Cloning and molecular characterization of human high affinity antibody fragments against Hepatitis C virus NS3 helicase.
Hepatitis C Virus (HCV) non-structural protein 3 (NS3) helicase is essential for viral replication. Cloning of human antibody fragments for binding and inhibiting HCV helicase intracellularly (intracellular immunization) was attempted. A phage display system was employed to isolate human sFv fragments. A large phagemid library was cloned from patients infected with HCV. Phages expressing human sFv fragments with binding activity against NS3 were highly enriched during affinity selection. Selected sFv antibody fragments showed high affinity to HCV helicase. The variable domains of the cloned antibody fragments were sequenced and their germ-line origin was determined. K(D) values describing affinity of sFv to NS3 were measured by competition-EIA. Bacterially expressed recombinant human high affinity antibodies can be used for diagnostic and therapeutic purposes. Further experiments will select antibody fragments inhibiting NS3 helicase. Employing vectors for transduction of the encoding cDNA into infected cells might be a novel gene therapy strategy for intracellular immunization against chronic HCV infection.
Tessmann
Kathi
K
Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstr. 5, 40225, Düsseldorf, Germany.
Erhardt
Andreas
A
Häussinger
Dieter
D
Heintges
Tobias
T
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005803:Genes, Immunoglobulin; D018937:Hepatitis C Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D019151:Peptide Library; D020365:RNA Helicases / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000276:immunology*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D018076:DNA, Complementary; D018937:Hepatitis C Antibodies; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D020365:RNA Helicases; D011993:Recombinant Fusion 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