Characterization and binding of intracellular antibody fragments to the hepatitis C virus core protein.
The monoclonal antibody C7-50 binds to the HCV core protein with high sensitivity and specificity. The coding sequences of the variable domains of the antibody were determined following cDNA cloning of the Fab and sFv fragments. Subsequently, intracellular expression and binding of these antibody fragments to the HCV core protein as a potential antiviral approach were studied. There was high specificity and sensitivity of binding of bacterially expressed, recombinant C7-50 Fab to HCV core as measured by EIA and immunoblot. For expression in mammalian cells, the C7-50 antibody was subcloned in the sFv format by the introduction of a (Gly(4)Ser)(3) linker spaced between light and heavy chains. Northern and Western blot analysis as well as confocal microscopy established the targeted expression of the C7-50 sFv antibody fragment in the endoplasmic reticulum of transfected cells. The colocalization and intracellular binding of the antibody fragment to HCV core protein was confirmed by immunoprecipitation and subsequent immunoblot analysis. This study demonstrates that gene delivery of cDNA coding sequences inducing intracellular expression of C7-50 antibody fragments leads to binding of the antibody fragment to the HCV core protein within the secretory compartment of transfected cells. Intracellular immunization represents a promising antiviral approach to interfere with the life cycle of HCV.
Heintges
T
T
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Boston, Massachusetts, 02129, USA.
zu Putlitz
J
J
Wands
J R
JR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000998:Antiviral Agents; D001483:Base Sequence; D003001:Cloning, Molecular; D005057:Eukaryotic Cells; D018390:Gene Targeting; D018937:Hepatitis C Antibodies / Q000235:genetics / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology*; D014758:Viral Core Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D018937:Hepatitis C Antibodies; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C055966:immunoglobulin Fv; C069130:nucleocapsid protein, Hepatitis C virus