Vaccines against amyloid-beta-peptide (Aβ) have been widely investigated as a potential immunotherapeutic approach for Alzheimer´s disease (AD) during the past years. However, previous vaccines tested in mice and humans have failed to address the major hallmarks of AD, such as senile plaque-like Aβ deposits, plaque-associated dystrophic neurites and glial proinflammatory cytokines, due to massive T-cell activation that resulted in a meningoencephalitis-like reaction. We have recently developed a vaccine (EB101), based on passive immunotherapy, that delivered Aβ1-42 in a novel immunogen-adjuvant consisting of sphingosine-1-phosphate (S1P)-containing niosomes, to APP/PS1 transgenic mice before and after detectable AD-like pathological effects. Quantitative analysis of amyloid burden, performed in the affected brain regions, showed a notable decrease in the mean area of Aβ plaques when EB101 was compared with other treatments, demonstrating that S1P plays a key role as a regenerative-neuroprotective agent during the development and consolidation periods of AD neuropathology. In the present study, we screened the efficacy of each component of our immunogenic vaccine EB101 in the APP/PS1 transgenic mouse model, in order to characterize the positive effect of S1P in the immunotherapeutic response. Complementary results demonstrated that EB101 vaccine significantly prevents and reverses the AD neuropathology hallmarks by clearing Aβ plaques, reducing dystrophic plaque neurites and minimizing neuroinflammation, while markedly reducing neuronal degeneration and thus prolonging lifespan in the APP/PS1 transgenic mouse model, paving the way for future relevant clinical trials.

Keywords: Alzheimer’s disease, Amyloid beta peptide, APP/PS1 transgenic mice, Immunotherapy, Vaccine

Abbreviations: AD: Alzheimer’s Disease; Aβ: β-Amyloid Protein; APP: Amyloid Precursor Protein; CA1: Field CA1 Hippocampus; CA3: Field CA3 Hippocampus; Cx: Cortex; DG: Dentate Gyrus; Ent: Entorhinal Cortex; GrDG: Granular Dentate Gyrus; IL: Interleukin; MODG: Molecular Dentate Gyrus; PoDG: Polymorph Dentate Gyrus; Tg: Transgenic; Th: T Helper.

Immunization procedures: 44 APPswe/PS1dE9 transgenic mice were inoculated intraperitoneally with 100 μl per injection of EB101 (group A), only liposome complex EB102 (group B) or PBS (group C), during seven months (9 injections) for each treatment period. The immunization protocol was systematically used in previous similar studies [4,5,6] and was followed strictly, with 3 injections every two weeks in the first month, and then one injection each following month. This immunization regimen was followed in the preventive and therapeutical periods.

Immunohistochemistry and imaging: Immunohistochemical analysis and imaging were prepared as previously reported [5,6].

Statistical analyses. All statistical parameters were performed with the use of SPSS (version 11.0; SPSS Inc, Chicago) and a P value <0.05 indicated statistical significance. The average range of Aβ plaque density and burden area were analyzed by using a two-factor repeated-measures analysis of variance (ANOVA) followed by a post hoc analysis when relevant. All data were expressed as the mean ± SEM.

EB101 vaccine prevents and reverse AD neuropathology as we recently demonstrated, by the marked reduction of Aβ plaques, plaque-associated dystrophic neurites and astrocytosis in affected mouse brain areas. Next, we conducted an EB101 component screening study in order to elucidate the effective role of each in the prevention of Aβ deposits in the APP/PS1 mouse brain. Aβ plaques and vascular amyloid loads were detected, counted and measured as the percentage of total surface stained by monoclonal anti-Aβ antibody in the hippocampal and cortical sections. At the first experimental phase (preventive treatment), the amyloid plaque load observed in the representative photomicrographs (Figure 2) showed a significant difference across the four experimental groups (ANOVA, F=3.15, P<0.001).

In this experiment we observed that EB101-immunized mice (Group A, Figure 2A) showed a significantly reduced hippocampal load when compared with the different vaccine components (Groups B-C, Figure 2B, 2C). In fact, the Aβ

load in these different vaccine component groups (B-C) was not significantly different from PBS-treated mice (Group D, Figure 2D), although there was a correlation between the presence of S1P+Aβ in treatment regime and the total Aβ burden. The total Aβ plaque density and size quantification also supported this data, being performed by image analysis software of anti-Aβ antibody-stained sections in the hippocampus (Figure 2A-2D). Statistical analysis of the data obtained in the present study demonstrates that the mean Aβ plaque burden (Figure 2A) of group A (25 ± 5 p/s) was significantly different from the other treated groups (63 ± 6 p/s in 2B; 65 ± 6 p/s in 2C; 68 ± 6 p/s in 2D), which represents a notable decrease in the mean percentage of Aβ plaque area of 30.1-38.2% in the EB101-immunized mouse group relative to other tested groups. Therefore, these data show that in the preventive treatment period (Figure 3), the clearance effect of the EB101 vaccine on Aβ plaques per section was about 64% (Figure 2A, Figure 3) compared to positive controls, while 10% was detected in Aβ42/Liposome without S1P (B) and 7% in Liposome with S1P-treated mice (C).

Efficacy of S1P-containing niosomes (EB101) in clearing Aβ plaque burden 

At the second experimental phase, (therapeutic treatment), we compared the effect of EB101 vaccine with different vaccine component groups in the reduction of Aβ in the brain after the plaques were established in the 35-week-old APP/PS1 transgenic mice (Figure 2E-2H). The results obtained showed that Aβ deposits were significantly reduced in the hippocampal brain sections of the EB101-treated mice (A), markedly different from the Aβ burden density observed in the different vaccine component groups (B-C).  Photomicrographs of EB101-treated mice showed a few compacted Aβ plaques located at the CA1 layer of the hippocampus (Figure 2E), while in the other experimental groups no reduction in plaque density was observed (Figure 2F-2H), numerous large compacted Aβ plaques being located in almost every layer of each brain section. The data obtained showed that the treatment groups without Aβ1-42, C (Figure 2G) and D (Figure 2H), were the section most densely populated with Aβ plaques, while a slight reduction in this hallmark was observed in mouse group B (Figure 2F). As observed in the preventive treatment, the mean burden of Aβ plaques in group A (35 ± 4; Figure 3) was significantly different from the other treated groups (65 ± 5 in B; 70 ± 5 in C; 80 ± 6 in D), while the Aβ plaque area in the EB101-immunized mouse group was reduced by 29.7-34.5%, relative to the other groups tested (Figure 3). In both preventive and therapeutic treatments, a reduced Aβ plaque staining intensity was detected in the EB101-immunized mouse group, as shown in the photomicrographs of figure 2A,2E, contrasting with the intense Aβ-immunoreactivity of Aβ plaques in the other experimental groups.

Immune response effects of S1P-containing niosomes (EB101) in APP/PS1 mouse models

In order to analyze astrocyte activation in the hippocampal regions of APP/PS1 mice, which is known to be implicated in neuroinflammation, amyloidogenesis and neuronal cell death in AD, we used immunohistochemical analysis to quantify and compare the GFAP-reactive cells in the affected regions after each treatment period (Figure 2I-P). At the end of the preventive treatment (Figure 2I-L), EB101 significantly reduced the density of GFAP-reactive cell clusters in the hippocampal and cortical sections as compared with other treatments, such as Aβ/Lip without S1P (B), Lip with S1P (C) and PBS (D). Only a few scattered GFAP-reactive clusters, mainly at the CA1 hippocampal layers, were observed in the transverse section of the mouse brains treated with EB101 (Figure 2I), markedly contrasting with the numerous dystrophic reactive astrocytes observed in different hippocampal areas of mouse brains in groups B-D (Figure 2J-2L). After therapeutic treatment, mouse brains treated with EB101 were similar to those of control mice, mostly devoid of reactive GFAP clusters, except a few scattered ones in the CA1 hippocampal layers (Figure 2M). In contrast, there was a moderate density in group B (Figure 2N) and extensive density in group C (Figure 2O), and group D mice (Figure 2P). No astrocytosis was observed in control mice of each group during preventive or therapeutic treatment periods.

DISCUSSION

Liposome-based vaccines have demonstrated great effective potential in preventing and treating AD [7]. Based on previous effectiveness of active immunotherapy in reducing Aβ plaque accumulation [8], we have developed a new vaccine cocktail to circumvent the previous vaccine failures, due to an extensive T-cell-mediated immune response [9,10], by judiciously selecting an adjuvant that addresses all the above targets while avoiding the massive autoimmune activation of T-cells. This novel immunogen-adjuvant configuration consists of a physiological matrix, niosomes composed of naturally-occurring phospholipids (phosphatidylglycerol, phosphatidylcholine, and cholesterol), that has proven to be safe and efficacious in other types of vaccines. In order to increase the immunogenic potential we added a biologically active sphingolipid, sphingosine-1-phosphate (S1P) to this phospholipidic configuration. S1P is a phosphorylated product of sphingosine, catalyzed by sphingosine kinase, which has been reported to be an important lipid mediator acting both inside and outside the cell membrane [11,12]. Due to its role in the regulation of neuronal function, S1P facilitates glutamate secretion in hippocampal neurons by secretion actions, being involved in regulatory mechanisms of synaptic transmission. In particular, S1P itself causes glutamate secretion from presynaptic sites and potentiates glutamate-induced transmitter secretion in primary hippocampal neurons [13], possibly facilitating the formation of a positive activation cycle in excitatory neurons such as glutaminergic neurons. As extracellular effects, S1P binds to members of GTP-binding protein (G-protein)-coupled S1P receptor family (S1P1-5), triggering active immunity, angiogenesis, cell motility, and neurite growth [14,15]. As intracellular effects, S1P modulates cellular calcium mobilization, cell growth, and the inhibition of apoptosis [16,17].

Recently, the potential effect of S1P in neuronal regeneration was demonstrated in detail, since intracellular S1P enhances nerve growth factor-induced excitability in the sensory neurons of rats [18,19], controls migration of neuronal stem cells toward a site of spinal cord injury [20], and induces cytoskeletal rearrangements through small G-protein Rac activation [21], which seems to facilitate synaptic vesicle fusion to plasma membranes, enhancing transmitter secretion. Taking advantage of its important role in neuronal regeneration, cell growth, suppression of apoptosis and glutamate secretion from presynaptic sites that are affected in AD neuropathology, we incorporated the S1P into the phospholipid liposome to form a phospholipid-S1P-liposome matrix used as an immunogen-adjuvant to deliver the active antigen, Aβ1-42 [5]. Thus, this combination added a regenerative and anti-inflammatory component to the vaccine, key elements to increase neuronal activity and prevent inflammation in the brains of APPswe/PS1dE9 transgenic mice, improving the immunotherapeutic results previously reported by the studies based on Aβ-immunization [22,23], while minimizing potential side effects.

EB101 vaccine was generated by using the hydration-rehydration method to efectively combine phospholipids, S1P and liposomes, forming niosomes against Aβ1-42 oligomers. This was used in other vaccines for an efficient liposomal-protein configuration, overcoming the immune pathological response encountered when using other types of adjuvants such as Freund’s adjuvant [24,25], Quil-A [26], QS-21 and the detergent polysorbate 80 to solubilize Aβ, which are believed to induce a proinflammatory Th1 response [10,27]. Over the last decade, Aβ immunotherapy studies in APP-tg mice have reported different Aβ antibody levels, depending on the mouse model used, the immunization methodology, and the type of adjuvant for the vaccine formulation, emphasizing the significant impact of the adjuvants on the immune response evoked [28]. Considering this, we strategically planned a novel adjuvant-liposomal vaccine formulation in which the phospholipid-S1P-liposomes represent the pivotal structure that provides an inmunotherapeutic response markedly different from previous vaccine formulations tested in AD-like mice. Moreover, the significant effect of EB101, obtained in mice after the establishment of Aβ plaques in the hippocampus/neocortex and subsequent neuropathological changes, shows that EB101 vaccine presents a tremendous potential as a therapeutic agent to reverse neuropathological hallmarks in the AD-like brain, which represent an effective immunotherapeutic approach [29,5,6]. The results obtained in the preventive and therapeutic treatments showed that EB101 vaccine is efficacious not only in preventing the development of AD-like pathology but also in reducing it once established. The APPswe/PS1dE9 transgenic mice showed tiny and compact nonfibrillary amyloid plaque accumulation in the initial deposition period mainly at the hippocampus/neocortex, consistent with previous reported studies [30,31], whereas at later stages sparse Aβ-fibrillar plaques are progressively observed also at nearby cortical areas. After the preventive immunization period with EB101 vaccine, a significant reduction in density of Aβ plaque burden was observed in APP/PS1 transgenic mouse regions, as well as reduced burden areas when compared with APP/PS1 mice treated with different EB101 vaccine components or PBS. Schenk and colleagues [4] reported similar preventive immunization results in PDAPP mice, although differences in immunization efficiency and vaccine conformation have been improved in the present study. During this preventive period, EB101 immunization also prevented the progressive development of other AD pathological effects such as dystrophic plaque neurites, astrocytosis and motor coordination deficits. The hindrance of massive glial activation in response to early development of Aβ deposits has been accepted by the scientific community as the main key in efficient preventive immunization [32-35], this being the case of our EB101 vaccine, which has demonstrated, when compared with different EB101 vaccine components or PBS-treated mouse groups, that it prevents the development of astrocyte activation. Since the activation of astrocytes and microglia has been reported to induce the degradation of Aβ [36,37], the near-absence of Aβ-related astrocytosis in cortex and hippocampus of mice treated with EB101 vaccine confirmed the preventive effect against the development of AD-like pathology in this mouse model.

ACKNOWLEDGEMENTS

This work was supported by the III Research Grant Program of the Camilo José Cela University 2014-15 (TransBioModel – 2014/36).

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