Incomplete freund s adjuvant

The first vaccine emulsion was prepared by mixing Complete Freund’s Adjuvant (Invivogen) and R-P4 (20 μM dissolved in sterile PBS) at a ratio of 1:1. For the first vaccination, 100 μL of the emulsion was injected (IP) into male (n = 12) and female (n = 12) C57BL6/J mice (obtained from Jackson Laboratories). Fourteen days after initial vaccination, mice were injected with 100 μL of freshly prepared vaccine emulsion in InComplete Freund’s Adjuvant (Invivogen). Adjuvant-only emulsions were made by mixing the appropriate adjuvant in sterile PBS at a ratio of 1:1. Adjuvant-only control mice were injected (IP) in male (n = 12) and female (n = 12) mice on the same schedule as analog-vaccinated mice.

Four-week-old male and female BALB/c mice (Jackson Laboratories, Bar Harbor, ME, USA) were immunized by injection of 50–60 μL vaccine mixture in the caudal thigh muscle. Vaccines contained 10 μg VLP suspended in sterile PBS, mixed 1:1 in Incomplete Freund’s Adjuvant (Invivogen, San Diego, CA, USA), or mock vaccinated with PBS alone. Four weeks later, mice were given an identical vaccination (boost). Serum for ELISAs was collected by retro-orbital bleed without euthanizing or at the time of euthanasia by cardiac puncture. Challenge experiments were performed eight weeks after boosting.