Structural and functional characterization of the single‐chain Fv fragment from a unique HCV E1E2‐specific monoclonal antibody. Issue 20 (7th September 2013)

• Record Type: Journal Article
• Authors: Fallecker, Catherine; Tarbouriech, Nicolas; Habib, Mohammed; Petit, Marie-Anne; Drouet, Emmanuel
• Is Part Of: FEBS letters. Volume 587:Issue 20(2013)
• Page Start: 3335
• Page End: 3340
• Publication Date: 2013-09-07
• Subjects: CDR -- complementarity-determining region -- DAAs -- direct-acting antivirals -- ELISA -- enzyme-linked immunoabsorbent assay -- FR -- framework region -- GT -- genotype -- HCV -- hepatitis C virus -- HCVsp -- serum-derived HCV particles -- HRP -- horseradish peroxidase -- IgG -- immunoglobulin G -- IMAC -- immobilized metal affinity chromatography -- IPTG -- isopropylthio-β-galactoside -- LB medium -- Leibovitz medium -- mAb -- monoclonal antibody -- MALLS -- multi-angle laser light scattering -- NDSB -- 3-(1-pyridino)-1-propanesulfonates -- NR -- non-reducing -- PCR -- polymerase chain reaction -- PEG-IFN -- pegylated interferon-α -- PVDF -- polyvinylidene difluoride -- RBV -- ribavirin -- R -- reducing -- scFv -- single chain antibody fragment -- SDS–PAGE -- sodium dodecylsulfate–polyacrylamide gel electrophoresis -- SOC -- standard of care -- TBS -- Tris buffer saline -- VH -- heavy chain variable region -- VL -- light chain variable region -- Hepatitis C virus -- E1E2 -- Antibody engineering -- Single chain Fv fragment -- Anti-HCV therapy; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1016/j.febslet.2013.07.057
• Languages: English
• ISSNs: 0014-5793

Abstract

Abstract : The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum‐derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single‐chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi‐angle laser light scattering. The scFv mimicked the antibody in binding to the native serum‐derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics. Abstract : A scFv from the anti‐HCV E1E2 D32.10 mAb was successfully expressed. This scFv consisted of functional monomers as shown by MALLS methodology. The scFv D32.10 mimicked the antibody in binding to patient‐derived HCV particles. The scFv D32.10 competed with the parental IgG for binding to antigen. This scFv is the basis of a drug discovery approach for tackling HCV reinfection.