Final Report Summary - HEPCENT (Molecular Analysis of Hepatitis C Virus Neutralization and Entry For the Development of Novel Antiviral Immunopreventive Strategies) Hepatitis C virus (HCV) infection is a major cause of chronic liver disease world-wide. With 180 million persistently infected people chronic hepatitis C infection represents a major public health problem of high socio-economic impact. Despite the ongoing development of novel HCV antivirals, it remains of outmost importance to develop a vaccine for prevention against HCV infection. HCV vaccine development is hampered by severe obstacles such as the high viral variability and several immune escape mechanisms that render antigen selection difficult. This requires efforts to identify conserved T cell as well as neutralization epitopes and to decipher neutralization mechanisms, aiming to discover the optimal targets of the HCV life cycle and how to counteract viral escape strategies. While most vaccine development programs are based on improving HCV cellular immunity, current knowledge suggests that is essential to include, in a same vaccine formulation, immunogens capable to induce both humoral neutralizing and cellular responses of broad spectrums. The investigation of the humoral response against HCV is central in the HEPCENT project. Viral attachment and entry - representing the first encounter of the virus with the host cell - is a major target of adaptive host cell defenses. Thus, aiming to characterize the entry steps of HCV into cells, from cell attachment to membrane fusion, we have i) generated novel assays to dissect the specific role of the four HCV entry co-factors, ii) characterized two crucial of these co-factors: the Claudin-1 tight-junction protein and the scavenger receptor BI (SR-BI) lipid transporter, and iii) investigated how E1E2-targeted antibodies block interaction of these HCV surface glycoproteins to HCV entry factors or membrane fusion. We have also proposed that rather than targeting the HCV particle surface, antibodies useful for HCV therapy could also target the entry factors themselves and we have developed Claudin-1 and SR-BI monoclonal antibodies that efficiently block HCV entry. These results, that have been obtained mostly with model viruses (HCVpp and HCVcc) in human hepatocarcinoma cells have been confirmed in relevant cell culture assays, using human primary hepatocytes, and in vivo, using human liver-humanized mouse models in which HCV can be grown. In a longitudinal analysis of six HCV-infected patients undergoing liver transplantation, we have found that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not selected after transplantation. These findings provide significant insights into the molecular mechanisms of viral evasion during HCV reinfection and confirmed that viral entry is a viable target for prevention of HCV reinfection of the liver graft. The extensive involvement of lipoprotein and lipoprotein receptors in HCV entry suggest that lipoproteins, eventually associated with HCV particles, modulate the entry pathway and affect neutralization. By using lipidomic analysis of affinity purified HCV virus particles, we found that the lipid composition of HCVcc particles is similar to that of very low- and low-density lipoprotein. The purified HCV particles were found to be heterogeneous, mostly of spherical structures, and to contain apoE on their surface as assessed by immuno-electron microscopy. We have also dissected intersections of the HCV early assembly steps with the lipid metabolism and characterized the interactions of host lipid droplets and apoE with some HCV proteins in these processes.Finally, we have elaborated new tools that, combined with the findings mentioned above, will contribute to ultimately lead to the development of immuno-preventive therapeutic strategies. They consist of i) vectors than can genetically transduce primary B cells, allowing to immortalize, clone or reprogram B cells; ii) novel immunization platforms, based on virus-like particles (VLPs), onto which specific antigenic formulations, such as HCV surface glycoproteins, can induce potent neutralizing antibody responses; iii) vectorization of these VLP-based platforms to enhance their efficiency in vivo.
