Improvements in Intranasal Amyloid-β (Aβ) Immunization in Mice

Previously, we showed that intranasal (i.n.) administration of Aβ1–40 in PDAPP mice for seven months led to a 58% decrease in cerebral Aβ burden (Weiner et al. 2000; Lemere et al. 2000). Serum anti-Aβ titers were low. Since then, we have focused on optimizing our i.n. Aβ immunization protocol using different routes of administration — a proven mucosal adjuvant, Escherichia coli heat-labile enterotoxin (LT), and various Aβ peptides as immunogens — in an effort to increase Aβ antibody titers. First, we compared three routes of Aβ immunization [intraperitoneal (i.p.) injection, i.n. administration and a combination thereof] in two strains of wild type (WT) mice. B6D2F1 mice were much more responsive to Aβ immunization than were C56BL/6 mice; i.p. Aβ immunization and the combination of i.p. with i.n. Aβ gave the highest titers. Second, when low doses of the mucosal adjuvant LT were given with Aβ i.n., there was a dramatic, 12-fold increase in Aβ antibody titers in B6D2F1 mice treated two times a week for eight weeks compared to those of mice receiving i.n. Aβ without adjuvant. A non-toxic, mutant form of LT, designated LT(R192G), showed even better adjuvanticity; anti-Aβ antibody titers were 16-fold higher than those seen in mice given i.n. Aβ without adjuvant. Third, in another study, B6D2F1 mice were given i.n. Aβ1–40/42 or Aβ1–15, each with LT(R192G) twice a week for six weeks. Mice receiving full-length Aβ + LT(R192G) generated serum anti-Aβ antibodies earlier and in much greater abundance than did mice given Aβ1β15 + LT(R192G). Mouse serum anti-Aβ anti-bodies consistently detected human Alzheimer’s disease (AD) plaques, had an epitope(s) within Aβ1–15, and were of IgG1 and IgG2b isotypes without adjuvant, but included IgG2a and low levels of IgA with adjuvant. Both forms of LT were well tolerated by the mice and showed no obvious toxic effects. Human Aβ peptide was not detected in any of the mouse brains. More recently, we immunized five PSAPP transgenic (tg) mice, an accelerated mouse model of AD, by giving a single i.p. injection of Aβ1–40/42 + Complete Freund Adjuvant (CFA) at the beginning of the study, followed by i.n. Aβ1–40/42 + LT twice weekly for eight weeks. Immunization was started at five weeks of age, prior to plaque formation. Serum anti-Aβ antibody titers were ~ nine-fold higher than those generated in our earlier study in PDAPP mice. As in the studies above in WT mice, the anti-Aβ antibodies had epitope(s) within Aβ1–15 and were of Ig isotypes IgG2b, IgG2 a and IgG1 in Aβ immunized mice. Serum anti-Aβ titers were not detected in control PSAPP mice. Aβ immunized mice showed significant decreases in cere-bral Aβ levels; a 75% decrease in plaque number and a 58% decrease in brain Aβx-42 by ELISA were observed. Gliosis and neuritic dystrophy were limited to the remaining, limited number of plaques. Vehicle controls showed no abnormal brain pathology, indicating the lack of overt inflammation due to treatment with LT. Morphological changes were absent from kidney, spleen and snout. A 28- fold increase in serum Aβ (total) protein levels was observed in Aβ immunized PSAPP mice compared to serum Aβ in control PSAPP mice; most of the Aβ in serum of Aβ immunized mice was complexed to antibodies. We conclude that prime/boost Aβ protocols with adjuvant lead to increased Aβ antibody titers and that the anti-Aβ antibodies may stabilize Aβ in the serum or clear it from the brain.