Elsevier

Journal of Hepatology

Volume 37, Issue 5, November 2002, Pages 660-668
Journal of Hepatology

Construction and characterization of an intracellular single-chain human antibody to hepatitis C virus non-structural 3 protein

https://doi.org/10.1016/S0168-8278(02)00270-2Get rights and content

Abstract

Background/Aims: We developed a single-chain antibody fragment (scFv) to the non-structural 3 protein (NS3) of hepatitis C virus (HCV) and tested its ability to interfere with the HCV replication cycle in infected hepatocytes.

Methods: The variable regions of the human monoclonal antibody CM3.B6 that recognizes a conformational epitope within the helicase domain of NS3 were introduced into adenoviral vectors for expression in mammalian hepatocytes. Expression and binding properties of the scFv were analyzed by immunological assays. Effects of intracellular expression of the scFv on HCV replication were assessed in primary hepatocytes isolated from explanted livers of patients with chronic HCV infection by reverse transcription-polymerase chain reaction.

Results: Transduction of HepG2 cells by the recombinant adenoviruses resulted in stable, efficient expression of scFv in the cytoplasm that was non-toxic to the cells. The scFv specifically bound to its cognate antigen. Significantly, intracellular expression of scFv resulted in a decrease in HCV genomic RNA in HCV infected hepatocytes.

Conclusions: These results indicate that specific binding of a scFv to NS3 may inhibit one or more functions of this essential viral protein thus interfering with the HCV replication cycle.

Introduction

Hepatitis C virus (HCV) is an enveloped virus with a plus-strand RNA genome encoding a polyprotein of approximately 3010 amino acids [1]. This polyprotein is cleaved co- and post-translationally into mature viral proteins by host cell signal peptidases and two viral enzymes designated the non-structural 2/3 (NS2/3) proteinase and the NS3/4A proteinase complex. The NS3 component of these proteinases is a 67 kDa multifunctional enzyme with three known catalytic activities segregated into two somewhat independent domains. The essential machinery of a serine-type protease is localized in the N-terminal one-third of the protein and nucleoside triphosphatase (NTPase) and helicase activities, presumably involved in the unwinding of the viral genome, reside in the remaining C-terminal region. The enzymatic functions of NS3 are essential for the virus replication cycle [2]. Therefore, NS3 presents an important target for antiviral therapy.

It is our hypothesis that binding of an antibody to the NS3 protein will block one or more functions of NS3. We previously generated a human monoclonal antibody, CM3.B6 (IgG1 kappa), that specifically recognizes an immunodominant conformational B-cell epitope (amino acids 1363–1454) within the helicase domain of HCV NS3 [3]. Recombinant Fab fragments generated from cells expressing CM3.B6 recognized the cognate antigen expressed in mammalian cells [4]. To explore the possibility that this antibody may have therapeutic potential, we constructed and characterized a single-chain form of this antibody (scFvNS3) for intracellular expression. To achieve high levels of scFvNS3 expression in hepatocytes, we employed recombinant adenovirus as gene vehicle. Most significantly, we demonstrate the potential of this scFv to inhibit HCV replication.

Section snippets

Cell culture

HepG2 and Hep3269 (HepG2 cells stably transfected with HCV complementary DNA (cDNA) representing the C-terminal half of NS2 to the N-terminal one-third of NS5 and constitutively expressing NS3) [5] cells were cultured in Eagle's minimal essential medium (MEM) (Invitrogen, Carlsbad, CA) supplemented with 1 mM sodium pyruvate, 10 μM non-essential amino acids, 50 U/ml penicillin/streptomycin, and 10% fetal bovine serum. Hep3269 cells were grown on plates coated with collagen I (Becton Dickinson,

Antibody mediated inhibition of NS3 helicase activity

Given the highly specific binding of CM3.B6 to an epitope within the helicase domain of NS3, we investigated whether binding of this antibody could inhibit NS3 helicase activity. Increasing amounts of CM3.B6 or an irrelevant human monoclonal antibody (IgG1) were added to recombinant full-length NS3 under optimal conditions for NS3 helicase activity as determined previously [6]. CM3.B6 inhibited NS3 helicase up to 50% in a dose dependent manner; whereas, the irrelevant antibody inhibited

Discussion

Selective gene ablation with intracellular scFv targeted to specific cellular compartments represents a novel approach to a gene therapy for viral infection [23], [24], [25], [26], [27], [28], [29]. Several recent studies report the construction and characterization of scFv's to HCV NS3 [30], [31], RNA-dependent RNA polymerase [20], core [32] or envelope protein [33]; however to date, none have demonstrated a biological consequence of scFv binding to HCV proteins. Here, we report the

Acknowledgements

We thank Dr Paola Gallinari for providing recombinant NS3, Dr T. Miyamura for providing Hep3269 cells and Chiron, Inc. for supplying rabbit polyclonal anti-NS3. This study was supported in part by NIH grants R01 CA68245 and R01 CA74242 to DTC; NIH grant R01 CA54576 to MAG and SD.

References (48)

  • M.U. Mondelli et al.

    Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody

    J Virol

    (1994)
  • G. Esposito et al.

    Recombinant human antibodies specific for hepatitis C virus proteins

    Arch Virol

    (1997)
  • T. Harada et al.

    Characterization of an established human hepatoma cell line constitutively expressing non-structural proteins of hepatitis C virus by transfection of viral cDNA

    J Gen Virol

    (1995)
  • P. Gallinari et al.

    Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A

    Biochemistry

    (1999)
  • Y. Gwack et al.

    DNA helicase activity of the hepatitis C virus non-structural protein 3

    Eur J Biochem

    (1997)
  • C.L. Tai et al.

    The helicase activity associated with hepatitis C virus non-structural protein 3 (NS3)

    J Virol

    (1996)
  • J.D. Marks et al.

    Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes

    Eur J Immunol

    (1991)
  • H.R. Hoogenboom et al.

    Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains

    Nucleic Acids Res

    (1991)
  • F.L. Graham et al.

    Manipulation of adenovirus vectors

  • J. Deshane et al.

    Intracellular single-chain antibody directed against erbB2 down-regulates cell surface erbB2 and exhibits a selective anti-proliferative effect in erbB2 overexpressing cancer cell lines

    Gene Ther

    (1994)
  • J. Brockman et al.

    Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation

    Mol Cell Biol

    (1995)
  • F. Ballet et al.

    Isolation, culture and characterization of adult human hepatocytes from surgical liver biopsies

    Hepatology

    (1984)
  • E.A. Roberts et al.

    Characterization of human hepatocyte lines derived from normal liver tissue

    Hepatology

    (1994)
  • A. Lieber et al.

    Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes

    J Virol

    (1996)
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