US20230183304A1 - Multi-Epitope Vaccine for the Treatment of Alzheimer's Disease - Google Patents

Multi-Epitope Vaccine for the Treatment of Alzheimer's Disease Download PDF

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US20230183304A1
US20230183304A1 US17/925,813 US202117925813A US2023183304A1 US 20230183304 A1 US20230183304 A1 US 20230183304A1 US 202117925813 A US202117925813 A US 202117925813A US 2023183304 A1 US2023183304 A1 US 2023183304A1
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polypeptide
peptide
tau
amino acid
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Robin Barbour
Gene Kinney
Wagner Zago
Tarlochan NIJJAR
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Prothena Biosciences Ltd
Othair Prothena Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the disclosure relates to the technical fields of immunology and medicine, and in particular to the treatment of Alzheimer's disease and other diseases of protein misfolding.
  • AD Alzheimer's disease
  • senile dementia a progressive disease resulting in senile dementia.
  • the disease falls into two categories: late onset, which occurs in old age (65+ years) and early onset, which develops well before the senile period, i.e., between 35 and 60 years.
  • late onset which occurs in old age (65+ years)
  • early onset which develops well before the senile period, i.e., between 35 and 60 years.
  • the pathology is the same, but the abnormalities tend to be more severe and widespread in cases beginning at an earlier age.
  • the disease is characterized by at least two types of lesions in the brain, neurofibrillary tangles and senile plaques.
  • Neurofibrillary tangles are intracellular deposits of microtubule associated tau protein consisting of two filaments twisted about each other in pairs.
  • Senile plaques amyloid plaques are areas of disorganized neuropil up to 150 ⁇ m across with extracellular amyloid deposits at the center which are visible by microscopic analysis of sections of brain tissue.
  • the accumulation of amyloid plaques within the central nervous system is also associated with Down's syndrome and other-cognitive disorders, Cerebral amyloid angiopathy (CAA), and the ocular disease Age-Related Macular Degeneration.
  • CAA Cerebral amyloid angiopathy
  • a principal constituent of the plaques is a peptide termed A ⁇ or ⁇ -amyloid peptide.
  • a ⁇ peptide is a 4-kDa internal fragment of 38-43 amino acids of a larger transmembrane glycoprotein named amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • a ⁇ is primarily found in both a short firm, 40 amino acids in length, and a long form, ranging from 42-43 amino acids in length.
  • Part of the hydrophobic transmembrane domain of APP is found at the carboxy end of A ⁇ , and may account for the ability of A ⁇ to aggregate into plaques, particularly in the case of the long form. Accumulation of amyloid plaques in the brain eventually leads to neuronal cell death.
  • the cognitive and physical symptoms associated with this type of neural deterioration characterize Alzheimer's disease.
  • tau Another protein reported to occur at increased levels in Alzheimer's patients relative to the general population is tau, the principal constituent of neurofibrillary tangles, which together with amyloid plaques are a hallmark characteristic of Alzheimer's disease.
  • Tau tangles constitute abnormal fibrils measuring 10 nm in diameter occurring in pairs wound in a helical fashion with a regular periodicity of 80 nm.
  • the tau within neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated) with phosphate groups attached to specific sites on the molecule.
  • Severe involvement of neurofibrillary tangles is seen in the layer II neurons of the entorhinal cortex, the CA1 and subicular regions of the hippocampus, the amygdala, and the clever layers (layers III, V, and superficial VI) of the neocortex in Alzheimer's disease. Tau pathologies are known to correlate to cognitive decline.
  • disclosure is directed to a polypeptide including a first peptide comprising 3-10 amino acids from residues 1-10 of SEQ ID NO:01 linked to a second peptide including 3-13 amino acids from residues 244-400 of SEQ ID NO:02.
  • the second peptide may be from the microtubule binding region (MTBR) of tau (residues 244-372 of SEQ ID NO:02).
  • the first peptide may be N-terminal to the second peptide or the first peptide may be C-terminal to the second peptide.
  • first peptide may include an amino acid sequence of one of SEQ ID NOS: 3 to 38 or SEQ ID NOS:1002 to 1057 and the second peptide may include an amino acid sequence of one of SEQ ID NOS: 39-56, 83-86, or 146-996.
  • the first polypeptide may be DAEFRHD (SEQ ID NO:06), DAEFR (SEQ ID NO:08), or EFRHD (SEQ ID NO:21), and the second polypeptide may be 5-13 amino acids, for example QIVYKPV (SEQ ID NO:39), EIVYKSV (SEQ ID NO:42), EIVYKSP (SEQ ID NO:43), EIVYKPV (SEQ ID NO:44), NIKHVP (SEQ ID NO:48), VKSKIGST (SEQ ID NO:801), SKIGSTEN (SEQ ID NO:817), TENLKHQP (SEQ ID NO:695), ENLKHQPG (SEQ ID NO:689), SKIGSTDNIKH (SEQ ID NO:985), SKIGSKDNIKH (SEQ ID NO:986), or SKIGSLDNIKH (SEQ ID NO:988).
  • QIVYKPV SEQ ID NO:39
  • EIVYKSV SEQ ID NO:
  • the first peptide and second peptide may be linked by a cleavable linker, which may be an amino acid sequence.
  • a cleavable peptide linker if present, can be 1-10 amino acids in length.
  • the linker comprises between about 1-10 amino acids, about 1-9 amino acids, about 1-8 amino acids, about 1-7 amino acids, about 1-6 amino acids, about 1-5 amino acids, about 1-4 amino acids, about 1-3 amino acids, about 2 amino acids, or one (1) amino acid.
  • the cleavable peptide linker is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, S amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids.
  • the linker may be arginine-arginine (Arg-Arg), arginine-valine-arginine-arginine (Arg-Val-Arg-Arg (SEQ ID NO:69)), (Val-Cit), valine-arginine (Val-Arg), valine-lysine (Val-Lys), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), glycine-alanine-glycine-alanine (Gly-Ala-Gly-Ala; SEQ NO:80), (SEQ ID NO:81), or Lys-Gly-Lys-Gly (SEQ ID NO:82).
  • the polypeptide may be DAEFRHDRRQIVYKPV (SEQ ID NO:57), DAEFRHDRREIVYKSV (SEQ ID NO:58), DAEFRHDRRVKSKIGSTGGC (SEQ ID NO:997), DAEFRHDRRSKIGSTENGGC (SEQ ID NO:998), DAEFRHDRRTENLKHQPGGC (SEQ ID NO:999), DAEFRHDRRENLKHQPGGGC (SEQ ID NO:1000), or DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO:1001).
  • the polypeptide may include a linker to a carrier at a C-terminal portion of the polypeptide, or at a N-terminal portion of the polypeptide.
  • a linker if present, can be 1-10 amino acids in length. In some embodiments, the linker comprises between about 1-10 amino acids, about 1-9 amino acids, about 1-8 amino acids, about 1-7 amino acids, about 1-6 amino acids, about 1.5 amino acids, about 1-4 amino acids, about 1-3 amino acids, about 2 amino acids, or one (1) amino acid. In some embodiments, the linker is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, amino acids, 9 amino acids, or 0.10 amino acids.
  • the linker may include an amino acid sequence of GG, GGG, AA, AAA, KK, KKK, SS and SSS.
  • the linker to the carrier if present at the C-terminus, may include a C-terminal cysteine (C).
  • the linker to the carrier if present at the N-terminus, may include a N-terminal cysteine (C).
  • the polypeptide may include the amino acid sequence of DAEFRHDRRQIVYKPVXXC (SEQ ID NO:70), wherein XX and C are independently optional and, if present, XX can be GG, AA, KK, SS, GAGA (SEQ ID NO:80), AGAG (SEQ ID NO:81), or KGKG (SEQ ID NO:82).
  • the polypeptide may include the amino acid sequence of DAEFRHDRREIVYKSVXXC (SEQ ID NO:79), wherein XX and C are independently optional and, if present, XX can be GG, AA, KK, SS, GAGA (SEQ ID NO:80), AGAG (SEQ ID NO: 81), and KGKG (SEQ ID NO:82).
  • the disclosure is directed to an immunotherapy composition including the polypeptides of the disclosure, wherein the polypeptide may be linked to a carrier.
  • the carrier may include serum albumins, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), a genetically modified cross-reacting material (CRM) of diphtheria toxin.
  • CRM197 meningococcal outer membrane protein complex (OMPC) and H. influenzae protein D (HiD), rEPA ( Pseudomonas aeruginosa exotoxin A), KLH (keyhole limpet hemoeyanin), and flagellin.
  • embodiments of the disclosure are directed to a pharmaceutical formulation includes the polypeptides or the immunotherapy compositions of the disclosure, and including at least one adjuvant.
  • the adjuvant may be aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3 De-O-acylated monophosphoryl lipid A (MPL), QS-21, QS-18, QS-17, QS-7, TQL1055, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), oil in water emulsions (such as squalene or peanut oil), CpG, polyglutamic acid, polylysine, AddaVaxTM, MF59®, and combinations thereof.
  • the formulation may include a liposomal formulation, a diluent, or a multiple antigen presenting system (MAP).
  • MAP may include one or more of a Lys-based dendritic scaffold, helper T-cell epitopes, immune stimulating lipophilic moieties, cell penetrating peptides, radical induced polymerization, self-assembling nanoparticles as antigen-presenting platforms and gold nanoparticles.
  • embodiments of the disclosure are directed to an immunotherapy composition including a first peptide sequence comprising 3-10 amino acid residues from the first ten N-terminal residues of SEQ ID NO:01 and a second peptide sequence comprising 3-13 amino acids from residues 244-400 of SEQ ID NO:02.
  • the first peptide may include an amino acid sequence of one of SEQ ID NOS: 3 to 38 or SEQ ID NOS:1002 to 1057
  • the second peptide may include an amino acid sequence of one of SEQ ID NOS: 39 to 56, SEQ ID NOS:83-86 or SEQ ID NOS:146-996.
  • Each of the first peptide and the second peptide may include a linker to a carrier at a C-terminal portion of the polypeptide, or at a N-terminal portion of the polypeptide.
  • the linker may include an amino acid sequence selected from GC, GGG, AA, AAA, KK, KKK, SS, SSS, GAGA (SEQ ID NO:80), AGAG (SEQ ID NO:81), and KGKG (SEQ ID NO:82), and may include a C-terminal cysteine (C).
  • the linker is an amino acid linker that does not have a N-terminal glycine (e.g., GG, GAGA (SEQ ID NO:80)).
  • the carrier may include serum albumins, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid (TT), diphtheria toxoid (DT), a genetically modified cross-reacting material (CRM) of diphtheria toxin, CRM197, meningococcal outer membrane protein complex (OMPC) and H. influenzae protein D TEPA ( Pseudomonas aeruginosa exotoxin A), KLH (keyhole limpet hemocyanin), and flagellin.
  • serum albumins serum albumins
  • immunoglobulin molecules thyroglobulin
  • ovalbumin ovalbumin
  • TT tetanus toxoid
  • DT diphtheria toxoid
  • CRM197 genetically modified cross-reacting material
  • OMPC meningococcal outer membrane protein complex
  • H. influenzae protein D TEPA Pseudomonas aeruginos
  • the immunotherapy composition may include at least one pharmaceutically acceptable diluent and/or a multiple antigen presenting system (MAP).
  • MAP may include one or more of a Lys-based dendritic scaffold, helper T-cell epitopes, immune stimulating lipophilic moieties, cell penetrating peptides, radical induced polymerization, self-assembling nanoparticles as antigen-presenting platforms and gold nanoparticles.
  • the immunotherapy composition may be included in a pharmaceutical composition including the immunotherapy composition and at least one adjuvant such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3 De-O-acylated monophosphoryl lipid A (MPL), QS-21, QS-18, QS-17, QS-7, TQL1055, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), oil in water emulsions (such as squalene or peanut oil), CpG, polyglutamic acid, polylysine, AddaVaxTM, MF59®, and combinations thereof.
  • adjuvant such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3 De-O-acylated monophosphoryl lipid A (MPL), QS-21, QS-18, QS-17, QS-7, TQL1055, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), oil in water emulsions (such as
  • Embodiments of the disclosure are also directed to nucleic acid sequences encoding the polypeptides and the immunotherapy compositions of the disclosure.
  • the nucleic acids may be included in a nucleic acid immunotherapy composition including the nucleic acid and at least one adjuvant.
  • embodiments of the disclosure are directed to a methods for treating or effecting prophylaxis of Alzheimer's disease in a subject, and methods for inhibiting or reducing aggregation of at least one of A ⁇ and tau in a subject having or at risk of developing Alzheimer's disease.
  • the methods include administrating to the subject an immunotherapy composition, a nucleic acids immunotherapy composition, or a pharmaceutical formulation of the disclosure.
  • the methods of the disclosure may include repeating the administering at least a second time, at least a third time, at least a fourth time, at least a fifth time, or at least a sixth time, and may include repeating the administering at an interval of about 21 to about 28 days.
  • methods of the disclosure are directed to inducing an immune response in an animal.
  • the methods include administering to the animal a polypeptide, an immunotherapy composition, a pharmaceutical formulation or a nucleic acid immunotherapy composition of the disclosure in a regimen effective to generate an immune response including antibodies that specifically bind to A ⁇ , tau, or both A ⁇ and tau.
  • the immune response may include antibodies that specifically bind to the N-terminal region of A ⁇ and/or the microtubule region of tau.
  • the disclosure is directed to an immunization kit including an immunotherapy composition of the disclosure and may include an adjuvant, wherein the immunotherapy composition may be in a first container and the adjuvant may be a second container.
  • kits including a nucleic acid immunotherapy composition of the disclosure and may include an adjuvant.
  • the nucleic acid may be in a first container and, the adjuvant may be in a second container.
  • FIG. 1 shows the results of an experiment comparing the geometric mean titers of Guinea Pig serum for immunogen DAEFRHDRRQIVYKPV (SEQ ID NO:57) on monomeric A ⁇ amino acids 1-28 (DAEFRHDSGYEVHHQKLFFAEDVGSNKG; SEQ ID NO:67), soluble aggregate species of A ⁇ (A ⁇ 42), full length tau, and tau MTBR epitope containing GGGSVQIVYKPVDLS (SEQ ID NO:68) containing an N-terminal biotin.
  • FIG. 2 A shows the results of an experiment comparing the titers of Guinea pig serum for a single peptide immunogen versus dual peptide immunogens of the disclosure on all forms of A ⁇ and full-length tau protein (DAEFRHD is SEQ ID NO:6; QIVYKPV is SEQ ID NO: 39; DAEFRHDRRQIVYKPV is SEQ ID NO:57).
  • DAEFRHD is SEQ ID NO:6
  • QIVYKPV is SEQ ID NO: 39
  • DAEFRHDRRQIVYKPV is SEQ ID NO:57.
  • FIG. 2 B shows the results of an experiment comparing the titers of Guinea pig serum for a single peptide immunogen versus dual peptide immunogens of the disclosure on all forms of A ⁇ and full-length tau protein (DAEFRHD is SEQ ID NO:6; QIVYKPV is SEQ ID NO:39; EIVYKSP is SEQ ID NO:43; DAEFRHDRRQIVYKPV is SEQ NO:57).
  • DAEFRHD is SEQ ID NO:6
  • QIVYKPV is SEQ ID NO:39
  • EIVYKSP is SEQ ID NO:43
  • DAEFRHDRRQIVYKPV is SEQ NO:57.
  • FIG. 3 A shows staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from a Guinea pig vaccinated with Immunogen 9 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59).
  • FIG. 3 B shows staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from a Guinea pig vaccinated with immunogen 9 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59).
  • FIG. 3 C shows staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using serum (1:1500 dilution) from a Guinea pig vaccinated with Immunogen 9 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59).
  • FIG. 3 D shows lack of staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using scrum (1:300 dilution) from an unvaccinated. Guinea pig.
  • FIG. 3 E shows lack of staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from an unvaccinated Guinea pig.
  • FIG. 3 F shows lack of staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using scrum (1:1500 dilution) from an unvaccinated Guinea pig.
  • FIG. 4 A shows that Guinea pig serum from animals vaccinated with a dual immunogen peptide, DAEFRHDRRQIVYKPVGGC (SEQ ID NO:59; Immunogen 9), inhibited Abeta soluble aggregates binding to primary neurons in a dose-dependent manner.
  • FIG. 4 B shows examples of Ab staining on primary neurons in the presence or absence of Guinea pig serum from animals vaccinated with a dual immunogen peptide, DAEFRHDRRQIVYKPVGGC (SEQ ID NO:59; Immunogen 9).
  • FIG. 5 A shows that mice vaccinated with dual peptide antigens with various linkers produce similar titers to A ⁇ and Tau.
  • FIG. 5 B shows that mice vaccinated with dual peptide antigens with the Tau sequence EIVYKSP (SEQ ID NO:43) produce titers to A ⁇ and Tau.
  • FIG. 6 A shows staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using serum (1:1000 dilution) from a mouse vaccinated with Immunogen 19.
  • FIG. 6 B shows staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using serum (1:1000 dilution) from a mouse vaccinated with immunogen 19.
  • FIG. 6 C shows lack of Staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using serum (1:1000 dilution) from an unvaccinated mouse.
  • FIG. 6 D shows staining of Tau pathology in fresh frozen human AD brain tissue using anti-Tau antibody m6H3 (0.1 ⁇ g/ml).
  • FIG. 6 E shows comparable staining of Tau pathology in fresh frozen human AD brain tissue using serum (1:1500 dilution) from a Guinea pig vaccinated with Immunogen 9 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59),
  • FIGS. 7 A and 7 B show titers of dual A ⁇ -tau immunogen in two injection paradigms. Titers to A ⁇ 1-28, full-length tau and the carrier CRM protein of the two injection schedules (( FIG. 7 A ) showing 0, 4, 12 and 24 weeks; ( FIG. 7 B ) showing 0, 8 and 24 weeks) over the experiment are expressed as mean titer +/ ⁇ SEM.
  • FIG. 8 A shows individual animal titers to A ⁇ and tau two weeks post each injection for the injection schedule of FIG. 7 A , i.e., at weeks 2, 6, 14 and 26.
  • FIG. 88 shows individual animal titers to A ⁇ and tau two weeks post each injection for the injection schedule of FIG. 7 B , i.e., at weeks 2, 14 and 26.
  • FIG. 9 A shows staining of A ⁇ and Tau pathology in fresh frozen human AD brain tissue using week 26 serum (1:300 dilution) from cynomolgus monkey #1001 vaccinated with Immunogen 15 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59) on a four injection vaccination schedule.
  • FIG. 9 B shows a zoomed view of the rectangle indicating staining of A ⁇ pathology in FIG. 9 A .
  • FIG. 9 C shows a shows a zoomed view of the rectangle indicating staining of Tau pathology in FIG. 9 A .
  • FIG. 10 A shows staining of A ⁇ pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from eynomolgus monkey #1003 vaccinated with immunogen 15 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59).
  • FIG. 10 B shows modest staining of Tau pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from cynomolgus monkey #1003.
  • FIG. 11 A shows staining of A ⁇ pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from cynomolgus monkey #1501 vaccinated with Immunogen 15 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59).
  • FIG. 11 B shows minimal staining of Tau pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from cynomolgus monkey #1501.
  • FIG. 12 A shows staining of A ⁇ pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from cynomolgus monkey #2501 vaccinated with Immunogen 15 (DAEFRHDRRQIVYKPVGGC; SEQ ID NO:59).
  • FIG. 12 B shows a lack of staining of Tau pathology in fresh frozen human AD brain tissue using serum (1:300 dilution) from eynomolgus monkey #2501.
  • FIG. 13 shows animal titers to A ⁇ and tau 9 weeks post injection at day 0, at 4 weeks and at 8 weeks for the dual Abeta-tau constructs DAEFRHDRRVKSKIGSTGGC (SEQ ID NO:997); DAEFRHDRRSKIGSTENGGC (SEQ ID NO:998); DAEFRHDRRTENLKHQPGGC (SEQ ID NO:999); DAEFRHDRRENLKHQPGGGC (SEQ ID NO:1000); and DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO:1001),
  • FIG. 14 shows blocking of tau binding to Heparin by sera from A ⁇ -tau constructs DAEFRHDRRVKSKIGSTGGC (SEQ ID NO:997); DAEFRHDRRSKIGSTENGGC (SEQ ID NO:998); DAEFRHDRRTENLKHQPGGC (SEQ ID NO:999); DAEFRHDRRENLKHQPGGGC (SEQ ID NO:1000); and DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO:1001).
  • FIG. 15 A shows competition of tau by sera from DAEFRHDRRVKSKIGSTGGC construct (SEQ ID NO:997).
  • FIG. 15 B shows competition of tau by sera from DAEFRHDRRSKIGSTENGGC construct (SEQ ID NO:998).
  • FIG. 15 C shows competition of tau by sera from DAEFRHDRRTENLKHQPGGC construct (SEQ ID NO:999).
  • FIG. 15 D shows competition of tau by sera from DAEFRHDRRENLKHQPGGGC construct (SEQ ID NO:1000).
  • FIG. 15 E shows competition of tau by sera from DAEFRHDRRSKIGSKDNIKHGGC construct (SEQ ID NO:1001).
  • FIG. 15 F shows competition of tau by sera from controls.
  • FIG. 15 G shows competition of tau by sera from DAEFRHDRRSKIGSKDNIKHGGC construct (SEQ ID NO:1001).
  • FIG. 16 A shows Abeta/Tau Dual Vaccine Mouse Serum 2.3_031521 (1:300) Example of staining with DAEFRHDRRVKSKIGSTGGC (SEQ IIS NO:997).
  • FIG. 16 B shows Abeta/Tau Dual Vaccine Mouse Serum 3.1 . . . 031521 (1:300) Example of staining with DAEFRHDRRSKIGSTENGGC (SEQ ID NO:998).
  • FIG. 16 C shows Abeta/Tau Dual Vaccine Mouse Serum 8.4_031521 (1:300) Example of staining with DAEFRHDRRTENLKHQPGGC (SEQ ID NO:999).
  • FIG. 16 D shows Abeta/Tau Dual Vaccine Mouse Serum 9.2_931521 (1:300) Example of staining with DAEFRHDRRENLKHQPGGGC (SEQ ID NO:1000),
  • FIG. 16 E shows Abeta/Tau Dual Vaccine Mouse Serum 11.1_031521 (1:300) Example of staining with DAEFRHDRRSKIGSKDNIKHGGC (SEQ ID NO: 1001).
  • the disclosure provides peptide compositions and immunotherapy compositions comprising an amyloid-beta (A ⁇ ) peptide and a tau peptide.
  • the disclosure also provides methods of treating or effecting prophylaxis of Alzheimer's disease or other diseases with beta-amyloid deposition in a subject, including methods of clearing and preventing formation of deposits, inhibiting or reducing aggregation of A ⁇ and/or tau, blocking the binding, and/or uptake of A ⁇ and/or tau by neurons, inhibiting transmission of tau species between cells, and inhibiting propagation of pathology between brain regions in a subject having or at risk of developing Alzheimer's disease or other diseases containing tau and/or amyloid-beta accumulations.
  • the methods include administering to such patients the compositions comprising an amyloid-beta (A ⁇ ) peptide and a tau peptide.
  • a compound or “at least one compound” can include a plurality of compounds, including mixtures thereof.
  • the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM) of a stated value.
  • a standard margin of error of measurement e.g., SEM
  • the term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration can encompass variations of +/ ⁇ 10% or less, +1-5% or less, or +/ ⁇ 1% or less or less of and from the specified value.
  • Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. As used herein, statistical significance means p ⁇ 0.05.
  • compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited.
  • a composition that “comprises” or “includes” a polypeptide sequence may contain the sequence alone or in combination with other sequences or ingredients.
  • An individual is at increased risk of a disease if the subject has at least one known risk-factor (e.g., age, genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor.
  • risk-factor e.g., age, genetic, biochemical, family history, and situational exposure
  • patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment, including treatment na ⁇ ve subjects.
  • the terms “subject” or “patient” refer to any single subject for which treatment is desired, including other mammalian subjects such as, humans, cattle, dogs, guinea pigs, rabbits, and so on. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.
  • disease refers to any abnormal condition that impairs physiological function.
  • the term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.
  • symptom refers to a subjective evidence of a disease, such as altered gait, as perceived by the subject.
  • a “sign” refers to objective evidence of a disease as observed by a physician.
  • treat and “treatment” refer to the alleviation or amelioration of one or more symptoms or effects associated with the disease, prevention, inhibition or delay of the onset of one or more symptoms or effects of the disease, lessening of the severity or frequency of one or more symptoms or effects of the disease, and/or increasing or trending toward desired outcomes as described herein.
  • prevention refers to contacting (for example, administering) the peptide(s) or immunotherapy compositions of the present disclosure with a subject before the onset of a disease, with or without A ⁇ and/or tau pathology already present (primary and secondary prevention), thereby delaying the onset of clinical symptoms and/or alleviating symptoms of the disease after the onset of the disease, compared to when the subject is not contacted with the peptide or immunotherapy compositions, and does not refer to completely suppressing the onset of the disease.
  • prevention may occur for limited time after administration of the peptide or immunotherapy compositions of the present disclosure. In other cases, prevention may occur for the duration of a treatment regimen comprising administering the peptide or immunotherapy compositions of the present disclosure.
  • reduction refers to decreasing the amount of A ⁇ and/or tau present in a subject or in tissue of the subject, or suppressing an increase in the amount of A ⁇ and/or tau present in a subject or in tissue of the subject, which encompasses decreasing or suppressing an increase in (e.g., decreasing the rate of increase) the amount of A ⁇ and/or tau present, accumulated, aggregated, or deposited in the subject or tissue in the subject, in certain embodiments, the decrease in or suppression of an increase in (e.g., decreasing the rate of increase) the amount of A ⁇ and/or tau present, accumulated, aggregated, or deposited in the subject refers to an amount of A ⁇ and/or tau present, accumulated, aggregated, or deposited in the central nervous system (CNS) of the subject.
  • CNS central nervous system
  • the decrease in or suppression of an increase in (e.g., decreasing the rate of increase) the amount of A ⁇ and/or tau present, accumulated, aggregated, or deposited in the subject refers to an amount of A ⁇ and/or tau present, accumulated, aggregated, or deposited in the periphery (e.g., peripheral circulatory system) of the subject.
  • the decrease in or suppression of an increase in (e.g., decreasing the rate of increase) the amount of A ⁇ and/or tau present, accumulated, aggregated, or deposited in the subject refers to an amount of A ⁇ and/or tau present, accumulated, aggregated, or deposited in the brain of the subject.
  • the A ⁇ and/or tau reduced is the pathological form(s) of the A ⁇ (e.g., extracellular plaque deposits of the ⁇ -amyloid peptide (A ⁇ ), neuritic amyloid plaques), and/or tau (e.g., neurofibrillary tangles of tau, dystrophic neurites).
  • pathological indicators of neurodegenerative disease are decreased.
  • epitopes refers to a site on an antigen to which B and/or T cells respond, or to a site on an antigen to which an antibody binds.
  • Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
  • an “immunogenic agent” or “immunogen” or “antigen” is capable of inducing an immunological response against itself or modified/processed versions of itself upon administration to an animal, optionally in conjunction with an adjuvant.
  • the terms “immunogenic anent” or “immunogen” or “antigen” refer to a compound or composition comprising a peptide, polypeptide or protein which is “antigenic” or “immunogenic” when administered in an appropriate amount (an “immunogenically effective amount”), i.e., capable of inducing, eliciting, augmenting or boosting a cellular and/or humoral immune response and of being recognized by the products of that response (T cells, antibodies).
  • An immunogen can be a peptide, or a combination of two or more same or different peptides, that includes at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acids in a liner or spatial conformation.
  • An immunogen may be effective when given alone or in combination, or linked to, or fused to, another substance (which can be administered at one time or Over several intervals).
  • An immunogenic agent or immunogen May include an antigenic peptide or polypeptide that is linked to a carrier as described herein.
  • a nucleic acid such as DNA or RNA that encodes an antigenic peptide, or polypeptide is referred to as a “DNA [or RNA] immunogen,” as the encoded peptide or polypeptide is expressed in vivo after administration of the DNA or RNA.
  • the peptide or polypeptide can be recombinantly expressed from a vaccine vector, which can be naked DNA or RNA that comprises the peptide or polypeptide coding sequence operably linked to a promoter, e.g., an expression vector or cassette as described herein.
  • peptide and “polypeptide” are used interchangeably herein and refer to a chain of two or more consecutive amino acids. If and when a distinction is made, context makes the meaning clear. For example, if two or more peptides described herein are joined to make a dimeric or multimeric peptide, polypeptide may be used to indicate “poly” or “more than one” peptide.
  • pharmaceutically acceptable means that the carrier, diluent, excipient, adjuvant, or auxiliary is compatible with the other ingredients of a pharmaceutical formulation and not substantially deleterious to the recipient thereof.
  • immunotherapy refers to the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against an A ⁇ and/or tau peptide in a recipient.
  • a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against an A ⁇ and/or tau peptide in a recipient can be an active response induced by administration of immunogen (e.g. an A ⁇ and/or tau peptide).
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4 + .
  • T helper cells and/or cytotoxic T cells are examples of T helper cells and/or cytotoxic T cells.
  • Amyloid Beta (A ⁇ ) Amyloid Beta (A ⁇ )
  • a ⁇ (also referred to herein as beta amyloid peptide or Abeta) peptide is about a 4-kDa internal fragment of 38-43 amino acids of APP (A ⁇ 39, A ⁇ 40, A ⁇ 41, A ⁇ 42, and A ⁇ 43).
  • a ⁇ 40 for example, consists of residues 672-711 of APP and A ⁇ 42 consists of residues 673-713 of APP.
  • a ⁇ is found in both a “short form”, 40 amino acids in length, and a “long form”, ranging from 42-43 amino acids in length.
  • Epitopes' or antigenic determinants are located within the N-terminus of the A ⁇ peptide and include: residues within amino acids 1-10 and 12-25 of A ⁇ , for example from residues 1-3, 1-4, 1-5, 1-6, 1-7, or 3-7, 2-4, 2-5, 2-6, 2-7, or 2-8 of A ⁇ , residues 3-5, 3-6, 3-7, 3-8, or 3-9 of A ⁇ , or residues 4-7.4-8, 4-9, or 4-10 residues 12-24, 12-23, 12-22, 13-25, 13-24, 13-23, 13-22, 14-25, 14-24, 14-23, 14-22, 15-25, 15-24, 15-23, or 15-22 of A ⁇ .
  • epitopes or antigenic determinants include residues 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24 or 17-25 of A ⁇ 42.
  • the A ⁇ peptide can include 3-10 amino acids from residues 1-10 or 12-25 of DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:01).
  • the A ⁇ peptide is selected from the following:
  • the A ⁇ peptide is DAEERHD (SEQ ID NO:06), DAEFR (SEQ ID NO:08) or EFRHD (SEQ ID NO:21).
  • the linker is an amino acid linker that does not have a N-terminal glycine (e.g., GG, GAGA (SEQ ID NO:80)).
  • the linker comprises an amino acid sequence any one of AA, AAA, KK, KKK, SS, SSS, AGAG (SEQ NO:81), GG, GGG, GAGA (SEQ ID NO:80), and KGKG (SEQ ID NO:82).
  • the tinker may be a cleavable linker.
  • cleavable linker refers to any linker between the antigenic peptides that promotes or otherwise renders the A ⁇ peptide and the tau peptide more susceptible to separation from each other by cleavage (for example, by endopeptidases, proteases, low pH or any other means that may occur within or around the antigen-presenting cell) and, thereby, processing by the antigen-presenting cell, than equivalent peptides lacking such a cleavable linker.
  • the cleavable linker comprises an amino acid sequence selected from the group consisting of arginine-arginine (Arg-Arg), arginine-valine-arginine-arginine (Arg-Val-Arg-Arg; SEQ ID NO:69), valine-citrulline (Val-Cit), valine-arginine (Val-Arg), valine-lysine (Val-Lys), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), GAGA (SEQ ID NO:80), AGAG (SEQ ID NO:81), and KGKG (SEQ ID NO:82).
  • the cleavable linker is arginine-arginine (Arg-Arg).
  • the dual A ⁇ /tau polypeptide is as follows:
  • tau peptide examples include any one of SEQ ID NOS: 39-56, 83-86, or 146-996.
  • Linker 2 is optional, and when present is a linker that couples the polypeptide to a carrier.
  • a linker if present, can be 1-10 amino acids in length.
  • the linker comprises between about 1-10 amino acids, about 1-9 amino acids, about 1-8 amino acids, about 1-7 amino acids, about 1-6 amino acids, about 1-5 amino acids, about 1-4 amino acids, about 1-3 amino acids, about 2 amino acids, or one (1) amino acid.
  • the linker is 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids.
  • the amino acid composition of a linker can mimic the composition of linkers found in natural multidomain proteins, where certain amino acids are overrepresented, underrepresented or equi-represented in natural linkers as compared to their abundance in whole protein.
  • threonine (Thr) serine (Seri, proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), arginine (Arg), phenylalanine (Phe), glutamic acid (Glu) and alanine (Ala) are overrepresented in natural linkers.
  • the amino acid composition of a linker can mimic the composition linkers commonly found in recombinant proteins, which can generally by classified as flexible or rigid linkers.
  • flexible linkers found in recombinant proteins are generally composed of small, non-polar (e.g. Gly) or polar (e.g.
  • a linker comprises stretches of Gly and Ser residues (“GS” linker).
  • Linkers can be rich in small or polar amino acids such as Gly and Ser but also contain additional amino adds such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility. See, e.g., Chen, X. et al., “Fusion Protein Linkers: Property. Design and Functionality” Adv Drug Rev., 15; 65(10): 1357-1369 (203).
  • the linker when present, can be an amino acid sequence selected from the group consisting of as GG, GGG, KK, KKK, AA, AAA, SS, SSS, G-A-G-A (SEQ ID NO:80), A-G-A-G (SEQ ID NO 81), and K-G-K-G (SEQ ID NO:82).
  • Cys is optional and can be helpful to conjugate the polypeptide to a carrier.
  • the Cys can be at the C-terminal portion of the polypeptide or at the N-terminal portion of the polypeptide.
  • Examples of the [first peptide]-[linker 1]-[second peptide]-[linker 2]-[Cys] dual A ⁇ /tau polypeptide of the disclosure include the following:
  • the A ⁇ peptide, the tau peptide and, the dual A ⁇ /tau polypeptide are immunogens in accordance with the disclosure.
  • the peptides and the dual A ⁇ -tau polypeptide can be linked to a suitable carrier to help elicit an immune response. Accordingly, one or more the peptides and dual A ⁇ -tau polypeptides of the disclosure can be linked to a carrier.
  • each of the A ⁇ peptide, tau peptide and the A ⁇ -tau polypeptide may be linked to the carrier with or without spacer amino acids Gly-Gly-Gly, Ala-Ala, Ala-Ala-Ala, Lys-Lys, Lys-Lys-Lys, Ser-Ser, Ser-Ser-Ser, (SEQ ID NO:80), Ala-Gly-Ala-Gly (SEQ ID NO:81), or Lys-Gly-Lys-Gly (SEQ ID NO:82)).
  • the dual A ⁇ -tau polypeptide can be linked to a suitable carrier using a C-terminal cysteine to provide a linker between the peptide(s) and the carrier or the dual A ⁇ /tau polypeptide and the carrier.
  • the dual A ⁇ -tau polypeptide can be linked to a suitable carrier using an N-terminal cysteine to provide a linker between the peptide(s) and the carrier.
  • Techniques for linking an immunogen to a carrier include the formation of disulfide linkages using N-succinimidyl 3-(2-pyridylthio)propionate (SPDP), and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue).
  • SPDP N-succinimidyl 3-(2-pyridylthio)propionate
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • These reagents create a disulfide linkage between themselves and peptide cysteine resides on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in other amino acids.
  • peptide immunogens can be linked to at least one artificial T-cell epitope capable of binding a large proportion of MHC Class II molecules, such as the pan DR epitope (“PADRE”).
  • Pan DR-binding peptides PADRE are described in U.S. Pat. No. 5,736,142, WO 95/07707, and Alexander, et al, Immunity. 1:751-761 (1994).
  • Active immunogens can be presented in multimeric form in which multiple copies of an immunogen (peptide of polypeptide) are presented on a carrier as a single covalent molecule.
  • the carrier includes various forms of the dual A ⁇ /tau polypeptide.
  • the dual A ⁇ /tau polypeptide of the immunogen can include polypeptides that have the A ⁇ antigen and the tau antigen in different orders, or may be present with or without an intrapeptide linker and/or a linker to a carrier.
  • a RNA vaccine can comprise trans-amplifying RNA (for example, see Beissert et al., Molecular Therapy January 2020 28(1):119-128).
  • RNA vaccines encode an A ⁇ peptide and a tau peptide as disclosed herein, and are capable of expressing the A ⁇ and a tau peptides, in particular if transferred into a cell such as an immature antigen presenting cell.
  • RNA may also contain sequences which encode other polypeptide sequences such as immune stimulating elements.
  • the RNA of a RNA vaccine can be modified RNA.
  • modified in the context of the RNA can include any modification of RNA which is not naturally present in RNA.
  • modified RNA can refer to RNA with a 5′-cap; however, RNA may comprise further modifications.
  • a 5′-cap can be modified to possess the ability to stabilize RNA when attached thereto.
  • a further modification may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5′- or 3′-untranslated regions (UTR).
  • the RNA e.g. or mRNA vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.
  • nucleic acids such as, e.g., generating mutations in sequences, sub-cloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausuhel, ed.
  • Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g fluid or gel precipitin reactions, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
  • Each of the peptides and immunogens described herein can be presented in a pharmaceutical composition that is administered with pharmaceutically acceptable adjuvants and pharmaceutically acceptable excipients.
  • the adjuvant increases the titer of induced antibodies and/or the binding affinity of induced antibodies relative to the situation if the peptide were used alone.
  • a variety of adjuvants can be used in combination with an immunogen of the disclosure to elicit an immune response. Some adjuvants augment the intrinsic response to an immunogen without causing conformational changes in die immunogen that affect the qualitative form of the response.
  • An adjuvant may be a natural compound, a modified version of or derivative of a natural compound, or a synthetic compound.
  • Some adjuvants include aluminum salts, such as aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPLTM) (see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Mont., now part of Corixa).
  • MPL refers to natural and synthetic versions of MPL. Examples of synthetic versions include PHAD®, 3D-PHAD® and 3D(6A)-PHAD® (Avanti Polar Lipids, Alabaster, Ala.).
  • QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja saponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, N Y, 1995)) QS-21 products include Stimulon® (Antigenics, Inc., New York, N.Y.; now Agenus, Inc. Lexington, Mass.) and QS-21 Vaccine Adjuvant (Desert King, San Diego, Calif.). QS-21 has been disclosed, characterized and evaluated in U.S. Pat. Nos. 5,057,540, and 8,034,348, the disclosures of which are herein incorporated by reference.
  • QS-21 is used in FDA approved vaccines including SHINGRIX.
  • SHINGRIX contains 50 mcg of QS-21. In certain embodiments, the amount of QS-21 is from about 10 ⁇ g to about 500 ⁇ g.
  • TQL1055 is an analogue of QS-21 (Adjuvance Technologies, Lincoln, Nebr.). The semi-synthetic TQL1055 has been characterized in comparison to QS-21 as having, high purity, increased stability, decreased local tolerability, decreased systemic tolerability. TQL1055 has been disclosed, characterized, and evaluated in US20180327436 A1, WO2018191598 A1, WO2018200656 A1, and WO2019079160 A1, the disclosures of which are herein incorporated by reference. US20180327436 A1 teaches that 2.5 fold more TQ1055 was superior to 20 ⁇ g QS-21 but there was not an improvement over 50 ⁇ g TQ1055.
  • the amount of TQL1055 is from about 10 ⁇ g to about 500 ⁇ g.
  • adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stowe et al., N. Engl. Med. 336, 86-91 (1997)), pluronic polymers, and killed mycobacteria.
  • Ribi adjuvants are oil-in-water emulsions.
  • Ribi contains a metabolizable oil (squalene) emulsified with saline containing Tween 80.
  • Ribi also contains refined mycobacterial products which act as immunostimulants and bacterial monophosphoryl lipid A.
  • adjuvants can be CpG oligonucleotides (see WO 98/40100), cytokines (e.g., IL-1, IL-1 alpha and ⁇ peptides, IL-2, ⁇ -INF, IL-10, GM-CSF), chemokines (e g., MIP1- ⁇ and ⁇ , and RANTES), saponins, RNA, and/or TLR agonists (for example, TLR4 agonists such as MPL and synthetic MPL molecules), aminoalkyl glucosaminide phosphate and other TLR agonists.
  • Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before concurrently with, or after administration of the therapeutic agent.
  • some embodiments of the disclosure can comprise a multiple antigen presenting system (MAP).
  • MAP multiple antigen presenting system
  • Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens), carrier proteins and cytotoxic adjuvants.
  • Two main approaches have been used to develop multiple antigen presenting peptide vaccine systems: (1) the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) synthetic approaches using size-defined nanomaterials, e.g., serf-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms.
  • MAP multiple antigenic peptide
  • a MAP system uses multiple copies of antigenic peptides to improve the sometimes poor immunogenicity of subunit peptide vaccines.
  • multiple copies of antigenic peptides are simultaneously bound to the a- and ⁇ -amino groups of a non-immunogenic Lys-based dendritic scaffold, helping to confer stability from degradation, thus enhancing molecular recognition by immune cells, and induction of stronger immune responses compared with small antigenic peptides alone.
  • the MAP comprises one or more of a Lys-based dendritic scaffold, helper T-cell epitopes, immune stimulating lipophilic moieties, cell penetrating peptides, radical induced polymerization, self-assembling nanoparticles as antigen-presenting platforms and gold nanoparticles.
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
  • Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
  • the peptides of the disclosure can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection).
  • the solution can contain formulators agents such as suspending, stabilizing and/or dispersing agents.
  • peptide compositions can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Peptides can also be administered in the form of a nucleic acid encoding the peptide(S) and expressed in situ in a subject.
  • a nucleic acid segment encoding an immunogen is typically linked to regulatory elements, such as a promoter and enhancer that allow expression of the DNA segment in the intended target cells of a subject.
  • regulatory elements such as a promoter and enhancer that allow expression of the DNA segment in the intended target cells of a subject.
  • promoter and enhancer elements from, for example, light or heavy chain immunoglobulin genes or the CMV major intermediate early promoter and enhancer are suitable to direct expression.
  • the linked regulatory elements and coding sequences are often cloned into a vector.
  • DNA and RNA can be delivered in naked form (i.e., without colloidal or encapsulating materials).
  • viral vector systems can be used including retroviral systems (sec, e.g., Boris-Lawrie and Teumin, Cur. Opin. Genet. Develop. 3(1): 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al, J. Virol. 67(10); 5911-21 (1993)); adeno-associated virus vectors (see, e.g., Thou et al., J. Exp. Med.
  • viral vectors from the pox family including vaccinia virus and the avian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, Dubensky et al., J. Virol. 70(1): 508-519 (1996)), Venezuelan equine encephalitis virus (see U.S. Pat. No.
  • rhabdoviruses such as vesicular stomatitis virus (see WO 96/34625) and papillomaviruses (WO 94/12629; Ohe et al., Human Gene Therapy 6(3):325-333 (1995); and Xiao & Brandsma, Nucleic Acids. Res. 24(13):2620-2622 (1996)).
  • Pharmaceutically acceptable carrier compositions can also include additives, including, but not limited to, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, carboxymethylcellulose sodium, sodium, polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerine, glycerine, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.
  • additives including, but not limited to, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, carboxymethylcellulose sodium, sodium, polyacrylate, sodium alginate,
  • a ⁇ plaques and/or neurofibrillary tangles has been found in several diseases including Alzheimer's disease, Down's syndrome, mild cognitive impairment, cerebral amyloid angiopathy, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistic, Pick's disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lcwy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease, progressive supranuclear palsy (PSP), dry age-related macular degeneration (AMD), and inclusion-body myositis.
  • CBD cortic
  • compositions and methods of the disclosure can be used in treatment or prophylaxis of any of these diseases. Because of the widespread association between neurological diseases and A ⁇ and/or tau, the compositions and methods of the disclosure can be used in treatment or prophylaxis of any subject showing elevated levels of A ⁇ and/or tau in the CSF) compared with a mean value in individuals without neurological disease. The compositions and methods of the disclosure can also be used in treatment or prophylaxis of neurological disease in individuals having a mutation in A ⁇ and/or tau associated with neurological disease. The methods are particularly suitable for treatment or prophylaxis of Alzheimer's disease.
  • Subjects amenable to treatment include individuals at risk of disease but not showing symptoms, as well as patients presently showing symptoms, including treatment na ⁇ ve subjects that have not been previous treated for disease.
  • Subjects at risk of disease include those in an aging population, asymptomatic subjects with A ⁇ and/or tau pathologies and having a known genetic risk of disease. Such individuals include those having relatives who have experienced this disease, and those whose risk is determined by analysis of genetic or biochemical markers.
  • Genetic markers of risk include mutations in A ⁇ and/or tau, as well as mutations in other genes associated with neurological disease. For example, the ApoE4 allele in heterozygous and even more so in homozygous form is associated with risk of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • markers of risk of Alzheimer's disease include mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations respectively, mutations in the presenilin genes, PS1 and PS2, a family history of AD, hypercholesterolemia or atherosclerosis.
  • Individuals presently suffering from Alzheimer's disease can be recognized by PET imaging, from characteristic dementia, as well as the presence of risk factors described above.
  • a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSR or blood tau or phospho-tau and A ⁇ 42 levels. Elevated tau or phospho-tau and decreased A ⁇ 42 levels signify the presence of AD.
  • Parkinson's disease for example, Ala30Pro or Ala53Thr, or mutations in other genes associated with Parkinson's disease such as leucine-rich repeat kinase (LARK2 or PARX8).
  • Subjects can also be diagnosed with any of the neurological diseases mentioned above by the criteria of the DSM IV TR.
  • treatment can begin at any age (e.g., 10, 20, 30, or more). Usually, however, it is not necessary to begin treatment until a subject reaches 20, 30, 40, 50, 60, 70, 80, or 90 years of age. Treatment typically entails multiple dosages over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response falls, a booster dosage is indicated. In the case of potential Down's syndrome patients, treatment can begin antenatally by administering therapeutic agent to the another or shortly after birth.
  • the disclosure provides methods of inhibiting or reducing aggregation of Abeta and/or tau in a subject having or at risk of developing a neurodegenerative disease (e.g., Alzheimer's disease).
  • the methods include administering to the subject the compositions as disclosed herein.
  • a therapeutically effective amount is a dosage that, when given for an effective period of time, achieves the desired immunological or clinical effect. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered at set intervals (e.g., weekly, monthly) or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • compositions described herein can be administered to a subject susceptible to, or otherwise at risk of a disease (e.g., Alzheimer's disease) in a regimen (dose, frequency and route of administration) effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of the disease.
  • a regimen dose, frequency and route of administration
  • the regimen is effective to inhibit or delay A ⁇ plaque formation and/or inhibit or delay tau or phospho-tau and paired filaments formed from it in the brain, and/or inhibit or delay its toxic effects and/or inhibit/or delay development of behavioral deficits.
  • compositions described herein are administered to a subject suspected of, or a patient already suffering from a disease (e.g., Alzheimer's disease) in a regimen (dose, frequency and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease.
  • a regimen dose, frequency and route of administration
  • the regimen is preferably effective to reduce or at least inhibit further increase of levels of A ⁇ plaques and/or tau, phosphor-tau, or paired filaments formed from it, associated toxicities and/or behavioral deficits.
  • a regimen is considered therapeutically or prophylactically effective if an individual treated achieves an outcome more favorable than the mean outcome in a control population of comparable subjects not treated by methods of the invention, or if a inure favorable outcome is demonstrated in treated subjects versus control subjects in a controlled clinical trial (e.g., a phase II, phase II/III or phase III trial) at the p ⁇ 0.05 or 0.01 or even 0.001 level.
  • a controlled clinical trial e.g., a phase II, phase II/III or phase III trial
  • Effective doses of vary depending on many different factors, such as means of administration, target site, physiological state of the patient, whether the patient is an ApoE carrier, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the effective amount is a total dose of 25 ⁇ g to 1000 ⁇ g, or 50 ⁇ g to 1000 ⁇ g. In some embodiments, the effective amount is a total dose of 100 ⁇ g. In some embodiments, the effective amount is a dose of 25 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 ⁇ g administered to the subject a total of two times. In some embodiments, a RNA (e.g., mRNA) vaccine is administered to a subject by intradermal, intramuscular injection, or by intranasal administration.
  • a RNA e.g., mRNA
  • a RNA vaccine is administered to a subject by intradermal, intramuscular injection, or by intranasal administration.
  • the amount of an agent for active immunotherapy varies from 1 to 1,000 micrograms ( ⁇ g), or from 0.1-500 ⁇ g, or front 10 to 500 ⁇ g, or from 50 to 250 ⁇ g per patient and can be from 1-100 or 1-10 ⁇ g per injection for human administration.
  • the timing of injections can vary significantly from once a day, to once a week, to once a month, to once a year, to once a decade.
  • a typical regimen consists of an immunization followed by booster injections at time intervals, such as 6 week intervals or two months.
  • Another regimen consists of an immunization followed by one or more booster injections 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months later.
  • Another regimen entails an injection every two months for life.
  • booster injections can be on an irregular basis as indicated by monitoring of immune response.
  • the frequency of administration may be once or more as long as the side effects are within a clinically acceptable range.
  • compositions or methods as disclosed herein comprise administering to a subject a nucleic acid vaccine comprising one or more DNA or RNA polynucleotides having an open reading frame encoding a first peptide and a second peptide wherein a dosage of between 10 ⁇ g/kg and 400 ⁇ g/kg of the nucleic acid vaccine is administered to the subject.
  • the dosage of the RNA polynucleotide is 1-5 ⁇ g, 5-10 ⁇ g, 10-15 ⁇ g, 15-20 ⁇ g, 10-25 ⁇ g, 20-25 ⁇ g, 20-50 ⁇ g, 30-50 ⁇ g, 40-50 ⁇ g, 40-60 ⁇ g, 60-80 ⁇ g, 60-100 ⁇ g, 50-100 ⁇ g, 80-120 ⁇ g, 40-120 ⁇ g, 40-150 ⁇ g, 50-150 ⁇ g, 50-200 ⁇ g, 80-200 ⁇ g, 100-200 ⁇ g, 120-250 ⁇ g, 150-250 ⁇ g, 180-280 ⁇ g, 200-300 ⁇ g, 50-300 ⁇ g, 80-300 ⁇ g, 100-300 ⁇ g, 40-300 ⁇ g, 50-350 ⁇ g, 100-350 ⁇ g, 200-350 ⁇ g, 300-350 ⁇ g, 320-400 ⁇ g, 40-380 ⁇ g, 40-100 ⁇ g, 100-400
  • the nucleic acid is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid is administered to the subject on day seven, or fourteen, or twenty one.
  • compositions described herein are preferably administered via a peripheral route (i.e., one in which the administered composition results in a robust immune response and/or the induced antibody population crosses the blood brain barrier to reach an intended site in the brain, spinal cord, or eye).
  • a peripheral route i.e., one in which the administered composition results in a robust immune response and/or the induced antibody population crosses the blood brain barrier to reach an intended site in the brain, spinal cord, or eye.
  • the induced antibodies leave the vasculature to reach the intended peripheral organs.
  • Routes of administration include oral, subcutaneous, intranasal, intradermal, or intramuscular. Some routes for active immunization are subcutaneous and intramuscular. Intramuscular administration and subcutaneous administration can be made at a single site or multiple sites. Intramuscular injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where deposits have accumulated.
  • An effective amount of a DNA or RNA encoded immunogen can be between about 1 nanogram and about 1 gram per kilogram of body weight of the recipient, or about between about 0.1 ⁇ g/kg and about 10 mg/kg, or about between about 1 ⁇ g/kg and about 1 mg/kg.
  • Dosage forms suitable for internal administration preferably contain (for the latter dose range) from about 0.1 ⁇ g to 100 ⁇ g of active ingredient per unit. The active ingredient may vary from 0.5 to 95% by weight based on the total weight of the composition.
  • an effective dose of dendritic cells loaded with the antigen is between about 10 4 and 10 8 cells. Those skilled in the art of immunotherapy will be able to adjust these doses without undue experimentation.
  • the composition may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • a material to prevent its inactivation.
  • an enzyme inhibitors of nucleases or proteases e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate and trasylol
  • liposomes including water-in-oil-in-water emulsions
  • conventional liposomes Strejan et al., J. Neuroimmunol 7(1):27-41, 1984.
  • the immunotherapeutic compositions disclosed herein may also be used in combination with other treatments for diseases associated with the accumulation of A ⁇ or tau, for example, anti-A ⁇ antibodies such as antibodies that specifically bind to any of the A ⁇ epitopes disclosed herein.
  • anti-A ⁇ antibodies such as antibodies that specifically bind to any of the A ⁇ epitopes disclosed herein.
  • aducanumab or any of the antibodies disclosed in, for example, U.S. Patent Publication No. 20100202968 and U.S. Pat. No. 8,906,367 and/or anti-tau antibodies such as antibodies that specifically bind to any of the tau epitopes disclosed herein, ABBV-8E12, gosuranemab, zagotenemab, RG-6100, BIIB076 or, any of the antibodies disclosed in WO2014/165271, U.S. Pat. No.
  • the patient receives passive immunotherapy prior to the active immunotherapy methods disclosed herein.
  • the patient receives passive and active immunotherapy during the same period of treatment.
  • patients may receive active immunotherapy prior to passive immunotherapy.
  • Combinations may also include small molecule therapies and non-immunogenic therapies such as RAZADYNE® (galantamine), EXELON® (rivastigmine), and ARICEPT® (donepezil) and other compositions that improve the function of nerve cells in the brain.
  • compositions of the disclosure may be used in the manufacture of medicaments for the treatment regimens described herein.
  • relative terms such as “improve,” “increase,” or “reduce” indicate values relative to a control, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control individual or, group.
  • a control individual is an individual afflicted with the same amyloid disease as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual are comparable), but who has not received treatment using the disclosed immunotherapy/vaccine formulations.
  • a control individual is a healthy individual, who is about the same age as the individual being treated. Changes or improvements in response to therapy are generally statistically significant and described by a p-value less than or equal to 0.1, less than 0.05, less than 0.01, less than 0.005, or less than 0.001 may be regarded as significant.
  • Effective doses of the compositions as disclosed herein, for the treatment of a subject vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, if any, and whether treatment is prophylactic or therapeutic. Treatment dosages can be titrated to optimize safety and efficacy.
  • the amount of immunogen can also depend on whether adjuvant is also administered, with higher dosages being required in the absence of adjuvant.
  • the amount of an immunogen for administration sometimes varies from 1-500 ⁇ g per patient and more usually from 5-500 ⁇ g per injection for human administration. Occasionally, a higher dose of 1-2 ⁇ g per dosage is used. Typically, about 10, 20, 50 or 100 ⁇ g is used for each human dosage.
  • the timing of dosages can vary significantly from once a day, to once a year, to once a decade. On any given day that a dosage of immunogen is given, the dosage is greater than 1 ⁇ g/patient and usually greater than 10 ⁇ g/patient if adjuvant is also administered, and greater than 10 ⁇ g/patient and usually greater than 100 ⁇ g/patient in the absence of adjuvant.
  • a typical regimen consists of an immunization followed by booster dosage(s) at 6-week intervals.
  • Another regimen consists of an immunization followed by booster dosage(s) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months later.
  • Another regimen entails dosage(s) every two months for life.
  • booster dosage(s) can be on an irregular basis as indicated by monitoring of immune response.
  • the second treatment can be administered according the product label or as necessary in view of the treatment with the compositions of the disclosure.
  • a second treatment for Alzheimer's disease such as, Razadyne® (galantamine), Exelon® (rivastigmine), and Aricept® (donepezil)
  • the second treatment can be administered according the product label or as necessary in view of the treatment with the compositions of the disclosure.
  • kits comprising the compositions disclosed herein and related materials, such as instructions for use (e.g., package insert).
  • the instructions for use may contain, for example, instructions for administration of the compositions and optionally one or MOM additional agents.
  • the containers of peptide and/or nucleic acid compositions may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses.
  • Each of the peptides, polypeptides, immunogens, and pharmaceutical compositions described herein may be for use in treating one or more of the diseases as described herein, in addition, each of the peptides, polypeptides, immunogens, and pharmaceutical compositions described herein may be for use in methods for treating one or more of the diseases as described herein.
  • Each of the peptides, polypeptides, immunogens, and pharmaceutical compositions described herein may be used in a method for manufacturing a medicament for treating or use in treating one or more of the diseases as described herein.
  • Guinea pigs were injected intramuscularly with 50 ⁇ g of a test immunogen, 25 ⁇ g QS21 in 200 ⁇ l of Addavax on day 0, 21, 49 and 77. Bleeds were done 7 days post immunization.
  • the peptides tested included DAEFRHD (SEQ ID NO:06), QIVYKPV (SEQ ID NO:39), and DAEFRHDRRQIVYKPV (SEQ 11) NO:57).
  • the specific immunogens were DAEFRHDC (SEQ 11) NO:71), QIVYKPVGGC (SEQ ID NO:72), and DAEFRHDRRQIVYKPVGGC (SEQ ID NO:59).
  • the peptides were coupled through the C-terminal cysteine to CRM-197 with a maleimide linkage.
  • Female Guinea Pits were at least 5 weeks old at the start of the study having an approximate body Weight of 350-500 g.
  • Appropriate animal housing and research procedures for animal husbandry and care were conducted in an accredited facility in accordance with the guidelines of the U.S. Department of Agriculture's (USDA) and the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.
  • USDA U.S. Department of Agriculture's
  • AALAC Assessment and Accreditation of Laboratory Animal Care
  • the immunogen concentration was 0.5 mg/ml. Prior to each administration of the test immunogen, approximately a 3 cm 2 area on each hind limb was shaved and wiped with ethanol for visualization of the injection site. Each animal received a test immunogen dose of 200 microliters (0.25 micrograms/microliter) divided into two separate sites each of 100 microliter per injection (i.e., animals received 50 ⁇ g of immunogen in 100 ⁇ l PBS+25 ⁇ g of QS21 in 100 ⁇ l MF59). A 25G-27G needle was inserted intramuscularly into the hind limb, approximately 0.25-0.5 cm deep, and injected at 100 microliters pet site. Injection sites were rotated each administration between four separate sites per hind limb and separated by at least 2 cm.
  • a soluble aggregate A ⁇ prep (HFIP film of A ⁇ 42 was resuspended and incubated overnight with shaking, then spun to remove insoluble aggregate) was coated on to the plate at 100 ⁇ l per well in PBS and incubated overnight at room temperature. Plates were blocked for 1 hour with 1% BSA in PBS. Plates were aspirated and to row A, 200 ⁇ l of 0.1% BSA in PBS Tween was added to 1-4. In 1 neg. GP serum was added a 1/100 while 2-4 contained 1/100 test serum. Rows B-H contained 100 ⁇ l of 0.1% BSA in PBS Tween.
  • Rows were serially diluted 1 ⁇ 2 down the plate giving dilution of 1/100 to 1/12800.
  • Wells were incubated. 2 boas at room temperature, then were washed and a 1/5000 dilution of anti Guinea Pig IgG HRP in 0.1% BSA in PBS Tween was prepared and then 100 ⁇ l added to the washed well. This incubated for 0.1 hour and was washed.
  • OPD substrate was prepared using Thermo-Fisher OPD tablets at 1 tablet per 10 mls. Thermo fisher substrate buffer was added at 1/10 and each well had 100 ⁇ l added and incubated for 15 minutes.
  • OPD substrate was prepared using Thermo-Fisher OPD tablets at 1 tablet per 10 mls. Thermo fisher substrate buffer was added at 1/10 and each well had 100 ⁇ l added and was incubated for 15 minutes. 50 ⁇ l of 2N H 2 SO 4 was added to stop the reaction and plates were read on a molecular devices spectromax at 490 nM. Titer was defined as the dilution giving 50% maximum OD and was extrapolated if it fell between dilutions.
  • a ⁇ 1-15 and A ⁇ 1-28 were both used at different parts of the study. Both of these will not form aggregates. 2 ⁇ g/ml A ⁇ monomers were coated at coated on to the plate 100 ⁇ l per well in PBS and incubated overnight at room temperature. Plates were blocked for 1 hour with 1% BSA in PBS. Plates were aspirated and to row A 200 ⁇ l of 0.1% BSA in PBS Tween was added. In column 1 neg. GP serum was added at 1/100 while the rest of the row contained 1/100 test serums. Rows were serially diluted 1 ⁇ 2 down the plate giving dilution of 1/100 to 1/12800.
  • OPD substrate was prepared using Thermo-Fisher OPD tablets at 1 tablet per 10 mls. Thermo fisher substrate buffer was added at 1/10 and each well had 100 ⁇ l added and was incubated for 15 minutes. 50 ⁇ l of 2N H 2 SO 4 was added to stop the reaction and plates were read on a molecular devices spectromax at 490 nM. Titer was defined as the dilution giving 50% maximum OD and was extrapolated if it fell between dilutions.
  • Thermofisher neutavidin plates were rehydrated with 0.05% Tween in TBS and aspirated.
  • Peptide GGGSVQIVYKPVDLS (SEQ ID NO:68) containing a biotin was made up in a 1/500 dilution in 0.1% BSA in PBS tween. Adding 100 ⁇ l per well for 1 hour and then washed. To row A of the plate 200 ⁇ l of 0.1% BSA in PBS Tween was added. In column 1 neg. GP-serum Was added at 1/100 while the rest of the rows contained 1/100 test serums. Rows were serially diluted 1 ⁇ 2 down the plate giving dilution of 1/100 to 1/12800.
  • OPD substrate was prepared using Thermo-Fisher OPD tablets at 1 tablet per 10 mls. Thermo fisher substrate buffer was added at 1/10 and each well had 100 ⁇ l added and was incubated for 15 minutes. 50 ⁇ l of 2N H 2 SO 4 was added to stop the reaction and plates were read on a molecular devices Spectromax at 490 nM. Titer defined as the dilution giving 50% maximum OD and was extrapolated if it fell between dilutions.
  • FIG. 1 shows the results comparing the geometric mean titers of Guinea Pig serum for immunogen DAEFRHDRRQIVYKPV (SEQ ID NO:57) titered on monomeric A ⁇ 1-28, soluble aggregates of A ⁇ , full length tau, and tau MTBR epitope containing GGGSVQIVYKPVDLS (SEQ ID NO:68),
  • FIG. 2 shows the results for a single peptide immunogen A ⁇ and tau peptides (DAEFRHD (SEQ ID NO:06) and QIVYKPV (SEQ ID NO:39)) versus dual peptide immunogen DAEFRHDRRQIVYKPV (SEQ ID NO:57) on A ⁇ 128 and full-length tau.
  • DAEFRHD single peptide immunogen A ⁇ and tau peptides
  • QIVYKPV SEQ ID NO:39
  • Example 3 Staining of Alzheimer's Brain Tissue with Sera from Guinea Pigs Immunized with a Vaccine as Disclosed Herein
  • Example 4 Serum from Vaccinated Animals Blocks Soluble A ⁇ Aggregates from Binding to Neurons
  • E18 primary rat hippocampal neurons were cultured as described previously (Zago, et eV. “Neutralization of Soluble, Syrtaptotoxic Amyloid ⁇ Species by Antibodies is Epitope Specific,” J Neurosci. 2012 Feb. 22; 32(8) 2696-2702). Soluble A ⁇ aggregate was pre-incubated with or without guinea pig vaccine serum on culture DIV14-21 to block soluble A ⁇ aggregate from neuritic binding. Guinea pig serum was isolated from animals vaccinated with dual immunogen peptide: DAEFRHDRRQIVYKPVGGC (SEQ ID NO:59; immunogen 9).
  • Fresh unlabeled, biotinylated or (9:1) soluble A ⁇ was prepared one day prior and incubated overnight at 4° C.
  • Each diluted serum sample (1:1000, 1:300, and 1:100) and soluble A ⁇ Solution was prepared at 2 ⁇ the final concentration in one-half of final treatment volume using NeuroBasal-no phenol red (NB-NPR) medium.
  • NB-NPR NeuroBasal-no phenol red
  • E18 neurons were rinsed with NB-NPR at 150 ⁇ L/well before adding binding treatment.
  • HCI High-content imaging
  • Example 5 Mice Vaccinated with Dual Peptide Antigens Produce Titers to A ⁇ and Tau
  • mice Female mice were injected on day 0, 14 and 28 with 25 ⁇ g of a dual peptide immunogen (Table 2) and 25 ⁇ g QS21 (Desert King) in PBS total 200 ⁇ l/injection. Each mouse received 200 ⁇ l subcutaneously. Mice were bled on day 21 and 35.
  • Table 2 a dual peptide immunogen
  • QS21 Desert King
  • the immunogens containing Tau peptide EIVYKSP (SEQ ID No:43); see FIG. 5 B ) showed greater variability overall in tau titers (with the exception of immunogen 23) than those with the tau MTBR sequence QIVYKPV (SEQ ID NO:39); therefore the base microtubule binding region (MTBR) peptide containing DAEFRHDRRQIVYKPV (SEQ ID NO:57) was also tested ( FIG. 5 A ). Titers of three immunogens with different linkers (no linker, AA and KK) compared to the GGC linker are shown in FIG. 5 .
  • Example 6 Serum from Vaccinated Animals Stains A ⁇ Plaques and Tau Pathologies in Human Brain Tissue
  • Fresh frozen human brain tissues from autopsied Alzheimer's disease donors or non-diseased controls was embedded in OCT, and cut in a cryostat to generate 10 ⁇ m frozen sections.
  • the tissue sections were incubated in a solution of glucose oxidase and beta D-glucose, in the presence of sodium azide, to block endogenous peroxidase.
  • the staining with Sera from vaccinated mice was performed using OCT, and cut in a cryostat to generate 10 ⁇ m frozen sections.
  • the tissue sections were incubated in a solution of glucose oxidase and beta D-glucose, in the presence of sodium azide, to block endogenous peroxidase.
  • the staining with Sera from vaccinated mice was performed incubated in a solution of glucose oxidase and beta D-glucose, in the presence of sodium azide, to block endogenous peroxidase.
  • mice Were vaccinated with the following dual antigen peptides: DAEFRHDRRQIVYKPVGGC (SEQ ID NO:59, Immunogen 9); DAEFRHDRRQIVYKPVC (SEQ ID NO:60, Immunogen 10); DAEFRHDRRQIVYKPVAAC (SEQ ID NO:61, Immunogen 18); and DAEFRHDRRQIVYKPVKKC (SEQ ID NO:62, Immunogen 19) or control mice. Staining was then carried out at 1:1000 dilution, in an automated Leica Bond Rx Stainer (Leica Biosystems). Antibody binding was detected using the Bond Polymer Refine Detection Kit (DS9800, Leica Biosystems).
  • Results demonstrate A ⁇ plaques and tau neurofibrillary tangles were identified based on their typical histopathological characteristics. Such pathologies were absent from tissues incubated with control mouse serum. Also, non-diseased tissue had no such pathological staining after incubation with the sera from vaccinated mice.
  • FIGS. 6 A-E and Table 6 summarize the results of A ⁇ and Tau staining using mouse sera from animals vaccinated with dual antigen peptides (1:1000 dilution of sera used to stain human brain tissue from AD patients).
  • Group 2 was injected at week 0, 8, 24 with bleeds taken every 2 weeks through week 38. Serum titer levels were determined against A ⁇ and full-length tau. Fresh frozen human AD or control brain sections were stained with sera from immunized and control animals. The activity of the guinea-pig immune sera was also assessed on soluble A ⁇ oligomer binding in primary rat hippocampal neurons.
  • Cynomolgus monkey bleeds were titered by enzyme-linked immunosorbent assay (ELISA). Plates were coated overnight at 2 ⁇ g/ml, with A ⁇ 1-28 (SEQ ID NO:67) in phosphate-buffered saline (PBS) and then blocked 1 hour with 1% bovine serum albumin (BSA) in PBS. Pre-bleed cynomolgus monkey was used as a negative control while known positive anti-scrum from previous mouse studies was used as a positive control at the same dilutions of test serum. Bleeds were diluted in PBS/0.1% BSA 0/1% Tween 20 (PBS/BSA/T) starting at 1/100 and serially diluted 1:2 down the plate.
  • ELISA enzyme-linked immunosorbent assay
  • Plates were washed with TBS/Tween 20 and goat anti-monkey immunoglobulin G (IgG) (heavy+light chains) horseradish peroxidase (HRP) (IgG [H+L] HRP; Invitrogen) was added and incubated 1 hour at room temperature. Plates were washed in TBS/Tween 20, and antibody binding was detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, Mass.) following manufacturer's instructions. Plates were read at 490 nM on a Molecular Devices Spectromax. Titer was defined as the dilution giving, 50% Maximum OD or 4 ⁇ background (defined in graphs and tables) extrapolation was used if it fell in between dilutions.
  • IgG immunoglobulin G
  • HRP IgG [H+L] HRP
  • HRP horseradish peroxidase
  • Cynomolgus monkey bleeds were titered by enzyme-linked immunosorbent assay (ELISA) against full-length recombinant tau (Proteus, Kalamazoo, Mich.; SEQ ID NO:02). Plates were coated overnight at 2 ⁇ g/ml . . . tau in phosphate-buffered saline (PBS) and then blocked 1 hour with 1% bovine serum albumin (BSA) in PBS. Pre-bleed guinea pig serum was used as a negative control while known positive anti-serum from previous mouse studies was used as a positive control at the same dilutions of test serum.
  • ELISA enzyme-linked immunosorbent assay
  • Bleeds were diluted in PBS/0.1% BSA/0/1% Tween 20 (PBS/BSA/T) starting at 1/100 and serially diluted 1:2 down the plate. Plates were washed with TBS/Tween 20 and goat anti-monkey immunoglobulin G (IgG) (heavy+light chains) horseradish peroxidase (HRP) (IgG [H+L] HRP; Invitrogen) was added and incubated 1 hour at room temperature. Plates were washed in TBS/Tween 20, and antibody binding was detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, Mass.) following Manufacturer's instructions.
  • IgG immunoglobulin G
  • HRP horseradish peroxidase
  • Cynomolgus monkey bleeds Were titered by enzyme-linked immunosorbent assay (ELISA) against CRM197 (FinaBio, Maryland) carrier protein. Plates were coated overnight at 2 ⁇ g/mL tau in phosphate-buffered saline (PBS) and then blocked 1 hour with 1% bovine serum albumin (BSA) in PBS. Pre-bleed guinea pig serum was used as a negative control while known positive anti-serum from previous mouse studies was used as a positive control at the same dilutions of test serum. Bleeds were diluted in PBS/0.1% BSA/0/1% Tween 20 (PBS/BSA/T) starting at 1/100 and serially diluted 1:2 down the plate.
  • PBS/BSA/T phosphate-buffered saline
  • Plates were washed with TBS/Tween 2.0 and goat anti-monkey immunoglobulin G (IgG) (heavy light chains) horseradish peroxidase (HRP) (IgG [H+L] HRP; Invitrogen) was added and incubated 1 hour at room temperature. Plates were washed in TBS/Tween 20, and antibody binding was detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, Mass.) following manufacturer's instructions. Plates were read at 490 nM on a Molecular Devices Spectromax. Titer was defined as the dilution giving 50% Maximum OD or 4 ⁇ background (defined in graphs and tables) extrapolation was used if it fell in between dilutions.
  • IgG immunoglobulin G
  • HRP horseradish peroxidase
  • Sera from immunized monkeys were evaluated for the ability to bind pathological A ⁇ plaques and tau tangles in human brain tissue from subjects with AD. Binding both A ⁇ plaques and tau tangles is expected reduce plaque burden and reduce mu transmission which in turn should reduce the signs and symptoms of AD.
  • Autopsy blocks of fresh frozen human brain tissue were embedded in optimal cutting temperature compound (OCT compound) and cut using a cryostat to generate 10 micron sections.
  • OCT compound optimal cutting temperature compound
  • the sections were placed into a solution of glucose oxidase and beta D-glucose, in the presence of sodium azide, to block endogenous peroxidase.
  • tissue sections were prepared, the staining with the specified eynomolgus immune sera was carried out at two dilutions (1:300 in 5% goat Serum with 0.25% Triton for 1 hour at RT).
  • Binding was detected with a mouse anti-monkey IgG secondary antibody purified unlabeled (Mybiosource 3 mg/mL) for 1 hour at RT, and a goat anti-mouse IgG secondary antibody (Jackson, 1:200) for 1 hour at RT, avidin-biotin complex (ABC; Vector PK-4000) and a DAKO DAB Detection Kit according to manufacturer instructions.
  • the staining was processed using an automated Leica Bond Stainer.
  • Table 7 shows a summary of the staining in the cynomolgus monkey study. All animal except 1002 had positive staining for A ⁇ . Only animal 1001 had Strong tau staining ( FIG. 9 A- 9 C ), with 1003 ( FIG. 10 A - FIG. 10 B ) and 1501 ( FIG. 11 A - FIG. 11 B ) demonstrating modest staining. Animal 2501, for example, did not demonstrate significant tau staining ( FIG. 12 A - FIG. 12 B ). There did not appear to be a correlation with tau staining and tau titers as evidenced by animal 1001, which did not have a strong tau titer but stained tau strongly.
  • T-cell reactivity was also investigated. Immune sera inhibited the binding of soluble A ⁇ aggregates to hippocampal neurons. Immunogens did not elicit cytotoxic T-cell responses to A ⁇ or tau.
  • Example 10 Animals Vaccinated with Dual Peptide Antigens Produce Titers to A ⁇ and Tau
  • the study described in this example was designed to assess dual A ⁇ and tau antigen Peptides in mice.
  • the dual A ⁇ -tau constructs in this example effectively demonstrated: high titers for both antigens; blocking of tau binding to heparin; and staining/binding to A ⁇ and tau peptides in brain tissue from human Alzheimer's patients.
  • titers against tau for an engineered tau immunogen (Dual #11) were comparable, and in some cases, better than other tau immunogens despite including a non-native tau sequence. This demonstrates that engineered immunogens are useful in vaccine constructs.
  • Immunogen preparation was 25 ⁇ g Immunogen, 25 ⁇ g QS21 and 150 ⁇ l of 0.02% Tween 80/PBS per injection. Each mouse received 200 ⁇ l subcutaneously. Mice were injected at day 0, at 4 weeks from day 0, and at 8 weeks from day 0, with bleeds taken for titer at 5 weeks from day 0 and animals sacrificed and terminal bleed collected at 9 weeks from day 0 (see FIG. 13 ).
  • Mouse serum was titered by enzyme-linked immunosorbent assay (ELISA). Plates were coated overnight at 2 ⁇ g/mL with either Abeta 1-28 (Anaspec, San Jose, Calif.), or recombinant tau (Proteos, Kalamazoo, Mich. in phosphate-buffered saline (PBS), and then blocked 1 hour with 1% bovine serum albumin (BSA) in PBS.
  • Abeta 1-28 Abeta 1-28
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • Bleeds were diluted in PBS/0.1% BSA/0.1% Tweet) 20 (PBS/BSA/7) starting at 1/100 and serially diluted 1:2 down the plate, Plates were washed with TBS/Tween 20 and goat anti-mouse immunoglobulin G (IgG) (heavy+light chains) horseradish peroxidase (HRP) (ThermoFisher) 1/5000 was added and incubated 1 hour at room temperature. Plates were washed in TBS/Tween 20, and antibody binding was detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, Mass.) following manufacturer's instructions.
  • IgG goat anti-mouse immunoglobulin G
  • HRP horseradish peroxidase
  • OPD o-phenylenediamine dihydrochloride
  • Antiserum is titered on four MTBR regions using peptides of MTBR 1-4 purchased from Anaspec (San Jose, Calif.)
  • MTBR peptide 1 (SEQ ID NO: 1058) QTAPVPMPDLKNVKSKIGSTENLKHQPGGGK MTBR peptide 2 (SEQ ID NO: 1059) VQINKKLDLSNVQSKCGSKDNIKHVPGGGS MTBR peptide 3 (SEQ ID NO: 1060) VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ MTBR peptide 4 (SEQ ID NO: 1061) VEVKSEKLDFKDRVQSKIGSLDNITHVPGGGN
  • Mouse serum is titered by enzyme-linked immunosorbent assay (ELISA). Plates are coated overnight at 2 ⁇ g/mL with each the various MTBR peptides in phosphate-buffered saline (PBS) and then blocked 1 hour with 1% bovine serum albumin (BSA) in PBS. Normal mouse serum is used as a negative control. Bleeds are diluted in PBS/0.1% BSA/0.1% Tween 20 (PBS/BSA/T) starting at 1/100 and serially diluted 1:2 down the plate.
  • PBS/BSA/T enzyme-linked immunosorbent assay
  • Plates are washed with TBS/Tween 20 and goat anti-mouse immunoglobulin G (IgG) (heavy H+light chains) horseradish peroxidase (HRP) (ThermoFisher) 1/5000 is added and incubated 1 hour at room temperature. Plates are washed in TBS/Tween 20, and antibody binding is detected with o-phenylenediamine dihydrochloride (OPD) substrate (Thermo Fisher Scientific, Waltham, Mass.) following manufacture's instructions. Plates are read at 490 nM on a Molecular Devices Spectromax. Titer is defined as the dilution giving 4 ⁇ background (defined in graphs and tables); extrapolation is used if it falls in between dilutions.
  • IgG immunoglobulin G
  • HRP horseradish peroxidase
  • OPD o-phenylenediamine dihydrochloride
  • E18 primary rat hippocampal neurons are cultured as described previously (Zago, el al. “Neutralization of Soluble, Synaptotoxic Amyloid ⁇ Species by Antibodies Is Epitope Specific,” J Neurosci, 2012 Feb. 22; 32(8): 2696-2702).
  • Soluble A ⁇ aggregate is pre-incubated with or without vaccine serum on culture DIV14-21 to block soluble A ⁇ aggregate from neuritic binding.
  • Fresh unlabeled, biotinylated or (9:1) soluble A ⁇ is prepared one day prior and incubated overnight at 4° C. Each diluted serum sample and soluble A ⁇ solution is prepared at 2 ⁇ the final concentration in one-half of final treatment volume using NeuroBasal-no phenol red (NB-NPR) medium.
  • NB-NPR NeuroBasal-no phenol red
  • E18 neurons are rinsed with NB-NPR at 150 ⁇ L/well before adding binding treatment. Serum from vaccinated animals/A ⁇ treatment to is added to E18 neurons at 60 ⁇ L/well, and then incubated for 30 minutes at 37° C. under normal incubator conditions (5% CO 2 ; 9% O 2 ). Cells are rinsed twice using 150 ⁇ L/well of NB-NPR, and then fixed in 4% paraformaldehyde in 1 ⁇ DPBS for 20 minutes.
  • Cells are permeabilized using 0.1 TX-100 for 5 minutes, and blocked using 10% normal goat serum (NGS) for 1 hour at room temperature (RT). Cells are incubated with MAP2 & NeuN primary antibodies in 1004/well, 1 ⁇ DPBS containing 1% BSA+1% NGS overnight at 4° C. The next day, cells are rinsed twice in 150 ⁇ L/well 1 ⁇ DPBS for 5 minutes each wash. Secondary antibodies are added for 1 hour at RT in 100 ⁇ L/well 1 ⁇ DPBS+1% BSA+1% NGS.
  • NGS normal goat serum
  • MCI High-content imaging
  • Immunogens were selected for evaluation in vaccine peptide constructs.
  • Constructs comprise A ⁇ immunogens and tau immunogens. Some immunogens comprise a tau peptide comprising 3-10 amino acids from tau. Other immunogens comprise an engineered tau immunogen.
  • Certain immunogenic peptides were designed and selected to (i) raise antibodies that bind Within the within microtubule binding repeats (MTBRs) of human Tau protein, (ii) be less likely to generate an unwanted T cell-mediated autoimmune response, and (iii) be less likely to raise antibodies that would cross-react with other human proteins.
  • MTBRs microtubule binding repeats
  • the engineered peptides were subjected to in silico analysis to predict MHC binding using the IEDB (Immune Epitope Database) from National Institute of Allergy and Infectious Diseases/La Jolla Immunology Institute. MHC class II binding is considered a good indicator of a sequence containing a T-cell epitope. A panel of alleles were used for MHC II binding prediction. Engineered peptides with a predicted half maximal inhibitory concentration (IC50) above a specified cutoff were considered to have a low probability of MHC II binding and were selected for further analysis.
  • IEDB Immunogen Epitope Database
  • the dual A ⁇ -tau constructs comprising an engineered tau immunogen demonstrated results that were comparable, and in some cases, better than other tau immunogens despite including a non-native tau sequence.
  • FIG. 13 shows high titers for both Abeta and tau antigens, including an engineered tau sequence.
  • FIG. 14 shows blocking of tau binding to heparin.
  • FIG. 15 G shows resulting tau antibody binding to MTBR region peptides.
  • FIG. 16 E shows staining/binding to A ⁇ and tau peptides in brain tissue from human Alzheimer's patients. This demonstrates that engineered immunogens are useful in vaccine constructs
  • Dual immunogen A ⁇ -tau vaccine constructs were developed and it was shown that these constructs raised balanced titers to and tau in mice, guinea-pigs, and cynomolgus monkeys.
  • the antibodies were immunoreactive with both A ⁇ plaques and neurofibrillary tau tangles in human AD brain sections and blocked the binding of soluble A ⁇ aggregates (oligomers) to neurons without eliciting T-cell responses for A ⁇ or tau.
  • the peptide may comprise, consist, or consist essentially of the recited sequences.
  • incorporated in this disclosure are the following sequences that can be part of the compositions comprising an amyloid-beta (AN peptide and a tau peptide as disclosed herein.
  • DAEFRHDRRQIVYKPV (SEQ ID NO: 57) DAEFRHDRREIVYKSV (SEQ ID NO: 58) DAEFRHDRRQIVYKPVGGC (SEQ ID NO: 59) DAEFRHDRRQIVYKPVC (SEQ ID NO: 60) DAEFRHDRRQIVYKPVAAC (SEQ ID NO: 61) DAEFRHDRRQIVYKPVKKC (SEQ ID NO: 62) DAEFRHDRREIVYKSPGGC (SEQ ID NO: 63) DAEFRHDRREIVYKSPAAC (SEQ ID NO: 64) DAEFRHDRREIVYKSPC (SEQ ID NO: 65) DAEFRHDRREIVYKSPKKC (SEQ ID NO: 66) DAEFRHDSGYEVHHQKLFFAEDVGSNKG (SEQ ID NO: 67) GGGSVQIVYKPVDL
  • DAEFRHDRREIVYKSVXXC (SEQ ID NO: 79) If present, XX can be GG or AA or KK or SS.
  • DAEFRHDC (SEQ ID NO: 71) QIVYKPVGGC (SEQ ID NO: 72) GGSQIVYKPVDLS (SEQ ID NO: 73) DAEFRHDRREIVYKSVGGC (SEQ ID NO: 74) DAEFRHDRREIVYKSVAAC (SEQ ID NO: 75) DAEFRHDRREIVYKSVC (SEQ ID NO: 76) DAEFRHDRREIVYKSVKKC (SEQ ID NO: 77) DAEFRHDRRNIKHVPGGC (SEQ ID NO: 78) DAEFRHDRRVKSKIGSTGGC (SEQ ID NO: 997) DAEFRHDRRSKIGSTENGGC (SEQ ID NO: 998) DAEFRHDRRTENLKHQPGGC (SEQ ID NO: 999) DAEFRHDRRENLK

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