Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen

Area of Science:

• Molecular Biology
• Virology
• Immunology

Background:

• Hepatitis C virus (HCV) infection remains a significant global health concern.
• Accurate identification of viral antigenic determinants is crucial for developing effective diagnostics and vaccines.
• Existing methods for mapping viral epitopes have limitations in identifying conformational determinants.

Purpose of the Study:

• To develop a novel phage display method for cloning and expressing HCV protein fragments.
• To identify and map antigenic determinants within HCV proteins.
• To create chimeric proteins for enhanced diagnostic and vaccine applications.

Main Methods:

• Utilized a novel method for cloning DNase I fragments into the bacteriophage display vector fUSE2.
• Created phage display libraries expressing hepatitis C virus (HCV) protein fragments.
• Selected antigenic determinants using panning with sera from HCV-seropositive individuals and enzyme-linked immunosorbent assay (ELISA).

Main Results:

• Identified antigenic determinants in HCV NS3 (aa 1,383-1,415), NS4 (aa 1,930-1,938), and NS5 (aa 2,088-2,104).
• Localized a major conformational determinant in NS3 that cannot be mimicked by short peptides.
• Co-expressed chimeric proteins, such as NS4P with an NS4N fragment, demonstrating improved detection of HCV positives compared to individual components.

Conclusions:

• The phage display approach effectively identifies conformational and linear antigenic regions of HCV.
• Co-expression of identified epitopes as chimeric proteins enhances diagnostic sensitivity and immunoreactivity.
• This method holds general applicability for developing improved diagnostic antigens and recombinant vaccines for infectious diseases.