2.4 Selection of VLPs dose for vaccine formulation
To select the VLPs vaccine dose, dilutions of VLPs in PBS containing: 8, 4, 2, 1, 0.5, 0.3, 0.15 or 0 µg in a final volume of 0.1 ml were prepared. Groups of mice (n=5) were subcutaneously (sc) inoculated at 0 and 21 dpv with these formulations. Animals were challenged with an ip injection of 102.5 TCID50/ml infectious FMDV, A2001 serotype, at 36 days post vaccination (dpv). Twenty-four h later, viremia was evaluated as described in Bidart et al (2020) (Bidart et al., 2020). Briefly, heparinized blood withdrawn at 24 h post infection was spread onto BHK-21 cell monolayers grown in 48-well plates and incubated at 37C in a 5% CO2 atmosphere. Then, cell monolayers were washed twice with sterile PBS. Fresh D-MEM supplemented with 2% fetal calf serum (FCS) was added and the cells were incubated for 48 h at 37C in 5% CO2. It was considered that animals were infected if the cell monolayer presented cytopathic effects after a blind passage, as established in previous studies (Gnazzo et al., 2020; V. Quattrocchi et al., 2014; V Quattrocchi et al., 2011; Zamorano et al., 2010). Percentages of protection were calculated as: 100 x [protected/challenged mice]. A dose of 0.5 µg VLPs induced 40% of protection and was thus selected for subsequent experiments, since it allows a good margin to observe the adjuvant effects.