Characterization Of The Hepatitis C Virus E2 Glycoprotein Back Layer Domain And Optimization To Inhibit Entry

Hepatitis C Virus(HCV)is a small,enveloped,positive sense single stranded RNA virus,belonging to the Flaviviridae family,and Hepacivirus genus.This blood-to-blood borne virus replicates specifically in the human hepatocyte liver cells which leads to the development of liver disease called hepatitis C.Worldwide,an estimated 185 million individuals are infected with HCV.While clearance of the virus is possible,initial HCV infections are most commonly followed by a chronic liver disease that may sometimes progress into a liver cirrhosis and hepatocellular carcinoma.The better understanding of the HCV's structure and life cycle has led to the development of direct-acting agents(DAAs)and numerous families of drugs that potently inhibit the HCV life cycle in vitro have been identified.However,their use still presents limits,and there is still,today,a pressing need to develop multitherapy strategies targeting different stages of HCV life cycle.The virus entry in hepatocyte is the first step of the virus life cycle and constitutes a favorable target for therapies.HCV entry is managed by multiple cellular and viral actors,amongst which HCV E1 and E2 envelope glycoproteins acts as a functional heterodimer on the virus surface.It is still unknown which of E1 and/or E2 is the protein leading membrane fusion.While E1's role remains enigmatic,E2 is known to bind receptors.The recent advances of HCV E2's structure revealed that E2 ectodomain exhibited a globular,non-extended fold divided into two distinct sheets: a front layer and central intergenotypic fold domain;and a back layer domain(Bld).A critical interaction between E1 and this E2-Bld has been identified.In order to test the critical role in the entry process of this interaction,E2-Bld polypeptides have been designed to inhibit this E1-E2-Bld interaction.In in vitro experiments,soluble E2-Bld polypeptide inhibits the replication of HCV virus in cell culture(HCVcc)and the entry of HCV pseudo-particles(HCVpp)that are a robust model to study HCV entry.Interestingly,this E2-Bld polypeptide was also able to inhibit the replication of HCV in mice harboring a humanized liver,a pertinent HCV replication model in vivo that reflect the complexity of HCV diversity.In this context,this project aimed to characterize the inhibition action of this E2-Bld polypeptide.In a first part,we constructed several E2-Bld in order to improve its purification and its functionality.To this aim we modified the length of the histidine tag,and we mutated unpaired cysteines to avoid aggregate formation,thus optimizing the E2-Bld's use.These plasmids were expressed in mammalian cells to produce the E2-Bld polypeptide,but this expression system did not seem optimal.To increase the yield,in a second part,some of these E2-Bld polypeptides were sub-cloned in order to express them in insect cells.Unfortunately,the productions of E2-Bld were not much improved.Therefore,in order to develop antibodies without injecting polypeptides,we decided to implement a DNA immunization strategy to avoid the purification steps.Mice were electroporated in their muscle with different plasmids allowing expression of E2-Bld.The sera of these mice were analyzed by neutralizing assay using HCV J6 stain.Interestingly,we observed that the mice developed and neutralizing response after immunization by different E2-Bld.Altogether,we hope these efforts could help contribute to the better understanding of HCV entry,and on the long run,could participate to the development of a vaccine solution against HCV infections.