Isolation Of Heavy Chain Antibodies To Support The design Of HCV Vaccine

Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments in antiviral therapeutics. An effective cross-genotype vaccine is needed to achieve the global elimination of HCV. HCV E2 envelope (Env) glycoprotein is the main target for neutralizing antibodies (nAbs), aiding HCV clearance and protection. The failure of B cell-based vaccination trials to prime a broad immune response highlighted that further optimization of the current vaccine candidates or the development of alternative vaccination approaches is urgently needed to control HCV spread. A molecular-level understanding of Ab neutralization responses against HCV is imperative for the rational design of cross-genotype vaccine antigens.

To this aim, we selected an alternative approach using HCV-specific heavy chain-only antibodies (HCAbs) to discover and characterize novel HCV neutralization epitopes. We immunized Llamas with E2 antigens and isolated a library of E2-specific HCAbs. The HCAbs were engineered to recombinant single-domain antigen-specific nanobodies (Nbs). We expressed and purified 53 Nbs, 24 of which succeeded in binding with a high affinity to E2 antigens and were able to block the binding of E2 to the host cell receptor CD81, suggesting the capability of the Nbs to inhibit viral entry to the host cells. HCV Pseudoparticle (HCVpp) neutralization assay confirms the Nbs neutralization capability. The subsequent phase of our study focused on mapping the neutralization epitopes. Competition assays indicated a diverse distribution of the Nbs, categorized into three groups, each associated with binding to a specific epitope on the antigen. The results were confirmed using Hydrogen Deuterium Exchange mass spectrometry assays. This advanced method allowed us to delve deeper into the interactions between E2-Nbs complexes. Finally, we are conducting crystallization experiments for high-resolution determination of the E2-HCAb complex’s structures. Our results advance the understanding of the neutralization response against HCV and promote the rational design of the HCV vaccine.