MER5101, a novel Aβ1-15:DT conjugate vaccine, generates a robust anti-Aβ antibody response and attenuates Aβ pathology and cognitive deficits in APPswe/PS1ΔE9 transgenic mice

Abstract

Active amyloid-β (Aβ) immunotherapy is under investigation to prevent or treat early Alzheimer's disease (AD). In 2002, a Phase II clinical trial (AN1792) was halted due to meningoencephalitis in ∼6% of the AD patients, possibly caused by a T-cell-mediated immunological response. Thus, generating a vaccine that safely generates high anti-Aβ antibody levels in the elderly is required. In this study, MER5101, a novel conjugate of Aβ1-15 peptide (a B-cell epitope fragment) conjugated to an immunogenic carrier protein, diphtheria toxoid (DT), and formulated in a nanoparticular emulsion-based adjuvant, was administered to 10-month-old APPswe/PS1ΔE9 transgenic (Tg) and wild-type (Wt) mice. High anti-Aβ antibody levels were observed in both vaccinated APPswe/PS1ΔE9 Tg and Wt mice. Antibody isotypes were mainly IgG1 and IgG2b, suggesting a Th2-biased response. Restimulation of splenocytes with the Aβ1-15:DT conjugate resulted in a strong proliferative response, whereas proliferation was absent after restimulation with Aβ1-15 or Aβ1-40/42 peptides, indicating a cellular immune response against DT while avoiding an Aβ-specific T-cell response. Moreover, significant reductions in cerebral Aβ plaque burden, accompanied by attenuated microglial activation and increased synaptic density, were observed in MER5101-vaccinated APPswe/PS1ΔE9 Tg mice compared with Tg adjuvant controls. Last, MER5101-immunized APPswe/PS1ΔE9 Tg mice showed improvement of cognitive deficits in both contextual fear conditioning and the Morris water maze. Our novel, highly immunogenic Aβ conjugate vaccine, MER5101, shows promise for improving Aβ vaccine safety and efficacy and therefore, may be useful for preventing and/or treating early AD.

Figure 1.

Plasma anti-Aβ antibody levels with and without low pH dissociation, and binding to human Aβ. A, Immunization with Aβ1-15:DT conjugate generated detectable plasma anti-Aβ antibody levels in both Wt and APPswe/PS1ΔE9 Tg mice by day 28. B, Low pH dissociation of antibodies and antigens in APPswe/PS1ΔE9 Tg mouse plasma resulted in enhanced detection of anti-Aβ antibodies at days 96 and 121 relative to levels detected before dissociation. C–F, Serial human AD brain sections were stained using plasma (1:10 dilution) from a MER5101-vaccinated APPswe/PS1ΔE9 Tg mouse (C), a Tg vehicle-control mouse (D), a MER5101-vaccinated Wt mouse (E), or a Wt vehicle-control mouse (F). Both vaccinated Wt and Tg mouse plasma, but not that of the vehicle-treated mice, immunolabeled Aβ plaques. G, A human AD brain section was stained using an anti-human Aβ monoclonal antibody, 6E10, as a positive control. H, Both monomeric and oligomeric Aβ in 7PA2 condition media were recognized by plasma antibodies from MER5101-vaccinated Wt and APPswe/PS1ΔE9 Tg mice but not from vehicle controls; 6E10 was used as a positive control. CHO media was used as negative control for all groups in the IP assay.

Figure 2.

Ig isotype and epitope mapping of plasma anti-Aβ antibodies. Ig isotyping of IgG1, IgG2a, IgG2b, and IgM anti-Aβ antibodies was measured by isotype-specific ELISA of plasma collected on day 121, the final bleed. A, IgG1 anti-Aβ antibodies were predominant in both vaccinated groups, followed by IgG2b anti-Aβ antibodies. Few IgG2a or IgM anti-Aβ antibodies were detected. B–D, Diluted plasma (1:100) from an immunized Wt mouse was used in combination with different isotype-specific secondary antibodies including goat-anti-mouse IgG1 (B), IgG2a (C), and IgG2b (D), respectively, to detect Aβ plaques on formalin-fixed paraffin-embedded AD brain tissues. Plasma with mouse IgG1 secondary antibody showed the clearest and most abundant staining of Aβ plaques (B). E, Epitope-mapping of antibodies was performed by pre-incubating mouse plasma overnight with short Aβ peptide fragments before using an ELISA to measure the ability of the antibodies to bind the plate-bound immunogen. Pre-incubation with Aβ1-7, Aβ1-15, Aβ3-9, and Aβ3-13 inhibited binding to the plate-bound immunogen, indicating that the vast majority of antibodies was directed against the Aβ1-15 sequence. The optical density (OD) levels are shown relative to the absence of peptide as a negative control. Data are shown as mean ± SEM.

Figure 3.

MER5101 induced a cellular immune response specific for the conjugate but not Aβ1-15 or Aβ1-40/42 peptides. Splenocytes from vehicle-treated Wt and APPswe/PS1ΔE9 Tg mice as well as MER5101-immunized Wt and APPswe/PS1ΔE9 Tg mice were isolated, pooled within each treatment group, and cultured in the presence of 0, 0.5, 5, or 50 μg/ml Aβ1-15:DT (A), Aβ1-15 (B), or Aβ1-40/42 (C) for 72 h, after which [³H]-thymidine was added for 18 h. The CPM was determined and the SI was calculated. Splenocytes isolated from MER5101-vaccinated Wt (line with solid circle) and APPswe/PS1ΔE9 (line with solid triangle) Tg mice showed a high SI in response to restimulation with the immunizing conjugate peptide, Aβ1-15:DT, but did not proliferate upon restimulation either Aβ1-15 or Aβ1-40/42 peptides. No proliferation of splenocytes from vehicle-treated Wt (line with open circle) or APPswe/PS1ΔE9 Tg mice (line with open triangle) was observed in response to any peptides.

Figure 4.

Cerebral Aβ protein load was significantly reduced in immunized APPswe/PS1ΔE9 Tg mice. A–F, Representative staining is shown for R1282-positive plaques in HC, frontal CTX, and CB in vehicle-treated APPswe/PS1ΔE9 Tg mice (A–C) and MER5101-vaccinated Tg mice (D–F). G, Quantitative image analysis demonstrated a significant reduction in R1282-positive Aβ plaque burden after MER5101 vaccination. H–M, Representative staining is shown for Thio S-positive fibrillar plaques in HC, frontal CTX, and CB in both vehicle-treated Tg (H–J) and MER5101-vaccinated Tg mice (K–M). N, Quantitative image analysis revealed that cerebral Thio S-positive plaques were significantly decreased in MER5101-vaccinated APPswe/PS1ΔE9 Tg mice versus vehicle-treated Tg controls. O–Q, The levels of Aβx-40 (O), Aβx-42 (P), and Aβ1-total (Q) in TBS-soluble and guanidine-soluble (TBS-insoluble) fractions of brain homogenates and plasma were measured by Aβ ELISA. Data are represented as mean ± SEM. *p < 0.05; **p < 0.01.

Figure 5.

Plaque-associated microglial activation but not astrocyte reactivity was decreased in the HC of MER5101-vaccinated APPswe/PS1ΔE9 Tg mice. R1282 Aβ (A, B), Iba-1 (C, D), CD45 (E, F), and GFAP (G, H) immunoreactivities were detected on brain sections of immunized and vehicle-treated APPswe/PS1dE9 Tg mice. The number of Iba-1- and CD45-immunopositive microglia was reduced in MER5101-vaccinated APPswe/PS1ΔE9 Tg mice (D and E, respectively). GFAP IR was similar between vaccinated and vehicle-treated Tg mice, with perhaps more clustering of astrocytes around plaques in the HC of vehicle Tg controls.

Figure 6.

Enhanced synaptic IR and reduced neuritic plaques were observed in the HC of MER5101-vaccinated APPswe/PS1ΔE9 Tg mice compared with vehicle Tg controls. The synaptic density was evaluated by staining with a presynaptic marker, SYP (A, B), and a postsynaptic marker, PSD-95 (E, F). Both SYP and PSD-95 immunoreactivities were increased in vaccinated APPswe/PS1ΔE9 Tg mice (B, F) compared with vehicle Tg controls (A, E). The optical density of SYP (C, D) and PSD-95 (G, H) IR was quantified in both the CA1 and CA3 regions of mouse HC for all treatment groups. Vehicle-treated APPswe/PS1ΔE9 Tg mice had significantly less SYP- and PSD-95-positive synaptic staining in HC compared with Wt mice. However, MER5101 vaccination restored synaptic staining close to Wt levels. Neuritic plaques, labeled by anti-human APP mAb, 22C11, were decreased in the HC of vaccinated APPswe/PS1ΔE9 Tg mice compared with vehicle controls. *p < 0.05; **p < 0.01.

Figure 7.

MER5101 vaccination resulted in attenuation of cognitive deficits in APPswe/PS1ΔE9 Tg mice. A, CFC showed that the vehicle-treated APPswe/PS1ΔE9 Tg mice froze less often than either vaccinated or vehicle-treated Wt mice, whereas MER5101-vaccinated APPswe/PS1ΔE9 Tg mice showed significantly more freezing behavior than the Tg vehicle controls (p = 0.04), indicating an improvement in fear conditioned memory in the vaccinated Tg mice. B, In the MWM, vehicle-treated Tg mice showed slower learning acquisition during days 3–5 of the hidden platform trials compared with Wt vehicle controls, indicating a spatial learning deficit. However, MER5101-vaccinated Tg mice showed faster learning acquisition during days 2–5 compared with vehicle-treated Tg mice. C, D, No group differences were observed in the first probe trial for short-term spatial memory (2 h after the last hidden platform trial on day 5). E, F, The second probe trial (24 h after the last hidden platform trial on day 5) revealed that vehicle-treated Tg mice had no preference for exploring the target quadrant (E), and had a lower and negative ACI (defined as average frequency of swims over sites in other quadrants of the pool; F) compared with the other groups of mice. However, MER5101-vaccinated Tg mice spent more time in the target quadrant searching for the platform (strong trend; p < 0.06), similar to the two groups of Wt mice and a positive ACI, indicating beneficial effects of MER5101 vaccination on spatial memory. *p < 0.05.