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

ABSTRACT: 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.

SUBMITTER: Gal-Tanamy M

PROVIDER: S-EPMC2614781 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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

Gal-Tanamy Meital M Keck Zhen-Yong ZY Yi MinKyung M McKeating Jane A JA Patel Arvind H AH Foung Steven K H SK Lemon Stanley M SM

Proceedings of the National Academy of Sciences of the United States of America 20081203 49

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 ...[more]

PMID: 19052239

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Project description:The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24-peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable.IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins.

Project description:Hepatitis C virus (HCV)-induced end-stage liver disease is the major indication for liver transplantation (LT). However, reinfection of the liver graft is still common, especially in patients with detectable viral load at the time of LT. Limited data are available on direct-acting antivirals in the transplant setting for prevention of graft infection. The human hepatitis C immunoglobulin (HCIG) Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing its safety and efficacy at preventing HCV recurrence after liver transplantation (LT) in the United States. Using well-characterized patient-derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) containing patient-derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after LT. The human hepatitis C immune globulin Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing safety and efficacy to prevent HCV recurrence after LT in the United States. Using well-characterized patient-derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles and cell culture-derived HCV containing patient-derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after liver transplantation. Additionally, we studied neutralization of different HCV genotypes and of direct-acting antiviral-resistant viruses. Our results indicate that Civacir potently, broadly, and dose-dependently neutralizes all tested patient variants in HCV pseudoparticles and cell culture-derived HCV assays including variants displaying resistance to host neutralizing antibodies and antiviral monoclonal antibodies. The half-maximal inhibitory concentrations were independent of the phenotype of the viral variant, indicating that virus neutralization by Civacir is not affected by viral selection. Furthermore, Civacir is equally active against tested direct-acting antiviral-resistant HCV isolates in cell culture.ConclusionCollectively, these results demonstrate broad neutralizing activity of Civacir against resistant viruses, likely due to synergy between anti-HCV antibodies derived from different plasma donors, and support its further clinical development for prevention of liver graft infection. (Hepatology 2016;64:1495-1506).

Project description:BackgroundGiven the side effects associated with intravenous injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable. Recently published clinical studies show that interferon-free combination therapies containing ribavirin are efficacious, suggesting that an interferon-free therapy could be adopted in the near future. Therefore, understanding HCV resistance to ribavirin could be of major importance. In an approach to understand the effect of ribavirin on HCV replication and HCV resistance, we have selected a ribavirin resistant mutant of HCV in vitro.MethodsWe serially passed the J6/JFH1 strain of HCV in Huh7D cells (a Huh7 cell derivative more permissive to HCV replication) in the presence of different concentrations of ribavirin. Virus replication was assessed by detection of HCV antigens by immunfluorscence of infected cells and titration of recovered virus present in the supernatant. cDNAs from virus RNA grown in 0 or 250 uM concentrations of ribavirin were synthesized by RT-PCR, and sequenced.ResultsA concentration of 125 uM of ribavirin did not have a dramatic effect on HCV replication, while 500 uM of ribavirin lead to viral extinction. Concentrations of 250 uM of ribavirin dramatically reduced virus replication which was sustained over six passages. At passage seven viral resurgence began and over two passages the level of virus reached that of the wild type virus grown without ribavirin. Virus recovered from these cultures were more resistant to 250 uM ribavirin than wild type virus, and showed no difference in replication relative to wild type virus when grown in the absence of ribavirin. The ribavirin resistant virus accumulated multiple synonymous and non-synonymous mutations that are presently being analyzed for their relationship to ribavirin resistance.ConclusionsIt is possible to select a ribavirin resistant mutant of HCV that can replicate to levels similar to wild type virus grown without ribavirin. Analysis of the mutations responsible for the ribavirin resistance may aid in understanding the mechanism of action of ribavirin.

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

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

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