PBMC from a patient with chronic hepatitis C virus (HCV) infection were immortalized with EBV and plated by limiting dilution. Cultures secreting antibodies reactive in a commercial HCV II generation ELISA, which incorporates Ag derived from the nucleocapsid, NS3, and NS4 regions, were repeatedly cloned in the presence of feeder cells and growth factors. Of 23 initially immunoreactive cultures, only one cloned line, designated B12.F8, secreted HCV nucleoprotein-specific IgG1(κ), whereas no reaction with recombinant polypeptides derived from NS3, NS4, and NS5 regions were documented. Human mAb (hmAb) B12.F8 was shown to recognize the native HCV nucleoprotein expressed in eukaryotic cells transfected with a core cDNA construct by immunofluorescence. The fine specificity of this hmAb was evaluated using synthetic oligopeptides covering the entire HCV nucleocapsid region. A weak but consistent reactivity was observed by PEPSCAN using a 12- mer encompassing residues 34-45 of the HCV-deduced amino acid sequence. Such weak reactivity is indicative for conformational epitopes and, in concurrence with this assumption, we found that longer peptides from the region containing residues 27-59 were more efficiently recognized and effectively inhibited binding of hmAb B12.F8 to recombinant nucleocapsid protein. Several overlapping immunoreactive fragments from the nucleocapsid region were selected from a random cDNA library consisting of DNase I fragments of recombinant core Ag. Best reactive recombinants were identified within residues 1-78 of the HCV sequence, in agreement with the results obtained using synthetic peptides. Comparative experiments on the fine specificity of sera from HCV-infected patients with anticore antibodies invariably showed recognition of peptides 8-40 and 27-59, as well as recombinant fragments spanning from residues 1 to 73, suggesting that hmAb B12.F8 identifies a major B cell epitope within the immunodominant nucleoprotein amino terminal subregion.
|Number of pages||11|
|Journal||Journal of Immunology|
|Publication status||Published - 1993|
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