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 (1231)PTGSGKSTK(1239) (EP05) or core motif (1373)IPFYGKAI(1380) (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.

Figure 1. Reactivity of mAbs to peptides and proteins of NS3 helicase.

MAbs reacted with 16mer peptides in Peptide-ELISA (A), denatured T1b-rNS3 in Western Blot (B), denatured FL1b-rNS3 expressing 293T cells (C. I) and denatured native NS3 of HCV JFH-1 (2a) infected Huh7.5.1 cells (D. I) in Western Blot, and non-denatured native NS3 of HCV JFH-1 infected Huh7.5.1 cells in IFS (E). C. II and D. II, un-transfected 293T or un-infected Huh7.5.1 cell controls, respectively; NC (negative control), an un-related mAb to BP26 protein of B. melitensis; (+), mAb C7–50 to HCV core as positive control; (−), un-infected Huh7.5.1 cells as negative control.

Figure 2. Epitope mapping for NS3 helicase recognized by mAbs.

(A) Peptides reactive to mAbs are localized to the corresponding aa sequences of NS3 helicase from HCV 1b. Aa sequence position is indicated by numbering either whole or NS3 protein sequence. Recognition of mAb to peptide is indicated by names of peptide and mAb. Recognitions of mAbs reactive to epitopes of FL1b-rNS3 expressing 293T cells are divided into positive (+) and negative (−) groups by IFS. (B and D) The binding of mAb 2E12 or 3E5 (0.5 µg/ml) to P05 or P21 coated plate was inhibited by the shortened peptides (P0501–0506 or P2101 to P2106). (C and E) MAb 2E12 or 3E5 bound to the plate coated with shortened peptides derived from P05 or P21. An un-related peptide from B. melitensis BP26 was used as a non-inhibitory control (NC) in Peptide-ELISA.

Figure 3. Specificity of two linear epitopes within NS3 helicases of HCV and other flaviviruses.

(A) Analysis of amino acid sequence corresponding to EP05 and EP21 regions within NS3 helicase cross HCV genotypes and other flaviviruses. Aa sequences (one-letter code) to epitopes of HCV genotype 1b are presented on the top. Positions at the beginning and end of sequences are indicated by numbers. Identities with the lead sequence are indicated by dashes. Representative sequences are retrieved from Genebank Database. Triangles labeling with EP05/2E12 or EP21/3E5 on the top indicate epitope sequences or corresponding sequences for mAb’s recognition. The aa residues GSGKS underlined in bold indicate the ATP binding site of motif I (Walker A) within NS3 helicase. n.a. indicates no corresponding sequence available from those viruses. (B and C) Reactivity of mAb 2E12 or 3E5 with mutant peptide corresponding to the defined epitope sequence in Peptide-ELISA.

Figure 4. Unwinding activity of HCV NS3 helicase inhibited by mAb 2E12.

(A) A representative of DNA oligonucleotides labeled with Fluorescein amidite (FAM) from unwound double-strand DNA substrates at various concentrations of NS3 helicase of HCV (FL4b-rNS3) in unwinding reactions. (B) The slope of the initial velocity curve calculated from 4 representative tests with mean±SD. (C) A representative of unwinding reactions with 75 nM NS3 helicase and 1 µg/ml mAb 2E12 or an unrelated control mAb to B. melitensis. (D) Percentages for unwinding activity measured as velocity from 4 representative tests of unwinding reactions with NS3 helicase and mAbs.