Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Abstract

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope 1231PTGSGKSTK1239 (EP05) or core motif 1373IPFYGKAI1380 (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.

A signal amplification system on a lateral flow immunoassay detecting for hepatitis e‐antigen in human blood samples

“…Ninety‐nine plasma samples with both HBsAg and HBeAg positive and 291 plasma samples with negative HBsAg were obtained from blood donors in Guangzhou Blood Center, which were routinely screened by ELISA and Utrio Plus nucleic acid testing (individual‐donation NAT, Grifols Diagnostic Solutions, San Diego, CA; limit of detection HBV DNA 3.4 IU/mL), respectively . The non‐HBV infected sera or plasmas of hepatitis C virus (HCV) infected patients, human immunodeficiency virus (HIV) infected patients or rabies vaccine immunized patients from the laboratory were used as controls for testing of assay specificity . This study was approved by the Medical Ethics Committees of Southern Medical University and Guangzhou Blood Center and followed the ethical guidelines of the 1975 Declaration of Helsinki.…”

Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies

“…Pre-immunization and immunized sera were collected and used as the negative or positive controls for screening mAbs, respectively. One mAb (IgG1 kappa) to recombinant non-structural protein-3 (NS3) of hepatitis C virus (HCV) was used as an unrelated negative control [ 29 ]. MAb isotyping was performed by IsoQuick Strips (Sigma-Aldrich, St Louis, Missouri, United States).…”

Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus

“…The formalin-fixed, paraffin-embedded liver tissues were tested for HCV NS2 to -4A protein expression by immunohistochemical (IHC) staining as described previously (22). Primary antibodies to HCV NS3 (clone 3E5) and to both HCV and GBV-B NS3 conserved epitopes (clone 2E12) were available in the laboratory (24). Antibodies to HCV core (clone C7-50) and alpha smooth muscle actin (␣-SMA) (clone 1A4) were purchased from Abcam (Cambridge, United Kingdom).…”

“…Twenty-nine HCV NS3 peptides (P1 to P29 16-mer with 7-mer overlap spanning 268 amino acids [aa] of HCV NS3 between aa 1192 and 1459) were commercially synthesized by the Chinese Peptide Company (Hangzhou, Zhejiang, China) (24). The peptides (4 g/ml each) were pooled in PBS.…”