Y whether this property is also shown when virions of hepatitis C are used, neutralization assays were performed using JFH1 virus. The virus was pre-incubated with different concentrations of the antibodies (EG89 H1H10) specific for HCV-LP (genotype 3a) for 1hr at 37uC before infection. An unrelated monoclonal antibody (F1G4) was used as negative control. Three days post infection, the effect of antibodies on HCV negative strand synthesis was measured by real time RT-PCR. Huh7.5 cells infected with JFH1 virus in the presence of 100 mg/ml E8G9 mAb showed nearly 65 reduction in intracellular HCV RNA level, while H1H10 showed a modest decrease of about 20 at the same concentration and non specific antibody did not show any inhibition (Fig. 3A). To further confirm that this inhibition of HCV negative strand synthesis by E8G9 antibody is due to inhibition of virus entry, weMonoclonal Antibodies Inhibiting HCV InfectionFigure 4. Epitope mapping of E8G9. (A) Schematic representation of different Nt, adult male and female BALC/c mice (6 of each per fragments of HCV E2 protein used for epitope mapping. (B) Western blot analysis of the recombinant proteins from five regions of E2 (region 3 specific for E8G9 is indicated using an arrow). (R1 5 denote different regions). doi:10.1371/journal.pone.0053619.gperformed in vitro neutralization assay and quantified the level of input positive strand three hours post infection using real time RTPCR 26001275 (Fig. 3B). A significant reduction in virus entry at 50 and 100 mg/ml was Title Loaded From File observed with E8G9 mAb suggesting it as a good candidate for 24272870 inhibiting HCV entry in cell culture system.Epitope Mapping of mAbsThe inhibition of binding of HCV-LPs to Huh 7 cells by E8G9 and not by D2H3 may be due to non-overlapping epitopic regions recognized by the two mAbs, E8G9 and D2H3. To delineate the specific epitopic regions, western blot analysis was carried out using different overlapping fragments of HCV E2 protein (Fig. 4A), expressed in E. coli. The entire E2 coding region of HCV was divided into five overlapping gene fragments (Fig. 4A), which were amplified, cloned and expressed in E. coli. All the five purified protein fragments were analyzed by western blot analysis with E8G9 and D2H3 mAbs. It was seen that E8G9 reacted with region 3 (555 to 646 aa) and region 4 (596 to 699 aa) whereas mAb D2H3 reacted with region 4 only (Fig. 4B). Results indicated that region 3 which is present between amino acids 555 to 646 may be involved in the inhibition of HCV-LP binding to Huh 7 cells. The epitope of mAb H1H10, could not be delineated because it recognizes a conformational epitope and thus fails to react in western blot analysis.DiscussionIn this work, we have reported for the first time the generation of recombinant HCV-LP for genotype 3a, which is prevalent in India. We have also generated the HCV-LP corresponding togenotype 1b prevalent worldwide for comparison. The HCV-LP corresponding to 1b appears to be polygonal in shape and 40 to 60 nm in size as reported earlier, whereas HCV-LP of 3a was found to be approximately 35?5 nm in size. Thus, structurally and morphologically the VLPs were distinct. This could be due to differences in the sequences and conformation of the envelop protein of the two different genotypes. Also it is possible that the amount of E2 protein incorporated in virus like particle could be relatively more in case of genotype 1b. The HCV-LP genotype 3a showed almost 80 binding to Huh 7 cells, whereas genotype 1b HCV-LP showed approximately 70 binding su.Y whether this property is also shown when virions of hepatitis C are used, neutralization assays were performed using JFH1 virus. The virus was pre-incubated with different concentrations of the antibodies (EG89 H1H10) specific for HCV-LP (genotype 3a) for 1hr at 37uC before infection. An unrelated monoclonal antibody (F1G4) was used as negative control. Three days post infection, the effect of antibodies on HCV negative strand synthesis was measured by real time RT-PCR. Huh7.5 cells infected with JFH1 virus in the presence of 100 mg/ml E8G9 mAb showed nearly 65 reduction in intracellular HCV RNA level, while H1H10 showed a modest decrease of about 20 at the same concentration and non specific antibody did not show any inhibition (Fig. 3A). To further confirm that this inhibition of HCV negative strand synthesis by E8G9 antibody is due to inhibition of virus entry, weMonoclonal Antibodies Inhibiting HCV InfectionFigure 4. Epitope mapping of E8G9. (A) Schematic representation of different fragments of HCV E2 protein used for epitope mapping. (B) Western blot analysis of the recombinant proteins from five regions of E2 (region 3 specific for E8G9 is indicated using an arrow). (R1 5 denote different regions). doi:10.1371/journal.pone.0053619.gperformed in vitro neutralization assay and quantified the level of input positive strand three hours post infection using real time RTPCR 26001275 (Fig. 3B). A significant reduction in virus entry at 50 and 100 mg/ml was observed with E8G9 mAb suggesting it as a good candidate for 24272870 inhibiting HCV entry in cell culture system.Epitope Mapping of mAbsThe inhibition of binding of HCV-LPs to Huh 7 cells by E8G9 and not by D2H3 may be due to non-overlapping epitopic regions recognized by the two mAbs, E8G9 and D2H3. To delineate the specific epitopic regions, western blot analysis was carried out using different overlapping fragments of HCV E2 protein (Fig. 4A), expressed in E. coli. The entire E2 coding region of HCV was divided into five overlapping gene fragments (Fig. 4A), which were amplified, cloned and expressed in E. coli. All the five purified protein fragments were analyzed by western blot analysis with E8G9 and D2H3 mAbs. It was seen that E8G9 reacted with region 3 (555 to 646 aa) and region 4 (596 to 699 aa) whereas mAb D2H3 reacted with region 4 only (Fig. 4B). Results indicated that region 3 which is present between amino acids 555 to 646 may be involved in the inhibition of HCV-LP binding to Huh 7 cells. The epitope of mAb H1H10, could not be delineated because it recognizes a conformational epitope and thus fails to react in western blot analysis.DiscussionIn this work, we have reported for the first time the generation of recombinant HCV-LP for genotype 3a, which is prevalent in India. We have also generated the HCV-LP corresponding togenotype 1b prevalent worldwide for comparison. The HCV-LP corresponding to 1b appears to be polygonal in shape and 40 to 60 nm in size as reported earlier, whereas HCV-LP of 3a was found to be approximately 35?5 nm in size. Thus, structurally and morphologically the VLPs were distinct. This could be due to differences in the sequences and conformation of the envelop protein of the two different genotypes. Also it is possible that the amount of E2 protein incorporated in virus like particle could be relatively more in case of genotype 1b. The HCV-LP genotype 3a showed almost 80 binding to Huh 7 cells, whereas genotype 1b HCV-LP showed approximately 70 binding su.
