Mondelli M U, Cerino A, Boender P, Oudshoorn P, Middeldorp J, Fipaldini C, La Monica N, Habets W
Istituto di Clinica delle Malattie Infettive, University of Pavia Medical School, I.R.C.C.S. Policlinico San Matteo, Italy.
J Virol. 1994 Aug;68(8):4829-36. doi: 10.1128/JVI.68.8.4829-4836.1994.
The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral 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 obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.
丙型肝炎病毒（HCV）的非结构蛋白NS3具有两个酶结构域，据认为这两个结构域对病毒生命周期至关重要：一个N端丝氨酸型蛋白酶，负责非结构多肽的加工；一个C端核苷三磷酸酶/解旋酶，可能参与病毒基因组的解旋。人类对NS3的抗体反应通常在HCV感染过程早期出现，且主要针对羧基末端部分；然而，其精细特异性和临床意义在很大程度上尚不清楚。我们从一名慢性HCV感染患者外周血获得的克隆B细胞系中产生了一种人单克隆抗体（hMAb），命名为CM3.B6，它能选择性识别在细菌中或用全长或NS3 cDNA转染的真核细胞中表达的纯化NS3蛋白。精细特异性研究表明，CM3.B6识别从NS3 DNA酶片段文库中选出的一个92个氨基酸的序列（克隆8，氨基酸1363至1454），但不能与从同一区域合成的12聚体肽结合，这表明识别的是一个构象性B细胞表位。使用克隆8的缺失突变体进行的实验以及使用一组NS3肽特异性鼠单克隆抗体进行的竞争性抑制研究表明，有限的N端和C端缺失会导致hMAb与克隆8的结合显著减少，从而确定了克隆8内的一个最小抗体结合结构域。竞争实验表明，44份HCV感染患者血清中的39份（89%）能有效抑制CM3.B6与NS3蛋白的结合，这表明该hMAb识别NS3区域内的一个免疫显性表位。更重要的是，CM3.B6定义的序列识别似乎能准确区分病毒血症和非病毒血症的抗HCV阳性血清，这表明在HCV感染的诊断和治疗中可能具有潜在的相关临床应用。
