Development of germline-targeting HCV E2 immunogens to drive neutralizing antibody evolution
Final Report Abstract
Developing a prophylactic HCV vaccine is a critical. As natural infection studies show that neutralizing antibodies (bnAb) play a key role in viral clearance but vaccine-induced antibody responses often lack sufficient breadth and potency. We investigated whether engineered, germline-targeting HCV glycoprotein immunogens can enhance broadly neutralizing antibody development by specifically stimulating germline B cell receptors. Based on a cohort of HCV patient sera, we enriched and sequenced patient samples. New infectious HCV test viruses were successfully generated, greatly expanding the available virus repertoire for antibody testing. Lentiviral vectors were used to express diverse HCV envelope proteins in Huh-7.5 cells, and antibody binding was validated by FACS. To evaluate antibody breadth and potency, a panel of mature and iGL antibodies was tested against 13 genetically and functionally diverse HCVcc chimeras. Results showed that certain iGL antibodies, could bind several E1/E2 antigens, but most envelope proteins bound only one or none of the tested iGL antibodies and a range of binding affinities was observed. No direct correlation between iGL binding and neutralization capacity was established. The immunogenicity and capacity for bnAb induction of various vaccine candidates were first evaluated in C57BL/6 mice and subsequently in a transgenic humanized mouse model. Neutralization assays and E2-binding analyses were used to assess broadly neutralizing antibody responses and binding efficiency. Several candidates elicited broadly neutralizing antibodies, although strong binding to iGL antibodies did not consistently correlate with superior neutralization. Ultimately, these studies identified a promising E2 antigen with robust immunogenicity. Eventually, we developed a structure-based pipeline to develop germline-targeting HCV immunogens using a crystal structure of the broadly neutralizing HCV antibody HC84.27. We bioinformatically designed and recombinantly expressed a panel comprising more than 15 potential germline-targeting immunogens. Binding of these germline-targeting immunogens to the corresponding mature and iGL antibody fragments was evaluated by biolayer interferometry and immunogens with highest binding affinity are currently evaluated in TRIANNI mice. These mice encode a fully humanized IgG locus and thus express human antibody germline segments acting as target for HCV germline-targeting immunogens.
