Monoclonal antibodies against Hepatitis C genotype 3a virus like particle inhibit virus entry in cell culture system

Abstract

The envelope protein (E1-E2) of Hepatitis C virus (HCV) is a major component of the viral structure. The glycosylated envelope protein is considered to be important for initiation of infection by binding to cellular receptor(s) and also known as one of the major antigenic targets to host immune response. The present study was aimed at identifying mouse monoclonal antibodies which inhibit binding of virus like particles of HCV to target cells. The first step in this direction was to generate recombinant HCV-like particles (HCV-LPs) specific for genotypes 3a of HCV (prevalent in India) using the genes encoding core, E1 and E2 envelop proteins in a baculovirus expression system. The purified HCV-LPs were characterized by ELISA and electron microscopy and were used to generate monoclonal antibodies (mAbs) in mice. Two monoclonal antibodies (E8G9 and H1H10) specific for the E2 region of envelope protein of HCV genotype 3a, were found to reduce the virus binding to Huh7 cells. However, the mAbs generated against HCV genotype 1b (D2H3, G2C7, E1B11) were not so effective. More importantly, mAb E8G9 showed significant inhibition of the virus entry in HCV JFH1 cell culture system. Finally, the epitopic regions on E2 protein which bind to the mAbs have also been identified. Results suggest a new therapeutic strategy and provide the proof of concept that mAb against HCV-LP could be effective in preventing virus entry into liver cells to block HCV replication.

Figure 1. Characterization of HCV-LPs.

(A) HCV-LPs corresponding to genotypes 3a and 1b were harvested on 4^th day post infection and purified as described in Materials and Methods. HCV-LPs were tested with different concentrations of anti-HCV-E1E2 antibody using ELISA. (B) Transmission electron microscopy of HCV-LPs of 1b and 3a as indicated. Scale bar: 200 nm for genotype 1b and 100 nm of genotype 3a; magnification: 10,000X. (Inset shows a single virus particle with 20,000× magnification).

Figure 2. Inhibition of HCV-LP and Huh7 cell binding by mAbs.

HCV-LPs of both genotypes 1b and 3a were incubated with increasing concentrations of mAbs as indicated. The Y-axis depicts the percentage activity representing both the percent binding (dark grey) and percent inhibition HCV-LP attachment (light grey).

Figure 3. Effect of mAbs on HCV infection.

(A) JFH1 virus was preincubated with increasing concentrations (0.5, 5, 50, 100 µg/ml) of mAbs (E8G9 and H1H10) or 100 µg/ml of non-specific antibody F1G4 (Non sp) for 1 hr at 37°C before infecting Huh7.5 cells. Three days post infection, total cellular RNA was isolated and HCV negative strand level was measured using real time RT-PCR. GAPDH was used as an internal control. (B) mAb E8G9 was used in increasing concentrations to inhibit the virus entry and the input viral RNA present inside the cells (positive strand) was estimated 3 h post infection by real time RT-PCR. GAPDH was used as an internal control.

Figure 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–R5 denote different regions).