WO2022067062A1 - Développement rapide d'un vaccin prophylactique à large spectre pour le sars-cov-2 à l'aide d'un système d'administration d'antigène à médiation par phage - Google Patents

Développement rapide d'un vaccin prophylactique à large spectre pour le sars-cov-2 à l'aide d'un système d'administration d'antigène à médiation par phage Download PDF

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WO2022067062A1
WO2022067062A1 PCT/US2021/051988 US2021051988W WO2022067062A1 WO 2022067062 A1 WO2022067062 A1 WO 2022067062A1 US 2021051988 W US2021051988 W US 2021051988W WO 2022067062 A1 WO2022067062 A1 WO 2022067062A1
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seq
phage
polypeptide
nos
aspects
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PCT/US2021/051988
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William Martin
Lenny MOISE
Biswajit Biswas
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Epivax, Inc.
Secretary Of The Navy
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Publication of WO2022067062A1 publication Critical patent/WO2022067062A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—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
    • 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

Definitions

  • This disclosure relates to the development of a rapid broad spectrum vaccine against SARS-CoV-2 viral infection.
  • the present disclosure provides a phage-produced vaccine for prevention of SARS-CoV-2 viral infection in human subjects.
  • these types of whole cell vaccines are good immune-stimulator due to their inherited nature of particulate structures which attract antigen presenting cells (APCs). Delivering foreign antigens as particulate is more promising as an immune stimulator because this presentation mimics the actual infection during normal disease manifestation process.
  • APCs antigen presenting cells
  • attenuated vaccines are not always ideal for mass scale vaccination due to reversal of virulence.
  • Formalin can also denature major immunogenic antigens.
  • empty capsid vaccines are prepared for imitating the particulate nature of pathogens.
  • several particulate vaccines are also prepared by using Ankara and Baculovirus vector systems where these recombinant virus act as a chimera of pathogens.
  • manufacturing process of these vaccines are time consuming and require expensive cell culture media, which are not ideal for large quantity production of vaccines during outbreaks.
  • Bacteriophage or phage are bacterial viruses which cannot infect eukaryotic cells. These bacterial-viruses, such as M13, T4, T7, lambda (X) etc., can be propagated using laboratory strains of E.coli with inexpensive bactrological 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 are recognized as displayed antigens and generate an immune response. Thus, such chimeric constructs can be used as vaccine agents.
  • LB Luria-Bertani
  • Individual phage particles can display multiple copies (in particular aspects, up to around 420 copies) of identical or combinational antigenic epitopes on its surface and act as a good immunogen when introduced into mammalian system.
  • the particulate nature of the phage specifically attracts antigen-presenting cells (APCs) and can provoke both cell mediated and humoral responses.
  • APCs antigen-presenting cells
  • phage can 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 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 selected from SEQ ID NO: 1, SEQ ID: 3 and/or SEQ ID NO: 5.
  • the at least one amino acid sequence includes SEQ ID NO: 1.
  • SEQ ID NO: 1 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 2.
  • the at least one amino acid sequence includes SEQ ID NO: 3, which in some aspects may be encoded by the nucleotide sequence as set forth in SEQ ID NO: 4.
  • the at least one amino acid sequence comprises SEQ ID NO: 5, which in further aspects may be encoded by the nucleotide sequence as set forth in SEQ ID NO: 6.
  • the bacteriophage is selected from Lambda, T4, T7 or M13/H. In other aspects, the bacteriophage is Lambda.
  • the present disclosure concerns a composition of three bacteriophage with one bacteriophage featuring SEQ ID NO: 1, a second bacteriophage featuring SEQ ID NO:
  • the present disclosure concerns a bacteriophage with a nucleic acid of at least 85% sequence identity to a nucleic acid sequence selected SEQ ID NO: 2, SEQ ID NO:
  • the nucleic acid is under the control of a promoter.
  • the promoter is a bacteriophage promoter.
  • the nucleic acid is fused in frame to a head protein 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 concerns a polypeptide consisting of an amino acid sequence selected from one or more of SEQ ID NOS: 1, 3, or 5 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 NOS: 1, 3, or 5.
  • the polypeptide consists essentially of an amino acid sequence selected from one or more of SEQ ID NOS: 1, 3, or 5 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 NOS: 1, 3, or 5.
  • the polypeptide includes an amino acid sequence selected from one or more of SEQ ID NOS: 1, 3, or 5 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 NOS: 1, 3, or 5.
  • the present disclosure concerns a nucleic acid encoding a polypeptide consisting of or consisting essentially of or including an amino acid sequence selected from one or more of SEQ ID NOS: 1, 3, or 5 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 NOS: 1, 3, or 5.
  • the nucleic acid is selected from one or more of SEQ ID NOS: 2, 4, or 6.
  • the present disclosure provides a vector including any nucleic acid disclosed herein.
  • 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 is selected from the group consisting of SEQ ID NO: 1, SEQ ID: 3 and SEQ ID NO: 5.
  • the present disclosure provides methods for inducing an immune response against SARS-CoV-2 in a subject in need thereof, by administering a therapeutically effective amount of one or more isolated T-cell epitopes or concatemers selected from SEQ ID NOS: 1, 3 or 5 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 NOS: 1, 3, or 5.
  • the immune response may be a result of treatment with at with at least one antigenic protein, or with a vaccine.
  • 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 or T-cell epitope as disclosed herein.
  • 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 nucleic acid as disclosed herein.
  • Figures 1 A-D show immunoinformatic reports for a sample epitope cluster sequence in one phage. Cluster Detail Reports for LGVYYHKNNKSWMESE are shown.
  • Figure 1A 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.
  • 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.
  • Figure 1C is the JanusMatrix report for identified MHC class II clusters and Figure ID is the JanusMatrix report for identified MHC class I clusters. *Count of HUMAN JanusMatrix matches found in the search database.
  • a Janus Matrix match is a 9-mer derived from the search database (e.g., the human genome) which is predicted to bind to the same allele as the EpiMatrix Hit and shares TCR facing contacts with the EpiMatrix Hit.
  • the search database e.g., the human genome
  • 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.
  • 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.
  • FIG. 2 shows PCR amplified products of NV106, NV107, and NV108.
  • the Integrated DNA Technologies (IDT) synthesize DNA fragments are PCR amplified for creating restriction sites to facilitate in-frame cloning with gpD sequence of lambda genomes.
  • the oligo sequence of each PCR primer is modified to produce Nhe I and Bssh II restriction sites at 5’ and 3’ end of amplified DNA fragments respectively.
  • Two pl of The PCR amplified products of each construct are electrophoresed on 1% agar gel for 1 hour at constant volt of 80. Agar gel is stained with ethidium bromide and visualized under ultra violet UV) light.
  • Lane 1 & 2 depict the PCR product amplified of NV 106.
  • Lane 3 & 4 depict the PCR product amplified of NV 107.
  • Lane 5 & 6 depict the PCR product amplified of NV 107.
  • Figure 3 shows cloning of NV-106 DNA fragment in donor plasmid containing gpD sequence of lambda phage.
  • DNA fragments are synthesized and PCR amplified for producing Nhel and BssHII sites at 5/ and 3/ end of the DNA fragments. Fragments are restriction digested with Nhel and BssHII and directionally cloned at the 3/ end of gpD sequence of plasmid vector specifically designated as donor plasmid.
  • Competent A coll cells are transformed by recombinant donor plasmid and plated on LB-Ampicillin (amp) plate for selections.
  • Recombinant plasmid DNA of these clones are harvested using GeneJet kit (Fisher Scientific, Waltham, MA) and subsequently restriction digested with Hindlll and EcoRI. Restriction digested recombinant plasmid DNA are electrophoresed on 1% agar gel at constant volt of 80. After electrophoresis gels are stained with ethidium bromide and visualized under ultraviolet (UV) light. Restriction digestion analysis of recombinant plasmid DNA extracted from several clones indicate that 2 colonies (lane# 2 & 4) are harboring NV-106 DNA fragment.
  • Figure 4 shows continued cloning of NV- 107 DNA fragment in donor plasmid containing gpD sequence of lambda phage.
  • DNA fragments are synthesized and PCR amplified for producing Nhel and BssHII sites at 5/ and 3/ end of the DNA fragments. Fragments are restriction digested with Nhel and BssHII and directionally cloned at the 3/ end of gpD sequence of plasmid vector specifically designated as donor plasmid.
  • Competent E. coli cells are transformed by recombinant donor plasmid and plated on LB-Ampicillin (amp) plate for selections.
  • Recombinant plasmid DNA of these clones are harvested using GeneJet kit (Fisher Scientific, Waltham, MA) and subsequently restriction digested with Hindlll and EcoRI. Restriction digested recombinant plasmid DNA are electrophoresed on 1% agar gel at constant volt of 80. After electrophoresis gels are stained with ethidium bromide and visualized under ultraviolet (UV) light. Restriction digestion analysis of recombinant plasmid DNA extracted from 6 separate clones indicate that all 6 colonies (lane# 1, 2, 3, 4, 5 & 6) are harboring NV-107 DNA fragment.
  • Figure 5 shows further cloning of NV-108 DNA fragment in donor plasmid containing gpD sequence of lambda phage.
  • DNA fragments are synthesized and PCR amplified for producing Nhel and BssHII sites at 5/ and 3/ end of the DNA fragments. Fragments are restriction digested with Nhel and BssHII and directionally cloned at the 3/ end of gpD sequence of plasmid vector specifically designated as donor plasmid.
  • Competent A. coli cells are transformed by recombinant donor plasmid and plated on LB-Ampicillin (amp) plate for selections.
  • Recombinant plasmid DNA of these clones are harvested using GeneJet kit (Fisher Scientific, Waltham, MA) and subsequently restriction digested with Hindlll and EcoRI. Restriction digested recombinant plasmid DNA are electrophoresed on 1% agar gel at constant volt of 80. After electrophoresis gels are stained with ethidium bromide and visualized under ultraviolet (UV) light. Restriction digestion analysis of recombinant plasmid DNA extracted from 6 separate clones indicate that all 6 colonies (lane# 1, 2, 3, 4, 5 & 6) are harboring NV-108 DNA fragment.
  • Figure 6 shows an overview of cloning of NV106, NV107, and NV108 peptide fragments in lambda phage genome. These peptide fragments are composed of linear array of predictive antigenic epitopes of SARS-CoV-2 virus. DNA sequence of corresponding peptide fragment of NV106, NV107, and NV 108 are chemically synthesized. These synthesized DNA fragments are PCR amplified using two modified primers for generating Nhel and BssHII restriction sites at 5/ and 3/ end of the fragments respectively. PCR amplified products are cleaned using PCR clean kit (Invitrogen, Waltham, MA) and restriction digested with Nhel and BssHII restriction enzymes.
  • PCR clean kit Invitrogen, Waltham, MA
  • Lambda phage infected Cre-expressing E. coll is 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 are spontaneously lyse the E. coll and released in culture media.
  • Figure 7 shows PCR amplification of lambda recombinant phage plaques for selections of phage which contains proper insertion of recombinant donor plasmid in lambda genome.
  • Lambda recombinant phage plaques of NV106 are amplified using XbaL5/ and XbaL3/ primers to confirm the proper integration of recombinant donor plasmid in lambda genome. Integration of recombinant donor plasmid in lambda produces an insertion of 3.77 kb new DNA segment in Xbal site of lambda genome.
  • PCR amplified products are electrophoresed on 1% agar gel at constant volt of 80.
  • Figure 8 shows PCR amplification of lambda recombinant phage plaques for selections of phage which contains proper insertion of recombinant donor plasmid in lambda genome.
  • Lambda recombinant phage plaques of NV107 are amplified using XbaL5/ and XbaL3/ primers to confirm the proper integration of recombinant donor plasmid in lambda genome. Integration of recombinant donor plasmid in lambda produces an insertion of 3.73 kb new DNA segment in Xbal site of lambda genome.
  • PCR amplified products are electrophoresed on 1% agar gel at constant volt of 80.
  • Figure 9 shows PCR amplification of lambda recombinant phage plaques for selections of phage which contains proper insertion of recombinant donor plasmid in lambda genome.
  • Lambda recombinant phage plaques of NV108 are amplified using XbaL5/ and XbaL3/ primers to confirm the proper integration of recombinant donor plasmid in lambda genome. Integration of recombinant donor plasmid in lambda produces an insertion of 3.75 kb new DNA segment in Xbal site of lambda genome.
  • PCR amplified products are electrophoresed on 1% agar gel at constant volt of 80.
  • Figure 10 shows the phage vaccine epitope cluster concatemers. Schematic representations of the three concatemers comprising the 34 epitope clusters in the vaccine are shown each linked to lambda phage gpD. Concatemers are similar in length. Amino acid and nucleotide lengths do not include gpD.
  • FIG 11 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.
  • CD4 T cells producing Thl cytokines IFNg, TNFa, IL-2
  • Figures 12 and 13 show ex vivo immune recall responses differentiate SARS-CoV-2 naive and experienced individuals and exhibit different COVID-19 immunotypes.
  • Figures 14 and 15 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5).
  • 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 NOS: 1, 3 and/or 5 (and/or fragments or variants thereof), and optional
  • Figure 16 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5), 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5
  • Figures 17 and 18 show 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5), stimulate or may stimulate higher IFN-y 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5
  • Figures 19 and 20 show 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5), 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5
  • Figure 21 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5), 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5
  • 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.
  • biological sample refers to any sample of tissue, cells, or secretions from an organism.
  • 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.
  • lymphocytes antigen presenting cells
  • phagocytic cells granulocytes
  • 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 autoimmun
  • B lymphocyte and T lymphocyte assays are well known, such as ELISAs, 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
  • PBL peripheral blood lymphocytes
  • tetramer assays cytokine production assays.
  • the term "effective amount”, “therapeutically effective amount”, or the like of a composition, including 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein; concatemeric polypeptides 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
  • 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 invention 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, 7 to 30 amino acids in length, and capable of specific binding to human leukocyte antigen (HL A) molecules and interacting with specific T cell receptors (TCRs).
  • HL A 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 The ability to interact with T-cell epitopes can be predicted by in silico methods (De Groot AS et al., (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.
  • 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 one or more of SEQ ID NOS: 1, 3 and/or 5 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%
  • a “concatemeric” peptide or polypeptide refers to a series of at least two peptides or polypeptides linked together. Such linkages may form of string-of-beads design.
  • 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.
  • 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.
  • 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 relates to bacteriophages that express and/or possess nucleic acids encoding the concatemeric SARS-CoV-2 peptides as set forth herein.
  • the phages express the concatemeric peptides disclosed herein.
  • the phages express the concatemers on its surface.
  • the phages express the concatemers 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 concatemers described herein.
  • the nucleic acids are integrated into the phage’s genome.
  • 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, HL A- 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 pM ICso, ⁇ 500 pM ICso, ⁇ 400 pM IC50, ⁇ 300 pM IC50, or ⁇ 200 pM IC50 in HLA binding assays based on soluble HLA molecules).
  • a moderate affinity e.g., in aspects, ⁇ 1000 pM ICso, ⁇ 500 pM ICso, ⁇ 400 pM IC50, ⁇ 300 pM IC50, or ⁇ 200 pM IC50 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 of achieving favorable presentation of peptides to a subject’s immune system.
  • Previous findings have revealed that recombinant bacteriophage can prime strong CD8+ T lymphocytes (CTLs) 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.
  • CTLs CD8+ T lymphocytes
  • 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 antigenic concatemers on the phage virion surface (also referred to as “phage vaccine particles” or “PVPs”).
  • PVP vaccines produced by cloning the concatemers 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 extra cellular 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, at least one antigenic epitope sequence (e.g., multiple copies).
  • the phage system may display between about one to about 420 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, and 400, 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 polypeptides sequences can be expressed on the surface of a lambda 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.
  • a phage such as a lambda 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 lambda phage.
  • Whole lambda phage particles possess numerous intrinsic characteristics which make them ideal as vaccine delivery vehicles.
  • Lambda phage gpD protein is a trimeric, 109 amino acid protein (excluding the initial methionine that is removed from the mature protein.
  • Lambda phage gpD protein is typically expressed in high copy number (approx. 420 copies/phage) on the surface of lambda phage.
  • Evidence in examining lambda phage surface expressed proteins suggest they maintain a natural tertiary structure, with function maintained as well.
  • the gpD protein is reported to decorate the exterior of the capsid, providing stability to the capsid shell (see, Lander et al., Structure 16(9): 1399-1406 (2008)).
  • 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). Therefor 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 which must be repeated with each vaccine batch. In aspects the instantly- disclosed phage vaccines do not require any additional adjuvant.
  • the present disclosure 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 here is to provoke a very strong long lasting cell mediated immunity by display of up to around 420 copies of SARS-CoV-2 virus specific selective linear epitopes as a lambda capsid protein fusions.
  • the antigenic polypeptides (T-cell epitopes) expressed on the surface of the phage 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, or 15 T-cell epitopes. As identified herein, 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 three assembled polypeptides, each polypeptide featuring between 8 to 13 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 which 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 al., 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 epitope concatemer may be further divided.
  • the 34 SARS-CoV-2 epitopes were split in three for production of three different recombinant viruses.
  • the three concatemers containing the identified 34 T cell epitope clusters are selected for inclusion in the vaccine using advanced immunoinformatics tools.
  • Each epitope cluster is 15 to 25 amino acids in length and contains multiple HLA class I and class Il-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.
  • epitope clusters may be randomly concatenated head-to-tail to facilitate production of epitopes as a single genetic construct.
  • the order of epitope cluster units may further be rearranged to reduce off-target junctional epitope potential.
  • Potential immunogenicity may also evaluated at the junction between the phage surface protein, e.g. lambda phage gpD, and the concatemer.
  • the concatemer polypeptides can be divided into multiple sequences for expression in separate phage.
  • the 34 epitopes identified suggested that the transgene length may constrain the limits of the phage platform and so the concatemer was split in three (see, e.g., Figure 10).
  • three concatemers linear polypeptides of assembled SARS-CoV-2 antigenic peptides
  • a schematic of the three T-cell epitope concatemers is shown in Figure 10.
  • the resulting vaccine may be composed of one, two or all three concatemer constructs as they collectively feature highly conserved and promiscuous SARS-CoV-2-specific T cell epitopes that broadly cover HLA diversity of the human population.
  • the present disclosure concerns the generation of three polypeptide sequences that are derived from the SARS-CoV-2 envelope, membrane, and spike proteins and designed for optimal expression and immunogenicity.
  • the three T-cell epitope polypeptide sequences can be used in isolation or in combinations.
  • the genome of a phage can be modified as described herein to express one or more of the three polypeptides, such that each phage may express one, two or three polypeptides on the surface of the phage.
  • phage expressing only one of the three polypeptides can be mixed with other phage expressing other of the polypeptides to provide a stoichiometric phage-display cocktail of all three polypeptide antigens, either in equimolar or non-equimolar ratios.
  • NV106 includes an amino acid sequence of:
  • NV107 includes an amino acid sequence of: TGRLQSLQTYVTQQLVEGFNCYFPLQSYGFQPTNGVGYQPYFLGVYYHKNNKSWMES EFRVYSSANNCTFEYVLDKYFKNHTSPDVDLPPAYTNSFTRGVYYPRTFLLKYNENGTI TDASRVKNLNSSRVPDTQSLLIVNNATNVVIKVGGNYNYLYRLFRKSNLKPFERDIMYS FVSEETGTLIVNFGGFNFSQILPDPSKPSKRS (SEQ ID NO: 3).
  • NV108 includes an amino acid sequence of: LACFVLAAVYRINWIGPGPGLLQYGSFCTQLNRALTGIAVEQISGINASVVNIQKEIDVVI GIVNNTVYDPLRGHLRIAGHHLGRCDLKKLLEQWNLVIGFLFLTWGPGPGTSNFRVQPT ESIVRFFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKIGNYKLNTDHSSS SDNILSYFIASFRLFARTRSMWSFNPETNILLNV (SEQ ID NO: 5).
  • the concatemers are 35 SARS-CoV-2 identified epitopes, assembled in a head-to fashion.
  • Tables 1-3 set out the 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:
  • 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 gpD in a lambda phage.
  • the epitope sequences of each concatemer are separated by a linker.
  • the linker may include an amino acid sequence of GPGPG.
  • each of SEQ ID NOS: 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, 69, 71, 73, and 75 may be arranged in any order, including as one continuous concatemer, or as two or more concatemers.
  • 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, SEQ ID NO: 3 and/or SEQ ID NO: 5.
  • the variants may share between about 85 to about 100 % identity with the sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5.
  • the variants may share 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 % identity with the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5.
  • 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. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Vai, Leu, Met, and He; 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 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.
  • the present disclosure further includes nucleic acids encoding the concatemer polypeptides derived from SARS-CoV-2.
  • 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 assemble to provide such.
  • nucleic acids encoding the concatemer polypeptides may be introduced into the genome of a phage.
  • nucleic acids encoding the antigenic concatemer polypeptides may be fused with nucleic acids encoding additional polypeptides or polypeptide sequences, including a linker polypeptide such as GPGPG (SEQ ID NO: 88) or a spacer.
  • a linker polypeptide such as GPGPG (SEQ ID NO: 88) 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, He by ATT, ATC or ATA, Met by ATG, Vai 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, AC A or ACG, Pro by CCT, CCC, CCA or CCG, His by CAT or CAC, Gin by CAA or
  • 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.
  • NV106, NV107 and/or NV108 can be surface displayed by inserting a concatemer nucleic acid into the genome of a M13, T7, fl, fd or T4 phage, as well as combinations thereof.
  • fusion proteins for surface display can similarly be utilized through placing a nucleic acid encoding a concatemer operably with a further surface protein, such as: the tail protein (gpV) (as well as gpD as discussed herein) in Lambda phage; gpIII, gpVI, gpVII, gpVIII and gpIX in M13; HOC and SOC in T4 phage; plOA, plOB, p8, pl 1, pl7 or pl2 in T7 phage and the like.
  • integration of the plasmid into the phage genome can be achieved by including recombination sequences directed to the desired genome insertion points in the plasmid.
  • the nucleic acids encoding the antigenic chimera include a surface display sequence and/or a restriction enzyme site.
  • the nucleic acids encode a gpD head protein fused to a SARS-CoV-2 derived antigenic concatemer polypeptide with further engineered restriction sites for ligation into the genome of a lambda phage.
  • the engineered restriction site of a 5’ Nhe I and a 3’ Bssh II allowed for ligation of the SARS-CoV-2 encoding nucleic acid into a plasmid that affixed in frame at the amino terminal of the antigenic polypeptide a gpD head protein sequence.
  • NV106 is encoded by the nucleic acid sequence as follows: GGTAGGGCCCGGTGGCAGCGGAGCTAGCGGGAAAGGCTATCACTTAATGAGCTTTC CTCAGTCTGCGCCTCACCAGACTTTGTTAGCGCTTCATCGTAGCTATTTGACACCGG GGGATTCATCCGTTCTTTCTTTCGAGCTGCTTCACGCACCTGCCACCGTATGTGGTCA AAAATTGATAGCTAATCAGTTTAACAGCGCCATTGGTAAGATCCAGGACAGCTTAC AGATACCGTTTGCGATGCAGATGGCCTATAGATTTAATGGGATAGGAGTCCCCAAG GAAATCACTGTGGCGACTAGCCGTACTTTGTCCTATTATCCGACTAATTTTACTATTT CAGTGACTACGGAGATTCTTCCGGTGATTTGTTTACTTCAGTTTGCGTACGCCAACC GCAATCGCTTTCTTTATATAAATGATGGGGTCTACTTCGCTTCAACTGAAAAGAGTA ACATTCGGACGGACGAAATGATAGCGCAG
  • nucleic acid sequence encoding NV107 is as follows: GGTAGGGCCCGGTGGCAGCGGAGCTAGC4G4GGTCGCTTACAGTCGTTACAAACAT ACGTTACACAACAACTGGTTGAAGGTTTTAATTGCTACTTCCCGCTGCAGAGTTACG GATTTCAACCGACAAATGGGGTTGGTTATCAGCCATATTTTTTAGGAGTGTATTATC ATAAGAACAACAAAAGTTGGATGGAGTCCGAATTTCGTGTGTGTACTCTAGCGCCAAT AACTGTACTTTTGAGTATGTTTTAGATAAGTATTTCAAGAACCATACTTCGCCGGAC GTTGACCTTCCACCTGCCTACACTAACTCATTTACGCGCGGCGTTTATTACCCTCGG ACGTTCCTGCTGAAGTATAATGAGAACGGAACCATAACCGATGCGAGCCGGGTGAA GAATCTTAACTCATCTAGAGTCCCAGATACTCAATCATTACTGATCGTGAACAATGC CACTAATGTTGTCATTAAAGTAGGAGGCAACTACAATT
  • the nucleic acids may include variants of the sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID NO: 6.
  • the variants may share between about 85 to about 100 % identity with the sequences as set forth in SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID NO: 6.
  • the variants may share 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 % identity with the amino acid sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID NO: 6.
  • the present disclosure includes concatemers of rearranged epitope sequences.
  • the concatemers of SEQ ID NOS: 1, 3 and 5 are an assembly of 34 identified epitopes from SARS-CoV-2 expressed proteins.
  • the nucleic acids encoding each epitope sequence are set for in SEQ ID NOS: 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, 72, 74, and 76.
  • a concatemer may include any arrange of the nucleic acids encoding each epitope peptide, including with a linker and/or an additional 1-12 amino acids inbetween each or some of the epitope sequences present therein.
  • the nucleic acids include a 5’ nucleic acid sequence that add additional amino acids between the gpD head protein of lambda phage and the antigenic concatemer polypeptide.
  • SEQ ID NOS: 2, 4 and 6 at the 5’ end is a sequence as set forth accordingly: GGTAGGGCCCGGTGGCAGCGGAGCTAGC (SEQ ID NO: 8).
  • each chimera may further include a spacer between the gpD and the SARS-CoV-2 derived antigenic polypeptide, wherein the spacer includes the amino acid sequence of: VGPGGSGAS (SEQ ID NO: 7).
  • 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.
  • 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).
  • APCs antigen presenting cells
  • the array of the linear epitopes of the construct NV106, NV107, and NV108 can be cloned inside the lambda 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 present disclosure also provides chimeric or fusion polypeptide compositions.
  • 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 phage secreted peptide including lambda gpD.
  • 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.
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 (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 NOS: 1, 3, and/or 5).
  • 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 with flanking amino acids of the heterologous polypeptide.
  • 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 NOS: 1, 3, and/or 5 (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 NOS: 1, 3, and/or 5), wherein said one or more of SEQ ID NOS: 1, 3, and/or 5 is not naturally included in the polypeptide and/or said of one or more of SEQ ID NOS: 1, 3, and/or 5 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.
  • expression and/or secretion of a polypeptide can be increased by using a heterologous signal sequence.
  • 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 e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1, 3, and/or 5 (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 NOS: 1, 3, and/or 5) 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 which 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 al., 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 NOs: 2, 4 and 6 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 gpD of lambda phage.
  • the resulting plasmid construct can then be introduced or transformed into a bacteria cell, such as E. coli, such asa recombinase expressing bacterial cell, to allow for the introduction of the gpD-antigenic fused nucleotide sequence in to the phage genome.
  • the transformed bacteria can then be infected with a lambda 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.
  • Cre recombination systems than Cre can be used, such as FLP, R, Lambda, HK101, and pSAM2.
  • At least one of three polypeptide sequences, NV106, NV107 and/or NV108, are expressed on the surface of a lambda phage.
  • two or three surface displayed polypeptides are utilized as inoculants, either expressed within the same phage or each expressed independently on separate phage mixed together in an inoculant cocktail.
  • the collective display of antigenic epitopes on lambda phage may provide unique vaccine nanoparticles that can provoke immunogenic responses (both T cell and B cells mediated) without any additional adjuvants.
  • the vaccine product is composed of the three recombinant phage vaccine components bearing all the epitopes identified by the in silico analysis. These 3 recombinant phage vaccines are designated as NV 106, NV 107, and NV 108.
  • the collective display of the original epitopes with subsequent modified sequences are set forth in SEQ ID NOs: 1, 3 and 5, with nucleic acids encoding such in SEQ ID NOs: 2, 4, and 6, respectively.
  • NV106, NV107, and NV108 concatemer polypeptides are introduced into a lambda phage and constructed in a plasmid that allows for recombination with such to alter the phage genome.
  • NV106, NV107 and/or NV108 can be surface displayed by a Ml 3, T7, fl, fd or T4 phage, as well as combinations thereof.
  • fusion proteins for surface display can similarly be utilized, such as: the tail protein (gpV) (as well as gpD as discussed herein) in Lambda phage; gpIII, gpVI, gpVII, gpVIII and gpIX in M13; HOC and SOC in T4 phage; plOA, plOB, p8, pl 1, pl7 or pl2 in T7 phage and the like.
  • integration of the plasmid into the phage genome can be achieved by including recombination sequences directed to the desired genome insertion points in the plasmid.
  • a lambda phage is selected for phage display.
  • a lambda phage can express larger polypeptides on its surface and further provides straightforward and efficient cloning efficiency.
  • a lambda phage further provides a vehicle for display of a high copy number of the antigenic polypeptide, typically displaying between about 200 to about 420 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 selective 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 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 NV106, NV107, NV108 can be cloned inside the lambda 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 which 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 previously 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5 (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, plasm
  • 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.
  • 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.
  • determining or titrating suitable dosages of a vaccine to find minimal effective dosages based on the age, weight of the animal subject, species (if nonhuman), 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 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 one or more of SEQ ID NOS: 1, 3 and/or 5 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 NOS: 1, 3, and/or 5 (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, re
  • 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 one or more of SEQ ID NOS: 2, 4 and/or 6, 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 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.
  • 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.
  • 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.
  • 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.
  • the vaccine compositions feature varying combinations and concentrations of the three identified phage constructs.
  • the three vaccine phage constructs i.e. NV106, NV107, and NV108
  • these three vaccine constructs could be mixed in an non- equimolar (different proportions) concentrations to achieve maximum immunogenicity against SARS-CoV-2.
  • these three vaccine constructs can be mixed and matched accordingly for preparation of final vaccine constituents.
  • vaccine #1 can be prepared by using NV106 andNV107 whereas Vaccine #2 can be prepared by mixing NV106 and NV108. Similarly, vaccine #3 can be prepared by mixing NV107 with NV108. In further aspects, the vaccine constructs may be used alone without mixing with each other.
  • 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 which may enhance the binding affinity of phage vaccine particles with epithelial cells for proper absorptions to enhance the efficacy of above described vaccination processes.
  • the present disclosure provides compositions with alternate presentation of phage vaccine particles to enhance immunogenic response in mammalian systems.
  • phage display or phage DNA 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.
  • 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 are essential for slow diffusion of phage vaccines from the site of inoculation to attract APCs for generating optimum immunogenic response of phage vaccination.
  • phage display and/or phage DNA vaccine 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 phage based compositions.
  • Microencapsulation of phage 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.
  • phage display or phage DNA vaccine compositions can be specifically designed for intradermal deposition of phage vaccines, such as with microneedles.
  • 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.
  • the SARS-CoV-2 vaccine bacteriophage constructs including concatemers of putative T-cell epitopes having an amino acid sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5, 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 glycol
  • 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-P, IFN-y, 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 coh. 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. [00141] 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.
  • 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 one or more of SEQ ID NOS: 1, 3 and/or 5 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 NOS: 1, 3 and/or 5; 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 more of e.g., phage display, phage DNA, peptides or polypeptid
  • “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 one or more of SEQ ID NOS: 1,3, and/or 5 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 NOS: 1,3, and/or 5
  • 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
  • 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.
  • Liposomes 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5 (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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • 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 vector
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5 (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, polypeptid
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5 (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
  • 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 com 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 com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening compound
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • 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, plasmid
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • 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, plasmi
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • 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
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • 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
  • 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 invention further concerns the design of a lambda phage system to display a combinations of SARS-CoV-2 specific linear epitopes fused as a concatemer chimera to the C terminus of the capsid protein gpD of phage lambda.
  • the identified 34 antigenic epitopes were divided into the three separate concatemer peptide fragments (i.e. SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5) to generate three separate phage vaccine constructs that are herein designated as NV106, NV107, and NV108.
  • the optimized SARS-CoV-2 concatemer polypeptides 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 gpD lambda 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 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • 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,
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5
  • 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,
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5 (in aspects, the polypeptides may be isolated, synthetic, or
  • 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 one or more of SEQ ID NOS: 1, 3, and/or 5 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 NOS: 1, 3, and/or 5 (in aspects, the polypeptides may be isolated, synthetic, or recombinant) as disclosed herein
  • 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 phagebased vaccine composition is warranted.
  • multiple administrations may include the development of a prime boosting strategy of vaccination using phage mediated display and/or DNA 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 NV106 construct and subsequent booster immunizations can be performed with phage DNA vaccine generated by, e.g., NV106 construct.
  • a regimen of priming the immune system with an immunogen can be followed by the boosting with a different immunogen (e.g. phage DNA vaccines generated by the NV 108 construct) or vice versa.
  • the initial priming may be achieved by administration of both a phage display and phage DNA of the same construct (e.g. NV106), followed by boosting with a different contruct (e.g. NV107 phage display and phage DNA compositions).
  • 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 S J, 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.
  • Epitopes 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: 90) spacer was inserted to eliminate immunogenicity potential.
  • GPGPG SEQ ID NO: 90
  • a total of 34 class I and class II peptides were selected for inclusion in the instantly- disclosed vaccine constructs, following immunoinformatic predictions. Selection was based on at least, high binding likelihood to HL A 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 peptides of SEQ ID NOS: 1, 3 and 5 include the below sequences from the identified SARS- CoV-2 proteins (indicated with the start amino acid number) in TABLES 4-6.
  • 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 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 an amino acid sequence of SEQ ID NOS: 1, 3 and 5 (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 NOS: 1, 3 and 5) over nine days at 37°C under a 5% CO2 atmosphere.
  • select 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 NOS: 1, 3 and 5 (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 NOS
  • a 48-well plate 5 x 10 A 6 cells in 150 pl 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 pl. 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.
  • FluoroSpot Assay Interferon-gamma (IFN-y) 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% CO2 atmosphere. Plates are developed according to the manufacturer’s directions using FITC- labeled anti-IFN-y detection antibody.
  • IFN-y Interferon-gamma
  • 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.
  • 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 NOS: 1, 3 and/or 5 (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 NOS: 1, 3 and/or 5).
  • 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-y 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.
  • differences between responses by pool in ex vivo and cultured assay may reflect variable phenotypes and/or proliferative capacities of epitopespecific 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 NOS: 1, 3, and/or 5 (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 NOS: 1, 3, and/or 5) 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 epitopespecific 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 NOS: 1, 3, and/or 5 (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 NOS: 1, 3, and/or 5) stimulate low frequency epitope-specific T cells following expansion in culture in naive and COVID-19 convalescent donors.
  • 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.
  • 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 NOS: 1, 3, and/or 5 (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 NOS: 1, 3, and/or 5) 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.
  • RNA sequences coding for concatemer peptide sequences of NV106, NV107, and NV108 were synthesized by Integrated DNA Technologies, Inc. (IDT) Coralville, Iowa. Using specific oligo primers, these synthesize DNA fragments were PCR amplified to provide restriction sites for in-frame cloning with gpD sequence (figure 2). The oligo sequence of each PCR primer is modified to produce Nhe I and Bssh II restriction sites in each end of amplified DNA fragments. After restriction digestion, these fragments were inserted separately at the Nhel- Bsshll site of the 3' end of a DNA fragment encoding gpD 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 was confirmed by restriction enzyme analysis (figures 3-5). Cre-expressing cells (E coli) were transformed with these recombinant donor plasmids and subsequently infected with a recipient lambda phage that carries a stuffer DNA fragment flanked by loxPwt and loxP511 sites. Lambda phage infected Cre-expressing E. coli is grown at LB Ampicillin (100 ug/ml) at 37° C. for four hours in presence of 0.2% maltose and 0.
  • Lambda cointegrates were used to produce lambda lysogens and were then selected on Luria Bartani (LB) ampicillin agar (100 ug/ml amp, 15% agar) plates. Briefly, cointegrates from spontaneously lysed E. coli culture were used to infect Cre-ve, suppressor-ve E. coli cells and spread on LB ampicillin agar plates. Plates were incubated at 30° C for 48 hours to obtain Ampr colonies which are actually recombinant lambda lysogens and carry a recombinant lambda phage genomes in their chromosome.
  • This cell free supernatant was used to infect E. coli cells and plated on solid LB agar (15%) plate to obtain phage plaques.
  • the resulting phage plaques were 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.
  • Example 5 Conformation of Lambda Phage Plaques Containing SARS-CoV-2 Fragments'.
  • Phage DNA containing cointegrates were subjected to complete genome sequencing and subsequent bioinformatics analysis to confirm the proper orientation and sequence of inserted DNA fragments in lambda genome as gpD fusion.
  • Initial analysis indicated frameshift of the inserted DNA sequences of these co-integrates. Therefore cloning procedures were repeated again for generating new phage co-integrates.
  • Phage DNA containing these new co-integrates were subjected for PCR amplification of the regions that harbored the inserted DNA fragments. Preliminary analysis of these PCR amplified products indicated that 4 out of 6 clones has proper insertion of the correct sequences in phage genomes. Data not shown. Additional sequencing and subsequent bioinformatics analysis of complete phage genomes generated from these new co-integrates will be conducted for further conformations of the inserted DNA sequences.
  • Example 6 Growth and Purification of Recombinant Phage Displaying SARS-CoV-2 Peptides'.
  • the materials include: Luria Broth, 20% Maltose in water, (filter sterilized), 1 M MgCh in water, (filter sterilized), an E. coli sup- host, flasks, shaking incubators, cuvettes, spectrophotometer, a L 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 MgCh 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 IM 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 600nm is obtained.
  • MOI multiplicity of infection
  • 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 2L preparation, complete lysis should take 4 to 6 hours post infection).
  • 1 mL of DNase I is added (Img/ml filter sterilized).
  • 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.
  • TMF tangential flow filtration
  • TFF purified phage vaccine preparations are extracted once with organic solvents octanol for removing lipopolysaccharide (Ips) 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 4L of 0.5M 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.
  • 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 i VAX-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 crossreactivity 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.
  • NV106, NV107 and NV108 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.
  • NV106, NV107 and NV108 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 (IL-2, IFNy, TNFa).
  • 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 5* 10 8 pfu of NV106 phage particles suspended in 500 pl of sterile PBS. Similarly group B and group C mice will receive same quantity of NV 107 and NV 108 phage particles respectively. Group D mice will receive equimolar mixture of all 3 phage constructs.
  • mice A fifth group of mice (group E) will receive recombinant SARS-CoV-2 antigen (50 pg/mice) 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. Sera from group D and group E animals will be saved at -70° C. freezer for further animal experiments. During experiment, all animals will be monitored for their health conditions. The immune response against various SARS-CoV-2-phage vaccines will be monitored by western immunoblot and ELISA.
  • mice/group Animal Study 2
  • a further study can use 3 separate groups (A, B, C - 15 mice/group) of male BALB/c mice (18-25 g each), each being immunized via intramuscular injection (i.m.) with the lambda phage display vaccine cocktail (i.e. SEQ ID NOS: 1, 3 and 5) constructs.
  • Group 1 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 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-y. Cytokines can be measuered via ELISA according to the manufacturer’s instructions. Antigen specific and lambda phage capsid gpD 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 statisitical plans, a 4-fold immune response with descriptive characterization across groups will be used.
  • the NV106, NV108 and NV109 phage display compositions can be administered to a suitable murine model in vivo, followed by two booster administrations each separated by two weeks.
  • Each phage can be administered alone as well as with an equimolar combination, wherein each mouse receives a similar total amount of phage composition of between 10 6 to 10 9 pfu.
  • a set of mice numbering at least three will be used, including sets that receive control lambda phage and vehicle only.
  • each set of mice will receive a challenge with live vaccine and then later euthanized to assess for protection and/or specific immune response. Similar studies can then be repeated in a higher mammal, preferable one such that exhibits high amino acid identity with human ACE2 receptor.
  • 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 NOS: 1, 3, and/or 5, or a phage DNA cocktail to provide SEQ ID NOS: 2, 4, and/or 6, or each T-cell epitope polypeptide or nucleic acid, are contemplated.
  • a clinical trial for a phage display cocktail to provide SEQ ID NOS: 1, 3, and/or 5 (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.
  • intradermal e.g., via microneedle
  • intramuscular subcutaneous, intravenous, or intraperitoneal
  • 3M TDM patch
  • Immunogenicty 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 NOS: 1, 3, 5).
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • an equimolar cocktail of NV106, NV107 and NV108 phage vaccines can be prepared and 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) to a small group of human subjects, with additional subjects receiving a vehicle placebo only. Blood samples can then be collected, challenged with SARS-CoV-2 and then assessed for the level of specific immune response.
  • 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 NV106, NV108 and NV109 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.
  • 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

La présente divulgation concerne un bactériophage recombinant conçu pour générer une réponse immunitaire chez un sujet pour assurer la reconnaissance du SARS-CoV-2 et/ou une protection contre le SARS-CoV-2. Les compositions de phage peuvent comprendre une exposition sur phage ou un ADN de phage avec des épitopes de lymphocytes T de SARS-CoV-2 optimisés par un algorithme pour interagir avec un large spectre de HLA dans la population humaine.
PCT/US2021/051988 2020-09-24 2021-09-24 Développement rapide d'un vaccin prophylactique à large spectre pour le sars-cov-2 à l'aide d'un système d'administration d'antigène à médiation par phage WO2022067062A1 (fr)

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WO2024026553A1 (fr) * 2022-08-03 2024-02-08 Centre Hospitalier De L'université De Montréal Nouvel épitope antigénique anti-sars-cov-2 et ses utilisations

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WO2024026553A1 (fr) * 2022-08-03 2024-02-08 Centre Hospitalier De L'université De Montréal Nouvel épitope antigénique anti-sars-cov-2 et ses utilisations
CN116987152A (zh) * 2023-09-27 2023-11-03 中国科学院微生物研究所 一种新冠病毒环肽抑制剂
CN116987152B (zh) * 2023-09-27 2024-01-02 中国科学院微生物研究所 一种结合沙贝冠状病毒s蛋白rbd结构域的环肽及其应用

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