Investigate The Use Of The Hepatitis C Virus E2 Glycoprotein Back Layer Domain As An Immunogen To Develop Neutralizing Antibody

Hepatitis C virus(HCV)is a small enveloped positive single-stranded RNA virus,belonging to the Flaviviridae family and Hepacivirus genus.Eight confirmed genotypes and 86 subtypes have so far been described.HCV infection has the potential to cause inflammation of the liver ranging from mild illness to cirrhosis and carcinomas.Hepatitis C compromises the quality of life of more than 350 million individuals worldwide.Approximately 40% to 95% of people with acute HCV infection will develop chronic HCV infection,that is,have persistent HCV RNA in their blood.Chronic hepatitis C virus infection appears thus as a global health problem.In 2016,WHO adopted a global hepatitis strategy to eliminate viral hepatitis as a public health threat by 2030.With the better understanding of the HCV’s structure and life cycle,a new multi-genotypic direct acting antivirals(DAA)in clinical practice has revolutionized HCV treatment.The DAA-based therapy targeting key HCV encoded proteins can cure over 90% of individuals for their infection including patients with advanced HCV induced liver disease.But the virological failures can still occur with development of resistance.More current difficulties remain in low income country with difficult access to diagnosis and expensive costs of treatments.Therefore,a prophylactic vaccine is necessary for global control of HCV.HCV has two envelope proteins,E1 and E2,which form heterodimers on viral surface and which mediate cell entry through their interactions with host cell receptors.It has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality,making the structural and functional characterization difficult.The comparison of the partial structural feature of HCV glycoproteins and the feature of other flavivirus envelope proteins,undermine that HCV E2 is a classical fusion protein and HCV E1 may constitute the HCV fusion protein.The structure of E2 ectodomain exhibits a globular,non-extended fold divided into two distinct sheets: a front sheet composed of a front layer and a central Ig-fold domain,and a back layer(BL).A critical interaction between E1 and this soluble E2-Back layer domain(sE2-BLd)has been identified.Consistently,the sE2-Bld is involved into significant conformational changes with E1 during the fusion of the HCV membrane with the cellular membrane.Previous data of our laboratory highlighted the inhibitory action of the back layer domain of HCV envelope glycoprotein sE2-Bld on HCV entry,provided in a soluble form.To study whether the E2-BLd polypeptide could induce a neutralizing response against HCV when used as an immunogen,we made several constructions that could be used for E2,E2-BLd expression.To optimize the E2-Bld’s use,we mutated the unpaired cysteines to avoid aggregate formation.After transfection into mammalian cells,the histidine-tagged(his-tagged)proteins were not easily purified.Therefore,to improve the quantity of the proteins,we generated glutathione S-transferase tag(GST-tag)tagged proteins and express different proteins in Escherichia coli DE3/BL21(E.coli DE3/BL21).The yield of proteins was enough for development of an Enzyme-linked immunosorbent assay(ELISA)assay.Finally,we decided to electroporated the mice intramuscularly with different plasmids allowing expression of E2 and E2-Bld directly.DNA immunization method take the advantages of both easy operation and high immunogenicity.Cell-culture grown HCV(HCVcc)offer the possibility to study virus attachment and entry in a replicating system.In order to validate the potential protective effect of E2-BLd antibodies,mice sera were extracted and analyzed by neutralizing assay using HCVcc virus.Unfortunately,we failed to detect specific neutralizing response after immunization by E2 and E2-Bld.However,the antibodies from the immunized mice sera specifically recognize antigens derived from HCV H77 and J6 strains by Western Blotting(WB)detection and ELISA detection.All together,we generated polyclonal antibodies against Core,E2 and E2-BLd(of HCV H77 stain)by mice DNA immunization which validate our protocol.These efforts provided a biological basis for further exploration about the inhibition of HCV entry by E2-BLd antibodies.