Dynopro nanostar

Uncoupled VLP, soluble proteins and proteins conjugated to VLP were adjusted to 0.5–1 mg/ml in PBS and spun at 15,000 g for 10 min. Seventy Microliter sample was loaded into a disposable Eppendorf Uvette cuvette (Sigma-Aldrich, USA) and measured at 25°C on a DynoPro NanoStar (WYATT Technology, USA) equipped with a 658 nm laser. Each sample was measured 20 times and intensity-average size and percentage polydispersity (PD) was estimated using Dynamic software (Version 7.5.0).

The CLP-HA_stem vaccine was assessed via Dynamic Light Scattering (DLS) as described previously [28 (link)]. In brief, the vaccine was spun at 15,000 g for 10 min, before being loaded into a Eppendorf Uvette cuvette (Sigma-Aldrich, St Louis, MO, USA) and measured 20 times at 25 °C on a DynoPro NanoStar (WYATT Technology, Santa Barbara, CA, USA) using a 658 nm laser. Dynamics software, Version 7.5.0 (WYATT Technology, Sanata Barbara, CA, USA) was used to estimate the intensity-average diameter (nm) and percentage polydispersity (%Pd). CLP-HA_stem was further visualized by negative stain Electron Microscopy as described in the subsequent section.

Synthetic binder was coupled to Acinetobacter bacteriophage AP205 virus-like particles (VLPs) displaying one N-terminal SpyCatcher per capsid subunit (35 (link)). VLPs were expressed in E. coli BL21 StarTM (DE3) cells (Thermo Fisher Scientific) and purified by ultracentrifugation using an OptiprepTM density gradient (Sigma). Assembled VLPs and synthetic binder antigen were mixed at a 1:1 molar ratio and incubated for 2 h at room temperature.
Assembled VLPs were quality assessed by dynamic light scattering (DLS). The vaccine was centrifuged at 15,000 × g for 10 min and the supernatant was loaded into a disposable Eppendorf Uvette cuvette (Sigma-Aldrich, USA) and measured 20 times at 25°C using a DynoPro NanoStar (WYATT Technology, USA) with a 658 nm wavelength laser. Intensity-average size (nm) and percentage polydispersity (%Pd) were estimated using Dynamic software (Version 7.5.0).
Groups of four rats were immunized with 19-kDa HB3var03 CIDRα1.4 STRPII-tagged protein (18 (link)), with synthetic binder coupled to VLPs or with synthetic binder alone. In all groups, rats received 20 μg protein in each immunization. The rats were immunized intramuscularly (i.m.) every third week in a prime boost setting with Freund’s incomplete adjuvant for a total of three immunizations.