SIGNIFICANCE OF THE IMMUNE-RESPONSE TO A MAJOR, CONFORMATIONAL B-CELLEPITOPE OS THE HEPATITIS-C VIRUS NS3 REGION DEFINED BY A HUMAN MONOCLONAL-ANTIBODY

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus lif e cycle: an N-terminal serine-type proteinase, responsible for the pro cessing of nonstructuraI polypeptides, and a C-terminal nucleoside tri phosphatase/helicase, presumably involved in the unwinding of the vira l genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B cell line obtaine d from the peripheral blood of a patient with chronic HCV infection, w hich selectively recognized the purified NS3 protein expressed in bact eria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-ac id sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 D Nase fragment library but failed to bind to 12-mer peptides synthesize d from the same region, suggesting recognition of a conformational B-c ell epitope. Experiments using deletion mutants of clone 8 and competi tive inhibition studies using a panel of NS3 peptide-specific murine M Abs indicated that limited N-terminal and C-terminal deletions resulte d in a significant reduction of hMAb binding to clone 8, thus identify ing a minimal antibody binding domain within clone 8. Competition expe riments showed that binding of CM3.B6 to the NS3 protein was efficient ly inhibited by 39 of 44 (89%) sera from HCV-infected patients, sugges ting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly recognition of the sequence defined by CM3.B 6 appeared to accurately discriminate between viremic and nonviremic a nti-HCV positive sera, suggesting potentially relevant clinical applic ations in the diagnosis and treatment of HCV infection.