Identification of a human epitope in hepatitis C virus (HCV) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-HCV-positive patient.
Healthy carriers of hepatitis C virus (HCV) infection exhibit a specific antibody response against all HCV antigens, which could play a role in disease control. Generation of panels of human antibodies may permit a thorough characterization of this response and further identify particular antibodies with potential clinical value. To this effect, we have established a human phage-display antibody library from a patient exhibiting a high antibody response against HCV antigens and no clinical symptoms of disease. This library was screened against a recombinant core antigen [amino acids (aa) 1-119] produced in E. coli. Two recombinant Fab-carrying phages (rFabCs) were isolated and characterized. Both rFabC3 and rFabC14 recognize aa 1-48 on core antigen, but rFabC14 is competed out by a synthetic peptide, C(2-20) (aa 1-20), at much lower concentrations than rFabC3. In order to identify more precisely the recognition sites of these antibodies, we produced soluble forms of the rFabs (sFabs), and used them to pan a random phage-display peptide library. A single peptide sequence, QLITKPL, was identified with sFabC3, while two equally represented sequences, HAFPHLH and SAPSSKN, were isolated using sFabC14. The QLITKPL sequence was partially localized between aa 8 and 14 of core protein, but no clear homology was found for the two sFabC14 peptides. However, we confirmed the specificity of these peptides by competition experiments with sFabC14.
Barban
V
V
Research Department, Pasteur Mérieux Connaught, 69290 Marcy l'Etoile, France. vbarban@fr.pmc-vacc.com
Fraysse-Corgier
S
S
Paranhos-Baccala
G
G
Petit
M
M
Manin
C
C
Berard
Y
Y
Prince
A M
AM
Mandrand
B
B
Meulien
P
P
D000328:Adult; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D002353:Carrier State / Q000276:immunology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology*; D006526:Hepatitis C / Q000235:genetics / Q000276:immunology; D018937:Hepatitis C Antibodies / Q000097:blood / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology
D000956:Antigens, Viral; D017931:DNA Primers; D000939:Epitopes; D018937:Hepatitis C Antibodies; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D014758:Viral Core Proteins