Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be mod- The role of immunogen conformation in the specificity and efficacy of the humoral immune response has been recognized in a number of recent therapeutic and prophylactic vaccines (1-4). Conformation-specific antibodies generated by immunization with conformationally restricted immunogens are also important tools for disease diagnosis as biochemical probes for establishing protein structure-function relationships (5, 6) and in affinity purification (7,8). Development of such peptide immunogens, however, has thus far been hindered by the lack of general design strategies to control the conformation of a given peptide sequence. In addition, most naked peptides have low inherent immunogenicity. Although design of peptide templates for the generation of antibodies that selectively recognize protein epitopes with an ␣-helical conformation has been reported (2, 4, 9, 10), general strategies to design short peptide fragments as immunogens for targeting other protein conformations are lacking.Protein aggregation resulting from aberrant folding is characterized by the formation of proteinaceous deposits called amyloid, which exhibit ␤-sheet structure (11). Misfolding is now associated with over 20 human diseases, including Creutzfeldt-Jakob disease, Alzheimer disease (AD), 2 Huntington disease, and Type II diabetes mellitus (12). Vaccines that can elicit antibodies against only the pathological ␤-sheet multimers of these targets would thus be considered advantageous from an efficacy perspective but also particularly from a safety perspective, where the non-folded protein has physiological relevance. In AD, ␤-amyloid peptides (A␤) A␤(1-40) and A␤(1-42) have been associated with disease progression over several decades of research, and the ␤-sheet aggregates of A␤ are one of the major pathological hallmarks of AD (13). Although the molecular pathology of AD has not yet been elucidated, a large variety of soluble and insoluble ␤-sheet A␤(1-40/42) oligomers with different degrees of polymerization have been identified in vitro or in AD brain as synaptotoxic and likely causal agents of cognitive impairment (14 -18).The discovery that active vaccination of transgenic mice overexpressing human amyloid precursor protein (APP) with A␤-derived peptides could be an effective means to modify disease progression has opened up new vistas in AD therapy (19 -21). The importance of immunogen aggregation state upon immune response specificity has been demonstrated by the generation of an...

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Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptidespecific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cellindependent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domaincontaining adapter inducing interferon-␤ (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are IntroductionMost peptide and protein antigens require T-cell help for B-cell activation and antibody production, 1 while polysaccharides and lipopolysaccharide (LPS) can stimulate antibody responses without T help. 2 In both T cell-independent (TI) and -dependent (TD) antibody responses, cognate antigens bind their B-cell receptor (BCR). TD antigens are internalized, processed, and presented in the context of MHC class II molecules to antigen-specific T-helper cells. 3 The activated T cell then facilitates B-cell responses and immunoglobulin isotype switching via costimulatory molecules, adhesive antigens, and cytokines. TI antigens typically stimulate transient IgM antibody production with little or no IgG, IgA, or IgE production. TI type 1 (TI-1) antigens are polyclonal B-cell activators such as LPS whereas TI type 2 (TI-2) antigens consist of repetitive biochemical structures, such as polysaccharides or glycoproteins. 4 Liposomes are submicron vesicles of phospholipids and allow integration of compounds within and on the lipid bilayer. Antigens can be packed on the surface to resemble TI-2 antigens; in nature, repetitive arrangement of a single protein or peptide on cells or microorganisms does not typically occur. In the present study, a peptide was palmitoylated and mixed with phospholipids and monophosphoryl lipid A (MPLA) to allow formation of liposomes with densely arranged peptides on the outer surface. The palmitoylated human ␤-amyloid (A␤, aa1-15) peptide adopts an aggregated ␤-sheet conformation on liposomes. 5 While previous reports have suggested that nonreplicating protein vaccines do not enable TI isotype switching, 6,7 the present study in mice demonstrates that switch is feasible with a correct structural assembly of antigen and adjuvant. This result could pave the way for vaccination of patients with T-cell deficiencies or elderly, or when T-cell involvement is associated with immunopathology or autoimmunity, f...

Sensitivity is the key: Dynamic nuclear polarization NMR spectroscopy provides structural information on liposomal vaccines targeting Alzheimer's disease. DMPC/DMPG/Cholesterol mainly stabilizes extended structures of the lipid‐anchored peptide, while in DMTAP/Cholesterol liposomes the peptide adopts a multitude of conformations including random‐coil and extended structures.

In Down syndrome (DS) or trisomy of chromosome 21, the β-amyloid (Aβ) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aβ as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aβ is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer’s disease (AD). At present, no treatment targets Aβ–related pathogenesis in people with DS. Herein we used a vaccine containing the Aβ 1–15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aβ vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aβ IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aβ without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aβ levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aβ as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aβ immunotherapeutic approach may act to target Aβ-related pathology in a mouse model of DS.

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Empfindlichkeit ist Trumpf: NMR‐Spektroskopie in Kombination mit dynamischer Kernpolarisation liefert Strukturinformation über liposomale Impfstoffe, die gegen Alzheimer gerichtet sind. DMPC/DMPG/Cholesterol‐Lipide stabilisieren hauptsächlich gestreckte Rückgratstrukturen des Lipid‐verankerten Peptids. Dagegen führen DMTAP/Cholesterol‐Liposomen zu verschiedenen Peptidkonformationen, die ungeordnete und gestreckte Faltungen enthalten.