WO2022174156A1 - Prophylactic broad spectrum vaccine for sars-cov-2 - Google Patents

Prophylactic broad spectrum vaccine for sars-cov-2 Download PDF

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Publication number
WO2022174156A1
WO2022174156A1 PCT/US2022/016341 US2022016341W WO2022174156A1 WO 2022174156 A1 WO2022174156 A1 WO 2022174156A1 US 2022016341 W US2022016341 W US 2022016341W WO 2022174156 A1 WO2022174156 A1 WO 2022174156A1
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seq
phage
aspects
vaccine
sars
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PCT/US2022/016341
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French (fr)
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Anne De Groot
William Martin
Subhendu BASU
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Epivax, Inc.
Adaptive Phage Therapeutics
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Publication of WO2022174156A1 publication Critical patent/WO2022174156A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • 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
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10141—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This disclosure relates to the development of a vaccine against SARS-CoV-2 viral infection.
  • the present disclosure provides in some aspects a phage-produced vaccine for prevention of SARS-CoV-2 viral infection in human subjects.
  • empty capsid vaccines have been prepared for imitating the particulate nature of pathogens.
  • particulate vaccines are also prepared by using Ankara and Baculovirus vector systems where these recombinant viruses act as a chimera of pathogens.
  • manufacturing processes required for these vaccines are time consuming and require expensive cell culture media, which are not ideal for rapid, large quantity production of vaccines during outbreaks.
  • Bacteriophage or phage are bacterial viruses that do not infect eukaryotic cells. These bacterial-viruses, such as M13, T4, T7, lambda (l) etc., can be propagated using laboratory strains of E.coli with inexpensive bacteriological media such as Luria-Bertani (LB) broth. Foreign antigen or peptides may be expressed by incorporating a corresponding nucleotide encoding such into the genome of the phage, often fused to a surface targeting sequence to ensure surface display. The resulting expression on the surface of bacteriophage when administered as a vaccine is recognized as displayed antigens and leads to immune response generation. Thus, such chimeric constructs may be considered valuable vaccine agents.
  • LB Luria-Bertani
  • Individual phage particles can display multiple copies (including in some aspects hundreds) of identical or combinational antigenic epitopes on its surface and act as a good immunogen when introduced into a mammalian system.
  • the particulate nature of the phage specifically attracts antigen-presenting cells (APCs) and may be able to provoke both cell mediated and humoral responses.
  • APCs antigen-presenting cells
  • phage may be used as a gene delivery system for DNA vaccination where genetic information is coded inside of phage genome under control of eukaryotic promoter.
  • the present disclosure relates to bacteriophage compositions to generate an immune response against SARS-CoV-2.
  • the bacteriophage systems include surface expression of concatemers of SARS-CoV-2 epitope peptides, as well as bacteriophage comprising nucleic acids encoding such concatemers within the genome thereof.
  • the present disclosure concerns a bacteriophage having at least one amino acid sequence of a concatemer of SARS-CoV-2 T cell epitopes.
  • the at least one amino acid sequence is SEQ ID NO: 1.
  • SEQ ID NO: 1 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 2.
  • the bacteriophage is selected from Lambda, T4, T7 or M13/fl. In other aspects, the bacteriophage is T4.
  • the present disclosure concerns a composition of a bacteriophage featuring SEQ ID NO: 1 and/or a nucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 1, such as that set forth in SEQ ID : 2.
  • the present disclosure concerns a bacteriophage with a nucleic acid of at least 85% sequence identity to a nucleic acid sequence encoding an amino acid sequences as set forth in SEQ ID NO: 1, such as 85% identity to the nucleic acid sequence set forth in SEQ ID NO: 2.
  • the nucleic acid is under the control of a promoter.
  • the promoter is a bacteriophage promoter.
  • the nucleic acid is fused in frame to one or more head proteins of the bacteriophage.
  • the promoter is a mammalian promoter, such as CMV, SV40, CAG or U6.
  • the instantly-disclosed phages are irradiated (e.g., after construction by prior to administration to a subject).
  • the present disclosure provides a vaccine of the bacteriophage as disclosed herein, including bacteriophage that express the concatemers disclosed herein, such as on the surface of the phage and/or include the nucleic acids disclosed herein with the genome of the phage.
  • the phages are irradiated (e.g., after construction by prior to administration to a subject).
  • the present disclosure provides a method of eliciting an immune response in a subject comprising administering an immune system stimulating amount of a bacteriophage comprising at least one amino acid sequence of a concatemer of SARS-CoV-2 T cell epitopes, wherein the at least one amino acid sequence includes the amino acid sequence as set forth in SEQ ID NO: 1.
  • the present disclosure provides methods for inducing immunity against 2019-nCoV in a subject in need thereof by administering to the subject a therapeutically effective amount of a phage as disclosed herein.
  • Figures 1 A shows immunoinformatic reports for a sample epitope cluster sequence in one phage.
  • Cluster Detail Reports for FLGVYYHKNNKSWMESE SEQ ID NO: 77 (a portion of SEQ ID NO: 1 is shown).
  • Figure 1 A is an EpiMatrix staircase report of the sequence for class II HLA supertypes.
  • Z-score indicates the potential of a 9-mer frame to bind to a given HLA allele; the strength of the score is indicated by the blue shading as noted in the respective Figures. All scores in the Top 5% (Z-Score > 1.64) are considered “Hits.” * Scores in the top 10% are considered elevated, other scores are grayed out for simplicity.
  • Figure IB is an EpiMatrix staircase report for identified MHC class I clusters of the presented peptide sequence of SARS-CoV-2.
  • Z-score indicates the potential of a 9-mer or 10- mer frame to bind to a given HLA allele; the strength of the score is indicated by the blue shading as noted in the respective Figure.
  • Figure 1C is the JanusMatrix report for identified MHC class II clusters. *Count of HUMAN JanusMatrix matches found in the search database. With respect to a given EpiMatrix Hit (a 9-mer contained within the input sequence which is predicted to bind to a specific allele), a Janus Matrix match is a 9-mer derived from the search database (e.g., the human genome) that is predicted to bind to the same allele as the EpiMatrix Hit and shares TCR facing contacts with the EpiMatrix Hit. ** Janus Homology Score represents the average depth of coverage in the search database for each EpiMatrix hit in the input sequence.
  • an input peptide with eight EpiMatrix hits has a Janus Homology Score of 1.
  • An input peptide with four EpiMatrix Hits has a Janus Homology Score of 2.
  • the JanusMatrix Homology Score considers all constituent 9-mers in any given peptide, including flanks. This peptide shows no class II HLA cross-conservation with the human proteome and insignificant class I HLA cross-conservation.
  • Figure ID is the JanusMatrix report for identified MHC class I clusters. * and ** are as per Figure 1C.
  • FIG. 2 shows elevated epitope-specific Thl cytokine production in HLA DR3 transgenic mice immunized with phage vaccine carrying H7N9 influenza effector CD4 + T cell epitopes.
  • Mice were primed with a plasmid DNA vaccine and boosted twice at two-week intervals with phage vaccine encoding H7N9 influenza class II HLA epitopes.
  • a matched group of control mice received vehicle (DNA and phage without epitopes).
  • splenic leukocytes were harvested and stimulated overnight with pools of vaccine-matched H7N9 HA and internal antigen peptides.
  • FIG. 1 shows ex vivo immune recall responses differentiate SARS-CoV-2 naive and experienced individuals and exhibit different COVID-19 immunotypes.
  • Figure 4 shows ex vivo immune recall responses differentiate SARS-CoV-2 naive and experienced individuals and exhibit different COVID-19 immunotypes.
  • Figure 5 demonstrates that strong ex vivo immune recall responses are found or may be found in SARS-CoV-2 experienced individuals using polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO; 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1).
  • polypeptides of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO; 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitope
  • Figure 6 demonstrates that strong ex vivo immune recall responses are found or may be found in SARS-CoV-2 experienced individuals using polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO; 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1).
  • polypeptides of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO; 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitope
  • Figure 7 shows polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate ex vivo immune recall response in natural SARS-CoV- 2 infection.
  • a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate
  • Figure 8 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate higher IFN-g responses in naive and COVID-19 convalescent donors following expansion in culture.
  • a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID
  • Figure 9 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate higher IFN-g responses in naive and COVID-19 convalescent donors following expansion in culture.
  • a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID
  • Figure 10 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors.
  • a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within
  • Figure 11 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors.
  • a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within
  • Figure 12 shows polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors.
  • a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within S
  • Figure 13 illustrates CD8+IFNy+ T-cell responses from mice immunized with a lipid vaccine including a plasmid housing SEQ ID NO: 1 for expression revealing that mice immunized with SEQ ID NO: 1 showed spike peptide (left side) or envelope/membrane peptide (right side) stimulation specific responses and demonstrating that following immunization both spike and envelope/membrane epitopes are sufficient to activate CD8 T-cells.
  • Figure 14 illustrates CD8+CD107a+ T-cell responses from mice immunized with a lipid vaccine including a plasmid housing SEQ ID NO: 1 for expression revealing that mice immunized with SEQ ID NO: 1 showed spike peptide (left side) or envelope/membrane peptide (right side) stimulation specific responses and demonstrating that following immunization both spike and envelope/membrane epitopes are sufficient to activate CD8 T-cells.
  • Figure 15 illustrates ability of cytolytic T lymphocytes from mice immunized with a lipid vaccine including a plasmid housing SEQ ID NO: 1 for expression to kill cells expressing either the concatemer peptide of SEQ ID NO: 1 or naturally known spike protein (Wuhan-Hu-1).
  • Figure 16 illustrates serum anti-spike antibody levels were measured using serum samples collected on day 21 following immunization with a lipid vaccine including a plasmid housing SEQ ID NO: 1 and demonstrating seroconversion and a dose dependent effect.
  • Figure 17 illustrates that the concatemer of SEQ ID NO: 1 at multiple doses tested in mice elicited potent neutralizing titers that were above titers found in SARS-CoV-2 convalescent patient samples.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. [0044] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order.
  • biological sample refers to any sample of tissue, cells, or secretions from an organism.
  • the term “medical condition” includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment and/or prevention is desirable, and includes previously and newly identified diseases and other disorders.
  • the term “immune response” refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, malignant melanoma, invading pathogens, cells or tissues infected with pathogens, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • B lymphocyte and T lymphocyte assays are well known, such as ELISA, cytotoxic T lymphocyte (CTL) assays, such as chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, and cytokine production assays.
  • CTL cytotoxic T lymphocyte
  • the term “effective amount”, “therapeutically effective amount”, or the like of a composition that is, includes or encodes such T-cell epitope compositions of the present disclosure is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount that results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated, such as SARS-CoV-2 infection or COVID-19 and/or related diseases or leads to a T-cell response in a subject above that of a non-treated control subject.
  • compositions of the present disclosure administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of pathogen and/or disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions of the present disclosure can also be administered in combination with each other or with one or more additional therapeutic compounds.
  • T-cell epitope means an MHC ligand or protein determinant, optionally 7 to 30 amino acids in length, and capable of specific binding to human leukocyte antigen (HLA) molecules and interacting with specific T-cell receptors (TCRs).
  • HLA human leukocyte antigen
  • TCRs T-cell receptors
  • T-cell epitopes are linear and do not express specific three-dimensional characteristics. T-cell epitopes are not affected by the presence of denaturing solvents.
  • T-cell epitopes may in the case of the present disclosure be predicted by in silico methods (De Groot AS et ah, (1997), AIDS Res Hum Retroviruses, 13(7):539-41; Schafer JR et al, (1998), Vaccine, 16( 19): 1880-4; De Groot AS et al, (2001), Vaccine, 19(31):4385-95; De Groot AR et al, (2003), Vaccine, 21(27-30):4486-504, all of which are herein incorporated by reference in their entirety.
  • T-cell epitope cluster refers to polypeptide that contains between about 4 to about 40 MHC binding motifs. In particular embodiments, the T-cell epitope cluster contains between about 5 to about 35 MHC binding motifs, between about 8 and about 30 MHC binding motifs; and between about 10 and 20 MHC binding motifs.
  • immune-stimulating T-cell epitope polypeptide refers to a molecule capable of inducing an immune response, e.g., a humoral, T cell-based, or innate immune response.
  • B-cell epitope means a protein determinant capable of specific binding to an antibody.
  • B-cell epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • MHC complex refers to a protein complex capable of binding with a specific repertoire of polypeptides known as HLA ligands and transporting said ligands to the cell surface.
  • MHC Ligand means a polypeptide capable of binding to one or more specific MHC alleles.
  • HLA ligand is interchangeable with the term “MHC Ligand”.
  • T-Cell Receptor or “TCR” refers to a protein complex expressed by T-cells that is capable of engaging a specific repertoire of MHC/Ligand complexes as presented on the surface of cells.
  • MHC Binding Motif refers to a pattern of amino acids in a protein sequence that predicts binding to a particular MHC allele.
  • subject refers to any living organism in which an immune response is elicited.
  • subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • polypeptide refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide.
  • a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
  • a polypeptide e.g., a polypeptide comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 53, SEQ ID NO: 65,
  • one or more T-cell epitopes of the present disclosure can be joined to, linked to, or inserted into another polypeptide wherein said one or more T-cell epitopes of the present disclosure is not naturally included in the polypeptide and/or said one or more T- cell epitopes of the present disclosure is not located at its natural position in the polypeptide.
  • a polypeptide When a polypeptide is recombinantly produced, it can also be substantially free of culture medium, for example, culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.
  • a “concatemeric” peptide or polypeptide refers to a series of at least two peptides or polypeptides linked together, optionally in a sequence not found in nature. Such linkages may in the form of “string-of-beads” design whereby each polypeptide is linked directly or via a linker to another, adjacent polypeptide of the same or differeing sequence.
  • a “variant” peptide or polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations, or a combination of any of these.
  • a variant peptide or polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these provided said variants retain MHC binding propensity and/or TCR specificity.
  • peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure can include, for example, modified forms of naturally occurring amino acids such as D-stereoisomers, non-naturally occurring amino acids; amino acid analogs; and mimetics.
  • peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure can include retro-inverso peptides of the instantly disclosed peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure, provided said peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure at least in part retain MHC binding propensity and/or TCR specificity.
  • the term “purpose built computer program” refers to a computer program designed to fulfill a specific purpose; typically to analyze a specific set of raw data and answer a specific scientific question.
  • z-score indicates how many standard deviations an element is from the mean.
  • Bacteriophage Provided herein are peptide, DNA, or RNA compositions that are or encode one or more T-cell epitopes as provided herein. Such may be used alone or as part of a delivery mechanism such as a naked peptide, DNA, or RNA molecule, or as part of a nanoparticle, viral particle, or other such delivery mechanism.
  • the present disclosure relates to bacteriophage (or “phage”) surface-expressed or surface-presented SARS-CoV-2 viral antigens (antigenic polypeptides), as well as to phage DNA vaccines comprised of nucleotides that encode the surface-expressed SARS-CoV-2 viral antigens and combinations thereof.
  • bacteriophage are presented hereing for exemplary purposes only and are not meant to be a limitation on the disclosure. It is equally appreciated that teaching related to a bacteriophage are equally attributable to other viral or non-viral organisms or delivery systems.
  • the bacteriophage vaccine system provides a well-tolerated and highly scalable system to immunize the general population against SARS-CoV-2 (and related diseases cause by SARS- Cov-2, including COVID-19) both effectively and inexpensively, with the ability to stimulate both cell mediated and humoral responses. Additional phage vaccine systems are described generally in US Patents 9,744,223 and 10,702,591, the contents of which are hereby incorporated by reference in their entirety.
  • the present disclosure in some aspects relates to bacteriophages that express and/or possess nucleic acids in nucleotide sequences encoding the concatemeric SARS-CoV-2 peptide(s) as set forth herein.
  • the phages express the concatemeric peptides as described herein.
  • cells or a subject express the concatemeri peptides as described herein.
  • the phages express the concatemer on its surface.
  • the phages express the concatemer on the surface via fusion to a phage surface expression polypeptide sequence that decorates to the outer capsid surface of the phage.
  • the present disclosure relates to nucleic acids encoding the concatemer described herein.
  • the nucleic acids are integrated into the phage’s genome.
  • the nucleic acids are operably linked to a nucleic acid sequence encoding part or all of a phage surface expression polypeptide sequence.
  • the instantly- disclosed phages are irradiated (e.g., after construction by prior to administration to a subject).
  • the concatemers of the present disclosure may provide T-cell epitopes to a host subject when expressed on the surface of a phage.
  • T-cell epitopes of the present disclosure are highly conserved among known variants of their source proteins (e.g., present in more than 10% of known variants).
  • T-cell epitopes of the present disclosure comprise at least one putative T-cell epitope as identified by EpiMatrixTM analysis.
  • EpiMatrixTM is a proprietary computer algorithm developed by EpiVax (Providence, Rhode Island), which is used to screen protein sequences for the presence of putative T-cell epitopes. The algorithm uses matrices for prediction of 9- and 10-mer peptides binding to MHC molecules.
  • Each matrix is based on position-specific coefficients related to amino acid binding affinities that are elucidated by a method similar to, but not identical to, the pocket profile method (Sturniolo, T. et al. , Nat. BiotechnoL, 17:555-561, 1999).
  • Input sequences are, for example, parsed into overlapping 9-mer frames or 10-mer where each frame overlaps the last by 8 or 9 amino acids, respectively.
  • Each of the resulting frames form the mutated peptide and the non-mutated peptide are then scored for predicted binding affinity with respect to MHC class I alleles (e.g., but not limited to, HLA-A and HLA-B alleles) and MHC class II alleles (e.g., but not limited to HLA-DRB1 alleles).
  • Raw scores are normalized against the scores of a large sample of randomly generated peptides.
  • the resulting “Z” scores are normally distributed and directly comparable across alleles.
  • the resulting “Z” score is reported.
  • any 9-mer or 10-mer peptide with an allele-specific EpiMatrixTM Z-score in excess of 1.64, theoretically the top 5% of any given sample is considered a putative T-cell epitope.
  • Peptides containing clusters of putative T-cell epitopes are more likely to test positive in validating in vitro and in vivo assays.
  • the results of the initial EpiMatrixTM analysis are further screened for the presence of putative T-cell epitope “clusters” using a second proprietary algorithm known as ClustimerTM algorithm.
  • the ClustimerTM algorithm identifies sub-regions contained within any given amino acid sequence that contains a statistically unusually high number of putative T cell epitopes.
  • Typical T-cell epitope “clusters” range from about 9 to roughly 30 amino acids in length and, considering their affinity to multiple alleles and across multiple 9-mer frames, can contain anywhere from about 4 to about 40 putative T-cell epitopes.
  • the JanusMatrix system (EpiVax, Buffalo, Rhode Island) is useful for screening peptide sequences for cross-conservation with a host proteome.
  • JanusMatrix is an algorithm that predicts the potential for cross-reactivity between peptide clusters and the host genome or proteome, based on conservation of TCR- facing residues in their putative MHC ligands.
  • the JanusMatrix algorithm first considers all the predicted epitopes contained within a given protein sequence and divides each predicted epitope into its constituent agretope and epitope. Each sequence is then screened against a database of host proteins.
  • Peptides with a compatible MHC-facing agretope i.e., the agretopes of both the input peptide and its host counterparty are predicted to bind the same MHC allele
  • the JanusMatrix Homology Score suggests a bias towards immune tolerance.
  • cross-conservation between autologous human epitopes and epitopes in the therapeutic may increase the likelihood that such a candidate will be tolerated by the human immune system.
  • cross-conservation between human epitopes and the antigenic epitopes may indicate that such a candidate utilizes immune camouflage, thereby evading the immune response and making for an ineffective vaccine.
  • the host is, for example, a human
  • the peptide clusters are screened against human genomes and proteomes, based on conservation of TCR-facing residues in their putative HLA ligands. The peptides are then scored using the JanusMatrix Homology Score.
  • peptides with a JanusMatrix Homology Score below 2.5 or below 3.0 indicate low tolerogenicity potential and may be useful for pharmaceutical formulations and vaccines for the treatment/prevention of SARS- CoV-2 infection and related diseases caused by SARS-CoV-2, including COVID-19, and in aspects may be included from the T cell epitope compositions and methods of the present disclosure.
  • peptides with a JanusMatrix Homology Score above 3.0 indicate high tolerogenicity potential and may not be useful for pharmaceutical formulations and vaccines for the treatment/prevention of SARS-CoV-2 infection and related diseases caused by SARS- CoV-2, including COVID-19, and in aspects may be excluded from the T cell epitope compositions and methods of the present disclosure.
  • T-cell epitopes of the present disclosure bind to at least one and preferably two or more common HLA class I and/or class II alleles with at least a moderate affinity (e.g., in aspects, ⁇ 1000 mM ICso, ⁇ 500 mM ICso , ⁇ 400 mM ICso, ⁇ 300 mM ICso, or ⁇ 200 mM ICso in HLA binding assays based on soluble HLA molecules).
  • a moderate affinity e.g., in aspects, ⁇ 1000 mM ICso, ⁇ 500 mM ICso , ⁇ 400 mM ICso, ⁇ 300 mM ICso, or ⁇ 200 mM ICso in HLA binding assays based on soluble HLA molecules.
  • T-cell epitopes of the present disclosure are capable of being presented at the cell surface by cells in the context of at least one and, in other aspects, two or more alleles of the HLA.
  • the epitope-HLA complex can be recognized by CD4+ or CD8+ T-cells (in aspects, including natural T-cells) having TCRs that are specific for the epitope-HLA complex and circulating in normal control subjects.
  • the recognition of the epitope-HLA complex can cause the matching T-cell to be activated and to secrete activating cytokines and chemokines.
  • Bacteriophage display provides a simple way, but not the only way provided in this disclosure, of achieving favorable presentation of peptides to a subject’s immune system.
  • Previous findings have revealed that recombinant bacteriophage can prime strong CD8+ T lymphocyte (CTL) responses both in vitro and in vivo against epitopes displayed in multiple copies on their surface, activate T-helper cells and elicit the production of specific antibodies all normally without adjuvant.
  • CTL CD8+ T lymphocyte
  • the instant disclosure provides a specific vaccine delivery system whereby relevant target antigenic concatemer peptides as disclosed herein are expressed within phage to produce recombinant phage displaying the antigenic concatemer on the phage virion surface (also referred to as “phage vaccine particles” or “PVPs”).
  • PVP vaccines produced by cloning the concatemer into a phage genome, can be propagated and purified rapidly using inexpensive Luria-Bertani (LB) bacteriological media.
  • phage display presents multiple copies of peptides on the same phage.
  • a further advantage is that once a first phage display is generated, subsequent production should be far easier and cheaper than the ongoing process of coupling peptides to carriers.
  • MHC major histocompatibility complex
  • phage display vaccines stimulate both cellular and humoral arms of the immune system (although as extracellular antigens, it is expected that the stronger response will be MHC class II biased and result in antibody production).
  • particulate antigens and phage in particular, can access the MHC I pathway through cross priming, and it is likely that it is this process that stimulates a cellular response.
  • Phage vaccines can also act as nonspecific immune stimulators. It is likely that a combination of the foreign DNA and the repeating peptide motif of the phage coat are responsible for any nonspecific immune stimulation (Adhya, S., C.R. Merril, and B. Biswas, Therapeutic and prophylactic applications of bacteriophage components in modern medicine. Cold Spring Harb Perspect Med, 2014. 4(1): p. a012518). Further, the peptide antigen displayed by the phage comes already covalently conjugated to an insoluble immunogenic carrier with natural adjuvant properties.
  • phage display vaccine immunogenic antigens (epitopes) are fused to the outer phage surface (i.e., capsid), typically by forming a chimera to a surface protein in the phage’s genome.
  • the displayed antigenic proteins or peptides can be selected for their specific binding affinity of antigen-presenting cells.
  • phage DNA vaccine foreign antigen genes are incorporated into the phage genome under the control of strong mammalian promoters.
  • the phage acts as a passive carrier to transfer the foreign DNA into mammalian cells (such as dendritic and Kupffer cells) when the antigen gene is expressed.
  • mammalian cells such as dendritic and Kupffer cells
  • both phage display and phage DNA vaccine platforms are utilized; the approach uses both phage that express the immunizing antigen on its surface (e.g., fusion antigen with its capsid protein) from the phage display approach and also incorporate foreign antigen genes into a phage to be then transduced into a host cell (antigen presenting cell “APC”) for expression.
  • APC antigen presenting cell
  • the surface expressed antigens are highly immunogenic and may provoke specific cell mediated responses in a subject, the result of which may provide long lasting immunity against SARS-CoV-2.
  • the present disclosure provides a rapidly producible and highly scalable vaccine to stop and contain the spread of this viral infection.
  • the compositions provided by the present disclosure further provide an opportunity to mitigate the possibility of antibody- dependent enhancement (ADE) related complications that may arise due to COVID-19 vaccination.
  • AD antibody- dependent enhancement
  • the present disclosure features, in some aspects, a phage display system to provide a SARS-CoV-2 vaccine through the presence of SARS-CoV-2 antigenic (T-cell epitope) polypeptides on the surface of the phage.
  • the phage system may display, or be capable thereof displaying, at least one antigenic epitope sequence (e.g., multiple copies).
  • the phage system may display between about one to about 900 copies of an antigenic epitope sequence, including about 2, 3,4 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 , 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, and 800 and any range or value therebetween.
  • the phage may display a polypeptide sequence of multiple SARS-CoV-2 epitopes.
  • the antigenic epitope sequence may feature a sequence of epitopes identified from the genome of the SARS-CoV-2 virus that are presented as linear epitopes to generate broad-spectrum immunity.
  • the polypeptide sequences can be expressed on the surface of a T4 phage and provided as an inoculant without the need of a further adjuvant, although in other aspects, such may be included.
  • the display of antigenic epitopes on a phage may provide unique vaccine nanoparticles (i.e. an antigenic peptide and phage combination chimera) that may provoke mammalian immune systems against SARS-CoV-2.
  • the copy number of the expressed concatemer may depend on the surface protein used as the chimera.
  • the concatemer in T4 phage, there are four different expressed head proteins that the concatemer can be co-expressed with: gp23, gp24, Hoc and Soc.
  • the copy numbers of each are 930, 55, 870 and 155, respectively.
  • the present disclosure provides a variety of possible levels of expression based on the phage protein selected.
  • a phage such as a T4 phage
  • the utilized antigenic polypeptides are specifically an aggregation of several linear epitopes (peptides) selected from all parts of the SARS-CoV-2 viral genomes to provoke broad-spectrum long lasting immunity.
  • simple spontaneous mutation in the viral spike proteins will not allow the SARS-CoV-2 virus to bypass the host’s immune system, a feature quite distinct from vaccines designed solely on the basis of spike protein sequence.
  • the present disclosure concerns use of a whole T4 phage.
  • Whole T4 phage particles possess numerous intrinsic characteristics that make them ideal as vaccine delivery vehicles.
  • T4 phage offers four surface proteins, gp23, gp24, Hoc, and Soc, with varying copy numbers capable of co-expressing the concatemer.
  • Bacteriophage expressing the concatemers disclosed herein may produce viral specific protective responses when administered to mammalian systems.
  • the particulate nature of phage means they are far easier and cheaper to purify than soluble recombinant proteins since a simple centrifugation/tangential flow filtration (TFF) /ultra-filtration steps are sufficient to remove the majority of soluble contaminants and associated toxins (mainly from the composition of bacterial cell walls). Therefore, this is very easy for scaling-up the phage based vaccine productions.
  • the vaccine production cost is much cheaper than conventional egg based or cell culture or whole cell inactivated vaccines.
  • the natural stability of phage vaccine particles provides easy storage and no cold chain is required for distribution.
  • the vaccine peptide antigens come already covalently attached to an insoluble immunogenic carrier (i.e. phage particles) with natural adjuvant properties, without the need for complex chemical conjugation and downstream purification processes that must be repeated with each vaccine batch.
  • the disclosed phage vaccines as provided herein do not require any additional adjuvant.
  • the present disclosure also in some aspects concerns the discovery of bacteriophage surface-expressed SARS-CoV-2 virus antigens that are highly immunogenic and specifically provoke cell-mediated response to neutralize the SARS-CoV-2 viral infection in human subjects.
  • the present disclosure provides for the development of a rapid vaccine to stop the epidemic related to SARS-CoV-2 virus infection by inducing cell-mediated immunity instead of simply humoral immunity.
  • a major advantage of the provided vaccine is to provoke a very strong long lasting cell mediated immunity by display of high copy numbers of SARS-CoV-2 virus specific selective linear epitopes as a T4 capsid protein fusions.
  • This specific presentation of linear epitopes on T4 phage head can skew the immunogenic response towards cell mediated response and as a result will reduce complications associated with antibody-dependent enhancement (ADE) related to SARS-CoV-2 vaccination.
  • ADE antibody-dependent enhancement
  • the major motivating reason for using the specific in silico systems described herein is that this approach allows for identification of conserved linear vaccine epitopes from the available sequence information of a given organism.
  • a phage based vaccine platform is also better suited for expressing linear epitopes (mainly provoked cell mediated responses) rather than conformational epitopes (provoke humoral/ antibody mediated responses).
  • the specific DNA sequences coding for the concatemer peptide sequence can be displayed on T4 phage using the protocol, for example, as described Zhu et ah, A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering bioRxiv, January 19, 2021 (herein incorporated by reference in its entirety).
  • engineered DNAs corresponding to the concatemer peptide sequence (or fragments thereof) can be incorporated into bacteriophage T4 genome as outlined in Figure 1 of Zhu et al. Each DNA is then introduced into E. coli as a donor plasmid and recombined into injected phage genome through CRISPR- targeted genome editing.
  • Different combinations of CoV-2 inserts can then be generated by simple phage infections and the recombinant phages in the progeny can be identified.
  • a pipeline of multiplex T4-concatemer peptide sequence and/or individual antigenic fragment vaccine phages can be rapidly constructed. Selected vaccine candidates can then be screened in a mouse model to identify the most potent vaccine.
  • the polypeptides or DNA or RNA nucleotide sequences that encode the polypetides may be delivered or presented by a lipid nanoparticle.
  • Lipid nanoparticles may include one or more ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest.
  • the lipid nanoparticles can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2021/029468; PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551 ; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.
  • LNPs may be used in some aspects to carry and/or deliver protein or nucleic acid cargo to a subject or a portion thereof such as a cell or cellular compartment.
  • DNA and RNA vaccines utilizing such LNPs share many similarities, but each target different cellular environments. For example, DNA vaccines target and are used in the nucleus of a cell, whereas RNA vaccines target and are expressed in the cytosol.
  • nucleic acid (DNA or RNA) compositions that are packaged in or on a lipid nanoparticle.
  • LNPs are typically an aquesou core that houses the nucleic acid of interest surrounded by a lipid bilayer of a combination of one or more lipids that may serve distinct functions as described by Li W, Szoka FC Jr, Pharm Res. 2007 Mar; 24(3):438- 49.
  • Cationic lipids and newer ionizable lipids may be used such as those described herein or as by Kanasty R, et al., Nat Mater. 2013 Nov; 12(11):967-77.
  • these LNPs provide unique advantages and allow alternative delivery systems for the peptides or DNA or RNA nucleic acids as described herein for presentation to a subject and development of immunity to SARS- CoV-2 as provided herein.
  • the polypeptides were designed and/or optimized using an in silico approach in combination with a proprietary algorithm.
  • a proprietary algorithm identified exclusive linear epitopes from an analysis of the complete genome sequence of SARS-CoV-2 virus.
  • the antigenic polypeptides are a concatemer of multiple identified SARS-CoV-2 epitopes.
  • the antigenic polypeptide is a concatemer of 2, 3, 4, 5, 6, 7 ,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 T-cell epitopes.
  • each T-cell epitope contemplated features between 9 and 10 amino acids in sequence.
  • the linear T-cell epitopes are then linked together as a concatemer to provide antigenic peptide sequences for generating broad spectrum immunity against SARS- CoV-2.
  • a unique long polypeptide is composed by assembly of various identified linear epitopes from SARS-CoV-2 virus. These epitopes were identified and selected from SARS-CoV-2 amino acid sequences (epitopes) in part based on the ability to interact with T-cells and thus provoke cell-mediated responses.
  • the T-cell epitope polypeptides of the present disclosure were designed by sequentially assembling identified HLA binding linear sequences into one or more unique polypeptide sequences. As disclosed herein in the examples, the resulting phage expressed a concatemer of 34 in silico identified SARS-CoV-2 epitopes assembled in a head-tail fashion to provide a single polypeptide (also referred to a concatemer). It will be apparent to those skilled in the art that the order of assembly can be rearranged and regrouped into further polypeptide sequences and placed as described herein in a phage vaccine system. For example, as set forth herein, 34 epitopes were identified using the proprietary in silico approach. Those skilled in the art will appreciate that each epitope can be expressed individually or as a doublet, triplet, quadruplet, quintuplet and so on, in any order and up to a single polypeptide of every of the identified epitopes.
  • the present disclosure concerns T-cell epitope polypeptide sequences of one or multiple SARS-CoV-2 epitope peptides.
  • SARS-CoV-2 envelope, membrane, and spike proteins can be analyzed for the presence of HLA class I and class II T cell epitopes. Specific regions where both HLA class I and class II T cell epitopes cluster can be thus identified and clusters with the highest predicted likelihood of human HLA class I and II binding, the broadest coverage of human HLA, highest SARS-CoV-2 conservation, and the lowest cross-conservation with the human proteome may then be selected.
  • a homology analysis tool may further eliminate sequences that could potentially elicit an undesired autoimmune or regulatory T cell response due to homology with the human genome.
  • a comprehensive description of the advanced set of tools used to identify such epitopes in silico can be further found in De Groot et ah, Better Epitope Discovery, Precision Immune Engineering, and Accelerated Vaccine Design Using Immunoinformatics Tools. Front Immunol. 2020 Apr 7;11 :442. doi: 10.3389/fimmu.2020.00442. PMID: 32318055; PMCID: PMC7154102. Resulting epitopes may then be concatenated head to tail and arranged in an order that minimizes potential T cell immunogenicity at epitope junctions.
  • the concatemer containing the identified 34 T-cell epitope clusters was selected for inclusion in the vaccine using advanced immunoinformatics tools.
  • Each epitope cluster is optioanlly 15 to 25 amino acids in length and contains multiple HLA class I and class II-restricted T-cell epitopes.
  • the 34 epitope clusters comprise hundreds of CTL and T helper epitopes.
  • the T-cell epitopes may be further selected to exclude human-like or related sequences.
  • the rationale for excluding human-like sequences is that T-cells that recognize antigen-derived epitopes sharing TCR contacts with epitopes derived from the human proteome may be deleted or rendered anergic before release into the periphery during thymic selection. Therefore, vaccine components targeting these T-cells may be ineffective.
  • vaccine-induced immune responses targeting cross-reactive epitopes may induce unwanted autoimmune responses targeting the human homologs of the cross-reactive epitopes identified by JanusMatrix. As a result, vaccine safety may be reduced.
  • the concatemer linear polypeptides of assembled SARS-CoV-2 antigenic peptides
  • the concatemer of up to 34 epitope clusters was designed. No class II junctional immunogenicity potential was found at the gpD/epitope interfaces.
  • the resulting vaccine collectively features highly conserved and promiscuous SARS-CoV-2-specific T cell epitopes that broadly cover HLA diversity of the human population.
  • the present disclosure concerns polypeptide sequences that are derived from the SARS-CoV-2 envelope, membrane, or spike proteins and designed for optimal expression and immunogenicity.
  • the genome of a phage can be modified as described herein to express the polypeptide, such that each T4 phage may express the 34 epitope clusters on the surface of the phage.
  • phage expressing the concatemer polypeptide can be mixed with other phage expressing other polypeptides to provide a stoichiometric phage- display cocktail, either in equimolar or non-equimolar ratios.
  • the concatemer polypeptide(s) of the present disclosure are set forth in the accompanying sequence listing and below.
  • the polypeptide includes an amino acid sequence of:
  • polypeptide includes the sequence of:
  • LNTDHSSSSDNIGSGSGLSYFIASFRLFARTRSMWSFNPETNILLNV (SEQ ID NO: 124).
  • the concatemers are 34 SARS-CoV-2 identified epitopes, assembled in a head-to fashion.
  • Table 1 sets out exemplary individual peptides, the protein from SARS-CoV-2 the peptide originates from (with starting amino acid) and the nucleic acids utilized in the constructs herein to encode such with Spike protein full sequence found at Genbank Accession No: YP 009724390.1, Envelope protein full sequence found at Genbank Accession No: YP 009724392.1, and Membrane protein full sequence found at Genbank Accession No: YP 009724393.1.
  • each of the polypeptide sequences are linear and continuous.
  • the polypeptides may include additional amino acids at either/or both terminals and/or internally within the sequences.
  • the polypeptides are fused to a surface phage protein, such as gp23, gp24, Hoc, and/or Soc in a T4 phage.
  • the epitope sequences of each concatemer are separated by a linker.
  • the linker may include an amino acid sequence of GPGPG (SEQ ID NO: 71).
  • each concatemer may include between 9, 10, 11, 12, or 13 epitope peptides.
  • the polypeptides introduced and/or expressed by the phage include variants of the sequences as set forth in SEQ ID NO: 1 or SEQ ID NO: 124.
  • the variants may share between about 80% to about 100% identity with the sequences as set forth in SEQ ID NO: 1, or SEQ ID NO: 124, or as within each of the above referenced T-cell epitopes contained in SEQ ID NO: 1 or SEQ ID NO: 124.
  • the variants may share 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the amino acid sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 124 or as any of the T-cell epitopes within SEQ ID NO: 1 or SEQ ID NO: 124.
  • the present disclosure also encompasses polypeptides (e.g., T-cell epitopes and T-cell epitope compositions as disclosed herein) having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide encoded by a nucleic acid molecule of the invention. Similarity is determined by conserved amino acid substitution or by deletion or insertion without significant change in physical characteristics of the polypeptide. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions or delection are likely to be phenotypically silent.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, Met, and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues His, Lys and Arg and replacements among the aromatic residues Trp, Phe and Tyr.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent are found (Bowie JU et al., (1990), Science, 247(4948):130610, which is herein incorporated by reference in its entirety).
  • T-cell epitopes of the present disclosure can include insertions, delections, allelic or sequence variants (“mutants”) or analogs thereof, or can include chemical modifications (e.g., pegylation, glycosylation).
  • a mutant retains the same functions performed by a polypeptide encoded by a nucleic acid molecule of the present disclosure, particularly MHC binding propensity and/or TCR (T-cell receptor) specificity.
  • a mutant can provide for enhanced binding to MHC molecules.
  • a mutant can lead to enhanced binding to TCRs.
  • a mutant can lead to a decrease in binding to MHC molecules and/or TCRs.
  • sequence variations that may be included in a peptide as provided herein are listed in Table 2. These peptides variants are also presented with a frequency percentage number representing the preference for selection of that particular mutation. A frequency percentage of 5% or greater represents most preferred sequence variations.
  • Table 2 the reference sequences are as listed in Table 1 with the start reference number relative to the full length SARS-CoV-2 original reference sequence and the N-terminal residue in the sequence being the number of the start reference amino acid. Variants with a dash are deletions of the reference amino acid residue. Amino acids that are more commonly mutated (e.g. more listed variants of that residue) or deleted in the listing in Table 2 are the most tolerant of mutation and maintenance of function.
  • Variants in SEQ ID NO: 1 or SEQ ID NO: 124 are optionally found in one or more of the individual T-cell epitopes of Table 1.
  • the number of T-cell epitopes with a variant include but are not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or all 34 T-cell epitopes have a variant, and optionally any of the listed spacers.
  • the number of variant amino acids (substitution, deletion, insertion) within each variant is optionally 6 or fewer, optionally 5 or fewer, optionally 4 or fewer, optionally 3 or fewer, optionally 2 or fewer, optionally 1, optionally zero.
  • a variant T-cell epitope within SEQ ID NO: 1 or SEQ ID NO: 124 may be any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 53, 65, 67, or 69, or any combination thereof, or in one or more linkers optionally of SEQ ID NO: 71, or both.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 47, optionally at residue T9.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 61, optionally at residue Q19.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 51, optionally at residue A63.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 37, optionally at residue P26.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 27, optionally at any one or more of residues H69, V70, A67, or delections at H69 and V70, or a substitution at A67 with deletions at H69 and V70.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 19, optionally at residue T95.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 33, optionally at one or more of residues G142, E156, F157, R158, Y144, G142, V143, and Y145, or any combination thereof.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 25, optionally at any one of residues N211 or L212, or both.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 65, optionally at any one of residues G339, R346, S371, S373, S375, or any combination therereof.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 45, optionally at any one of residues L452, G446, or any combination therereof.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 31, optionally at any one of residues N501, E484, Q493, G496, Q498, Y505, or any combination therereof.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 23, optionally at residue T547.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 15, optionally at residue T716.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 53, optionally at residue N764.
  • a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 55, optionally at residue VI 176.
  • the present disclosure further includes nucleic acids encoding the concatemer polypeptides derived from SARS-CoV-2 optionally encoding SEQ ID NO: 1 or SEQ ID NO: 124 or any T-cell epitope or linker therein.
  • nucleic acid sequences may be derived from a SARS-CoV-2 genome or synthesized based on the desired amino acid sequence with appropriate codon sequences assembled to provide such.
  • nucleic acids encoding the concatemer polypeptide may be introduced into the genome of a T4 phage.
  • the nucleic acids may be packaged into a plasmid or other nucleotide sequence for delivery by a lipid particle.
  • nucleotide sequence(s) that are able to generate that protein sequence.
  • nucleic acid sequences encoding the amino acid sequences as set forth herein are readily apparent to those skilled in the art based on the codon coding options known in the art.
  • Ala residues can be encoded by a DNA sequence of GCT, GCC, GCA or GCG
  • Phe residues can be encoded by TTT or TTC
  • Ser residues by TCT, TCC, TCA, TCG, AGT or AGG Tyr residues by TAT or TAC
  • Trp by TGG Leu by CTT, CTC, CTA, or CTG, lie by ATT, ATC or ATA, Met by ATG, Val by GTT, GTC, GTA or GTG, Gly by GGT, GGC, GGA or GGG, Asp by GAT or GAC, Glu by GAA or GAG, Lys by AAA or AAG, Thr by ACT, ACC, ACA or ACG
  • Pro by CCT, CCC, CCA or CCG His by CAT or CAC, Gin by CAA or CAG, Asn by AAT or AAC, Arg by AGA, AGG, CGG, CGA, CGC or CGT, Cys by TGT or TGC and STOP
  • nucleotide sequence that may be included in the genome of a phage to produce upon transcription and translation the concatemers peptide of SEQ ID NO: 1 or any variant thereof is well understood in the art and explicitly understood as presented within this specification for any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 53, 65, 67, or 69, or any variant thereof, or any combination thereof, or in one or more linkers optionally of SEQ ID NO: 71, or both.
  • nucleic acids encoding the antigenic concatemer polypeptides may be fused with nucleic acids encoding additional polypeptides or polypeptide sequences, including by a linker encoding polypeptide such as GPGPG (SEQ ID NO: 71), AAY, or a spacer.
  • a linker encoding polypeptide such as GPGPG (SEQ ID NO: 71), AAY, or a spacer.
  • Nucleic acid sequences encoding the amino acid sequences as set forth herein are readily apparent to those skilled in the art based on the codon coding options known in the art.
  • Ala residues can be encoded by a DNA sequence of GCT, GCC, GCA or GCG
  • Phe residues can be encoded by TTT or TTC
  • Ser residues by TCT, TCC, TCA, TCG, AGT or AGG Tyr residues by TAT or TAC
  • Trp by TGG Leu by CTT, CTC, CTA, or CTG, lie by ATT, ATC or ATA, Met by ATG, Val by GTT, GTC, GTA or GTG, Gly by GGT, GGC, GGA or GGG, Asp by GAT or GAC, Glu by GAA or GAG, Lys by AAA or AAG, Thr by ACT, ACC, ACA or ACG
  • Pro by CCT, CCC, CCA or CCG His by CAT or CAC, Gin by CAA or CA
  • the nucleic acid sequences may encode the antigenic concatemer polypeptide fused to a further polypeptide sequence.
  • the nucleic acids encode a fusion or chimera of an antigenic concatemer polypeptide and an additional sequence, typically fused at the amino or carboxyl termini.
  • the additional sequence may include a spacer and/or a surface expression polypeptide sequence.
  • the co-expressed surface expression polypeptide sequence allows for the display of the antigenic polypeptide on the surface of the phage, such as a structural or decorative protein on the capsid of the phage.
  • a T4 phage the presence of a gp23, gp24, Hoc, and/or Soc of phage T4 is fused at the amino terminus of the antigenic polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO: 124.
  • the nucleic acids encoding the antigenic chimera include a surface display sequence and/or a restriction enzyme site.
  • the nucleic acids encode a capsid head protein, such as gp23, gp24, Hoc, and/or Soc, fused to the SARS-CoV-2 derived antigenic concatemer polypeptide with further engineered restriction sites for ligation into the genome of a T4 phage.
  • the concatemer polypeptide is encoded by the nucleic acid sequence as follows:
  • AACGTTTAA SEQ ID NO: 2 (the stop TAA in bold) or
  • the nucleic acids may include variants of the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 125.
  • the variants may share between about 80% to about 100% identity with the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 125.
  • the variants may share 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 125.
  • other concatemers are envisioned.
  • Any one or more of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 70 may be arranged in any order, including as one continuous concatemer, or as two or more concatemers, or optionally as separated by a linker.
  • the nucleic acids include a 5’ nucleic acid sequence that add additional amino acids between the head protein of T4 phage and the antigenic concatemer polypeptide.
  • the instant disclosure is directed to a nucleic acid (e.g., DNA or RNA, including mRNA) encoding one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as described herein.
  • a nucleic acid e.g., DNA or RNA, including mRNA
  • the activation or promoter sequence of the surface protein drives the production and expression of the concatemer.
  • the nucleic acids encoding the concatemer may be under the control of a eukaryotic promoter, such as CMV, SV40, CAG, U6 etc. Such a promoter may be introduced by cloning inside a nonessential region of a phage genome to produce the phage based DNA vaccine.
  • this phage vaccine when presented in a mammalian system, such as by injection, this phage vaccine may act as a DNA vaccine and induce potent immune response by expressing foreign antigen inside of antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • the array of the linear epitopes of the construct i.e. SEQ ID NO: 2
  • SEQ ID NO: 2 can be cloned inside the T4 phage genome under a strong eukaryotic promoter. This may allow for optimum expression of antigenic epitopes inside APCs like macrophages, dendritic cells, Langerhans cells and Kupffer cells to provoke strong immunogenic response against SARS-CoV-2.
  • a nucleotide sequence may be in the form of DNA or RNA.
  • a nucleotide sequence encoding a SARS-CoV-2 T-cell epitope or concatemer of two or more such epitopes may include mRNA.
  • mRNA is understood in the art as a single- stranded ribonucleic acid copy of a gene, including pre-mRNA and mature mRNA, a spliced mRNA, a 5’ capped mRNA, an edited mRNA and a polyadenylated mRNA.
  • mRNA can include single stranded RNA nucleotide sequences encoding one or more T-cell epitopes as provided herein and optionally marked by a 5’ cap, such as an RNA 7-methylguanosine cap or an RNA m 7 G cap.
  • An mRNA encoding one or more T-cell epitopes as provided herein may include a start codon of the trimer ATG sequence of bases toward the 5’ end of the molecule to signify the initiation for translation of the mRNA segment of interest to a protein and may further include a stop codon of UAA, UAG or UGA that is in frame with the start codon to signify the end of the coding region or the point at which translation is to cease.
  • An mRNA may further include an untranslated region (UTR) following a stop codon and can further include a polyadenylated (poly A) tail after the 3’ untranslated region (UTR) of the single stranded molecule.
  • a polyA tail can be provided by the template DNA or by the use of a polyA polymerase. Those skilled in the art will appreciate that the exact length of adenosine in the poly A tail need not be exact but may generally fall within the range of about 100 to about 200 adenosine residues.
  • the mRNA may be optimized to avoid a double-stranded secondary or tertiary structures and/or purified to remove any double-stranded variants (see, e.g., Kariko et al. Nucleic Acids Res. 39: el42 (2011)).
  • mRNA sequences that encode one or more SARS-CoV-2 T-cell epitopes as provided herein, optionally encoding one or more of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
  • an mRNA includes a sequence of ribonucleic acids that are 80% or greater identical to one or more of the following where G is guanine, A is adenine, U is uracil, and C is cytosine: GGGAAAGGCUAU C ACUUAAU GAGCUUUCCUC AGU CU GCGCCUC AC (SEQ ID NO: 88) encoding Spike 1044;
  • AAC AAGU GU GUUAAUUUUAAUUU C AACGGGUUAAC AGGGACGGGGGU GUU GA CAGAA (SEQ ID NO: 98) encoding Spike 536;
  • ACU C AAU C AUUACUGAUCGU GAAC A AU GCC ACUAAU GUU GU C AUUAAAGUA (SEQ ID NO: 108) encoding Spike 114;
  • ACUU C AAAUUUUAG AGUU C
  • AGCCUAC AGAGAGC AUU GUACGUUU C (SEQ ID NO: 120) encoding Spike 315;
  • a mRNA sequence is a concatemer of 2 or more of SEQ ID NOs: 88-123.
  • a mRNA sequence is a concatemer of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 of any of SEQ ID NOs: 88-123 in any order.
  • a mRNA is a sequence including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 of any of SEQ ID NOs: 88-123 in any order.
  • a mRNA is a concatemer of all of SEQ ID NOs: 88-123 in any order, optionally in the order of SEQ ID NO: 88 to SEQ ID NO: 124.
  • an mRNA sequence comprises
  • a nucleic acid encoding one or more T-cell epitopes or polypeptides as provided herein may be packaged for delivery to a subject with one or more ionic lipids.
  • lipid nanoparticles (LNPs) or lipid-like nanoparticles (LLNs) are synthesized to contain at least one nucleotide sequence (e.g. mRNA or DNA) contained therein.
  • a “lipid particle” is defined as a single or double layer membrane defining a particle. It is appreciated that the lipid particle is formed of one or more ionic lipids.
  • ionic lipids include phosphtidyl serine (PS), phosphatidyl choline (PC), protamine, cholesterol, or polysaccharide, among others.
  • PS phosphtidyl serine
  • PC phosphatidyl choline
  • protamine protamine
  • cholesterol cholesterol
  • polysaccharide polysaccharide
  • the nucleic acid or plasmid or other expression construct including the nucleotide sequence is optionally encapsulated within an LNP or LLN of two or more lipids, such as three, four, five or more.
  • Ionic lipids as provided herein may optionally include three sections including an amine head, a linker and a hydrophobic tail thereby defining general overall structure of exemplary ionic lipids. More specific examples of ionic lipids include but are not limited to heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), DLinDMA, and DLin-KC2-DMA).
  • the LNP may include an ionic lipid, a polyethylene glycol and a cholesterol.
  • the LNP may include a combination of an ionic lipid with polyethylene glycol (PEG), cholesterol and/or distearoyl phosphocholine.
  • PEG polyethylene glycol
  • Specific constructs of LNPs suitable for use may be found in: Pardi, N, et ah, Nat Rev Drug Discov, 17, 261-279 (2016) (and references cited therein); Sabnis et ah, Mol. Ther. 26: 1509- 1519 (2016); Pardi et al. J. Exp. Med. 215:1571-1588 (2016); and, Pardi et al. J. Control. Release 217: 345-351 (2015)).
  • a nanoparticle housing or bound to a nucleotide sequence as provided herein further includes one or more helper lipids.
  • a helper lipid may include l,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and/or dioleoylphosphatidylethanolamine (DOPE) and/or lipofectamine and/or dioleoylphosphatidylcholine (DOPC) and/or phosphatidylethanolamine (dioleoyl PE) and/or 3p-[N-(N’,N’-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) (see, e.g., Du et al. Scientific Reports 4: 7107 (2014) and Cheng et al. Advanced Drug Delivery Reviews 99(A): 129-137 (2016)).
  • DC-Chol 3p-[N-(N’,N’-dimethylaminoethane)
  • the present disclosure provides chimeric or fusion polypeptide compositions (which in aspects may be isolated, synthetic, or recombinant) wherein one or more of the instantly-disclosed T-cell epitopes is a part thereof, such as a fusion of one or more of the concatemers disclosed herein with a T4 phage secreted peptide including gp23, gp24, Hoc and/or Soc.
  • a chimeric or fusion polypeptide composition comprises one or more polypeptides of the present disclosure joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide.
  • heterologous polypeptide is intended to mean that the one or more T-cell epitopes of the instant disclosure are heterologous to, or not included naturally, in the heterologous polypeptide.
  • one or more of the instantly-disclosed polypeptides may be inserted into the heterologous polypeptide (e.g., through recombinant techniques, mutagenesis, or other known means in the art), may be added to the C-terminus (with or without the use of linkers, as is known in the art), and/or added to the N-terminus (with or without the use of linkers, as is known in the art) of the heterologous polypeptide.
  • protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on.
  • chimeric or fusion polypeptides comprise one or more of the instantly-disclosed polypeptides operatively linked to a heterologous polypeptide. “Operatively linked” indicates that the one or more of the instantly-disclosed polypeptides and the heterologous polypeptide are fused in-frame or chemically-linked or otherwise bound.
  • the one or more polypeptides of the present disclosure have a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124).
  • the one or more peptides or polypeptides of the instant disclosure may be joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide as a whole, although it may be made up from a joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted amino acid sequence, together
  • a chimeric or fusion polypeptide composition comprises a polypeptide of the instant disclosure (which, in aspects, may be an isolated, synthetic, or recombinant) having a sequence comprising one or more of SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124), wherein said one or more of SEQ ID NO: 1 or SEQ ID NO: 124 is not naturally included in the polypeptide and/or said of one or more of SEQ ID NO: 1 or SEQ ID NO: 124 is not located at its natural position in the polypeptide.
  • the one or more peptide or polypeptides of the present disclosure can be joined, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into the heterologous polypeptide.
  • chimeric or fusion polypeptide compositions comprise one or more of the instantly-disclosed T-cell epitopes operatively linked to a heterologous polypeptide having an amino acid sequence not substantially homologous to the T-cell epitope.
  • the chimeric or fusion polypeptide does not affect function of the T-cell epitope per se.
  • the fusion polypeptide can be a GST-fusion polypeptide in which the T-cell epitope sequences are fused to the C-terminus of the GST sequences.
  • Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions.
  • Such fusion polypeptides, particularly poly-His fusions or affinity tag fusions can facilitate the purification of recombinant polypeptide.
  • host cells e.g.
  • the chimeric or fusion polypeptide contains a heterologous signal sequence at its N-terminus.
  • the heterologous polypeptide or polypeptide comprises a biologically active molecule.
  • the biologically active molecule is selected from the group consisting of an immunogenic molecule, a T cell epitope, a viral protein, and a bacterial protein.
  • the one or more peptides or polypeptides of the instant disclosure can be joined or linked to (e.g., fused in-frame, chemically-linked, or otherwise bound) a small molecule, drug, or drug fragment.
  • one or more peptides or polypeptides of the instant disclosure can be joined or linked to (e.g., fused in- frame, chemically-linked, or otherwise bound) a drug or drug fragment that is binds with high affinity to defined SLAs.
  • the chimeric or fusion polypeptide compositions can be recombinant, isolated, and/or synthetic.
  • a chimeric or fusion polypeptide composition can be produced by standard recombinant DNA or RNA techniques as are known in the art. For example, DNA or RNA fragments coding for the different polypeptide sequences may be ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR polymerase chain reaction
  • anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence
  • one or more polypeptides of the present disclosure can be inserted into a heterologous polypeptide or inserted into a non-naturally occurring position of a polypeptide through recombinant techniques, synthetic polymerization techniques, mutagenesis, or other standard techniques known in the art.
  • protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et ah, 2012, Protein Engineering Methods and Applications, which are herein incorporated by reference in their entirety).
  • fusion moiety e.g., a GST protein
  • a nucleic acid molecule encoding a T-cell epitope of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the at least one T-cell epitope.
  • the present disclosure further provides for vaccine systems of phage that encode the antigenic chimera protein and/or phage that express/display the antigenic fusion proteins, referred to also as phage DNA and phage display.
  • the vaccine systems include a phage with a genome altered such that it is capable of expressing (displaying) the antigenic polypeptides derived from SARS- CoV-2 as set forth herein.
  • the sequences as set forth in SEQ ID NO: 2 or SEQ ID NO: 125, or of any other combination of nucleic acids as provided herein can be introduced into a plasmid DNA construct.
  • the nucleic acid sequences are fused (in frame) at the 3’ end of a surface phage protein, such as gp23, gp24, Hoc and/or Soc of T4 phage.
  • a surface phage protein such as gp23, gp24, Hoc and/or Soc of T4 phage.
  • the resulting plasmid construct can then be introduced or transformed into a bacterial cell, such as E. coli, a recombinase expressing bacterial cell, to allow for the introduction of the gp23/gp24/Hoc/Soc- antigenic fused nucleotide sequence in to the phage genome.
  • the transformed bacteria can then be infected with a T4 phage.
  • the plasmid and the phage may further contain recombination sites for integration into the phage genome, such as Lox recombination domains, thereby allowing for production of phage with the antigenic encoding nucleic acids and phage expressing the antigenic polypeptides.
  • recombination sites for integration into the phage genome, such as Lox recombination domains, thereby allowing for production of phage with the antigenic encoding nucleic acids and phage expressing the antigenic polypeptides.
  • Cre recombination systems than Cre can be used, such as FLP, R, Lambda, HK101, and pSAM2.
  • the vaccine product is composed of the recombinant phage vaccine component bearing the desired epitopes identified by the in silico analysis.
  • the collective display of the original epitopes with subsequent modified sequences are set forth in SEQ ID NO: 1 or SEQ ID NO: 124 or variants thereof, with a nucleic acid encoding such in SEQ ID NO: 2 or SEQ ID NO: 125 or variants thereof.
  • a T4 phage is selected for phage display.
  • a T4 phage can express large polypeptides on its surface and further provides straightforward and efficient cloning efficiency.
  • a T4 phage further provides a vehicle for display of a high copy number of the antigenic polypeptide, typically displaying hundreds of copies per phage.
  • the phage vaccine may be a DNA vaccine, to be used either alone or in combination with a phage display vaccine.
  • a phage DNA vaccine may be generated using the nucleic acids encoding the concatemer of collected linear epitopes.
  • the antigen genes may be under the control of a eukaryotic promoter, such as CMV, SV40, CAG, U6 etc. Such a promoter may be introduced by cloning inside a nonessential region of a phage genome to produce the phage based DNA vaccine.
  • this phage vaccine when presented in a mammalian system, such as by injection, this phage vaccine may act as a DNA vaccine and induce potent immune response by expressing foreign antigen inside of antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • the array of the linear epitopes of the construct set forth in SEQ ID NO: 2 or SEQ ID NO: 125 can be cloned inside the T4 phage genome under a strong eukaryotic promoter. This may allow for optimum expression of antigenic epitopes inside APCs like macrophages, dendritic cells, Langerhans cells and Kupffer cells to provoke strong immunogenic response against SARS-CoV-2.
  • the phage of the vaccine product/system is irradiated (e.g., after construction by prior to administration to a subject).
  • the term “vaccine” as used herein includes an agent that may be used to cause, stimulate or amplify the immune system of animals (e.g., humans) against a pathogen. Vaccines of the invention are able to cause or stimulate or amplify immunity against a SARS-CoV-2 infection.
  • the term “immunization” includes the process of delivering an immunogen to a subject. Immunization may, for example, enable a continuing high level of antibody and/or cellular response in which T-lymphocytes can kill or suppress the pathogen in the immunized subject, such as human, which is directed against a pathogen or antigen to which the subject has been exposed.
  • Vaccines of the present disclosure comprise an immunologically effective amount of a T-cell epitope composition (including one or more of e.g., phage display or polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T- cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed as disclosed
  • animals may become at least partially or completely immune to SARS- CoV-2, SARS, MERS or similar coronavirus infections, or resistant to developing moderate or severe SARS-CoV-2 infections and/or SARS-CoV-2 related diseases.
  • the instantly-disclosed SARS-CoV-2 vaccines may be used to elicit a humoral and/or a cellular response, including CD4+ and CD8+ T effector cell responses.
  • SARS-CoV-2 infections or associated diseases include, for example, COVID-19.
  • a human is protected to an extent to which one to all of the adverse physiological symptoms or effects of SARS-CoV-2, SARS, MERS or similar coronavirus infections are significantly reduced, ameliorated or prevented.
  • an immunologically effective dose may vary from subject to subject depending on factors such as the age and general condition of the subject, the nature of the formulation and the mode of administration.
  • An appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. For instance, methods are known in the art for determining or titrating suitable dosages of a vaccine to find minimal effective dosages based on the age, weight of the animal subject, species (if non- human), medical condition of the subject to be treated, concentration of the vaccine, and other typical factors.
  • the vaccine comprises a unitary dose of between 0.1-50 micrograms (pg), preferably between 0.1 and 25, even more preferably of between 1 and 15 pg, typically approx. 10 pg, of phage display, phage DNA, polypeptide or nucleic acid antigen of the invention.
  • the dosage of the vaccine, concentration of components therein and timing of administering the vaccine, which elicit a suitable immune response can be determined by methods such as by antibody titrations of sera, e.g., by ELISA and/or seroneutralization assay analysis and/or by vaccination challenge evaluation.
  • the vaccine comprises a T cell epitope composition (including one or more of e.g., phage display or polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 124 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptides, or chimeric of
  • the vaccine comprises a nucleic acid as defined above, optionally in combination with any suitable excipient, carrier, adjuvant, and/or additional protein antigen.
  • the vaccine comprises a viral vector or phage containing a nucleic acid as defined above.
  • the vaccine comprises one or more plasmid vectors or phage genomes.
  • the one or more plasmid vectors or phage genome contain a nucleic acid sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or any one or more of SEQ ID NOs: 88-123, or variants or fragments thereof.
  • the vaccine compositions are in aspects designated for administration to human subjects to provide an immune response, ideally a protective immune response. It will be appreciated that the vaccine compositions may be most effective by coming into contact with a human subject’s immune system. It will be appreciated by those skilled in the art that the phage display and phage DNA vaccines, LNP vaccines, mRNA vaccines, or peptide antiben vaccines may be prepared with additional excipients, adjuvants and pharmaceutically acceptable carriers based on the route of administration desired. Such are described in further detail in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 22nd Ed., 2012; and Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, 10th Ed., Philadelphia, PA, 2013.
  • compositions described herein are in some aspects referred to as vaccines. It will be appreciated by those skilled in the art that reference to such refers to stimulating or provoking or providing an immune response within a subject that receives the phage-based compositions described herein.
  • the vaccine compositions may provide an innate immune response, a humoral immune response, a cell-mediated immune response or combinations thereof.
  • the compositions may, in some aspects, provide immunity to a subject from exposure to SARS-CoV-2. In other aspects, the compositions may provide partial immunity.
  • compositions may provide varying lengths of an immune response, and such responses may further vary between individual subjects.
  • the compositions may provide temporary immunity or partial immunity.
  • the compositions may provide long-lasting immunity or partial immunity. It will be appreciated by those skilled in the art, therefore, that reference to the compositions as vaccines refers generally to provoking, stimulating or providing an immune response, the level of which may vary.
  • the vaccine compositions feature varying combinations and concentrations of the three identified phage constructs.
  • the vaccine phage construct may be mixed with other SARS-CoV-2 antigenic compounds in equimolar concentration (same proportion) to produce a mixed vaccine for generating strong immunogenic response against SARS-COV-2.
  • the vaccine construct could be mixed in a non-equimolar (different proportions) concentrations to achieve maximum immunogenicity against SARS-CoV-2.
  • the vaccine construct can be mixed and matched accordingly for preparation of final vaccine constituents.
  • phage based SARS-CoV-2 phage DNA vaccines can be altered on the surface to display specific peptides to enhance the binding affinity of phage particles to APCs for accelerating the uptake of phage DNA vaccines by APCs.
  • the phage based DNA vaccines could be decorated with selective linear peptides that may enhance the binding affinity of phage vaccine particles with epithelial cells for proper absorptions to enhance the efficacy of above described vaccination processes.
  • compositions with alternate presentation of vaccine particles or protein components to enhance immunogenic response in mammalian systems could be mixed with adjuvant like aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate to increase the “depot effect” during intramuscular route of vaccinations.
  • adjuvant like aluminum salts such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate to increase the “depot effect” during intramuscular route of vaccinations.
  • Depot effects may be essential for slow diffusion of phage vaccines from the site of inoculation to attract APCs for generating optimum immunogenic response of phage vaccination.
  • compositions to SARS-CoV-2 can include Bacillus calmette- guerin (BCG) to increase the adjuvant effect of phage, as well as provoking very strong cell mediated responses.
  • BCG Bacillus calmette- guerin
  • the present disclosure further considers microencapsulation of the compositions.
  • Microencapsulation of vaccine particles using biodegradable polymer microspheres may also increase the stability and immunogenicity of phage vaccines during transportation in austere locations.
  • Microencapsulation of phage vaccine particles may also help to prevent proteolytic disintegration of phage vaccine particles during long storage.
  • vaccine compositions can be specifically designed for intradermal deposition of vaccines, such as with microneedles.
  • Intradermal inoculation route for SARS- CoV-2 vaccine using microneedle is highly desirable to enhance immune response, resulting in a potential reduction of the antigen dose, decreased anxiety and pain.
  • Microneedles can be engineered as small patches to deliver vaccines without any complications.
  • the SARS-CoV-2 vaccine constructs including concatemers of putative T-cell epitopes having an amino acid sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124, and nucleic acids (e.g., RNA mRNA, DNA, cDNA) encoding such concatemeric peptides; and chimeric or fusion polypeptide compositions as disclosed herein.
  • nucleic acids e.g., RNA mRNA, DNA, cDNA
  • the vaccine compositions may initiate a strong T-cell mediated immune response but may not always induce a humoral immune response. Therefore, aspects of a vaccine against SARS-CoV-2, SARS, MERS or other similar coronavirus contains a combination of the putative T-cell epitopes together with either live attenuated virus (LAV) or inactivated virus.
  • This vaccine composition (including both the putative T-cell epitopes and an LAV or inactivated virus) upon administration to a subject may induce both cellular and humoral immune responses, thereby conferring comprehensive immunity against SARS-CoV- 2, SARS, MERS or other similar coronavirus.
  • Vaccine compositions may comprise other ingredients, known per se by one of ordinary skill in the art, such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze drying stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives, depending on the route of administration.
  • pharmaceutically acceptable carriers such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze drying stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives, depending on the route of administration.
  • Examples of pharmaceutically acceptable carriers, excipients or diluents include, but are not limited to: demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as light liquid paraffin oil, or heavy liquid paraffin oil; squalene; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium salt, or hydroxypropyl methylcellulose; lower alkanols, for example ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene
  • the carrier or carriers will form from 10% to 99.9% by weight of the vaccine composition and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.
  • Adjuvants suitable for use with the compositions and vaccines of the instant disclosure are familiar to one of skill in the art and are available from a variety of commercial vendors.
  • adjuvants include, but are not limited to, oil in water emulsions, aluminum hydroxide (alum), immunostimulating complexes, non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-a, IFN-b, IFN-g, etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP) and the like.
  • MFA monophosphoryl lipid A
  • MDP muramyl dipeptides
  • Suitable adjuvants include, for example, aluminum potassium sulfate, heat-labile or heat-stable enterotoxin(s) isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc.
  • Toxin- based adjuvants such as diphtheria toxin, tetanus toxin, cholera toxin, and pertussis toxin which may be inactivated prior to use, for example, by treatment with formaldehyde.
  • Further adjuvants include, for example,: glycolipids; chemokines; compounds that induce the production of cytokines and chemokines; interferons; inert carriers such as alum, bentonite, latex, and cyclic particles; pluronic block polymers; depot formers; surface active materials such as saponin, lysolecithin, retinal, liposomes, and pluronic polymer formulations; macrophage stimulators such as bacterial lipopolysaccharide; alternative pathway complement activators such as insulin, zymosan, endotoxin, and levamisole; non-ionic surfactants; poly(oxyethylen)-poly(oxypropylene) tri-block copolymers; trehalsoe dimycolate (TDM); cell wall skeleton (CWS); macrophage colony stimulating factor (M-CSF); tumor necrosis factor (TNF); 3-O-deacylated MPL; CpG oligonucleotides; poly
  • freeze-drying stabilizer may be for example carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, and derivatives thereof.
  • the vaccine compositions of the instant disclosure may be liquid formulations such as an aqueous solution, water-in-oil or oil-in-water emulsion, syrup, an elixir, a tincture, or a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.
  • Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems.
  • Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents.
  • the route of administration can be oral, sublingual, intranasal, transdermal (i.e., applied on or at the skin surface for systemic absorption), ocular, percutaneous, via mucosal administration, or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal).
  • Vaccine compositions according to the present disclosure may be administered alone, or can be co-administered or sequentially administered with other treatments or therapies.
  • the dosage of the vaccines of the present invention will depend on various factors such as the age, size, vaccination history, and health status of the subject to be vaccinated, as well as the route of administration.
  • the vaccines of the instant disclosure can be administered as single doses or in repeated doses.
  • the vaccines of the instant disclosure can be administered alone, or can be administered simultaneously or sequentially administered with one or more further compositions, or vaccine compositions. Where the compositions are administered at different times, the administrations may be separate from one another or overlapping in time.
  • the vaccine compositions of the present disclosure are administered to a subject susceptible to or otherwise at risk for SARS-CoV-2, SARS, MERS or similar coronavirus infection to enhance the subject’s own immune response capabilities.
  • the present disclosure also includes pharmaceutically acceptable salts of the T-cell epitope compositions (including one or more of e.g., phage display, phage DNA, peptides or polypeptides as disclosed herein, which may be isolated, synthetic, or recombinant, such as polypeptides having an amino acid sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, or SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124; concatemeric peptides as disclosed herein; chimeric or fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant)).
  • pharmaceutically acceptable salts of the T-cell epitope compositions including one or
  • “Pharmaceutically acceptable salt” of a T-cell epitope composition means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent peptide or polypeptide (e.g., peptides, polypeptides, concatermic peptides, and/or chimeric or fusion polypeptides as disclosed herein).
  • pharmaceutically acceptable salt refers to derivative of the instantly-disclosed peptides or polypeptides, wherein such compounds are modified by making acid or base salts thereof.
  • Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric
  • the present disclosure also provides a container comprising an immunologically effective amount of a phage, polypeptide, nucleic acid or vaccine as described above.
  • the present disclosure also provides vaccination kits comprising an optionally sterile container comprising an immunologically effective amount of the vaccine, means for administering the vaccine to animals, and optionally an instruction manual including information for the administration of the immunologically effective amount of the composition for treating and/or preventing SARS-CoV-2, SARS, MERS or similar coronavirus associated diseases.
  • the T-cell epitope compositions of the present disclosure including one or more of e.g., phage display, phage DNA, polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or
  • compositions or formulations generally comprise a T-cell epitope composition of the present disclosure and a pharmaceutically-acceptable carrier and/or excipient.
  • said pharmaceutical compositions are suitable for administration.
  • Pharmaceutically-acceptable carriers and/or excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the instantly-disclosed T-cell epitope compositions (see, e.g., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 22nd Ed., 2012)).
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • compositions, carriers, excipients, and reagents are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • pharmaceutically-acceptable excipient means, for example, an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.
  • excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • a person of ordinary skill in the art would be able to determine the appropriate timing, sequence and dosages of administration for particular phage or T-cell epitope compositions of the present disclosure.
  • preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin.
  • Fiposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art.
  • any conventional media or compound is incompatible with the T-cell epitope compositions of the present disclosure and as previously described above (including one or more of e.g., phage or polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassette
  • phage or T-cell epitope compositions or concatemers of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1, or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of
  • T-cell epitope compositions of the present disclosure can be administered by parenteral, topical, intravenous, oral, subcutaneous, sublingual, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; vaginally; intramuscular route or as inhalants.
  • T-cell epitope compositions of the present disclosure can be injected directly into a particular tissue where deposits have accumulated, e.g., intracranial injection.
  • intramuscular injection or intravenous infusion may be used for administration of T-cell epitope compositions of the present disclosure.
  • T-cell epitope compositions of the present disclosure are administered as a sustained release composition or device, such as but not limited to a MedipadTM device.
  • phage or T-cell epitope compositions or concatemers of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of
  • solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include, but are not limited to, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial compounds such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfit
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • excipients can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, water, ethanol, DMSO, glycol, propylene, dried skim milk, and the like.
  • the composition can also contain pH buffering reagents, and wetting or emulsifying agents.
  • compositions or formulations suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition is sterile and should be fluid to the extent that easy syringeability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • T-cell epitope formulations may include aggregates, fragments, breakdown products and post-translational modifications, to the extent these impurities bind SLA and present the same TCR face to cognate T cells they are expected to function in a similar fashion to pure T-cell epitopes.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol ( e.g ., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound that delays absorption, e.g., aluminum monostearate and gelatin.
  • sterile injectable solutions can be prepared by incorporating the phage or T-cell epitope compositions of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric
  • dispersions are prepared by incorporating the binding agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • phage or T-cell epitope compositions of the present disclosure can be administered in the form of a depot injection or implant preparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • oral compositions generally include an inert diluent or an edible carrier and can be enclosed in gelatin capsules or compressed into tablets.
  • the binding agent can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening compound such as
  • phage or T-cell epitope compositions or concatemers of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or
  • systemic administration of the phage or T-cell epitope compositions or concatemers of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides,
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the T-cell epitope may be formulated into ointments, salves, gels, or creams and applied either topically or through transdermal patch technology as generally known in the art.
  • the phage or T-cell epitope compositions or concatemers of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric
  • the phage or T-cell epitope compositions or concatemers of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric
  • Biodegradable, biocompatible polymers can be used, such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art (U.S. Pat. No. 4,522,811, which is herein incorporated by reference in its entirety). In aspects, the T-cell epitope compositions of the present disclosure can be implanted within or linked to a biopolymer solid support that allows for the slow release of the phage or T-cell epitope compositions to the desired site.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of binding agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the instant disclosure are dictated by and directly dependent on the unique characteristics of the binding agent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such phage or T- cell epitope compositions for the treatment of a subject.
  • the present disclosure further concerns the design of a T4 phage system to display a combinations of SARS-CoV-2 specific linear epitopes fused as a concatemer chimera to the C terminus of a capsid protein gp23, gp24, Hoc and/or Soc of phage T4.
  • the identified 34 antigenic epitopes SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
  • phage vaccine constructs were assembled into the single concatemer peptide fragment (i.e. SEQ ID NO: 1 or SEQ ID NO: 124) to generate the phage vaccine constructs that are herein.
  • the details of the cloning steps are set out below, but briefly the optimized SARS-CoV-2 concatemer polypeptide were reverse translated into a synthetic polynucleotide that would encode such and then affixed with suitable restriction sequences to allow for in frame insertion into the 3 ’ end (C-terminus) of a gp23/gp24/Hoc/Soc T4 head genome in a plasmid.
  • the resulting constructs were transformed into Cre expressing E. coli and then infected with a lambda phage.
  • Recombination sites e.g. lox
  • the phage is irradiated (e.g., after construction by prior to administration to a subject).
  • the specific DNA sequences coding for the concatemer peptide sequence can be displayed on T4 phage using the protocol, for example, as described Zhu et al., “A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS- CoV-2 Vaccine Candidates by CRISPR Engineering bioRxiv, January 19, 2021 (herein incorporated by reference in its entirety).
  • engineered DNAs corresponding to the concatemer peptide sequence (or fragments thereof) can be incorporated into bacteriophage T4 genome as outlined in Figure 1 of Zhu et al. Each DNA is then introduced into E.
  • coli as a donor plasmid and recombined into injected phage genome through CRISPR- targeted genome editing.
  • Different combinations of CoV-2 inserts can then be generated by simple phage infections and the recombinant phages in the progeny can be identified.
  • a pipeline of multiplex T4-concatemer peptide sequence and/or individual antigenic fragment vaccine phages can be rapidly constructed. Selected vaccine candidates can then be screened in a mouse model to identify the most potent vaccine.
  • the present disclosure concerns, in some aspects, methods of using the phage vaccines disclosed herein.
  • the vaccine compositions are in aspects designated for administration to human subjects to provide an immune response, ideally a protective immune response. It will be appreciated that the vaccine compositions may be most effective by coming into contact with a human subject’s immune system. It may be therefore generally desirable to administer the compositions by a route that allows for such.
  • the route of inoculation of phage based vaccines can be altered or varied to provoke desirable immunogenicity.
  • phage based display or phage DNA vaccines can be introduced through intranasal or sublingual routes instead of intramuscular route to activate production of secretory antibody like IgA.
  • phage display or phage DNA vaccines can be introduced via microneedles specifically designed for intradermal deposition of phage vaccines.
  • This intradermal inoculation route for SARS-CoV-2 phage vaccine using microneedle is highly desirable to enhance immune response, resulting in a potential reduction of the antigen dose, decreased anxiety and pain.
  • Microneedles can be engineered as small patches to deliver vaccines without any complications.
  • T-cells with phage or T-cell epitope compositions of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or
  • T cells activated by the phage or T-cell epitope compositions of the present disclosure including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or
  • the present disclosure is directed to a method of stimulating, inducing, and/or expanding an immune response, e.g., against SARS-CoV-2 infection (or a closely related virus such as Severe Acute Respiratory Syndrome (SARS) or Middle East respiratory syndrome coronavirus (MERS-CoV)) and/or related diseases caused by SARS-CoV-2, including COVID-19, in a subject in need thereof by administering to the subject a therapeutically effect amount of a phage or T-epitope composition (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO:
  • the present disclosure is directed to a method of preventing, treating, or ameliorating a disease caused by SARS-CoV-2 infection (or a closely related virus such as Severe Acute Respiratory Syndrome (SARS) or Middle East respiratory syndrome coronavirus (MERS-CoV))) such as COVID-19, in a subject in need thereof by administering to the subject a therapeutically effect amount of a phage display, phage DNA or T-cell epitope composition of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (
  • the present disclosure includes multiple rounds of administration of the phage-based vaccine compositions. Such are known in the art to improve or boost the immune system to improve protection against the pathogen. Additionally, the present disclosure may also include assessing a subject’s immune system to determine if further administrations of a phage-based vaccine composition is warranted.
  • multiple administrations may include the development of a prime boosting strategy of vaccination using phage mediated display and/or DNA or RNA vaccines. Such may provide an opportunity to produce sequential immunogenic responses against SARS- CoV-2.
  • phage mediated display and phage mediated DNA vaccinations can be achieved in an alternative manner to provide a regimen of immunization with the same immunogen presented in different fashions to mammalian immune system.
  • the initial priming of the immune system can be accomplished with phage display vaccine generated by, e.g., the construct set forth in SEQ ID NO: 2, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, or variant thereof and subsequent booster immunizations can be performed with phage DNA vaccine generated by the corresponding peptide construct, i.e. SEQ ID NO: 1 or SEQ ID NO: 124.
  • the EpiVax proprietary iVAX vaccine design software platform was used to prospectively identify conserved epitope clusters using the first SARS-CoV-2 virus sequence submitted to GenBank on January 17, 2020 as a reference and other publicly available SARS- CoV-2 genomes.
  • a comprehensive description of the advanced set of tools used to develop this vaccine was recently published (De Groot AS, Moise L, Terry F, Gutierrez AH, Hindocha P, Richard G, Hoft DF, Ross TM, Noe AR, Takahashi Y, Kotraiah V, Silk SE, Nielsen CM, Minassian AM, Ashfield R, Ardito M, Draper SJ, Martin WD.
  • T cells that recognize antigen-derived epitopes sharing TCR contacts with epitopes derived from the human proteome may be deleted or rendered anergic before release into the periphery during thymic selection. Therefore, vaccine components targeting these T-cells may be ineffective.
  • vaccine- induced immune responses targeting cross-reactive epitopes may induce unwanted autoimmune responses targeting the human homologs of the cross-reactive epitopes identified by JanusMatrix. As a result, vaccine safety may be reduced.
  • Epitope clusters were randomly concatenated head-to-tail to facilitate production of epitopes as a single genetic construct. To avoid generating non-SARS-CoV-2 epitopes at epitope cluster junctions, the order of epitope cluster units was rearranged using the VaxCAD algorithm that reduces off-target junctional epitope potential. Potential immunogenicity was also evaluated at the junction between lambda phage gpD and the concatemer. A concatemer containing all 34 epitopes clusters was generated with no class II junctional immunogenicity aside from one junction where a GPGPG (SEQ ID NO: 71) spacer was inserted to eliminate immunogenicity potential.
  • GPGPG SEQ ID NO: 71
  • a total of 34 class I and class II peptides were selected for inclusion in the instantly- disclosed vaccine construct, following immunoinformatic predictions. Selection was based on at least, high binding likelihood to HLA class I and class II alleles, and low tolerogenicity potential. Putative class I epitopes were in the top 1% of predicted ligands, and had Janus Matrix Homology Scores below 2. Putative class II epitopes, were predicted to bind to four or more HLA alleles, and had JanusMatrix Homology Scores below 2.
  • the selected epitope clusters for HLA Class I and Class II used to produce the concatemeric peptide of SEQ ID NO: 1 include the below sequences from the identified SARS-CoV-2 proteins (indicated with the start amino acid number) in TABLE 3.
  • the concatemer was assessed for potential to form a transmembrane domain using the TMHMM 2.0 prediction tool. Insertion of the concatemer into a membrane could hamper vaccine production. No transmembrane domains were predicted within the concatemer.
  • Example 2 Memory T cell responses to SARS-CoV-2 polypeptides in COVID-19 Convalescents
  • SARS-CoV-2 convalescent donors are recruited by Sanguine Biosciences, a clinical services group that identifies, consents and enrolls participants. Inclusion criteria includes subjects (i) willing and able to provide written informed consent and photo identification, (ii) aged 18-60, both male or female, (iii) confirmed COVID- 19 diagnosis (recovered) with date of diagnosis a minimum of 30 days from blood collection, and (iv) positive COVID-19 PCR based-kit documented by time-stamped medical record and/or diagnostic test report and test kit used identified.
  • Exclusion criteria includes subjects who (i) are pregnant or nursing, (ii) have a known history of HIV, hepatitis or other infectious diseases, (iii) have autoimmune diseases, (iv) in vulnerable patient population (prisoners, mentally impaired), (v) have medical conditions impacting their ability to donate blood (i.e. anemia, acute illness) (vi) received immunosuppressive therapy or steroids within the last 6 months, (vii) received an investigational product in the last 30 days, (viii) experienced excess blood loss including blood donation defined as 250 mL in the last month or 500 mL in the last two months, or (ix) had a positive COVID-19 PCR test, but were asymptomatic. Samples are collected in accordance with NIH regulations and with IRB approval.
  • Samples are obtained from leukocyte reduction filters from the Rhode Island Blood Center for unrelated studies prior to the SARS-CoV-2 outbreak in December 2019. Samples are collected in accordance with NIH regulations and with IRB approval.
  • PBMC culture Thawed whole PBMCs (normal healthy donors) are rested overnight and expanded by antigen stimulation (including select polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1) over nine days at 37°C under a 5% C02 atmosphere.
  • select polypeptides of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 over nine days at 37°C under a 5% C02 atmosphere.
  • a 48-well plate 5 x 10 A 6 cells in 150 m ⁇ RPMI medium supplemented with human AB serum are stimulated with pools of peptides at 10 pg/ml on Day 1. Three days later, IL-2 is added at 10 ng/ml and the culture volume raised to 300 m ⁇ . On Day 7, cells are supplemented with 10 ng/ml IL-2 by half media replacement. Two days later, PBMCs are collected and washed in preparation to measure immune recall responses.
  • IFN-g Interferon-gamma Fluorospot assays are performed ex vivo and following culture using kits purchased from Mabtech and performed according to the manufacturer’s specifications. Peptides are added individually at 10 pg/ml and pooled at 10 pg/ml (8 peptides, 1.25 pg/mL) to triplicate wells containing 250,000 PBMCs (ex vivo) or 100,000 PBMCs (cultured) in RPMI medium supplemented with 10% human AB serum. Triplicate wells are plated with ConA (10 pg/ml) as a positive control, and six wells containing no antigen stimulus are used for background determination. Cells are incubated for 40-48 hours at 37 °C under a 5% C02 atmosphere. Plates are developed according to the manufacturer’s directions using FITC-labeled anti-IFN-g detection antibody.
  • Raw spot counts are recorded by ZellNet Consulting, Inc. using a FluoroSpot reader system (iSpot Spectrum, AID, Strassberg, Germany) with software version 7.0, build 14790, where fluorescent spots are counted utilizing separate filters for FITC, Cy3, and Cy5. Camera exposure and gain settings are adapted for each filter to obtain high quality spot images preventing over- or underexposure. Fluorophore-specific spot parameters are defined using spot size, spot intensity and spot gradient (fading of staining intensity from center to periphery of spot), and a spot separation algorithm is applied for optimal spot detection.
  • Fluorophore-specific spot parameters are defined using spot size, spot intensity and spot gradient (fading of staining intensity from center to periphery of spot), and a spot separation algorithm is applied for optimal spot detection.
  • Results are calculated as the average number of spots in the peptide wells, adjusted to spots per one million cells. Responses meeting the following criteria are positive when the number of spots is (i) at least twice background, (ii) greater than 50 spot forming cells per well above background (1 response per 20,000 PBMCs), and (iii) statistically different (p ⁇ 0.05) from the media-only control by the Student’s t test.
  • peptide 1 (or rank 1) is SEQ ID NO: 45; peptide 2 (or rank 2) is SEQ ID NO: 17; peptide 3 (or rank 3) is SEQ ID NO: 5; peptide 4 (or rank 4, which is I ASFRLF ARTRSM W SFN (SEQ ID NO: 76)) is a segment of SEQ ID NO: 69; peptide 5 (or rank 5, which is FLGVYYHKNNKSWMESE (SEQ ID NO: 77)) is a segment of SEQ ID NO: 33; peptide 6 (or rank 6, which is ESEFRV Y S S ANNCTFE YV (SEQ ID NO: 78)) is a segment of SEQ ID NO: 33; peptide 7 (or rank 7, which is LSYFI
  • ex vivo immune recall responses differentiate SARS- CoV-2 naive and experienced individuals and exhibit different COVID-19 immunotypes.
  • Robust and failed immune responses in convalescent donors may represent different immunotypes characterized in a deep immune profiling study of SARS-CoV-2 experienced humans (Giles et al. Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications. Science. 2020 Jul 15:eabc8511. doi: 10.1126/science. abc8511. PMID: 32669297, herein incorporated by reference in its entirety).
  • polypeptides included in the concatemers of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1).
  • polypeptides included in the concatemers of the instant disclosure may stimulate ex vivo immune recall response in natural SARS-CoV-2 infection.
  • polypeptides included in the concatemers of the instant disclosure may stimulate higher IFN-g responses in naive and COVID-19 convalescent donors following expansion in culture.
  • polypeptides included in the concatemers of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1) stimulate or may stimulate low frequency epitope-specific T-cells following expansion in culture in naive and COVID-19 convalescent donors. Differences between responses to spike and membrane peptides in ex vivo and cultured assay may reflect variable phenotypes and/or proliferative capacities of epitope-specific T-cells when they are put into culture.
  • polypeptides included in the concatemers of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1) stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors. Of 32 tested peptides, 27 peptides demonstrated positive responses, as shown in green, in at least one donor.
  • polypeptides of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1) are recognized by T cells raised in natural infection, stimulate Thl cytokine production, may stimulate pre-existing immunity to common cold coronaviruses, and memory may boost immunity in clinical trials.
  • polypeptides of the instant disclosure e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide
  • the specific DNA sequences coding for the concatemer peptide sequence can be synthesized. Using specific oligo primers, the synthesized DNA fragments can be PCR amplified to provide restriction sites for in-frame cloning with a head protein sequence. The oligo sequence of each PCR primer is next modified to produce restriction sites in each end of amplified DNA fragments. After restriction digestion, these fragments were inserted separately at the 3 ' end of a DNA fragment encoding the head protein under the control of the lac promoter. The constructs were created in a plasmid vector (donor plasmid), which also carries loxPwt and loxP511 sequences.
  • donor plasmid also carries loxPwt and loxP511 sequences.
  • Cre-expressing cells E . coli
  • Cre-expressing cells can then be transformed with these recombinant donor plasmids and subsequently infected with a recipient T4 phage that carries a stuffer DNA fragment flanked by loxPwt and loxP511 sites.
  • T4 phage infected Cre-expressing E. coli is then grown at LB Ampicillin (100 ug/ml) at 37° C. for four hours in presence of 0.2% maltose and 0.1M CaC12. Recombination occurs in vivo at the lox sites and Ampr cointegrates are formed, which then spontaneously lyse the E. coli and release in culture media (figure 6).
  • T4 cointegrates can be used to produce T4 lysogens and then selected on Luria Bartani (LB) ampicillin agar (100 ug/ml amp, 15% agar) plates. Briefly, cointegrates from spontaneously lysed E. coli culture are used to infect Cre-ve, suppressor-ve E. coli cells and spread on LB ampicillin agar plates. Plates are incubated at 30 °C for 48 hours to obtain Ampr colonies which are actually recombinant T4 lysogens and carry a recombinant T4 phage genome in their chromosome.
  • LB Luria Bartani
  • phage plaques are then harvested from the plate and single plaques are purified three times on E. coli by the standard procedures described by Russell, J.S.a.D., Molecular Cloning: A Laboratory Manual. 4th Edition ed., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Approximately 220 copies of ARS- COV-2 virus specific peptides are displayed on a single phage head.
  • All phage plaques, containing T4 cointegrates, which are used for SARS-CoV-2 phage vaccine production can then be verified by PCR. In this process the presence of each cloned inserts in T4 phage are confirmed by PCR amplification of insert DNA. The specific primers flank the T4 genome and the insert. Phage DNA containing co-integrates are then subjected to complete genome sequencing and subsequent bioinformatics analysis to confirm the proper orientation and sequence of inserted DNA fragments in T4 genome as gp23/gp24/Hoc/Soc fusion.
  • the materials include: Luria Broth, 20% Maltose in water (filter sterilized), 1 M gCL in water (filter sterilized), an E. coli sup- host, flasks, shaking incubators, cuvettes, spectrophotometer, a T4 phage construct, micropipetters and pipette tips, serological pipettes, 1 mg/ml DNase I stock in water (filter sterilized), and filter membranes (0.8 pm, 0.45 pm, and 0.22 pm). Briefly, an overnight culture of E.
  • coli sup- host strain in Luria Broth with 0.2% Maltose and 10 mM MgCk is prepared along with a suitable media of 1:100 20% Maltose and 1:100 1 M MgCh to Luria Broth (e.g. for 2 Liters LB media add 20 ml 20% Maltose and 20 ml 1M MgCh).
  • a 1 mL aliquot of the prepared media for an optical density standard is transferred to a cuvette.
  • the prepared media is then inoculated at a 1:100 with the overnight E. coli sup- culture and mixed.
  • a 1 ml aliquot of the inoculated culture is transfer to a cuvette and the optical density (OD) at 600nm is measured to obtain a starting time point of the culture.
  • the E. coli culture is then incubated at 37 °C in a shaking incubator and the OD of the culture monitored until an optical density of 0.090 to 0.100 at 600 nanometers (nm) is obtained.
  • MOI multiplicity of infection
  • TFF purified phage vaccine preparations are extracted once with organic solvents octanol for removing lipopolysaccharide (lps) from vaccine preparations.
  • the materials include: 50 ml conical tubes, Lipid Removal Adsorbent (Supelco Cat# 13360-U), 1 -Octanol (Fisher Chemical Cat# A402-500), ethanol (Fisher Chemical Cat# BP28184), Float- A-Lyzer G2, 10 kD, 10 ml (Repligen Cat# G235067), Thermo Scientific BupH Phosphate Buffered Saline Packs (Fisher Cat# PI28372), Steriflip vacuum filter (Millipore Sigma Cat# SE1M179M6), syringes and 18 gauge needles, 4 L container, and a magnetic stir bar.
  • the steps include starting with a phage lysate sample that has been previously purified through tangential flow filtration which are achieved by concentrating 5 L of phage lysate down to 100 ml total volume. After concentration, media is replaced by dia- filtration against 4 L of 0.5 molar (M) Phosphate Buffered Saline (PBS). 50 ml of purified phage lysate is then even split between two 50 ml conical tubes and 15 mg/ml Lipid Removal Adsorbent (LRA) is added to each conical tube.
  • M 0.5 molar
  • PBS Phosphate Buffered Saline
  • the LRA is next mixed into solution by inverting the tube (do not mix too harshly as the LRA may damage the phage) and once the LRA is in solution, the 50 ml conical tubes are placed on a rocker at room temperature to be rocked vigorously for about 30 minutes. The conical tubes are then removed and the LRA is allowed to settle for 5 minutes. The phage lysate with LRA is then passed through a 0.22 pm Steri-flip vacuum filter to remove LRA and a 0.4 volume of 1-Octanol is added to each LRA- treated phage lysate, followed by mixing by inversion. The tubes are then placed on the rocker at room temperature and rocked vigorously for 1 hour.
  • the tubes are then incubated on ice for 15 minutes, followed by centrifugation at 4000 x g for 10 minutes at 4 °C.
  • the bottom of the tubes is then pierced using a syringe and needle and the lower aqueous portion is removed without disturbing the top 1-Octanol layer (contains endotoxin) and transferred to a clean 50 ml conical tube. A small amount of the aqueous layer behind may be left behind to prevent disturbing the top layer.
  • Dialysis tubing is pre-moistened in 25% ethanol in distilled water for 1 minute and then loaded with 10 ml of 1-Octanol treated per tube and dialyzed in 4 L of 25% ethanol in distilled water overnight at 4 °C with slow agitation from a magnetic stir bar.
  • Dialysis chambers are then transferred to 4 L of 0.5 M PBS to dialyze for 6 to 8 hours, followed by further dialysis overnight in fresh 4 L of 0.5 M PBS.
  • the dialysis chambers are then transferred to fresh 4 L of 0.5 M PBS to dialyze for another 6 to 8 hours.
  • the lysate is then removed, transferred to clean 50 ml conical tubes and sterilized through 0.22 pm Steri-flip vacuum filter. The resulting phage lysate can then be stored at 4 °C.
  • H7N9 influenza vaccine displaying concatenated iVAX-identified CD4+ T cell epitopes stimulates de novo epitope-specific Thl responses in HLA-DR3 transgenic mice.
  • H7N9 class II HLA epitopes with low human cross reactivity potential were identified in H7N9 HA and internal antigens using the EpiMatrix, ClustiMer, and JanusMatrix algorithms.
  • Epitopes were concatenated in an arrangement that minimized off-target immunogenicity at epitope junctions using the VaxCAD algorithm.
  • a synthetic gene encoding the epitope concatemer was produced, subcloned into a plasmid DNA vector and lamba phage capsid, and DNA and phage vaccines were produced.
  • HLA-DR3 mice were primed with pDNA and boosted twice with phage at two week intervals. Two weeks following the final immunization, splenic leukocytes were harvested and stimulated overnight with pools of vaccine-matched HA and internal antigen peptides to determine frequencies of Thl cytokine-producing CD4+ T cells by flow cytometry.
  • Thl cytokine-producing CD4+ T cells A similar experimental study can be used to determine frequencies of Thl cytokine- producing CD4+ T cells.
  • the T4 phage display constructs and phage DNA constructs can be primed in murine models with phage DNA (pDNA), followed by additional phage boosters.
  • Splenic leukocytes may then be harvested and stimulated O/N with pools of vaccine-matched SARS-CoV-2 and internal antigen peptides.
  • Thl cytokine producing CD4+ T cells can be identified by flow cytometry and cytokine levels determined by intracellular staining.
  • T4 phage display constructs and phage DNA constructs used in such a prime-boost setting will result in elevated frequencies of cytokine + CD4 + T cells specific for SARS-CoV-2 and internal antigen peptides epitopes as determined by intracellular cytokine staining (IF-2, IFNy, TNFa).
  • Splenocytes were collected from mice on day 28 and stimulated with pools of overlapping spike peptides or envelope, or membrane peptides spanning the full length of the antigens. Interferon-y-producing CD8+ T cells were enumerated by flow cytometry. IFNy is the signature cytokine of type 1 helper T cells. As is observed in Figure Splenocytes stimulated with each peptide pool demonstrate significantly higher frequencies of CD8+IFNy+ T-cells over unstimulated cells in mice immunized with SEQ ID NO: 1. No significant differences were observed in mice that received saline or empty vector. Frequencies of CD8+IFNy+ T- cells in peptide stimulations were significantly elevated in mice that received SEQ ID NO: 1 over those that received saline or empty vector.
  • CD8+ T-cells expressing surface CD107a were enumerated by flow cytometry.
  • CD 107a expression at the cell surface is a marker of cytolytic granule release.
  • splenocytes stimulated with both spike and envelope/membrane peptide pool demonstrate significantly higher frequencies of CD8+CD107a+ T-cells over unstimulated cells in mice immunized with SEQ ID NO: 1. No significant differences were observed in mice that received saline or empty vector. Frequencies of CD8+CD107a+ T-cells in peptide stimulations were significantly elevated in mice that received SEQ ID NO: 1 over those that received saline or empty vector.
  • cytolytic T lymphocytes from mice immunized with SEQ ID NO: 1 kill cells expressing the vaccine-matched epitopes (SEQ ID NO: 1; left side) or spike protein (right side) significantly more than cytolytic T lymphocytes from mice that received saline or empty vector.
  • Serum anti-spike antibody levels were measured using serum samples collected on day 21 following immunization with a lipid vaccine including a plasmid housing SEQ ID NO: 1 as above. As is illustrated in Figure 16, all mice that received the concatemer of SEQ ID NO: 1 seroconverted. A dose-dependent effect was observed with mice that received the 100 pg dose showing higher anti-spike antibody fold increases over baseline (prior to immunization) than those that received the 25 pg dose. Both SEQ ID NO: 1 doses elicited significantly higher fold increases in anti-spike antibody over mice that received saline.
  • mice The purpose of this experiment is to determine the efficacy of the phage vaccines to elicit antibody response in BALB/c female mice.
  • Four separate groups of mice (Group A, Group B, Group C, 5 mice in each group and Group D, 40 mice) will be injected subcutaneously (s/c) with various phage constructs. Briefly, group A mice will receive 5xlO s pfu of T4 phage particles suspended in 500 m ⁇ of sterile PBS.
  • a control group of mice (group B) will receive recombinant SARS-CoV-2 antigen (50 pg/mouse) suspended in sterile PBS.
  • mice After primary inoculation, mice will receive 1st and 2nd booster (dose will be the same as primary inoculation) of corresponding antigens at 2 weeks interval. All animals will be bled prior primary inoculation. Serum samples will be collected before every booster to monitor progression of immune response against SARS-CoV-2 antigens. After 21 days, animals will be euthanized for final bleeding through cardiac puncture. Finally animals will be sacrificed by spinal dislocations. The immune response against various SARS-CoV-2-phage vaccines will be monitored by western immunoblot and ELISA. [00232] Animal Study 3
  • mice A further study can use male BALB/c mice (18-25 g each), each being immunized via intramuscular injection (i.m.) with the T4 phage display vaccine construct.
  • Group A receives a single dose
  • B receives 2 (days 0 and 14)
  • C receives 3 (days 0, 14, and 21).
  • Blood is collected before each dose and at 2, 4, and 8 weeks post all vaccinations.
  • 50 pL of blood will also be collected 7 hours after challenge to compare vaccine efficacy.
  • the dose will be a total of 10 9 pfu of the phage cocktail.
  • mice At day 56, mice will be euthanized and tissue and blood collected therefrom. Serum, lungs, spleen and lymph nodes will be analyzed immunologically and histopathologically.
  • Blood will be clotted for collection of serum and sera will be pooled and analyzed to determine levels of various cytokines including TNF-a, IL- la, IL-6 and IFN-g. Cytokines can be measured via ELISA according to the manufacturer’s instructions. Antigen specific and T4 phage capsid specific antibody titers of mouse sera before and after vaccinations will also be monitored by ELISA and western blot to enumerate humoral immune response to the vaccine. Histopathology of various organs will also be analyzed to determine the extent of inflammatory tissue damage, particularly with respect to any cytotoxic effect of phage vaccines. For statistical plans, a 4-fold immune response with descriptive characterization across groups will be used.
  • the therapeutic vaccines of the present disclosure are designed to elicit antibodies that may disrupt the lifecycle of SARS-CoV-2, as well as SARS, MERS and/or similar coronaviruses.
  • the vaccines are designed to present viral T-cell epitopes to elicit such response.
  • Clinical trials with the therapeutic vaccines employing a dosage form including an immunogenic composition of the present disclosure against SARS-CoV-2, such as a phage display cocktail to provide SEQ ID NO: 1, or a phage DNA cocktail to provide SEQ ID NO: 2, or each T-cell epitope polypeptide or nucleic acid, are contemplated.
  • a clinical trial for a phage display cocktail to provide SEQ ID NO: 1 (which may be administered, e.g., orally, sublingually, intranasal, transdermal (i.e., applied on or at the skin surface for systemic absorption), ocularly, percutaneous, via mucosal administration, or via a parenteral route (intradermal (e.g., via microneedle), intramuscular, subcutaneous, intravenous, or intraperitoneal) in a phase I study.
  • SEQ ID NO: 1 which may be administered, e.g., orally, sublingually, intranasal, transdermal (i.e., applied on or at the skin surface for systemic absorption), ocularly, percutaneous, via mucosal administration, or via a parenteral route (intradermal (e.g., via microneedle), intramuscular, subcutaneous, intravenous, or intraperitoneal) in a phase I study.
  • 3M TDM patch
  • Immunogenicity will be assessed through blood samples and obtaining antibody and T-cell responses at baseline and then at weeks 1, 2, 4, 8 and 12 after each vaccination, and at 3, 6, and 12 months following final vaccination.
  • Whole blood will be processed to obtain peripheral blood mononuclear cells (PBMCs) for determination of cell mediated immune response (CD4 and CD8 T-cell responsiveness to the T-cell epitope polypeptides (SEQ ID NO: 1) ⁇
  • PBMCs peripheral blood mononuclear cells
  • CD4 and CD8 T-cell responsiveness to the T-cell epitope polypeptides SEQ ID NO: 1
  • Participants will include subjects between 18-50 yrs old including male and female subjects. Informed consent will be obtained for each. Subjects with a history of immunosuppressive or autoimmune disease will not be considered. Subjects already known to have had a prior SARS-CoV-2 infection or that test positive for infection prior to commencing vaccination will also not be considered.
  • the clinical model may be also adapted.
  • a series of vaccine applications may also be applied to a set of human subjects.
  • the set can be divided into a control group, a group that receives alternating phage display and phage DNA compositions, a group that receives only phage display and a group that receives only phage DNA.
  • Subgroups may be further established to study a cocktail of all three constructs in comparison to one construct only. Further groups may also be established to assess administration of a cocktail of all three in comparison to sequential administration of different phage constructs. Following the final administration, blood samples may be collected and challenged with SARS-CoV-2 virus, followed by determination of specific immune response to the challenge.
  • the T4 phage concatemer can be further prepared as an equimolar oral or transdermal vaccine with appropriate carriers and excipients to assist in administration.
  • the vaccine can be applied as a single dose or followed up later with a further booster of the same vaccine. In other instances, a booster may include phage DNA.
  • the efficacy of the route of administration can also be determined.
  • compositions and methods described herein are presently representative of particular embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

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Abstract

The present disclosure concerns bacteriophages that express T-cell epitopes for SARS-CoV-2 designed to generate an immune response in a subject to provide for recognition and/or protection against SARS-CoV-2. The compositions can include phage display or phage DNA with algorithm optimized SARS-CoV-2 T-cell epitopes to interact with a broad spectrum of HLA in the human population.

Description

Prophylactic Broad Spectrum Vaccine for SARS-CoV-2
Cross reference to related applications
[0001] This application depends from and claims priority to U.S. Provisional Application No: 63/148,702 filed February 12, 2021, the entire contents of which are incorporated herein by reference.
Sequence Listing
[0002] The instant application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 11, 2022, is named EPV0048WO_ST25.txt and is 52,472 bytes in size.
Field
[0003] This disclosure relates to the development of a vaccine against SARS-CoV-2 viral infection. The present disclosure provides in some aspects a phage-produced vaccine for prevention of SARS-CoV-2 viral infection in human subjects.
Background
[0004] The art of making vaccines is continuously evolving to compensate the production cost without compromising the quality of vaccine. In that sense, production of a large quantity of infectious organism using highly selective media, cell culture or egg based system is not always cost effective for making conventional vaccines. As an easy alternative several other modes of vaccine preparations have been attempted where subunit protein antigens of an organism are expressed and purified by using various recombinant technologies. Unfortunately, majority of these recombinant antigens alone cannot be used as a vaccine until a potent mixture of adjuvant is created for delivery along with these protein antigens.
[0005] In the past, production of vaccines using formalin treated or attenuated organism was always considered as better choice for stimulating mammalian immune system. Generally, these types of whole cell vaccines are good immune-stimulators due to their inherited nature of particulate structures that attract antigen presenting cells (APCs). Delivering foreign antigens as particulate is more promising as an immune stimulator relative to recombinant antigens because this presentation more accurately mimics the actual infection during normal disease manifestation process. Unfortunately, attenuated vaccines are not always ideal for mass scale vaccination due to possible reversal of virulence, complexity and time required for production. Formalin can also denature major immunogenic antigens.
[0006] As an alternate approach, empty capsid vaccines have been prepared for imitating the particulate nature of pathogens. Currently several particulate vaccines are also prepared by using Ankara and Baculovirus vector systems where these recombinant viruses act as a chimera of pathogens. Unfortunately, manufacturing processes required for these vaccines are time consuming and require expensive cell culture media, which are not ideal for rapid, large quantity production of vaccines during outbreaks.
[0007] Bacteriophage or phage are bacterial viruses that do not infect eukaryotic cells. These bacterial-viruses, such as M13, T4, T7, lambda (l) etc., can be propagated using laboratory strains of E.coli with inexpensive bacteriological media such as Luria-Bertani (LB) broth. Foreign antigen or peptides may be expressed by incorporating a corresponding nucleotide encoding such into the genome of the phage, often fused to a surface targeting sequence to ensure surface display. The resulting expression on the surface of bacteriophage when administered as a vaccine is recognized as displayed antigens and leads to immune response generation. Thus, such chimeric constructs may be considered valuable vaccine agents.
[0008] Individual phage particles can display multiple copies (including in some aspects hundreds) of identical or combinational antigenic epitopes on its surface and act as a good immunogen when introduced into a mammalian system. The particulate nature of the phage specifically attracts antigen-presenting cells (APCs) and may be able to provoke both cell mediated and humoral responses.
[0009] The rate and ease of phage growth are very robust, with a high production rate of plaque forming units (pfu) of phage particles per liter of culture being achievable through routine fermentation processes. The phage particles are further non-pathogenic to humans, can effectively deliver all relevant information about target antigens to the immune system and the do not require an accompanying adjuvant to be effective. As a further approach, phage may be used as a gene delivery system for DNA vaccination where genetic information is coded inside of phage genome under control of eukaryotic promoter.
[0010] The advent of the phage vaccine systems remains relatively new, yet features such as safety and scalability of production have attracted significant interest in developing numerous vaccines. Accordingly, when the pandemic of COVID-19 disease, caused by the SARS-CoV-2 (or 2019-nCoV) coronavirus, rapidly swept over the globe, it was immediately apparent there was a need for a new vaccine formulations against the SARS-CoV-2 virus.
Summary
[0011] The present disclosure relates to bacteriophage compositions to generate an immune response against SARS-CoV-2. The bacteriophage systems include surface expression of concatemers of SARS-CoV-2 epitope peptides, as well as bacteriophage comprising nucleic acids encoding such concatemers within the genome thereof.
[0012] In some aspects, the present disclosure concerns a bacteriophage having at least one amino acid sequence of a concatemer of SARS-CoV-2 T cell epitopes. In further aspects, the at least one amino acid sequence is SEQ ID NO: 1. In further aspects, SEQ ID NO: 1 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 2.
[0013] In some aspects, the bacteriophage is selected from Lambda, T4, T7 or M13/fl. In other aspects, the bacteriophage is T4.
[0014] In some aspects, the present disclosure concerns a composition of a bacteriophage featuring SEQ ID NO: 1 and/or a nucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 1, such as that set forth in SEQ ID : 2.
[0015] In further aspects, the present disclosure concerns a bacteriophage with a nucleic acid of at least 85% sequence identity to a nucleic acid sequence encoding an amino acid sequences as set forth in SEQ ID NO: 1, such as 85% identity to the nucleic acid sequence set forth in SEQ ID NO: 2. In some aspects, the nucleic acid is under the control of a promoter. In further aspects, the promoter is a bacteriophage promoter. In further aspects, the nucleic acid is fused in frame to one or more head proteins of the bacteriophage. In other aspects, the promoter is a mammalian promoter, such as CMV, SV40, CAG or U6.
[0016] In some aspects, the instantly-disclosed phages are irradiated (e.g., after construction by prior to administration to a subject).
[0017] In further aspects, the present disclosure provides a vaccine of the bacteriophage as disclosed herein, including bacteriophage that express the concatemers disclosed herein, such as on the surface of the phage and/or include the nucleic acids disclosed herein with the genome of the phage. In some aspects, the phages are irradiated (e.g., after construction by prior to administration to a subject).
[0018] In further aspects, the present disclosure provides a method of eliciting an immune response in a subject comprising administering an immune system stimulating amount of a bacteriophage comprising at least one amino acid sequence of a concatemer of SARS-CoV-2 T cell epitopes, wherein the at least one amino acid sequence includes the amino acid sequence as set forth in SEQ ID NO: 1.
[0019] In further aspects, the present disclosure provides methods for inducing immunity against 2019-nCoV in a subject in need thereof by administering to the subject a therapeutically effective amount of a phage as disclosed herein.
Brief Description of the Drawings
[0020] Figures 1 A shows immunoinformatic reports for a sample epitope cluster sequence in one phage. Cluster Detail Reports for FLGVYYHKNNKSWMESE (SEQ ID NO: 77) (a portion of SEQ ID NO: 1 is shown). Figure 1 A is an EpiMatrix staircase report of the sequence for class II HLA supertypes. Z-score indicates the potential of a 9-mer frame to bind to a given HLA allele; the strength of the score is indicated by the blue shading as noted in the respective Figures. All scores in the Top 5% (Z-Score > 1.64) are considered “Hits.” * Scores in the top 10% are considered elevated, other scores are grayed out for simplicity. Frames containing 4 or more alleles scoring above 1.64 are referred to as EpiBars and are highlighted in yellow. These frames have an increased likelihood of binding to HLA. Flanking amino acids, added to stabilize the cluster during in vitro testing, are presented in blue typeface and underlined. [0021] Figure IB is an EpiMatrix staircase report for identified MHC class I clusters of the presented peptide sequence of SARS-CoV-2. Z-score indicates the potential of a 9-mer or 10- mer frame to bind to a given HLA allele; the strength of the score is indicated by the blue shading as noted in the respective Figure. All scores in the Top 5% (Z-Score > 1.64) are considered “Hits.” This 17-mer Spike sequence contains 14 distinct T helper epitopes (‘hits’) and 6 CTL epitopes for a total of 20 high-quality T cell epitopes covering 95% of the human population.
[0022] Figure 1C is the JanusMatrix report for identified MHC class II clusters. *Count of HUMAN JanusMatrix matches found in the search database. With respect to a given EpiMatrix Hit (a 9-mer contained within the input sequence which is predicted to bind to a specific allele), a Janus Matrix match is a 9-mer derived from the search database (e.g., the human genome) that is predicted to bind to the same allele as the EpiMatrix Hit and shares TCR facing contacts with the EpiMatrix Hit. ** Janus Homology Score represents the average depth of coverage in the search database for each EpiMatrix hit in the input sequence. For example, an input peptide with eight EpiMatrix hits, all of which have one match in the search database, has a Janus Homology Score of 1. An input peptide with four EpiMatrix Hits, all of which have two matches in the search database, has a Janus Homology Score of 2. The JanusMatrix Homology Score considers all constituent 9-mers in any given peptide, including flanks. This peptide shows no class II HLA cross-conservation with the human proteome and insignificant class I HLA cross-conservation.
[0023] Figure ID is the JanusMatrix report for identified MHC class I clusters. * and ** are as per Figure 1C.
[0024] Figure 2 shows elevated epitope-specific Thl cytokine production in HLA DR3 transgenic mice immunized with phage vaccine carrying H7N9 influenza effector CD4+ T cell epitopes. Mice were primed with a plasmid DNA vaccine and boosted twice at two-week intervals with phage vaccine encoding H7N9 influenza class II HLA epitopes. A matched group of control mice received vehicle (DNA and phage without epitopes). Two weeks following the final immunization, splenic leukocytes were harvested and stimulated overnight with pools of vaccine-matched H7N9 HA and internal antigen peptides. CD4 T cells producing Thl cytokines (IFNy, TNFa, IL-2) were measured by flow cytometry. [0025] Figure 3 shows ex vivo immune recall responses differentiate SARS-CoV-2 naive and experienced individuals and exhibit different COVID-19 immunotypes.
[0026] Figure 4 shows ex vivo immune recall responses differentiate SARS-CoV-2 naive and experienced individuals and exhibit different COVID-19 immunotypes.
[0027] Figure 5 demonstrates that strong ex vivo immune recall responses are found or may be found in SARS-CoV-2 experienced individuals using polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO; 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1).
[0028] Figure 6 demonstrates that strong ex vivo immune recall responses are found or may be found in SARS-CoV-2 experienced individuals using polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO; 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1).
[0029] Figure 7 shows polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate ex vivo immune recall response in natural SARS-CoV- 2 infection.
[0030] Figure 8 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate higher IFN-g responses in naive and COVID-19 convalescent donors following expansion in culture.
[0031] Figure 9 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate higher IFN-g responses in naive and COVID-19 convalescent donors following expansion in culture.
[0032] Figure 10 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors.
[0033] Figure 11 demonstrates polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors.
[0034] Figure 12 shows polypeptides of SARS-CoV-2, including those of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes within SEQ ID NO: 1), stimulate or may stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors.
[0035] Figure 13 illustrates CD8+IFNy+ T-cell responses from mice immunized with a lipid vaccine including a plasmid housing SEQ ID NO: 1 for expression revealing that mice immunized with SEQ ID NO: 1 showed spike peptide (left side) or envelope/membrane peptide (right side) stimulation specific responses and demonstrating that following immunization both spike and envelope/membrane epitopes are sufficient to activate CD8 T-cells.
[0036] Figure 14 illustrates CD8+CD107a+ T-cell responses from mice immunized with a lipid vaccine including a plasmid housing SEQ ID NO: 1 for expression revealing that mice immunized with SEQ ID NO: 1 showed spike peptide (left side) or envelope/membrane peptide (right side) stimulation specific responses and demonstrating that following immunization both spike and envelope/membrane epitopes are sufficient to activate CD8 T-cells.
[0037] Figure 15 illustrates ability of cytolytic T lymphocytes from mice immunized with a lipid vaccine including a plasmid housing SEQ ID NO: 1 for expression to kill cells expressing either the concatemer peptide of SEQ ID NO: 1 or naturally known spike protein (Wuhan-Hu-1).
[0038] Figure 16 illustrates serum anti-spike antibody levels were measured using serum samples collected on day 21 following immunization with a lipid vaccine including a plasmid housing SEQ ID NO: 1 and demonstrating seroconversion and a dose dependent effect.
[0039] Figure 17 illustrates that the concatemer of SEQ ID NO: 1 at multiple doses tested in mice elicited potent neutralizing titers that were above titers found in SARS-CoV-2 convalescent patient samples.
Detailed Description
[0040] The following description of particular embodiment(s) is merely exemplary in nature and is in no way intended to limit the scope of the invention, its application, or uses, which may, of course, vary. The invention is described with relation to the non-limiting definitions and terminology included herein. These definitions and terminology are not designed to function as a limitation on the scope or practice of the invention but are presented for illustrative and descriptive purposes only. While the compositions and methods are described as using specific materials or a specific order of individual steps, it is appreciated that materials or steps may be interchangeable such that the description of the invention may include multiple parts or steps arranged in many ways as is readily appreciated by one of skill in the art.
[0041] The terminology used herein is for describing particular aspects only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and/or.” As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof. It is to be further understood that where descriptions of various embodiments use the term “comprising,” and/or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.” The term “or a combination thereof’ means a combination including at least one of the foregoing elements.
[0042] It should be understood that every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[0043] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. [0044] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred. Any recited single or multiple feature or aspect in any one claim can be combined or permuted with any other recited feature or aspect in any other claim or claims.
[0045] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Definitions
[0046] To further facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0047] As used herein, the term “biological sample” as refers to any sample of tissue, cells, or secretions from an organism.
[0048] As used herein, the term “medical condition” includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment and/or prevention is desirable, and includes previously and newly identified diseases and other disorders. [0049] As used herein, the term “immune response” refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, malignant melanoma, invading pathogens, cells or tissues infected with pathogens, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. One of ordinary skill would know various assays to determine whether an immune response against a peptide, polypeptide, or related composition was generated. Various B lymphocyte and T lymphocyte assays are well known, such as ELISA, cytotoxic T lymphocyte (CTL) assays, such as chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, and cytokine production assays.
[0050] As used herein, the term “effective amount”, “therapeutically effective amount”, or the like of a composition that is, includes or encodes such T-cell epitope compositions of the present disclosure is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount that results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated, such as SARS-CoV-2 infection or COVID-19 and/or related diseases or leads to a T-cell response in a subject above that of a non-treated control subject. The amount of a composition of the present disclosure administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of pathogen and/or disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions of the present disclosure can also be administered in combination with each other or with one or more additional therapeutic compounds.
[0051] As used herein, the term “T-cell epitope” means an MHC ligand or protein determinant, optionally 7 to 30 amino acids in length, and capable of specific binding to human leukocyte antigen (HLA) molecules and interacting with specific T-cell receptors (TCRs). Generally, T-cell epitopes are linear and do not express specific three-dimensional characteristics. T-cell epitopes are not affected by the presence of denaturing solvents. The ability to interact with T-cell epitopes may in the case of the present disclosure be predicted by in silico methods (De Groot AS et ah, (1997), AIDS Res Hum Retroviruses, 13(7):539-41; Schafer JR et al, (1998), Vaccine, 16( 19): 1880-4; De Groot AS et al, (2001), Vaccine, 19(31):4385-95; De Groot AR et al, (2003), Vaccine, 21(27-30):4486-504, all of which are herein incorporated by reference in their entirety.
[0052] As used herein, the term “T-cell epitope cluster” refers to polypeptide that contains between about 4 to about 40 MHC binding motifs. In particular embodiments, the T-cell epitope cluster contains between about 5 to about 35 MHC binding motifs, between about 8 and about 30 MHC binding motifs; and between about 10 and 20 MHC binding motifs.
[0053] As used herein, the term “immune-stimulating T-cell epitope polypeptide” refers to a molecule capable of inducing an immune response, e.g., a humoral, T cell-based, or innate immune response.
[0054] As used herein, the term “B-cell epitope” means a protein determinant capable of specific binding to an antibody. B-cell epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
[0055] As used herein, the term “MHC complex” refers to a protein complex capable of binding with a specific repertoire of polypeptides known as HLA ligands and transporting said ligands to the cell surface.
[0056] As used herein, the term “MHC Ligand” means a polypeptide capable of binding to one or more specific MHC alleles. The term “HLA ligand” is interchangeable with the term “MHC Ligand”.
[0057] As used herein, the term “T-Cell Receptor” or “TCR” refers to a protein complex expressed by T-cells that is capable of engaging a specific repertoire of MHC/Ligand complexes as presented on the surface of cells.
[0058] As used herein, the term “MHC Binding Motif’ refers to a pattern of amino acids in a protein sequence that predicts binding to a particular MHC allele. [0059] The term “subject” as used herein refers to any living organism in which an immune response is elicited. The term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
[0060] The term “polypeptide” refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. As used herein, a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized. A polypeptide (e.g., a polypeptide comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 53, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69 or variants and fragments thereof, which in aspects may be isolated, synthetic, or recombinant) of the present disclosure, however, can be joined to, linked to, or inserted into another polypeptide (e.g., a heterologous polypeptide) with which it is not normally associated in a cell and still be “isolated” or “purified”. Additionally, one or more T-cell epitopes of the present disclosure can be joined to, linked to, or inserted into another polypeptide wherein said one or more T-cell epitopes of the present disclosure is not naturally included in the polypeptide and/or said one or more T- cell epitopes of the present disclosure is not located at its natural position in the polypeptide. When a polypeptide is recombinantly produced, it can also be substantially free of culture medium, for example, culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation. [0061] As used herein, a “concatemeric” peptide or polypeptide refers to a series of at least two peptides or polypeptides linked together, optionally in a sequence not found in nature. Such linkages may in the form of “string-of-beads” design whereby each polypeptide is linked directly or via a linker to another, adjacent polypeptide of the same or differeing sequence.
[0062] A “variant” peptide or polypeptide (including a variant T-cell epitope) can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations, or a combination of any of these. In some aspects, a variant peptide or polypeptide (including a variant T-cell epitope) can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these provided said variants retain MHC binding propensity and/or TCR specificity. For the purposes of the present disclosure, peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure can include, for example, modified forms of naturally occurring amino acids such as D-stereoisomers, non-naturally occurring amino acids; amino acid analogs; and mimetics. Further, in aspects, peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure can include retro-inverso peptides of the instantly disclosed peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure, provided said peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure at least in part retain MHC binding propensity and/or TCR specificity.
[0063] As used herein, the term “purpose built computer program” refers to a computer program designed to fulfill a specific purpose; typically to analyze a specific set of raw data and answer a specific scientific question.
[0064] As used herein, the term “z-score” indicates how many standard deviations an element is from the mean. A z-score can be calculated from the following formula: z = (X - m) / s; where z is the z-score, X is the value of the element, m is the population mean, and s is the standard deviation.
[0065] Bacteriophage [0066] Provided herein are peptide, DNA, or RNA compositions that are or encode one or more T-cell epitopes as provided herein. Such may be used alone or as part of a delivery mechanism such as a naked peptide, DNA, or RNA molecule, or as part of a nanoparticle, viral particle, or other such delivery mechanism. In some aspets, the present disclosure relates to bacteriophage (or “phage”) surface-expressed or surface-presented SARS-CoV-2 viral antigens (antigenic polypeptides), as well as to phage DNA vaccines comprised of nucleotides that encode the surface-expressed SARS-CoV-2 viral antigens and combinations thereof. Such bacteriophage are presented hereing for exemplary purposes only and are not meant to be a limitation on the disclosure. It is equally appreciated that teaching related to a bacteriophage are equally attributable to other viral or non-viral organisms or delivery systems. The bacteriophage vaccine system provides a well-tolerated and highly scalable system to immunize the general population against SARS-CoV-2 (and related diseases cause by SARS- Cov-2, including COVID-19) both effectively and inexpensively, with the ability to stimulate both cell mediated and humoral responses. Additional phage vaccine systems are described generally in US Patents 9,744,223 and 10,702,591, the contents of which are hereby incorporated by reference in their entirety.
[0067] The present disclosure in some aspects relates to bacteriophages that express and/or possess nucleic acids in nucleotide sequences encoding the concatemeric SARS-CoV-2 peptide(s) as set forth herein. In some aspects, the phages express the concatemeric peptides as described herein. In other aspects, cells or a subject express the concatemeri peptides as described herein. In certain aspects, the phages express the concatemer on its surface. In further aspects, the phages express the concatemer on the surface via fusion to a phage surface expression polypeptide sequence that decorates to the outer capsid surface of the phage. In yet further aspects, the present disclosure relates to nucleic acids encoding the concatemer described herein. In some aspects, the nucleic acids are integrated into the phage’s genome. In further aspects, the nucleic acids are operably linked to a nucleic acid sequence encoding part or all of a phage surface expression polypeptide sequence. In some aspects, the instantly- disclosed phages are irradiated (e.g., after construction by prior to administration to a subject).
[0068] In some aspects, the concatemers of the present disclosure may provide T-cell epitopes to a host subject when expressed on the surface of a phage. T-cell epitopes of the present disclosure are highly conserved among known variants of their source proteins (e.g., present in more than 10% of known variants). T-cell epitopes of the present disclosure comprise at least one putative T-cell epitope as identified by EpiMatrix™ analysis. EpiMatrix™ is a proprietary computer algorithm developed by EpiVax (Providence, Rhode Island), which is used to screen protein sequences for the presence of putative T-cell epitopes. The algorithm uses matrices for prediction of 9- and 10-mer peptides binding to MHC molecules. Each matrix is based on position-specific coefficients related to amino acid binding affinities that are elucidated by a method similar to, but not identical to, the pocket profile method (Sturniolo, T. et al. , Nat. BiotechnoL, 17:555-561, 1999). Input sequences are, for example, parsed into overlapping 9-mer frames or 10-mer where each frame overlaps the last by 8 or 9 amino acids, respectively. Each of the resulting frames form the mutated peptide and the non-mutated peptide are then scored for predicted binding affinity with respect to MHC class I alleles (e.g., but not limited to, HLA-A and HLA-B alleles) and MHC class II alleles (e.g., but not limited to HLA-DRB1 alleles). Raw scores are normalized against the scores of a large sample of randomly generated peptides. The resulting “Z” scores are normally distributed and directly comparable across alleles. The resulting “Z” score is reported. In aspects, any 9-mer or 10-mer peptide with an allele-specific EpiMatrix™ Z-score in excess of 1.64, theoretically the top 5% of any given sample, is considered a putative T-cell epitope.
[0069] Peptides containing clusters of putative T-cell epitopes are more likely to test positive in validating in vitro and in vivo assays. In aspects, the results of the initial EpiMatrix™ analysis are further screened for the presence of putative T-cell epitope “clusters” using a second proprietary algorithm known as Clustimer™ algorithm. The Clustimer™ algorithm identifies sub-regions contained within any given amino acid sequence that contains a statistically unusually high number of putative T cell epitopes. Typical T-cell epitope “clusters” range from about 9 to roughly 30 amino acids in length and, considering their affinity to multiple alleles and across multiple 9-mer frames, can contain anywhere from about 4 to about 40 putative T-cell epitopes.
[0070] The JanusMatrix system (EpiVax, Providence, Rhode Island) is useful for screening peptide sequences for cross-conservation with a host proteome. JanusMatrix is an algorithm that predicts the potential for cross-reactivity between peptide clusters and the host genome or proteome, based on conservation of TCR- facing residues in their putative MHC ligands. The JanusMatrix algorithm first considers all the predicted epitopes contained within a given protein sequence and divides each predicted epitope into its constituent agretope and epitope. Each sequence is then screened against a database of host proteins. Peptides with a compatible MHC-facing agretope (i.e., the agretopes of both the input peptide and its host counterparty are predicted to bind the same MHC allele) and exactly the same TCR-facing epitope are returned. The JanusMatrix Homology Score suggests a bias towards immune tolerance. In the case of a therapeutic protein, cross-conservation between autologous human epitopes and epitopes in the therapeutic may increase the likelihood that such a candidate will be tolerated by the human immune system. In the case of a vaccine, cross-conservation between human epitopes and the antigenic epitopes may indicate that such a candidate utilizes immune camouflage, thereby evading the immune response and making for an ineffective vaccine. When the host is, for example, a human, the peptide clusters are screened against human genomes and proteomes, based on conservation of TCR-facing residues in their putative HLA ligands. The peptides are then scored using the JanusMatrix Homology Score. In aspects, peptides with a JanusMatrix Homology Score below 2.5 or below 3.0 indicate low tolerogenicity potential and may be useful for pharmaceutical formulations and vaccines for the treatment/prevention of SARS- CoV-2 infection and related diseases caused by SARS-CoV-2, including COVID-19, and in aspects may be included from the T cell epitope compositions and methods of the present disclosure. In aspects, peptides with a JanusMatrix Homology Score above 3.0 indicate high tolerogenicity potential and may not be useful for pharmaceutical formulations and vaccines for the treatment/prevention of SARS-CoV-2 infection and related diseases caused by SARS- CoV-2, including COVID-19, and in aspects may be excluded from the T cell epitope compositions and methods of the present disclosure.
[0071] In aspects, T-cell epitopes of the present disclosure (e.g., those included in the instantly-disclosed concatemer) bind to at least one and preferably two or more common HLA class I and/or class II alleles with at least a moderate affinity (e.g., in aspects, <1000 mM ICso, <500 mM ICso, <400 mM ICso, <300 mM ICso, or <200 mM ICso in HLA binding assays based on soluble HLA molecules). In aspects, T-cell epitopes of the present disclosure are capable of being presented at the cell surface by cells in the context of at least one and, in other aspects, two or more alleles of the HLA. In this context, the epitope-HLA complex can be recognized by CD4+ or CD8+ T-cells (in aspects, including natural T-cells) having TCRs that are specific for the epitope-HLA complex and circulating in normal control subjects. In aspects, the recognition of the epitope-HLA complex can cause the matching T-cell to be activated and to secrete activating cytokines and chemokines.
[0072] Bacteriophage display provides a simple way, but not the only way provided in this disclosure, of achieving favorable presentation of peptides to a subject’s immune system. Previous findings have revealed that recombinant bacteriophage can prime strong CD8+ T lymphocyte (CTL) responses both in vitro and in vivo against epitopes displayed in multiple copies on their surface, activate T-helper cells and elicit the production of specific antibodies all normally without adjuvant. In some aspects, the instant disclosure provides a specific vaccine delivery system whereby relevant target antigenic concatemer peptides as disclosed herein are expressed within phage to produce recombinant phage displaying the antigenic concatemer on the phage virion surface (also referred to as “phage vaccine particles” or “PVPs”). PVP vaccines, produced by cloning the concatemer into a phage genome, can be propagated and purified rapidly using inexpensive Luria-Bertani (LB) bacteriological media.
[0073] One of the many advantages provided by a phage display is presentation of multiple copies of peptides on the same phage. A further advantage is that once a first phage display is generated, subsequent production should be far easier and cheaper than the ongoing process of coupling peptides to carriers. There is also good evidence that due to particulate nature, phage- displayed peptides can access both the major histocompatibility complex (MHC) I and MHC II pathways, suggesting phage display vaccines stimulate both cellular and humoral arms of the immune system (although as extracellular antigens, it is expected that the stronger response will be MHC class II biased and result in antibody production). It has been shown that particulate antigens, and phage in particular, can access the MHC I pathway through cross priming, and it is likely that it is this process that stimulates a cellular response. Phage vaccines can also act as nonspecific immune stimulators. It is likely that a combination of the foreign DNA and the repeating peptide motif of the phage coat are responsible for any nonspecific immune stimulation (Adhya, S., C.R. Merril, and B. Biswas, Therapeutic and prophylactic applications of bacteriophage components in modern medicine. Cold Spring Harb Perspect Med, 2014. 4(1): p. a012518). Further, the peptide antigen displayed by the phage comes already covalently conjugated to an insoluble immunogenic carrier with natural adjuvant properties. [0074] There are three main approaches for utilizing phage as vaccine delivery systems. The first being a “phage display vaccine”, the second being a “phage DNA vaccine” and the third being a combination of the two. In the phage display vaccine, immunogenic antigens (epitopes) are fused to the outer phage surface (i.e., capsid), typically by forming a chimera to a surface protein in the phage’s genome. The displayed antigenic proteins or peptides can be selected for their specific binding affinity of antigen-presenting cells. In a “phage DNA vaccine”, foreign antigen genes are incorporated into the phage genome under the control of strong mammalian promoters. Similar to other vectors exploited for transferring genetic material, the phage acts as a passive carrier to transfer the foreign DNA into mammalian cells (such as dendritic and Kupffer cells) when the antigen gene is expressed. In the combinatorial phage vaccines approach, both phage display and phage DNA vaccine platforms are utilized; the approach uses both phage that express the immunizing antigen on its surface (e.g., fusion antigen with its capsid protein) from the phage display approach and also incorporate foreign antigen genes into a phage to be then transduced into a host cell (antigen presenting cell “APC”) for expression.
[0075] The surface expressed antigens are highly immunogenic and may provoke specific cell mediated responses in a subject, the result of which may provide long lasting immunity against SARS-CoV-2. The present disclosure provides a rapidly producible and highly scalable vaccine to stop and contain the spread of this viral infection. The compositions provided by the present disclosure further provide an opportunity to mitigate the possibility of antibody- dependent enhancement (ADE) related complications that may arise due to COVID-19 vaccination.
[0076] The present disclosure features, in some aspects, a phage display system to provide a SARS-CoV-2 vaccine through the presence of SARS-CoV-2 antigenic (T-cell epitope) polypeptides on the surface of the phage. The phage system may display, or be capable thereof displaying, at least one antigenic epitope sequence (e.g., multiple copies). In other aspects, the phage system may display between about one to about 900 copies of an antigenic epitope sequence, including about 2, 3,4 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 , 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, and 800 and any range or value therebetween. In some aspects, the phage may display a polypeptide sequence of multiple SARS-CoV-2 epitopes. The antigenic epitope sequence may feature a sequence of epitopes identified from the genome of the SARS-CoV-2 virus that are presented as linear epitopes to generate broad-spectrum immunity. In some aspects, the polypeptide sequences can be expressed on the surface of a T4 phage and provided as an inoculant without the need of a further adjuvant, although in other aspects, such may be included. The display of antigenic epitopes on a phage may provide unique vaccine nanoparticles (i.e. an antigenic peptide and phage combination chimera) that may provoke mammalian immune systems against SARS-CoV-2.
[0077] In some aspects, the copy number of the expressed concatemer may depend on the surface protein used as the chimera. For example, in T4 phage, there are four different expressed head proteins that the concatemer can be co-expressed with: gp23, gp24, Hoc and Soc. The copy numbers of each are 930, 55, 870 and 155, respectively. As such, the present disclosure provides a variety of possible levels of expression based on the phage protein selected.
[0078] As disclosed herein, vaccination with a phage (such as a T4 phage) that displays the optimized collective antigenic epitopes of SARS-CoV-2 virus (as disclosed herein) provides a number of advantages. The utilized antigenic polypeptides are specifically an aggregation of several linear epitopes (peptides) selected from all parts of the SARS-CoV-2 viral genomes to provoke broad-spectrum long lasting immunity. As such, simple spontaneous mutation in the viral spike proteins will not allow the SARS-CoV-2 virus to bypass the host’s immune system, a feature quite distinct from vaccines designed solely on the basis of spike protein sequence.
[0079] In some aspects, the present disclosure concerns use of a whole T4 phage. Whole T4 phage particles possess numerous intrinsic characteristics that make them ideal as vaccine delivery vehicles. T4 phage offers four surface proteins, gp23, gp24, Hoc, and Soc, with varying copy numbers capable of co-expressing the concatemer. Bacteriophage expressing the concatemers disclosed herein may produce viral specific protective responses when administered to mammalian systems.
[0080] For use as phage display vaccines, the particulate nature of phage means they are far easier and cheaper to purify than soluble recombinant proteins since a simple centrifugation/tangential flow filtration (TFF) /ultra-filtration steps are sufficient to remove the majority of soluble contaminants and associated toxins (mainly from the composition of bacterial cell walls). Therefore, this is very easy for scaling-up the phage based vaccine productions. The vaccine production cost is much cheaper than conventional egg based or cell culture or whole cell inactivated vaccines. Further, the natural stability of phage vaccine particles provides easy storage and no cold chain is required for distribution. Additionally, the vaccine peptide antigens come already covalently attached to an insoluble immunogenic carrier (i.e. phage particles) with natural adjuvant properties, without the need for complex chemical conjugation and downstream purification processes that must be repeated with each vaccine batch. In some aspects, the disclosed phage vaccines as provided herein do not require any additional adjuvant.
[0081] The present disclosure also in some aspects concerns the discovery of bacteriophage surface-expressed SARS-CoV-2 virus antigens that are highly immunogenic and specifically provoke cell-mediated response to neutralize the SARS-CoV-2 viral infection in human subjects. The present disclosure provides for the development of a rapid vaccine to stop the epidemic related to SARS-CoV-2 virus infection by inducing cell-mediated immunity instead of simply humoral immunity. A major advantage of the provided vaccine is to provoke a very strong long lasting cell mediated immunity by display of high copy numbers of SARS-CoV-2 virus specific selective linear epitopes as a T4 capsid protein fusions. This specific presentation of linear epitopes on T4 phage head, as one non-limiting example, can skew the immunogenic response towards cell mediated response and as a result will reduce complications associated with antibody-dependent enhancement (ADE) related to SARS-CoV-2 vaccination. The major motivating reason for using the specific in silico systems described herein is that this approach allows for identification of conserved linear vaccine epitopes from the available sequence information of a given organism. A phage based vaccine platform is also better suited for expressing linear epitopes (mainly provoked cell mediated responses) rather than conformational epitopes (provoke humoral/ antibody mediated responses).
[0082] The specific DNA sequences coding for the concatemer peptide sequence can be displayed on T4 phage using the protocol, for example, as described Zhu et ah, A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering bioRxiv, January 19, 2021 (herein incorporated by reference in its entirety). Specifically, engineered DNAs corresponding to the concatemer peptide sequence (or fragments thereof) can be incorporated into bacteriophage T4 genome as outlined in Figure 1 of Zhu et al. Each DNA is then introduced into E. coli as a donor plasmid and recombined into injected phage genome through CRISPR- targeted genome editing. Different combinations of CoV-2 inserts can then be generated by simple phage infections and the recombinant phages in the progeny can be identified. By repeating this process, a pipeline of multiplex T4-concatemer peptide sequence and/or individual antigenic fragment vaccine phages can be rapidly constructed. Selected vaccine candidates can then be screened in a mouse model to identify the most potent vaccine.
Lipid nanoparticles
[0083] In some aspect of this disclosure, the polypeptides or DNA or RNA nucleotide sequences that encode the polypetides may be delivered or presented by a lipid nanoparticle. Lipid nanoparticles may include one or more ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2021/029468; PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551 ; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.
[0084] LNPs may be used in some aspects to carry and/or deliver protein or nucleic acid cargo to a subject or a portion thereof such as a cell or cellular compartment. DNA and RNA vaccines utilizing such LNPs share many similarities, but each target different cellular environments. For example, DNA vaccines target and are used in the nucleus of a cell, whereas RNA vaccines target and are expressed in the cytosol. This makes mRNA vaccines easier to deliver, yet both may capitalize on the success of recent advances in LNP formulations and sometimes other modifications to the nucleic acid cargo itself that may improve overall function, briefly as described in Reichumth, et ah, Ther Deliv, 2016 May; 7(5): 319-334, the entire contents of which are incorporated herein by reference.
[0085] As such, provided are nucleic acid (DNA or RNA) compositions that are packaged in or on a lipid nanoparticle. Such LNPs are typically an aquesou core that houses the nucleic acid of interest surrounded by a lipid bilayer of a combination of one or more lipids that may serve distinct functions as described by Li W, Szoka FC Jr, Pharm Res. 2007 Mar; 24(3):438- 49. Cationic lipids and newer ionizable lipids may be used such as those described herein or as by Kanasty R, et al., Nat Mater. 2013 Nov; 12(11):967-77. Overall, these LNPs provide unique advantages and allow alternative delivery systems for the peptides or DNA or RNA nucleic acids as described herein for presentation to a subject and development of immunity to SARS- CoV-2 as provided herein.
Polypeptides and Nucleic Acids
[0086] In some aspects of the present disclosure, the polypeptides (T-cell epitopes) were designed and/or optimized using an in silico approach in combination with a proprietary algorithm. In some aspects, a proprietary algorithm identified exclusive linear epitopes from an analysis of the complete genome sequence of SARS-CoV-2 virus. In further aspects, the antigenic polypeptides are a concatemer of multiple identified SARS-CoV-2 epitopes. In certain aspects, the antigenic polypeptide is a concatemer of 2, 3, 4, 5, 6, 7 ,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 T-cell epitopes. As identified herein, each T-cell epitope contemplated features between 9 and 10 amino acids in sequence.
[0087] In some aspects, the linear T-cell epitopes are then linked together as a concatemer to provide antigenic peptide sequences for generating broad spectrum immunity against SARS- CoV-2. In some aspects of the present disclosure, a unique long polypeptide is composed by assembly of various identified linear epitopes from SARS-CoV-2 virus. These epitopes were identified and selected from SARS-CoV-2 amino acid sequences (epitopes) in part based on the ability to interact with T-cells and thus provoke cell-mediated responses.
[0088] The T-cell epitope polypeptides of the present disclosure were designed by sequentially assembling identified HLA binding linear sequences into one or more unique polypeptide sequences. As disclosed herein in the examples, the resulting phage expressed a concatemer of 34 in silico identified SARS-CoV-2 epitopes assembled in a head-tail fashion to provide a single polypeptide (also referred to a concatemer). It will be apparent to those skilled in the art that the order of assembly can be rearranged and regrouped into further polypeptide sequences and placed as described herein in a phage vaccine system. For example, as set forth herein, 34 epitopes were identified using the proprietary in silico approach. Those skilled in the art will appreciate that each epitope can be expressed individually or as a doublet, triplet, quadruplet, quintuplet and so on, in any order and up to a single polypeptide of every of the identified epitopes.
[0089] The present disclosure concerns T-cell epitope polypeptide sequences of one or multiple SARS-CoV-2 epitope peptides. Using the EpiVax proprietary “iVAX” vaccine design platform, the SARS-CoV-2 envelope, membrane, and spike proteins can be analyzed for the presence of HLA class I and class II T cell epitopes. Specific regions where both HLA class I and class II T cell epitopes cluster can be thus identified and clusters with the highest predicted likelihood of human HLA class I and II binding, the broadest coverage of human HLA, highest SARS-CoV-2 conservation, and the lowest cross-conservation with the human proteome may then be selected. A homology analysis tool may further eliminate sequences that could potentially elicit an undesired autoimmune or regulatory T cell response due to homology with the human genome. A comprehensive description of the advanced set of tools used to identify such epitopes in silico can be further found in De Groot et ah, Better Epitope Discovery, Precision Immune Engineering, and Accelerated Vaccine Design Using Immunoinformatics Tools. Front Immunol. 2020 Apr 7;11 :442. doi: 10.3389/fimmu.2020.00442. PMID: 32318055; PMCID: PMC7154102. Resulting epitopes may then be concatenated head to tail and arranged in an order that minimizes potential T cell immunogenicity at epitope junctions.
[0090] In some aspects, and as described herein, the concatemer containing the identified 34 T-cell epitope clusters was selected for inclusion in the vaccine using advanced immunoinformatics tools. Each epitope cluster is optioanlly 15 to 25 amino acids in length and contains multiple HLA class I and class II-restricted T-cell epitopes. Altogether, the 34 epitope clusters comprise hundreds of CTL and T helper epitopes.
[0091] In some aspects, the T-cell epitopes may be further selected to exclude human-like or related sequences. The rationale for excluding human-like sequences is that T-cells that recognize antigen-derived epitopes sharing TCR contacts with epitopes derived from the human proteome may be deleted or rendered anergic before release into the periphery during thymic selection. Therefore, vaccine components targeting these T-cells may be ineffective. In addition, vaccine-induced immune responses targeting cross-reactive epitopes may induce unwanted autoimmune responses targeting the human homologs of the cross-reactive epitopes identified by JanusMatrix. As a result, vaccine safety may be reduced. [0092] In some aspects, the concatemer (linear polypeptides of assembled SARS-CoV-2 antigenic peptides) of up to 34 epitope clusters was designed. No class II junctional immunogenicity potential was found at the gpD/epitope interfaces. The resulting vaccine collectively features highly conserved and promiscuous SARS-CoV-2-specific T cell epitopes that broadly cover HLA diversity of the human population.
[0093] In certain aspects, the present disclosure concerns polypeptide sequences that are derived from the SARS-CoV-2 envelope, membrane, or spike proteins and designed for optimal expression and immunogenicity. In the instances where the peptide is intended to be expressed or delivered by a phage, the genome of a phage can be modified as described herein to express the polypeptide, such that each T4 phage may express the 34 epitope clusters on the surface of the phage. It will be apparent that phage expressing the concatemer polypeptide can be mixed with other phage expressing other polypeptides to provide a stoichiometric phage- display cocktail, either in equimolar or non-equimolar ratios.
[0094] In some aspects, the concatemer polypeptide(s) of the present disclosure are set forth in the accompanying sequence listing and below. In some aspects, the polypeptide includes an amino acid sequence of:
[0095]
GKGYHLMSFPQSAPHQTLLALHRSYLTPGDSSVLSFELLHAPATVCGQKLIANQFNS
AIGKIQDSLQIPFAMQMAYRFNGIGVPKEITVATSRTLSYYPTNFTISVTTEILPVICLL
QFAYANRNRFLYINDGVYFASTEKSNIIRTDEMIAQYTSALLANKCVNFNFNGLTGT
GVLTEDGYFKIYSKHTPINLQDLFLPFFSNVTWFHAIHVSGTNGTTGRLQSLQTYVTQ
QL VEGFN C YFPLQS Y GF QPTN GV GY QP YFLGV YYHKNNKS WMESEFRV Y S S ANN C
TFEYVLDKYFKNHTSPDVDLPPAYTNSFTRGVYYPRTFLLKYNENGTITDASRVKNL
NSSRVPDTQSLLIVNNATNVVIKVGGNYNYLYRLFRKSNLKPFERDIMYSFVSEETGT
LIVNFGGFNFSQILPDPSKPSKRSLACFVLAAVYRINWIGPGPGLLQYGSFCTQLNRAL
TGIAVEQISGINASVVNIQKEIDVVIGIVNNTVYDPLRGHLRIAGHHLGRCDLKKLLEQ
WNLVIGFLFLTWGPGPGTSNFRVQPTESIVRFFGEVFNATRFASVYAWNRKRISNCV
AD Y S VL YN S ASF STFKIGNYKLNTDHS S S SDNILS YFI ASFRLF ARTRSM WSFNPETNI
LLNV (SEQ ID NO: 1). [0096] Optionally, the polypeptide includes the sequence of:
GKGYHLMSFPQSAPHGSGSGQTLLALHRSYLTPGDSSGSGSGVLSFELLHAPATVCG
GSGSGQKLIANQFNSAIGKIQDSLGSGSGQIPFAMQMAYRFNGIGVGSGSGPKEITVA
TSRTLSYYGSGSGPTNFTISVTTEILPVGSGSGICLLQFAYANRNRFLYIGSGSGNDGV
YFASTEKSNIIRGSGSGTDEMIAQYTSALLAGSGSGNKCVNFNFNGLTGTGVLTEGSG
SGDGYFKIYSKHTPINLGSGSGQDLFLPFFSNVTWFHAIHVSGTNGTGSGSGTGRLQS
LQTYVTQQLGSGSGVEGFNCYFPLQS Y GF QPTNGV GY QPY GSGSGFLGVYYHKNNK
SWMESEFRVYSSANNCTFEYVGSGSGLDKYFKNHTSPDVDLGSGSGPPAYTNSFTRG
VYYGSGSGPRTFLLKYNENGTITDAGSGSGSRVKNLNSSRVPDGSGSGTQSLLIVNN
ATNVVIKVGSGSGGGNYNYLYRLFRKSNLKPFERDIGSGSGMYSFVSEETGTLIVNG
SGSGFGGFNFSQILPDPSKPSKRSGSGSGLACFVLAAVYRINWIGPGPGLLQYGSFCT
QLNRALTGIAVEQGSGSGISGINASVVNIQKEIGSGSGDVVIGIVNNTVYDPLGSGSGR
GHLRIAGHHLGRCDGSGSGLKKLLEQWNLVIGFLFLTWGPGPGTSNFRVQPTESIVR
FGSGSGFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKGSGSGIGNYK
LNTDHSSSSDNIGSGSGLSYFIASFRLFARTRSMWSFNPETNILLNV (SEQ ID NO: 124).
[0097] In further aspects, the concatemers are 34 SARS-CoV-2 identified epitopes, assembled in a head-to fashion. Table 1 sets out exemplary individual peptides, the protein from SARS-CoV-2 the peptide originates from (with starting amino acid) and the nucleic acids utilized in the constructs herein to encode such with Spike protein full sequence found at Genbank Accession No: YP 009724390.1, Envelope protein full sequence found at Genbank Accession No: YP 009724392.1, and Membrane protein full sequence found at Genbank Accession No: YP 009724393.1.
[0098] TABLE 1
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
[0099] In some aspects, each of the polypeptide sequences are linear and continuous. In other aspects, the polypeptides may include additional amino acids at either/or both terminals and/or internally within the sequences. In some aspects, the polypeptides are fused to a surface phage protein, such as gp23, gp24, Hoc, and/or Soc in a T4 phage. [00100] In other aspects, the epitope sequences of each concatemer are separated by a linker. In some instance, the linker may include an amino acid sequence of GPGPG (SEQ ID NO: 71).
[00101] In further aspects, other concatemers are envisioned. Each of SEQ ID NOS: 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, and 69 may be arranged in any order and in any number, including as one continuous concatemer, or as two or more concatemers. In other aspects, each concatemer may include between 9, 10, 11, 12, or 13 epitope peptides.
[00102] In some aspects, the polypeptides introduced and/or expressed by the phage include variants of the sequences as set forth in SEQ ID NO: 1 or SEQ ID NO: 124. The variants may share between about 80% to about 100% identity with the sequences as set forth in SEQ ID NO: 1, or SEQ ID NO: 124, or as within each of the above referenced T-cell epitopes contained in SEQ ID NO: 1 or SEQ ID NO: 124. For example, the variants may share 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the amino acid sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 124 or as any of the T-cell epitopes within SEQ ID NO: 1 or SEQ ID NO: 124. In aspects, the present disclosure also encompasses polypeptides (e.g., T-cell epitopes and T-cell epitope compositions as disclosed herein) having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide encoded by a nucleic acid molecule of the invention. Similarity is determined by conserved amino acid substitution or by deletion or insertion without significant change in physical characteristics of the polypeptide. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions or delection are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, Met, and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues His, Lys and Arg and replacements among the aromatic residues Trp, Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found (Bowie JU et al., (1990), Science, 247(4948):130610, which is herein incorporated by reference in its entirety). In some aspects, T-cell epitopes of the present disclosure can include insertions, delections, allelic or sequence variants (“mutants”) or analogs thereof, or can include chemical modifications (e.g., pegylation, glycosylation). In some aspects, a mutant retains the same functions performed by a polypeptide encoded by a nucleic acid molecule of the present disclosure, particularly MHC binding propensity and/or TCR (T-cell receptor) specificity. In aspects, a mutant can provide for enhanced binding to MHC molecules. In aspects, a mutant can lead to enhanced binding to TCRs. In another instance, a mutant can lead to a decrease in binding to MHC molecules and/or TCRs. Also contemplated is a mutant that binds, but does not allow signaling via the TCR.
[00103] As particular examples and not as limitation, sequence variations that may be included in a peptide as provided herein are listed in Table 2. These peptides variants are also presented with a frequency percentage number representing the preference for selection of that particular mutation. A frequency percentage of 5% or greater represents most preferred sequence variations. In Table 2, the reference sequences are as listed in Table 1 with the start reference number relative to the full length SARS-CoV-2 original reference sequence and the N-terminal residue in the sequence being the number of the start reference amino acid. Variants with a dash are deletions of the reference amino acid residue. Amino acids that are more commonly mutated (e.g. more listed variants of that residue) or deleted in the listing in Table 2 are the most tolerant of mutation and maintenance of function.
[00104] Table 2:
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
[00105] Variants in SEQ ID NO: 1 or SEQ ID NO: 124 are optionally found in one or more of the individual T-cell epitopes of Table 1. Optionally, the number of T-cell epitopes with a variant include but are not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or all 34 T-cell epitopes have a variant, and optionally any of the listed spacers. The number of variant amino acids (substitution, deletion, insertion) within each variant is optionally 6 or fewer, optionally 5 or fewer, optionally 4 or fewer, optionally 3 or fewer, optionally 2 or fewer, optionally 1, optionally zero.
[00106] A variant T-cell epitope within SEQ ID NO: 1 or SEQ ID NO: 124 may be any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 53, 65, 67, or 69, or any combination thereof, or in one or more linkers optionally of SEQ ID NO: 71, or both. In some aspects, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 47, optionally at residue T9. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 61, optionally at residue Q19. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 51, optionally at residue A63. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 37, optionally at residue P26. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 27, optionally at any one or more of residues H69, V70, A67, or delections at H69 and V70, or a substitution at A67 with deletions at H69 and V70. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 19, optionally at residue T95. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 33, optionally at one or more of residues G142, E156, F157, R158, Y144, G142, V143, and Y145, or any combination thereof. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 25, optionally at any one of residues N211 or L212, or both. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 65, optionally at any one of residues G339, R346, S371, S373, S375, or any combination therereof. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 45, optionally at any one of residues L452, G446, or any combination therereof. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 31, optionally at any one of residues N501, E484, Q493, G496, Q498, Y505, or any combination therereof. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 23, optionally at residue T547. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 15, optionally at residue T716. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 53, optionally at residue N764. Optionally, a variant T-cell epitope within SEQ ID NO: 1 may be within SEQ ID NO: 55, optionally at residue VI 176.
[00107] In some aspects, the present disclosure further includes nucleic acids encoding the concatemer polypeptides derived from SARS-CoV-2 optionally encoding SEQ ID NO: 1 or SEQ ID NO: 124 or any T-cell epitope or linker therein. Such nucleic acid sequences may be derived from a SARS-CoV-2 genome or synthesized based on the desired amino acid sequence with appropriate codon sequences assembled to provide such. As set forth herein, nucleic acids encoding the concatemer polypeptide may be introduced into the genome of a T4 phage. Alternatively, the nucleic acids may be packaged into a plasmid or other nucleotide sequence for delivery by a lipid particle. One of ordinary skill in the art understands from a protein sequence the nucleotide sequence(s) that are able to generate that protein sequence. For example, nucleic acid sequences encoding the amino acid sequences as set forth herein are readily apparent to those skilled in the art based on the codon coding options known in the art. For example, Ala residues can be encoded by a DNA sequence of GCT, GCC, GCA or GCG, Phe residues can be encoded by TTT or TTC, Ser residues by TCT, TCC, TCA, TCG, AGT or AGG, Tyr residues by TAT or TAC, Trp by TGG, Leu by CTT, CTC, CTA, or CTG, lie by ATT, ATC or ATA, Met by ATG, Val by GTT, GTC, GTA or GTG, Gly by GGT, GGC, GGA or GGG, Asp by GAT or GAC, Glu by GAA or GAG, Lys by AAA or AAG, Thr by ACT, ACC, ACA or ACG, Pro by CCT, CCC, CCA or CCG, His by CAT or CAC, Gin by CAA or CAG, Asn by AAT or AAC, Arg by AGA, AGG, CGG, CGA, CGC or CGT, Cys by TGT or TGC and STOP by TAA, TAG or TGA. Thus, from any given protein sequence, the nucleotide sequence that may be included in the genome of a phage to produce upon transcription and translation the concatemers peptide of SEQ ID NO: 1 or any variant thereof is well understood in the art and explicitly understood as presented within this specification for any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 53, 65, 67, or 69, or any variant thereof, or any combination thereof, or in one or more linkers optionally of SEQ ID NO: 71, or both.
[00108] In some aspects, the nucleic acids encoding the antigenic concatemer polypeptides may be fused with nucleic acids encoding additional polypeptides or polypeptide sequences, including by a linker encoding polypeptide such as GPGPG (SEQ ID NO: 71), AAY, or a spacer. Those skilled in the art will appreciate that such fusion may further require the presence of further additional nucleic acids such as to maintain the reading frame for accurate translation or for other purposes such as introducing restriction enzyme cleavage sites to allow for cleaving and ligating to achieve a desired construct for introduction into the phage genome. Nucleic acid sequences encoding the amino acid sequences as set forth herein are readily apparent to those skilled in the art based on the codon coding options known in the art. For example, Ala residues can be encoded by a DNA sequence of GCT, GCC, GCA or GCG, Phe residues can be encoded by TTT or TTC, Ser residues by TCT, TCC, TCA, TCG, AGT or AGG, Tyr residues by TAT or TAC, Trp by TGG, Leu by CTT, CTC, CTA, or CTG, lie by ATT, ATC or ATA, Met by ATG, Val by GTT, GTC, GTA or GTG, Gly by GGT, GGC, GGA or GGG, Asp by GAT or GAC, Glu by GAA or GAG, Lys by AAA or AAG, Thr by ACT, ACC, ACA or ACG, Pro by CCT, CCC, CCA or CCG, His by CAT or CAC, Gin by CAA or CAG, Asn by AAT or AAC, Arg by AGA, AGG, CGG, CGA, CGC or CGT, Cys by TGT or TGC and STOP by TAA, TAG or TGA.
[00109] The nucleic acid sequences may encode the antigenic concatemer polypeptide fused to a further polypeptide sequence. Those skilled in the art will appreciate that to achieve expression of both successfully, the reading frame will have to be maintained. In some instances, the nucleic acids encode a fusion or chimera of an antigenic concatemer polypeptide and an additional sequence, typically fused at the amino or carboxyl termini. In some aspects, the additional sequence may include a spacer and/or a surface expression polypeptide sequence. In some aspects, the co-expressed surface expression polypeptide sequence allows for the display of the antigenic polypeptide on the surface of the phage, such as a structural or decorative protein on the capsid of the phage. For example, as described herein, with a T4 phage, the presence of a gp23, gp24, Hoc, and/or Soc of phage T4 is fused at the amino terminus of the antigenic polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO: 124.
[00110] In some aspects, the nucleic acids encoding the antigenic chimera include a surface display sequence and/or a restriction enzyme site. As set forth in the examples herein, in some instances, the nucleic acids encode a capsid head protein, such as gp23, gp24, Hoc, and/or Soc, fused to the SARS-CoV-2 derived antigenic concatemer polypeptide with further engineered restriction sites for ligation into the genome of a T4 phage. In certain aspects, the concatemer polypeptide is encoded by the nucleic acid sequence as follows:
GGGAAAGGCTATCACTTAATGAGCTTTCCTCAGTCTGCGCCTCACCAGACTTTGT
TAGCGCTTCATCGTAGCTATTTGACACCGGGGGATTCATCCGTTCTTTCTTTCGAG
CTGCTTCACGCACCTGCCACCGTATGTGGTCAAAAATTGATAGCTAATCAGTTTA
ACAGCGCCATTGGTAAGATCCAGGACAGCTTACAGATACCGTTTGCGATGCAGA
TGGCCTATAGATTTAATGGGATAGGAGTCCCCAAGGAAATCACTGTGGCGACTA
GCCGTACTTTGTCCTATTATCCGACTAATTTTACTATTTCAGTGACTACGGAGATT
CTTCCGGTGATTTGTTTACTTCAGTTTGCGTACGCCAACCGCAATCGCTTTCTTTA
TATAAATGATGGGGTCTACTTCGCTTCAACTGAAAAGAGTAACATTATTCGGACG
GACGAAATGATAGCGCAGTATACTAGTGCGTTATTGGCTAACAAGTGTGTTAATT
TTAATTTCAACGGGTTAACAGGGACGGGGGTGTTGACAGAAGACGGATACTTCA
AGATATATAGCAAACATACCCCGATCAATCTTCAAGACCTGTTCCTGCCCTTCTT
TTCTAATGTTACTTGGTTTCACGCAATACATGTTTCTGGCACCAATGGCACAGCG
CGCHCHGGTCGCTTACAGTCGTTACAAACATACGTTACACAACAACTGGTTGAAG GTTTTAATTGCTACTTCCCGCTGCAGAGTTACGGATTTCAACCGACAAATGGGGT
TGGTTATCAGCCATATTTTTTAGGAGTGTATTATCATAAGAACAACAAAAGTTGG
ATGGAGTCCGAATTTCGTGTGTACTCTAGCGCCAATAACTGTACTTTTGAGTATG
TTTTAGATAAGTATTTCAAGAACCATACTTCGCCGGACGTTGACCTTCCACCTGC
CTACACTAACTCATTTACGCGCGGCGTTTATTACCCTCGGACGTTCCTGCTGAAG
TATAATGAGAACGGAACCATAACCGATGCGAGCCGGGTGAAGAATCTTAACTCA
TCTAGAGTCCCAGATACTCAATCATTACTGATCGTGAACAATGCCACTAATGTTG
TCATTAAAGTAGGAGGCAACTACAATTATTTGTACCGTTTGTTCCGCAAAAGCAA
TCTTAAACCGTTCGAACGGGACATAATGTACAGCTTTGTCAGTGAAGAAACGGG
TACATTAATTGTTAATTTTGGTGGCTTCAATTTTTCACAGATATTACCAGACCCGT
CAAAACCGTCTAAGCGGAGTGCGCGC7TNGCATGTTTCGTGCTTGCTGCAGTGTA
TCGTATAAATTGGATAGGCCCTGGACCTGGTTTGTTACAATATGGCTCTTTCTGCA
CACAGCTTAACCGTGCTCTTACGGGGATCGCTGTGGAGCAAATATCAGGGATTA
ATGCCTCGGTTGTCAATATACAAAAAGAAATTGACGTGGTTATTGGTATAGTTAA
CAATACTGTCTATGACCCTTTGAGAGGGCATCTGCGGATCGCGGGTCACCATTTG
GGCCGGTGTGACCTTAAAAAGTTGTTGGAACAATGGAACTTGGTCATAGGATTCT
TGTTTCTGACTTGGGGTCCCGGACCGGGCACTTCAAATTTTAGAGTTCAGCCTAC
AGAGAGCATTGTACGTTTCTTCGGCGAAGTTTTTAACGCGACGCGCTTCGCATCC
GTCTACGCATGGAATCGTAAACGTATCTCGAATTGCGTCGCCGATTATTCCGTCC
TTTATAACAGCGCATCGTTTTCAACGTTCAAGATCGGGAACTACAAATTGAATAC
CGACCATAGCTCCTCGTCGGACAATATATTATCGTACTTTATTGCGTCTTTCAGAC
TTTTCGCCCGCACTCGTAGTATGTGGTCGTTTAACCCGGAGACGAATATACTTCTT
AACGTTTAA (SEQ ID NO: 2) (the stop TAA in bold) or
GGCAAAGGCTATCATCTGATGAGCTTTCCGCAGAGCGCGCCGCATGGCAGCGGC
AGCGGCCAGACCCTGCTGGCGCTGCATCGCAGCTATCTGACCCCGGGCGATAGC
AGCGGC AGCGGC AGCGGCGT GCT GAGCTTTGAACT GCT GCAT GCGCCGGCGACC
GTGTGCGGCGGCAGCGGCAGCGGCCAGAAACTGATTGCGAACCAGTTTAACAGC
GCGATTGGCAAAATTCAGGATAGCCTGGGCAGCGGCAGCGGCCAGATTCCGTTT
GCGATGCAGATGGCGTATCGCTTTAACGGCATTGGCGTGGGCAGCGGCAGCGGC
CCGAAAGAAATTACCGTGGCGACCAGCCGCACCCTGAGCTATTATGGCAGCGGC
AGCGGCCCGACCAACTTTACCATTAGCGTGACCACCGAAATTCTGCCGGTGGGC
AGCGGCAGCGGCATTTGCCTGCTGCAGTTTGCGTATGCGAACCGCAACCGCTTTC
TGTATATTGGCAGCGGCAGCGGCAACGATGGCGTGTATTTTGCGAGCACCGAAA AAAGCAACATTATTCGCGGCAGCGGCAGCGGCACCGATGAAATGATTGCGCAGT
ATACCAGCGCGCTGCTGGCGGGCAGCGGCAGCGGCAACAAATGCGTGAACTTTA
ACTTTAACGGCCTGACCGGCACCGGCGTGCTGACCGAAGGCAGCGGCAGCGGCG
ATGGCTATTTTAAAATTTATAGCAAACATACCCCGATTAACCTGGGCAGCGGCAG
CGGCCAGGATCTGTTTCTGCCGTTTTTTAGCAACGTGACCTGGTTTCATGCGATTC
ATGTGAGCGGCACCAACGGCACCGGCAGCGGCAGCGGCACCGGCCGCCTGCAGA
GCCTGCAGACCTATGTGACCCAGCAGCTGGGCAGCGGCAGCGGCGTGGAAGGCT
TTAACTGCTATTTTCCGCTGCAGAGCTATGGCTTTCAGCCGACCAACGGCGTGGG
CTATC AGCCGTAT GGC AGCGGC AGCGGCTTTCT GGGCGT GTATTATC ATAAAAAC
A AC A A A AGC T GG AT GG A A AGCG A ATTTC GCGTGTAT AGC AGC GC G A AC A AC TGC
ACCTTTGAATATGTGGGCAGCGGCAGCGGCCTGGATAAATATTTTAAAAACCAT
ACCAGCCCGGATGTGGATCTGGGCAGCGGCAGCGGCCCGCCGGCGTATACCAAC
AGCTTTACCCGCGGCGTGTATTATGGCAGCGGCAGCGGCCCGCGCACCTTTCTGC
TGAAATATAACGAAAACGGCACCATTACCGATGCGGGCAGCGGCAGCGGCAGCC
GCGT GAAAAACCT GAACAGC AGCCGCGT GCCGGAT GGCAGCGGC AGCGGC ACCC
AGAGCCTGCTGATTGTGAAC AACGCGACC AACGT GGT GATTAA AGT GGGC AGCG
GCAGCGGCGGCGGCAACTATAACTATCTGTATCGCCTGTTTCGCAAAAGCAACCT
GAAACCGTTTGAACGCGATATTGGCAGCGGCAGCGGCATGTATAGCTTTGTGAG
CGAAGAAACCGGCACCCTGATTGTGAACGGCAGCGGCAGCGGCTTTGGCGGCTT
TAACTTTAGCCAGATTCTGCCGGATCCGAGCAAACCGAGCAAACGCAGCGGCAG
CGGCAGCGGCCTGGCGTGCTTTGTGCTGGCGGCGGTGTATCGCATTAACTGGATT
GGCCCGGGCCCGGGCCTGCTGCAGTATGGCAGCTTTTGCACCCAGCTGAACCGC
GCGCTGACCGGCATTGCGGTGGAACAGGGCAGCGGCAGCGGCATTAGCGGCATT
AACGCGAGCGTGGTGAACATTCAGAAAGAAATTGGCAGCGGCAGCGGCGATGTG
GTGATTGGCATTGTGAACAACACCGTGTATGATCCGCTGGGCAGCGGCAGCGGC
CGCGGCCATCTGCGCATTGCGGGCCATCATCTGGGCCGCTGCGATGGCAGCGGC
AGCGGCCTGAAAAAACTGCTGGAACAGTGGAACCTGGTGATTGGCTTTCTGTTTC
TGACCTGGGGCCCGGGCCCGGGCACCAGCAACTTTCGCGTGCAGCCGACCGAAA
GCATTGTGCGCTTTGGCAGCGGCAGCGGCTTTGGCGAAGTGTTTAACGCGACCCG
CTTTGCGAGCGTGTATGCGTGGAACCGCAAACGCATTAGCAACTGCGTGGCGGA
TTATAGCGTGCTGTATAACAGCGCGAGCTTTAGCACCTTTAAAGGCAGCGGCAGC
GGCATTGGCAACTATAAACTGAACACCGATCATAGCAGCAGCAGCGATAACATT
GGCAGCGGCAGCGGCCTGAGCTATTTTATTGCGAGCTTTCGCCTGTTTGCGCGCA CCCGCAGCATGTGGAGCTTTAACCCGGAAACCAACATTCTGCTGAACGTG (SEQ ID NO: 125).
[00111] In other aspects, the nucleic acids may include variants of the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 125. The variants may share between about 80% to about 100% identity with the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 125. For example, the variants may share 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 125. In further aspects, other concatemers are envisioned. Any one or more of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 70 may be arranged in any order, including as one continuous concatemer, or as two or more concatemers, or optionally as separated by a linker.
[00112] In some aspects, the nucleic acids include a 5’ nucleic acid sequence that add additional amino acids between the head protein of T4 phage and the antigenic concatemer polypeptide.
[00113] In aspects, the instant disclosure is directed to a nucleic acid (e.g., DNA or RNA, including mRNA) encoding one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as described herein.
[00114] In some aspects, the activation or promoter sequence of the surface protein, such as the gp23, gp24, Hoc, and/or Soc gene within T4 phage, drives the production and expression of the concatemer. In other aspects, the nucleic acids encoding the concatemer may be under the control of a eukaryotic promoter, such as CMV, SV40, CAG, U6 etc. Such a promoter may be introduced by cloning inside a nonessential region of a phage genome to produce the phage based DNA vaccine. Accordingly, when presented in a mammalian system, such as by injection, this phage vaccine may act as a DNA vaccine and induce potent immune response by expressing foreign antigen inside of antigen presenting cells (APCs). For example, the array of the linear epitopes of the construct (i.e. SEQ ID NO: 2) can be cloned inside the T4 phage genome under a strong eukaryotic promoter. This may allow for optimum expression of antigenic epitopes inside APCs like macrophages, dendritic cells, Langerhans cells and Kupffer cells to provoke strong immunogenic response against SARS-CoV-2. [00115] In some aspects, a nucleotide sequence may be in the form of DNA or RNA. Illustratively, a nucleotide sequence encoding a SARS-CoV-2 T-cell epitope or concatemer of two or more such epitopes may include mRNA. mRNA is understood in the art as a single- stranded ribonucleic acid copy of a gene, including pre-mRNA and mature mRNA, a spliced mRNA, a 5’ capped mRNA, an edited mRNA and a polyadenylated mRNA. mRNA can include single stranded RNA nucleotide sequences encoding one or more T-cell epitopes as provided herein and optionally marked by a 5’ cap, such as an RNA 7-methylguanosine cap or an RNA m7G cap. An mRNA encoding one or more T-cell epitopes as provided herein may include a start codon of the trimer ATG sequence of bases toward the 5’ end of the molecule to signify the initiation for translation of the mRNA segment of interest to a protein and may further include a stop codon of UAA, UAG or UGA that is in frame with the start codon to signify the end of the coding region or the point at which translation is to cease. An mRNA may further include an untranslated region (UTR) following a stop codon and can further include a polyadenylated (poly A) tail after the 3’ untranslated region (UTR) of the single stranded molecule. A polyA tail can be provided by the template DNA or by the use of a polyA polymerase. Those skilled in the art will appreciate that the exact length of adenosine in the poly A tail need not be exact but may generally fall within the range of about 100 to about 200 adenosine residues. Optionally, the mRNA may be optimized to avoid a double-stranded secondary or tertiary structures and/or purified to remove any double-stranded variants (see, e.g., Kariko et al. Nucleic Acids Res. 39: el42 (2011)).
As provided herein are one or more mRNA sequences that encode one or more SARS-CoV-2 T-cell epitopes as provided herein, optionally encoding one or more of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,
SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 53, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, and / or SEQ ID NO: 124. In some aspects, an mRNA includes a sequence of ribonucleic acids that are 80% or greater identical to one or more of the following where G is guanine, A is adenine, U is uracil, and C is cytosine: GGGAAAGGCUAU C ACUUAAU GAGCUUUCCUC AGU CU GCGCCUC AC (SEQ ID NO: 88) encoding Spike 1044;
CAGACUUUGUUAGCGCUUCAUCGUAGCUAUUUGACACCGGGGGAUUCAUCC (SEQ ID NO: 89) encoding Spike 239;
GUU CUUUCUUUCG AGCUGCUU C ACGC ACCUGCC ACCGUAU GU GGU (SEQ ID NO: 90) encoding Spike 512;
C AAAAAUU G AUAGCUAAU C AGUUUAAC AGCGCC AUU GGUAAG AUCC AGGAC A GCUUA (SEQ ID NO: 91) encoding Spike 920;
C AGAUACCGUUU GCGAU GC AGAU GGCCU AUAGAUUUAAU GGG AUAGGAGU C (SEQ ID NO: 92) encoding Spike 895;
CCC AAGGAAAU C ACUGU GGCGACUAGCCGUACUUU GUCCUAUUAU (SEQ ID NO: 93) encoding Membrane 165;
CCGACUAAUUUUACUAUUU C AGU GACUACGGAGAUUCUUCCGGU G (SEQ ID NO: 94) encoding Spike 715;
AUUUGUUUACUUCAGUUUGCGUACGCCAACCGCAAUCGCUUUCUUUAUAUA (SEQ ID NO: 95) encoding Membrane 32;
AAU GAU GGGGUCUACUUCGCUU C AACU G AAAAGAGUAAC AUUAUUCGG (SEQ ID NO: 96) encoding Spike 87;
ACGGACGAAAUGAUAGCGCAGUAUACUAGUGCGUUAUUGGCU (SEQ ID NO: 97) encoding Spike 866;
AAC AAGU GU GUUAAUUUUAAUUU C AACGGGUUAAC AGGGACGGGGGU GUU GA CAGAA (SEQ ID NO: 98) encoding Spike 536;
GACGGAUACUUCAAGAUAUAUAGCAAACAUACCCCGAUCAAUCUU (SEQ ID NO: 99) encoding Spike 198;
C AAGACCU GUUCCU GCCCUUCUUUU CUA AU GUUACUU GGUUU C ACGC AAUAC A UGUUUCUGGCACCAAUGGCACA (SEQ ID NO: 100) encoding Spike 52; ACAGGUCGCUUACAGUCGUUACAAACAUACGUUACACAACAACUG (SEQ ID NO: 101) encoding Spike 998;
GUU GAAGGUUUUAAUU GCUACUUCCCGCU GC AGAGUU ACGGAUUU C AACCGA C AAAU GGGGUU GGUUAU C AGCC AUAU (SEQ ID NO: 102) encoding Spike 483; UUUUUAGGAGU GUAUUAU C AUAAGAAC AAC AAAAGUU GGAUGGAGUCCGAAU UUCGU GU GUACUCUAGCGCC AAU AACU GUACUUUU GAGUAU GUU (SEQ ID NO: 103) encoding Spike 140;
UUAGAUAAGUAUUUCAAGAACCAUACUUCGCCGGACGUUGACCUU (SEQ ID NO: 104) encoding Spike 1152);
CCACCUGCCUACACUAACUCAUUUACGCGCGGCGUUUAUUAC (SEQ ID NO: 105) encoding Spike 25;
CCUCGGACGUUCCUGCUGAAGUAUAAUGAGAACGGAACCAUAACCGAUGCG (SEQ ID NO: 106) encoding Spike 272;
AGCCGGGU GAAGAAU CUUAACUC AUCUAGAGUCCC AGAU (SEQ ID NO: 107) encoding Env 60;
ACU C AAU C AUUACUGAUCGU GAAC A AU GCC ACUAAU GUU GU C AUUAAAGUA (SEQ ID NO: 108) encoding Spike 114;
GGAGGCAACUACAAUUAUUUGUACCGUUUGUUCCGCAAAAGCAAUCUUAAAC CGUUCGAACGGGACAUA (SEQ ID NO: 109) encoding Spike 446;
AU GUAC AGCUUU GU C AGU GAAGA AACGGGUAC AUUAAUU GUUAAU (SEQ ID NO: 110) encoding Env 1;
TTTGGTGGCTTCAATTTTTCACAGATATTACCAGACCCGTCAAAACCGTCTAAGC GGAGT (SEQ ID NO: 111) encoding Spike 797;
UUAGC AU GUUUCGU GCUU GCUGC AGU GUAUCGUAUAA AUU GG AUA (SEQ ID NO: 112) encoding membrane 62;
CU GGCCU GCUUCGU GCU GGCCGCCGU GU AC AGGAU C AACUGG AU C (SEQ ID NO: 113) encoding linker;
UU GUUAC AAUAU GGCUCUUU CU GC AC AC AGCUUAACCGU GCU CUUACGGGGAU CGCUGUGGAGCAA (SEQ ID NO: 114) encoding Spike 753;
AUAU C AGGG AUUAAU GCCUCGGUU GU C AAUAUAC AAA AAGAA AUU (SEQ ID NO: 115) encoding Spike 1169);
GACGU GGUU AUU GGUAUAGUUAAC AAUACU GUCUAUGACCCUUU G (SEQ ID NO: 116) encoding Spike 1127);
AGAGGGC AUCUGCGGAUCGCGGGU C ACC AUUU GGGCCGGU GU GAC (SEQ ID NO: 117) encoding Membrane 146;
CUUAAAAAGUU GUU GGAAC AAUGGAACUU GGU C AUAGGAUUCUU GUUUCUGA CUUGG (SEQ ID NO: 118) encoding Membrane 13; GGUCCCGGACCGGGC (SEQ ID NO: 119) encoding Linker;
ACUU C AAAUUUUAG AGUU C AGCCUAC AGAGAGC AUU GUACGUUU C (SEQ ID NO: 120) encoding Spike 315;
UUCGGCGAAGUUUUUAACGCGACGCGCUUCGC AUCCGUCUACGC AU GGAAUCG UAAACGUAUCUCG AAUU GCGUCGCCGAUUAUUCCGUCCUUUAUAAC AGCGC AU CGUUUUCAACGUUCAAG (SEQ ID NO: 121) encoding Spike 338; AUCGGGAACUACAAAUUGAAUACCGACCAUAGCUCCUCGUCGGACAAUAUA (SEQ ID NO: 122) encoding Membrane 201; and/or
UUAUCGUACUUUAUU GCGUCUUU C AGACUUUUCGCCCGC ACUCGUAGUAU GU G GUCGUUUAACCCGGAGACGAAUAUACUUCUUAACGUU (SEQ ID NO: 123) encoding Membrane 93; or any combination thereof. Optionally, a mRNA sequence is a concatemer of 2 or more of SEQ ID NOs: 88-123. Optionally, a mRNA sequence is a concatemer of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 of any of SEQ ID NOs: 88-123 in any order. In some aspects, a mRNA is a sequence including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 of any of SEQ ID NOs: 88-123 in any order. Optionally, a mRNA is a concatemer of all of SEQ ID NOs: 88-123 in any order, optionally in the order of SEQ ID NO: 88 to SEQ ID NO: 124. Optionally, an mRNA sequence comprises
GGGAAAGGCUAU C ACUUAAU GAGCUUUCCUC AGU CU GCGCCUC ACC AGACUUU GUUAGCGCUU C AUCGUAGCUAUUU GAC ACCGGGGGAUU C AUCCGUU CUUUCUU UCGAGCUGCUUCACGCACCUGCCACCGUAUGUGGUCAAAAAUUGAUAGCUAAU CAGUUUAACAGCGCCAUUGGUAAGAUCCAGGACAGCUUACAGAUACCGUUUGC GAUGCAGAUGGCCUAUAGAUUUAAUGGGAUAGGAGUCCCCAAGGAAAUCACU GU GGCGACUAGCCGUACUUU GUCCUAUU AUCCGACUAAUUUU ACUAUUU C AG U GACUACGGAGAUU CUUCCGGU GAUUU GUUUACUU C AGUUUGCGUACGCC AA CCGC AAUCGCUUUCUUUAUAUAAAU GAU GGGGU CUACUUCGCUU C AACU GAA AAGAGUAACAUUAUUCGGACGGACGAAAUGAUAGCGCAGUAUACUAGUGCGU UAUU GGCUA AC AAGU GU GUUAAUUUUAAUUU C AACGGGUUAAC AGGGACGGG GGUGUUGACAGAAGACGGAUACUUCAAGAUAUAUAGCAAACAUACCCCGAUC AAU CUU C AAGACCUGUUCCU GCCCUU CUUUU CUAAU GUUACUU GGUUU C ACGC AAUAC AU GUUU CUGGC ACC AAU GGC AC AGCGCGC AC AGGUCGCUUAC AGUCGU UAC AAAC AUACGUUAC AC AAC AACU GGUU GAAGGUUUUAAUU GCUACUUCCC GCU GC AGAGUUACGGAUUU C AACCGAC A AAU GGGGUU GGUUAU C AGCC AUAU UUUUUAGGAGU GUAUUAU C AUAAGAAC AAC AAAAGUU GGAUGGAGUCCGAAU UUCGU GU GUACUCUAGCGCC AAU AACU GUACUUUU GAGUAU GUUUUAG AUAA GUAUUU C AAGAACC AUACUUCGCCGGACGUU GACCUUCC ACCUGCCUAC ACUA ACUCAUUUACGCGCGGCGUUUAUUACCCUCGGACGUUCCUGCUGAAGUAUAAU GAGAACGGAACCAUAACCGAUGCGAGCCGGGUGAAGAAUCUUAACUCAUCUA GAGUCCC AGAUACUC AAU C AUUACUGAU CGU GAAC AAU GCC ACUAAU GUU GU C AUUAAAGUAGGAGGCAACUACAAUUAUUUGUACCGUUUGUUCCGCAAAAGCA AU CUU A A AC CGUU C GAAC GGG AC AU AAU GU AC AGCUUU GU C AGU G A AG A A AC GGGUAC AUU AAUU GUUAAUUUU GGU GGCUU C AAUUUUU C AC AGAUAUUACC A GACCCGU C AAAACCGU CUAAGCGGAGUGCGCGCUUAGC AU GUUUCGU GCUU GC U GC AGU GUAUCGUAUAAAUU GGAUAGGCCCUGGACCU GGUUU GUUAC AAUAU GGCUCUUU CU GC AC AC AGCUUAACCGU GCUCUUACGGGGAUCGCUGU GGAGC A AAUAU C AGGGAUUA AU GCCUCGGUU GU C AAUAUAC AA AAAGA AAUU GACGU G GUU AUU GGUAU AGUUAAC AAUACU GU CU AU GACCCUUU GAGAGGGC AUCUGC GGAUCGCGGGU C ACC AUUU GGGCCGGU GU GACCUUAAAA AGUU GUU GG AAC A AU GGAACUU GGU C AUAGGAUUCUU GUUU CU GACUUGGGGUCCCGGACCGGGC ACUU C AAAUUUUAG AGUU C AGCCUAC AGAGAGC AUU GUACGUUU CUUCGGCG AAGUUUUUAACGCGACGCGCUUCGCAUCCGUCUACGCAUGGAAUCGUAAACGU AUCUCGAAUUGCGUCGCCGAUUAUUCCGUCCUUUAUAACAGCGCAUCGUUUUC AACGUUCAAGAUCGGGAACUACAAAUUGAAUACCGACCAUAGCUCCUCGUCGG AC AAUAU AUUAUCGUACUUU AUU GCGUCUUU C AGACUUUUCGCCCGC ACUCGU AGUAUGUGGUCGUUUAACCCGGAGACGAAUAUACUUCUUAACGUUUAA (SEQ ID NO: 126) or
GGC AAAGGCUAU C AUCUGAU GAGCUUUCCGC AGAGCGCGCCGC AU GGC AGCGG
CAGCGGCCAGACCCUGCUGGCGCUGCAUCGCAGCUAUCUGACCCCGGGCGAUA
GCAGCGGCAGCGGCAGCGGCGUGCUGAGCUUUGAACUGCUGCAUGCGCCGGCG
ACCGUGUGCGGCGGCAGCGGCAGCGGCCAGAAACUGAUUGCGAACCAGUUUAA
CAGCGCGAUUGGCAAAAUUCAGGAUAGCCUGGGCAGCGGCAGCGGCCAGAUUC
CGUUU GCGAU GC AGAU GGCGUAUCGCUUUAACGGC AUUGGCGU GGGC AGCGG
CAGCGGCCCGAAAGAAAUUACCGUGGCGACCAGCCGCACCCUGAGCUAUUAUG
GCAGCGGCAGCGGCCCGACCAACUUUACCAUUAGCGUGACCACCGAAAUUCUG
CCGGUGGGCAGCGGCAGCGGCAUUUGCCUGCUGCAGUUUGCGUAUGCGAACCG
C AACCGCUUU CU GU AUAUU GGC AGCGGC AGCGGC AACGAUGGCGU GUAUUUU
GCGAGCACCGAAAAAAGCAACAUUAUUCGCGGCAGCGGCAGCGGCACCGAUGA
AAUGAUUGCGCAGUAUACCAGCGCGCUGCUGGCGGGCAGCGGCAGCGGCAACA
AAUGCGUGAACUUUAACUUUAACGGCCUGACCGGCACCGGCGUGCUGACCGAA GGCAGCGGCAGCGGCGAUGGCUAUUUUAAAAUUUAUAGCAAACAUACCCCGA
UUAACCUGGGC AGCGGC AGCGGCC AGGAU CU GUUU CU GCCGUUUUUUAGC AAC
GU GACCUGGUUU C AU GCGAUU C AU GU GAGCGGC ACC AACGGC ACCGGC AGCGG
CAGCGGCACCGGCCGCCUGCAGAGCCUGCAGACCUAUGUGACCCAGCAGCUGG
GCAGCGGCAGCGGCGUGGAAGGCUUUAACUGCUAUUUUCCGCUGCAGAGCUAU
GGCUUUCAGCCGACCAACGGCGUGGGCUAUCAGCCGUAUGGCAGCGGCAGCGG
CUUUCUGGGCGUGUAUUAUCAUAAAAACAACAAAAGCUGGAUGGAAAGCGAA
UUUCGCGUGUAUAGCAGCGCGAACAACUGCACCUUUGAAUAUGUGGGCAGCG
GCAGCGGCCUGGAUAAAUAUUUUAAAAACCAUACCAGCCCGGAUGUGGAUCU
GGGCAGCGGCAGCGGCCCGCCGGCGUAUACCAACAGCUUUACCCGCGGCGUGU
AUUAUGGCAGCGGCAGCGGCCCGCGCACCUUUCUGCUGAAAUAUAACGAAAAC
GGCACCAUUACCGAUGCGGGCAGCGGCAGCGGCAGCCGCGUGAAAAACCUGAA
CAGCAGCCGCGUGCCGGAUGGCAGCGGCAGCGGCACCCAGAGCCUGCUGAUUG
UGAACAACGCGACCAACGUGGUGAUUAAAGUGGGCAGCGGCAGCGGCGGCGGC
AACUAUAACUAUCUGUAUCGCCUGUUUCGCAAAAGCAACCUGAAACCGUUUGA
ACGCGAUAUUGGCAGCGGCAGCGGCAUGUAUAGCUUUGUGAGCGAAGAAACC
GGCACCCUGAUUGUGAACGGCAGCGGCAGCGGCUUUGGCGGCUUUAACUUUAG
CCAGAUUCUGCCGGAUCCGAGCAAACCGAGCAAACGCAGCGGCAGCGGCAGCG
GCCUGGCGU GCUUU GU GCUGGCGGCGGU GUAUCGC AUUA ACU GGAUU GGCCCG
GGCCCGGGCCUGCUGCAGUAUGGCAGCUUUUGCACCCAGCUGAACCGCGCGCU
GACCGGCAUUGCGGUGGAACAGGGCAGCGGCAGCGGCAUUAGCGGCAUUAACG
CG AGC GU GGU G A AC AUU C AG A A AG A A AUU GGC AGCGGC AGC GGC G AU GU GGU
G AUU GGC AUU GU G A AC A AC ACC GU GU AU G AU CCGCUGGGCAGC GGC AGC GGCC
GCGGCC AUCUGCGC AUU GCGGGCC AU C AU CU GGGCCGCUGCG AU GGC AGCGGC
AGCGGCCU GAAAA AACUGCU GGAAC AGU GGAACCU GGU G AUU GGCUUU CU GU
UUCUGACCUGGGGCCCGGGCCCGGGCACCAGCAACUUUCGCGUGCAGCCGACC
GAAAGC AUU GU GCGCUUU GGC AGCGGC AGCGGCUUU GGCGA AGU GUUU AACG
CGACCCGCUUUGCGAGCGUGUAUGCGUGGAACCGCAAACGCAUUAGCAACUGC
GUGGCGGAUUAUAGCGUGCUGUAUAACAGCGCGAGCUUUAGCACCUUUAAAG
GCAGCGGCAGCGGCAUUGGCAACUAUAAACUGAACACCGAUCAUAGCAGCAGC
AGCGAUAACAUUGGCAGCGGCAGCGGCCUGAGCUAUUUUAUUGCGAGCUUUC
GCCUGUUUGCGCGCACCCGCAGCAUGUGGAGCUUUAACCCGGAAACCAACAUU
CUGCUGAACGUG (SEQ ID NO: 127). [00116] In some aspects, a nucleic acid encoding one or more T-cell epitopes or polypeptides as provided herein may be packaged for delivery to a subject with one or more ionic lipids. Optionally, lipid nanoparticles (LNPs) or lipid-like nanoparticles (LLNs) are synthesized to contain at least one nucleotide sequence (e.g. mRNA or DNA) contained therein. As is used in this disclosure, a “lipid particle” is defined as a single or double layer membrane defining a particle. It is appreciated that the lipid particle is formed of one or more ionic lipids. Illustrative non-limiting examples of such ionic lipids include phosphtidyl serine (PS), phosphatidyl choline (PC), protamine, cholesterol, or polysaccharide, among others. Optionally, the nucleic acid or plasmid or other expression construct including the nucleotide sequence is optionally encapsulated within an LNP or LLN of two or more lipids, such as three, four, five or more.
[00117] Ionic lipids as provided herein may optionally include three sections including an amine head, a linker and a hydrophobic tail thereby defining general overall structure of exemplary ionic lipids. More specific examples of ionic lipids include but are not limited to heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), DLinDMA, and DLin-KC2-DMA). Optionally, the LNP may include an ionic lipid, a polyethylene glycol and a cholesterol. In further aspects, the LNP may include a combination of an ionic lipid with polyethylene glycol (PEG), cholesterol and/or distearoyl phosphocholine. Specific constructs of LNPs suitable for use may be found in: Pardi, N, et ah, Nat Rev Drug Discov, 17, 261-279 (2018) (and references cited therein); Sabnis et ah, Mol. Ther. 26: 1509- 1519 (2018); Pardi et al. J. Exp. Med. 215:1571-1588 (2018); and, Pardi et al. J. Control. Release 217: 345-351 (2015)).
[00118] Optionally, a nanoparticle housing or bound to a nucleotide sequence as provided herein further includes one or more helper lipids. Illustrative, non-limiting examples of a helper lipid may include l,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and/or dioleoylphosphatidylethanolamine (DOPE) and/or lipofectamine and/or dioleoylphosphatidylcholine (DOPC) and/or phosphatidylethanolamine (dioleoyl PE) and/or 3p-[N-(N’,N’-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) (see, e.g., Du et al. Scientific Reports 4: 7107 (2014) and Cheng et al. Advanced Drug Delivery Reviews 99(A): 129-137 (2016)).
[00119] In aspects, the present disclosure provides chimeric or fusion polypeptide compositions (which in aspects may be isolated, synthetic, or recombinant) wherein one or more of the instantly-disclosed T-cell epitopes is a part thereof, such as a fusion of one or more of the concatemers disclosed herein with a T4 phage secreted peptide including gp23, gp24, Hoc and/or Soc. In aspects, a chimeric or fusion polypeptide composition comprises one or more polypeptides of the present disclosure joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide. As previously described, with respect to the one or more T-cell epitopes of the instant disclosure, the term “heterologous polypeptide” is intended to mean that the one or more T-cell epitopes of the instant disclosure are heterologous to, or not included naturally, in the heterologous polypeptide. In aspects, one or more of the instantly-disclosed polypeptides may be inserted into the heterologous polypeptide (e.g., through recombinant techniques, mutagenesis, or other known means in the art), may be added to the C-terminus (with or without the use of linkers, as is known in the art), and/or added to the N-terminus (with or without the use of linkers, as is known in the art) of the heterologous polypeptide. For example, protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et ah, 2012, Protein Engineering Methods and Applications, which are herein incorporated by reference in their entirety). In aspects, chimeric or fusion polypeptides comprise one or more of the instantly-disclosed polypeptides operatively linked to a heterologous polypeptide. “Operatively linked” indicates that the one or more of the instantly-disclosed polypeptides and the heterologous polypeptide are fused in-frame or chemically-linked or otherwise bound. In aspects of the above isolated, synthetic, or recombinant chimeric or fusion polypeptide compositions, the one or more polypeptides of the present disclosure have a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124). In aspects of the chimeric or fusion polypeptide compositions, the one or more peptides or polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124) may be joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide as a whole, although it may be made up from a joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted amino acid sequence, together with flanking amino acids of the heterologous polypeptide. In aspects, a chimeric or fusion polypeptide composition comprises a polypeptide of the instant disclosure (which, in aspects, may be an isolated, synthetic, or recombinant) having a sequence comprising one or more of SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124), wherein said one or more of SEQ ID NO: 1 or SEQ ID NO: 124 is not naturally included in the polypeptide and/or said of one or more of SEQ ID NO: 1 or SEQ ID NO: 124 is not located at its natural position in the polypeptide. In aspects, the one or more peptide or polypeptides of the present disclosure can be joined, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into the heterologous polypeptide. In aspects, chimeric or fusion polypeptide compositions comprise one or more of the instantly-disclosed T-cell epitopes operatively linked to a heterologous polypeptide having an amino acid sequence not substantially homologous to the T-cell epitope. In aspects, the chimeric or fusion polypeptide does not affect function of the T-cell epitope per se. For example, the fusion polypeptide can be a GST-fusion polypeptide in which the T-cell epitope sequences are fused to the C-terminus of the GST sequences. Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Such fusion polypeptides, particularly poly-His fusions or affinity tag fusions, can facilitate the purification of recombinant polypeptide. In certain host cells (e.g. , mammalian host cells), expression and/or secretion of a polypeptide can be increased by using a heterologous signal sequence. Therefore, in aspects, the chimeric or fusion polypeptide contains a heterologous signal sequence at its N-terminus. In aspects of the above chimeric or fusion polypeptide compositions, the heterologous polypeptide or polypeptide comprises a biologically active molecule. In aspects, the biologically active molecule is selected from the group consisting of an immunogenic molecule, a T cell epitope, a viral protein, and a bacterial protein. In aspects, the one or more peptides or polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124) can be joined or linked to (e.g., fused in-frame, chemically-linked, or otherwise bound) a small molecule, drug, or drug fragment. For example, one or more peptides or polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124) can be joined or linked to (e.g., fused in- frame, chemically-linked, or otherwise bound) a drug or drug fragment that is binds with high affinity to defined SLAs. In aspects of the above-described chimeric or fusion polypeptide compositions, the chimeric or fusion polypeptide compositions can be recombinant, isolated, and/or synthetic.
[00120] A chimeric or fusion polypeptide composition can be produced by standard recombinant DNA or RNA techniques as are known in the art. For example, DNA or RNA fragments coding for the different polypeptide sequences may be ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, polymerase chain reaction (PCR) amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, (2nd, 1992), FM Asubel et al. (eds), Green Publication Associates, New York, NY (Publ), ISBN: 9780471566355, which is herein incorporated by reference in its entirety). Further, one or more polypeptides of the present disclosure (e.g., one or more T-cell epitopes of the present disclosure having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 124 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124) can be inserted into a heterologous polypeptide or inserted into a non-naturally occurring position of a polypeptide through recombinant techniques, synthetic polymerization techniques, mutagenesis, or other standard techniques known in the art. For example, protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et ah, 2012, Protein Engineering Methods and Applications, which are herein incorporated by reference in their entirety).
[00121] Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A nucleic acid molecule encoding a T-cell epitope of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the at least one T-cell epitope.
Vaccine Systems, Vaccine Compositions and Pharmaceutical Compositions
[00122] The present disclosure further provides for vaccine systems of phage that encode the antigenic chimera protein and/or phage that express/display the antigenic fusion proteins, referred to also as phage DNA and phage display.
[00123] In some aspects, the vaccine systems include a phage with a genome altered such that it is capable of expressing (displaying) the antigenic polypeptides derived from SARS- CoV-2 as set forth herein. To achieve such, the sequences as set forth in SEQ ID NO: 2 or SEQ ID NO: 125, or of any other combination of nucleic acids as provided herein (illustratively SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or variants thereof) can be introduced into a plasmid DNA construct. The nucleic acid sequences are fused (in frame) at the 3’ end of a surface phage protein, such as gp23, gp24, Hoc and/or Soc of T4 phage. The resulting plasmid construct can then be introduced or transformed into a bacterial cell, such as E. coli, a recombinase expressing bacterial cell, to allow for the introduction of the gp23/gp24/Hoc/Soc- antigenic fused nucleotide sequence in to the phage genome. The transformed bacteria can then be infected with a T4 phage. The plasmid and the phage may further contain recombination sites for integration into the phage genome, such as Lox recombination domains, thereby allowing for production of phage with the antigenic encoding nucleic acids and phage expressing the antigenic polypeptides. It will be apparent to those skilled in the art that other recombination systems than Cre can be used, such as FLP, R, Lambda, HK101, and pSAM2.
[00124] In some aspects, the vaccine product is composed of the recombinant phage vaccine component bearing the desired epitopes identified by the in silico analysis. The collective display of the original epitopes with subsequent modified sequences are set forth in SEQ ID NO: 1 or SEQ ID NO: 124 or variants thereof, with a nucleic acid encoding such in SEQ ID NO: 2 or SEQ ID NO: 125 or variants thereof.
[00125] In other aspects, a T4 phage is selected for phage display. A T4 phage can express large polypeptides on its surface and further provides straightforward and efficient cloning efficiency. A T4 phage further provides a vehicle for display of a high copy number of the antigenic polypeptide, typically displaying hundreds of copies per phage.
[00126] In some aspects, the phage vaccine may be a DNA vaccine, to be used either alone or in combination with a phage display vaccine. A phage DNA vaccine may be generated using the nucleic acids encoding the concatemer of collected linear epitopes. In such as system, the antigen genes may be under the control of a eukaryotic promoter, such as CMV, SV40, CAG, U6 etc. Such a promoter may be introduced by cloning inside a nonessential region of a phage genome to produce the phage based DNA vaccine. Accordingly, when presented in a mammalian system, such as by injection, this phage vaccine may act as a DNA vaccine and induce potent immune response by expressing foreign antigen inside of antigen presenting cells (APCs). For example, the array of the linear epitopes of the construct set forth in SEQ ID NO: 2 or SEQ ID NO: 125 can be cloned inside the T4 phage genome under a strong eukaryotic promoter. This may allow for optimum expression of antigenic epitopes inside APCs like macrophages, dendritic cells, Langerhans cells and Kupffer cells to provoke strong immunogenic response against SARS-CoV-2.
[00127] In some aspects, the phage of the vaccine product/system is irradiated (e.g., after construction by prior to administration to a subject).
[00128] The term “vaccine” as used herein includes an agent that may be used to cause, stimulate or amplify the immune system of animals (e.g., humans) against a pathogen. Vaccines of the invention are able to cause or stimulate or amplify immunity against a SARS-CoV-2 infection. [00129] The term “immunization” includes the process of delivering an immunogen to a subject. Immunization may, for example, enable a continuing high level of antibody and/or cellular response in which T-lymphocytes can kill or suppress the pathogen in the immunized subject, such as human, which is directed against a pathogen or antigen to which the subject has been exposed.
[00130] Vaccines of the present disclosure comprise an immunologically effective amount of a T-cell epitope composition (including one or more of e.g., phage display or polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T- cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, phage (including lambda phage) or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2 or SEQ ID NO: 125 as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) as described above, and in aspects in a pharmaceutically acceptable vehicle and optionally with additional excipients and/or an adjuvant. As a result of the vaccination with a composition of the present disclosure, animals may become at least partially or completely immune to SARS- CoV-2, SARS, MERS or similar coronavirus infections, or resistant to developing moderate or severe SARS-CoV-2 infections and/or SARS-CoV-2 related diseases. The instantly-disclosed SARS-CoV-2 vaccines may be used to elicit a humoral and/or a cellular response, including CD4+ and CD8+ T effector cell responses. SARS-CoV-2 infections or associated diseases include, for example, COVID-19. Preferably, a human is protected to an extent to which one to all of the adverse physiological symptoms or effects of SARS-CoV-2, SARS, MERS or similar coronavirus infections are significantly reduced, ameliorated or prevented.
[00131] In practice, the exact amount required for an immunologically effective dose may vary from subject to subject depending on factors such as the age and general condition of the subject, the nature of the formulation and the mode of administration. An appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. For instance, methods are known in the art for determining or titrating suitable dosages of a vaccine to find minimal effective dosages based on the age, weight of the animal subject, species (if non- human), medical condition of the subject to be treated, concentration of the vaccine, and other typical factors.
[00132] In aspects, the vaccine comprises a unitary dose of between 0.1-50 micrograms (pg), preferably between 0.1 and 25, even more preferably of between 1 and 15 pg, typically approx. 10 pg, of phage display, phage DNA, polypeptide or nucleic acid antigen of the invention.
[00133] The dosage of the vaccine, concentration of components therein and timing of administering the vaccine, which elicit a suitable immune response, can be determined by methods such as by antibody titrations of sera, e.g., by ELISA and/or seroneutralization assay analysis and/or by vaccination challenge evaluation.
[00134] In aspects, the vaccine comprises a T cell epitope composition (including one or more of e.g., phage display or polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 124 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, phage or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2 or SEQ ID NO: 125 or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) of the instant disclosure in purified form, optionally in combination with any suitable excipient, carrier, adjuvant, and/or additional protein antigen. The present disclosure also relates to a combination vaccine comprising a polypeptide, nucleic acid or cell of the invention in combination with at least one additional protein antigen.
[00135] In aspects, the vaccine comprises a nucleic acid as defined above, optionally in combination with any suitable excipient, carrier, adjuvant, and/or additional protein antigen. In aspects, the vaccine comprises a viral vector or phage containing a nucleic acid as defined above. In aspects, the vaccine comprises one or more plasmid vectors or phage genomes. In aspects, the one or more plasmid vectors or phage genome contain a nucleic acid sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or any one or more of SEQ ID NOs: 88-123, or variants or fragments thereof.
[00136] The vaccine compositions are in aspects designated for administration to human subjects to provide an immune response, ideally a protective immune response. It will be appreciated that the vaccine compositions may be most effective by coming into contact with a human subject’s immune system. It will be appreciated by those skilled in the art that the phage display and phage DNA vaccines, LNP vaccines, mRNA vaccines, or peptide antiben vaccines may be prepared with additional excipients, adjuvants and pharmaceutically acceptable carriers based on the route of administration desired. Such are described in further detail in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 22nd Ed., 2012; and Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, 10th Ed., Philadelphia, PA, 2013. Further processing or packaging may further be applied in order to optimize the phage stimulating the immune system of the subject. [00137] The compositions described herein are in some aspects referred to as vaccines. It will be appreciated by those skilled in the art that reference to such refers to stimulating or provoking or providing an immune response within a subject that receives the phage-based compositions described herein. In some aspects, the vaccine compositions may provide an innate immune response, a humoral immune response, a cell-mediated immune response or combinations thereof. The compositions may, in some aspects, provide immunity to a subject from exposure to SARS-CoV-2. In other aspects, the compositions may provide partial immunity. It will be further apparent to those skilled in the art that the compositions may provide varying lengths of an immune response, and such responses may further vary between individual subjects. In some aspects, the compositions may provide temporary immunity or partial immunity. In other aspects, the compositions may provide long-lasting immunity or partial immunity. It will be appreciated by those skilled in the art, therefore, that reference to the compositions as vaccines refers generally to provoking, stimulating or providing an immune response, the level of which may vary.
[00138] In some aspects, the vaccine compositions feature varying combinations and concentrations of the three identified phage constructs. In some aspects, the vaccine phage construct may be mixed with other SARS-CoV-2 antigenic compounds in equimolar concentration (same proportion) to produce a mixed vaccine for generating strong immunogenic response against SARS-COV-2. In other aspects, the vaccine construct could be mixed in a non-equimolar (different proportions) concentrations to achieve maximum immunogenicity against SARS-CoV-2. In other aspects, the vaccine construct can be mixed and matched accordingly for preparation of final vaccine constituents.
[00139] In further aspects, phage based SARS-CoV-2 phage DNA vaccines can be altered on the surface to display specific peptides to enhance the binding affinity of phage particles to APCs for accelerating the uptake of phage DNA vaccines by APCs. In some aspects, the phage based DNA vaccines could be decorated with selective linear peptides that may enhance the binding affinity of phage vaccine particles with epithelial cells for proper absorptions to enhance the efficacy of above described vaccination processes.
[00140] In some aspects, the present disclosure provides compositions with alternate presentation of vaccine particles or protein components to enhance immunogenic response in mammalian systems. As for example, vaccines could be mixed with adjuvant like aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate to increase the “depot effect” during intramuscular route of vaccinations. Depot effects may be essential for slow diffusion of phage vaccines from the site of inoculation to attract APCs for generating optimum immunogenic response of phage vaccination.
[00141] In further aspects, compositions to SARS-CoV-2 can include Bacillus calmette- guerin (BCG) to increase the adjuvant effect of phage, as well as provoking very strong cell mediated responses.
[00142] In other aspects, the present disclosure further considers microencapsulation of the compositions. Microencapsulation of vaccine particles using biodegradable polymer microspheres may also increase the stability and immunogenicity of phage vaccines during transportation in austere locations. Microencapsulation of phage vaccine particles may also help to prevent proteolytic disintegration of phage vaccine particles during long storage.
[00143] In some aspects, vaccine compositions can be specifically designed for intradermal deposition of vaccines, such as with microneedles. Intradermal inoculation route for SARS- CoV-2 vaccine using microneedle is highly desirable to enhance immune response, resulting in a potential reduction of the antigen dose, decreased anxiety and pain. Microneedles can be engineered as small patches to deliver vaccines without any complications.
[00144] The SARS-CoV-2 vaccine constructs including concatemers of putative T-cell epitopes having an amino acid sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124, and nucleic acids (e.g., RNA mRNA, DNA, cDNA) encoding such concatemeric peptides; and chimeric or fusion polypeptide compositions as disclosed herein. Upon administration to a subject, the vaccine compositions may initiate a strong T-cell mediated immune response but may not always induce a humoral immune response. Therefore, aspects of a vaccine against SARS-CoV-2, SARS, MERS or other similar coronavirus contains a combination of the putative T-cell epitopes together with either live attenuated virus (LAV) or inactivated virus. This vaccine composition (including both the putative T-cell epitopes and an LAV or inactivated virus) upon administration to a subject may induce both cellular and humoral immune responses, thereby conferring comprehensive immunity against SARS-CoV- 2, SARS, MERS or other similar coronavirus.
[00145] Vaccine compositions may comprise other ingredients, known per se by one of ordinary skill in the art, such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze drying stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives, depending on the route of administration.
[00146] Examples of pharmaceutically acceptable carriers, excipients or diluents include, but are not limited to: demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as light liquid paraffin oil, or heavy liquid paraffin oil; squalene; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium salt, or hydroxypropyl methylcellulose; lower alkanols, for example ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 -butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum acacia; and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the vaccine composition and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.
[00147] Adjuvants suitable for use with the compositions and vaccines of the instant disclosure are familiar to one of skill in the art and are available from a variety of commercial vendors. Examples of adjuvants include, but are not limited to, oil in water emulsions, aluminum hydroxide (alum), immunostimulating complexes, non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-a, IFN-b, IFN-g, etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP) and the like. Other suitable adjuvants include, for example, aluminum potassium sulfate, heat-labile or heat-stable enterotoxin(s) isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc. Toxin- based adjuvants, such as diphtheria toxin, tetanus toxin, cholera toxin, and pertussis toxin which may be inactivated prior to use, for example, by treatment with formaldehyde. Further adjuvants include, for example,: glycolipids; chemokines; compounds that induce the production of cytokines and chemokines; interferons; inert carriers such as alum, bentonite, latex, and cyclic particles; pluronic block polymers; depot formers; surface active materials such as saponin, lysolecithin, retinal, liposomes, and pluronic polymer formulations; macrophage stimulators such as bacterial lipopolysaccharide; alternative pathway complement activators such as insulin, zymosan, endotoxin, and levamisole; non-ionic surfactants; poly(oxyethylen)-poly(oxypropylene) tri-block copolymers; trehalsoe dimycolate (TDM); cell wall skeleton (CWS); macrophage colony stimulating factor (M-CSF); tumor necrosis factor (TNF); 3-O-deacylated MPL; CpG oligonucleotides; polyoxyetylene ethers; aluminum; poly[di(carboxylatophenoxy)phosphazene] (PCPP); QS-21; and formyl methionyl peptide.
[00148] Examples of freeze-drying stabilizer may be for example carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, and derivatives thereof.
[00149] The vaccine compositions of the instant disclosure may be liquid formulations such as an aqueous solution, water-in-oil or oil-in-water emulsion, syrup, an elixir, a tincture, or a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems. Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents.
[00150] The route of administration can be oral, sublingual, intranasal, transdermal (i.e., applied on or at the skin surface for systemic absorption), ocular, percutaneous, via mucosal administration, or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal). Vaccine compositions according to the present disclosure may be administered alone, or can be co-administered or sequentially administered with other treatments or therapies. The dosage of the vaccines of the present invention will depend on various factors such as the age, size, vaccination history, and health status of the subject to be vaccinated, as well as the route of administration. The vaccines of the instant disclosure can be administered as single doses or in repeated doses. The vaccines of the instant disclosure can be administered alone, or can be administered simultaneously or sequentially administered with one or more further compositions, or vaccine compositions. Where the compositions are administered at different times, the administrations may be separate from one another or overlapping in time.
[00151] In one aspect, the vaccine compositions of the present disclosure are administered to a subject susceptible to or otherwise at risk for SARS-CoV-2, SARS, MERS or similar coronavirus infection to enhance the subject’s own immune response capabilities.
[00152] In aspects, the present disclosure also includes pharmaceutically acceptable salts of the T-cell epitope compositions (including one or more of e.g., phage display, phage DNA, peptides or polypeptides as disclosed herein, which may be isolated, synthetic, or recombinant, such as polypeptides having an amino acid sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, or SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124; concatemeric peptides as disclosed herein; chimeric or fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant)). “Pharmaceutically acceptable salt” of a T-cell epitope composition means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent peptide or polypeptide (e.g., peptides, polypeptides, concatermic peptides, and/or chimeric or fusion polypeptides as disclosed herein). As used herein, “pharmaceutically acceptable salt” refers to derivative of the instantly-disclosed peptides or polypeptides, wherein such compounds are modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.
[00153] The present disclosure also provides a container comprising an immunologically effective amount of a phage, polypeptide, nucleic acid or vaccine as described above. The present disclosure also provides vaccination kits comprising an optionally sterile container comprising an immunologically effective amount of the vaccine, means for administering the vaccine to animals, and optionally an instruction manual including information for the administration of the immunologically effective amount of the composition for treating and/or preventing SARS-CoV-2, SARS, MERS or similar coronavirus associated diseases.
[00154] In aspects, the T-cell epitope compositions of the present disclosure (including one or more of e.g., phage display, phage DNA, polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, phage, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein) may be comprised in a pharmaceutical composition or formulation. In aspects, pharmaceutical compositions or formulations generally comprise a T-cell epitope composition of the present disclosure and a pharmaceutically-acceptable carrier and/or excipient. In aspects, said pharmaceutical compositions are suitable for administration. Pharmaceutically-acceptable carriers and/or excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the instantly-disclosed T-cell epitope compositions (see, e.g., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 22nd Ed., 2012)). In aspects, the pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
[00155] The terms “pharmaceutically-acceptable,” “physiologically-tolerable,” and grammatical variations thereof, as they refer to compositions, carriers, excipients, and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” means, for example, an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. A person of ordinary skill in the art would be able to determine the appropriate timing, sequence and dosages of administration for particular phage or T-cell epitope compositions of the present disclosure.
[00156] In aspects, preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. Fiposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the T-cell epitope compositions of the present disclosure and as previously described above (including one or more of e.g., phage or polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein), use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[00157] In aspects, phage or T-cell epitope compositions or concatemers of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1, or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) are formulated to be compatible with its intended route of administration. The T-cell epitope compositions of the present disclosure can be administered by parenteral, topical, intravenous, oral, subcutaneous, sublingual, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; vaginally; intramuscular route or as inhalants. In aspects, T-cell epitope compositions of the present disclosure can be injected directly into a particular tissue where deposits have accumulated, e.g., intracranial injection. In other aspects, intramuscular injection or intravenous infusion may be used for administration of T-cell epitope compositions of the present disclosure. In some methods, T-cell epitope compositions of the present disclosure are administered as a sustained release composition or device, such as but not limited to a Medipad™ device.
[00158] In aspects, phage or T-cell epitope compositions or concatemers of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124 or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) can optionally be administered in combination with other agents that are at least partly effective in treating various medical conditions as described herein. For example, in the case of administration into the central nervous system of a subject, phage or T-cell epitope compositions of the present disclosure can also be administered in conjunction with other agents that increase passage of the agents of the invention across the blood-brain barrier.
[00159] In aspects, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include, but are not limited to, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Examples of excipients can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, water, ethanol, DMSO, glycol, propylene, dried skim milk, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.
[00160] In aspects, pharmaceutical compositions or formulations suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition is sterile and should be fluid to the extent that easy syringeability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. In aspects, T-cell epitope formulations may include aggregates, fragments, breakdown products and post-translational modifications, to the extent these impurities bind SLA and present the same TCR face to cognate T cells they are expected to function in a similar fashion to pure T-cell epitopes. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol ( e.g ., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound that delays absorption, e.g., aluminum monostearate and gelatin.
[00161] In aspects, sterile injectable solutions can be prepared by incorporating the phage or T-cell epitope compositions of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the binding agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Further, phage or T-cell epitope compositions of the present disclosure can be administered in the form of a depot injection or implant preparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. [00162] In aspects, oral compositions generally include an inert diluent or an edible carrier and can be enclosed in gelatin capsules or compressed into tablets. In aspects, for the purpose of oral therapeutic administration, the binding agent can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition. In aspects, the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate or orange flavoring.
[00163] For administration by inhalation, phage or T-cell epitope compositions or concatemers of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) can be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[00164] In aspects, systemic administration of the phage or T-cell epitope compositions or concatemers of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the T-cell epitope may be formulated into ointments, salves, gels, or creams and applied either topically or through transdermal patch technology as generally known in the art.
[00165] In aspects, the phage or T-cell epitope compositions or concatemers of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) can also be prepared in the form of suppositories ( e.g ., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[00166] In aspects, the phage or T-cell epitope compositions or concatemers of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein)are prepared with carriers that protect the T-cell epitope compositions against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art (U.S. Pat. No. 4,522,811, which is herein incorporated by reference in its entirety). In aspects, the T-cell epitope compositions of the present disclosure can be implanted within or linked to a biopolymer solid support that allows for the slow release of the phage or T-cell epitope compositions to the desired site.
[00167] In aspects, it is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of binding agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the instant disclosure are dictated by and directly dependent on the unique characteristics of the binding agent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such phage or T- cell epitope compositions for the treatment of a subject.
[00168] T4 Phage Vaccine Construction
[00169] In some aspects, the present disclosure further concerns the design of a T4 phage system to display a combinations of SARS-CoV-2 specific linear epitopes fused as a concatemer chimera to the C terminus of a capsid protein gp23, gp24, Hoc and/or Soc of phage T4. As an example, the identified 34 antigenic epitopes (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, and 69) were assembled into the single concatemer peptide fragment (i.e. SEQ ID NO: 1 or SEQ ID NO: 124) to generate the phage vaccine constructs that are herein. The details of the cloning steps are set out below, but briefly the optimized SARS-CoV-2 concatemer polypeptide were reverse translated into a synthetic polynucleotide that would encode such and then affixed with suitable restriction sequences to allow for in frame insertion into the 3 ’ end (C-terminus) of a gp23/gp24/Hoc/Soc T4 head genome in a plasmid. The resulting constructs were transformed into Cre expressing E. coli and then infected with a lambda phage. Recombination sites (e.g. lox) allowed for the plasmid to insert the construct into the lambda phage genome. In some aspects, the phage is irradiated (e.g., after construction by prior to administration to a subject).
[00170] As previously explained, the specific DNA sequences coding for the concatemer peptide sequence can be displayed on T4 phage using the protocol, for example, as described Zhu et al., “A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS- CoV-2 Vaccine Candidates by CRISPR Engineering bioRxiv, January 19, 2021 (herein incorporated by reference in its entirety). Specifically, engineered DNAs corresponding to the concatemer peptide sequence (or fragments thereof) can be incorporated into bacteriophage T4 genome as outlined in Figure 1 of Zhu et al. Each DNA is then introduced into E. coli as a donor plasmid and recombined into injected phage genome through CRISPR- targeted genome editing. Different combinations of CoV-2 inserts can then be generated by simple phage infections and the recombinant phages in the progeny can be identified. By repeating this process, a pipeline of multiplex T4-concatemer peptide sequence and/or individual antigenic fragment vaccine phages can be rapidly constructed. Selected vaccine candidates can then be screened in a mouse model to identify the most potent vaccine.
[00171] Methods of Use
[00172] The present disclosure concerns, in some aspects, methods of using the phage vaccines disclosed herein. The vaccine compositions are in aspects designated for administration to human subjects to provide an immune response, ideally a protective immune response. It will be appreciated that the vaccine compositions may be most effective by coming into contact with a human subject’s immune system. It may be therefore generally desirable to administer the compositions by a route that allows for such. [00173] In another aspects, the route of inoculation of phage based vaccines can be altered or varied to provoke desirable immunogenicity. As for example phage based display or phage DNA vaccines can be introduced through intranasal or sublingual routes instead of intramuscular route to activate production of secretory antibody like IgA.
[00174] In another aspect, phage display or phage DNA vaccines can be introduced via microneedles specifically designed for intradermal deposition of phage vaccines. This intradermal inoculation route for SARS-CoV-2 phage vaccine using microneedle is highly desirable to enhance immune response, resulting in a potential reduction of the antigen dose, decreased anxiety and pain. Microneedles can be engineered as small patches to deliver vaccines without any complications.
[00175] Stimulating T-cells with phage or T-cell epitope compositions of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1 SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2 or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) can stimulate, induce, and/or expand corresponding naturally occurring immune response to SARS-CoV-2, SARS, MERS or similar coronavirus, including CD4+ and CD8+ T cell response, and in aspects results in increased secretion of one or more cytokines and chemokines. [00176] In aspects, T cells activated by the phage or T-cell epitope compositions of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2 pr SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) stimulate cell-mediated immunity against SARS-CoV-2, SARS, MERS or similar coronavirus in a subject.
[00177] In aspects, the present disclosure is directed to a method of stimulating, inducing, and/or expanding an immune response, e.g., against SARS-CoV-2 infection (or a closely related virus such as Severe Acute Respiratory Syndrome (SARS) or Middle East respiratory syndrome coronavirus (MERS-CoV)) and/or related diseases caused by SARS-CoV-2, including COVID-19, in a subject in need thereof by administering to the subject a therapeutically effect amount of a phage or T-epitope composition (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein) of the present disclosure.
[00178] In aspects, the present disclosure is directed to a method of preventing, treating, or ameliorating a disease caused by SARS-CoV-2 infection (or a closely related virus such as Severe Acute Respiratory Syndrome (SARS) or Middle East respiratory syndrome coronavirus (MERS-CoV))) such as COVID-19, in a subject in need thereof by administering to the subject a therapeutically effect amount of a phage display, phage DNA or T-cell epitope composition of the present disclosure (including one or more of e.g., polypeptides having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 1, SEQ ID NO: 124, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1, or SEQ ID NO: 124, or between T-cell epitopes in SEQ ID NO: 1 or SEQ ID NO: 124 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, or recombinant); nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells (all of which in aspects may be isolated, synthetic, or recombinant) which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, phage, or cells (all of which in aspects may be isolated, synthetic, or recombinant) having a sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 2, SEQ ID NO: 125, or variants thereof as disclosed herein; vaccine compositions or formulations; and/or pharmaceutical compositions or formulations as disclosed herein). In aspects, the immune response is the result of one or more therapeutic treatments with at least one therapeutic protein, treatment with a vaccine (particularly in situations in which an adverse event results from the vaccination), or treatment with at least one antigen.
[00179] In other aspects, the present disclosure includes multiple rounds of administration of the phage-based vaccine compositions. Such are known in the art to improve or boost the immune system to improve protection against the pathogen. Additionally, the present disclosure may also include assessing a subject’s immune system to determine if further administrations of a phage-based vaccine composition is warranted.
[00180] In some aspects, multiple administrations may include the development of a prime boosting strategy of vaccination using phage mediated display and/or DNA or RNA vaccines. Such may provide an opportunity to produce sequential immunogenic responses against SARS- CoV-2. In some aspects, phage mediated display and phage mediated DNA vaccinations can be achieved in an alternative manner to provide a regimen of immunization with the same immunogen presented in different fashions to mammalian immune system. In such instances, the initial priming of the immune system can be accomplished with phage display vaccine generated by, e.g., the construct set forth in SEQ ID NO: 2, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, or variant thereof and subsequent booster immunizations can be performed with phage DNA vaccine generated by the corresponding peptide construct, i.e. SEQ ID NO: 1 or SEQ ID NO: 124.
[00181] Further aspects and advantages of the invention are provided in the following section, which should be considered as illustrative only.
Examples
[00182] Example 1. SARS-CoV- 2 Antigen design
[00183] The EpiVax proprietary iVAX vaccine design software platform was used to prospectively identify conserved epitope clusters using the first SARS-CoV-2 virus sequence submitted to GenBank on January 17, 2020 as a reference and other publicly available SARS- CoV-2 genomes. A comprehensive description of the advanced set of tools used to develop this vaccine was recently published (De Groot AS, Moise L, Terry F, Gutierrez AH, Hindocha P, Richard G, Hoft DF, Ross TM, Noe AR, Takahashi Y, Kotraiah V, Silk SE, Nielsen CM, Minassian AM, Ashfield R, Ardito M, Draper SJ, Martin WD. Better Epitope Discovery, Precision Immune Engineering, and Accelerated Vaccine Design Using Immunoinformatics Tools. Front Immunol. 2020 Apr 7;11:442. doi: 10.3389/fimmu.2020.00442. PMID: 32318055; PMCID: PMC7154102).
[00184] Using the iVAX platform, the SARS-CoV-2 Envelope, Membrane, and Spike proteins were evaluated for the presence of HLA class I and class II T cell epitopes using the EpiMatrix algorithm. Regions where conserved HLA class I and class II T cell epitopes cluster where then identified using the ClustiMer and JanusMatrix algorithms. These sequences were analyzed using JanusMatrix for potential T cell cross-reactivity with human proteins. An example of iVAX reports for one epitope cluster is shown in Figure 1.
[00185] The rationale for excluding human-like sequences is that T cells that recognize antigen-derived epitopes sharing TCR contacts with epitopes derived from the human proteome may be deleted or rendered anergic before release into the periphery during thymic selection. Therefore, vaccine components targeting these T-cells may be ineffective. In addition, vaccine- induced immune responses targeting cross-reactive epitopes may induce unwanted autoimmune responses targeting the human homologs of the cross-reactive epitopes identified by JanusMatrix. As a result, vaccine safety may be reduced.
[00186] Epitopes demonstrating (i) class II HLA binding potential in the top 5th percentile across multiple supertype alleles (>4), (ii) class I HLA binding potential in the top 5th percentile, (iii) conservation in SARS-CoV-2 isolates (>90%), and (iv) lowest potential to stimulate Tregs educated on human antigens (JanusMatrix score <2) were selected. Sixty-four epitope clusters met the first three criteria; forty-two met ah criteria. Overlapping sequences among the forty-two were consolidate to finally yield 34 epitope clusters.
[00187] Epitope clusters were randomly concatenated head-to-tail to facilitate production of epitopes as a single genetic construct. To avoid generating non-SARS-CoV-2 epitopes at epitope cluster junctions, the order of epitope cluster units was rearranged using the VaxCAD algorithm that reduces off-target junctional epitope potential. Potential immunogenicity was also evaluated at the junction between lambda phage gpD and the concatemer. A concatemer containing all 34 epitopes clusters was generated with no class II junctional immunogenicity aside from one junction where a GPGPG (SEQ ID NO: 71) spacer was inserted to eliminate immunogenicity potential. No class II junctional immunogenicity potential was found at the gpD/epitope interface. Class I immunogenicity potential could not be eliminated from all junctions. Concatemer designs with GSGSG (SEQ ID NO: 74) and GSGSGSG (SEQ ID NO: 75) spacers at all epitope cluster junctions reduced class I junctional immunogenicity potential but were counter-indicated for gene synthesis because they introduce several repeat sequences.
[00188] A total of 34 class I and class II peptides were selected for inclusion in the instantly- disclosed vaccine construct, following immunoinformatic predictions. Selection was based on at least, high binding likelihood to HLA class I and class II alleles, and low tolerogenicity potential. Putative class I epitopes were in the top 1% of predicted ligands, and had Janus Matrix Homology Scores below 2. Putative class II epitopes, were predicted to bind to four or more HLA alleles, and had JanusMatrix Homology Scores below 2. The selected epitope clusters for HLA Class I and Class II used to produce the concatemeric peptide of SEQ ID NO: 1 (and associated nucleic acid constructs encoding such) include the below sequences from the identified SARS-CoV-2 proteins (indicated with the start amino acid number) in TABLE 3.
[00189] TABLE 3
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
[00190] In addition, the concatemer was assessed for potential to form a transmembrane domain using the TMHMM 2.0 prediction tool. Insertion of the concatemer into a membrane could hamper vaccine production. No transmembrane domains were predicted within the concatemer.
[00191] Example 2. Memory T cell responses to SARS-CoV-2 polypeptides in COVID-19 Convalescents
[00192] Materials and Methods
[00193] Peptide synthesis. Synthetic peptides were and can be manufactured using 9- fluoronylmethoxycarbonyl (Fmoc) chemistry by 21st Century Biochemicals (Marlboro, MA). Peptide purity was >90% as ascertained by analytical reversed phase HPLC. Peptide mass was confirmed by tandem mass spectrometry.
[00194] SARS-CoV-2 convalescent donors. Convalescent patients are recruited by Sanguine Biosciences, a clinical services group that identifies, consents and enrolls participants. Inclusion criteria includes subjects (i) willing and able to provide written informed consent and photo identification, (ii) aged 18-60, both male or female, (iii) confirmed COVID- 19 diagnosis (recovered) with date of diagnosis a minimum of 30 days from blood collection, and (iv) positive COVID-19 PCR based-kit documented by time-stamped medical record and/or diagnostic test report and test kit used identified. Exclusion criteria includes subjects who (i) are pregnant or nursing, (ii) have a known history of HIV, hepatitis or other infectious diseases, (iii) have autoimmune diseases, (iv) in vulnerable patient population (prisoners, mentally impaired), (v) have medical conditions impacting their ability to donate blood (i.e. anemia, acute illness) (vi) received immunosuppressive therapy or steroids within the last 6 months, (vii) received an investigational product in the last 30 days, (viii) experienced excess blood loss including blood donation defined as 250 mL in the last month or 500 mL in the last two months, or (ix) had a positive COVID-19 PCR test, but were asymptomatic. Samples are collected in accordance with NIH regulations and with IRB approval.
[00195] Healthy unexposed donors. Samples are obtained from leukocyte reduction filters from the Rhode Island Blood Center for unrelated studies prior to the SARS-CoV-2 outbreak in December 2019. Samples are collected in accordance with NIH regulations and with IRB approval.
[00196] PBMC culture. Thawed whole PBMCs (normal healthy donors) are rested overnight and expanded by antigen stimulation (including select polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1) over nine days at 37°C under a 5% C02 atmosphere. In a 48-well plate, 5 x 10A6 cells in 150 mΐ RPMI medium supplemented with human AB serum are stimulated with pools of peptides at 10 pg/ml on Day 1. Three days later, IL-2 is added at 10 ng/ml and the culture volume raised to 300 mΐ. On Day 7, cells are supplemented with 10 ng/ml IL-2 by half media replacement. Two days later, PBMCs are collected and washed in preparation to measure immune recall responses.
[00197] FluoroSpot Assay. Interferon-gamma (IFN-g) Fluorospot assays are performed ex vivo and following culture using kits purchased from Mabtech and performed according to the manufacturer’s specifications. Peptides are added individually at 10 pg/ml and pooled at 10 pg/ml (8 peptides, 1.25 pg/mL) to triplicate wells containing 250,000 PBMCs (ex vivo) or 100,000 PBMCs (cultured) in RPMI medium supplemented with 10% human AB serum. Triplicate wells are plated with ConA (10 pg/ml) as a positive control, and six wells containing no antigen stimulus are used for background determination. Cells are incubated for 40-48 hours at 37 °C under a 5% C02 atmosphere. Plates are developed according to the manufacturer’s directions using FITC-labeled anti-IFN-g detection antibody.
[00198] Raw spot counts are recorded by ZellNet Consulting, Inc. using a FluoroSpot reader system (iSpot Spectrum, AID, Strassberg, Germany) with software version 7.0, build 14790, where fluorescent spots are counted utilizing separate filters for FITC, Cy3, and Cy5. Camera exposure and gain settings are adapted for each filter to obtain high quality spot images preventing over- or underexposure. Fluorophore-specific spot parameters are defined using spot size, spot intensity and spot gradient (fading of staining intensity from center to periphery of spot), and a spot separation algorithm is applied for optimal spot detection.
[00199] Results are calculated as the average number of spots in the peptide wells, adjusted to spots per one million cells. Responses meeting the following criteria are positive when the number of spots is (i) at least twice background, (ii) greater than 50 spot forming cells per well above background (1 response per 20,000 PBMCs), and (iii) statistically different (p<0.05) from the media-only control by the Student’s t test.
[00200] Results:
[00201] Results with some identical and other similar SARS-CoV-2 peptides that are included in the concatemers of the instant disclosure using the experimental design disclosed above are herein referenced as a guide for expected results. For FIGS. 3-12, peptide 1 (or rank 1) is SEQ ID NO: 45; peptide 2 (or rank 2) is SEQ ID NO: 17; peptide 3 (or rank 3) is SEQ ID NO: 5; peptide 4 (or rank 4, which is I ASFRLF ARTRSM W SFN (SEQ ID NO: 76)) is a segment of SEQ ID NO: 69; peptide 5 (or rank 5, which is FLGVYYHKNNKSWMESE (SEQ ID NO: 77)) is a segment of SEQ ID NO: 33; peptide 6 (or rank 6, which is ESEFRV Y S S ANNCTFE YV (SEQ ID NO: 78)) is a segment of SEQ ID NO: 33; peptide 7 (or rank 7, which is LSYFIASFRLFARTR (SEQ ID NO: 79)) is a segment of SEQ ID NO: 69; peptide 8 (or rank 8, which is VEGFNCYFPLQSYGFQPT (SEQ ID NO: 80)) is a segment of SEQ ID NO: 31; peptide 9 (or rank 9) is SEQ ID NO: 21; peptide 10 (or rank 10) is TLSYYKLGASQRVAGD (SEQ ID NO: 81); peptide 11 (or rank 11, which is ANQFNSAIGKIQDSL (SEQ ID NO: 82) is a segment of SEQ ID NO: 9; peptide 12 representing residues 4 to 15 of SEQ ID NO: 67 (or rank 12); peptide 13 (or rank 13) is SEQ ID NO: 37; peptide 14 (or rank 14) is SEQ ID NO: 35; peptide 15 (or rank 15, which is NKC VNFNFN GLTGT (SEQ ID NO: 128) is a segment of SEQ ID NO: 23; peptide 16 (or rank 16) is SEQ ID NO: 29; peptide 17 (or rank 17) is SEQ ID NO: 55; peptide 18 (or rank 18) is SEQ ID NO: 11; peptide 19 (or rank 19) is SEQ ID NO: 47; peptide 20 (or rank 20) is SEQ ID NO: 13; peptide 21 (or rank 21) is SEQ ID NO: 39; peptide 22 (or rank 22, which is FASVYAWNRKRISNSVAD (SEQ ID NO: 83)) is a segment of SEQ ID NO: 65; peptide 23 (or rank 23) is SEQ ID NO: 59; peptide 24 (or rank 24) is SEQ ID NO: 19; peptide 25 (or rank 25) is SEQ ID NO: 63; peptide 26 (or rank 26) is SEQ ID NO: 49; peptide 27 (or rank 27, which is LQS YGFQPTN GV GY QP Y (SEQ ID NO: 84)) is a segment of SEQ ID NO: 31; peptide 28 (or rank 28, which is QKLIANQFNSAIGKI (SEQ ID NO: 85)) is a segment of SEQ ID NO: 9; peptide 29 (or rank 29) is SEQ ID NO: 3; peptide 30 (or rank 30, which is LKKLLEQWNLVIGFL (SEQ ID NO: 86)) is a segment of SEQ ID NO: 61; peptide 31 (or rank 31, which is FGEVFNATRFASVYA (SEQ ID NO: 87)) is a segment of SEQ ID NO: 65; and peptide 32 (or rank 32) is SEQ ID NO: 25. Additionally, pool A includes peptides 1-8, pool B includes peptides 9-16, pool C includes peptides 11-18, and pool D includes peptides 19-26.
[00202] As shown in FIG. 3 and FIG. 4, ex vivo immune recall responses differentiate SARS- CoV-2 naive and experienced individuals and exhibit different COVID-19 immunotypes. Robust and failed immune responses in convalescent donors may represent different immunotypes characterized in a deep immune profiling study of SARS-CoV-2 experienced humans (Giles et al. Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications. Science. 2020 Jul 15:eabc8511. doi: 10.1126/science. abc8511. PMID: 32669297, herein incorporated by reference in its entirety).
[00203] As shown in Figures 5 and 6, strong ex vivo immune recall responses are found or may be found in SARS-CoV-2 experienced individuals using polypeptides included in the concatemers of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1).
[00204] As shown in Figure 7, polypeptides included in the concatemers of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1) may stimulate ex vivo immune recall response in natural SARS-CoV-2 infection. [00205] As shown in Figures 8 and 9, polypeptides included in the concatemers of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1) may stimulate higher IFN-g responses in naive and COVID-19 convalescent donors following expansion in culture. Response in naive donors may suggest such polypeptides of the instant disclosure expand low frequency cold coronavirus cross-reactive T cells. Further, differences between responses by pool in ex vivo and cultured assay may reflect variable phenotypes and/or proliferative capacities of epitope-specific T cells when they are put into culture.
[00206] As shown in Figures 10 and 11, polypeptides included in the concatemers of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1) stimulate or may stimulate low frequency epitope-specific T-cells following expansion in culture in naive and COVID-19 convalescent donors. Differences between responses to spike and membrane peptides in ex vivo and cultured assay may reflect variable phenotypes and/or proliferative capacities of epitope-specific T-cells when they are put into culture.
[00207] As shown in Figure 12, polypeptides included in the concatemers of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1) stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors. Of 32 tested peptides, 27 peptides demonstrated positive responses, as shown in green, in at least one donor. Further, predicted spike epitope cross conservation with common cold coronaviruses were confirmed in naive donors. [00208] As such, the data in Figures 3-12 demonstrates that polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NO: 1 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NO: 1 or between T-cell epitopes in SEQ ID NO: 1) are recognized by T cells raised in natural infection, stimulate Thl cytokine production, may stimulate pre-existing immunity to common cold coronaviruses, and memory may boost immunity in clinical trials.
[00209] Example 3. Cloning
[00210] The specific DNA sequences coding for the concatemer peptide sequence can be synthesized. Using specific oligo primers, the synthesized DNA fragments can be PCR amplified to provide restriction sites for in-frame cloning with a head protein sequence. The oligo sequence of each PCR primer is next modified to produce restriction sites in each end of amplified DNA fragments. After restriction digestion, these fragments were inserted separately at the 3 ' end of a DNA fragment encoding the head protein under the control of the lac promoter. The constructs were created in a plasmid vector (donor plasmid), which also carries loxPwt and loxP511 sequences. Presence of each inserted DNA fragments in recombinant donor plasmid can be confirmed by restriction enzyme analysis. Cre-expressing cells ( E . coli ) can then be transformed with these recombinant donor plasmids and subsequently infected with a recipient T4 phage that carries a stuffer DNA fragment flanked by loxPwt and loxP511 sites. T4 phage infected Cre-expressing E. coli is then grown at LB Ampicillin (100 ug/ml) at 37° C. for four hours in presence of 0.2% maltose and 0.1M CaC12. Recombination occurs in vivo at the lox sites and Ampr cointegrates are formed, which then spontaneously lyse the E. coli and release in culture media (figure 6).
[00211] Example 4. Selection of T4 Lysogens and Production of Recombinant Lambda Phage which display SARS-COV-2 virus specific linear epitopes:
[00212] T4 cointegrates can be used to produce T4 lysogens and then selected on Luria Bartani (LB) ampicillin agar (100 ug/ml amp, 15% agar) plates. Briefly, cointegrates from spontaneously lysed E. coli culture are used to infect Cre-ve, suppressor-ve E. coli cells and spread on LB ampicillin agar plates. Plates are incubated at 30 °C for 48 hours to obtain Ampr colonies which are actually recombinant T4 lysogens and carry a recombinant T4 phage genome in their chromosome. Individual Ampr colonies of T4 lysogens are subjected to PCR amplification using primers to confirm that T4 lysogens are recombined properly with donor plasmid and successful transfer of the recombinant plasmid in the T4 genome. The PCR confirmed Ampr colonies containing the lambda cointegrate are grown separately at LB Ampicillin (100 ug/ml) at 37° C. for four hours in presence of 0.2% maltose and 0.1M CaC12. Lambda lysogens are spontaneously induced in these cultures and result in complete lysis. This cell free supernatant can be used to infect E. coli cells and plated on solid LB agar (15%) plate to obtain phage plaques. The resulting phage plaques are then harvested from the plate and single plaques are purified three times on E. coli by the standard procedures described by Russell, J.S.a.D., Molecular Cloning: A Laboratory Manual. 4th Edition ed., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Approximately 220 copies of ARS- COV-2 virus specific peptides are displayed on a single phage head.
[00213] Example 5. Conformation of T4 Phage Plaques Containing SARS-CoV-2 Fragments:
[00214] All phage plaques, containing T4 cointegrates, which are used for SARS-CoV-2 phage vaccine production can then be verified by PCR. In this process the presence of each cloned inserts in T4 phage are confirmed by PCR amplification of insert DNA. The specific primers flank the T4 genome and the insert. Phage DNA containing co-integrates are then subjected to complete genome sequencing and subsequent bioinformatics analysis to confirm the proper orientation and sequence of inserted DNA fragments in T4 genome as gp23/gp24/Hoc/Soc fusion.
[00215] Example 6. Growth and Purification of Recombinant Phage Displaying SARS-CoV- 2 Peptides:
[00216] Growth of the plaque purified phage is performed in two steps. The steps are designated as plate lysate method and large scale liquid lysate method. The detail of these procedures are described in Russell, J.S.a.D., Molecular Cloning: A Laboratory Manual. 4th Edition ed., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. The resulting lysed culture is chilled at stored at room temperature for further purification by tangential flow filtration system (TFF). Due to the lysogenic nature of T4 phage, a few modifications of phage amplification process are required for optimal growth of phage production (phage titer of 108pfu/ml to 109pfu/ml).
[00217] For the plate lysate steps, the materials include: Luria Broth, 20% Maltose in water (filter sterilized), 1 M gCL in water (filter sterilized), an E. coli sup- host, flasks, shaking incubators, cuvettes, spectrophotometer, a T4 phage construct, micropipetters and pipette tips, serological pipettes, 1 mg/ml DNase I stock in water (filter sterilized), and filter membranes (0.8 pm, 0.45 pm, and 0.22 pm). Briefly, an overnight culture of E. coli sup- host strain in Luria Broth with 0.2% Maltose and 10 mM MgCk is prepared along with a suitable media of 1:100 20% Maltose and 1:100 1 M MgCh to Luria Broth (e.g. for 2 Liters LB media add 20 ml 20% Maltose and 20 ml 1M MgCh). A 1 mL aliquot of the prepared media for an optical density standard is transferred to a cuvette. The prepared media is then inoculated at a 1:100 with the overnight E. coli sup- culture and mixed. A 1 ml aliquot of the inoculated culture is transfer to a cuvette and the optical density (OD) at 600nm is measured to obtain a starting time point of the culture. The E. coli culture is then incubated at 37 °C in a shaking incubator and the OD of the culture monitored until an optical density of 0.090 to 0.100 at 600 nanometers (nm) is obtained. The liquid culture is then infected with the l construct phage at a multiplicity of infection (MOI) of 0.05 (e.g. a 2 liter culture at an OD = 0.1 would have approximately 2x1011 colony forming units of E. coli. This would require lxl 010 plaque forming units (pfu) of phage for a successful lysate production). The infected culture is then incubated in a shaking incubator at 38.5 °C, with the optical density at 600 nm being measured every hour after infection (e.g. for a 2 liter (L) preparation, complete lysis should take 4 to 6 hours post infection). Once visible debris begins to form (around 3 to 4 hours post infection) 1 milliliter (mL) of DNase I is added (1 mg/ml filter sterilized). After complete lysis of the culture, the contents are transferred to containers suitable for centrifugation at 10,000 x g for 10 minutes at 4 °C. The resulting lysate is then sterilized through filter membranes sequentially from 0.8 pm, 0.45 pm, and 0.22 pm and stored at 4 °C for further purification through tangential flow filtration (TFF) system.
[00218] For large scale purification, TFF purified phage vaccine preparations are extracted once with organic solvents octanol for removing lipopolysaccharide (lps) from vaccine preparations. The materials include: 50 ml conical tubes, Lipid Removal Adsorbent (Supelco Cat# 13360-U), 1 -Octanol (Fisher Chemical Cat# A402-500), ethanol (Fisher Chemical Cat# BP28184), Float- A-Lyzer G2, 10 kD, 10 ml (Repligen Cat# G235067), Thermo Scientific BupH Phosphate Buffered Saline Packs (Fisher Cat# PI28372), Steriflip vacuum filter (Millipore Sigma Cat# SE1M179M6), syringes and 18 gauge needles, 4 L container, and a magnetic stir bar. Briefly, the steps include starting with a phage lysate sample that has been previously purified through tangential flow filtration which are achieved by concentrating 5 L of phage lysate down to 100 ml total volume. After concentration, media is replaced by dia- filtration against 4 L of 0.5 molar (M) Phosphate Buffered Saline (PBS). 50 ml of purified phage lysate is then even split between two 50 ml conical tubes and 15 mg/ml Lipid Removal Adsorbent (LRA) is added to each conical tube. The LRA is next mixed into solution by inverting the tube (do not mix too harshly as the LRA may damage the phage) and once the LRA is in solution, the 50 ml conical tubes are placed on a rocker at room temperature to be rocked vigorously for about 30 minutes. The conical tubes are then removed and the LRA is allowed to settle for 5 minutes. The phage lysate with LRA is then passed through a 0.22 pm Steri-flip vacuum filter to remove LRA and a 0.4 volume of 1-Octanol is added to each LRA- treated phage lysate, followed by mixing by inversion. The tubes are then placed on the rocker at room temperature and rocked vigorously for 1 hour. The tubes are then incubated on ice for 15 minutes, followed by centrifugation at 4000 x g for 10 minutes at 4 °C. The bottom of the tubes is then pierced using a syringe and needle and the lower aqueous portion is removed without disturbing the top 1-Octanol layer (contains endotoxin) and transferred to a clean 50 ml conical tube. A small amount of the aqueous layer behind may be left behind to prevent disturbing the top layer. Dialysis tubing is pre-moistened in 25% ethanol in distilled water for 1 minute and then loaded with 10 ml of 1-Octanol treated per tube and dialyzed in 4 L of 25% ethanol in distilled water overnight at 4 °C with slow agitation from a magnetic stir bar. Dialysis chambers are then transferred to 4 L of 0.5 M PBS to dialyze for 6 to 8 hours, followed by further dialysis overnight in fresh 4 L of 0.5 M PBS. The dialysis chambers are then transferred to fresh 4 L of 0.5 M PBS to dialyze for another 6 to 8 hours. The lysate is then removed, transferred to clean 50 ml conical tubes and sterilized through 0.22 pm Steri-flip vacuum filter. The resulting phage lysate can then be stored at 4 °C.
[00219] Example 7. Immunogenicity
[00220] It was previously demonstrated that a phage H7N9 influenza vaccine displaying concatenated iVAX-identified CD4+ T cell epitopes stimulates de novo epitope-specific Thl responses in HLA-DR3 transgenic mice. H7N9 class II HLA epitopes with low human cross reactivity potential were identified in H7N9 HA and internal antigens using the EpiMatrix, ClustiMer, and JanusMatrix algorithms. Epitopes were concatenated in an arrangement that minimized off-target immunogenicity at epitope junctions using the VaxCAD algorithm. A synthetic gene encoding the epitope concatemer was produced, subcloned into a plasmid DNA vector and lamba phage capsid, and DNA and phage vaccines were produced. HLA-DR3 mice were primed with pDNA and boosted twice with phage at two week intervals. Two weeks following the final immunization, splenic leukocytes were harvested and stimulated overnight with pools of vaccine-matched HA and internal antigen peptides to determine frequencies of Thl cytokine-producing CD4+ T cells by flow cytometry. Immunization elevated frequencies of cytokine+ CD4+ T cells specific for HA and internal antigen epitopes as determined by intracellular cytokine staining (IL-2, IFNy, TNFa) (Figure 2). In addition to demonstrating the importance of selecting CD4+ T cell epitopes that can drive effector T cell responses, this study also shows the effectiveness of epitope-driven, phage-vectored vaccine designs for stimulation of Thl cytokine production, which is needed to support an effective response to SARS-CoV-2 and avoid Th2 responses associated with enhanced respiratory disease.
[00221] A similar experimental study can be used to determine frequencies of Thl cytokine- producing CD4+ T cells. The T4 phage display constructs and phage DNA constructs can be primed in murine models with phage DNA (pDNA), followed by additional phage boosters. Splenic leukocytes may then be harvested and stimulated O/N with pools of vaccine-matched SARS-CoV-2 and internal antigen peptides. Thl cytokine producing CD4+ T cells can be identified by flow cytometry and cytokine levels determined by intracellular staining. It is expected that the use of the T4 phage display constructs and phage DNA constructs used in such a prime-boost setting will result in elevated frequencies of cytokine+ CD4+ T cells specific for SARS-CoV-2 and internal antigen peptides epitopes as determined by intracellular cytokine staining (IF-2, IFNy, TNFa).
[00222] Example 8. Vaccinations
[00223] Animal Study 1 [00224] Studies of T-cell responses in C57BL/6 mice were performed as an art acceptable model predicting efficacy in humans and other organisms. SEQ ID NO: 1 was subcloned into a plasmid DNA vaccine vector (Nature Technology Corporation, Lincon, NE). Plasmid was formulated in a proteinaceous lipid vesicle (FUSOGENIX, Entos Pharmaceuticals, Edmonton, Alberta, Canada) for administration to C57BL/6 mice to evaluate vaccine immunogenicity. Mice received a 25 pg or 100 pg dose of encapsulated DNA vaccine intramuscularly on day 1 and day 14. Controls received saline or encapsulated empty vector.
[00225] Splenocytes were collected from mice on day 28 and stimulated with pools of overlapping spike peptides or envelope, or membrane peptides spanning the full length of the antigens. Interferon-y-producing CD8+ T cells were enumerated by flow cytometry. IFNy is the signature cytokine of type 1 helper T cells. As is observed in Figure Splenocytes stimulated with each peptide pool demonstrate significantly higher frequencies of CD8+IFNy+ T-cells over unstimulated cells in mice immunized with SEQ ID NO: 1. No significant differences were observed in mice that received saline or empty vector. Frequencies of CD8+IFNy+ T- cells in peptide stimulations were significantly elevated in mice that received SEQ ID NO: 1 over those that received saline or empty vector.
[00226] From the same splenocyte collection, CD8+ T-cells expressing surface CD107a were enumerated by flow cytometry. CD 107a expression at the cell surface is a marker of cytolytic granule release. As illustrated in Figure 14, splenocytes stimulated with both spike and envelope/membrane peptide pool demonstrate significantly higher frequencies of CD8+CD107a+ T-cells over unstimulated cells in mice immunized with SEQ ID NO: 1. No significant differences were observed in mice that received saline or empty vector. Frequencies of CD8+CD107a+ T-cells in peptide stimulations were significantly elevated in mice that received SEQ ID NO: 1 over those that received saline or empty vector.
[00227] Functional cytolytic cells among the splenocytes as collected at day 28 as above were enumerated in a cytolytic T lymphocyte assay using B 16-OVA cells transduced with vectors expressing SEQ ID NO: 1 or full length spike (Wuhan-Hu-1; Genbank Accession No: YP 009724390.1). Splenocytes were mixed with target cells and target cell viability was measured after 24 hours. As is illustrated in Figure 15, cytolytic T lymphocytes from mice immunized with SEQ ID NO: 1 kill cells expressing the vaccine-matched epitopes (SEQ ID NO: 1; left side) or spike protein (right side) significantly more than cytolytic T lymphocytes from mice that received saline or empty vector.
[00228] Serum anti-spike antibody levels were measured using serum samples collected on day 21 following immunization with a lipid vaccine including a plasmid housing SEQ ID NO: 1 as above. As is illustrated in Figure 16, all mice that received the concatemer of SEQ ID NO: 1 seroconverted. A dose-dependent effect was observed with mice that received the 100 pg dose showing higher anti-spike antibody fold increases over baseline (prior to immunization) than those that received the 25 pg dose. Both SEQ ID NO: 1 doses elicited significantly higher fold increases in anti-spike antibody over mice that received saline.
[00229] Day 21 neutralizing titers of the above paragraph were also measured in a pseudotyped virus neutralization assay using spike pseudotyped lentivirus. Both SEQ ID NO: 1 vaccine doses elicited potent neutralizing titers significantly greater than mice that received empty vector. Neutralizing titers in SEQ ID NO: 1 immunized mice exceed titers found in SARS-CoV-2 convalescent patient samples measured in the pseudotyped virus neutralization assay.
[00230] Animal Study 2
[00231] The purpose of this experiment is to determine the efficacy of the phage vaccines to elicit antibody response in BALB/c female mice. Four separate groups of mice (Group A, Group B, Group C, 5 mice in each group and Group D, 40 mice) will be injected subcutaneously (s/c) with various phage constructs. Briefly, group A mice will receive 5xlOspfu of T4 phage particles suspended in 500 mΐ of sterile PBS. A control group of mice (group B) will receive recombinant SARS-CoV-2 antigen (50 pg/mouse) suspended in sterile PBS. After primary inoculation, mice will receive 1st and 2nd booster (dose will be the same as primary inoculation) of corresponding antigens at 2 weeks interval. All animals will be bled prior primary inoculation. Serum samples will be collected before every booster to monitor progression of immune response against SARS-CoV-2 antigens. After 21 days, animals will be euthanized for final bleeding through cardiac puncture. Finally animals will be sacrificed by spinal dislocations. The immune response against various SARS-CoV-2-phage vaccines will be monitored by western immunoblot and ELISA. [00232] Animal Study 3
[00233] A further study can use male BALB/c mice (18-25 g each), each being immunized via intramuscular injection (i.m.) with the T4 phage display vaccine construct. Group A receives a single dose, B receives 2 (days 0 and 14) and C receives 3 (days 0, 14, and 21). Blood is collected before each dose and at 2, 4, and 8 weeks post all vaccinations. 50 pL of blood will also be collected 7 hours after challenge to compare vaccine efficacy. The dose will be a total of 109 pfu of the phage cocktail. At day 56, mice will be euthanized and tissue and blood collected therefrom. Serum, lungs, spleen and lymph nodes will be analyzed immunologically and histopathologically. Blood will be clotted for collection of serum and sera will be pooled and analyzed to determine levels of various cytokines including TNF-a, IL- la, IL-6 and IFN-g. Cytokines can be measured via ELISA according to the manufacturer’s instructions. Antigen specific and T4 phage capsid specific antibody titers of mouse sera before and after vaccinations will also be monitored by ELISA and western blot to enumerate humoral immune response to the vaccine. Histopathology of various organs will also be analyzed to determine the extent of inflammatory tissue damage, particularly with respect to any cytotoxic effect of phage vaccines. For statistical plans, a 4-fold immune response with descriptive characterization across groups will be used.
[00234] Clinical Studies
[00235] The therapeutic vaccines of the present disclosure, including phage display and/or phage DNA, are designed to elicit antibodies that may disrupt the lifecycle of SARS-CoV-2, as well as SARS, MERS and/or similar coronaviruses. The vaccines are designed to present viral T-cell epitopes to elicit such response. Clinical trials with the therapeutic vaccines employing a dosage form including an immunogenic composition of the present disclosure against SARS-CoV-2, such as a phage display cocktail to provide SEQ ID NO: 1, or a phage DNA cocktail to provide SEQ ID NO: 2, or each T-cell epitope polypeptide or nucleic acid, are contemplated. For example, a clinical trial for a phage display cocktail to provide SEQ ID NO: 1 (which may be administered, e.g., orally, sublingually, intranasal, transdermal (i.e., applied on or at the skin surface for systemic absorption), ocularly, percutaneous, via mucosal administration, or via a parenteral route (intradermal (e.g., via microneedle), intramuscular, subcutaneous, intravenous, or intraperitoneal) in a phase I study. Participants (n=25/group) will be administered the phage display cocktail at a fixed dosing level using a TDM patch (3M) with 1 mL of phage 106 pfu in a single dose, 2 dose (14 days apart) or 3 dose (14 days apart). Participants will be monitored for adverse effects using a toxicity grading scale and pain will be assessed at a minimum of 30 minutes post- vaccination.
[00236] Immunogenicity will be assessed through blood samples and obtaining antibody and T-cell responses at baseline and then at weeks 1, 2, 4, 8 and 12 after each vaccination, and at 3, 6, and 12 months following final vaccination. Whole blood will be processed to obtain peripheral blood mononuclear cells (PBMCs) for determination of cell mediated immune response (CD4 and CD8 T-cell responsiveness to the T-cell epitope polypeptides (SEQ ID NO: 1)·
[00237] Participants will include subjects between 18-50 yrs old including male and female subjects. Informed consent will be obtained for each. Subjects with a history of immunosuppressive or autoimmune disease will not be considered. Subjects already known to have had a prior SARS-CoV-2 infection or that test positive for infection prior to commencing vaccination will also not be considered.
[00238] The clinical model may be also adapted. For example, a series of vaccine applications may also be applied to a set of human subjects. The set can be divided into a control group, a group that receives alternating phage display and phage DNA compositions, a group that receives only phage display and a group that receives only phage DNA. Subgroups may be further established to study a cocktail of all three constructs in comparison to one construct only. Further groups may also be established to assess administration of a cocktail of all three in comparison to sequential administration of different phage constructs. Following the final administration, blood samples may be collected and challenged with SARS-CoV-2 virus, followed by determination of specific immune response to the challenge.
[00239] The T4 phage concatemer can be further prepared as an equimolar oral or transdermal vaccine with appropriate carriers and excipients to assist in administration. The vaccine can be applied as a single dose or followed up later with a further booster of the same vaccine. In other instances, a booster may include phage DNA. The efficacy of the route of administration can also be determined. [00240] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.
[00241] The compositions and methods described herein are presently representative of particular embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

Claims

Claims We claim:
1. A bacteriophage comprising at least one amino acid sequence of a concatemer of SARS-CoV-2 T-cell epitopes, wherein the at least one amino acid sequence is SEQ ID NO:
1.
2. The bacteriophage of claim 1, wherein SEQ ID NO: 1 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 2.
3. The bacteriophage of claim 1, wherein the bacteriophage is T4.
4. A bacteriophage comprising a nucleic acid with at least 85% sequence identity to a nucleic acid sequence as set forth in SEQ ID NO: 2, wherein the nucleic acid is under the control of a promoter.
5. The bacteriophage of claim 4, wherein the promoter is a bacteriophage promoter.
6. The bacteriophage of claim 5, wherein the nucleic acid is fused in frame to a head protein of the bacteriophage.
7. The bacteriophage of claim 4, wherein the promoter is a mammalian promoter.
8. The bacteriophage of claim 7, wherein the mammalian promoter is selected from the group consisting of CMV, SV40, CAG or U6.
9. A method of eliciting an immune response in a subject comprising administering a therapeutically effective amount of the bacteriophage of any one of claims 1-8 to the subject.
10. A method for inducing immunity against SARS-CoV-2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bacteriophage of any one of claims 1-8.
11. A method for inducing immunity against 2019-nCoV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a phage as disclosed herein.
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