A comparative evaluation of a novel vaccine in APP/PS1 mouse models of Alzheimer's disease

Abstract

Immunization against amyloid-beta-peptide (Aβ) has been widely investigated as a potential immunotherapeutic approach for Alzheimer's disease (AD). With the aim of developing an active immunogenic vaccine without need of coadjuvant modification for human trials and therefore avoiding such side effects, we designed the Aβ 1-42 vaccine (EB101), delivered in a liposomal matrix, that based on our previous studies significantly prevents and reverses the AD neuropathology, clearing Aβ plaques while markedly reducing neuronal degeneration, behavioral deficits, and minimizing neuroinflammation in APP/PS1 transgenic mice. Here, the efficacy of our immunogenic vaccine EB101 was compared with the original immunization vaccine cocktail Aβ 42 + CFA/IFA (Freund's adjuvant), in order to characterize the effect of sphingosine-1-phosphate (S1P) in the immunotherapeutic response. Quantitative analysis of amyloid burden showed a notable decrease in the neuroinflammation reaction against Aβ plaques when S1P was compared with other treatments, suggesting that S1P plays a key role as a neuroprotective agent. Moreover, EB101 immunized mice presented a protective immunogenic reaction resulting in the increase of Aβ-specific antibody response and decrease of reactive glia in the affected brain areas, leading to a Th2 immunological reaction.

Figure 1

Biophysical characterization of the EB101 vaccine and experimental design. Biophysical structure of Aβ ₄₂/S1P-containing liposome (EB101) and schedule of experiments.

Figure 2

Comparative effect of EB101 vaccine on beta amyloid deposits. Comparative photomicrographs of Aβ immunoreactivity were taken in the hippocampus ((a)–(e), (k)–(o)) and cortical ((f)–(j), (p)–(t)) brain regions of transgenic mice with different treatments before Aβ plaques development, preventive treatment ((a)–(j)) and after the Aβ plaques developed, therapeutic treatment ((k)–(t)). Preventive treatment: transverse brain sections of 11-month-old mice show reduced number of Aβ deposits in the dentate gyrus, hippocampal subregion CA1 (a), and entorhinal cortex (f) following EB101 vaccine immunization (Group A), contrasting with the numerous Aβ immunoreactive plaques in the correspondent mouse brain sections of Groups B ((b), (g)), C ((c), (h)), D ((d), (i)), and E ((e), (j)). Immunoreactive Aβ in the brain sections of wild-type (WT) mice is absent (inserts in panels (e), (o)). Therapeutic treatment: transverse brain sections of 18-month-old mice are shown in panels (k)–(t). Treatment with EB101 almost completely cleared Aβ load in the dentate gyrus and reduced notably the density in hippocampal subregion CA1 (k) and entorhinal cortex (p) compared to the same areas of mouse Groups B ((l), (q)), C ((m),(r)), D ((n), (s)), and E ((o), (t)), as determined by number of plaques and staining intensity area. Scale bar: 100 μm.

Figure 3

Comparative analysis of Aβ plaque burden in the brains of APP/PS1 mice. Mean of Aβ plaque burden in the hippocampal and cortical regions of APPswe/PS1ΔE9 mice in the five treatment groups. The mean of the Aβ burden is significantly reduced (P < 0.05) in Group A (EB101) when compared with Groups B–E during preventive treatment and markedly reduced in the therapeutic treatment period. Data are presented as mean ± standard error of the mean (SEM). Density mean of Aβ plaques in Tg mice hippocampus.

Figure 4

Comparative quantification of Aβ burden area in the brain of APP/PS1 mice. Quantitative analysis of Aβ burden area in the hippocampal and cortical regions of APPswe/PS1ΔE9 mice treated with EB101, Aβ/liposomes, S1P/liposomes, Aβ ₄₂ + FA/IFA, and PBS, represented by the number of pixels inside the stained area of each Aβ plaque. This graphic shows that Aβ plaques of EB101-treated mice (Group A) are significantly smaller in size (P < 0.05) than those of the other four treatment groups. Data are presented as mean ± SEM. Mean of Aβ plaques area in Tg mice hippocampus.

Figure 5

Comparative effect of EB101 vaccine on astrocytosis. Comparative photomicrographs of GFAP immunoreactivity in the hippocampus ((a)–(e), (k)–(o)) and cortical ((f)–(j), (p)–(t)) brain regions at the preventive treatment phase ((a)–(j)) and therapeutic treatment group ((k)–(t)). Preventive treatment: transverse brain sections of 11-month-old mice treated with EB101 show an almost complete prevention of astrocytosis in the detailed sections of the dentate gyrus (a), contrasting with numerous dystrophic reactive astrocytes, typical of an inflammatory reaction, observed in different hippocampal corresponding areas of mouse brains in Group B (Aβ/Lip-treated mice; (b)), Group C (Lip/S1P-treated mice; (c)), Group D (Aβ ₄₂ + CFA/IFA-treated mice; (d)), and Group E (PBS-treated mice, (e)). Immunoreactive astrocytosis in the brain sections of WT mice is absent (data not shown). Detailed sections of the parietal cortex of treated mice show a gradient density of reactive astrocytes immunoreactive to GFAP, indicating a neuroinflammation pattern. Comparative cortical sections show numerous astrocytosis areas in mice Groups C and E ((h), (j)) and wide reactive astrocytes clusters in mice Groups B and D ((g), (i)), while just a few of them are observed in Group A (f). Therapeutic treatment: transverse brain sections of 18-month-old mice of Group A show a notable reduction of astrocytosis in the hippocampal region (k) after EB101 vaccine immunization. Note the astrocytosis contrast density between EB101-treated mice and the other treated groups ((l)–(o)), where numerous immunoreactive GFAP clusters are observed in the correspondent brain sections. A few immunoreactive GFAP clusters are observed in the parietal cortex of EB101 treated mice group, while a moderate ((q), (s)) to high ((r), (t)) density of these neuropathological inflammation clusters is apparent in the other treatment groups (Groups B–E). For abbreviations, see list. Scale bar: 100 μm.

Figure 6

Quantification of specific anti-β-amyloid IgG in immunized APP/PS1 mice. The presence of anti-β-amyloid antibodies measured in the sera of vaccinated and control mice groups during the preventive and therapeutic treatments indicates that EB101 liposome vaccine was able to induce the highest production of specific IgG antibodies. Compared to the Group D immunized mice, the level of antibodies observed in the Group A was also higher, suggesting a more effective immune response, (P < 0.01 versus Group D; P < 0.001 versus Groups B, C, and E). Quantification of specific anti-β-amyloid IgG in immunized Tg mice.

Figure 7

Detection of Th1 and Th2 cytokine profile. Measurements of the effect of EB101 and Aβ ₄₂ + CFA/IFA vaccines on Th1 and Th2 cytokines profile during preventive and therapeutic treatments. ((a)–(c)) A significant increase of Th1 cytokine profile was observed in preventive treatment in Group D with respect to all other groups, while a tendency to a reduction in Th1 secretion was observed in Group A with respect to all other groups in both preventive and therapeutic treatment. ((d)–(g)) The changes in Th2 protein levels were observed in the IL-4, IL-5, and IL-10 cytokine secretions of Group A (P < 0.05 versus Group D; P < 0.01 versus Group E).

Figure 8

Analysis of motor coordination and balance. (a) Rotarod results of motor coordination and balance test after the preventive and therapeutic treatment showing improved effect of EB101 and Aβ ₄₂ + CFA/IFA-vaccine compared to other treated mice groups (P < 0.05) and (P < 0.02) versus control mice. Latency to fall on the rotarod apparatus was significantly improved in EB101 immunized mice. Values represent means of all six trials of a group/day. (b) Spatial learning and memory tested by fear conditioning-active avoidance test. The mean response time of each treatment group to reach the platform is represented. EB101 and Aβ ₄₂ + CFA/IFA immunized mice had significantly lower latencies than the other treatment groups (P < 0.01) versus control mice.