Hepatitis B core VLP-based mis-disordered tau vaccine elicits strong immune response and alleviates cognitive deficits and neuropathology progression in Tau.P301S mouse model of Alzheimer’s disease and frontotemporal dementia

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory loss [1], intracellular neurofibrillary tangles (NFTs) composed of microtubule-associated protein tau, and extracellular amyloid plaques formed by amyloid-β (Aβ) aggregates in the brain [2]. Unlike Aβ plaques, the amount and extent of NFT pathology positively correlate with the severity of the cognitive deficit of AD [3, 4]. Tau inclusions are also found in other tauopathies that lack Aβ pathology, such as corticobasal degeneration, Pick’s disease, and progressive supranuclear palsy [5]. Notably, mutations in the tau gene (such as P301S, P301L) cause some forms of frontotemporal dementia (FTD), indicating that tau dysfunction alone is sufficient to cause neurodegeneration [6]. Current pharmacological treatment of AD is based on cholinesterase inhibitors and memantine. However, this treatment could not halt the disease’s progress [7].

Previous reports suggest that cerebrospinal fluid tau in patients with AD and patients with mild cognitive impairment comprises primarily truncated forms of tau (151–391/2N4R, sequence corresponding to full-length 2N4R tau), which are conformationally different from normal healthy tau and essential for pathologic tau-tau interaction [8‐10]. During the development of AD, the mis-disordered truncated tau aggregates to tau tangles and oligomers and drives AD-like neurofibrillary degeneration accompanied by microglial and astroglial activation in the brain [8, 9]. Therefore, therapy targeting mis-disordered truncated tau may be an effective treatment strategy [10]. In the past few years, the field of anti-tau immunotherapies has been galvanized by hundreds of studies trying to optimize the approach for treating AD. Tau-targeted immunotherapies have shown potential for AD treatment, some of which have paved the way to clinical trials [11‐14]. Compared with passive immunotherapy, active immunotherapy can provide persistent effects because it uses the immune system to produce long-lasting antibody. Because active immunization with full-length Aβ induced meningitis in clinical trials by a cell-mediated type 1 helper T cell (Th1) immune response [15, 16], the second generation of AD vaccines was developed by conjugating a B-cell epitope of tau or Aβ with a carrier [17, 18]. Previous studies demonstrated that tau_294–305 is a structural determinant of the truncated tau protein for the pathological tau-tau interaction, and it contains a motif, “HXPGGG,” which localizes not only in tau_299–304 (within microtubule-binding region 2 [MTBR2]) but also in tau_268–273 (within MTBR1), tau_330–335 (within MTBR3), and tau_362–367 (within MTBR4). A tau_294–305-targeting approach reduced tau pathology and associated behavioral deficits in transgenic tau rats [17].

Virus-like particles (VLPs) are multimeric nanoparticles that are assembled from viral structural proteins and are biosafe because they lack a viral genome [19]. VLPs contain repetitive high-density viral surface proteins and are an excellent platform from which to present heterologous antigens. Tau_294–305 is a short peptide with low immunogenicity. To develop a vaccine with tau_294–305 as an immunogen to induce high antibody titers, tau_294–305 should be combined with a carrier such as keyhole limpet hemocyanin. Hepatitis B virus core protein (HBc) VLPs are promising carriers because of their high capacity for foreign insertions, high-level generation, and efficient self-assembly in virtually all known homologous and heterologous expression systems, including bacteria and yeast [20‐22]. The major immunodominant region (MIR) in HBc is generally used for the insertion of foreign B-cell epitopes to maximally expose these epitopes on the VLP surface and consequently provide the most efficient immunogenic activity. In this study, we fused tau_294–305 to HBc MIR and assessed its effect on tauopathies in Tau.P301S transgenic mice.

Both hyperphosphorylated and truncated tau play a critical role in AD pathogenesis. Authors of many reports selectively targeted phosphorylated tau (phospho-tau) epitopes, including phospho-Ser396/phospho-Ser404 [32‐34], phospho-Ser422 [35], or phospho-Thr231/phospho-Ser235 [36]. However, most  phosphoepitopes of tau such as phospho-Ser404 are present in healthy human brains [37], which raises concerns about the safety of immunotherapies targeting those phospho-tau species. Truncated tau is a pathogenic tau present in AD brains but not in normal human brains; thus, targeting truncated tau may be a more promising approach [17]. In this study, we chose tau_294–305 as our targeting epitope because it is a structural determinant of the truncated tau protein for the pathological tau-tau interaction. This epitope contains a motif, “HXPGGG,” that localizes not only in tau_299–304 (within MTBR2) but also in tau_268–273 (within MTBR1), tau_330–335 (within MTBR3), and tau_362–367 (within MTBR4) [17, 38]. Therefore, the antibodies induced by tau_294–305 may simultaneously bind to MTBR1–4.

HBc VLP is a widely used carrier to generate putative vaccines. HBc-based malaria and influenza vaccines have entered into clinical trials, and they were well tolerated. To avoid T-cell autoimmunity likely induced by full-length Aβ, we developed a tau vaccine (T294-HBc VLP vaccine) by genetically fusing a B-cell epitope of tau (tau_294–305) to HBc MIR. In our tau vaccine, the Th epitopes were derived exclusively from the VLP carrier protein, and the vaccine was formulated with Alum adjuvant that promoted the Th2 immune response. As expected, the antibody induced by T294-HBc VLPs was predominantly the IgG1 isotype, which indicates that the immune response to the vaccine mainly involved the Th2 phenotype and that T294-HBc VLPs may be a safe vaccine type. Moreover, T294-HBc VLPs formed uniform nanoparticles with a diameter of approximately 33.55 nm and elicited robust and specific antibodies against mis-disordered tau in mice.

FTD, the second most common form of dementia before the age of 65, is caused by P301S/L mutated tau. A previous report showed that tau-targeting vaccine AADvac1 improved cognition and reduced tauopathy in a transgenic rat model expressing human truncated tau [17]. Although this transgenic rat model displayed some tau pathology and motor and behavioral deficits, it still could not mimic some incidence and progression of tauopathy in AD and FTD. Moreover, AADvac1 induced antibodies against tau_294–305, but whether the vaccine has a beneficial effect on an FTD model with P301S/L mutation remains unknown. To explore the effects of T294-HBc VLP vaccine on the animal models of FTD and AD, we applied the vaccine to the Tau.P301S transgenic mouse model, which is widely used to mimic the incidence and progression of FTD and AD and recapitulates the essential molecular and cellular features of the human tauopathies, including truncated tau generation, hyperphosphorylation, tau filament formation, and neurodegeneration [31, 39]. Woerman et al.’s report showed that the PS19 mouse model exhibited great variability in pathology onset at ages over 31 weeks, but it was relatively uniform before age 30 weeks [40]; the mice at 22 weeks of age that we used conformed the latter age range. Our results showed that T294-HBc vaccine improved cognition and memory of Tau.P301S mice and reduced the levels of truncated tau monomer, oligomer, and hyperphosphorylated tau. Our observations are in agreement with previous findings that passive immunization with tau antibodies against pathological tau forms improved cognition of AD mice and reduced the levels of hyperphosphorylated tau [41, 42]. Although the antibodies induced by T294-HBc VLPs recognized tau_294–305 (P301S) with lower affinity, they could also bind to tau_294–305, tau_263–274, tau_325–336, and tau_357–368, resulting in beneficial effects on Tau.P301S mice.

The accumulation of NFTs can cause inflammation and synapse loss in Tau.P301S mice [31]. However, the T294-HBc VLP-immunized mice exhibited lower levels of astrogliosis and microgliosis and higher levels of synaptophysin than adjuvant-treated mice, leading to attenuation of cognitive deficits and neuropathology in the transgenic mice. Several mechanisms could explain the antibody-mediated clearance of the mis-disordered tau in vivo [35, 43]. The direct targeting mechanism proposes that a small amount of serum antibodies can cross the blood-brain barrier, bind to the mis-disordered tau, and then induce the phagocytosis of the antigen-antibody complexes via the Fc portion of the antibody [44]. The pathologic tau can propagate from cell to cell and induce the aggregation of pathologic tau in the other neurons [45], and the clearance of mis-disordered tau by our vaccine results in the inhibition of the NFT formation.

In summary, our study indicates that the HBc VLP-based T294-HBc vaccine exerted favorable effects on cognition and neuropathology in the Tau.P301S transgenic mouse model by inducing high titers of antibodies against truncated tau; decreasing the levels of truncated tau monomer, oligomer, and hyperphosphorylated tau; increasing synaptophysin levels; and suppressing microgliosis and astrogliosis in mouse brains. Moreover, T294-HBc VLP-induced antibodies could simultaneously bind to MTBR 1–4 [tau_263–274, tau_294–305, tau_325–336, tau_357–368, and tau_294–305(P301S)], indicating that this vaccine has promising therapeutic potential for the treatment of FTD and AD.