Artificial NS4 mosaic antigen of hepatitis C virus

An artificial antigen composed of 17 small antigenic regions derived from t he NS4-protein of hepatitis C virus (HCV) genotypes 1 through 5 was designe d and constructed. Eleven antigenic regions were derived from the 5-1-1 reg ion, and 6 others were derived from the C-terminus of the NS4-protein of di fferent genotypes. The gene encoding for this artificial antigen was assemb led from synthetic oligonucleotides by a new approach designated as restric tion enzyme-assisted ligation (REAL). The full-length synthetic gene was ex pressed in Escherichia coli as a fusion protein with glutathione S-transfer ase. By the use of site-specific antibodies raised against synthetic peptid es, it was shown that all regions for which sequence-specific antibodies we re obtained were accessible to antibody binding. The diagnostic relevance o f the NS4 artificial antigen was demonstrated by testing this antigen with 4 HCV seroconversion panels and a panel of previously tested and stored ser um specimens. The artificial antigen was found to specifically detect anti- NS4 antibodies in a number of specimens that were previously found to be an ti-NS4 negative. Furthermore, this antigen detected anti-NS4 activity earli er in 2 of 4 seroconversion panels than did the antigen used in a commercia lly available supplemental assay. Equally important is the observation that the artificial NS4 antigen demonstrated equivalent anti-NS4 immunoreactivi ty with serum specimens obtained from patients infected with different HCV genotypes, whereas the NS4 recombinant protein derived from genotype 1, use d in the commercial supplemental test, was less immunoreactive with serum s pecimens containing HCV genotypes 2, 3, and 4. Collectively, these data sup port the significant diagnostic potential of the NS4 mosaic antigen. The st rategy employed in this study may be applied to the design and construction of other artificial antigens with improved diagnostically pertinent proper ties. (C) 1999 Wiley-Liss, Inc.