Humanized-VHH transbodies that inhibit HCV protease and replication

Area of Science:

• Virology
• Immunology
• Biotechnology

Background:

• Hepatitis C virus (HCV) presents a challenge due to genetic diversity and mutations.
• Existing anti-HCV agents require improvement for broad efficacy and safety.
• Developing novel therapeutic strategies is crucial for HCV eradication.

Purpose of the Study:

• To develop safe and broadly effective anti-HCV agents.
• To create cell-penetrable nanobodies targeting HCV serine protease.
• To evaluate the efficacy of these nanobodies in reducing viral load and restoring host immunity.

Main Methods:

• Production of humanized-camel VHHs targeting genotype 3a HCV serine protease.
• Molecular linkage of VHHs to a cell-penetrating peptide (Penetratin - PEN).
• Treatment of HCV genotype 2a-infected human hepatic (Huh7) cells with PEN-VHH transbodies.
• Intracellular and extracellular HCV RNA quantification, HCV foci, and core antigen measurement.
• Analysis of host innate immune gene expression (TRIF, TRAF3, IRF3, IL-28B, IFN-β).
• Computerized intermolecular docking to determine VHH binding sites on the protease.

Main Results:

• PEN-VHH transbodies significantly reduced intracellular and extracellular HCV RNA.
• Reduced HCV foci and core antigen levels were observed in treated cells.
• Up-regulation of host innate immune response genes (TRIF, TRAF3, IRF3, IL-28B, IFN-β) was noted.
• VHHs demonstrated binding to critical residues of the HCV serine protease, including the catalytic triad and NS4A binding site.
• The nanobodies rescued host innate immune response from HCV-mediated suppression.

Conclusions:

• Cell-penetrable nanobodies (transbodies) are effective against HCV in vitro.
• These transbodies demonstrate potential for broad efficacy and tolerance to viral mutations.
• The developed transbodies represent a promising candidate for future anti-HCV therapeutic development.
• Restoration of host innate immunity is a key mechanism of action.