Experimental Protocols

Tg2576 mice in this study were chosen from two different cohorts. Cohort I was used in the first experimental set (preventive study versus therapeutic study), and cohort II was used in the second experimental set (analysis of β-amyloid in mice with or without pre-existing memory T cells). In each experimental set, all mice were allocated randomly among experimental and control groups.

In preventive studies, two groups of 6–6.5-month-old mice were injected nine times with 100 μg/mouse adjuvanted EV (n = 8) or adjuvant alone (n = 6) as we described previously (Figure 1A).37, 38, 40 In parallel, we conducted therapeutic studies by immunization of 16–19-month-old Tg2576 mice (n = 6). Control animals of the same age were injected with adjuvant alone (n = 5). Blood was collected before injections (pre-bleed) or 10 days after each immunization. Mice were terminated at the ages of 15–15.5 months for preventive and 21–24 months for therapeutic studies, and the neuropathological changes were analyzed in their brains (Figure 1B).

To generate mice with pre-existing Th memory cells, the first group of 6–6.5-month-old Tg2576 mice (group 3, n = 8) was injected with carrier Th epitopes as we previously reported.40 Mice from control groups were injected with the same Th epitopes (group 1, n = 9) or adjuvant only (group 2, n = 5). After 4 months of resting, period mice from group 1 were injected with adjuvant only and mice from groups 2 and 3 were immunized with adjuvanted EV (Figure 4A). Blood was collected before priming and boosting (pre-bleeds) or 10 days after each boost. The behavioral study was performed after the third boost, when mice were 14–14.5 months old. At the age of 15–15.5 months, mice were terminated and the neuropathological changes were analyzed in the brains.