Isolation and epitope characterization of human monoclonal antibodies to hepatitis C virus core antigen

Area of Science:

• Immunology
• Virology
• Molecular Biology

Background:

• Hepatitis C virus (HCV) infection is a major global health concern.
• The HCV nucleocapsid protein (core, p22) is a key target for antiviral therapies.
• Developing specific monoclonal antibodies is crucial for understanding HCV and for therapeutic development.

Purpose of the Study:

• To establish hybridoma cell lines secreting human monoclonal antibodies against the HCV core protein.
• To characterize the specificity and epitope mapping of these antibodies.
• To identify a linear epitope on the HCV nucleocapsid protein recognized by a specific antibody.

Main Methods:

• Isolation of B lymphocytes from an anti-HCV positive blood donor.
• Infection of B lymphocytes with Epstein-Barr virus (EBV) to create lymphoblastoid cell clones.
• Fusion of B-cell cultures with a mouse:human heteromyeloma cell line (K6H6/B5) to generate stable hybridomas.
• Characterization of antibody isotypes (IgG1/kappa and IgM/kappa).
• Epitope mapping using overlapping peptides and immunoblot assays.

Main Results:

• Two stable hybridomas, U1/F10 (IgG1/kappa) and U1/F11 (IgM/kappa), were established, secreting antibodies specific to the recombinant HCV core protein p22.
• The U1/F10 antibody was found to recognize a linear epitope on the nucleocapsid protein.
• The specific epitope recognized by U1/F10 was identified as the amino acid sequence N-VYLLPR-C (amino acids 34-39).

Conclusions:

• The study successfully generated human monoclonal antibodies against the HCV core protein.
• The U1/F10 antibody provides a valuable tool for studying HCV, potentially aiding in diagnostics and therapeutics.
• The identification of a specific linear epitope offers insights into the structural and functional aspects of the HCV nucleocapsid protein.