Virus-like particle (VLP)-based vaccine targeting tau phosphorylated at Ser396/Ser404 (PHF1) site outperforms phosphorylated S199/S202 (AT8) site in reducing tau pathology and restoring cognitive deficits in the rTg4510 mouse model of tauopathy

Abstract

Tauopathies, including Alzheimer's disease (AD) and Frontotemporal Dementia (FTD), are histopathologically defined by the aggregation of hyperphosphorylated pathological tau (pTau) as neurofibrillary tangles in the brain. Site-specific phosphorylation of tau occurs early in the disease process and correlates with progressive cognitive decline, thus serving as targetable pathological epitopes for immunotherapeutic development. Previously, we developed a vaccine (Qβ-pT181) displaying phosphorylated Thr181 tau peptides on the surface of a Qβ bacteriophage virus-like particle (VLP) that induced robust antibody responses, cleared pathological tau, and rescued memory deficits in a transgenic mouse model of tauopathy. Here we report the characterization and comparison of two additional Qβ VLP-based vaccines targeting the dual phosphorylation sites Ser199/Ser202 (Qβ-AT8) and Ser396/Ser404 (Qβ-PHF1). Both Qβ-AT8 and Qβ-PHF1 vaccines elicited high-titer antibody responses against their pTau epitopes. However, only Qβ-PHF1 rescued cognitive deficits, reduced soluble and insoluble pathological tau, and reactive microgliosis in a 4-month rTg4510 model of FTD. Both sera from Qβ-AT8 and Qβ-PHF1 vaccinated mice were specifically reactive to tau pathology in human AD post-mortem brain sections. These studies further support the use of VLP-based immunotherapies to target pTau in AD and related tauopathies and provide potential insight into the clinical efficacy of various pTau epitopes in the development of immunotherapeutics.

Fig 1.. Qβ-AT8 and Qβ-PHF1 VLP conjugation, treatment timeline, and antibody titer responses.

a. A 21-mer pTau peptide containing the Ser199/Ser202 phosphorylation sites and a modified Gly-Gly-Cys (GGC) C-terminal sequence (AT8 peptide) and a 25-mer pTau peptide containing the Ser396/Ser404 phosphorylation sites and a modified GGC C-terminal sequence (PHF1 peptide) were individually conjugated to surface exposed Lys residues of coat proteins on Qβ bacteriophage VLPs using SMPH cross-linker. b. 2-month-old rTg4510 mice received three bi-weekly intramuscular vaccinations with either unconjugated Qβ Control VLP (n=5), Qβ-AT8 VLP (n=7), or Qβ-PHF1 VLP (n=8) and were evaluated at 4.5-months for vaccine efficiency. c. An upward mobility shift on a 10% SDS denaturing gel shows the number of AT8 or PHF1 peptides conjugated per Qβ coat protein monomer; Qβ-PHF1 VLP shows a higher conjugation efficiency than Qβ-AT8 VLPs. d. Qβ-AT8 and Qβ-PHF1 VLP vaccines elicited significantly elevated serum IgG antibody titers at 6-weeks post-vaccination compared to Qβ Control. Graph displays mean ± SEM. p<0.05*; one-way ANOVA with Dunnett’s multiple comparisons. Qβ Control VLP (n=5), Qβ-AT8 VLP (n=7), or Qβ-PHF1 VLP (n=7).

Fig 2.. Qβ-PHF1 vaccination rescues delay-dependent memory deficits while Qβ-AT8 vaccination does not.

a. In the Novel Object Recognition (NOR) test, no difference in the time spent with each object on the sample day. b. Twenty-four hours later (Test Day), non-transgenic (B6) mice spent significantly more time with the novel object. There was no difference in time spent with the novel object and familiar object for Qβ Control and Qβ-AT8 vaccinated rTg4510 mice, but this impairment was significantly rescued in Qβ-PHF1 vaccinated rTg4510 mice. c. Hidden platform trials of the Morris Water Maze (MWM) test show that B6 mice learned the location of the platform faster (shorter latency) than all rTg4510 vaccine groups. All rTg4510 vaccine groups exhibited hyperactivity based on distance traveled and velocity compared to B6 mice. d-e. During the probe trial (platform removed), B6 mice spent significantly more time exploring the target quadrant. Qβ Control rTg4510 mice spent significantly more time in the wrong quadrants. Qβ-AT8 and Qβ-PHF1 vaccinated rTg4510 spent equal time in all quadrants indicating a potentially milder impairment than Qβ Control mice but incomplete rescue of spatial memory. All graphs display mean ± SEM. Two-way ANOVA with Šidák correction (a, b). Two-way ANOVA with Dunnett’s correction comparing each group to the B6 group on each day (c). Student’s t-test (e). p<0.05*, p<0.01**, p<0.001***. B6 (n=9), Qβ Control (n=7), Qβ-AT8 (n=7), Qβ-PHF1 (n=8).

Fig 3.. Qβ-PHF1 vaccination reduces soluble pathological tau in the brains of rTg4510 mice while Qβ-AT8 vaccination does not.

a. Western blot of soluble hippocampal lysates from Qβ Control and Qβ-PHF1 vaccinated rTg4510 mice was evaluated for markers of phosphorylated (AT8, AT180, PHF1) and total tau (Tau5). All samples were run on the same gel. b. Compared to Qβ Control vaccinated mice, Qβ-PHF1 vaccinated mice showed significant reduction in pathological AT180 tau without any reduction in total physiologic tau. c. Immunohistochemistry of brain sections from Qβ Control and Qβ-PHF1 vaccinated rTg4510 mice was performed to validate the changes observed in AT180 tau where an obvious reduction in AT180 neuropathology was observed in both the cerebral cortex and CA1 hippocampus of Qβ-PHF1 vaccinated mice compared to the Qβ Control group. d-e. Western blot of soluble hippocampal lysates from Qβ Control and Qβ-AT8 vaccinated rTg4510 mice showed no reductions in pathological tau. All graphs display mean ± SEM. Student’s t-test (b, e). p<0.05*. Qβ Control (n=5), Qβ-AT8 (n=7), Qβ-PHF1 (n=8).

Fig 4.. Qβ-PHF1 vaccination preferentially reduces Sarkosyl insoluble tau in rTg4510 mice and elicits antibodies specific to human pathological tau.

a-b. Western blot (a) and quantification (b) showing a significant reduction in the ratio of Sarkosyl insoluble to soluble AT8 and Tau12 positive tau in the hippocampus of Qβ-PHF1 vaccinated rTg4510 mice compared to Qβ Control vaccinated mice. c-d. Gallyas silver impregnation (c) and quantification (d) showing reduction in insoluble tau and dramatic clearance of NFTs from the cerebral cortex and CA1 hippocampus of Qβ-PHF1 vaccinated mice compared to the Qβ Control group. e. Qβ-PHF1 immune sera stained somatodendritic NFTs, pre-tangles, neuritic plaques, neuropil threads, and ghost tangles in human autopsy AD, but not non-AD, brain tissue. Qβ Control sera did not show any specific staining. AT8 antibody staining was used as positive control. All graphs show mean ± SEM. Student’s t-test (b, d). p<0.05*, p<0.01**. b. Qβ Control (n=5), Qβ-PHF1 (n=8). d. Qβ Control (n=5), Qβ-PHF1 (n=5).

Fig 5.

Qβ-PHF1 vaccination reduces reactive microgliosis in rTg4510 mice. a. IHC of Qβ Control and Qβ-PHF1 vaccinated rTg4510 mouse brain sections using Iba1 showed a reduction in amoeboid microglial morphology and overall Iba1 intensity in both the cerebral cortex and CA1 hippocampus of Qβ-PHF1 vaccinated mice compared to the Qβ Control group. b. Qβ-PHF1 vaccinated mice also exhibited reductions in CD45 intensity, a marker of microglial activation, in both the cerebral cortex and CA1 hippocampus compared to the Qβ Control group.