In vitro selection of a neutralization-resistant hepatitis C virus escape mutant.
Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.
Gal-Tanamy
Meital
M
Center for Hepatitis Research, Institute for Human Infections & Immunity, and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1073, USA.
Keck
Zhen-Yong
ZY
Yi
MinKyung
M
McKeating
Jane A
JA
Patel
Arvind H
AH
Foung
Steven K H
SK
Lemon
Stanley M
SM
D000914:Antibodies, Viral / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000821:virology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development / Q000276:immunology*; D006526:Hepatitis C / Q000276:immunology* / Q000821:virology*; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D009500:Neutralization Tests; D013601:T-Lymphocytes / Q000166:cytology / Q000821:virology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology; D014761:Viral Hepatitis Vaccines / Q000276:immunology
D000914:Antibodies, Viral; D014759:Viral Envelope Proteins; D014761:Viral Hepatitis Vaccines