the AP33 epitope. HCV, a member of the Flaviviridae family, has a 9-kb genome encoding a polyprotein precursor that is cleaved to yield three structural proteins, core, E1, and E2, together with at least six non-structural proteins. E1 and E2 are highly glycosylated membrane-anchored proteins that mediate viral entry. 2-6 E2 has been shown to bind to a number of cell surface molecules. 7-10 Whereas the exact mechanism of viral entry is unknown, mounting evidence indicates that CD81 and SR-BI are key molecules. 2,[4][5][6][11][12][13][14] In an infected individual, HCV exists as a viral quasispecies. 15 HCV can be classified into at least six major genotypes that exhibit extensive genetic variability, particularly in E1 and E2. 16 E1 and E2 are the principle targets for neutralizing antibodies, and identification of protective epitopes conserved across different strains of HCV is a major challenge for vaccine design. 17 A number of antibodies that are capable of blocking E2 binding to cells or cell receptors have been described, 18-23 some of which neutralize HCV entry in animal or in vitro models. 6,[24][25][26] The first hypervariable region of E2 contains potent neutralizing epitopes, and antibodies raised against

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

“…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 majority of the substitutions were tolerated by MAb AP33, although binding to mutants L413A, N415A, G418A, and W420A was reduced by at least 50% compared to the wild-type protein. We have recently shown that these residues form part of the AP33 epitope (47). Recognition of the mutant proteins by three conformation-depen-FIG.…”

In vitro selection of a neutralization-resistant hepatitis C virus escape mutant

“…These mutations were not found in virus passaged in parallel without AP33 neutralization. Although N415 is within a domain identified previously as comprising the AP33 epitope 413-420 (22,23), E655 is distant in the linear E2 sequence and has not been implicated previously in recognition of HCV by AP33. To validate the role of these mutations in conferring neutralization escape, they were engineered both singly and together into pHJ3-5 plasmid DNA, and the cognate viruses rescued after RNA transfection of naive cells.…”

“…Importantly, immunization of mice and rats with recombinant E2 also elicits mAbs that recognize this E2 segment (20)(21)(22). One such mAb, AP33, is of particular interest because it demonstrates broad and potent, cross-genotypic neutralizing activity.…”

“…One such mAb, AP33, is of particular interest because it demonstrates broad and potent, cross-genotypic neutralizing activity. Prior studies using synthetic peptides, recombinant antigens, and HCVpp suggest that AP33 binds a highly conserved, linear epitope spanning residues 413-420 (22,23). Here, we describe the use of a cell culture-infectious, intergenotypic chimeric HCV with genotype 1a structural proteins (24,25) to further define the AP33 epitope by analyzing mutants selected for the ability to escape neutralization by this mAb.…”