Purification and Stabilization of Plant-Derived NDV VLPs

Six days after infiltration, 18g of infiltrated leaves was harvested and homogenized in PBS as previously described. ND VLPs were partially purified using density gradient ultracentrifugation (70%/20% sucrose), whereafter the NDV F/HN VLPs in sucrose fractions 2 to 4 were pooled, dialyzed in 1 x PBS overnight using a 3500 MWCO Slide-A-Lyzer protein dialysis cassette (Thermo Fisher Scientific), and trehalose (15% [w/v]) was added as stabilising agent. Partially-purified NDV F/HN VLPs were tested by hemagglutination assay (see serological testing) and stored at 4°C until use. The vaccine dose of 25 µl partially-purified ND F/HN VLPs was calculated to correspond to an HA titre of 10 log₂ or 1024 HAU. On the day of vaccination, the partially purified plant extract containing the ND VLPs (25 µl) were either diluted in 1 x PBS to a final volume of 250 µl (non-adjuvanted treatment group; supplemented with 1% [v/v] enrofloxacin (Baytril^® 100, Bayer Animal Health, South Africa), or diluted in 1 x PBS and mixed with a commercial oil-in-water adjuvant Emulsigen^®-P (MVP adjuvants^®, Phibro Animal Health Corporation, United States; 20% [v/v]) plus 1% [v/v] enrofloxacin (Baytril^® 100, Bayer Animal Health, South Africa) to a final volume of 250 µl (adjuvanted treatment group). The Emulsigen-P commercial oil-in water adjuvant was selected due its effectiveness in a previous study conducted by the UP/CSIR research group involving the testing of a plant-produced VLP vaccine in poultry (Abolnik et al., 2022 (link)).