JCI - Nasal vaccination with a proteosome-based adjuvant and glatiramer acetate clears β-amyloid in a mouse model of Alzheimer disease

Induction of EAE in APP-Tg mice results in a decrease of A β levels in the brain. Based on the finding that some AD patients immunized with Aβ developed meningoencephalitis (19), which in some respects resembled EAE, we wished to determine whether Aβ-producing mice were more susceptible to EAE than their non-Tg littermates. We immunized aged APP-Tg mice with robust amyloid pathology (J20 mice) (24) with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 subcutaneously in CFA plus pertussis toxin (PT). As controls, APP-Tg mice were immunized with BSA or synthetic human Aβ 1–40. We found that EAE developed to an identical degree in APP-Tg animals and non-Tg littermates (data not shown), and no EAE was observed in APP-Tg mice immunized with Aβ 1–40 or BSA.

Unexpectedly, when we examined the brains neuropathologically, we discovered that there was markedly less Aβ deposition in mice that had developed EAE. We quantified the amount of staining for Aβ fibrils in the hippocampus using thioflavin S staining and found a 92% reduction in the MOG-immunized mice versus controls (P < 0.001) and a 73% reduction compared with mice immunized with Aβ 1–40 (P = 0.03; Table 1 and Figure 1). We also quantified the total brain Aβ content by ELISA and found a 94% reduction in Aβ in MOG immunized animals compared with controls (P < 0.001) and an 86% reduction compared with Aβ 1–40–immunized mice (P = 0.03; Table 1 and Figure 1). No changes were observed in animals immunized with BSA in CFA plus PT. We thus used untreated and BSA-treated animals as controls.

In order to determine whether the dramatic effect we observed was unique to MOG-induced EAE, we induced EAE with myelin proteolipid protein (PLP) peptide 139–151 in CFA plus PT. To induce EAE with PLP, we used aged APP-transgenic mice (Tg2576; average age 16 months) described by Hsiao et al. (25), which are on a B6/SJL background and thus susceptible to PLP-induced EAE. We found a 76% reduction of thioflavin S staining for Aβ fibrils (P < 0.002) and a 70% reduction in total brain Aβ levels in animals with PLP-induced EAE (P < 0.02) versus controls (Table 2). Our results with Tg2576 mice were similar to those obtained when we immunized J20 mice with MOG to induce EAE (Table 1). These results demonstrate that our observation was not related to either the antigen used for EAE induction or the animal model of AD studied.

A β levels are reduced in B cell–deficient (Ig μ–null) mice with MOG-induced EAE. In previous studies involving mouse models of AD in which animals were immunized with Aβ in CFA, anti-amyloid antibodies were shown to have a key role both in vitro (11, 12, 17) and in vivo (4–9) in reducing amyloid load. We therefore measured levels of antibodies against Aβ in mice immunized with MOG or PLP peptides to determine whether any humoral cross-reactivity to Aβ had occurred. As shown in Tables 1 and 2, we could not detect anti-Aβ antibodies in animals immunized with MOG or PLP peptides. To definitively establish that antibodies were not playing a role in the EAE-induced clearance of Aβ, we immunized aged J20 mice bred to B cell–deficient mice with MOG 35–55 in CFA. As shown in Table 1 and Figure 1, B cell–deficient APP-Tg mice showed a more than 90% reduction in amyloid load compared with untreated mice, as measured by either thioflavin S staining (P < 0.001) or ELISA for total brain Aβ (P < 0.001). These results establish that the reduction of Aβ following MOG immunization occurs by an antibody-independent mechanism.

Clearance of A β following immunization with GA. Because we were interested in the potential application of our findings to the treatment of AD in human subjects, we next addressed the question of whether we could immunize APP-Tg mice in a manner that would result in Aβ clearance without causing EAE. To answer this question, we tested the effect of immunization of APP-Tg mice with GA, which is a random amino acid copolymer of alanine, lysine, glutamic acid, and tyrosine that is effective in suppression of EAE and is an approved and widely used treatment for relapsing forms of MS (23). There is some evidence that GA may have cross-reactivity with myelin basic protein (MBP) (26), although it has never been reported to induce EAE in animals, even when given with CFA. We thus investigated whether immunization of APP-Tg mice with GA in a fashion identical to that by which we induced EAE would reduce amyloid load in APP-Tg mice without inducing clinical disease. Mice were immunized with 100 μg GA in CFA, and immediately thereafter and at 48 hours, they received an i.p. injection of 150 ng of PT. Fifty days after immunization, the brains were examined. We found that GA immunization led to a 92% reduction in thioflavin S staining for amyloid fibrils in the hippocampal region versus that in controls (P < 0.01) and a 70% reduction in total Aβ levels (P < 0.01; Table 1 and Figure 1). As expected, there was no clinical or neuropathological evidence of EAE in mice immunized with GA/CFA plus PT.

Clearance of A β by nasal vaccination of GA plus a proteosome-based adjuvant. Our laboratory has been interested in vaccination strategies for noninfectious diseases using the mucosal immune system, an approach that is less invasive than parenteral administration and that is clinically applicable (27–29). We have previously demonstrated that nasal vaccination with Aβ 1–40 peptide resulted in a 50–60% reduction in cerebral Aβ burden in APP-Tg mice (5). To investigate the effect of nasal vaccination with GA in a mouse model of AD, we treated animals with nasal GA alone or together with a mucosal adjuvant. For the latter, we used a proteosome-based mucosal adjuvant comprising purified outer membrane proteins of Neisseria meningitides and LPS (IVX-908; Protollin; ID Biomedical Corp.) that has been used safely in both humans (22) and mice (30, 31). APP-Tg mice received 4 nasal treatments the first week and then were boosted on a weekly basis for the next 5 weeks, after which neuropathological analysis was performed. As controls, APP-Tg mice were nasally treated with IVX-908 plus BSA, IVX-908 alone, or GA alone. We found that nasal administration of GA plus IVX-908 (GA+IVX-908) resulted in an 84% reduction in thioflavin S–positive fibrillar amyloid in the hippocampus compared with control (P < 0.001) and a 70% reduction compared with treatment with IVX-908 alone (P < 0.01; Table 3 and Figure 2). In terms of total brain Aβ levels, we observed a 73% reduction following nasal administration of GA+IVX-908 (P < 0.001) compared with control and a 45% reduction compared with treatment with IVX-908 alone (P < 0.002; Table 3 and Figure 2). Nasal administration of GA alone did not affect Aβ fibrils or total Aβ levels in the brain (Table 3 and Figure 2). Nasal administration of BSA plus IVX-908 or IVX-908 alone resulted in an approximately 50% reduction in total Aβ levels (P < 0.02 vs. control), although there was no effect on fibrillar Aβ staining. None of the animals developed clinical EAE.

Of note, there can be variability in the amount of amyloid deposition in the J20 strain depending on the age of the animal, especially in animals between the ages of 4 and 10 months (24). Nonetheless, we studied animals of the J20 strain that were older than 11 months of age and did not observe substantial increases in Aβ deposition linked to age in the cohort we studied (Aβ deposition values as determined both by ELISA and thioflavin S staining for individual mice in each treatment group and controls are presented in Supplemental Tables 1–3; supplemental material available online with this article; doi:10.1172/JCI23241DS1). Our experience is consistent with the reports of other investigators who did not find a large increase in Aβ levels in animals between the ages of 12 and 15 months (32–34). Also, when we exactly age-matched our control group (13.3 months) with MOG-EAE animals and animals treated nasally with GA+IVX-908, we found a significant effect of treatment on Aβ levels measured by 2 methods (P < 0.001; Supplemental Table 4). To definitively rule out an effect of age or sex and to test the effect of chronic administration of GA+IVX-908 given at an early stage of amyloid deposition, we tested age- and sex-matched littermates that received nasal treatment with GA+IVX-908 versus controls beginning at age 5 months. As shown in Figure 3, Table 4, and Supplemental Table 5, we found a highly significant reduction in the level of amyloid fibril (83%) in the brains of GA+IVX-908–treated mice versus control animals (P < 0.0001). Thus, the marked decrease in total Aβ load we observed was related to treatment, not to the age or sex of the animals, and also occurred when treatment was begun at an earlier stage of amyloid deposition.

Activated microglia colocalize with A β fibrils and correlate with A β clearance. To investigate the potential mechanisms by which Aβ reduction occurred, we performed immunohistochemical analyses to assess microglial activation, T cell infiltration, and cytokine patterns in the brains of the immunized mice. We also investigated the types of cells in the hippocampus and their correlation with Aβ fibrils.

Previous studies have suggested that activated microglia may play an important role in clearing Aβ in vivo (9, 20, 35). To investigate the potential role of microglial activation in the Aβ clearance we observed after subcutaneous or nasal immunization of APP-Tg mice, we stained the brains of mice with CD11b, a marker of activated microglia. As shown in Figures 4 and 5 and Table 5, immunostaining of the hippocampus revealed increased numbers of activated microglia following MOG immunization in APP-Tg mice compared with control mice (P < 0.02). The activated microglia we observed tended to colocalize with Aβ plaques. Furthermore, increased levels of microglia activation were also observed in B cell–deficient mice (P < 0.001) (Table 5). In addition, we found activated microglia surrounding the amyloid plaques following GA immunization (Figure 4).

We then investigated whether the microglial activation we observed in immunized APP-Tg mice was also observed in wild-type (non-Tg) mice immunized with GA or IVX-908. We found no microglial activation in non-Tg littermates in any of the immunization protocols: parenterally with CFA/PT plus GA, nasally with GA+IVX-908 (see Figure 4B), or nasally with IVX-908 alone. These results suggest that the activation of microglia by GA or IVX-908 immunization was dependent on the presence of amyloid deposition, which primed endogenous microglia for further activation.

Although there is no known cross-reactivity between GA and Aβ and we did not observe anti-Aβ antibodies in either GA-treated or EAE animals, it is possible that immunization with GA+ IVX-908 could have resulted in priming of Aβ-reactive T cells. We measured T cell proliferative responses and production of cytokines (IL-2, IFN-γ, IL-6) after 6 weeks of weekly treatment with GA+IVX-908 (at which time the experiment was terminated) by stimulating splenic T cells with Aβ 1–40. We found no priming of Aβ reactive T cells as measured by proliferation: counts per minute in response to Aβ for untreated, 3,315 ± 1,682; for GA+IVX-908, 4,566 ± 1,412 (background counts were 100–300 cpm). The stimulation index (GA+IVX-908–treated/untreated) was 1.37; a minimal stimulation index of greater than 2.5 is considered positive. Furthermore, we did not find secretion of IL-2, IFN-γ, or IL-6 above background in these cultures. This lack of T cell response to Aβ is consistent with the fact that we did not detect anti-Aβ antibodies, as T cell help is required for production of antibodies. Similarly, we did not find priming in response to Aβ in EAE animals. We also examined the immune response to GA in the spleen 10 days after nasal GA+IVX-908 treatment and found proliferative responses to GA (stimulation index, 6.4; 11,440 ± 1,171 cpm) plus secretion of IFN-γ (4,990 pg/ml), IL-10 (156 pg/ml), and IL-2 (966 pg/ml).

To examine the effect of GA+IVX-908 treatment on other brain sites besides the hippocampus, we investigated the olfactory bulb and the cerebellum. We stained the olfactory bulb for Aβ, CD11b, and fibrinogen and obtained results similar to those observed in the hippocampus (Figure 6). Following nasal administration of GA+IVX-908, we found an increased number of activated microglia compared with control. The activation also occurred in animals with EAE, but was associated with leakage in the blood-brain barrier (BBB), as measured by staining for fibrinogen (Figure 7).

When we examined the cerebellum, we did not find increased activation of microglial staining in GA+IVX-908–treated animals, which suggests that the increased activation is restricted to areas with Aβ deposition. Furthermore, no activation of microglia was observed anywhere in the brains of non-Tg littermates following GA+IVX-908 treatment, which further demonstrates that the increased activation is restricted to areas with Aβ deposition (see Figure 4B).

To better understand the mechanism of the clearance we observed, we stained for CD68, which is highly expressed on activated macrophages from the periphery as opposed to brain microglia. As shown in Figure 8, we obtained higher staining for CD68 in animals with EAE compared with those treated with GA+IVX-908. This pattern of staining shows the migration of macrophages from the choroid plexus to the surrounding brain parenchyma including the cerebellum and cortex. With GA+IVX-908 treatment, there was increased expression of CD68, primarily in the choroid plexus space. This suggests that the CD11b⁺ cells responsible for clearance of Aβ in animals with EAE migrate to the CNS from the periphery and are associated with neuronal toxicity, whereas the CD11b⁺ cells in GA+IVX-908–treated animals are primarily endogenous microglial cells and are associated with clearance of Aβ without evidence of direct toxicity. As further support for this interpretation, we found that there was increased expression of CD68⁺ cells in the cerebellum of animals with EAE but not GA+IVX-908–treated or untreated animals (data not shown). Moreover, activated CD11b⁺ cells following GA+IVX-908 treatment were only found in regions where there was accumulation of amyloid.

As shown in Table 5 and Figure 5, the reduction of Aβ fibrils in the hippocampus was strongly correlated with increased numbers of both activated microglial cells, as shown by CD11b staining (r = –0.7, CD11b vs. Aβ fibrils), and IFN-γ–secreting cells (r = –0.8, IFN-γ vs. Aβ fibrils). There was a strong correlation between CD11b cells and IFN-γ–secreting cells (r = 0.9). In addition, as with CD11b and IFN-γ cells, we observed increased numbers of microglia immunoreactive for M-CSF receptor (M-CSFR) in treated animals compared with controls (P < 0.02; Table 5). We observed a reduction in TGF-β expression in MOG- (P < 0.02) and GA+IVX-908–treated mice (P < 0.001) compared with controls (Table 5). Furthermore, there was a strong correlation between reduction in TGF-β expression and the percentage of Aβ fibril in the hippocampus region (r = 0.91). No significant changes were observed in levels of IL-10–immunoreactive cells between control and GA+IVX-908–treated animals, although animals with EAE had lower levels of IL-10 than controls.

Reduction in astrocytosis and neurotoxicity following nasal vaccination with GA+IVX-908. In order to investigate toxicity and potential negative effects of therapy after 6 weeks of treatment, we examined the following: (a) glial fibrillary acidic protein (GFAP), a marker of astrocytosis that occurs in response to neuronal damage; (b) SMI32, a marker for phosphorylation of neurofilaments, which increases with neuronal damage; (c) TUNEL, a marker of the apoptotic death cascade; (d) iNOS, an enzyme that is upregulated when there is stress to neuronal cells.

Astrocytosis (determined as the area of activated astrocytes as indicated by the presence of GFAP⁺ cells) occurred in untreated control animals (Figure 9). Astrocytosis was reduced when GA+IVX-908 was given nasally (3.1%; P = 0.039 vs. control). It was not reduced in EAE animals (even though there was clearance of Aβ in EAE animals). Thus, clearance of Aβ by GA+IVX-908 is associated with reduced astrocytosis.

SMI32-positive cells associated with neuritic plaques that have an abnormal ovoid morphology were observed in control animals (Figure 10). In EAE mice, although there were no neuritic plaques, there was increased SMI32 cells with abnormal ovoid morphology throughout the brain in association with inflammation. In GA+IVX-908–treated animals, there was a reduction in SMI32-positive cells with abnormal ovoid morphology in neuritic plaques. Thus, GA+IVX 908 treatment is not associated with toxicity as measured by SMI32.

We found no TUNEL staining in control animals and increased TUNEL staining in the cortex of EAE animals (Figure 10). No TUNEL staining was observed in GA+IVX-908–treated animals. A staining pattern similar to that of TUNEL was observed with iNOS, viz, upregulation in EAE mice and no upregulation in GA+IVX-908–treated animals. Furthermore, we examined BBB integrity by staining for the serum protein fibrinogen (36). We found no damage to the integrity of the BBB in GA+IVX-908–treated mice in either the hippocampus or the olfactory bulb (Figures 6 and 7). As would be expected, BBB breakdown was observed in EAE mice. The GA+IVX-908–treated animals exhibited no changes in the behavior, as measured by body weight, eating habits, tail tone, or mobility, that indicated toxicity. Such changes were, however, observed in EAE animals. GA+IVX-980 treatment did not lead to mortality in any of the animals studied. Of note, in an ongoing, long-term experiment, we have observed no behavioral changes in animals that have been treated weekly with GA+IVX-908 for 8 months as described in Figure 3.