Struktur-basiertes Design einer epitop-fokussierten Hepatitis C virus Vakzine
Strukturbiologie
Zusammenfassung der Projektergebnisse
Within the course of this project, we in silico designed immunogens displaying specific HCV neutralization epitopes within the two HCV glycoproteins E1 and E2 based on available three-dimensional structures of complexes containing neutralizing antibody fragments (Fabs) and their respective cognate antigens. We extensively characterized the resulting immunogens biochemically and with respect to interaction with the corresponding antibody and thereby identified several promising immunogens. Using novel computation methods these immunogens were subsequently stabilized, expressed and subjected to an exhaustive pipeline of characterization. This characterization included biochemical, biophysical, structural analyses and allowed for the selection of two scaffolds presenting two distinct HCV neutralization epitopes. These results suggested that the selected scaffolds are suitable for presentation on multivalent nanoparticles, which further improve the immunogenicity of the scaffolds when compared to monomeric soluble scaffolds. Several self-assembling nanoparticle platforms were tested without success, likely due to the hydrophobic nature of the HCV neutralization epitopes that leads to insoluble nanoparticles and therefore minimal expression levels. Ultimately, nanoparticles based on Hepatitis B virus capsids carrying either of two distinct HCV neutralization epitopes were produced and used for immunization of mice alongside a soluble E2 ectodomain (sE2) used as control. The antibody response elicited by these nanoparticles in mice was evaluated using a single dose neutralization assays against a panel of four different cell culture-derived HCV (HCVcc). A significant cross-neutralizing activity within the sera of mice immunized with nanoparticles, but not in mice immunized with sE2 was observed. This potent polyclonal humoral immune response indicates that our nanoparticles represent a promising novel candidate to be developed into an effective B cell vaccine. Our results demonstrate that structure-based epitope-focusing immunogen design can be successfully applied to HCV and offers a proof-of-concept for the development of potent epitope-focused immunogens.
