Significance Of Monoclonal Antibodies Against The Conserved Epitopes Within Non-structural Protein3Helicase Of Hepatitis C Virus

Hepatitis C caused by the infection of hepatitis C virus (HCV) is a major liver disease that jeopardizes the world’s population. More than170million people are infected with hepatitis C virus (HCV) worldwide. Approximately30%of HCV infected individuals spontaneously clear the virus and70%become chronically infected, the latter being at risk of developing chronic liver disease including liver failure, liver cirrhosis and hepatocellular carcinoma possibly requiring liver transplantation.The hepatitis C virus (HCV) is now the main etiological agent of posttransfusion non-A, non-B hepatitis, one of members in the virus family Flaviviridae. The viral genome is a positive-stranded RNA of approximately9.6kb and has one large open reading frame that encodes for a polyprotein of3,011amino acids, containing structural protein core (C), envelope1and2(E1,E2), p7, and nonstructural protein NS2, NS3, NS4A, NS4B, NS5A, NS5B.Nonstructural protein3(NS3) provides protease, helicase and NTPase enzymatic activities that play a crucial role in viral replication and constitute a suitable target for antiviral therapy, vaccination and diagnosis in HCV infection. The N-terminal third of NS3contains a serine-protease domain responsible for processing the nonstructural polyprotein of HCV, while the C-terminal two-thirds encode for an adenosine triphosphatase (ATPase)/helicase capable of unwinding duplex RNA. Previous studies revealed that the NS3helicase contains immunodominant B-cell epitopes eliciting high levels of antibodies in HCV infected individuals. The human and murine humoral immune responses to HCV NS3protein are almost exclusively targeting the ATPase/helicase domain, which appears to be serologically reactive during the early phase of HCV infections and is routinely used in clinical diagnostic HCV antibody immunoassays.In recent years, the NS3helicase has become a popular potential target for exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3inhibitors. This study explored extensively monoclonal antibodies to antigenic epitopes of NS3helicase and their potential applications to diagnosis and antiviral drugs in HCV infection.A cDNA fragment of nonstructural protein2-4A was cloned in a plasmid pMD20NS2-4A from an HCV genotype lb strain. The truncated recombinant NS3protein with a polyhistidine tag (T-rNS3) covering the functional part of amino acid (aa) sequence (aa1192-1459for whole protein) within HCV NS3helicase was expressed in E. coli with pET-32a vector. Soluble T-rNS3was purified by Ni-NTA agarose according to the manufacturer’s instructions and analyzed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The purity of T-rNS3was over90%. As the antigen T-rNS3immunized BALB/c mice, and hybridoma cells by fusing mouse spleen and myeloma cells were used to produce monoclonal antibodies (mAbs) specific to HCV NS3.The full-length recombinant HCV NS3protein (FL-rNS3, aa1-631or aa1027-1657) was expressed with lentiviral construct pTY-CMV-NS3in293T cells. HCV was generated by transfection of an infectious RNA of HCV genotype2a (JFH-1clone) to Huh7.5.1cells by Lipofectamin2000. HCV was inoculated to the fresh Huh7.5.1cells for viral culturing and passaging. The native NS3in HCV infected cells was expressed. A full-length recombinant NS3of HCV genotype4b (FL4b-rNS3) produced in E. coli was purchased from CUSABIO. The purity of FL4b-rNS3was over95%.A panel of47peptides was commercially synthesized. Twenty-nine of16mer peptides with7mer overlap were designated as PO1to P29spanning268amino acid of HCV NS3between aa1192and1459. Twelve6-11mer peptides from P05and P21were shortened and designated P0501to P0506or P2101to P2106. Five peptides were designated VatP2101-03corresponding to P05or P21derived from HCV variants or GB virus C with aa substitutions. One peptide derived from BP26protein of Brucella melitensis strain was used as a negative control (NC).Reactivity of mAbs were tested to a panel of HCV NS3peptides, FL-rNS3, FL4b-rNS3,and native HCV NS3in ELISA, Western-Blot and Immunofluorescence staining (IFS). The epitopes recognized by mAbs were identified by overlapping peptides, shortened peptides and mutated peptides in Peptide-ELISA. In order to determine the conservation of two linear epitopes, amino acid sequences corresponding to the regions of epitopes within HCV NS3helicase and relevant flaviruses were randomly selected from GenBank database and compared with EP05orEP21.The study was undertaken to explore the antibody of HCV infection in blood donors from Beijing and Guangdong in China, to determine the reactivity of linear epitope peptides and the compare with a combination of two different anti-HCV El As with recombinant core and NS3-5antigens and nucleic acid test. According the results of the two anti-HCV EIAs and PCR for confirming of viral load, HCV infected blood were classified into recovery infections (RNA-/Ab+) and chronic infections (RNA+/Ab+). One hundred and eight plasmas from healthy blood donors were used as negative controls. The cutoffs of peptides reactive to negative control samples were calculated as mean+2SD with95%confidence interval (CI). Blood antibody responses to epitope peptides were analyzed in line with the results obtained by combination of standard tests, their ROC curves were plotted, and optimal cutoff values were defined. Optimal cutoff values were defined using the highest sum of sensitivity and specificity. For each optimal cutoff value, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated. P value was obtained for comparing the differences between the areas under ROC curve (AUC) and reference (0.5) by the Kolmogorov-Smirnov Z analysis. Pearson Chi-Square test was used for comparison of difference between the peptides reacting with chronic and resolved HCV infected sample groups. Moreover, the fluorescence helicase unwinding assay using dsDNA substrate was performed as described to test the inhibition of monoclonal antibody. One-way ANONA test and Dunnett’s T3were used to compare the difference of NS3helicase activity between experimental and control groups. P value<0.05was considered significant.A total of29mAbs against HCV NS3were prepared by lymphocyte hybridoma technique. To classify mAb epitope recognition,29mAbs were tested for reactivity with a panel of2916mer peptides, FL-rNS3(1b) and native NS3(2a), respectively. Six mAbs reacted with8of16mer overlapping peptides. MAb1C11recognized peptides P13and P14that shared a putative epitope sequence, while mAb1A10bound to two non-overlapping peptides P09and P16that may constitute a discontinuous epitope. Altogether, using synthetic peptides as target, mAbs recognized five putative linear and1discontinuous epitope sequences from overlapping peptide. Twenty-seven mAbs reacted with the denatured T-rNS3and eight mAbs reacted with the denatured FL-rNS3(1b) expressing cells, while only three of them cross-reacted with the denatured native NS3in Western-blot. Ten mAb reacted with the non-denatured FL-rNS3in293T cells, and seven (6of10and1extra) cross-reacted with the non-denatured native NS3from HCV JFH-1infected cells in immunofluorescence staining (IFS). According to mAb recognition, NS3epitopes were classified as six linear, three semi-conformational and three conformational, respectively. Two mAbs cross-reacted with the exposed linear epitopes of native NS3in HCV JFH-1(2a) infected cells, mAb2E12reacting with the sequence corresponding to the ATP binding site, and mAb3E5with a sequence close to the nucleotide binding region of NS3helicase. In order to further define those two exposed linear epitopes, peptides P05or P21were shortened and tested for direct and competitive binding to mAbs2E12and3E5in Peptide-ELISA, respectively. Antibody2E12reacted only with P0501or P0502coated plates, suggesting that the minimal amino acid residues of the linear epitope (EP05,205PTGSGKSTK213) recognized by mAb2E12was located at position205-213of the NS3protein of HCV1b. The binding of mAb3E5to P21(16mers) coated plate was competitively inhibited by the shorter peptides P2101-P2104and P21itself, but3E5did not bind any shorter peptide coated plate, suggesting that the epitope of P21(EP21) was the longer amino acid residues346EIPFYGKAIPIETIKG361and its core motif was347IPFYGKAI354in NS3.To analyze diversity or similarity of the two identified linear epitopes, the corresponding amino acid consensus sequences from each genotype of HCV and other human flaviviruses were compared with the sequences of epitope EP05or core motifs of epitope EP21within NS3helicases. EP05sequence205PTGSGKSTK213recognized by mAb2E12was totally conserved across genotypes, which was also present in the non-human flavivirus GBV-B. MAb2E12did not react with peptide GP05’when S208A and K213R substitutions corresponding to the GBV-C sequence were present, suggesting that the amino acid Serine208was critical for epitope EP05specificity to HCV. The core motif of EP21347IPFYGKAI354recognized by mAb3E5was highly conserved and amino acid differences were found in genotype3b (I354L), genotype4f,4r,5a,6p and6o (K352R) and genotype6k and6v (I347V). MAb3E5did not react with peptide VatP2101(I347V) or peptide VatP2102(K352R), weakly reacted with peptide VatP2103(I354L) corresponding to epitope EP21derived from HCV variants, but strongly reacted when substitution I347V or K352R was present in FL4b-rNS3(genotype4b) or native NS3(genotype2a), respectively. MAb3E5did not bind to the corresponding peptide GP21’(K352H and A353G) derived from GBV-C. Even though mAb3E5did not bind to the linear peptides VatP2101and VatP2102with substitution I247V or K352R corresponding to the core motif sequence of epitope EP21, it still strongly reacted with FL4b-rNS3(genotype4b) or native NS3(genotype2a) carrying the identical mutants, suggesting that the substitution of I247V or K352R within the core motif of EP21did not alter the epitope conformation presented in NS3helicase proteins but differed in linear peptide form. Both mAbs2E12and3E5were non-reactive with Dengue virus infected cells by immunofluorescence staining. To evaluate the potential capacity of the two highly conserved epitopes (P05and P21) to elicit antibody response in HCV infected blood donors,136HCV infected and128healthy plasmas from Chinese blood donors were tested for reactivity with epitopes EP05or EP21mimicked by peptides P05and P21using Peptide-ELISA. Of264plasmas,86were from individuals with chronic HCV infection,50from individuals who spontaneously resolved HCV infection and128non-infected individuals (Ab-/RNA-). The cutoffs of P05or P21reactive to negative control samples were calculated as mean+2SD with over95%confidence interval, which was0.267and0.298, respectively. Peptides P05and P21reacted strongly with79.1%or59.3%chronic HCV infected plasmas, which were more active than58%or30%weakly reactive resolved HCV infected plasmas from blood donors (P<0.001). Blood antibody response to epitope peptides P05or P21were analyzed in line with the results obtained by standard tests, and their ROC curves were plotted (0.782-0.930) and cutoffs were defined (0.193-0.234). Results showed that the detection of reactive antibodies in the order by P05or P21in ELISA was closer to the result by a combination of standard tests (P<0.001). Two linear epitope peptide conjugates were reactive to73-88%sera in peptide-ELISA consistent with the standard tests (P＜0.001), which indicated that both linear epitopes were dominant in HCV.Monoclonal antibody2E12recognize an epitope covering the ATP binding site in domain1of the helicase raising the possibility of functional interference of this antibody in the helicase function. To investigate this possibility, mAb2E12and an unrelated control were added to a full-length recombinant protein HCV NS3genotype4b (FL4b-rNS3) in an unwinding assay. FL4b-rNS3exhibited helicase unwinding activity, that was inhibited by mAb2E12(P=0.003) but not by an irrelevant mAb control (P=0.239).In the present study, mAbs2E12interacted to win the50%dysfunction of unwinding activity for recombinant FL4b-rNS3in vitro, suggesting that2E12had potential for inhibiting the enzymatic activity of NS3helicase. The inhibitory function of mAb2E12to NS3helicase might be under estimated in this study due to the relatively low purity of FL4b-rNS3protein with host cell helicase activity contamination. In a prospective study, recombinant mAb2E12within a viral vector will be transduced into Huh7.5.1cells for further evaluating its intracellular capacity for inhibiting helicase activity and HCV replication in vivo.In summary, we generated and characterized two interested mAbs2E12and3E5, which strongly and specifically bound to fine defined and highly conserved epitope of HCV NS3helicase, respectively. The binding of mAbs to the ATP binding site at motif I (Walker A) of domain1or the close to nucleotide binding region of domain2within NS3helicase might affect the enzymatic activity of helicase in HCV replication. The present data suggested that mAbs2E12and3E5might become the most universal antibodies for diagnosis and antiviral therapy in chronic HCV infection.