Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization

Brian G. Pierce; Zhen-Yong Keck; Ruixue Wang; Patrick Lau; Kyle Garagusi; Khadija Elkholy; Eric A. Toth; Richard A. Urbanowicz; Johnathan D. Guest; Pragati Agnihotri; Melissa C. Kerzic; Alexander Marin; Alexander K. Andrianov; Jonathan K. Ball; Roy A. Mariuzza; Thomas R. Fuerst; Steven K.H. Foung

Following confirmation of antigenicity, E2 designs were tested in vivo for immunogenicity, to assess elicitation of antibodies that demonstrate potency and neutralization breadth. CD1 mice (6 per group) were immunized with H77C sE2 and designs, using with Day 0 prime followed by three biweekly boosts. Sera were obtained at Day 56 (two weeks after the final boost) and tested for binding to H77C sE2 and key conserved epitopes (AS412/Domain E, AS434/Domain D) ( Figure 4 ). Peptide epitopes were confirmed for expected monoclonal antibody specificity using ELISA ( Figure 4B ). Endpoint titers demonstrated that sera from mice immunized with E2 designs maintained recognition of sE2 and tested epitopes. Intra-group variability resulted in lack of statistically significant differences in serum binding between immunized groups, however mean titers from ΔΗVR1 group were moderately lower than the wild-type sE2 group, and other mutants yielded moderately higher serum binding to the tested epitopes. Notably, design H445P elicited antibodies that robustly cross-reacted with the wild type AS434/Domain D epitope. To assess differential binding to conformational epitopes on E2, serum binding competition with selected HMAbs was performed ( Figure S1 ) but did not show major differences between immunized groups.