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

Thermal melting curves for monomeric E2 proteins were acquired using a MicroCal PEAQ-DSC automated system (Malvern Panalytical). Purified monomeric E2 proteins were dialyzed into PBS prior to analysis and the dialysis buffer was used as the reference in the experiments. Samples were diluted to 10 M in PBS prior to analysis. Thermal melting was probed at a scan rate of 90 °C•h -1 over a temperature range of 25 to 115 °C. All data analyses including estimation of the melting temperature were performed using standard protocols that are included with the PEAQ-DSC software. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.044073 doi: bioRxiv preprint iodoacetamide (room temperature for 30 min). Samples were then buffer exchanged into 1 M urea in 0.1 M Tris, pH 7.8 for digestion. Sequential digestion was performed using trypsin (1/50 enzyme/protein ratio, w/w) for 18 hours at 37 °C, followed by chymotrypsin (1/20 enzyme:protein, w/w) overnight at room temperature. Samples were then absorbed onto Sep-Pak tC18 columns to remove proteolytic digestion buffer, eluted with 50% acetonitrile/0.1% trifluoroacetic acid (TFA) buffer and concentrated to dryness in a centrifugal vacuum concentrator. The samples were then The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.044073 doi: bioRxiv preprint antigen solution at 1:1 (prime) or 1:5 (w/w) (boost immunization) antigen:adjuvant ratio to provide for 50 mcg PCPP dose per animal. The absence of aggregation in adjuvanted formulations was confirmed by dynamic light scattering (DLS): single peak, z-average hydrodynamic diameter -60 nm. The formation of sE2 antigen -PCPP complex was proven by asymmetric flow field flow fractionation (AF4) as described previously (62) . On scheduled vaccination days, groups of 6 female mice, age 7-9 weeks, were injected via the intraperitoneal (IP) route with a 50 µg sE2 prime (day 0) and boosted with 10 µg sE2 on days 7, 14, 28, and 42. Blood samples were collected prior to each injection with a terminal bleed on day 56. The collected samples were processed for serum by centrifugation and stored at -80°C until analysis was performed.