WO2021226520A1 - Compositions de peptides pour le traitement d'infections pathogènes - Google Patents

Compositions de peptides pour le traitement d'infections pathogènes Download PDF

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Publication number
WO2021226520A1
WO2021226520A1 PCT/US2021/031387 US2021031387W WO2021226520A1 WO 2021226520 A1 WO2021226520 A1 WO 2021226520A1 US 2021031387 W US2021031387 W US 2021031387W WO 2021226520 A1 WO2021226520 A1 WO 2021226520A1
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WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
polypeptide
composition
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PCT/US2021/031387
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English (en)
Inventor
Maurizio Chiriva-Internati
Leonardo MIRANDOLA
Scott Dahlbeck
Gianluca ROTINO
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Kiromic BioPharma, Inc.
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Publication of WO2021226520A1 publication Critical patent/WO2021226520A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

Definitions

  • the technology relates in part to peptide compositions for the treatment of a disease or condition caused by a pathogen, such as a virus, to methods of selecting peptides for preparing the compositions, and to methods of treatment, including prophylactic treatment, using the compositions.
  • a pathogen such as a virus
  • Vaccines as preventive means, offer a more attractive alternative as development costs and time frames are relatively lower. Vaccines arguably have been the most successful biomedical advance in the prophylactic treatment of pathogenic diseases or conditions. Each year, over 100 million children globally receive vaccinations to prevent diseases that were once widespread and linked to serious medical conditions or even death.
  • Attenuated vaccines also known as “live-attenuated” vaccines
  • live-attenuated vaccines are created by altering the genome, such that they have low pathogenicity or are harmless.
  • Vaccines against measles, mumps, rubella, and others have been created this way and have had a good success rate, but they have sometimes reverted to pathogenic (virulent) status through mutation.
  • Another conventional vaccine is the inactivated vaccine, which is produced by killing the original pathogen (e.g., virus) through heat or chemicals and then introducing the remaining shell (e.g., virus shell) into the host body. The shell, when properly manufactured, retains enough of the original pathogen to elicit an immune response.
  • VLP viral-like particle
  • VLP vaccines are constructed out of surface proteins that can self-assemble to a virus-like structure, which mimics the original virus structure and can elicit a strong immune response with adjuvants.
  • VLP vaccines have been manufactured for the hepatitis B virus, the human papillomavirus (the main cause of cervical cancer), and the hepatitis E virus.
  • Vaccine compositions containing peptides as active agents can be produced, and peptides can be generated by chemical synthesis, approaches suitable for large-scale and cost-effective production (e.g., solid-phase peptide synthesis approaches).
  • Peptides can be modified (e.g., with lipids, carbohydrates, phosphate, acetyl, and amide groups) to increase stability, immunogenicity, and solubility.
  • composition that includes:
  • polypeptides each independently containing an amino acid sequence consisting of, or 95% or more identical to, an amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; and
  • 95% or more identical to means any percentage, as a whole number or fraction thereof, between 95% and 100%, such as, for example, about or equal to 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%
  • the pharmaceutical composition contains 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID N0:4 or SEQ ID NO:5.
  • the pharmaceutical composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the pharmaceutical composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO:1, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5.
  • the pharmaceutical composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5.
  • compositions provided herein can be formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration.
  • the pharmaceutical compositions provided herein are formulated for oral administration.
  • the pharmaceutical compositions can be formulated as a gel, ointment, liquid, suspension, aerosol, tablet, pill, powder or lyophile.
  • the pharmaceutical compositions provided herein are formulated as microparticles.
  • the microparticle contains a sustained-release polymeric matrix.
  • the microparticle contains b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).
  • the pharmaceutical compositions containing the polypeptides provided herein are formulated as microparticles that include between about or equal to 10% to about or equal to 20% w/w ethylcellulose, such as about or equal to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 16%, 17%, 18%, 19% or 20% w/w cellulose, based on the weight of the microparticles.
  • the microparticle contains about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles.
  • the microparticle contains about or equal to 20% w/w ethylcellulose, based on the weight of the microparticles.
  • the pharmaceutical compositions containing the polypeptides provided herein are formulated as microparticles that include between about or equal to 50% to about or equal to 70% w/w b- cyclodextrin, such as about or equal to 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% w/w b-cyclodextrin, based on the weight of the microparticles.
  • the microparticle contains about or equal to 60% w/w b-cyclodextrin, based on the weight of the microparticles.
  • the pharmaceutical compositions provided herein contain an agent that protects the components of the composition against degradation in the acidic environment of the stomach.
  • the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS) and in certain aspects, the composition is formulated as a microparticle.
  • the composition is a microparticle and the microparticle includes between about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles, such as about or equal to 20%,
  • the microparticle contains about or equal to 30% w/w HPMCAS, based on the weight of the microparticles. In certain aspects, the microparticle contains about or equal to 20% w/w HPMCAS, based on the weight of the microparticles.
  • the pharmaceutical compositions provided herein include microparticles of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).
  • any of the pharmaceutical compositions provided herein are formulated as microparticles and the microparticles include a lectin.
  • the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.
  • the lectin is AAL.
  • the pharmaceutical compositions containing the polypeptides provided herein are formulated as microparticles that include between about or equal to 0.1% to about or equal to 0.5% w/w lectin, based on the weight of the microparticles, such as about or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% w/w lectin, based on the weight of the microparticles.
  • the microparticles include about or equal to 0.25% w/w lectin, based on the weight of the microparticles.
  • the lectin is AAL.
  • compositions provided herein include about or equal to 1% to about or equal to 10% w/w of the total amount of polypeptides, such as about or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w polypeptides, based on the total weight of the composition.
  • compositions provided herein include about or equal to 1% to about or equal to 10% w/w of each polypeptide in the composition, such as about or equal to 1%, 2%,
  • the pharmaceutical compositions are formulated as microparticles and the percent weight of the polypeptides is based on the weight of the microparticles.
  • the pharmaceutical compositions provided herein include equimolar amounts of each polypeptide in the composition. In aspects, the pharmaceutical compositions provided herein include about or equal to 1% w/w of each polypeptide in the composition.
  • the pharmaceutical compositions provided herein of are formulated for prophylactic treatment.
  • the pharmaceutical compositions provided herein are formulated as vaccines.
  • the pharmaceutical compositions include one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • the pharmaceutical compositions are for treatment of a disease or condition caused by a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the disease or condition is COVID-19.
  • Also provided herein is a method of selecting a polypeptide for treatment of a disease or condition caused by a pathogen in a subject, which includes:
  • the one or more HLA alleles include an HLA Class 1 allele or an HLA Class 2 allele.
  • the binding affinity values for each of the amino acid subsequences are computed for two or more HLA alleles.
  • at least one HLA allele is an HLA Class 1 allele and at least one HLA allele is an HLA Class 2 allele.
  • At least one HLA allele is selected from among HLA-A*11:01, HLA-DRB1*01:01, HLA-B*18:01 and HLA-B*58:01.
  • at least one selected amino acid subsequence binds to an HLA Class 2 allele.
  • the polypeptide selection methods provided herein additionally include, in (e): analyzing the accessibility of the at least one amino acid subsequence on the protein surface as an epitope; and based additionally on the accessibility of the at least one amino acid subsequence on the protein surface as an epitope, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).
  • two or more amino acid subsequences are selected in (e) and two or more polypeptides are selected in (f).
  • at least one polypeptide binds to an HLA Class 1 allele and at least one polypeptide binds to an HLA Class 2 allele.
  • at least one polypeptide binds to an HLA Class 1 allele and also binds to an HLA Class 2 allele.
  • polypeptide selection methods 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid subsequences are selected in (e) and 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more polypeptides are selected in (f). In aspects, 5 amino acid subsequences are selected in (e) and 5 polypeptides are selected in (f).
  • (e) includes, additionally: identifying whether the at least one amino acid subsequence is conserved among species of the pathogen; and based additionally on identifying the at least one amino acid subsequence as being conserved among species of the pathogen, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).
  • (e) includes, additionally: identifying whether the at least one amino acid subsequence is resistant to mutations; and based additionally on identifying the at least one amino acid subsequence as being resistant to mutations, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).
  • the pathogen is a virus or a bacterium.
  • the protein that is expressed by the pathogen is associated with entry of the pathogen into the subject.
  • the pathogen is a virus.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the protein is selected from among a spike protein, a membrane protein and an envelope protein.
  • the protein is a spike protein.
  • the at least one amino acid subsequence is selected from among SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • the treatment of a disease or condition caused by the pathogen in the subject is a prophylactic treatment.
  • at least one of the polypeptides selected in (f) is immunogenic.
  • the polypeptide selection methods provided herein further include preparing a polypeptide composition containing the polypeptide(s) selected in (f) for treatment of a disease or condition caused by the pathogen in the subject.
  • the composition contains at least one polypeptide that includes an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains at least one polypeptide having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or an amino acid sequence 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains at least one polypeptide whose sequence is the sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition includes 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains the following 5 polypeptides: a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:1, a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:2, a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:3, a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide that includes the amino acid sequence set forth in SEQ ID NO:5.
  • the composition includes the following 5 polypeptides: a polypeptide whose sequence is the amino acid sequence set forth in SEQ I D NO: 1 , a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:2, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:3, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:5.
  • the compositions prepared by the methods provided herein include a microparticle.
  • the microparticle includes a sustained-release polymeric matrix.
  • the microparticle includes b- cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).
  • the composition is formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration.
  • the composition is formulated for oral administration.
  • the composition includes microparticles that are formulated for oral administration.
  • the composition or microparticle includes an agent that protects the components of the composition or microparticle against degradation in the acidic environment of the stomach.
  • the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS).
  • the composition is a microparticle of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).
  • the compositions prepared by the methods provided herein include a lectin.
  • the composition is formulated as a microparticle.
  • the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.
  • the lectin is AAL.
  • the composition is formulated for prophylactic treatment.
  • the composition is formulated as a vaccine.
  • the composition includes one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • compositions prepared by any of the methods provided herein are compositions prepared by any of the methods provided herein.
  • the composition is formulated for prophylactic treatment.
  • the composition is formulated as a vaccine.
  • the composition includes one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • the composition is formulated for oral administration.
  • at least one polypeptide is encoded by a coronavirus.
  • at least one polypeptide includes an amino acid sequence that is, or is 95% or more identical to, an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition includes 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently includes an amino acid sequence that is, or is 95% or more identical to, an amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition includes 5 polypeptides each independently including an amino acid sequence that is, or is 95% or more identical to, an amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition includes the following 5 polypeptides: a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:2, a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:3, a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide whose sequence includes the amino acid sequence set forth in SEQ ID NO:5.
  • the composition includes the following 5 polypeptides: a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:1, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:2, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:3, a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide whose sequence is the amino acid sequence set forth in SEQ ID NO:5.
  • the composition includes a microparticle.
  • the microparticle includes a sustained-release polymeric matrix.
  • the microparticle includes b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).
  • compositions prepared by the methods provided herein are formulated as microparticles that include between about or equal to 10% to about or equal to 20% w/w ethylcellulose, such as about or equal to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 16%, 17%, 18%, 19% or 20% w/w cellulose, based on the weight of the microparticles.
  • the microparticle contains about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles.
  • compositions are formulated as microparticles that include between about or equal to 50% to about or equal to 70% w/w b-cyclodextrin, such as about or equal to 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% w/w b- cyclodextrin, based on the weight of the microparticles.
  • the microparticle contains about or equal to 60% w/w b-cyclodextrin, based on the weight of the microparticles.
  • compositions prepared by the methods provided herein contain an agent that protects the components of the composition against degradation in the acidic environment of the stomach.
  • the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS) and in certain aspects, the composition is formulated as a microparticle.
  • the composition is a microparticle and the microparticle includes between about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles, such as about or equal to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% w/w b-cyclodextrin, based on the weight of the microparticles.
  • the microparticle contains about or equal to 30% w/w HPMCAS, based on the weight of the microparticles.
  • the compositions include microparticles of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).
  • any of the compositions prepared by the methods provided herein are formulated as microparticles and the microparticles include a lectin.
  • the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.
  • the lectin is AAL.
  • compositions are formulated as microparticles that include between about or equal to 0.1% to about or equal to 0.5% w/w lectin, based on the weight of the microparticles, such as about or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% w/w lectin, based on the weight of the microparticles.
  • the compositions prepared by the methods provided herein include about or equal to 1% to about or equal to 10% w/w of the total amount of polypeptides, such as about or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w polypeptides, based on the total weight of the composition.
  • the compositions include about or equal to 1% to about or equal to 10% w/w of each polypeptide in the composition, such as about or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% w/w of each polypeptide in the composition, based on the total weight of the composition.
  • the compositions are formulated as microparticles and the percent weight of the polypeptides is based on the weight of the microparticles.
  • compositions prepared by the methods provided herein contain 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5.
  • the composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5.
  • isolated polypeptides selected by the methods provided herein.
  • the isolated polypeptides are derived from the protein, e.g., by digestion with proteases.
  • the isolated polypeptides obtained by digestion are subjected to further purification prior to isolation.
  • the isolated polypeptides are synthesized.
  • the isolated polypeptides obtained by digestion are subjected to further purification prior to isolation.
  • the isolated polypeptide is encoded by a virus or a bacterium.
  • at least one isolated polypeptide of a composition containing two or more polypeptides is encoded by a virus or a bacterium.
  • the isolated polypeptide or at least one isolated polypeptide is encoded by a virus.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the isolated polypeptide is derived from a protein is selected from among a spike protein, a membrane protein and an envelope protein.
  • the protein is a spike protein.
  • the isolated polypeptide includes an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the isolated polypeptide has the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • polynucleotides encoding any of the isolated polypeptides provided herein.
  • the polynucleotide is an expression vector or expression plasmid.
  • the polynucleotide is a DNA plasmid or vector, or RNA plasmid or vector.
  • the polynucleotide is a DNA plasmid or vector and a portion of the DNA plasmid or vector includes a DNA virus or portion thereof.
  • the DNA virus is a herpesvirus, an adenovirus or a poxvirus.
  • the polynucleotide is a RNA plasmid or vector and a portion of the RNA plasmid or vector includes a RNA virus.
  • the RNA virus is a retrovirus or a ssRNA virus.
  • any of the isolated polypeptides provided herein, including isolated polypeptides in compositions containing two or more isolated polypeptides, is encoded by a virus.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the protein from which an isolated polypeptide is derived is selected from among a spike protein, a membrane protein and an envelope protein.
  • the protein is a spike protein.
  • the isolated polypeptide is a portion of the S1 subunit of the spike protein.
  • compositions containing two or more isolated polypeptides provided herein contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • a composition that includes 2 or more isolated polypeptides as provided herein contains 2, 3, 4 or 5 isolated polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5.
  • the composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5.
  • a vaccine composition that includes an isolated polypeptide or polynucleotide provided herein, and further including one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • composition that includes an antigen presenting cell (APC) and an isolated polypeptide or polynucleotide encoding the polypeptide as provided herein.
  • APC antigen presenting cell
  • the composition includes a polynucleotide and the polynucleotide resides within the APC.
  • the polypeptide or a portion thereof is presented on the surface of the APC.
  • the APC is a monocyte.
  • the APC is a dendritic cell.
  • the composition includes two or more isolated polypeptides or one or more polynucleotides encoding the two or more isolated polypeptides.
  • at least one polypeptide contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • a composition that includes 2 or more isolated polypeptides and an APC as provided herein contains 2, 3, 4 or 5 isolated polypeptides whose sequences differ from each other and each of which independently contains an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains 5 polypeptides each independently containing an amino acid sequence that is, or is 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the composition contains 5 polypeptides that include: a polypeptide containing the amino acid sequence set forth in SEQ ID NO:1, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:2, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:3, a polypeptide containing the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide containing the amino acid sequence set forth in SEQ ID NO:5.
  • the composition contains 5 polypeptides that include: a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:5.
  • the terms “having” and “whose sequence is” are used interchangeably in reference to any of the aspects provided herein.
  • a device that contains any of the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein.
  • the contents of the device is/are in unit dosage form, liquid dosage form, solid dosage form, oral dosage form, a tablet, a capsule, a topical patch, a syringe, an inhaler, a dosage cup, a dropper, a pump, a spray bottle, an aerosol container, a wound dressing, or an applicator for administering the contents.
  • kits that contains any of the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein and a device for administration of the contents of the kit other than the device.
  • the contents of the kit other than the device are contained in the device.
  • the contents of the kit other than the device are present as a separate component that is distinct from the device.
  • the device is a dressing, a topical patch, a pump, a spray bottle, an aerosol container, a syringe, an inhaler, a dosage cup, a dropper, or an applicator.
  • the pathogen is a virus.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the treatment is a prophylactic treatment.
  • the disease or condition is COVID-19.
  • the method further includes obtaining polyclonal antibodies from the subject and/or antiserum that immunospecifically binds to a polypeptide, or a polypeptide encoded by, the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein.
  • the method further includes: isolating spleen cells from the subject, and combining the spleen cells with myeloma cells under conditions that produce monoclonal antibody generating hybridomas.
  • the method further includes screening the hybridomas for those that produce monoclonal antibodies that immunospecifically bind to a polypeptide, or a polypeptide encoded by, the compositions, pharmaceutical compositions, isolated polypeptides or polynucleotides provided herein. In aspects, the method further includes isolating or otherwise obtaining the monoclonal antibodies identified by screening the hybridomas.
  • CAR chimeric antigen receptor
  • the pathogen is a virus.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the disease or condition is COVID-19.
  • Figure 1 depicts the cytotoxic effect of microparticles at various given concentrations.
  • Figure 2 depicts uptake of the microparticles in mice.
  • Figure 2A depicts uptake in the small intestine.
  • Figure 2B depicts uptake in Peyer’s Patches (PP).
  • Figure 3 depicts flow-cytometry results of the immunophenotype of human dendritic cells exposed to the microparticles or to control stimuli.
  • Figure 4 depicts a study of the immune stimulant function of the microparticles.
  • Figures 5A & 5B illustrate 3D mapping of the CPFGEVFNATRFASV epitope (SEQ ID NO: 1 ), with wire frame (Figure 5A) and sphere (Figure 5B) models. Darker shading depicts the location of the epitope.
  • Figures 6A & 6B illustrate 3D mapping of the GEVFNATRF epitope (SEQ ID NO:2), with wire frame ( Figure 6A) and sphere ( Figure 6B) models. Darker shading depicts the location of the epitope.
  • Figures 7 A & 7B illustrate 3D mapping of the ASVYAWNRK epitope (SEQ ID NO:3), with wire frame ( Figure 7A) and sphere ( Figure 7B) models. Darker shading depicts the location of the epitope.
  • Figures 8A & 8B illustrate 3D mapping of the VGGNYNYLYRLFRKS epitope (SEQ ID NO:4), with wire frame ( Figure 8A) and sphere ( Figure 8B) models. Darker shading depicts the location of the epitope.
  • Figures 9A & 9B illustrate 3D mapping of the VGGNYNYLY epitope (SEQ ID NO:5), with wire frame ( Figure 9A) and sphere ( Figure 9B) models. Darker shading depicts the location of the epitope.
  • Figure 10 depicts the potency of BSK-02 microparticles in a dendritic cell (iDC) model.
  • Figure 11 depicts immunogenicity testing of the polypeptides having the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • Figure 12 depicts T-cell mediated selective killing of antigen presenting cells (APCs) that present the polypeptides having the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • APCs antigen presenting cells
  • Infectious disease vaccines aim to induce a protective immune response in a naive host by exposing the immune system to epitopes contained on the pathogen prior to exposure to the infectious agent itself.
  • the major challenges that confront infectious disease vaccines stem from the nature of the epitopes against which the immune response is directed; in some cases, immunodominant epitopes arising from natural infection may not be those that are most desirable (e.g., susceptible to neutralization and/or highly conserved).
  • vaccines targeting diseases that involve “self” antigens e.g., cancer or neurodegenerative disease
  • Viral diseases affect millions of people worldwide. Annually, dengue virus disease affects about 50 to 100 million people globally with 9000+ fatalities, rotavirus infects about two million children under five years of age, of whom about 527,000 die, seasonal influenza epidemics cause severe illness in three to five million people and a quarter to a half million deaths, and the most recent SARS-CoV-2 virus has caused over a million deaths within the space of just a few months, not to mention countless cases of serious illness with lingering long-term effects such as organ damage and cardiac disease.
  • SARS-CoV-2 virus has caused over a million deaths within the space of just a few months, not to mention countless cases of serious illness with lingering long-term effects such as organ damage and cardiac disease.
  • compositions containing the immunogenic polypeptides so identified, or polynucleotides that encode the immunogenic polypeptides so identified are for oral administration, such as an oral vaccine.
  • the composition is formulated as a microparticle.
  • the microparticle is for oral administration.
  • DIAMONDTM is a computational platform and a neural network that can identify novel immunologic targets for T- and B-lymphocytes. It uses proprietary SpliceDiffTM software, which is part of an integrated bioinformatic and artificial intelligence (A. I.) system such as that described in PCT application PCT/US20/35183, filed on May 29, 2020, the contents of which are expressly incorporated by reference herein and the contents of which are outlined herein.
  • This proprietary platform can use public and proprietary databases to identify target polypeptides for the treatment of diseases or conditions caused by pathogens, such as infectious diseases and can expand into the tumor or the infectious disease target space.
  • the platform addresses several challenges in today’s clinical pipeline: target identification and the selection of immunologically significant peptides for activating T and B lymphocytes, e.g., for prophylactic treatment or for generating antibodies for treatment.
  • the A. I. system used to identify and select polypeptides for the treatment of diseases or conditions associated with or caused by pathogens is an Immunotherapy Builder System (IBS) that includes multiple modules.
  • IBS Immunotherapy Builder System
  • An IBS can narrow a multitude of amino acid sequence variants to a subset of predicted disease-associated variants, or immunogenic variants.
  • an IBS can narrow a multitude of amino acid subsequences in an input amino acid sequence to a subset identified as having immunogenic potential (e.g., for an immunotherapy).
  • An IBS can facilitate in silico (i) discovery of novel disease-associated targets, and/or (ii) narrowing of a large number of targets to a significantly smaller subset of targets having high immunogenic potential, thereby facilitating resource-efficient development of novel immunotherapies.
  • Portions of disease- associated targets e.g., amino acid subsequences
  • portions of disease-associated targets identified by systems and processes described herein as having immunogenic potential, and/or longer amino acid sequences each containing one or more of such portions, can be considered predicted disease-associated antigens.
  • Portions of predicted disease-associated targets can be identified by systems and processes described herein as having immunogenic potential according to assessment of major histocompatibility complex (MHC) interaction and/or T-cell receptor (TCR) interaction and/or B-cell receptor (BCR) interaction, for example.
  • MHC major histocompatibility complex
  • TCR T-cell receptor
  • BCR B-cell receptor
  • An IBS can include two or more of the following modules: a Differential Expression Module (DEM), a MHC Allele Affinity Determination Module (MAAM), a MHC Composite Feature Module (MCFM), a MHC Fragment Locator Module (MFLM), a T-Cell Receptor Immunogenicity Determination Module (TIM); and a B-Cell Receptor Epitope Determination Module (BEM).
  • An IBS can include a Sequence Acquisition Interface (SAI), which can be implemented to acquire an amino acid sequence of interest.
  • SAI Sequence Acquisition Interface
  • a MAAM, MCFM, MFLM, BEM and/or a DEM can be configured to receive an amino acid sequence from an SAI.
  • An IBS can be implemented to (i) identify a disease-associated amino acid sequence variant or an immunogenic amino acid subsequence (e.g., by implementation of a DEM or a TIM) among variants or subsequences of a particular gene/protein; and/or (ii) compute MHC binding affinity values for amino acid subsequences within an amino acid sequence of interest (e.g., by implementation of a MAAM, MCFM and/or a MFLM); and/or (iii) compute a T-cell receptor (TCR) immunogenicity score for each of a plurality of amino acid subsequences having an estimated MHC binding affinity value above or below a threshold (e.g., by implementation of a TIM); and/or (iv) identify B-cell receptor (BCR) epitopes in an amino acid sequence of interest (e.g., by implementation of a BEM), for example.
  • a MAAM can compute a MHC binding affinity value for amino acid subsequences of an input amino acid sequence, for one or more MHC alleles or MHC supertypes, by implementation of a convolutional neural network (CNN) that contains a plurality of virtual neurons arranged in capsules.
  • CNN convolutional neural network
  • a MAAM can compute a MHC binding affinity value with an advantageously low error rate, and can narrow amino acid subsequences to a subset predicted to exhibit strong and/or intermediate MHC binding affinity. These features are useful for identifying a subset of amino acid subsequences of high immunogenic potential for an immunotherapy, for example.
  • a MFLM can output a graphic representation of the amino acid subsequences, or subset thereof, mapped to an input amino acid sequence.
  • a MCFM can compute a composite MHC binding affinity value for amino acid subsequences of an input amino acid sequence, for one or more MHC alleles or MHC supertypes, where the composite binding affinity value is based on (i) a proteasome cleavage score for the amino acid subsequence, and/or (ii) a transporter affinity score for the amino acid subsequence, and/or (iii) a MHC allele or MHC supertype binding affinity value for the amino acid subsequence (e.g., a normalized MHC allele or MHC supertype binding affinity value).
  • a composite binding affinity value is useful for narrowing amino acid subsequences to a subgroup having high immunogenic potential for an immunotherapy, for example.
  • a TIM can compute a T-cell receptor (TCR) immunogenicity score based on estimation of interaction of amino acid subsequences of an input amino acid with a TCR.
  • TCR T-cell receptor
  • a TIM often is implemented after a subset of amino acid subsequences characterized by high immunogenicity potential and/or high multiple MHC allele-binding potential is identified by one or more modules that assess MHC interaction (/.e., a MAAM, a MFLM and/or a MCFM).
  • Immunogencity scores computed by a TIM are useful for narrowing amino acid subsequences to a smaller subset of high T-cell- mediated immunogenic potential, for example.
  • a BEM can compute a B-cell receptor (BCR) epitope score for each amino acid in an input amino acid sequence, where the score is indicative of the probability that the amino acid exists within a BCR epitope scores computed by a BEM are useful for narrowing amino acid subsequences to a smaller subset of high B-cell-mediated immunogenic potential, for example.
  • BCR B-cell receptor
  • a DEM can identify a disease-associated amino acid sequence variant among variants encoded by a particular gene based on an analysis of expression level of the variant in disease samples and non-disease samples from multiple tissues.
  • a DEM is useful for identifying disease-associated alternatively-spliced variants.
  • a disease-associated alternatively-spliced variant includes an insert of an amino acid or two or more consecutive amino acids relative to other variants encoded by a gene, for example.
  • a DEM is useful for identifying disease-associated variants that can be targeted by an immunotherapeutic, for example.
  • An amino acid sequence of a disease-associated variant identified by a DEM, or portion thereof, can be utilized as an input amino acid sequence for one or more of a MAAM, a MFLM, a MCFM, a TIM and a BEM, for example.
  • the IBS can generate a multi-sequence alignment (MSA) to identify the possible presence of emerging non-synonymous mutational hotspots in the immunogenic epitopes (subsequences) that are identified, or to identify immunogenic epitopes that are conserved between multiple species of the same pathogen, e.g., two or more coronaviruses, to further identify immunogenic epitopes that are resistant to mutations and/or offer cross protection of multiple species in immunogenic therapy (e.g., vaccines).
  • a MSA often aligns a variant amino acid sequence with an amino acid sequence of at least one other variant encoded by the same gene.
  • a MSA can be generated using any suitable sequence alignment algorithm, non-limiting examples of which include Clustal (e.g., ClustalW, ClustalW2, Clustal Omega), Multiple Alignment using Fast Fourier Transform (MAFFT), T- COFFEE, M-COFFEE, LALIGN, PSAIign, PRRN, PRRP, DIALIGN, MUSCLE, MergeAlign, Partial- Order Alignment (POA), Sequence Alignment and Modeling System (SAM), HMMER, PRANK, PAGAN, ProGraphMSA, MEME, MAST and EDNA.
  • a DEM can generate a MSA based on a gene identifier, which can involve synching amino acid sequence databases having disparate gene identifier information.
  • ANN artificial neural networks
  • DIAMONDTM is equipped with a state-of-the-art capsule neural network (CNN), which applies an iterative routing-by-agreement mechanism, and therefore when multiple capsules agree, the probability of correct prediction is expected to be higher than that obtained with other network architectures.
  • CNN state-of-the-art capsule neural network
  • the methods provided herein can be used to identify and select polypeptides for the treatment of any disease or condition caused by or associated with a pathogen, include, but are not limited to, diseases caused by pathogens such as viruses, bacteria, fungi, protozoa, and parasites.
  • Infectious diseases can be caused by viruses including coronaviruses, adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus, (HIV), human papilloma virus (HPV), influenza, measles, mumps, papova virus, polio, respiratory syncytial virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox and viral meningitis.
  • viruses including coronaviruses, adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus, (HIV), human
  • Infectious diseases can also be caused by bacteria including Bacillus anthracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diphtheria, Escherichia coli, Legionella, Helicobacter pylori, Mycobacterium rickettsia, Mycobacterium tuberculosis, Mycoplasma Neisseria, Pertussis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus, Staphylococcus, Vibrio cholerae and Yersinia pestis.
  • bacteria including Bacillus anthracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diphtheria, Escherichi
  • Infectious diseases can also be caused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei. Infectious diseases can also be caused by protozoa and parasites such as chlamydia, kokzidiose, leishmania, malaria, rickettsia, and trypanosoma.
  • fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei.
  • Infectious diseases can also be caused by protozoa and parasites such as chlamydia, kokzidiose, leishmania, malaria, rickettsi
  • the immunology prediction modules from the DIAMONDTM artificial neural network can be applied to select immunogenic epitopes of a protein expressed by any pathogen associated with a disease or condition.
  • the protein that is analyzed using the DIAMONDTM artificial neural network is identified based on its resistance to mutations, or based on whether it is conserved between species of the pathogen, e.g., conserved between strains of the influenza virus or between strains of coronaviruses.
  • the protein that is identified for analysis is associated with entry of the pathogen into the target host.
  • the selected immunogenic epitopes include amino acid sequences that are subsequences of a protein associated with entry of a pathogen into a target host.
  • the immunogenic epitopes containing amino acid sequences that are subsequences of a protein associated with entry of a pathogen into a target host can neutralize entry of the pathogen into the host.
  • the pathogen is a virus.
  • the protein identified for analysis using selection methods provided herein is selected from among a spike protein, a membrane protein and an envelope protein. In certain aspects, the protein is a spike protein.
  • the selection methods provided herein can, in certain aspects, select immune-dominant polypeptides that are predicted to bind to the most common HLA class I and class II alleles and would be most likely to elicit neutralizing antibodies. From the predicted immune-dominant polypeptides, in some aspects, the polypeptides that are selected include one or more polypeptides that are accessible on the protein surface. In aspects, immune hotspot regions can be identified as follows:
  • compositions provided herein that include immunogenic polypeptides (epitopes) selected according to the methods provided herein, and/or that include polynucleotides that encode the immunogenic polypeptides (epitopes) selected according to the methods provided herein.
  • immunogenic polypeptides selected by the methods provided herein, and related compositions that include or encode capsid surface antigens of a pathogen.
  • the immunogenic polypeptides and related compositions are for treatment, including prophylactic treatment, of a disease or condition caused by or associated with a pathogen.
  • the immunogenic polypeptides and related compositions can cross protect against more than one strain of a pathogen.
  • the immunogenic polypeptides selected by the methods provided herein are predicted by the method as being the most immunogenic. In aspects, the immunogenic polypeptides are predicted to bind to the most common HLA class I and class II alleles that would be most likely to elicit neutralizing antibodies. In aspects, at least one of the selected the immunogenic polypeptides is accessible on the surface of the pathogen.
  • immunogenic polypeptides polynucleotides encoding the polypeptides or compositions containing the immunogenic polypeptides and/or polynucleotides for treating a disease or condition caused by a coronavirus.
  • Novel coronaviruses are part of a family of beta coronaviruses that can cause illnesses such as the common cold, or create more life threatening conditions such as SARS-CoV (Severe Acute Respiratory Syndrome) or MERS-CoV (Middle East Respiratory Syndrome).
  • SARS-CoV severe Acute Respiratory Syndrome
  • MERS-CoV Middle East Respiratory Syndrome
  • SARS-CoV severe Acute Respiratory Syndrome
  • MERS-CoV Middle East Respiratory Syndrome
  • SARS-CoV-2 is an enveloped positive single strand RNA virus that the WHO (World Health Organization) has declared to have caused a global pandemic, causing world-wide distress and economic hardship.
  • COVID-19 disease SARS-CoV-2
  • can present with flu like symptoms such as fever, cough, shortness of breath, breathing difficulties, nausea, diarrhea, pneumonia, severe acute respiratory syndrome, coagulation dysfunction, septic shock, kidney failure, and even death.
  • corona protein spikes situated at the exterior surface of the virus are the binding point for the Angiotensin-converting enzyme 2 (ACE2) receptor in lung tissue.
  • ACE2 receptor mediated antigenic stimuli may therefore increase the risk of the corresponding inflammation-related adverse effects associated with COVID-19 infections. Consequently, pneumonia, acute myocardial injury, and/or chronic damage to the cardiovascular system are potential severe complications of this viral disease, and the symptoms of the infection may be more pronounced in patients with hypertension and diabetes. Therefore, affected patients may be at an increased risk of more severe disease outcomes mediated by infection-induced endothelial dysfunction.
  • SARS-CoV-2 virus is highly polymorphic, both on HLA and on Single Nucleotides (SNP).
  • SNP Single Nucleotides
  • the 47 key point mutations are likely host dependent (host-dependent RNA editing in the transcriptome), with 910 identified Single Nucleotide Variations (SNVs) in addition to other novel missense mutations. This creates complexity in developing an effective vaccine. A low or absent immune response is therefore possible, and the risk of severe adverse effects is high.
  • APCs antigen-presenting cells
  • DCs dendritic cells
  • APCs are capable of presenting viral peptide antigens (VPAs), eliciting adaptive immune responses against VPA infected cells.
  • VPAs viral peptide antigens
  • the DiamondTM artificial intelligence antigen discovery platform used in the selection methods provided herein has revealed key conserved peptide domains within the SARS-CoV-2 protein spikes, membrane, and viral envelope proteins that are most likely to induce a potentially immunogenic response.
  • immunogenic polypeptides selected by the methods provided herein, and related compositions, that include or encode capsid surface antigens of a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the immunogenic polypeptides and related compositions are for treatment, including prophylactic treatment of COVID-19.
  • the immunogenic polypeptides and related compositions can cross protect against more than one strain of coronavirus.
  • the immunogenic polypeptides selected by the methods provided herein are predicted by the method as being the most immunogenic. In aspects, the immunogenic polypeptides are predicted to bind to the most common HLA class I and class II alleles that would be most likely to elicit neutralizing antibodies. In aspects, at least one of the selected the immunogenic polypeptides is accessible on the surface of the viral protein.
  • the immunogenic polypeptides selected by the methods provided herein are derived from a coronavirus glycoprotein.
  • the protein is a SARS-CoV-2 surface glycoprotein.
  • the glycoprotein is a spike glycoprotein.
  • SARS-CoV-2 uses the spike glycoprotein (S), the main target for neutralization antibody, to bind to its receptor, and mediate membrane fusion and virus entry.
  • S spike glycoprotein
  • the immunogenic polypeptides are derived from the coronavirus S protein, the risk for off-target immunotoxicity is extremely low as this protein is not found on human cells.
  • Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively.
  • the immunogenic polypeptides selected by the methods provided herein are in (and are derived from) the S1 subunit of the spike protein (first 600 amino acids).
  • the immunogenic polypeptides are accessible to neutralizing antibodies.
  • the immunogenic polypeptides contain or have a sequence selected from among those set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or a sequence that is 95% or more identical to a sequence selected from among those set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • binding molecules that specifically bind to a polypeptide having the sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or to a sequence that is 95% or more identical to SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • the binding molecules provided herein are antibodies, or antigen-binding fragments thereof.
  • the VH and VL domains of the antibodies provided herein are humanized and/or deimmunized so as to exhibit a reduced immunogenicity upon administration to recipient subjects.
  • the binding molecules provided herein can include, but are not limited to, bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules and the like that include: (i) Variable Domains (VH and VL) and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell.
  • compositions that contain any of the binding molecules provided herein, and methods involving the use of any of such binding molecules in the treatment of diseases or conditions associated with a coronavirus, such as COVID-19.
  • the binding molecules are monoclonal antibodies.
  • the binding molecules are chimeric antigen receptors (CARs).
  • antibody refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab’) fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and epitope-binding fragments of any of the above.
  • antibody includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an epitope-binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, lgG2, lgG3, lgG4, IgAi and lgA 2 ) or subclass.
  • Antibodies are capable of “immunospecifically binding” to a polypeptide or protein or a non-protein molecule (or of binding to such molecule in an “immunospecific manner”) due to the presence on such molecule of a particular domain or moiety or conformation (an “epitope”).
  • the terms “immunospecific” or “immunospecifically binding” are used interchangeably herein with “specific” or “specifically binding,” respectively.
  • An epitope-containing molecule can have immunogenic activity, such that it elicits an antibody production response in an animal; such molecules are termed “antigens”.
  • Examples of epitopes in the SARS-CoV-2 spike protein include those having the sequences set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • an antibody, diabody or other epitope-binding molecule is said to “immunospecifically” bind a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with that epitope relative to alternate epitopes.
  • an antibody (or moiety or epitope) that immunospecifically binds to a first target may or may not bind to a second target.
  • immunospecific binding does not necessarily require (although it can include) exclusive binding.
  • reference to antibody (or CAR molecule) binding means “immunospecific” binding.
  • monoclonal antibody refers to a homogeneous antibody population wherein the monoclonal antibody contains amino acids (naturally occurring or non-naturally occurring) that are involved in the selective binding of an antigen. Monoclonal antibodies are specific, being directed against a single epitope (or antigenic site or determinant).
  • antibody or “monoclonal antibody,” as used herein, encompass not only intact antibodies / monoclonal antibodies and full-length antibodies / monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) binding molecules, mutants thereof, fusion proteins comprising an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that contains an antigen recognition site of the required specificity and the ability to bind to an antigen.
  • fragments thereof such as Fab, Fab', F(ab')2, Fv, etc.
  • scFv single-chain binding molecules
  • antibody It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).
  • the term also includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody.”
  • Antibodies such as polyclonal antibodies and monoclonal antibodies, can be prepared using standard methods (see, e.g., Kohler et al., Nature 256:495-497 (1975); Kohler et al., Eur. J. Immunol. 6:511-519 (1976); and WO 02/46455).
  • a host animal such as mice, rats or rabbits
  • Blood from the host animal is then collected and the serum fraction containing the secreted antibodies is separated from the cellular fraction, using methods known to those of skill in the art.
  • an animal is immunized by standard methods to produce antibody-secreting somatic cells. These cells then are removed from the immunized animal for fusion to myeloma cells.
  • Somatic cells that can produce antibodies can be used for fusion with a myeloma cell line. These somatic cells can be derived from the lymph nodes, spleens and peripheral blood of primed animals.
  • Specialized myeloma cell lines have been developed from lymphocytic tumors for use in hybridoma-producing fusion procedures (Kohler and Milstein, Eur. J. Immunol.
  • binding molecules provided herein can be assayed for the ability to bind to their cognate immunogenic polypeptide epitopes by any method known to those of skill in the art. Binding assays can be performed in solution, suspension or on a solid support. Negative controls also can be included in such assays as a measure of background binding. Binding affinities can be determined using quantitative ELISA, Scatchard analysis (Munson et aL, (1980) Anal. Biochem., 107:220), surface plasmon resonance, isothermal calorimetry, or other methods known to one of skill in the art (e.g., Liliom et aL (1991) J. Immunol Methods. 143(1 ): 119-25).
  • Such assays also can be performed, for example, in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), on beads (Lam (1991) Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et aL (1992) Proc. Natl. Acad. Sci.
  • the binding can be detected using a method that is capable of being quantified such that the level of activity can be assessed.
  • methods of quantitation include, but are not limited to, spectrophotometric, fluorescent and radioactive methods. Such methods measure, for example, colorimetric signals, chemiluminescent signals, chemifluorescent signals or radioactive signals.
  • the binding molecules provided herein can be labeled with a detectable moiety or tag to facilitate detection and determination of binding activity. The skilled artisan can select an appropriate detectable moiety or tag for use in the assays described or known in the art. Linkage can be at the N- or C-terminus of the therapeutic antibody. Examples of tags and moieties are provided in Table 13 hereafter.
  • Solution binding assays can be used to assess binding activity including equilibrium dialysis, competitive binding assays (e.g., Myers eta!., (1975) Proc. Natl. Acad. Sci. USA), radiolabeled binding assays (e.g., Feau et a!., (2009) J. Biomol. Screen. 14(1):43-48), calorimetry, including isothermal titration calorimetry (ITC) and differential scanning calorimetry (e.g., Alvarenga et al. (2012) Anal. Biochem 421 ( 1 ) : 138- 151, Perozzo et al., (2004) J. Recept Signal. Transduct Res. 24(1-2): 1-52; Holdgate (2001)
  • ITC isothermal titration calorimetry
  • differential scanning calorimetry e.g., Alvarenga et al. (2012) Anal. Biochem 421 ( 1 ) : 138- 151, Perozzo e
  • Immunoassays include competitive and non-competitive assay systems using techniques such as, but not limited to, western blots or immunoblots, such as quantitative western blots; radioimmunoassays; ELISA (enzyme linked immunosorbent assay); Meso Scale Discovery (MSD, Gaithersburg, Maryland); "sandwich” immunoassays; immunoprecipitation assays; ELISPOT; precipitin reactions; gel diffusion precipitin reactions; immunodiffusion assays; agglutination assays; complement-fixation assays; immunoradiometric assays; fluorescent immunoassays; protein A immunoassays; immunohistochemistry; immuno-electron microscopy or liposome immunoassays (LIA).
  • Such assays are routine and well-known in the art (see, e.g., Ausubel etal., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc
  • immunohistochemistry and/or immunofluorescence can be used to assess binding in animal models. For example, antibody binding to xenograft tumors in a rodent or other animal model can be analyzed. In other examples, immunohistochemistry can be used to assess antibody binding to skin, such as primate skin. In other examples, immunohistochemistry can be used to assess binding to xenograft tumors and primate skin grafts, ex vivo, for example to visually or quantitatively compare binding preferences of the antibody and to determine if the tested antibody exhibits selective or specific binding.
  • an animal model containing a xenograft tumor or skin graft can be administered a binding molecule, such as an antibody, provided herein, such as by systemic administration., to assess in vivo binding of the antibody.
  • the tissue can be harvested at particular time(s) to assess binding ex vivo by immunohistochemistry or immunofluorescence as described above.
  • the administered binding molecule is conjugated to a fluorophore, such as an infrared fluorophore (e.g., DyLight 755 ), which is capable of transmitting fluorescence through the skin.
  • antibody binding can be visualized in vivo using a fluorescent imaging system such as the I VIS Caliper imaging system, and antibody binding to xenograft tumors and/or primate skin grafts can be assessed. Tissue can subsequently be harvested for ex vivo confirmational immunohistochemical analysis.
  • a fluorescent imaging system such as the I VIS Caliper imaging system
  • absolute binding can be represented, for example, in terms of optical density (OD), such as from densitometry or spectrophotometry measurements; arbitrary fluorescent units (AFU), such as from fluorescence measurements; or lumens, such as from chemiluminescence measurements.
  • OD optical density
  • AFU arbitrary fluorescent units
  • lumens such as from chemiluminescence measurements.
  • the specific activity is calculated by dividing the absolute binding signal by the antibody protein concentration.
  • the specific activity is normalized to give a normalized specific activity (NSA) for each antibody by dividing the specific activity of the antibody by the specific activity of a reference antibody, such as an antibody that is not specific for any of the immunogenic polypeptides provided herein (e.g., SEQ ID NOS:1-5), or is a parental antibody from which the antibody of interest is derived.
  • NSA normalized specific activity
  • Binding activity also can be measured in terms of binding affinity, which can be determined in terms of binding kinetics, such as measuring rates of association (k a or k on ) and/or dissociation (k d or /c off ), half maximal effective concentration (EC50) values, and/or thermodynamic data (e.g., Gibbs free energy, enthalpy, entropy, and/or calculating association (KA) or dissociation (KD) constants.
  • KA association
  • KD dissociation
  • polypeptides including immunogenic polypeptides, provided herein can be prepared using the methods provided herein.
  • the terms peptides and polypeptides are used interchangeably herein and can refer to a single peptide unit or to two or more peptide units (e.g., peptide units independently obtained e.g., by recombinant method or by chemical synthesis and then linked together by a peptide bond or other linker).
  • polypeptides referred to herein such as immunogenic peptides or immunogenic polypeptides, or peptide vaccines or polypeptide vaccines, also can, in certain aspects, refer to peptides or polypeptides that are conjugated to additional entities, such as polymers, e.g., to increase half-life or stability.
  • a polypeptide generally refers to a polymer, linked by peptide bonds, that has a sequence of amino acids encoded by a polynucleotide. Proteins or portions thereof (e.g., a subunit of a protein) are generally made up of polypeptides.
  • a peptide generally refers to a portion or fragment of a larger polypeptide. In some instances, a peptide refers to a polymer containing between about 2 amino acids to about 10 amino acids, 2 amino acids to about 20 amino acids, or about 2 amino acids to about 30 amino acids.
  • Peptides can include, for example, dipeptides, tripeptides, tetrapeptides, and oligopeptides.
  • amino acids that have been incorporated into peptides and/or polypeptides may be referred to as residues.
  • Peptides and polypeptides typically have an N-terminal (amine group) residue at one end and C-terminal (carboxyl group) residue at the opposite end, and amino acid sequences are typically read in the N-terminal to C-terminal direction.
  • the immunogenic polypeptides selected by the methods provided herein can be produced synthetically, such as using solid phase or solution phase peptide synthesis.
  • the immunogenic polypeptides can be produced by direct peptide synthesis using solid-phase techniques (see e.g., Stewart etal. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J (1963) J Am Chem Soc., 85:2149-2154).
  • In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City CA) in accordance with the instructions provided by the manufacturer.
  • the immunogenic polypeptides provided herein can be obtained using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries for the identification of polynucleotides that encode the immunogenic polypeptides can be used, including nucleic acid hybridization screening, antibody-based screening and activity-based screening.
  • a nucleic acid containing material can be used as a starting material from which a desired polypeptide-encoding polynucleotide can be isolated.
  • DNA and mRNA preparations, cell extracts, tissue extracts, fluid samples (e.g., blood, serum, saliva), samples from healthy and/or diseased subjects can be used in amplification methods.
  • the source can be from any eukaryotic species including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources.
  • Nucleic acid libraries also can be used as a source of starting material.
  • Primers can be designed to amplify a desired polypeptide.
  • primers can be designed based on expressed sequences from which a desired polypeptide is generated.
  • Additional polynucleotide sequences can be joined to a polypeptide-encoding nucleic acid molecule, including linker sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the core protein coding DNA sequences.
  • additional nucleotide sequences specifying functional DNA elements can be operatively linked to a polypeptide-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and secretion sequences, for example heterologous signal sequences, designed to facilitate protein secretion. Such sequences are known to those of skill in the art.
  • tags or other moieties can be added, for example, to aid in detection or affinity purification of the polypeptide.
  • additional nucleotide residue sequences such as sequences of bases specifying an epitope tag or other detectable marker also can be linked to enzyme-encoding nucleic acid molecules. Examples of such sequences include nucleic acid sequences encoding a His tag or Flag Tag.
  • the identified and isolated nucleic acids can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene, La Jolla, CA). Other expression vectors include the HZ24 expression vector exemplified herein (see e.g., SEQ ID NOS:4 and 5).
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. Insertion can be effected using, for example, TOPO cloning vectors (Invitrogen, Carlsbad, CA). Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of polynucleotide are generated, and large quantities of the immunogenic polypeptides expressed.
  • the immunogenic polypeptides can be conjugated to polymers.
  • Exemplary polymers that can be conjugated to the polypeptides include natural and synthetic homopolymers, such as polyols (/.e., poly-OH), polyamines (/.e., poly-Nhy and polycarboxylic acids (/.e., poly- COOH), and further heteropolymers, /.e., polymers containing one or more different coupling groups, e.g., hydroxyl groups and amine groups.
  • suitable polymeric molecules include polymeric molecules selected from among polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co- maleic acid anhydride, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose, hydrolys
  • the immunogenic polypeptides selected by the methods provided herein can be produced synthetically, or can be produced by standard recombinant DNA techniques known to one of skill in the art.
  • nucleic acid encoding one or more immunogenic polypeptides can be incorporated into an expression vector and then introduced into a host cell to be expressed heterologously.
  • polynucleotides encoding the immunogenic polypeptides provided herein can be formulated as any of the compositions provided herein.
  • polynucleotide molecules encoding any of the immunogenic polypeptides provided herein are also provided herein.
  • polynucleotides encoding the immunogenic polypeptides provided herein are synthesized by methods known to those of skill in the art. In aspects, the polynucleotides encoding the immunogenic polypeptides provided herein are isolated.
  • compositions that include any of the polypeptides, or nucleotides encoding the polypeptides, provided herein, and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutical composition provided herein can be formulated as a gel, ointment, liquid, suspension, aerosol, tablet, pill, powder or lyophile, and/or can be formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration.
  • a pharmaceutical composition provided herein can be formulated as a microparticle.
  • the microparticle is for oral administration.
  • a pharmaceutical composition provided herein can be formulated for single dosage administration or for multiple dosage administration.
  • a pharmaceutical composition provided herein can be a sustained release formulation.
  • a pharmaceutical composition provided herein is for prophylactic treatment.
  • a pharmaceutical composition provided herein is formulated as a vaccine.
  • the pharmaceutical compositions provided herein can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition that is effective for treating a disease, such as COVID-19, by administration of an immunogenic polypeptide or a cocktail of 2, 3, 4, 5 or more immunogenic polypeptides, or polynucleotides encoding the polypeptide(s), and a label that indicates the infection, disease or disorder that the polypeptide(s) or polynucleotide(s) is/are to be used for.
  • the pharmaceutical compositions can be packaged in unit dosage forms containing an amount of the pharmaceutical composition for a single dose or multiple doses.
  • the packaged compositions can contain a lyophilized powder of the pharmaceutical compositions, which can be reconstituted (e.g., with water or saline) prior to administration.
  • Kits can optionally include one or more components such as instructions for use, devices and additional reagents (e.g., sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods.
  • additional reagents e.g., sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein
  • components such as tubes, containers and syringes for practice of the methods.
  • kits can include one or more immunogenic polypeptides as provided herein, or polynucleotides encoding the one or more polypeptides as provided herein, or a microparticle containing the one or more immunogenic polypeptides as provided herein, and can optionally include instructions for use, a device for administering the antibody to a subject, a device for detecting the immunogenic polypeptides or an antibody response elicited by the immunogenic polypeptides in a subject or in samples obtained from a subject, and a device for administering an additional therapeutic agent to a subject.
  • the kit optionally, can include instructions.
  • Instructions typically include a tangible expression describing the one or more immunogenic polypeptides, or polynucleotides encoding the one or more polypeptides, or a microparticle containing the one or more immunogenic polypeptides, and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, dosing regimens, and the proper administration method for administering the one or more immunogenic polypeptides, or polynucleotides encoding the one or more polypeptides, or a microparticle containing the one or more immunogenic polypeptides, . Instructions also can include guidance for monitoring the subject over the duration of the treatment time.
  • the dosage and the frequency of administration can vary.
  • the compositions can be administered in a single dose, in multiple separate administrations, or by continuous infusion.
  • the treatment can be repeated until a desired suppression of disease symptoms occurs, the desired improvement in the patient's condition is achieved, or the desired level of immune response is elicited.
  • Repeated administrations can include increased or decreased amounts of the compositions, depending on the progress and/or the desired result.
  • compositions can be administered to animal or human subjects at a dosage of about or equal to 0.1 mg to about or equal to 10 g or more, such as, for example, about or equal to 0.5 mg to about or equal to 5 g, about or equal to 5 g to about or equal to 50 g, about or equal to 1 mg to about or equal to 20 g, about or equal to 1 g to about or equal to 10 g, about or equal to 1 g to about or equal to 5 g, about or equal to 1 mg to about or equal to 100 mg, about or equal to 5 mg to about or equal to 8 g, about or equal to 10 g to about or equal to 80 g, or about or equal to 50 mg to about or equal to 100 mg, about or equal to 0.1 mg/kg to about or equal to 100 mg/kg, such as, for example, about or equal to 0.5 mg/kg to about or equal to 50 mg/kg, about or equal to 5 mg/kg to about or equal to 50 mg/kg, about or equal to 1 mg/kg to about or or more
  • compositions or kits provided herein can include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of a Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered.
  • adjuvant e.g., Freund’s adjuvant (complete and incomplete)
  • excipient e.g., incomplete and incomplete
  • vehicle e.g., a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • a pharmaceutical composition sometimes is provided as a pharmaceutical pack or kit containing one or more containers filled with a therapeutic composition of cells prepared by a method described herein, alone or with such pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit.
  • a pharmaceutical pack or kit may include one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • a pharmaceutical pack or kit sometimes includes one or more other prophylactic and/or therapeutic agents useful for the treatment of a disease, in one or more containers.
  • compositions that are formulated as microparticles.
  • the microparticles provided herein can be made by spray drying methods by mixing the various matrix and active ingredients, in an aqueous medium spraying in droplets of controlled size and drying the droplets to form microparticles, nanoparticles or both microparticles and nanoparticles.
  • Spray drying is a drying method that was firstly described more than 140 years ago as an improvement in drying and concentrating liquids. But it was not until the beginning of the 20th century that the level of sophistication and knowledge of the process allowed its industrial use.
  • Spray drying involves the atomization of a liquid feed into very small droplets within a hot drying gas leading to flash drying of the droplets into solid particles. The particles are then separated from the drying gas, using a cyclone and/or a filter bag, as a final spray dried product.
  • the feed can be a solution, a suspension or an emulsion and the resulting product can be classified as a powder, granules or agglomerates.
  • spray drying therefore converts a liquid feedstock into a powder with well-defined properties.
  • Properties such as level of moisture or residual solvent in the powder, particle morphology or size and powder density can be manipulated to a great extent to target levels.
  • the remarkable flexibility in tailoring the properties of the final powder, the gentleness of the process and its economics when compared with competing technologies such as freeze drying led to its proliferation in multiple industrial applications including cosmetics, fine chemicals, detergents, polymers, excipients and pharmaceuticals.
  • microparticles as provided herein are described in part in WO 2010/037142 and WO 2016/081,783, the contents of which are incorporated in their entirety by reference herein.
  • the microparticles can be spray-dried, e.g., using a Buchi 191 Spray Dryer.
  • the spray drying procedure aerosolizes the vaccine antigen-polymer matrix, where optimum water removal from the droplet results in nanoparticles containing the vaccine antigen in a polymer matrix.
  • the particle size and zeta potential can be determined, e.g., using a Malvern Zeta Sizer. Physical characteristics of the particles, such as size morphology, distribution and zeta potential and any other characteristic desired to be assessed in the pharmaceutical API or microparticles, can be achieved by any known methods.
  • the microparticle compositions provided herein are for oral administration.
  • GALT gut-associated lymphoid tissue
  • the murine and human GALT is defined by the presence of highly interactive and specialized lympho-epithelial and lymphoid units known as follicle-associated epithelium (FAE).
  • FAE is arranged in clusters, known as Peyer’s patches (PPs), or isolated lymphoid follicles (I LFs), distributed throughout the small and large intestines.
  • PPs Peyer’s patches
  • I LFs isolated lymphoid follicles
  • FAE contains specialized microfold cells (M cells) that sample and expose luminal antigens to TLR-regulated and GALT-associated multifunctional DCs.
  • M cells play a critical role in delivering luminal antigens for further processing and immune presentation.
  • M cell targeting agent to increase antigen delivery and processing by GALT APCs.
  • a lectin can be used.
  • the lectin is Aleuria aurantia lectin (AAL) (10).
  • the AAL in the formulation can function as an immune adjuvant.
  • the microparticles provided herein can include an immune adjuvant other than AAL.
  • the adjuvant other than AAK is selected from the group consisting of CpG ODN, MF59, alum, flagellin, R848, monophosphoryl lipid A, ODN 1826, and combinations thereof.
  • the immune adjuvant is CpG ODN.
  • the microparticles provided herein include AAL and no immune adjuvant other than AAL.
  • the microparticles provided herein include AAL and an immune adjuvant other than AAL.
  • the microparticles include one or more immune adjuvants at a total concentration, or, independently, at a concentration of between about or equal to 0.1% to about or equal to 0.5%, such as about or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% w/w.
  • the ingredients for the microparticle matrix include a water-soluble polymer such as beta cyclodextrin and enteric coating materials such as ethyl cellulose (EC) and/or hydroxypropylmethyl cellulose acetate succinate (HPMCAS).
  • the microparticles provided herein have immunogenic peptides (e.g., VPAs) associated with the microparticle.
  • the VPAs are encapsulated by the microparticle.
  • M cells act as sampling ports for any foreign molecules encountered in the small intestine and have been shown to house numerous dendritic cells and immune cells. Following the sampling of the oral vaccine by M cells, the particle is processed by a dendritic/antigen presenting cell (APC) and presented on major histocompatibility complex (MHC) class I or MHC class II molecules.
  • APC dendritic/antigen presenting cell
  • MHC major histocompatibility complex
  • CpG oligodeoxynucleotides which have been shown to provide signals for the dendritic cell and T- cell activation can be used as an adjuvant in the formulation. In certain aspects, CpG has also been shown to enhance the efficacy of weak tumor antigens and to promote T cell responses.
  • microparticle formulations are described and/or provided herein:
  • OroVAX refers to a microparticle formulation that includes HPMCAS, b-cyclodextrin, EC, AAL and the five polypeptides (VPAs) having the sequences set forth in SEQ ID NOS: 1-5, in the proportions and percentage ranges described herein.
  • the OroVAX formulations provided herein contain equimolar amounts of the five polypeptides (VPAs) having the sequences set forth in SEQ ID NOS: 1-5.
  • the OroVAX formulations provided herein contain, as w/w based on the weight of the microparticles, 20% HPMCAS, 60% b-cyclodextrin, 20% EC, 0.25% AAL and 1% of each of the five polypeptides (VPAs) having the sequences set forth in SEQ ID NOS:1-5.
  • the terms “BSK” or OroVAX without VPAs,” as used interchangeably herein refer to a microparticle formulation that includes HPMCAS, b-cyclodextrin, EC and AAL, in the proportions and percentage ranges described herein.
  • the BSK formulations provided herein contain, as w/w based on the weight of the microparticles, 20% HPMCAS, 60% b- cyclodextrin, 15% EC and 0.25% AAL.
  • the terms “BSK02” or “BSK-02,” as used interchangeably herein, refer to a microparticle formulation that includes HPMCAS, b-cyclodextrin, EC, AAL and the SP17 protein or a SP17 polypeptide, in the proportions and percentage ranges described herein.
  • the SP17 protein (GenBank Accession No. CAA88459.1) has the sequence set forth below as SEQ ID NO:6:
  • the SP17 polypeptide (amino acids 111-142 of the SP17 protein) has the sequence set forth below as SEQ ID NO:7:
  • the BSK02 formulations can contain, as w/w based on the weight of the microparticles, 20% HPMCAS, 60% b-cyclodextrin, 15% EC, 0.25% AAL and 5% SP17 protein or SP17 polypeptide.
  • the BSK02 formulations can additionally contain CpG as an immune adjuvant.
  • the CpG immune adjuvant is present in a weight ratio of 1:500, relative to the weight of the microparticles (e.g., 10 pg CpG / 5 mg dose of microparticles).
  • a key step to start the chain of reaction of activating the immune system begins with antigen presenting cells (APCs), such as dendritic cells (DCs).
  • APCs antigen presenting cells
  • DCs dendritic cells
  • S-protein coronavirus protein spike
  • S-protein coronavirus protein spike
  • ACE-II receptor on lung tissue (4).
  • Al Artificial Intelligence
  • the DCs Upon absorption into the Gl tract, the DCs digest the VPAs derived from the S-protein; thus, beginning the chain reaction of immune system activation. Reactogenicity is also a concern with any type of vaccination for Covid-19 based treatment of Covid-19. However, this risk is much greater for vaccinations that are administered by injection versus a more physiologic oral route, since injections are not only more invasive to the patient, but they are also not the normal pathway through which SARS-Cov-2 is transmitted and thus an injectable route might generate a more abrupt/rapid immune response that may be more likely to facilitate more adverse effects.
  • OroVAX is delivered orally, it will be absorbed and processed by the patient’s immune system through the mucous membranes of the gastrointestinal system, and will hence more closely mirror the physiologic absorption pathway of SARS-CoV-2 as compared to an injectable formulation, resulting in a more gradual increase in the activity of the immune system and yielding a safer and more durable immunologic response with less expected long-term exhaustion of T-cell immunity.
  • OroVAX incorporates only the key immunogenic proteins as identified by the Diamond Al antigen discovery platform, and thus it is a non-infectious oral vaccine.
  • OroVAX is a biodegradable and biocompatible multifunctional polymer microparticle
  • the adverse effects will likely be minimal at the doses prescribed.
  • it is also ideal for low- and middle- income countries (4), as well as medically underserved rural sites in the US. For example, since it can be stored at room temperature, it is easily manufactured and shipped, and requires minimal training of health care providers world-wide, making it an ideal vaccine for LMICs, and for world-wide military distribution and support.
  • OroVAX is a biodegradable and biocompatible multifunctional polymer microparticle vehicle suitable for the immunotherapy-based treatment of SARS-CoV-2 targeting Viral Peptide Antigens (VPA).
  • the microparticles are composed of b-cyclodextrin, hydroxypropyl-methyl cellulose acetate succinate (HPMCAS), and ethyl cellulose (EC).
  • HPMCAS hydroxypropyl-methyl cellulose acetate succinate
  • EC ethyl cellulose
  • OroVAX manufacturing is more time-and cost-effective compared with other vaccines under development and meets the requirement for broad and rapid deployment.
  • the virus Spike surface protein was selected as the target, due to its role in viral entry and because it is less likely to undergo mutations that would make the vaccine less effective.
  • OroVAX Within the protein spikes that are present on the surface of the virus, are stable highly immunogenic genomic sequences (e.g., SEQ ID NOS:1-5) that are important for the immune system to be able to effectively fight the Covid-19 pandemic, and it is these peptide sequences which have been incorporated into OroVAX. OroVAX manufacturing is more time-and cost-effective compared with other vaccines under development and meets the requirement for broad and rapid deployment.
  • Parameters such as cost of production, scalability, and storage at room temperature are fundamental for a vaccine that, if effective, will have to be deployed as widely as possible in a short time, in order to make an impact on the world-wide consequences of a pandemic such as COVID- 19.
  • OroVAX is manufactured in a single-step spray drying process, which allows it to 1) contain production costs, 2) facilitate production scale-up from a few hundred to several million doses, and 3) facilitate transportation and storage, due to its stability at room temperature.
  • OroVAX unlike other vaccine strategies currently being evaluated in a clinical setting, has fewer chances of eliciting a non-neutralizing immune response, since it contains only selected epitopes instead of the full-length Spike protein, making OroVAX the first rationally designed anti-COVID-19 vaccine developed for human use.
  • the immunogenic polypeptides, polynucleotides encoding the immunogenic polypeptides and compositions provided herein can be used to treat any disease or condition associated with a pathogen.
  • the treatment is a prophylactic treatment.
  • methods of eliciting an immune response by administering, to a subject in need thereof, any one of the immunogenic polypeptides, polynucleotides encoding the immunogenic polypeptides and compositions provided herein.
  • the disease or condition is associated with a virus.
  • the virus is a coronavirus.
  • the coronavirus is SARS-CoV-2.
  • the disease or condition is COVID-19.
  • a method of selecting a polypeptide for treatment of a disease or condition caused by a pathogen in a subject comprising:
  • A5. The method of any one of embodiments A1-A4, wherein at least one HLA allele is selected from among HLA-A*11:01, HLA-DRB1*01:01, HLA-B*18:01 and HLA-B*58:01.
  • A6 The method of any one of embodiments A1-A5, wherein at least one selected amino acid subsequence binds to an HLA Class 2 allele.
  • A7 The method of any one of embodiments A1-A6, wherein (e) comprises, additionally: analyzing the accessibility of the at least one amino acid subsequence on the protein surface as an epitope; and based additionally on the accessibility of the at least one amino acid subsequence on the protein surface as an epitope, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).
  • A8 The method of any one of embodiments A1-A7, wherein two or more amino acid subsequences are selected in (e) and two or more polypeptides are selected in (f).
  • A9. The method of embodiment A8, wherein at least one polypeptide binds to an HLA Class 1 allele and at least one polypeptide binds to an HLA Class 2 allele.
  • A10 The method of any one of embodiments A1-A9, wherein at least one polypeptide binds to an HLA Class 1 allele and binds to an HLA Class 2 allele.
  • A11 The method of any one of embodiments A1-A10, wherein 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid subsequences are selected in (e) and 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more polypeptides are selected in (f).
  • A14 The method of any one of embodiments A1-A13, wherein (e) comprises, additionally: identifying whether the at least one amino acid subsequence is resistant to mutations; and based additionally on identifying the at least one amino acid subsequence as being resistant to mutations, selecting the at least one amino acid subsequence for subsequent selection of the polypeptide in (f).
  • A14 The method of any one of embodiments A1-A13, wherein the pathogen is a virus or a bacterium.
  • A15 The method of any one of embodiments A1-A14, wherein the protein that is expressed by the pathogen is associated with entry of the pathogen into the subject.
  • A16 The method of any one of embodiments A1-A15, wherein the pathogen is a virus.
  • A19 The method of any one of embodiments A16-A18, wherein the protein is selected from among a spike protein, a membrane protein and an envelope protein.
  • A20 The method of any one of embodiments A16-A19, wherein the protein is a spike protein.
  • A21. The method of embodiment A20, wherein the at least one amino acid subsequence is selected from among SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • A22 The method of any one of embodiments A1-A21 , wherein the treatment of a disease or condition caused by the pathogen in the subject is a prophylactic treatment.
  • A23 The method of any one of embodiments A1-A22.1 , further comprising, preparing a polypeptide composition comprising the polypeptide(s) selected in (f) for treatment of a disease or condition caused by the pathogen in the subject.
  • composition comprises at least one polypeptide comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition comprises at least one polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or an amino acid sequence 95% or more identical to the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition comprises at least one polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition comprises 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition comprises 5 polypeptides comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:1, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.
  • composition comprises 5 polypeptides comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.
  • A27 The method of any one of embodiments A23-A26, wherein the composition comprises a microparticle.
  • microparticle comprises a sustained-release polymeric matrix.
  • microparticle comprises b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).
  • composition is formulated for systemic, parenteral, topical, oral, mucosal, intranasal, subcutaneous, aerosolized, intravenous, bronchial, pulmonary, vaginal, vulvovaginal, esophageal, or oroesophageal administration.
  • A32 The method of any one of embodiments A27-A29, wherein the microparticle is formulated for oral administration.
  • composition or microparticle comprises an agent that protects the components of the microparticle against degradation in the acidic environment of the stomach.
  • composition is a microparticle of a size between about or equal to 1 pm and about or equal to 5 pm in diameter, as determined by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • A36 The method of any one of embodiments A27-A29 and A32-A35, wherein the microparticle comprises a lectin.
  • A37 The method of embodiment A36, wherein the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.
  • A38 The method of embodiment A37, wherein the lectin is AAL.
  • composition is formulated for prophylactic treatment.
  • composition is formulated as a vaccine.
  • composition comprises one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • composition of embodiment B1 that is formulated for prophylactic treatment.
  • composition of embodiment B1 or B2 that is formulated as a vaccine is formulated as a vaccine.
  • composition of embodiment B3 comprising one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • composition of any one of embodiments B1-B4 that is formulated for oral administration is formulated for oral administration.
  • composition of embodiment B5, wherein at least one polypeptide is encoded by a coronavirus is encoded by a coronavirus.
  • composition of embodiment B6 comprising at least one polypeptide comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment B7 comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment B8, comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment B9 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO: 1 , a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.
  • composition of embodiment B9.1 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.
  • composition B11 The composition of any one of embodiments B1-B10, wherein the composition comprises a microparticle.
  • composition of embodiment B11, wherein the microparticle comprises a sustained- release polymeric matrix.
  • composition of embodiment B11 or B12, wherein the microparticle comprises b- cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).
  • composition of embodiment B13, wherein the microparticle comprises about or equal to 10% to about or equal to 20% w/w ethylcellulose, based on the weight of the microparticles.
  • composition of embodiment B13 or B14, wherein the microparticle comprises about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles.
  • composition of embodiment B16, wherein the microparticle comprises about or equal to 60% w/w b- cyclodextrin, based on the weight of the microparticles.
  • composition of any one of embodiments B1-B17 that is formulated for oral administration is formulated for oral administration.
  • composition of embodiment B18 comprising an agent that protects the components of the microparticle against degradation in the acidic environment of the stomach.
  • composition of embodiment B19, wherein the agent is hydroxypropyl-methyl cellulose acetate succinate (HPMCAS).
  • HPMCAS hydroxypropyl-methyl cellulose acetate succinate
  • composition of embodiment B20 wherein the composition is a microparticle and the microparticle comprises about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles.
  • B22 The composition of embodiment B21 , wherein the microparticle comprises about or equal to 30% w/w HPMCAS, based on the weight of the microparticles.
  • DLS dynamic light scattering
  • composition of embodiment B24, wherein the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.
  • AAL Aleuria aurantia lectin
  • wheatgerm agglutinin Aleuria aurantia lectin
  • composition of embodiment B25, wherein the lectin is AAL.
  • composition of embodiment B28, wherein the composition comprises a microparticle is provided.
  • composition of embodiment B29 comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment B30 comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment B31 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:1, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.
  • composition of embodiment B32 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.
  • composition comprising two or more isolated polypeptides or polynucleotides encoding the two or more isolated polypeptides, wherein the polypeptides are selected by a method of any one of embodiments A1-A22.1.
  • C11.1 The isolated polypeptide or polynucleotide of embodiment C11, wherein the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • C12. The polynucleotide of any one of embodiments C2, C4 and C6-C11.1, wherein the polynucleotide is an expression vector or expression plasmid.
  • polynucleotide of embodiment C12 wherein the polynucleotide is a DNA plasmid or vector, or RNA plasmid or vector.
  • polynucleotide of embodiment C13 wherein the polynucleotide is a DNA plasmid or vector and a portion of the DNA plasmid or vector comprises a DNA virus or portion thereof.
  • polynucleotide of embodiment C13 wherein the polynucleotide is in a RNA plasmid or vector and a portion of the RNA plasmid or vector comprises a RNA virus.
  • RNA virus is a retrovirus or a ssRNA virus.
  • composition of embodiment C5, wherein the isolated polypeptide is encoded by a virus is provided.
  • composition of embodiment C18, wherein the virus is a coronavirus.
  • composition of embodiment C19, wherein the coronavirus is SARS-CoV-2.
  • composition of any one of embodiments C18-C22, wherein at least one polypeptide comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment C23 comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment C24 comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment C25 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO: 1 , a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.
  • composition of embodiment C26 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.
  • a vaccine composition comprising an isolated polypeptide or polynucleotide of any one of embodiments C1, C2, C4 and C6-C17, and comprising one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • composition comprising an antigen presenting cell (APC) and an isolated polypeptide or polynucleotide of any one of embodiments C1 , C2, C4 and C6-C17.
  • APC antigen presenting cell
  • composition of embodiment C32, wherein at least one polypeptide comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment C33 comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment C34 comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • C36 The composition of embodiment C34, comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment C35 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO: 1 , a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.
  • composition of embodiment C36 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.
  • a pharmaceutical composition comprising:
  • polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; and
  • composition of embodiment D1 comprising 2, 3, 4 or 5 polypeptides whose sequences differ from each other and each of which independently comprises an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment D2 comprising 5 polypeptides each independently comprising an amino acid sequence consisting of, or 95% or more identical to, the amino acid sequence set forth SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • composition of embodiment D3 comprising 5 polypeptides, comprising: a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:1, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3, a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4, and a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:5.
  • a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:1 a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:2
  • a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:3 a polypeptide comprising an amino acid sequence consisting of the sequence set forth in SEQ ID NO:4
  • composition of embodiment D4 comprising 5 polypeptides, comprising: a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 , a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5.
  • composition of any one of embodiments D1-D6 that is formulated as a gel, ointment, liquid, suspension, aerosol, tablet, pill, powder or lyophile.
  • composition of any one of embodiments D1-D7, wherein the composition comprises a microparticle.
  • composition of embodiment D8, wherein the microparticle comprises a sustained-release polymeric matrix.
  • microparticle comprises b-cyclodextrin, ethyl cellulose (EC) or b-cyclodextrin and ethyl cellulose (EC).
  • microparticle comprises about or equal to 10% to about or equal to 20% w/w ethylcellulose, based on the weight of the microparticles.
  • composition of embodiment D10 or D11, wherein the microparticle comprises about or equal to 15% w/w ethylcellulose, based on the weight of the microparticles.
  • microparticle comprises about or equal to 60% w/w b-cyclodextrin, based on the weight of the microparticles.
  • D15 The pharmaceutical composition of any one of embodiments D8-D14, wherein the microparticle is formulated for oral administration.
  • D16 The pharmaceutical composition of any one of embodiments D1-D15, wherein the composition or microparticle comprises an agent that protects the components of the composition or microparticle against degradation in the acidic environment of the stomach.
  • composition of embodiment D17 wherein the composition is a microparticle and the microparticle comprises about or equal to 20% to about or equal to 40% w/w HPMCAS, based on the weight of the microparticles.
  • microparticle comprises about or equal to 30% w/w HPMCAS, based on the weight of the microparticles.
  • DLS dynamic light scattering
  • composition of any one of embodiments D8-D20, wherein the composition comprises a microparticle and the microparticle comprises a lectin.
  • composition of embodiment D21, wherein the lectin is selected from among Aleuria aurantia lectin (AAL), wheatgerm agglutinin and Ulex europaeus- 1.
  • AAL Aleuria aurantia lectin
  • wheatgerm agglutinin Aleuria aurantia lectin
  • microparticle comprises about or equal to 0.1% to about or equal to 0.5% w/w lectin, based on the weight of the microparticles.
  • composition of any one of embodiments D1-D24 comprising about or equal to 1% to about or equal to 10% w/w polypeptides.
  • composition of embodiment D26 wherein the composition is formulated as a vaccine.
  • composition of embodiment D27 wherein the composition comprises one or more suitable pharmaceutically acceptable adjuvants and/or one or more suitable pharmaceutically acceptable carriers.
  • E1. A device, comprising the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28.
  • the device of embodiment D1 that is in unit dosage form, liquid dosage form, solid dosage form, oral dosage form, a tablet, a capsule, a topical patch, a syringe, an inhaler, a dosage cup, a dropper, a pump, a spray bottle, an aerosol container, a wound dressing, or an applicator for administering the pharmaceutical composition.
  • a kit comprising the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 and a device for administration of the composition.
  • kits of embodiment F1 wherein the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 is contained in the device for administration.
  • kit of embodiment F1 wherein the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 is present as a separate component that is distinct from the device.
  • a method for treating a disease or condition associated with a pathogen in a subject comprising administering the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 to a subject in need thereof, in an amount sufficient to induce an immune response.
  • a method for treating a disease or condition associated with a pathogen in a subject comprising administering a therapeutically effective amount of the composition of any one of embodiments B1-B32, the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36, or the pharmaceutical composition of any one of embodiments D1-D28 to a subject in need thereof.
  • G6 The method of any one of embodiments G1-G6, wherein the treatment is a prophylactic treatment.
  • G7 The method of any one of embodiments G1-G6, wherein the disease or condition is COVID-19.
  • a method of inducing an immune response in a subject comprising administering the composition of any one of embodiments B1-B32 or the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36 in an amount sufficient to induce an immune response.
  • embodiment H2 further comprising obtaining polyclonal antibodies from the subject and/or antiserum that immunospecifically binds to a polypeptide, or a polypeptide encoded by, the composition of any one of embodiments B1-B32 or the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36.
  • invention H3 further comprising: isolating spleen cells from the subject, and combining the spleen cells with myeloma cells under conditions that produce monoclonal antibody generating hybridomas.
  • embodiment H4 further comprising screening the hybridomas for those that produce monoclonal antibodies that immunospecifically bind to a polypeptide, or a polypeptide encoded by, the composition of any one of embodiments B1-B32 or the isolated polypeptide, polynucleotide or composition of any one of embodiments C1-C36.
  • a method of treating a disease or condition associated with a pathogen comprising administering, to a subject in need thereof, the monoclonal antibody of embodiment H5.
  • Example 1 Selection of Immunogenic Polypeptide Epitopes of the SARS-CoV-2 Coronavirus
  • DIAMONDTM is a computational platform and a neural network that can identify immunologic targets for T- and B-lymphocytes, which then can be used for the development of prophylactic treatments, such as vaccines, or to generate antibodies (e.g., monoclonal antibodies) for the treatment of diseases or conditions caused by pathogens.
  • SpliceDiffTM is a proprietary software module for the identification of immunogenic polypeptide epitopes and is part of an integrated bioinformatics and artificial intelligence (Al) system as described herein and as described in PCT Application No. PCT/US20/35183, filed on May 29, 2020, the contents of which are expressly incorporated by reference herein. This example describes the use of this platform to identify immunogenic polypeptide epitopes of the SARS-CoV-2 coronavirus, which causes COVID-19.
  • DIAMONDTM was deployed to focus on the S1 sub-unit of the SARS-CoV-2 surface glycoprotein, also known as Spike (S) protein, which contains the receptor binding-domain (RBD) for ACE2, as it is essential for the virus to enter host cells. It was expected that anti-S1 antibodies directed against epitopes located in the RBD could be protective by neutralizing virus entry. Combined B and T cell activation are required for an effective immune response, hence peptides predicted to bind to HLA class I and class II molecules were included.
  • S S protein
  • RBD receptor binding-domain
  • ANN artificial neural networks
  • CNN state-of-the-art capsule neural network
  • the DIAMONDTM platform was used to select immune-dominant peptides from the S1 subunit of the Spike protein that were predicted to bind to the most common HLA class I and class II alleles and would be most likely to elicit neutralizing antibodies. From the predicted immune-dominant peptides, those that are accessible on the S1 protein surface were selected using a proprietary 3D rendering tool. A Phase I clinical study was conducted to evaluate the tolerability and the seroconversion rates in healthy individuals. The platform analyzed the sequence of the S glycoprotein to identify peptides in immune hotspots. The immune hotspot regions were defined as follows:
  • the positions of the overlapping peptides were determined and further selected based on whether they were accessible on the surface of the S protein, thereby maximizing the chance that antibodies elicited by the vaccine will specifically neutralize the virus, thus minimizing the risk of antibody-dependent enhancement (ADE), which is driven by non-neutralizing antibodies.
  • ADE antibody-dependent enhancement
  • the selected peptides might provide cross-protection against coronaviruses other than SARS-CoV-2. It was found that the SARS-CoV-2 sequence where three of the 5 epitopes are located (one HLA-A*11:01- and one HLA-B*18:01- restricted peptide, and one Ab epitope overlapping with the HLA-DRB1*01:01 restricted peptide) shares an 86% homology with that of the SARS-CoV virus, with only two amino acid substitutions.
  • the remaining 2 epitopes are located in a region which shares 88% homology to that of the SARS-CoV virus.
  • the differences in the sequence did not alter the predicted B cell epitope localization, nor did they significantly reduce the affinity for HLA-DRB1*01:01, and HLA:18:01, indicating that a vaccine formulation containing these peptides might afford for a level of cross-protection against other coronaviruses, especially those that use ACE2 as a cellular receptor due to the tendency to conserve the RBD sequence.
  • MSA multi-sequence alignment tool
  • Example 2 Preparation, Characterization and Cytotoxicity of Microparticles for Formulation of a SARS-CoV-2 Vaccine
  • the 5 viral peptide antigens (VPAs) identified as discussed in Example 1 above can be incorporated into a microparticle formulation that can be administered orally, e.g., as an oral vaccine.
  • Microparticles containing immunogenic polypeptides, such as the VPAs can be prepared as described, for example in WO 2010/037142 and WO 2016/081783, the contents of which are incorporated in their entirety by reference herein.
  • the polymer matrix of the microparticles is loaded with specific components to increase target specificity and immunogenicity.
  • the benefits of using MP for immunotherapy include uptake by antigen presenting cells (APC) such as dendritic cells (DCs) and macrophages, targeting, shielded trafficking of cargo, co-delivery of immunomodulatory agents and antigen(s), and sustained release of antigen(s), eliminating the need for repeated doses of the vaccine (7).
  • APC antigen presenting cells
  • DCs dendritic cells
  • macrophages targeting, shielded trafficking of cargo, co-delivery of immunomodulatory agents and antigen(s), and sustained release of antigen(s), eliminating the need for repeated doses of the vaccine (7).
  • the microparticles are composed of b-cyclodextrin, hydroxypropyl-methyl cellulose acetate succinate (HPMCAS), and ethyl cellulose (EC).
  • HPMCAS hydroxypropyl-methyl cellulose acetate succinate
  • EC ethyl cellulose
  • the HPMCAS component is used for its enteric properties to protect proteins from the harsh pH of the stomach, while the b-cyclodextrin and ethyl cellulose function as a sustained-release polymeric matrix.
  • This formulation has previously been shown as ideal to deliver immune-stimulatory agents to the Gut-Associated Lymphoid Organs (GALT), in a murine model of ovarian cancer treated with an analogue oral vaccine (5).
  • GALT Gut-Associated Lymphoid Organs
  • the vaccine formulation also contains microfold (M)-cell targeting ligand Aleuria Aurantia lectin (AAL), which has been shown to improve the targeting of the particles to the Peyer's Patche
  • microparticles are formulated using a single-step spray drying method (8).
  • OroVAX with VPAs will be formulated under cGMP constraints at Kiromic’s GMP facility located at 7707 Fannin St., Suite 140, Houston, TX 77054.
  • Kiromic GMP facility located at 7707 Fannin St., Suite 140, Houston, TX 77054.
  • a bio-degradable polymer matrix consisting of 20%
  • HPMCAS Hydroxyl propyl methyl cellulose acetate succinate
  • 60% beta-cyclodextrin 60% beta-cyclodextrin
  • 20% ethyl cellulose is prepared in a sterile aqueous alkaline solution (1 mM NaOH in RNase-free H 2 0).
  • the M cell-targeting ligand AAL 0.25% w/w loading relative to total mass of polymers
  • the peptide mix 1% w/w loading per each peptide.
  • This feeding solution is sprayed, resulting in an aerosolized peptide/AAL/polymer matrix mixture.
  • Optimal water removal from the aerosolized droplets results in polymeric microparticles containing the encapsulated peptide (VPAs)/AAL.
  • Formulation details form part of a US and International Patent filed in 2009 entitled “A one-step method of formulating bioactive nano-particles with the use of a spray drying technique”, and a provisional patent filed in 2014 entitled “Method of Production and Use of a Novel Nanoparticle-Based Vaccine Targeting Cancer Testes Antigens in Solid and Hematologic Malignancies” (No. 62/081,825).
  • the encapsulation efficiency of the VPAs can be determined by ELISA and MS following their extraction from a particle sample dissolved in a slightly basic medium (Trizma®, pH 8.3), while the potency of the OroVAX batch can be determined by an immature dendritic cell (iDC) activation assay (21).
  • iDC immature dendritic cell
  • Biologic characterization of the final spray-dried product can be conducted for each OroVAX batch to ensure the desired properties and biological efficacy.
  • the characterization includes induction of maturation markers in immature dendritic cells (iDC) generated from adherent peripheral blood monocytes in the presence of IL4 and GM-CSF.
  • iDC immature dendritic cells
  • a passing result for the biological release assays is defined as DCs expressing 360% DC80, 365% CD83, 350% CD86, and 390% MHC class I (20).
  • the components of the microparticles are generally recognized as safe (GRAS) for human use by the FDA and have shown no toxicity, even at very high concentrations, in cultured cells and primates.
  • OroVAX microparticles without VPAs w/w composition, based on the weight of the microparticles, is 20% HPMCAS, 60% b-cyclodextrin, 15% EC and 0.25% AAL
  • Percent cytotoxicity is relative to negative control of cells treated with complete DMEM media only. Positive control of benzalkonium chloride was found to be cytotoxic, with 21% cell viability (Kiromic unpublished results).
  • Example 3 M Cell Targeting, Uptake and Immune Adjuvant Properties of Microparticle Formulations Containing AAL
  • AAL allows for microparticle anti-gravitational uptake by M cells in vitro in inverted orientation (11, 12). At the doses used, AAL optimizes M cell targeting, while not harming intestinal cells and it is therefore very likely that it will be safe and suitable for application also in humans (10, 11).
  • Roth-Walter F. et al. (10-12) they reported no toxicity in Balb/C mice of oral microparticles carrying a 80-times higher dose of AAL than that of the amount of AAL in OroVAX.
  • neuraminidases act as a virulence factors for several pathogens that invade the human body through Peyer's Patch M-cells.
  • AAL Aleuria Aurantia lectin
  • AAL might also target human M-cells (11, 12)
  • PBMCs proliferated in response to AAL and secreted the cytokines, IL-2, IFN-gamma, IL-10 and IL- 5 in a concentration-dependent manner, without signs of toxicity.
  • Figure 2 demonstrates preliminary uptake studies using fluorescent-labeled (fluorescein isothiocyanate-labelled bovine serum albumin; FITC-BSA) and AAL blank microparticles in mice; previous studies with human M-cells have demonstrated excellent uptake into PPs within 60 minutes following oral administration. Mice were dosed with the microparticles, euthanized shortly thereafter and microparticle localization determined by fluorescence analysis. The results are presented in Figure 2. Particle distribution (green dots, represented in grayscale) in small intestine ( Figure 2A) and uptake of labeled microparticles in Peyer’s patches ( Figure 2B) microvilli are shown (Kiromic unpublished results).
  • iDC immature dendritic cells
  • microparticle formulations described in a recent publication (16) and in WO 2016/081783 include, in certain aspects, the TLR9 agonist, CpG ODN, as immune adjuvant.
  • CpG ODN CpG ODN
  • OroVAX-CpG microparticles were manufactured and tested for their ability to activate HEK-BlueTM cells (InVivogen).
  • HEK- BlueTM-hTLR9 cells were obtained by co-transfection of the human TLR9 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene into HEK293 cells.
  • the SEAP gene was placed under the control of the IFN-b minimal promoter fused to five NF-KB and AP-1- binding sites (16). Stimulation with a TLR9 ligand activates NF-KB and AP-1, which induces the production of SEAP.
  • Cells were exposed to OroVAX, OroVAX-CpG, or free CpG ODN for 26 hours, before reading the optical density (OD) at 655 nm to assess reporter activity.
  • OD optical density
  • Figure 4 which illustrates measurement of CpG ODN-dependent TLR9 activation before and after spray drying (16)
  • CpG ODN does not show any residual activity after the spray-drying process, indicating that the immune stimulant function of OroVAX is exerted by AAL (16).
  • AAL serves both as an M-cell targeting agent and as an immune adjuvant, collectively enhancing the immunogenicity of the peptides loaded into OroVAX.
  • the potency of the OroVAX particles can be inferred based on potency studies that were performed using BSK-02 microparticle formulations (Composition, as w/w based on the weight of the microparticles, is 20% HPMCAS, 60% b-cyclodextrin, 15% EC, 0.25% AAL and 5% SP17 polypeptide (SEC ID NO:7)).
  • Figure 10 illustrates a potency assay for BSK-02 microparticles incubated at 37 °C for 7 days or 14 days, as measured by activation of iDC. iDC were generated and tested as indicated in Example 2 above. There was no significant difference between BSK-02 stored at -20 °C for 7 days or 14 days and thawed prior to use and BSK-02 stored at 37 °C for up to 14 days (Kiromic unpublished data).
  • BSK02-1 is microparticles stored for 7 days at -20 °C
  • BSK02-2 is microparticles stored for 7 days at 37 °C
  • BSK02-3 is microparticles stored for 7 days at -20 °C
  • BSK02-4 is microparticles stored for 7 days at 37 °C.
  • the control is freshly prepared BSK-02 microparticles.
  • Example 4 Immunogenicity of the VP As, and Ability to Elicit T-cell Mediated Killing
  • Monocytes were used as antigen presenting cells (APCs) to present the immunogenic polypeptides identified as described in Example 1 (SEC ID NOS:1-5).
  • APCs antigen presenting cells
  • in vitro immunogenicity testing indicated that these peptides are presented by APC in selected HLA type donors and elicit cytotoxic T cell responses and Interferon g release.
  • Figure 11 depicts light micrographs of APCs co-cultured with autologous peripheral blood lymphocytes (PBL), and IFNy and Turbofectin release in the medium, as measured by ELISA. T- cell mediated cytotoxicity is visible in APCs pulsed with the peptides of SEQ ID NOS: 1-5.
  • CTLs cytotoxic T cells activated by APCs pulsed with the 5 SARS-CoV-2 peptides (SEQ ID NOS: 1-5).
  • CTLs cytotoxic T cells
  • APCs autologous monocytes
  • Apoptosis was detected as increase of fluorescence following the activation of Caps3/7 Green Reagent (Incucyte®).
  • Figures 12 A and 12B depict T-cell mediated cytotoxicity of target cells pulsed with SARS-CoV-2 peptides.
  • Cells from two donors bearing the HLA Types HLA Class I A* 11 :01 , HLA Class II DRB1 *01 :01 (A), and HLA-B*58:01 (B) were utilized in this assay.
  • Autologous monocytes were utilized as targets with an effectors to targets ratio of 1 :1.
  • Apoptosis was detected as increase of fluorescence following the activation of Caps3/7 Green Reagent (Incucyte®).
  • Figure 12B depicts the kinetics of apoptosis of monocytes enriched with 10 ug/mL equimolar solution of each of the SARS-CoV-2 peptides as indicated, as well as a cocktail of all 5 peptides.
  • This phase I pilot study is an open-label, multi-site trial in 10 healthy male and non-pregnant female subjects, ages 18-55 years of age, who are in good health and meet all inclusion and exclusion eligibility criteria. Each subject will receive one 5mg dose and will be followed as per the study calendar (Appendix 1) for one year after the vaccination.
  • This study is designed to rapidly evaluate the safety and efficacy of a novel oral vaccination delivery mechanism (OroVAX) designed to prevent the development of Covid-19 without generating cytokine mediated toxicity such as ARDS.
  • the trial is designed to meet 2 key objectives. First, it will evaluate the safety and tolerability of this novel oral vaccine delivery platform.
  • OroVAX will assess the ability of OroVAX to generate an immune response, by quantifying the IgG and IgM antibody titer values (ATV) to the selected coronavirus peptide antigens.
  • ATV antibody titer values
  • the ATV generated from OroVAX will also be used to determine the percentage of those subjects who seroconvert (a surrogate endpoint for protection against the coronavirus).
  • OroVAX will elicit an immune response as measured by peptide-specific IgG/lgM in the serum of treated subjects. Seroconversion is defined as a 4X increase in the ATV above baseline.
  • the study population will be drawn from patients at various clinical institutions following contractual agreements.
  • the study population will consist of 10 patients.
  • Patients must have baseline evaluations performed prior to administration of the first dose of OroVAX and must meet all inclusion and exclusion criteria. In addition, the patient must be thoroughly informed about all aspects of the study, including the study visit schedule, required evaluations, and all regulatory requirements for informed consent. The written informed consent must be obtained from the patient prior to enrollment.
  • Each clinical trial site will have the option to enroll patients for a particular indication and/or stage as allowed by the protocol, if that indication and/or stage is more prevalent, accruable, or clinically relevant to that particular institution, as long as the patient meets all the inclusion and exclusion criteria.
  • CBC with differential, serum creatinine (Scr), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (TB), lipase, prothrombin time (PT), and partial thromboplastin time (PTT) are within normal reference range limits of the laboratory processing the results 7 days
  • Negative urine or serum pregnancy test 7 days (+/- 3 days) or less prior to enrollment. In women of child-bearing age, and must agree to use accepted contraception methods for at least 3 months after the final vaccination. 4. Either a female or male of reproductive capacity (females not of childbearing potential are post-menopausal as defined by having a history of amenorrhea for at least one year, or are surgically sterile via hysterectomy, bilateral oophorectomy, or tubal ligation/salpingectomy) wishing to participate in this study must be using, or agree to use, one or more types of birth control during the entire study and for
  • birth control methods may include condoms, diaphragms, birth control pills, anti-gonadotropin injections/implants/inserts, intrauterine devices (IUD), or surgical sterilization. All post-pubertal males are considered to be of reproductive capacity unless vasectomy for 180 days or more prior to first vaccination or bilateral orchiectomy. Women of reproductive capacity will be required to undergo a urine pregnancy test before completion of the post screening informed consent process. Accordingly, males agree to decline sperm donations until greater than 3 months after the final vaccination.
  • Subject is breast feeding or plans to breast feed within 3 months after the final vaccination.
  • ulcerative colitis including prescription or OTC medications for alleviation of gastritis, celiac disease, and/or any other type of intestinal malabsorption syndrome related disease such as pancreatic insufficiency
  • a subject withdrawn from the study may be followed indefinitely, or until resolution of the event(s) which caused the withdrawal.
  • the primary endpoints for this Phase I pilot trial are feasibility, safety, and toxicity of a single 5mg OroVAX vaccination.
  • An adverse event is defined as any untoward medical occurrence associated with the use of a drug in humans, whether considered drug related. More specifically, an adverse event can be any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the use of a drug, without any judgment about causality. An adverse event can arise from any use of the drug (e.g., off-label use, use in combination with another drug) and from any route of administration, formulation, or dose, including an overdose.
  • An adverse reaction is defined as any adverse event caused by a drug. Adverse reactions are a subset of all suspected adverse reactions for which there is reason to conclude that the drug caused the event.
  • a suspected adverse reaction is defined as any adverse event for which there is a reasonable possibility that the drug caused the adverse event.
  • “reasonable possibility” indicates that there is evidence to suggest a causal relationship between the drug and the adverse event.
  • a suspected adverse reaction implies less certainty about causality than an adverse reaction.
  • An adverse event or suspected adverse reaction is considered unexpected if it is not listed in the investigator brochure or package insert(s) or is not listed at the specificity or severity that has been observed, or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application.
  • Adverse events that would be anticipated to occur as part of the disease process are considered unexpected for the purposes of reporting because they would not be listed in the investigator brochure. For example, a certain number of non-acute deaths in a cancer trial would be anticipated as an outcome of the underlying disease, but such deaths would generally not be listed as a suspected adverse reaction in the investigator brochure.
  • An adverse event or suspected adverse reaction is considered serious if, in the view of either the investigator or sponsor, it results in any of the following outcomes: a. Death b. Life-threatening adverse event c. Inpatient hospitalization or prolongation of existing hospitalization d. A persistent or significant incapacity or substantial disruption of the ability to conduct normal life function e. Congenital anomaly/birth defect f. Important medical events that may not result in death, or require hospitalization yet may be considered serious when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.
  • An adverse event or suspected adverse reaction is considered life-threatening if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death.
  • the AE is possibly related to the vaccination
  • the Investigator will assess all AE and determine reportability requirements to the Food and Drug Administration (FDA), if it meets the FDA reporting criteria.
  • FDA Food and Drug Administration
  • Grade III or higher allergic reaction is defined as symptomatic bronchospasm requiring medication, edema or angioedema, and Grade IV is defined as anaphylaxis.
  • Grade II or higher autoimmune reaction is defined as an autoimmune reaction involving a non-essential organ or function requiring treatment.
  • Grade III or higher hematologic or non-hematologic toxicity including fever (> 40°C for > 24 hours).
  • IR Immune Response
  • Immune Response Endpoint will be determined as follows: Peptide-specific IgG and IgM in the serum of patients will be analyzed by ELISA using 5-fold dilutions of serum and a standard colorimetric measurement. Immune response tests will be performed at Kiromic laboratories or by third-party contractors with ISO certification, if available.
  • DLTs Dose limiting toxicities
  • IUD intrauterine devices
  • ATV test will be obtained as a baseline (+/- 3 days), and 7, 14, and 28 days after the vaccination (+/- 3 days).
  • SARS-Cov-2 RT-PCR will be obtained 7 days or less (+/- 3 days) prior to the first vaccination and again on days 12, 42, 194 and 379 to confirm the patient does not develop Covid-19 during the study. 4.
  • Patient enrollment Six (6) patients will be treated to evaluate for potential toxicity and reactogenicity and if no more than 1 DLT is noted in either disease cohort, then the study may continue to accrue patients until accrual is complete.
  • Laboratory tests at the final study visit will include hepatic and renal profiles, and CBC with differential. See Appendix 2 for laboratory adverse effect (AE) monitoring criteria.
  • the micro particulate vaccine will contain a maximum of 1% weight ratio of VPAs mix.
  • Peptides will be synthesized with 395% purity, TFA-free, and prior to the spray-drying process they will be resuspended at 10-20 mg/mL in sterile water for injection (WFI), or DMSO, or sterile DPBS, depending on their solubility.
  • WFI sterile water for injection
  • DMSO DMSO
  • DPBS sterile DPBS
  • peptides from the subunit 1 of the SARS-Cov-2 S glycoprotein (YP_009724390.1 surface glycoprotein, Severe acute respiratory syndrome coronavirus 2) that are predicted to generate a protective immune response in the majority of the world’s population, basing on in silico binding predictions to HLA-DRB1-*01:01, HLA-A*01:01, HLA-B*18:01, HLA-B*58:01, and to antibodies.
  • the following figures show the mapping of the selected epitopes in the 3D space highlighted in red.
  • the S protein is shown in gray in a vertical orientation, with the selected epitope in red at the top outer edge of the S-protein, where it is most likely to interact with the ACE2 cellular receptor.
  • the estimated population coverage represents the percentage of individuals within the population that are likely to elicit an immune response to the indicated epitope.
  • Population coverages were computed using the tool provided by the Immune Epitope Database (IEDB) (http://tools.iedb.org/population/). This tool uses the distribution of MHC alleles (with at least 4-digit resolution, e.g., A*02:01) within a defined population (obtained from http://www.allelefrequencies.net/) to estimate the population coverage for a given epitope.
  • IEDB Immune Epitope Database
  • Microparticle polymers a. b-cyclodextrin: International Chemical Identifier: 1 S/C42H70O35/c43- 1-8-29- 15(50)22(57)36(64-8)72-30-9(2-44)66-38(24(59)17(30)52)74-32-11(4-46)68- 40(26(61)19(32)54)76-34-13(6-48)70-42(28(63)21(34)56)77-35-14(7-49)69- 41(27(62)20(35)55)75-33-12(5-47)67-39(25(60)18(33)53)73-31-10(3-45)65- 37 (71 -29)23(58) 16(31 )51/h8-63H , 1 -7H2/t8-,9-, 10-, 11-,12
  • b-cyclodextrin Food-grade b-cyclodextrin is obtained from Sigma-Aldrich (catalogue number W402826). b- cyclodextrin is “generally recognized as safe” (GRAS) by the FDA. b. Hydroxypropyl-methyl cellulose acetate succinate: International Chemical
  • HPMCAS is “generally recognized as safe” (GRAS) by the FDA.
  • Ethyl cellulose International Chemical Identifier: 1S/C20H38O11/c1-6-26-10- 12-16(17(27-7-2) 18(28-8-3)20(25-5)30- 12)31-19-14(23) 13(22) 15(24-4) 11 (9- 21)29-19/h11-23H,6-10H2,1-5H3
  • Ethyl cellulose Aqueous Dispersion (Aquacoat® ECD, NF, JPE) is obtained from DuPont Nutrition USA, Inc. (catalogue number 422002-15).
  • Ethyl cellulose is “generally recognized as safe” (GRAS) by the FDA.
  • OroVAX will be formulated under cGMP constraints at Kiromic’s GMP facility located at 7707 Fannin St., Suite 140, Houston, TX 77054.
  • a bio-degradable polymer matrix consisting of 20% Hydroxyl propyl methyl cellulose acetate succinate (HPMCAS), 60% beta- cyclodextrin, and 20% ethyl cellulose will be prepared in a sterile aqueous alkaline solution (1 mM NaOH in RNase-free H 2 0).
  • the M cell targeting ligand AAL 0.25% w/w loading relative to total mass of polymers
  • the peptide mix 1% w/w loading per each peptide).
  • This feeding solution will be sprayed resulting in an aerosolized peptide/AAL/polymer matrix mixture.
  • Optimal water removal from the aerosolized droplets will result in polymeric microparticles containing the encapsulated peptide/AAL.
  • Formulation details form part of a US and International Patent filed in 2009 entitled “A one-step method of formulating bioactive nano particles with the use of a spray drying technique”, and a provisional patent filed in 2014 entitled “Method of Production and Use of a Novel Nanoparticle-Based Vaccine Targeting Cancer Testes Antigens in Solid and Hematologic Malignancies” (No. 62/081,825).
  • OroVAX microparticles will be stored refrigerated (+4 °C) in a dry form, for up to -600 days, or at room temperature (+24 °C), for up to -90 days.
  • OroVAX will be administered for one dose on Day 0.
  • Peptides incorporated into the formulation will be at least 95% pure, and they will not contain TFA.
  • the OroVAX batch will be discarded. Treatment may proceed if it is possible to prepare a new batch. Otherwise, the patient will be withdrawn from the study.
  • the sterility of the final spray dried product will be assessed by performing bacteria/fungi culture prior to batch release.
  • IPRC Institutional Protocol Review Committee
  • IRB Institutional Review Board
  • GRAS generally recognized as safe
  • Kiromic may apply for a patent covering the potential use of some peptide antigens in cancer therapeutics and diagnostics.
  • the investigators will declare this potential conflict of interest in the Patient Consent Form so that all patients recruited to this study will be aware that some or all data obtained may be used to support future application that may result in commercial value of the microparticle immunotherapy.
  • a panel of independent physicians/investigators will serve as an external Independent Clinical and Scientific Review Board monitor team (ICSRB) that will review the clinical and immunologic data generated in the study to guarantee the un-biased evaluation of study results.
  • ICSRB Independent Clinical and Scientific Review Board monitor team
  • the listing includes all ranges between any two of the values listed (e.g., the listing of "80%, 90% or 95%” includes ranges of "80% to 90%, “ “80% to 95%” and “90% to 95%”).

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Abstract

La technologie concerne en partie des compositions de peptides, comprenant des compositions de vaccins peptidiques, pour le traitement d'une maladie ou d'une affection provoquée par un pathogène, tel qu'un virus, y compris des coronavirus tels que le SARS-CoV-2. La technologie concerne également en partie des procédés de sélection de peptides pour la préparation de compositions, et des méthodes de traitement, y compris un traitement prophylactique, faisant appel aux compositions.
PCT/US2021/031387 2020-05-08 2021-05-07 Compositions de peptides pour le traitement d'infections pathogènes WO2021226520A1 (fr)

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