Structural analysis of HCV E1E2 glycoproteins

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ABSTRACT – PROJECT 4 Despite extensive research efforts to understand the biology of hepatitis C virus (HCV), it remains a major human pathogen without a vaccine. In addition, there is a remarkable absence of information regarding the structure of the virion and organization of its component proteins. We intend to use our extensive structural biology experience with the related flaviviruses, to focus on determining the structure of the viral glycoproteins, the virus particle, and complexes of potent neutralizing antibodies bound to the surface glycoproteins. This project will rely heavily on and interact extensively with Project 1 to interrogate the structure and provide guidance into the features of the glycoproteins that are important for eliciting broad and potent neutralizing antibodies. Iterative analysis and re-engineering of the E1E2 glycoproteins will be conducted to guide the antibody component of a dual B and T cell HCV vaccine, the focus of this U19 proposal. This specific project will use three aims to determine the structure of the E1E2 heterodimer, the virus particle, and neutralizing antibody Fabs bound to the glycoproteins. Based on biophysical and immunological assays, computational modeling and structure analyses, the E1E2 glycoproteins will be engineered to present epitopes that elicit strong neutralizing and broadly reactive antibodies and minimize the presentation of decoy non-productive epitopes. Furthermore, the organization of the glycoproteins and the structure of the virus particle will be determined. The tools of molecular virology, biophysics, computational modeling, x-ray crystallography and cryo-electron microscopy will be used to produce atomic-level details of the epitope landscape of HCV. The gained structural information will be utilized to design novel antigens in Project 1, determine the structures of designed immunogens experimentally in Project 4 and tested in vivo in Projects 1, 2 and 3. We will guide Project 1 at various times during the project to allow them to utilize the optimal E1E2 immunogen for studies in guinea pigs and, ultimately, a lead candidate for an optimized E1E2 immunogen for nonhuman primate studies. This information will be invaluable in deciphering the nature of neutralizing antibodies and their mechanisms of action against HCV and will shed light on the assembly and entry of hepatitis C virions.