BACKGROUND/AIMS: We developed a single-chain antibody fragment (scFv) to 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 th ehelicase domain of NS3 were introduced into adenoviral vectors for expression in mammallian hepatocytes. Expression and binding properties of the scFv were analyzed by immunological assays. Effects of intracellullar expression of the scFV on HCV replication were assessed in primary hepatocytes isolated from explanted livers of patients with chronic HCV infection by reverse trasncription-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.

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

MONDELLI, MARIO UMBERTO;
2002

Abstract

BACKGROUND/AIMS: We developed a single-chain antibody fragment (scFv) to 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 th ehelicase domain of NS3 were introduced into adenoviral vectors for expression in mammallian hepatocytes. Expression and binding properties of the scFv were analyzed by immunological assays. Effects of intracellullar expression of the scFV on HCV replication were assessed in primary hepatocytes isolated from explanted livers of patients with chronic HCV infection by reverse trasncription-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.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11571/13153
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