RU2810774C2 - Peptide immunogens from c-terminal end of alpha-synuclein protein and compositions based on them for treatment of synucleonopathies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to peptide immunogenic constructs of alpha-synuclein (α-Syn), and can be used medically to treat brain disease in an individual with synucleinopathy. Proposed peptide immunogenic constructs of α-Syn contain a B-cell epitope of α-Syn bound to a heterologous T-helper cell (Th) epitope directly or via a heterologous spacer.

EFFECT: invention ensures the production of peptide immunogenic constructs of α-Syn, which stimulate the formation of highly specific antibodies that cross-react with β-leaf of α-Syn in the form of monomers, oligomers and fibrils, but with not naturally occurring α-spiral of α-Syn, providing therapeutic immune responses in patients at risk of developing synucleinopathies.

17 cl, 28 dwg, 15 tbl, 17 ex

Description

This application is a PCT international application that claims priority to US provisional application Serial No. 62/521287, filed June 16, 2017, which is incorporated herein by reference in its entirety.

Field of technology

This disclosure relates to peptide immunogenic constructs based on the C-terminal end of the alpha-synuclein protein (α-Syn) and compositions based thereon for the treatment of synucleinopathies.

State of the art

Synuclein proteins (reviewed at: en.wikipedia.org/wiki/Synuclein) are a family of soluble proteins native to vertebrates that are expressed primarily in nervous tissue and certain tumors. The synuclein family includes three known proteins: alpha-synuclein (reviewed at: en.wikipedia.org/wiki/Alpha-synuclein), beta-synuclein (reviewed at: en.wikipedia.org/wiki/Beta- synuclein) and gamma-synuclein. All synucleins have in common the presence of a highly conserved alpha-helical lipid-binding motif with similarities to the class A2 lipid-binding domains of interchangeable apolipoproteins. Normal cellular functions have not been determined for any of the synuclein proteins, although some evidence suggests a role in regulating membrane stability and metabolism.

The full-length alpha-synuclein protein (α-Syn) is a 140 amino acid protein (Accession No. NP_000336) and is encoded by the SNCA gene. At least three isoforms of α-Syn are generated by alternative splicing. The main form is full-length protein. Other isoforms are α-Syn-126, which lacks residues 41-54 as a result of loss of exon 3; and α-Syn-112, which lacks residues 103–130 as a result of the loss of exon 5.

The primary structure of α-Syn is generally divided into three distinct domains: (1) residues 1-60: an amphiphilic N-terminal region dominated by four 11-residue repeats containing the KTKEGV consensus sequence, which has the structural property of an alpha helix, similar to apolipoprotein binding domains; (2) residues 61-95: a central hydrophobic region that contains a region of non-amyloid β-component (NAC - non-amyloid-(component) that is involved in protein aggregation; and (3) residues 96-140: a highly acidic and proline-rich region , which does not have a distinct structural property. A 35 amino acid fragment of α-Syn, the NAC region, was found to be present with A( in the amyloid-rich fraction. NAC was later demonstrated to be a fragment of its precursor protein, NACP ( non-amyloid-(component protein), which was later determined to be a full-length homolog of synuclein from the California stingray (Torpedo californica), now designated human α-Syn.

The use of high-resolution ion-mobility mass spectrometry (IMS-MS) on HPLC-purified α-Syn in vitro showed that α-Syn is autoproteolytic (self-proteolytic), leading to the formation of a number of low molecular weight fragments mass during incubation. The full-length 14.46 kDa protein was found to produce numerous smaller fragments, including a 12.16 kDa fragment (amino acids 14-133) and a 10.44 kDa fragment (amino acids 40-133). 140), formed as a result of C- and N-terminal truncations, as well as a fragment with a molecular weight of 7.27 kDa (amino acids 72-140). The 7.27 kDa fragment, which contains most of the NAC region, was demonstrated to aggregate significantly faster than full-length α-Syn. It is possible that these autoproteolytic products play a role as mediators or cofactors in α-Syn aggregation.

α-Syn is present in large quantities in the human brain, accounting for up to 1% of all proteins in the cytosol of brain cells and glial cells. α-Syn is highly expressed in the neocortex, hippocampus, dentate gyrus, olfactory bulb, striatum, thalamus, and cerebellum. It is also highly expressed in hematopoietic cells, including B, T and NK cells, as well as monocytes and platelets. Smaller amounts of α-Syn are found in the heart, muscle, and other tissues. In the brain, α-Syn occurs primarily at the tips of nerve cells (neurons) in specialized structures called presynaptic terminals. Within these structures, α-Syn interacts with phospholipids and proteins. Presynaptic terminals release chemical messengers called neurotransmitters, such as dopamine, from compartments known as synaptic vesicles. The release of neurotransmitters leads to the transmission of signals between neurons and is essential for normal brain function, including cognitive performance.

α-Syn in solution is considered an intrinsically disordered protein in the sense that it lacks a single stable 3D structure. It has been demonstrated that α-Syn interacts significantly with tubulin and that α-Syn may have activity as a potential microtubule-associated protein such as tau. α-Syn has classically been considered an unstructured soluble protein; unmutated α-Syn forms a stably folded tetramer that is resistant to aggregation. Despite this, α-Syn can aggregate to form insoluble fibrils under pathological conditions characterized by Lewy bodies. These disorders are known as synucleinopathies (described on the website: en.wikipedia.org/wiki/Synucleinopathies).

Synucleinopathies are a diverse group of neurodegenerative disorders that share a common pathological characteristic: on neuropathological studies, characteristic lesions containing abnormal aggregates of insoluble α-Syn are present in selectively susceptible populations of neurons and glial cells. The most common synucleinopathies include Lewy body disorders (LBD), such as Parkinson's disease (PD), Parkinson's disease with dementia (PDD), and dementia with Lewy bodies (DLB). Lewy bodies), as well as multiple system atrophy (MSA - Multiple System Atrophy) or neurodegeneration with brain iron accumulation type I (NBIA (Neurodegeneration with Brain Iron Accumulation) type I). Current treatments for these diseases include symptomatic medications such as levodopa, anticholinergic drugs, and monoamine oxidase inhibitors. At the same time, all currently existing treatment options lead only to symptomatic normalization, but do not induce a long-term modifying effect on the disease in patients.

LBDs are progressive neurodegenerative disorders characterized by tremor, rigidity, bradykinesia, and loss of dopaminergic neurons in the brain. In the case of DLB and PDD, symptoms also include cognitive impairment. Up to 2% of the population over 60 years of age in Western countries develop typical symptoms of PD/LBD. Apparently, genetic predisposition and environmental factors are involved in the development of this disease. Patients suffering from this disease develop characteristic intracellular inclusions, called Lewy bodies (LB - Lewy bodies), in the cortical and subcortical areas of the brain, especially in the case of areas with a high content of dopaminergic neurons or neuronal processes. In LBD, α-Syn accumulates in the LB throughout affected brain regions. In addition, it could be demonstrated that point mutations as well as duplications or multiplications in the α-Syn gene are associated with rare familial forms of parkinsonism.

Multiple system atrophy (MSA - Multiple System Atrophy) is a sporadic neurodegenerative disorder characterized by symptoms of levodopa-resistant parkinsonism, cerebellar ataxia and dysautonomia. Patients suffering from multisystem neuronal loss will be affected in various areas of the brain, including the striatum, substantia nigra, cerebellum, pons, inferior olive, and spinal cord. MSA is characterized by α-Syn-positive glial cytoplasmic (GCI) and sparse neuronal inclusions throughout the central nervous system.

Other neural disorders, such as various neuroaxonal dystrophies, also have α-Syn pathologies, in which α-Syn is a major structural component of Lewy body fibrils. Lewy bodies sometimes contain tau protein; however, α-Syn and tau are two distinct subsets of filaments in the same inclusion bodies. α-Syn pathology is also found in both sporadic and familial cases of Alzheimer's disease.

The mechanism of α-Syn aggregation is unclear. Monomeric α-Syn is naturally unfolded in solution but can also associate with membranes in an α-helical form. Unfolded monomer can aggregate first into small oligomeric molecules, which can be stabilized by β-sheet-like interactions, and then into insoluble molecules of higher molecular weight. α-Syn exists as a mixture of unstructured conformers with a high content of alpha helices and beta sheets in equilibrium. Mutations or buffer conditions known to normalize aggregation result in a significant increase in the beta conformer population, thus indicating that this may represent a conformation associated with pathogenic aggregation. Evidence suggests the presence of a high-beta structured mediator that may represent a precursor to aggregation and ultimately Lewy bodies.

Several physiological factors can lead to modification of α-Syn, leading to the formation of aggregates, including (1) phosphorylation by one or more kinases, (2) truncation by proteases such as calpains; and (3) nitration by nitric oxide (NO) or other reactive nitrogen species that are present during inflammation. Transport in the ER and Golgi apparatus, synaptic vesicles, mitochondria, lysosomes, and other proteolytic machinery are some of the proposed cellular targets for α-Syn-mediated toxicity resulting from such aggregation.

Among the treatment strategies for synucleinopathies are compounds that inhibit α-Syn aggregation. The small molecule cuminaldehyde has been demonstrated to inhibit α-Syn fibrillation. In addition to small molecule therapy, a recent report indicates that α-Syn aggregates can be targeted by immunotherapy (described by Lee JS and Lee S-J, 2016). At the same time, this report highlights several potential issues or concerns that exist in the development of α-Syn immunotherapy, including (1) potential undesirable effects on the normal physiological function of α-Syn; (2) the difficulty of delivering an antibody drug to the brain parenchyma; and (3) the effectiveness of immunotherapy.

Today, there is still an unmet need for the development of site-directed peptide immunogens and formulations based on them for the low-cost treatment of patients suffering from synucleinopathies.

Literary sources:

1. “Alpha-synuclein,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php?title=Alpha-synuclein&oldid=781366541 (accessed May 30, 2017).

2. “Synucleinopathies,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php?title=Synucleinopathies&oldid=686287116 (accessed May 30, 2017).

3. “Beta-synuclein,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php?title=Beta-synuclein&oldid=763171134 (accessed May 30, 2017).

4. “Synucleinopathies,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php?title=Synucleinopathies&oldid=686287116 (accessed May 30, 2017).

5. LEE, J.S., et al., “Mechanism of Anti-α-Synuclein Immunotherapy,” J Mov Disord.; 9(1):14-19 (2016)

6. TRAGGIAI, E., et al. “An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus,” Nat Med.; 10(8):871-875 (2004)

7. WANG, C., et al. “Versatile Structures of α-Synuclein,” Front Mol Neurosci. 9:48 (2016)

Brief description of the ESSENCE of the invention

This disclosure relates to alpha-synuclein protein (α-Syn) peptide immunogenic constructs. This disclosure also relates to compositions containing peptide immunogenic constructs, methods of making and using said peptide immunogenic constructs, and antibodies generated by said peptide immunogenic constructs.

The disclosed peptide immunogenic constructs comprise a B cell epitope from α-Syn linked to a heterologous T helper (Th) cell epitope directly or via an optional heterologous spacer. The B cell epitope region of the peptide immunogenic constructs contains from about 10 to about 25 amino acid residues from the C-terminal region of α-Syn, corresponding to the sequence from approximately glycine at amino acid position 111 (G111) to approximately asparagine at amino acid position 135 (D135) of full-length α -Syn (SEQ ID NO: 1). The heterologous Th epitope region of peptide immunogenic constructs is derived from amino acid sequences derived from pathogenic proteins. The B cell epitope and Th epitope regions of the peptide immunogenic constructs function together when administered to a host to stimulate the production of antibodies that specifically recognize and bind to the B cell epitope region of the α-Syn constructs.

In some embodiments, the α-Syn peptide immunogenic construct comprises: (a) a B cell epitope comprising from about 10 to about 25 amino acid residues from the C-terminal fragment of α-Syn, corresponding to about amino acid G111 to about amino acid D135 SEQ ID NO : 1; (b) a T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 70-98; and (c) an optional heterologous spacer selected from the group consisting of amino acid, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys ( SEQ ID NO: 148), wherein the B cell epitope is covalently linked to the T helper epitope directly or through an optional heterologous spacer. In specific embodiments, the α-Syn peptide immunogenic construct comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

This disclosure also relates to compositions containing the disclosed peptide immunogenic constructs, including pharmaceutical compositions. The disclosed pharmaceutical compositions are capable of inducing an immune response and the production of antibodies against the disclosed peptide immunogenic constructs in the host. The disclosed compositions may contain one or a mixture of more than one of the disclosed peptide immunogenic compositions. In some embodiments, the compositions contain the disclosed peptide immunogenic constructs together with additional components, including carriers, adjuvants, buffers and other suitable reagents. In certain embodiments, the compositions comprise the disclosed peptide immunogenic constructs of a stabilized immunostimulatory complex with a CpG oligomer, which is optionally supplemented with an adjuvant.

In some embodiments, the compositions comprise an α-Syn peptide immunogenic construct that contains the amino acid sequence consisting of SEQ ID NO: 107, 108, 111-113, 115-147. In certain embodiments, the composition is a pharmaceutical composition comprising an α-Syn peptide immunogenic construct selected from the group consisting of SEQ ID NO: 107, 108, 111-113, 115-147, and a pharmaceutically acceptable carrier or adjuvant.

This disclosure also relates to antibodies that are produced by a host that is immunized with the disclosed peptide immunogenic constructs. The disclosed antibodies specifically recognize and bind to the B cell epitope region of α-Syn peptide immunogenic constructs. The disclosed antibodies to α-Syn have an unexpectedly high cross-reactivity towards the β-sheet of α-Syn in the form of monomers, oligomers or fibrils. Based on their unique characteristics and properties, the disclosed antibodies are capable of providing an immunotherapeutic approach to target, identify and treat synucleinopathies.

In specific embodiments, the antibody or epitope-binding fragment thereof specifically binds to a B cell epitope of an α-Syn peptide immunogenic construct selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, 115-147.

This disclosure also relates to methods for making and using the disclosed peptide immunogenic constructs, antibodies and compositions. The disclosed methods offer low-cost production and quality control of peptide immunogenic constructs and compositions containing these constructs, which can be used in methods of preventing and treating synopathies.

This disclosure also includes methods for treating and/or preventing synucleinopathies using peptide immunogenic constructs and/or antibodies directed against peptide immunogenic constructs. In some embodiments, methods of treating and/or preventing synucleinopathies include administering to the host a disclosed peptide immunogenic construct. In certain embodiments, the compositions used in the methods contain the disclosed peptide immunogenic construct in the form of a stable immunostimulatory complex with negatively charged oligonucleotides, such as CpG oligomers, resulting from electrostatic association, which complexes are then supplemented, optionally, with mineral salts or oil as an adjuvant, to administration to patients with synucleinopathies. The disclosed methods also include dosage regimens, dosage forms, and routes of administration of peptide immunogenic constructs to a host at risk of developing or having synucleinopathies.

In various embodiments, methods of using an α-Syn peptide immunogenic construct and/or antibodies generated by the α-Syn peptide immunogenic construct are described. In specific embodiments, methods for producing antibodies, inhibiting α-Syn aggregation, reducing the number of α-Syn aggregates, and identifying α-Syn aggregates of various sizes are described. Various methods include administering a pharmacologically effective amount of α-Syn peptide immunogen to a host in need thereof.

Brief description of graphic materials

In FIG. 1 shows a graph depicting the level of aggregation of α-Syn in vitro after 6 days in the presence of antibodies directed against the C-terminal end of α-Syn (samples 1-4) or in the presence of a vehicle control (sample 5). In particular, α-Syn aggregation was performed in the presence of anti-α-Syn antibodies, the formation of which is caused by: α-Syn _111-132 (sample 1); α-Syn _121-135 (sample 2); α-Syn _123-135 (sample 3); α-Syn _126-135 (sample 4); or vehicle control (sample 5). The level of α-Syn aggregation was measured by thioflavin-T (ThT) staining of the aggregates. Samples 1-4 were normalized to the vehicle control sample 5. Error bars represent the SEM (standard error of the mean) of each assay in duplicate.

In FIG. Figure 2 shows a graph showing the level of dissociation of preformed α-Syn aggregates in vitro after incubation of the aggregates for 3 days in the presence of antibodies directed against the C-terminal end of α-Syn (samples 1-3) or preimmune control serum ( sample 4). In particular, preformed α-Syn aggregates were incubated with anti-α-Syn antibodies, the formation of which is caused by: α-Syn _111-132 (sample 1); α-Syn _126-135 (sample 2); a combination of antibodies, the formation of which is caused by α-Syn _111-132 and α-Syn _126-135 (sample 3); or preimmune control serum (sample 4). The level of α-Syn aggregation was measured by thioflavin-T (ThT) staining of the aggregates. Samples 1-3 were normalized to the preimmune control serum of sample 4. Error bars represent the SEM (standard error of the mean) of each assay in duplicate.

In FIG. Figure 3 shows a graph showing the levels of α-Syn aggregation and α-Syn disaggregation in PC12 cells overexpressing α-Syn incubated with nerve growth factor (NGF) in the presence of antibodies directed against the C-terminal end of α-Syn (samples 1 -4) or vehicle control (sample 5). In particular, PC12 cells were incubated with anti-α-Syn antibodies, the formation of which is caused by: α-Syn _111-132 (sample 1); α-Syn _121-135 (sample 2); α-Syn _123-135 (sample 3); α-Syn _126-135 (sample 4); or vehicle control (sample 5). Samples 1-4 were normalized to the vehicle control of sample 5. Error bars represent the SD (standard deviation) of each study in triplicate.

In FIG. 4 is a graph depicting the levels of TNF-α and IL-6 release mediated by α-Syn aggregates from cells incubated in the presence of antibodies directed against the C-terminal end of α-Syn (samples 1-4) or vehicle control ( sample 5). In particular, microglial cells were incubated with anti-α-Syn antibodies, the formation of which is caused by: α-Syn _111-132 (sample 1); α-Syn _121-135 (sample 2); α-Syn _123-135 (sample 3); α-Syn _126-135 (sample 4); or vehicle control (sample 5). Samples 1-4 were normalized to vehicle control sample 5. Error bars represent the SD (standard deviation) of each study in three repetitions.

In FIG. 5A-5C show graphs that illustrate the effect of anti-α-Syn antibodies in an in vitro model of neurodegeneration using exogenous preformed α-Syn aggregates in NGF-induced PC12 cells differentiated into neurons. In FIG. Figure 5A shows an estimate of neurite length in PC12 cells treated with NGF alone (dark solid line); NGF with exogenous preformed α-Syn aggregates (dotted line); NGF with preimmune sera (light solid line); and NGF, both with exogenous preformed α-Syn aggregates and preimmune sera (dashed line). In FIG. Figure 5B shows an estimate of neurite length in PC12 cells treated with NGF along with vehicle (dark solid line); NGF with exogenous preformed α-Syn aggregates (dotted line); NGF with anti-α-Syn antibodies induced by α-Syn _111-132 (SEQ ID NO:113) (light solid line); and NGF, both with exogenous preformed α-Syn aggregates and anti-α-Syn antibodies induced by α-Syn _111-132 (SEQ ID NO:113) (dashed line). In FIG. Figure 5C shows an estimate of neurite length in PC12 cells treated with vehicle-only NGF (dark solid line); NGF with exogenous preformed α-Syn aggregates (dotted line); NGF with anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO:112) (light solid line); and NGF, both with exogenous preformed α-Syn aggregates and anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO:112) (dashed line).

In FIG. 6A-6B show graphs that illustrate the effect of anti-α-Syn antibodies on cell number and neurite length in an in vitro model of neurodegeneration using NGF-induced wild-type PC12 cells overexpressing α-Syn differentiated into neurons. Cells were treated with vehicle control (sample 1); anti-α-Syn antibodies, the formation of which is caused by α-Syn _101-132 (sample 2), α-Syn _111-132 (sample 3), α-Syn _121-135 (sample 4), α-Syn _123-135 ( sample 5), α-Syn _126-135 (sample 6), a combination of anti-α-Syn antibodies, the formation of which is caused by α-Syn _111-132 and α-Syn _126-135 (sample 7); or pre-immune control serum (sample 8). In FIG. 6A shows an assessment of the respective protective effects of each sample on PC12 cell count recovery. In FIG. Figure 6B shows an estimate of the neurite length of cells treated with each sample. Samples 1-8 were normalized to NGF-induced wild-type PC12 cells differentiating into neurons. The t-test was used to examine significance (p-value less than 0.05 was defined as statically significant and indicated with an asterisk (*)).

In FIG. 7A-7B demonstrate the ability of anti-α-Syn antibodies to recognize and bind to α-Syn aggregates of various sizes using Western blotting. In FIG. 7A shows a Western blot image comparing the commercially available anti-α-Syn antibody, Syn211 (lane 1); preimmune control serum (lane 2); anti-α-Syn antibody raised by Syn _111-132 (lane 3); anti-α-Syn antibody raised by Syn _111-135 (lane 4); anti-α-Syn antibody raised by Syn _121-135 (lane 5); anti-α-Syn antibody, the production of which is caused by Syn _123-135 (lane 6); and anti-α-Syn antibody, the production of which is caused by α-Syn _126-135 (lane 7). In FIG. 7B shows a bar graph depicting the relative ability of each antibody to bind to α-Syn molecular complexes of various sizes (including monomers, dimers, trimers, tetramers, and oligomers). The chemiluminescent signals of the Western blot bands shown in FIG. 7A were quantified and indicated in the bar graph of FIG. 7B .

In FIG. 8A-8C show dot blot images that illustrate that antibodies directed only against the C-terminal end of α-Syn recognize and bind to various α-Syn molecules (i.e., α-helical monomers, β-sheet monomers , β-sheet oligomers and β-sheet fibrils) rather than with the same molecules of other amyloidogenic proteins (i.e., Aβ1-42 and Tau441). In FIG. 8A shows a control sample demonstrating that antibodies purified from preimmune sera from guinea pigs did not exhibit detectable levels against any of all protein molecules analyzed. In FIG. 8B shows an assessment of the ability of the anti-α-Syn antibody raised by α-Syn _111-132 (SEQ ID NO:113) to recognize and bind to various α-Syn molecules, Aβ1-42 and Tau441 proteins. In FIG. 8C shows an assessment of the ability of the anti-α-Syn antibody raised by α-Syn _126-135 (SEQ ID NO:112) to recognize and bind to various α-Syn molecules, Aβ1-42 and Tau441 proteins.

In FIG. 9 shows a table showing the comparative binding affinities of antibodies directed against the C-terminal end of α-Syn to intracellular α-Syn in various PC12 cell lines as measured by positive signals in an immunohistochemical (IHC) assay. Specifically, the comparative binding affinities of anti-α-Syn antibodies raised by α-Syn _111-132 , α-Syn _121-135 , α-Syn _126-135 , or control preimmune serum were assessed in parental PC12 cells, blank control PC12 cells , wild-type PC12 cells overexpressing α-Syn, and PC12 cells overexpressing α-Syn with the A53T mutation upon NGF treatment.

In FIG. 10A-10C demonstrate that antibodies directed only against the C-terminal end of α-Syn bind to α-Syn in PD brain slices but not in healthy brain slices. In FIG. 10A demonstrates that α-Syn antibodies raised by α-Syn peptide immunogenic constructs and preimmune antibodies did not exhibit detectable immunoreactivity in a panel of normal human tissues, including brain sections. In FIG. 10B demonstrates immunoreactivity of antibodies directed against α-Syn aggregates in PD thalamic slices, indicated by an arrow. In FIG. 10C shows a table describing the immunoreactivity of antibodies directed against the C-terminal end of α-Syn and preimmune control serum to α-Syn aggregates in PD brain sections, as well as in healthy brain sections, determined by counting positive stains by microscopic observation.

In FIG. 11A-11B show graphs representing serum anti-α-Syn IgG levels in mouse models of PD after three immunizations with adjuvant alone (open circle) or peptide immunogens containing α-Syn _111-132 (open box); α-Syn _126-135 (filled circle); or a combination of α-Syn _111-132 and α-Syn _126-135 (shaded rectangle). In FIG. 11A demonstrates IgG levels in a mouse model with MPP ⁺ induction. In FIG. 11B demonstrates IgG levels in a mouse model inoculated with fibrillar α-Syn.

In FIG. 12A-12B show graphs representing the level of α-Syn in the peripheral circulation in mouse models of PD after three immunizations with adjuvant alone (open circle) or peptide immunogens containing α-Syn _111-132 (open box); α-Syn _126-135 (filled circle); or a combination of α-Syn _111-132 and α-Syn _126-135 (shaded rectangle). In FIG. 12A demonstrates α-Syn levels in a mouse model with MPP ⁺ induction. In FIG. 12B demonstrates α-Syn levels in a mouse model inoculated with fibrillar α-Syn.

In FIG. 13A-13B demonstrate the level of oligomeric α-Syn in brain samples from untreated healthy mouse models (lane 1) or PD mouse models (lanes 2-3) receiving three immunizations with either adjuvant alone (lane 2) or peptide immunogens, containing α-Syn _111-132 (lane 3). Untreated Balb/c mice are a healthy mouse model, while MPP+-induced mice are a PD mouse model. In FIG. 13A shows a Western blot showing the level of oligomeric α-Syn as well as GAPDH as a protein loading control in the samples. In FIG. 13B shows a graph comparing the relative levels of oligomeric α-Syn shown in the Western blot of FIG. 13A, after protein levels were normalized to GAPDH and the lysate ratio in the untreated healthy mouse model was further standardized to 1.00 for comparison.

In FIG. 14A-14G demonstrate oligomeric α-Syn and tyrosine hydroxylase levels in brain samples from untreated healthy mouse models (lane 1) or PD mouse models (lanes 2-4) receiving three immunizations with either adjuvant alone (lane 2) or peptide immunogens containing α-Syn _111-132 (lane 3); or α-Syn _126-135 (lane 4). Untreated FVB mice represent a healthy mouse model, while mice inoculated with fibrillar α-Syn represent a PD mouse model. In FIG. 14A shows a Western blot showing the levels of oligomeric α-Syn and tyrosine hydroxylase, as well as GAPDH as a protein loading control in ipsilateral substantia nigra lysates. In FIG. 14B shows a graph comparing the relative levels of oligomeric α-Syn shown in the Western blot of FIG. 14A , after protein levels were normalized to GAPDH levels. In FIG. 14C shows a graph comparing the relative levels of tyrosine hydroxylase protein shown in the Western blot of FIG. 14A , after protein levels were normalized to GAPDH levels. In FIG. 14D shows a Western blot showing the level of oligomeric α-Syn as well as GAPDH as a protein loading control in ipsilateral striatal lysates. In FIG. 14E shows a graph comparing the relative levels of oligomeric α-Syn shown in the Western blot of FIG. 14C after protein levels were normalized to GAPDH levels. In FIG. 14F shows a Western blot showing the level of oligomeric α-Syn as well as GAPDH as a protein loading control in contralateral striatal lysates. In FIG. 14G shows a graph comparing the relative levels of oligomeric α-Syn shown in the Western blot of FIG. 14E , after protein levels were normalized to GAPDH levels.

In FIG. 15A-15C show graphs depicting assessment of mouse motor function as measured by CatWalk™ XT in healthy mouse models (lanes 1-2), treated with saline (lane 1) or adjuvant alone (lane 2); or PD mouse models (lanes 3–5) immunized with either adjuvant alone (lane 3) or peptide immunogens containing α-Syn _126–135 (lane 4) or α-Syn _111–132 (lane 5). The t-test was used to examine significance (p-value less than 0.05 was defined as statically significant and indicated with an asterisk “*”). In FIG. 15A demonstrates evaluation of left hindlimb strut(s) in treated mice, with untreated FVB mice representing a healthy mouse model and mice inoculated with fibrillar α-Syn representing a PD mouse model. In FIG. 15B shows an assessment of running duration in treated mice, with untreated FVB mice representing a healthy mouse model and mice inoculated with fibrillar α-Syn representing a PD mouse model. In FIG. 15C shows an assessment of running duration in treated mice, with untreated Balb/c mice representing a healthy mouse model, while MPP+ induced mice representing a PD mouse model.

Fig. 16A-16H . In FIG. 16A demonstrates that PD-021514 (α-Syn _85-140 , weeks post immunization 08) recognizes α-Syn strain fibrils with maximum affinity. High binding to ribbons and fibrils-91 of the specified strain is observed. There is weak binding to oligomers and fibrils-65. There is weak binding to the α-Syn monomer and to fibrils lacking the C-terminal 30 amino acid residues (Fib-110). In FIG. 16B demonstrates that PD-021522 (α-Syn _85-140 , wk 13) binds to all strains/oligomers but not to monomers, with no clear concentration-dependent increase in signal observed. The antibody binds to fibrils lacking the C-terminal 30 amino acid residues (Fib-110). Thus, the epitope is not located in this region. In FIG. 16C demonstrates that PD-100806 (α-Syn _126-135 , wk post immunization 09) binds to all strains, with maximum affinity for ribbons. It binds native oligomeric α-Syn with lower efficiency. Almost no binding was observed with glutaraldehyde, dopamine cross-linked oligomers and monomeric a-syn. The antibody is likely directed against the C-terminal amino acid residues of a-syn 30, since it does not bind fibrils lacking the C-terminal 30 amino acid residues (Fib-110). In FIG. 16D demonstrates that the commercial Syn1 antibody (clone 42, BD bioscience) binds to all α-Syn strains and to oligomers except glutaraldehyde cross-links. It also binds to monomeric α-Syn. Its epitope is described to be located over residues 91-96/99. Accordingly, it binds fibrils lacking the C-terminal 30 amino acid residues (Fib-110). In FIG. 16E demonstrates that PRX002 recognizes fibrillar α-Syn with slightly higher affinity compared to monomeric α-Syn. In FIG. 16F shows a control for the antibody background generated in guinea pigs. In FIG. 16G shows a control for the Syn1 antibody background. In FIG. 16H shows a control for the PRX002 antibody background.

In FIG. 17A-17D demonstrate IHC analysis of the specificity of UNS antibodies for α-Syn in the basal ganglia of patients with dementia with Lewy bodies (DLB). The average percentage area of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 mm ² in the globus pallidus ( Figure 17A ), internal capsule ( Figure 17B ) and insular cortex ( Figure 17C ). Exemplary microscopic images obtained from immunostaining in the globus pallidus with each antibody are shown in FIG. 17D . UNS antibodies resulted in the detection of a higher percentage of α-Syn aggregate area in the globus pallidus (F(3,7)=1.550, p=0.284 by ANOVA), internal capsule (F(3,7)=1.356, p=0.332 s using ANOVA) and insular cortex (F(3,8)=2.050, p=0.195 using ANOVA). P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean+SD (error bars).

In FIG. 18A-18D demonstrate IHC analysis of the specificity of UNS antibodies for α-Syn in the basal ganglia of patients with Parkinson's disease dementia (PD). The average percentage area of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 mm ² in the globus pallidus ( Figure 18A ), internal capsule ( Figure 18B ) and insular cortex ( Figure 18C ) based on three PD cases. Exemplary microscopic images obtained from immunostaining are shown in FIG. 18D for globus pallidus. UNS antibodies resulted in the detection of a higher percentage of α-Syn aggregate area in the globus pallidus (F(3,18)=4.152, p=0.047 by ANOVA), internal capsule (F(3,8)=1.995, p=0, 1934 by ANOVA) and insular cortex (F(3,8)=0.4044, p=0.754 by ANOVA). A significantly higher percentage of α-Syn area was detected in PD100806 compared with NCL-L-ASYN (p=0.023 in PD100806 vs. NCL-L-ASYN; n=3). P<0.05 (*); P<0.01 (**); P<0.001 (***). Dunnett's test was used after one-way ANOVA. Data are shown as mean+SD (error bars).

Fig. 19A-19C: IHC analysis of the specificity of UNS antibodies for α-Syn in the basal ganglia of patients with multiple system atrophy (MSA). The average percentage area of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 mm² in the globus pallidus (Fig. 19A) and internal capsule (Fig. 19B) in three MSA cases. No pathology was detected in the insular cortex of patients with MSA and thus was not quantified. UNS antibodies resulted in the detection of a higher percentage of α-Syn aggregate area in the globus pallidus (F(3,8)=1.56, p=0.273 by ANOVA) and internal capsule (F(3,8)=1.126, p= 0.395 using ANOVA). Illustrative microscopic images obtained from immunostaining with each antibody are shown inFig. 19C in the case of the globus pallidus. P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean+SD (error bars).

In FIG. 20A-20E demonstrate IHC analysis of the specificity of UNS antibodies for α-Syn in the midbrain ganglia of patients with various synucleinopathies. The average percentage area of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 ^mm2 in the substantia nigra of patients with PD ( Figure 20A ), DLB ( Figure 20A). 20B ) and MSA ( Fig. 20C ). The percentage of area stained by each antibody was compared with the diagnostic antibody, NCL-L-ASYN. UNS antibodies resulted in the detection of a higher percentage of α-Syn aggregate area in the substantia nigra of MSA patients (F(3,8)=0.830, p=0.51 by ANOVA); DLB (F(3,7)=2.493, p=0.144 by ANOVA) and PD (F(3,7)=0.189, p=0.900 by ANOVA). Exemplary microscopic images obtained from immunostaining with each antibody are shown in FIG. 20D (MSA) and FIG. 20E (DLB). P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean+SD (error bars).

In FIG. 21A-21F demonstrate IHC analysis of the specificity of UNS antibodies for α-Syn in the white and gray matter of the temporal cortex of patients with various synucleinopathies. The average percentage area of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 mm ² in the cortical gray matter and subcortical white matter of patients with PD ( FIGS. 21A and 21D ), DLB ( FIGS. 21B and 21E ) and MSA ( FIGS. 21C and 21F ). The percentage of area stained by each antibody was compared with the diagnostic antibody, NCL-L-ASYN. P<0.05 (*); P<0.01 (**); P<0.001 (***). Dunnett's test was used after one-way ANOVA. Data are shown as mean+SD (error bars).

In FIG. 22A-22C demonstrate IHC analysis of the specificity of UNS antibodies for α-Syn in the cerebellum of patients with various synucleinopathies. The average percentage area of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 mm ² in the cerebellar white matter of patients with PD ( Figure 22A ), DLB ( Figure 22A). 22B ) and MSA ( Fig. 22C ). UNS antibodies resulted in the detection of a higher percentage of α-Syn aggregate area in MSA (F(3,8)=0.929, p=0.469 by ANOVA); DLB (F(3,6)=1.426, p=0.325 by ANOVA) and PD (F(3,6)=2.509, p=0.157 by ANOVA). The percentage of area stained by each antibody was compared with the diagnostic antibody, NCL-L-ASYN. P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean+SD (error bars).

In FIG. 23A-23B show exemplary images of immunostaining with each antibody of the substantia nigra ( FIG. 23A ) and globus pallidus ( FIG. 23B ) from the brains of control patients without disease. None of the UNS antibodies resulted in detection of any α-Syn pathology compared with the NCL-L-ASYN diagnostic antibody.

In FIG. 24A-24D demonstrate IHC analysis of the specificity of UNS antibodies against LB in the insular cortex of the basal ganglia of patients with DLB or PD. The average percentage area of immunopositive LBs detected with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 mm ² in the insular cortex of patients with PD ( Figure 24A ) and DLB ( Figure 24A). 24B ). The percentage of LB area is presented as the fraction of total α-Syn detected by each antibody. UNS antibodies resulted in a lower proportion of LB (or higher proportion of LN (Lewy neurites)) in the insular cortex of patients with DLB (F(3,7)=0.836, p=0.516 by ANOVA) and PD (F(3, 4)=0.913, p=0.510 using ANOVA). The percentage of area stained by each antibody was compared with the diagnostic antibody, NCL-L-ASYN. Exemplary microscopic images obtained from immunostaining with each antibody are shown in FIG. 24C (PD) and FIG. 24D (DLB). P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean+SD (error bars).

In FIG. 25A-25D demonstrate IHC analysis of the specificity of UNS antibodies for LB in the gray matter of the temporal cortex of patients with DLB or PD. The average percentage area of immunopositive LBs detected with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of 7.5 mm ² in the gray matter of patients with PD ( Figure 25A ) and DLB ( Figure 25A). 25B ). The percentage of LB area is presented as the proportion of total alpha-synuclein detected by each antibody. UNS antibodies resulted in the detection of a lower proportion of LB (or higher proportion of LN) in the gray matter of patients with PD (F(2,3)=1.983, p=0.282 by ANOVA) and DLB (F(3,7)=1.906 , p=0.217 using ANOVA). The percentage of area stained by each antibody was compared with the diagnostic antibody, NCL-L-ASYN. Exemplary microscopic images obtained from immunostaining with each antibody are shown in FIG. 25C (PD) and Fig. 25D (DLB). P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean+SD (error bars).

In FIG. 26A-26B show exemplary images of immunostaining with UNS and NCL-L-ASYN antibodies in the substantia nigra of the midbrain of patients with DLB ( Fig. 26A ) and PD ( Fig. 26B ). There is a higher detection of LN using UNS antibodies compared to NCL-L-ASYN.

In FIG. 27A-27C demonstrate cell-specific aggregation of α-Syn. Maximum projection resulted in overlay of confocal images of α-Syn aggregates obtained from the basal ganglia and midbrain of clinical cases with PD ( Figure 27A ), DLB ( Figure 27B ), and MSA ( Figure 27C ). Shown are α-Syn aggregates (PD062205, red) in neurons (HuD, green) in PD and DLB cases, but not MSA. α-Syn (PD062205) and HuD are indicated in the grayscale figures filed with this application; however, color copies are available upon request. Scale bars: 10 µM.

In FIG. 28A-28C demonstrate cell-specific aggregation of α-Syn. Maximum projection resulted in overlay of confocal images of α-Syn aggregates obtained from clinical cases of PD ( Figure 28A ), DLB ( Figure 28B ), and MSA ( Figure 28C ). α-Syn aggregates (PD062205, red) were rarely located in oligodendrocytes (Olig2, green) in MSA but not PD or DLB cases. α-Syn (PD062205) and Olig2 are indicated in the grayscale figures filed with this application; however, color copies are available upon request. Scale bars: 10 µM.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure relates to alpha-synuclein protein (α-Syn) peptide immunogenic constructs. This disclosure also relates to compositions containing peptide immunogenic constructs, methods of making and using said peptide immunogenic constructs, and antibodies generated by the peptide immunogenic constructs.

The disclosed peptide immunogenic constructs comprise a B cell epitope from α-Syn linked to a heterologous T helper (Th) cell epitope directly or via an optional heterologous spacer. The B cell epitope region of the peptide immunogenic constructs contains from about 10 to about 25 amino acid residues from the C-terminal end of α-Syn, corresponding to the sequence from approximately glycine at amino acid position 111 (G111) to approximately asparagine at amino acid position 135 (D135) of full-length α -Syn (SEQ ID NO: 1). The heterologous Th epitope region of peptide immunogenic constructs is derived from amino acid sequences derived from pathogenic proteins. The B cell epitope and Th epitope regions of the peptide immunogenic constructs function together when administered to a host to stimulate the production of antibodies that specifically recognize and bind to the B cell epitope region of the α-Syn constructs.

This disclosure also relates to compositions containing the disclosed peptide immunogenic constructs, including pharmaceutical compositions. The disclosed pharmaceutical compositions are capable of inducing an immune response and the production of antibodies against the disclosed peptide immunogenic constructs in the host. The disclosed compositions may contain one or a mixture of more than one of the disclosed peptide immunogenic compositions. In some embodiments, the compositions contain the disclosed peptide immunogenic constructs together with additional components, including carriers, adjuvants, buffers and other suitable reagents. In certain embodiments, the compositions comprise the disclosed peptide immunogenic constructs of a stabilized immunostimulatory complex with a CpG oligomer, which is optionally supplemented with an adjuvant.

This disclosure also relates to antibodies that are produced by a host that is immunized with the disclosed peptide immunogenic constructs. The disclosed antibodies specifically recognize and bind to the B cell epitope region of α-Syn peptide immunogenic constructs. The disclosed antibodies to α-Syn have an unexpectedly high cross-reactivity towards the β-sheet of α-Syn in the form of monomers, oligomers or fibrils. Based on their unique characteristics and properties, the disclosed antibodies are capable of providing an immunotherapeutic approach to target, identify and treat synucleinopathies.

This disclosure also relates to methods for making and using the disclosed peptide immunogenic constructs, antibodies and compositions. The disclosed methods offer low-cost production and quality control of peptide immunogenic constructs and compositions containing these constructs, which can be used in methods of preventing and treating synopathies.

This disclosure also includes methods for treating and/or preventing synucleinopathies using peptide immunogenic constructs and/or antibodies directed against peptide immunogenic constructs. In some embodiments, methods of treating and/or preventing synucleinopathies include administering to the host a disclosed peptide immunogenic construct. In certain embodiments, the compositions used in the methods contain the disclosed peptide immunogenic construct in the form of a stable immunostimulatory complex with negatively charged oligonucleotides, such as CpG oligomers, resulting from electrostatic association, which complexes are then supplemented, optionally, with mineral salts or oil as an adjuvant, to administration to patients with synucleinopathies. The disclosed methods also include dosage regimens, dosage forms, and routes of administration of peptide immunogenic constructs to a host at risk of developing or having synucleinopathies.

The section titles used herein are for organizational purposes only and are not intended to limit the subject matter described. All references or portions of references cited in this application are expressly incorporated by reference herein in their entirety for any purpose.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. Singular forms of a noun include plural forms unless the context clearly indicates otherwise. Likewise, the expression “or” is intended to include “and” unless the context clearly indicates otherwise. Thus, "comprising A or B" means including A or B, or A and B. It is also understood that all amino acid sizes and all molecular weights or molecular weights given for polypeptides are approximate and are intended to be descriptions. Although methods and materials similar or equivalent to the methods or materials described herein may be useful in the practice or investigation of the disclosed method, suitable methods and materials are described below. All publications, patent applications, patents and other literature references herein are incorporated by reference in their entirety. In the event of a conflict, this description, including explanations of terms, will control. In addition, the materials, methods and examples are for illustrative purposes only and are not intended to be limiting.

Peptide immunogenic constructs α-Syn

This disclosure provides peptide immunogenic constructs comprising a B cell epitope from α-Syn covalently linked to a heterologous T helper (Th) cell epitope directly or via an optional heterologous spacer.

The phrase "α-Syn peptide immunogenic construct" as used herein refers to a peptide containing (a) a B cell epitope having from about 10 to about 25 amino acid residues from the C-terminal end of α-Syn corresponding to the sequence from about glycine at amino acid position 111 (G111) to approximately asparagine at amino acid position 135 (D135) of full-length α-Syn (SEQ ID NO: 1); (b) heterologous Th epitope; and (c) an optional heterologous spacer.

In certain embodiments, the peptide immunogenic construct may be represented by the formulas:

(Th) _m -(A) _n -(C-terminal fragment of α-Syn)-X

or

(C-terminal fragment α-Syn)-(A) _n -(Th) _m -X,

Where

Th is a heterologous T helper epitope;

A is a heterologous spacer;

(α-Syn C-terminal fragment) is a B cell epitope having from about 10 to about 25 amino acid residues from the C-terminal end of α-Syn;

X represents α-COOH or α-CONH ₂ amino acids;

m is from 1 to about 4; And

n ranges from 0 to approximately 10.

The various components of the disclosed α-Syn peptide immunogenic construct are described below.

a. α-Syn and C-terminal fragments of α-Syn.

The term “α-Syn,” “alpha-synuclein,” “α-synuclein,” and the like, as used herein, refers to (a) the full-length α-Syn protein and/or (b) fragments thereof from any organism , which expresses α-Syn. α-Syn is characterized by extreme conformational diversity, which adapts to different conditions in the membrane-bound, cytosolic, and amyloid aggregation states, and performs various functions. In some embodiments, the α-Syn protein is human-derived. In certain embodiments, the full-length human α-Syn protein has 140 amino acids (Accession No. NP_000336) (SEQ ID NO: 1).

The phrase "C-terminal region" or "C-terminal end" of α-Syn as used herein refers to any amino acid sequence from the carboxy-terminal portion of α-Syn. In certain embodiments, the C-terminal region or C-terminal end of α-Syn refers to the amino acid sequence between residues 96-140, or fragments thereof, of α-Syn. The C-terminal region of α-Syn has a high content of prolines and negatively charged residues, which are common characteristics found in intrinsically disordered proteins, in order to maintain solubility. The C-terminal region of α-Syn is typically present in a random helical structure due to its hydrophobicity and high net negative charge. In vitro studies have shown that α-Syn aggregation can be induced by lowering the pH, resulting in the neutralization of these negative charges.

The phrase "C-terminal fragment of α-Syn" or "B cell epitope from the C-terminal end of α-Syn" as used herein refers to a portion of the full-length α-Syn sequence that contains from about 10 to about 25 amino acid residues from the C-terminal end of α-Syn, corresponding to the sequence from approximately glycine at amino acid position 111 (G111) to approximately asparagine at amino acid position 135 (D135) of full-length α-Syn. The C-terminal fragment of α-Syn is also referred to herein as the G111-D135 α-Syn peptide and fragments thereof. The various C-terminal α-Syn fragments described herein are designated by their amino acid positions relative to the full-length α-Syn sequence represented by SEQ ID NO: 1.

The amino acid sequences of the C-terminal fragments of α-Syn used in the α-Syn peptide immunogenic constructs were selected based on the number of design principles. Some of these principles will include the use of the α-Syn peptide sequence, which:

(i) does not share significant sequence homology with beta-synuclein (β-Syn) to prevent the formation of antibodies that cross-react with β-Syn, since β-Syn can bind to α-Syn and prevent its aggregation;

(ii) lacks the autologous T helper epitope in α-Syn to prevent the activation of autologous T cells that can lead to brain inflammation leading to meningococcal encephalitis, as previously described in clinical studies with the AN1792 vaccine targeting Aβ1 -42, for the treatment of Alzheimer's disease;

(iii) is contained in a region of α-Syn that is susceptible to conformational changes relative to its native form;

(iv) itself is non-immunogenic, since it is a molecule of the body's own;

(v) can be made immunogenic using a protein carrier or effective T-helper epitope(s);

(vi) when performing immunogenic and administering to the host:

(a) causes the formation of a high titer of antibodies directed against the α-Syn peptide sequence (B-cell epitope), and not against the protein carrier or effective T-helper epitope(s);

(b) causes the formation of a high titer of antibodies that react with the denatured β-sheet of α-Syn, in the form of monomers, oligomers or fibrils, in order to allow such antibodies to prevent aggregation of α-Syn, cause disaggregation of any α-Syn aggregates and lead to to remove toxic oligomers, aggregates and/or fibrils of α-Syn, thereby reducing or preventing the load of α-Syn aggregates in the brain;

(c) does not induce the formation of antibodies that react with native α-Syn, which would pose a major safety concern since native α-Syn is a major cellular protein with significant tissue distribution.

Taking into account these design principles, the C-terminal region of α-Syn was chosen as a target for the development of peptide immunogens. In addition, the C-terminal region of α-Syn was chosen because, based on its structural characteristics, this region appears to be most susceptible to modulation by antibody or other physical factors compared to other regions of α-Syn.

Evaluation of numerous peptide sequences derived from α-Syn, described below in the Examples section, led to the identification and selection of numerous α-Syn peptides that satisfy the design principles described above. In particular, sequences that satisfy the design principles include peptides having from about 10 to about 25 amino acid residues from the C-terminal region of α-Syn, corresponding to the sequence from about glycine at amino acid position 111 (G111) to about asparagine at position amino acid 135 (D135) of full-length α-Syn.

In some embodiments, the α-Syn C-terminal fragment is the 25 amino acid α-Syn peptide G111-D135 represented by SEQ ID NO: 12. In other embodiments, the α-Syn C-terminal fragment comprises approximately 10 contiguous α-peptide amino acids. Syn G111-D135, represented by SEQ ID NO: 12. In certain embodiments, the C-terminal fragment of α-Syn contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids of the G111-D135 peptide represented by SEQ ID NO: 12. In specific embodiments, the C-terminal fragment of α-Syn has the amino acid sequence represented by SEQ ID NO: 12-15, 17, or 49-64, as demonstrated in Table. 1 .

The C-terminal α-Syn fragment of this disclosure also includes immunologically functional analogs or homologues of the G111-D135 α-Syn peptide and fragments thereof. Functional immunological analogues or homologues of the G111-D135 α-Syn peptide and fragments thereof include variants that retain substantially the same immunogenicity as the parent peptide. Immunologically functional analogues may have a conservative substitution at an amino acid position; change in total charge; a covalent bond with another fragment; or addition, insertion or deletion of amino acids; and/or any combination thereof.

Conservative substitutions are performed when one amino acid residue is replaced by another amino acid residue with similar chemical properties. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparaginine and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Immunologically functional analogues include amino acid sequences that contain conservative substitutions, additions, deletions or insertions of one to about four amino acid residues that elicit immune responses that cross-react with the G111-D135 α-Syn peptide. Conservative substitutions, additions and insertions can be made using natural or non-naturally occurring amino acids. Non-naturally occurring amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, tyrosine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitrotyrosine, pyroglutamic acid, etc. Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryprophan, tyrosine and valine.

In one embodiment, a functional immunological analogue of a particular peptide contains the same amino acid sequence as the parent peptide and additionally contains three lysine residues (Lys-Lys-Lys) added to the amino terminus of the G111-D135 α-Syn peptide and B cell fragments thereof. epitope. In this embodiment, the inclusion of three lysine residues in the sequence of the parent peptide results in a change in the overall charge of the parent peptide, but does not change the function of the parent peptide.

In certain embodiments, the functional analogue of the C-terminal fragment of α-Syn has at least 50% identity to the parent amino acid sequence. In other embodiments, said functional analog has at least 80% identity to the parent amino acid sequence. In yet other embodiments, said functional analog has at least 85% identity to the parent amino acid sequence. In yet other embodiments, said functional analog has at least 90% or at least 95% identity to the parent amino acid sequence.

b. Heterologous T helper epitopes (Th epitopes)

This disclosure provides peptide immunogenic constructs comprising a B cell epitope from α-Syn covalently linked to a heterologous T helper (Th) cell epitope directly or via an optional heterologous spacer.

The heterologous Th epitope in the α-Syn peptide immunogenic construct results in enhanced immunogenicity of the C-terminal fragment of α-Syn, which facilitates the production of high titer specific antibodies directed against the optimized B cell epitope (i.e., the C-terminal fragment of α-Syn). Syn) with rational development.

The term “heterologous” as used herein refers to an amino acid sequence that is derived from an amino acid sequence that is not part of or homologous to the wild-type α-Syn sequence. Thus, a heterologous Th epitope is a Th epitope derived from an amino acid sequence that does not naturally occur in α-Syn (ie, the Th epitope is not the same as α-Syn). Because the Th epitope is heterologous to α-Syn, the naturally occurring amino acid sequence of α-Syn does not extend in either the N-terminal or C-terminal directions if the heterologous Th epitope is covalently linked to the C-terminal fragment of α-Syn. Syn.

A heterologous Th epitope as used herein can be any Th epitope that does not have an amino acid sequence naturally occurring in α-Syn. The Th epitope may have an amino acid sequence originating from any species (eg, human, pig, bovine, dog, rat, mouse, guinea pig, etc.). The Th epitope may also have a non-selective binding motif to MHC class II molecules of many species. In certain embodiments, the Th epitope contains multiple non-selective MHC class II binding motifs to ensure maximum activation of T helper cells, leading to the initiation and regulation of immune responses. The Th epitope is preferably immunosilent in nature, i.e., little or no antibodies generated by α-Syn peptide immunogenic constructs will be directed against the Th epitope, thereby providing a very focused immune response directed to the targeted B-cell epitope of the C-terminal fragment of α-Syn.

The Th epitopes of this disclosure include, but are not limited to, amino acid sequences derived from foreign pathogens, as exemplified in Table. 2 (SEQ ID NO: 70-98). In addition, Th epitopes include idealized artificial Th epitopes and combined idealized Th epitopes (eg, SEQ ID NOs: 71 and 78-84). Heterologous Th epitope peptides, presented as a combinatorial sequence (eg, SEQ ID NOs: 79-82), contain a mixture of amino acid residues presented at specific positions in the peptide backbone based on the variable residues of the homologs for that particular peptide. The assembly of combinatorial peptides can occur in a single process by adding a mixture of designed protective amino acids, instead of one specific amino acid, at a specific position during the synthesis process. The assembly of such peptides of combinatorial heterologous Th epitopes can provide broad coverage of Th epitopes for animals having different genetic backgrounds. Exemplary combinatorial peptide sequences of heterologous Th epitopes include SEQ ID NOs: 79-82, which are shown in Table. 2 . The Th epitope peptides of this invention provide significant reactivity and immunogenicity in animals and patients from genetically diverse populations.

α-Syn peptide immunogenic constructs containing Th epitopes are generated simultaneously in a single solid-phase peptide synthesis in tandem with the C-terminal fragment of α-Syn. Th epitopes also include immunological analogues of Th epitopes. Immunological Th analogs include immunostimulatory analogs, cross-reacting analogs, and segments of any of these Th epitopes that are sufficient to enhance or stimulate an immune response to the C-terminal fragments of α-Syn.

Functional immunological analogues of Th epitope peptides are also effective and are included as part of this invention. Functional immunological Th analogues may include conservative substitutions, additions, deletions and insertions of one to about five amino acid residues in the Th epitope, which do not necessarily result in a modification of the Th stimulatory function of the Th epitope. Conservative substitutions, additions and insertions can be made using natural or non-naturally occurring amino acids, as described above for the C-terminal fragments of α-Syn. In Table. 2, another variation of the functional analogue of the Th epitope peptide is defined. In particular, SEQ ID NOs: 71 and 78 MvF1 and Th MvF2 are functional analogues of SEQ ID NOs: 81 and 83 MvF4 and MvF5 in that they differ in the amino acid backbone by deletion (SEQ ID NOs: 71 and 78) or inclusion ( SEQ ID NO: 81 and 83) of two amino acids, each located at the N- and C-termini. Differences between these two series of similar sequences will not affect the function of the Th epitopes contained in the indicated sequences. Thus, functional immunological Th analogues include several versions of the Th epitope derived from the measles virus fusion protein MvF1-4 Ths (SEQ ID NOs: 71, 78, 79, 81 and 83) and from the hepatitis virus surface protein HBsAg 1 -3 Ths (SEQ ID NO: 80, 82 and 84).

The Th epitope in the α-Syn peptide immunogenic construct may be covalently linked to either the N- or C-terminal end of the C-terminal α-Syn peptide. In some embodiments, the Th epitope is covalently linked to the N-terminal end of the C-terminal α-Syn peptide. In other embodiments, the Th epitope is covalently linked to the C-terminal end of the C-terminal α-Syn peptide. In certain embodiments, more than one Th epitope is covalently linked to the C-terminal fragment of α-Syn. If more than one Th epitope is associated with the C-terminal fragment of α-Syn, each Th epitope may have the same amino acid sequence or different amino acid sequences. In addition, if more than one Th epitope is associated with the C-terminal fragment of α-Syn, then the Th epitopes can be arranged in any order. For example, Th epitopes may be sequentially linked to the N-terminal end of the C-terminal fragment of α-Syn, or sequentially linked to the C-terminal end of the C-terminal fragment of α-Syn, or the Th epitope may be covalently linked to the N-terminal end of the C-terminal fragment of α-Syn, while a separate Th epitope is covalently linked to the C-terminal end of the C-terminal fragment of α-Syn. There is no restriction on the structure of Th epitopes with respect to the C-terminal fragment of α-Syn.

In some embodiments, the Th epitope is covalently linked to the C-terminal fragment of α-Syn directly. In other embodiments, the Th epitope is covalently linked to the C-terminal fragment of α-Syn using a heterologous spacer, described in detail below.

c. Heterologous spacer

The disclosed α-Syn peptide immunogenic constructs optionally contain a heterologous spacer that covalently links a B cell epitope from α-Syn to a heterologous T helper (Th) epitope.

As discussed above, the term “heterologous” refers to an amino acid sequence that is derived from an amino acid sequence that is not part of or homologous to the wild-type α-Syn sequence. Thus, the naturally occurring amino acid sequence of α-Syn does not continue in either the N-terminal or C-terminal directions if the heterologous spacer is covalently linked to a B cell epitope from α-Syn, since the spacer is heterologous to the α sequence -Syn.

A spacer is any molecule or chemical structure capable of linking two amino acids and/or peptides together. The spacer may vary in length or polarity depending on the application. The spacer attachment can be via an amide or carboxyl bond, but other functional groups are also possible. The spacer may include a chemical compound, a naturally occurring amino acid, or a non-naturally occurring amino acid.

The spacer may provide structural properties to the α-Syn peptide immunogenic construct. Structurally, the spacer provides physical separation of the Th epitope from the B-cell epitope of the C-terminal fragment of α-Syn. Physical separation by a spacer may disrupt any artificial secondary structures created by binding of the Th epitope to the B cell epitope. In addition, physical separation of epitopes using a spacer may eliminate interference between Th cell and/or B cell responses. In addition, a spacer can be designed to create or modify the secondary structure of a peptide immunogenic construct. For example, a spacer may be designed to function as a flexible hinge to enhance separation of a Th epitope and a B cell epitope. The flexible hinge spacer can also provide more efficient interactions between the presented peptide immunogen and the corresponding Th cells and B cells to enhance immune responses to the Th epitope and B cell epitope. Examples of sequences encoding flexible hinges are found in the hinge region of immunoglobulin heavy chains, which often have a high proline content. One particularly useful flexible hinge that can be used as a spacer is represented by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 148), where Xaa is any amino acid, and preferably aspartic acid.

The spacer may also provide functionality to the α-Syn peptide immunogenic construct. For example, a spacer may be designed to change the overall charge of the α-Syn immunogenic peptide construct, which may affect the solubility of the immunogenic peptide construct. In addition, a change in the net charge of the α-Syn peptide immunogenic construct may affect the ability of the peptide immunogenic construct to associate with other compounds and reagents. As discussed in detail below, the α-Syn peptide immunogenic construct can be formed as a stable immunostimulatory complex with a highly charged oligonucleotide, such as CpG oligomers, via electrostatic association. The overall charge of the α-Syn immunogenic peptide construct is important for the formation of these stable immunostimulatory complexes.

Chemical compounds that can be used as a spacer include, but are not limited to, (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), 8-amino -3,6-dioxaoctanoic acid (AEEA, mini-PEG1), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (mini- PEG3), trioxatridecanesuccinic acid (Ttd), 12-aminododecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), etc.

Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryprophan, tyrosine and valine.

Non-naturally occurring amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, tyrosine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitrotyrosine, pyroglutamic acid, etc.

The spacer in the α-Syn peptide immunogenic construct may be covalently linked to either the N- or C-terminal end of the Th epitope and the C-terminal α-Syn peptide. In some embodiments, the spacer is covalently linked to the C-terminal end of the Th epitope and the N-terminal end of the C-terminal α-Syn peptide. In other embodiments, the spacer is covalently linked to the C-terminal end of the C-terminal α-Syn peptide and the N-terminal end of the Th epitope. In certain embodiments, more than one spacer may be used, for example, if more than one Th epitope is present in the peptide immunogenic construct. If more than one spacer is used, each spacer may be the same as the others or different. In addition, if more than one Th epitope is present in a peptide immunogenic construct, the Th epitopes may be separated by a spacer, which may be the same or different from the spacer used to separate the Th epitope from the B cell epitope. There is no restriction on the spacer structure with respect to the Th epitope or the C-terminal fragment of α-Syn.

In certain embodiments, the heterologous spacer is a naturally occurring amino acid or a non-naturally occurring amino acid. In other embodiments, the spacer contains more than one naturally occurring or non-naturally occurring amino acid. In specific embodiments, the spacer is Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

d. Specific options for implementing the α-Syn peptide immunogenic construct

The α-Syn peptide immunogenic construct can be represented by the formulas:

(Th) _m -(A) _n -(C-terminal fragment of α-Syn)-X

or

(C-terminal fragment α-Syn)-(A) _n -(Th) _m -X,

Where

Th is a heterologous T helper epitope;

A is a heterologous spacer;

(α-Syn C-terminal fragment) is a B cell epitope having from about 10 to about 25 amino acid residues from the C-terminal end of α-Syn;

X represents α-COOH or α-CONH ₂ amino acids;

m is from 1 to about 4; And

n ranges from 0 to approximately 10.

In certain embodiments, the heterologous Th epitope in the α-Syn peptide immunogenic construct has an amino acid sequence selected from any of SEQ ID NOs: 70-98 or combinations thereof shown in Table. 2 . In specific embodiments, the Th epitope has an amino acid sequence selected from any of SEQ ID NOs: 78-84. In certain embodiments, the α-Syn peptide immunogenic construct contains more than one Th epitope.

In certain embodiments, the optional heterologous spacer is selected from any of Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148 ) and their combinations. In specific embodiments, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

In certain embodiments, the C-terminal fragment of α-Syn has from about 10 to about 25 amino acid residues from the C-terminal end of α-Syn corresponding to the sequence from about glycine at amino acid position 111 (G111) to about asparagine at amino acid position 135 (D135 ) full-length α-Syn. In specific embodiments, the C-terminal fragment of α-Syn has the amino acid sequence represented by SEQ ID NO: 12-15, 17, or 49-64, as shown in Table. 1 .

In certain embodiments, the α-Syn peptide immunogenic construct has an amino acid sequence selected from any of SEQ ID NOs: 107-108, 111-113, and 115-147, as shown in Table. 3 . In specific embodiments, the α-Syn peptide immunogenic construct has an amino acid sequence selected from any of SEQ ID NOs: 107-108 and 111-113.

Compositions

This disclosure also provides compositions containing the disclosed α-Syn immunogenic peptide construct.

a. Peptide compositions

Compositions containing the disclosed α-Syn peptide immunogenic construct may be in liquid or solid form. Liquid compositions may include water, buffers, solvents, salts and/or any other suitable reagent that does not alter the structural or functional properties of the α-Syn peptide immunogenic construct. Peptide compositions may contain one or more of the disclosed α-Syn peptide immunogenic constructs.

b. Pharmaceutical compositions

This disclosure also relates to pharmaceutical compositions containing the disclosed α-Syn peptide immunogenic construct.

Pharmaceutical compositions may contain carriers and/or other excipients in a pharmaceutically acceptable delivery system. Accordingly, pharmaceutical compositions may contain a pharmaceutically effective amount of an α-Syn peptide immunogenic construct together with a pharmaceutically acceptable carrier, adjuvant and/or other excipients such as diluents, excipients, stabilizing agents, preservatives, solubilizing agents, buffers and the like.

The pharmaceutical compositions may contain one or more adjuvants that function to accelerate, prolong or enhance the immune response to the α-Syn peptide immunogenic construct without exerting any specific antigenic effect of its own. Adjuvants used in the pharmaceutical composition may include oils, aluminum salts, virosomes, aluminum phosphate (eg, ADJU-PHOS®), aluminum hydroxide (eg, ALHYDROGEL®), liposin, saponin, squalene, L121, Emulsigen®, monophosphoryl -lipid A (MPL), QS21, ISA 35, ISA 206, ISA50V, ISA51, ISA 720, as well as other adjuvants and emulsifiers.

In some embodiments, the pharmaceutical composition contains Montanide™ ISA 51 (an oil adjuvant composition consisting of vegetable oil and mannide oleate to produce water-in-oil emulsions), Tween(80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotide and/or any combination thereof.In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (ie, w/o/w) emulsion with emulsigen or emulsigen D as an adjuvant.

Pharmaceutical compositions can be formulated as immediate release or sustained release formulations. In addition, pharmaceutical compositions can be formulated to induce systemic or local mucosal immunity through immunogen capture and coadministration with microparticles. Such delivery systems are readily determined by one skilled in the art.

Pharmaceutical compositions can be prepared as injectable preparations, either as liquid solutions or suspensions. Liquid carriers containing the α-Syn peptide immunogenic construct may also be prepared prior to injection. The pharmaceutical composition may be administered by any suitable route of administration, for example, i.d., i.v., i.p., i.m., intranasally, orally, subcutaneously, etc., in any suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other routes of administration, including oral and intranasal routes, can also be prepared.

Pharmaceutical compositions can be formulated as immediate release or sustained release formulations. In addition, pharmaceutical compositions can be formulated to induce systemic or local mucosal immunity through immunogen capture and coadministration with microparticles. Such delivery systems are readily determined by one skilled in the art.

The pharmaceutical compositions can be readily formulated in a suitable unit dosage form. In some embodiments, the pharmaceutical compositions contain from about 0.5 μg to about 1 mg of α-Syn peptide immunogenic construct per kg of body weight. Effective doses of pharmaceutical compositions vary depending on many factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically the patient is a human, but non-human mammals, including transgenic mammals, may also receive treatment. When delivered in multiple doses, the pharmaceutical compositions can be divided into a suitable amount per unit dosage form in a standard manner. The dose administered will depend on the age, weight and general health of the subject, as is well known in the therapeutic fields.

In some embodiments, the pharmaceutical composition contains more than one α-Syn peptide immunogenic construct. A pharmaceutical composition containing a mixture of more than one α-Syn peptide immunogenic construct may provide a synergistic enhancement of the immunological efficacy of the constructs. Pharmaceutical compositions containing more than one α-Syn peptide immunogenic construct may be more effective in a more diverse genetic population due to broad MHC class II coverage, thereby providing an enhanced immune response to the α-Syn peptide immunogenic construct.

In some embodiments, the pharmaceutical composition contains an α-Syn peptide immunogenic construct selected from SEQ ID NOs: 107-108, 111-113, 115-147, as well as homologues, analogs and/or combinations thereof. In specific embodiments, the pharmaceutical compositions comprise an α-Syn immunogenic peptide construct selected from SEQ ID NOs: 107-108, 111-113, and any combination thereof.

Pharmaceutical compositions containing the α-Syn immunogenic peptide construct can be used to induce an immune response and result in the production of antibodies when administered to a host.

c. Immunostimulating complexes

This disclosure also relates to pharmaceutical compositions containing the α-Syn peptide immunogenic construct in the form of an immunostimulatory complex with a CpG oligonucleotide. Such immunostimulatory complexes are specifically adapted to function as an adjuvant and as a stabilizer of a peptide immunogen. The immunostimulatory complexes are in the form of a granule that can effectively present the α-Syn peptide immunogen to cells of the immune system to elicit an immune response. Immunostimulating complexes can be formulated as a suspension for parenteral administration. Immunostimulatory complexes can also be formulated as w/o emulsions, suspensions in combination with a mineral salt, or with an in-situ gelling polymer to effectively deliver the α-Syn peptide immunogen to host immune cells following parenteral administration. Immunostimulatory complexes are capable of leading to an immune response against the β-sheet of α-Syn (eg, Figures 8A, 8B and 8C from Example 13 ) with prophylactic/therapeutic effect.

The stabilized immunostimulatory complex can be formed by complexing the α-Syn peptide immunogenic construct with an anionic molecule, oligonucleotide, polynucleotide, or combinations thereof via electrostatic association. The stabilized immunostimulating complex can be included in a pharmaceutical composition in the form of an immunogen delivery system.

In certain embodiments, the α-Syn peptide immunogenic construct is designed to contain a cationic moiety that is positively charged at a pH in the range of 5.0 to 8.0. The net charge of the cationic portion of an α-Syn peptide immunogenic construct or mixture of constructs is calculated by assigning a charge of +1 for each lysine (K), arginine (R), or histidine (H), a charge of -1 for each aspartic acid (D), or glutamic acid (E) and a charge of 0 in the case of another amino acid in the sequence. The charges are summed in the cationic portion of the α-Syn immunogenic peptide construct and are expressed as the total average charge. A suitable peptide immunogen has a cationic moiety with a net positive charge of +1. Preferably, the peptide immunogen has a net positive charge in the range that is greater than +2. In some embodiments, the cationic portion of the α-Syn peptide immunogenic construct is a heterologous spacer. In certain embodiments, the cationic portion of the α-Syn peptide immunogenic construct has a charge of +4 when the spacer sequence is (α,ε-N)Lys,ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

The term "anionic molecule" as described herein refers to any molecule that is negatively charged at a pH in the range of 5.0-8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge of an oligomer or polymer is calculated by assigning a charge of -1 to each phosphodiester or phosphorothioate group in the oligomer. A suitable anionic oligonucleotide is a single-stranded DNA molecule of 8-64 nucleotide bases, the number of CpG motif repeats ranging from 1 to 10. Preferably, immunostimulatory single-stranded CpG DNA molecules contain 18-48 nucleotide bases, with the number of CpG motif repeats is in the range from 3 to 8.

More preferably, the anionic oligonucleotide is represented by the formula: 5(X ¹ CGX ² 3', wherein C and G are unmethylated; X ¹ is selected from the group consisting of A (adenine), G (guanine) and T (thymine); and X ² is is C (cytosine) or T (thymine). Or the anionic oligonucleotide is represented by the formula: 5((X ³ ) ₂ CG(X ⁴ ) ₂ 3', where C and G are unmethylated; X ³ is selected from the group consisting of A , T or G; and X ⁴ represents C or T.

The resulting immunostimulatory complex is in the form of particles with a size typically in the range of 1-50 microns and is a function of many factors, including the stoichiometry of the relative charge and the molecular weight of the interacting molecules. Granular immunostimulatory complex has the advantage of providing adjuvant and stimulating immune responses in vivo. In addition, the stabilized immunostimulatory complex is suitable for the preparation of pharmaceutical compositions through various processes, including water-in-oil emulsions, mineral salt suspensions and polymer gels.

Antibodies

This disclosure also provides antibodies raised by the α-Syn immunogenic peptide construct.

C-terminal α-Syn fragments having from about 10 to about 25 amino acid residues from the C-terminal end of α-Syn corresponding to the sequence from approximately glycine at amino acid position 111 (G111) to approximately asparagine at amino acid position 135 (D135) of full-length α -Syn are non-immunogenic or weakly immunogenic by nature. At the same time, the disclosed α-Syn peptide immunogenic constructs containing the C-terminal fragment of α-Syn, a heterologous Th epitope and an optional heterologous spacer are capable of inducing an immune response and leading to the formation of antibodies when administered to a host. The development of α-Syn peptide immunogenic constructs can break tolerance to intrinsic α-Syn and induce the formation of site-specific antibodies that recognize conformational, but not linear, epitopes.

Surprisingly, antibodies generated using α-Syn peptide immunogenic constructs do not bind to the naturally occurring alpha helix of the α-Syn monomer in its native form. In contrast, antibodies generated using α-Syn peptide immunogenic constructs recognize and bind to the denatured β-sheet of α-Syn in the forms of monomers, oligomers and fibrils. In addition, antibodies generated using α-Syn peptide immunogenic constructs do not bind to similar structures of other amyloidogenic proteins (ie, Aβ1-42 and Tau441). Thus, specific development of the α-Syn peptide immunogenic construct (containing the α-Syn C-terminal fragment, a heterologous Th epitope, and an optional heterologous spacer) appears to have altered the conformation of the various α-Syn C-terminal fragments so that to provide a β-sheet-like conformation.

In many functional assays, a variety of comparisons have been made between antibodies derived from immune sera from animals immunized with α-Syn peptide immunogenic constructs. These comparisons demonstrated that the antibodies bind to α-Syn in PC12 cells treated with nerve growth factor (NGF) with high specificity only to β-sheet monomers and oligomers of α-Syn, but not to other amyloidogenic protein molecules (see Example 9 ).

Antibodies raised by α-Syn peptide immunogenic constructs can unexpectedly prevent α-Syn aggregation (anti-aggregation activity) and can lead to dissociation of preformed α-Syn aggregates (disaggregation activity). Additionally, in an unexpected manner, the antibodies may result in a reduction in microglial cell-induced TNF-alpha and IL6 production, indicating that these antibodies may result in an effective reduction in α-Syn aggregates or fibril-induced microglial activation. These antibodies were also found to result in a reduction in neurodegeneration caused by both exogenous α-Syn aggregates and endogenous α-Syn aggregates in cells overexpressing α-Syn. In addition, such antibodies recognize and specifically bind to pathological oligomeric aggregates or fibrils of α-Syn, but do not react to non-pathological α-Syn. Specifically, the antibodies react with Lewy bodies from brain slices taken from alpha-synucleinopathies patients with Parkinson's disease, but not with normal human tissue.

Surprisingly, it was found that two mouse models of Parkinson's disease (MPP+ induction mouse model and α-Syn fibril inoculation mouse model) administered compositions containing α-Syn peptide immunogenic constructs (a) resulted in the formation of antibodies that were highly cross-reactive to the β-sheet of α-Syn, (b) had decreased serum levels of α-Syn, (c) had decreased levels of oligomeric α-Syn in the brain, and (d) had decreased neuropathology, resulting in restoration of motor function.

The resulting immune responses from animals immunized with the α-Syn peptide immunogenic constructs of this invention demonstrated the ability of the constructs to generate effective site-directed antibodies that react with the denatured β-sheet of α-Syn in the forms of monomers, oligomers and fibrils, but non-random helical structure of the C-terminal region of α-Syn in its native form.

In vitro functional assays

Antibodies generated by α-Syn peptide immunogenic constructs can be used in in vitro functional assays. These functional assays include, but are not limited to:

(a) inhibition of aggregation of recombinant α-Syn in vitro; and disaggregation of preformed aggregates of recombinant α-Syn (see Example 8 );

(b) inhibition of cellular α-Syn aggregation, and dissociation of preformed α-Syn aggregates in cells (see Example 9 );

(c) decreased secretion of TNF-alpha and IL6 in microglia (see Example 10 );

(d) reducing neurodegeneration caused by exogenous α-Syn aggregates (see Example 11 );

(e) reduction of neurodegeneration in cells overexpressing α-Syn (see Example 12 );

(f) evidence of in vivo efficacy in a mouse model of Parkinson's disease with fibrillar α-Syn inoculation and MPP+ induction, showing decreased serum α-Syn levels, decreased brain oligomeric α-Syn levels, decreased neuropathology, and restoration of motor activity ( see Example 15 ).

Methods

This disclosure also relates to methods of making and using α-Syn peptide immunogenic constructs, compositions and pharmaceutical compositions.

a. Methods for obtaining α-Syn peptide immunogenic construct

The α-Syn peptide immunogenic constructs of this disclosure can be prepared using chemical synthesis methods well known to one of ordinary skill in the art (see, e.g., Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, NY, 1992, p.77). α-Syn peptide immunogenic constructs can be synthesized using automated Merrifield solid-phase synthesis techniques using α-NH ₂ protected by either the t-Boc or F-moc chemical structure, using side-chain protected amino acids, e.g. Applied Biosystems Model 430A or 431 Peptide Synthesizer. The preparation of α-Syn peptide immunogenic constructs containing combinatorial library peptides for Th epitopes can be accomplished by obtaining a mixture of alternative amino acids for binding at a specific varying position.

Once the desired α-Syn peptide immunogenic construct is fully assembled, the resin can be processed according to standard procedures to separate the peptide from the resin and the functional groups on the amino acid side chains can be unlocked. The free peptide can be purified by HPLC and characterized biochemically, for example by amino acid analysis or sequencing. Methods for purifying and characterizing peptides are well known to one skilled in the art.

The quality of the peptides produced by this chemical process can be controlled and determined, and as a result, the reproducibility of α-Syn peptide immunogenic constructs, immunogenicity, and yield can be guaranteed. A detailed description of the preparation of the α-Syn peptide immunogenic construct using solid-phase peptide synthesis is demonstrated in Example 1.

It has been found that the range of structural variability that allows for retention of putative immunological activity is much more appropriate than the range of structural variability allowed for maintaining drug-specific activity with a small molecule drug or the desired activities and unwanted toxicities found in large molecules , which are formed together with drugs obtained biologically. Thus, peptide analogues, either intentionally designed or inevitably resulting from synthetic process errors as a mixture of deleted sequence byproducts that have chromatographic and immunological properties similar to the intended peptide, are often as effective as a purified preparation of the desired peptide. Designed analogues and unintended mixtures of analogues are effective as long as a QC procedure for recognition is developed to monitor both the manufacturing process and the product evaluation process to ensure the reproducibility and effectiveness of the final product using these peptides.

α-Syn peptide immunogenic constructs can also be produced using recombinant DNA technology, including nucleic acid molecules, vectors and/or host cells. Thus, nucleic acid molecules encoding the α-Syn immunogenic peptide construct and their immunologically functional analogs are also covered by this disclosure as part of the invention. Likewise, vectors, including expression vectors containing nucleic acid molecules, as well as host cells containing these vectors, are also covered by this disclosure as part of the present invention.

Various exemplary embodiments also cover methods for producing an α-Syn peptide immunogenic construct and immunologically functional analogues of peptide immunogenic constructs derived from the G111-D135 fragment of α-Syn. For example, the methods may include the step of incubating a host cell containing an expression vector containing a nucleic acid molecule encoding an α-Syn peptide immunogenic construct and/or an immunologically functional analog thereof under conditions such that the peptide and/or analog is expressed. Longer synthetic peptide immunogens can be synthesized using well-known recombinant DNA techniques. Such techniques are presented in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of the present invention, the amino acid sequence is reverse translated to produce a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimal for the organism in which the gene is to be expressed. The synthetic gene is then typically produced by synthesizing oligonucleotides that encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under appropriate conditions suitable for the selected expression system and host. The peptide is purified and characterized using standard methods.

b. Methods for obtaining immunostimulating complexes

Various exemplary embodiments also cover methods for preparing immunostimulatory complexes containing α-Syn peptide immunogenic constructs and a CpG oligodeoxynucleotide (ODN) molecule. Stabilized immunostimulatory complexes (ISCs) are derived from the cationic portion of the α-Syn peptide immunogenic construct and the polyanionic CpG ODN molecule. The self-assembling system is activated by electrostatic charge neutralization. The stoichiometry of the molar charge ratio of the cationic moiety of the α-Syn immunogenic peptide construct relative to the anionic oligomer determines the degree of association. The non-covalent electrostatic association of the α-Syn peptide immunogenic construct and the CpG ODN is a completely reproducible process. The peptide/CpG ODN immunostimulatory complex aggregates, which facilitate presentation to the “professional” antigen presenting cells (APCs) of the immune system, thus further enhance the immunogenicity of the complexes. These complexes are easily characterized for quality control upon receipt. Peptide/CpG ISC is well tolerated in vivo. This novel bead system containing CpG ODN and peptide immunogenic constructs derived from the G111-D135 fragment of α-Syn was developed to take advantage of the generalized B cell mitogenicity associated with the use of CpG ODN while promoting balanced Th-1/Th-2 type responses.

The CpG ODN in the disclosed pharmaceutical compositions is 100% bound to the immunogen in a process mediated by electrostatic neutralization of the opposing charge, resulting in the formation of micron-sized particles. The granular form allows for a significant reduction in CpG dosage compared to standard CpG adjuvants, reduces the potential for adverse innate immune reactions, and facilitates alternative immunogen processing pathways, including antigen presenting cells (APCs). Therefore, such formulations are novel in concept and offer potential benefits by promoting stimulation of immune responses through alternative mechanisms.

With. Methods for preparing pharmaceutical compositions

Various illustrative embodiments also include pharmaceutical compositions containing α-Syn peptide immunogenic constructs. In certain embodiments, the pharmaceutical compositions employ water-in-oil emulsions and suspensions with mineral salts.

With the aim of administering the pharmaceutical composition to a large population and with the aim of preventing α-Syn aggregation also being part of the purpose of administration, safety becomes another important factor to consider. Despite the use of oil-in-water emulsions in humans for many formulations in clinical studies, alum remains the primary adjuvant for use in formulations due to its safety. Alum or its mineral salts aluminum phosphate (ADJUPHOS) are thus often used as adjuvants when prepared for clinical applications.

d. Methods of using pharmaceutical compositions

This disclosure also includes methods of using pharmaceutical compositions containing α-Syn peptide immunogenic constructs.

In certain embodiments, pharmaceutical compositions containing α-Syn peptide immunogenic constructs can be used to:

(a) inhibiting α-Syn aggregation in the host;

(b) inducing disaggregation of preformed α-Syn aggregates in the host;

(c) reducing the secretion of TNF-alpha and IL6 in microglia in the host;

(d) reducing neurodegeneration caused by exogenous α-Syn aggregates in the host;

(e) reducing neurodegeneration in cells overexpressing α-Syn;

(f) reducing serum α-Syn levels in the host;

(g) reducing the level of oligomeric α-Syn in the host brain;

(h) reducing neuropathology and restoring motor activity in the host’s body, etc.

The above methods include administering a pharmaceutical composition containing a pharmacologically effective amount of an α-Syn peptide immunogenic construct to a host in need thereof.

Specific Embodiments

Specific embodiments of the present invention include, but are not limited to, the following.

(1) An alpha-synuclein (α-Syn) peptide immunogenic construct containing:

A B cell epitope comprising from about 10 to about 25 amino acid residues from the C-terminal fragment of α-Syn, corresponding to about amino acid G111 to about amino acid D135 SEQ ID NO: 1;

T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 70-98; And

an optional heterologous spacer selected from the group consisting of amino acid, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148),

wherein the B cell epitope is covalently linked to the T helper epitope directly or via an optional heterologous spacer.

(2) The α-Syn peptide immunogenic construct of (1) , wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 12-15, 17 and 49-63.

(3) The α-Syn peptide immunogenic construct of (1) , wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 81, 83 and 84.

(4) The α-Syn peptide immunogenic construct of (1) , wherein the optional heterologous spacer is (α, ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

(5) The α-Syn peptide immunogenic construct from (1) , where the T helper epitope is covalently linked to the amino terminus of the B cell epitope.

(6) The α-Syn peptide immunogenic construct of (1) , wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope via an optional heterologous spacer.

(7) The α-Syn peptide immunogenic construct from (1) containing the following formula:

(Th) _m -(A) _n -(C-terminal fragment of α-Syn)-X

or

(C-terminal fragment α-Syn)-(A) _n -(Th) _m -X,

Where

Th is a T helper epitope;

A is a heterologous spacer;

(C-terminal fragment of α-Syn) is a B-cell epitope;

X represents α-COOH or α-CONH ₂ amino acids;

m is from 1 to about 4; And

n ranges from 1 to approximately 10.

(8) The α-Syn peptide immunogenic construct of (1) , comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113 and 115-147.

(9) The α-Syn peptide immunogenic construct of (1) , comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108 and 111-113.

(10) A composition containing the α-Syn peptide immunogenic construct from (1) .

(11) A composition comprising more than one α-Syn peptide immunogenic construct from (1) .

(12) The composition of (11) , wherein the α-Syn peptide immunogenic construct has the amino acid sequences of SEQ ID NOs: 112 and 113.

(13) A pharmaceutical composition containing the peptide immunogenic construct α-Syn from (1) and a pharmaceutically acceptable delivery vehicle and/or adjuvant.

(14) Pharmaceutical composition from (13) where

a. the α-Syn peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113 and 115-147; And

b. the adjuvant is an aluminum mineral salt selected from the group consisting of Al(OH) ₃ or AlPO ₄ .

(15) Pharmaceutical composition from (13) where

a. the α-Syn peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113 and 115-147; And

b. the α-Syn immunogenic peptide construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

(16) An isolated antibody or epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogenic construct of (1) .

(17) An isolated antibody or epitope-binding fragment thereof from (16) bound to an α-Syn peptide immunogenic construct.

(18) An isolated antibody or epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogenic construct from (9) .

(19) A composition containing an isolated antibody or an epitope-binding fragment thereof from (16) .

(20) A composition containing an isolated antibody or an epitope-binding fragment thereof from (18) .

(21) A composition of (20) containing a mixture

a. an isolated antibody or epitope-binding fragment thereof that specifically binds to the B cell epitope of SEQ ID NO: 112; And

b. an isolated antibody or epitope-binding fragment thereof that specifically binds to the B cell epitope of SEQ ID NO: 113.

(22) A method for producing antibodies that recognize α-Syn in a host, comprising administering to the host a composition containing the α-Syn peptide immunogen from (1) and a delivery vehicle and/or an adjuvant.

(23) A method of inhibiting α-Syn aggregation in an animal, comprising administering a pharmacologically effective amount of the α-Syn peptide immunogen from (1) to the animal.

(24) A method of reducing the number of α-Syn aggregates in an animal, comprising administering a pharmacologically effective amount of the α-Syn peptide immunogen from (1) to the animal.

(25) A method for identifying α-Syn aggregates of various sizes in a biological sample, comprising:

a. exposing said biological sample to an antibody or epitope-binding fragment thereof according to (16) under conditions that allow the antibody or epitope-binding fragment thereof to bind to α-Syn aggregates; And

b. detection of the amount of antibody or its epitope-binding fragment associated with α-Syn aggregates in a biological sample.

A detailed description of the procedures used is offered in the next Examples section.

Example 1

Synthesis of peptides associated with alpha-synuclein and preparation of compounds based on them

a. Synthesis of C-terminal fragments of α-Syn

Methods for the synthesis of the developed C-terminal fragments of α-Syn, which were included in the program for the development of peptide immunogenic constructs of α-Syn, are described. Peptides were synthesized in small quantities that are suitable for serological assays, laboratory pilot and field studies, as well as in large (kilogram) quantities that are suitable for industrial/commercial production of pharmaceutical compositions. A large repertoire of α-Syn-related antigenic peptides, having sequences ranging from about 10 to 40 amino acids, was developed to screen and select the most optimal peptide constructs for use in an effective α-Syn peptide immunogenic construct.

Exemplary full-length α-Syn (SEQ ID NO:1) and β-Syn (SEQ ID No: 2), α-Syn segments such as α-Syn _111-132 , α-Syn _126-135 , 10-mer peptides and etc. used for epitope mapping in various serological assays are identified in Table. 1 (SEQ ID NO: 1 and 3-69). Selected α-Syn fragments were made into α-Syn peptide immunogenic constructs by synthetic binding to a carefully designed T helper (Th) epitope derived from pathogenic proteins, including the measles virus fusion protein (MVF), the surface antigenic protein of the hepatitis B virus (HBsAg), influenza virus protein, Clostridum tetani protein and Epstein-Barr virus (EBV) protein identified in Table. 2 (SEQ ID NO: 70-98). Th epitopes were used either in single sequences (SEQ ID NOs: 70-78 and 83-98) or in a combinatorial library (SEQ ID NOs: 79-82) to enhance the immunogenicity of their corresponding α-Syn peptide immunogenic constructs.

Exemplary α-Syn peptide immunogenic constructs selected from over 100 peptide constructs are identified in Table. 3 (SEQ ID NO: 99-147). All peptides used for immunogenicity studies or corresponding serological tests for detection and/or measurement of anti-α-Syn antibodies were synthesized in small quantities by F-moc chemical synthesis using Applied BioSystems Models 430A, 431, and/or 433 peptide synthesizers. Each peptide was prepared by independent synthesis on a solid phase support, with F-moc protection at the N-terminus and trifunctional amino acid side-chain protecting groups. The finished peptides were separated from the solid support and the side chain protecting groups were removed with 90% trifluoroacetic acid (TFA). Synthetic peptide formulations were assessed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to ensure appropriate amino acid content. Each synthetic peptide was also assessed using reverse phase HPLC (RP-HPLC) to confirm the synthetic profile and drug concentrations. Despite tight control of the synthesis process (including stepwise monitoring of binding efficiency), peptide analogues have also been produced as a result of unintended events during extension cycles, including insertions, deletions, amino acid substitutions, and premature termination. Thus, the synthesized drugs typically included numerous peptide analogs along with the target peptide. Despite the inclusion of such unintended peptide analogues, the resulting synthesized peptide preparations were nonetheless suitable for use in immunological applications, including immunodiagnostics (as antigen capture antibodies) and pharmaceutical compositions (as peptide immunogens) . Typically, such peptide analogues, either specifically designed or formed during the synthesis process as a mixture of by-products, are often as effective as a purified preparation of the desired peptide if the QC procedure for recognition is designed to be monitored as a manufacturing process , and the product evaluation process to ensure the reproducibility and effectiveness of the final product using the specified peptides. Peptide synthesis on a large scale in quantities of many kilograms was performed on a custom-made automatic peptide synthesizer UBI2003 or similar at levels from 15 mmol to 50 mmol. For the active ingredients used in the final pharmaceutical composition for clinical studies, α-Syn peptide constructs were purified by preparative RP-HPLC using a surface gradient in the eluent and characterized by MALDI-TOF mass spectrometry, amino acid analysis and RP-HPLC for purity and identity.

b. Preparation of compositions containing α-Syn peptide immunogenic constructs

Formulations were prepared using emulsions and suspensions such as oil in water with mineral salts. With the purpose of administering the developed pharmaceutical composition to a large population and for prophylactic purposes also being part of the purpose of administration, safety becomes another important factor to consider. Despite the use of oil-in-water emulsions in humans for many pharmaceutical formulations in clinical trials, alum remains the primary adjuvant for use in pharmaceutical formulations due to its safety. Alum or its mineral salts ADJUPHOS (aluminum phosphate) are thus often used as adjuvants when prepared for clinical applications.

Briefly, the formulations identified in each of the study arms described below generally contained all types of α-Syn peptide immunogenic constructs developed. Over 100 developed α-Syn peptide immunogenic constructs were initially evaluated in guinea pigs for their relative immunogenicity with the corresponding peptide representative of the α-Syn B epitope peptide immunogen, and to assess serological cross-reactivity patterns among various homologous peptides by plate-based ELISA. , coated with various peptides selected from peptides with SEQ ID NO: 1-153.

α-Syn peptide immunogenic constructs were prepared (i) in an oil-in-water emulsion with Seppic Montanide™ ISA 51 as an approved oil for human use, or (ii) mixed with ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum) mineral salts. in varying amounts of peptide constructs as described. The compositions are typically prepared by dissolving α-Syn peptide immunogenic constructs in water at about 20 to 800 μg/ml and formulated with Montanide™ ISA 51 in oil-in-water emulsions (1:1 v/v) or with mineral salts or ALHYDROGEL (alum) (1:1 by volume). The compositions were stored at room temperature for approximately 30 minutes and vortexed for approximately 10 to 15 seconds prior to immunization. Some animals were immunized with 2-3 doses of a specific composition, which was administered at time 0 (priming) and 3 weeks after the initial immunization (wk post-immunization) (boosting), optionally after 5 or 6 weeks. after immunization in the case of the second boost, using the intramuscular route. These immunized animals were then tested with the selected B epitope peptide(s) to evaluate the immunogenicity of the various α-Syn peptide immunogenic constructs present in the formulation, as well as their cross-reactivity with the corresponding target peptides or proteins. α-Syn peptide immunogenic constructs with high immunogenicity in the initial screening in guinea pigs were then further studied in both water-in-oil emulsion, mineral salts, and alum-based formulations in primates with respect to dosing regimens over a period of time according to immunization protocols.

Only the most promising α-Syn peptide immunogenic constructs were further carefully evaluated prior to inclusion in final formulations for immunogenicity, duration of use, toxicity, and efficacy studies in GLP-controlled preclinical studies in preparation of investigational new drug applications and clinical studies in patients with synucleinopathies.

Example 2

Preparation of recombinant alpha-synuclein protein

Cloning of the α-Syn gene in the pGEX-4T1 vector was previously described in Neurotoxicology and teratology 2004, 26 (3): 397-406. The target sequence (SEQ ID NO: 1) was included in the pGEX-4T1 vector between the BamHI and XhoI restriction sites. The indicated fragment was obtained by polymerase chain reaction (PCR) using KAPA HiFi DNA polymerase (Kapa Biosystems, Inc., Woburn, MA, USA). The primer sequences were as follows: forward primer, 5'-cgggatccgatgtgtttatgaaaggtctgag-3((SEQ ID NO: 149); reverse primer, 5'-ggaattccgatgtgtttatgaaaggtctgag-3((SEQ ID NO: 150). PCR condition was as follows: denaturation at 94° C for 1 minute followed by 30 cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds and extension at 68°C for 2 minutes, then termination after another 5 minutes at 68°C. Site-directed mutagenesis of A53T α-Syn was performed using the Q5 Site-Directed Mutagenesis Kit (New England BioLabs, Beverly, MA, USA).The primer sequences for mutant α-Syn were as follows: forward primer, 5′-tcatggtgtgaccaccgttgcag-3(( SEQ ID NO: 151); reverse primer, 5'-accacgccttctttggttttg-3((SEQ ID NO: 152).

α-Syn cloned into the pGEX-4T1 GST vector was transformed into E. coli BL21 (DE3) for protein expression. E. coli was cultured in LB broth at 37°C and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 4 mM when the OD600 value reached 0.8. After 4 hours of incubation, cells were collected by centrifugation at 5000 xg for 20 minutes at 4°C. The collected cells were resuspended in PBS, disrupted by sonication on ice, and then centrifuged at 5000 x g for 20 minutes. The supernatant fraction was loaded onto a glutathione Sepharose-4B column (GE Healthcare) equilibrated with PBS. After washing three times with PBS, 1 ml of thrombin (20 U/ml in PBS) was added for overnight digestion at 4°C to release GST from the fusion protein. The tag-free α-Syn was then eluted, followed by thrombin removal using a HiTrap FF benzamidine column (GE Healthcare). Dialyzed α-Syn was immediately frozen at -80°C. Purified α-Syn with an MW of 14 kDa was identified by Western blotting using an anti-α-Syn antibody (1:2000, Millipore, targeting α-Syn _111-131 ) after separation by 10% SDS-PAGE.

Example 3

Serological tests and reagents

Serological assays and reagents for assessing the functional immunogenicity of synthetic peptide constructs and formulations based on them are described in detail below.

a. Peptide ELISA for antibody specificity analysis

An ELISA for evaluating the immune serum samples described in the following Examples was developed and described below. Wells of 96-well plates were coated separately for 1 hour at 37°C with 100 μl of α-Syn target protein fragments with peptide A85-A140, A91-A140, A101-A140, A111-A140, D121-A140, E126-A140, K97-D135 , G101-D135, G111-D135, D121-D135, E123-D135, E126-D135, G101-132 and G111-G132 (SEQ ID NO: 4-17) at a concentration of 2 µg/ml (unless otherwise stated), in 10 mM NaHCO ₃ buffer, pH 9.5 (unless otherwise stated).

b. Evaluation of antibody reactivity to Th peptide using Th peptide ELISA

Peptide-coated wells (SEQ ID No: 70-98) were incubated with 250 μl of 3% by weight gelatin in PBS at 37°C for 1 hour to block nonspecific protein binding sites, followed by three washes with PBS containing 0 .05% by volume TWEEN(20, and dried. Sera to be analyzed were diluted 1:20 (unless otherwise stated) with PBS containing 20% by volume normal goat serum, 1% by weight gelatin and 0.05% by volume TWEEN(20). One hundred microliters (100 μl) of diluted samples (eg, serum, blood plasma) was added to each of the wells and allowed to react for 60 minutes at 37°C. The wells were then washed six times with 0.05% volume of TWEEN(20 in PBS to remove unbound antibodies. Horseradish peroxidase (HRP)-conjugated species-specific (e.g., mouse, guinea pig, or human) goat anti-IgG was used as a tracer to bind to the antigen antibody/peptide complex formed in positive wells. One hundred microliters of peroxidase-labeled goat anti-IgG at pre-titrated optimal dilution and 1% v/v normal goat serum with 0.05% v/v TWEEN(20 in PBS) was added to each well and incubated at 37°C for an additional 30 minutes. Wells were washed six times with 0.05% by volume TWEEN(20 in PBS to remove unbound antibody and reacted with 100 μl of a substrate mixture containing 0.04% by weight 3', 3', 5', 5- tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for an additional 15 minutes. This substrate mixture was used to detect the peroxidase label by producing a colored product. The reactions were stopped by adding 100 μl of 1.0 MH ₂ SO ₄ and absorbance was determined at 450 nm (A ₄₅₀ ). To determine the antibody titers of immunized animals that received various peptide immunogens derived from α-Syn, 10-fold serial dilutions of sera from 1:100 to 1:10,000 were tested and the titer of the test serum, expressed as Log ₁₀ , was calculated using linear regression A ₄₅₀ analysis with the A ₄₅₀ threshold set to 0.5.

c. Detailed specificity analysis and epitope mapping of α-Syn fragments by ELISA of 10-mer B cell epitope cluster peptides

A detailed analysis of the specificity of anti-α-Syn antibodies in immunized hosts was determined using epitope mapping. Briefly, wells of 96-well plates were coated with individual 10-mer α-Syn peptides (SEQ ID NO: 18-69) at a concentration of 0.5 μg per 0.1 ml per well, followed by 100 μl of serum samples (1:100 dilution in PBS) were incubated in duplicate wells of 10-mer plates according to the steps of the ELISA method described above. The B cell epitope of the α-Syn peptide immunogenic construct and the anti-α-Syn antibody immune sera from the corresponding detailed specificity assays in immunized hosts were also examined with the corresponding α-Syn peptides (SEQ ID Nos: 99, 102, 108, 110, 112, 113) or a fragment thereof without spacer and Th sequences or with β-Syn (SEQ ID NO: 153) to confirm additional reactivity and specificity.

d. Immunogenicity assessment

Preimmune and immune serum samples from animals were collected according to immunization protocols and heated at 56°C for 30 minutes to inactivate serum complement factors. Following administration of the pharmaceutical composition, blood samples were obtained in accordance with the protocols and their immunogenicity at the specific target site(s) was assessed. Serially diluted sera were assayed and positive titers were expressed as Log ₁₀ reciprocal dilution. The immunogenicity of a particular pharmaceutical composition was assessed by its ability to induce a high titer B cell antibody response directed against the specificity of the desired epitope in the target antigen, while maintaining low or negligible antibody reactivity towards the “T helper epitopes” used to enhance the required B -cellular responses.

e. Immunological analysis of α-Syn levels in mouse immune sera

Serum α - Syn levels in mice treated with α-Syn-derived peptide immunogens were measured by sandwich ELISA (Cloud-clon, SEB222Mu) using anti-α-Syn antibody as capture antibody and biotin-labeled anti-α-Syn antibody. Syn antibody as a detector antibody. Briefly, the antibody was immobilized on 96-well plates at a concentration of 100 ng/well in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6) and incubated at 4°C overnight. Coated wells were blocked with 200 μg/well of assay diluents (0.5% BSA, 0.05% TWEEN®-20, 0.02% ProClin 300 in PBS) at room temperature for 1 hour. The plates were washed 3 times with 200 μl/well wash buffer (PBS with 0.05% TWEEN®-20). Purified recombinant α-Syn was used to generate a standard curve (range 156 to 1250 ng/ml at 2-fold serial dilution) in assay diluent with 5% mouse serum. 50 μl of diluted sera (1:20) and standards were added to coated wells. Incubation was carried out at room temperature for 1 hour. All wells were aspirated and washed 6 times with 200 μl/well wash buffer. Captured human α-Syn was incubated with 100 μl of detection antibody solution (50 ng/ml biotin-labeled HP6029 in assay diluent) at room temperature for 1 h. Bound biotin-HP6029 was then detected using streptavidin poly-HRP (1:10,000 dilution, Thermo Pierce) for 1 hour (100 μl/well). All wells were aspirated and washed 6 times with 200 μl/well wash buffer, and the reaction was stopped by adding 100 μl/well 1 M H ₂ SO ₄ . A standard curve was generated using SoftMax Pro software (Molecular Devices) with four-parameter logistic curve fitting and was used to calculate α-Syn concentrations in all study samples. Student's t-tests were used to compare data using Prism software.

f. Preparation of α-Syn aggregates with recombinant α-Syn

To prepare aggregated α-Syn, purified wild-type or A53T mutant α-Syn [0.1 μg/μl in 100 μl PBS/KCl aggregation buffer (2.5 mM MgCl ₂ , 50 mM HEPES, and 150 mM KCl in 1 x PBS, pH 7.4)] were incubated at 37°C in 1.5 ml Eppendorf tubes for 7 days in a thermomixer (Eppendorf) without shaking. Aggregated α-Syn was immediately frozen at -80°C for later use.

g. Purification of anti-α-Syn antibodies

Anti-α-Syn antibodies were purified from sera collected 3-15 weeks post-injection (wk p.i.) from guinea pigs immunized with α-Syn peptide immunogenic constructs containing peptides of various sequences (SEQ ID NO: 99-121) with using an affinity column (Thermo Scientific, Rockford). Briefly, after equilibration of the buffer (0.1 M phosphate and 0.15 M sodium chloride, pH 7.2), 400 μl of serum was added to the protein G Nab spin column, followed by bottom-up and top-bottom mixing for 10 minutes and centrifugation at 5800 x g for 1 minute. The column was washed three times with binding buffer (400 μl). Elution buffer (400 μL, 0.1 M glycine, pH 2.0) was then added to the spin column to elute the antibodies after centrifugation at 5800 x g for 1 minute. The eluted antibodies were mixed with neutralization buffer (400 μl, 0.1 M Tris, pH 8.0) and the concentrations of these purified antibodies were measured using Nan-Drop at OD280, using BSA (bovine serum albumin) as the standard.

h. Specificity of anti-α-Syn antibodies purified from guinea pig antisera immunized with various α-Syn peptide immunogenic constructs of various sizes

Western blot was used to screen anti-α-Syn antibodies from guinea pig antisera immunized with various α-Syn peptide immunogenic constructs for binding specificity to different sizes of α-Syn molecular complex. 20 μM α-Syn was resolved on 12% Tris-glycine SDS-PAGE and transferred to a nitrocellulose (NC) membrane before photoinduced cross-linking (PICUP) treatment. The membrane was incubated with anti-α-Syn antibodies purified from guinea pig antisera at a concentration of 1 μg/ml and then incubated with HRP-conjugated donkey anti-guinea pig antibody (706-035-148, Jackson). The blot was visualized using ECL Pro Western Lightning chemiluminescent reagent (PerkinElmer). As a result, monomeric α-Syn (MW 14460 Da) was blotted at a size of about 14 kDa, while the dimer, trimer or oligomers had their molecular weights several times larger than the 14 kDa monomeric α-Syn. A commercial antibody capable of detecting various oligomer molecules, such as dimers, trimers, and larger oligomers, Syn211 (Abcam), was used as a positive control.

i. Dot blot analysis using various amyloidogenic protein molecules

The preparation of α-helical monomers, β-sheet monomers, β-sheet oligomers and β-sweet fibrils of Aβ _1-42 , Tau and α-Syn is described below.

1. α -Helical Monomers Aβ _1-42 : 20 µg β-sheet monomers Aβ _1-42 (50 µl) was added to 1x PBS containing 20% trifluoroacetic acid and 20% hexafluoroisopropanol (10 µl) and incubated at 4°C within 24 hours to form α-helical monomers.

2. Aβ _1-42 β -pleated monomers : 60 μg Aβ _1-42 in 120 μl 1x PBS containing 5% TFA aggregated at 37°C for 24 hours, transferred to a 10 kDa cutoff filter (Millipore) and recovered β-sheet monomers.

3. β -Fold oligomers of Aβ _1-42 : 60 μg of Aβ _1-42 in 120 μl of 1x PBS aggregated at 37°C for 3 days, sonicated on ice and transferred to 10 and 30 kDa cut-off filters (Millipore) with by extracting β-sheet oligomeric fibrils less than 35 kDa.

4. Aβ _1-42 β -pleated fibrils : 60 μg Aβ _1-42 in 120 μl 1x PBS aggregated at 37°C for 3 days, sonicated on ice and transferred to 30 kDa cut-off filters (Millipore) to isolate β - folded fibrils.

5. α-helical α -Syn monomers : 40 μg of freshly prepared α-Syn was dissolved in 100 μl of cold 1x PBS at 4°C and immediately transferred to a 10 kDa cut-off filter (Millipore) to recover the α-helical monomers.

6. α -Syn β -sheet monomers : 40 μg α-Syn incubated in 100 μl PBS/KCl buffer at 37°C for 24 hours was transferred to a 10 kDa cut-off filter (Millipore) to recover β-sheet monomers.

7. β -Sheet oligomers of α -Syn : 40 μg of α-Syn aggregated in 100 μl of PBS/KCl buffer at 37°C for 8 days, sonified on ice and then transferred to 30 and 100 kDa cut-off filters (Millipore) with extraction of β-sheet oligomers.

8. α -Syn β -pleated fibrils : 40 μg α-Syn aggregated in 100 μl PBS/KCl buffer at 37°C for 8 days, sonified on ice and then transferred to 30 and 100 kDa cut-off filters (Millipore) with by isolating β-pleated fibrils. α- helical monomers Tau441 : 60 μg Tau in 100 μl 1x PBS at 4°C and transferred to a 100 kDa cut-off filter (Millipore) to recover α-helical monomers.

9. β -pleated Tau441 monomers : 60 μg Tau aggregated in 100 μl 1x PBS with 10 U/ml heparin at 25°C for 48 hours, transferred to a 100 kDa cutoff filter (Millipore) at 4°C to recover β -folded monomers.

10. Tau441 β-pleated oligomers : 60 μg Tau aggregated in 100 μl 1x PBS with 10 U/ml heparin at 37°C for 48 hours, transferred to 100 and 300 kDa cut-off filters (Pall) at 4°C with extraction of β-sheet oligomers.

11. Tau441 β-pleated fibrils : 60 μg Tau aggregated in 100 μl 1x PBS with 10 U/ml heparin at 37°C for 6 days, transferred to 300 kDa cut-off filters (Pall) at 4°C to release β - folded fibrils.

These monomers and oligomers were verified using thioflavin-T fluorescence (ThT, Sigma) or PAGE (polyacrylamide gel electrophoresis). Amyloidogenic protein concentrations were measured by Nano-Drop using a commercial amyloidogenic working solution Aβ _1-42 as a standard. These monomers and oligomers were determined individually on PVDF membranes with an amount of 3 μg in the case of Aβ _1-42 , 4 μg in the case of α-Syn and 7 μg in the case of Tau. Membranes were incubated with anti-α-Syn antibodies purified from guinea pig antisera (1:1000 dilution) as the primary antibody, followed by hybridization with an HRP-conjugated secondary anti-guinea pig antibody (1:5000; Vector Laboratories). Membranes were treated with Luminata Western HRP substrates (Bio-Rad, Hercules, CA, USA) and signals were recorded using a ChemiDoc-It 810 digital imaging system (UVP Inc., Upland, CA, USA).

j. Specificity of binding to aggregated α-Syn in PC12 cells overexpressing α-Syn upon treatment with nerve growth factor (NGF)

Immunocytochemical analysis (ICC) using anti-α-Syn antibodies purified from guinea pig antisera collected after 8 or 9 weeks. after immunization against parental PC12 cells, empty control PC12 cells, and PC12 cells overexpressing α-Syn after NGF treatment were performed to assess the binding affinity of antibodies induced following immunization. Cell nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Photographs were taken using a fluorescence microscope, and the ratio of the number of positively stained cells to the total number of cells was categorically assessed using -, +, ++ and +++, which is <1%, 1-15%, 16-50%, >50 %.

Example 4

Cells and animals used in immunogenicity and efficacy studies

a. PC12 cells overexpressing α-Syn

Plasmid pZD/XOL-L-α-Syn was constructed by inserting a cDNA sequence encoding full-length wild-type human α-Syn or α-Syn with the A53T mutation into the pZD/XOL-L vector with the CMV promoter. Constructs were transfected into PC12 cells using Lipofectamine LTX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's procedure. 2.5 μl of transfection mixture, 500 μl of Opti-MEM medium, 2.5 μl of PLUS reagent and 8.75 μl of Lipofectamine LTX were mixed and then incubated for 25 minutes at room temperature. After replacing the culture medium with 1.5 ml of RMPI 1640 cell culture medium, 500 μl of the transfection mixture was added directly to each well, followed by incubation at 37°C for one day. Transfection efficiency was confirmed by PCR and Western blotting.

b. Guinea pigs

Immunogenicity studies were performed on mature, naive, adult male and female Duncan-Hartley guinea pigs (300-350 g/body weight). A minimum of 3 guinea pigs per group were used in the experiments. Protocols using Duncan-Hartley guinea pigs (8–12 weeks of age; Covance Research Laboratories, Denver, PA, USA) were performed in accordance with approved IACUC applications in a contract vivarium, as well as with UBI as the sponsor.

c. Mouse model of Parkinson's disease inoculated with fibrillar α-Syn

Female FVB mice (weight range 25–30 g) were maintained on a 12-h light:12-h dark cycle and cared for according to approved AAALAC guidelines. Fibrillar α-Syn was prepared by incubating α-Syn peptides (5 mg/ml) at 37°C in 0.1% PBS containing NaN ₃ /high-concentration KCl buffer without shaking for 7 days. Fibrillation was monitored by measuring ThT fluorescence and confirmation was made when the signal increased more than 3-fold compared to the original α-Syn monomer. Western blotting was also used to confirm α-Syn aggregation prior to inoculation into the substantia nigra on one side (antero-posterior; -3.0 mm; medial-lateral: -1.3 mm; dorsal-ventral: -4.7 mm from bregma and dura mater) and dorsal neostriatum (antero-posterior; +0.2 mm; medial-lateral: -2 mm; dorsal-ventral: -3.2 mm from bregma and dura mater) of animals anesthetized with isoflurane.

d. Mouse model of Parkinson's disease with MPP+ induction

Female Balb/c mice (weight ranging from 18–20 g) were maintained on a 12-hour light:12-hour dark cycle and cared for according to approved AAALAC guidelines. MPP+iodide (Sigma, St. Louis, MO) was dissolved in saline and injected with 10 μl of a solution containing 18 μg MPP+iodide (0.8 mg/kg) into the ventricle on one side of the anesthetized animal. The stereotaxic coordinates of the injection site were: bregma -1.0 mm, lateral 1.0 mm, depth 2.0 mm.

Example 5

Principle of development, screening, identification and optimization of multicomponent pharmaceutical compositions including alpha-synuclein peptide immunogenic constructs

a. Development history

Each peptide immunogenic construct or α-Syn immunotherapy product requires its own individual targeting and approach based on the specific disease mechanism and target protein(s) required for intervention. The targets that are subsequently modeled for development may include cellular proteins involved in a disease pathway or an infectious agent that may involve multiple proteins from the pathogen. The process from research to commercialization is very lengthy, typically taking one or more decades to complete.

Once a target molecule is selected, a significant serological validation process is required. The identification and distribution of B cell and T cell epitopes in the target molecule is important for the development of the α-Syn peptide immunogenic construct. Once the target B cell epitope is recognized, sequential immunogenicity pilot studies are conducted in small animals to evaluate the functional properties of antibodies induced by pharmaceutical formulations of the designed peptides. This serological application is then carried out in animals of the target species to further test the immunogenicity of the α-Syn peptide immunogenic construct and the functional properties of the antibodies that are induced. All studies are conducted in multiple parallel groups with sera collected from immunized hosts for evaluation. Early immunogenicity studies in target species or non-human primates for pharmaceutical compositions for human use are also conducted to further validate immunogenicity and guide development. The target peptides are then prepared in various mixtures to evaluate the subtle differences in functional property associated with the respective interactions between the peptide constructs when used in combinations to obtain appropriate formulation designs. After additional evaluations, the final peptide constructs, peptide compositions and formulations based on them are established, along with the corresponding physical parameters of the formulations, leading to the final product development process.

b. Development and validation of peptide immunogenic constructs derived from α-Syn for pharmaceutical compositions with the potential to treat patients with synucleinopathies

In order to obtain the most effective peptide constructs for inclusion in pharmaceutical compositions, a significant repertoire of non-selective T helper epitopes derived from various pathogens or artificial T helper epitopes further developed from the sequence of the Measles Virus Fusion protein (MVF) or protein surface antigen of the hepatitis B virus (HBsAg - Hepatitis B Surface Antigen protein) was included in immunogenicity studies in guinea pigs. Illustrative study of peptide constructs derived from α-Syn _126-140 , α-Syn _121-140 , α-Syn _111-140 , α-Syn _101-140 , α-Syn _91-140 , α-Syn _85-140 , α -Syn _121-135 , α-Syn _111-135 , α-Syn _101-135 , α-Syn _97-135 , α-Syn _123-135 , α-Syn _126-135 , α-Syn _111-132 and α- Syn _101-132 , demonstrated in Table. 3 (SEQ ID NO: 99-121), wherein the α-Syn peptide was linked using εK and/or KKK as spacer(s) to individual non-selective T helper epitopes.

i) Selection of the C-terminal part of α-Syn as a target for the development of peptide immunogens.

α-Syn is an intrinsically disordered protein. It consists of 140 amino acids and is divided into three sections. The N-terminal region (residues 1–60) is capable of forming an amphipathic helix, which is a typical conformation for membrane recognition and association. The central region containing residues 61–95 is well known as the non-amyloid β-component (NAC), first identified in AD senile plaques. This region has a high propensity to form a β-sheet-rich conformation and is extremely susceptible to aggregation. Different types of post-translational modifications in this region show different effects on modulating α-Syn aggregation. The C-terminal region from residues 96-140 has a high content of proline and negatively charged residues, which are a common characteristic found in intrinsically disordered proteins, in order to maintain solubility. This C-terminal domain is present in a random helical structure due to its hydrophobicity and high net negative charge. In vitro studies have shown that α-Syn aggregation can be induced by lowering the pH, which neutralizes these negative charges. α-Syn exhibits extreme conformational diversity that adapts to different conditions in membrane-bound, cytosolic, and amyloid aggregation states and performs diverse functions. With considerable consideration, the C-terminal random loop and intrinsically disordered region, important for maintaining protein solubility, was chosen as a target for peptide immunogen development because this region was most susceptible to modulation by antibody or other physical factors, compared to the N-terminal amphipathic helix and central regions with a high content of β-sheets.

ii) Identification of autologous Th epitopes for exclusion in the development of the α-Syn B epitope.

Preliminary immunogenicity analysis confirmed the presence of a structural feature of the T helper epitope(s) at the C terminus of α-Syn, where deletion of a peptide sequence from the N terminus of the α-Syn sequence rendered α-Syn peptides _126-140 (SEQ ID NO: 9), α-Syn _121-140 (SEQ ID NO: 8), α-Syn _111-140 (SEQ ID NO: 7) are completely non-immunogenic, while some mild immunogenicity was observed in the case of α-Syn peptides _101-140 (SEQ ID NO: 6), α-Syn _91-140 (SEQ ID NO: 5) and α-Syn _85-140 (SEQ ID NO: 4) ( Table 4 ), indicating the presence of potential autologous Th -like structure in the C-terminal sequence. Inclusion of such a sequence in the design of the B epitope(s) could potentially induce brain inflammation during boost immunization through activation of autologous T cells, as in the previous AN1792 patent for an Alzheimer's disease vaccine. Thus, this result requires the development of α-Syn peptide immunogenic constructs with B cell epitope(s) starting at amino acid residue G111 to eliminate any possibility of inclusion of autologous T cell epitope(s) (epitopes) when developing the B-epitope.

iii) Ranking of heterologous T helper epitopes and their incorporation into α-Syn peptide immunogenic constructs to restore and enhance the immunogenicity of the selected α-Syn B epitope peptide.

In Table. 2 presents a total of 29 heterologous Th epitopes (SEQ ID NO: 70-98) that have been studied in our group for their relative potency in many species such as mice, rats, guinea pigs, baboons, macaques, etc. ., to enhance the immunogenicity of B-cell epitopes. As demonstrated in Table. 5 , both T cell epitopes UBITh1 (SEQ ID NO: 83) and UBITh2 (SEQ ID NO: 84), derived from the MvF protein, can potentiate the non-immunogenic peptide α-Syn101-140 (SEQ ID NO: 6) to significant and moderate immunogenicity, respectively. Extensive research on numerous peptide immunogenic constructs derived from α-Syn was performed to provide a ranking of the relative immunogenicity among these immunogenic constructs. Similar immunopotentiating activity occurs with UBITh3 (SEQ ID NO: 81) when it is covalently linked via a spacer to various C-terminal α-Syn peptides (SEQ ID NO: 4-9), as demonstrated in Table 1. 6 , when tested in ELISA using a plate coated with the long peptide α-Syn A91-A140 (SEQ ID NO: 5).

iv) Evaluation of the immunogenicity of α-Syn C-terminal peptide immunogenic constructs in relation to their antibody reactivity with the corresponding α-Syn and β-Syn.

The synuclein family includes three known proteins: α-Syn, β-Syn, and gamma-synuclein. All synucleins share as a common property a highly conserved alpha-helical lipid-binding motif with similarity to the class A2 lipid-binding domains of interchangeable apolipoproteins. β-Syn is highly homologous to α-Syn. β-Syn is thought to be an inhibitor of α-Syn aggregation, which occurs in neurodegenerative diseases such as Parkinson's disease. Thus, β-Syn may protect the central nervous system from the neurotoxic effects of α-Syn. Thus, it is preferred that α-Syn peptide immunogenic constructs elicit antibodies that preferentially react with α-synuclein rather than the corresponding anti-aggregation β-Syn. In a study of six peptide immunogenic constructs, all of which were C-terminated with A140, all of the antibodies derived from the immune sera of these constructs showed significant cross-reactivity with β-Syn of the appropriate size, as demonstrated in Table. 6 . After careful examination of the sequence homology between α-Syn and β-Syn (SEQ ID NO: 1 and 2), it was demonstrated that the sequence corresponding to the C-terminal five amino acids of YEPEA was identical for the two proteins. Thus, it is desirable to develop a B epitope(s) excluding the sequence containing the five amino acids of YEPEA. Thus, the result based on the immunogenicity studies shown in Table. 6 resulted in the deletion of YEPEA (Y136 to A140) during the development of the B epitope(s). By incorporating a spacer sequence and, for example, an artificial T helper peptide UBITh1 (SEQ ID NO: 83) into an α-Syn peptide immunogenic construct using B cell epitope sequences excluding the YEPEA tail, as demonstrated with α-Syn peptide immunogens (SEQ ID NO: 107-114) in Table. 7 , all became highly immunogenic when evaluated with the long peptide α-Syn K97-A140 (SEQ ID NO: 110). None of the immune sera reacted with β-Syn. Based on the data obtained from Table. 6 and 7 , B-epitope development for peptide immunogenic constructs will thus be limited to G111-D135 α-Syn and fragments thereof.

v) Antibodies elicited by α-Syn peptide immunogenic constructs reacted exclusively with the beta-sheet monomer, oligomer, or fibril, but not with the α-helical monomer.

Although we used sound principles in our development of the α-Syn peptide immunogens, it was unexpected to find that antibodies derived from the designed α-Syn peptide immunogenic constructs with B epitopes having their sequences starting at G111 and ending at D135, or their fragments, antibodies, the formation of which was induced, specifically react with the β-sheet of the α-Syn monomer, oligomer and fibril; and does not react with the β-sheet of Aβ _1-42 or Tau1-441, thus offering ideal candidates for the α-Syn peptide immunogenic construct, as illustrated by the α-Syn peptide immunogenic constructs (SEQ ID NOs: 112 and 113) in Fig. 8 .

vi) Expanding MHC coverage using α-Syn peptide immunogenic constructs with various non-selective T helper epitopes.

When developing a pharmaceutical composition for the treatment of patients with different genetic backgrounds, it is important that this development covers the maximum population with different genetic backgrounds. Therefore, the synergistic immunogenicity of α-Syn-derived peptide immunogenic constructs was investigated for this combination. Since non-selective T helper epitopes derived from MVF or HBsAg are among the most effective in providing this enhancement of immunogenicity, a combination of peptide constructs containing a T helper epitope was developed for this study. It was found that a mixture of two peptide immunogenic constructs with the same B-epitopes induce a significant immune response compared to that caused by the corresponding individual peptide construct.

Example 6

Targeted antibody response generated by α-SYN peptide immunogenic constructs to the target B-cell epitope only

It is well known that all carrier proteins (e.g., keyhole limpet hemocyanin (KLH) or other carrier proteins such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins) are used to enhance the immune response directed against the target epitope peptide B cells, by chemical conjugation of such a B cell epitope peptide to the corresponding carrier protein, will produce more than 90% of antibodies directed against the potentiating carrier protein and less than 10% of antibodies again directed against the target B cell epitope in immunized hosts. In this regard, it is of interest to evaluate the specificity of the α-Syn peptide immunogenic constructs of this invention. To assess immunogenicity, a series of eight α-Syn peptide immunogenic constructs (SEQ ID NO: 107-114) with B-cell epitopes of varying lengths, which are linked via a spacer sequence to the heterologous T-cell epitope UBITh1 (SEQ ID NO: 83), were prepared. . UBITh1 (T helper peptide used for B epitope immunopotentiation) was coated on plates and guinea pig immune sera were used to examine cross-reactivities with the UBITh1 peptide used for immunopotentiation. In contrast to the high immunogenicity of these constructs towards the corresponding target B epitopes, as evidenced by the high titers of antibodies generated against the B epitope(s), as demonstrated in Table. 6 and 7 , it was found that most, if not all, of the immune sera were non-reactive with the UBITh1 peptide, as demonstrated in Table. 8.

Thus, the simple design of an immunogen comprising a targeted B cell epitope linked to a carefully selected T helper epitope allows for a targeted and pure immune response targeting only the B cell epitope of α-Syn. In the case of developing a pharmaceutical composition, the more specific the immune response it produces, the higher the safety profile required for the composition. Thus, the α-Syn peptide immunogenic constructs of this invention are highly specific, but at the same time highly effective against their target.

Example 7

Epitope mapping for detailed specificity analysis using immune sera (9 weeks post-immunization) against various alpha-synuclein peptide immunogenic constructs

In a detailed epitope mapping study ( Table 9 ), to identify antibody binding site(s) with specific residues in the C-terminal region of α-Syn, 52 overlapping 10-mers (SEQ ID NO: 18-69) were synthesized that cover the amino acid sequence of α-Syn (K80-A140). Two longer peptides (97-135, SEQ ID No: 10) and (111-132, SEQ ID No: 17) were used as positive controls. These 10-mer peptides and two longer peptides were separately applied to the wells of 96-well microtiter plates as solid-phase immunoabsorbents. Pooled guinea pig antisera were added at a 1:100 dilution in sample dilution buffer to plate wells coated with 2.0 μg/ml 10-mer peptide and then incubated for one hour at 37°C. After washing the plate wells with wash buffer, horseradish peroxidase-conjugated protein A/G was added and incubated for 30 minutes. After repeated washings with PBS, the substrate was added to the wells to measure absorbance at 450 nm using an ELISA plate reader, samples of which were analyzed in duplicate. Binding of antisera to the corresponding long α-Syn peptide of the immunogenic B-epitope construct represents maximum binding.

As demonstrated in Table. 9 , pooled guinea pig sera after 9 weeks. after immunization, obtained respectively from six α-Syn peptide immunogenic constructs [(K97-D135, SEQ ID No: 110), (G111-D135, SEQ ID No: 108), (G111-G132, SEQ ID No: 113) , (E126-D135, SEQ ID No: 112), (G101-A140, SEQ ID No: 104) and (E126-A140, SEQ ID No: 99)] were selected for detailed epitope mapping. These six fragments of B-epitopes of varying length completely cover the sequence 97-140 C-terminal region of α-synuclein. The ELISA results showed that all six immune sera reacted significantly with the exemplary α-Syn long peptide (97-135, SEQ ID No: 10). In the case of the detailed 10-mer epitope mapping study, results showed coverage of the immunogenic epitope near the region AA114 to 125 (peptides 114-123, 115-124, 116-125 SEQ ID Nos: 52, 53 and 54) and the highly immunogenic region at C- terminal end, represented by peptide 131-140 (SEQ ID NO: 69). Interestingly, most immune sera derived from the C-terminal α-Syn peptide immunogenic constructs elicited antibodies that recognize conformational rather than linear epitopes, with the exception of one, which is located at the C-terminal α-Syn with the sequence EGYQDYEPEA (SEQ ID NO: 69) and is responsible for cross-reactivity with the β-Syn protein.

The discovery of said epitope was less expected, but highly correlated with the finding that said antibodies derived from the α-Syn peptide immunogenic constructs represented by 111-132 α-Syn (SEQ ID NO: 113) and 126-135 α-Syn (SEQ ID NO: 112) from the C-terminal random loop region of α-Syn, which are associated with the structure of heterologous Th epitopes, resulted in the formation of a conformational structure reminiscent of the denatured β-sheet of α-Syn, and did not cross-react with the native α-helix α -Syn.

Example 8

Antibodies, the formation of which was induced by α-SYN peptide immunogenic constructs and compositions based on them: anti-aggregation and disaggregation effects on the recombinant alpha-synuclein protein

The effect of α-Syn peptide immunogenic constructs was assessed in in vitro anti-aggregation and disaggregation assays using anti-α-Syn antibodies purified from guinea pig antisera against recombinant α-Syn.

a. Inhibition of α-Syn aggregation

An initial screening analysis of various anti-α-Syn antibodies purified from guinea pigs immunized with various α-Syn peptide immunogenic constructs for potential anti-aggregation capacity was performed by quantifying the level of changes in α-Syn aggregation using thioflavin T measurement as described in Example 3 . Recombinant α-Syn, prepared in PBS at a concentration of 100 μM, was then incubated in 40 μl of PBS/KCl buffer (2.5 mM MgCl2, 50 mM HEPES and 150 mM KCl in 1x PBS, pH 7.4) at a concentration of 5 μM in 384-well plate for 6 days to induce aggregation. Various concentrations (0.05, 0.5 or 5 μg/ml) of anti-α-Syn antibodies purified from antisera from guinea pigs immunized with various α-Syn peptide immunogenic constructs collected at different time points were added to the incubation mixture for evaluation respectively effects on inhibition of α-Syn aggregation. At the end of incubation, the level of aggregation was determined using ThT assays. The readings obtained at each study interval were normalized by taking the level of aggregation in the vehicle control as 100% and taking the readings obtained in the absence of α-Syn as 0%.

As demonstrated in Table. 10 , three anti-α-Syn antibodies, the formation of which was caused by α-Syn _111-132 , α-Syn _121-135 or α-Syn _126-135 , collected after 9 weeks. after immunization and more, showed more effective and concentration-dependent inhibition of α-Syn aggregation. Of all the analyzed anti-α-Syn antibodies, four selected antibodies, the formation of which were caused by α-Syn _111-132 (SEQ ID NO:113), α-Syn _121-135 (SEQ ID NO:107), α-Syn _123-135 (SEQ ID NO:111) or α-Syn _126-135 (SEQ ID NO:112) (collected 9 weeks after immunization), demonstrated an inhibitory effect on α-Syn aggregation by almost 40% ( Figure 1 ) compared to vehicle control aggregation of 100%.

b. Dissociation of preformed α-Syn aggregates

Based on the above studies, it was noted that anti-α-Syn antibodies purified from antisera from guinea pigs immunized with certain α-Syn peptide immunogenic constructs had effects on inhibiting α-Syn aggregation. To further evaluate whether antibodies induced by α-Syn peptide immunogenic constructs remained effective in dissociating preformed α-Syn aggregates, in vitro disaggregation assays were performed using anti-α-Syn antibodies purified from guinea pig antisera.

α-Syn was aggregated in 200 μL of PBS/KCl buffer at a concentration of 5 μM for 3 days. After centrifugation (13,000 xg, 4°C, 30 min), α-Syn aggregates were collected and confirmed by ThT assays. The preformed α-Syn aggregates were then incubated in 100 μl of PBS/KCl buffer with or without anti-α-Syn antibodies purified from guinea pig antisera (5 μg/ml) for 3 days. After incubation, aggregates were collected after centrifugation at 13,000 x g at 4°C for 30 minutes and then quantified using the ThT assay as described in Example 3 . Residual α-Syn aggregates after spontaneous dissociations in vehicle control were normalized to 100%.

Two selected anti-α-Syn antibodies produced by α-Syn _111-132 (SEQ ID NO:113) or α-Syn _126-135 (SEQ ID NO:112), and the combination of α-Syn _111-132 (SEQ ID NO:113), the formation of which was caused by α-Syn _126-135 (SEQ ID NO:112), the formation of which was caused by anti-α-Syn antibodies, was studied by the indicated in vitro disaggregation assay. As a result, anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) showed a dissociating effect on preformed α-Syn aggregates almost 50% compared to the 100% vehicle control, while other anti-α-Syn antibodies and antibodies purified from pre-immunized animals did not show comparable effects ( Figure 2 ).

Example 9

Antibodies induced by α-SYN peptide immunogenic constructs and formulations based on them: anti-aggregation and disaggregation effects on α-SYN aggregation kinetics in cells overexpressing α-SYN

α-Syn aggregation is known to accelerate during neuronal differentiation. To evaluate the effect of α-Syn peptide immunogenic constructs on either the inhibition of α-Syn aggregation or the dissociation of preformed α-Syn aggregates under on-cell conditions, anti-α-Syn antibodies were purified from antisera from guinea pigs immunized with various peptide immunogenic constructs. α-Syn was assessed by anti-aggregation and disaggregation assays in NGF-treated PC12 cells overexpressing α-Syn differentiated into neurons.

a. Inhibition of α-Syn aggregation

PC12 cells overexpressing α-Syn were seeded onto poly-D-lysine pre-coated 96-well plates and then treated with nerve growth factor (NGF) (100 ng/ml) along with anti-α-Syn antibodies purified from guinea pigs. immunized with various α-Syn peptide immunogenic constructs (0 or 0.5 μg/ml) for 4 days to confirm anti-aggregation activity.

Treated cells were lysed and 20 μg of cell lysates were separated by SDS-PAGE and then detected with anti-α-Syn antibody (BD). The amount of detectable α-Syn signals in the higher molecular weight region was quantified and then normalized to the vehicle control as 100%. As shown in FIG. 3 , inhibitory effects on the amount of aggregated α-Syn up to 80-90% were observed among all four selected anti-α-Syn antibodies, the formation of which was caused by the peptide immunogenic constructs α-Syn α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO:107), α-Syn _123-135 (SEQ ID NO:111) or α-Syn _126-135 (SEQ ID NO:112), compared to the amount of aggregated α-Syn in the control vehicle.

b. Dissociation of preformed α-Syn aggregates

To confirm disaggregation activity against preformed α-Syn aggregates, PC12 cells overexpressing α-Syn were treated with NGF (100 ng/ml) for 3 days for neuronal differentiation to initiate α-Syn aggregation before further treatment anti-α-Syn antibodies purified from guinea pigs immunized with various α-Syn peptide immunogenic constructs (0 or 0.5 μg/ml) for an additional 4 days.

Treated cells were lysed and 20 μg of cell lysates were separated by SDS-PAGE and then detected with anti-α-Syn antibody (BD). The amount of detectable α-Syn signals in the higher molecular weight region was quantified and then normalized to the vehicle control as 100%. As also demonstrated in FIG. 3 , a 50-60% reduction in the amount of aggregated α-Syn was observed when using anti-α-Syn antibodies, the formation of which was induced by peptide immunogenic constructs α-Syn _121-135 (SEQ ID NO:107), α-Syn _123-135 ( SEQ ID O:111) or α-Syn _126-135 (SEQ ID NO:112), while anti-α-Syn antibodies, the formation of which was caused by α-Syn _111-132 (SEQ ID NO: 113), demonstrated more than 90% reduction in the amount of aggregated α-Syn.

Example 10

Antibodies, the formation of which was induced by α-SYN peptide immunogenic constructs and compositions based on them: the effect of reducing the secretion of TNF-(and IL-6) in microglia

Injury to nigral neurons is thought to result in the release of aggregated α-Syn into the substantia nigra, which activates microglia and produces proinflammatory mediators, thereby leading to persistent and progressive nigral neurodegeneration in PD. To evaluate the effects of reducing microglial activation by anti-α-Syn antibodies purified from guinea pigs immunized with various α-Syn peptide immunogenic constructs, the proinflammatory mediators TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6) were measured. , released by microglia upon treatment with α-Syn aggregates in the presence or absence of various anti-α-Syn antibodies.

Mouse BV2 cells or human SVG p12 cells were seeded at 5000 cells/well in RPMI 1640 medium supplemented with 1% FBS. Cells were treated with 1 µM α-Syn and incubated at 37°C, 5% CO ₂ in a humidified atmosphere for 24 hours. After this, the cell culture medium was collected, centrifuged, and supernatants were isolated. Concentrations of IL-6 secreted by BV2 cells and TNF-α secreted by SVG p12 cells in the supernatants were analyzed in triplicate using mouse IL-6 or human TNF-α ELISA kits (Thermofisher), respectively. Signal was normalized to vehicle control as 100%.

Data showed that anti-α-Syn antibodies induced by α-Syn _111-132 (SEQ ID NO: 113) and α-Syn _123-135 (SEQ ID NO: 111) reduced α-Syn aggregate-mediated TNF release - (by SVG p12 cells by 30-50%, while anti-α-Syn antibodies, the formation of which was caused by α-Syn _123-135 (SEQ ID NO: 111), reduced the release of IL-6 by SVGP12 cells by approximately 30% ( Figure 4 ) The results indicated that anti-α-Syn antibodies induced by α-Syn _123-135 (SEQ ID NO: 111) were more effective than other anti-α-Syn antibodies, investigated in reducing α-Syn aggregate-mediated microglial activation.

Example 11

Antibodies, the formation of which was induced by α-SYN peptide immunogenic constructs and compositions based on them: effect on reducing neurodegeneration caused by exogenous alpha-synuclein

To evaluate the neuroprotective effects of anti-α-Syn antibodies purified from antisera from guinea pigs immunized with various α-Syn peptide immunogenic constructs, an in vitro model of neurodegeneration using exogenous, preformed α-Syn aggregates was adopted against NGF-treated PC12 differentiated cells. into neurons.

PC12 cells were treated with NGF (100 ng/ml) for 6 days to induce neuronal differentiation. The morphology of the neuron-differentiating cells was confirmed and analyzed using an InCell High Content Image Analysis System (GE Healthcare). The neurotrophic effects of NGF were reflected in neurite outgrowth, and the number of cells differentiating into neurons was quantified. The levels of neurite outgrowth and the number of differentiating cells were shown as a percentage (mean (SEM) after normalization. The length of neurites of PC12 cells with and without NGF treatment was taken as 100% and 0%, respectively. The number of PC12 cells differentiating into neurons 6 days after NGF treatment was normalized up to 100%.

Neurodegeneration was observed by adding exogenous, preformed α-Syn aggregates to neuronally differentiated PC12 cells. In the presence of preformed α-Syn aggregates, neurite length shortened and the number of cells in PC12 cells differentiating into neurons decreased. This neurodegeneration caused by α-Syn aggregates was proportional to the amount of exogenous α-Syn aggregates added and could be blocked by curcumin, widely known for its neuroprotective effects against the neurotoxicity of α-Syn aggregates, in a concentration-dependent manner. Commercially available anti-α-Syn antibodies (BD bioscience), but not antibodies purified from naïve guinea pigs, attenuated neurodegeneration caused by α-Syn aggregates. This model was used as a screening platform to determine which anti-α-Syn antibodies purified from antisera from guinea pigs immunized with various α-Syn peptide immunogenic constructs had neuroprotective effects in restoring neurite outgrowth as well as neuronal survival depending on on concentration ( Tables 11 and 12 ).

As a result, anti-α-Syn antibodies purified from antisera from guinea pigs immunized with more than half of the different α-Syn peptide immunogenic constructs restored neurite concentration in a concentration-dependent manner ( Table 11 ), and anti-α-Syn antibodies, purified from antisera from guinea pigs immunized with almost all of the different α-Syn peptide immunogenic constructs protected PC12 cells differentiating into neurons from neuronal death caused by α-Syn aggregates ( Table 12 ). Taking these two different parameters together, almost a third of the anti-α-Syn antibodies analyzed were found to have effects on both neurite length and cell survival against the neurotoxicity of α-Syn aggregates. Anti-neurodegenerative effects of anti-α-Syn antibodies, the formation of which was caused by α-Syn _111-132 (SEQ ID NO: 113), α-Syn _126-135 (SEQ ID NO: 112), and preimmune antibodies from naive guinea pigs, were observed and neurite length and cell number were quantified with calcein AM (Life Technologies), a fluorescent dye for labeling living cells. It was demonstrated that in PC12 cells differentiating into neurons, with a high content of neurites, anti-α-Syn antibodies, the formation of which was caused by α-Syn _111-132 (SEQ ID NO: 113) ( Fig. 5B ) and α-Syn _126-135 (SEQ ID NO: 112) ( Fig. 5C ), but not preimmune antibodies purified from naïve guinea pigs ( Fig. 5A ), showed protective effects on α-Syn aggregate-mediated neurite length shortening.

Example 12

Antibodies, the formation of which was induced by peptide immunogenic constructs of α-SYN, and compositions based on them: effect on reducing neurodegeneration in cells overexpressing α-SYN

To evaluate the neuroprotective effects of anti-α-Syn antibodies purified from antisera from guinea pigs immunized with various α-Syn peptide immunogenic constructs, in vitro models of neurodegeneration were adopted using wild-type PC12 cells overexpressing α-Syn and PC12 mutant cells. A53T, overexpressing α-Syn.

After incubation with NGF, empty control cells (transfected with the plasmid vector) elongated long neurites and increased the number of cells similar to the parental wild-type PC12 cells, whereas wild-type PC12 cells overexpressing α-Syn and PC12 cells overexpressing α-Syn , with the A53T mutation, failed to produce comparable neurite extension or increase in cell number, confirming the neurodegenerative effects accompanied by aggregated α-Syn upon NGF treatment. In characterizing overexpressed α-Syn in wild-type PC12 cells overexpressing α-Syn upon NGF treatment, Western blot and ThT assays were performed using cell lysates of wild-type PC12 cells overexpressing α-Syn upon NGF treatment. Western blot results demonstrated that α-Syn overexpressed in cell lysates of wild-type PC12 cells overexpressing α-Syn when treated with NGF was oligomeric, and ThT assay results showed that α-Syn in cell lysates of wild-type PC12 cells overexpressing α -Syn, when treated with NGF, had a β-sheet structure (i.e., increased ThT fluorescence signals). Compared with the results of Western blotting and ThT analysis of wild-type PC12 cells overexpressing α-Syn without NGF treatment, it was suggested that the α-helix to β-sheet structural transition of overexpressed α-Syn occurs in NGF-induced neuronal differentiation, which could subsequently cause neurodegenerative diseases. β-sheet effects of oligomeric α-Syn. Additionally, compared with wild-type PC12 cells overexpressing α-Syn, overexpressed A53T mutant α-Syn resulted in greater neurodegenerative effects, reflected by both shortened neurite length and decreased cell number upon NGF treatment, indicating that that A53T mutant α-Syn caused stronger neurodegenerative effects than wild-type α-Syn in PC12 cells overexpressing α-Syn.

Anti-α-Syn antibodies, the formation of which was caused by α-Syn _101-132 (SEQ ID NO:114), α-Syn _111-132 (SEQ ID NO:113), α-Syn _121-135 (SEQ ID NO:107 ), α-Syn _123-135 (SEQ ID NO:111) or α-Syn _126-135 (SEQ ID NO:112), and a combination of anti-α-Syn antibodies induced by α-Syn _111-132 (SEQ ID NO: 113) and (-Syn _126-135 (SEQ ID NO: 112), were analyzed in an in vitro model of neurodegeneration using wild-type PC12 cells overexpressing α-Syn to evaluate their individual protective effects against neurodegeneration. PC12 cells wild type overexpressing α-Syn were treated with NGF for 3 days to initiate neuronal differentiation, before incubation with both anti-α-Syn antibodies (final concentration 5 μg/ml) and NGF for an additional 3 days. cells by the end of the incubation period showed restored neurite length and increased cell number when co-incubated with selected anti-α-Syn antibodies compared to vehicle control. Quantification of neurite length and cell number was performed using readings from parental PC12 cells treated with NGF for 6 days, normalized to 100%. As a result, anti-α-Syn, the formation of which was caused by α-Syn _101-132 (SEQ ID NO:114), α-Syn _111-132 (SEQ ID NO:113) or α-Syn _123-135 (SEQ ID NO: 111), and the combination of anti-α-Syn, the formation of which was caused by α-Syn _111-132 (SEQ ID NO: 113) and α-Syn _126-135 (SEQ ID NO: 112), showed a significantly higher number of cells, while while anti-(-Syn antibodies, the formation of which was caused by α-Syn _101-132 (SEQ ID NO:114), α-Syn _111-132 (SEQ ID NO:113), α-Syn _123-135 (SEQ ID NO :111) or α-Syn _126-135 (SEQ ID NO:112), and a combination of anti-α-Syn antibodies produced by α-Syn _111-132 (SEQ ID NO: 115) and α-Syn _126-135 (SEQ ID NO: 114) showed significantly longer neurite length compared to vehicle control ( Figures 6A and 6B ).

Example 13

Antibodies, the formation of which was caused by peptide immunogenic constructs α-SYN, and compositions based on them: specificity in relation to the beta-sheet oligomeric and fibrillar protein alpha-synuclein

In order to more effectively characterize the specificity of anti-α-Syn antibodies purified from antisera of guinea pigs immunized with various α-Syn peptide immunogenic constructs, a series of in vitro analyzes were performed on the α-Syn molecular complex of various sizes, various amyloidogenic proteins, including α-Syn. Syn, A (both tau protein and aggregated α-Syn in PC12 cells overexpressing α-Syn upon NGF treatment.

a. Specificity for larger molecular α-Syn complexes

Western blotting of α-Syn molecular complexes of different sizes was performed using anti-α-Syn antibodies purified from guinea pig antisera immunized with various α-Syn peptide immunogenic constructs as primary antibodies. The results showed that all anti-α-Syn reacted significantly with larger sized molecular α-Syn complexes, including dimers, trimers, tetramers, and oligomers, in addition to smaller sized monomeric α-Syn. Compared to the commercially available anti-α-Syn antibody, Syn211 (Abcam), the anti-α-Syn antibodies produced by α-Syn _111-132 (SEQ ID NO:113), α-Syn _121-135 ( SEQ ID NO:107), α-Syn _123-135 (SEQ ID NO:111) and α-Syn _126-135 (SEQ ID NO:112), showed a higher signal ratio of larger sized α-Syn molecular complexes (in including dimers, trimers, tetramers, and oligomers) to the smaller monomeric α-Syn signal ( Figures 7A and 7B ), indicating that the anti-α-Syn antibodies had specificity for the larger molecular α-Syn complexes.

b. Specificity for α-Syn among various amyloidogenic proteins

Dot blot analysis using various molecules (i.e., α-helical monomers, β-sheet monomers, β-sheet oligomers, and β-sheet fibrils) of various amyloidogenic proteins (i.e., α-Syn, Aβ _1-42 and Tau441), prepared as described in Example 3 , were performed using anti-α-Syn purified from guinea pig antisera immunized with various α-Syn peptide immunogenic constructs as primary antibodies. The results showed that anti-α-Syn antibodies, the formation of which was caused by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113), specifically reacted to all β-sheets. forms of α-Syn (monomeric, oligomeric and fibrillar molecules), but not into α-helical monomers ( Figures 8A, 8B and 8C ). In addition, anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) responded more strongly to β-sheet fibrils and β-sheet α-Syn oligomers than β-sheet α-Syn monomers. In contrast, anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) did not show detectable reactivity towards to β-Syn or various molecules (i.e., α-helical monomers, β-sheet monomers, β-sheet oligomers, and β-sheet fibrils) of the amyloidogenic proteins Aβ _1-42 and Tau441 ( Figures 8A, 8B, and 8C ) . The results obtained indicated that the anti-α-Syn antibodies produced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) were specific for to α-Syn β-sheet monomeric, β-sheet oligomeric and β-sheet fibrillar forms.

c. Specificity of binding to aggregated α-Syn in PC12 cells overexpressing α-Syn upon NGF treatment

Immunocytochemical analysis (ICC) using anti-α-Syn antibodies purified from antisera from guinea pigs immunized with various α-Syn peptide immunogenic constructs was performed on parental PC12 cells, PC12 blank control cells, PC12 cells overexpressing wild-type α-Syn, and A53T mutant PC12 cells overexpressing α-Syn to evaluate the binding affinity of antibodies to aggregated α-Syn upon NGF treatment as described in Example 3 . As shown by the quantification result in FIG. 9 , anti-α-Syn antibodies, the formation of which was caused by α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107) or α-Syn _126-135 (SEQ ID NO : 112) showed stronger reactivity in wild-type PC12 cells overexpressing α-Syn and in A53T mutant PC12 cells overexpressing α-Syn than in parental PC12 cells or empty control PC12 cells when treated with NGF. Since aggregation of overexpressed α-Syn was induced by NGF treatment, the results indicated that the anti-α-Syn antibodies produced by α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107) or α-Syn _126-135 (SEQ ID NO: 112), were specific for aggregated α-Syn in wild-type PC12 cells overexpressing α-Syn and A53T mutant PC12 cells overexpressing α-Syn , when treated with NGF.

Example 14

Immunohistochemical staining of the human brain in Parkinson's disease to assess the tissue specificity of α-SYN peptide immunogenic constructs and compositions based on them

Immunohistopathological study using preimmune anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113), and a 1:1 combination of both anti -α-Syn antibodies were performed on normal human tissues to monitor the specificity and unwanted autoreactivity of the antibodies. A panel of human tissues (Pantomics) was deparaffinized with xylene, rehydrated in ethanol and then treated with 0.25% trypsin with 0.5% CaCl ₂ in PBS for 30 minutes and incubated in 1% hydrogen peroxide in methanol to block endogenous peroxidase activity, and then incubated with 10% Block Ace (Sigma) in PBS before using anti-α-Syn antibodies from guinea pigs immunized with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113), and a 1:1 combination of both antibodies (1:300 dilution). Sections were visualized using 3-3′-diaminobenzidine (DAB) and counterstained with hematoxylin before microscopic examination. In contrast to the positive reactivity of commercial anti-α-Syn (BD, 610708), anti-α-Syn antibodies purified from guinea pigs immunized with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) and a 1:1 combination of both antibodies showed negative reactivity to normal human tissues, which was comparable to the pattern of preimmune antibodies from naïve guinea pigs ( Figure 10A ).

Another immunohistopathological study using preimmune anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113), and a 1:1 combination of both anti-α-Syn antibodies were tested to test their reactivity against human Parkinson's disease brain. Tissue sections from three regions (i.e., cerebellum, corpus callosum, and thalamus) were analyzed (BioChain). As a result, anti-α-Syn antibodies produced by α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113), and a 1:1 combination of both anti-α- Syn antibodies showed positive reactivity (indicated by arrows) to PD brain sections in all three regions compared to negative reactivity in normal brain sections ( Figures 10B and 10C ). Quantification of reactivity to α-Syn aggregates in PD brain sections was performed by counting positive spots by microscopic observation. The results showed that anti-α-Syn antibodies produced by α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113), and a 1:1 combination of both anti- α-Syn antibodies were strongly positive in PD brain sections compared with healthy brain sections. All three different anti-α-Syn antibodies analyzed, induced by α-Syn _111-132 (SEQ ID NO: 113), had the strongest immunoreactivity towards α-Syn aggregates in PD brain sections.

Example 15

Evidence of the effectiveness of α-SYN peptide immunogenic constructs and formulations based on them in animal models

a. Immunization and blood/brain tissue collection

Mouse models of Parkinson's disease ( PD ) were generated as described in Example 4 . Two weeks after MPP ⁺ injection or 7 weeks after fibrillar α-Syn inoculation, mice were randomly divided into three groups including peptide, including UBITh1-related peptide α-Syn _111-132 (SEQ ID NO: 113), associated with UBITh1 peptide α-Syn _126-135 (SEQ ID NO: 112) and a combination of both peptides, in addition to the adjuvant group (immunized with adjuvants and solvent used in the preparation of the compositions (ISA 51 VG, CpG3, 0.2% TWEEN®-80) ). Intramuscular (IM) immunization was administered three times with an interval of 3 weeks at a dose of 40 μg. The administration and blood collection regimens were performed in accordance with Table. 13 .

At each time point, 200 μl of blood was collected by drawing a blood sample from the facial vein. Blood dripping from the punctured submandibular vein was collected in a microtube, and blood serum was obtained by centrifugation at 300 rpm. for 10 minutes. After killing the animals, brain tissue samples were collected for Western blotting.

b. Immune responses in mouse models of PD treated with compositions containing α-Syn _111-132 (SEQ ID NO: 113) and/or α-Syn _126-135 (SEQ ID NO: 112) peptide immunogenic constructs

Pooled serum samples from each treatment group were diluted in 1% BSA (in PBST) and then applied to an ELISA plate coated with 200 μl of full-length α-Syn peptide (Cloud-clone) in 0.1 M sodium bicarbonate (α-Syn concentration 4, 4 µg/µl, pH 9.6). After 2 hours of incubation at room temperature and after three washes with PBST, 100 μl of HRP-conjugated anti-mouse IgG antibody diluted 1:3000 with 1% BSA was added to react for 2 hours at room temperature. The plates were then washed three times with PBST and incubated with 100 μl of 3,3,5,5-tetramethylbenzidine (TMB) for 10 minutes in the dark. Then 100 μl of 2M H ₂ SO ₄ was applied and incubated for 15-30 minutes, after which the optical density (OD) value was measured at 450 nm using multi-mode detection using a SpectraMax i3x (Molecular Devices).

Two mouse models of PD immunized with formulated α-Syn _111-132 (SEQ ID NO: 113), formulated α-Syn _126-135 (SEQ ID NO: 112) or a combination of both peptide immunogenic constructs had an optical density (OD) value of anti -α-Syn antibodies greater than 3.0 after the second immunization, which remained elevated at the end of the study 15 or 19 weeks after the initial immunization, in the MPP ⁺ induction model ( Fig. 11A ) or in the fibrillar α-Syn inoculation model ( Fig. 11B ) respectively, while adjuvanted animals did not mount a measurable immune response against anti-α-Syn antibodies.

It was noted that in the fibrillar α-Syn inoculation model, the α-Syn _111-132 construct induced a stronger immune response than the α-Syn _126-135 construct ( Figure 11B ), while no difference in immunogenicity was observed in the by induction of MPP ⁺ ( Fig. 11A ).

c. Decreased serum α-Syn levels

Pooled serum α-Syn levels from animals in each group were assessed using an ELISA kit (SEB222Mu, USCN) that could detect both the alpha-helical and β-sheet α-Syn described in Example 3 .

The quantitative α-Syn ELISA was designed to investigate whether the anti-α-Syn antibody response of immunized groups was associated with reduced amounts of peripheral α-Syn compared to untreated animals. It was demonstrated that immunization with α-Syn _126-135 (SEQ ID NO: 112), α-Syn _111-132 (SEQ ID NO: 113), or a combination of these constructs resulted in a decrease in the optical density (OD) values of α-Syn levels by compared to adjuvanted animals in both the MPP ⁺ induction model ( Figure 12A ) and the fibrillar α-Syn inoculation model ( Figure 12B ). The results indicated that when an anti-α-Syn antibody response was obtained upon immunization with the α-Syn peptide immunogenic constructs, the amount of α-Syn in the peripheral blood was correspondingly reduced.

d. Decreased levels of oligomeric α-Syn in the brain

After the animals were sacrificed, brain tissue samples were collected for Western blotting. In MPP+-induced mice, the brain was removed and homogenized, while in mice inoculated with fibrillar α-Syn, the striatal and substantia nigra regions were first isolated and then homogenized. Brain tissue lysate was prepared by adding lysis buffer (Amresco) and 1x proteinase inhibitor (Roche) to the homogenate. The lysate was then separated by 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated overnight with 5% milk in PBS. To detect high levels of dopaminergic neurons, membranes were incubated with anti-tyrosine hydroxylase antibody (1:1000 dilution, Abcam) and then hybridized with a secondary antibody, HRP-conjugated goat anti-rabbit IgG (H+L) antibody (1:5000 dilution, Jackson Immunoresearch). Luminata Western HRP substrates were used for imaging and the resulting signal was captured by a ChemiDoc-It 810 digital imaging system. Quantification of oligomeric α-Syn levels was performed by normalization to GAPDH levels, and the undisturbed lysate ratio was further standardized to 100% for comparison.

In the MPP ⁺ induction model, a decrease in the proportion of oligomeric α-Syn was shown in animals immunized with the α-Syn _111-132 peptide immunogenic construct ( Figure 13A ). Similarly, in mice inoculated with fibrillar α-Syn, Western blot analysis was performed using lysates from the substantia nigra as well as the ipsilateral striatum when inoculated with fibrillar α-Syn ( Figures 14A and 14D ) and using lysates from the contralateral striatum when inoculated with fibrillar α-Syn. α-Syn ( Figure 14F ) showed that the 2- to 3-fold increase in oligomeric α-Syn levels observed in adjuvanted control mice was reduced following treatment with formulated α-Syn _111-132 (SEQ ID NO:113) and construct α-Syn _126-135 (SEQ ID NO:112). Quantification of Western blot results is presented in FIG. 13B, 14B, 14C, 14D and 14G .

e. Reduced neuropathology

In mfs inoculated with fibrillar α-Syn, areas of the substantia nigra were first isolated and then homogenized. Tissue lysate was prepared by adding lysis buffer (Amresco) and 1x proteinase inhibitor (Roche) to the homogenate. The lysate was then separated by 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated overnight with 5% milk in PBS. To detect high levels of dopaminergic neurons, membranes were incubated with anti-tyrosine hydroxylase antibody (1:1000 dilution, Abcam) and then hybridized with a secondary antibody, HRP-conjugated goat anti-rabbit IgG (H+L) antibody (1:5000 dilution, Jackson Immunoresearch). Luminata Western HRP substrates were used for imaging and the resulting signal was captured by a ChemiDoc-It 810 digital imaging system. The expression level of α-Syn was standardized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as a protein loading control.

The results demonstrated that immunization with the α-Syn _111-132 construct restored the amount of tyrosine hydroxylase to levels equivalent to those in unaffected normal animals ( Figures 14C-14D ), suggesting a neuroprotective effect of the α-Syn peptide immunogenic constructs against neurotoxicity associated with aggregated α-Syn inoculated into mice.

f. Restoration of motor activity

CatWalk™ XT (Noldus information Technology, Wageningen, The Netherlands) is a video-based analysis system used to objectively measure various aspects of gait in a dynamic manner based on the position, pressure and surface area of each step. All mice were trained to consistently cross the track at least three times daily before the experiment. A successful run was defined as the animal running along the track without interruption or hesitation, and mice that did not complete the training were excluded from the study.

On average, 5 intersections per mouse were analyzed. Because fibrillar α-Syn inoculation was performed on the right side of the brain, time to stand on the left hind paw was considered a reference parameter, along with run time.

In the fibrillar α-Syn inoculation model, a significant difference in left hindlimb stance time measurements was observed following treatment with compositions containing α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) ( Fig. 15A ). Meanwhile, in both the fibrillar α-Syn inoculation model and the MPP+ induction model, a significant difference in travel time measurements was observed after treatment with compositions containing α-Syn _111-132 (SEQ ID NO:113) ( Figure 15B and 15C ). The results indicated an association of treatment with α-Syn peptide immunogenic constructs formulated α-Syn _126-135 (SEQ ID NO:112) or formulated α-Syn _111-132 (SEQ ID NO:113) and improved motor function in two mouse models of PD.

Example 16

Reactivities of antibodies generated using α-SYN peptide immunogenic constructs with different α-SYN species found in neurodegenerative diseases

α-Syn causes Parkinson's disease and other synucleinopathies. The α-Syn protein is capable of forming different types of aggregates that have different sizes and structures, and different effects on cells, such that each of these diseases is caused by one or more different types of aggregates. α-Syn aggregates of different shapes can cause different patterns of damage in the brain and can even cause different brain diseases. This study was designed to evaluate how antibodies generated by α-Syn peptide immunogenic constructs interact with various α-Syn species found in neurodegenerative diseases.

Dr. Ronald Melki participated in this study. Various types of α-Syn aggregates have been prepared in the laboratory, which include (a) a fibril, a long, twisted, strand of α-Syn proteins joined together; (b) a ribbon, wider, flatter structure, and (c) α-Syn (O550) oligomers stabilized by dopamine (ODA) and glutaraldehyde (OGA).

Antibodies raised in guinea pigs by the various α-Syn peptide immunogenic constructs disclosed herein were examined for their relative affinities. Illustrative samples from PD-021514 (α-Syn _85-140 , wk 08), PD-021522 (α-Syn _85-140 , wk 13), PD-100806 (α-Syn _{126- 135} , weeks after immunization 09), PRX002 and the commercial monoclonal antibody Syn1 (clone 42) were examined for individual α-Syn structures, including: fibrils, ribbons, fibrils 65, fibrils 91, fibrils 110, for α oligomers -Syn fibrillar assembly pathway (O550), dopamine-stabilized (ODA), and glutaraldehyde-stabilized (OGA) oligomers, along with a control monomer using a filter trap assay.

Methods and materials

a. Assembly of α-Syn into fibrils and ribbons

To form fibrils, soluble α-Syn WT was incubated in buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl) at 37°C with continuous shaking in an Eppendorf thermomixer set at 600 rpm. Assembly was monitored continuously in a Cary Eclipse spectrofluorometer (Varian Inc., Palo Alto, CA, USA) in the presence of thioflavin T (15 μM) in 1 cm cuvettes with stirring (100 rpm) using a magnetic stirrer ( 6 (3 mm) with excitation wavelength set to 440 nm and emission wavelength set to 440 nm and 480 nm, and averaging time equal to 1 s. To form the α-Syn ribbon, DT was dialyzed for 16 hours against 1000 volume of buffer B (5 mM Tris-HCl, pH 7.5) at 4°C, then incubated at 37°C with continuous shaking in an Eppendorf thermomixer set at 600 rpm Assembly was monitored by measuring scattered light at 440 Alternatively, the amount of protein remaining in the supernatant after precipitation at 35,000 x g was determined by measuring absorbance at 280 nm on a Hewlett Packard 8453 Diode Array Spectrophotometer. The nature of the oligomeric molecules was assessed using a Jeol 1400 TEM (Jeol Ltd.) after adsorption samples on carbon-coated 200 mesh grids and negative staining with 1% uranyl acetate. Images were recorded using a Gatan Orius CCD camera (Gatan). The ability of α-Syn structures to bind Congo red was assessed as follows: α-Syn fibrils and ribbons were incubated for 1 hour with 100 μM Congo red (Sigma-Aldrich, St. Louis, MO, USA) in 20 mM Tris buffer (pH 7, 5). The polymers were then pelleted at 20°C in a TL100 Tabletop Beckman ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA) at 25,000 g for 30 minutes. The beads were washed four times using an equal volume of water. After resuspension of the beads, an aliquot was placed on a glass coverslip and immediately imaged or allowed to dry. Samples were viewed under bright field and cross-polarization light using polarization microscopy using a Leica microscope (MZ12.5) equipped with cross-polarizers (Leica Microsystems, Ltd., Heerbrugg, Switzerland).

b. Determination of concentrations of α-Syn fibrils and ribbons

Length heterogeneity of α-Syn fibrils and ribbons was reduced by sonication for 20 min on ice in 2-ml Eppendorf tubes in a VialTweeter running on a UIS250v ultrasound processor (250 W, 2.4 kHz; Hielscher Ultrasonic, Tetlow, Germany) installed at 75% amplitude, with pulses of 0.5 s. The sedimentation rates of α-Syn fibrils and ribbons were measured. Sedimentation boundaries were analyzed using Sedfit software using ls - g* (s) least squares modeling, which is best suited for heterogeneous mixtures of large particles. This resulted in a distribution of particles with sedimentation coefficients ranging from 50 to 150 S for α-Syn ribbons, from 100 to 1000 S for α-Syn fibrils, focusing on molecules that have sedimentation coefficients of ~90 S and 375 S for α ribbons and fibrils -Syn respectively, corresponding to particles that have a molecular mass of ~11,500 kDa, e.g., composed of ~800 α-Syn molecules (12,000 kDa/14.5 kDa) for α-Syn ribbons, ~102,000 kDa, e.g., composed of ~7,000 α-Syn molecules (102,000 kDa/14.5 kDa) for α-Syn fibrils. Thus, at a working concentration of 20 µM, the total particle concentrations of α-Syn ribbons and fibrils are 20 µM/~800=~0.02 µM, 20 µM/~7000=~0.003 µM for α-Syn ribbons and fibrils, respectively, taking into account that 100% of α-Syn assembles in ribbons or fibrils at steady state, since 100% of the protein is in the granule fraction when samples are centrifuged.

c. Assessment of endothelial affinity for various α-Syn fibrils and ribbons

The affinity of antibodies generated by the α-Syn peptide immunogenic constructs disclosed herein was assessed for various α-Syn constructs using a filter trap assay with the antibody as reference. The structures of α-Syn (fibrils, ribbons, fibrils 65, fibrils 91, fibrils 110, α-Syn fibrillar assembly pathway oligomers (O550), dopamine-stabilized (ODA) and glutaraldehyde-stabilized (OGA) oligomers) are described in Bousset L. et al. , 2013 Nat Commun 4:2575; Makky A. et al., 2016 Sci Rep 6:37970; and Pieri L. et al, 2016 Sci. Rep 6:24526. A control monomeric α-Syn was also used.

Increasing amounts of fibrillar, oligomeric, or monomeric α-Syn ranging from 20 pg to 200 ng were detected on nitrocellulose filters using a slot filtration device. The filters were then blocked with skim milk, incubated with the PRX002 or Syn1 antibody or the GP test antibodies of this invention at the indicated dilution. After extensive washing, a second anti-human or anti-guinea pig IgG-HRP was used to detect primary antibody binding profiles. A control using only the secondary antibody was also examined. Super Signal ECL (Pierce #34096) was used on the blots, and the blots were then imaged on a BioRad scanner (Chemidoc MP imaging system/BioRad imagelab software). Exposure time and dynamic range are shown in Fig. 16A-16H . In this set of measurements, a human brain homogenate with a case of DLB was detected on the membrane.

d. results

Antibody affinity of guinea pigs (GP) PD-021514 (α-Syn _85-140 , weeks after immunization 08), PD-021522 (α-Syn _85-140 , weeks after immunization 13), PD-100806 (α -Syn _126-135 , wk 09) from immunized GP, PRX002, and commercial Syn1 antibodies (clone 42) were compared for different α-Syn structures using a filter trap assay. α-Syn structures used included fibrils, ribbons, fibrils 65, fibrils 91, fibrils 110, α-Syn fibrillar assembly pathway oligomers (O550), dopamine-stabilized (ODA) and glutaraldehyde-stabilized (OGA) oligomers, as well as a control monomeric α -Syn.

In FIG. 16A-16H demonstrate that the reference antibody, PRX002, recognizes fibrillar α-Syn with slightly higher affinity compared to monomeric α-Syn; whereas PD-100806 and PD-021514, both directed against the α-Syn peptide construct _126-135 of this disclosure, have a much higher affinity for fibrillar α-Syn compared to monomeric α-Syn, indicating that that both have preferential binding to fibrillar α-Syn. The affinities of PRX002 for oligomeric and fibrillar α-Syn were found to be similar. Monoclonal antibody Syn1 bound to fibrillar α-Syn, as well as oligomeric and monomeric α-Syn, without significant differential preference.

Example 17

Immunohistochemical study of antibodies derived from α-SYN peptide immunogenic constructs using brain sections from patients with Parkinson's disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB)

Antibodies generated by immunization of guinea pigs with the exemplary α-Syn _126-135 peptide immunogenic construct of the present invention were used in an immunochemical assay to characterize their ability to bind to α-Syn present in brain sections obtained from patients with alpha-synucleinopathies. The study was carried out in collaboration with Prof. Roxana Carare. The ability of antibodies to bind α-Syn present in brain sections obtained from patients with PD, LBD, and MSA was assessed. Healthy tissues were included in the study as a negative control. NCL-L-ASYN, a commercially available monoclonal antibody used for post-mortem diagnosis of alpha-synucleinopathies, was included as a positive control. This study offers evidence of positive immunoreactivity of antibodies directed against the peptide immunogenic construct α-Syn _126-135 in brain tissue sections obtained from patients with PD, LBD and MSA. Binding was particularly noticeable in the brains of patients with synucleopathies, but not in the brains of nonpatients, and binding was greater with the study antibody than with the commercial diagnostic antibody.

Methods and materials

a. Description of the reagents used and their suppliers

Antibodies obtained from immunization of guinea pigs with the exemplary α-Syn _126-135 peptide immunogenic construct were used at a 1:100 dilution. PD062220-09-1-2-Syn; PD062205-09-1-2-Syn; PD100806-09-1-2-Syn was provided by United NeuroScience (UNS), NCL-L-ASYN (mouse monoclonal antibody used at 1:100 dilution) was provided by Leica Biosystems, HuD (EI) (mouse monoclonal antibody used at 1:100 dilution ) was provided by Santa Cruz Biotechnology, Olig2 (rabbit antibody at 1:100 dilution) was provided by Millipore, Alexa Flour 594 (goat-anti-guinea pig at 1:200 dilution), Alexa Flour 488 (goat-anti-mouse at 1:200) and Alexa Flour 488 (goat-rabbit diluted 1:200) were provided by Molecular Probes life technologies.

b. Human brain tissue

Micron-thick sections were obtained from the UCL brain bank used in this study. All samples were collected and obtained in accordance with approved National Research Ethics Service protocols.

Tissue was obtained from subjects ( Table 15 ) with primary α-Syn pathologies including multiple system atrophy (MSA; n=3), dementia with Lewy bodies (DLB; n=3), and Parkinson's disease (PD; n=3). Subjects were diagnosed postmortem according to published criteria**.

c. Immunohistochemical analysis of human subjects with synucleinopathies

Immunohistochemical analysis (IHC) of human subjects with three different synucleinopathies (MSA, DLB, and PD) was performed to quantitatively compare the specificity for α-Syn aggregates of three antibodies obtained by United Neuroscience (UNS). The specificity of UNS antibodies (PD062220, PD062205, and PD100806) for α-Syn aggregates was compared with that of a commercially available diagnostic antibody (NCL-L-ASYN). Antibody specificity was analyzed in the following four brain regions in each patient and each disease type (1) globus pallidus, internal capsule, and insular cortex; (2) midbrain: substantia nigra; (3) temporal cortex: gray matter of the cerebral cortex; and (4) cerebellum: white matter of the subcortex; white matter of the cerebellum.

These brain regions are known to be susceptible to α-Syn aggregation to varying degrees and at different stages of disease progression in each disease type. Typically, the basal ganglia and midbrain are affected in the early stages of DLB, PD, and MSA and also have the highest burden of aggregates. The temporal cortex and cerebellum are affected in later stages of the disease, with very few aggregates in the cerebellum present in PD and DLB. A negative control (no primary antibody) was used along with each IHC protocol to confirm the absence of nonspecific binding of the secondary antibody. Paraffin-embedded microslides were dewaxed in an oven at 60°C for 15-20 minutes and then immersed in xylene I and II for 5 minutes each. The tissue was rehydrated in 4 dilutions of IMS from 100% to 50% for 5 minutes each. The tissue was washed 3 times for 5 minutes in 1x PBS and then incubated for 3 minutes in 100% formic acid for antigen retrieval. The tissue was washed thoroughly in 1x PBS before quenching endogenous peroxidase activity with 3% H ₂ O ₂ for 10 minutes. The tissue was allowed to cool and washed 3 more times in 1x PBS (5 minutes each) before microwave treatment in citrate buffer (15 mM Tris- sodium citrate, TWEEN, pH 6) at medium temperature for 25 minutes to ensure even microwave processing per cycle, 3 racks of microslides in 3 containers were turned on each time. Slides were allowed to cool and washed three times in 1x PBS (5 minutes) before blocking nonspecific binding sites with 15% normal goat serum. Tissue was incubated in primary antibody (1:100 in 0.1% TBS/t) overnight at 4°C. Tissue was washed 3 (5 minutes in 1x PBS and incubated for 1 hour (RT) in biotinylated secondary antibody solution. ABC was prepared 30 minutes before its use. After washing the tissue for 3 x 5 min with 1x PBS, it was incubated in ABC for 1 hour at RT. VIP peroxidase substrate was prepared using the ImmPACTVIP peroxidase kit as detailed in the manufacturer's instructions. VIP peroxidase substrate was added for 7 minutes at RT and washed in dH ₂ O. Before mounting in DPX, the tissue was dehydrated for 2 minutes each time in IMS 50%, 70%, 95%, 100%, 100% and xylenes I and II. For double immunofluorescence staining tissue was not quenched with 3% H ₂ O ₂ before application of the primary antibody. After application of the first primary and equivalent secondary antibodies, the tissue was blocked with 15% normal goat serum for 30 minutes and incubated with the second primary and secondary antibody as described previously. After final application fluorescently labeled secondary antibodies, tissue was incubated in 1% Sudan black for 5 minutes to quench autofluorescence, washed in 0.1% TBS/T, and immediately placed in Mowiol Cituflour. Fluorescently stained tissue was stored at 4°C until image acquisition.

d. Image analysis and statistical analysis

Slides were scanned for analysis with a x20 objective using an Olympus VS110 high-performance virtual microscopy system or an Olympus dot slide virtual microscopy system. Thirty images (each 500 μm ² ) were obtained from the scanned image using Olympus VS software from equivalent areas of each section from each object (see Figs. 17A-17D, 18A-18D, 19A-19C, 20A-20E, 21A-21F , 22A-22C, 24A-24D and 25A-25D ). This allowed the analysis of a total area of 7.5 mm ² in each brain region. Fiji windows-64 version of ImageJ software was used to quantify α-Syn immunoreactivity of each image.

To analyze the total amount of α-Syn detected by each antibody, immunoreactivity was described as a percentage of the total image area. The threshold used to select α-Syn positive immunoreactivity was adjusted for each brain region analyzed to account for differences in background staining that could influence the results. The average percentage of area covered by α-Syn positive aggregates was calculated for each antibody and brain region analyzed.

To analyze the relative specificity of each antibody for LB or LN, Fiji software was used to quantify LB immunoreactivity based on size and roundness parameters to distinguish them from LN (see Figures 24A-24D, 25A-25D and 26A-26B ). Brain regions with clear LB and LN morphology were selected for this analysis to avoid false-positive results and included the insular cortex of the basal ganglia and the cortical gray matter of the temporal cortex. LB immunoreactivity was expressed as a percentage of total α-Syn immunoreactivity.

Statistical analysis was performed using GraphPad Prism v7.01 software and presented as mean+SD (unless otherwise stated). Results were analyzed using one-way analysis of variance (ANOVA) followed by post hoc analysis with Dunnett's corrections as appropriate. Differences were considered significant at p<0.05 (*). Numbers (n) refer to the number of subjects used for each experiment.

Qualitative analysis of the location of α-Syn in neurons or glia was performed using double immunofluorescence staining as previously described. Microspecimens were viewed using a Leica SP8 laser scanning confocal microscope. Images with superimposed maximum projections were obtained with a ×40 objective sequentially. These images contained a series of z-slide images stacked together with both color channels superimposed to show their relative positions.

e. Antibodies to α-Syn _126-135 resulted in the detection of a distinct pattern of α-Syn aggregates compared to NCL-L-ASYN

The cell type and subcellular localization of α-Syn aggregates vary in different synucleinopathies. MSA is characterized by glial cytoplasmic inclusions (GCI), while in DLB and PD α-Syn aggregation occurs in neuronal bodies (LB) and axonal processes (LN). Analysis of the percentage stained area quantified the total α-Syn aggregates detected by each antibody. At the same time, differences in the type or subcellular location of the identified aggregates were not taken into account. The distinct pattern of α-Syn aggregates in cell bodies and neurites in PD and DLB cases allowed us to determine the relative sensitivity of the UNS antibodies of this disclosure against these different types of α-Syn aggregates.

To investigate this, the proportion of aggregates detected in cell bodies was assessed for each antibody in DLB and PD cases. Using FIJI software, aggregates in cell bodies were selected based on their size and roundness. The average percentage area of cell body aggregates was then calculated as a fraction of the total α-Syn detected, and the results are shown in Fig. 24A-24D and 25A-25D . The difference in area percentage of total α-Syn and α-Syn cell bodies was attributed to α-Syn axonal aggregates (LNs) based on qualitative tissue analysis. A decrease in the percentage of detection of α-Syn cell bodies corresponds to an increase in LN detection. This analysis was performed in the gray matter of the temporal cortex and insular cortex, as these regions showed both LB and LN-like pathology. LNs were very sparse and unevenly distributed throughout the globus pallidus and capsule, and therefore these regions of the basal ganglia were not selected for this analysis. A similar correlation was observed in the substantia nigra of the midbrain ( Fig. 26A and 26B ) with the UNS antibodies of this disclosure, which resulted in the detection of higher levels of LN compared to NCL-L-ASYN in DLB and PD. At the same time, due to the complex morphology of LN and LB, it was not possible to reliably distinguish and quantify them using the same method.

Results in Fig. 24A-24D show that of the total α-Syn detected by each antibody, the proportion of aggregates detected in cell bodies was reduced by UNS antibodies compared to NCL-L-ASYN. This means that the ratio of inclusions in cell bodies relative to LN was reduced, and a higher proportion of LN was detected in the case of UNS antibodies. Of the UNS antibodies, PD062205 was robust in DLB and PD, detecting high proportions of LN in the insular cortex ( Figures 17A-17D and 18A-18D ). In contrast, all antibodies to α-Syn _126-135 resulted in the detection of higher proportions of cell body aggregates compared to NCL-L-ASYN in the gray matter of the temporal cortex of DLB and PD cases ( Figures 25A-25B ).

f. α-Syn Aggregation is Cell Type Specific

Aggregates containing α-Syn are a characteristic pathogenic feature of synucleinopathies, including MSA, DLB, and PD. Although α-Syn aggregation is associated with the major proteins implicated in synucleinopathies, the pattern of aggregation and the cell types that are susceptible to aggregate formation differ between specific disease subtypes. The clinical characteristic of MSA, DLB and PD is described as the accumulation of α-Syn in the cell bodies and neurite processes of neurons in both DLB and PD, but in MSA it occurs mainly in glial cells and oligodendrocytes.

To determine the selectivity of α-Syn _126-135 antibodies for cell-specific α-Syn aggregates, a dual immunofluorescence assay was performed using PD062205 and either neuronal (HuD) or oligodendrocyte (Olig2) markers.

Results in Fig. 27A-27C show that α-Syn identified by PD062205 colocalizes with neuronal cell bodies in the basal ganglia and midbrain (areas of high pathology) in PD and DLB, but not in MSA. Use of oligodendrocyte markers (Olig2) in Fig. 28A-28C show that in MSA, but not in PD or DL, α-Syn aggregates in glial cells. These results demonstrate that antibodies to α-Syn _126–135 are consistent with the clinical characteristics of these synucleinopathies and confirm the specificity of these antibodies in the case of pathological α-Syn aggregates.

results

a. Quantitative analysis of antibodies generated by immunization in guinea pigs with an exemplary peptide immunogenic construct α-Syn _126-135 for the purpose of immunotherapy

To investigate the use of novel anti-α-Syn antibodies for immunotherapy, the relative specificity of each α-Syn antibody was quantified by immunohistochemistry (IHC) in cases of three human synucleinopathies (MSA, DLB, and PD).

b. Antibodies generated by immunization in guinea pigs with an exemplary α-Syn peptide immunogenic construct _126-135 are more sensitive than commercially used diagnostic antibodies when binding to α-Syn aggregates

To examine the relative antigenicity of the disclosed α-Syn antibodies _126-135 , the α-Syn load detected by each antibody was compared with a commercially available diagnostic antibody for synucleinopathies (NCL-L-ASYN). Having initially examined the overall picture of the results shown in FIG. 17A-D-22A-22C , it can be seen that there is a marked increase in the average percentage of α-Syn area detected by α-Syn antibodies _126-135 compared to NCL-L-ASYN. This trend is consistent across each brain region and disease type and suggests that the disclosed α-Syn _126-135 antibodies are more sensitive or selective in binding to aggregated α-Syn than NCL-L-ASYN. Although the sample size in this study was relatively small (n=3), a clear trend can still be seen in the data. The specificity of the disclosed α-Syn antibodies _126-135 for α-Syn was confirmed in the same brain regions as in the brains of control patients without disease. These results, which are demonstrated in Fig. 23A-23B show the absence of any immunopositive staining with each antibody, including NCL-L-ASYN. These data indicate that the disclosed antibodies to α-Syn _126-135 are specific for pathological forms of α-Syn.

c. The higher levels of α-Syn detected with α-Syn antibodies _126-135 indicate their increased sensitivity and specificity compared to commercial antibodies

Antibodies to α-Syn_126-135 according to this disclosure lead to the detection of more α-Syn compared to NCL-L-ASYN, which indicates that the disclosed antibodies are more preferable for use in immunotherapy to facilitate the clearance of these α-Syn aggregates.

The first step in selecting an appropriate antibody for use as an immunotherapy reagent is to determine the selectivity of the antibody for the target antigen (α-Syn) in human brain tissue with primary α-Syn pathology. The various synucleinopathies differ in the mechanisms and neuroanatomical pattern of α-Syn aggregation, as well as in the susceptibility of certain cell types to aggregation.

It is important to evaluate the selectivity of α-Syn antibodies _126-135 over α-Syn in various synucleinopathies with different neuropathologies in order to explore the use of the reagent as an immunotherapy for synucleinopathies in general. Clinically confirmed cases of PD, DLB and MSA were selected for this purpose. PD and DLB are the second most common forms of dementia and are primarily caused by the accumulation of α-Syn in neurons (LB and LN). In contrast to PD, amyloid beta and tau pathologies are known to contribute to neurodegeneration in DLB2. A different pattern of α-Syn aggregation is observed in MSA, where aggregates are primarily formed in glial cells rather than neurons ( Figures 27A-27C and 28A-28B ). Additionally, the progression of α-Syn pathology varies between disease types, with the midbrain and basal ganglia being common areas of early pathology. Examining the antigenicity of each antibody in areas of the brain affected at different stages of the disease will provide insight into which antibody may be more effective in treating early stages of the disease.

d. Antibodies to α-Syn _126-135 (PD062220, PD062205 and PD100806) are able to specifically bind to pathological α-Syn aggregates in human brain tissue obtained from PD, DLB and MSA (Fig. 17A-D-22A-22C) without detection any synuclein pathology in healthy controls (Figures 23A-23B).

Detection of α-Syn using the disclosed α-Syn antibodies _126-135 was performed with the same cell type specificity that has been described in clinical neuropathology ( Figures 27A-27B and 28A-28B ). Importantly, the disclosed antibodies to α-Syn _126-135 did not demonstrate equal antigenicity against all forms of human α-Syn.

The specificity of PD062205 and PD100806 was further supported by the ability of each antibody to result in detection of a higher proportion of LN than NCL-L-ASYN in the basal ganglia ( Figures 24A-24D ). This was also observed visually in the midbrain ( Figures 26A-26B ). Taken together, with the higher percentage of α-Syn area detected by PD062205 and PD100806, these results indicate that the additional α-Syn detected by disclosed α-Syn antibodies _126–135 may be due in part to increased specificity these antibodies against LN. These results are relevant for immunotherapy because in early stages of the disease, LNs represent the predominant form of α-Syn aggregation in the basal ganglia. Other reagents for the treatment of synucleinopathies that are in preclinical development do not provide IHC detection of LN. Thus, the disclosed peptide immunogenic constructs and antibodies to α-Syn _126-135 formed from the peptide immunogenic constructs have unique properties and characteristics compared to other commercially available products.

This study used IHC to analyze the sensitivity of antibodies to α-Syn _126-135 generated by the disclosed peptide immunogenic constructs by measuring the average number of α-Syn aggregates in affected brain regions. This study, which quantified the mean percentage area of α-Syn in brain samples, demonstrated that the disclosed α-Syn antibodies _126-135 were very sensitive to detecting α-Syn earlier in disease progression compared to MSA, DLB and PD compared to a commercially available antibody.

The higher sensitivity found in this study may be due to the higher specificity of the disclosed antibodies against LN compared to the diagnostic antibody NCL-L-ASYN. These results suggest that the disclosed antibodies to α-Syn _126–135 are likely the most effective candidates for investigating antibody-mediated clearance of α-Syn aggregates in synucleinopathies.

Table 1

α-Syn amino acid sequences and fragments thereof used in serological assays

Amino acid positions SEQ ID NO: Subsequence α-Synuclein 1-140 1 MDVFM KGLSK AKEGV VAAAE KTKQG VAEAA GKTKE GVLYV GSKTK EGVVH GVATV AEKTK EQVTN VGGAV VTGVT AVAQK TVEGA GSIAA ATGFV KKDQL GKNEE GAPQE GILED MPVDP DNEAY EMPSE EGYQD YEPEA α-Synuclein 80-140 3 KTVEG AGSIA AATGF VKKDQ LGKNE EGAPQ EGILE DMPVD PDNEA YEMPS EEGYQ DYEPE A α-Synuclein 85-140 4 AGSIA AATGF VKKDQ LGKNE EGAPQ EGILE DMPVD PDNEA YEMPS EEGYQ DYEPEA α-Synuclein 91-140 5 ATGFV KKDQL GKNEE GAPQE GILED MPVDP DNEAY EMPSE EGYQD YEPEA α-Synuclein 101-140 6 GKNEE GAPQE GILED MPVDP DNEAY EMPSE EGYQD YEPEA α-Synuclein 111-140 7 GILED MPVDP DNEAY EMPSE EGYQD YEPEA α-Synuclein 121-140 8 DNEAY EMPSE EGYQD YEPEA α-Synuclein 126-140 9 EMPSE EGYQD YEPEA α-Synuclein 97-135 10 KDQLG KNEEG APQEG ILEDM PVDPD NEAYE MPSEE GYQD α-Synuclein 101-135 eleven GKNEE GAPQE GILED MPVDP DNEAY EMPSE EGYQD α-Synuclein 111-135 12 GILED MPVDP DNEAY EMPSE EGYQD α-Synuclein 121-135 13 DNEAY EMPSE EGYQD α-Synuclein 123-135 14 EAYEM PSEEG YQD α-Synuclein 126-135 15 EMPSE EGYQD α-Synuclein 101-132 16 GKNEE GAPQE GILED MPVDP DNEAY EMPSE EG α-Synuclein 111-132 17 GILED MPVDP DNEAY EMPSE EG α-Synuclein 80-89 18 KTVEG AGSIA α-Synuclein 81-90 19 TVEGA GSIAA α-Synuclein 82-91 20 VEGAG SIAAA α-Synuclein 83-92 21 EGAGS IAAAT α-Synuclein 84-93 22 GAGSI AAATG α-Synuclein 85-94 23 AGSIA AATGF α-Synuclein 86-95 24 GSIAA ATGFV α-Synuclein 87-96 25 SIAAA TGFVK α-Synuclein 88-97 26 IAAAT GFVKK α-Synuclein 89-98 27 AAATG FVKKD α-Synuclein 90-99 28 AATGF VKKDQ α-Synuclein 91-100 29 ATGFV KKDQL α-Synuclein 92-101 thirty TGFVK KDQLG α-Synuclein 93-102 31 GFVKK DQLGK α-Synuclein 94-103 32 FVKKD QLGKN α-Synuclein 95-104 33 VKKDQ LGKNE α-Synuclein 95-105 34 KKDQL GKNEE α-Synuclein 97-106 35 KDQLG KNEEG α-Synuclein 98-107 36 DQLGK NEEGA α-Synuclein 99-108 37 QLGKN EEGAP

Table 1 (continued)

Amino acid positions SEQ ID
NO: Subsequence α-Synuclein 100-109 38 LGKNE EGAPQ α-Synuclein 101-110 39 GKNEE GAPQE α-Synuclein 102-111 40 KNEEG APQEG α-Synuclein 103-112 41 NEEGA PQEGI α-Synuclein 104-113 42 EEGAP QEGIL α-Synuclein 105-114 43 EGAPQ EGILE α-Synuclein 106-115 44 GAPQE GILED α-Synuclein 107-117 45 APQEG ILEDM α-Synuclein 108-117 46 PQEGI LEDMP α-Synuclein 109-118 47 QEGIL EDMPV α-Synuclein 110-119 48 EGILE DMPVD α-Synuclein 111-120 49 GILED MPVDP α-Synuclein 112-121 50 ILEDM PVDPD α-Synuclein 113-122 51 LEDMP VDPDN α-Synuclein 114-123 52 EDMPV DPDNE α-Synuclein 115-124 53 DMPVD PDNEA α-Synuclein 116-125 54 MPVDP DNEAY α-Synuclein 117-126 55 PVDPD NEAYE α-Synuclein 118-127 56 VDPDN EAYEM α-Synuclein 119-128 57 DPDNE AYEMP α-Synuclein 120-129 58 PDNEA YEMPS α-Synuclein 121-130 59 DNEAY EMPSE α-Synuclein 122-131 60 NEAYE MPSEE α-Synuclein 123-132 61 EAYEM PSEEG α-Synuclein 124-133 62 AYEMP SEEGY α-Synuclein 125-134 63 YEMPS EEGYQ α-Synuclein 126-135 64 EMPSE EGYQD α-Synuclein 127-136 65 MPSEE GYQDY α-Synuclein 128-137 66 PSEEG YQDYE α-Synuclein 129-138 67 SEEGY QDYEP α-Synuclein 130-139 68 EEGYQ DYEPE α-Synuclein 131-140 69 EGYQD YEPEA

table 2

Amino acid sequences of Th epitopes derived from pathogenic proteins, including idealized artificial Th epitopes for use in the development of α -Syn peptide immunogenic constructs

Description SEQ ID
NO: Subsequence Th Clostridium tetani1 70 KKQYIKANSKFIGITEL Th MvF1 71 LSEIKGVIVHRLEGV Th Bordetella pertussis 72 GAYARCPNGTRALTVAELRGNAEL Th Clostridium tetani2 73 WVRDIIDDFTNESSQKT Th diphtheria 74 DSETADNLEKTVAALSILPGHGC Th Plasmodium falciparum 75 DHEKKHAKMEKASSVFNVVNS Th Schistosoma mansoni 76 KWFKTNAPNGVDEKHRH Th cholera toxin 77 ALNIWDRFDVFCTLGATTGYLKGNS Th MvF2 78 ISEIKGVIVHKIEGI Th KKKMvF3 79 KKKISISEIKGVIVHKIEGILF
T RT TR T Th HBsAg1 80 KKKLFLLTKLLTLPQSLD
RRRIKII RII IL IR
VRVV VV VIV
F FF FF FVF
F Th MvF4 (UBITh®3) 81 ISISEIKGVIVHKIETILF
T RT TR Th HBsAg2 82 KKKIITITRIITIPQSLD
FFLL LITTI Th MvF5 (UBITh®1) 83 ISITEIKGVIVHRIETILF Th HBsAg3 (UBITh®2) 84 KKKIITITRIITIITTID Th influenza virus MP1_1 85 FVFTLTVPSER Th influenza virus MP1_2 86 SGPLKAEIAQRLEDV Th influenza virus NSP1 87 DRLRRDQKS Th EBV BHRF1 88 AGLTLSLLVICSYLFISRG Th Clostridium tetani TT1 89 QYIKANSKFIGITEL Th EBV EBNA-1 90 PGPLRESIVCYFMVFLQTHI Th Clostridium tetani TT2 91 FNNFTVSFWLRVPKVSASHLE Th Clostridium tetani TT3 92 KFIIKRYTPNNEIDSF Th Clostridium tetani TT4 93 VSIDKFRIFCKALNPK Th EBV CP 94 VPGLYSPCRAFFNKEELL Th HCMV IE1 95 DKREMWMACIKELH Th EBV GP340 96 TGHGARTSTEPTTDY Th EBV BPLF1 97 KELKRQYEKKLRQ Th EBV EBNA-2 98 TVFYNIPPMPL

Table 3

Amino acid sequences of α -Syn peptide immunogenic constructs

Description of peptides Seq ID
NO: Subsequence UBITh3-εK-KKK-α-synuclein 99 UBITh3-εk-kkk-EMPSEEGYQDYEPEA UBITh3-εK-KKK-α-synuclein 100 UBITh3-εk-kkk-DNEAYEMPSEEGYQDYEPEA UBITh3-εK-KKK-α-synuclein 101 UBITh3-εk-kkk-GILEDMPVDPDNEAYEMPSEEGYQDYEPEA UBITh3-εK-KKK-α-synuclein 102 UBITh3-εk-kkk-GKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA UBITh1-εK-KKK-α-synuclein 103 UBITh1-εk-kkk-GKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA UBITh2-εK-KKK-α-synuclein 104 UBITh2-εk-kkk-GKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA UBITh3-εK-KKK-α-synuclein 105 UBITh3-εk-kkk-ATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA UBITh3-εK-KKK-α-synuclein 106 UBITh3-εk-kkk-AGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA UBITh1-εK-KKK-α-synuclein 107 UBITh1-εk-kkk-DNEAYEMPSEEGYQD UBITh1-εK-KKK-α-synuclein 108 UBITh1-εk-kkk-GILEDMPVDPDNEAYEMPSEEGYQD UBITh1-εK-KKK-α-synuclein 109 UBITh1-εk-kkk-GKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQD UBITh1-εK-KKK-α-synuclein 110 UBITh1-εk-kkk-KDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQD UBITh1-εK-KKK-α-synuclein 111 UBITh1-εk-kkk-EAYEMPSEEGYQD UBITh1-εK-KKK-α-synuclein 112 UBITh1-εk-kkk-EMPSEEGYQD UBITh1-εK-KKK-α-synuclein 113 UBITh1-εk-kkk-GILEDMPVDPDNEAYEMPSEEG UBITh1-εK-KKK-α-synuclein 114 UBITh1-εk-kkk-GKNEEGAPQEGILEDMPVDPDNEAYEMPSEEG UBITh1-εK-KKK-mouse analogue of α-synuclein 115 UBITh1-εk-kkk-GILEDMPVDPGSEAYEMPSEEG UBITh3-εK-KKK-α-synuclein 116 UBITh3-εk-kkk-EMPSEEGYQD UBITh3-εK-KKK-α-synuclein 117 UBITh3-εk-kkk-GILEDMPVDPDNEAYEMPSEEG UBITh1-εK-α-synuclein 118 UBITh1-εk-EMPSEEGYQD UBITh1-εK-α-synuclein 119 UBITh1-εk-GILEDMPVDPDNEAYEMPSEEG UBITh2-εK-α-synuclein 120 UBITh2-εk-EMPSEEGYQD UBITh2-εK-α-synuclein 121 UBITh2-εk-GILEDMPVDPDNEAYEMPSEEG Th Clostridium tetani1-εK-α-Syn 122 KKQYIKANSKFIGITEL-εk-GILEDMPVDPDNEAYEMPSEEG Th MvF1-εK-α-synuclein 123 LSEIKGVIVHRLEGV-εk-GILEDMPVDPDNEAYEMPSEEG Th Bordetella pertussis-εK-α-Syn 124 GAYARCPNGTRALTVAELRGNAEL-εk-GILEDMPVDPDNEAYEMPSEEG Th Clostridium tetani2-εK-α-Syn 125 WVRDIIDDFTNESSQKT-εk-GILEDMPVDPDNEAYEMPSEEG Th diphtheria-εK-α-Syn 126 DSETADNLEKTVAALSILPGHGC-εk-GILEDMPVDPDNEAYEMPSEEG Th Plasmodium falciparum-εK-α-Syn 127 DHEKKHAKMEKASSVFNVVNS-εk-GILEDMPVDPDNEAYEMPSEEG Th Schistosoma mansoni-εK-α-Syn 128 KWFKTNAPNGVDEKHRH-εk-GILEDMPVDPDNEAYEMPSEEG Th cholera toxin-εK-α-Syn 129 ALNIWDRFDVFCTLGATTGYLKGNS-εk-GILEDMPVDPDNEAYEMPSEEG Th MvF2-εK-α-Syn 130 ISEIKGVIVHKIEGI-εk-GILEDMPVDPDNEAYEMPSEEG

Table 3 (continued)

Description of peptides Seq ID
NO: Subsequence Th KKKMvF3-εK-α-Syn _111-132 131 KKKISISEIKGVIVHKIEGILF-εk-GILEDMPVDPDNEAYEMPSEEG
T RT TR T Th HBsAg1-εK-α-Syn 132 KKKLFLLTKLLTLPQSLD-εk-GILEDMPVDPDNEAYEMPSEEG
RRRIKII RII IL IR
VRVV VV VIV
F FF FF FVF
F Th HBsAg2-εK-α-Syn 133 KKKIITITRIITIPQSLD-εk-GILEDMPVDPDNEAYEMPSEEG
FFLL LITTI Th influenza virus MP1_1-εK-α-Syn 134 FVFTLTTVPSER-εk-GILEDMPVDPDNEAYEMPSEEG Th virus MP1_2-εK-α-Syn 135 SGPLKAEIAQRLEDV-εk-GILEDMPVDPDNEAYEMPSEEG Th influenza virus NSP1-εK-α-Syn 136 DRLRRDQKS-εk-GILEDMPVDPDNEAYEMPSEEG Th EBV BHRF1-εK-α-Syn 137 AGLTLSLLVICSYLFISRG-εk-GILEDMPVDPDNEAYEMPSEEG Th Clostridium tetani TT1-εK-α-Syn 138 QYIKANSKFIGITEL-εk-GILEDMPVDPDNEAYEMPSEEG Th EBV EBNA-1-εK-α-Syn 139 PGPLRESIVCYFMVFLQTHI-εk-GILEDMPVDPDNEAYEMPSEEG Th Clostridium tetani TT2-εK-α-Syn 140 FNNFTVSFWLRVPKVSASHLE-εk-GILEDMPVDPDNEAYEMPSEEG Th Clostridium tetani TT3-εK-α-Syn 141 KFIIKRYTPNNEIDSF-εk-GILEDMPVDPDNEAYEMPSEEG Th Clostridium tetani TT4-εK-α-Syn 142 VSIDKFRIFCKALNPK-εk-GILEDMPVDPDNEAYEMPSEEG Th EBV CP-εK-α-Syn 143 VPGLYSPCRAFFNKEELL-εk-GILEDMPVDPDNEAYEMPSEEG Th HCMV IE1-εK-α-Syn 144 DKREMWMACIKELH-εk-GILEDMPVDPDNEAYEMPSEEG Th EBV GP340-εK-α-Syn 145 TGHGARTSTEPTTDY-εk-GILEDMPVDPDNEAYEMPSEEG Th EBV BPLF1-εK-α-Syn 146 KELKRQYEKKLRQ-εk-GILEDMPVDPDNEAYEMPSEEG Th EBV EBNA-2-εK-α-Syn 147 TVFYNIPPMPL-εk-GILEDMPVDPDNEAYEMPSEEG

Table 4

Evaluation of the immunogenicity of C-terminal peptide fragments of α -Syn for the identification of autologous Th epitopes in guinea pigs

Description of peptides Seq ID NO: Animal ID α-Syn _(A85-A140) (SEQ ID NO: 4), Log ₁₀ ELISA titer 0 weeks after immunization 3 weeks after immunization 6 weeks after immunization 8 weeks after immunization α-Synuclein (E126-A140) 9 5413 0.075 0.000 0.000 0.000 5414 0.086 0.000 0.000 0.000 5415 0.079 0.000 0.000 0.000 Average 0.080 0.000 0.000 0.000 α-Synuclein (D121-A140) 8 5416 0.056 0.000 0.000 0.000 5417 0.091 0.000 0.000 0.000 5418 0.066 0.000 0.000 0.000 Average 0.071 0.000 0.000 0.000 α-Synuclein (G111-A140) 7 5419 0.060 0.000 0.000 0.000 5420 0.089 0.000 0.000 1.026 5421 0.092 0.139 0.000 0.000 Average 0.081 0.046 0.000 0.342 α-Synuclein (G101-A140) 6 5422 0.084 0.000 1,997 3,096 5423 0.072 0.000 0.000 0.000 5424 0.077 0.000 0.000 0.000 Average 0.078 0.000 0.666 1.032 α-Synuclein (A91-A140) 5 5425 0.082 0.000 0.000 0.000 5426 0.079 0.294 3,007 2.765 5427 0.093 0.000 2,840 3.355 Average 0.084 0.098 1,949 2,040 α-Synuclein (A85-A140) 4 5428 0.082 3,059 3.628 4,349 5429 0.082 0.000 0.000 0.000 5430 0.073 0.000 3.005 2,894 Average 0.079 1,020 2,211 2,414

Table 5

Ranking of immunogenicity of α-Syn peptide immunogenic constructs in guinea pigs

Group no. Peptide immunogenic construct of α-synuclein Seq ID NO: Animal ID α-Syn _(G101-A140) (SEQ ID NO: 6), Log ₁₀ ELISA titer 0 weeks after immunization 3 weeks after immunization 6 weeks after immunization 8 weeks after immunization 1 UBITh1-εK-KKK-α-synuclein (G101-A140) 103 5431 0.167 4,740 4.938 4,912 5432 0.111 4,787 4,979 4,819 5433 0.110 4,799 4,920 4,924 Average 0.129 4.775 4,946 4.885 2 UBITh2-εK-KKK-α-synuclein (G101-A140) 104 5434 0.101 0.000 3,095 3.172 5435 0.100 2,743 4,439 4,052 5436 0.097 0.967 1,790 1.952 Average 0.099 1.237 3.108 3,059

Table 6

Evaluation of the immunogenicity of α-Syn peptide immunogenic constructs in guinea pigs

Group no. Immunogen Seq ID NO: Animal no. α-Syn _(A91-A140) (SEQ ID NO: 5), Log ₁₀ ELISA titer β-Syn _(103-134) (SEQ ID NO: 153), Log ₁₀ ELISA titer 0 weeks 3 weeks 6 weeks 8 weeks 13 weeks 0 weeks 3 weeks 6 weeks 8 weeks 13 weeks 1 UBITh3-εk-kkk-α-synuclein (E126-A140) 99 5334 0.1 5.1 5.3 6.6 5.0 0.1 2.9 4.4 4.9 4.7 5335 0.1 5.3 5.5 5.4 5.0 0.1 3.9 4.8 4.9 4.7 5336 0.1 6.9 11.0 12.5 8.3 0.1 3.8 5.2 5.7 5.4 Average 0.1 5.8 7.3 8.2 6.1 0.1 3.5 4.8 5.2 4.9 2 UBITh3-εk-kkk-α-synuclein (D121-A140) 100 5337 0.1 5.1 4.9 5.1 4.9 0.1 2.5 4.3 4.6 4.5 5338 0.2 4.5 4.6 4.7 4.4 0.1 1.3 3.3 3.8 3.3 5339 0.1 4.7 4.9 5.1 4.7 0.1 2.0 4.4 4.6 4.3 Average 0.1 4.8 4.8 5.0 4.7 0.1 1.9 4.0 4.4 4.1 3 UBITh3-εk-kkk-α-synuclein (G111-A140) 101 5340 0.2 5.1 5.0 5.1 4.6 0.1 2.2 3.9 4.4 3.5 5341 0.1 7.1 7.8 9.2 6.2 0.1 3.6 4.9 5.0 4.9 5342 0.1 4.9 5.2 5.8 5.2 0.1 2.0 4.6 4.8 4.8 Average 0.1 5.7 6.0 6.7 5.4 0.1 2.6 4.5 4.7 4.4 4 UBITh3-εk-kkk-α-synuclein (G101-A140) 102 5343 0.2 6.0 8.5 12.0 7.3 0.1 4.3 5.2 >5.00 5.8 5344 0.3 6.6 5.7 6.0 5.3 0.1 4.0 4.7 4.8 4.6 5345 0.2 5.9 6.2 9.4 5.9 0.1 4.0 5.0 5.5 5.2 Average 0.2 6.2 6.8 9.1 6.1 0.1 4.1 4.9 5.1 5.2 5 UBITh3-εk-kkk-α-synuclein (A91-A140) 105 5362 0.2 5.5 6.6 8.0 5.5 0.1 3.6 4.8 5.0 4.8 5363 0.1 5.1 5.7 5.7 5.4 0.1 2.8 4.4 4.5 4.5 5364 0.2 4.8 4.9 4.9 4.9 0.1 0.0 3.0 3.5 3.6 Average 0.1 5.2 5.7 6.2 5.3 0.1 2.1 4.1 4.3 4.3 6 UBITh3-εk-kkk-α-synuclein (A85-A140) 106 5365 0.1 5.1 5.0 5.3 5.1 0.1 3.2 3.9 4.3 3.6 5366 0.2 5.4 4.9 4.9 4.8 0.1 3.2 3.2 3.1 3.1 5367 0.1 5.1 5.3 5.3 5.3 0.1 1.1 4.7 4.7 4.6 Average 0.1 5.2 5.1 5.2 5.1 0.1 2.1 3.9 4.0 3.7

Table 7

Evaluation of the immunogenicity of α-Syn peptide immunogenic constructs in guinea pigs

Group no. Immunogen Seq ID NO: Animal no. α-Syn _(K97-D135) (SEQ ID NO: 10), Log ₁₀ ELISA titer β-Syn _(103-134) (SEQ ID NO: 153), Log ₁₀ ELISA titer 0 weeks 3 weeks 6 weeks 9 weeks 12 weeks 0 weeks 3 weeks 6 weeks 9 weeks 12 weeks 1 UBITh1-εk-kkk-α-synuclein (K97-D135) 110 5616 0.055 4,814 5.132 4.823 4,776 0.051 0.000 0.000 0.000 0.000 5617 0.049 3,394 4,464 4.323 4,292 0.050 0.000 0.000 0.000 0.000 5618 0.052 4,420 4,864 4,673 4,598 0.051 0.000 0.000 0.000 0.000 Average 0.052 4,209 4,820 4,606 4.555 0.051 0.000 0.000 0.000 0.000 2 UBITh1-εk-kkk-α-synuclein (G101-D135) 109 5613 0.056 4,738 4,882 4,848 4.855 0.056 0.000 0.000 0.000 0.000 5614 0.052 4,391 4,708 4.565 4,674 0.053 0.000 0.000 0.000 0.000 5615 0.058 4,789 5,050 4.956 4,904 0.055 0.000 0.000 0.000 0.000 Average 0.055 4.639 4,880 4,790 4,811 0.055 0.000 0.000 0.000 0.000 3 UBITh1-εk-kkk-α-synuclein (G101-G132) 114 5628 0.049 4,290 4,794 4.426 4.537 0.053 0.000 0.000 0.000 0.000 5629 0.069 4,502 4,939 4,764 4.645 0.067 0.000 0.000 0.000 0.000 5630 0.053 2,978 3.695 4,092 4,274 0.056 0.000 0.000 0.000 0.000 Average 0.057 3.923 4,476 4.427 4.485 0.059 0.000 0.000 0.000 0.000 4 UBITh1-εk-kkk-α-synuclein (G111-D135) 108 5545 0.051 4,941 4,919 4,842 4.735 0.069 0.000 0.000 0.000 0.000 5546 0.056 3.229 4,866 4,912 4,843 0.063 0.000 0.000 0.000 0.000 5547 0.053 5.075 5.237 5,033 4.954 0.065 0.000 0.000 0.000 0.000 Average 0.053 4.415 5,007 4,929 4,844 0.066 0.000 0.000 0.000 0.000 5 UBITh1-εk-kkk-α-synuclein (G111-G132) 113 5625 0.056 2,906 4,541 4,346 4.114 0.069 0.000 0.000 0.000 0.000 5626 0.051 2,596 4,087 3,504 3.655 0.053 0.000 0.000 0.000 0.000 5627 0.052 3.471 4.633 4.333 4.415 0.056 0.000 0.000 0.000 0.000 Average 0.053 2,991 4,420 4,061 4,061 0.059 0.000 0.000 0.000 0.000 6 UBITh1-εk-kkk-α-synuclein (D121-D135) 107 5542 0.067 3,042 4,214 4.121 3,989 0.062 0.000 0.000 0.000 0.000 5543 0.054 4,733 4,948 4.832 4,862 0.062 0.000 0.000 0.000 0.000 5544 0.060 2,943 4,306 4,249 4.222 0.065 0.000 0.000 0.000 0.000 Average 0.060 3,573 4,489 4,401 4,358 0.063 0.000 0.000 0.000 0.000 7 UBITh1-εk-kkk-α-synuclein (E123-D135) 111 5619 0.074 4.538 4.923 4,792 4,750 0.053 0.000 0.000 0.000 0.000 5620 0.052 4,880 5,930 5,069 5,046 0.054 0.000 0.000 0.000 0.000 5621 0.058 4,073 4.932 4,898 4,940 0.058 0.000 0.000 0.000 0.000 Average 0.061 4,497 5,262 4,920 4,912 0.055 0.000 0.000 0.000 0.000 8 UBITh1-εk-kkk-α-synuclein (E126-D135) 112 5622 0.051 4,820 5.156 5.015 5,018 0.055 0.000 0.000 0.000 0.000 5623 0.054 4,190 5.035 4,990 4.958 0.058 0.000 0.000 0.000 0.000 5624 0.048 4,906 6,747 5,630 5,602 0.063 0.000 0.000 0.000 0.000 Average 0.051 4.639 5,646 5.212 5,193 0.059 0.000 0.000 0.000 0.000

Table 8

Evaluation of immunogenicity against the Th epitope region of α -Syn peptide immunogenic constructs in guinea pigs

Group No. Immunogen SEQ ID NO: Animal ID UBITh1 (SEQ ID NO: 83), Log ₁₀ ELISA titer 0 weeks 3 weeks 6 weeks 9 weeks 12 weeks 1 UBITh1-εk-kkk-α-synuclein (K97-D135) 110 5616 0.065 0.000 0.616 1,746 2,023 5617 0.052 0.000 0.000 0.000 0.000 5618 0.058 0.000 0.000 0.000 0.000 Average 0.058 0.000 0.205 0.582 0.674 2 UBITh1-εk-kkk-α-synuclein (G101-D135) 109 5613 0.057 0.000 0.000 0.000 0.000 5614 0.054 0.000 0.000 0.000 0.000 5615 0.063 0.000 0.000 1.527 1.462 Average 0.058 0.000 0.000 0.509 0.487 3 UBITh1-εk-kkk-α-synuclein (G101-G132) 114 5628 0.052 0.000 0.000 0.000 0.000 5629 0.062 0.000 0.000 0.000 0.000 5630 0.058 0.000 0.000 0.000 0.000 Average 0.057 0.000 0.000 0.000 0.000 4 UBITh1-εk-kkk-α-synuclein (G111-D135) 108 5545 0.065 0.000 0.000 0.000 0.000 5546 0.069 0.000 0.000 0.000 0.000 5547 0.060 0.000 0.095 1.105 1.175 Average 0.065 0.000 0.032 0.368 0.392 5 UBITh1-εk-kkk-α-synuclein (G111-G132) 113 5625 0.062 0.000 0.000 0.000 0.000 5626 0.057 0.000 0.000 0.000 0.000 5627 0.058 0.000 0.000 0.000 0.000 Average 0.059 0.000 0.000 0.000 0.000 6 UBITh1-εk-kkk-α-synuclein (D121-D135) 107 5542 0.078 0.000 0.000 0.000 0.000 5543 0.069 0.000 2,468 2,349 2,980 5544 0.082 0.000 0.000 0.000 0.000 Average 0.076 0.000 0.823 0.783 0.993 7 UBITh1-εk-kkk-α-synuclein (E123-D135) 111 5619 0.058 0.000 0.000 0.662 1.887 5620 0.056 0.000 2,892 3.138 2,910 5621 0.062 0.000 0.000 1.321 0.000 Average 0.059 0.000 0.964 1,707 1,599 8 UBITh1-εk-kkk-α-synuclein (E126-D135) 112 5622 0.058 0.000 2,878 2,959 3,059 5623 0.063 0.000 0.000 0.000 0.000 5624 0.053 1.437 2,933 2,996 2,940 Average 0.058 0.479 1.937 1.985 2,000

Table 10

Inhibition of α -Syn aggregation by antibodies from animals receiving α -Syn peptide immunogenic constructs

Description of peptides SEQ ID NO weeks after immunization Aggregation inhibition (%) IgG (µg/ml) 0.05 0.5 5 UBITh3-εK-KKK-α-synuclein _85-140 106 3 33 49 45 8 51 72 76 13 47 50 43 UBITh3-εK-KKK-α-synuclein _91-140 105 3 40 42 54 8 65 75 92 13 56 41 55 UBITh3-εK-KKK-α-synuclein _101-140 102 3 45 45 53 8 55 73 70 13 41 51 48 UBITh3-εK-KKK-α-synuclein _111-140 101 3 36 40 49 8 66 60 59 13 77 66 70 UBITh3-εK-KKK-α-synuclein _121-140 100 3 41 44 46 8 51 77 76 13 40 47 54 UBITh3-εK-KKK-α-synuclein _126-140 99 3 49 54 48 8 65 50 63 13 110 73 84 UBITh1-εK-KKK-α-synuclein _97-135 110 6 51 54 83 9 27 74 77 12 44 41 55 UBITh1-εK-KKK-α-synuclein _101-135 109 6 105 98 68 9 70 65 95 12 57 76 85 UBITh1-εK-KKK-α-synuclein _111-135 108 6 55 84 82 9 52 70 82 12 56 58 87 UBITh1-εK-KKK-α-synuclein _121-135 107 6 29 38 51 9 42 48 69 12 87 64 64 15 74 74 76 UBITh1-εK-KKK-α-synuclein _123-135 111 6 34 45 60 9 42 thirty 48 12 58 55 59 15 56 64 75 UBITh1-εK-KKK-α-synuclein _126-135 112 6 17 45 54 9 49 49 59 12 58 68 56 15 70 76 62 UBITh1-εK-KKK-α-synuclein _101-132 114 6 79 83 87 9 61 66 87 12 48 55 51 UBITh1-εK-KKK-α-synuclein _111-132 113 6 43 46 57 9 24 57 46 12 28 44 51

Table 11

Evaluation of neuroprotective potential against neurodegeneration caused by α -Syn aggregates by quantifying neurite length through a multiparametric screen using antibodies from animals receiving α -Syn peptide immunogenic constructs

Description of peptides SEQ ID NO weeks after immunization Neurite length (%) IgG (µg/ml) 0.05 0.5 5 UBITh3-εK-KKK-α-synuclein _85-140 106 3 6 10 25 8 8 eleven 17 13 9 8 26 UBITh3-εK-KKK-α-synuclein _91-140 105 3 17 4 29 8 9 14 15 13 14 12 12 UBITh3-εK-KKK-α-synuclein _101-140 102 3 12 9 27 8 12 eleven 14 13 10 10 18 UBITh3-εK-KKK-α-synuclein _111-140 101 3 13 16 21 8 12 18 31 13 10 15 23 UBITh3-εK-KKK-α-synuclein _121-140 100 3 10 8 23 8 9 15 29 13 5 18 19 UBITh3-εK-KKK-α-synuclein _126-140 99 3 13 24 26 8 12 24 48 13 12 17 35 UBITh1-εK-KKK-α-synuclein _97-135 110 6 13 15 17 9 8 12 16 12 13 14 23 UBITh1-εK-KKK-α-synuclein _101-135 109 6 9 10 12 9 12 8 17 12 13 10 12 UBITh1-εK-KKK-α-synuclein _111-135 108 6 eleven 14 19 9 eleven 14 27 12 9 16 26 UBITh1-εK-KKK-α-synuclein _121-135 107 6 15 22 31 9 13 17 34 12 eleven 16 26 15 9 16 15 UBITh1-εK-KKK-α-synuclein _123-135 111 6 14 13 31 9 eleven 21 29 12 10 12 22 15 8 8 15 UBITh1-εK-KKK-α-synuclein _126-135 112 6 13 26 55 9 13 20 46 12 12 12 22 15 eleven 10 14 UBITh1-εK-KKK-α-synuclein _101-132 114 6 eleven 18 27 9 12 29 64 12 12 22 50 UBITh1-εK-KKK-α-synuclein _111-132 113 6 10 15 31 9 14 26 55 12 14 21 59

Table 12

Neuroprotective assessment in neurodegenerative neurons caused by α -Syn aggregates by quantifying neuronal numbers through multiparameter screening using antibodies from animals receiving α -Syn peptide immunogenic constructs

Description of peptides SEQ ID NO weeks after immunization Neuronal survival (%) IgG (µg/ml) 0.05 0.5 5 UBITh3-εK-KKK-α-synuclein _85-140 106 3 18 23 22 8 19 18 25 13 20 23 20 UBITh3-εK-KKK-α-synuclein _91-140 105 3 26 29 31 8 22 27 31 13 24 23 21 UBITh3-εK-KKK-α-synuclein _101-140 102 3 eleven 14 17 8 16 20 23 13 17 18 20 UBITh3-εK-KKK-α-synuclein _111-140 101 3 23 21 31 8 20 34 43 13 24 26 28 UBITh3-εK-KKK-α-synuclein _121-140 100 3 25 28 35 8 22 34 39 13 21 38 43 UBITh3-εK-KKK-α-synuclein _126-140 99 3 25 32 41 8 22 37 42 13 16 28 25 UBITh1-εK-KKK-α-synuclein _97-135 110 6 23 19 24 9 22 26 27 12 16 24 thirty UBITh1-εK-KKK-α-synuclein _101-135 109 6 18 23 27 9 25 21 22 12 22 26 29 UBITh1-εK-KKK-α-synuclein _111-135 108 6 23 37 42 9 28 45 65 12 24 34 46 UBITh1-εK-KKK-α-synuclein _121-135 107 6 19 26 49 9 24 22 31 12 19 26 28 15 20 19 22 UBITh1-εK-KKK-α-synuclein _123-135 111 6 20 26 29 9 24 21 31 12 19 24 32 15 20 36 49 UBITh1-εK-KKK-α-synuclein _126-135 112 6 20 36 49 9 26 thirty 35 12 28 thirty 36 15 25 20 31 UBITh1-εK-KKK-α-synuclein _101-132 114 6 22 thirty 43 9 26 37 57 12 25 34 55 UBITh1-εK-KKK-α-synuclein _111-132 113 6 24 34 34 9 22 39 50 12 21 31 38

Table 15

List of cases received from UCL and their diagnosis after autopsy

Case ID Age Floor Diagnosis PD505 TBC TBC M.S.A. PD363 TBC TBC M.S.A. PD300 TBC TBC M.S.A. PD294 TBC TBC DLB PD330 TBC TBC DLB PD385 TBC TBC DLB PD451 TBC TBC P.D. PD458 TBC TBC P.D. PD413 TBC TBC P.D. PDC87 TBC TBC CONTROL

--->

LIST OF SEQUENCES

<110>United Neuroscience

Wang, Chang Yi

<120>PEPTIDE IMMUNOGENS FROM THE C-TERMINAL END OF ALPHA-SYNUCLEIN PROTEIN AND COMPOSITIONS BASED ON THEM FOR THE TREATMENT OF SYNUCLEONOPATHIES

<130>UNS1001-US

<140>TBD

<141>2018-06-15

<150>US 62/521,287

<151>2017-06-16

<160>153

<170>PatentIn version 3.5

<210>1

<211>140

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(140)

<223>alpha-Synuclein 1-140

<400>1

Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val

1 5 10 15

Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys

20 25 30

Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val

35 40 45

Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr

50 55 60

Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys

65 70 75 80

Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys

85 90 95

Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile

100 105 110

Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

115 120 125

Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

130 135 140

<210>2

<211>134

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(134)

<223>beta-Synuclein 1-134

<400>2

Met Asp Val Phe Met Lys Gly Leu Ser Met Ala Lys Glu Gly Val Val

1 5 10 15

Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys

20 25 30

Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Arg Glu Gly Val

35 40 45

Val Gln Gly Val Ala Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Ser

50 55 60

His Leu Gly Gly Ala Val Phe Ser Gly Ala Gly Asn Ile Ala Ala Ala

65 70 75 80

Thr Gly Leu Val Lys Arg Glu Glu Phe Pro Thr Asp Leu Lys Pro Glu

85 90 95

Glu Val Ala Gln Glu Ala Ala Glu Glu Pro Leu Ile Glu Pro Leu Met

100 105 110

Glu Pro Glu Gly Glu Ser Tyr Glu Asp Pro Pro Gln Glu Glu Tyr Gln

115 120 125

Glu Tyr Glu Pro Glu Ala

130

<210>3

<211>61

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(61)

<223>alpha-Synuclein 80-140

<400>3

Lys Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val

1 5 10 15

Lys Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly

20 25 30

Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met

35 40 45

Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

50 55 60

<210>4

<211>56

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(56)

<223>alpha-Synuclein 85-140

<400>4

Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys Lys Asp Gln Leu

1 5 10 15

Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp Met

20 25 30

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

Tyr Gln Asp Tyr Glu Pro Glu Ala

50 55

<210>5

<211>50

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(50)

<223>alpha-Synuclein 91-140

<400>5

Ala Thr Gly Phe Val Lys Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly

1 5 10 15

Ala Pro Gln Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn

20 25 30

Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro

35 40 45

Glu Ala

50

<210>6

<211>40

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(40)

<223>alpha-Synuclein 101-140

<400>6

Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp Met

1 5 10 15

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

20 25 30

Tyr Gln Asp Tyr Glu Pro Glu Ala

35 40

<210>7

<211>30

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(30)

<223>alpha-Synuclein 111-140

<400>7

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

1 5 10 15

Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

20 25 30

<210>8

<211>20

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(20)

<223>alpha-Synuclein 121-140

<400>8

Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr

1 5 10 15

Glu Pro Glu Ala

20

<210>9

<211>15

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(15)

<400>9

Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

1 5 10 15

<210>10

<211>39

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(39)

<223>alpha-Synuclein 97-135

<400>10

Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile

1 5 10 15

Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

20 25 30

Ser Glu Glu Gly Tyr Gln Asp

35

<210>11

<211>35

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(35)

<223>alpha-Synuclein 101-135

<400>11

Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp Met

1 5 10 15

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

20 25 30

Tyr Gln Asp

35

<210>12

<211>25

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(25)

<223>alpha-Synuclein 111-135

<400>12

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

1 5 10 15

Met Pro Ser Glu Glu Gly Tyr Gln Asp

20 25

<210>13

<211>15

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>alpha-Synuclein 121-135

<400>13

Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

1 5 10 15

<210>14

<211>13

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(13)

<223>alpha-Synuclein 123-135

<400>14

Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

1 5 10

<210>15

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 126-135

<400>15

Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

1 5 10

<210>16

<211>32

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(32)

<223>alpha-Synuclein 101-132

<400>16

Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp Met

1 5 10 15

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

20 25 30

<210>17

<211>22

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(22)

<223>alpha-Synuclein 111-132

<400>17

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

1 5 10 15

Met Pro Ser Glu Glu Gly

20

<210>18

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 80-89

<400>18

Lys Thr Val Glu Gly Ala Gly Ser Ile Ala

1 5 10

<210>19

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 81-90

<400>19

Thr Val Glu Gly Ala Gly Ser Ile Ala Ala

1 5 10

<210>20

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 82-91

<400>20

Val Glu Gly Ala Gly Ser Ile Ala Ala Ala

1 5 10

<210>21

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 83-92

<400>21

Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr

1 5 10

<210>22

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 84-93

<400>22

Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly

1 5 10

<210>23

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 85-94

<400>23

Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe

1 5 10

<210>24

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 86-95

<400>24

Gly Ser Ile Ala Ala Ala Thr Gly Phe Val

1 5 10

<210>25

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 87-96

<400>25

Ser Ile Ala Ala Ala Thr Gly Phe Val Lys

1 5 10

<210>26

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 88-97

<400>26

Ile Ala Ala Ala Thr Gly Phe Val Lys Lys

1 5 10

<210>27

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 89-98

<400>27

Ala Ala Ala Thr Gly Phe Val Lys Lys Asp

1 5 10

<210>28

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 90-99

<400>28

Ala Ala Thr Gly Phe Val Lys Lys Asp Gln

1 5 10

<210>29

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 91-100

<400>29

Ala Thr Gly Phe Val Lys Lys Asp Gln Leu

1 5 10

<210>30

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 92-101

<400>30

Thr Gly Phe Val Lys Lys Asp Gln Leu Gly

1 5 10

<210>31

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 93-102

<400>31

Gly Phe Val Lys Lys Asp Gln Leu Gly Lys

1 5 10

<210>32

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 94-103

<400>32

Phe Val Lys Lys Asp Gln Leu Gly Lys Asn

1 5 10

<210>33

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 95-104

<400>33

Val Lys Lys Asp Gln Leu Gly Lys Asn Glu

1 5 10

<210>34

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 96-105

<400>34

Lys Lys Asp Gln Leu Gly Lys Asn Glu Glu

1 5 10

<210>35

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 97-106

<400>35

Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly

1 5 10

<210>36

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 98-107

<400>36

Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala

1 5 10

<210>37

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 99-108

<400>37

Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro

1 5 10

<210>38

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 100-109

<400>38

Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln

1 5 10

<210>39

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 101-110

<400>39

Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu

1 5 10

<210>40

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 102-111

<400>40

Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly

1 5 10

<210>41

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 103-112

<400>41

Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile

1 5 10

<210>42

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 104-113

<400>42

Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu

1 5 10

<210>43

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 105-114

<400>43

Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu

1 5 10

<210>44

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 106-115

<400>44

Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp

1 5 10

<210>45

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 107-116

<400>45

Ala Pro Gln Glu Gly Ile Leu Glu Asp Met

1 5 10

<210>46

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 108-117

<400>46

Pro Gln Glu Gly Ile Leu Glu Asp Met Pro

1 5 10

<210>47

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 109-118

<400>47

Gln Glu Gly Ile Leu Glu Asp Met Pro Val

1 5 10

<210>48

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 110-119

<400>48

Glu Gly Ile Leu Glu Asp Met Pro Val Asp

1 5 10

<210>49

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 111-120

<400>49

Gly Ile Leu Glu Asp Met Pro Val Asp Pro

1 5 10

<210>50

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 112-121

<400>50

Ile Leu Glu Asp Met Pro Val Asp Pro Asp

1 5 10

<210>51

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha1-Synuclein 113-122

<400>51

Leu Glu Asp Met Pro Val Asp Pro Asp Asn

1 5 10

<210>52

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 114-123

<400>52

Glu Asp Met Pro Val Asp Pro Asp Asn Glu

1 5 10

<210>53

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 115-124

<400>53

Asp Met Pro Val Asp Pro Asp Asn Glu Ala

1 5 10

<210>54

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 116-125

<400>54

Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

1 5 10

<210>55

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 117-126

<400>55

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

1 5 10

<210>56

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 118-127

<400>56

Val Asp Pro Asp Asn Glu Ala Tyr Glu Met

1 5 10

<210>57

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 119-128

<400>57

Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

1 5 10

<210>58

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 120-129

<400>58

Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser

1 5 10

<210>59

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 121-130

<400>59

Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu

1 5 10

<210>60

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 122-131

<400>60

Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu

1 5 10

<210>61

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 123-132

<400>61

Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

1 5 10

<210>62

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 124-133

<400>62

Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr

1 5 10

<210>63

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 125-134

<400>63

Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln

1 5 10

<210>64

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 126-135

<400>64

Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

1 5 10

<210>65

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 127-136

<400>65

Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr

1 5 10

<210>66

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 128-137

<400>66

Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu

1 5 10

<210>67

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 129-138

<400>67

Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro

1 5 10

<210>68

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 130-139

<400>68

Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu

1 5 10

<210>69

<211>10

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(10)

<223>alpha-Synuclein 131-140

<400>69

Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

1 5 10

<210>70

<211>17

<212>PROTEIN

<213>Clostridium tetani

<220>

<221>PEPTIDE

<222>(1)..(17)

<223>Clostridium tetani 1 Th

<400>70

Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu

1 5 10 15

Leu

<210>71

<211>15

<212>PROTEIN

<213>Measles virus

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>MvF1 Th

<400>71

Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val

1 5 10 15

<210>72

<211>24

<212>PROTEIN

<213>Bordetella pertussis

<220>

<221>PEPTIDE

<222>(1)..(24)

<223>Bordetella pertussis Th

<400>72

Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val Ala

1 5 10 15

Glu Leu Arg Gly Asn Ala Glu Leu

20

<210>73

<211>17

<212>PROTEIN

<213>Clostridium tetani

<220>

<221>PEPTIDE

<222>(1)..(17)

<223>Clostridium tetani 2 Th

<400>73

Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys

1 5 10 15

Thr

<210>74

<211>23

<212>PROTEIN

<213>diphtheria bacillus

<220>

<221>PEPTIDE

<222>(1)..(23)

<223>Diphtheria Th

<400>74

Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val Ala Ala Leu Ser

1 5 10 15

Ile Leu Pro Gly His Gly Cys

20

<210>75

<211>21

<212>PROTEIN

<213>Plasmodium falciparum

<220>

<221>PEPTIDE

<222>(1)..(21)

<223>Plasmodium falciparum Th

<400>75

Asp His Glu Lys Lys His Ala Lys Met Glu Lys Ala Ser Ser Val Phe

1 5 10 15

Asn Val Val Asn Ser

20

<210>76

<211>17

<212>PROTEIN

<213>Schistosoma mansoni

<220>

<221>PEPTIDE

<222>(1)..(17)

<223>Schistosoma mansoni Th

<400>76

Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp Glu Lys His Arg

1 5 10 15

His

<210>77

<211>25

<212>PROTEIN

<213>Cholera toxin

<220>

<221>PEPTIDE

<222>(1)..(25)

<223>Cholera toxin Th

<400>77

Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala

1 5 10 15

Thr Thr Gly Tyr Leu Lys Gly Asn Ser

20 25

<210>78

<211>15

<212>PROTEIN

<213>Measles virus

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>MvF 2 Th

<400>78

Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile Glu Gly Ile

1 5 10 15

<210>79

<211>22

<212>PROTEIN

<213>Measles virus

<220>

<221>PEPTIDE

<222>(1)..(22)

<223>KKKMvF 3 Th

<220>

<221>SITE

<222>(7)..(7)

<223>S or T

<220>

<221>SITE

<222>(10)..(10)

<223>K or R

<220>

<221>SITE

<222>(11)..(11)

<223>G or T

<220>

<221>SITE

<222>(15)..(15)

<223>H or T

<220>

<221>SITE

<222>(16)..(16)

<223>K or R

<220>

<221>SITE

<222>(19)..(19)

<223>G or T

<400>79

Lys Lys Lys Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa

1 5 10 15

Ile Glu Xaa Ile Leu Phe

20

<210>80

<211>18

<212>PROTEIN

<213>Hepatitis B virus

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg 1 Th

<220>

<221>SITE

<222>(1)..(1)

<223>K or R

<220>

<221>SITE

<222>(2)..(2)

<223>K or R

<220>

<221>SITE

<222>(3)..(3)

<223>K or R

<220>

<221>SITE

<222>(4)..(4)

<223>L or I or V or F

<220>

<221>SITE

<222>(5)..(5)

<223>F or K or R

<220>

<221>SITE

<222>(6)..(6)

<223>L or I or V or F

<220>

<221>SITE

<222>(7)..(7)

<223>L or I or V or F

<220>

<221>SITE

<222>(9)..(9)

<223>K or R

<220>

<221>SITE

<222>(10)..(10)

<223>L or I or V or F

<220>

<221>SITE

<222>(11)..(11)

<223>L or I or V or F

<220>

<221>SITE

<222>(13)..(13)

<223>L or I or V or F

<220>

<221>SITE

<222>(15)..(15)

<223>Q or L or I or V or F

<220>

<221>SITE

<222>(17)..(17)

<223>L or I or V or F

<220>

<221>SITE

<222>(18)..(18)

<223>D or R

<400>80

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Thr Xaa Pro Xaa Ser

1 5 10 15

Xaa Xaa

<210>81

<211>19

<212>PROTEIN

<213>Measles virus

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<400>81

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe

<210>82

<211>18

<212>PROTEIN

<213>Hepatitis B virus

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg 2 Th

<220>

<221>SITE

<222>(4)..(4)

<223>I or F

<220>

<221>SITE

<222>(5)..(5)

<223>I or F

<220>

<221>SITE

<222>(6)..(6)

<223>T or L

<220>

<221>SITE

<222>(7)..(7)

<223>I or L

<220>

<221>SITE

<222>(11)..(11)

<223>I or L

<220>

<221>SITE

<222>(14)..(14)

<223>P or I

<220>

<221>SITE

<222>(15)..(15)

<223>Q or T

<220>

<221>SITE

<222>(16)..(16)

<223>S or T

<220>

<221>SITE

<222>(17)..(17)

<223>L or I

<400>82

Lys Lys Lys Xaa Xaa Xaa Xaa Thr Arg Ile Xaa Thr Ile Xaa Xaa Xaa

1 5 10 15

Xaa Asp

<210>83

<211>19

<212>PROTEIN

<213>Measles virus

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 5 Th

<400>83

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe

<210>84

<211>18

<212>PROTEIN

<213>Hepatitis B virus

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg 3 Th

<400>84

Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr Ile Ile Thr Thr

1 5 10 15

Ile Asp

<210>85

<211>11

<212>PROTEIN

<213>Influenza virus

<220>

<221>PEPTIDE

<222>(1)..(11)

<223>Influenza virus matrix protein 1 _1 Th

<220>

<221>PEPTIDE

<222>(1)..(11)

<223>Influenza virus matrix protein 1_1 Th

<400>85

Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg

1 5 10

<210>86

<211>15

<212>PROTEIN

<213>Influenza virus

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>Influenza virus matrix protein 1_2 Th

<400>86

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val

1 5 10 15

<210>87

<211>9

<212>PROTEIN

<213>Influenza virus

<220>

<221>PEPTIDE

<222>(1)..(9)

<223>Nonstructural protein 1 of influenza virus Th

<400>87

Asp Arg Leu Arg Arg Asp Gln Lys Ser

15

<210>88

<211>19

<212>PROTEIN

<213>Epstein-Bar virus (EBV)

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>EBV BHRF1 Th

<400>88

Ala Gly Leu Thr Leu Ser Leu Leu Val Ile Cys Ser Tyr Leu Phe Ile

1 5 10 15

Ser Arg Gly

<210>89

<211>15

<212>PROTEIN

<213>Clostridium tetani

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>Clostridium tetani TT1 Th

<400>89

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210>90

<211>20

<212>PROTEIN

<213>Epstein-Bar virus

<220>

<221>PEPTIDE

<222>(1)..(20)

<223>EBV EBNA-1 Th

<400>90

Pro Gly Pro Leu Arg Glu Ser Ile Val Cys Tyr Phe Met Val Phe Leu

1 5 10 15

Gln Thr His Ile

20

<210>91

<211>21

<212>PROTEIN

<213>Clostridium tetani

<220>

<221>PEPTIDE

<222>(1)..(21)

<223>Clostridium tetani TT2 Th

<400>91

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

Ala Ser His Leu Glu

20

<210>92

<211>16

<212>PROTEIN

<213>Clostridium tetani

<220>

<221>PEPTIDE

<222>(1)..(16)

<223>Clostridium tetani TT3 Th

<400>92

Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe

1 5 10 15

<210>93

<211>16

<212>PROTEIN

<213>Clostridium tetani

<220>

<221>PEPTIDE

<222>(1)..(16)

<223>Clostridium tetani TT4 Th

<400>93

Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1 5 10 15

<210>94

<211>18

<212>PROTEIN

<213>Epstein-Bar virus

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>EBV CP Th

<400>94

Val Pro Gly Leu Tyr Ser Pro Cys Arg Ala Phe Phe Asn Lys Glu Glu

1 5 10 15

Leu Leu

<210>95

<211>14

<212>PROTEIN

<213>Human cytomegalovirus (HCMV)

<220>

<221>PEPTIDE

<222>(1)..(14)

<223>HCMV IE1 Th

<400>95

Asp Lys Arg Glu Met Trp Met Ala Cys Ile Lys Glu Leu His

1 5 10

<210>96

<211>15

<212>PROTEIN

<213>Epstein-Bar virus

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>EBV GP340 Th

<400>96

Thr Gly His Gly Ala Arg Thr Ser Thr Glu Pro Thr Thr Asp Tyr

1 5 10 15

<210>97

<211>13

<212>PROTEIN

<213>Epstein-Bar virus

<220>

<221>PEPTIDE

<222>(1)..(13)

<223>EBV BPLF1 Th

<400>97

Lys Glu Leu Lys Arg Gln Tyr Glu Lys Lys Leu Arg Gln

1 5 10

<210>98

<211>11

<212>PROTEIN

<213>Epstein-Bar virus

<220>

<221>PEPTIDE

<222>(1)..(11)

<223>EBV EBNA-2 Th

<400>98

Thr Val Phe Tyr Asn Ile Pro Pro Met Pro Leu

1 5 10

<210>99

<211>38

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(38)

<223>alpha-Synuclein 126-140

<400>99

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Glu Met Pro Ser Glu Glu Gly Tyr Gln

20 25 30

Asp Tyr Glu Pro Glu Ala

35

<210>100

<211>43

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(43)

<223>alpha-Synuclein 121-140

<400>100

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Asp Asn Glu Ala Tyr Glu Met Pro Ser

20 25 30

Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

35 40

<210>101

<211>53

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(53)

<223>alpha-Synuclein 111-140

<400>101

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Ile Leu Glu Asp Met Pro Val Asp

20 25 30

Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

35 40 45

Tyr Glu Pro Glu Ala

50

<210>102

<211>63

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(63)

<223>alpha-Synuclein 126-140

<400>102

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Lys Asn Glu Glu Gly Ala Pro Gln

20 25 30

Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

35 40 45

Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

50 55 60

<210>103

<211>63

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(63)

<223>alpha-Synuclein 101-140

<400>103

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Lys Asn Glu Glu Gly Ala Pro Gln

20 25 30

Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

35 40 45

Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

50 55 60

<210>104

<211>62

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg3 Th

<220>

<221>SITE

<222>(19)..(19)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(19)..(22)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(23)..(62)

<223>alpha-Synuclein 101-140

<400>104

Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr Ile Ile Thr Thr

1 5 10 15

Ile Asp Lys Lys Lys Lys Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu

20 25 30

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

35 40 45

Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

50 55 60

<210>105

<211>73

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(73)

<223>alpha-Synuclein 91-140

<400>105

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ala Thr Gly Phe Val Lys Lys Asp Gln

20 25 30

Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp

35 40 45

Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu

50 55 60

Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

65 70

<210>106

<211>79

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(79)

<223>alpha-Synuclein 85-140

<400>106

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ala Gly Ser Ile Ala Ala Ala Thr Gly

20 25 30

Phe Val Lys Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln

35 40 45

Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

50 55 60

Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

65 70 75

<210>107

<211>38

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(38)

<223>alpha-Synuclein 121-135

<400>107

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Asp Asn Glu Ala Tyr Glu Met Pro Ser

20 25 30

Glu Glu Gly Tyr Gln Asp

35

<210>108

<211>48

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(48)

<223>alpha-Synuclein 111-135

<400>108

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Ile Leu Glu Asp Met Pro Val Asp

20 25 30

Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

35 40 45

<210>109

<211>58

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(58)

<223>alpha-Synuclein 101-135

<400>109

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Lys Asn Glu Glu Gly Ala Pro Gln

20 25 30

Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

35 40 45

Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

50 55

<210>110

<211>62

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(62)

<223>alpha-Synuclein 97-135

<400>110

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Asp Gln Leu Gly Lys Asn Glu Glu

20 25 30

Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp

35 40 45

Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

50 55 60

<210>111

<211>36

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(36)

<223>alpha-Synuclein 123-135

<400>111

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Glu Ala Tyr Glu Met Pro Ser Glu Glu

20 25 30

Gly Tyr Gln Asp

35

<210>112

<211>33

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(33)

<223>alpha-Synuclein 126-135

<400>112

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Glu Met Pro Ser Glu Glu Gly Tyr Gln

20 25 30

Asp

<210>113

<211>45

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(45)

<223>alpha-Synuclein 111-132

<400>113

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Ile Leu Glu Asp Met Pro Val Asp

20 25 30

Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

<210>114

<211>55

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(55)

<223>alpha-Synuclein 101-132

<400>114

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Lys Asn Glu Glu Gly Ala Pro Gln

20 25 30

Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

35 40 45

Glu Met Pro Ser Glu Glu Gly

50 55

<210>115

<211>45

<212>PROTEIN

<213>Mus musculus

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(45)

<223>Mouse alpha-Synuclein 111-132

<400>115

Ile Ser Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Ile Leu Glu Asp Met Pro Val Asp

20 25 30

Pro Gly Ser Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

<210>116

<211>33

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(33)

<223>alpha-Synuclein 126-135

<400>116

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Glu Met Pro Ser Glu Glu Gly Tyr Gln

20 25 30

Asp

<210>117

<211>45

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF 4 Th

<220>

<221>SITE

<222>(4)..(4)

<223>S or T

<220>

<221>SITE

<222>(7)..(7)

<223>K or R

<220>

<221>SITE

<222>(8)..(8)

<223>G or T

<220>

<221>SITE

<222>(12)..(12)

<223>H or T

<220>

<221>SITE

<222>(13)..(13)

<223>K or R

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon-K

<220>

<221>PEPTIDE

<222>(20)..(23)

<223>epsilon K-KKK as a spacer

<220>

<221>PEPTIDE

<222>(24)..(45)

<223>alpha-Synuclein 111-132

<400>117

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Gly Ile Leu Glu Asp Met Pro Val Asp

20 25 30

Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

<210>118

<211>30

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(21)..(30)

<223>alpha-Synuclein 126-135

<400>118

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

20 25 30

<210>119

<211>42

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>MvF5 Th

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(21)..(42)

<223>alpha-Synuclein 111-132

<400>119

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn

20 25 30

Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>120

<211>29

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg3 Th

<220>

<221>SITE

<222>(19)..(19)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(20)..(29)

<223>alpha-Synuclein 126-135

<400>120

Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr Ile Ile Thr Thr

1 5 10 15

Ile Asp Lys Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

20 25

<210>121

<211>41

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg3 Th

<220>

<221>SITE

<222>(19)..(19)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(20)..(41)

<223>alpha-Synuclein 111-132

<400>121

Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr Ile Ile Thr Thr

1 5 10 15

Ile Asp Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu

20 25 30

Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>122

<211>40

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(17)

<223>Clostridium tetani1 Th

<220>

<221>SITE

<222>(18)..(18)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(19)..(40)

<223>alpha-Synuclein 111-132

<400>122

Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu

1 5 10 15

Leu Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala

20 25 30

Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>123

<211>38

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>MvF1 Th

<220>

<221>SITE

<222>(16)..(16)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(17)..(38)

<223>alpha-Synuclein 111-132

<400>123

Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Lys

1 5 10 15

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

20 25 30

Met Pro Ser Glu Glu Gly

35

<210>124

<211>47

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(24)

<223>Bordetella pertussis Th

<220>

<221>SITE

<222>(25)..(25)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(26)..(47)

<223>alpha-Synuclein 111-132

<400>124

Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val Ala

1 5 10 15

Glu Leu Arg Gly Asn Ala Glu Leu Lys Gly Ile Leu Glu Asp Met Pro

20 25 30

Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

<210>125

<211>40

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(17)

<223>Clostridium tetani2 Th

<220>

<221>SITE

<222>(18)..(18)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(19)..(40)

<223>alpha-Synuclein 111-132

<400>125

Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys

1 5 10 15

Thr Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala

20 25 30

Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>126

<211>46

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(23)

<223>Diphtheria Th

<220>

<221>SITE

<222>(24)..(24)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(25)..(46)

<223>alpha-Synuclein 111-132

<400>126

Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val Ala Ala Leu Ser

1 5 10 15

Ile Leu Pro Gly His Gly Cys Lys Gly Ile Leu Glu Asp Met Pro Val

20 25 30

Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

<210>127

<211>44

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(21)

<223>Plasmodium falciparum Th

<220>

<221>SITE

<222>(22)..(22)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(23)..(44)

<223>alpha-Synuclein 111-132

<400>127

Asp His Glu Lys Lys His Ala Lys Met Glu Lys Ala Ser Ser Val Phe

1 5 10 15

Asn Val Val Asn Ser Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro

20 25 30

Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>128

<211>40

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(17)

<223>Schistosoma mansoni Th

<220>

<221>SITE

<222>(18)..(18)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(19)..(40)

<223>alpha-Synuclein 111-132

<400>128

Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp Glu Lys His Arg

1 5 10 15

His Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala

20 25 30

Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>129

<211>48

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(25)

<223>Cholera toxin Th

<220>

<221>SITE

<222>(26)..(26)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(27)..(48)

<223>alpha-Synuclein 111-132

<400>129

Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala

1 5 10 15

Thr Thr Gly Tyr Leu Lys Gly Asn Ser Lys Gly Ile Leu Glu Asp Met

20 25 30

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

<210>130

<211>38

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>MvF2 Th

<220>

<221>SITE

<222>(16)..(16)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(17)..(38)

<223>alpha-Synuclein 111-132

<400>130

Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile Glu Gly Ile Lys

1 5 10 15

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

20 25 30

Met Pro Ser Glu Glu Gly

35

<210>131

<211>45

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(22)

<223>KKKMvF3 Th

<220>

<221>SITE

<222>(7)..(7)

<223>S or T

<220>

<221>SITE

<222>(10)..(10)

<223>K or R

<220>

<221>SITE

<222>(11)..(11)

<223>G or T

<220>

<221>SITE

<222>(15)..(15)

<223>H or T

<220>

<221>SITE

<222>(16)..(16)

<223>K or R

<220>

<221>SITE

<222>(19)..(19)

<223>G or T

<220>

<221>SITE

<222>(23)..(23)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(24)..(45)

<223>alpha-Synuclein 111-132

<400>131

Lys Lys Lys Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa

1 5 10 15

Ile Glu Xaa Ile Leu Phe Lys Gly Ile Leu Glu Asp Met Pro Val Asp

20 25 30

Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40 45

<210>132

<211>41

<212>PROTEIN

<213>Hepatitis B virus

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg 1 Th

<220>

<221>SITE

<222>(1)..(1)

<223>K or R

<220>

<221>SITE

<222>(2)..(2)

<223>K or R

<220>

<221>SITE

<222>(3)..(3)

<223>K or R

<220>

<221>SITE

<222>(4)..(4)

<223>L or I or V or F

<220>

<221>SITE

<222>(5)..(5)

<223>F or K or R

<220>

<221>SITE

<222>(6)..(6)

<223>L or I or V or F

<220>

<221>SITE

<222>(7)..(7)

<223>L or I or V or F

<220>

<221>SITE

<222>(9)..(9)

<223>K or R

<220>

<221>SITE

<222>(10)..(10)

<223>L or I or V or F

<220>

<221>SITE

<222>(11)..(11)

<223>L or I or V or F

<220>

<221>SITE

<222>(13)..(13)

<223>L or I or V or F

<220>

<221>SITE

<222>(15)..(15)

<223>Q or L or I or V or F

<220>

<221>SITE

<222>(17)..(17)

<223>L or I or V or F

<220>

<221>SITE

<222>(18)..(18)

<223>D or R

<220>

<221>SITE

<222>(19)..(19)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(20)..(41)

<223>alpha-Synuclein 111-132

<400>132

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Thr Xaa Pro Xaa Ser

1 5 10 15

Xaa Xaa Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu

20 25 30

Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>133

<211>41

<212>PROTEIN

<213>Hepatitis B virus

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>HBsAg 2 Th

<220>

<221>SITE

<222>(4)..(4)

<223>I or F

<220>

<221>SITE

<222>(5)..(5)

<223>I or F

<220>

<221>SITE

<222>(6)..(6)

<223>T or L

<220>

<221>SITE

<222>(7)..(7)

<223>I or L

<220>

<221>SITE

<222>(11)..(11)

<223>I or L

<220>

<221>SITE

<222>(14)..(14)

<223>P or I

<220>

<221>SITE

<222>(15)..(15)

<223>Q or T

<220>

<221>SITE

<222>(16)..(16)

<223>S or T

<220>

<221>SITE

<222>(17)..(17)

<223>L or I

<220>

<221>SITE

<222>(19)..(19)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(20)..(41)

<223>alpha-Synuclein 111-132

<400>133

Lys Lys Lys Xaa Xaa Xaa Xaa Thr Arg Ile Xaa Thr Ile Xaa Xaa Xaa

1 5 10 15

Xaa Asp Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu

20 25 30

Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>134

<211>34

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(11)

<223>Influenza virus MP1_1 Th

<220>

<221>SITE

<222>(12)..(12)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(13)..(34)

<223>alpha-Synuclein 111-132

<400>134

Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Lys Gly Ile Leu Glu

1 5 10 15

Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu

20 25 30

Glu Gly

<210>135

<211>38

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>Influenza virus MP1_2 Th

<220>

<221>SITE

<222>(16)..(16)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(17)..(38)

<223>alpha-Synuclein 111-132

<400>135

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Lys

1 5 10 15

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

20 25 30

Met Pro Ser Glu Glu Gly

35

<210>136

<211>32

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(9)

<223>Influenza virus NSP1 Th

<220>

<221>SITE

<222>(10)..(10)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(11)..(32)

<223>alpha-Synuclein 111-132

<400>136

Asp Arg Leu Arg Arg Asp Gln Lys Ser Lys Gly Ile Leu Glu Asp Met

1 5 10 15

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

20 25 30

<210>137

<211>42

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(19)

<223>EBV BHRF1 Th

<220>

<221>PEPTIDE

<222>(20)..(20)

<223>epsilon K as a spacer

<220>

<221>SITE

<222>(20)..(20)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(21)..(42)

<223>alpha-Synuclein 111-132

<400>137

Ala Gly Leu Thr Leu Ser Leu Leu Val Ile Cys Ser Tyr Leu Phe Ile

1 5 10 15

Ser Arg Gly Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn

20 25 30

Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>138

<211>38

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>Clostridium tetani TT1 Th

<220>

<221>SITE

<222>(16)..(16)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(17)..(38)

<223>alpha-Synuclein 111-132

<400>138

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys

1 5 10 15

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

20 25 30

Met Pro Ser Glu Glu Gly

35

<210>139

<211>43

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(20)

<223>EBV EBNA-1 Th

<220>

<221>SITE

<222>(21)..(21)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(22)..(43)

<223>alpha-Synuclein 111-132

<400>139

Pro Gly Pro Leu Arg Glu Ser Ile Val Cys Tyr Phe Met Val Phe Leu

1 5 10 15

Gln Thr His Ile Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp

20 25 30

Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>140

<211>44

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(21)

<223>Clostridium tetani TT2 Th

<220>

<221>SITE

<222>(22)..(22)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(23)..(44)

<223>alpha-Synuclein 111-132

<400>140

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

Ala Ser His Leu Glu Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro

20 25 30

Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>141

<211>39

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(16)

<223>Clostridium tetani TT3 Th

<220>

<221>SITE

<222>(17)..(17)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(18)..(39)

<223>alpha-Synuclein 111-132

<400>141

Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe

1 5 10 15

Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

20 25 30

Glu Met Pro Ser Glu Glu Gly

35

<210>142

<211>39

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(16)

<223>Clostridium tetani TT4 Th

<220>

<221>SITE

<222>(17)..(17)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(18)..(39)

<223>alpha-Synuclein 111-132

<400>142

Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1 5 10 15

Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr

20 25 30

Glu Met Pro Ser Glu Glu Gly

35

<210>143

<211>41

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(18)

<223>EBV CP Th

<220>

<221>SITE

<222>(19)..(19)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(20)..(41)

<223>alpha-Synuclein 111-132

<400>143

Val Pro Gly Leu Tyr Ser Pro Cys Arg Ala Phe Phe Asn Lys Glu Glu

1 5 10 15

Leu Leu Lys Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu

20 25 30

Ala Tyr Glu Met Pro Ser Glu Glu Gly

35 40

<210>144

<211>37

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(14)

<223>HCMV IE1 Th

<220>

<221>SITE

<222>(15)..(15)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(16)..(37)

<223>alpha-Synuclein 111-132

<400>144

Asp Lys Arg Glu Met Trp Met Ala Cys Ile Lys Glu Leu His Lys Gly

1 5 10 15

Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met

20 25 30

Pro Ser Glu Glu Gly

35

<210>145

<211>38

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(15)

<223>EBV GP340 Th

<220>

<221>SITE

<222>(16)..(16)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(17)..(38)

<223>alpha-Synuclein 111-132

<400>145

Thr Gly His Gly Ala Arg Thr Ser Thr Glu Pro Thr Thr Asp Tyr Lys

1 5 10 15

Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

20 25 30

Met Pro Ser Glu Glu Gly

35

<210>146

<211>36

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(13)

<223>EBV BPLF1 Th

<220>

<221>SITE

<222>(14)..(14)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(15)..(36)

<223>alpha-Synuclein 111-132

<400>146

Lys Glu Leu Lys Arg Gln Tyr Glu Lys Lys Leu Arg Gln Lys Gly Ile

1 5 10 15

Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

20 25 30

Ser Glu Glu Gly

35

<210>147

<211>34

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(11)

<223>EBV EBNA-2 Th

<220>

<221>SITE

<222>(12)..(12)

<223>epsilon K as a spacer

<220>

<221>PEPTIDE

<222>(13)..(34)

<223>alpha-Synuclein 111-132

<400>147

Thr Val Phe Tyr Asn Ile Pro Pro Met Pro Leu Lys Gly Ile Leu Glu

1 5 10 15

Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu

20 25 30

Glu Gly

<210>148

<211>4

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(4)

<223>epsilon K-KKK as a spacer

<220>

<221>SITE

<222>(1)..(1)

<223>epsilon-K

<400>148

Lys Lys Lys Lys

1

<210>149

<211>31

<212>DNA

<213>Homo sapiens

<220>

<221>primer_bind

<222>(1)..(31)

<223>alpha-synuclein forward primer sequence

<400>149

cgggatccga tgtgtttatg aaaggtctga g 31

<210>150

<211>31

<212>DNA

<213>Homo sapiens

<220>

<221>primer_bind

<222>(1)..(31)

<223>reverse primer for alpha-synuclein

<400>150

ggaattccga tgtgtttatg aaaggtctga g 31

<210>151

<211>23

<212>DNA

<213>Homo sapiens

<220>

<221>primer_bind

<222>(1)..(23)

<223>Primer sequences for mutant alpha-synuclein

<400>151

tcatggtgtg accaccgttg cag 23

<210>152

<211>21

<212>DNA

<213>Homo sapiens

<220>

<221>primer_bind

<222>(1)..(21)

<223>reverse primer for mutant alpha-synuclein

<400>152

accacgcctt ctttggtttt g 21

<210>153

<211>32

<212>PROTEIN

<213>Homo sapiens

<220>

<221>PEPTIDE

<222>(1)..(32)

<223>beta-synuclein 103-134

<400>153

Ala Glu Glu Pro Leu Ile Glu Pro Leu Met Glu Pro Glu Gly Glu Ser

1 5 10 15

Tyr Glu Asp Pro Pro Gln Glu Glu Tyr Gln Glu Tyr Glu Pro Glu Ala

20 25 30

<---

Claims (31)

1. Peptide immunogenic construct of alpha-synuclein (α-Syn) for the treatment of brain disease in an individual with synucleinopathy, containing the following formula: (Th) _m -(A) _n -(C-terminal fragment of α-Syn)-X or (C-terminal fragment α-Syn)-(A) _n -(Th) _m -X, where (C-terminal fragment of α-Syn) is a B-cell epitope containing a fragment of amino acids G111-D135 of the sequence SEQ ID NO:1; Th is a T helper epitope containing an amino acid sequence selected from the group consisting of SEQ ID NO:70-98; And A is an optional heterologous spacer selected from the group consisting of amino acid, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO:148), X represents α-COOH or α-CONH ₂ amino acids; m is from 1 to about 4; And n is from 1 to about 10; wherein the B cell epitope is covalently linked to the T helper epitope directly or via an optional heterologous spacer. 2. The α-Syn peptide immunogenic construct of claim 1, wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 12-15, 17 and 49-63. 3. The α-Syn peptide immunogenic construct according to claim 1, wherein the T helper epitope is selected from the group consisting of SEQ ID NO:81, 83 and 84. 4. The α-Syn peptide immunogenic construct of claim 1, wherein the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO:148). 5. Peptide immunogenic construct α-Syn according to claim 1, where the T-helper epitope is covalently linked to the amino terminus of the B-cell epitope. 6. The α-Syn peptide immunogenic construct of claim 1, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope via an optional heterologous spacer. 7. Peptide immunogenic construct α-Syn according to claim 1, containing an amino acid sequence selected from the group consisting of SEQ ID NO: 107, 108, 111-113 and 115-147. 8. Peptide immunogenic construct α-Syn according to claim 1, containing an amino acid sequence selected from the group consisting of SEQ ID NO: 107, 108 and 111-113. 9. A composition for the treatment of a brain disease in an individual with synucleinopathy, containing an effective amount of the peptide immunogenic construct α-Syn according to claim 1 as the active agent of the composition. 10. A composition for treating a brain disease in an individual with synucleinopathy, comprising an effective amount of more than one α-Syn peptide immunogenic construct according to claim 1 as an active agent of the composition. 11. The composition according to claim 10, wherein the α-Syn peptide immunogenic construct has the amino acid sequences of SEQ ID NO: 112 and 113. 12. A pharmaceutical composition for the treatment of a brain disease in an individual with synucleinopathy, containing the peptide immunogenic construct α-Syn according to claim 1 and a pharmaceutically acceptable delivery vehicle and/or adjuvant. 13. Pharmaceutical composition according to claim 13, where a. the α-Syn peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113 and 115-147; And b. the adjuvant is an aluminum mineral salt selected from the group consisting of Al(OH) ₃ or AlPO ₄ . 14. Pharmaceutical composition according to claim 12, where a. the α-Syn peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113 and 115-147; And b. the peptide immunogenic construct α-Syn is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex. 15. A method for producing antibodies that recognize α-Syn in the body of an animal, including administering to the animal a composition containing the peptide immunogen α-Syn according to claim 1 and a delivery vehicle and/or an adjuvant. 16. A method of inhibiting α-Syn aggregation in an animal, including administering a pharmacologically effective amount of the α-Syn peptide immunogen according to claim 1 to the animal. 17. A method for reducing the number of α-Syn aggregates in an animal, including administering a pharmacologically effective amount of the α-Syn peptide immunogen according to claim 1 to the animal.

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