Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice

Abstract

We investigated the therapeutic effects of two different versions of Abeta(1-15 (16)) liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetra-palmitoylated amyloid 1-15 peptide (palmAbeta(1-15)), or with amyloid 1-16 peptide (PEG-Abeta(1-16)) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmAbeta(1-15) liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-Abeta(1-16) had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmAbeta(1-15), whereas those elicited by PEG-Abeta(1-16) were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly beta-sheet conformation of palmAbeta(1-15) and random coil of PEG-Abeta(1-16). We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a "conformational" disease, implying that antibodies against amyloid sequences in the beta-sheet conformation are preferred as potential therapeutic agents.

Fig. 1.

Design and biophysical characterization of the two liposomal vaccines containing peptide immunogens with the first 16 (ACI-01, Aβ1–16) and 15 (ACI-24, Aβ1–15) amino acids of the full length Aβ1–42 peptide. (A) ACI-01 contains Aβ1–16 flanked with one PEGylated lysine residue on each side that utilizes DSPE as liposomal anchor of the PEG chain. For ACI-24, two terminal palmitoylated lysine residues were covalently linked at each end of Aβ1–15 to reconstitute and anchor the antigen into the liposome. (B) Sequence of the peptides integrated into the liposomal vaccines ACI-01 and ACI-24. (C) CD spectra of ACI-01 at 10 and 20 μM peptide concentration (Upper) and ACI-24 at 20 μM (Lower).

Fig. 2.

ELISA analysis of anti-β-amyloid antibody response after immunization with PEGylated (ACI-01) or palmitoylated (ACI-24) liposomal antigens. (A) Analysis of amyloid-specific titers [IgG, (Left) and IgM (Right)] in the sera of APPxPS1 mice immunized (arrows) with PEGylated (ACI-01) or palmitoylated (ACI-24) liposomal antigens compared with mice immunized with empty liposomes (control). (B) IgG isotype analysis after 9-week treatment of ACI-01 or ACI-24. (C) Epitope-mapping of serum antibodies generated by ACI-01 or ACI-24 taken 1 week after the final boost. Results are shown as means ± SEM obtained in groups of 7–8 immunized mice.

Fig. 3.

ELISA analysis of Aβ1–42 fiber-specific antibody titers in the sera of APPxPS1 mice immunized with PEGylated (ACI-01) or palmitoylated (ACI-24) antigens in liposomes compared with mice immunized with empty liposomes (control). Data are expressed in mean ± SEM and ∗, P < 0.05 and ∗∗ with P < 0.01 by ANOVA (Turkey–Kramer multiple comparison test obtained in groups of 7–8 immunized mice).

Fig. 4.

Effect of immunization of APPxPS-1 mice with ACI-01 and ACI-24 on memory capacity and brain amyloid load. (A) Analysis of cognition assessed by ORT of 6-month-old APPxPS-1 mice immunized with PEGylated (ACI-01) and palmitoylated (ACI-24) liposomal vaccines. Data are expressed in mean ± SEM in groups of 5–8 mice. (B) Analysis of individual correlation (nonlinear regression, order of three) of anti Aβ1–42-specific IgG antibody titer (Left) and of anti Aβ1–42-specific IgM antibody titer (Right) with ORT Index (ORT individual − ORT mean of control). (C) Analysis of soluble and insoluble Aβ1–40 and Aβ1–42 of brain homogenates of ACI-24 immunized APPxPS-1 mice compared with empty liposome-immunized control group by Aβ1–40- and Aβ1–42-specific ELISA. Data are percent means ± SD of the values of 7–8 mice. ∗, P < 0.05; ∗∗, P < 0.01 by ANOVA (Turkey–Kramer multiple-comparison test).