A MultiTEP platform-based epitope vaccine targeting the phosphatase activating domain (PAD) of tau: therapeutic efficacy in PS19 mice

Abstract

Pathological tau correlates well with cognitive impairments in Alzheimer's disease (AD) patients and therefore represents a promising target for immunotherapy. Targeting an appropriate B cell epitope in pathological tau could in theory produce an effective reduction of pathology without disrupting the function of normal native tau. Recent data demonstrate that the N-terminal region of tau (aa 2-18), termed the "phosphatase activation domain (PAD)", is hidden within native Tau in a 'paperclip'-like conformation. Conversely, PAD is exposed in pathological tau and plays an essential role in the inhibition of fast axonal transport and tau polymerization. Thus, we hypothesized that anti-tau2-18 antibodies may safely and specifically reduce pathological tau and prevent further aggregation, which in turn would neutralize tau toxicity. Therefore, we evaluated the immunogenicity and therapeutic efficacy of our MultiTEP platform-based vaccine targeting tau2-18 formulated with Advax^CpG adjuvant (AV-1980R/A) in PS19 tau transgenic mice. The AV-1980R/A induced extremely high antibody responses and the resulting sera recognized neurofibrillary tangles and plaque-associated dystrophic neurites in AD brain sections. In addition, under non-denaturing conditions AV-1980R/A sera preferentially recognized AD-associated tau. Importantly, vaccination also prevented age-related motor and cognitive deficits in PS19 mice and significantly reduced insoluble total and phosphorylated tau species. Taken together, these findings suggest that predominantly targeting misfolded tau with AV-1980R/A could represent an effective strategy for AD immunotherapy.

Figure 1

Schematic representations of Protein vaccine construct and experimental design in PS19 transgenic mice. (a) Schematic representation of AV-1980 construct encoding 3 copies of tau2-18 fused to MultiTEP, one universal synthetic Th epitope, PADRE and eleven foreign promiscuous Th epitopes from infectious agents. (b) Design of experimental protocol in PS19 transgenic mice vaccinated with AV-1980R/A and injected with Advax^CpG adjuvant only (control group).

Figure 2

The AV-1980R/A vaccine induced high titers of antibodies in PS19 mice. (a) Concentration of anti-tau antibodies in sera was measured by binding to tau_2-18 peptide and to full length recombinant tau protein in ELISA. (b) Isotypes of anti-tau antibodies were analyzed in sera at dilution 1:500 by ELISA. Error bars represent average ± SEM (n = 10 for Advax^CpG injected group; n = 9 for AV-1980R/A immunized group).

Figure 3

AV-1980R/A vaccine induced functional antibodies recognizing human pathological tau. (a) Immune sera, but not sera from adjuvant injected mice bound to the 50 µm brain sections of cortical tissues from two AD cases (scale = 10 µm). (b–d) Co-staining of AD brain sections with immune sera and total htau and Amylo-Glo (b, scale = 20 µm), pS199/202 (c, scale = 40 µm, merge-40x = 10 µm) and pS396/404 (d, scale = 50 µm, merge-40x = 10 µm) demonstrated co-localization of tau recognized by anti-tau_2-18 sera and these antibodies. (e) Immune sera, but not sera from adjuvant injected mice also bound to different forms of tau in soluble extracts from four AD cases detected by Western blot. Sera were used at dilution 1:1000. Sera bound full-length recombinant tau, but not tau lacking aa 2-18. Lane 1- Recombinant tau 2N4R; Lane 2 – Recombinant tau 2N4R with deleted aa2-18 (ΔTau2-18); Lane 3-severe AD, tangle stage 6; Lane 4- moderate AD, tangle stage 5; Lane 5-mild AD tangle stage 5, Lane 6 – MCI, tangle stage 2; Lane 7-Control non-AD, stage 2. (f) Using non-denaturing Dot Blots, AV-1980R immunized sera as well as commercial TNT-1 mAb specific to N-terminus of Tau (epitope aa 7–12) preferentially bound to soluble tau in AD brain lysates but showed minimal immunoreactivity in controls. Of note, the HT7 Ab which detects total tau, recognized non-denatured tau in both control and AD brains.

Figure 4

Vaccination with AV-1980R/A improved both motor and cognitive function in PS19 mice. Motor skills of mice were tested in Fixed rotarod (a) and accelerated speed rotarod (b). Cognitive functions of mice were tested in Y maze alternation test (c) and in novel object recognition (d) and novel place recognition (e) test. Error bars represent average ± SEM (n = 10 for Advax^CpG injected group; n = 9 for AV-1980R/A immunized group and n = 8 for non-injected littermate). Statistical significances were calculated using ANOVA test (*P < 0.05, **P < 0.01 and ****P < 0.0001).

Figure 5

Effect of AV-1980R/A vaccination on tau proteins in PS19 mice. Levels of human total tau protein (a,e) and several phosphorylated tau species (b–d,f–h) in brain soluble (a–d) and insoluble (e–h) extracts were analyzed by ELISA. Error bars represent average ± SEM (n = 9 for both groups).

Figure 6

Vaccination with AV-1980R/A significantly decreased PAD-positive Tau and astrogliosis in brains of PS19 mice. The levels of PAD-positive tau protein, GFAP, IBA-1 and CD45 proteins in the soluble fraction of the brain extracts were analyzed by Western blotting and quantitatively determined by densitometric analysis with normalization against β-actin. The relative protein level in the brains of vaccinated mice is presented as a percentage of the protein level in the brains of mice injected with Advax^CpG. Error bars represent average ± SEM of (n = 9 for both groups).