Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies.
The hepatitis C virus (HCV) NS3 protein possesses both protease and helicase activities and is essential for virus replication and maturation. Specific inhibition of NS3 enzymatic activity can be achieved by antibody binding. Transduction of hepatocytes with encoding cDNA leading to intracellular expression of antibody fragments is expected to terminate HCV replication in infected cells. The objective of the present study was the generation of human antibody fragments that neutralize the viral NS3 helicase activity for gene therapeutic applications and drug design. A human immunoglobulin phage-display library cloned from bone marrow aspirate of patients infected with HCV was used for affinity selection against HCV NS3 helicase. Antibody fragments with high affinity to HCV helicase were isolated. To evaluate the inhibitory potential of isolated single-chain antibody fragments, a helicase-mediated, DNA-unwinding enzymatic assay was developed in ELISA format. Recombinant protein comprising the full-length HCV NS3 helicase domain was expressed in the baculovirus expression system. Recombinant antibodies that inhibit the HCV helicase at nanomolar concentrations, with efficacies ranging from 20 % to complete abrogation of enzymatic unwinding activity, were identified. These antibody fragments may be useful for novel gene therapeutic strategies that employ intracellular immunization and may provide new insights into the design of small molecule inhibitors of essential HCV proteins.
Artsaenko
Olga
O
Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany.
Tessmann
Kathi
K
Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany.
Sack
Markus
M
Department of Molecular Biotechnology, University of Aachen, Germany.
Häussinger
Dieter
D
Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany.
Heintges
Tobias
T
Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany.
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D016367:Baculoviridae / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015316:Genetic Therapy; D016174:Hepacivirus / Q000201:enzymology* / Q000276:immunology; D006526:Hepatitis C / Q000628:therapy; D018937:Hepatitis C Antibodies / Q000096:biosynthesis / Q000494:pharmacology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000494:pharmacology; D008969:Molecular Sequence Data; D019151:Peptide Library; D020365:RNA Helicases / Q000037:antagonists & inhibitors* / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology; D014161:Transduction, Genetic
D018937:Hepatitis C Antibodies; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D020365:RNA Helicases