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WO2020047107A1 - Synthetic carrier compositions for peptide vaccines - Google Patents

Synthetic carrier compositions for peptide vaccines Download PDF

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Publication number
WO2020047107A1
WO2020047107A1 PCT/US2019/048585 US2019048585W WO2020047107A1 WO 2020047107 A1 WO2020047107 A1 WO 2020047107A1 US 2019048585 W US2019048585 W US 2019048585W WO 2020047107 A1 WO2020047107 A1 WO 2020047107A1
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WO
WIPO (PCT)
Prior art keywords
seq
peptide
composition
antigen
alpha
Prior art date
Application number
PCT/US2019/048585
Other languages
French (fr)
Inventor
Christopher H. Clegg
David F. ZEIGLER
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Tria Bioscience Corp.
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Publication of WO2020047107A1 publication Critical patent/WO2020047107A1/en

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    • 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
    • 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
    • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61K2039/55555—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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/605—MHC molecules or ligands thereof
    • 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • vaccine products that provide broad protection and that may be inexpensive to produce and easily distributable worldwide create a high demand for indications including infectious as well as chronic diseases.
  • influenza virus is a highly contagious pathogen and, due to its rapid mutation rates, creates a demand for vaccines that provide a more effective and sustained protection.
  • compositions and methods of use for peptide-based vaccines are provided.
  • the present disclosure provides a composition comprising: (a) an alpha-helical peptide domain; (b) at least one CD4 T cell epitope peptide; and (c) at least one B cell epitope peptide selected from an immunogenic protein or peptide, carbohydrate, or lipid that mediates a physiological condition or disease including neural degenerative diseases, allergy, and autoimmunity, wherein each of the alpha-helical peptide domain, T cell epitope peptide, and B cell epitope peptide are associated.
  • the B cell epitope peptide is selected from SEQ ID NO: 183 - SEQ ID NO: 270.
  • the present disclosure provides a composition comprising: (a) an alpha-helical peptide domain; (b) at least one CD4 T cell epitope peptide selected from SEQ ID NO: 66 - SEQ ID NO: 182; and (c) at least one B cell epitope peptide, wherein each of the alpha- helical peptide domain, T cell epitope peptide, and B cell epitope peptide are associated.
  • the alpha-helical peptide domain comprises at least one heptad repeat with an amino acid sequence according to the general formula: [X 1 X 2 X 3 X 4 X 5 X 6 X 7 ] n (SEQ ID NO: 295), wherein X 1 and X 4 are each independently selected from I, L, V, A, F, Y, W, N, and Q; X 2 , X 3 , X 6 are independently selected from the amino acids K, R, E, D, H, S, N, Q, A, T, and C; X 5 , X 7 are independently selected from the amino acids K, R, E, D, and H; and n is any number from 1 to 10.
  • the alpha-helical peptide domain can comprise an amino acid sequence according to any one of the following general formulas:
  • the alpha-helical peptide domain comprises an amino acid sequence with at least 80% identity to IKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 18),
  • the alpha- helical peptide domain comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
  • the alpha- helical peptide domain comprises an amino acid sequence with 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
  • composition comprising: (a) an alpha-helical peptide domain comprising an amino acid sequence with at least 80% identity to, at least 82.5% identity to, at least 85% identity to, at least 87.5% identity to, at least 90% identity to, at least 92.5% identity to, at least 95% identity to, at least 97.5% identity to, or at least 99% identity to identity to IKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 18),
  • DEIEERIEEIEERIEEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 45); (b) at least one T cell epitope peptide or a B cell epitope peptide, or a combination thereof; and (c) a target antigen, wherein the alpha-helical peptide domain, T cell epitope peptide, and B cell epitope are associated.
  • the alpha-helical peptide domain comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
  • the alpha-helical peptide domain comprises an amino acid sequence with 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
  • the at least one CD4 T cell epitope peptide is an immunogenic peptide fragment selected from the group consisting of diphtheria toxoid peptide epitopes, measles morbillivirus fusion glycoprotein F peptide epitopes, a pan DR epitope (PADRE) peptide, influenza-derived epitope peptides, hepatitis B and C virus epitope peptides, tetanus toxoid peptide epitopes, P.
  • immunogenic peptide fragment selected from the group consisting of diphtheria toxoid peptide epitopes, measles morbillivirus fusion glycoprotein F peptide epitopes, a pan DR epitope (PADRE) peptide, influenza-derived epitope peptides, hepatitis B and C virus epitope peptides, tetanus toxoid peptide epitopes, P.
  • falciparum peptide epitopes falciparum peptide epitopes, gamma 2ab peptide epitopes, GAD65 peptide epitopes, plasmodium peptide epitopes, polio peptide epitopes, Pseudomonas peptide epitopes, Vaccinia peptide epitopes, Streptococcus peptide epitopes, Yellow Fever peptide epitopes, Coxiella peptide epitopes, Yrsenia pestis peptide epitopes, RSV peptide epitopes, SSP2.61 peptide epitopes, ESAT6 peptide epitopes, tuberculosis peptide epitopes, ebola peptide epitopes, HPV peptide epitopes, anthrax peptide epitopes, varicella peptide epitopes, HSV peptide epitopes, and
  • the at least one CD4 T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, at least two CD4 T cell epitope peptides are linked in tandem. In some aspects, the at least one CD4 T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182.
  • the at least one CD4 epitope peptide has a sequence set forth in SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 157, SEQ ID NO: 164, SEQ ID NO: 165, or SEQ ID NO: 166, or a sequence having at least 90%, 95%
  • the B cell epitope peptide is a foreign antigen comprised of an immunogenic protein or peptide, carbohydrate, lipid, or small molecule; a host-derived antigen comprised of an immunogenic protein or peptide, carbohydrate, or lipid that mediates a physiological condition or disease including infectious diseases, neural degenerative diseases, allergy, autoimmunity, and cancer.
  • the B cell epitope peptide is selected from SEQ ID NO: 183 - SEQ ID NO: 270, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the at least two CD4 T cell epitope peptides are linked in tandem.
  • the at least one CD4 epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the at least one CD4 epitope peptide has a sequence of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:
  • SEQ ID NO: 70 SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 157, SEQ ID NO: 164, SEQ ID NO: 165, or SEQ ID NO: 166, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the target antigen is a small molecule, a peptide, a polysaccharide, a glycolipid, or a lipid.
  • the small molecule is nicotine.
  • the N-terminus of the at least one T cell epitope peptide is linked to the C-terminus of the alpha-helical peptide monomer.
  • the N-terminus of the at least one B cell epitope peptide is linked to the C-terminus of the at least one T cell epitope peptide or the at least one B cell epitope peptide is linked to the N-terminus of the peptide carrier, or any combination thereof.
  • the N-terminus of the at least one B cell epitope peptide is linked to the C-terminus of the at least one T cell epitope peptide or the at least one B cell epitope peptide is linked to the N-terminus of the peptide carrier, or is linked to the alpha-helical peptide monomer, or any combination thereof.
  • the at least one B cell epitope peptide is linked along the length of the alpha-helical peptide domain via a non-terminal amino acid.
  • the at least one B cell epitope peptide is linked to the N-terminus, C-terminus, or along the length of the alpha-helical peptide domain via an amino acid.
  • the at least one B cell epitope peptide is linked to the alpha-helical peptide domain via an unnatural amino acid. In some aspects, the at least one B cell epitope peptide is linked to the alpha-helical peptide domain via a non-amino acid chemical functionality. In some aspects, the alpha-helical peptide domain comprises at least 1 heptad repeat. In some aspects, the alpha-helical peptide domain comprises at least 2 heptad repeats. In some aspects, the alpha-helical peptide domain comprises at least 3 heptad repeats.
  • the at least one T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the at least one CD4 T cell epitope peptides is selected from SEQ ID NO: 66 - SEQ ID NO: 182.
  • the at least one T cell epitope peptide comprises the amino acid sequence set forth in SEQ ID NO: 81 - SEQ ID NO: 87, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the at least one B cell epitope peptide is an immunogenic fragment of a microbial antigen.
  • the microbial antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a protozoan antigen. In some aspects, the microbial antigen is a conserved antigen within a divergent family of bacteria, fungi, or protozoan antigen.
  • the viral antigen is an influenza virus antigen. In some aspects, the influenza virus antigen is a hemagglutinin antigen, an M2 ectodomain antigen, a neuraminidase antigen, or a nucleoprotein antigen. In some aspects, the hemagglutinin antigen is a conserved influenza hemagglutinin Helix A epitope peptide.
  • influenza hemagglutinin Helix A epitope peptide comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 183 - SEQ ID NO: 191, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • influenza M2 ectodomain antigen comprises all or part of the amino acid sequence set forth in SEQ ID NO: 222 - SEQ ID NO: 227, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • influenza hemagglutinin antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 192 - SEQ ID NO: 207, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • influenza neuraminidase antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 208 - SEQ ID NO: 221, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the at least one B cell epitope peptide is an immunogenic peptide or a peptide fragment of a hormone antigen.
  • the hormone antigen is a GnRH antigen.
  • the GnRH antigen comprises the amino acid sequence set forth in SEQ ID NO: 263.
  • the at least one B cell epitope peptide is an immunogenic peptide fragment of a neurodegenerative disease antigen.
  • the neurodegenerative disease antigen is an Alzheimer’s disease antigen, a
  • the Alzheimer’s disease antigen is a Tau antigen or an amyloid beta (Ab) antigen.
  • the Tau antigen comprises the amino acid sequence set forth in SEQ ID NO: 262.
  • the amyloid beta (Ab) antigen comprises the amino acid sequence set forth in SEQ ID NO: 261.
  • the Parkinson’s disease antigen is an alpha-synuclein antigen.
  • the at least one B cell epitope peptide is an immunogenic peptide fragment of a tumor antigen.
  • the tumor antigen is derived from a member of the receptor tyrosine kinase family or a member of the human epidermal growth factor receptor family.
  • the at least one B cell epitope peptide is an immunogenic peptide fragment derived from an immunoglobulin E (IgE).
  • the IgE is human IgE.
  • the at least one B cell epitope peptide is an immunogenic peptide fragment derived from the Ce3 domain of human IgE.
  • the at least one B cell epitope peptide comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 264-267, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the at least one T cell epitope peptide is linked to the alpha-helical peptide monomer via a linker.
  • the at least one B cell epitope peptide is linked to the at least one T cell epitope peptide or the alpha-helical peptide domain via a linker.
  • the linker comprises a chain of amino acids, a synthetic linker, a PEG moiety, or a cleavable linker.
  • the chain of amino acids comprises 1-10 amino acids.
  • the linker comprises a serine, alanine, threonine, aspartic acid, lysine, glutamic acid, lysine, glutamine, asparagine, arginine, proline, tryptophan, or glycine linker, or a combination thereof.
  • a peptide vaccine herein comprises a peptide carrier with an amino acid sequence having at least 90%, 95%, 99%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 271 - SEQ ID NO: 294, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a peptide carrier comprising: (a) an alpha-helical peptide domain; (b) at least one T cell epitope peptide; and (c) at least one B cell epitope peptide, wherein the at least one T cell epitope peptide and the at least one B cell epitope peptide are linked in tandem via the C-terminus to the alpha-helical peptide monomer, and wherein the net surface charge of the alpha-helical peptide monomer matches the net surface charge of the B cell epitope peptide, which induces an electrostatic repulsion between the alpha- helical peptide monomer and the at least one B cell epitope peptide resulting in an improved vaccine performance.
  • the present disclosure provides a peptide carrier comprising: (a) an alpha-helical peptide domain; (b) at least one T cell epitope peptide; and (c) at least one B cell epitope peptide, wherein the at least one T cell epitope peptide and the alpha-helical peptide monomer are linked in tandem, and wherein the B cell epitope peptide is attached by solid phase synthesis at specific locations along the length of the alpha-helical peptide monomer using amino acid-linked building blocks resulting in an improved vaccine performance.
  • the present disclosure provides an immunogenic composition comprising at least one, at least two, or at least three of the peptide carriers described herein and a pharmaceutically acceptable carrier.
  • such immunogenic composition comprising the at least one, at least two, or at least three peptide carrier described herein is capable of inducing an immune response in a subject (e.g., a rodent or a human).
  • the present disclosure provides an immunogenic composition capable of self-assembling into polymeric, coiled-coil nanoparticles.
  • the size of the polymeric, coiled-coil nanoparticles ranges from about 2 nm to about 30 nm. In some aspects, the size of the polymeric, coiled-coil nanoparticles ranges from about 30 nm to about 100 nm. In some aspects, the size of the polymeric, coiled-coil nanoparticles ranges from about 100 nm to about 1 pm. In some aspects, the size of the polymeric, coiled-coil nanoparticles ranges from about 1 pm to about 10 pm. In some aspects, such immunogenic composition can further comprise an adjuvant.
  • the present disclosure provides a method for inducing an immune response in a subject specific for a target antigen comprising administering to the subject the immunogenic composition according to the present disclosure (e.g., comprising any of the peptide carriers with SEQ ID NOs: 271-306, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto).
  • the immunogenic composition according to the present disclosure e.g., comprising any of the peptide carriers with SEQ ID NOs: 271-306, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for inducing an immune response against a microbial antigen in a subject, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 183-227, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for inducing an immune response against most or all members of a diverged family of microbes in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 183-227, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the microbial antigen against which the immune response is induced is an influenza virus antigen.
  • the present disclosure provides a method for inducing an immune response against a Human papillomavirus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 229-231, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for inducing an immune response against a Herpes simplex virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 232-244, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for inducing an immune response against a Dengue virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 245-247, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for inducing an immune response against a Hepatitis C virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 248-258, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for inducing an immune response against a Respiratory syncytial virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 259-260, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for inducing an immune response against a hormone antigen in a subject, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising an epitope peptide with a sequence of SEQ ID NO: 263, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • hormone antigen can be a GnRH antigen.
  • the immune response to a GnRH antigen is used to inhibit sex hormone production in a host mammal.
  • the immune response to a GnRH antigen is used to inhibit sex hormone production in humans for treatment of cancer, hyperproliferative, and post-menopausal disorders.
  • the present disclosure provides a method for inducing an immune response against a neurodegenerative disease antigen in a subject, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising an epitope peptide capable of inducing an immune response to the neurodegenerative disease antigen in the subject.
  • the neurodegenerative disease antigen against which the immune response is induced is an Alzheimer’s disease antigen.
  • such peptide carrier comprises an Alzheimer’s disease antigen with an amino acid sequence set forth in SEQ ID NO: 261 or 262, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for treating a
  • neurodegenerative disease in a subject in need thereof, the method comprising administering a composition comprising an alpha-helical peptide carrier and at least one neurodegenerative disease antigen.
  • the neurodegenerative disease is Alzheimer’s disease
  • any of the methods described herein can comprise a composition comprising an alpha-helical peptide carrier comprising an alpha-helical peptide domain.
  • the peptide carrier can further comprise at least one T cell epitope peptide with a sequence set forth in any one of SEQ ID NOs: 66-182, or 300, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the present disclosure provides a method for treating an IgE-mediated hypersensitivity disorder in a subject in need thereof, the method comprising administering a composition comprising an alpha-helical peptide carrier and at least one IgE antigen.
  • the at least one IgE antigen is derived from human IgE.
  • the at least one IgE antigen is derived from the Ce3 domain of human IgE.
  • the at least one IgE antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 264 to SEQ ID NO: 267, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • the composition further comprises an alpha-helical peptide carrier described herein, e.g., those with a sequence set forth in SEQ ID NO: 1 - SEQ ID NO: 65 or SEQ ID NO: 295 - SEQ ID NO: 299, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • such peptide carrier further comprises at least one T cell epitope peptide with a sequence set forth in any one of SEQ ID NOs: SEQ ID NO: 66 - SEQ ID NO: 182, or SEQ ID NO: 300, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
  • an immunogenic peptide carrier monomer of the present disclosure comprising an alpha-helical domain comprising four heptad repeats (e.g., IKKIEKR, SEQ ID NO: 7) that is linked via its C-terminus and/or N-terminus to one or more CD4 T cell epitope (TCE) peptides (e.g., PADRE) in tandem.
  • TCE CD4 T cell epitope
  • the peptide carrier of the present disclosure can further comprise one or more B cell epitope (BCE) peptides linked to the N- terminus and/or C-terminus and/or main chain of the peptide carrier.
  • BCE B cell epitope
  • FIG. 2 illustrates the physical characterization of peptide carrier monomers having SEQ ID NO: 271 and comprising the alpha-helical domain with SEQ ID NO: 20 linked in series to a PADRE T cell epitope peptide (SEQ ID NO: 71) and a T cell epitope peptide from influenza H5N1 hemagglutinin (SEQ ID NO: 73) form stable trimeric coiled-coil complexes.
  • SEQ ID NO: 71 PADRE T cell epitope peptide
  • SEQ ID NO: 73 T cell epitope peptide from influenza H5N1 hemagglutinin
  • FIG. 2A illustrates circular dichroism (CD) spectra of the peptide carrier monomer having SEQ ID NO: 271 dissolved in PBS and subjected to increasing temperatures from 5 °C to 95 °C, followed by a return to 5 °C (grey line).
  • CD circular dichroism
  • FIG. 2B illustrates an analytical ultracentrifugation (AUC) spectrum of the alpha-helical peptide having SEQ ID NO: 271 showing monomer (0.76 S), trimer (1.78 S) and higher order assemblies (>3 S).
  • AUC analytical ultracentrifugation
  • FIG. 2C illustrates intensity-weighted dynamic light scattering (DLS) spectrum of the alpha-helical peptide having SEQ ID NO: 271 showing major self-assembly at 7 nm
  • FIG. 3 illustrates size analyses for the peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 275 and SEQ ID NO: 277.
  • FIG. 3A illustrates AUC analysis of peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 275.
  • FIG. 3B illustrates DLS analysis of peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 275.
  • FIG. 3C depicts the DLS analysis of peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 277.
  • FIG. 3D shows a transmission electron microscopy (TEM) image of self-assembled coiled-coil peptide carriers with SEQ ID NO: 277 showing circular assemblies of approximately 10-20 nanometers (nm) in size.
  • TEM transmission electron microscopy
  • FIG. 4 illustrates the comparison of the antibody responses to three peptide-based nicotine vaccines comprising either a single TCE or fusion peptide of two TCEs linked in tandem.
  • the nicotine hapten (average 4 nicotine haptens/peptide) was linked to the alpha-helical peptide domain of the peptide monomer via a linker that was covalently attached to the 1’ position of the nicotine molecule (see e.g., FIG. 9A).
  • Antibody responses to the nicotine hapten were induced by using peptide carriers with SEQ ID NO: 273, SEQ ID NO: 274, and SEQ ID NO: 272 comprising either a PADRE CD4 T cell epitope peptide (SEQ ID NO: 71), a Diphtheria CD4 T cell epitope peptide (SEQ ID NO: 72), or a fusion T cell epitope comprising both the Diphtheria and PADRE CD4 T cell epitopes (SEQ ID NO: 81).
  • Outbred CD-l mice were then injected with 5 pg of peptide carrier or PBS as a control on days 0, 21, and 42.
  • Antibody titers were determined by ELISA 56 days after administration of the peptide carrier. The asterisk indicates a significant difference between groups where PO.OOOl.
  • FIG. 5 illustrates predicted and empirical immune responses induced by a hapten containing a linker attached to the 3’ position of nicotine attached to peptide carriers with SEQ ID NO: 272, SEQ ID NO: 275, SEQ ID NO: 276, and SEQ ID NO: 277 comprising the lysine- rich heptad sequence with SEQ ID NO: 19 (5 heptad repeats) and the following combinations of CD4 T cell epitope peptides as shown in FIG. 2: Diphtheria + PADRE (SEQ ID NO: 81),
  • Measles V F2 + Hepatitis (Hep) B (SEQ ID NO: 83), tetanus toxoid + Influenza hemagglutinin (SEQ ID NO: 82), and tetanus toxoid (SEQ ID NO: 67).
  • FIG. 5A illustrates the number of predicted high affinity binding CD4 TCEs that can be generated from the four different peptide vaccines comprising peptide carriers with SEQ ID NO: 272, SEQ ID NO: 275, SEQ ID NO: 276, and SEQ ID NO: 277 following a search using the Immune Epitope Database (http://www.iedb.org/). This analysis predicts that the
  • Measles/Hepatitis B T cell epitope combination with SEQ ID NO: 83 may have the best activity across polymorphic animal and human populations in comparison to tetanus toxoid (947-967) (SEQ ID NO: 67), the Diphtheria toxin (332-346)/PADRE (SEQ ID NO: 81), or the tetanus toxoid/HA (307-319) (SEQ ID NO: 82) fusion epitope peptides.
  • FIG. 5B illustrates a graph of antibody titers induced by a nicotine hapten attached to the peptide carriers via the 3’ position of nicotine in outbred mice by the tested CD4 T cell epitope peptides.
  • Mice were injected with 4 pg of peptide carrier or PBS as a control on days 0, 21, and 42.
  • Antibody titers were determined by ELISA 56 days after administration of the peptide carrier. Animals immunized with the peptide carrier having SEQ ID NO: 275 expressed the best titer.
  • FIG. 5C shows a graph of antibody affinity induced by a nicotine hapten attached to the peptide carriers via the 3’ position of nicotine in outbred mice by the tested CD4 T cell epitope peptides in outbred mice by the tested CD4 T cell epitope peptides.
  • Mice were injected with 4 pg of peptide carrier or PBS as a control on days 0, 21, and 42.
  • Antibody titers were determined by ELISA 56 days after administration of the peptide carrier. Animals immunized with the peptide carrier having SEQ ID NO: 275 expressed the best affinity.
  • FIG. 6 illustrates that the activity of CD4 TCEs in a given species can be predicted, verified and enhanced by in vitro and in silico methods.
  • CD4 TCEs were chosen from pertinent publications and analyzed using the Immune Epitope Database and Analysis Resource (iedb.org) MHC Class II binding predictor to identify CD4 T cell epitopes of interest. This set of epitopes was further culled based on activity using a predictive ELISPOT-based in vitro assay.
  • PBMCs or splenocytes from a host species of interest were isolated and co-cultured with interleukin-2 and the desired T cell epitope for 10-14 days. These cultures were added to
  • T cell epitopes selected using this in vitro screen can be further combined as fusions of 2 or more TCEs in various orientations and tested for activity (e.g., using the Immune Epitope Database and Analysis Resource, in vitro ELISPOT methods, etc.). Combinations showing the best activity using this algorithm can be selected to build vaccines for in vivo testing. This shows that this class of peptide carrier vaccines can be rationally designed by ranking CD4 TCE activities through in silico and in vitro methods.
  • FIG. 7 shows that nicotine hapten incorporation by solid phase protein synthesis induces superior antibody responses relative to traditional conjugation technology (i.e.,“wet” synthesis).
  • FIG. 7A illustrates the structure of a hapten 6 nicotine-lysine building block (BB or 6HA) comprised of a nicotine hapten attached via the 6 position to the lysine sidechain that is used to incorporate nicotine haptens into a peptide carrier by solid-phase protein synthesis.
  • BB or 6HA hapten 6 nicotine-lysine building block
  • FIG. 7B illustrates the building block insertion points in the a-helical domain of the peptide carrier with SEQ ID NO: 275 during solid phase protein synthesis to yield SEQ ID NO: 275 x 1 BB (also referred to herein as“SEQ ID NO: 275 x 1/6HA”) with a single nicotine hapten-lysine insertion and SEQ ID NO: 275 x 3 BB (also referred to herein as“SEQ ID NO:
  • FIG. 7C illustrates that the antibody titer in the sera of mice immunized with the peptide having SEQ ID NO: 275 x 3 BB is superior to peptides containing 1 building block per monomer (SEQ ID NO: 275 x 1 BB) and also superior to a hapten peptide made using traditional (i.e., “wet”) conjugation technology (average 3 haptens/peptide) (SEQ ID NO: 275 wet).
  • FIG. 7D illustrates the antibody avidity in the sera of mice immunized with peptides containing 3 building blocks per monomer is superior to peptides containing 1 building block per monomer and also superior to a hapten peptide (i.e., SEQ ID NO: 275 wet) made using traditional (i.e.,“wet”) conjugation technology (average 3 haptens/peptide).
  • a hapten peptide i.e., SEQ ID NO: 275 wet
  • FIG. 7E illustrates the total nicotine binding capacity of antibodies in the sera of mice immunized with the peptide having SEQ ID NO: 275 x 3 BB is superior to peptides containing 1 building block per monomer (SEQ ID NO: 275 x 1 BB) and also superior to a hapten peptide (e.g., SEQ ID NO: 275 wet) made using traditional (i.e.,“wet”) conjugation technology (average 4 haptens/peptide).
  • a hapten peptide e.g., SEQ ID NO: 275 wet
  • FIG. 8 shows that inclusion of two nicotine-lysine building blocks generates antibody responses to both molecules.
  • FIG. 8A illustrates the placement of 6 nicotine haptemlysine building blocks along the peptide with SEQ ID NO: 275 following solid-phase peptide synthesis to yield a peptide having SEQ ID NO: 275 x 3/3’+3/6HA.
  • Three haptens contain a linker attached to the 3’ position of the nicotine molecule and the other three haptens contain a linker attached to the 6 position of nicotine.
  • FIG. 8B shows that this peptide induces antibody titers specific to both haptens.
  • mice were immunized with peptides synthesized with only the 3’ hapten (SEQ ID NO: 275 x 3/3’) or the 6HA hapten (SEQ ID NO: 275 x 3/6HA). Serum was collected on day 35 and assayed by ELISA using BSA conjugated with the 6HA hapten or the 3’ hapten as coating antigens.
  • the peptide with SEQ ID NO: 275 x 3/3’+3/6HA induced antibody titers that were equivalent to the positive control peptide with SEQ ID NO: 275 x 3/6HA and greater than the negative control SEQ ID NO: 275 x 3/3’.
  • the converse was also true when antisera was assayed in the 3’ hapten ELISA.
  • FIG. 9 shows the additive antibody responses achieved with multivalent peptide vaccines.
  • FIG. 9A illustrates three different hapten structures attached at the 1’, 3’, and 6’ position on nicotine molecule used for this analysis.
  • FIG. 9B illustrates the total nicotine binding capacity of antibodies in the sera of mice immunized with monovalent (3’-SEQ ID NO: 275), admixed bivalent (l’,3’-SEQ ID NO: 275), and admixed trivalent (l’,3’, 6’-SEQ ID NO: 275) nicotine peptide conjugate vaccines using nicotine haptens G, 3’, and 6’ attached to the peptide carrier peptide using“wet” chemistry.
  • the nicotine binding capacity of mice injected with a trivalent vaccine was highest, showing the utility of multivalent peptide formulations.
  • FIG. 10 illustrates the design and protective functionality of the peptide carrier with SEQ ID NO: 278 comprising a conserved influenza helix A epitope peptide (SEQ ID NO: 183) at its N-terminus.
  • the self-assembling peptide-based influenza vaccine particles that were used in this study comprised peptide monomers comprising 4 lysine-rich heptad sequences in the coiled-coil domain (SEQ ID NO: 18), HBV and measles-derived TCE (SEQ ID NO: 83) linked to its C- terminus, and the conserved influenza helix A epitope peptide (SEQ ID NO: 183) linked to the N-terminus of the alpha-helical peptide domain.
  • FIG. 10A shows the alpha helix A epitope sequence expressed by H1N1 viruses as compared to the alpha helix A epitope expressed by drifted H7N4, H3N2, H5N1 and Type B influenza viruses to show amino acid conservation.
  • the bold underlined amino acids identify residues within viral hemagglutinin helix A that are outward-facing and recognized by broadly neutralizing monoclonal antibodies, such as monoclonal antibody (mAh) CR9114.
  • the conserved H1N1 influenza helix A epitope peptide (SEQ ID NO: 183) that was used in this study contained all of the underlined amino acid residues that can be recognized by broadly neutralizing monoclonal antibodies (see, e.g., TABLE 3).
  • the position of the epitope within the HA2 domain of HA is denoted by the residue numbers shown above the H1N1 sequence. conserveed residues are indicated by a dash.
  • FIG. 10B shows a model confirming that the influenza hemagglutinin helix A epitope maintains its helical secondary structure following its attachment in register to the alpha-helical N-terminus of the monomer peptide with SEQ ID NO: 278.
  • the neutralizing residues (shown as blue spheres in the top and side view) are outward facing antibody contact residues, which mimics the helix A orientation in native hemagglutinin.
  • FIG. 10C shows the DLS analysis of the peptide construct with SEQ ID NO: 278.
  • FIG. 10D illustrates the antibody titers 35 days after mice were immunized on days 0 and 21 with the H1N1 virus-derived helix A epitope peptide vaccine having SEQ ID NO: 278. These antibodies recognized Hl hemagglutinin and showed cross-reactivity to drifted Helix A epitopes in H7, H3, and H5 hemagglutinin.
  • FIG. 10E shows that the peptide construct with SEQ ID NO: 278 induces strong CD4+ T cell activity.
  • FIG. 10F shows antisera from d35 were assayed by ELISA for titers to recombinant Hl. Titers of total IgG, IgGl, and IgG2a are indicated.
  • FIG. 10G shows the ratio between IgG2a and IgGl isotypes. Values greater than 1 are indicative of a Thl response, which is induced by the TLR4 agonist GLA.
  • FIG. 10H shows the survival of mice immunized with PBS (naive) or with the H1N1 virus-derived Helix A epitope peptide vaccine (SEQ ID NO: 278) after challenge with H1N1 influenza virus.
  • Mice were monitored for survival with a weight loss cutoff of 80%.
  • Mice were challenged on day 40 with 10 times the LD50 concentration (also denoted as IOLD50) of the influenza virus subtype H1N1 (A/California/07/2009) and were monitored for survival with a weight loss cutoff of 75%.
  • IOLD50 also denoted as IOLD50
  • a significant difference in survival 80% survival vs. 0% at day 14 was observed for those animals that received the peptide vaccine (i.e., SEQ ID NO: 278).
  • FIG. 11 illustrates that matching the electrostatic charge of the alpha-helical domain of the peptide carrier with the BCE improves vaccine performance, due to electrostatic repulsion between the alpha-helical domain and the BCE and a resulting decrease in steric hindrance of the BCE interactions with, e.g., antibodies. It shows the antibody titer from animals immunized with a peptide-based influenza vaccine as disclosed herein wherein the peptide monomers with SEQ ID NO: 279 and SEQ ID NO: 280 (see e.g., FIG.
  • M2ectodomain (M2e) epitope SEQ ID NO: 94
  • a peptide carrier comprising either a glutamic acid-rich alpha-helical peptide domain (SEQ ID NO: 280) or a lysine-rich (SEQ ID NO: 279) alpha-helical peptide domain followed by the tetanus toxoid TCE (SEQ ID NO: 67).
  • Mice were immunized with 10 pg of peptide carrier on days 0 and 21.
  • FIG. 11A illustrates the amino acid sequences and net surface charge distribution of peptide carrier with SEQ ID NO: 279 and SEQ ID NO: 280.
  • FIG. 11B shows antibody titers against the M2 ectodomain epitope 35 days after outbred CD-l mice were injected with PBS control or 10 pg of peptide carrier on days 0 and 21. Titers were significantly higher in animals that were injected with a peptide-vaccine comprising peptide monomers with identical charges on the surfaces of the peptide carrier and BCE (SEQ ID NO: 280), compared to mice that were injected with peptide-vaccine comprising peptide monomers with opposite charges on the surfaces of the peptide carrier and BCE (SEQ ID NO: 279).
  • SEQ ID NO: 280 a peptide-vaccine comprising peptide monomers with identical charges on the surfaces of the peptide carrier and BCE
  • 11B illustrates that the peptide-based influenza vaccine comprising SEQ ID NO: 148 comprising the M2 ectodomain epitope peptide elicits strong antibody responses against the M2 ectodomain epitope, and that mice that were immunized with this peptide-based influenza vaccine were protected against influenza infections compared to control cohort.
  • FIG. 11C shows the survival curves of control (PBS) and peptide carriers with SEQ ID NO: 280 immunized outbred CD-l mice following virus challenge. Mice were monitored for survival with a weight loss cutoff of 80%.
  • FIG. 12 illustrates that peptide carrier with SEQ ID NO: 282 that contains two M2e BCEs (SEQ ID NO: 222) attached along the length of the alpha-helical coiled-coil peptide domain (SEQ ID NO: 26) that were incorporated during solid phase protein synthesis can induce a strong antibody response.
  • FIG. 12A illustrates the attachment locations of 2 M2e BCEs (SEQ ID NO: 222) to an alpha-helical coiled-coil peptide monomer (SEQ ID NO: 26) to yield peptide carrier SEQ ID NO: 282.
  • FIG. 12B illustrates the antibody titers measured 35 days after outbred CD-l mice were injected with PBS control or 10 pg of the peptide carrier (SEQ ID NO: 282) on days 0 and 21.
  • FIG. 13 illustrates that the peptide carrier can be used for BCE discovery by empirically testing the activity of novel linear B cell epitopes identified using protein structural and sequence information.
  • FIG. 13A shows a representative X-ray diffraction crystal structure of the B/Hong Kong/8/l973 hemagglutinin (Wang, Q et al. Crystal structure of unliganded influenza B virus hemagglutinin. 2008 J. Virol. 82:3011-3020).
  • Two surface BCEs available for antibody binding are denoted (circles with cross-hatching denotes HA27-39BCE (SEQ ID NO: 201) and circles with diagonal lines denotes HA231-241 BCE (SEQ ID NO: 207)).
  • the sequence homology of hemagglutinin proteins for two influenza B and two influenza A viruses has been demonstrated (see e.g., Terajima M et al.
  • FIG. 13B illustrates that vaccination with peptides containing novel influenza epitopes HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284) leads to antibodies that can recognize recombinant hemagglutinin.
  • mice were bled and titers characterized by ELISA.
  • Antibody recognition of a peptide-BSA conjugate is compared to recognition of recombinant influenza B hemagglutinin (rHA(B) from B/Malaysia/2506/04).
  • FIG. 13C shows that mice immunized with peptides containing novel influenza epitopes HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284) are protected from mortality against lethal viral challenge.
  • mice were challenged with 5xLD50 of B/Florida/04/06 and monitored for survival with a weight loss cutoff of 80%. Immunized mice were partially (HA231- 24i , SEQ ID NO: 284) or fully (HA27-39 , SEQ ID NO: 283) protected against virus challenge.
  • FIG. 13D shows that mice immunized with peptides containing novel influenza epitopes HA 27-39 (SEQ ID NO: 283) or HA 231-241 (SEQ ID NO: 284) experience minimal weight loss following lethal viral challenge. This experiment confirms that these novel influenza epitopes can be identified using in silico methods and incorporated into the peptide to generate protective immune responses to native influenza proteins.
  • FIG. 14 illustrates that these peptide carriers can induce protective antiviral responses to a variety of influenza BCEs, including M2e from IAV (SEQ ID NO: 222), NA1 (SEQ ID NO: 208), NA2 (SEQ ID NO: 221), HxA from H1N1 (SEQ ID NO: 183), M2e from IBV (SEQ ID NO: 224), HxA from IBV (SEQ ID NO: 190), HA27-39 (SEQ ID NO: 201) or HA 23 1-241 (SEQ ID NO: 207).
  • M2e from IAV SEQ ID NO: 222
  • NA1 SEQ ID NO: 208
  • NA2 SEQ ID NO: 221
  • HxA from H1N1
  • M2e from IBV SEQ ID NO: 224
  • HxA from IBV SEQ ID NO: 190
  • HA27-39 SEQ ID NO: 201
  • HA 23 1-241 SEQ ID NO: 207
  • Peptides were made with two copies of M2e from IAV (SEQ ID NO: 282), NA1 (SEQ ID NO: 285), NA2 (SEQ ID NO: 286), M2e from IBV (SEQ ID NO: 287), HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284). Peptides were also synthesized with a single N-terminus copy of either the H1N1 HxA BCE (SEQ ID NO: 278) or the influenza B HxA BCE (SEQ ID NO: 288).
  • Female CD-l mice were immunized with one of the peptides (10 pg) adjuvanted with GLA-SE on day 0 and day 21. On day 40, mice were challenged with 5xLD50 of
  • FIG. 15 illustrates that antiviral responses can be broadened and enhanced by targeting multiple surface epitopes using multivalent vaccines.
  • FIG. 15A shows that a trivalent, peptide-based influenza vaccine yields improved protection compared to monovalent and bivalent peptide-based influenza vaccines.
  • M2e SEQ ID NO: 222
  • NA SEQ ID NO: 208
  • HxA SEQ ID NO: 183
  • BCEs were used to make peptides with two copies of M2e (SEQ ID NO: 282) or NA (SEQ ID NO: 285), or a single N- terminus copy of HxA (SEQ ID NO: 278).
  • mice Female CD-l mice were immunized with either M2e, NA, or HxA peptides singly (10 pg) or admixtures of M2e+NA or M2e+NA+HxA peptides (10 pg each peptide) adjuvanted with GLA-SE on day 0 and day 21. On day 40, mice were challenged with 5xLD50 of A/California/07/2009(HlNl) and monitored for survival with a weight loss cutoff of 80%. The monovalent vaccines led to partial survival while the multivalent vaccines led to complete survival against the virus.
  • FIG. 15B illustrates that the trivalent vaccine minimized least weight loss.
  • FIG. 16 illustrates the full length human Tau protein (2N4R, 441 amino acids) and 5 isoforms (1N4R, 0N4R, 2N3R, 1N3R, and 0N3R) produced in the central nervous system by alternative splicing.
  • the repeat (R) domain contains two sequences that allow aggregation of the Tau protein and that flank the regulatory domain 294 KDNIKHVPGGGS 305 (SEQ ID NO: 262) that) which can be used in combination with the compositions and methods of the present disclosure.
  • FIG. 17 illustrates DLS analyses of four particles comprising peptide carrier monomers with SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, and SEQ ID NO: 292 comprising either a Tau epitope peptide (SEQ ID NO: 262) or an amyloid-beta epitope peptide (SEQ ID NO: 261) that is linked to the alpha-helical peptide carrier via the N- or C-terminus, respectively, as described below.
  • SEQ ID NO: 262 Tau epitope peptide
  • SEQ ID NO: 261 amyloid-beta epitope peptide
  • FIG. 17A illustrates the DLS analysis for the alpha-helical peptide with the Tau epitope peptide linked to its N-terminus (SEQ ID NO: 289).
  • FIG. 17B illustrates the DLS analysis for the alpha-helical peptide with the Tau epitope peptide linked to its C-terminus (SEQ ID NO: 290).
  • FIG. 17C illustrates the DLS analysis for the alpha-helical peptide with the amyloid-beta epitope peptide linked to its N-terminus (SEQ ID NO: 291).
  • FIG. 17D illustrates the DLS analysis for the alpha-helical peptide with the amyloid-beta epitope peptide linked to its C-terminus (SEQ ID NO: 292).
  • FIG. 18 illustrates that peptide carriers with SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, and SEQ ID NO: 292 comprising the Tau epitope peptide (SEQ ID NO: 262) and the amyloid-beta epitope peptide (SEQ ID NO: 261) induce comparable antibody responses when the BCE is linked to the peptide carrier via the N- or C-terminus.
  • FIG. 18A shows the measured antibody titers for the alpha-helical peptide with N- terminal Tau epitope peptide (SEQ ID NO: 289).
  • FIG. 18B shows the measured antibody titers for the alpha-helical peptide with C- terminal Tau epitope peptide (SEQ ID NO: 290).
  • FIG. 18C shows the measured antibody titers for the alpha-helical peptide with N- terminal amyloid-beta epitope peptide (SEQ ID NO: 291).
  • FIG. 18D shows the measured antibody titers for the alpha-helical peptide with C- terminal amyloid-beta epitope peptide (SEQ ID NO: 292).
  • FIG. 19 illustrates the physiological responses in male mice immunized with the alpha- helical peptide-based vaccine having SEQ ID NO: 293 and comprising a GnRH B cell epitope peptide (SEQ ID NO: 263) at the C-terminus of the carrier peptide with SEQ ID NO: 275.
  • CD-l male mice (5/group) were immunized (day 0, day 21, day 42) with PBS or 10 pg of the peptide vaccine formulated in GLA-SE adjuvant and then monitored for the following activities.
  • FIG. 19A shows the anti-GnRH antibody response following administration of the peptide carrier having the sequence set forth in SEQ ID NO: 293. Titers were determined by ELISA using plates coated with a GnRH-BSA conjugate.
  • FIG. 19B shows the number of pregnancies and embryos procreated by male mice that either received PBS (control) or peptide vaccine having SEQ ID NO: 293.
  • Male mice from both groups were housed with 4 female mice between day 40 through day 80, and the number of pregnancies and resulting embryos were counted.
  • FIG. 19C shows the testosterone production in immunized mice, which was assayed by ELISA using day 63 serum.
  • FIG. 19D shows testis weights of immunized mice, which were measured on day 100.
  • FIG. 20 illustrates testis architecture in PBS control and mice immunized with a peptide vaccine having SEQ ID NO: 293.
  • FIG. 20A shows Hematoxylin and eosin stained cross-section from PBS-immunized mice.
  • Normal tissue architecture includes interstitial Leydig cells surrounding tubules which contain subcapsular Sertoli cells and orderly arranged layers of maturing germ cells and spermatozoa.
  • FIG. 20B shows representative cross-section from immunized mice using the peptide vaccine with SEQ ID NO: 293. Presumptive interstitial Leydig cells appear clustered with fragmented nuclei. Tubules lined with Sertoli cells are significantly reduced in size, contain degenerating germ cells and are devoid of spermatids and spermatozoa.
  • FIG. 21 shows the measured antibody titers, antibody affinity, and antibody specificity for the polypeptide with SEQ ID NO: 294 and comprising a murine-specific C-terminal Peptide Y epitope (SEQ ID NO: 264) corresponding to the Cs3 domain of human immunoglobulin E (IgE). This is the same epitope recognized by the commercial anti-IgE monoclonal, XolairTM
  • FIG. 21A shows the antibody titers elicited by the peptide having SEQ ID NO: 294 measured by ELISA using plates coated with IgE.
  • FIG. 21B shows that the anti-IgE antibodies in d35 immunized mouse sera contains nanomolar affinities, as measured by a competition ELISA assay using IgE.
  • FIG. 21C shows the results of a competition ELISA where antibodies induced in d35 mouse sera specifically bound IgE and not IgG and IgM immunoglobulin proteins.
  • FIG. 22 shows the antibody titers measured by ELISA using plates coated with IgE that were generated using the peptide carrier vaccine with the sequence set forth in SEQ ID NO: 294 comprising the murine Ce3 sequence with SEQ ID NO: 302.
  • the different symbols used in A, C, and D represent antisera from individual animals in each group.
  • FIG. 22A shows that the anti-IgE antibodies described in FIG. 21 were able to reduce the concentration of free IgE ⁇ l0-fold in 6 of 10 mice and ⁇ l 000-fold in 4 of 10 mice.
  • FIG. 22B shows that the peptide carrier with SEQ ID NO: 294 could also be used to inhibit acute IgE-mediated anaphylaxis, confirming its therapeutic function.
  • FIG. 22C shows that the peptide vaccine having the amino acid sequence set forth in SEQ ID NO: 294 forms nanoparticles with an average DLS diameter of -15 nm.
  • Vaccines are among the most effective interventions in modem medicine and have resulted in the eradication of disease worldwide. For example, smallpox claimed over 375 million lives in the 20 th century alone. Since the development of effective and widely available smallpox vaccines, not a single person has died from smallpox since 1978. Today, more than 70 vaccine medications are available against approximately 30 microbes. Despite their success, current vaccine approaches have outstanding issues and barriers. For example, currently available vaccines are frequently used in less responsive or immunocompromised populations reducing their overall efficacy and response rate. New approaches, including advanced delivery systems, may help to improve the outcome in those populations. A major challenge for combating infectious disease is the high mutation rate and antigenic variation of pathogenic microbes.
  • HLA Class I- and Class Il-presented peptides that trigger adaptive immune responses are termed T-cell epitopes (TCEs), and their use is required for the development of epitope-based vaccines and immunotherapies against infections, tumors, and autoimmune diseases.
  • TCEs T-cell epitopes
  • HLA genes exhibit a high level of polymorphism (>10,000 alleles identified to date), and their distribution in the human population varies with ethnicity and region. This extreme
  • polymorphism represents a natural barrier for the development of peptide-based vaccines, which would require hundreds or thousands of peptides to maximize population coverage.
  • HLA alleles may share similar binding specificities, which permits their grouping into supertypes, each of which bind a repertoire of related peptides.
  • promiscuous or universal CD4 T-cell epitopes as disclosed herein that bind various HLA supertypes may have great potential in the development of vaccines with wide population coverage.
  • a number of databases such as SYFPEITHI, MHCBN, AntiJen, IEDB, and HLAsupE have been constructed for identifying T cell epitopes based on binding predictions to common and rare MHC II alleles (Schuler et al. SYFPEITHI: database for searching and T-cell epitope prediction. Methods Mol Biol. 2007;409:75-93; Lata et al. MHCBN 4.0: A database of MHC/TAP binding peptides and T-cell epitopes.
  • the synthetic peptide vaccines contemplated herein comprising effective promiscuous TCEs may be candidates for next-generation vaccines to address the above-mentioned drawbacks of currently marketed vaccines.
  • the synthetic peptide vaccines of the present disclosure can contain solely the antigen or fragments thereof to exert an immune response.
  • Peptide-based vaccines of the present disclosure can be produced in whole or at least in part using automated peptide synthesizers, viruses, or cell-based production systems.
  • the antigens either in whole or in part as specific epitope peptides can be customized to elicit immune responses against a myriad of different antigens enabling their use in a wide variety of disease areas.
  • peptide epitope sequences that are highly conserved within divergent strains of bacteria and viruses, which in turn, alleviates the major road block for creating broader protection against any given pathogen family.
  • a vaccine containing a peptide carrier as disclosed herein may target a single epitope, or alternatively, be manufactured as a multivalent vaccine for inducing antibodies against multiple epitope targets.
  • the synthetic peptide-based vaccines of the present disclosure can be comprised of nanoparticles that are formed from a plurality of monomeric peptide carrier by, for example, self-assembly to mono-, di-, tri-, tetrameric or higher order assemblies, i.e., self-assembled peptide nanoparticle (SAPN)-based vaccines (see e.g., FIG. 2, FIG. 3, FIG. 17).
  • SAPN self-assembled peptide nanoparticle
  • the SAPN-based vaccines of the present disclosure can comprise a peptide carrier to enhance the immunogenicity of otherwise weak antigens.
  • the peptide carrier monomers of the present disclosure can comprise several peptide domains, including an alpha-helical coiled-coil domain comprising an alpha-helical coiled-coil peptide, T cell epitope (TCE) domain comprising one or more CD4 TCE peptides, and B cell epitope (BCE) domains comprising one or more BCE peptides.
  • TCE T cell epitope
  • BCE B cell epitope
  • a TCE peptide or a BCE peptide can be linked to the N-, C-, or both termini, or along the length of the alpha-helical coiled-coil peptide monomer.
  • peptide-based vaccines including, but not limited to those comprising a peptide carrier that can elicit antibody responses in a mammal against viral pathogens like influenza, HPV, HSV, DENV, HCV, and RSV, as well as induce immune responses to host proteins, such as those involved with Alzheimer’s disease-related proteins, IgE- mediated allergies, cancer, as well as neuropeptides involved in fertility and the development and maintenance of sexual organs.
  • HA-fp highly conserved influenza A and B virus hemagglutinin fusion peptide
  • HxA hemagglutinin helix A epitope
  • NA neuraminidase peptides
  • M2e matrix 2 ectodomain epitope peptid
  • Host protein targets for treating various indications and conditions include Tau protein derived epitope peptides, and amyloid-beta (Ab) protein derived epitope peptides, IgE derived epitope peptides, cancer-related epitopes and gonadotropin-releasing hormone (GnRH) derived epitope peptide.
  • Tau protein derived epitope peptides and amyloid-beta (Ab) protein derived epitope peptides
  • IgE derived epitope peptides IgE derived epitope peptides
  • cancer-related epitopes cancer-related epitopes
  • GnRH gonadotropin-releasing hormone
  • L-enantiomeric amino acids are conventional and are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn);
  • Xaa can indicate any amino acid.
  • X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).
  • the term“antigen” or“Ag,” and its grammatical equivalents as used herein, can refer to a molecule that provokes the immune response. This immune response can involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • the skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
  • the term“immunoglobulin” or“Ig,” as used herein, can refer to a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the chimeric antigen receptor or antigen receptor.
  • IgA immunoglobulin
  • IgG is the most common circulating antibody. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses.
  • percent (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • the % sequence identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence.
  • the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
  • T cell epitope includes a plurality of T cell epitopes, including combinations thereof.
  • treatment includes the therapeutic and/or prophylactic treatment of a condition or disorder.
  • target antigen includes any antigen that can elicit an immune response.
  • Target antigens can include haptens, T cell epitope peptides, or B cell epitope peptides.
  • Polypeptides of the present disclosure can comprise at least two distinct domains, e.g., an alpha-helical coiled-coil domain and at least one epitope domain.
  • the polypeptides of the present disclosure may be referred to as“peptide carriers” or“alpha-helical peptide carriers.”
  • Peptide carriers of the present disclosure can be single chain peptide monomers, and the at least one epitope domain can be linked to a coiled-coil domain (e.g., an alpha-helical peptide) via the N-, C-, or both termini, or along the length of the coiled-coil domain.
  • the coiled-coil domain of a peptide carrier can comprise an alpha-helical peptide monomer.
  • An epitope domain of a peptide carrier as disclosed herein can comprise at least one epitope peptide (e.g., one or more TCEs or one or more BCEs or any combination thereof).
  • a peptide carrier may contain additional (e.g., covalently bound) components, such as tags, linkers, markers, haptens, or bioactive molecules.
  • the peptide carrier of the present disclosure can comprise epitope peptides that are linked (e.g., covalently linked) to the alpha-helical peptide monomer.
  • the alpha-helical peptide monomer can be linked to one or more epitope peptides, such as one or more T cell epitope (TCE) peptides or one or more B cell epitope (BCE) peptides, or any combination thereof.
  • TCE T cell epitope
  • BCE B cell epitope
  • the one or more BCE and/or TCE peptides can be linked in tandem to the alpha-helical peptide via the N-terminus.
  • the one or more BCE and/or TCE peptides can be linked in tandem to the alpha- helical peptide via the C-terminus. In some embodiments, the one or more BCE and/or TCE peptides can be linked in tandem to the alpha-helical peptide via both the N-terminus and C- terminus. In some embodiments, the one more BCE and/or TCE peptides are linked to the peptide carrier via an amino acid residue side chain along the length of the alpha-helical peptide domain. In some embodiments, the one or more BCE and/or TCE peptides can be linked to the peptide carrier via both termini and along the length of the alpha-helical peptide domain.
  • a peptide carrier comprises two epitope domains each comprising at least one epitope peptide (e.g., one or more BCEs and/or TCEs), wherein one epitope domain is linked to the N-terminus of the alpha-helical peptide monomer, and one epitope domain is linked to the C-terminus of the same alpha-helical peptide monomer (FIG. 1).
  • epitope peptide e.g., one or more BCEs and/or TCEs
  • the one or more BCE epitope peptides are linked directly to the alpha-helical peptide monomer. In other embodiments, the one or more BCE epitope peptides are directly linked to a TCE peptide epitope, which can be directly linked to the alpha-helical peptide monomer. In some embodiments, the one or more TCE epitope peptides are linked directly to the alpha-helical peptide monomer. In other embodiments, the one or more TCE epitope peptides are directly linked to a BCE peptide epitope, which is directly linked to the alpha-helical peptide monomer.
  • the peptide carrier of the present disclosure comprises an epitope domain comprising two or more repeats of the same TCE linked in tandem to the alpha-helical peptide monomer at the N-terminus, C-terminus, both termini, along the length of the alpha- helical domain or any combination thereof.
  • the peptide carrier of the present disclosure comprise an epitope domain comprising two or more different TCEs (e.g., PADRE, Diphtheria toxin) linked in tandem to the N-, C-, both termini, along the length of the alpha-helical domain or any combination thereof.
  • the peptide carrier of the present disclosure comprise an epitope domain comprising one or more TCE peptide epitopes with the same or different amino acid sequences and one more BCE with the same or different amino acid sequences or any combination thereof that are linked individually or in tandem to the N-, C-, both termini, along the length of the alpha-helical peptide domain or any combination thereof.
  • the peptide carrier monomers of the present disclosure comprising a linear, alpha-helical coiled-coil peptide domain are capable of forming oligomeric complexes comprising two, three, four, or more peptide carrier monomers (See e.g., Grigoryan et al. Structural specificity in coiled- coil interactions. Curr Opin Struct Biol. 2008 Aug; l8(4):477-83).
  • the peptide carrier monomers assemble in a coiled-coil structure, with peptide monomers forming parallel bundles and each monomer having the same helical orientation.
  • the peptide carrier monomers can assemble in an antiparallel coiled-coil complex, wherein the peptide carrier monomers have an antiparallel helical orientation.
  • a peptide carrier as described herein may be further modified at the termini (i.e., the N- and/or C-terminus) and/or the amino acids along the polypeptide chain.
  • the N-terminus of the peptide carrier may be modified by acetylation.
  • the C-terminus of the peptide carrier may be modified by amidation.
  • a terminal modification may increase the stability of the peptide carrier.
  • a terminal modification may increase the biological activity of the peptide carrier.
  • acetylation of the N-terminus of a peptide carrier may be formulated as“Ac” at the N-terminus of the peptide carrier.
  • acetylation of a terminal lysine-rich heptad repeat e.g., IKKIEKR, SEQ ID NO: 7
  • the N-terminus of an alpha-helical peptide can comprise an aspartate residue (“D”).
  • the alpha-helical peptide monomers of the present disclosure can comprise an“alpha- helical coiled-coil domain.”
  • the alpha-helical coiled-coil domain as described herein can comprise two or more repeats of a heptad sequence (i.e.,“heptad” or“heptad repeat”) in tandem (FIG. 1).
  • the heptad sequences may trigger the formation of multimeric coiled-coil complexes by forming non-covalent (e.g., ionic, van der Waals, or hydrogen) bonds between heptad sequences of two or more alpha-helical peptide monomers.
  • the heptad sequences can be lysine-rich heptad sequences, wherein multiple heptad sequences can be linked in tandem (e.g., SEQ ID NO: 26 - SEQ ID NO: 35).
  • the heptad sequences can be glutamic acid-rich heptad sequence, wherein multiple heptad sequences can be linked in tandem (e.g., SEQ ID NO: 36 - SEQ ID NO: 47).
  • the alpha-helical peptides can comprise heptad repeats with an amino acid sequence according to the general formula:
  • X 1 and X 4 are each independently selected from I, L, V, A, F, Y, W, N, and Q;
  • X 2 , X 3 , X 6 are independently selected from the amino acids K, R, E, D, H, S, N, Q, T, C and A;
  • X 5 , X 7 are independently selected from the amino acids K, R, E, D, and H; and
  • n is any number greater than or equal to 1, preferably a number from 1 to 20, most preferably from 1 to 10.
  • the alpha-helical peptides can comprise an amino acid sequence according to any one of the following general formulas:
  • each Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , and Y 7 is independently selected from the amino acids I, L, V, A, K, R, E, S, T, P, G, C, N, Q, W, and D;
  • each X 5 , X 7 are independently selected from the amino acids K, R, E, D, H; and each n is independently any number greater than or equal to 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, etc.).
  • each heptad sequence can have hydrophobic isoleucines in the X 1 and X 4 position to form a hydrophobic core, the lysine residues at the X 2 , X 3 , and X 6 positions can then become solvent exposed, and the glutamic acid and arginine in the X 5 and X 7 positions can form salt bridges between adjacent helices that stabilize the coiled-coil structure.
  • the heptad sequence can create a dense array of surface exposed lysines along the carrier that can be used for hapten (e.g., nicotine derivative) conjugation.
  • the heptad repeats form an alpha-helical secondary structure that can form a coiled coil structural motif.
  • the surface of the alpha-helix has mainly hydrophilic amino acids and the opposite face has mainly hydrophobic or lipophilic amino acids.
  • the amino acid sequence of the peptide carrier alternates between hydrophilic and hydrophobic residues every 3 to 4 residues, with an alpha-helical turn every 3.6 residues.
  • the peptide carrier of the present disclosure can comprise at least two heptad repeats in tandem (e.g., ([IKKIEKR]2, SEQ ID NO: 16).
  • a peptide carrier can comprise at least three heptad repeats in tandem (e.g., [IKKIEKR]3, SEQ ID NO: 17).
  • a peptide carrier can comprise at least four heptad repeats in tandem (e.g.,
  • a peptide carrier can comprise at least five heptad repeats in tandem (e.g., [IKKIEKR] 5, SEQ ID NO: 19).
  • a peptide carrier can comprise incomplete heptad repeat sequences at the N- or C-terminus, or at both termini (see, e.g, SEQ ID NO: 20 - SEQ ID NO: 25).
  • a peptide carrier may comprise three or four full heptad repeats of n KIEKR (SEQ ID NO: 7) in tandem, and split a respective fourth or fifth heptad repeat sequence between the N-terminus and C-terminus while still preserving the continuity of the repeating heptad pattern.
  • a peptide carrier can have a portion of the heptad repeat denoted by g 2 g 3 g 4 g 5 g 6 g 7 (i e, KKTEKR (SEQ ID NO: 307)) at the N-terminus and a portion of the heptad repeat denoted by Y 1 (i.e., I) at the C-terminus.
  • a variety of N- and C-terminal sequences may be constructed by varying the length of the portion of the heptad repeat sequence used at each terminus from 1 to 7 amino acids.
  • the alpha- helical domain of a peptide carrier may comprise an amino acid sequence selected from:
  • TABLE 1 shows the amino acid sequences of various representative heptad sequences and the sequences of exemplary full-length alpha-helical peptide domains.
  • the polypeptide sequences comprise amino acid linker(s) at the C-, N-, or both termini.
  • an alpha-helical carrier contemplated herein can comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one or more of SEQ ID NO: 1 - SEQ ID NO: 65 or SEQ ID NO: 295 - SEQ ID NO: 299, or a functional fragment thereof.
  • a peptide carrier consists of, consists essentially of, or comprises a lysine-rich, an arginine-rich, a glutamic acid-rich, an aspartic acid-rich, a serine-rich, or a threonine-rich alpha-helical domain.
  • a peptide carrier consists of, consists essentially of, or comprises an alpha-helical domain with a combination of Lys and/or Arg and/or Glu and/or Asp and/or Ser and/or Thr residues interspersed with each other (e.g., in a pattern or randomly) in outward-facing heptad positions (see e.g., SEQ ID NO: 10, SEQ ID NO: 12 - SEQ ID NO: 14, SEQ ID NO: 60 - SEQ ID NO: 65).
  • Such alpha-helical domain can consist of, consist essentially of, or comprise an alpha-helical domain with the amino acid sequence set forth in any one of SEQ ID NO: 296 - SEQ ID NO: 299, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.
  • Such alpha-helical peptides can allow two or more peptide carriers to form multimeric structures, such as homodimers, homotrimers, etc.
  • Such higher order structures can be nanoparticles.
  • the size and/or structure of such particles can be determined using, e.g., dynamic light scattering (DLS) as described in EXAMPLE 2.
  • Peptide carriers provided herein can comprise an alpha-helical domain that can be linked or coupled to an epitope (e.g., a BCE or TCE) peptide directly (e.g., directly coupled to a heptad repeat) or indirectly.
  • An alpha-helical domain can be indirectly coupled to an epitope peptide via a linker.
  • Linkers can be used to change the physical properties and function of the peptide carrier. These changes include, but are not limited to, improved solubility, stability, B cell epitope presentation, and introduction of protease sites.
  • the linker is composed of residues bearing the same charge as the prevailing charge on the heptad repeats.
  • the linker is composed of residues to improve B cell epitope presentation through flexibility and/or rigidity.
  • the linker is an amino acid linker comprising 1 to 3 amino acids, 1 to 5 amino acids, or 5 to 10 amino acids, or greater than 10 amino acids.
  • the amino acids used herein as linker moieties can include serine (S), alanine (A), threonine (T), aspartic acid (D), lysine (K), glutamic acid (E), glutamine (Q), asparagine (N), arginine (R), proline (P), tryptophan (W), or glycine (G) (e.g., P, G, GGP, PGG, WWP, or WP linkers).
  • S serine
  • A alanine
  • T threonine
  • D aspartic acid
  • K glutamic acid
  • E glutamine
  • Q asparagine
  • N arginine
  • R proline
  • P tryptophan
  • W glycine
  • Proline and tryptophan are reported to terminate the extension of a-helical domains within proteins. Tryptophan may also be used in small peptides to quantitate peptide concentration by UV spectrometry.
  • Glycine, alanine and serine are used to increase the length and flexibility between the rigid a-helical domain and the T- and B-cell epitope domains.
  • Serine, threonine, aspartic acid, arginine, glutamic acid, lysine, glutamine, asparagine can be used to improve solubility.
  • the linker is a cleavable linker.
  • Cleavable linkers of the present disclosure can include, for example, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH-sensitive linkers, hypoxia-sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.
  • the linker is not a cleavable linker.
  • Linker residues can be used to facilitate manufacturing of longer peptide monomers.
  • these linkers join the heptad domain to T or B cell epitopes.
  • these linkers join two peptide carriers via the N-terminus, C-terminus, both termini, along the main chain, or any combination thereof.
  • these linkers can lie within the heptad, B cell epitope, or T cell epitope sequences.
  • two peptide fragments are joined.
  • three or more peptide fragments are joined. Any of a variety of methods can be used to associate a linker with a peptide carrier.
  • click chemistry can be used to associate a linker- containing moiety with a peptide carrier.
  • Staudinger ligation expressed protein ligation, split inteins, isopeptide bond formation, sortase ligation, liquid or solid phase fragment condensation can be used to associate a linker with a peptide carrier.
  • Ligation reactions can lead to the introduction of linker residues, for example, Cys, Ser, Thr, Ala, or His linkers.
  • a peptide carrier of the present disclosure may further comprise 1 or more additional amino acids at the N-terminus, C-terminus, or at both termini.
  • one, two, three, four, five, six, seven, eight, nine or ten, or more additional amino acids are added to the N- terminus, the C-terminus and/or both termini of the peptide carrier.
  • the additional amino acids may act as linkers to additional components of the peptide carrier (e.g., CD4 T cell epitope peptide, B cell epitope peptide).
  • the additional amino acids may act as linkers to bond one or more peptide carriers to other peptide carriers with the same or different amino acid sequences via the N-terminus, C-terminus, both termini, along the main chain, or any combination thereof.
  • the peptide carrier can comprise a “WP” dipeptide at the N- or C-terminus, or both termini, that link the alpha-helical peptide domain with an epitope peptide and act as a linker.
  • the peptide carrier can comprise GGP or PGG linkers that link epitope peptides to the N- or C-terminus of the alpha- helical peptide monomer, respectively (see e.g., SEQ ID NO: 64 and SEQ ID NO: 65).
  • a peptide carrier as disclosed herein comprises a proline (P) linker that links an epitope domain to the N-, or C-, or both termini of the alpha-helical peptide monomer.
  • the peptide carrier comprises a“DK” dipeptide or“SP” dipeptide at the N- terminus of the four heptad repeats.
  • The“DK” or“SP” at the N-terminus may facilitate a-helix formation of the polypeptide.
  • the peptide carrier may comprise a“DK” or“SP” at the N-terminus of [IKKIEKRJ4 (SEQ ID NO: 18), KKIEKR[IKKIEKR]3l (SEQ ID NO: 20), KIEKR[IKKIEKR] 3IK (SEQ ID NO: 21), IEKR[IKKIEKR] 3 IKK (SEQ ID NO: 22),
  • the peptide carrier comprises DKIEKRIKKIEKRIKKIEKRIKKIEKRIKK (SEQ ID NO: 26).
  • the peptide carrier of the present disclosure can comprise an alpha-helical peptide monomer comprising a coiled-coil domain, one or more CD4 T cell epitope (TCE) peptides, and one or more B cell epitope (BCE) peptides that can be linked in multiple orientations and locations to form said peptide carrier.
  • TCE CD4 T cell epitope
  • BCE B cell epitope
  • one or both termini and/or the main chain of the alpha-helical peptide monomers comprising the heptad sequences can be linked directly or indirectly to one, two, three, or more CD4 T cell epitopes (TCEs) with the same or different amino acid sequences and/or one, two, three, or more B cell epitopes (BCEs) with the same or different amino acid sequences.
  • TCEs CD4 T cell epitopes
  • BCEs B cell epitopes
  • the one, two, three, or more CD4 T cell epitopes (TCEs) with the same or different amino acid sequences and/or one, two, three, or more B cell epitopes (BCEs) with the same or different amino acid sequences are connected in series.
  • the one, two, three, or more CD4 T cell epitopes (TCEs) with the same or different amino acid sequences and/or one, two, three, or more B cell epitopes (BCEs) with the same or different amino acid sequences are interspersed randomly, in a pattern, and/or in series at one or both termini and/or along the main chain of the alpha-helical peptide monomer.
  • TCEs can be isolated from common pathogens (i.e., tetanus, measles, hepatitis B, influenza) and can bind a broad repertoire of MHC class II alleles in rodents, pigs, monkeys, and humans.
  • one or more TCE peptides are linked to the alpha-helical peptide monomer via the C-terminus.
  • one or more TCE peptides are linked to the alpha-helical peptide monomer via the N-terminus.
  • one or more TCE peptides are linked to the N- and C-terminus of the same alpha-helical peptide monomer.
  • two or more TCE peptides are linked in tandem to either the C-terminus or the N- terminus or to both termini of the alpha-helical peptide monomer. In other embodiments, multiple copies of the same or different TCE peptides are included at one or both termini, or along the length of the peptide.
  • one or more BCE peptides are linked to a fusion polypeptide comprising an alpha-helical peptide and one or more TCE peptides linked to the alpha-helical peptide monomer via the C-terminus.
  • At least one BCE peptide is linked in tandem to the C-terminus of a fusion peptide comprising an alpha-helical peptide monomer and at least one TCE peptide linked to the peptide carrier in tandem via one or both termini, and/or along the main chain of the alpha-helical peptide monomer.
  • one of more copies of a BCE peptide is linked to the N-, C-, or both termini, or attached to amino acid side chains along the length of the same alpha-helical peptide monomer or fusion polypeptide comprising an alpha-helical peptide monomer and at least one TCE that is linked to the alpha- helical peptide monomer via one or both termini, and/or attached to amino acid side chains along the length of the same alpha-helical peptide monomer.
  • one or more copies of one or more BCE peptides are linked to the C-terminus or N-terminus, or attached to amino acid side chains along the length of a fusion polypeptide comprising an alpha-helical peptide monomer and at least one TCE that is linked to the alpha-helical peptide monomer via the C-terminus, N-terminus, both termini, or along the length of the alpha-helical peptide monomer.
  • the fusion peptides of the present disclosure may be used as immunogenic
  • compositions for inducing an antibody response in a subject to one or more specific target BCEs are provided.
  • broad MHC Class II coverage across a variety of target populations can be achieved using these approaches (see e.g., FIG. 6).
  • the coiled- coil peptide-based vaccines as disclosed herein can induce a high titer of functional antibodies (Abs), and increasing concentrations of functional monoclonal Abs can be achieved using bivalent, trivalent, and multivalent vaccines containing one or more TCEs or BCEs, and/or additional haptens (e.g., nicotine).
  • Fusion polypeptides of the present disclosure can comprise an alpha-helical peptide monomer and a CD4 + T cell epitope (TCE) peptide that is linked to the alpha-helical peptide monomer N- or C-terminus.
  • TCE T cell epitope
  • a TCE peptide refers to a short peptide sequence of about 9-40 amino acids in length that are presented by major histocompatibility complex molecules (MHC) Class II molecules to CD4 + T cells (helper T cells) and evoke a specific immune response from the CD4 + T cells.
  • Major histocompatibility complex molecules are typically glycoproteins that deliver peptide antigens to a cell surface.
  • MHC class II molecules deliver peptide antigens originating in the vesicular system to the cell surface, where they are recognized by CD4 + T cells.
  • MHC molecules in humans may also be referred to as human leukocyte antigen (HLA).
  • HLA human leukocyte antigen
  • a fusion polypeptide monomer of the present disclosure may comprise one or more TCE peptides that are linked in series and can be selected from TABLE 2.
  • a fusion polypeptide monomer of the present disclosure may comprise two or more TCE peptides with the same or different amino acid sequences that are linked in series and/or independently to different sites on the alpha-helical peptide monomer.
  • the two or more TCE peptides with the same or different amino acid sequences that are linked in series and/or independently to different sites on the alpha-helical peptide monomer can be selected from TABLE 2.
  • the C-terminal TCE of the two or more TCEs that are linked in series is the Hepatitis B surface antigen, as shown in TABLE 2 (see e.g., SEQ ID NOs: 83-87).
  • the one or more TCEs can be linked to an alpha-helical peptide monomer via the N-, C-, both termini, or along the length of the alpha-helical peptide domain to yield peptide carriers using conventional and/or solid-phase peptide synthesis techniques, e.g., automated (solid phase) peptide synthesizers.
  • the peptide carriers of the present disclosure are produced via cell-based expression systems.
  • a linear TCE is a peptide sequence of at least 5-10 contiguous amino acids in length. In other embodiments, the linear TCE is a peptide sequence of at least 5-20 contiguous amino acids in length.
  • the linear TCE is a peptide sequence of at least 5-30 contiguous amino acids in length. In other embodiments, the linear TCE is a peptide sequence of at least 10-40 contiguous amino acids in length. In other embodiments, the linear TCE is a peptide sequence of at least 10-100 contiguous amino acids in length.
  • the presence of the CD4 + T cell epitope peptide may cause helper T cells to promote the
  • TCE peptides may enhance the immune response against certain antigens, more copies of a single TCE may further enhance the strength of the response, and a greater number of TCEs may broaden vaccine responses across diverse populations.
  • TABLE 2 contains examples of CD4 T cell epitope peptide sequences that can be used in combination with the compositions and methods of the present disclosure.
  • a peptide carrier contemplated herein can comprise a CD4 T cell epitope peptide, wherein such CD4 T cell epitope peptide can comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one or more of SEQ ID NO: 66 - SEQ ID NO: 182 and SEQ ID NO: 300, or a functional fragment thereof.
  • a peptide carrier consists of, consists essentially of, or comprises a TCE with the amino acid sequence set forth in any one of SEQ ID NO: 66 - SEQ ID NO: 94, any one of SEQ ID NO: 112 - SEQ ID NO: 125, any one of SEQ ID NO: 135 - SEQ ID NO: 146, any one of SEQ ID NO: 159 - SEQ ID NO: 167, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.
  • TCE can be used to enhance the immune response of a peptide carrier in a subject (e.g., a rodent or a human).
  • Such TCE’s can be used in combination with one or more B cell epitopes (BCE’s), resulting in a peptide carrier comprising an alpha-helical peptide linked to a TCE and a BCE.
  • BCE B-cell epitopes
  • a BCE may be linear (i.e., a linear sequence of adjacent (contiguous) amino acids) or conformational (i.e., non- sequential amino acids or segments of the antigen that are brought together in spatial proximity when the corresponding antigen or a portion of the antigen is folded).
  • a linear BCE is a peptide sequence of at least 5-10 contiguous amino acids in length. In other embodiments, the linear BCE is a peptide sequence of at least 5-20 contiguous amino acids in length.
  • the linear BCE is a peptide sequence of at least 5-30 contiguous amino acids in length. In other embodiments, the linear BCE is a peptide sequence of at least 10-40 contiguous amino acids in length. In some embodiments, the conformational epitope is at least 5-10 contiguous amino acids in length and has helical secondary structure. In other embodiments, the conformational epitope is at least 5-20 contiguous amino acids in length and has helical secondary structure. In other embodiments, the conformational epitope is at least 5-30 contiguous amino acids in length and has helical secondary structure. In other embodiments, the conformational epitope is at least 10-40 contiguous amino acids in length and has helical secondary structure. In some embodiments, the conformational epitope is at least 5-40 noncontiguous amino acids in length and represents a non-helical conformational surface BCE.
  • the BCE may be a contiguous peptide sequence from an endogenous protein that can be used to generate therapeutic antibodies capable of binding an endogenous protein or peptide (see e.g., FIG. 18, FIG. 19, FIG. 21).
  • the BCE may be a contiguous or noncontiguous peptide sequence from a pathogenic protein that is highly conserved within a diverging family of microbes, which in turn, can be used as a universal vaccine for broadening protection against infection (see e.g., FIG. 10-FIG. 15).
  • the BCE may be a contiguous or noncontiguous peptide sequence conserved within a specific pathogenic strain, subtype, or clade that can be used as a vaccine for protection against infection by the same strain, subtype or clade of pathogenic organism (see e.g., FIG. 10, FIG. 13A).
  • the BCE may assume a secondary structure (e.g., alpha helical) that can be incorporated in register at the N-, or C-terminus of the alpha-helical peptide carrier and represents a noncontiguous surface epitope (see e.g., FIG 10).
  • thermodynamic favorability of heptad helix formation and/or coiled-coil assembly may provide an impetus for these BCEs to assume a helical shape.
  • the BCE may be presented in a helix conformation to lymphocytes thereby generating antibodies specific to the helical conformation instead of (or in addition to) the linear sequence.
  • the face of the helix that is presented to lymphocytes may be dictated by how the linear BCE amino acid sequence aligns with the heptad repeat pattern.
  • the BCE sequence (“STQNAIDEITNKVN” (SEQ ID NO: 183)) comes from an area of the influenza hemagglutinin protein that is well conserved amongst divergent viruses, is known to elicit broadly protective antibody responses, and folds as an alpha helix in the surface of the hemagglutinin protein.
  • this BCE fused to the N-terminus of the alpha-helical peptide carrier and is aligned with the heptad pattern such that certain residues of the BCE face outward to mimic how the BCE is presented in the native hemagglutinin, thereby eliciting antibodies that can bind hemagglutinin on the surface of influenza virus (see, e.g., FIG. 10B).
  • the peptide carriers of the present disclosure can comprise one or more BCEs.
  • the one or more BCEs can be linked to an alpha-helical peptide monomer via the N-, C-, or both termini, or along the length of the same alpha-helical peptide monomer or fusion polypeptide (see e.g., FIG. 1 and FIG. 12A) using conventional synthetic methodologies, e.g., automated peptide synthesizers.
  • peptides containing one or more linear BCEs are mixed to generate antibody responses to multiple targets and enhance vaccine activity (see e.g., FIG. 15).
  • peptides containing one or more linear BCEs and one or more conformational BCEs are mixed to generate antibody responses to multiple targets (see e.g., FIG. 15). In other embodiments, peptides containing one or more conformational BCEs are mixed to generate antibody responses to multiple targets. In some embodiments, peptides containing one or more linear and/or conformational BCEs and/or TCEs are mixed to generate antibody responses to multiple targets.
  • peptides containing one or more linear and/or conformational BCEs and/or TCEs are covalently linked to each other using a variety of methods known to those skilled in the art (e.g., through cysteine chemistry, unnatural amino acids, etc.) via one or both termini, and/or along the main chain of the peptide carrier.
  • covalently linking multiple immunogenic peptide carriers may improve solubility, stability, and/or immunogenicity.
  • TABLE 3 contains examples of B cell epitope peptides that can be used in combination with the compositions and methods of the present disclosure.
  • a peptide carrier contemplated herein can comprise a B cell epitope peptide.
  • B cell epitope peptide can comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 183 - SEQ ID NO: 270, SEQ ID NO: 301 - SEQ ID NO: 302, or a functional fragment thereof [0150]
  • a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 183 - SEQ ID NO: 228.
  • Such peptide carriers can elicit immune response in a subject (e.g., a rodent or a human) that can protect the subject from infection through influenza virus, thus, vaccines comprising such peptide carriers can be used as influenza peptide vaccines and prevent and/or treat an influenza virus infection.
  • a subject e.g., a rodent or a human
  • vaccines comprising such peptide carriers can be used as influenza peptide vaccines and prevent and/or treat an influenza virus infection.
  • a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 232 - SEQ ID NO: 244.
  • Such peptide carriers can protect a subject against Herpes Simplex Virus by eliciting immune responses against gB, gD, or gH surface proteins.
  • a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 259 - SEQ ID NO: 260. Such peptide carriers can protect a subject against Respiratory Syncytial Virus by eliciting responses to the conserved F protein. [0153] In some instance, a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 261 - SEQ ID NO: 262. Such peptide carriers can protect a subject against Alzheimer’s disease by eliciting immune responses against Amyloid-beta and/or Tau protein.
  • a peptide carrier herein can comprise a B cell epitope peptide having a sequence of SEQ ID NO: 263. Such peptide carrier can be used as a vaccine for
  • immunocastration in a subject e.g., a rodent, a pig, a cow, or another farm animal.
  • a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 264 - SEQ ID NO: 267.
  • Such peptide carrier can be used to treat or prevent IgE-related diseases in a subject (e.g., a rodent or a human), such as IgE- mediated allergic reactions or inflammation.
  • incorporation of the BCE can occur at pre-specified locations within (i.e., along the length of) the alpha-helical domain of the peptide monomer by automated peptide synthesizers or solution based chemical reactions.
  • the BCE can be a peptide epitope of varying length where the peptide is incorporated into a Lysine-, Glu-, or Asp-rich alpha-helical peptide domain via isopeptide or other covalent bonds (See, e.g., FIG. 11 A, FIG. 12A).
  • the BCE can be a small molecule hapten, such as a nicotine derivative, that is manufactured as a Lys, Glu, or Asp building block in order to be incorporated into, for example, a Lysine-, Glu-, or Asp-rich alpha-helical peptide domain during solid phase peptide synthesis (FIG. 7, FIG. 8A).
  • the BCE can be a carbohydrate, polysaccharide or lipid building block that is incorporated in a similar fashion into a Lysine-, Glu-, or Asp-rich alpha-helical peptide domain.
  • Cys residues are inserted at specific sites in the peptide monomer that are then used to selectively install the BCE via thiol-specific chemistries.
  • unnatural amino acids are inserted at specifics sites in the peptide monomer with specific functional groups (e.g., iodoacetamide-, azide-, alkyne-, maleimide-containing chemical moieties, etc.) that link to complementary functional groups in the BCE using click chemistry or other chemical reactions.
  • chemical functionalities e.g., iodoacetamide-, azide-, alkyne-, maleimide- containing chemical moieties, etc.
  • chemical functionalities are inserted at specifics sites in the peptide monomer that can be used to, e.g., link complementary to, e.g., functional groups unnatural amino acids in the BCE using click chemistry or other chemical reactions.
  • the peptide carriers of the present disclosure can exhibit different net surface charges in different regions of the peptide carrier.
  • a coiled-coil domain comprising multiple lysine (Lys)-rich heptad repeats e.g., SEQ ID NO: 16 - SEQ ID NO: 35
  • a coiled-coil domain comprising multiple glutamic acid (Glu)-rich heptad repeats e.g., SEQ ID NO: 36 - SEQ ID NO: 49
  • Glu glutamic acid
  • a coiled-coil domain comprising multiple serine (Ser)-rich heptad repeats (e.g., SEQ ID NO: 59 - SEQ ID NO: 60) can have a neutral net surface charge due to the neutral serine residues at physiologic pH.
  • Ser serine
  • embodiments will have varied net surface charge that depends upon their amino acid sequence.
  • the epitope domain of a peptide carrier can likewise exhibit an epitope-specific net surface charge distribution depending on the amino acid sequence of the epitope peptide.
  • the peptide-based vaccines of the present disclosure can elicit an enhanced immune response when the net surface charge of the coiled-coil domain matches the net surface charge of the BCE.
  • SEQ ID NO: 280 comprising a coiled-coil domain and BCE with matching surface charge elicited an enhanced immune response compared to SEQ ID NO: 279 which comprises a coiled-coil domain with opposite surface charge to the BCE (see e g , FIG. 11A-FIG. 11C)
  • the enhanced activity of the peptide vaccine comprising peptide carriers having SEQ ID NO: 280 may be due to a repulsion of the M2e epitope peptide from the coiled-coil domain of the peptide carrier.
  • the BCE may be more available to cross-link with antigen receptors on B cells.
  • the negative charges in the M2e epitope can hydrogen bond with the positively-charged surface lysine residues, which buries the epitope in the peptide nanoparticle and reduces its ability to bind B cells.
  • TABLE 4 contains examples of amino acid sequences of peptide carriers as described in the present disclosure comprising an alpha-helical coiled-coil domain, one or more TCE peptides and, optionally, one or more BCE peptides.
  • a peptide carrier contemplated herein can consist of, consist essentially of, or comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 271 - SEQ ID NO: 294 or SEQ ID NO: 303 - SEQ ID NO: 306, or a functional fragment thereof.
  • a peptide carrier consists of, consists essentially of, or comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 288, or SEQ ID NO: 306.
  • Lys side chains on the peptide carrier e.g., SEQ ID NO: 308 are modified with one or more amino acid sequences set forth in SEQ ID NO: 191 - SEQ ID NO:
  • Such peptide carriers can be used as influenza vaccines by eliciting immune responses in a subject that provide protection against influenza virus. Due to the universality of the influenza B cell epitope peptide, the vaccines may provide broad protection against multiple influenza viruses.
  • a peptide carrier consists of, consists essentially of, or comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 293 or SEQ ID NO: 305.
  • Such peptide carriers can be used for immunocastration of a subject (e.g., a rodent, a pig, a cow, other farm animals, or other wild/feral animals).
  • a peptide carrier consists of, consists essentially of, or comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 290 - SEQ ID NO: 292.
  • Such peptide carriers can be used for treating or preventing Alzheimer’s disease (e.g., as a vaccine) by eliciting immune response in a subject (e.g., a rodent or a human) directed against the Amyloid-beta and/or Tau protein by enabling the generation of antibodies directed against such disease-promoting proteins.
  • the peptide-based vaccines can comprise one or more haptens (i.e., antigens).
  • haptens are small molecules that are covalently attached to the alpha-helical peptide domain of the peptide carriers (i.e., immunogenic compositions). Small molecules that are weak immunogens but become more immunogenic when attached to a larger molecule can be characterized as haptens.
  • a hapten may be a small organic molecule, a monosaccharide, disaccharide, or oligosaccharide, a lipid, glycolipid, nucleic acid, or an oligopeptide, for example.
  • hapten may be capable of binding to a B cell receptor, immunization with a hapten does not usually provoke an antibody response.
  • immunogenicity may be achieved by covalently attaching (i.e., linkingjoining, conjugating) a hapten to a larger molecule, called the carrier. Databases that describe thousands of haptens are available in the art.
  • a peptide carrier of the present disclosure can comprise a peptide monomer linked to one or more haptens.
  • the hapten is linked to a lysine, glutamic acid, aspartic acid, or cysteine residues residue of the alpha-helical peptide domain.
  • at least two haptens are linked to the alpha-helical peptide domain.
  • at least four haptens are linked to the alpha-helical peptide domain.
  • haptens are linked to lysine, glutamic acid, aspartic acid, or cysteine residues of the alpha-helical peptide domain and/or the TCE domain.
  • a hapten is a drug of abuse, for example, nicotine, ethyl alcohol, opiates, cannabinoids, amphetamines, barbiturates, glutethimide, methyprylon, chloral hydrate, methaqualone, benzodiazepines, LSD, anticholinergic drugs, antipsychotic drugs, tryptarine, other psychomirnetic drugs, sedatives, tranquilizers, cough suppressants, hallucinogens, stimulants, phencyclidine, psilocybine, volatile nitrite, benzodiazepine, other drugs inducing physical dependence and/or psychological dependence, or analogs of each of the drugs.
  • nicotine for example, nicotine, ethyl alcohol, opiates, cannabinoids, amphetamines, barbiturates, glutethimide, methyprylon, chloral hydrate, methaqualone, benzodiazepines, LSD, anticholinergic drugs, antipsychotic drugs, tryptarine,
  • a hapten is nicotine (i.e., (S)-3[l-Methylpyrrolidin-2-yl]pyridine, a stereoisomer thereof, an analog thereof, or a structurally distinct nicotine hapten.
  • Nicotine is parasympathomimetic alkaloid found in Solanaceae plants (e.g., tobacco) that acts as a potent stimulant.
  • the one or more haptens can be linked directly or indirectly (e.g., via a linker) to the alpha-helical peptide domain and/or TCE domain of the peptide-based vaccines as described herein.
  • Any suitable linker can be used in accordance with the present disclosure.
  • Linkers may be used to form amide linkages, ester linkages, disulfide linkages, etc.
  • Linkers may contain carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.).
  • linkers are 1 to 50 atoms long, 1 to 40 atoms long, 1 to 30 atoms long, 1 to 20 atoms long, 1 to 15 atoms long, or 1 to 10 atoms long.
  • Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic het- erocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. As would be appreciated by one of ordinary skill, each of these groups may in turn be substituted.
  • a linker is an aliphatic or heteroaliphatic linker.
  • the linker is a polyalkyl linker. In certain embodiments, the linker is a polyether linker. In certain embodiments, the linker is a polyethylene linker. In certain specific embodiments, the linker is a polyethylene glycol (PEG) linker.
  • SAPN-based vaccines of the present disclosure are capable of inducing an immune response in mammals, including humans.
  • SAPN-based vaccines show a microbial pathogen-like structure due to size and repetitive antigen structure that may be critical for B cell activation and facilitation of transport, uptake and processing in lymphoid cells and tissues.
  • the SAPNs of the present disclosure possess a size of about 2 to 20 nm. In some cases, the SANPs have a size of about 5 to 50 nm. In some cases, the SANPs have a size of about 20 to 100 nm. In some cases, the SANPs have a size of about 50 to 500 nm. In other cases, the SAPNs have a size of about 500 nm to >10 pm. In some cases, the size and shape of nanoparticle-based vaccines can be used to determine immunogenicity and function.
  • the coiled-coil domain comprising the heptad repeats may be beneficial to attach relatively hydrophobic or relatively hydrophilic BCE and/or TCE sequences to the coiled-coil domain comprising the heptad repeats to influence the physicochemical properties of the nanoparticles, including their solubility and assembly size.
  • the peptide carriers of the present disclosure can be chemically synthesized by manual techniques or by automated procedures (e.g., automated peptide synthesizers), including solid phase polypeptide synthesis.
  • automated peptide synthesizers e.g., automated peptide synthesizers
  • the equipment for the automated synthesis of peptides is commercially available (e.g., Perkin-Elmer, Inc.; Applied BioSystems Division, Foster City,
  • Solid phase polypeptide synthesis has been performed since the early l960s. Numerous improvements to synthesis methods have been developed, and many methods have been automated and chemistries have been developed to protect terminal ends and other reactive groups. There are a number of methods to attach BCE peptides and haptens to the peptide both during and after peptide synthesis (e.g., isopeptide bonds, thiol-specific reactions, click chemistry, etc.).
  • the synthetic peptide monomers of the present disclosure may also be obtained from any number of different custom peptide synthesizing manufacturers. If advantageous, the synthesized peptides or polypeptides of the present disclosure may be purified using any number of methods routinely practiced in the art, such as preparative reversed phase chromatography, partition
  • synthesized peptides or polypeptides of the present disclosure may be analyzed using any number of methods routinely practiced in the art, such as analytical reversed phase chromatography, nuclear magnetic resonance (NMR) spectroscopy, or mass spectrometry or other methods used in the art.
  • analytical reversed phase chromatography nuclear magnetic resonance (NMR) spectroscopy
  • mass spectrometry or other methods used in the art.
  • the peptide carriers of the present disclosure may be recombinantly produced using methods routinely practiced in the molecular biology art. Selection of the appropriate vector and expression control sequences (e.g., a promoter) and preparation of certain recombinant expression constructs is well within the level of ordinary skill in the art.
  • the expression vector also comprises expression control sequences, such as a promoter, enhancer, initiation site, and the like that are selected depending on the vector and host cell used to produce the peptide monomer.
  • the nucleotide sequence encoding a peptide in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis.
  • a polynucleotide that encodes a peptide carrier of the present disclosure may be incorporated into a recombinant expression vector for production of the respective peptide in a host cell.
  • Host cells containing recombinant expression constructs may be genetically engineered (transduced, transformed, or transfected) with the vectors and/or expression constructs (for example, a cloning vector, a shuttle vector, or an expression construct).
  • the vector or construct may be in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying particular genes or encoding-nucleotide sequences.
  • the host cell is one that can be adapted to sustained propagation in culture to yield a stable cell line that can express sufficient amount of the peptide monomer.
  • expression control sequences include LTR or SV40 promoter, E. coli lac or trp , the phage lambda PL promoter, and other promoters that were reported to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • promoter regions can be selected from any gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Particular bacterial promoters can include lad, lacZ, T3, T5, T7, gpt, lambda PR, PL, and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruses, and mouse metallothionein-I. Selection of the appropriate vector and promoter and preparation of certain recombinant expression constructs comprising at least one promoter or regulated promoter operatively linked to a polynucleotide described herein is well within the level of ordinary skill in the art.
  • Useful bacterial expression constructs can be prepared by inserting into an expression vector a structural DNA sequence encoding the specific peptide monomer together with suitable translation initiation and termination signals in an operative reading phase with a functional promoter.
  • the construct may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector construct and, if desirable, to provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli , Bacillus subtilis , Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • Suitable eukaryotic hosts for transformation include yeast (e.g., Pichia pastoris ), mammalian, insect and algal systems, although others may also be employed as a matter of choice. Any other plasmid or vector may be used as long as the plasmid or vector is replicable and viable in the host.
  • peptide monomers can be manufactured using a mixture of synthetic and recombinant systems. In some cases, the peptide monomer can be created entirely through solid phase peptide synthesis. In other cases, all peptide fragments are expressed recombinantly. In yet other cases, peptide fragments manufactured synthetically are linked to other recombinantly expressed peptide fragments.
  • SAPN-based vaccines of the present disclosure can comprise peptide carriers comprising a wide variety of different epitope peptides. Consequently, the peptide carriers of the present disclosure can be useful for applications in a wide variety of disease areas, including infectious and chronic diseases. Below are described some exemplary but non-limiting indications for which the peptide vaccines of the present disclosure can be used for.
  • Influenza virus diversity and antigenic drift requires the annual manufacture of hundreds of millions of vaccines that are primarily made using antiquated egg-based
  • Influenza infection is an acute global health issue and new technologies are needed that can maximize vaccine coverage, ease manufacturing constraints and reduce the price of goods.
  • Influenza virus encodes 2 essential surface glycoproteins called hemagglutinin and neuraminidase.
  • Hemagglutinin (HA) mediates cell binding and internalization, and
  • NA neuraminidase releases budding virus from the cell surface.
  • the Influenza A virus has diverged into an increasingly large number of strains that are categorized by their expression of different HA and NA proteins. Influenza A is the dominant pathogen responsible for most seasonal and pandemic infections. Influenza type B virus shares limited homology with A viruses and is responsible for widespread epidemics every 3 to 4 years and a disproportionately large frequency of infections in children. The Influenza B genome is less much less mutable than Influenza A and is classified into Victoria and Yamagata lineages based on antigenic variation of HA.
  • the peptide-based vaccines are capable of inducing an immune response in mammals against several different Influenza strains or subtypes or types through immunization with B cell epitopes (e.g., SEQ ID NO: 222) that are highly conserved among different Influenza strains or subtypes or types as shown in FIG. 10-FIG. 15 and described in EXAMPLE 9-EXAMPLE 14, and EXAMPLE 18.
  • B cell epitopes e.g., SEQ ID NO: 222
  • Hemagglutinin fusion peptide (HA-fp) epitope Hemagglutinin hydrophobic pocket peptide (HA- hp) epitope, hemagglutinin helix A epitopes, neuraminidase (NA)Neuraminidase epitopes, M2 ectodomain (M2e) epitopes, and nucleoprotein (NP) epitopes.
  • these peptides can present BCEs predicted by three-dimensional images (e.g., X-ray crystallography) of proteins from influenza or other viruses or pathogens.
  • X-ray crystallography images of representative influenza B hemagglutinins can be used to identify areas of the hemagglutinin protein sequence on the protein surface and available for antibody binding (see e.g., FIG. 13A). Conservation within these areas can be identified by sequence homology. For example, surface BCEs that are conserved across Yamagata/Victoria lineages (SEQ ID NO: 201 to SEQ ID NO: 207) and an area of NA is conserved across both A and B viruses (SEQ ID NO: 219 to SEQ ID NO: 221) can be identified in this manner.
  • these BCEs When these BCEs are incorporated into the peptide sequence using one of the methods described herein, they elicit antibodies that bind recombinant hemagglutinin and protect mice from viral challenge (see e.g., FIG 13, FIG. 14 and EXAMPLE 12). Additionally, multivalent combinations of peptides containing these and other BCEs lead to 100% protection and improved weight loss (see e.g., FIG. 15 and EXAMPLE 14). In this fashion, these and other peptides and/or conserved epitopes can be used and combined to formulate a universal vaccine for the prevention of all influenza A and B viruses, all B viruses, or one or more subtypes or strains of A or B viruses. This strategy can be used to formulate vaccines to inhibit pathogenic infection across a variety of indications (see e.g., EXAMPLES 17-20).
  • the HA2 stalk subunit is highly conserved between strains capable of inducing cross-reactive HA Abs.
  • One of the most functionally significant regions is the first ca. 100 amino acid (AA) residues of the HA2 subunit.
  • the fusion peptide (residues 1-23) mediates virus entry by inserting into the host endosomal membrane.
  • residues 1-11 (SEQ ID NO: 199 and SEQ ID NO: 200) are identical between A and B viruses, except for 2 conserved substitutions.
  • residues 16-23 contain a hydrophobic pocket (HA-hp, SEQ ID NOS: 192-198) and residues 42-75 contain Helix A (HxA, SEQ ID NOS: 183-191), both of which are highly conserved within influenza A and B viruses and are recognized by broadly cross-reactive mAbs.
  • HA-hp hydrophobic pocket
  • HxA Helix A
  • the HA-derived B cell epitope peptide of the present disclosure is selected from SEQ ID NO: 183 - SEQ ID NO: 207.
  • HA-derived B cell epitope peptides including SEQ ID NO: 183 - SEQ ID NO: 207 can be linked to a peptide carrier or fusion polypeptide via either the N- or C-terminus or both termini, or along the length of the alpha-helical peptide domain, or any combination thereof.
  • NA Neuraminidase
  • NA is a validated therapeutic target for influenza infection.
  • Influenza A and B viruses share 8 out of 9 identical residues near the active site of the enzyme and immunization with a peptide using the type A sequence induced cross-reactive type A and B Abs.
  • mAbs that bind this epitope neutralize 9 different NA virus families in vitro and cross-protect mice after a lethal challenge with most Influenza A viruses.
  • the NA-derived epitope peptide is SEQ ID NO: 208 - SEQ ID NO: 209.
  • NA Another sequence in NA that is 90-100% conserved, respectively, within influenza A and influenza B viruses is the N-terminal 1-12 amino acids (SEQ ID NOS: 210-213). Another area of NA (SEQ ID NOS: 214-221) is largely conserved across A and B viruses and can protect mice from viral challenge as described in FIG 14.
  • NA-derived B cell epitope peptides including SEQ ID NO: 208 - SEQ ID NO: 221 can be linked to a peptide carrier or fusion peptide via either the N- or C-terminus or both termini, and/or along the length of the alpha-helical peptide domain.
  • the M2 ectodomain is an essential trans-membrane ion channel.
  • the M2 extracellular domain, M2e is 24 AA in length.
  • the N-terminal epitope SLLTEVETPT (residues 2-11, SEQ ID NO: 222) is 100% identical for all type A viruses.
  • M2e-directed vaccines confer protection in animal models, including mice, ferrets, and swine, and a phase I clinical vaccine candidate was shown to be safe and immunogenic in humans. While M2e sequence identity is very low between A and B viruses, this region is also highly conserved in B viruses and mAbs that bind this 9 AA epitope also have antiviral activity (see e.g., FIG. 14). In some cases, the M2e-derived B cell epitope peptide is selected from SEQ ID NO: 222 - SEQ ID NO: 227.
  • an M2e-derived B cell epitope peptide including SEQ ID NO: 222 - SEQ ID NO: 227 can be linked to a peptide carrier or fusion peptide via the N-, C-, both termini, along the length of the alpha-helical peptide domain or any combination thereof.
  • NP nucleoprotein
  • the nucleoprotein (NP) is an internal protein involved in the transcription and replication of the virus genome and it is highly conserved between strains. (Fujimoto et al. Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection. J Gen Virol. 2016 Sep;97(9):2l04-l6).
  • Evidence that NP represents a bone-fide Ab target is based on studies showing the presence of anti-NP Abs in the sera of infected people, the discovery that NP is readily detected on the surface of infected cells, and the fact that anti-NP antibodies protect against homologous and heterologous type A viruses.
  • NP-derived B cell epitope peptide has SEQ ID NO: 228.
  • NP-derived B cell epitope peptide including SEQ ID NO:
  • 228 can be linked to a peptide carrier or fusion peptide via either the N- or C-terminus or both termini, and/or along the length of the alpha-helical peptide domain.
  • HPVs Human papillomaviruses.
  • HPVs Human papillomaviruses
  • Three prophylactic vaccines e.g., Cervarix, Gardasil-4 and Gardasil-9 have been approved to protect against certain types of HPV infections.
  • Cervarix Cervarix
  • Gardasil-4 Gardasil-4
  • Gardasil-9 Three prophylactic vaccines
  • HPV-directed peptide vaccines of the present disclosure include multivalent vaccines comprising a collection of peptides with strain-specific sequences, and consensus sequences comprised of the most commonly used residues.
  • the N-terminal region of the surface L2 protein is highly conserved among diverse HPV serotypes and have three regions of strong homology; residue 17-31, 69-86, and 108-122.
  • the consensus sequence of HPV peptide vaccines can comprise SEQ ID NO: 229 (domain 17-31), SEQ ID NO: 230 (domain 69-86) or SEQ ID NO: 231 (domain 108-122).
  • Herpes Simplex Virus Herpes simplex virus types 1 (HSV-l) and 2 (HSV-2) are the most common sexually transmitted infection and cause severe infections in newborns. HSV-2 is the leading cause of infectious blindness in the western world (Sandgren et al. Understanding natural herpes simplex virus immunity to inform next-generation vaccine design. Clin Trans Immunol 20l6,5:e94). The virus infects nerve endings and establishes persistent ganglia infection. HSV has several surface glycoproteins. Glycoprotein B (gB) mediates viral fusion with host membranes. Efficient fusion is aided by glycoproteins D, H, L and K (gD, gH, gL, gK).
  • virions that lack gK enter cells with lower efficiency and are unable to establish latency in neuronal cells. Additionally, deletion of amino acids 31-68 of gK inhibits viral fusion entirely. Neutralizing antibodies and CD4 T cell responses to natural infection are focused on gB and gD. However, a long-lasting fully-protective HSV-l/HSV-2 vaccine remains elusive. For 50 years, attempts at HSV vaccines have met with failure.
  • the cross-reactive peptide vaccines against HSV-l and HSV-2 of the present disclosure utilize conserved linear B cell epitopes and CD8 T cell epitopes that have been identified to be present in both HSV types.
  • a multivalent vaccine formulation can be envisioned that contains both B and CD8 T cell epitopes.
  • Potential B cell targets include four gD
  • HNPGASALL SEQ ID NO:242
  • THTPLPRGIGY SEQ ID NO:244
  • the peptide-based vaccines of the present disclosure can also include conserved sequences with overlapping promiscuous CD8 T cell epitopes from gB
  • a gH epitope with both B and CD8 T cell activity (“AEFPRDPGQLLY”/aa96-l08, SEQ ID NO: 236) can be used in the presently described peptide vaccines.
  • epitope peptides that show an immune activity can be combined into multivalent peptide-based vaccines.
  • DENV Dengue Virus
  • Flavivirus is part of the genus Flavivirus and is the most prevalent anthropod-transmitted viral infection worldwide, causing 25,000 deaths and up to 1 million severe infections annually (Flipse at al. The complexity of a dengue vaccine: a review of the human antibody response. PLoS Negl Trop Dis. 20l5,9:e0003749). DENV comprises 50 closely-related viruses including yellow fever, West Nile, chikungunya and Japanese encephalitis viruses.
  • DENV1 antigenically distinct DENV serotypes
  • DENV2 deoxyribonucleic acid
  • DENV3 antigenically distinct DENV serotypes
  • Most immune responses to DENV are to the envelope (E) protein, with lesser responses to the pre-membrane (prM) and non- structural 1 (NS1) proteins. Serum NS1 levels correlate to disease severity suggesting that NS1 may act as a viral toxin.
  • the first DENV vaccine (CYD-TDV) was licensed in 2015 but has shown inconsistent efficacy across populations.
  • This vaccine consists of four recombinant viruses with a yellow fever vaccine backbone expressing the pre-membrane (prM) and envelope (E) proteins of each of the four DENV serotypes.
  • prM pre-membrane
  • E envelope proteins of each of the four DENV serotypes.
  • a Phase III trial showed that the protective efficacy of the vaccine varied across serotypes from 35% (DENV2) to 72% (DENV4) and did not correlate to antibody titers. This limited protection greatly undermines the utility of this vaccine.
  • Most other DENV vaccines in development are based on chimeric or attenuated virus.
  • the peptide carriers of the present disclosure provide for an effective DENV vaccine and can offer high protection against the four DENV serotypes, regardless of exposure history or age.
  • the genetic similarity between DENV and other flaviviruses may enable targeting multiple viruses with a single vaccine.
  • Cross-reactive epitopes have been found in the E protein.
  • the E protein contains linear epitopes with strong conservation across all flaviviruses (e.g., E35-50, E98- 120, and E250-270) as well as DENV-specific epitopes (e.g., E197-214 and E309-326).
  • the peptide carriers of the present disclosure comprise epitopes from the E protein (SEQ ID NO: 245), original NS1-WD (SEQ ID NO: 246) or modified NS1- WD (SEQ ID NO: 247) epitope peptides derived from the non-structural protein 1 (NS1), which is the only surface NS protein.
  • NS1 non-structural protein 1
  • the wing domain (WD) loop at the C-terminus of the protein is an exposed antigenic site that is conserved across all four DENV serotypes.
  • Hepatitis C Virus Hepatitis C Virus
  • HCV Hepatitis C Virus
  • HCV infections such as interferon/ribavirin combination treatment and direct-acting antivirals.
  • Direct-acting antivirals have shown the most promise by inhibiting the function of essential viral proteins.
  • Current treatment regimens have been able to cure 30-97% of patients, depending on the HCV subtype.
  • Vaccines to treat HCV are currently unavailable, though there has been substantial research into therapeutic and prophylactic HCV vaccines based on recombinant protein, peptide- based, virus-like particle (VLP), and DNA Recombinant, VLP and DNA vaccines are capable of eliciting strong humoral immune responses to HCV in preclinical and clinical trials, however, these immune responses do not correlate with protection. Moreover, humoral responses tend to focus on regions of viral proteins with high mutational rates leading to the generation of escape mutants and limited cross-protection against heterologous HCV subtypes. These vaccines also stimulate solely a weak long-term protection.
  • the peptide-based HCV vaccines of the present disclosure comprise conserved neutralizing B cell epitopes and CD8 T cell epitopes of HCV that can lead to broad protection against many HCV subtypes.
  • HCV possesses two surface envelope proteins - El and E2 - that are associated with viral entry to host cells. Both have regions of strong conservation and linear neutralizing antibody epitopes have been identified.
  • the peptide carriers comprise residues 192-206 (YEVRNSSGLYHVTND (SEQ ID NO: 248)) and 313-330
  • QNIQLINTNGSWHINRTALN (SEQ ID NO: 250)
  • 523-536 GAPTYNW GENETD V (SEQ ID NO: 251)
  • 626-639 FKVRM YV GGVEHRL (SEQ ID NO: 252)
  • CD8 T cell epitopes have been reported from the HCV core protein (YLLPRRGPRL, residues 35-44 (SEQ ID NO: 253)), El (VYEAADMIM, residues 213-221 (SEQ ID NO: 254)), E2 (HYAPRPCGI, residues 488-496 (SEQ ID NO: 255)), non- structural region 3 (VYHGAGSKTL, residues 1081-1090 (SEQ ID NO: 256)), and non- structural region 5 (RYAPACKPL, residues 2132-2140 (SEQ ID NO: 257)).
  • excerpts of the above described epitope sequences may be used as disclosed herein, e.g., a l2-residue excerpt (QLINTN GS WHIN (SEQ ID NO: 258) in lieu of the entire E2/409-238 epitope
  • the epitope peptides with SEQ ID NO: 248 - SEQ ID NO: 258 can be incorporated into multivalent peptide vaccines according to the present disclosure.
  • Those multivalent vaccines are capable of stimulating humoral and cellular immunity and offer a broad protection against multiple HCV genotypes.
  • Respiratory syncytial virus is currently the leading cause of lower respiratory tract illness in infants, but has minimal treatment options.
  • RSV is classified as Type A (RSVA) or B (RSVB), where Type A is more prevalent.
  • RSVA Type A
  • RSVB RSVB
  • Current treatment options for RSV include Ribavarin, only small-molecule therapeutic used to treat RSV infections, and palivizumab, a monoclonal antibody administered prophylactically to high-risk infants.
  • RSV vaccines available. Attempts to develop anti-RSV vaccines with attenuated viruses have shown safety, but with limited efficacy.
  • a biomarker for protection is the presence of RSV-specific CD8 T cell responses and neutralizing antibodies (nAbs) that target the attachment (G) or fusion (F) glycoproteins.
  • the G protein is highly glycosylated and heterogeneous across HSV strains and generates weaker neutralizing responses than the F protein.
  • vaccine development has focused on the F protein, which is 90% conserved between RSVA and RSVB.
  • the F protein mediates viral entry into host cells and its major neutralizing antigenic sites have been mapped in its pre- and post fusion states. To prevent viral entry, the pre-fusion state can be targeted by focusing on the antigenic sites 0, II, and IV that are highly conserved neutralizing epitopes accessible to antibodies.
  • conformational change between the pre- and post-fusion states can obscure and sterically modify pre-fusion epitopes.
  • the pre-fusion state can be metastable and readily undergo conversion to post-fusion form as a recombinant protein.
  • pre-fusion F protein has several regions of conservation. Specifically, residues 62-75 and 196-209 of site 0, 254-277 (palivizumab epitope) of site II, and 173-182 of site II.
  • the multivalent peptide vaccines of the present disclosure can comprise either consensus sequences or strain-specific sequences with pending cross-reactivity between RSV strains or any combination thereof.
  • site 0 conserved sequence for RSVA
  • RSVB consensus sequence KNYINNQLLPIVNQ (SEQ ID NO: 260)
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD dementias
  • NFTs neurofibrillary tangles
  • Tau pathology is composed of a range of aberrant tau forms that arise through post-translational modifications and form a range of aggregates.
  • Therapeutic vaccines for Alzheimer’s disease have focused on conjugating short peptides found in the plaque-forming N- terminus of amyloid-beta to various carriers including KLH, CRM 197, and Qb phage VLPs.
  • the peptide-based Alzheimer’s vaccines of the present disclosure can comprise a 12 AA Tau protein (TAU) derived epitope peptide.
  • TAU Tau protein
  • the peptide-based Alzheimer’s vaccines of the present disclosure can comprise a 12 AA Tau protein (TAU) derived epitope peptide.
  • TAU Tau protein
  • Alzheimer’ s vaccine of the present disclosure can comprise an amyloid-beta (Ab) (SEQ ID NO: 261) epitope peptide or a Tau (SEQ ID NO: 262) epitope peptide.
  • an amyloid-beta epitope peptide or a Tau epitope peptide can be liked to a peptide carrier or fusion peptide via either the N- or C-terminus of the peptide, or along the length of the alpha-helical peptide domain, or any combination thereof.
  • the peptide-based Tau and Ab vaccines of the present disclosure can comprise alpha-helical peptide monomers with SEQ ID NO: 286 - SEQ ID NO: 289.
  • the peptide carriers of the Tau and Ab vaccines can comprise one or more Tau and Ab derived epitope peptides linked in tandem to the N- or C-terminus, or along the length of the alpha-helical peptide, or any combination thereof.
  • a universal vaccine for neurodegenerative diseases can comprise either a combination of peptide carriers comprising Tau and Ab epitope peptides, respectively, or a peptide carrier that comprises an epitope domain comprising the Tau and the Ab epitope, thereby eliciting immune responses against several antigens at the same time.
  • Contraceptive vaccines are a methodology for fertility control that represents a valuable alternative to surgical sterilization. Contraception vaccines, which can provide a long-term effect without health hazards, are being used in numerous ways for wild, zoo, farm, and domestic animal populations.
  • GnRH Gonadotropin-releasing hormone
  • a 10 AA neuropeptide that acts as a molecular regulator for sexual development and function plays a prominent role in regulating sexual development and function.
  • GnRH stimulates the synthesis and release of follicle-stimulating hormone and luteinizing hormone from the pituitary, which in turn, control androgen and estrogen production.
  • Other GnRH conjugate vaccines using different carrier proteins are being marketed for animal reproduction and husbandry purposes.
  • GnRH conjugate vaccines that employ KLH and Diphtheria toxoid carriers are being used to inhibit reproduction of domestic and wild animals, and as an immunocastration method for controlling meat quality in pigs and aggressive behavior patterns in horses and cattle.
  • epitope peptides derived from the follicle-stimulating hormone receptor (FSHR) antigen can be used for immunocastration.
  • GnRH antagonists are being marketed or developed for several indications in humans. For instance, testosterone promotes growth of many prostate tumors and the elimination of testosterone through castration has been a major treatment goal for men with advanced prostate cancer. GnRH antagonists are also being investigated for the treatment of benign prostatic hyperplasia, and for women, hormone-sensitive breast cancer, endometriosis, and uterine fibroids.
  • the peptide-based vaccine of the present disclosure having SEQ ID NO: 293 can elicit a targeted Ab production against GnRH (FIG. 19-FIG. 20 and EXAMPLE 16).
  • peptide-based GnRH vaccines of the present disclosure can comprise alpha-helical peptide monomers with SEQ ID NO: 293 or SEQ ID NO: 305.
  • the peptide carriers of the GnRH vaccine can comprise one or more GnRH derived epitope peptides linked in tandem to the N- or C-terminus, or along the length of the alpha-helical peptide, or any combination thereof.
  • IgE immunoglobulin E
  • the first monoclonal approved for the treatment of severe allergic asthma and urticaria (hives) was Omalizumab (XolairTM), which binds the Cs3 domain on the heavy chain of IgE.
  • Omalizumab XolairTM
  • several issues have limited its utility including the dosing regimen, required administration in the clinic, and its potential to induce acute anaphylaxis, which necessitated the FDA’s issuance of a black-box warning on its label.
  • the peptide-based vaccines of the present disclosure can comprise two IgE peptide antigens that induce anti-IgE antibodies (see e.g., FIG. 21) that bind free IgE or deplete IgE secreting B cells.
  • the peptide vaccines may not interact with IgE bound to cell surface Fc receptors, thus avoid triggering the release of inflammatory mediators through IgE receptor cross-linking.
  • peptides were derived from different loops of the Ce3 domain of IgE can be used to induce an immune response.
  • the peptides comprising SEQ ID NO: 264 and SEQ ID NO: 265 corresponds to the epitope recognized by monoclonal antibody omalizumab (SEQ ID NO: 301), whereas the peptide comprising SEQ ID NO: 266 (Peptide P) targets a different loop on IgE Ce3 (see e.g., Champion et al., US Patent No. US 8,722,053).
  • the peptide carrier can comprise a BCE with SEQ ID NO: 267 (CsmX, SVNPGLAGGSAQSQRAPDRVL) which sequence corresponds to the CsmX domain located between the CH4 domain and the C-terminal membrane anchor of the e chain of membrane bound IgE (mlgE) on human B cells.
  • Antibodies that are generated by a vaccine comprising SEQ ID NO: 267 may bind to CsmX which in turn may reduce disease-associated concentrations of IgE by inducing antibody-dependent cellular cytotoxicity and apoptosis of mlgE expressing B cells.
  • the antigenic targets mediate inflammatory disease including;
  • atopy asthma
  • an autoimmune disease an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behcet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus
  • Cancer is currently an important health problem in developed countries where it is the second leading cause of death.
  • the peptide-based vaccines of the present disclosure can comprise three HER2 peptide antigens that induce anti-HER2 antibodies.
  • the peptide vaccines comprise SEQ ID NO: 268, SEQ ID NO: 269, or SEQ ID NO: 270.
  • these peptides can be coformulated into a multivalent vaccine targeting all three epitopes.
  • the peptide-based cancer vaccines of the current disclosure can comprise peptide carriers comprising epitope peptides that are derived from a member of the receptor tyrosine kinase family, a member of the epidermal growth factor receptor family, WT1, p53, Brachyury, brachyury (TIVS7-2, polymorphism), brachyury (IVS7 T/C polymorphism), T brachyury, T, hTERT, hTRT, iCE, HPV E6, HPV E7, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE- A 10, MAGE-A12, BAGE, DAM-6, -10, GAGE-l, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-l, MART-l, MC1R, GplOO, PSA, PSA, PS
  • ETV6-AML1 fusion protein FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase fusion protein, HLA-A2d, HLA-A1 ld, hsp70-2, KIAAO205, MART2, ME1, MUM-lf, MUM-2, MUM-
  • neo-PAP Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1- or -SSX2 fusion protein, TGF- betaRII, triosephosphate isomerase, BAGE-l, GAGE-l, 2, 8, Gage 3, 4, 5, 6, 7, GnTVf, HERV- K-MEL, KK-LC-l, KM-HN-l, LAGE-l, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE- A 10, MAGE-A12, MAGE-C2, mucink, NA-88, NY-ESO- l/LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, T
  • RAB 38/NY -MEL- 1 TRP-l/gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BCMA, BING-4, CPSF, cyclin Dl, DKK1, ENAH (hMena), EP-CAM, EphA3, EZH2, FGF5, G250/MN/CAIX, HER-2/neu, ILl3Ralpha2, intestinal carboxyl esterase, alpha fetoprotein, M-CSFT, MCSP, mdm-2, MMP-2, MUC1, p53, PBF, PRAME, PSMA, RAGE-l, RGS5, RNF43, RU2AS, secernin 1, SOX10, STEAP1, survivin, Telomerase, VEGF, BRCA1, or a modified variant, a splice variant thereof.
  • Tumor-associated antigens can be antigens not normally expressed by the host; they can be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they can be identical to molecules normally expressed but expressed at abnormally high levels; or they can be expressed in a context or environment that is abnormal.
  • Tumor-associated antigens can be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, other biological molecules, or any combinations thereof.
  • compositions of the present disclosure including
  • immunogenic compositions can comprise one or more compositions (i.e., one or more peptide carriers) of the present disclosure and a pharmaceutically acceptable excipient.
  • the immunogenic composition comprises at least one of the compositions of the present disclosure for the prevention or treatment of one or more indications.
  • the immunogenic composition comprises at least two of different compositions of the present disclosure for the prevention or treatment of one or more indications including influenza, HSV, HPV, HCV, or neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease.
  • an immunogenic composition of the present disclosure can comprise a universal vaccine comprising one or more identical or different epitope peptides against a variety of conserved epitopes.
  • an immunogenic composition can comprise a peptide carrier comprising a plurality of influenza-derived BCE peptides that can elicit the production of broadly neutralizing antibodies in a subject after administration of said immunogenic composition.
  • an immunogenic composition of the present disclosure can comprise two or more different compositions (e.g., peptide carriers) to vaccinate a subject against one or more indications simultaneously.
  • the immunogenic composition may be a sterile aqueous or non- aqueous solution, suspension or emulsion containing the compositions of the present disclosure, which additionally comprises a pharmaceutically acceptable carrier (physiologically acceptable excipient or pharmaceutically suitable excipient or carrier; i.e., a non-toxic material that does not interfere with the activity of the active ingredient).
  • a pharmaceutically acceptable carrier physiologically acceptable excipient or pharmaceutically suitable excipient or carrier; i.e., a non-toxic material that does not interfere with the activity of the active ingredient.
  • an effective amount or therapeutically effective amount refers to an amount of the immunogen administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a specific therapeutic effect.
  • an immunogenic composition can comprise a plurality of fusion polypeptides.
  • the plurality of fusion polypeptides may be the same.
  • the plurality of fusion polypeptides is composed of two or more different fusion polypeptides.
  • the two or more different fusion polypeptides may target the same B cell antigen or may target different B cell antigens.
  • Subjects may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which assays will be familiar to those having ordinary skill in the art and are described herein.
  • the level of an immunogenic composition that is administered to a subject may be monitored by determining the level of the immunogenic composition, in a biological fluid, for example, in the blood, blood fraction (e.g., serum), and/or in the urine, and/or other biological sample from the subject. Any method practiced in the art to detect the immunogenic composition may be used to measure the level of immunogenic composition during the course of an immunization regimen.
  • an immunogenic composition described herein for evoking a specific immune response may depend upon the subject’s condition, that is, stage of the addiction or disease if present, severity of symptoms caused by the addiction or disease, general health status, as well as age, gender, and weight, and other factors apparent to a person skilled in the medical art.
  • Immunogenic compositions may be administered in a manner appropriate to the addiction, disease or disorder to be treated or prevented as determined by persons skilled in the medical arts.
  • An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the s’s addiction or disease, the particular form of the active ingredient, and the method of
  • Optimal doses of an immunogenic composition may generally be determined using experimental models and/or clinical trials. In some cases, the optimal dose may depend upon the body mass, weight, or blood volume of the subject. The use of the minimum dose that is sufficient to provide an effective immune response is usually preferred. Design and execution of pre-clinical and clinical studies for an agent (including when administered for prophylactic benefit) described herein are well within the skill of a person skilled in the relevant art.
  • an immunogenic composition may be administered at a dose between 0.01 mg/kg and 1000 mg/kg (e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10 to 50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000 mg/kg) body weight.
  • an immunogen may be administered at a dose of between 1 and 500 pg.
  • an immunogen may be administered at a dose of about 1 pg to 10 pg, about 10 pg to 50 pg, about 50 pg to 100 pg, or about 100 pg to 500 pg.
  • the immunogenic compositions of the present disclosure may be administered to a subject in need thereof by any one of several routes that effectively deliver an effective amount of the immunogen.
  • such administrative routes include, for example, oral, topical, parenteral, enteral, rectal, intranasal, buccal, sublingual, intramuscular, transdermal, vaginal, rectal, or by intracranial injection, or any combination thereof.
  • such compositions may be in the form of a solid, liquid, or gas (aerosol).
  • the administrative route is also determined by the type of immunogenic composition being administered.
  • the immunogenic composition is administered intramuscularly.
  • compositions i.e., non-toxic materials that do not interfere with the activity of the active ingredient
  • pharmaceutical acceptable excipients are practiced in the pharmaceutical art and described, for example, in Rowe et ah, Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5 th Ed., 2006, and in Remington: The Science and Practice of Pharmacy (Gennaro, 2l st Ed. Mack Pub. Co., Easton, PA (2005)).
  • exemplary pharmaceutically acceptable excipients include sterile saline and phosphate buffered saline at physiological pH. In some cases, preservatives, stabilizers, dyes, buffers, and the like may be provided in the immunogenic composition.
  • compositions described herein may be formulated as a lyophilizate, or the immunogenic composition may be encapsulated within liposomes using established technology. In some cases, an immunogenic composition may be formulated for any appropriate manner of administration described herein and in the art.
  • a composition may be in the form of a liquid.
  • a liquid immunogenic composition may include, for example, one or more of the following: a sterile diluent such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils that may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents; antioxidants; chelating agents; buffers and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils that may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents
  • antibacterial agents antioxidants
  • antioxidants chelating agents
  • buffers and agents for the adjustment of tonicity
  • a parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • physiological saline is preferred, and an injectable pharmaceutical composition is preferably sterile.
  • an immunogenic composition described in the present disclosure can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, and, optionally, with diluents, buffering agents, moistening agents, preservatives, coloring agents, and flavoring agents.
  • an immunogenic composition included in the compositions may be formulated for oral delivery with a buffering agent, flavoring agent, e.g., in a liquid, solid or semi-solid formulation and/or with an enteric coating.
  • a composition of the present disclosure can comprise any one of the immunogenic compositions described herein, and may be formulated for sustained or slow release.
  • such compositions may generally be prepared using elsewhere reported technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the particular target site.
  • sustained-release formulations may contain the immunogen dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Excipients for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
  • the amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.
  • Kits with unit doses of an immunogen described herein, usually in oral or injectable doses are provided.
  • such kits may include a container containing the unit dose, an informational package insert describing the use and attendant benefits of the immunogen or antibody in treating pathological condition of interest, and optionally an appliance or device for delivery of the composition.
  • the immunogenic compositions of the present disclosure may comprise a
  • an adjuvant is intended to enhance (or improve, augment) the immune response to the immunogens described herein, the peptide monomer conjugates, peptide dimer conjugates, or trimeric coiled-coil peptide conjugates (i.e., increase the level of the specific immune response in a statistically, biologically, or clinically significant manner compared with the level of the specific immune response in the absence of administering the adjuvant).
  • Vaccine adjuvants can control the magnitude and quality of adaptive T and B cell responses by facilitating antigen/plasmid uptake into antigen presenting cells and stimulating innate pathways that control leukocyte recruitment to the site of injection (see, e.g., Carter and Reed, 2010, Curr. Opin. HIV AIDS 5:409-13).
  • the only licensed adjuvant in the United States was aluminum-based mineral salts. Since then squalene-based oil-in-water emulsions like MF59, AS03, and AF03 have been used extensively in marketed influenza vaccines (Wilkins AL et al. AS03- and MF59-Adjuvanted Influenza Vaccines in Children. Front Immunol.
  • TLRs Toll-like receptors
  • TLR4 LPS
  • TLR1, TLR2, TLR6 lipoproteins
  • TLR5 flagellin
  • TLR7, TLR8, TLR3 intracellular pathogens
  • TLR9 single-stranded RNA
  • TLR9 double stranded RNA
  • a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies.
  • suitable adjuvants augment the response to the immunogen without causing conformational changes in the immunogen that might adversely affect the qualitative immune response.
  • suitable adjuvants include aluminum salts, such as alum (potassium aluminum sulfate), or other aluminum containing adjuvants such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate.
  • Other pharmaceutically suitable adjuvants include nontoxic lipid A-related adjuvants such as, by way of non-limiting example, nontoxic monophosphoryl lipid A (see, e.g., Persing et al., Trends Microbiol. !0:s32-s37 (2002)), for example, 3 De-O-acylated monophosphoryl lipid A (MPL) (see, e.g., United Kingdom Patent Application No. GB
  • Other useful adjuvants include QS21 and QuilA that comprise a triterpene glycoside or saponin isolated from the bark of the Quillaja saponaria Molina tree found in South America (see, e.g., Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell and Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540).
  • Other suitable adjuvants include oil in water emulsions, optionally in combination with immune stimulants, such as monophosphoryl lipid A.
  • Other suitable adjuvants include polymeric or monomeric amino acids such as polyglutamic acid or polylysine, liposomes, and CpG. (See e.g., U.S. Patent No.
  • TLR based adjuvants include, for example, glucopyranosyl lipid A-stable emulsion (GLA- SE). (See e.g., U.S. Patent No. 8,609,114); AS04.
  • the immunogenic compositions of the present disclosure comprise an adjuvant that is squalene-based oil-in-water emulsion, and/or a toll-like receptor (TLR) agonist.
  • the adjuvant is a glucopyranosyl Lipid A-Stable Emulsion (GLA-SE) adjuvant.
  • the adjuvant is an aluminum based adjuvant.
  • Methods of the present disclosure for inducing a humoral immune response specific for target antigen comprising administering to the subject any of the immunogenic compositions described herein.
  • a subject includes a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a horse, a non-human primate, a pig, and a human.
  • a subject is a human.
  • the immunogenic compositions of the present disclosure may be administered to a subject by a parenteral (e.g., intravenous), intraperitoneal, intramuscular, intradermal, intraocular, or subcutaneous route.
  • the immunogenic composition may further comprise a suitable adjuvant to enhance the immune response to the immunogen.
  • Adjuvants typically used for immunization of non-human animals include but are not limited to Freund’s complete adjuvant, Freund’s incomplete adjuvant, montanide ISA, Ribi Adjuvant System (RAS) (GlaxoSmithKline,
  • subjects receive one or more booster immunizations according to a preferred schedule that may vary according to, inter alia, the immunogen, the adjuvant (if any) and/or the particular species of subject.
  • the B cell immune response may be monitored by periodically bleeding the subject, separating the sera from the collected blood, and analyzing the sera in an immunoassay, such as an ELISA or Ouchterlony diffusion assay, or the like, to determine the specific antibody titer. When an adequate antibody titer is established, the subject may be bled periodically to accumulate the polyclonal antisera.
  • Monitoring the immune response of an immunized host during pre-clinical studies in subjects includes obtaining sera from the subjects before the first dose (i.e., pre-immune sera) and after the final boosting dose. In some cases, sera may also be obtained after any one or more of the boosting doses between the primary dose and final boosting dose. For monitoring the immune response of an immunized host during clinical studies or during post-marketing studies, sera may also be obtained from humans before the first immunization and after one or more administrations of the immunogenic compositions.
  • Production of antigen-specific antibodies in an immunized host may include production of any class of immunoglobulin, including IgG, IgA, IgM, and/or IgE, and isotypes within the classes.
  • the presence of specific IgG, IgM, IgE, and IgA may be detected in a biological sample (e.g., serum, nasal wash, lung lavage, or other tissues) obtained from an immunized host.
  • a biological sample e.g., serum, nasal wash, lung lavage, or other tissues
  • the biological sample may be permitted to interact with or contact an antigen that is purified, isolated, partially isolated, or a functional fragment thereof.
  • Production of antigen specific T cells in an immunized host may include production of any type of T cells, including CD4 + T cells and CD8+ T cells.
  • the presence of specific CD4 + or CD8+ T cells may be detected in a biological sample obtained from an immunized host.
  • Assays for measurement of T cell responses are well established in the art and include, for example, T cell proliferation assays, cytokine based assays (e.g., ELISA, cytokine ELISPOT, intracellular cytokine staining), flow cytometry, cytometry by time of flight (CyTOF) mass spectroscopy, MHC tetramer staining, and cytotoxicity assays.
  • T cell proliferation assays e.g., ELISA, cytokine ELISPOT, intracellular cytokine staining
  • flow cytometry cytometry by time of flight (CyTOF) mass spectroscopy
  • MHC tetramer staining cyto
  • immunogenicity of immunogens described herein may also be characterized by any number of assays and techniques practiced in the art, including immunoassays to evaluate binding and the capability of the immunogen to induce an immune response.
  • immunoassays include ELISA, immunoblot, radioimmunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), Ouchterlony immunodiffusion, proliferation assays, cytotoxicity assays, MHC peptide tetramer staining, intracellular cytokine staining, cytokine ELIspot, and the like.
  • Conditions for in vitro assays include temperature, buffers (including salts, cations, media), and other components that maintain the integrity of any cell used in the assay and the compound, which a person skilled in the art will be familiar and/or which can be readily determined. A person skilled in the art also readily appreciates that appropriate controls can be designed and included when performing the in vitro methods and in vivo methods described herein.
  • In vitro assay methods used for the immunogenic compositions of the present disclosure typically comprise contacting the biological sample with at least one source of the antigens described above and herein under conditions and for a time sufficient for an antibody in the sample to interact with the antigen source (i.e., mixing, combining, or in some manner permitting the biological sample and the antigen to interact).
  • An antibody present in the biological sample that specifically binds to the antigen can be detected using any one of the exemplary detection methods described herein and in the art for detecting antibody-antigen binding.
  • antibody-bound to the antigen may be detected using a reagent specific for a conserved region of the antibody, such as the Fc portion of the antibody, which reagent is typically selected depending on the source of the antibody (i.e., whether the antibody is from an animal, such as a mouse, rat, goat, or sheep, etc. or whether the antibody is from a human).
  • reagents typically comprise a detectable label, for example an enzyme, fluorescent label, luminescent label, or radioactive label.
  • Additional exemplary reagents include those that detect a specific isotype or class of antibody. Many such reagents may be obtained from commercial sources.
  • Protocol #2015-11 was approved by the Institutional Animal Care and Use Committees of the Infectious Disease Research Institute which operates under a currently approved Assurance #A4337-0l and USDA certificate #9l-R-006l.
  • Peptides of the present disclosure can be synthesized at Bio-Synthesis Inc (Lewiston, TX).
  • a peptide carrier of the present disclosure can be synthesized to comprise 5 IKKIEKR heptad repeats (e.g., SEQ ID NO: 19) in the coiled-coil domain followed by the PADRE TCE (SEQ ID NO: 71) and a TCE isolated from influenza H5N1 hemagglutinin (SEQ ID NO: 73).
  • a peptide carrier can comprise the same 5 heptad repeats followed by a TCE selected from Measles virus F2 protein (SEQ ID NO: 75) and Hepatitis B surface antigens (SEQ ID NO: 76).
  • peptide carrier of the present disclosure can comprise three overlapping TCEs isolated from tetanus toxoid (SEQ ID NO: 67).
  • Peptides synthesized with linear BCEs may comprise 4 IKKIEKR heptad sequences (e.g., SEQ ID NO: 18) and 2 TCEs (see e.g., FIG. 12).
  • Peptides containing BCEs covalently bound to Lys residues in the carrier sequence may be synthesized through selective deprotection of pre-specified Lys residues, which allows the side chain amine to react with the carboxylic acid at the C-terminus of the BCE peptide using standard amino acid coupling chemistry.
  • the amyloid-b BCE (SEQ ID NO: 261) can be linked to the N- and C- termini of a peptide monomer with Gly-Gly-Pro or Pro-Gly-Gly linkers, respectively.
  • the Tau BCE (SEQ ID NO: 262) can be linked to the N- and C-termini of a peptide monomer with a Pro linker.
  • the GnRH BCE (SEQ ID NO: 263) can be linked to the C-terminus of a peptide monomer with a Gly linker.
  • the M2e for IAV (SEQ ID NO: 222), M2e for IBV (SEQ ID NO: 224), NA1 (SEQ ID NO: 208), NA2 (SEQ ID NO: 220), HA27-39 (SEQ ID NO: 201) and HA231-241 (SEQ ID NO: 207) can either be linked to the C-terminus or to Lys side-chains of a carrier peptide having alpha helical domain SEQ ID NO: 26 and TCE domain SEQ ID NO: 83.
  • HxA for H1N1 (SEQ ID NO: 183) and HxA for IBV (SEQ ID NO: 190) BCEs can be included on the N-terminus of a fusion polypeptide monomer having SEQ ID NO: 26 fused to SEQ ID NO: 83.
  • Circular Dichroism Circular Dichroism analyses of the immunogenic compositions (e.g., peptide-based vaccines) may be recorded from 190-270 nm on a Jasco J720
  • Peptide stock solutions (ca. 50 mM) may be prepared for CD Spectroscopy using PBS. Temperatures ranged from 5 to 95 °C in increments of 10 °C.
  • Dynamic Light Scattering analysis of the immunogenic compositions (e.g., peptide-based vaccines) for determining size distributions of assembled peptides may be measured by dynamic light scattering using a Zetasizer Nano (Malvern).
  • Analytical Ultracentrifugation can be performed by Alliance Protein Laboratories (San Diego, CA).
  • the peptide-based vaccine particles can be dissolved in PBS to ⁇ l mg/mL and in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (100 mM, 50 mM NaCl, pH 7.5) to concentration of about 0.5 mg/mL.
  • MOPS 3-(N-morpholino)propanesulfonic acid
  • Samples can be filtered through a 0.2 pm nylon membrane.
  • AUC can be performed by Alliance Protein Laboratories (San Diego, CA).
  • the samples can be loaded into a Beckman-Coulter ProteomeLab XL-A analytical ultracentrifuge. After equilibration at 20 °C the rotor can be brought to 60,000 rpm. Scans can be recorded every 4 min for -10 h. The data may be analyzed using SEDFIT (version 11.3). The resultant size distributions may be graphed and the peaks integrated using OriginLab Origin® version 9.0 (Northampton, MA).
  • Serum Ab titers can be determined by ELISA as reported. (See e.g., Clegg et al. Adjuvant solution for pandemic influenza vaccine production. Proc Natl Acad Sci U S A (2012) 109: 17585-17590. Serum samples may be serially diluted 3-fold from 1/100 in blocking buffer. Midpoint titers at half maximal absorbance can be calculated using GraphPad Prism (GraphPad Software, San Diego, CA).
  • Amyloid-beta-directed, GnRH-directed and influenza-directed (M2e, NA1, NA2, HA27-39 and HA231-241) Abs can be measured using cysteine- terminated synthetic peptides conjugated onto BSA using maleimide crosslinking chemistry. Titers to certain HA proteins (e.g., Helix A, HA27-39 and HA231-241) may be measured using recombinant influenza hemagglutinin.
  • Tau epitope-directed Abs can be measured using a 441 AA recombinant isoform of human Tau protein (rPeptide, Watkinsville GA, e.g., FIG. 16) and mouse testosterone can be measured using a commercial kit purchased from Abeam (Cambridge MA).
  • Binding capacity Serum can be pooled from each immunization group and aliquots (100 pL) may be spiked with serially diluted nicotine to achieve final nicotine concentrations of 0.01- 10000 mM. These samples were then subjected to equilibrium dialysis against an equal volume of IX phosphate-buffer saline (PBS) for 4 h (37 °C) using an HTD96b equilibrium dialysis setup (HTDialysis, Gales Ferry, CT). Aliquots from the sera and buffer sides of the dialysis membranes can be removed and analyzed by liquid chromatography-tandem mass spectrometry (LC- MS/MS) (Alturas, Moscow, ID). Unbound nicotine can be quantified by comparing peak intensities to an internal standard of d4-nicotine and a standard curve generated with a nicotine standard.
  • LC- MS/MS liquid chromatography-tandem mass spectrometry
  • Theoretical MHC II Population Coverage of Epitope Combinations The number of predicted high affinity binding CD4 TCEs that can be generated from peptide carriers comprising various TCE and/or BCE combinations (e.g., SEQ ID NO: 68 - SEQ ID NO: 74) can be calculated using the Immune Epitope Database (http://www.iedb.org/). A larger number of entries at a percentile ⁇ 1 implies an improved MHCII binding across a representative set of human haplotypes.
  • PBMCs or splenocytes from a host species of interest can be isolated and co-cultured with species-specific interleukin-2 and the desired T cell epitope for 10-14 days. These cultures may then be added to ELISPOT plates and co-incubated with the same T cell epitope for a three-day stimulation period.
  • the ELISPOT plates can be developed and the number of spots counted and compared to background.
  • the spots generated in the assay are predicted to correspond to the immunogenicity of the T cell epitope, and allow down-selection of the T cell epitopes with the best empirical performance.
  • the T cell epitopes selected using this in vitro screen can be input into the IEDB MHC Class II binding predictor as fusions of 2 or more TCEs in various orientations. These fusions can also be screened in vitro (e.g., ELISPOT). Combinations showing the best activity using this algorithm can be selected to build vaccines for in vivo testing.
  • Antibody Assays Serum samples are serially diluted 3-fold from 1/100 in blocking buffer (3% BSA in PBST) and assayed by ELISA using standard methodologies. Midpoint titers at half maximal absorbance are calculated using GraphPad Prism (GraphPad Software, San Diego, CA). Amyloid-beta and GnRH Abs are measured using cysteine-terminated synthetic peptides conjugated onto BSA using maleimide crosslinking chemistry. Tau antibodies (Abs) are measured using a 441 AA recombinant isoform of human Tau protein (rPeptide, Watkinsville GA). Mouse testosterone can be detected with a commercial kit purchased from Abeam
  • IgE Ab titers can be measured using mouse IgE (Thermo-Fisher) as a coating reagent and goat anti-mouse IgG Fc-HRP (Southern Biotech, Birmingham AL) as the secondary. Specificity of Ab binding and relative avidity to IgE can be measured by competition ELISA. Serial dilutions of murine IgE, IgG, or IgM can be preincubated with antisera for 1 hour and then added to wells coated with these Ab isotypes for an additional hour. Goat anti-mouse IgG Fc- HRP can be used for detection. ELISA signals can be fit with an inhibition regression algorithm and IC50S determined for each group using GraphPad Prism.
  • Free IgE in mice, or the amount of IgE unbound by anti-IgE Abs in serum, is measured by ELISA for its ability to bind receptor, where mFcsRI (NBS-C Bioscience, Austria) and goat anti-mlgE (Southern Biotech;
  • Helix A titers can be measured using recombinant HA from H1N1 A/California/07/2009, H3N2 A/Wisconsin/67/2005, H5N1
  • T Cell ELISPOT IFN-g (R&D Systems, Minneapolis, MN, USA) ELISPOT analyses can be conducted according to the manufacturer’s instructions. Briefly, splenocytes from immunized and unimmunized mice can be resuspended and serially diluted in RPMI media supplemented with 10% Fetal Bovine Serum (FBS) and L-glutamine.
  • FBS Fetal Bovine Serum
  • Resuspended splenocytes can be stimulated with 20 pg/mL peptide (e.g., peptide-dimer, peptide-trimer, etc.), media alone or Phorbol l2-myristate l3-acetate (PMA) and ionomycin for 48 h prior to development.
  • Spot images can be collected using ImmunoCapture 6.4 and analyzed with ImmunoSpot 5.0 on an automated ELISPOT plate reader (C.T.L. Seri3A Analyzer; Cellular Technology, Shaker Heights, OH, USA).
  • This example shows the determination of the physicochemical properties including the size and shape of the peptide carriers.
  • the helical structure of the peptide carrier (SEQ ID NO: 271) comprising an alpha-helical peptide with five heptad sequences in the coiled-coil domain (SEQ ID NO: 19) followed by the PADRE TCE (SEQ ID NO: 71) and a TCE isolated from influenza H5N1 hemagglutinin (SEQ ID NO: 73) linked to its C-terminus was determined using circular dichroism (CD).
  • Peptide stock solutions (ca. 50 mM) were prepared for CD Spectroscopy using PBS.
  • CD spectra were recorded in the wavelength range of 190-270 nm on a Jasco J720 spectropolarimeter using 10 mm path length cells (FIG. 2).
  • the peptide carrier showed a clear alpha-helical structure as indicated by the signal minima at 208 nm and 222 nm (FIG. 2A). At temperature above 65 °C the ratio of signal intensity at 222 nm and 208 nm indicated the formation of stable coiled-coil conformations.
  • the size distribution of the formed coiled-coil oligomers was determined using analytical ultracentrifugation (AUC). Three main species were found to sediment at 0.76, 1.77, and >3.0+ Svedberg (S) units, corresponding to peptide assemblies of 14% monomers ( ⁇ 7.5 kDa), 70% trimers ( ⁇ 22 kDa), and 16% higher-order assemblies that likely correspond to hexamers, nanomers, dodecamers, etc. (FIG. 2B). [0257] Thus, the peptide carrier possessed an alpha-helical secondary peptide structure, and assembled primarily into stable trimeric coiled-coil structures.
  • This example shows the determination of size distribution of the formed peptide oligomers.
  • the Zetasizer Nano instrument uses a 4 mW He-Ne laser (633 nm) and a fixed detection angle (173 °C).
  • the particle size distribution of the peptide assemblies using the peptide carrier having SEQ ID NO: 271 showed maximum peak intensities that correspond to particles sizes of 7, 22, and 350 nm, respectively.
  • the 7 nm assembly corresponded to the trimeric coiled-coil peptide, whereas the 22 and 350 nm assemblies corresponded to higher aggregates (FIG. 2C).
  • the particle size distribution of the peptide assemblies using the peptide carrier having SEQ ID NO: 275 and SEQ ID NO: 277 showed maximum peak intensities that correspond to particles sizes of 20 nm and 15 nm, respectively (FIG. 3B and FIG. 3C). These correspond to higher-order assemblies.
  • This nanoparticle size distribution was verified for the peptide carrier having SEQ ID NO: 275 using AUC, which exhibited an absence of the trimeric peak at 1.72S (FIG. 3A).
  • the nicotine hapten (average 4 nicotine haptens/peptide) was linked to the alpha-helical peptide domain of the three peptide carrier peptides (SEQ ID NO: 272-274) via a linker that was covalently attached to the G position of the nicotine molecule (see e.g., FIG. 9A).
  • Antibody responses to the nicotine hapten were induced by nicotine-carrying peptides with SEQ ID NO: 272, SEQ ID NO: 273, and SEQ ID NO: 274 comprising either one PADRE CD4 T cell epitope peptide (SEQ ID NO: 71), one diphtheria CD4 T cell epitope peptide (SEQ ID NO: 72), or a fusion epitope peptide (SEQ ID NO: 81) of both the diphtheria and the PADRE CD4 T cell epitope.
  • SEQ ID NO: 272 SEQ ID NO: 273, and SEQ ID NO: 274 comprising either one PADRE CD4 T cell epitope peptide (SEQ ID NO: 71), one diphtheria CD4 T cell epitope peptide (SEQ ID NO: 72), or a fusion epitope peptide (SEQ ID NO: 81) of both the diphtheria and the PADRE CD4 T cell epitope.
  • This example demonstrates activity of the indicated peptides which comprise TCE domains having SEQ ID NO: 83, SEQ ID NO: 67, SEQ ID NO: 81, and SEQ ID NO: 82 can be predicted from the high affinity binding modes using the Immune Epitope Database
  • FIG. 5A shows calculation results that predicted that the TCE domain with SEQ ID NO: 83 may have the best activity across a polymorphic human population in comparison to the peptide carriers with SEQ ID NO: 67, SEQ ID NO: 81, and SEQ ID NO: 82.
  • FIG. 5B illustrates a graph of antibody titers induced in outbred mice by the tested CD4 T cell epitope peptides. Animals immunized with peptide carrier having SEQ ID NO: 275 expressed the best titer.
  • FIG. 5C shows a graph of antibody affinity induced in outbred mice by the tested CD4 T cell epitope peptides. Animals immunized with peptide carrier having SEQ ID NO: 275 also expressed the best affinity.
  • FIG. 6 shows the activity of a variety of CD4 TCE sequences selected through in silico methods (SEQ ID NOS: 66-80, 88-102, 112-136, 141-167, 83, 75, 76) in naive cynomolgus macaque peripheral blood mononuclear cells (PBMCs) in a predictive ELISPOT assay used to build a vaccine for cynomolgus macaques.
  • PBMCs peripheral blood mononuclear cells
  • ELISPOT assay used to build a vaccine for cynomolgus macaques.
  • CD4 TCEs were chosen from pertinent publications. These sequences were entered into the Immune Epitope Database and Analysis Resource (iedb.org) MHC Class II binding predictor.
  • results from this in silico screen were used to identify CD4 T cell epitopes of interest (SEQ ID NOs: 66-80, 88-102, 112-136, 141-167, 83, 75, 76).
  • This set of epitopes was further culled based on activity using an ELISPOT-based in vitro assay.
  • PBMCs or splenocytes from a host species of interest were isolated and co cultured with interleukin-2 and the desired T cell epitope for 10-14 days. These cultures were added to ELISPOT plates and co-incubated with the same T cell epitope for a three-day stimulation period. The ELISPOT plates were developed and the number of spots counted and compared to background.
  • the spots generated in the assay were predicted to correspond to the immunogenicity of the T cell epitope, and allow down-selection of the T cell epitopes with the best empirical performance.
  • the T cell epitopes selected using this in vitro screen can be input into the IEDB MHC Class II binding predictor as fusions of 2 or more TCEs in various orientations. Combinations showing the best activity using this algorithm can be tested using another in vitro screen (e.g., ELISPOT) or selected to build vaccines for in vivo testing. This shows that this class of peptide carrier vaccines can be rationally designed by ranking CD4 TCE activities through in silico and in vitro methods.
  • FIG. 7A shows that peptide carriers with SEQ ID NO: 275 that were synthesized by solid phase with 3 copies of a lysine-nicotine building block hapten (FIG. 7A, SEQ ID NO: 275 x 3/6HA) positioned along the alpha-helical heptad repeat (FIG. 7B) elicited stronger Ab responses in mice than a peptide carrier with 1 copy of the lysine-nicotine building block (SEQ ID NO: 275 x 1/6HA) and a vaccine prepared by conventional chemistry methods (SEQ ID NO: 275 wet, FIG. 7C-FIG. 7E).
  • FIG. 7C-FIG. 7E show that the peptide vaccine that was synthesized by solid phase chemistry (both SEQ ID NO: 275 x 1 BB and SEQ ID NO: 275 x 3 BB) generated higher antibody responses and exhibited higher binding capacities than the peptide vaccines that were synthesized using conventional synthesis techniques.
  • This example shows that peptide carriers with SEQ ID NO: 275 that were synthesized by solid phase with 3 copies of a 3’ lysine-nicotine building block hapten and 3 copies of a 6HA lysine-nicotine building block hapten (FIG. 8, SEQ ID NO: 275 x 3/3’+3/6HA) positioned along the alpha-helical heptad repeat elicit Ab responses in mice specific to both the 3’ and 6HA haptens.
  • FIG. 8A illustrates the placement of 6 nicotine hapten-lysine building blocks along the peptide following solid-phase peptide synthesis.
  • Three haptens contained a linker attached to the 3’ position of the nicotine molecule and 3 haptens contained a linker attached to the 6 position of nicotine.
  • FIG. 8B shows that this peptide induced antibody titers specific to both the 6HA hapten and the 3’ hapten.
  • mice were immunized with peptides synthesized with just the 3’ hapten (SEQ ID NO: 275 x 3/3’) or the 6HA hapten (SEQ ID NO: 275 x 3/6HA). Serum was collected on day 35 and assayed by ELISA using BSA conjugated with the 6HA hapten or the 3’ hapten as coating antigens.
  • the peptide having SEQ ID NO: 275 x 3/3’+3/6HA induced antibody titers that were equivalent to the positive control peptide (SEQ ID NO: 275 x 3/6HA) and greater than the negative control peptide (SEQ ID NO: 275 x 3/3’).
  • SEQ ID NO: 275 x 3/6HA positive control peptide
  • SEQ ID NO: 275 x 3/3 negative control peptide
  • This example shows that a multivalent, peptide-based nicotine vaccine yields improved nicotine binding capacity compared to a monovalent and bivalent peptide-based nicotine vaccine.
  • the peptide vaccine was created by conjugating three structurally distinct nicotine haptens that activate different populations of B cells (FIG. 9A) to the peptide carrier with SEQ ID NO: 275 via linkers at the G, 3’ and 6 position of nicotine to yield three peptide-nicotine conjugates: l’-SEQ ID NO: 275, l’,3’- SEQ ID NO: 275, and l’,3’,6HA- SEQ ID NO: 275. Each peptide monomer was conjugated to 3-4 nicotine hapten copies.
  • mice Female CD-l mice were immunized with the peptide vaccine adjuvanted with GLA-SE on day 0, day 21, and day 42. As shown in FIG. 9B, the nicotine binding capacities increase from the monovalent (2000 ng/mL) to the bivalent (3500 ng/mL) to the trivalent (5200 ng/mL). These results establish the utility of using multivalent peptide vaccines to target multiple disparate B-cell epitopes.
  • An epitope recognized by several broadly neutralizing mAbs includes Helix A, the first alpha helical domain within the HA stem.
  • Helix A the first alpha helical domain within the HA stem.
  • This example demonstrates that the secondary structure of the Helix A epitope was successfully recreated by appending the linear Helix A H1N1 sequence on the helical N-terminus of an alpha-helical peptide domain (e.g., SEQ ID NO: 299) with two universal CD4 TCEs (e.g., SEQ ID NO: 83) attached to its C-terminus.
  • the example shows the construction, characterization and in vivo evaluation of a SAPN-based vaccine according to the present disclosure containing a Helix A epitope derived from H1N1 influenza virus that is recognized by broadly neutralizing monoclonal antibodies.
  • FIG. 10A shows the Helix A epitope sequence expressed by H1N1 viruses as compared to the alpha helix A epitope expressed by drifted H7N4, H3N2, H5N1 and Type B influenza viruses.
  • the bold underlined amino acids identify residues within viral hemagglutinin helix A that are outward-facing and recognized by broadly neutralizing monoclonal antibodies.
  • the conserved H1N1 influenza helix A epitope peptide (SEQ ID NO: 183) that was used in this study contained all of the underlined amino acid residues that can be recognized by broadly
  • the peptide carrier having SEQ ID NO: 278 comprised of 4 heptad sequences in the coiled-coil domain fused to TCEs from Measles virus F2 protein (SEQ ID NO: 75) and Hepatitis B surface antigens (SEQ ID NO: 76) via its C-terminus, and the conserved H1N1 influenza helix A epitope peptide (SEQ ID NO: 183) was linked to the N-terminus. The construction of this peptide did not involve a linker sequence to ensure that the helicity of the carrier would not be interrupted and would continue into the BCE sequence, forcing the BCE into a helix.
  • the peptide carrier (SEQ ID NO: 278) was synthesized and characterized by DLS, which showed a diameter of ⁇ 27 nm (FIG. 10C).
  • FIG. 10E Further analysis established that the vaccine induced a strong Thl CD4 T cell response, which is a property of TLR4 activation by GLA adjuvant (FIG. 10E - FIG. 10G).
  • Titers of total IgG, IgGl, and IgG2a are indicated (FIG. 10F), as well as the ratio between IgG2a and IgGl isotypes (FIG. 10G). Values greater than 1 are indicative of a Thl response, which is induced by the TLR4 agonist GLA.
  • influenza challenge experiments were performed by infecting mice intranasally with 1 OxLD o dose of A/C A/07/09 in 50 pL PBS. Mice were monitored for weight loss and other signs of virus induced morbidity daily and sacrificed if weight loss exceeded 20% of initial body weight. As shown in FIG. 10H,
  • FIG. 11 A The sequences of the peptide carriers with SEQ ID NO: 279 and SEQ ID NO: 280 are shown in FIG. 11 A.
  • the alpha-helical domain with outward-facing negatively charged glutamic acid residues (E) e.g., SEQ ID NO: 46
  • SEQ ID NO: 67 the tetanus toxoid TCE
  • SEQ ID NO: 222 the Influenza A M2e epitope
  • the alpha-helical domain with outward-facing positively charged lysines (e.g., SEQ ID NO: 34) is followed by the tetanus toxoid T cell epitope (SEQ ID NO: 67) and the Influenza A M2e epitope (SEQ ID NO: 222).
  • K alpha-helical domain with outward-facing positively charged lysines
  • SEQ ID NO: 34 tetanus toxoid T cell epitope
  • SEQ ID NO: 222 Influenza A M2e epitope
  • the negative charges in the M2e epitope can hydrogen bond with the positively-charged surface lysine residues, which buries the epitope in the peptide nanoparticle and reduces its ability to bind B cells.
  • a peptide-based vaccine containing M2e BCEs (e.g., SEQ ID NO: 222) attached along the length of the alpha-helical coiled-coil peptide domain can induce strong antibody responses.
  • the peptide carrier with SEQ ID NO: 282 was synthesized using automated peptide synthesizers. Specified Lys residues were selectively deprotected for attachment with the M2e BCE peptide. The C-terminus carboxylic acid of the BCE peptide was attached to the amine in the Lys side chains through standard amino acid coupling chemistry.
  • FIG. 12A illustrates the attachment locations of 2 M2e BCEs (SEQ ID NO: 222) on the peptide monomer with SEQ ID NO: 282.
  • FIG. 12B illustrates the antibody titers measured 30 days after outbred CD-l mice were injected with PBS control or 5 pg of peptide carrier.
  • This example illustrates that a peptide-based vaccine containing novel hemagglutinin BCEs attached along the length of the alpha-helical coiled-coil peptide domain can induce antibodies that bind recombinant hemagglutinin and protect mice from viral challenge.
  • FIG. 13B illustrates the antibody titers to a BSA conjugate and recombinant influenza B hemagglutinin (B/Malaysia/2506/04) measured 30 days after outbred CD-l mice were injected with PBS control or 10 pg of peptide carrier, showing that structural and sequence information can be used to predict BCE activity and BCE activity can be verified by determining whether antisera binds native protein.
  • mice were challenged with 5xLD50 of B/Florida/04/06 and monitored for survival with a weight loss cutoff of 80%. As shown in FIG. 13C-FIG. 13D, immunized mice were successfully protected against virus challenge. Given the strong conservation in these sequences, these vaccines may be able to prevent infection from all Influenza B viruses. These results also demonstrate that these peptide carriers can be used for B cell epitope discovery by empirically verifying the activity of BCEs predicted using protein sequence and structure information.
  • Peptides were made with two copies of M2e from IAV (SEQ ID NO: 282), NA1 (SEQ ID NO: 285), NA2 (SEQ ID NO: 286), M2e from IBV (SEQ ID NO: 287), HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284). Peptides were synthesized with a single N-terminus copy of either the H1N1 HxA BCE (SEQ ID NO: 278) or the influenza B HxA BCE (SEQ ID NO: 288).
  • Female CD-l mice were immunized with one peptide (10 pg) adjuvanted with GLA-SE on day 0 and day 21.
  • mice were challenged with 5xLD50 of A/California/07/2009(HlNl) or B/Florida/04/06 (depending on whether the BCE was IAV-specific, IBV-specific, or universal) and monitored for survival with a weight loss cutoff of 80%.
  • Immunized mice were successfully protected against virus challenge (FIG. 14), showing that a variety of linear and helical epitopes can successfully be presented with this class of peptides. Given the strong conservation in these sequences, these vaccines may elicit broad protection against all A or B Type influenza strains.
  • Peptide carriers with SEQ ID NO: 278, 282 and 285 were synthesized using automated peptide synthesizers using the methodologies described elsewhere herein.
  • the peptides contained the Measles/Hepatitis B fusion TCE domain (SEQ ID NO: 83) and two copies of the M2eA sequence (SEQ ID NO: 222), two copies of the NA peptide sequence (SEQ ID NO: 208) or a single copy of the Hl Helix A (HxA) peptide fused to the N-terminus of the carrier peptide (SEQ ID NO: 183).
  • mice Female CD-l mice were immunized with either the M2e (SEQ ID NO: 282), NA (SEQ ID NO: 285), or HxA (SEQ ID NO: 278) peptides singly (10 pg) or admixtures of M2e+NA or M2e+NA+HxA peptides (10 pg each peptide) adjuvanted with GLA-SE on day 0 and day 21.
  • mice were challenged with 5xLD50 of A/California/07/2009(HlNl) and monitored for survival with a weight loss cutoff of 80%. As shown in FIG.
  • This example describes the construction and in vivo evaluation of a peptide-based vaccine for Alzheimer’s disease. These functional vaccines were tested in murine CD-l models.
  • Tau protein (SEQ ID NO: 261) (FIG. 16) or Amyloid-beta (SEQ ID NO: 262) epitopes were linked to either the N- or C-terminus of an alpha-helical peptide resulting in peptide carriers with SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, and SEQ ID NO: 292.
  • DLS analysis demonstrated that appending terminal BCEs to the alpha-helical peptide had marginally affected nanoparticle size (FIG. 17A-FIG. 17D), although the peak intensities of the N-Tau protein (20 nm; FIG. 17A) and C-Tau protein (12.5 nm; FIG.
  • the peptide vaccines were formulated with GLA-SE adjuvant and injected into outbred CD-l mice. Both pairs of amyloid-b and Tau peptides successfully induced strong antibody titers after the first injection of the peptide vaccine on day 35 (FIG. 18A-FIG. 18D). There were no significant differences in response observed between the placement of the BCE at either the N- or C-termini. These results confirm that nanoparticle-forming peptides synthesized with clinical candidate BCE’s can induce Ab responses in mice. Importantly, both the Tau and amyloid-beta peptides induced strong antibody responses in outbred mice with no apparent difference in immunogenicity when the BCE was inserted at either terminus. These results show that the universal T cell epitopes in the vaccine are effective in mice with a heterogeneous MHC background and that the Ab epitopes are readily accessible for B cell engagement, regardless of their attachment point.
  • the peptide vaccine was synthesized using the alpha-helical peptide having SEQ ID NO: 293 comprising a 10 amino acid gonadotropin-releasing hormone (GnRH, SEQ ID NO: 263) sequence via its C-terminus.
  • the peptides formed nanoparticles with an average size of about 20 nm.
  • mice Male CD-l mice were immunized with the peptide vaccine adjuvanted with GLA-SE on day 0, day 21, and day 42. As shown in FIG. 19A, anti-GnRH Ab titers reached maximal levels following the first boost of the peptide carrier having the sequence set forth in SEQ ID NO: 293 and remained high throughout the course of the experiment. To confirm that these antibodies may prevent normal testes function, their fertility was measured by breeding each male mouse with 4 female mice.
  • This example shows multivalent, peptide-based vaccines that offer protection against the four DENV serotypes DENV1, DENV2, DENV3, and DENV4.
  • the peptide carriers used for this study comprise an alpha-helical peptide domain (e.g., SEQ ID NO: 16 - SEQ ID NO: 65), two TCEs (e.g, SEQ ID NO: 66 - SEQ ID NO: 182), and a BCE peptide that is either an E protein epitope peptide (SEQ ID NO: 245), anon- structural protein 1 (NS1) epitope peptide (SEQ ID NO:246) or a modified derivative of this non- structural protein 1 (NS1) epitope peptide (SEQ ID NOs: 247).
  • SAPN anon- structural protein 1
  • SAPN a modified derivative of this non- structural protein 1
  • SANP -based vaccines comprising the BCE with SEQ ID NO: 14 - SEQ ID NO: 16 are tested for their ability to induce antibody responses against the four DENV serotypes DENV1, DENV2, DENV3, and DENV4 in vivo.
  • This example describes a multivalent, peptide-based vaccine that offers broad protection against the seven reported HCV genotypes.
  • Peptide carriers comprising an alpha-helical peptide domain (e.g., SEQ ID NO: 16 - SEQ ID NO: 65) and an HCV-derived epitope peptides comprising an epitope peptide selected from SEQ ID NO: 248 - SEQ ID NO: 258.
  • the resulting peptide-based vaccines are tested for their ability to induce antibody responses against the seven reported HCV genotypes in vivo.
  • This example shows a multivalent, peptide-based vaccine that offers simultaneous protection against HSV-l and HSV-2 utilizing B cell epitope peptides that are conserved between both strains.
  • Peptide carriers comprising the alpha-helical peptide monomer (e.g., SEQ ID NO: 16 - SEQ ID NO: 65), a tetanus toxoid TCE (e.g., SEQ ID NO: 66 - SEQ ID NO: 70), a combination of the HSV-l and HSV-2 specific glycoprotein epitope peptides (SEQ ID NO: 232 - SEQ ID NO: 244) are tested for their ability to induce antibody responses against those glycoprotein epitopes in vivo.
  • alpha-helical peptide monomer e.g., SEQ ID NO: 16 - SEQ ID NO: 65
  • a tetanus toxoid TCE e.g., SEQ ID NO: 66 - SEQ ID NO: 70
  • SEQ ID NO: 232 - SEQ ID NO: 244 a combination of the HSV-l and HSV-2 specific glycoprotein epitope peptides
  • This example shows a multivalent, peptide-based vaccine that offers protection against RSV Type A (RSVA) and B (RSVB) utilizing B cell epitope peptides that are conserved between both strains.
  • RSVA RSV Type A
  • RSVB B cell epitope peptides
  • Peptide carriers comprising the alpha-helical peptide monomer (e.g., SEQ ID NO: 16 - SEQ ID NO: 65), a tetanus toxoid TCE (e.g, SEQ ID NO: 66 - SEQ ID NO: 70), and a RSVA and RSVB specific epitope peptide with either SEQ ID NO: 259 (KNYIDKQLLPIVNK) or SEQ ID NO: 260 (KNYINNQLLPIVNQ) are tested for their ability to induce antibody responses against those glycoprotein epitopes in vivo.
  • alpha-helical peptide monomer e.g., SEQ ID NO: 16 - SEQ ID NO: 65
  • a tetanus toxoid TCE e.g, SEQ ID NO: 66 - SEQ ID NO: 70
  • RSVA and RSVB specific epitope peptide with either SEQ ID NO: 259 (KNYIDKQLLPIVNK
  • a peptide carrier comprising an alpha-helical domain comprising 5 IKKIEKR heptad repeats (e.g., SEQ ID NO: 19), and a TCE domain comprising the Measles virus F2 protein TCE (SEQ ID NO: 75) and the Hepatitis B surface TCE (SEQ ID NO: 76) was further modified by linking a murine-specific Peptide Y BCE (SEQ ID NO: 264) to the C-terminus of the peptide carrier (SEQ ID NO: 294).
  • the peptide Y epitope peptide was derived from the Cs3 domain of murine IgE, which serves as an analogue for human IgE in this animal model.
  • FIG. 22A shows that the anti-IgE antibodies described in FIG. 21 were able to reduce the concentration of free IgE ⁇ l0-fold in 6 of 10 mice and ⁇ l 000-fold in 4 of 10 mice.
  • FIG. 22B shows that the polypeptide with SEQ ID NO: 294 could also be used to inhibit acute IgE-mediated anaphylaxis, confirming its therapeutic potential.
  • FIG. 22C shows that the peptide vaccine having the amino acid sequence set forth in SEQ ID NO: 294 forms nanoparticles with an average DLS diameter of -15 nm.
  • Peptide stock solutions were prepared for CD Spectroscopy using 50 mM phosphate- buffered saline (PBS). Spectra were recorded from 190-270 nm on a Jasco J720
  • spectropolarimeter Easton, MD using 10 mm path length cells. Temperatures ranged from 5 to 95 °C in increments of 10 °C.
  • Analytical ultracentrifugation was performed by Alliance Protein Laboratories (San Diego, CA). The peptide was dissolved in PBS to ⁇ l mg/mL and the peptide was dissolved in aqueous MOPS (100 mM, 50 mM NaCl, pH 7.5) to ⁇ 0.5 mg/mL. Samples were filtered through a 0.2 pm nylon membrane. The samples were loaded into a Beckman-Coulter ProteomeLab XL-A analytical ultracentrifuge (Brea, CA).
  • the structure of the peptide with SEQ ID NO: 275 was constructed, analyzed, and rendered with Chimera software using the trimeric coiled-coil construct without T cell epitopes as a template.
  • the T-cell epitopes with the sequence set forth in SEQ ID NO: 83 were modeled using the PEP-FOLD3 44 server and coupled to the trimeric coiled-coil carrier at the C-terminus using the structure building tools in Chimera to form peptide bonds.
  • the Qwik-MD tool in VMD was used for molecular dynamics setup, solvation/ionization, and a 1,000 step equilibration.

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Abstract

Disclosed herein are compositions of self-assembling nanoparticle-based vaccines comprising carrier polypeptide monomers that can comprise an alpha-helical domain, at least one T cell epitope peptide, and at least one B cell epitope peptide. Further disclosed herein are methods of using these peptide-based vaccines for the prophylaxis and treatment of a variety of infectious, chronic, and autoimmune diseases including influenza, neurodegenerative diseases, cancer, and allergies.

Description

SYNTHETIC CARRIER COMPOSITIONS FOR PEPTIDE VACCINES
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application Nos. 62/864,024 filed June 20, 2019, and 62/723,909 filed August 28, 2018, which applications are incorporated herein by reference in their entirety for all purposes.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under DA041162 awarded by the National Institutes of Health and 6 R43IP001108-01 awarded by the Centers of Disease Control and Prevention. The government has certain rights in the invention.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted
electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 23, 2019, is named 53723-702_60l_SL.txt and is 956,380 bytes in size.
BACKGROUND
[0004] Despite recent advances in vaccine development, there remains a global need for novel, safe, and effective vaccines for a variety of diseases. In particular, vaccine products that provide broad protection and that may be inexpensive to produce and easily distributable worldwide create a high demand for indications including infectious as well as chronic diseases. For instance, the influenza virus is a highly contagious pathogen and, due to its rapid mutation rates, creates a demand for vaccines that provide a more effective and sustained protection.
Furthermore, chronic degenerative diseases such as Alzheimer’s disease currently lack appropriate prophylactic and therapeutic treatment options. Consequently, novel and innovative vaccine platforms may be beneficial to address the existing global health needs.
SUMMARY
[0005] The present disclosure provides compositions and methods of use for peptide-based vaccines.
[0006] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. [0007] In various aspects, the present disclosure provides a composition comprising: (a) an alpha-helical peptide domain; (b) at least one CD4 T cell epitope peptide; and (c) at least one B cell epitope peptide selected from an immunogenic protein or peptide, carbohydrate, or lipid that mediates a physiological condition or disease including neural degenerative diseases, allergy, and autoimmunity, wherein each of the alpha-helical peptide domain, T cell epitope peptide, and B cell epitope peptide are associated. In some aspects, the B cell epitope peptide is selected from SEQ ID NO: 183 - SEQ ID NO: 270.
[0008] In various aspects, the present disclosure provides a composition comprising: (a) an alpha-helical peptide domain; (b) at least one CD4 T cell epitope peptide selected from SEQ ID NO: 66 - SEQ ID NO: 182; and (c) at least one B cell epitope peptide, wherein each of the alpha- helical peptide domain, T cell epitope peptide, and B cell epitope peptide are associated. In some aspects, the alpha-helical peptide domain comprises at least one heptad repeat with an amino acid sequence according to the general formula: [X1X2X3X4X5X6X7]n (SEQ ID NO: 295), wherein X1 and X4 are each independently selected from I, L, V, A, F, Y, W, N, and Q; X2, X3, X6 are independently selected from the amino acids K, R, E, D, H, S, N, Q, A, T, and C; X5, X7 are independently selected from the amino acids K, R, E, D, and H; and n is any number from 1 to 10. In some aspects, the alpha-helical peptide domain can comprise an amino acid sequence according to any one of the following general formulas:
Figure imgf000004_0001
Y 1 Y 2[X1X2X3X4X5X6X7]nY 3 Y 4 Y 5 Y 6 Y 7 (SEQ ID NO: 5); and
Y1[X1X2X3X4X5X6X7]nY 2 Y 3 Y 4 Y 5 Y 6 Y 7 (SEQ ID NO: 6),
wherein: each Y1, Y2, Y3, Y4, Y5, Y6, and Y7 is independently selected from the amino acids I, L, V, A, K, R, E, S, T, P, G, C, N, Q, W, and D; each X1, X4 are independently selected from the amino acids I, L, V, A, F, Y, W, N, Q; each X2, X3, X6 are independently selected from the amino acids K, R, E, D, H, S, N, Q, A, T, C; each X5, X7 are independently selected from the amino acids K, R, E, D, H; and each n is independently any number from 1 to 10. In some aspects, the alpha-helical peptide domain comprises an amino acid sequence with at least 80% identity to IKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 18),
IKKIEKRIKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 19),
KKIEKRIKKIEKRIKKIEKRIKKIEKRI (SEQ ID NO: 30),
KIEKRIKKIEKRIKKIEKRIKKIEKRIK (SEQ ID NO: 27), IEKRIKKIEKRIKKIEKRIKKIEKRIKK (SEQ ID NO: 28),
EKRIKKIEKRIKKIEKRIKKIEKRIKKI (SEQ ID NO: 31),
KRIKKIEKRIKKIEKRIKKIEKRIKKIE (SEQ ID NO: 32),
RIKKIEKRIKKIEKRIKKIEKRIKKIEK (SEQ ID NO: 33),
DEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 44), or
DETEER TEETEER TEETEER TEETEER TEETEER TEE (SEQ ID NO: 45). In some aspects, the alpha- helical peptide domain comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45. In some aspects, the alpha- helical peptide domain comprises an amino acid sequence with 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
[0009] In various aspects, the present disclosure provides a composition comprising: (a) an alpha-helical peptide domain comprising an amino acid sequence with at least 80% identity to, at least 82.5% identity to, at least 85% identity to, at least 87.5% identity to, at least 90% identity to, at least 92.5% identity to, at least 95% identity to, at least 97.5% identity to, or at least 99% identity to identity to IKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 18),
IKKIEKRIKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 19),
KKIEKRIKKIEKRIKKIEKRIKKIEKRI (SEQ ID NO: 30),
KIEKRIKKIEKRIKKIEKRIKKIEKRIK (SEQ ID NO: 27),
IEKRIKKIEKRIKKIEKRIKKIEKRIKK (SEQ ID NO: 28),
EKRIKKIEKRIKKIEKRIKKIEKRIKKI (SEQ ID NO: 31),
KRIKKIEKRIKKIEKRIKKIEKRIKKIE (SEQ ID NO: 32),
RIKKIEKRIKKIEKRIKKIEKRIKKIEK (SEQ ID NO: 33),
DEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 44), or
DEIEERIEEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 45); (b) at least one T cell epitope peptide or a B cell epitope peptide, or a combination thereof; and (c) a target antigen, wherein the alpha-helical peptide domain, T cell epitope peptide, and B cell epitope are associated. In some aspects, the alpha-helical peptide domain comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45. In some aspects, the alpha-helical peptide domain comprises an amino acid sequence with 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45. In some aspects, the at least one CD4 T cell epitope peptide is an immunogenic peptide fragment selected from the group consisting of diphtheria toxoid peptide epitopes, measles morbillivirus fusion glycoprotein F peptide epitopes, a pan DR epitope (PADRE) peptide, influenza-derived epitope peptides, hepatitis B and C virus epitope peptides, tetanus toxoid peptide epitopes, P. falciparum peptide epitopes, gamma 2ab peptide epitopes, GAD65 peptide epitopes, plasmodium peptide epitopes, polio peptide epitopes, Pseudomonas peptide epitopes, Vaccinia peptide epitopes, Streptococcus peptide epitopes, Yellow Fever peptide epitopes, Coxiella peptide epitopes, Yrsenia pestis peptide epitopes, RSV peptide epitopes, SSP2.61 peptide epitopes, ESAT6 peptide epitopes, tuberculosis peptide epitopes, ebola peptide epitopes, HPV peptide epitopes, anthrax peptide epitopes, varicella peptide epitopes, HSV peptide epitopes, and VEEV peptide epitopes. In some aspects, the at least one CD4 T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, at least two CD4 T cell epitope peptides are linked in tandem. In some aspects, the at least one CD4 T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182. In some aspects, the at least one CD4 epitope peptide has a sequence set forth in SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 157, SEQ ID NO: 164, SEQ ID NO: 165, or SEQ ID NO: 166, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the B cell epitope peptide is a foreign antigen comprised of an immunogenic protein or peptide, carbohydrate, lipid, or small molecule; a host-derived antigen comprised of an immunogenic protein or peptide, carbohydrate, or lipid that mediates a physiological condition or disease including infectious diseases, neural degenerative diseases, allergy, autoimmunity, and cancer. In some aspects, the B cell epitope peptide is selected from SEQ ID NO: 183 - SEQ ID NO: 270, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the at least two CD4 T cell epitope peptides are linked in tandem. In some aspects, the at least one CD4 epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the at least one CD4 epitope peptide has a sequence of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:
70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 157, SEQ ID NO: 164, SEQ ID NO: 165, or SEQ ID NO: 166, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the target antigen is a small molecule, a peptide, a polysaccharide, a glycolipid, or a lipid. In some aspects, the small molecule is nicotine. In some aspects, the N-terminus of the at least one T cell epitope peptide is linked to the C-terminus of the alpha-helical peptide monomer. In some aspects, the N-terminus of the at least one B cell epitope peptide is linked to the C-terminus of the at least one T cell epitope peptide or the at least one B cell epitope peptide is linked to the N-terminus of the peptide carrier, or any combination thereof. In some aspects, the N-terminus of the at least one B cell epitope peptide is linked to the C-terminus of the at least one T cell epitope peptide or the at least one B cell epitope peptide is linked to the N-terminus of the peptide carrier, or is linked to the alpha-helical peptide monomer, or any combination thereof. In some aspects, the at least one B cell epitope peptide is linked along the length of the alpha-helical peptide domain via a non-terminal amino acid. In some aspects, the at least one B cell epitope peptide is linked to the N-terminus, C-terminus, or along the length of the alpha-helical peptide domain via an amino acid. In some aspects, the at least one B cell epitope peptide is linked to the alpha-helical peptide domain via an unnatural amino acid. In some aspects, the at least one B cell epitope peptide is linked to the alpha-helical peptide domain via a non-amino acid chemical functionality. In some aspects, the alpha-helical peptide domain comprises at least 1 heptad repeat. In some aspects, the alpha-helical peptide domain comprises at least 2 heptad repeats. In some aspects, the alpha-helical peptide domain comprises at least 3 heptad repeats. In some aspects, the at least one T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the at least one CD4 T cell epitope peptides is selected from SEQ ID NO: 66 - SEQ ID NO: 182. In some aspects, the at least one T cell epitope peptide comprises the amino acid sequence set forth in SEQ ID NO: 81 - SEQ ID NO: 87, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the at least one B cell epitope peptide is an immunogenic fragment of a microbial antigen. In some aspects, the microbial antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a protozoan antigen. In some aspects, the microbial antigen is a conserved antigen within a divergent family of bacteria, fungi, or protozoan antigen. In some aspects, the viral antigen is an influenza virus antigen. In some aspects, the influenza virus antigen is a hemagglutinin antigen, an M2 ectodomain antigen, a neuraminidase antigen, or a nucleoprotein antigen. In some aspects, the hemagglutinin antigen is a conserved influenza hemagglutinin Helix A epitope peptide. In some aspects, the influenza hemagglutinin Helix A epitope peptide comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 183 - SEQ ID NO: 191, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the influenza M2 ectodomain antigen comprises all or part of the amino acid sequence set forth in SEQ ID NO: 222 - SEQ ID NO: 227, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the influenza hemagglutinin antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 192 - SEQ ID NO: 207, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the influenza neuraminidase antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 208 - SEQ ID NO: 221, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the at least one B cell epitope peptide is an immunogenic peptide or a peptide fragment of a hormone antigen. In some aspects, the hormone antigen is a GnRH antigen. In some aspects, the GnRH antigen comprises the amino acid sequence set forth in SEQ ID NO: 263. In some aspects, the at least one B cell epitope peptide is an immunogenic peptide fragment of a neurodegenerative disease antigen. In some aspects, the neurodegenerative disease antigen is an Alzheimer’s disease antigen, a
Parkinson’s disease antigen, or a Huntington’s disease antigen. In some aspects, the Alzheimer’s disease antigen is a Tau antigen or an amyloid beta (Ab) antigen. In some aspects, the Tau antigen comprises the amino acid sequence set forth in SEQ ID NO: 262. In some aspects, the amyloid beta (Ab) antigen comprises the amino acid sequence set forth in SEQ ID NO: 261. In some aspects, the Parkinson’s disease antigen is an alpha-synuclein antigen. In some aspects, the at least one B cell epitope peptide is an immunogenic peptide fragment of a tumor antigen. In some aspects, the tumor antigen is derived from a member of the receptor tyrosine kinase family or a member of the human epidermal growth factor receptor family. In some aspects, the at least one B cell epitope peptide is an immunogenic peptide fragment derived from an immunoglobulin E (IgE). In some aspects, the IgE is human IgE. In some aspects, the at least one B cell epitope peptide is an immunogenic peptide fragment derived from the Ce3 domain of human IgE. In some aspects, the at least one B cell epitope peptide comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 264-267, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the at least one T cell epitope peptide is linked to the alpha-helical peptide monomer via a linker. In some aspects, the at least one B cell epitope peptide is linked to the at least one T cell epitope peptide or the alpha-helical peptide domain via a linker. In some aspects, the linker comprises a chain of amino acids, a synthetic linker, a PEG moiety, or a cleavable linker. In some aspects, the chain of amino acids comprises 1-10 amino acids. In some aspects, the linker comprises a serine, alanine, threonine, aspartic acid, lysine, glutamic acid, lysine, glutamine, asparagine, arginine, proline, tryptophan, or glycine linker, or a combination thereof. In some aspects, a peptide vaccine herein comprises a peptide carrier with an amino acid sequence having at least 90%, 95%, 99%, or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 271 - SEQ ID NO: 294, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0010] In various aspects, the present disclosure provides a peptide carrier comprising: (a) an alpha-helical peptide domain; (b) at least one T cell epitope peptide; and (c) at least one B cell epitope peptide, wherein the at least one T cell epitope peptide and the at least one B cell epitope peptide are linked in tandem via the C-terminus to the alpha-helical peptide monomer, and wherein the net surface charge of the alpha-helical peptide monomer matches the net surface charge of the B cell epitope peptide, which induces an electrostatic repulsion between the alpha- helical peptide monomer and the at least one B cell epitope peptide resulting in an improved vaccine performance.
[0011] In various aspects, the present disclosure provides a peptide carrier comprising: (a) an alpha-helical peptide domain; (b) at least one T cell epitope peptide; and (c) at least one B cell epitope peptide, wherein the at least one T cell epitope peptide and the alpha-helical peptide monomer are linked in tandem, and wherein the B cell epitope peptide is attached by solid phase synthesis at specific locations along the length of the alpha-helical peptide monomer using amino acid-linked building blocks resulting in an improved vaccine performance.
[0012] In various aspects, the present disclosure provides an immunogenic composition comprising at least one, at least two, or at least three of the peptide carriers described herein and a pharmaceutically acceptable carrier. In some aspects, such immunogenic composition comprising the at least one, at least two, or at least three peptide carrier described herein is capable of inducing an immune response in a subject (e.g., a rodent or a human).
[0013] In various aspects, the present disclosure provides an immunogenic composition capable of self-assembling into polymeric, coiled-coil nanoparticles. In some aspects, the size of the polymeric, coiled-coil nanoparticles ranges from about 2 nm to about 30 nm. In some aspects, the size of the polymeric, coiled-coil nanoparticles ranges from about 30 nm to about 100 nm. In some aspects, the size of the polymeric, coiled-coil nanoparticles ranges from about 100 nm to about 1 pm. In some aspects, the size of the polymeric, coiled-coil nanoparticles ranges from about 1 pm to about 10 pm. In some aspects, such immunogenic composition can further comprise an adjuvant. [0014] In various aspects, the present disclosure provides a method for inducing an immune response in a subject specific for a target antigen comprising administering to the subject the immunogenic composition according to the present disclosure (e.g., comprising any of the peptide carriers with SEQ ID NOs: 271-306, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto).
[0015] In various aspects, the present disclosure provides a method for inducing an immune response against a microbial antigen in a subject, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 183-227, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0016] In various aspects, the present disclosure provides a method for inducing an immune response against most or all members of a diverged family of microbes in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 183-227, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the microbial antigen against which the immune response is induced is an influenza virus antigen.
[0017] In various aspects, the present disclosure provides a method for inducing an immune response against a Human papillomavirus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 229-231, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0018] In various aspects, the present disclosure provides a method for inducing an immune response against a Herpes simplex virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 232-244, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0019] In various aspects, the present disclosure provides a method for inducing an immune response against a Dengue virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 245-247, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. [0020] In various aspects, the present disclosure provides a method for inducing an immune response against a Hepatitis C virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 248-258, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0021] In various aspects, the present disclosure provides a method for inducing an immune response against a Respiratory syncytial virus in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising one or more epitope peptides with a sequence of any one of SEQ ID NOs: 259-260, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0022] In various aspects, the present disclosure provides a method for inducing an immune response against a hormone antigen in a subject, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising an epitope peptide with a sequence of SEQ ID NO: 263, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. Such hormone antigen can be a GnRH antigen. In some aspects, the immune response to a GnRH antigen is used to inhibit sex hormone production in a host mammal. In some aspects, the immune response to a GnRH antigen is used to inhibit sex hormone production in humans for treatment of cancer, hyperproliferative, and post-menopausal disorders.
[0023] In various aspects, the present disclosure provides a method for inducing an immune response against a neurodegenerative disease antigen in a subject, the method comprising administering to the subject an immunogenic composition comprising a peptide carrier comprising an epitope peptide capable of inducing an immune response to the neurodegenerative disease antigen in the subject. In some aspects, the neurodegenerative disease antigen against which the immune response is induced is an Alzheimer’s disease antigen. In some aspects, such peptide carrier comprises an Alzheimer’s disease antigen with an amino acid sequence set forth in SEQ ID NO: 261 or 262, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0024] In various aspects, the present disclosure provides a method for treating a
neurodegenerative disease in a subject in need thereof, the method comprising administering a composition comprising an alpha-helical peptide carrier and at least one neurodegenerative disease antigen. In some aspects, the neurodegenerative disease is Alzheimer’s disease,
Parkinson’s disease, or a Huntington’s disease. In some aspects, the neurodegenerative disease is Alzheimer’s disease. In some aspects, the neurodegenerative disease is Parkinson’s disease. In some aspects, the Parkinson’s disease antigen is an alpha-synuclein antigen. In some aspects, any of the methods described herein can comprise a composition comprising an alpha-helical peptide carrier comprising an alpha-helical peptide domain. In some aspects, the peptide carrier can further comprise at least one T cell epitope peptide with a sequence set forth in any one of SEQ ID NOs: 66-182, or 300, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
[0025] In various aspects, the present disclosure provides a method for treating an IgE-mediated hypersensitivity disorder in a subject in need thereof, the method comprising administering a composition comprising an alpha-helical peptide carrier and at least one IgE antigen. In some aspects, the at least one IgE antigen is derived from human IgE. In some aspects, the at least one IgE antigen is derived from the Ce3 domain of human IgE. In some aspects, the at least one IgE antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 264 to SEQ ID NO: 267, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, the composition further comprises an alpha-helical peptide carrier described herein, e.g., those with a sequence set forth in SEQ ID NO: 1 - SEQ ID NO: 65 or SEQ ID NO: 295 - SEQ ID NO: 299, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto. In some aspects, such peptide carrier further comprises at least one T cell epitope peptide with a sequence set forth in any one of SEQ ID NOs: SEQ ID NO: 66 - SEQ ID NO: 182, or SEQ ID NO: 300, or a sequence having at least 90%, 95%, 99%, or 100% sequence identity thereto.
INCORPORATION BY REFERENCE
[0026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [0028] FIG. 1 illustrates an exemplary depiction of an immunogenic peptide carrier monomer of the present disclosure comprising an alpha-helical domain comprising four heptad repeats (e.g., IKKIEKR, SEQ ID NO: 7) that is linked via its C-terminus and/or N-terminus to one or more CD4 T cell epitope (TCE) peptides (e.g., PADRE) in tandem. The peptide carrier of the present disclosure can further comprise one or more B cell epitope (BCE) peptides linked to the N- terminus and/or C-terminus and/or main chain of the peptide carrier. These TCEs and BCEs can have the same or different amino acid sequences.
[0029] FIG. 2 illustrates the physical characterization of peptide carrier monomers having SEQ ID NO: 271 and comprising the alpha-helical domain with SEQ ID NO: 20 linked in series to a PADRE T cell epitope peptide (SEQ ID NO: 71) and a T cell epitope peptide from influenza H5N1 hemagglutinin (SEQ ID NO: 73) form stable trimeric coiled-coil complexes.
[0030] FIG. 2A illustrates circular dichroism (CD) spectra of the peptide carrier monomer having SEQ ID NO: 271 dissolved in PBS and subjected to increasing temperatures from 5 °C to 95 °C, followed by a return to 5 °C (grey line).
[0031] FIG. 2B illustrates an analytical ultracentrifugation (AUC) spectrum of the alpha-helical peptide having SEQ ID NO: 271 showing monomer (0.76 S), trimer (1.78 S) and higher order assemblies (>3 S).
[0032] FIG. 2C illustrates intensity-weighted dynamic light scattering (DLS) spectrum of the alpha-helical peptide having SEQ ID NO: 271 showing major self-assembly at 7 nm
corresponding to the trimeric coiled-coil peptide complex, and two minor assemblies (25 and 350 nm).
[0033] FIG. 3 illustrates size analyses for the peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 275 and SEQ ID NO: 277.
[0034] FIG. 3A illustrates AUC analysis of peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 275.
[0035] FIG. 3B illustrates DLS analysis of peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 275.
[0036] FIG. 3C depicts the DLS analysis of peptide-based nanoparticles comprising peptide carrier monomers having SEQ ID NO: 277.
[0037] FIG. 3D shows a transmission electron microscopy (TEM) image of self-assembled coiled-coil peptide carriers with SEQ ID NO: 277 showing circular assemblies of approximately 10-20 nanometers (nm) in size.
[0038] FIG. 4 illustrates the comparison of the antibody responses to three peptide-based nicotine vaccines comprising either a single TCE or fusion peptide of two TCEs linked in tandem. The nicotine hapten (average 4 nicotine haptens/peptide) was linked to the alpha-helical peptide domain of the peptide monomer via a linker that was covalently attached to the 1’ position of the nicotine molecule (see e.g., FIG. 9A). Antibody responses to the nicotine hapten were induced by using peptide carriers with SEQ ID NO: 273, SEQ ID NO: 274, and SEQ ID NO: 272 comprising either a PADRE CD4 T cell epitope peptide (SEQ ID NO: 71), a Diphtheria CD4 T cell epitope peptide (SEQ ID NO: 72), or a fusion T cell epitope comprising both the Diphtheria and PADRE CD4 T cell epitopes (SEQ ID NO: 81). Outbred CD-l mice were then injected with 5 pg of peptide carrier or PBS as a control on days 0, 21, and 42. Antibody titers were determined by ELISA 56 days after administration of the peptide carrier. The asterisk indicates a significant difference between groups where PO.OOOl.
[0039] FIG. 5 illustrates predicted and empirical immune responses induced by a hapten containing a linker attached to the 3’ position of nicotine attached to peptide carriers with SEQ ID NO: 272, SEQ ID NO: 275, SEQ ID NO: 276, and SEQ ID NO: 277 comprising the lysine- rich heptad sequence with SEQ ID NO: 19 (5 heptad repeats) and the following combinations of CD4 T cell epitope peptides as shown in FIG. 2: Diphtheria + PADRE (SEQ ID NO: 81),
Measles V F2 + Hepatitis (Hep) B (SEQ ID NO: 83), tetanus toxoid + Influenza hemagglutinin (SEQ ID NO: 82), and tetanus toxoid (SEQ ID NO: 67).
[0040] FIG. 5A illustrates the number of predicted high affinity binding CD4 TCEs that can be generated from the four different peptide vaccines comprising peptide carriers with SEQ ID NO: 272, SEQ ID NO: 275, SEQ ID NO: 276, and SEQ ID NO: 277 following a search using the Immune Epitope Database (http://www.iedb.org/). This analysis predicts that the
Measles/Hepatitis B T cell epitope combination with SEQ ID NO: 83 may have the best activity across polymorphic animal and human populations in comparison to tetanus toxoid (947-967) (SEQ ID NO: 67), the Diphtheria toxin (332-346)/PADRE (SEQ ID NO: 81), or the tetanus toxoid/HA (307-319) (SEQ ID NO: 82) fusion epitope peptides.
[0041] FIG. 5B illustrates a graph of antibody titers induced by a nicotine hapten attached to the peptide carriers via the 3’ position of nicotine in outbred mice by the tested CD4 T cell epitope peptides. Mice were injected with 4 pg of peptide carrier or PBS as a control on days 0, 21, and 42. Antibody titers were determined by ELISA 56 days after administration of the peptide carrier. Animals immunized with the peptide carrier having SEQ ID NO: 275 expressed the best titer.
[0042] FIG. 5C shows a graph of antibody affinity induced by a nicotine hapten attached to the peptide carriers via the 3’ position of nicotine in outbred mice by the tested CD4 T cell epitope peptides in outbred mice by the tested CD4 T cell epitope peptides. Mice were injected with 4 pg of peptide carrier or PBS as a control on days 0, 21, and 42. Antibody titers were determined by ELISA 56 days after administration of the peptide carrier. Animals immunized with the peptide carrier having SEQ ID NO: 275 expressed the best affinity.
[0043] FIG. 6 illustrates that the activity of CD4 TCEs in a given species can be predicted, verified and enhanced by in vitro and in silico methods. CD4 TCEs were chosen from pertinent publications and analyzed using the Immune Epitope Database and Analysis Resource (iedb.org) MHC Class II binding predictor to identify CD4 T cell epitopes of interest. This set of epitopes was further culled based on activity using a predictive ELISPOT-based in vitro assay. In brief, PBMCs or splenocytes from a host species of interest were isolated and co-cultured with interleukin-2 and the desired T cell epitope for 10-14 days. These cultures were added to
ELISPOT plates and co-incubated with the same T cell epitope for a three-day stimulation period. The ELISPOT plates were developed and the number of spots counted and compared to background. The T cell epitopes selected using this in vitro screen can be further combined as fusions of 2 or more TCEs in various orientations and tested for activity (e.g., using the Immune Epitope Database and Analysis Resource, in vitro ELISPOT methods, etc.). Combinations showing the best activity using this algorithm can be selected to build vaccines for in vivo testing. This shows that this class of peptide carrier vaccines can be rationally designed by ranking CD4 TCE activities through in silico and in vitro methods.
[0044] FIG. 7 shows that nicotine hapten incorporation by solid phase protein synthesis induces superior antibody responses relative to traditional conjugation technology (i.e.,“wet” synthesis).
[0045] FIG. 7A illustrates the structure of a hapten 6 nicotine-lysine building block (BB or 6HA) comprised of a nicotine hapten attached via the 6 position to the lysine sidechain that is used to incorporate nicotine haptens into a peptide carrier by solid-phase protein synthesis.
[0046] FIG. 7B illustrates the building block insertion points in the a-helical domain of the peptide carrier with SEQ ID NO: 275 during solid phase protein synthesis to yield SEQ ID NO: 275 x 1 BB (also referred to herein as“SEQ ID NO: 275 x 1/6HA”) with a single nicotine hapten-lysine insertion and SEQ ID NO: 275 x 3 BB (also referred to herein as“SEQ ID NO:
275 x 3/6HA”) with three nicotine hapten-lysine insertions.
[0047] FIG. 7C illustrates that the antibody titer in the sera of mice immunized with the peptide having SEQ ID NO: 275 x 3 BB is superior to peptides containing 1 building block per monomer (SEQ ID NO: 275 x 1 BB) and also superior to a hapten peptide made using traditional (i.e., “wet”) conjugation technology (average 3 haptens/peptide) (SEQ ID NO: 275 wet).
[0048] FIG. 7D illustrates the antibody avidity in the sera of mice immunized with peptides containing 3 building blocks per monomer is superior to peptides containing 1 building block per monomer and also superior to a hapten peptide (i.e., SEQ ID NO: 275 wet) made using traditional (i.e.,“wet”) conjugation technology (average 3 haptens/peptide).
[0049] FIG. 7E illustrates the total nicotine binding capacity of antibodies in the sera of mice immunized with the peptide having SEQ ID NO: 275 x 3 BB is superior to peptides containing 1 building block per monomer (SEQ ID NO: 275 x 1 BB) and also superior to a hapten peptide (e.g., SEQ ID NO: 275 wet) made using traditional (i.e.,“wet”) conjugation technology (average 4 haptens/peptide).
[0050] FIG. 8 shows that inclusion of two nicotine-lysine building blocks generates antibody responses to both molecules.
[0051] FIG. 8A illustrates the placement of 6 nicotine haptemlysine building blocks along the peptide with SEQ ID NO: 275 following solid-phase peptide synthesis to yield a peptide having SEQ ID NO: 275 x 3/3’+3/6HA. Three haptens contain a linker attached to the 3’ position of the nicotine molecule and the other three haptens contain a linker attached to the 6 position of nicotine.
[0052] FIG. 8B shows that this peptide induces antibody titers specific to both haptens. CD-l female mice (h=10) were immunized on days 0, 21 and 35 with 10 pg of this single peptide (376HA-3-B10) with the two indicated nicotine haptens adjuvanted with GLA-SE. As a control, mice were immunized with peptides synthesized with only the 3’ hapten (SEQ ID NO: 275 x 3/3’) or the 6HA hapten (SEQ ID NO: 275 x 3/6HA). Serum was collected on day 35 and assayed by ELISA using BSA conjugated with the 6HA hapten or the 3’ hapten as coating antigens. As measured in the 6HA ELISA, the peptide with SEQ ID NO: 275 x 3/3’+3/6HA induced antibody titers that were equivalent to the positive control peptide with SEQ ID NO: 275 x 3/6HA and greater than the negative control SEQ ID NO: 275 x 3/3’. The converse was also true when antisera was assayed in the 3’ hapten ELISA.
[0053] FIG. 9 shows the additive antibody responses achieved with multivalent peptide vaccines.
[0054] FIG. 9A illustrates three different hapten structures attached at the 1’, 3’, and 6’ position on nicotine molecule used for this analysis.
[0055] FIG. 9B illustrates the total nicotine binding capacity of antibodies in the sera of mice immunized with monovalent (3’-SEQ ID NO: 275), admixed bivalent (l’,3’-SEQ ID NO: 275), and admixed trivalent (l’,3’, 6’-SEQ ID NO: 275) nicotine peptide conjugate vaccines using nicotine haptens G, 3’, and 6’ attached to the peptide carrier peptide using“wet” chemistry. The nicotine binding capacity of mice injected with a trivalent vaccine was highest, showing the utility of multivalent peptide formulations. CD-l female mice (n=5) were immunized on days 0, 21 and 42 with 5 pg of each of the indicated nicotine hapten peptide conjugates adjuvanted with GLA-SE. Serum was collected on day 56, and binding capacity assayed using equilibrium dialysis.
[0056] FIG. 10 illustrates the design and protective functionality of the peptide carrier with SEQ ID NO: 278 comprising a conserved influenza helix A epitope peptide (SEQ ID NO: 183) at its N-terminus. The self-assembling peptide-based influenza vaccine particles that were used in this study comprised peptide monomers comprising 4 lysine-rich heptad sequences in the coiled-coil domain (SEQ ID NO: 18), HBV and measles-derived TCE (SEQ ID NO: 83) linked to its C- terminus, and the conserved influenza helix A epitope peptide (SEQ ID NO: 183) linked to the N-terminus of the alpha-helical peptide domain. Antibody responses against homologous and heterologous drifted strains of influenza hemagglutinin (HA) proteins, T cell responses to the TCE domain (SEQ ID NO: 83), and survival against a homologous H1N1 challenge were characterized.
[0057] FIG. 10A shows the alpha helix A epitope sequence expressed by H1N1 viruses as compared to the alpha helix A epitope expressed by drifted H7N4, H3N2, H5N1 and Type B influenza viruses to show amino acid conservation. The bold underlined amino acids identify residues within viral hemagglutinin helix A that are outward-facing and recognized by broadly neutralizing monoclonal antibodies, such as monoclonal antibody (mAh) CR9114. The conserved H1N1 influenza helix A epitope peptide (SEQ ID NO: 183) that was used in this study contained all of the underlined amino acid residues that can be recognized by broadly neutralizing monoclonal antibodies (see, e.g., TABLE 3). The position of the epitope within the HA2 domain of HA is denoted by the residue numbers shown above the H1N1 sequence. Conserved residues are indicated by a dash.
[0058] FIG. 10B shows a model confirming that the influenza hemagglutinin helix A epitope maintains its helical secondary structure following its attachment in register to the alpha-helical N-terminus of the monomer peptide with SEQ ID NO: 278. The neutralizing residues (shown as blue spheres in the top and side view) are outward facing antibody contact residues, which mimics the helix A orientation in native hemagglutinin.
[0059] FIG. 10C shows the DLS analysis of the peptide construct with SEQ ID NO: 278.
[0060] FIG. 10D illustrates the antibody titers 35 days after mice were immunized on days 0 and 21 with the H1N1 virus-derived helix A epitope peptide vaccine having SEQ ID NO: 278. These antibodies recognized Hl hemagglutinin and showed cross-reactivity to drifted Helix A epitopes in H7, H3, and H5 hemagglutinin. [0061] FIG. 10E shows that the peptide construct with SEQ ID NO: 278 induces strong CD4+ T cell activity. Splenocytes (n=4) that were harvested from the vaccinated and naive groups on d28 for T cell analysis were stimulated with the TCE dimer (SEQ ID NO: 83) contained within the vaccine peptide immunogen (i.e., SEQ ID NO: 278) and then assayed for activity by IFN-g ELISPOT.
[0062] FIG. 10F shows antisera from d35 were assayed by ELISA for titers to recombinant Hl. Titers of total IgG, IgGl, and IgG2a are indicated.
[0063] FIG. 10G shows the ratio between IgG2a and IgGl isotypes. Values greater than 1 are indicative of a Thl response, which is induced by the TLR4 agonist GLA.
[0064] FIG. 10H shows the survival of mice immunized with PBS (naive) or with the H1N1 virus-derived Helix A epitope peptide vaccine (SEQ ID NO: 278) after challenge with H1N1 influenza virus. Mice were monitored for survival with a weight loss cutoff of 80%. Mice were challenged on day 40 with 10 times the LD50 concentration (also denoted as IOLD50) of the influenza virus subtype H1N1 (A/California/07/2009) and were monitored for survival with a weight loss cutoff of 75%. A significant difference in survival (80% survival vs. 0% at day 14) was observed for those animals that received the peptide vaccine (i.e., SEQ ID NO: 278).
[0065] FIG. 11 illustrates that matching the electrostatic charge of the alpha-helical domain of the peptide carrier with the BCE improves vaccine performance, due to electrostatic repulsion between the alpha-helical domain and the BCE and a resulting decrease in steric hindrance of the BCE interactions with, e.g., antibodies. It shows the antibody titer from animals immunized with a peptide-based influenza vaccine as disclosed herein wherein the peptide monomers with SEQ ID NO: 279 and SEQ ID NO: 280 (see e.g., FIG. 11A), which, in this case possessed an M2 ectodomain (M2e) epitope (SEQ ID NO: 94) linked in series to the C-terminus of a peptide carrier comprising either a glutamic acid-rich alpha-helical peptide domain (SEQ ID NO: 280) or a lysine-rich (SEQ ID NO: 279) alpha-helical peptide domain followed by the tetanus toxoid TCE (SEQ ID NO: 67). Mice were immunized with 10 pg of peptide carrier on days 0 and 21.
[0066] FIG. 11A illustrates the amino acid sequences and net surface charge distribution of peptide carrier with SEQ ID NO: 279 and SEQ ID NO: 280.
[0067] FIG. 11B shows antibody titers against the M2 ectodomain epitope 35 days after outbred CD-l mice were injected with PBS control or 10 pg of peptide carrier on days 0 and 21. Titers were significantly higher in animals that were injected with a peptide-vaccine comprising peptide monomers with identical charges on the surfaces of the peptide carrier and BCE (SEQ ID NO: 280), compared to mice that were injected with peptide-vaccine comprising peptide monomers with opposite charges on the surfaces of the peptide carrier and BCE (SEQ ID NO: 279). FIG. 11B illustrates that the peptide-based influenza vaccine comprising SEQ ID NO: 148 comprising the M2 ectodomain epitope peptide elicits strong antibody responses against the M2 ectodomain epitope, and that mice that were immunized with this peptide-based influenza vaccine were protected against influenza infections compared to control cohort.
[0068] FIG. 11C shows the survival curves of control (PBS) and peptide carriers with SEQ ID NO: 280 immunized outbred CD-l mice following virus challenge. Mice were monitored for survival with a weight loss cutoff of 80%.
[0069] FIG. 12 illustrates that peptide carrier with SEQ ID NO: 282 that contains two M2e BCEs (SEQ ID NO: 222) attached along the length of the alpha-helical coiled-coil peptide domain (SEQ ID NO: 26) that were incorporated during solid phase protein synthesis can induce a strong antibody response.
[0070] FIG. 12A illustrates the attachment locations of 2 M2e BCEs (SEQ ID NO: 222) to an alpha-helical coiled-coil peptide monomer (SEQ ID NO: 26) to yield peptide carrier SEQ ID NO: 282.
[0071] FIG. 12B illustrates the antibody titers measured 35 days after outbred CD-l mice were injected with PBS control or 10 pg of the peptide carrier (SEQ ID NO: 282) on days 0 and 21.
[0072] FIG. 13 illustrates that the peptide carrier can be used for BCE discovery by empirically testing the activity of novel linear B cell epitopes identified using protein structural and sequence information.
[0073] FIG. 13A shows a representative X-ray diffraction crystal structure of the B/Hong Kong/8/l973 hemagglutinin (Wang, Q et al. Crystal structure of unliganded influenza B virus hemagglutinin. 2008 J. Virol. 82:3011-3020). Two surface BCEs available for antibody binding are denoted (circles with cross-hatching denotes HA27-39BCE (SEQ ID NO: 201) and circles with diagonal lines denotes HA231-241 BCE (SEQ ID NO: 207)). The sequence homology of hemagglutinin proteins for two influenza B and two influenza A viruses has been demonstrated (see e.g., Terajima M et al. Cross-reactive human B cell and T cell epitopes between influenza A and B viruses. Virol. J. 2013, 10:244). The locations of the HA27-39 (SEQ ID NO: 201) and HA231-241 BCEs (SEQ ID NO: 207) within the hemagglutinin sequence show their conservation within influenza B viruses.
[0074] FIG. 13B illustrates that vaccination with peptides containing novel influenza epitopes HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284) leads to antibodies that can recognize recombinant hemagglutinin. Outbred CD-l mice (n=8) received a prime-boost injection with either PBS or 10 pg of either peptide. At day 30, mice were bled and titers characterized by ELISA. Antibody recognition of a peptide-BSA conjugate is compared to recognition of recombinant influenza B hemagglutinin (rHA(B) from B/Malaysia/2506/04). These results demonstrate that activity of predicted BCEs can be ascertained using these peptide carriers.
[0075] FIG. 13C shows that mice immunized with peptides containing novel influenza epitopes HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284) are protected from mortality against lethal viral challenge. On day 40, mice were challenged with 5xLD50 of B/Florida/04/06 and monitored for survival with a weight loss cutoff of 80%. Immunized mice were partially (HA231- 24i, SEQ ID NO: 284) or fully (HA27-39, SEQ ID NO: 283) protected against virus challenge.
[0076] FIG. 13D shows that mice immunized with peptides containing novel influenza epitopes HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284) experience minimal weight loss following lethal viral challenge. This experiment confirms that these novel influenza epitopes can be identified using in silico methods and incorporated into the peptide to generate protective immune responses to native influenza proteins.
[0077] FIG. 14 illustrates that these peptide carriers can induce protective antiviral responses to a variety of influenza BCEs, including M2e from IAV (SEQ ID NO: 222), NA1 (SEQ ID NO: 208), NA2 (SEQ ID NO: 221), HxA from H1N1 (SEQ ID NO: 183), M2e from IBV (SEQ ID NO: 224), HxA from IBV (SEQ ID NO: 190), HA27-39 (SEQ ID NO: 201) or HA231-241 (SEQ ID NO: 207). Peptides were made with two copies of M2e from IAV (SEQ ID NO: 282), NA1 (SEQ ID NO: 285), NA2 (SEQ ID NO: 286), M2e from IBV (SEQ ID NO: 287), HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284). Peptides were also synthesized with a single N-terminus copy of either the H1N1 HxA BCE (SEQ ID NO: 278) or the influenza B HxA BCE (SEQ ID NO: 288). Female CD-l mice were immunized with one of the peptides (10 pg) adjuvanted with GLA-SE on day 0 and day 21. On day 40, mice were challenged with 5xLD50 of
A/California/07/2009(HlNl) or B/Florida/04/06 and monitored for survival with a weight loss cutoff of 80%. Percent survival following challenge is listed for each epitope, showing that a variety of linear and helical epitopes can successfully be presented with this class of peptides.
[0078] FIG. 15 illustrates that antiviral responses can be broadened and enhanced by targeting multiple surface epitopes using multivalent vaccines.
[0079] FIG. 15A shows that a trivalent, peptide-based influenza vaccine yields improved protection compared to monovalent and bivalent peptide-based influenza vaccines. M2e (SEQ ID NO: 222), NA (SEQ ID NO: 208), and HxA (SEQ ID NO: 183) BCEs were used to make peptides with two copies of M2e (SEQ ID NO: 282) or NA (SEQ ID NO: 285), or a single N- terminus copy of HxA (SEQ ID NO: 278). Female CD-l mice were immunized with either M2e, NA, or HxA peptides singly (10 pg) or admixtures of M2e+NA or M2e+NA+HxA peptides (10 pg each peptide) adjuvanted with GLA-SE on day 0 and day 21. On day 40, mice were challenged with 5xLD50 of A/California/07/2009(HlNl) and monitored for survival with a weight loss cutoff of 80%. The monovalent vaccines led to partial survival while the multivalent vaccines led to complete survival against the virus.
[0080] FIG. 15B illustrates that the trivalent vaccine minimized least weight loss. These results establish the utility of using multivalent peptide vaccines to target multiple pathogenic B-cell epitopes and thereby enhance antibody-mediated protection.
[0081] FIG. 16 illustrates the full length human Tau protein (2N4R, 441 amino acids) and 5 isoforms (1N4R, 0N4R, 2N3R, 1N3R, and 0N3R) produced in the central nervous system by alternative splicing. The repeat (R) domain contains two sequences that allow aggregation of the Tau protein and that flank the regulatory domain 294KDNIKHVPGGGS305 (SEQ ID NO: 262) that) which can be used in combination with the compositions and methods of the present disclosure.
[0082] FIG. 17 illustrates DLS analyses of four particles comprising peptide carrier monomers with SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, and SEQ ID NO: 292 comprising either a Tau epitope peptide (SEQ ID NO: 262) or an amyloid-beta epitope peptide (SEQ ID NO: 261) that is linked to the alpha-helical peptide carrier via the N- or C-terminus, respectively, as described below.
[0083] FIG. 17A illustrates the DLS analysis for the alpha-helical peptide with the Tau epitope peptide linked to its N-terminus (SEQ ID NO: 289).
[0084] FIG. 17B illustrates the DLS analysis for the alpha-helical peptide with the Tau epitope peptide linked to its C-terminus (SEQ ID NO: 290).
[0085] FIG. 17C illustrates the DLS analysis for the alpha-helical peptide with the amyloid-beta epitope peptide linked to its N-terminus (SEQ ID NO: 291).
[0086] FIG. 17D illustrates the DLS analysis for the alpha-helical peptide with the amyloid-beta epitope peptide linked to its C-terminus (SEQ ID NO: 292).
[0087] FIG. 18 illustrates that peptide carriers with SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, and SEQ ID NO: 292 comprising the Tau epitope peptide (SEQ ID NO: 262) and the amyloid-beta epitope peptide (SEQ ID NO: 261) induce comparable antibody responses when the BCE is linked to the peptide carrier via the N- or C-terminus. CD-l female mice (n=5) were immunized on days 0, 21 and 42 with 10 pg of peptide adjuvanted with GLA-SE. Serum was collected on day 56, and antibody titers were assayed by ELISA.
[0088] FIG. 18A shows the measured antibody titers for the alpha-helical peptide with N- terminal Tau epitope peptide (SEQ ID NO: 289). [0089] FIG. 18B shows the measured antibody titers for the alpha-helical peptide with C- terminal Tau epitope peptide (SEQ ID NO: 290).
[0090] FIG. 18C shows the measured antibody titers for the alpha-helical peptide with N- terminal amyloid-beta epitope peptide (SEQ ID NO: 291).
[0091] FIG. 18D shows the measured antibody titers for the alpha-helical peptide with C- terminal amyloid-beta epitope peptide (SEQ ID NO: 292).
[0092] FIG. 19 illustrates the physiological responses in male mice immunized with the alpha- helical peptide-based vaccine having SEQ ID NO: 293 and comprising a GnRH B cell epitope peptide (SEQ ID NO: 263) at the C-terminus of the carrier peptide with SEQ ID NO: 275. CD-l male mice (5/group) were immunized (day 0, day 21, day 42) with PBS or 10 pg of the peptide vaccine formulated in GLA-SE adjuvant and then monitored for the following activities.
[0093] FIG. 19A shows the anti-GnRH antibody response following administration of the peptide carrier having the sequence set forth in SEQ ID NO: 293. Titers were determined by ELISA using plates coated with a GnRH-BSA conjugate.
[0094] FIG. 19B shows the number of pregnancies and embryos procreated by male mice that either received PBS (control) or peptide vaccine having SEQ ID NO: 293. Male mice from both groups were housed with 4 female mice between day 40 through day 80, and the number of pregnancies and resulting embryos were counted.
[0095] FIG. 19C shows the testosterone production in immunized mice, which was assayed by ELISA using day 63 serum.
[0096] FIG. 19D shows testis weights of immunized mice, which were measured on day 100.
[0097] FIG. 20 illustrates testis architecture in PBS control and mice immunized with a peptide vaccine having SEQ ID NO: 293.
[0098] FIG. 20A shows Hematoxylin and eosin stained cross-section from PBS-immunized mice. Normal tissue architecture includes interstitial Leydig cells surrounding tubules which contain subcapsular Sertoli cells and orderly arranged layers of maturing germ cells and spermatozoa.
[0099] FIG. 20B shows representative cross-section from immunized mice using the peptide vaccine with SEQ ID NO: 293. Presumptive interstitial Leydig cells appear clustered with fragmented nuclei. Tubules lined with Sertoli cells are significantly reduced in size, contain degenerating germ cells and are devoid of spermatids and spermatozoa.
[0100] FIG. 21 shows the measured antibody titers, antibody affinity, and antibody specificity for the polypeptide with SEQ ID NO: 294 and comprising a murine-specific C-terminal Peptide Y epitope (SEQ ID NO: 264) corresponding to the Cs3 domain of human immunoglobulin E (IgE). This is the same epitope recognized by the commercial anti-IgE monoclonal, Xolair™
[0101] FIG. 21A shows the antibody titers elicited by the peptide having SEQ ID NO: 294 measured by ELISA using plates coated with IgE. CD-l female mice (n=8) were immunized on days 0, 21 with 10 pg of peptide adjuvanted with GLA-SE. Serum was collected on day 35.
[0102] FIG. 21B shows that the anti-IgE antibodies in d35 immunized mouse sera contains nanomolar affinities, as measured by a competition ELISA assay using IgE.
[0103] FIG. 21C shows the results of a competition ELISA where antibodies induced in d35 mouse sera specifically bound IgE and not IgG and IgM immunoglobulin proteins.
[0104] FIG. 22 shows the antibody titers measured by ELISA using plates coated with IgE that were generated using the peptide carrier vaccine with the sequence set forth in SEQ ID NO: 294 comprising the murine Ce3 sequence with SEQ ID NO: 302. CD-l female mice (n=5) were immunized on days 0 and 21 with 10 pg of peptide adjuvanted with GLA-SE. Serum was collected on day 35. The different symbols used in A, C, and D represent antisera from individual animals in each group.
[0105] FIG. 22A shows that the anti-IgE antibodies described in FIG. 21 were able to reduce the concentration of free IgE ~l0-fold in 6 of 10 mice and ~l 000-fold in 4 of 10 mice. Day 35 antisera from immunized (h=10) and control (h=10) animals were assayed for free IgE in a competition ELISA that measured IgE binding to murine Fee receptor I (mFceRI) receptor (unpaired t-test,* p<0.002).
[0106] FIG. 22B shows that the peptide carrier with SEQ ID NO: 294 could also be used to inhibit acute IgE-mediated anaphylaxis, confirming its therapeutic function. Vaccinated (n=4) and naive mice (n=4) were anesthetized and injected (intraorbital) with 0.5 pg of 2,4- dinitrophenol (DNP)-specific IgE and then DNP-HAS 24 hours later. Anaphylaxis was measured by a reduction in body temperature. Anesthesia alone caused a drop in temperature as in naive mice not injected with IgE (grey circles). No significant difference in body temps were observed between the vaccinated and naive (-IgE) mice except at 20 min (p<0.05). Differences in body temps between naive (+IgE) and vaccinated (+IgE) were significantly different after the first 10 minutes (l-way ANOVA; p<0.05).
[0107] FIG. 22C shows that the peptide vaccine having the amino acid sequence set forth in SEQ ID NO: 294 forms nanoparticles with an average DLS diameter of -15 nm.
DETAILED DESCRIPTION
[0108] Vaccines are among the most effective interventions in modem medicine and have resulted in the eradication of disease worldwide. For example, smallpox claimed over 375 million lives in the 20th century alone. Since the development of effective and widely available smallpox vaccines, not a single person has died from smallpox since 1978. Today, more than 70 vaccine medications are available against approximately 30 microbes. Despite their success, current vaccine approaches have outstanding issues and barriers. For example, currently available vaccines are frequently used in less responsive or immunocompromised populations reducing their overall efficacy and response rate. New approaches, including advanced delivery systems, may help to improve the outcome in those populations. A major challenge for combating infectious disease is the high mutation rate and antigenic variation of pathogenic microbes. For example, more than 90 different strains of Streptococcus pneumoniae bacteria have been identified. Likewise, disease-causing viruses like Influenza Virus (IV), Fluman Papilloma Vims (HPV), Hepatitis C Vims (PICV), Respiratory Syncytial Virus (RSV), Dengue Virus (DENV) and Herpes Simplex Vims (HSV) have all evolved into multiple sub-strains and lineages.
Currently, no technology exists for creating a universal vaccine that can prevent infection fro antigenicaliy diverse microbial pathogens, although limited success has been achieved using of multivalent vaccines that combine 23 different surface antigens of the estimated 90 Streptococcus serotypes in a single formulation, or as in case of influenza vims, constantly updating the vaccine antigens annually in order to remain effective. Another challenge for vaccines that attempt to treat certain conditions or chronic ailments like Alzheimer’s disease, allergy, autoimmunity, and cancer is that the target antigens are often“self’ proteins that are immunologically ignored or tolerated by the host. The new approaches in vaccine design as disclosed herein may be able to address these challenges and further strive to lower production costs and ease worldwide distribution for improved access to life-saving vaccines.
[0109] HLA Class I- and Class Il-presented peptides that trigger adaptive immune responses are termed T-cell epitopes (TCEs), and their use is required for the development of epitope-based vaccines and immunotherapies against infections, tumors, and autoimmune diseases. However, HLA genes exhibit a high level of polymorphism (>10,000 alleles identified to date), and their distribution in the human population varies with ethnicity and region. This extreme
polymorphism represents a natural barrier for the development of peptide-based vaccines, which would require hundreds or thousands of peptides to maximize population coverage. HLA alleles may share similar binding specificities, which permits their grouping into supertypes, each of which bind a repertoire of related peptides.
[0110] Thus, promiscuous or universal CD4 T-cell epitopes as disclosed herein that bind various HLA supertypes may have great potential in the development of vaccines with wide population coverage. At present, a number of databases, such as SYFPEITHI, MHCBN, AntiJen, IEDB, and HLAsupE have been constructed for identifying T cell epitopes based on binding predictions to common and rare MHC II alleles (Schuler et al. SYFPEITHI: database for searching and T-cell epitope prediction. Methods Mol Biol. 2007;409:75-93; Lata et al. MHCBN 4.0: A database of MHC/TAP binding peptides and T-cell epitopes. BMC Res Notes. 2009;2:6l; Toseland et al. AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data. Immunome Res. 2005; 1 :4; Fleri et al. The Immune Epitope Database: How Data Are Entered and Retrieved. J Immunol Res. 20l7;20l7:5974574; Wang et al. HLAsupE: an integrated database of HLA supertype-specific epitopes to aid in the
development of vaccines with broad coverage of the human population. BMC Immunol. 2016 Jun 16; 17(1): 17). Importantly, these databases can also be used to help improve vaccine activity by predicting the broadest MHC II haplotype binding contributed by the combination of universal TCEs, including the collection of new >10 amino acid (AA) segments that are processed and presented along the length of the fused TCEs.
[0111] The synthetic peptide vaccines contemplated herein comprising effective promiscuous TCEs may be candidates for next-generation vaccines to address the above-mentioned drawbacks of currently marketed vaccines. In contrast to classic vaccines that utilize live or attenuated microorganisms either in whole or in part to elicit an immune response, the synthetic peptide vaccines of the present disclosure can contain solely the antigen or fragments thereof to exert an immune response. Peptide-based vaccines of the present disclosure can be produced in whole or at least in part using automated peptide synthesizers, viruses, or cell-based production systems. Furthermore, the antigens either in whole or in part as specific epitope peptides can be customized to elicit immune responses against a myriad of different antigens enabling their use in a wide variety of disease areas. Also disclosed herein is the use of peptide epitope sequences that are highly conserved within divergent strains of bacteria and viruses, which in turn, alleviates the major road block for creating broader protection against any given pathogen family. For all intended uses, a vaccine containing a peptide carrier as disclosed herein may target a single epitope, or alternatively, be manufactured as a multivalent vaccine for inducing antibodies against multiple epitope targets.
[0112] Further described herein are novel, synthetic peptide-based vaccines, and methods of using these vaccines for the prophylaxis and treatment of a variety of common diseases that cause unmet clinical and public needs. The synthetic peptide-based vaccines of the present disclosure can be comprised of nanoparticles that are formed from a plurality of monomeric peptide carrier by, for example, self-assembly to mono-, di-, tri-, tetrameric or higher order assemblies, i.e., self-assembled peptide nanoparticle (SAPN)-based vaccines (see e.g., FIG. 2, FIG. 3, FIG. 17). Without being bound by any theory, the SAPN-based vaccines of the present disclosure can comprise a peptide carrier to enhance the immunogenicity of otherwise weak antigens. The peptide carrier monomers of the present disclosure can comprise several peptide domains, including an alpha-helical coiled-coil domain comprising an alpha-helical coiled-coil peptide, T cell epitope (TCE) domain comprising one or more CD4 TCE peptides, and B cell epitope (BCE) domains comprising one or more BCE peptides. A TCE peptide or a BCE peptide can be linked to the N-, C-, or both termini, or along the length of the alpha-helical coiled-coil peptide monomer.
[0113] Described herein are peptide-based vaccines, including, but not limited to those comprising a peptide carrier that can elicit antibody responses in a mammal against viral pathogens like influenza, HPV, HSV, DENV, HCV, and RSV, as well as induce immune responses to host proteins, such as those involved with Alzheimer’s disease-related proteins, IgE- mediated allergies, cancer, as well as neuropeptides involved in fertility and the development and maintenance of sexual organs. Examples for epitope peptides that represent candidates for a pathogen-specific universal vaccine include: the highly conserved influenza A and B virus hemagglutinin fusion peptide (HA-fp), the hemagglutinin helix A epitope (HxA) peptide, neuraminidase peptides (NA) and matrix 2 ectodomain (M2e) epitope peptides; highly conserved HPV L2 protein derived epitope peptides; the highly conserved glycoprotein B (gB) and H (gH) derived epitope peptides from HSV; HCV derived epitope peptides and DENV-related peptides. Host protein targets for treating various indications and conditions include Tau protein derived epitope peptides, and amyloid-beta (Ab) protein derived epitope peptides, IgE derived epitope peptides, cancer-related epitopes and gonadotropin-releasing hormone (GnRH) derived epitope peptide.
[0114] Additional aspects and advantages of the present disclosure will become apparent to those skilled in this art from the following detailed description, wherein illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
[0115] As used herein, the abbreviations for the natural L-enantiomeric amino acids are conventional and are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn);
aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, lie); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Typically, Xaa can indicate any amino acid. In some embodiments, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).
[0116] The term“antigen” or“Ag,” and its grammatical equivalents as used herein, can refer to a molecule that provokes the immune response. This immune response can involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. The term“immunoglobulin” or“Ig,” as used herein, can refer to a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the chimeric antigen receptor or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE, of which IgG is the most common circulating antibody. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses.
[0117] The term“percent (%) sequence identity,” as used herein in the context of amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. In various embodiments, the % sequence identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
[0118] As used herein, the singular forms“a,”“an,” and“the” include plural references unless the context clearly indicates otherwise. For example, the term“a T cell epitope” includes a plurality of T cell epitopes, including combinations thereof.
[0119] The term“treatment” or“treating” as used herein includes the therapeutic and/or prophylactic treatment of a condition or disorder.
[0120] The term“target antigen” as used herein includes any antigen that can elicit an immune response. Target antigens can include haptens, T cell epitope peptides, or B cell epitope peptides. Peptide Carriers
[0121] Polypeptides of the present disclosure can comprise at least two distinct domains, e.g., an alpha-helical coiled-coil domain and at least one epitope domain. The polypeptides of the present disclosure may be referred to as“peptide carriers” or“alpha-helical peptide carriers.” Peptide carriers of the present disclosure can be single chain peptide monomers, and the at least one epitope domain can be linked to a coiled-coil domain (e.g., an alpha-helical peptide) via the N-, C-, or both termini, or along the length of the coiled-coil domain. The coiled-coil domain of a peptide carrier can comprise an alpha-helical peptide monomer. An epitope domain of a peptide carrier as disclosed herein can comprise at least one epitope peptide (e.g., one or more TCEs or one or more BCEs or any combination thereof). In some cases, a peptide carrier may contain additional (e.g., covalently bound) components, such as tags, linkers, markers, haptens, or bioactive molecules.
[0122] In some embodiments, the peptide carrier of the present disclosure can comprise epitope peptides that are linked (e.g., covalently linked) to the alpha-helical peptide monomer. In preferred embodiments, the alpha-helical peptide monomer can be linked to one or more epitope peptides, such as one or more T cell epitope (TCE) peptides or one or more B cell epitope (BCE) peptides, or any combination thereof. In some embodiments, the one or more BCE and/or TCE peptides can be linked in tandem to the alpha-helical peptide via the N-terminus. In some embodiments, the one or more BCE and/or TCE peptides can be linked in tandem to the alpha- helical peptide via the C-terminus. In some embodiments, the one or more BCE and/or TCE peptides can be linked in tandem to the alpha-helical peptide via both the N-terminus and C- terminus. In some embodiments, the one more BCE and/or TCE peptides are linked to the peptide carrier via an amino acid residue side chain along the length of the alpha-helical peptide domain. In some embodiments, the one or more BCE and/or TCE peptides can be linked to the peptide carrier via both termini and along the length of the alpha-helical peptide domain. In various embodiments, a peptide carrier comprises two epitope domains each comprising at least one epitope peptide (e.g., one or more BCEs and/or TCEs), wherein one epitope domain is linked to the N-terminus of the alpha-helical peptide monomer, and one epitope domain is linked to the C-terminus of the same alpha-helical peptide monomer (FIG. 1).
[0123] In some embodiments, the one or more BCE epitope peptides are linked directly to the alpha-helical peptide monomer. In other embodiments, the one or more BCE epitope peptides are directly linked to a TCE peptide epitope, which can be directly linked to the alpha-helical peptide monomer. In some embodiments, the one or more TCE epitope peptides are linked directly to the alpha-helical peptide monomer. In other embodiments, the one or more TCE epitope peptides are directly linked to a BCE peptide epitope, which is directly linked to the alpha-helical peptide monomer.
[0124] In some embodiments, the peptide carrier of the present disclosure comprises an epitope domain comprising two or more repeats of the same TCE linked in tandem to the alpha-helical peptide monomer at the N-terminus, C-terminus, both termini, along the length of the alpha- helical domain or any combination thereof. In some embodiments, the peptide carrier of the present disclosure comprise an epitope domain comprising two or more different TCEs (e.g., PADRE, Diphtheria toxin) linked in tandem to the N-, C-, both termini, along the length of the alpha-helical domain or any combination thereof. In some embodiments, the peptide carrier of the present disclosure comprise an epitope domain comprising one or more TCE peptide epitopes with the same or different amino acid sequences and one more BCE with the same or different amino acid sequences or any combination thereof that are linked individually or in tandem to the N-, C-, both termini, along the length of the alpha-helical peptide domain or any combination thereof.
[0125] The peptide carrier monomers of the present disclosure comprising a linear, alpha-helical coiled-coil peptide domain are capable of forming oligomeric complexes comprising two, three, four, or more peptide carrier monomers (See e.g., Grigoryan et al. Structural specificity in coiled- coil interactions. Curr Opin Struct Biol. 2008 Aug; l8(4):477-83). In some embodiments, the peptide carrier monomers assemble in a coiled-coil structure, with peptide monomers forming parallel bundles and each monomer having the same helical orientation. In some embodiments, the peptide carrier monomers can assemble in an antiparallel coiled-coil complex, wherein the peptide carrier monomers have an antiparallel helical orientation.
[0126] In some embodiments of the present disclosure, a peptide carrier as described herein may be further modified at the termini (i.e., the N- and/or C-terminus) and/or the amino acids along the polypeptide chain. In some embodiments, the N-terminus of the peptide carrier may be modified by acetylation. In some embodiments, the C-terminus of the peptide carrier may be modified by amidation. In some embodiments, a terminal modification may increase the stability of the peptide carrier. In some cases, a terminal modification may increase the biological activity of the peptide carrier. In some instances, acetylation of the N-terminus of a peptide carrier may be formulated as“Ac” at the N-terminus of the peptide carrier. For example, acetylation of a terminal lysine-rich heptad repeat (e.g., IKKIEKR, SEQ ID NO: 7) may be formulated as Ac- IKKIEKR (SEQ ID NO: 7). In some cases, the N-terminus of an alpha-helical peptide can comprise an aspartate residue (“D”). Alpha-helical Peptide Monomers
[0127] The alpha-helical peptide monomers of the present disclosure can comprise an“alpha- helical coiled-coil domain.” The alpha-helical coiled-coil domain as described herein can comprise two or more repeats of a heptad sequence (i.e.,“heptad” or“heptad repeat”) in tandem (FIG. 1). Without being bound to any theory, the heptad sequences may trigger the formation of multimeric coiled-coil complexes by forming non-covalent (e.g., ionic, van der Waals, or hydrogen) bonds between heptad sequences of two or more alpha-helical peptide monomers. In some embodiments of the present disclosure, the heptad sequences can be lysine-rich heptad sequences, wherein multiple heptad sequences can be linked in tandem (e.g., SEQ ID NO: 26 - SEQ ID NO: 35). In some embodiments, the heptad sequences can be glutamic acid-rich heptad sequence, wherein multiple heptad sequences can be linked in tandem (e.g., SEQ ID NO: 36 - SEQ ID NO: 47). In some embodiments of the present disclosure, the alpha-helical peptides can comprise heptad repeats with an amino acid sequence according to the general formula:
[X1X2X3X4X5X6X7]n(SEQ ID NO: 295), wherein X1 and X4 are each independently selected from I, L, V, A, F, Y, W, N, and Q; X2, X3, X6 are independently selected from the amino acids K, R, E, D, H, S, N, Q, T, C and A; X5, X7 are independently selected from the amino acids K, R, E, D, and H; and n is any number greater than or equal to 1, preferably a number from 1 to 20, most preferably from 1 to 10.
[0128] In some embodiments of the present disclosure, the alpha-helical peptides can comprise an amino acid sequence according to any one of the following general formulas:
Figure imgf000030_0001
wherein:
each Y1, Y2, Y3, Y4, Y5, Y6, and Y7 is independently selected from the amino acids I, L, V, A, K, R, E, S, T, P, G, C, N, Q, W, and D;
each X1, X4 are independently selected from the amino acids I, L, V, A, F, Y, W, N, Q; each X2, X3, X6 are independently selected from the amino acids K, R, E, D, H, S, N, Q,
T, C, A;
each X5, X7 are independently selected from the amino acids K, R, E, D, H; and each n is independently any number greater than or equal to 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, etc.).
[0129] In some embodiments, each heptad sequence can have hydrophobic isoleucines in the X1 and X4 position to form a hydrophobic core, the lysine residues at the X2, X3, and X6 positions can then become solvent exposed, and the glutamic acid and arginine in the X5 and X7 positions can form salt bridges between adjacent helices that stabilize the coiled-coil structure. In some cases, the heptad sequence can create a dense array of surface exposed lysines along the carrier that can be used for hapten (e.g., nicotine derivative) conjugation. In some cases, the heptad repeats form an alpha-helical secondary structure that can form a coiled coil structural motif. In some cases, the surface of the alpha-helix has mainly hydrophilic amino acids and the opposite face has mainly hydrophobic or lipophilic amino acids. In some cases, the amino acid sequence of the peptide carrier alternates between hydrophilic and hydrophobic residues every 3 to 4 residues, with an alpha-helical turn every 3.6 residues.
[0130] the peptide carrier of the present disclosure can comprise at least two heptad repeats in tandem (e.g., ([IKKIEKR]2, SEQ ID NO: 16). In some embodiments, a peptide carrier can comprise at least three heptad repeats in tandem (e.g., [IKKIEKR]3, SEQ ID NO: 17). In some embodiments, a peptide carrier can comprise at least four heptad repeats in tandem (e.g.,
[IKKIEKR]4, SEQ ID NO: 18). In some embodiments, a peptide carrier can comprise at least five heptad repeats in tandem (e.g., [IKKIEKR]5, SEQ ID NO: 19). In some embodiments, a peptide carrier can comprise incomplete heptad repeat sequences at the N- or C-terminus, or at both termini (see, e.g, SEQ ID NO: 20 - SEQ ID NO: 25).
[0131] In some embodiments, a peptide carrier may comprise three or four full heptad repeats of n KIEKR (SEQ ID NO: 7) in tandem, and split a respective fourth or fifth heptad repeat sequence between the N-terminus and C-terminus while still preserving the continuity of the repeating heptad pattern. In some cases, a peptide carrier can have a portion of the heptad repeat denoted by g2g3g4g5g6g7 (i e, KKTEKR (SEQ ID NO: 307)) at the N-terminus and a portion of the heptad repeat denoted by Y1 (i.e., I) at the C-terminus. In other instances, a variety of N- and C-terminal sequences may be constructed by varying the length of the portion of the heptad repeat sequence used at each terminus from 1 to 7 amino acids. By way of example, the alpha- helical domain of a peptide carrier may comprise an amino acid sequence selected from:
[IKKIEKR]4 (SEQ ID NO: 18)
KKIEKR[IKKIEKR] 3I (SEQ ID NO: 20);
KIEKR[IKKIEKR] 3IK (SEQ ID NO: 21);
IEKR[IKKIEKR] 3IKK (SEQ ID NO: 22); EKR[IKKIEKR] 3IKKI (SEQ ID NO: 23);
KR[IKKIEKR]3lKKIE (SEQ ID NO: 24); and
R[IKKIEKR] 3IKKIEK (SEQ ID NO: 25).
A. Exemplary Alpha-helical Peptide Domain Sequences
[0132] TABLE 1 shows the amino acid sequences of various representative heptad sequences and the sequences of exemplary full-length alpha-helical peptide domains. In some instances, the polypeptide sequences comprise amino acid linker(s) at the C-, N-, or both termini.
TABLE 1 - Amino acid sequences of exemplary alpha-helical peptides
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
[0133] Thus, an alpha-helical carrier contemplated herein can comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one or more of SEQ ID NO: 1 - SEQ ID NO: 65 or SEQ ID NO: 295 - SEQ ID NO: 299, or a functional fragment thereof.
[0134] In some instances, a peptide carrier consists of, consists essentially of, or comprises a lysine-rich, an arginine-rich, a glutamic acid-rich, an aspartic acid-rich, a serine-rich, or a threonine-rich alpha-helical domain. In other instances, a peptide carrier consists of, consists essentially of, or comprises an alpha-helical domain with a combination of Lys and/or Arg and/or Glu and/or Asp and/or Ser and/or Thr residues interspersed with each other (e.g., in a pattern or randomly) in outward-facing heptad positions (see e.g., SEQ ID NO: 10, SEQ ID NO: 12 - SEQ ID NO: 14, SEQ ID NO: 60 - SEQ ID NO: 65). Such alpha-helical domain can consist of, consist essentially of, or comprise an alpha-helical domain with the amino acid sequence set forth in any one of SEQ ID NO: 296 - SEQ ID NO: 299, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.
[0135] Such alpha-helical peptides can allow two or more peptide carriers to form multimeric structures, such as homodimers, homotrimers, etc. Such higher order structures can be nanoparticles. The size and/or structure of such particles can be determined using, e.g., dynamic light scattering (DLS) as described in EXAMPLE 2.
B. Linkers
[0136] Peptide carriers provided herein can comprise an alpha-helical domain that can be linked or coupled to an epitope (e.g., a BCE or TCE) peptide directly (e.g., directly coupled to a heptad repeat) or indirectly. An alpha-helical domain can be indirectly coupled to an epitope peptide via a linker. Linkers can be used to change the physical properties and function of the peptide carrier. These changes include, but are not limited to, improved solubility, stability, B cell epitope presentation, and introduction of protease sites. In some embodiments, the linker is composed of residues bearing the same charge as the prevailing charge on the heptad repeats. In other embodiments, the linker is composed of residues to improve B cell epitope presentation through flexibility and/or rigidity. In some embodiments, the linker is an amino acid linker comprising 1 to 3 amino acids, 1 to 5 amino acids, or 5 to 10 amino acids, or greater than 10 amino acids. In preferred embodiments, the amino acids used herein as linker moieties can include serine (S), alanine (A), threonine (T), aspartic acid (D), lysine (K), glutamic acid (E), glutamine (Q), asparagine (N), arginine (R), proline (P), tryptophan (W), or glycine (G) (e.g., P, G, GGP, PGG, WWP, or WP linkers). Proline and tryptophan are reported to terminate the extension of a-helical domains within proteins. Tryptophan may also be used in small peptides to quantitate peptide concentration by UV spectrometry. Glycine, alanine and serine are used to increase the length and flexibility between the rigid a-helical domain and the T- and B-cell epitope domains. Serine, threonine, aspartic acid, arginine, glutamic acid, lysine, glutamine, asparagine can be used to improve solubility. In some embodiments, the linker is a cleavable linker. Cleavable linkers of the present disclosure can include, for example, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH-sensitive linkers, hypoxia-sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers. In some embodiments, the linker is not a cleavable linker.
[0137] Linker residues can be used to facilitate manufacturing of longer peptide monomers. In some embodiments, these linkers join the heptad domain to T or B cell epitopes. In other embodiments, these linkers join two peptide carriers via the N-terminus, C-terminus, both termini, along the main chain, or any combination thereof. In other embodiments, these linkers can lie within the heptad, B cell epitope, or T cell epitope sequences. In some cases, two peptide fragments are joined. In other cases, three or more peptide fragments are joined. Any of a variety of methods can be used to associate a linker with a peptide carrier. General strategies include passive adsorption (e.g., via electrostatic interactions), multivalent chelation, high affinity non- covalent binding between members of a specific binding pair, covalent bond formation, etc. (See e.g., Gao et ah, In vivo molecular and cellular imaging with quantum dots. Curr Opin Biotechnol. 2005 Feb;l6(l):63-72). In some embodiments, click chemistry can be used to associate a linker- containing moiety with a peptide carrier. In some embodiments, Staudinger ligation, expressed protein ligation, split inteins, isopeptide bond formation, sortase ligation, liquid or solid phase fragment condensation can be used to associate a linker with a peptide carrier. Ligation reactions can lead to the introduction of linker residues, for example, Cys, Ser, Thr, Ala, or His linkers.
[0138] A peptide carrier of the present disclosure may further comprise 1 or more additional amino acids at the N-terminus, C-terminus, or at both termini. In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten, or more additional amino acids are added to the N- terminus, the C-terminus and/or both termini of the peptide carrier. In some instances, the additional amino acids may act as linkers to additional components of the peptide carrier (e.g., CD4 T cell epitope peptide, B cell epitope peptide). In other instances, the additional amino acids may act as linkers to bond one or more peptide carriers to other peptide carriers with the same or different amino acid sequences via the N-terminus, C-terminus, both termini, along the main chain, or any combination thereof. In some embodiments, the peptide carrier can comprise a “WP” dipeptide at the N- or C-terminus, or both termini, that link the alpha-helical peptide domain with an epitope peptide and act as a linker. In some embodiments, the peptide carrier can comprise GGP or PGG linkers that link epitope peptides to the N- or C-terminus of the alpha- helical peptide monomer, respectively (see e.g., SEQ ID NO: 64 and SEQ ID NO: 65). In some embodiments, a peptide carrier as disclosed herein comprises a proline (P) linker that links an epitope domain to the N-, or C-, or both termini of the alpha-helical peptide monomer. In certain embodiments, the peptide carrier comprises a“DK” dipeptide or“SP” dipeptide at the N- terminus of the four heptad repeats. The“DK” or“SP” at the N-terminus may facilitate a-helix formation of the polypeptide. For example, the peptide carrier may comprise a“DK” or“SP” at the N-terminus of [IKKIEKRJ4 (SEQ ID NO: 18), KKIEKR[IKKIEKR]3l (SEQ ID NO: 20), KIEKR[IKKIEKR] 3IK (SEQ ID NO: 21), IEKR[IKKIEKR] 3IKK (SEQ ID NO: 22),
EKR[IKKIEKR] 3IKKI (SEQ ID NO: 23), KR[IKKIEKR] 3IKKIE (SEQ ID NO: 24), and R[IKKIEKR]3lKKIEK (SEQ ID NO: 25). ). In a specific embodiment, the peptide carrier comprises DKIEKRIKKIEKRIKKIEKRIKKIEKRIKK (SEQ ID NO: 26).
Epitope Peptides
[0139] The peptide carrier of the present disclosure can comprise an alpha-helical peptide monomer comprising a coiled-coil domain, one or more CD4 T cell epitope (TCE) peptides, and one or more B cell epitope (BCE) peptides that can be linked in multiple orientations and locations to form said peptide carrier. In some embodiments, one or both termini and/or the main chain of the alpha-helical peptide monomers comprising the heptad sequences can be linked directly or indirectly to one, two, three, or more CD4 T cell epitopes (TCEs) with the same or different amino acid sequences and/or one, two, three, or more B cell epitopes (BCEs) with the same or different amino acid sequences. In some embodiments, the one, two, three, or more CD4 T cell epitopes (TCEs) with the same or different amino acid sequences and/or one, two, three, or more B cell epitopes (BCEs) with the same or different amino acid sequences are connected in series. In other embodiments, the one, two, three, or more CD4 T cell epitopes (TCEs) with the same or different amino acid sequences and/or one, two, three, or more B cell epitopes (BCEs) with the same or different amino acid sequences are interspersed randomly, in a pattern, and/or in series at one or both termini and/or along the main chain of the alpha-helical peptide monomer.
In some cases, TCEs can be isolated from common pathogens (i.e., tetanus, measles, hepatitis B, influenza) and can bind a broad repertoire of MHC class II alleles in rodents, pigs, monkeys, and humans. In some embodiments, one or more TCE peptides are linked to the alpha-helical peptide monomer via the C-terminus. In some embodiments, one or more TCE peptides are linked to the alpha-helical peptide monomer via the N-terminus. In certain embodiments, one or more TCE peptides are linked to the N- and C-terminus of the same alpha-helical peptide monomer. In some embodiments, two or more TCE peptides are linked in tandem to either the C-terminus or the N- terminus or to both termini of the alpha-helical peptide monomer. In other embodiments, multiple copies of the same or different TCE peptides are included at one or both termini, or along the length of the peptide. In some embodiments, one or more BCE peptides are linked to a fusion polypeptide comprising an alpha-helical peptide and one or more TCE peptides linked to the alpha-helical peptide monomer via the C-terminus. In other cases, at least one BCE peptide is linked in tandem to the C-terminus of a fusion peptide comprising an alpha-helical peptide monomer and at least one TCE peptide linked to the peptide carrier in tandem via one or both termini, and/or along the main chain of the alpha-helical peptide monomer. In some cases, one of more copies of a BCE peptide is linked to the N-, C-, or both termini, or attached to amino acid side chains along the length of the same alpha-helical peptide monomer or fusion polypeptide comprising an alpha-helical peptide monomer and at least one TCE that is linked to the alpha- helical peptide monomer via one or both termini, and/or attached to amino acid side chains along the length of the same alpha-helical peptide monomer. In certain embodiments, one or more copies of one or more BCE peptides are linked to the C-terminus or N-terminus, or attached to amino acid side chains along the length of a fusion polypeptide comprising an alpha-helical peptide monomer and at least one TCE that is linked to the alpha-helical peptide monomer via the C-terminus, N-terminus, both termini, or along the length of the alpha-helical peptide monomer. The fusion peptides of the present disclosure may be used as immunogenic
compositions for inducing an antibody response in a subject to one or more specific target BCEs.
[0140] In some embodiments, broad MHC Class II coverage across a variety of target populations can be achieved using these approaches (see e.g., FIG. 6). In some cases, the coiled- coil peptide-based vaccines as disclosed herein can induce a high titer of functional antibodies (Abs), and increasing concentrations of functional monoclonal Abs can be achieved using bivalent, trivalent, and multivalent vaccines containing one or more TCEs or BCEs, and/or additional haptens (e.g., nicotine).
1. CD4 T Cell Epitope Peptides
[0141] Fusion polypeptides of the present disclosure can comprise an alpha-helical peptide monomer and a CD4+ T cell epitope (TCE) peptide that is linked to the alpha-helical peptide monomer N- or C-terminus. As used herein, a TCE peptide refers to a short peptide sequence of about 9-40 amino acids in length that are presented by major histocompatibility complex molecules (MHC) Class II molecules to CD4+ T cells (helper T cells) and evoke a specific immune response from the CD4+ T cells. Major histocompatibility complex molecules (MHC molecules) are typically glycoproteins that deliver peptide antigens to a cell surface. MHC class II molecules deliver peptide antigens originating in the vesicular system to the cell surface, where they are recognized by CD4+ T cells. MHC molecules in humans may also be referred to as human leukocyte antigen (HLA). In some instances, a fusion polypeptide monomer of the present disclosure may comprise one or more TCE peptides that are linked in series and can be selected from TABLE 2. In other instances, a fusion polypeptide monomer of the present disclosure may comprise two or more TCE peptides with the same or different amino acid sequences that are linked in series and/or independently to different sites on the alpha-helical peptide monomer. In some embodiments, the two or more TCE peptides with the same or different amino acid sequences that are linked in series and/or independently to different sites on the alpha-helical peptide monomer can be selected from TABLE 2. In some embodiments, the C-terminal TCE of the two or more TCEs that are linked in series is the Hepatitis B surface antigen, as shown in TABLE 2 (see e.g., SEQ ID NOs: 83-87). In some embodiments, the one or more TCEs can be linked to an alpha-helical peptide monomer via the N-, C-, both termini, or along the length of the alpha-helical peptide domain to yield peptide carriers using conventional and/or solid-phase peptide synthesis techniques, e.g., automated (solid phase) peptide synthesizers. In some embodiments, the peptide carriers of the present disclosure are produced via cell-based expression systems. In some embodiments, a linear TCE is a peptide sequence of at least 5-10 contiguous amino acids in length. In other embodiments, the linear TCE is a peptide sequence of at least 5-20 contiguous amino acids in length. In other embodiments, the linear TCE is a peptide sequence of at least 5-30 contiguous amino acids in length. In other embodiments, the linear TCE is a peptide sequence of at least 10-40 contiguous amino acids in length. In other embodiments, the linear TCE is a peptide sequence of at least 10-100 contiguous amino acids in length. The presence of the CD4+ T cell epitope peptide may cause helper T cells to promote the
development of memory B cells and high affinity antibody responses. Without being bound by any theory, the presence of a TCE peptide in the fusion peptide may enhance the immune response against certain antigens, more copies of a single TCE may further enhance the strength of the response, and a greater number of TCEs may broaden vaccine responses across diverse populations.
[0142] TABLE 2 contains examples of CD4 T cell epitope peptide sequences that can be used in combination with the compositions and methods of the present disclosure.
Table 2 - Examples of CD4 T cell epitopes that promiscuously bind MHC Class II molecules
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
[0143] Thus, a peptide carrier contemplated herein can comprise a CD4 T cell epitope peptide, wherein such CD4 T cell epitope peptide can comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one or more of SEQ ID NO: 66 - SEQ ID NO: 182 and SEQ ID NO: 300, or a functional fragment thereof.
[0144] In some instances, a peptide carrier consists of, consists essentially of, or comprises a TCE with the amino acid sequence set forth in any one of SEQ ID NO: 66 - SEQ ID NO: 94, any one of SEQ ID NO: 112 - SEQ ID NO: 125, any one of SEQ ID NO: 135 - SEQ ID NO: 146, any one of SEQ ID NO: 159 - SEQ ID NO: 167, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. Such TCE’s can be used to enhance the immune response of a peptide carrier in a subject (e.g., a rodent or a human). Such TCE’s can be used in combination with one or more B cell epitopes (BCE’s), resulting in a peptide carrier comprising an alpha-helical peptide linked to a TCE and a BCE.
2. B-Cell Epitope Peptides
[0145] Antibodies specifically bind to an antigen at portions of the antigen referred to as B-cell epitopes (BCE). In some embodiments of the present disclosure, a BCE may be linear (i.e., a linear sequence of adjacent (contiguous) amino acids) or conformational (i.e., non- sequential amino acids or segments of the antigen that are brought together in spatial proximity when the corresponding antigen or a portion of the antigen is folded). In some embodiments, a linear BCE is a peptide sequence of at least 5-10 contiguous amino acids in length. In other embodiments, the linear BCE is a peptide sequence of at least 5-20 contiguous amino acids in length. In other embodiments, the linear BCE is a peptide sequence of at least 5-30 contiguous amino acids in length. In other embodiments, the linear BCE is a peptide sequence of at least 10-40 contiguous amino acids in length. In some embodiments, the conformational epitope is at least 5-10 contiguous amino acids in length and has helical secondary structure. In other embodiments, the conformational epitope is at least 5-20 contiguous amino acids in length and has helical secondary structure. In other embodiments, the conformational epitope is at least 5-30 contiguous amino acids in length and has helical secondary structure. In other embodiments, the conformational epitope is at least 10-40 contiguous amino acids in length and has helical secondary structure. In some embodiments, the conformational epitope is at least 5-40 noncontiguous amino acids in length and represents a non-helical conformational surface BCE.
[0146] In some embodiments, the BCE may be a contiguous peptide sequence from an endogenous protein that can be used to generate therapeutic antibodies capable of binding an endogenous protein or peptide (see e.g., FIG. 18, FIG. 19, FIG. 21). In some embodiments, the BCE may be a contiguous or noncontiguous peptide sequence from a pathogenic protein that is highly conserved within a diverging family of microbes, which in turn, can be used as a universal vaccine for broadening protection against infection (see e.g., FIG. 10-FIG. 15). In other embodiments, the BCE may be a contiguous or noncontiguous peptide sequence conserved within a specific pathogenic strain, subtype, or clade that can be used as a vaccine for protection against infection by the same strain, subtype or clade of pathogenic organism (see e.g., FIG. 10, FIG. 13A). In other embodiments, the BCE may assume a secondary structure (e.g., alpha helical) that can be incorporated in register at the N-, or C-terminus of the alpha-helical peptide carrier and represents a noncontiguous surface epitope (see e.g., FIG 10). Without being bound by any theory, when a helical BCE is fused to the N-, C- or both termini of the alpha-helical peptide carrier, the thermodynamic favorability of heptad helix formation and/or coiled-coil assembly may provide an impetus for these BCEs to assume a helical shape. As a result, the BCE may be presented in a helix conformation to lymphocytes thereby generating antibodies specific to the helical conformation instead of (or in addition to) the linear sequence. Moreover, the face of the helix that is presented to lymphocytes may be dictated by how the linear BCE amino acid sequence aligns with the heptad repeat pattern. For example, in the case of SEQ ID NO: 183, the BCE sequence (“STQNAIDEITNKVN” (SEQ ID NO: 183)) comes from an area of the influenza hemagglutinin protein that is well conserved amongst divergent viruses, is known to elicit broadly protective antibody responses, and folds as an alpha helix in the surface of the hemagglutinin protein. In SEQ ID 275, this BCE fused to the N-terminus of the alpha-helical peptide carrier and is aligned with the heptad pattern such that certain residues of the BCE face outward to mimic how the BCE is presented in the native hemagglutinin, thereby eliciting antibodies that can bind hemagglutinin on the surface of influenza virus (see, e.g., FIG. 10B).
[0147] In some embodiments, the peptide carriers of the present disclosure can comprise one or more BCEs. In some cases, the one or more BCEs can be linked to an alpha-helical peptide monomer via the N-, C-, or both termini, or along the length of the same alpha-helical peptide monomer or fusion polypeptide (see e.g., FIG. 1 and FIG. 12A) using conventional synthetic methodologies, e.g., automated peptide synthesizers. In some embodiments, peptides containing one or more linear BCEs are mixed to generate antibody responses to multiple targets and enhance vaccine activity (see e.g., FIG. 15). In other embodiments, peptides containing one or more linear BCEs and one or more conformational BCEs are mixed to generate antibody responses to multiple targets (see e.g., FIG. 15). In other embodiments, peptides containing one or more conformational BCEs are mixed to generate antibody responses to multiple targets. In some embodiments, peptides containing one or more linear and/or conformational BCEs and/or TCEs are mixed to generate antibody responses to multiple targets. In other embodiments, peptides containing one or more linear and/or conformational BCEs and/or TCEs are covalently linked to each other using a variety of methods known to those skilled in the art (e.g., through cysteine chemistry, unnatural amino acids, etc.) via one or both termini, and/or along the main chain of the peptide carrier. Without being bound by theory, covalently linking multiple immunogenic peptide carriers may improve solubility, stability, and/or immunogenicity.
[0148] TABLE 3 contains examples of B cell epitope peptides that can be used in combination with the compositions and methods of the present disclosure.
TABLE 3 - Examples of amino acid sequences of B-Cell Epitope Peptides
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
[0149] Thus, a peptide carrier contemplated herein can comprise a B cell epitope peptide. Such B cell epitope peptide can comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 183 - SEQ ID NO: 270, SEQ ID NO: 301 - SEQ ID NO: 302, or a functional fragment thereof [0150] In various instances, a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 183 - SEQ ID NO: 228. Such peptide carriers can elicit immune response in a subject (e.g., a rodent or a human) that can protect the subject from infection through influenza virus, thus, vaccines comprising such peptide carriers can be used as influenza peptide vaccines and prevent and/or treat an influenza virus infection.
[0151] In some instance, a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 232 - SEQ ID NO: 244. Such peptide carriers can protect a subject against Herpes Simplex Virus by eliciting immune responses against gB, gD, or gH surface proteins.
[0152] In some instance, a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 259 - SEQ ID NO: 260. Such peptide carriers can protect a subject against Respiratory Syncytial Virus by eliciting responses to the conserved F protein. [0153] In some instance, a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 261 - SEQ ID NO: 262. Such peptide carriers can protect a subject against Alzheimer’s disease by eliciting immune responses against Amyloid-beta and/or Tau protein.
[0154] In some instance, a peptide carrier herein can comprise a B cell epitope peptide having a sequence of SEQ ID NO: 263. Such peptide carrier can be used as a vaccine for
immunocastration in a subject (e.g., a rodent, a pig, a cow, or another farm animal).
[0155] In some instance, a peptide carrier herein can comprise a B cell epitope peptide having a sequence of any one of SEQ ID NO: 264 - SEQ ID NO: 267. Such peptide carrier can be used to treat or prevent IgE-related diseases in a subject (e.g., a rodent or a human), such as IgE- mediated allergic reactions or inflammation.
A. B cell epitope attachments to the alpha-helical domain of the peptide monomer
[0156] In addition to the N- and C-termini, incorporation of the BCE can occur at pre-specified locations within (i.e., along the length of) the alpha-helical domain of the peptide monomer by automated peptide synthesizers or solution based chemical reactions. In some embodiments, the BCE can be a peptide epitope of varying length where the peptide is incorporated into a Lysine-, Glu-, or Asp-rich alpha-helical peptide domain via isopeptide or other covalent bonds (See, e.g., FIG. 11 A, FIG. 12A). In some embodiments, the BCE can be a small molecule hapten, such as a nicotine derivative, that is manufactured as a Lys, Glu, or Asp building block in order to be incorporated into, for example, a Lysine-, Glu-, or Asp-rich alpha-helical peptide domain during solid phase peptide synthesis (FIG. 7, FIG. 8A). In some embodiments, the BCE can be a carbohydrate, polysaccharide or lipid building block that is incorporated in a similar fashion into a Lysine-, Glu-, or Asp-rich alpha-helical peptide domain. In other embodiments, Cys residues are inserted at specific sites in the peptide monomer that are then used to selectively install the BCE via thiol-specific chemistries. In other embodiments, unnatural amino acids are inserted at specifics sites in the peptide monomer with specific functional groups (e.g., iodoacetamide-, azide-, alkyne-, maleimide-containing chemical moieties, etc.) that link to complementary functional groups in the BCE using click chemistry or other chemical reactions. In some embodiments, chemical functionalities (e.g., iodoacetamide-, azide-, alkyne-, maleimide- containing chemical moieties, etc.) are inserted at specifics sites in the peptide monomer that can be used to, e.g., link complementary to, e.g., functional groups unnatural amino acids in the BCE using click chemistry or other chemical reactions. B. Peptide Surface Charge Distribution
[0157] The peptide carriers of the present disclosure can exhibit different net surface charges in different regions of the peptide carrier. For example, a coiled-coil domain comprising multiple lysine (Lys)-rich heptad repeats (e.g., SEQ ID NO: 16 - SEQ ID NO: 35) can have a positive net surface charge due to the positively charged lysine residues at physiologic pH. Similarly, a coiled-coil domain comprising multiple glutamic acid (Glu)-rich heptad repeats (e.g., SEQ ID NO: 36 - SEQ ID NO: 49) can have a negative net surface charge due to the negatively charged glutamic acid residues at physiologic pH. In this fashion, a coiled-coil domain comprising multiple serine (Ser)-rich heptad repeats (e.g., SEQ ID NO: 59 - SEQ ID NO: 60) can have a neutral net surface charge due to the neutral serine residues at physiologic pH. Other
embodiments will have varied net surface charge that depends upon their amino acid sequence. Moreover, the epitope domain of a peptide carrier can likewise exhibit an epitope-specific net surface charge distribution depending on the amino acid sequence of the epitope peptide.
[0158] In some embodiments, the peptide-based vaccines of the present disclosure can elicit an enhanced immune response when the net surface charge of the coiled-coil domain matches the net surface charge of the BCE. In some aspects, SEQ ID NO: 280 comprising a coiled-coil domain and BCE with matching surface charge elicited an enhanced immune response compared to SEQ ID NO: 279 which comprises a coiled-coil domain with opposite surface charge to the BCE (see e g , FIG. 11A-FIG. 11C)
[0159] Without being bound by any theory, the enhanced activity of the peptide vaccine comprising peptide carriers having SEQ ID NO: 280 may be due to a repulsion of the M2e epitope peptide from the coiled-coil domain of the peptide carrier. As a result, the BCE may be more available to cross-link with antigen receptors on B cells. In the case of SEQ ID NO: 279, the negative charges in the M2e epitope can hydrogen bond with the positively-charged surface lysine residues, which buries the epitope in the peptide nanoparticle and reduces its ability to bind B cells.
Exemplary Peptide Carriers
[0160] TABLE 4 contains examples of amino acid sequences of peptide carriers as described in the present disclosure comprising an alpha-helical coiled-coil domain, one or more TCE peptides and, optionally, one or more BCE peptides.
TABLE 4 - Examples of amino acid sequences of peptide carriers
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
[0161] Thus, a peptide carrier contemplated herein can consist of, consist essentially of, or comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 271 - SEQ ID NO: 294 or SEQ ID NO: 303 - SEQ ID NO: 306, or a functional fragment thereof.
[0162] In some instances, a peptide carrier consists of, consists essentially of, or comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 288, or SEQ ID NO: 306. In other instances, Lys side chains on the peptide carrier (e.g., SEQ ID NO: 308) are modified with one or more amino acid sequences set forth in SEQ ID NO: 191 - SEQ ID NO:
228 (e.g., using isopeptide bonds or other covalent attachment methods). Such peptide carriers can be used as influenza vaccines by eliciting immune responses in a subject that provide protection against influenza virus. Due to the universality of the influenza B cell epitope peptide, the vaccines may provide broad protection against multiple influenza viruses.
[0163] In some instances, a peptide carrier consists of, consists essentially of, or comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 293 or SEQ ID NO: 305. Such peptide carriers can be used for immunocastration of a subject (e.g., a rodent, a pig, a cow, other farm animals, or other wild/feral animals).
[0164] In some instances, a peptide carrier consists of, consists essentially of, or comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 290 - SEQ ID NO: 292. Such peptide carriers can be used for treating or preventing Alzheimer’s disease (e.g., as a vaccine) by eliciting immune response in a subject (e.g., a rodent or a human) directed against the Amyloid-beta and/or Tau protein by enabling the generation of antibodies directed against such disease-promoting proteins.
Haptens
[0165] The peptide-based vaccines, as described herein, can comprise one or more haptens (i.e., antigens). In some embodiments of the present disclosure, haptens are small molecules that are covalently attached to the alpha-helical peptide domain of the peptide carriers (i.e., immunogenic compositions). Small molecules that are weak immunogens but become more immunogenic when attached to a larger molecule can be characterized as haptens. A hapten may be a small organic molecule, a monosaccharide, disaccharide, or oligosaccharide, a lipid, glycolipid, nucleic acid, or an oligopeptide, for example. Although a hapten may be capable of binding to a B cell receptor, immunization with a hapten does not usually provoke an antibody response. As described herein, immunogenicity may be achieved by covalently attaching (i.e., linkingjoining, conjugating) a hapten to a larger molecule, called the carrier. Databases that describe thousands of haptens are available in the art.
[0166] In some embodiments, a peptide carrier of the present disclosure can comprise a peptide monomer linked to one or more haptens. In some embodiments, the hapten is linked to a lysine, glutamic acid, aspartic acid, or cysteine residues residue of the alpha-helical peptide domain. In some embodiments, at least two haptens are linked to the alpha-helical peptide domain. In some embodiments, at least four haptens are linked to the alpha-helical peptide domain. In some embodiments, haptens are linked to lysine, glutamic acid, aspartic acid, or cysteine residues of the alpha-helical peptide domain and/or the TCE domain. In various embodiment, a hapten is a drug of abuse, for example, nicotine, ethyl alcohol, opiates, cannabinoids, amphetamines, barbiturates, glutethimide, methyprylon, chloral hydrate, methaqualone, benzodiazepines, LSD, anticholinergic drugs, antipsychotic drugs, tryptarine, other psychomirnetic drugs, sedatives, tranquilizers, cough suppressants, hallucinogens, stimulants, phencyclidine, psilocybine, volatile nitrite, benzodiazepine, other drugs inducing physical dependence and/or psychological dependence, or analogs of each of the drugs. In a more specific embodiment, a hapten is nicotine (i.e., (S)-3[l-Methylpyrrolidin-2-yl]pyridine, a stereoisomer thereof, an analog thereof, or a structurally distinct nicotine hapten. Nicotine is parasympathomimetic alkaloid found in Solanaceae plants (e.g., tobacco) that acts as a potent stimulant.
[0167] Generally, the one or more haptens can be linked directly or indirectly (e.g., via a linker) to the alpha-helical peptide domain and/or TCE domain of the peptide-based vaccines as described herein. Any suitable linker can be used in accordance with the present disclosure. Linkers may be used to form amide linkages, ester linkages, disulfide linkages, etc. Linkers may contain carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.). Typically, linkers are 1 to 50 atoms long, 1 to 40 atoms long, 1 to 30 atoms long, 1 to 20 atoms long, 1 to 15 atoms long, or 1 to 10 atoms long. Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic het- erocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. As would be appreciated by one of ordinary skill, each of these groups may in turn be substituted. In some embodiments, a linker is an aliphatic or heteroaliphatic linker. In some embodiments, the linker is a polyalkyl linker. In certain embodiments, the linker is a polyether linker. In certain embodiments, the linker is a polyethylene linker. In certain specific embodiments, the linker is a polyethylene glycol (PEG) linker.
Self-assembled Peptide Nanoparticle (SAPNVbased Vaccines
[0168] Disclosed herein are peptide carrier monomers that can comprise an alpha-helical coiled- coil domain and one or more epitope peptides that can form self-assembled peptide nanoparticles (SAPNs), such as dimeric, trimeric, or higher order coiled-coil complexes (see e.g., FIG. 2, FIG. 3, FIG. 17). The SAPN-based vaccines of the present disclosure are capable of inducing an immune response in mammals, including humans. In some embodiments, SAPN-based vaccines show a microbial pathogen-like structure due to size and repetitive antigen structure that may be critical for B cell activation and facilitation of transport, uptake and processing in lymphoid cells and tissues.
[0169] In some embodiments, the SAPNs of the present disclosure possess a size of about 2 to 20 nm. In some cases, the SANPs have a size of about 5 to 50 nm. In some cases, the SANPs have a size of about 20 to 100 nm. In some cases, the SANPs have a size of about 50 to 500 nm. In other cases, the SAPNs have a size of about 500 nm to >10 pm. In some cases, the size and shape of nanoparticle-based vaccines can be used to determine immunogenicity and function.
[0170] In some cases, it may be beneficial to attach relatively hydrophobic or relatively hydrophilic BCE and/or TCE sequences to the coiled-coil domain comprising the heptad repeats to influence the physicochemical properties of the nanoparticles, including their solubility and assembly size. In some embodiments, it may be beneficial to attach specific linkers to the peptide or increase the number of heptad repeats in the alpha helical coiled-coil domain comprising the heptad repeats to influence the physicochemical properties of the nanoparticles, including solubility and assembly size in order to improve immunogenicity and/or stability.
Peptide Synthesis
[0171] The peptide carriers of the present disclosure can be chemically synthesized by manual techniques or by automated procedures (e.g., automated peptide synthesizers), including solid phase polypeptide synthesis. The equipment for the automated synthesis of peptides is commercially available (e.g., Perkin-Elmer, Inc.; Applied BioSystems Division, Foster City,
CA), and may be operated according to the manufacturer's instructions. Solid phase polypeptide synthesis has been performed since the early l960s. Numerous improvements to synthesis methods have been developed, and many methods have been automated and chemistries have been developed to protect terminal ends and other reactive groups. There are a number of methods to attach BCE peptides and haptens to the peptide both during and after peptide synthesis (e.g., isopeptide bonds, thiol-specific reactions, click chemistry, etc.). The synthetic peptide monomers of the present disclosure may also be obtained from any number of different custom peptide synthesizing manufacturers. If advantageous, the synthesized peptides or polypeptides of the present disclosure may be purified using any number of methods routinely practiced in the art, such as preparative reversed phase chromatography, partition
chromatography, gel filtration, gel electrophoresis, or ion-exchange chromatography or other methods used in the art.
[0172] The synthesized peptides or polypeptides of the present disclosure may be analyzed using any number of methods routinely practiced in the art, such as analytical reversed phase chromatography, nuclear magnetic resonance (NMR) spectroscopy, or mass spectrometry or other methods used in the art.
[0173] The peptide carriers of the present disclosure may be recombinantly produced using methods routinely practiced in the molecular biology art. Selection of the appropriate vector and expression control sequences (e.g., a promoter) and preparation of certain recombinant expression constructs is well within the level of ordinary skill in the art. The expression vector also comprises expression control sequences, such as a promoter, enhancer, initiation site, and the like that are selected depending on the vector and host cell used to produce the peptide monomer. The nucleotide sequence encoding a peptide in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis.
[0174] A polynucleotide that encodes a peptide carrier of the present disclosure may be incorporated into a recombinant expression vector for production of the respective peptide in a host cell. Host cells containing recombinant expression constructs may be genetically engineered (transduced, transformed, or transfected) with the vectors and/or expression constructs (for example, a cloning vector, a shuttle vector, or an expression construct). The vector or construct may be in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying particular genes or encoding-nucleotide sequences.
Selection and maintenance of culture conditions for particular host cells, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan. In general, the host cell is one that can be adapted to sustained propagation in culture to yield a stable cell line that can express sufficient amount of the peptide monomer.
[0175] Representative examples of such expression control sequences include LTR or SV40 promoter, E. coli lac or trp , the phage lambda PL promoter, and other promoters that were reported to control expression of genes in prokaryotic or eukaryotic cells or their viruses. In some cases, promoter regions can be selected from any gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Particular bacterial promoters can include lad, lacZ, T3, T5, T7, gpt, lambda PR, PL, and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruses, and mouse metallothionein-I. Selection of the appropriate vector and promoter and preparation of certain recombinant expression constructs comprising at least one promoter or regulated promoter operatively linked to a polynucleotide described herein is well within the level of ordinary skill in the art.
[0176] Useful bacterial expression constructs can be prepared by inserting into an expression vector a structural DNA sequence encoding the specific peptide monomer together with suitable translation initiation and termination signals in an operative reading phase with a functional promoter. The construct may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector construct and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli , Bacillus subtilis , Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. Suitable eukaryotic hosts for transformation include yeast (e.g., Pichia pastoris ), mammalian, insect and algal systems, although others may also be employed as a matter of choice. Any other plasmid or vector may be used as long as the plasmid or vector is replicable and viable in the host.
[0177] Those skilled in the art will understand that peptide monomers can be manufactured using a mixture of synthetic and recombinant systems. In some cases, the peptide monomer can be created entirely through solid phase peptide synthesis. In other cases, all peptide fragments are expressed recombinantly. In yet other cases, peptide fragments manufactured synthetically are linked to other recombinantly expressed peptide fragments.
Indications
[0178] The SAPN-based vaccines of the present disclosure can comprise peptide carriers comprising a wide variety of different epitope peptides. Consequently, the peptide carriers of the present disclosure can be useful for applications in a wide variety of disease areas, including infectious and chronic diseases. Below are described some exemplary but non-limiting indications for which the peptide vaccines of the present disclosure can be used for.
[0179] Influenza. Influenza virus diversity and antigenic drift requires the annual manufacture of hundreds of millions of vaccines that are primarily made using antiquated egg-based
technologies. Furthermore, the risk in choosing the wrong vaccine strain leads to sharp drops in efficacy and increased infection rates. Influenza infection is an acute global health issue and new technologies are needed that can maximize vaccine coverage, ease manufacturing constraints and reduce the price of goods.
[0180] Influenza virus encodes 2 essential surface glycoproteins called hemagglutinin and neuraminidase. Hemagglutinin (HA) mediates cell binding and internalization, and
neuraminidase (NA) releases budding virus from the cell surface. The Influenza A virus has diverged into an increasingly large number of strains that are categorized by their expression of different HA and NA proteins. Influenza A is the dominant pathogen responsible for most seasonal and pandemic infections. Influenza type B virus shares limited homology with A viruses and is responsible for widespread epidemics every 3 to 4 years and a disproportionately large frequency of infections in children. The Influenza B genome is less much less mutable than Influenza A and is classified into Victoria and Yamagata lineages based on antigenic variation of HA.
[0181] In some embodiments of the present disclosure, the peptide-based vaccines are capable of inducing an immune response in mammals against several different Influenza strains or subtypes or types through immunization with B cell epitopes (e.g., SEQ ID NO: 222) that are highly conserved among different Influenza strains or subtypes or types as shown in FIG. 10-FIG. 15 and described in EXAMPLE 9-EXAMPLE 14, and EXAMPLE 18. Examples include
Hemagglutinin fusion peptide (HA-fp) epitope, Hemagglutinin hydrophobic pocket peptide (HA- hp) epitope, hemagglutinin helix A epitopes, neuraminidase (NA)Neuraminidase epitopes, M2 ectodomain (M2e) epitopes, and nucleoprotein (NP) epitopes. Additionally, these peptides can present BCEs predicted by three-dimensional images (e.g., X-ray crystallography) of proteins from influenza or other viruses or pathogens. For example, X-ray crystallography images of representative influenza B hemagglutinins (Research Collaboratory for Structural Informatics, Protein Data Bank, rcsb.org) can be used to identify areas of the hemagglutinin protein sequence on the protein surface and available for antibody binding (see e.g., FIG. 13A). Conservation within these areas can be identified by sequence homology. For example, surface BCEs that are conserved across Yamagata/Victoria lineages (SEQ ID NO: 201 to SEQ ID NO: 207) and an area of NA is conserved across both A and B viruses (SEQ ID NO: 219 to SEQ ID NO: 221) can be identified in this manner. When these BCEs are incorporated into the peptide sequence using one of the methods described herein, they elicit antibodies that bind recombinant hemagglutinin and protect mice from viral challenge (see e.g., FIG 13, FIG. 14 and EXAMPLE 12). Additionally, multivalent combinations of peptides containing these and other BCEs lead to 100% protection and improved weight loss (see e.g., FIG. 15 and EXAMPLE 14). In this fashion, these and other peptides and/or conserved epitopes can be used and combined to formulate a universal vaccine for the prevention of all influenza A and B viruses, all B viruses, or one or more subtypes or strains of A or B viruses. This strategy can be used to formulate vaccines to inhibit pathogenic infection across a variety of indications (see e.g., EXAMPLES 17-20).
[0182] In contrast to the antigenic variability of the HA1 globular head subunit, the HA2 stalk subunit is highly conserved between strains capable of inducing cross-reactive HA Abs. One of the most functionally significant regions is the first ca. 100 amino acid (AA) residues of the HA2 subunit. The fusion peptide (residues 1-23) mediates virus entry by inserting into the host endosomal membrane. Importantly, residues 1-11 (SEQ ID NO: 199 and SEQ ID NO: 200) are identical between A and B viruses, except for 2 conserved substitutions. Additionally, residues 16-23 contain a hydrophobic pocket (HA-hp, SEQ ID NOS: 192-198) and residues 42-75 contain Helix A (HxA, SEQ ID NOS: 183-191), both of which are highly conserved within influenza A and B viruses and are recognized by broadly cross-reactive mAbs. Other linear and
conformational areas of HA that are conserved across subtypes or types and are appropriate for antibody targeting can be identified using protein sequence and structure information. In some cases, the HA-derived B cell epitope peptide of the present disclosure is selected from SEQ ID NO: 183 - SEQ ID NO: 207.
[0183] In preferred embodiments, a variety of HA-derived B cell epitope peptides including SEQ ID NO: 183 - SEQ ID NO: 207 can be linked to a peptide carrier or fusion polypeptide via either the N- or C-terminus or both termini, or along the length of the alpha-helical peptide domain, or any combination thereof.
[0184] Neuraminidase (NA) cleaves sialic acid receptors and releases new virus particles from host cells. NA is a validated therapeutic target for influenza infection. Influenza A and B viruses share 8 out of 9 identical residues near the active site of the enzyme and immunization with a peptide using the type A sequence induced cross-reactive type A and B Abs. In addition, mAbs that bind this epitope neutralize 9 different NA virus families in vitro and cross-protect mice after a lethal challenge with most Influenza A viruses. In some cases, the NA-derived epitope peptide is SEQ ID NO: 208 - SEQ ID NO: 209. Another sequence in NA that is 90-100% conserved, respectively, within influenza A and influenza B viruses is the N-terminal 1-12 amino acids (SEQ ID NOS: 210-213). Another area of NA (SEQ ID NOS: 214-221) is largely conserved across A and B viruses and can protect mice from viral challenge as described in FIG 14.
[0185] In preferred embodiments, a variety of NA-derived B cell epitope peptides including SEQ ID NO: 208 - SEQ ID NO: 221 can be linked to a peptide carrier or fusion peptide via either the N- or C-terminus or both termini, and/or along the length of the alpha-helical peptide domain. [0186] The M2 ectodomain is an essential trans-membrane ion channel. The M2 extracellular domain, M2e, is 24 AA in length. The N-terminal epitope SLLTEVETPT (residues 2-11, SEQ ID NO: 222) is 100% identical for all type A viruses. M2e-directed vaccines confer protection in animal models, including mice, ferrets, and swine, and a phase I clinical vaccine candidate was shown to be safe and immunogenic in humans. While M2e sequence identity is very low between A and B viruses, this region is also highly conserved in B viruses and mAbs that bind this 9 AA epitope also have antiviral activity (see e.g., FIG. 14). In some cases, the M2e-derived B cell epitope peptide is selected from SEQ ID NO: 222 - SEQ ID NO: 227.
[0187] In preferred embodiments, an M2e-derived B cell epitope peptide including SEQ ID NO: 222 - SEQ ID NO: 227 can be linked to a peptide carrier or fusion peptide via the N-, C-, both termini, along the length of the alpha-helical peptide domain or any combination thereof.
[0188] The nucleoprotein (NP) is an internal protein involved in the transcription and replication of the virus genome and it is highly conserved between strains. (Fujimoto et al. Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection. J Gen Virol. 2016 Sep;97(9):2l04-l6). Evidence that NP represents a bone-fide Ab target is based on studies showing the presence of anti-NP Abs in the sera of infected people, the discovery that NP is readily detected on the surface of infected cells, and the fact that anti-NP antibodies protect against homologous and heterologous type A viruses. It was recently shown that a panel of mAbs were shown to bind a surface exposed linear epitope on NP whose sequence is 98% identical in type A viruses. (Gui et al. Identification of a highly conserved and surface exposed B-cell epitope on the nucleoprotein of influenza A virus. 2014 Jun;86(6):995-l002). In some cases, the NP-derived B cell epitope peptide has SEQ ID NO: 228.
[0189] In preferred embodiments, a NP-derived B cell epitope peptide including SEQ ID NO:
228 can be linked to a peptide carrier or fusion peptide via either the N- or C-terminus or both termini, and/or along the length of the alpha-helical peptide domain.
[0190] Human papillomaviruses. Human papillomaviruses (HPVs) are the most common sexually transmitted infections, of which there are approximately 40 different strains. Three prophylactic vaccines (e.g., Cervarix, Gardasil-4 and Gardasil-9) have been approved to protect against certain types of HPV infections. However, none of the currently available HPV vaccines offers protection against all types of HPV.
[0191] The HPV-directed peptide vaccines of the present disclosure include multivalent vaccines comprising a collection of peptides with strain-specific sequences, and consensus sequences comprised of the most commonly used residues. For instance, the N-terminal region of the surface L2 protein is highly conserved among diverse HPV serotypes and have three regions of strong homology; residue 17-31, 69-86, and 108-122. In some embodiments, the consensus sequence of HPV peptide vaccines can comprise SEQ ID NO: 229 (domain 17-31), SEQ ID NO: 230 (domain 69-86) or SEQ ID NO: 231 (domain 108-122).
[0192] Herpes Simplex Virus. Herpes simplex virus types 1 (HSV-l) and 2 (HSV-2) are the most common sexually transmitted infection and cause severe infections in newborns. HSV-2 is the leading cause of infectious blindness in the western world (Sandgren et al. Understanding natural herpes simplex virus immunity to inform next-generation vaccine design. Clin Trans Immunol 20l6,5:e94). The virus infects nerve endings and establishes persistent ganglia infection. HSV has several surface glycoproteins. Glycoprotein B (gB) mediates viral fusion with host membranes. Efficient fusion is aided by glycoproteins D, H, L and K (gD, gH, gL, gK). For example, virions that lack gK enter cells with lower efficiency and are unable to establish latency in neuronal cells. Additionally, deletion of amino acids 31-68 of gK inhibits viral fusion entirely. Neutralizing antibodies and CD4 T cell responses to natural infection are focused on gB and gD. However, a long-lasting fully-protective HSV-l/HSV-2 vaccine remains elusive. For 50 years, attempts at HSV vaccines have met with failure.
[0193] There are vaccine candidates currently under development to prophylactically and therapeutically treat HSV infection. Most of these are subunit-based and focused on gD and, to a lesser extent, gB (Stanfield B et al. Herpes simplex vaccines: Prospects of live-attenuated HSV vaccines to combat genital and ocular infections. Curr Clin Microbiol Rep 2015,2: 125-136). While live attenuated HSV vaccines have shown promise in animal models, there are concerns about recombination with wild type virus and residual pathogenic potential. There have been attempts to utilize peptide concatemers of reported T cell epitopes to elicit CD4 and CD8 T cell immunity, yet merely one (HerpV) showed any efficacy by eliciting strong T cell responses. While these attempts have exhibited some efficacy in clinical subgroup analyses, their protection is inconsistent and short-lived. A long-lasting fully-protective HSV-l/HSV-2 vaccine remains elusive.
[0194] The cross-reactive peptide vaccines against HSV-l and HSV-2 of the present disclosure utilize conserved linear B cell epitopes and CD8 T cell epitopes that have been identified to be present in both HSV types. A multivalent vaccine formulation can be envisioned that contains both B and CD8 T cell epitopes. Potential B cell targets include four gD
(QIPPNWHIP S IQD/ aa289-301 (SEQ ID NO: 232), KMADPNRFRGK/aa 10-20 (SEQ ID NO: 233), NATPELVPEDP/aa262-272 (SEQ ID NO: 234), PEDPEDSALL/aa270-279, (SEQ ID NO: 235)), two gB (EDRAP VPFEE/ aa 186-196 (SEQ ID NO: 309), HIKVGQPQYYL/aa433-443, (SEQ ID NO: 310)), and two gH (HNPTASVLL/aal45-l55 (SEQ ID NO: 241), THSPLPRGIGY/aa676-686 (SEQ ID NO: 243)) epitopes (Du et al. A novel glycoprotein D- specific monoclonal antibody neutralizes herpes simplex virus. Antiviral Res 2017,147: 131-141). The gH sequence is HSV-l specific and, pending cross-reactivity, HSV-2 sequences
(HNPGASALL (SEQ ID NO:242) or THTPLPRGIGY (SEQ ID NO:244)) or may also need to be used (Cairns et al. Epitope mapping of herpes simplex virus type 2 gH/gL definse distinct antigenic sites, including some associated with biological function. J Virol 2006,80:2596-2608). The peptide-based vaccines of the present disclosure can also include conserved sequences with overlapping promiscuous CD8 T cell epitopes from gB
(KENIAP YKFK ATM YYKD VT V/aa 151-170 (SEQ ID NO: 237) and
QVWFGHRYSQFMGIF/aal72-l85 (SEQ ID NO: 238)). Furthermore, a gH epitope with both B and CD8 T cell activity (“AEFPRDPGQLLY”/aa96-l08, SEQ ID NO: 236) can be used in the presently described peptide vaccines. In some embodiments, epitope peptides that show an immune activity can be combined into multivalent peptide-based vaccines.
[0195] Dengue Virus. Dengue Virus (DENV) is part of the genus Flavivirus and is the most prevalent anthropod-transmitted viral infection worldwide, causing 25,000 deaths and up to 1 million severe infections annually (Flipse at al. The complexity of a dengue vaccine: a review of the human antibody response. PLoS Negl Trop Dis. 20l5,9:e0003749). DENV comprises 50 closely-related viruses including yellow fever, West Nile, chikungunya and Japanese encephalitis viruses. However, while these other flaviviruses have one circulating strain, there are four antigenically distinct DENV serotypes (DENV1, DENV2, DENV3, DENV4) with approximately 40% divergence in amino acid sequences between them, making DENV vaccine development challenging. Most immune responses to DENV are to the envelope (E) protein, with lesser responses to the pre-membrane (prM) and non- structural 1 (NS1) proteins. Serum NS1 levels correlate to disease severity suggesting that NS1 may act as a viral toxin.
[0196] The first DENV vaccine (CYD-TDV) was licensed in 2015 but has shown inconsistent efficacy across populations. This vaccine consists of four recombinant viruses with a yellow fever vaccine backbone expressing the pre-membrane (prM) and envelope (E) proteins of each of the four DENV serotypes. A Phase III trial showed that the protective efficacy of the vaccine varied across serotypes from 35% (DENV2) to 72% (DENV4) and did not correlate to antibody titers. This limited protection greatly undermines the utility of this vaccine. Most other DENV vaccines in development are based on chimeric or attenuated virus.
[0197] The peptide carriers of the present disclosure provide for an effective DENV vaccine and can offer high protection against the four DENV serotypes, regardless of exposure history or age. The genetic similarity between DENV and other flaviviruses may enable targeting multiple viruses with a single vaccine. Cross-reactive epitopes have been found in the E protein. The E protein contains linear epitopes with strong conservation across all flaviviruses (e.g., E35-50, E98- 120, and E250-270) as well as DENV-specific epitopes (e.g., E197-214 and E309-326).
[0198] In some embodiments, the peptide carriers of the present disclosure comprise epitopes from the E protein (SEQ ID NO: 245), original NS1-WD (SEQ ID NO: 246) or modified NS1- WD (SEQ ID NO: 247) epitope peptides derived from the non-structural protein 1 (NS1), which is the only surface NS protein. Based on structural studies of NS1, the wing domain (WD) loop at the C-terminus of the protein is an exposed antigenic site that is conserved across all four DENV serotypes.
[0199] Hepatitis C Virus. Hepatitis C Virus (HCV) currently affects over 170 million people and leads to cirrhosis and liver cancer, making HCV the leading cause of liver transplantation. (See e.g., Taherkhani et al. Global elimination of hepatitis C virus infection: Progresses and the remaining challenges. World J Hepatol 2017,9: 1239-1252). There are seven HCV genotypes comprising 67 subtypes, which can have different pathogenicity and response to treatment.
Several therapeutics currently exist to treat HCV infections, such as interferon/ribavirin combination treatment and direct-acting antivirals. Direct-acting antivirals have shown the most promise by inhibiting the function of essential viral proteins. Current treatment regimens have been able to cure 30-97% of patients, depending on the HCV subtype.
[0200] Vaccines to treat HCV are currently unavailable, though there has been substantial research into therapeutic and prophylactic HCV vaccines based on recombinant protein, peptide- based, virus-like particle (VLP), and DNA Recombinant, VLP and DNA vaccines are capable of eliciting strong humoral immune responses to HCV in preclinical and clinical trials, however, these immune responses do not correlate with protection. Moreover, humoral responses tend to focus on regions of viral proteins with high mutational rates leading to the generation of escape mutants and limited cross-protection against heterologous HCV subtypes. These vaccines also stimulate solely a weak long-term protection. Additionally, these strategies have focused on either cellular (using internal HCV proteins) or humoral (using surface proteins) immunity; an efficacious vaccine will likely need to generate both cellular and humoral responses. Peptide vaccines have in the past focused on eliciting therapeutic cytotoxic T cell responses, however, these attempts have not been fully successful. Subjects with this phenotype show strong CD4 and CD8 T cell responses as well as cross-neutralizing antibodies.
[0201] The peptide-based HCV vaccines of the present disclosure comprise conserved neutralizing B cell epitopes and CD8 T cell epitopes of HCV that can lead to broad protection against many HCV subtypes. HCV possesses two surface envelope proteins - El and E2 - that are associated with viral entry to host cells. Both have regions of strong conservation and linear neutralizing antibody epitopes have been identified. In some embodiments, the peptide carriers comprise residues 192-206 (YEVRNSSGLYHVTND (SEQ ID NO: 248)) and 313-330
(ITGHRMAWDMMMNW SPT (SEQ ID NO: 249)) of El and 409-428
QNIQLINTNGSWHINRTALN (SEQ ID NO: 250)), 523-536 (GAPTYNW GENETD V (SEQ ID NO: 251)), and 626-639 (FKVRM YV GGVEHRL (SEQ ID NO: 252)) of E2 can engage neutralizing monoclonal antibodies. Conserved CD8 T cell epitopes have been reported from the HCV core protein (YLLPRRGPRL, residues 35-44 (SEQ ID NO: 253)), El (VYEAADMIM, residues 213-221 (SEQ ID NO: 254)), E2 (HYAPRPCGI, residues 488-496 (SEQ ID NO: 255)), non- structural region 3 (VYHGAGSKTL, residues 1081-1090 (SEQ ID NO: 256)), and non- structural region 5 (RYAPACKPL, residues 2132-2140 (SEQ ID NO: 257)). Moreover, excerpts of the above described epitope sequences may be used as disclosed herein, e.g., a l2-residue excerpt (QLINTN GS WHIN (SEQ ID NO: 258) in lieu of the entire E2/409-238 epitope
(QNIQLINTNGSWHINRTALN (SEQ ID NO: 250)).
[0202] In some embodiments, the epitope peptides with SEQ ID NO: 248 - SEQ ID NO: 258 can be incorporated into multivalent peptide vaccines according to the present disclosure. Those multivalent vaccines are capable of stimulating humoral and cellular immunity and offer a broad protection against multiple HCV genotypes.
[0203] Respiratory syncytial virus. Respiratory syncytial virus (RSV) is currently the leading cause of lower respiratory tract illness in infants, but has minimal treatment options. RSV is classified as Type A (RSVA) or B (RSVB), where Type A is more prevalent. There are 16 RSVA and 22 RSVB clades, respectively. Current treatment options for RSV include Ribavarin, only small-molecule therapeutic used to treat RSV infections, and palivizumab, a monoclonal antibody administered prophylactically to high-risk infants. However, there are currently no licensed RSV vaccines available. Attempts to develop anti-RSV vaccines with attenuated viruses have shown safety, but with limited efficacy.
[0204] A biomarker for protection is the presence of RSV-specific CD8 T cell responses and neutralizing antibodies (nAbs) that target the attachment (G) or fusion (F) glycoproteins. The G protein is highly glycosylated and heterogeneous across HSV strains and generates weaker neutralizing responses than the F protein. Thus, vaccine development has focused on the F protein, which is 90% conserved between RSVA and RSVB. The F protein mediates viral entry into host cells and its major neutralizing antigenic sites have been mapped in its pre- and post fusion states. To prevent viral entry, the pre-fusion state can be targeted by focusing on the antigenic sites 0, II, and IV that are highly conserved neutralizing epitopes accessible to antibodies. In some cases, conformational change between the pre- and post-fusion states can obscure and sterically modify pre-fusion epitopes. Moreover, the pre-fusion state can be metastable and readily undergo conversion to post-fusion form as a recombinant protein.
Attempts have been made to stabilize the pre-fusion F protein in various ways, including grafting pre-fusion epitopes onto other proteins, disulfide stabilization, cavity-filling hydrophobic substitutions and point mutations. However, current vaccine candidates do not specifically target conserved epitopes, potentially limiting their cross-reactivity against other RSV strains. The pre fusion F protein has several regions of conservation. Specifically, residues 62-75 and 196-209 of site 0, 254-277 (palivizumab epitope) of site II, and 173-182 of site II.
[0205] The multivalent peptide vaccines of the present disclosure can comprise either consensus sequences or strain-specific sequences with pending cross-reactivity between RSV strains or any combination thereof. For example, for site 0 (residues 196-209), the conserved sequence for RSVA (KNYIDKQLLPIVNK (SEQ ID NO: 259)) may yield antibodies with limited RSVB cross-neutralization, in which case the RSVB consensus sequence (KNYINNQLLPIVNQ (SEQ ID NO: 260)) can also be used in a multivalent vaccine approach.
[0206] Alzheimer’s disease. Alzheimer's disease (AD) and related dementias currently affects more than 44 million people worldwide. AD is a neurodegenerative disease defined by pathological accumulation of amyloid b (Ab) protein into extracellular plaques in the brain parenchyma and in the vasculature and abnormally phosphorylated tau protein that accumulates intraneuronally forming neurofibrillary tangles (NFTs). (See e.g., Nelson PT et al. Correlation of Alzheimer's disease neuropathologic changes with cognitive status: a review of the literature. JNEN. 2012; 71 :362— 381). Tau pathology is composed of a range of aberrant tau forms that arise through post-translational modifications and form a range of aggregates. Therapeutic vaccines for Alzheimer’s disease have focused on conjugating short peptides found in the plaque-forming N- terminus of amyloid-beta to various carriers including KLH, CRM 197, and Qb phage VLPs.
[0207] The peptide-based Alzheimer’s vaccines of the present disclosure can comprise a 12 AA Tau protein (TAU) derived epitope peptide. In some embodiments, the peptide-based
Alzheimer’ s vaccine of the present disclosure can comprise an amyloid-beta (Ab) (SEQ ID NO: 261) epitope peptide or a Tau (SEQ ID NO: 262) epitope peptide. In preferred embodiments, an amyloid-beta epitope peptide or a Tau epitope peptide can be liked to a peptide carrier or fusion peptide via either the N- or C-terminus of the peptide, or along the length of the alpha-helical peptide domain, or any combination thereof.
[0208] The peptide-based Ab and Tau vaccines of the present disclosure having SEQ ID NOs:
261 and SEQ ID NO: 262 can elicit a production of broadly recognizing antibodies against the Tau and Ab proteins, respectively (see e.g., FIG. 18). In some embodiments, the peptide-based Tau and Ab vaccines of the present disclosure can comprise alpha-helical peptide monomers with SEQ ID NO: 286 - SEQ ID NO: 289. In some embodiments, the peptide carriers of the Tau and Ab vaccines can comprise one or more Tau and Ab derived epitope peptides linked in tandem to the N- or C-terminus, or along the length of the alpha-helical peptide, or any combination thereof. In some cases, a universal vaccine for neurodegenerative diseases can comprise either a combination of peptide carriers comprising Tau and Ab epitope peptides, respectively, or a peptide carrier that comprises an epitope domain comprising the Tau and the Ab epitope, thereby eliciting immune responses against several antigens at the same time.
[0209] Immunocastration. Controlling animal fertility may be needed due to overpopulation of several animal species, the increasing need for meat production, and growing ethical concerns for animal welfare and the avoidance of animal culling. Contraceptive vaccines are a methodology for fertility control that represents a valuable alternative to surgical sterilization. Contraception vaccines, which can provide a long-term effect without health hazards, are being used in numerous ways for wild, zoo, farm, and domestic animal populations.
[0210] Gonadotropin-releasing hormone (GnRH), a 10 AA neuropeptide that acts as a molecular regulator for sexual development and function, plays a prominent role in regulating sexual development and function. Produced within the hypothalamus, GnRH stimulates the synthesis and release of follicle-stimulating hormone and luteinizing hormone from the pituitary, which in turn, control androgen and estrogen production. Other GnRH conjugate vaccines using different carrier proteins are being marketed for animal reproduction and husbandry purposes. GnRH conjugate vaccines that employ KLH and Diphtheria toxoid carriers are being used to inhibit reproduction of domestic and wild animals, and as an immunocastration method for controlling meat quality in pigs and aggressive behavior patterns in horses and cattle. However, there exists an unmet need for single-shot vaccines that can be produced and distributed efficiently. In some embodiments, epitope peptides derived from the follicle-stimulating hormone receptor (FSHR) antigen can be used for immunocastration.
[0211] Small molecule GnRH antagonists are being marketed or developed for several indications in humans. For instance, testosterone promotes growth of many prostate tumors and the elimination of testosterone through castration has been a major treatment goal for men with advanced prostate cancer. GnRH antagonists are also being investigated for the treatment of benign prostatic hyperplasia, and for women, hormone-sensitive breast cancer, endometriosis, and uterine fibroids. [0212] The peptide-based vaccine of the present disclosure having SEQ ID NO: 293 can elicit a targeted Ab production against GnRH (FIG. 19-FIG. 20 and EXAMPLE 16). In some embodiments, peptide-based GnRH vaccines of the present disclosure can comprise alpha-helical peptide monomers with SEQ ID NO: 293 or SEQ ID NO: 305. In some embodiments, the peptide carriers of the GnRH vaccine can comprise one or more GnRH derived epitope peptides linked in tandem to the N- or C-terminus, or along the length of the alpha-helical peptide, or any combination thereof.
[0213] Allergies. The incidence of immunoglobulin E (IgE)-mediated allergic conditions, including allergic asthma and rhinitis, has significantly increased over the last few decades, resulting in significant morbidity and mortality and associated costs. (See e.g., World Health Organization. 2007. Global surveillance, prevention and control of chronic respiratory diseases: a comprehensive approach). Despite the availability of multiple treatment modalities, there remains a significant unmet medical need for new therapeutic approaches to treat and potentially modify allergic diseases.
[0214] For instance, the first monoclonal approved for the treatment of severe allergic asthma and urticaria (hives) was Omalizumab (Xolair™), which binds the Cs3 domain on the heavy chain of IgE. Despite its success, several issues have limited its utility including the dosing regimen, required administration in the clinic, and its potential to induce acute anaphylaxis, which necessitated the FDA’s issuance of a black-box warning on its label.
[0215] The peptide-based vaccines of the present disclosure can comprise two IgE peptide antigens that induce anti-IgE antibodies (see e.g., FIG. 21) that bind free IgE or deplete IgE secreting B cells. In some embodiments, the peptide vaccines may not interact with IgE bound to cell surface Fc receptors, thus avoid triggering the release of inflammatory mediators through IgE receptor cross-linking. In some embodiments of the present disclosure, peptides were derived from different loops of the Ce3 domain of IgE can be used to induce an immune response. In some embodiments, the peptides comprising SEQ ID NO: 264 and SEQ ID NO: 265 (Peptide Y) corresponds to the epitope recognized by monoclonal antibody omalizumab (SEQ ID NO: 301), whereas the peptide comprising SEQ ID NO: 266 (Peptide P) targets a different loop on IgE Ce3 (see e.g., Champion et al., US Patent No. US 8,722,053).
[0216] In other embodiments, the peptide carrier can comprise a BCE with SEQ ID NO: 267 (CsmX, SVNPGLAGGSAQSQRAPDRVL) which sequence corresponds to the CsmX domain located between the CH4 domain and the C-terminal membrane anchor of the e chain of membrane bound IgE (mlgE) on human B cells. Antibodies that are generated by a vaccine comprising SEQ ID NO: 267 may bind to CsmX which in turn may reduce disease-associated concentrations of IgE by inducing antibody-dependent cellular cytotoxicity and apoptosis of mlgE expressing B cells.
[0217] In some embodiments, the antigenic targets mediate inflammatory disease including;
allergy, atopy, asthma, an autoimmune disease, an autoinflammatory disease, a hypersensitivity, pediatric allergic asthma, allergic asthma, inflammatory bowel disease, Celiac disease, Crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnant loop syndrome, chronic persistent diarrhea, intractable diarrhea of infancy, Traveler's diarrhea, immunoproliferative small intestinal disease, chronic prostatitis, postenteritis syndrome, tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis, Behcet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.
[0218] Cancer. Cancer is currently an important health problem in developed countries where it is the second leading cause of death.
[0219] The peptide-based vaccines of the present disclosure can comprise three HER2 peptide antigens that induce anti-HER2 antibodies. In some embodiments, the peptide vaccines comprise SEQ ID NO: 268, SEQ ID NO: 269, or SEQ ID NO: 270. In some embodiments, these peptides can be coformulated into a multivalent vaccine targeting all three epitopes.
[0220] In some embodiments, the peptide-based cancer vaccines of the current disclosure can comprise peptide carriers comprising epitope peptides that are derived from a member of the receptor tyrosine kinase family, a member of the epidermal growth factor receptor family, WT1, p53, Brachyury, brachyury (TIVS7-2, polymorphism), brachyury (IVS7 T/C polymorphism), T brachyury, T, hTERT, hTRT, iCE, HPV E6, HPV E7, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE- A 10, MAGE-A12, BAGE, DAM-6, -10, GAGE-l, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-l, MART-l, MC1R, GplOO, PSA, PSMA, PSCA, STEAP, PAP, Tyrosinase, TRP-l, TRP-2, ART-4, CAMEL, Cyp-B, EGFR, HER1, HER2/neu, HER3, HER4, hTERT, hTRT, iCE, mucin 1 (MUC1), MUC1 (VNTR polymorphism), MUCl-c, MUCl-n, MUC2, PRAME, P15, RU1, RU2, SART-l, SART-3, WT1, AFP, b-catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-l, MUM-2, MUM-3, Myosin/m, RAGE, S ART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML1, carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 HER2/neu), human epidermal growth factor receptor 3 (HER3), Human papillomavirus (HPV), MUC1, Prostate-specific antigen (PSA), alpha-actinin-4, ARTC1, CAR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta- catenin, Cdc27, CDK4, CDKN2A, COA-l, dek-can fusion protein, EFTUD2, Elongation factor
2, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase fusion protein, HLA-A2d, HLA-A1 ld, hsp70-2, KIAAO205, MART2, ME1, MUM-lf, MUM-2, MUM-
3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1- or -SSX2 fusion protein, TGF- betaRII, triosephosphate isomerase, BAGE-l, GAGE-l, 2, 8, Gage 3, 4, 5, 6, 7, GnTVf, HERV- K-MEL, KK-LC-l, KM-HN-l, LAGE-l, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE- A 10, MAGE-A12, MAGE-C2, mucink, NA-88, NY-ESO- l/LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, TRAG-3, TRP2-INT2g, XAGE-lb, gpl00/Pmell7, Kallikrein 4, mammaglobin-A, Mel an- A/M ART- 1, NY-BR-l, OA1, PSA,
RAB 38/NY -MEL- 1 , TRP-l/gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BCMA, BING-4, CPSF, cyclin Dl, DKK1, ENAH (hMena), EP-CAM, EphA3, EZH2, FGF5, G250/MN/CAIX, HER-2/neu, ILl3Ralpha2, intestinal carboxyl esterase, alpha fetoprotein, M-CSFT, MCSP, mdm-2, MMP-2, MUC1, p53, PBF, PRAME, PSMA, RAGE-l, RGS5, RNF43, RU2AS, secernin 1, SOX10, STEAP1, survivin, Telomerase, VEGF, BRCA1, or a modified variant, a splice variant thereof. Tumor-associated antigens can be antigens not normally expressed by the host; they can be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they can be identical to molecules normally expressed but expressed at abnormally high levels; or they can be expressed in a context or environment that is abnormal. Tumor-associated antigens can be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, other biological molecules, or any combinations thereof.
Pharmaceutical Compositions
[0221] Pharmaceutically acceptable compositions of the present disclosure, including
immunogenic compositions (i.e., vaccines), can comprise one or more compositions (i.e., one or more peptide carriers) of the present disclosure and a pharmaceutically acceptable excipient. In some embodiments, the immunogenic composition comprises at least one of the compositions of the present disclosure for the prevention or treatment of one or more indications. In some embodiments, the immunogenic composition comprises at least two of different compositions of the present disclosure for the prevention or treatment of one or more indications including influenza, HSV, HPV, HCV, or neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease. In some embodiments, an immunogenic composition of the present disclosure can comprise a universal vaccine comprising one or more identical or different epitope peptides against a variety of conserved epitopes. For example, an immunogenic composition can comprise a peptide carrier comprising a plurality of influenza-derived BCE peptides that can elicit the production of broadly neutralizing antibodies in a subject after administration of said immunogenic composition. In some embodiments, an immunogenic composition of the present disclosure can comprise two or more different compositions (e.g., peptide carriers) to vaccinate a subject against one or more indications simultaneously.
[0222] In some embodiments, the immunogenic composition may be a sterile aqueous or non- aqueous solution, suspension or emulsion containing the compositions of the present disclosure, which additionally comprises a pharmaceutically acceptable carrier (physiologically acceptable excipient or pharmaceutically suitable excipient or carrier; i.e., a non-toxic material that does not interfere with the activity of the active ingredient). In some embodiments, an effective amount or therapeutically effective amount refers to an amount of the immunogen administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a specific therapeutic effect.
[0223] In some embodiments, an immunogenic composition can comprise a plurality of fusion polypeptides. In certain embodiments, the plurality of fusion polypeptides may be the same. In certain embodiments, the plurality of fusion polypeptides is composed of two or more different fusion polypeptides. In further embodiments, the two or more different fusion polypeptides may target the same B cell antigen or may target different B cell antigens.
[0224] Subjects may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which assays will be familiar to those having ordinary skill in the art and are described herein. The level of an immunogenic composition that is administered to a subject may be monitored by determining the level of the immunogenic composition, in a biological fluid, for example, in the blood, blood fraction (e.g., serum), and/or in the urine, and/or other biological sample from the subject. Any method practiced in the art to detect the immunogenic composition may be used to measure the level of immunogenic composition during the course of an immunization regimen.
[0225] The dose of an immunogenic composition described herein for evoking a specific immune response may depend upon the subject’s condition, that is, stage of the addiction or disease if present, severity of symptoms caused by the addiction or disease, general health status, as well as age, gender, and weight, and other factors apparent to a person skilled in the medical art. Immunogenic compositions may be administered in a manner appropriate to the addiction, disease or disorder to be treated or prevented as determined by persons skilled in the medical arts. An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the s’s addiction or disease, the particular form of the active ingredient, and the method of
administration. Optimal doses of an immunogenic composition may generally be determined using experimental models and/or clinical trials. In some cases, the optimal dose may depend upon the body mass, weight, or blood volume of the subject. The use of the minimum dose that is sufficient to provide an effective immune response is usually preferred. Design and execution of pre-clinical and clinical studies for an agent (including when administered for prophylactic benefit) described herein are well within the skill of a person skilled in the relevant art. In some cases, an immunogenic composition may be administered at a dose between 0.01 mg/kg and 1000 mg/kg (e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10 to 50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000 mg/kg) body weight. In another example, an immunogen may be administered at a dose of between 1 and 500 pg. In certain embodiments, an immunogen may be administered at a dose of about 1 pg to 10 pg, about 10 pg to 50 pg, about 50 pg to 100 pg, or about 100 pg to 500 pg.
[0226] The immunogenic compositions of the present disclosure may be administered to a subject in need thereof by any one of several routes that effectively deliver an effective amount of the immunogen. In some cases, such administrative routes include, for example, oral, topical, parenteral, enteral, rectal, intranasal, buccal, sublingual, intramuscular, transdermal, vaginal, rectal, or by intracranial injection, or any combination thereof. In some cases, such compositions may be in the form of a solid, liquid, or gas (aerosol). In other cases, the administrative route is also determined by the type of immunogenic composition being administered. In certain embodiments, the immunogenic composition is administered intramuscularly.
[0227] Pharmaceutical acceptable excipients (i.e., non-toxic materials that do not interfere with the activity of the active ingredient) are practiced in the pharmaceutical art and described, for example, in Rowe et ah, Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5th Ed., 2006, and in Remington: The Science and Practice of Pharmacy (Gennaro, 2lst Ed. Mack Pub. Co., Easton, PA (2005)). Exemplary pharmaceutically acceptable excipients include sterile saline and phosphate buffered saline at physiological pH. In some cases, preservatives, stabilizers, dyes, buffers, and the like may be provided in the immunogenic composition. In addition, antioxidants and suspending agents may also be used. In general, the type of excipient is selected based on the mode of administration, as well as the chemical composition of the active ingredient(s). Alternatively, compositions described herein may be formulated as a lyophilizate, or the immunogenic composition may be encapsulated within liposomes using established technology. In some cases, an immunogenic composition may be formulated for any appropriate manner of administration described herein and in the art.
[0228] A composition (e.g., for oral administration or delivery by injection) may be in the form of a liquid. In some cases, a liquid immunogenic composition may include, for example, one or more of the following: a sterile diluent such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils that may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents; antioxidants; chelating agents; buffers and agents for the adjustment of tonicity such as sodium chloride or dextrose. In some cases, a parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some cases, the use of physiological saline is preferred, and an injectable pharmaceutical composition is preferably sterile.
[0229] For oral formulations, an immunogenic composition described in the present disclosure can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, and, optionally, with diluents, buffering agents, moistening agents, preservatives, coloring agents, and flavoring agents. In some cases, an immunogenic composition included in the compositions may be formulated for oral delivery with a buffering agent, flavoring agent, e.g., in a liquid, solid or semi-solid formulation and/or with an enteric coating.
[0230] A composition of the present disclosure can comprise any one of the immunogenic compositions described herein, and may be formulated for sustained or slow release. In some cases, such compositions may generally be prepared using elsewhere reported technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the particular target site. In some cases, sustained-release formulations may contain the immunogen dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Excipients for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. In some cases, the amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.
[0231] In some embodiments, Kits with unit doses of an immunogen described herein, usually in oral or injectable doses, are provided. In some cases, such kits may include a container containing the unit dose, an informational package insert describing the use and attendant benefits of the immunogen or antibody in treating pathological condition of interest, and optionally an appliance or device for delivery of the composition.
Adjuvants
[0232] The immunogenic compositions of the present disclosure may comprise a
pharmaceutically acceptable adjuvant. In some cases, an adjuvant is intended to enhance (or improve, augment) the immune response to the immunogens described herein, the peptide monomer conjugates, peptide dimer conjugates, or trimeric coiled-coil peptide conjugates (i.e., increase the level of the specific immune response in a statistically, biologically, or clinically significant manner compared with the level of the specific immune response in the absence of administering the adjuvant).
Vaccine adjuvants can control the magnitude and quality of adaptive T and B cell responses by facilitating antigen/plasmid uptake into antigen presenting cells and stimulating innate pathways that control leukocyte recruitment to the site of injection (see, e.g., Carter and Reed, 2010, Curr. Opin. HIV AIDS 5:409-13). Until 2009, the only licensed adjuvant in the United States was aluminum-based mineral salts. Since then squalene-based oil-in-water emulsions like MF59, AS03, and AF03 have been used extensively in marketed influenza vaccines (Wilkins AL et al. AS03- and MF59-Adjuvanted Influenza Vaccines in Children. Front Immunol. 2017 Dec l3;8: 1760). Additional evidence suggests that adjuvant formulations that bind innate pattern recognition receptors on APC may provide an equivalent immune response as alum or a greater immune response than alum. Molecules that may be useful as adjuvants include a Toll-like receptor agonists. Toll-like receptors (TLRs) bind molecules characteristic of extracellular pathogens, such as LPS (TLR4), lipoproteins (TLR1, TLR2, TLR6), and flagellin (TLR5), as well intracellular pathogens, such as single-stranded RNA (TLR7, TLR8), double stranded RNA (TLR3) and CpG motif DNA (TLR9).
[0233] For administration in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies.
Preferred adjuvants augment the response to the immunogen without causing conformational changes in the immunogen that might adversely affect the qualitative immune response. In some cases, suitable adjuvants include aluminum salts, such as alum (potassium aluminum sulfate), or other aluminum containing adjuvants such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate. Other pharmaceutically suitable adjuvants include nontoxic lipid A-related adjuvants such as, by way of non-limiting example, nontoxic monophosphoryl lipid A (see, e.g., Persing et al., Trends Microbiol. !0:s32-s37 (2002)), for example, 3 De-O-acylated monophosphoryl lipid A (MPL) (see, e.g., United Kingdom Patent Application No. GB
2220211). Other useful adjuvants include QS21 and QuilA that comprise a triterpene glycoside or saponin isolated from the bark of the Quillaja saponaria Molina tree found in South America (see, e.g., Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell and Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540). Other suitable adjuvants include oil in water emulsions, optionally in combination with immune stimulants, such as monophosphoryl lipid A. Other suitable adjuvants include polymeric or monomeric amino acids such as polyglutamic acid or polylysine, liposomes, and CpG. (See e.g., U.S. Patent No.
7,402,572; European Patent No. 772 619). Other suitable adjuvants include toll-like receptor agonists that bind to TLR4, TLR1, TLR2, TLR6, TLR5, TLR7, TLR8, TLR3, and TLR9. (See, e.g., Mifsud et al., 2014, Front. Immunol. 5:79; Steinhagen et al., 2011, Vaccine 29:3341-3355). Such TLR based adjuvants include, for example, glucopyranosyl lipid A-stable emulsion (GLA- SE). (See e.g., U.S. Patent No. 8,609,114); AS04. In certain embodiments, the immunogenic compositions of the present disclosure comprise an adjuvant that is squalene-based oil-in-water emulsion, and/or a toll-like receptor (TLR) agonist. In a specific embodiment, the adjuvant is a glucopyranosyl Lipid A-Stable Emulsion (GLA-SE) adjuvant. In other embodiments, the adjuvant is an aluminum based adjuvant.
Administration
[0234] Methods of the present disclosure for inducing a humoral immune response specific for target antigen, comprising administering to the subject any of the immunogenic compositions described herein. A subject includes a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a horse, a non-human primate, a pig, and a human. In some embodiments, a subject is a human.
[0235] The immunogenic compositions of the present disclosure may be administered to a subject by a parenteral (e.g., intravenous), intraperitoneal, intramuscular, intradermal, intraocular, or subcutaneous route. The immunogenic composition may further comprise a suitable adjuvant to enhance the immune response to the immunogen. Adjuvants typically used for immunization of non-human animals include but are not limited to Freund’s complete adjuvant, Freund’s incomplete adjuvant, montanide ISA, Ribi Adjuvant System (RAS) (GlaxoSmithKline,
Hamilton, MT), and nitrocellulose-adsorbed antigen. In general, after the first injection, subjects receive one or more booster immunizations according to a preferred schedule that may vary according to, inter alia, the immunogen, the adjuvant (if any) and/or the particular species of subject. In some cases, the B cell immune response may be monitored by periodically bleeding the subject, separating the sera from the collected blood, and analyzing the sera in an immunoassay, such as an ELISA or Ouchterlony diffusion assay, or the like, to determine the specific antibody titer. When an adequate antibody titer is established, the subject may be bled periodically to accumulate the polyclonal antisera.
[0236] Monitoring the immune response of an immunized host during pre-clinical studies in subjects includes obtaining sera from the subjects before the first dose (i.e., pre-immune sera) and after the final boosting dose. In some cases, sera may also be obtained after any one or more of the boosting doses between the primary dose and final boosting dose. For monitoring the immune response of an immunized host during clinical studies or during post-marketing studies, sera may also be obtained from humans before the first immunization and after one or more administrations of the immunogenic compositions.
[0237] Production of antigen-specific antibodies in an immunized host (including a human host) may include production of any class of immunoglobulin, including IgG, IgA, IgM, and/or IgE, and isotypes within the classes. The presence of specific IgG, IgM, IgE, and IgA may be detected in a biological sample (e.g., serum, nasal wash, lung lavage, or other tissues) obtained from an immunized host. For detection of antibodies in an immunoassay, the biological sample may be permitted to interact with or contact an antigen that is purified, isolated, partially isolated, or a functional fragment thereof.
[0238] Production of antigen specific T cells in an immunized host (including a human host) may include production of any type of T cells, including CD4+ T cells and CD8+ T cells. The presence of specific CD4+ or CD8+ T cells may be detected in a biological sample obtained from an immunized host. Assays for measurement of T cell responses are well established in the art and include, for example, T cell proliferation assays, cytokine based assays (e.g., ELISA, cytokine ELISPOT, intracellular cytokine staining), flow cytometry, cytometry by time of flight (CyTOF) mass spectroscopy, MHC tetramer staining, and cytotoxicity assays. The
immunogenicity of immunogens described herein may also be characterized by any number of assays and techniques practiced in the art, including immunoassays to evaluate binding and the capability of the immunogen to induce an immune response. By way of non-limiting example, immunoassays include ELISA, immunoblot, radioimmunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), Ouchterlony immunodiffusion, proliferation assays, cytotoxicity assays, MHC peptide tetramer staining, intracellular cytokine staining, cytokine ELIspot, and the like. Conditions for in vitro assays include temperature, buffers (including salts, cations, media), and other components that maintain the integrity of any cell used in the assay and the compound, which a person skilled in the art will be familiar and/or which can be readily determined. A person skilled in the art also readily appreciates that appropriate controls can be designed and included when performing the in vitro methods and in vivo methods described herein.
[0239] In vitro assay methods used for the immunogenic compositions of the present disclosure typically comprise contacting the biological sample with at least one source of the antigens described above and herein under conditions and for a time sufficient for an antibody in the sample to interact with the antigen source (i.e., mixing, combining, or in some manner permitting the biological sample and the antigen to interact). An antibody present in the biological sample that specifically binds to the antigen can be detected using any one of the exemplary detection methods described herein and in the art for detecting antibody-antigen binding. By way of non limiting example, antibody-bound to the antigen may be detected using a reagent specific for a conserved region of the antibody, such as the Fc portion of the antibody, which reagent is typically selected depending on the source of the antibody (i.e., whether the antibody is from an animal, such as a mouse, rat, goat, or sheep, etc. or whether the antibody is from a human). Such reagents typically comprise a detectable label, for example an enzyme, fluorescent label, luminescent label, or radioactive label. Additional exemplary reagents include those that detect a specific isotype or class of antibody. Many such reagents may be obtained from commercial sources.
Methods
[0240] General. All experiments were carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Protocol #2015-11 was approved by the Institutional Animal Care and Use Committees of the Infectious Disease Research Institute which operates under a currently approved Assurance #A4337-0l and USDA certificate #9l-R-006l.
[0241] Data analyses shown and described herein report the mean +/- SD (standard deviation) and statistical differences between groups is determined by unpaired t-test or one-way ANOVA (P<0.05).
[0242] Synthesis. Peptides of the present disclosure can be synthesized at Bio-Synthesis Inc (Lewiston, TX). For example, a peptide carrier of the present disclosure can be synthesized to comprise 5 IKKIEKR heptad repeats (e.g., SEQ ID NO: 19) in the coiled-coil domain followed by the PADRE TCE (SEQ ID NO: 71) and a TCE isolated from influenza H5N1 hemagglutinin (SEQ ID NO: 73). In addition, a peptide carrier can comprise the same 5 heptad repeats followed by a TCE selected from Measles virus F2 protein (SEQ ID NO: 75) and Hepatitis B surface antigens (SEQ ID NO: 76). Furthermore, peptide carrier of the present disclosure can comprise three overlapping TCEs isolated from tetanus toxoid (SEQ ID NO: 67). Peptides synthesized with linear BCEs may comprise 4 IKKIEKR heptad sequences (e.g., SEQ ID NO: 18) and 2 TCEs (see e.g., FIG. 12). Peptides containing BCEs covalently bound to Lys residues in the carrier sequence (see e.g., SEQ ID NO: 281) may be synthesized through selective deprotection of pre-specified Lys residues, which allows the side chain amine to react with the carboxylic acid at the C-terminus of the BCE peptide using standard amino acid coupling chemistry. The amyloid-b BCE (SEQ ID NO: 261) can be linked to the N- and C- termini of a peptide monomer with Gly-Gly-Pro or Pro-Gly-Gly linkers, respectively. The Tau BCE (SEQ ID NO: 262) can be linked to the N- and C-termini of a peptide monomer with a Pro linker. The GnRH BCE (SEQ ID NO: 263) can be linked to the C-terminus of a peptide monomer with a Gly linker. The M2e for IAV (SEQ ID NO: 222), M2e for IBV (SEQ ID NO: 224), NA1 (SEQ ID NO: 208), NA2 (SEQ ID NO: 220), HA27-39 (SEQ ID NO: 201) and HA231-241 (SEQ ID NO: 207) can either be linked to the C-terminus or to Lys side-chains of a carrier peptide having alpha helical domain SEQ ID NO: 26 and TCE domain SEQ ID NO: 83. The HxA for H1N1 (SEQ ID NO: 183) and HxA for IBV (SEQ ID NO: 190) BCEs can be included on the N-terminus of a fusion polypeptide monomer having SEQ ID NO: 26 fused to SEQ ID NO: 83.
[0243] Circular Dichroism. Circular Dichroism analyses of the immunogenic compositions (e.g., peptide-based vaccines) may be recorded from 190-270 nm on a Jasco J720
spectropolarimeter using 10 mm path length cells. Peptide stock solutions (ca. 50 mM) may be prepared for CD Spectroscopy using PBS. Temperatures ranged from 5 to 95 °C in increments of 10 °C.
[0244] Dynamic Light Scattering. Dynamic Light Scattering analysis of the immunogenic compositions (e.g., peptide-based vaccines) for determining size distributions of assembled peptides may be measured by dynamic light scattering using a Zetasizer Nano (Malvern
Instruments, UK). Stock solutions (ca. 50 pM) were prepared for DLS Spectroscopy using MOPS (100 mM, 50 mM NaCl, pH 7.5). Prior to the measurement, the peptide can be filtered through a 0.2 pm nylon membrane and loaded into a plastic microcuvette. DLS studies may be carried out in general purpose mode. Measurement parameters can be as follows: material setting was protein (refractive index = 1.440), dispersant setting was water (viscosity = 0.8872 cP, refractive index = 1.330), 10 cycles averaged per measurement, and 30 second temperature equilibration at 25 °C. The Zetasizer Nano instrument uses a 4 mW He-Ne laser (633 nm) and a fixed detection angle (173 °C). [0245] Analytical Ultracentrifugation. Analytical Ultracentrifugation (AUC) can be performed by Alliance Protein Laboratories (San Diego, CA). The peptide-based vaccine particles can be dissolved in PBS to ~l mg/mL and in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (100 mM, 50 mM NaCl, pH 7.5) to concentration of about 0.5 mg/mL. Samples can be filtered through a 0.2 pm nylon membrane. AUC can be performed by Alliance Protein Laboratories (San Diego, CA). Briefly, the samples can be loaded into a Beckman-Coulter ProteomeLab XL-A analytical ultracentrifuge. After equilibration at 20 °C the rotor can be brought to 60,000 rpm. Scans can be recorded every 4 min for -10 h. The data may be analyzed using SEDFIT (version 11.3). The resultant size distributions may be graphed and the peaks integrated using OriginLab Origin® version 9.0 (Northampton, MA).
[0246] Antibody titers. Serum Ab titers can be determined by ELISA as reported. (See e.g., Clegg et al. Adjuvant solution for pandemic influenza vaccine production. Proc Natl Acad Sci U S A (2012) 109: 17585-17590. Serum samples may be serially diluted 3-fold from 1/100 in blocking buffer. Midpoint titers at half maximal absorbance can be calculated using GraphPad Prism (GraphPad Software, San Diego, CA). Amyloid-beta-directed, GnRH-directed and influenza-directed (M2e, NA1, NA2, HA27-39 and HA231-241) Abs can be measured using cysteine- terminated synthetic peptides conjugated onto BSA using maleimide crosslinking chemistry. Titers to certain HA proteins (e.g., Helix A, HA27-39 and HA231-241) may be measured using recombinant influenza hemagglutinin. Tau epitope-directed Abs can be measured using a 441 AA recombinant isoform of human Tau protein (rPeptide, Watkinsville GA, e.g., FIG. 16) and mouse testosterone can be measured using a commercial kit purchased from Abeam (Cambridge MA).
[0247] Binding capacity. Serum can be pooled from each immunization group and aliquots (100 pL) may be spiked with serially diluted nicotine to achieve final nicotine concentrations of 0.01- 10000 mM. These samples were then subjected to equilibrium dialysis against an equal volume of IX phosphate-buffer saline (PBS) for 4 h (37 °C) using an HTD96b equilibrium dialysis setup (HTDialysis, Gales Ferry, CT). Aliquots from the sera and buffer sides of the dialysis membranes can be removed and analyzed by liquid chromatography-tandem mass spectrometry (LC- MS/MS) (Alturas, Moscow, ID). Unbound nicotine can be quantified by comparing peak intensities to an internal standard of d4-nicotine and a standard curve generated with a nicotine standard.
[0248] Theoretical MHC II Population Coverage of Epitope Combinations. The number of predicted high affinity binding CD4 TCEs that can be generated from peptide carriers comprising various TCE and/or BCE combinations (e.g., SEQ ID NO: 68 - SEQ ID NO: 74) can be calculated using the Immune Epitope Database (http://www.iedb.org/). A larger number of entries at a percentile < 1 implies an improved MHCII binding across a representative set of human haplotypes.
[0249] In vitro characterization of MHC II activity of T cell epitopes. The anticipated activity of CD4 TCEs may be determined in a specific species using a predictive ELISPOT-based assay. PBMCs or splenocytes from a host species of interest can be isolated and co-cultured with species-specific interleukin-2 and the desired T cell epitope for 10-14 days. These cultures may then be added to ELISPOT plates and co-incubated with the same T cell epitope for a three-day stimulation period. The ELISPOT plates can be developed and the number of spots counted and compared to background. The spots generated in the assay are predicted to correspond to the immunogenicity of the T cell epitope, and allow down-selection of the T cell epitopes with the best empirical performance. The T cell epitopes selected using this in vitro screen can be input into the IEDB MHC Class II binding predictor as fusions of 2 or more TCEs in various orientations. These fusions can also be screened in vitro (e.g., ELISPOT). Combinations showing the best activity using this algorithm can be selected to build vaccines for in vivo testing.
[0250] Antibody Assays. Serum samples are serially diluted 3-fold from 1/100 in blocking buffer (3% BSA in PBST) and assayed by ELISA using standard methodologies. Midpoint titers at half maximal absorbance are calculated using GraphPad Prism (GraphPad Software, San Diego, CA). Amyloid-beta and GnRH Abs are measured using cysteine-terminated synthetic peptides conjugated onto BSA using maleimide crosslinking chemistry. Tau antibodies (Abs) are measured using a 441 AA recombinant isoform of human Tau protein (rPeptide, Watkinsville GA). Mouse testosterone can be detected with a commercial kit purchased from Abeam
(Cambridge MA). IgE Ab titers can be measured using mouse IgE (Thermo-Fisher) as a coating reagent and goat anti-mouse IgG Fc-HRP (Southern Biotech, Birmingham AL) as the secondary. Specificity of Ab binding and relative avidity to IgE can be measured by competition ELISA. Serial dilutions of murine IgE, IgG, or IgM can be preincubated with antisera for 1 hour and then added to wells coated with these Ab isotypes for an additional hour. Goat anti-mouse IgG Fc- HRP can be used for detection. ELISA signals can be fit with an inhibition regression algorithm and IC50S determined for each group using GraphPad Prism. Free IgE in mice, or the amount of IgE unbound by anti-IgE Abs in serum, is measured by ELISA for its ability to bind receptor, where mFcsRI (NBS-C Bioscience, Austria) and goat anti-mlgE (Southern Biotech;
Birmingham, AL) are the capture and detection reagents. Helix A titers can be measured using recombinant HA from H1N1 A/California/07/2009, H3N2 A/Wisconsin/67/2005, H5N1
A/Vietnam/l 203/2004, H7N9 A/Anhui/l/20l3 or B/Malaysia/2506/2004 (Protein Sciences). [0251] T Cell ELISPOT. IFN-g (R&D Systems, Minneapolis, MN, USA) ELISPOT analyses can be conducted according to the manufacturer’s instructions. Briefly, splenocytes from immunized and unimmunized mice can be resuspended and serially diluted in RPMI media supplemented with 10% Fetal Bovine Serum (FBS) and L-glutamine. Resuspended splenocytes can be stimulated with 20 pg/mL peptide (e.g., peptide-dimer, peptide-trimer, etc.), media alone or Phorbol l2-myristate l3-acetate (PMA) and ionomycin for 48 h prior to development. Spot images can be collected using ImmunoCapture 6.4 and analyzed with ImmunoSpot 5.0 on an automated ELISPOT plate reader (C.T.L. Seri3A Analyzer; Cellular Technology, Shaker Heights, OH, USA).
EXAMPLES
[0252] The following examples are included to further describe some aspects of the present disclosure, and should not be used to limit the scope of the disclosure.
EXAMPLE 1
Determination of Size and Shape of SAPN-based Vaccines
[0253] This example shows the determination of the physicochemical properties including the size and shape of the peptide carriers.
[0254] The helical structure of the peptide carrier (SEQ ID NO: 271) comprising an alpha-helical peptide with five heptad sequences in the coiled-coil domain (SEQ ID NO: 19) followed by the PADRE TCE (SEQ ID NO: 71) and a TCE isolated from influenza H5N1 hemagglutinin (SEQ ID NO: 73) linked to its C-terminus was determined using circular dichroism (CD). Peptide stock solutions (ca. 50 mM) were prepared for CD Spectroscopy using PBS. CD spectra were recorded in the wavelength range of 190-270 nm on a Jasco J720 spectropolarimeter using 10 mm path length cells (FIG. 2).
[0255] The peptide carrier showed a clear alpha-helical structure as indicated by the signal minima at 208 nm and 222 nm (FIG. 2A). At temperature above 65 °C the ratio of signal intensity at 222 nm and 208 nm indicated the formation of stable coiled-coil conformations.
[0256] The size distribution of the formed coiled-coil oligomers was determined using analytical ultracentrifugation (AUC). Three main species were found to sediment at 0.76, 1.77, and >3.0+ Svedberg (S) units, corresponding to peptide assemblies of 14% monomers (~7.5 kDa), 70% trimers (~22 kDa), and 16% higher-order assemblies that likely correspond to hexamers, nanomers, dodecamers, etc. (FIG. 2B). [0257] Thus, the peptide carrier possessed an alpha-helical secondary peptide structure, and assembled primarily into stable trimeric coiled-coil structures.
EXAMPLE 2
Comparison of Nanoparticle Size Distributions
[0258] This example shows the determination of size distribution of the formed peptide oligomers.
[0259] Stock solutions of peptide monomers having SEQ ID NO: 271, SEQ ID NO: 275 and SEQ ID NO: 277 (ca. 50 mM) were prepared for dynamic light scattering (DLS) Spectroscopy using 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (100 mM, 50 mM NaCl, pH 7.5). Size distributions of assembled peptides were measured by DLS using a Zetasizer Nano
(Malvern Instruments, UK). Prior to the measurement, the peptide was filtered through a 0.2 pm nylon membrane and loaded into a plastic microcuvette. DLS studies were carried out in general purpose mode. Measurement parameters were as follows: material setting was protein (refractive index = 1.440), dispersant setting was water (viscosity = 0.8872 cP, refractive index = 1.330), 10 cycles averaged per measurement, and 30 second temperature equilibration at 25 °C. The Zetasizer Nano instrument uses a 4 mW He-Ne laser (633 nm) and a fixed detection angle (173 °C).
[0260] The particle size distribution of the peptide assemblies using the peptide carrier having SEQ ID NO: 271 showed maximum peak intensities that correspond to particles sizes of 7, 22, and 350 nm, respectively. The 7 nm assembly corresponded to the trimeric coiled-coil peptide, whereas the 22 and 350 nm assemblies corresponded to higher aggregates (FIG. 2C).
Conversely, the particle size distribution of the peptide assemblies using the peptide carrier having SEQ ID NO: 275 and SEQ ID NO: 277 showed maximum peak intensities that correspond to particles sizes of 20 nm and 15 nm, respectively (FIG. 3B and FIG. 3C). These correspond to higher-order assemblies. This nanoparticle size distribution was verified for the peptide carrier having SEQ ID NO: 275 using AUC, which exhibited an absence of the trimeric peak at 1.72S (FIG. 3A). Peptides (SEQ ID NO: 289-292, see e.g., FIG. 17, FIG. 17A-FIG.
17D for DLS analysis of these peptide carriers) with tau and amyloid-beta BCEs on the N- or C- terminus of the carrier peptide having alpha helical domain SEQ ID NO: 26 and TCE domain SEQ ID NO: 67 showed a similar size distribution to the unmodified carrier peptide having SEQ ID NO: 277. EXAMPLE 3
Two TCEs Linked in Tandem Elicit Synergistic Effects
[0261] This example illustrates that two TCEs linked in tandem elicit superior immune responses compared to either TCE alone (see e.g., FIG. 4).
[0262] The nicotine hapten (average 4 nicotine haptens/peptide) was linked to the alpha-helical peptide domain of the three peptide carrier peptides (SEQ ID NO: 272-274) via a linker that was covalently attached to the G position of the nicotine molecule (see e.g., FIG. 9A). Antibody responses to the nicotine hapten were induced by nicotine-carrying peptides with SEQ ID NO: 272, SEQ ID NO: 273, and SEQ ID NO: 274 comprising either one PADRE CD4 T cell epitope peptide (SEQ ID NO: 71), one diphtheria CD4 T cell epitope peptide (SEQ ID NO: 72), or a fusion epitope peptide (SEQ ID NO: 81) of both the diphtheria and the PADRE CD4 T cell epitope. The antibody response of all three peptide-based nicotine vaccines were compared to PBS as the internal control.
[0263] Outbred CD-l mice were injected with 5 pg of peptide carrier. Antibody titers were determined by ELISA 56 days after administration of the peptide carriers. The asterisk indicates a significant difference between groups where PO.OOOl. The results show demonstrate that the use of multiple TCEs results in synergistic effects, making peptide-based vaccines even more effective compared to the use of single TCEs.
EXAMPLE 4
Theoretical MHC II Population Coverage of TCE Combinations
[0264] This example demonstrates activity of the indicated peptides which comprise TCE domains having SEQ ID NO: 83, SEQ ID NO: 67, SEQ ID NO: 81, and SEQ ID NO: 82 can be predicted from the high affinity binding modes using the Immune Epitope Database
(http://www.iedb.org/) (FIG. 5).
[0265] FIG. 5A shows calculation results that predicted that the TCE domain with SEQ ID NO: 83 may have the best activity across a polymorphic human population in comparison to the peptide carriers with SEQ ID NO: 67, SEQ ID NO: 81, and SEQ ID NO: 82.
[0266] Evaluation of these TCEs in peptide carrier-based vaccines in vivo confirmed that the peptide carrier with SEQ ID NO: 275 exhibits the strongest activity across a polymorphic human population. FIG. 5B illustrates a graph of antibody titers induced in outbred mice by the tested CD4 T cell epitope peptides. Animals immunized with peptide carrier having SEQ ID NO: 275 expressed the best titer. FIG. 5C shows a graph of antibody affinity induced in outbred mice by the tested CD4 T cell epitope peptides. Animals immunized with peptide carrier having SEQ ID NO: 275 also expressed the best affinity.
EXAMPLE 5
Activity of TCE Combinations can be guided by in vitro and in silico methods.
[0267] This example demonstrates that the activity of established as well as novel and synthetic CD4 TCEs in a given species can be predicted, verified and enhanced by in vitro and in silico methods.
[0268] FIG. 6 shows the activity of a variety of CD4 TCE sequences selected through in silico methods (SEQ ID NOS: 66-80, 88-102, 112-136, 141-167, 83, 75, 76) in naive cynomolgus macaque peripheral blood mononuclear cells (PBMCs) in a predictive ELISPOT assay used to build a vaccine for cynomolgus macaques. CD4 TCEs were chosen from pertinent publications. These sequences were entered into the Immune Epitope Database and Analysis Resource (iedb.org) MHC Class II binding predictor. Results from this in silico screen were used to identify CD4 T cell epitopes of interest (SEQ ID NOs: 66-80, 88-102, 112-136, 141-167, 83, 75, 76). This set of epitopes was further culled based on activity using an ELISPOT-based in vitro assay. In brief, PBMCs or splenocytes from a host species of interest were isolated and co cultured with interleukin-2 and the desired T cell epitope for 10-14 days. These cultures were added to ELISPOT plates and co-incubated with the same T cell epitope for a three-day stimulation period. The ELISPOT plates were developed and the number of spots counted and compared to background. The spots generated in the assay were predicted to correspond to the immunogenicity of the T cell epitope, and allow down-selection of the T cell epitopes with the best empirical performance. The T cell epitopes selected using this in vitro screen can be input into the IEDB MHC Class II binding predictor as fusions of 2 or more TCEs in various orientations. Combinations showing the best activity using this algorithm can be tested using another in vitro screen (e.g., ELISPOT) or selected to build vaccines for in vivo testing. This shows that this class of peptide carrier vaccines can be rationally designed by ranking CD4 TCE activities through in silico and in vitro methods.
EXAMPLE 6
Peptide Vaccine Activity Using Three BCE Haptens Attached to the Alpha-Helical Domain
[0269] This example shows that peptide carriers with SEQ ID NO: 275 that were synthesized by solid phase with 3 copies of a lysine-nicotine building block hapten (FIG. 7A, SEQ ID NO: 275 x 3/6HA) positioned along the alpha-helical heptad repeat (FIG. 7B) elicited stronger Ab responses in mice than a peptide carrier with 1 copy of the lysine-nicotine building block (SEQ ID NO: 275 x 1/6HA) and a vaccine prepared by conventional chemistry methods (SEQ ID NO: 275 wet, FIG. 7C-FIG. 7E).
[0270] Outbred CD-l mice (n=5) received a prime-boost injection with either PBS or 10 pg of a peptide carrier synthesized by either solid phase peptide synthesis or conventional chemistry methods. Midpoint titer, relative avidity, and binding capacities of antibodies against the nicotine hapten were determined in serum 35 days after administration. FIG. 7C-FIG. 7E show that the peptide vaccine that was synthesized by solid phase chemistry (both SEQ ID NO: 275 x 1 BB and SEQ ID NO: 275 x 3 BB) generated higher antibody responses and exhibited higher binding capacities than the peptide vaccines that were synthesized using conventional synthesis techniques.
EXAMPLE 7
Vaccine Activity of Peptides with Combinations of Different Nicotine Haptens attached to the Alpha-Helical Domain of a Carrier Peptide.
[0271] This example shows that peptide carriers with SEQ ID NO: 275 that were synthesized by solid phase with 3 copies of a 3’ lysine-nicotine building block hapten and 3 copies of a 6HA lysine-nicotine building block hapten (FIG. 8, SEQ ID NO: 275 x 3/3’+3/6HA) positioned along the alpha-helical heptad repeat elicit Ab responses in mice specific to both the 3’ and 6HA haptens.
[0272] FIG. 8A illustrates the placement of 6 nicotine hapten-lysine building blocks along the peptide following solid-phase peptide synthesis. Three haptens contained a linker attached to the 3’ position of the nicotine molecule and 3 haptens contained a linker attached to the 6 position of nicotine.
[0273] FIG. 8B shows that this peptide induced antibody titers specific to both the 6HA hapten and the 3’ hapten. CD-l female mice (h=10) were immunized on days 0, 21 and 35 with 5, 10,
20, or 40 pg of this single peptide containing both the two indicated nicotine haptens having SEQ ID NO: 275 x 3/3’+3/6HA adjuvanted with GLA-SE. As a control, mice were immunized with peptides synthesized with just the 3’ hapten (SEQ ID NO: 275 x 3/3’) or the 6HA hapten (SEQ ID NO: 275 x 3/6HA). Serum was collected on day 35 and assayed by ELISA using BSA conjugated with the 6HA hapten or the 3’ hapten as coating antigens. As measured in the 6HA ELISA, the peptide having SEQ ID NO: 275 x 3/3’+3/6HA induced antibody titers that were equivalent to the positive control peptide (SEQ ID NO: 275 x 3/6HA) and greater than the negative control peptide (SEQ ID NO: 275 x 3/3’). The converse was also true when antisera was assayed in the 3’ hapten ELISA.
EXAMPLE 8
A Multivalent Peptide Nicotine Vaccine
[0274] This example shows that a multivalent, peptide-based nicotine vaccine yields improved nicotine binding capacity compared to a monovalent and bivalent peptide-based nicotine vaccine.
[0275] The peptide vaccine was created by conjugating three structurally distinct nicotine haptens that activate different populations of B cells (FIG. 9A) to the peptide carrier with SEQ ID NO: 275 via linkers at the G, 3’ and 6 position of nicotine to yield three peptide-nicotine conjugates: l’-SEQ ID NO: 275, l’,3’- SEQ ID NO: 275, and l’,3’,6HA- SEQ ID NO: 275. Each peptide monomer was conjugated to 3-4 nicotine hapten copies.
[0276] Female CD-l mice were immunized with the peptide vaccine adjuvanted with GLA-SE on day 0, day 21, and day 42. As shown in FIG. 9B, the nicotine binding capacities increase from the monovalent (2000 ng/mL) to the bivalent (3500 ng/mL) to the trivalent (5200 ng/mL). These results establish the utility of using multivalent peptide vaccines to target multiple disparate B-cell epitopes.
EXAMPLE 9
A Functional, Single SAPN-based Vaccine Against Multiple Influenza Helix A Domains
[0277] An epitope recognized by several broadly neutralizing mAbs (e.g., CR9114) includes Helix A, the first alpha helical domain within the HA stem. This example demonstrates that the secondary structure of the Helix A epitope was successfully recreated by appending the linear Helix A H1N1 sequence on the helical N-terminus of an alpha-helical peptide domain (e.g., SEQ ID NO: 299) with two universal CD4 TCEs (e.g., SEQ ID NO: 83) attached to its C-terminus. The example shows the construction, characterization and in vivo evaluation of a SAPN-based vaccine according to the present disclosure containing a Helix A epitope derived from H1N1 influenza virus that is recognized by broadly neutralizing monoclonal antibodies.
[0278] FIG. 10A shows the Helix A epitope sequence expressed by H1N1 viruses as compared to the alpha helix A epitope expressed by drifted H7N4, H3N2, H5N1 and Type B influenza viruses. The bold underlined amino acids identify residues within viral hemagglutinin helix A that are outward-facing and recognized by broadly neutralizing monoclonal antibodies. The conserved H1N1 influenza helix A epitope peptide (SEQ ID NO: 183) that was used in this study contained all of the underlined amino acid residues that can be recognized by broadly
neutralizing monoclonal antibodies. [0279] Although non-linear epitopes may be challenging to recreate in conjugate vaccines, it was assumed that the alpha-helical heptad domain of an alpha-helical carrier could be used to constrain the Helix A primary sequence to its native helical state with CR9114 contact residues facing outward. The peptide carrier having SEQ ID NO: 278 comprised of 4 heptad sequences in the coiled-coil domain fused to TCEs from Measles virus F2 protein (SEQ ID NO: 75) and Hepatitis B surface antigens (SEQ ID NO: 76) via its C-terminus, and the conserved H1N1 influenza helix A epitope peptide (SEQ ID NO: 183) was linked to the N-terminus. The construction of this peptide did not involve a linker sequence to ensure that the helicity of the carrier would not be interrupted and would continue into the BCE sequence, forcing the BCE into a helix. Indeed, in silico modeling affirmed that correct positioning of CR9114 contact residues in outward-facing heptad positions ensures proper presentation of the secondary structure of the epitope, thereby mimicking the Helix A orientation in native H1N1 hemagglutinin (FIG. 10B, CR9114 contact residues shown as blue spheres).
[0280] The peptide carrier (SEQ ID NO: 278) was synthesized and characterized by DLS, which showed a diameter of ~27 nm (FIG. 10C).
[0281] Outbred CD-l mice (n=l5) were prime boosted using GLA-SE adjuvanted doses (10 pg) or PBS (naive). 35 days after administration, sera were tested for antibody binding to the homologous recombinant Hl hemagglutinin protein as well as the drifted, H7, H3, and H5 proteins (FIG. 10D). The data confirm that anti-sera from vaccinated mice recognized all four helix A domains, although lower amounts were detected for the genetically drifted H7, H3, and H5 epitopes.
[0282] Further analysis established that the vaccine induced a strong Thl CD4 T cell response, which is a property of TLR4 activation by GLA adjuvant (FIG. 10E - FIG. 10G). Splenocytes (n=4) that were harvested from the group on d28 for T cell analysis were stimulated with the TCE dimer (SEQ ID NO: 83) of the construct with SEQ ID NO: 278 and then assayed for activity by IFN-g ELISPOT. Titers of total IgG, IgGl, and IgG2a are indicated (FIG. 10F), as well as the ratio between IgG2a and IgGl isotypes (FIG. 10G). Values greater than 1 are indicative of a Thl response, which is induced by the TLR4 agonist GLA.
[0283] On day 40 after administration of the peptide vaccine, influenza challenge experiments were performed by infecting mice intranasally with 1 OxLD o dose of A/C A/07/09 in 50 pL PBS. Mice were monitored for weight loss and other signs of virus induced morbidity daily and sacrificed if weight loss exceeded 20% of initial body weight. As shown in FIG. 10H,
immunized mice induced a robust anti-A helix domain Ab titer and were successfully protected against virus challenge. EXAMPLE 10
Matching the Electrostatic Charge Between the Coiled-coil Domain and the B Cell Epitope
Improves Vaccine Performance
[0284] This example shows that the vaccine performance of a peptide-based vaccine can be improved if the electronic net charges of the peptide carrier and the epitope peptide match (FIG. 11)
[0285] The sequences of the peptide carriers with SEQ ID NO: 279 and SEQ ID NO: 280 are shown in FIG. 11 A. The alpha-helical domain with outward-facing negatively charged glutamic acid residues (E) (e.g., SEQ ID NO: 46) is followed by the tetanus toxoid TCE (SEQ ID NO: 67) and the Influenza A M2e epitope (SEQ ID NO: 222), which contains 2 negatively charged glutamic acid amino acids. The alpha-helical domain with outward-facing positively charged lysines (K) (e.g., SEQ ID NO: 34) is followed by the tetanus toxoid T cell epitope (SEQ ID NO: 67) and the Influenza A M2e epitope (SEQ ID NO: 222). A WP linker was used to fuse the alpha-helical and TCE domains, while a G linker was used to fuse the TCE and BCE domains.
[0286] Outbred CD-l mice (n=5) received a prime-boost injection with PBS, or 10 pg of either SEQ ID NO: 279 (Glu-rich, FIG. 11A top) or SEQ ID NO: 280 (Lys-rich, FIG. 11A bottom). On day 35, sera were assayed for anti-M2e Abs using an M2e-BSA ELISA reagent. The data demonstrated that Ab responses (FIG. 11B) are significantly improved (P<0.000l; Students T test) when the carrier’s negative surface charge matches the negative charge of the M2e epitope and that this Glu-rich analogue of the carrier peptide confers protection against an influenza challenge with A/California/07/2009(HlNl) (FIG. 11C) . Without being bound to any theory, the results suggest that the M2e epitope peptide is repelled from associating with the Glu-rich peptide carrier, making it more available to cross-link antigen receptors on B cells. In the case of Lysine-rich carrier, the negative charges in the M2e epitope can hydrogen bond with the positively-charged surface lysine residues, which buries the epitope in the peptide nanoparticle and reduces its ability to bind B cells.
EXAMPLE 11
Peptide Vaccine Performance using two BCE Peptides Attached Along the Length of the
Alpha-Helical Domain
[0287] This example illustrates that a peptide-based vaccine containing M2e BCEs (e.g., SEQ ID NO: 222) attached along the length of the alpha-helical coiled-coil peptide domain can induce strong antibody responses. [0288] The peptide carrier with SEQ ID NO: 282 was synthesized using automated peptide synthesizers. Specified Lys residues were selectively deprotected for attachment with the M2e BCE peptide. The C-terminus carboxylic acid of the BCE peptide was attached to the amine in the Lys side chains through standard amino acid coupling chemistry. Thereafter, the remaining amino acids of the M2e BCE and alpha-helical carrier (containing the Measles/Hepatitis B fusion TCE domain with SEQ ID NO: 83) peptide chains were deprotected. FIG. 12A illustrates the attachment locations of 2 M2e BCEs (SEQ ID NO: 222) on the peptide monomer with SEQ ID NO: 282.
[0289] Outbred CD-l mice (n=5) received a prime-boost injection with either PBS or 10 pg of the peptide carrier with SEQ ID NO: 282. FIG. 12B illustrates the antibody titers measured 30 days after outbred CD-l mice were injected with PBS control or 5 pg of peptide carrier. These results demonstrate that BCEs attached along the length of the alpha-helical domain of a peptide carrier elicit strong antibody responses against the BCE even better than to those observed for linear peptide-based vaccines.
EXAMPLE 12
Protective peptide vaccines containing novel influenza B BCEs found using hemagglutinin structural and sequence information
[0290] This example illustrates that a peptide-based vaccine containing novel hemagglutinin BCEs attached along the length of the alpha-helical coiled-coil peptide domain can induce antibodies that bind recombinant hemagglutinin and protect mice from viral challenge.
[0291] X-ray crystallography images (Research Collaboratory for Structural Informatics, Protein Data Bank, rcsb.org) of representative influenza B hemagglutinins from Yamagata and Victoria lineages were used to identify areas of the hemagglutinin protein sequence on the protein surface and available for antibody binding (FIG. 13A). Two of these sequence domains (SEQ ID NO:
201 and SEQ ID NO: 207) were verified to be conserved across both Yamagata/Victoria lineages using sequence homology (NCBI protein BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi). When these BCEs were incorporated into the alpha-helical carrier (containing the Measles/Hepatitis B fusion TCE domain with SEQ ID NO: 83) using the method described herein, they elicited antibodies that bind recombinant hemagglutinin and protect mice from viral challenge (FIG. 13B-13D).
[0292] Outbred CD-l mice (n=8) received a prime-boost injection with either PBS or 10 pg of one of the peptides (SEQ ID NO: 283 or SEQ ID NO: 284). FIG. 13B illustrates the antibody titers to a BSA conjugate and recombinant influenza B hemagglutinin (B/Malaysia/2506/04) measured 30 days after outbred CD-l mice were injected with PBS control or 10 pg of peptide carrier, showing that structural and sequence information can be used to predict BCE activity and BCE activity can be verified by determining whether antisera binds native protein. On day 40, mice were challenged with 5xLD50 of B/Florida/04/06 and monitored for survival with a weight loss cutoff of 80%. As shown in FIG. 13C-FIG. 13D, immunized mice were successfully protected against virus challenge. Given the strong conservation in these sequences, these vaccines may be able to prevent infection from all Influenza B viruses. These results also demonstrate that these peptide carriers can be used for B cell epitope discovery by empirically verifying the activity of BCEs predicted using protein sequence and structure information.
EXAMPLE 13
Functional SAPN-based Vaccines Against Influenza B Cell Epitopes
[0293] This example demonstrates that epitope-targeting influenza vaccines according to the present disclosure induced robust antibody titers and successfully protected mice against virus challenge.
[0294] Peptides were made with two copies of M2e from IAV (SEQ ID NO: 282), NA1 (SEQ ID NO: 285), NA2 (SEQ ID NO: 286), M2e from IBV (SEQ ID NO: 287), HA27-39 (SEQ ID NO: 283) or HA231-241 (SEQ ID NO: 284). Peptides were synthesized with a single N-terminus copy of either the H1N1 HxA BCE (SEQ ID NO: 278) or the influenza B HxA BCE (SEQ ID NO: 288). Female CD-l mice were immunized with one peptide (10 pg) adjuvanted with GLA-SE on day 0 and day 21. On day 40, mice were challenged with 5xLD50 of A/California/07/2009(HlNl) or B/Florida/04/06 (depending on whether the BCE was IAV-specific, IBV-specific, or universal) and monitored for survival with a weight loss cutoff of 80%. Immunized mice were successfully protected against virus challenge (FIG. 14), showing that a variety of linear and helical epitopes can successfully be presented with this class of peptides. Given the strong conservation in these sequences, these vaccines may elicit broad protection against all A or B Type influenza strains.
[0295]
EXAMPLE 14
A Multivalent Peptide Influenza Vaccine Enhances Protection
[0296] This example shows that a trivalent peptide-based influenza vaccine yields improved protection compared to monovalent and bivalent peptide-based influenza vaccines.
[0297] Peptide carriers with SEQ ID NO: 278, 282 and 285 were synthesized using automated peptide synthesizers using the methodologies described elsewhere herein. The peptides contained the Measles/Hepatitis B fusion TCE domain (SEQ ID NO: 83) and two copies of the M2eA sequence (SEQ ID NO: 222), two copies of the NA peptide sequence (SEQ ID NO: 208) or a single copy of the Hl Helix A (HxA) peptide fused to the N-terminus of the carrier peptide (SEQ ID NO: 183).
[0298] Female CD-l mice were immunized with either the M2e (SEQ ID NO: 282), NA (SEQ ID NO: 285), or HxA (SEQ ID NO: 278) peptides singly (10 pg) or admixtures of M2e+NA or M2e+NA+HxA peptides (10 pg each peptide) adjuvanted with GLA-SE on day 0 and day 21. On day 40, mice were challenged with 5xLD50 of A/California/07/2009(HlNl) and monitored for survival with a weight loss cutoff of 80%. As shown in FIG. 15A, the monovalents led to partial survival while the multivalent vaccines led to full protection against the virus and as shown in FIG. 15B, the trivalent vaccine minimized least weight loss. These results establish the utility of using multivalent peptide vaccines to target multiple pathogenic B-cell epitopes and thereby enhance antibody-mediated protection.
EXAMPLE 15
Functional SAPN-based Vaccines for Alzheimer’s Disease
[0299] This example describes the construction and in vivo evaluation of a peptide-based vaccine for Alzheimer’s disease. These functional vaccines were tested in murine CD-l models.
[0300] Tau protein (SEQ ID NO: 261) (FIG. 16) or Amyloid-beta (SEQ ID NO: 262) epitopes were linked to either the N- or C-terminus of an alpha-helical peptide resulting in peptide carriers with SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, and SEQ ID NO: 292. DLS analysis demonstrated that appending terminal BCEs to the alpha-helical peptide had marginally affected nanoparticle size (FIG. 17A-FIG. 17D), although the peak intensities of the N-Tau protein (20 nm; FIG. 17A) and C-Tau protein (12.5 nm; FIG. 17B) were somewhat smaller than N-amyloid- beta (28 nm; FIG. 17C) and C-amyloid-beta (20 nm; FIG. 17D), despite their similar lengths. Regardless of the epitope, the peak intensities were slightly larger when the epitope was attached to the N- versus the C-terminus. Thus, the BCE sequence and location influences particle shape and size, although not to the degree seen with the TCE sequence (FIG. 2-FIG. 3).
[0301] The peptide vaccines were formulated with GLA-SE adjuvant and injected into outbred CD-l mice. Both pairs of amyloid-b and Tau peptides successfully induced strong antibody titers after the first injection of the peptide vaccine on day 35 (FIG. 18A-FIG. 18D). There were no significant differences in response observed between the placement of the BCE at either the N- or C-termini. These results confirm that nanoparticle-forming peptides synthesized with clinical candidate BCE’s can induce Ab responses in mice. Importantly, both the Tau and amyloid-beta peptides induced strong antibody responses in outbred mice with no apparent difference in immunogenicity when the BCE was inserted at either terminus. These results show that the universal T cell epitopes in the vaccine are effective in mice with a heterogeneous MHC background and that the Ab epitopes are readily accessible for B cell engagement, regardless of their attachment point.
EXAMPLE 16
Functional SAPN-based Vaccines for Immunocastration
[0302] This example demonstrates that the peptide vaccine of the present disclosure significantly reduces the fertility of mammals.
[0303] The peptide vaccine was synthesized using the alpha-helical peptide having SEQ ID NO: 293 comprising a 10 amino acid gonadotropin-releasing hormone (GnRH, SEQ ID NO: 263) sequence via its C-terminus. The peptides formed nanoparticles with an average size of about 20 nm.
[0304] Male CD-l mice were immunized with the peptide vaccine adjuvanted with GLA-SE on day 0, day 21, and day 42. As shown in FIG. 19A, anti-GnRH Ab titers reached maximal levels following the first boost of the peptide carrier having the sequence set forth in SEQ ID NO: 293 and remained high throughout the course of the experiment. To confirm that these antibodies may prevent normal testes function, their fertility was measured by breeding each male mouse with 4 female mice.
All 5 PBS-injected mice impregnated each female successfully and collectively produced 285 embryos. On the contrary, 4 of 5 mice peptide-immunized with the peptide vaccine having SEQ ID NO: 293 failed to breed and the fifth mouse impregnated 2 of 4 females, resulting in a total of 25 embryos (FIG. 19B). Additional evidence that vaccine-induced GnRH Abs effectively bound and neutralized the endogenous hormone was corroborated by undetectable testosterone levels in day 63 sera (FIG. 19C) and a lO-fold reduction in testis weight (FIG. 19D). Corresponding to the loss of testosterone production and testis function, histological analysis of male mice immunized with the peptide vaccine having SEQ ID NO: 293 demonstrated a marked change in interstitial Leydig cell morphology (FIG. 20) and severe tubular degeneration (FIG. 20A and FIG. 20B). These results establish the functionality of a new SAPN-based vaccine platform for therapeutic vaccines targeting self-proteins. EXAMPLE 17
A Multivalent Peptide Vaccine for Dengue Virus
[0305] This example shows multivalent, peptide-based vaccines that offer protection against the four DENV serotypes DENV1, DENV2, DENV3, and DENV4.
[0306] The peptide carriers used for this study comprise an alpha-helical peptide domain (e.g., SEQ ID NO: 16 - SEQ ID NO: 65), two TCEs (e.g, SEQ ID NO: 66 - SEQ ID NO: 182), and a BCE peptide that is either an E protein epitope peptide (SEQ ID NO: 245), anon- structural protein 1 (NS1) epitope peptide (SEQ ID NO:246) or a modified derivative of this non- structural protein 1 (NS1) epitope peptide (SEQ ID NOs: 247). SAPN), and that are linked to the C- terminus of the peptide carrier. SANP -based vaccines comprising the BCE with SEQ ID NO: 14 - SEQ ID NO: 16 are tested for their ability to induce antibody responses against the four DENV serotypes DENV1, DENV2, DENV3, and DENV4 in vivo.
[0307] Outbred CD-l mice (n=5) receive a prime-boost injection with PBS, 10 pg of one peptide vaccine, or all three peptides admixed. On day 35, sera are assayed for anti -DENV Abs using an peptide-BSA ELISA assays. The data show that the three peptide carriers comprising SEQ ID NO: 245, SEQ ID NO: 246 and SEQ ID NO: 247 elicit strong Ab responses when admixed against all four DENV serotypes DENV1, DENV2, DENV3, and DENV4. This demonstrates that the multivalent peptide vaccine approach of the present disclosure allows for the in vivo production of broadly neutralizing anti -DENV antibodies in animal models.
EXAMPLE 18
A Multivalent Peptide Vaccine for Hepatitis C Virus
[0308] This example describes a multivalent, peptide-based vaccine that offers broad protection against the seven reported HCV genotypes.
[0309] Peptide carriers comprising an alpha-helical peptide domain (e.g., SEQ ID NO: 16 - SEQ ID NO: 65) and an HCV-derived epitope peptides comprising an epitope peptide selected from SEQ ID NO: 248 - SEQ ID NO: 258. The resulting peptide-based vaccines are tested for their ability to induce antibody responses against the seven reported HCV genotypes in vivo.
[0310] Outbred CD-l mice (n=5) receive a prime-boost injection with PBS, or 10 pg of above described peptide-based vaccines containing BCEs with SEQ ID NO: 248 - SEQ ID NO: 258, or admixtures thereof. On day 35, sera are assayed for monoclonal antibodies against these epitopes using ELISA assays. The data show that some admixtures of the peptide carriers comprising epitope peptides with SEQ ID NO: 248 - SEQ ID NO: 258 elicit strong Ab responses against all HCV genotypes. Thus, the results demonstrate that the multivalent peptide-based vaccine approach of the present disclosure allows for broad HCV protection by eliciting a strong production of broadly neutralizing anti -HCV antibodies in animal models.
EXAMPLE 19
A Multivalent Peptide Vaccine for Herpes Simplex Virus 1 and 2
[0311] This example shows a multivalent, peptide-based vaccine that offers simultaneous protection against HSV-l and HSV-2 utilizing B cell epitope peptides that are conserved between both strains.
[0312] Peptide carriers comprising the alpha-helical peptide monomer (e.g., SEQ ID NO: 16 - SEQ ID NO: 65), a tetanus toxoid TCE (e.g., SEQ ID NO: 66 - SEQ ID NO: 70), a combination of the HSV-l and HSV-2 specific glycoprotein epitope peptides (SEQ ID NO: 232 - SEQ ID NO: 244) are tested for their ability to induce antibody responses against those glycoprotein epitopes in vivo.
[0313] Outbred CD-l mice (n=5) receive a prime-boost injection with PBS, or 10 pg of a peptide carrier comprising BCEs with SEQ ID NO: 232 - SEQ ID NO: 244, or admixtures thereof. On day 35, sera are assayed for antibodies against these epitopes using ELISA assays. The data show that some admixtures of the peptide carriers comprising BCEs with SEQ ID NO: 232 - SEQ ID NO: 244 elicit strong Ab responses against the HSV epitope peptides. This demonstrates that the multivalent peptide-based vaccine approach of the present disclosure allows for the in vivo production of broadly neutralizing anti -HSV antibodies in animal models.
EXAMPLE 20
A Multivalent Peptide Vaccine against Respiratory Syncytial Virus
[0314] This example shows a multivalent, peptide-based vaccine that offers protection against RSV Type A (RSVA) and B (RSVB) utilizing B cell epitope peptides that are conserved between both strains.
[0315] Peptide carriers comprising the alpha-helical peptide monomer (e.g., SEQ ID NO: 16 - SEQ ID NO: 65), a tetanus toxoid TCE (e.g, SEQ ID NO: 66 - SEQ ID NO: 70), and a RSVA and RSVB specific epitope peptide with either SEQ ID NO: 259 (KNYIDKQLLPIVNK) or SEQ ID NO: 260 (KNYINNQLLPIVNQ) are tested for their ability to induce antibody responses against those glycoprotein epitopes in vivo.
[0316] Outbred CD-l mice (n=5) receive a prime-boost injection with PBS, or 10 pg of a peptide carrier comprising BCEs with SEQ ID NO: 259 and SEQ ID NO: 260, or a bivalent mixture of the two peptides. On day 35, sera are assayed for antibodies against these epitopes using ELISA assays. The data show that the peptide carriers comprising BCEs with SEQ ID NO: 259 and SEQ ID NO: 260 elicit strong Ab responses against the RSV epitope peptides and that the bivalent mixture generates antibodies to both targets. This demonstrates that the multivalent peptide vaccine approach of the present disclosure allows for the in vivo production of broadly neutralizing anti-RSV antibodies in animal models.
EXAMPLE 21
A Peptide Vaccine against IgE-mediated hypersensitivity
[0317] This example shows that strong antibody responses against IgE can be elicited using the peptide vaccines as described herein.
[0318] A peptide carrier comprising an alpha-helical domain comprising 5 IKKIEKR heptad repeats (e.g., SEQ ID NO: 19), and a TCE domain comprising the Measles virus F2 protein TCE (SEQ ID NO: 75) and the Hepatitis B surface TCE (SEQ ID NO: 76) was further modified by linking a murine-specific Peptide Y BCE (SEQ ID NO: 264) to the C-terminus of the peptide carrier (SEQ ID NO: 294). The peptide Y epitope peptide was derived from the Cs3 domain of murine IgE, which serves as an analogue for human IgE in this animal model.
[0319] Outbred CD-l mice (n=5) receive a prime-boost injection with PBS, or 10 pg of the peptide carrier comprising the peptide Y BCE with SEQ ID NO: 294. (FIG. 21). On day 35, sera are assayed for antibodies against these epitopes using ELISA assays. The data show that the peptide carriers that comprise the Peptide Y BCE elicited strong Ab responses that have nanomolar affinities that are specific for IgE and not other immunoglobulin proteins like IgG or IgM. This demonstrates that the peptide vaccine approach of the present disclosure allows for the in vivo production of broadly neutralizing anti-IgE antibodies in animal models (FIG. 21 A - FIG. 21C, FIG. 22)
[0320] FIG. 22A shows that the anti-IgE antibodies described in FIG. 21 were able to reduce the concentration of free IgE ~l0-fold in 6 of 10 mice and ~l 000-fold in 4 of 10 mice. Day 35 antisera from immunized (h=10) and control (h=10) animals were assayed for free IgE in a competition ELISA that IgE binding to mFceRI receptor (unpaired t-test,* p<0.002).
[0321] FIG. 22B shows that the polypeptide with SEQ ID NO: 294 could also be used to inhibit acute IgE-mediated anaphylaxis, confirming its therapeutic potential. Vaccinated (n=4) and naive mice (n=4) were anesthetized and injected (intraorbital) with 0.5 pg of 2,4-dinitrophenol (DNP)- specific IgE and then DNP-HAS 24 hours later. Anaphylaxis was measured by a reduction in body temperature. Anesthesia alone caused a drop in temperature as in naive mice not injected with IgE (grey circles). No significant difference in body temps were observed between the
-ca vaccinated and naive (-IgE) mice except at 20 min (p<0.05). Differences in body temps between naive (+IgE) and vaccinated (+IgE) were significantly different after the first 10 minutes (l-way ANOVA; p<0.05).
[0322] FIG. 22C shows that the peptide vaccine having the amino acid sequence set forth in SEQ ID NO: 294 forms nanoparticles with an average DLS diameter of -15 nm.
EXAMPLE 22
General Peptide Analytics and Peptide Modeling Procedures
[0323] This example demonstrates the general analytical evaluation of the peptide carrier vaccines described herein as well as procedures for modeling their three-dimensional structure.
[0324] All peptides described herein were synthesized using automated solid-phase peptide synthesizers.
Peptide Analytics
[0325] Peptide stock solutions were prepared for CD Spectroscopy using 50 mM phosphate- buffered saline (PBS). Spectra were recorded from 190-270 nm on a Jasco J720
spectropolarimeter (Easton, MD) using 10 mm path length cells. Temperatures ranged from 5 to 95 °C in increments of 10 °C. Analytical ultracentrifugation was performed by Alliance Protein Laboratories (San Diego, CA). The peptide was dissolved in PBS to ~l mg/mL and the peptide was dissolved in aqueous MOPS (100 mM, 50 mM NaCl, pH 7.5) to ~0.5 mg/mL. Samples were filtered through a 0.2 pm nylon membrane. The samples were loaded into a Beckman-Coulter ProteomeLab XL-A analytical ultracentrifuge (Brea, CA). After equilibration at 20 °C the rotor was brought to 60,000 rpm. Scans were recorded every 4 min for -10 h. The data were analyzed using SEDFIT (version 11.3). The resultant size distributions were graphed, and the peaks were integrated using OriginLab Origin® version 9.0 (Northampton, MA). DLS Spectroscopy was performed using a Zetasizer Nano (Malvern Instruments, UK) with a 4 mW He-Ne laser (633 nm) and a fixed detection angle (173°). Prior to measurement, peptides were raised in PBS or MOPS (100 mM, 50 mM NaCl, pH 7.5), filtered through a 0.2 pm nylon membrane and loaded into a plastic microcuvette. Measurements were carried out in general purpose model with the following parameters: material setting was protein (refractive index = 1.440), dispersant setting was water (viscosity = 0.8872 cP, refractive index = 1.330), 10 cycles averaged per measurement, and 30 second temperature equilibration at 25°C. For transmission electron microscopy, peptides (0.05 mg/ml in PBS) were adsorbed onto carbon coated grids, washed and negatively stained with 0.5% uranyl acetate (Charles River, Durham NC). Electron micrographs were taken on a JEOL 1400 Transmission Electron Microscope. Molecular Modeling of the peptide carrier having the sequence set forth in SEQ ID NO:
275
[0326] The structure of the peptide with SEQ ID NO: 275 was constructed, analyzed, and rendered with Chimera software using the trimeric coiled-coil construct without T cell epitopes as a template. The T-cell epitopes with the sequence set forth in SEQ ID NO: 83 were modeled using the PEP-FOLD344 server and coupled to the trimeric coiled-coil carrier at the C-terminus using the structure building tools in Chimera to form peptide bonds. The Qwik-MD tool in VMD was used for molecular dynamics setup, solvation/ionization, and a 1,000 step equilibration.
[0327] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A composition comprising:
(a) an alpha-helical peptide domain;
(b) at least one CD4 T cell epitope peptide; and
(c) at least one B cell epitope peptide selected from an immunogenic protein or peptide, carbohydrate, or lipid that mediates a physiological condition or disease including neural degenerative diseases, allergy, and
autoimmunity,
wherein each of the alpha-helical peptide domain, T cell epitope peptide, and B cell epitope peptide are associated.
2. The composition of claim 1, wherein the B cell epitope peptide is selected from SEQ ID NO: 183 - SEQ ID NO: 270.
3. A composition comprising:
(a) an alpha-helical peptide domain;
(b) at least one CD4 T cell epitope peptide selected from SEQ ID NO: 66 - SEQ ID NO: 182; and
(c) at least one B cell epitope peptide,
wherein each of the alpha-helical peptide domain, T cell epitope peptide, and B cell epitope peptide are associated.
4. The composition of any one of claims 1-3, wherein the alpha-helical peptide domain comprises at least one heptad repeat with an amino acid sequence according to the general formula: [X1X2X3X4X5X6X7]n(SEQ ID NO: 295), wherein X1 and X4 are each independently selected from I, L, V, A, F, Y, W, N, and Q; X2, X3, X6 are independently selected from the amino acids K, R, E, D, H, S, N, Q, A, T, and C; X5, X7 are independently selected from the amino acids K, R, E, D, and H; and n is any number from 1 to 10.
5. The composition of any one of claims 1-4, wherein the alpha-helical peptide domain can comprise an amino acid sequence according to any one of the following general formulas:
Figure imgf000096_0001
Y1Y2Y3[X1X2X3X4X5X6X7]nY4Y5Y6Y7 (SEQ ID NO: 4);
Y1Y2[X1X2X3X4X5X6X7]nY3Y4Y5Y6Y7 (SEQ ID NO: 5); and
Y1[X1X2X3X4X5X6X7]nY2Y3Y4Y5Y6Y7 (SEQ ID NO: 6),
wherein:
each Y1, Y2, Y3, Y4, Y5, Y6, and Y7 is independently selected from the amino acids I, L, V, A, K, R, E, S, T, P, G, C, N, Q, W, and D;
each X1, X4 are independently selected from the amino acids I, L, V, A, F, Y, W, N, Q; each X2, X3, X6 are independently selected from the amino acids K, R, E, D, H, S, N, Q, A, T, C;
each X5, X7 are independently selected from the amino acids K, R, E, D, H; and each n is independently any number from 1 to 10.
6. The composition of any one of claims 1-5, wherein the alpha-helical peptide domain comprises an amino acid sequence with at least 80% identity to
IKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 18),
IKKIEKRIKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 19),
KKIEKRIKKIEKRIKKIEKRIKKIEKRI (SEQ ID NO: 30),
KIEKRIKKIEKRIKKIEKRIKKIEKRIK (SEQ ID NO: 27),
IEKRIKKIEKRIKKIEKRIKKIEKRIKK (SEQ ID NO: 28),
EKRIKKIEKRIKKIEKRIKKIEKRIKKI (SEQ ID NO: 31),
KRIKKIEKRIKKIEKRIKKIEKRIKKIE (SEQ ID NO: 32),
RIKKIEKRIKKIEKRIKKIEKRIKKIEK (SEQ ID NO: 33),
DEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 44), or
DEIEERIEEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 45).
7. The composition of claim 6, wherein the alpha-helical peptide domain comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 18, SEQ ID NO:
19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
8. The composition of claim 6, wherein the alpha-helical peptide domain comprises an amino acid sequence with 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
9. A composition comprising:
(a) an alpha-helical peptide domain comprising an amino acid sequence with at least 80% identity to, at least 82.5% identity to, at least 85% identity to, at least 87.5% identity to, at least 90% identity to, at least 92.5% identity to, at least 95% identity to, at least 97.5% identity to, or at least 99% identity to identity to IKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 18), IKKIEKRIKKIEKRIKKIEKRIKKIEKRIKKIEKR (SEQ ID NO: 19), KKIEKRIKKIEKRIKKIEKRIKKIEKRI (SEQ ID NO: 30),
KIEKRIKKIEKRIKKIEKRIKKIEKRIK (SEQ ID NO: 27),
IEKRIKKIEKRIKKIEKRIKKIEKRIKK (SEQ ID NO: 28),
EKRIKKIEKRIKKIEKRIKKIEKRIKKI (SEQ ID NO: 31),
KRIKKIEKRIKKIEKRIKKIEKRIKKIE (SEQ ID NO: 32),
RIKKIEKRIKKIEKRIKKIEKRIKKIEK (SEQ ID NO: 33),
DEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 44), or
DEIEERIEEIEERIEEIEERIEEIEERIEEIEERIEE (SEQ ID NO: 45);
(b) at least one T cell epitope peptide or a B cell epitope peptide, or a
combination thereof; and
(c) a target antigen,
wherein the alpha-helical peptide domain, T cell epitope peptide, and B cell epitope are associated.
10. The composition of claim 9, wherein the alpha-helical peptide domain comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO: 18, SEQ ID NO:
19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
11. The composition of claim 9, wherein the alpha-helical peptide domain comprises an amino acid sequence with 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31 - SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 45.
12. The composition according to claim 1 or 9, wherein the at least one CD4 T cell epitope peptide is an immunogenic peptide fragment selected from the group consisting of diphtheria toxoid peptide epitopes, measles morbillivirus fusion glycoprotein F peptide epitopes, a pan DR epitope (PADRE) peptide, influenza-derived epitope peptides, hepatitis B and C virus epitope peptides, tetanus toxoid peptide epitopes, P. falciparum peptide epitopes, gamma 2ab peptide epitopes, GAD65 peptide epitopes, plasmodium peptide epitopes, polio peptide epitopes, Pseudomonas peptide epitopes, Vaccinia peptide epitopes, Streptococcus peptide epitopes, Yellow Fever peptide epitopes, Coxiella peptide epitopes, Yrsenia pestis peptide epitopes, RSV peptide epitopes, SSP2.61 peptide epitopes, ESAT6 peptide epitopes, tuberculosis peptide epitopes, ebola peptide epitopes, HPV peptide epitopes, anthrax peptide epitopes, varicella peptide epitopes, HSV peptide epitopes, and VEEV peptide epitopes.
13. The composition according to claim 12, wherein the at least one CD4 T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182.
14. The composition of any one of claims 12-13, wherein the at least two CD4 T cell epitope peptides are linked in tandem.
15. The composition of claim 14, wherein the at least one CD4 T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182.
16. The composition of claim 15, wherein the at least one CD4 epitope peptide has SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 157, SEQ ID NO: 164, SEQ ID NO: 165, or SEQ ID NO: 166.
17. The composition of any one of claims 3-16, wherein the B cell epitope peptide is a foreign antigen comprised of an immunogenic protein or peptide, carbohydrate, lipid, or small molecule; a host-derived antigen comprised of an immunogenic protein or peptide, carbohydrate, or lipid that mediates a physiological condition or disease including infectious diseases, neural degenerative diseases, allergy, autoimmunity, and cancer.
18. The composition of claim 17, wherein the B cell epitope peptide is selected from SEQ ID NO: 183 - SEQ ID NO: 270.
19. The composition of any one of claims 1-18, wherein the at least two CD4 T cell epitope peptides are linked in tandem.
20. The composition of claim 19, wherein the at least one CD4 epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182.
21. The composition of claim 20, wherein the at least one CD4 epitope peptide has SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 133, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 157, SEQ ID NO: 164, SEQ ID NO: 165, or SEQ ID NO: 166..
22. The composition of any one of claims 1-21, wherein the target antigen is a small molecule, a peptide, a polysaccharide, a glycolipid, or a lipid.
23. The composition of claim 22, wherein the small molecule is nicotine.
24. The composition of any one of claims 1-23, wherein the N-terminus of the at least one T cell epitope peptide is linked to the C-terminus of the alpha-helical peptide monomer.
25. The composition of any one of claims 1-24, wherein the N-terminus of the at least one B cell epitope peptide is linked to the C-terminus of the at least one T cell epitope peptide or the at least one B cell epitope peptide is linked to the N-terminus of the peptide carrier, or any combination thereof.
26. The composition of any one of claims 1-25, wherein the N-terminus of the at least one B cell epitope peptide is linked to the C-terminus of the at least one T cell epitope peptide or the at least one B cell epitope peptide is linked to the N-terminus of the peptide carrier, or is linked to the alpha-helical peptide monomer, or any combination thereof.
27. The composition of any one of claims 1-26, wherein the at least one B cell epitope peptide is linked along the length of the alpha-helical peptide domain via a non-terminal amino acid.
28. The composition of any one of claims 1-27, wherein the at least one B cell epitope peptide is linked to the N-terminus, C-terminus, or along the length of the alpha-helical peptide domain via an amino acid.
29. The composition of any one of claims 1-28, wherein the at least one B cell epitope peptide is linked to the alpha-helical peptide domain via an unnatural amino acid.
30. The composition of any one of claims 1-29, wherein the at least one B cell epitope peptide is linked to the alpha-helical peptide domain via a non-amino acid chemical functionality.
31. The composition of any one of claims 1-30, wherein the alpha-helical peptide domain comprises at least 1 heptad repeat.
32. The composition of any one of claims 1-31, wherein the alpha-helical peptide domain comprises at least 2 heptad repeats.
33. The composition of any one of claims 1-32, wherein the alpha-helical peptide domain comprises at least 3 heptad repeats.
34. The composition of any one of claims 1-33, wherein the at least one T cell epitope peptide is selected from SEQ ID NO: 66 - SEQ ID NO: 182.
35. The composition of any one of claims 1-34, wherein the at least one CD4 T cell epitope peptides is selected from SEQ ID NO: 66 - SEQ ID NO: 182.
36. The composition of any one of claims 1-35, wherein the at least one T cell epitope peptide comprises the amino acid sequence set forth in SEQ ID NO: 81 - SEQ ID NO: 87.
37. The composition of any one of claims 1-36, wherein the at least one B cell epitope peptide is an immunogenic fragment of a microbial antigen.
38. The composition of claim 37, wherein the microbial antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a protozoan antigen.
39. The composition of claim 38, wherein the microbial antigen is a conserved antigen within a divergent family of bacteria, fungi, or protozoan antigen.
40. The composition of claim 38, wherein the viral antigen is an influenza virus antigen.
41. The composition of claim 40, wherein the influenza virus antigen is a
hemagglutinin antigen, an M2 ectodomain antigen, a neuraminidase antigen, or a nucleoprotein antigen.
42. The composition of claim 41, wherein the hemagglutinin antigen is a conserved influenza hemagglutinin Helix A epitope peptide.
43. The composition of claim 42, wherein the influenza hemagglutinin Helix A epitope peptide comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 183 - SEQ ID NO: 191.
44. The composition of claim 41, wherein the influenza M2 ectodomain antigen comprises all or part of the amino acid sequence set forth in SEQ ID NO: 222 - SEQ ID NO: 227.
45. The composition of claim 41, wherein the influenza hemagglutinin antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 192 - SEQ ID NO: 207.
46. The composition of claim 41, wherein the influenza neuraminidase antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 208 - SEQ ID NO: 221.
47. The composition of any one of claims 1-36, wherein the at least one B cell epitope peptide is an immunogenic peptide or a peptide fragment of a hormone antigen.
48. The composition of claim 47, wherein the hormone antigen is a GnRH antigen.
49. The composition of claim 48, wherein the GnRH antigen comprises the amino acid sequence set forth in SEQ ID NO: 263.
50. The composition of any one of claims 1-36, wherein the at least one B cell epitope peptide is an immunogenic peptide fragment of a neurodegenerative disease antigen.
51. The composition of claim 50, wherein the neurodegenerative disease antigen is an Alzheimer’s disease antigen, a Parkinson’s disease antigen, or a Huntington’s disease antigen.
52. The composition of claim 51, wherein the Alzheimer’s disease antigen is a Tau antigen or an amyloid beta (Ab) antigen.
53. The composition of claim 52, wherein the Tau antigen comprises the amino acid sequence set forth in SEQ ID NO: 262.
54. The composition of claim 52, wherein the amyloid beta (Ab) antigen comprises the amino acid sequence set forth in SEQ ID NO: 261.
55. The composition of claim 51, wherein the Parkinson’s disease antigen is an alpha- synuclein antigen.
56. The composition of any one of claims 1-36, wherein the at least one B cell epitope peptide is an immunogenic peptide fragment of a tumor antigen.
57. The composition of claim 56, wherein the tumor antigen is derived from a member of the receptor tyrosine kinase family or a member of the human epidermal growth factor receptor family.
58. The composition of any one of claims 1-36, wherein the at least one B cell epitope peptide is an immunogenic peptide fragment derived from an immunoglobulin E (IgE).
59. The composition of claim 58, wherein the IgE is human IgE.
60. The composition of claim 59, wherein the at least one B cell epitope peptide is an immunogenic peptide fragment derived from the Ce3 domain of human IgE.
61. The composition of claim 60, wherein the at least one B cell epitope peptide comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 264- 267.
62. The composition of any one of claims 1-61, wherein the at least one T cell epitope peptide is linked to the alpha-helical peptide monomer via a linker.
63. The composition of any one of claims 1-62, wherein the at least one B cell epitope peptide is linked to the at least one T cell epitope peptide or the alpha-helical peptide domain via a linker.
64. The composition of any one of claims 1-63, wherein the linker comprises a chain of amino acids, a synthetic linker, a PEG moiety, or a cleavable linker.
65. The composition of claim 64, wherein the chain of amino acids comprises 1-10 amino acids.
66. The composition of any one of claims 1-65, wherein the linker comprises a serine, alanine, threonine, aspartic acid, lysine, glutamic acid, lysine, glutamine, asparagine, arginine, proline, tryptophan, or glycine linker, or a combination thereof.
67. The composition of any one of claims 1-66 comprising one of the amino acid sequences set forth in SEQ ID NO: 271 - SEQ ID NO: 294.
68. A peptide carrier comprising:
a) an alpha-helical peptide domain;
b) at least one T cell epitope peptide; and
c) at least one B cell epitope peptide,
wherein the at least one T cell epitope peptide and the at least one B cell epitope peptide are linked in tandem via the C-terminus to the alpha-helical peptide monomer, and wherein the net surface charge of the alpha-helical peptide monomer matches the net surface charge of the B cell epitope peptide, which induces an electrostatic repulsion between the alpha- helical peptide monomer and the at least one B cell epitope peptide resulting in an improved vaccine performance.
69. A peptide carrier comprising:
a) an alpha-helical peptide domain;
b) at least one T cell epitope peptide; and
c) at least one B cell epitope peptide,
wherein the at least one T cell epitope peptide and the alpha-helical peptide monomer are linked in tandem, and wherein the B cell epitope peptide is attached by solid phase synthesis at specific locations along the length of the alpha-helical peptide monomer using amino acid-linked building blocks resulting in an improved vaccine performance.
70. An immunogenic composition comprising at least one of the compositions according to any one of claims 1-69 and a pharmaceutically acceptable carrier.
71. An immunogenic composition comprising at least two of the compositions according to any one of claims 1-69 and a pharmaceutically acceptable carrier.
72. An immunogenic composition comprising at least three of the compositions according to any one of claims 1-69 and a pharmaceutically acceptable carrier.
73. An immunogenic composition comprising the composition according to any one of claims 70-72 and a pharmaceutically acceptable carrier, and wherein the composition is capable of inducing an immune response.
74. An immunogenic composition comprising the compositions according to claims 37-41 and a pharmaceutically acceptable carrier, and wherein the composition is capable of inducing an immune response.
75. An immunogenic composition comprising the composition according to claim 47 and a pharmaceutically acceptable carrier, and wherein the composition is capable of inducing an immune response.
76. An immunogenic composition comprising the composition according to claim 50 and a pharmaceutically acceptable carrier, and wherein the composition is capable of inducing an immune response.
77. An immunogenic composition comprising the composition according to claim 56 and a pharmaceutically acceptable carrier, and wherein the composition is capable of inducing an immune response.
78. An immunogenic composition comprising the composition according to claim 58 and a pharmaceutically acceptable carrier, and wherein the composition is capable of inducing an immune response.
79. The immunogenic compositions of any one of claims 68-78, wherein the compositions are capable of self-assembling into polymeric, coiled-coil nanoparticles.
80. The immunogenic composition of claim 79, wherein the size of the polymeric, coiled-coil nanoparticles ranges from about 2 nm to about 30 nm.
81. The immunogenic composition of claim 79, wherein the size of the polymeric, coiled-coil nanoparticles ranges from about 30 nm to about 100 nm.
82. The immunogenic composition of claim 79, wherein the size of the polymeric, coiled-coil nanoparticles ranges from about 100 nm to about 1 pm.
83. The immunogenic composition of claim 79, wherein the size of the polymeric, coiled-coil nanoparticles ranges from about 1 pm to about 10 pm.
84. The immunogenic composition of any one of claims 70-83, further comprising an adjuvant.
85. A method for inducing an immune response in a subject specific for a target antigen comprising administering to the subject the immunogenic composition according to any one of claims 70-84.
86. A method for inducing an immune response against a microbial antigen in a subject, the method comprising administering to the subject an immunogenic composition according to claim 74.
87. A method for inducing an immune response against most or all members of a diverged family of microbes in a subject using one or more conserved B cell epitopes, the method comprising administering to the subject an immunogenic composition according to claim 74.
88. The method of claim 86, wherein the microbial antigen against which the immune response is induced is an influenza virus antigen.
89. A method for inducing an immune response against a hormone antigen in a subject, the method comprising administering to the subject an immunogenic composition according to claim 75.
90. The method of claim 89, wherein the hormone antigen against which the immune response is induced is a GnRH antigen.
91. The method of claim 90, wherein the immune response to a GnRH antigen is used to inhibit sex hormone production in a host mammal.
92. The method of claim 90, wherein the immune response to a GnRH antigen is used to inhibit sex hormone production in humans for treatment of cancer, hyperproliferative, and post-menopausal disorders.
93. A method for inducing an immune response against a neurodegenerative disease antigen in a subject, the method comprising administering to the subject an immunogenic composition according to claim 76.
94. The method of claim 93, wherein the neurodegenerative disease antigen against which the immune response is induced is an Alzheimer’s disease antigen.
95. A method for treating a neurodegenerative disease in a subject in need thereof, the method comprising administering a composition comprising an alpha-helical peptide carrier and at least one neurodegenerative disease antigen.
96. The method of claim 95, wherein the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, or a Huntington’s disease.
97. The method of any one of claims 95-96, wherein the neurodegenerative disease is Alzheimer’s disease.
98. The method of any one of claims 95-96, wherein the neurodegenerative disease is Parkinson’s disease.
99. The method of any one of claims 95-96, wherein the neurodegenerative disease is a Huntington’s disease.
100. The method of any one of claims 93-96,-98 wherein the composition comprises at least one neurodegenerative disease antigen according to claim 50.
101. The method of any one of claims 93-96, wherein the composition comprises at least one Alzheimer’s disease antigen according to claims 94-100.
102. The method of any one of claims 92-101, wherein the composition comprises an alpha-helical peptide carrier comprising an alpha-helical peptide domain according to claims 1- 11
103. The method of any one of claims 92-102, wherein the composition comprises an alpha-helical peptide carrier comprising at least one T cell epitope peptide according to claims 12-16.
104. A method for inducing an immune response in a subject against a hormone antigen, the method comprising administering to the subject a composition comprising an alpha- helical peptide carrier and at least one hormone antigen.
105. The method of claim 104, wherein the at least one hormone antigen is a GnRH antigen.
106. The method of any one of claims 104-105, wherein the GnRH antigen comprises the amino acid sequence set forth in SEQ ID NO: 263.
107. The method of any one of claims 104-106, wherein the composition comprises an alpha-helical peptide carrier comprising an alpha-helical peptide domain according to claims 1- 11
108. The method of any one of claims 104-107, wherein the composition comprises an alpha-helical peptide carrier comprising at least one T cell epitope peptide according to claims 12-16.
109. The method of any one of claims 104-108, wherein the immune response against the hormone antigen is used to inhibit sex hormone production in the subject.
110. A method for treating an infectious disease in a subject in need thereof, the method comprising administering a composition comprising an alpha-helical peptide carrier and at least one infectious disease antigen.
111. The method of claim 110, wherein the infectious disease is caused by an influenza virus.
112. The method of any one of claims 110-111, wherein the at least one infectious disease antigen is an influenza virus antigen according to claims 40-46.
113. The method of any one of claims 110-112, wherein the composition comprises an alpha-helical peptide carrier comprising an alpha-helical peptide domain according to claims 1- 11
114. The method of any one of claims 110-113, wherein the composition comprises an alpha-helical peptide carrier comprising at least one T cell epitope peptide according to claims 12-16.
115. A method for treating an IgE-mediated hypersensitivity disorder in a subject in need thereof, the method comprising administering a composition comprising an alpha-helical peptide carrier and at least one IgE antigen.
116. The method of claim 115, wherein the at least one IgE antigen is derived from human IgE.
117. The method of any one of claims 115-116, wherein the at least one IgE antigen is derived from the Ce3 domain of human IgE.
118. The method of any one of claims 115-117, wherein the at least one IgE antigen comprises all or part of at least one of the amino acid sequences set forth in SEQ ID NO: 264 to SEQ ID NO: 267.
119. The method of any one of claims 115-118, wherein the composition comprises an alpha-helical peptide carrier comprising an alpha-helical peptide domain according to claims 1- 11
120. The method of any one of claims 115-119, wherein the composition comprises an alpha-helical peptide carrier comprising at least one T cell epitope peptide according to claims 12-16.
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WO2022072550A3 (en) * 2020-09-29 2022-06-09 Academia Sinica Clec2 fusion protein and uses thereof
CN117164673A (en) * 2023-08-04 2023-12-05 东北农业大学 Anti-enzymolysis antibacterial peptide IK3-A and preparation method and application thereof
CN117164673B (en) * 2023-08-04 2024-03-29 东北农业大学 Anti-enzymolysis antibacterial peptide IK3-A and preparation method and application thereof

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