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Human recombinant antibodies specific for hepatitis C virus core and envelope E2 peptides from an immune phage
1Medical Research Council (MRC) Molecular Immunopathology Unit, MRC Centre, Cambridge, UK. swc21@cam.ac.uk
Insights
Researchers developed phage display technology to create human antibodies targeting Hepatitis C virus (HCV) proteins. These single-chain variable fragments (scFvs) show promise for detecting and potentially treating HCV infection.
Area of Science:
- Virology and Immunology
- Biotechnology and Molecular Biology
Background:
- Hepatitis C virus (HCV) causes significant global health issues, including chronic infections leading to cirrhosis and liver cancer.
- HCV's genetic variability complicates serological detection and vaccine development.
- Phage display technology offers a novel approach for generating specific antibodies.
Purpose of the Study:
- To generate a phage display library for cloning human anti-HCV antibodies.
- To isolate and characterize single-chain variable fragments (scFvs) targeting HCV core and envelope proteins.
- To assess the therapeutic potential of these generated scFvs.
Main Methods:
- Construction of a phage display library using V(H) genes from an HCV-infected patient and V(L) genes from non-immune individuals.
- Selection of specific IgG single-chain Fvs (scFvs) by screening against synthetic peptides from HCV core and E2 envelope proteins.
- Validation of scFv specificity using ELISA and inhibition ELISA assays with homologous peptides and patient sera.
Main Results:
- Successfully generated scFvs that specifically recognize viral core and envelope proteins.
- Demonstrated scFv specificity through binding assays and competitive inhibition with homologous peptides.
- Showed that anticore scFv binding could be blocked by HCV-positive human sera, indicating epitope recognition.
Conclusions:
- Phage display is effective for generating specific human antibodies against HCV proteins.
- The isolated scFvs have potential diagnostic and therapeutic applications for Hepatitis C.
- The findings suggest scFvs recognize epitopes similar to those targeted by natural polyclonal antibodies.
Abstract:
Hepatitis C virus (HCV) is the aetiological agent responsible for most cases of non-A non-B hepatitis. Hepatitis C is a disease of clinical importance because of its high infection rate in blood donors and its persistence as chronic infections which may lead to cirrhosis and hepatocellular carcinoma in the long term. The variability of the HCV genome has posed difficulties in serological detection and vaccine design. The recent advance in phage technology offers a means of cloning human anti-HCV antibodies of a defined specificity that may have potential therapeutic use. We now report the generation of a phage display library using the V(H) genes of a HCV-infected patient and the V(L) genes of two non-immune individuals. From this library we were able to obtain specific IgG single-chain Fvs (scFvs) that recognize viral core and envelope proteins by selection on synthetic peptides derived from the core sequence PKARRPEGRTWAQPG and the envelope E2 sequence RPIDDFDQGWGPITY. The specificity of the scFvs was demonstrated by their specific reactions with homologous peptides in ELISA and the specific blocking of scFv binding by homologous peptides, in a dose-dependent manner, in inhibition ELISA. The binding of the anticore 4c2 to homologous peptide was blocked by HCV-positive human sera in an antibody-concentration-dependent manner, suggesting that the scFv recognizes a similar if not identical epitope to those of one or more of the polyclonal antibodies present in the sera.