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.

Identification of a novel epitope in the C terminus of hepatitis C virus-E2 protein that induces potent and cross-reactive neutralizing antibodies

“…: core, GU172376; E1E2, GU172375) and HCV-LP generated as described earlier [45,46]. Similarly, the core-E1-E2 of gt1b was amplified from replicon Con 1FL (accession no.…”

“…Similarly, the core-E1-E2 of gt1b was amplified from replicon Con 1FL (accession no. AJ238799) [47] and HCV-LP for gt1b was generated as mentioned earlier [45,46]. To generate HCV-LPs, Sf21 cells were infected with recombinant baculovirus (at an m.o.i.…”

“…After 72 h, cell pellets were washed three times with PBS (50 mM phosphate buffer, pH 7.2, containing 150 mM NaCl) and were resuspended in lysis buffer (50 mM NaCl, 50 mM Tris, 0.5 mM EDTA, 1 mM PMSF, 0.1 % Nonidet P-40 and 0.25 % protease inhibitors) using a homogenizer. The lysate was centrifuged and HCV-LPs were purified using sucrose gradient ultracentrifugation [45,46]. The HCV-LPs (both gt3a and gt1b) were characterized by ELISA and confirmed by electron microscopy [45,46].…”

“…The lysate was centrifuged and HCV-LPs were purified using sucrose gradient ultracentrifugation [45,46]. The HCV-LPs (both gt3a and gt1b) were characterized by ELISA and confirmed by electron microscopy [45,46].…”

“…Binding of the VLPs was monitored by flow cytometry as described earlier [45,46]. Briefly, HCV-LPs of both genotypes were labelled with Alexa Fluor 488 using a gel exclusion chromatography column (Sigma Aldrich).…”