Construction and characterization of an intracellular single-chain human antibody to hepatitis C virus non-structural 3 protein.
OBJECTIVE
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.
Sullivan
Deborah E
DE
Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA. dsulliva@tulane.edu
Mondelli
Mario U
MU
Curiel
David T
DT
Krasnykh
Viktor
V
Mikheeva
Gallina
G
Gaglio
Paul
P
Morris
Cindy B
CB
Dash
Srikanta
S
Gerber
Michael A
MA
D000256:Adenoviridae / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000235:genetics; D006528:Carcinoma, Hepatocellular; D015316:Genetic Therapy; D006526:Hepatitis C / Q000628:therapy*; D018937:Hepatitis C Antibodies / Q000276:immunology*; D022781:Hepatocytes / Q000166:cytology / Q000821:virology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000276:immunology*; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000911:Antibodies, Monoclonal; D018937:Hepatitis C Antibodies; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases