WO2021198999A1 - Epitope-based vaccines for treatment of coronavirus associated diseases - Google Patents

Epitope-based vaccines for treatment of coronavirus associated diseases Download PDF

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WO2021198999A1
WO2021198999A1 PCT/IB2021/052781 IB2021052781W WO2021198999A1 WO 2021198999 A1 WO2021198999 A1 WO 2021198999A1 IB 2021052781 W IB2021052781 W IB 2021052781W WO 2021198999 A1 WO2021198999 A1 WO 2021198999A1
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seq
peptide
polypeptide
sars
protein
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PCT/IB2021/052781
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French (fr)
Inventor
Norbert ZILKA
Branislav Kovacech
Eva Kontsekova
Rostislav SKRABANA
Peter Filipcik
Andrej KOVAC
Michal Novak
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Axon Neuroscience Se
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Publication of WO2021198999A1 publication Critical patent/WO2021198999A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • SARS severe acute respiratory syndrome
  • Coronaviruses are large, enveloped, positive-strand ribonucleic acid (RNA) viruses, having the largest genomes among all RNA viruses. They are roughly spherical and moderately pleiomorphic and contain crown-shape peplomers that are 80- 160 nM in size (Sahin et al., 2020).
  • Coronaviruses have a highly conserved genomic organization, with a large replicase gene (encoded by approximately two thirds of the genome) preceding structural and accessory genes; express many non-structural genes by ribosomal frameshifting; develop constant transcription errors and RNA-Dependent RNA Polymerase jumps; display several unique or unusual enzymatic activities encoded within the large replicase-transcriptase polyprotein; and expression of structural and accessory downstream genes by synthesis of 3' nested sub-genomic micro-ribonucleic acids (mRNAs) (Fehr and Perlman, 2015; Sahin et al., 2020).
  • mRNAs micro-ribonucleic acids
  • Coronaviruses have a high substitution rate, with an average of roughly 10- 4 nucleotide substitutions per site per year (Su et al., 2016). They cause a variety of respiratory, enteric and neurologic diseases in birds and mammals. Infections are transmitted mainly via deposition of infected droplets or aerosols on the respiratory epithelium or via fecal-oral routes (Masters, 2006). [0004] There are four genera of coronaviruses: Alphacoronavirus (aCoV), Betacoronavirus (pCoV), Deltacoronavirus (bCoV) and Gammacoronavirus (yCoV).
  • aCoV Alphacoronavirus
  • pCoV Betacoronavirus
  • bCoV Deltacoronavirus
  • yCoV Gammacoronavirus
  • Bats and rodents are the sources of most aCoVs and bOo /s, while avian species are the sources of most bCoVs and yCoVs (Chan et al., 2013).
  • Seven human coronaviruses have been identified, two of them belonging to the a-coronavirus subgroup, HCoV-229E, HCoV-NL63 and another five are part of the b-coronavirus subgroup, HCoV-OC43, HC0V-HKUI , SARS-CoV and Middle East respiratory syndrome (MERS) -CoV (Fehr and Perlman, 2015) and SARS-Cov2 (Dong et al., 2020, Lu et al., 2020).
  • MERS Middle East respiratory syndrome
  • SARS first emerged in the human population in November 2002.
  • MERS was first reported in Saudi Arabia in 2012.
  • the coronaviral genome encodes four canonical structural proteins, the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein.
  • N is a phosphoprotein which binds to the CoV RNA genome and makes up the nucleocapsid and participates in viral transcription.
  • the M glycoprotein - the most abundant structural protein - coordinates virion assembly and release.
  • the envelope protein is a short, integral membrane protein, which is abundantly expressed inside the infected cell, but only a small portion is incorporated into the virion envelope. Recent studies have expanded on its functions as an ion-channelling viroporin.
  • the hydrophobic transmembrane domain contains at least one predicted amphipathic a-helix that oligomerizes to form an ion-conductive pore in membranes (Schoeman and Fielding, 2019).
  • the transmembrane S glycoprotein forms homotrimers and mediates receptor attachment and subsequent fusion between the viral and host cell membranes to facilitate viral entry into the host cell.
  • the S protein assembles into trimers to form the distinctive surface spikes of coronaviruses (Song et al., 2004).
  • the interaction between the S protein and receptor represents the key determinant of coronavirus host species range and tissue tropism (Masters, 2006).
  • the S protein comprises two functional subunits: S1 is responsible for binding to the host cell receptor and S2 orchestrates the fusion of the viral and cellular membranes.
  • the S protein is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently bound in the prefusion conformation.
  • the S protein harbors a furin cleavage site at the S1/S2 boundary.
  • the S protein is further cleaved by host proteases at the so-called S2 site located immediately upstream of the fusion peptide. This cleavage was shown to activate the protein for membrane fusion via extensive irreversible conformational changes (Walls et al., 2020).
  • the S1 domain is the most divergent region of the molecule and can vary extensively across and within the coronavirus subgroups, while the S2 portion of the ectodomain, which is heavily glycosylated, is the most conserved part of the molecule across the coronaviruses (Masters, 2006).
  • the SARS- CoV and Cov-2 spike glycoproteins showed large sequence difference ( ⁇ 55% identity) in the S1 domain, while the S2 domain region displays ⁇ 91% identity (Song et al., 2018; Vandakari and Wilce, 2020).
  • ACE2 angiotensin-converting enzyme 2
  • ACE2 is a monocarboxypeptidase that catalyses cleavage of the Angiotensin II - the major vasoactive peptide in renin- angiotensin system, to produce Angiotensin I (1 -7). It has been shown that ACE inhibiting drugs have an antihypertensive effect (Francis, 2000; Clarke and Turner, 2012).
  • SARS-CoV-2 A predictive study of the SARS-CoV-2 showed that 8 out of these 14 positions were conserved in SARS-CoV- 2, whereas 6 positions were (semi)conservatively substituted: R426 SARS-CoV, N439 SARS-CoV-2; Y442 SARS-CoV, L455 SARS-CoV-2; L472 SARS-CoV , F486 SARS- CoV-2; N479 SARS-CoV, Q493 SARS-CoV-2; Y484 SARS-CoV Q498 SARS-CoV-2; and T487 SARS-CoV, N501 SARS-CoV-2 (Walls et al., 2020).
  • the receptor-mediated conformational change in S1 and the dissociation of S1 from S2 are thought to initiate a major rearrangement in the remaining S2 trimer.
  • This rearrangement exposes a fusion peptide that interacts with the host cellular membrane, and it brings together the two heptad repeats in each monomer so as to form an antiparallel, six-helix “trimer-of-dimers” bundle.
  • the trimer of dimers is extremely stable, forming a rod-like, protease-resistant complex (Bosch et al., 2004, 2005).
  • the S trimer in SARS-CoV2 exists in multiple, distinct conformational states.
  • S glycoprotein trimers are found to exist in partially opened states in SARS-CoV2, while they remain largely closed in less pathogenic human coronaviruses (Wrapp et al., 2020; Walls et al., 2020).
  • RNA-based vaccine mRNA encapsulated in lipid nanoparticles
  • viral vector- based vaccine Addenovirus type V vector
  • RNA-based vaccines DNA vaccines, recombinant protein-based vaccines, viral vector-based vaccines, live attenuated vaccines, inactivated virus vaccine, subunit vaccines and peptide- based vaccines (Dhama et al. , 2020).
  • Another important aspect of the vaccine development is safety.
  • vaccines against SARS-CoV such as recombinant spike protein-based vaccines, attenuated and whole inactivated vaccines as well as vectored vaccines were shown to protect animals from challenge with SARS-CoV.
  • vaccination with the live virus results in complications, including lung damage and infiltration of eosinophils in the mouse model (Bolles et al., 2011 ; Tseng et al., 2012).
  • RNA and DNA vaccines, recombinant protein-based vaccines, viral vector- based vaccines, live attenuated vaccines, inactivated virus vaccines and subunit vaccines against SARS-Cov2 virus will contain hundreds of antigenic epitopes - all of which are not necessary, and some may even be detrimental and induce allergenic or reactogenic responses.
  • the development of peptide vaccines containing only epitopes capable of inducing a desirable T cell and B cell mediated immune response may prevent unwanted side effects of the abovementioned vaccines.
  • the peptide vaccines are considered sufficient to activate the appropriate cellular and humoral responses, while eliminating allergenic or reactogenic responses. Taking into the consideration these findings, peptide vaccines represent an effective and safe therapeutic and preventive approach for the SARS-Cov2 virus.
  • the present disclosure is based, in part, on the development of peptides for immunotherapy of coronavirus-associated diseases or disorders.
  • the disease is Covid-19.
  • Embodiment 1 An isolated polypeptide, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of one or more of the following sequences:
  • DSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ SEQ ID NO:10; LQSYGFQPTNGVGYQ, SEQ ID NO:11 ;
  • RDISTEIYQAGSTP SEQ ID NO: 121 ;
  • RGDEVRQIAPGQTGK SEQ ID NO: 123;
  • DNGVEGFNGYFPLQSYG SEQ ID NO: 124;
  • polypeptide, fragment, variant, or derivative thereof comprising a contiguous sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of said sequences, wherein the polypeptide, fragment, variant, or derivative thereof is at least 7 amino and at most 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues-long; and wherein the polypeptide, or a fragment, variant, or derivative thereof is neither a fragment of SARS Cov2 Spike protein of NC_045512.2 nor a fragment of any other naturally-existing coronavirus Spike protein, optionally wherein the polypeptide, fragment, variant, or derivative thereof comprises at least one modification to any one of the peptides of SEQ ID NOs: 8 through 16, SEQ ID NOs:118 through 124, or SEQ ID NOs. 42 through 116; and SEQ ID Nos. 125 through 227; and a poly
  • Embodiment 2 The isolated polypeptide of embodiment 1 , or a fragment, variant, or derivative thereof wherein the polypeptide is selected from:
  • Peptide (1) 442- CGSGDSKVGGNYNKLYRLFE -456, SEQ ID NO:1 ;
  • Peptide (2) 442- CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ -506, SEQ ID NO:2;
  • Peptide (3) 469-SGSTEIYQAGSTPCNGVEGFNCYFAK-490, SEQ ID NO:3;
  • Peptide (6) 919-CNQKLIANQFNSAIGKIQDSLSS-940, SEQ ID NO:6;
  • Peptide (7) 695-CYTMSLGAENSVAYS -708, SEQ ID NO:7;
  • Peptide (8) 438-CSNNLDSKVGGNYN-450, SEQ ID NO: 83;
  • Peptide (13) 403-CSRGDEVRQIAPGQTGK-417, SEQ ID NO: 88;
  • Peptide (14) 481 -CDNGVEGFNGYFPLQSYG-496, SEQ ID NO: 89;
  • a polypeptide, fragment, variant, or derivative thereof comprising a contiguous sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of said peptides, wherein the numbering of the amino acids in the Peptides is relative to the sequence of SARS CoV2 S protein, and the differences between the two are underlined; wherein the polypeptide is neither a fragment of SARS Cov2 S protein of NC_045512.2 nor a fragment of any other naturally-existing coronavirus S protein.
  • Embodiment 3 The isolated polypeptide of any one of embodiments 1 and 2, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of a continguos sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NO:1 through SEQ ID NO:16, SEQ ID NO: 83 through SEQ ID NO:116, or SEQ ID NO: 140 through SEQ ID NO:175, optionally wherein the polypeptide, fragment, variant, or derivative thereof comprises one or more conservative amino acid substitutions.
  • Embodiment 4 The isolated polypeptide of any one of embodiments 1 through 3, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof differs from the polypeptides of SEQ ID NO:1 through 16, SEQ ID NO: 83 through 116, or SEQ ID NO: 140 through 175 in that it has an addition, deletion, or insertion that comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,
  • Embodiment 5 The isolated polypeptide of any one of embodiments 1 through 4, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof is at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least
  • At least 20 at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41 , at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51 , at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58 residues, at least 59, at least 60, at least 61 , at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79
  • Embodiment 6 The isolated polypeptide of any one of embodiments 1 through 5, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof, is at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21 , at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31 , at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41 , at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51 , at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58 residues, at most 59, at most 60, at most 61 ,
  • Embodiment 7 The isolated polypeptide of any one of embodiments 1 through 6, or a fragment, variant, or derivative thereof, wherein the polypeptide further comprises a moiety or a moiety and a linker, and/or a peptide corresponding to Peptides (1) through Peptide (14) without a N-terminal Cys.
  • Embodiment 8 The isolated polypeptide of any one of embodiments 1 through 7, or a fragment, variant, or derivative thereof, wherein the moiety is a carrier protein selected from any one or more of a Cys residue, an Asp residue, a Ser residue, Keyhole Limpet Hemocyanin (KLH) functional unit, Cys-KLH, a tetanus toxin heavy chain C fragment, a diphteria toxin, a diphtheria toxin variant CRM197, an H influenzae protein D, a Meningococcal outer membrane protein complex protein, an Outer-membrane lipoprotein carrier protein, or a Cholera toxin B subunit, a virus-like particle, biotin, avidin, streptavidin, neutravidin, serum albumin, an enzyme, a metallic nanomaterial, CRM197 and an outer membrane protein mixture from N.
  • a carrier protein selected from any one or more of a Cys residue, an Asp residue, a Ser residue, Keyhole Limpet
  • OMP meningitidis
  • biodegradable polymers including polylactic, polycaproic, polyglycolic, polymalic, polybutyric acids and their combination and modifications; hydrogels of polyethyleneglycol and its modifications; hyaluronic acid, dextran, chitosan, liposome- polymer hybrid carrier, and a fragment or derivative or combination thereof.
  • Embodiment 9 The isolated polypeptideof any one of embodiments 1 through 8, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of one or more of the polypeptides of SEQ ID NO:1 through SEQ ID NO:16, SEQ ID NO:118 through SEQ ID NO:124, SEQ ID NO:83 through SEQ ID NO: 89, and/or SEQ ID NO: 140 through 175 connected through a linker.
  • Embodiment 10 The isolated polypeptide of any one of embodiments 1 through 9, or a fragment, variant, or derivative thereof, wherein the linker is selected from GSG, GPAD (SEQ ID NO: 17), and SG.
  • Embodiment 11 The isolated polypeptide of any one of embodiments 1 through 10, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of a peptide of SEQ ID Nos: 3, 5, 6, 7, 9, 11 , 12, 14, 15, 16, 83 through 89, or 118 through 124.
  • Embodiment 12 The isolated polypeptide of any one of embodiments 1 through 11 , or a fragment, variant, or derivative thereof, wherein the polypeptide further comprises one or more chemical modifications selected from an internal bridge, short- range cyclization, methylation, amidation, acetylation or substitution with other chemical groups cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, which can be N-terminal, C-terminal, or internal.
  • Embodiment 13 The isolated polypeptide of any one of embodiments 1 through 12, or a fragment, variant, or derivative thereof, wherein
  • Embodiment 14 A nucleic acid encoding one or more of the polypeptides, fragments, variants, or derivatives thereof of any one of embodiments 1 through 13.
  • Embodiment 15 The nucleic acid of embodiment 14, wherein the nucleic acid is DNA, RNA, or modified RNA.
  • Embodiment 16 A vector comprising one or more of the nucleic acids of any one of embodiments 14 and 15.
  • Embodiment 17 The vector of embodiment 16, wherein the vector is a retroviral vector, lentiviral vector, adenoviral vector, poxvirus vector, plasmid, or bacterial vector.
  • Embodiment 18 A recombinant cell comprising a polypeptide, or a fragment, variant, or derivative thereof, of any one of embodiments 1 through 13, a nucleic acid according to any one of embodiments 14 and 15, or a vector of any one of embodiments 16 and 17.
  • Embodiment 19 The recombinant cell of embodiment 18, wherein the cell is a prokaryotic cell (e.g. E. coli) or an eukaryotic cell (e.g. Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NS0, Sp2/0), a monkey kidney cell (COS-7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TRI cell, an MRC 5 cell, a FS4, or a MDCK cell.
  • a prokaryotic cell e.g. E. coli
  • an eukaryotic cell e.g. Chinese ham
  • Embodiment 20 An immunogenic composition comprising one or more of the polypeptides of any one of embodiments 1 through 13, or a fragment, variant, or derivative thereof, nucleic acids of embodiments 14 and 15, vector of embodiments 16 and 17, or cell of embodiments 18 and 19.
  • Embodiment 21 The immunogenic composition of embodiment 20, comprising one or more of the polypeptides of any one of embodiments 1 through 13, or a fragment, variant, or derivative thereof, and an adjuvant.
  • Embodiment 22 The immunogenic composition of embodiment 21 , comprising a combination of any two of Peptides (1 ) through Peptide (14).
  • Embodiment 23 The immunogenic composition of embodiment 22, wherein the composition comprises a combination of Peptide (1) and (2), (1) and (3), (1) and (4), (1) and (5), 1 and (6), (1 ) and (7), (2) and (3), or any one of the 21 possible two-peptide combinations of these seven Peptides; or any one of the 21 possible two-peptide combinations of Peptides (8) through (14); or any one of the 91 two-peptide combinations, 364 three-peptide combinations, and 1001 four-peptide combinations of Peptides (1) through (14), or any one of the 1456 combinations of Peptides (1) through (14).
  • Embodiment 24 The immunogenic composition of embodiment 23, comprising a combination of any three of Peptides (1) through Peptide (14).
  • Embodiment 25 The immunogenic composition of embodiment 24, comprising a combination of Peptides (1), (2) and (3); (1), (2) and (4); (1), (3), and (4); and (2), (3), (4) or any one of the 35 possible three-peptide combinations of these seven Peptides; or any one of the 35 three-peptide combinations of Peptides (8) through (14); or any one of the 364 three-peptide combinations of Peptides (1) through (14); or any one of the 1001 four-peptide combinations of Peptides (1) through (14).
  • Embodiment 26 The immunogenic composition of any one of embodiments 19 through 25, wherein the immunogenic composition is a vaccine.
  • Embodiment 27 The immunogenic composition of any one of embodiments 19 through 26, wherein the adjuvant is aluminum hydroxide or one of its salts.
  • Embodiment 28 A method of preventing or treating severe acute respiratory syndrome or other SARS-CoV-related disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one of embodiments 20 through 27, or of a polypeptide of any one of embodiments 1 through 13.
  • Embodiment 29 The method of embodiment 28, wherein the efficacy is measured by measuring a reduced viral load or delays or prevention of a further increase in viral load.
  • Embodiment 30 The method of embodiment 29, wherein the efficacy is measured by elimination, reduction, or decrease in the intensity or frequency of one or more symptoms associated with SARS such as fever, cough, shortness of breath, organ failure (e.g. kidneys) and/or septic shock.
  • Embodiment 31 The method of any one of embodiments 28 through 30, wherein the subject has an underlying disorder such as diabetes, cancer, chronic lung disease, heart disease, or generally weakened immune system.
  • an underlying disorder such as diabetes, cancer, chronic lung disease, heart disease, or generally weakened immune system.
  • Embodiment 32 The method of any one of embodiments 28 through 31 , wherein the immunogenic composition induces a humoral response, such as an increased level of neutralizing antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine, or as compared to the same subject prior to administration of the immunogenic composition.
  • a humoral response such as an increased level of neutralizing antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine, or as compared to the same subject prior to administration of the immunogenic composition.
  • Embodiment 33 The method of any one of embodiments 28 through 32, wherein the immunogenic composition induces a cellular immune response, such as a CD8+T cell response, (including the production of cytokines such as interferon- gamma (TFN-g), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or any combinations thereof.
  • a cellular immune response such as a CD8+T cell response
  • cytokines such as interferon- gamma (TFN-g), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or any combinations thereof.
  • Embodiment 34 The method of any one of embodiments 28 through 33, wherein the therapeutically effective amount is prophylactic.
  • Embodiment 35 The method of any one of embodiments 28 through 34, wherein the immunogenic composition or polypeptide is administered by any route typically used for vaccination, including topical, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, inhalation, or combinations thereof.
  • Embodiment 36 The method of any one of embodiments 28 through 35, wherein the immunogenic composition or polypeptide is administered in a single-dose vaccination schedule.
  • Embodiment 37 The method of any one of embodiments 28 through 36, further comprising the administration of another treatment for SARS.
  • Embodiment 38 The method of any one of embodiments 28 through 37, further comprising the administration of remdesivir, azithromycin, hydroxychloroquire, chloroquine, standard of care, or combinations thereof.
  • Embodiment 39 The method of any one of embodiments 28 through 38, further comprising the administration of hydroxychloroquine combination (200 mg X 3 per days for 10 days) with Azithromycin (500 mg on the 1st day then 250 mg per day for 5 more days);
  • Anti-inflammatory therapy such as tocilizumab and favipiravir, tocilizumab, sarilumab, leronlimab, rintatolimod, BPI-002, REGN3048 and REGN 3051 , monoclonal antibody designed to bind SARS-CoV-2;
  • Anti-Viral therapy such as remdesivir, lopinavir and ritonavir, danoprevir and ritonavir, favipiravir, darunavir and cobicistat, umifenovir, galidesivir, linebacker and equivir, compounds that inhibit the virus interaction with the receptor ACE2; or Immunotherapy such as RNA vaccines (e.g
  • Embodiment 40 The method of any one of embodiments 28 through 39, wherein the disease is Covid-19.
  • Embodiment 41 A method of using a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, as a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
  • Embodiment 42 An immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19 for manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
  • Embodiment 43 Use of an immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, or a recombinant cell of any one of embodiments 18 and 19 for the manufacture of a medicament for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
  • Embodiment 44 An immunogenic composition of any one of embodiments
  • Embodiment 45 Use of an immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19, or an antibody against a polypeptide of any one of embodiments 1 through 13, for the manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
  • Embodiment 46 An immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19 for manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
  • FIG. 1 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:1.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.65 corresponds to sensitivity of 0.01969and specificity of 0.99272.
  • FIG. 2 shows the hydrophobicity prediction of the peptide SEQ ID NO:1 using the Kyte-Doolittle algorithm.
  • FIG. 3 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:2.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of two B-cell epitopes.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116.
  • FIG.4 shows the hydrophobicity prediction of the peptide SEQ ID NO:2 using the Kyte-Doolittle algorithm.
  • FIG. 5 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:3.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116.
  • FIG.6 shows the hydrophobicity prediction of the peptide SEQ ID NO:3 using the Kyte-Doolittle algorithm.
  • FIG. 7 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:4.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116.
  • FIG. 8 shows the hydrophobicity prediction of the peptide SEQ ID NO:4 using the Kyte-Doolittle algorithm.
  • FIG. 9 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:5.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.60 corresponds to sensitivity of 0.09559 and specificity of 0.95116.
  • FIG. 10 shows the hydrophobicity prediction of the peptide SEQ ID NO:5 using the Kyte-Doolittle algorithm.
  • FIG. 11 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:6.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
  • FIG. 12 shows the hydrophobicity prediction of the peptide SEQ ID NO:6 using the Kyte-Doolittle algorithm.
  • FIG. 13 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:7.
  • the table (A) contains the score of each of the amino acid
  • the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116.
  • FIG. 14 shows the hydrophobicity prediction of the peptide SEQ ID NO:7 using the Kyte-Doolittle algorithm.
  • FIG. 15 List of epitopes in immunogenic polypeptides (e.g.SEQ ID NO: 1 through 16).
  • the figure discloses SEQ ID NOS 1 , 42-47, 2, 44, 48-56, 3, 57-67, 4, 68, 51 - 56, 69-72, 5, 73-74, 6, 75-80, 7, 81-83, 125, 42, 84, 69-71 , 85, 126, 86, 127, 87, 128-131 , 88, 132-135, 89 and 136-139, respectively, in order of appearance.
  • FIG. 16 shows the prediction of the B-cell epitope in the Peptide (8) SEQ ID NO: 83.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0. 60 corresponds to sensitivity of 0.09559 and specificity of 0.95116.
  • FIG. 17 shows the hydrophobicity prediction of the peptide SEQ ID NO: 83 using the Kyte-Doolittle algorithm.
  • FIG. 18 shows the prediction of the B-cell epitope in the Peptide (9) SEQ
  • the table (A) contains the score of each of the amino acid, the graph (A) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
  • FIG. 19 shows the hydrophobicity prediction of the peptide SEQ ID NO: 84 using the Kyte-Doolittle algorithm
  • FIG. 20 shows the prediction of the B-cell epitope in the Peptide (10) SEQ ID NO: 85.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.60 corresponds to sensitivity of 0.09559 and specificity of 0.95116.
  • FIG. 21 shows the hydrophobicity prediction of the peptide SEQ ID NO: 85 using the Kyte-Doolittle algorithm.
  • FIG. 22 shows the prediction of the B-cell epitope in the Peptide (11) SEQ ID NO: 86.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
  • FIG. 23 shows the hydrophobicity prediction of the peptide SEQ ID NO: 86 using the Kyte-Doolittle algorithm
  • FIG. 24 shows the prediction of the B-cell epitope in the Peptide (12) SEQ ID NO: 87.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
  • FIG. 25 shows the hydrophobicity prediction of the peptide SEQ ID NO: 87 using the Kyte-Doolittle algorithm.
  • FIG. 26 shows the prediction of the B-cell epitope in the Peptide (13) SEQ ID NO: 88.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
  • FIG. 27 shows the hydrophobicity prediction of the peptide SEQ ID NO: 88 using the Kyte-Doolittle algorithm
  • FIG. 28 shows the prediction of the B-cell epitope in the Peptide (14) SEQ ID NO: 89.
  • the table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope.
  • the amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158.
  • Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
  • FIG. 29 shows the hydrophobicity prediction of the peptide SEQ ID NO: 89 using the Kyte-Doolittle algorithm.
  • FIG. 30 shown predicted T cell epitopes (MFIC binding). Detailed Description
  • nucleotides includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73,
  • consisting of is defined as "closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith.
  • a claim which depends from a claim which "consists of” the recited elements or steps cannot add an element or step.
  • the terms “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of” may mean within one or more than one standard deviation per the practice in the art. “About” or “comprising essentially of” may mean a range of up to 10% (i.e., ⁇ 10%).
  • “about” may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value.
  • about 5 mg may include any amount between 4.5 mg and 5.5 mg.
  • the terms may mean up to an order of magnitude or up to 5-fold of a value.
  • any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one- hundredth of an integer), unless otherwise indicated.
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • Polypeptides, nucleic acids and host cells of the present description, and immunogenic compositions and vaccines thereof may be administered to a subject in need thereof by routes known in the art, and may vary depending on the use. Routes of administration include, for example, local administration (e.g., to the lungs) and parenteral administration such as subcutaneous, intraperitoneal, intramuscular, intravenous, intraportal and intrahepatic.
  • polypeptides, nucleic acids, composiitions, vaccines or host cells of the present disclosure, or immunogenic compositions thereof are administered to a subject by local infusion, for example using an infusion pump and/or catheter system.
  • routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the polypeptides, nucleic acids, composiitions, vaccines or host cells of the present disclosure, or immunogenic compositions thereof is administered via a non- parenteral route, e.g., orally.
  • a non-parenteral route include a topical, epidermal, or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering may also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • amino acid/s or an “amino acid residue/s” may be a natural or non natural amino acid residue/s linked by peptide bonds or bonds different from peptide bonds.
  • the amino acid residues may be in D- configuration or L -configuration (referred to herein as D- or L- enantiomers).
  • An amino acid residue comprises an amino terminal part (NH3) and a carboxy terminal part (COOH) separated by a central part (R group) comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group.
  • NH3 refers to the amino group present at the amino terminal end of an amino acid or peptide
  • COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide.
  • the generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature are listed in 37 C.F.R. 1.822(b)(2). Examples of non-natural amino acids are also listed in 37 C.F.R. 1.822(b)(4), other non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues. Naturally occurring amino acids may be further modified, e.g.
  • an antibody includes, without limitation, a glycoprotein immunoglobulin, which binds specifically to an antigen.
  • an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three constant domains, CH1 , CH2, and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprises one constant domain, CL.
  • the VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-id) antibodies (including, e.g., anti-anti-ld antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes
  • An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human lgG1 , lgG2, lgG3 and lgG4.
  • “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG 1 ) that is encoded by the heavy chain constant region genes.
  • antibody includes, by way of example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or nonhuman Abs; wholly synthetic Abs; and single chain Abs.
  • a nonhuman Ab may be humanized by recombinant methods to reduce its immunogenicity in man.
  • the term “antibody” also includes an antigen-binding fragment or an antigen binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab.
  • an “antigen binding molecule,” “antigen binding portion,” or “antibody fragment” refers to any molecule that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived.
  • An antigen binding molecule may include the antigenic complementarity determining regions (CDRs).
  • Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules.
  • Peptibodies i.e. , Fc fusion molecules comprising peptide binding domains
  • suitable antigen binding molecules are another example of suitable antigen binding molecules.
  • the antigen binding molecule binds to an epitope present in the surface of a viral particle. In some embodiments, the antigen binding molecule binds to an epitope in the SARS coronavirus S protein. In certain embodiments, the antigen binding molecule binds to any one of polypeptides of the disclosure. In further embodiments, the antigen binding molecule is an antibody fragment that specifically binds to the antigen, including one or more of the complementarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of avimers.
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • the variability in sequence is concentrated in those regions called CDRs while the more highly conserved regions in the variable domain are called framework regions (FR).
  • variable region is a human variable region.
  • variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • FRs human framework regions
  • the variable region is a primate (e.g., non-human primate) variable region.
  • the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).
  • an antigen binding molecule, an antibody, or an antigen binding molecule thereof, or a polypeptide of the dislosure “cross-competes” with a reference antibody or an antigen binding molecule thereof or polypeptide of the disclosure if the interaction between an antigen and the first binding molecule, an antibody, or an antigen binding molecule or polypeptide of the disclosure blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, an antigen binding molecule thereof, or polypeptide of the disclosure to interact with the antigen or with the coronavirus S protein.
  • Cross competition may be complete, e.g., binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind the antigen, or it may be partial, e.g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind the antigen.
  • an antigen binding molecule that cross- competes with a reference antigen binding molecule binds the same or an overlapping epitope as the reference antigen binding molecule.
  • the antigen binding molecule that cross-competes with a reference antigen binding molecule binds a different epitope as the reference antigen binding molecule.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid phase direct biotin-avidin EIA Karlland et al., 1986, J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1 -125 label (Morel et al., 1988, Molec. Immunol. 25:7-15), solid phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). These methods are also examples of methods for measuring the affinity of the peptides of the disclosure for the antibodies or coronaviral S protein of the disclosure.
  • an “antigen” refers to any molecule that provokes an immune response or is capable of being bound by an antibody or an antigen binding molecule (e.g., a TCR).
  • the immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • An antigen may be endogenously expressed, i.e. expressed by genomic DNA, or may be recombinantly expressed.
  • An antigen may be specific to a certain tissue, such as a cancer cell, or it may be broadly expressed.
  • fragments of larger molecules may act as antigens.
  • the antigens are any one of the polypeptides of the disclosure.
  • a “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a therapeutic agent, e.g., polypeptide or antibody of the disclosure, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • the ability of a therapeutic agent to promote disease regression may be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays. Dosages of the molecules of the present disclosure may vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated. A physician will ultimately determine appropriate dosages to be used.
  • a “patient” or ’’subject” as used herein includes any human or animal who is afflicted with coronavirus-related disease or disorder.
  • the terms “subject” and “patient” are used interchangeably herein.
  • an in vitro cell refers to any cell, which is cultured ex vivo.
  • an in vitro cell may include a T cell.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • Treatment refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity, or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.
  • treatment or “treating” includes a partial remission. In another embodiment, “treatment” or “treating” includes a complete remission.
  • GCG program package which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.).
  • GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
  • sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span,” as determined by the algorithm.)
  • a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.
  • identity may be determined as percentage of identity using known computer algorithms such as the “PASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.
  • similarity and “similar” and grammatical variations thereof, as used herein, mean that an amino acid sequence contains a limited number of conservative amino acid substitutions compared to a peptide reference sequence, e.g. the variant peptide versus the parent peptide as defined herein.
  • a variety of criteria can be used to indicate whether amino acids at a particular position in a peptide are similar.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • substitutions may be conservative or non-conservative amino acid substitutions.
  • a "conservative substitution” is the replacement of one amino acid by a biologically, chemically or structurally similar residue.
  • Biological similarity means that the substitution does not destroy a biological activity, e.g. T ceil reactivity or HLA coverage.
  • Structural similarity means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size.
  • Chemical similarity means that the residues have the same charge, or are both either hydrophilic or hydrophobic.
  • a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain, for example amino acids with basic side chains (e.g., lysine, arginine, histidine) ; acidic side chains (e.g., aspartic acid, glutamic acid) ; uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine) ; nonpolar side chains
  • alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
  • Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, serine for threonine, and the like.
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., Leu, Val, He, and Ala).
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • S1 ectodomain subunit of SARS-CoV-2 spike (S) glycoprotein binds to the cellular receptor angiotensin-converting enzyme 2 (ACE2) present on permissive host cells.
  • ACE2 angiotensin-converting enzyme 2
  • the membrane-proximal subunit S2 of SARS-CoV-2 is responsible for the fusion of the virus with the plasma membrane of host cell. Since the S protein is cleaved during egress of the virus from the host cells, the two subunits are held together only by non- covalent interactions.
  • suitable immunogenic peptides with potential for protective therapeutic function were derived from the S1 subunit (SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4) and from the S2 subunit (SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7).
  • B-cell epitope probability prediction BepiPred-2.0 server (Jespersen et al., 2017) run at http://t00ls.iedb.0rg/bcell// was used to evaluate the physical and chemical properties of the designed peptides by Random Forest algorithm trained on epitopes and non-epitope amino acids determined from crystal structures.
  • KLH is a highly immunogenic T-cell dependent antigen, xenogeneic to mammalian immune system, elicits strong immune response and promotes generation of antibodies against haptens.
  • the peptides may also be conjugated to other molecules via other methods, including click-chemistry (e.g., Peptide (3)).
  • the conjugation linker may be attached to the N-terminal or C-terminal end of the peptides.
  • the vaccines carrying the peptides derived from the S1 subunit conjugated to KLH will elicit antibodies that will bind and inhibit SARS-CoV-2 S1-Ace2 interaction and binding to host permissive cells.
  • the vaccines containing the peptides derived from S2 subunit conjugated to KLH can induce antibodies preventing the conformational change and fusion of the virus with membrane of the host cell.
  • KLH-conjugated peptides derived from S1 subunit (SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89 will be combined with KLH-conjugated peptides derived from S2 subunit (SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7).
  • This approach allows to obtain a defined pool of vaccine induced antibodies with a combined mode of action, preventing the first step of the infection - viral binding to a host cell, and also preventing the second step - fusion of the virus with the plasma membrane of the host cell.
  • All proposed peptides can also be produced with amide at its C-terminus. This might increase the binding capacity (crossreactivity) of vaccine induced antibodies with desired (therapeutic) epitopes on native surface S protein.
  • the amino acid numbers are according to full length SARS-CoV-2 Spike protein of the virus isolate under the NCBI Reference Sequence NC_045512.2 (Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 , complete genome).
  • the disclosure is directed to isolated or purified polypeptides encompassing at least one epitope from a coronavirus Spike protein, including the SARS CoV2 Spike protein.
  • isolated or purified mean modified “by the hand of humans” from the natural state; in other words if an object exists in nature, it is said to be isolated or purified if it is modified or extracted from its natural environment or both.
  • a polynucleotide or a protein/peptide naturally present in a living organism is neither isolated nor purified; on the other hand, the same polynucleotide or protein/peptide separated from coexisting molecules in its natural environment, obtained by cloning, amplification and/or chemical synthesis is isolated for the purposes of the present disclosure.
  • a polynucleotide or a protein/peptide which is introduced into an organism by transformation, genetic manipulation or by any other method is “isolated” even if it is present in said organism.
  • the term purified as used in the present disclosure means that the proteins/peptides according to the disclosure is essentially free from, contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • isolated polypeptide does not exclude the presence of the same polypeptide in alternative physical forms, such as dinners or alternatively glycosylated or derivatized forms.
  • isolated peptides are also non-naturally occurring, synthetic peptides.
  • the SARS-CoV-2 spike (S) polypeptide is the peptide disclosed in NCBI access number NC_045512.2 (Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 , complete genome).
  • the S polypeptide has 1273 amino acids. It contains a large N-terminal ectodomain consisting of two subunits S1 and S2, one transmembrane domain close to the C-terminus and a short intraviral Cys-rich domain.
  • the S1 ectodomain subunit of SARS-CoV-2 spike (S) glycoprotein binds to the cellular receptor angiotensin-converting enzyme 2 (ACE2) present on permissive host cells.
  • ACE2 angiotensin-converting enzyme 2
  • the membrane-proximal subunit S2 of SARS-CoV-2 is responsible for the fusion of the virus with the plasma membrane of the host cell.
  • the peptides below are numbered relatively to the sequence of NCBI access number NC_045512.2.
  • the SARS-CoV-2 polypeptide is a peptide selected from the following:
  • Peptide (2) 442- CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ -506 (SEQ ID NO: 2)
  • Peptide (3) 469-SGSTEIYQAGSTPCNGVEGFNCYFAK-490 (SEQ ID NO: 3)
  • Peptide (4) 483-CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE-516 (SEQ ID NO: 4)
  • the SARS-CoV-2 polypeptide is a peptide selected from the following:
  • Peptide (5) 804-QILPDPSKPSKRSC-816 (SEQ ID NO: 5)
  • Peptide (6) 919-CNQKLIANQFNSAIGKIQDSLSS-940 (SEQ ID NO: 6)
  • Peptide (7) 695-CYTMSLGAENSVAYS -708 (SEQ ID NO: 7)
  • the SARS-CoV-2 peptide is a peptide selected from the following: [00153] Peptide (8): 438-CSNNLDSKVGGNYN-450, SEQ ID NO: 83
  • each of Peptides (1) through (14), if not already there, may have a N-terminal Cysteine to facilitate conjugation to KLH.
  • the N- terminal (or C-terminal) Cys in all of the peptides is used for conjugation and may or may not be present.
  • the peptides may be conjugated to CRM197ln
  • Peptide (1) is derived from the S1 subunit, modified by adding GSG linker sequence.
  • N-terminal C may be used for conjugation to KLH.
  • Peptide (1) may be combined with Peptide (3) and Peptides (5), (6), and/or (7) from the S2 subunit.
  • additional changes (K instead of Y451) and C-terminal E may be added to stabilize the peptide structure.
  • Peptide (2) is derived from the S1 subunit and is generated by fusing two separated sequences: 442-454 and 492-506. The aim is to mimic a compact 3D structure similar to that present on native Spike protein, which makes contacts with ACE2 receptor on human epithelial cells.
  • the linker sequence (GPAD (SEQ ID NO: 17)) may form a beta turn was generated by using the Motivated proteins web site facility.
  • the N-terminal C may be used for conjugation to KLH.
  • the peptide may be produced with amide at its C-terminus.
  • Peptide (3) is derived from the S1 subunit, and the last two amino acids Ala(A) and Lys(K) may be added in order to force the peptide to generate a compact structure by contacting the added K with E471. This may mimic the 3D structure of the native S protein contact site with ACE2.
  • the two internal cysteines (underlined) may be oxidized to form internal disulfide bond.
  • the N-terminus of the peptide is modified (e.g. by a chemoselective reactive group like azide which can bind selectively to alkyne or phosphine) and may be used for conjugation to KLH.
  • an SG may be used as a spacer.
  • the peptide may also be produced with amide at its C-terminus.
  • Peptide (4) derived from S1 subunit, internal Cys (C488) is modified to Ala(A) to prevent dimerization, may change the 3D peptide structure.
  • N-terminal C may be used for conjugation to KLH.
  • the peptide may be produced with amide at its C-terminus.
  • Peptide (5) will induce antibodies that prevent access of the protease to the S2’ cleavage site, and thus prevent a conformational change to induce ‘fusion peptide’ interaction with cellular membrane.
  • the C- terminal C may be used for conjugation to KLH.
  • the peptide may be produced with amide at its N-terminus.
  • Peptide (5) may be used in combination with peptides derived from the S1 subunit.
  • Peptide (6) will induce antibodies against S2 subunit of S protein, that should inhibit its conformational changes and prevent fusion with cellular plasma membrane.
  • the N-terminal C may be used for conjugation to KLH.
  • the peptide may be produced with amide at its C-terminus.
  • Peptide (6) may be used in combination with peptides derived from the S1 subunit.
  • Peptide (7) will induce antibodies against S2 subunit of S protein, that should inhibit its conformational changes and prevent fusion with cellular plasma membrane.
  • the N-terminal C may be used for conjugation to KLH.
  • the peptide may be produced with amide at its C-terminus.
  • Peptide (7) may be used in combination with peptides derived from the S1 subunit.
  • Peptide (8) is derived from the surface of the S1 subunit.
  • the designed peptide contains one B cell epitope located in the middle part of the peptide and two contact residues (Spike protein positions N439, Y449), which may be important for binding the virus to the ACE2 receptor.
  • the peptide- induced antibody may affect binding of virus to hACE2, thus inhibiting of entry into a host cell.
  • N-terminal cysteine (C) is added for conjugation to KLH (the carrier protein) through a maleimide linker.
  • Peptide (8) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
  • Peptide (9) is derived from the surface of the S1 subunit.
  • the designed peptide contains one B cell epitope located in middle part of the peptide.
  • the peptide carries several contact residues (Spike protein positions T500, N501 , G502, Y505), which may play important roles in binding the virus to its receptor (ACE2). Blocking these residues by peptide-induced antibodies may be therapeutically effective to prevent the S1 subunit - ACE2 interaction.
  • N-terminal cysteine (C) is used for conjugation to KLH as a carrier protein using a maleimide linker
  • aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid.
  • Peptide (9) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
  • Peptide (10) is derived from the surface amino acids of the S1 subunit.
  • the peptide contains one B cell epitope located in middle region and two contact residues (Spike protein positions L455, F456), which may play important roles in binding the virus to receptor of host cell.
  • the peptide-induced antibodies may block binding of virus to hACE2 and entry of virus into the cell.
  • the N- terminal cysteine (C) is added for conjugation to KLH (as a carrier protein) through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid.
  • Peptide (10) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
  • Peptide (11) is derived from surface S1 subunit.
  • the peptide contains one B cell epitope located in middle region.
  • Alanine located at the position 10 (A475 of the Spike protein) of the peptide is one of the contact residues with ACE2, YQAGS residues (SEQ ID NO: 117) form a part of the contact surface of S1 with ACE2, which the virus can utilize to enter into the host cell.
  • the peptide-induced antibodies could have neutralizing activity and thus effectively block the virus from binding to the receptor of permissive cells.
  • the N-terminal cysteine (C) is added for conjugation to KLH (as a carrier protein) through a maleimide linker.
  • Peptide (11 ) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
  • the designed peptide contains one B cell epitope located in central part of peptide. Threonine located at position 5 (the Spike protein position T415) and lysine at position 7 (the Spike protein position K417) were identified as the contact residues, which the virus may exploit to enter the host cell.
  • the binding of the peptide-induced antibodies to these strategic residues or near these residues may lead to the lowering number of virus -infected cells.
  • N-terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker.
  • Peptide (12) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
  • Peptide (13) is derived from the surface of the S1 subunit.
  • the designed peptide contains one B cell epitope located in medium part of peptide.
  • the residues R3, D5, E6, Q9, T14 and K16 of the peptide were identified as part the S1 protein contact surface with ACE2, which the virus exploits to enter the host cell.
  • the blocking of the positions important for binding to human ACE2 by peptide-induced antibodies can lead to the lower affinity between the viral RBD and host ACE2 in the initial viral attachment step. In the end this lowering of affinity can lead to lowering of virus efficiency.
  • the N-terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker.
  • Peptide (13) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
  • Peptide (14) is derived from the surface of the S1 subunit.
  • the peptide contains one B cell epitope located in medium part of peptide and four residues which were identified as important contact sites which virus exploit for binding to host cell receptor.
  • the identified amino acids residues at the Spike protein positions F486 (phenylalanine), N487 (asparagine), Y489 (tyrosine) and Q493 (glutamine) enhance viral attachment to human ACE2.
  • the occupation (blocking) of these strategic residues by peptide-induced antibodies may be therapeutically effective.
  • N-terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid.
  • Peptide (14) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
  • the polypeptide comprises one or more peptides of the S1 protein and/or the S2 protein. In one embodiment, the polypeptide comprises one or more epitopes from those disclosed on FIG. 15.
  • the peptides may be linked directly as one sequence. In other embodiments, the peptides may be linked through a linker. In one embodiment, the polypeptide is Peptide (2).
  • Linkers may be used to link peptides and/or to conjugate the peptides to other molecules.
  • Linkers may have one or more properties that may include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain.
  • Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence.
  • the linker is GSG.
  • the linker is GPAD (SEQ ID NO: 17).
  • the linker is selected from a GS-short linker GGGGSG (SEQ ID NO: 18), a GS-medium linker, and a GS-long linker GGGGSGGGGSGGGG (SEQ ID NO: 19).
  • Linkers may be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, may be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage.
  • the linker is a cleavable linker, which may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition.
  • the cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met-X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp-Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole.
  • protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used. The precise sequences are available in the art and the skilled person will have no difficulty in selecting a suitable cleavage site.
  • the protease cleavage region targeted by Factor Xa is I E G R (SEQ ID NO: 20).
  • the protease cleavage region targeted by Enterokinase is D D D D D (SEQ ID NO: 21).
  • the protease cleavage region targeted by Thrombin is L V P R G (SEQ ID NO: 22).
  • the cleavable linker region may be one that is targeted by endocellular proteases.
  • Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage. Additional linkers include:
  • AEA SEQ ID NO: 32
  • EDVVCCISMSY (SEQ ID NO: 33)
  • GGIEGRIGS SEQ ID NO: 34
  • TRHRQPRIGWE SEQ ID NO: 35
  • GFLGI SEQ ID NO: 38
  • the peptides may comprise and Aspartic acid (D) at the N-terminus (e.g., after Cys), which may be placed before, within, or after an SG spacer.
  • D Aspartic acid
  • Such peptides may be cleavable by hot formic acid after the D, which generates a peptide- specific fragment suitable for HPLC-UV or HPLC-MS/MS identification and/or quantification.
  • Peptides (1) through (14) may be conjugated to other peptides, which may be, in some instances, B or T cell epitopes. In one embodiment, they are arranged as PADRE constructs.
  • the disclosure also provides for variants of the specific parent polypeptides identified above namely those of SEQ ID NOS 1 -16, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NOS 90-139, or those comprising one or more peptide epitopes disclosed in FIG. 15.
  • a variant which is also termed a "variant peptide” or "modified peptide” herein, is a peptide, that is derived from but not identical to a parent peptide as defined herein.
  • a variant peptide may include a number of variations compared to the parent peptide as defined herein, for example to increase or decrease physical or chemical properties of the parent peptide as defined herein, for example to decrease its ability to resist oxidation, to improve or increase solubility in aqueous solution, to decrease aggregation, to decrease synthesis problems, etc.
  • a variant peptide may comprise one or more of the same B or T cell epitopes as the parent peptide as defined herein.
  • this may be determined by the ability of the variant peptide to induce or stimulate in-vitro T cell proliferation using cultured PBMCs (peripheral blood monocytes) compared to the parent peptide as defined herein, optionally using same test conditions, or by the ability of the variant peptide to induce or stimulate production of cytokines, (e.g. cytokines, IL-5, IL-13 and/or IL-10) from T cells (obtained from cultured PBMC's) compared to the parent peptide as defined herein.
  • cytokines e.g. cytokines, IL-5, IL-13 and/or IL-10
  • the epitopes of FIG. 15 are predicted to be B cell epitopes.
  • the epitopes of FIG. 15 may include B cell epitopes and the epitopes of FIG. 30 may include T cell epitopes. The latter were predicted using IEDB analysis resource Consensus tool in www.iedb.org; Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B. 2008.
  • the polypeptide is modified.
  • modification means that the polypeptide is not a fragment of the SARS Cov2 S protein and is different from the polypeptides whose sequence is specifically described in this disclosure by at least one amino acid residue either in sequence and/or in chemical modification.
  • an amide group is added at the 3' end of the peptide of the disclosure.
  • a Cys residue or a D residue, or an S residue is added at either the C terminus and/or N-terminus (or removed, in the case of Peptide (1) through Peptide (14)).
  • the Cys residue is added through a linker.
  • the peptide can be modified by a chemoselective group, which allows coupling to a carrier (e.g. azide binding to phosphine or alkyne).
  • the polypeptide may be modified by an internal bridge, short-range cyclization, methylation, amidation, acetylation or substitution with other chemical groups, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation.
  • a disulfide linkage may be present or absent in the peptides of the disclosure.
  • the polypeptide is modified by manosylation, glycosylation, amidation (specifically C-terminal amides), carboxylation or phosphorylation. In one embodiment, these modifications may preserve the biological activity of the original molecule.
  • Functional derivatives of the peptides are also included in the present disclosure. Functional derivatives are meant to include peptides which differ in one or more amino acids in the overall sequence, which have deletions, substitutions, inversions or additions but preserve or improve the biological activity of the original peptide. In one embodiment, amino acid substitutions which may work not to essentially alter biological and immunological activities may be found.
  • Amino acid replacements between related amino acids or replacements which may have occurred frequently in evolution include, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, lle/Val.
  • Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence may provide advantageous physical, chemical, biochemical, and pharmacological properties, such as enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others.
  • the substitutions are located at the end of the amino acid chain.
  • the substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical nature, such as where leucine is replaced by isoleucine. Certain amino acid substitutions are more often tolerated than others, and these often show correlation with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement. Chemical and biological effects are not totally predictable and substitutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles. In some embodiments, the substitutions provide unexpected results.
  • the polypeptide further comprises one or more moieties.
  • the moiety is a Cys residue.
  • the moiety is a carrier protein selected from any one of a Keyhole Limpet Hemocyanin (KLH) functional unit, a tetanus toxin heavy chain C fragment, a diphteria toxin, a diphtheria toxin variant CRM197, an H influenzae protein D, a Meningococcal outer membrane protein complex protein, an Outer-membrane lipoprotein carrier protein, or a Cholera toxin B subunit, a virus-like particle; biotin, avidin, streptavidin, neutravidin, serum albumin, keyhole limpet hemocyanin (KLH), an enzyme, a metallic nanomaterial, CRM197 and an outer membrane protein mixture from N.
  • KLH Keyhole Limpet Hemocyanin
  • MMP meningitidis
  • micro- and nano-particles of biodegradable polymers including polylactic, polycaproic, polyglycolic, polymalic, polybutyric acids and their combination and modifications; hydrogels of polyethyleneglycol and its modifications; hyaluronic acid, dextran, chitosan, liposome- polymer hybrid carrier, or a derivative or combination thereof.
  • the moiety is a Cys and a KLH.
  • the moiety is an azidopentanoyl moiety, which may or may not be conjugated to KLH.
  • the moiety or modification is an alkyne modification, a cyclooctyne derivative [e.g., BCN (Bicyclo[6.1 0]non-4- yne)-9-methanol; DBCO Dibenzocyclooctyne (11 ,12-didehydro-5, 6-dihydro-dibenz[b,f] azocine derivatives); DIFO 2-[(6,6-difluoro-4- cyclooctyn-1-yl)oxy] acetic acid; MOFO 2-(4-carboxybenzyl)- 2-fluorocyclooctyne.
  • BCN Bicyclo[6.1 0]non-4- yne
  • DBCO Dibenzocyclooctyne 11 ,12-didehydro-5, 6-dihydro-dibenz[b,f] azocine derivatives
  • the variant may for instance include one or more deletions of amino acid residues from the N- and/or C- terminal end of the parent peptide as defined herein one or more additions of amino acid residues to the N- and/or C- terminal of the parent peptide as defined herein and/or one or more amino acid substitutions, additions or deletions within the amino acid sequence of the parent peptide as defined herein.
  • One type of variant is a "derivative", where chemical modifications are introduced, for instance in the side-chains of one of more of the amino acid residues of the parent peptide's amino acid sequence (thus effectively resulting in a peptide that includes an amino acid residue substitution relative to the parent peptide as defined herein).
  • a derivative can also include a chemical modification that involves the N-terminal amino group and/or the C-terminal COOH group.
  • Derivatives are described in more detail herein. It is important to note that some derivatives of the parent peptides as defined herein, are those that could be obtained by substituting an amino acid residue with another naturally occurring amino acid residue, whereas other derivatives involve chemical modifications that result in the provision of peptides that could not be encoded by a nucleic acid sequence.
  • a longer variant of the parent peptide as defined herein may be up to 60 amino acids in length, for example up to 55, 50, 45 amino acids in length. More typically, a longer variant peptide is up to about 100 amino acids in length, such as up to 70 amino acids in length.
  • the longer variant may comprise the amino acid sequence of a parent peptide disclosed herein, or an amino acid sequence having at least 85% identity or similarity over the length of the amino acid sequence of the parent peptide as defined herein, or a fragment thereof, such as over at least 12 contiguous amino acids, for example over at least 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein.
  • the longer variant comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity or similarity over the length of the amino acid sequence of the parent peptide as defined herein, or over at least 12 contiguous amino acids, for example over at least 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein.
  • a variant of the parent peptide as defined herein is a longer peptide up to 50 amino acid residues in length that comprises one or more additional amino acid residues at the N- and/or C- terminal end of the parent peptide as defined herein, or comprises an amino acid sequence having at least 80%, such as at least 85%, 90% or 95% identity or similarity over at least 14 contiguous amino acids of the parent peptide as defined herein, such as over at least 15, 16, 17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein.
  • a variant of the parent peptide may also include a fragment of a parent peptide as defined herein disclosed herein.
  • a fragment of the parent peptide can have one or more amino acids less than the parent peptide as defined herein, either comprising deletions from within the amino acid sequence of the parent peptide as defined herein and/or amino acid deletions from the N- and/or C- terminus of the parent peptide as defined herein.
  • a fragment will have a length of at least 12 amino acids, for example at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 amino acids, and will have at least 65% identity or similarity over the length of the fragment, or over the length of at least 12 contiguous amino acids, when aligned with the parent peptide as defined herein.
  • the percentage identity or similarity is at least 70%, 75%, 80%, 85%, 90% or 95% over the length of the fragment, or over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein.
  • a variant thereof may be a shorter peptide comprising an amino acid sequence having at least 80%, such as at least 85%, 90% or 95% identity or similarity over at least 14 contiguous amino acids of the parent peptide as defined herein, such as over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide.
  • the variant is a peptide consisting of 25- 45 amino acids and comprises an amino acid sequence having at least 80%, such as at least 85%, 90% or 95% identity or similarity over at least 25 or 30 contiguous amino acids of the parent sequence as defined here.
  • the variant is a peptide consisting of 25-45 amino acids and comprises an amino acid sequence having at least 80% identity or similarity over at least 20-30 or 30-35 contiguous amino acids with the sequence of a parent peptide as defined herein.
  • the variant is a peptide consisting of 15-25 amino acids and comprises an amino acid sequence having at least 80%, such as at least 85%, or at least 90%, or at least 95%, or at least 98% identity or similarity over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide sequence as defined herein.
  • the variant is a peptide consisting of 16-25 amino acids and comprises an amino acid sequence having at least 80% identity or similarity over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids with the sequence of a parent as defined herein
  • the at least 80% identity or similarity is over at least 16, 17, 18 or 19 contiguous amino acids with the sequence of a parent peptide as defined herein and the percent identity or similarity may be at least is 80%, such as at least 85%, 90% or 95% over at least 16, 17, 18 or 19 contiguous amino acids with the sequence of a parent peptide .
  • identity and “identical” and grammatical variations thereof, as used herein, mean that two or more referenced entities are the same (e.g., amino acid sequences). Thus, where two peptides are identical, they have the same amino acid sequence.
  • the identity can be over a defined area, e.g. over at least 12, 13, 14, 15 or 16 contiguous amino acids with the sequence of a parent peptide as defined herein, optionally wherein the alignment is the best fit with gaps permitted.
  • the variant peptide may be aligned with the parent peptide as defined herein and the percent identity calculated with respect to the identical amino acid residues found within the amino acid sequence of the variant peptide that overlaps with the 15 contiguous amino acids with the sequence of a parent peptide as defined herein.
  • the disclosure provides an isolated polypeptide comprising one or more peptides that differ from the peptides of amino acid sequences of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 , SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NOS 90-140 by 1 , 2, 3, 4,
  • a variant of a parent peptide as defined herein may comprise additional amino acids or may consist of a fragment of the parent peptide as defined herein.
  • the polypeptide further comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the polypeptide is at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21 , at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31 , at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41 , at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51 , at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58 residues, at most 59, at most 60, at most 61 , at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70
  • the polypeptide is between about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, and 90 to 100 amino acid residues in length.
  • the polypeptide is at least, about, or no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or similar to any one of the polypeptides whose specific sequence is disclosed herein (SEQ ID NOs: 1 through 16), by at least one method, provided that the polypeptide is not a fragment of the SARS Cov2 S protein in that it comprises at least different amino acid residue in sequence and/or chemical modification.
  • a variant of a parent peptide as defined herein comprises: a) one or more (e.g. 1 , 2, or 3) amino acid substitutions in the sequence of the parent peptide as defined herein, for example a glutamate residue at the N-terminus of the parent peptide as defined herein may be replaced with pyroglutamate and/or one or more cysteine residues in the parent peptide as defined herein may be replaced with serine or 2-aminobutyric acid; and/or b) one or more amino acid additions (e.g.
  • variants 1 , 2, 3, 5, 4, 6, 7, 8 to the sequence of a parent peptide as defined herein , for example wherein the variant comprises one or more (e.g. 1 , 2, 3, or 4) lysine residue(s) and/or one or more (e.g.
  • a variant of a parent peptide as defined herein may comprise one, two, three or more lysine or arginine amino acid residue(s) added to the N- and/or C-terminus of the parent peptide as defined herein that have been extended with one or more, e.g. 1 , 2, 3, 4, or 5 amino acid residues.
  • a parent peptide as defined herein may be modified to contain "non-natural" modifications. Such peptides are also referred to as variants herein and more specifically they are referred to as derivative peptides or derivatives.
  • the term derivative refers to a chemically modified form of a peptide disclosed herein. Typically, a derivative is formed by reacting a functional side group of an amino acid (e.g. amino, sulfhydryl or carboxy- group) with another molecule to form a covalent or non- covalent attachment of any type of molecule (naturally occurring or designed), such as a sugar moiety.
  • an amino acid e.g. amino, sulfhydryl or carboxy- group
  • a covalent or non- covalent attachment of any type of molecule (naturally occurring or designed), such as a sugar moiety such as a sugar moiety.
  • Specific examples of derivatives of a peptide include glycosylation, acylation (e.g.
  • acetylation phosphorylation
  • amidation formylation
  • ubiquitination phosphorylation
  • derivatization by protecting/blocking groups and any of numerous chemical modifications.
  • Additional specific non- limiting examples are tagged peptides, fusion peptides, chimeric peptides including peptides having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide.
  • a derivative comprises one or more modifications, for example selected from any of: (a) N-terminal acylation (e.g. acetylation or formylation); (b) C-terminal amidation (e.g.
  • the peptides are amidated at the C-terminal end.
  • Peptides are typically provided in the form of a salt, for example as a pharmaceutically acceptable and/or a physiologically acceptable salt.
  • the salt may be an acid addition salt with an inorganic acid, an acid addition salt with an organic acid, a salt with a basic inorganic acid, a salt with a basic organic acid, a salt with an acidic or basic amino acid or a mixture thereof.
  • an acid addition salts with an inorganic acid are selected from any of the salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, or the like.
  • An acid salt with an organic acid may be selected from any of the salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or the like.
  • Salts with an inorganic base may be selected from a salt of an alkali metal salts such as sodium salts and potassium salts; alkali earth metal salts such as calcium salts and magnesium salts; and aluminum salts and ammonium salts.
  • Salts with a basic organic base may be selected from any salt with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triet hanolamine, dicyclohexylamine, N,N-dibenzylethylenediamine, caffeine, piperidine, and pyridine.
  • Salts with a basic amino acid may be selected from any salt with arginine, lysine, ornithine, or the like.
  • Salts with an acidic amino acid may be selected from any salt with aspartic acid, glutamic acid, or the like.
  • the peptide is a TFA salt.
  • An isolated and purified peptide of the disclosure and fragments and variants thereof may be produced synthetically or recombinantly.
  • a polypeptide that contains an epitope that induces an immune response against coronavirus may be synthesized by standard chemical methods, including synthesis by automated procedure.
  • the polypeptide may be produced recombinantly.
  • the polypeptide may be expressed from a polynucleotide that is operably linked to an expression control sequence, such as a promoter, in a nucleic acid expression construct.
  • the polypeptide may be expressed in mammalian cells, yeast, bacteria, insect or other cells under the control of appropriate expression control sequences.
  • Cell-free translation systems may also be employed to produce such coronavirus proteins using nucleic acids, including RNAs, and expression constructs.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are routinely used by persons skilled in the art, and may include plasmids, cosmids, shuttle vectors, viral vectors, and vectors comprising a chromosomal origin of replication as disclosed therein.
  • a nucleotide sequence encoding a coronavirus S polypeptide or variant thereof may differ from the sequences presented herein due to, for example, the degeneracy of the genetic code.
  • a nucleotide sequence that encodes a coronavirus polypeptide variant includes a sequence that encodes a homologue or strain variant or other variant. Variants may result from natural polymorphisms or may be synthesized by recombinant methodology, for example to introduce an amino acid mutation, or chemical synthesis, and may differ from wild-type polypeptides by one or more amino acid substitutions, insertions, deletions, and the like. [00213] The current disclosure also provides nucleic acid molecules encoding any one or more of the polypeptides disclosed herein.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA or modified RNA.
  • the nucleic acid sequence is codon optimized to improve expression of the polypeptide.
  • the disclosure provides vectors comprising the nucleic acids of the disclosure.
  • the vector is an expression vector.
  • the vector is a retroviral vector, lentiviral vector, adenoviral vector, poxvirus vector, plasmid, bacterial vector.
  • the vector is a recombinant vaccinia virus.
  • the disclosure provides recombinant host cells comprising a nucleic acid and/or vector of the disclosure.
  • the recombinant host cell is selected from a prokaryotic cell (e.g. E. coli) or an eukaryotic cell (e.g.
  • CHO Chinese hamster ovary
  • myeloma cell e.g.,Y0, NS0, Sp2/0
  • COS-7 monkey kidney cell
  • COS-7 monkey kidney cell
  • BHK baby hamster kidney cell
  • TM4 mouse Sertoli cell
  • VERO-76 African green monkey kidney cell
  • HELA human cervical carcinoma cell
  • W138 human lung cell
  • Hep G2 human liver cell
  • TRI cell a mouse mammary tumor cell
  • TRI cell a MRC 5 cell
  • a FS4, and MDCK cell a mouse mammary tumor cell.
  • the disclosure provides immunogenic compositions that are useful in the prevention or treatment of severe acute respiratory syndrome (SARS) or other SARS-CoV-related disease, including Covid-19.
  • the immunogenic composition comprises one or more of the isolated or purified polypeptides disclosed in this disclosure, including, for example, Peptides (1) through (7), Peptides (8) through (14), fragments, variants, and/or derivatives of the same.
  • the composition may comprise one or more of any other polypeptides described in this disclosure.
  • an “immunogenic composition,” as used in the present disclosure refers to a composition that comprises an immunogenic component capable of provoking an immune response in an individual, such as a human, or an animal (such as a mouse or a rat), optionally when suitably formulated with an adjuvant.
  • the disclosure provides an immunogenic composition comprising an immunogenic SARS coronavirus S (spike) polypeptide, or a fragment or variant thereof, and an adjuvant.
  • the immunogenic composition of the disclosure is a vaccine, i.e. the immunogenic composition is capable of provoking a protective immune response against a SARS-CoV infection.
  • the immunogenic composition of the present disclosure comprises one or more immunogenic SARS coronavirus S (spike) polypeptides, including fragments and variants thereof.
  • the immunogenic S polypeptides may comprise any portion of an S protein that has an epitope capable of eliciting an immune response, for example an epitope capable of eliciting production of a neutralizing antibody and/or stimulating a cell-mediated immune response, against a SARS-CoV infection.
  • the immunogenic composition is a vaccine.
  • a “vaccine” is an immunogenic composition capable of provoking a protective immune response against a SARS-CoV infection.
  • epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope.
  • different antibodies may bind to different areas on an antigen and may have different biological effects.
  • epitope also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody.Exemplary epitopes identified in this disclosure can be found at FIG.
  • Induction of an immune response in a subject or host (human or non-human animal) by a SARS-CoV S polypeptide, fragment, or variant described herein may be determined and characterized by methods described herein and routinely practiced in the art. These methods include in vivo assays, such as animal immunization studies, for example, using a rabbit, mouse, ferret, civet cat, African green monkey, or rhesus macaque model, and any one of a number of in vitro assays, such as immunochemistry methods for detection and analysis of antibodies, including Western immunoblot analysis, ELISA, immunoprecipitation, radioimmunoassay, and the like, and combinations thereof.
  • in vivo assays such as animal immunization studies, for example, using a rabbit, mouse, ferret, civet cat, African green monkey, or rhesus macaque model
  • immunochemistry methods for detection and analysis of antibodies including Western immunoblot analysis, ELISA, immunoprecipit
  • neutralization assays such as a plaque reduction assay or an assay that measures cytopathic effect (CPE) or any other neutralization assay practiced by persons skilled in the art.
  • CPE cytopathic effect
  • S protein immunogens and variants thereof that have at least one epitope that elicits a protective humoral or cell-mediated immune response against SARS coronavirus.
  • the statistical significance of the results obtained in the various assays may be calculated and understood according to methods routinely practiced by persons skilled in the relevant art.
  • the SARS-CoV-2 polypeptide is a peptide selected from the following:
  • Peptide (2) 442- CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ -506 (SEQ ID NO: 2)
  • Peptide (3) 469-SGSTEIYQAGSTPCNGVEGFNCYFAK-490 (SEQ ID NO: 3)
  • Peptide (4) 483-CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE-516 (SEQ ID NO: 4)
  • the SARS-CoV-2 polypeptide is a peptide selected from the following:
  • Peptide (5) 804-QILPDPSKPSKRSC-816 (SEQ ID NO: 5)
  • Peptide (6) 919-CNQKLIANQFNSAIGKIQDSLSS-940 (SEQ ID NO: 6)
  • Peptide (7) 695-CYTMSLGAENSVAYS -708 (SEQ ID NO: 7):
  • Peptide (8) 438-CSNNLDSKVGGNYN-450, SEQ ID NO: 83;
  • Peptide (9) 496- CDGFQPTNGVGYQPYR-509, SEQ ID NO: 84;
  • Peptide (10) 454-CDRLFRKSNLKPFE-465, SEQ ID NO: 85;
  • Peptide (13) 403-CSRGDEVRQIAPGQTGK-417, SEQ ID NO: 88;
  • Peptide (14) 481 -CDNGVEGFNGYFPLQSYG-496, SEQ ID NO: 89; Peptides
  • the peptide comprises, consists of, or consists essentialy of one or more of the following sequences:
  • Peptide (4A) 483-VEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFEC-
  • Peptide (2E) DSKVGGNYNYLYRXLQSYGFQPTNGVGYQC, SEQ ID NO:
  • Peptide (4B) 483-VEGFNXYFPLQSYGFQPTNGVGYQPYRVVVLSFE-
  • NNLDSKVGGNYNSC SEQ ID NO: 160
  • NNLDSKVGGNYNSDC SEQ ID NO: 161
  • CDGRLFRKSNLKPFE SEQ ID NO: 167
  • RGDEVRQIAPGQTGKSDC SEQ ID NO: 172
  • NGVEGFNGYFPLQSYGDC SEQ ID NO: 173
  • CSYTMSLGAENSVAYS SEQ ID NO: 174
  • CDSYTMSLGAENSVAYS (SEQ ID NO: 175)
  • GFQPTNGVGYQPHR SEQ ID NO: 190
  • NGVEDFNGYFPLQSYG SEQ ID NO: 191
  • NGVKDVNGYFPLQSYG (SEQ ID NO: 193)
  • the composition comprises one or more fragments, variants, or derivatives of any one of Polypeptides (1 ) through (7).
  • the composition comprises one or more immunogenic SARS coronavirus S (spike) polypeptides consisting of Peptides (1) through (7), with or without an additional Cys residue at either the N- or C- terminus, and an adjuvant.
  • the composition comprises two of the listed polypeptides described herein. For example, any combination of peptide (1) and (2), (1) and (3), (1 ), and (5), 1 and (6), (1) and (7), (2) and (3), and any other one of the 21 possible two-peptide combinations of the seven listed peptides is within the scope of the disclosure.
  • the composition comprises three polypeptides described herein. Exemplary combinations include that combination of peptides (1), (2) and (3); (1 ), (2) and (4); (1), (3), and (4); and (2), (3), (4)) and any other one of the 35 combination of three peptides that may be made with the seven specifically listed peptides in one embodiment, the composition comprises four of the peptides described herein in one embodiment, the composition comprises five, six, seven, eight, nine, ten, or any other combination of the peptides disclosed herein. Peptide combinations where one or more peptides have been replaced by modified forms of the same peptides (variants) are also included in this disclosure.
  • polypeptides are linked to one another, directly or via a linker.
  • polypeptides exist in the composition as separate peptides.
  • an adjuvant or an adjuvant component in the broadest sense is typically a (e.g. pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine.
  • a (e.g. pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine.
  • the term refers in the context of the disclosure to a compound or composition that serves as a carrier or auxiliary substance for immunogens and/or other pharmaceutically active compounds.
  • the term is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or co-administered with an adjuvant in question.
  • an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present disclosure.
  • an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines.
  • an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent.
  • an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
  • adjuvant or “adjuvant component” has the same meaning and may be used mutually.
  • Adjuvants may be divided, e.g., into immunopotentiators, antigenic delivery systems or even combinations thereof.
  • the adjuvant is selected from the group consisting of mineral substances, aluminum hydroxide, aluminum phosphate, bacterial extracts (e.g., bacterial liposaccharides, Freund's adjuvants, and/or MDP), oily emulsions, saponin, squalene, potassium aluminium sulfate, calcium phosphate hydroxide, a TLR agonist, CRM197, Montanide®, Freund's incomplete adjuvant, MF59, a CpG oligonucleotide, iscoms, iscom matrix, ISCOMATRIXTM adjuvant, Matrix MTM adjuvant, Matrix CTM adjuvant, Matrix QTM adjuvant, AblSCQ®-100 adjuvant, AblSC()®-300 adjuvant, ISCOPREPTM, an ISCOPREPTM derivative, adjuvant containing I
  • the immunogenic composition comprises in addition to the peptide combination, therapeutically inactive ingredients, such as a pharmaceutically acceptable or physiologically acceptable excipient and/or carrier, which are well-known to the person skilled in the art and may include, but are not limited to, solvents, emulsifiers, wetting agents, plasticizers, solubilizers (e.g. solubility enhancing agents) coloring substances, fillers, preservatives, anti-oxidants, anti-microbial agents, viscosity adjusting agents, buffering agents, pH adjusting agents, isotonicity adjusting agents, mucoadhesive substances, and the like. Examples of formulation strategies are well- known to the person skilled in the art).
  • the immunogenic composition may be formulated for parenteral administration, such as formulated for injection, e.g. subcutaneous and/or intradermal injection. Therefore, in some embodiments, the immunogenic composition may be a liquid (i.e. formulated as a liquid), including a solution, a suspension, a dispersion, and a gelled liquid.
  • a liquid immunogenic composition may be formed by dissolving a powder, granulate or lyophilizate of a peptide combination described herein in a suitable solvent and then administering to a subject.
  • Suitable solvents may be any solvent having physiologically acceptable properties and able to dissolve the peptide combination in desired concentrations.
  • a desired concentration may depend on the aliquot to be administered (i.e. to be injected) and the desired single dose.
  • the aliquot is in the range of about 10 to 500 microliters, e.g. 50 to 300 microliters or less and a desired single dose is within range of 1 to 1000 nanomole. Therefore, a suitable solvent should be able to dissolve any peptide of the combination to achieve a final concentration of about 1 to 1000 mM for each of the peptides.
  • a liquid composition comprises each of the peptides of the combination in a concentration of 10 to 800 pM, for Example 20 to 500 pM or 20 to 300 pM.
  • the concentration of each peptide is the same, such as in an equimolar concentration, but each peptide of the composition may be present in different concentrations.
  • the solvent is an aqueous solution, optionally mixed with other solvents.
  • a solvent may comprise at least 60% w/w of water, e.g. at least 65% w/w, 70% w/w, 75% w/w, 80% w/w , 85% w/w, 90% w/w or 95% w/w, 99% w/w of water, such as distilled water, such as sterile water.
  • the solvent is sterile distilled water, e.g. water for injection.
  • An aqueous solution may comprise other solvents than water, for example DMSO (dimethylsulfoxide), glycerol, ethanol, acetonitrile, vegetable or synthetic oils.
  • DMSO dimethylsulfoxide
  • glycerol glycerol
  • ethanol glycerol
  • acetonitrile vegetable or synthetic oils.
  • the pH of the aqueous phase of the solvent may be in a physiological acceptable range, typically in the range of 3 to 9, such as in the range of pH 3 to 8, such as in the range of pH 4 to 8, such as in the range of 5 to 8, such as in the range of 6 to 8.
  • the liquid formulation may comprise a pH controlling agent or buffering agent
  • the pH may be adjusted with dilutions of strong base (e.g. sodium hydroxide or the like) and/or dilutions of strong acids (e.g. hydrochloric acid).
  • strong base e.g. sodium hydroxide or the like
  • strong acids e.g. hydrochloric acid
  • the liquid formulation is isotonic, and optionally sterile. Therefore, in some embodiments, the formulation comprises saline, such as isotonic saline.
  • the liquid may contain additional excipients, such as another solvent, a solubilizing enhancing agent (e.g. polyoxyethylene (20) sorbitan monolaurate (Tween® 20), ionic and non-ionic emulsifiers (e.g.
  • Non- limiting illustrative examples of solvents include water, saline, DMSO, glycerol, ethanol, acetonitrile, vegetable or synthetic oil.
  • a immunogenic composition may be formulated to contain only a limited amount of water or aqueous solution, e.g. containing less than 10% w/w of water or aqueous solution, such as less than 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5% w/w of water or aqueous solution.
  • aqueous solution e.g. containing less than 10% w/w of water or aqueous solution, such as less than 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5% w/w of water or aqueous solution.
  • immunogenic compositions with limited levels of water may include granulates, powders, for example lyophilizates, i.e. freeze-dried powders.
  • the freeze-dried composition may be dissolved before use, for example dissolved in an aqueous, optionally sterile, solution, for example a solution having a pH in the range of 3-9, such as pH in the range of 3 to 8, such as pH in the range of 4 to 8.
  • an aqueous, optionally sterile, solution for example a solution having a pH in the range of 3-9, such as pH in the range of 3 to 8, such as pH in the range of 4 to 8.
  • a lyophilizate may contain additional ingredients, e.g. bulking agents and lyoprotectants e.g. sucrose, lactose, trehalose, mannose, mannitol, sorbitol, glucose, raffinose, glycine, histidine or mixtures thereof), buffering agents (e.g. sodium citrate, sodium phosphate, disodium phosphate, sodium hydroxide, Tris base, Tris acetate, Tris HCI or mixtures thereof), antioxidants, antimicrobial agents, solubilizers (e.g. polyoxyethylene (20) sorbitan monolaurate (Tween® 20))
  • buffering agents e.g. sodium citrate, sodium phosphate, disodium phosphate, sodium hydroxide, Tris base, Tris acetate, Tris HCI or mixtures thereof
  • antioxidants e.g. polyoxyethylene (20) sorbitan monolaurate (Tween® 20)
  • kits comprising a compartment and instructions, wherein the compartment comprises a immunogenic composition as described herein for single, sequential or simultaneous administration, and wherein the instructions are for use in treating allergy to grass, such as grass.
  • a kit may further comprise packaging material comprising corrugated fiber, glass, plastic, foil, ampules, vials, blister pack, preloaded syringes or tubes, optionally that maintain sterility of the components.
  • a kit may further comprise labels or inserts comprising printed matter or computer readable medium optionally including identifying components, dose amounts, clinical pharmacology and instructions for the clinician or for a subject using one or more of the kit components, prophylactic or therapeutic benefits, adverse side effects or manufacturer information.
  • the kit additionally comprises a container comprising a solvent for dissolving the composition before use. Examples of suitable solvents are described supra.
  • the kit may also comprise a device for use in parenteral injection, e.g. for injecting the composition (e.g. dissolved composition) to a subcutaneous or intradermal tissue.
  • a device may be any suitable device for that purpose, such as a needle or microneedle adapted for intradermal or subcutaneous delivery of the composition.
  • the device may be a microneedle or a device comprising a plurality of microneedles designed for intradermal delivery of liquids.
  • the kit comprises a means for delivery of the immunogenic composition via inhalation.
  • compositions comprising one of those antibodies and, optionally, an adjuvant. These antibodies may be used alone or in any of the other combinations described herein, including combination with a polypeptide. The combination may be in the same composition. The use of the combination may be separate administrations. In one embodiment, the antibodies are used as diagnostic agents.
  • the antibodies are used method of preventing, treating or ameliorating at least one symptom of SARS-Cov infection, or of decreasing the frequency or severity of at least one symptom of SARS-Cov infection, the method comprising administering an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to the S1 or S2 subunit of SARS-Cov2 S protein prepared using one or more of the polypeptides of the disclosure.
  • the term “recombinant”, as used herein, refers to antibodies or antigen binding fragments thereof of the invention created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression.
  • the term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.
  • the antibody or the polypeptide of the disclosure is used to detect a SARS-associated coronavirus infection.
  • the detection is carried out by ELISA.
  • the disclosure also provides a SARS-Cov associated coronavirus detection kit comprising an antibody according to the disclosure.
  • the antibodies of the disclosure can be full-length (for example, an lgG1 or lgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab')2 or scFv fragment), and may be modified to affect functionality, e.g., to increase persistence in the host or to eliminate residual effector functions.
  • an antigen-binding portion for example, a Fab, F(ab')2 or scFv fragment
  • the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to SARS-Cov and/or a SARS-Cov S protein, wherein the antibody or antigen-binding fragment thereof neutralizes SARS-Cov in vitro with an IC50 less than or equal to 10-9M and wherein the antibody or antigen-binding fragment thereof demonstrates a protective effect in vivo in a SARS-Cov infected animal.
  • the antibodies of the disclosure are neutralizing antibodies.
  • neutralizing antibody as used herein (or an “antibody that neutralizes SARS-Cov2 activity” or “antagonist antibody”), is intended to refer to an antibody whose binding to SARS-Cov2 results in inhibition of at least one biological activity of SARS-Cov2.
  • an antibody of the invention may prevent or block SARS-Cov2 attachment to, fusion with, and/or entry into a host cell.
  • a “neutralizing antibody” is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host.
  • neutralizing antibody and “an antibody that neutralizes” or “antibodies that neutralize” are used interchangeably herein. These antibodies can be used, alone or in combination, as prophylactic or therapeutic agents with other anti-viral agents upon appropriate formulation, or in association with active vaccination, or as a diagnostic tool.
  • the subject in need thereof may be administered at least one anti-SARS-Cov S protein antibody of the invention or an antigen-binding fragment thereof, or a pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof of the invention in combination with a second therapeutic agent.
  • the second therapeutic agent may be selected from the group consisting of an anti-viral drug, an anti-inflammatory drug (such as corticosteroids, and non-steroidal anti-inflammatory drugs, such as antibodies to TNF, a different antibody to SARS-Cov, a vaccine for SARS-Cov, and/or interferons (alpha/beta/or lambda).
  • an anti-viral drug such as corticosteroids, and non-steroidal anti-inflammatory drugs, such as antibodies to TNF, a different antibody to SARS-Cov, a vaccine for SARS-Cov, and/or interferons (alpha/beta/or lambda).
  • S protein immunogens, fragments, and variants thereof described herein contain an epitope that elicits or induces an immune response, for instance a protective immune response, which may be a humoral response and/or a cell-mediated immune response.
  • a protective immune response may be manifested by at least one of the following: preventing infection of a host by a coronavirus; modifying or limiting the infection; aiding, improving, enhancing, or stimulating recovery of the host from infection; and generating immunological memory that will prevent or limit a subsequent infection by a SARS coronavirus.
  • the protective immune response may be assessed for instance by the viral load in lungs and upper respiratory tract, the score of pulmonary inflammation and lesions, the scores of viral antigen loads in lungs, the presence of neutralizing antibodies, the CD4+ T cell responses in PBMC, spleen and the cytokine secretion from spleen.
  • a humoral response may include production of antibodies that neutralize infectivity, lyse the virus and/or infected cell, facilitate removal of the virus by host cells (for example, facilitate phagocytosis), and/or bind to and facilitate removal of viral antigenic material.
  • a humoral response may also include a mucosal response, which comprises eliciting or inducing a specific mucosal IgA response.
  • the host is a human.
  • the disclosure provides method of preventing or treating severe acute respiratory syndrome or other SARS-CoV-related disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one or more of the polypeptides of the disclosure.
  • the efficacy is measured by measuring a reduced viral load or delays or prevention of a further increase in viral load.
  • efficacy is measured by an elimination, reduction, or decrease in the intensity or frequency of one or more symptoms associated with SARS such as fever, cough, shortness of breath, organ failure (e.g. kidneys) and/or septic shock.
  • the subject has an underlying disorder such as diabetes, cancer, chronic lung disease, or generally weakened immune system.
  • the SARS is Covid-19.
  • the immunogenic composition induces a humoral response, such as an increased level of neutralizing antibodies associated with the subject administered the immunogenic composition as compared to a subject not administered the immunogenic composition.
  • the immunogenic composition induces a cellular immune response, such as a CD8+T cell response, (including the production of cytokines such as interferon- gamma (TFN-g), tumor necrosis factor alpha (TNF-alpha), interleukin- 2 (IL-2), or any combinations thereof.
  • the immunogenic composition is a vaccine.
  • the therapeutically effective amount is prophylactic.
  • the immunogenic composition or polypeptide is administered by any route typically used for vaccination, including topical, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, inhalation, or combinations thereof.
  • the immunogenic composition or polypeptide is administered in a single-dose vaccination schedule.
  • each vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and the type and amount of adjuvant used. An optimal amount for a particular vaccine may be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Generally, it is expected that each dose will comprise 1-2000 or 1-1000pg of protein, for example 1- 200pg, or 10-100pg. This amount may relate to the peptide with or without any modification. An exemplary dose will contain 10-50pg, for example 15- 25 pg, suitably about 20pg of protein.
  • the dose may contain 40 pg or 160 pg of each peptide, conjugated to approximately 300 pg of KLFI (conjugated to KLH), or a total of 160 pg of peptide plus 1200 pg of KLFI, adding up to a total dose of 1360 pg of protein per dose, when the vaccine comprises a combination of four peptides.
  • a "dose-sparing" approach may be used, for example in a pandemic situation. This is based on the finding that it is possible to provide the same protective effect using lower doses of antigen, due to the presence of an effective adjuvant.
  • each human dose may contain a significantly lower quantity of polypeptide, for example from 0.1 to 10pg, or 0.5 to 5pg, or 1 to 3pg, suitably 2pg polypeptide per dose.
  • human dose is meant a dose which is in a volume suitable for human use. Generally, this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml.
  • subjects typically receive a boost after a 2 to 4-week interval, for example a 3 week interval, optionally followed by repeated boosts for as long as a risk of infection exists.
  • a single-dose vaccination schedule is provided, whereby one dose of S protein polypeptides in combination with adjuvant is sufficient to provide protection against the SARS CoV2, without the need for any boost after the initial vaccination.
  • the vaccination schedule includes more than one dose, and each dose may be different.
  • the repeated doses are administered a week apart or less, between more than a week apart and a month apart or less; more than a month apart; more than 2 months apart, more than 3 months apart and other monthly regimens as suited.
  • the method further comprises the administration of another treatment for SARS or other coronavirus-related disease or disorder.
  • the method further comprises the administration of remdesivir, azithromycin, hydroxychloroquire, chloroquine, or combinations thereof.
  • the method further comprises the administration of hydroxychloroquine combination (200 mg X 3 per days for 10 days) with Azithromycin (500 mg on the 1st day then 250 mg per day for 5 more days).
  • the treatment with the polypeptides, nucleic acids, vectors, and immunogenic compositions of the disclosure is combined with one of the following:
  • Anti-inflammatory therapy such as tocilizumab and favipiravir, tocilizumab, sarilumab, leronlimab, rintatolimod, BPI-002, REGN3048 and REGN 3051 , monoclonal antibody designed to bind SARS-CoV-2;
  • Anti-Viral therapy such as remdesivir, lopinavir and ritonavir, danoprevir and ritonavir, favipiravir, darunavir and cobicistat, umifenovir, galidesivir, linebacker and equivir, compounds that inhibit the virus interaction with the receptor ACE2;
  • RNA vaccines e.g., targeting Spike protein
  • DNA vaccines e.g., targeting Spike protein
  • recombinant protein vaccines e.g., targeting Spike protein
  • viral-vector based vaccines e.g., targeting Spike protein
  • live attenuated vaccines e.g., targeting the whole virion
  • inactivated vaccines e.g., targeting the whole virion
  • subunit vaccine e.g., targeting the whole virion
  • peptide vaccines e.g., targeting Spike protein
  • the disclosure is directed to the use of the immunogenic compositions of the disclosure for the manufacture of a medicament for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
  • an immunogenic composition of the disclosure for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
  • the disclosure is related to methods of using the polypeptides and/or antibodies of the disclosure as diagnostic agents in diagnostic methods.
  • the polypeptides and/or antibodies of the disclosure are used in the detection of coronavirus-related diseases and viruses such as those in SARS and Covid-19.
  • the polypeptides are labeled for detection by immunohistochemisty, ELISA, and other methods known in the art for using polypeptides in diagnostic methods.
  • the polypeptides and/or antibodies of the disclosure are used to identify other immunogenic polypeptides and antibodies that bind to the same region of the S protein or to a complex of the S protein with the ACE2 protein at the surface of target cells.
  • the polypeptides and/or antibodies of the disclosure are used in competition assays.
  • Immunogenic peptide SEQ ID NO:1 (CGSGDSKVGGNYNKLYRLFE) is derived from the ACE2-contacting surface of the S1 subunit (SARS-CoV-2 Spike protein aa 442-456).
  • the peptide contains the contact residues Y449, Y453, L455 and F456.
  • GSG linker sequence was added to the N terminus. Additional changes as lysine (K) instead of tyrosine (Y) at position 451 and C-terminal glutamic acid (E) were added with the aim to stabilize the peptide structure in a conformation similar to that present in the native S protein.
  • N-terminal cysteine (C) was used for conjugation of the peptide to the KLH carrier protein.
  • C N-terminal cysteine
  • the peptide sequence was analyzed by BepiPred-2.0 server (FIG.1A, 1 B).
  • the designed peptide contains one B cell epitope located at the N-terminal part of the peptide.
  • CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ (aa 442-506) of the Spike protein was derived from the parts of the ACE2-contacting surface of the S1 subunit.
  • the peptide was designed by fusing two peptides from of the S1 glycoprotein, aa 442-454 which include the contact residues Y449 and Y453, and aa 492-506 which include the contact residues Q493, T500, N501 , G502 and Y505.
  • the immunogenic peptide SEQ ID NO:3 (SGSTEIYQAGSTPCNGVEGFNCYFAK) was derived from the surface of the S1 subunit contacting ACE2 receptor, amino acids 469-490. Designed peptide spans the ACE2 contact site with the following contact residues: A475, F486, N487, Y489 and Q493.
  • A Alanine
  • Lysine K
  • This modification can generate a compact structure by contacting the added Lysine (K) with Glutamic acid (E) at position 471.
  • N-terminus of the peptide is modified (e.g. by a chemoselective reactive group like azide which can bind selectively to alkyne or phosphine) and is used for conjugation to KLH. Because of the better accessibility of the B-cell epitope to the immune system, the short SG sequence was used as a spacer. Alternatively, an N-terminal or C-terminal Cys may be added for conjugation to KLH, with or without the N-terminal spacer.
  • the immunogenic peptide SEQ ID NO:4 CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE was derived from the ACE2- contacting surface of the S1 subunit, amino acids 483-516.
  • the peptide contains the following ACE2 contact sites: F486, N487, Y489, Q493, T500, N501 , G502 and Y505.
  • Internal Cysteine (C) at position 488 is modified to Alanine (A) to prevent dimerization, which will change the 3D peptide structure.
  • N-terminal cysteine (C) is used for conjugation to KLFI.
  • the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 7A, 7B).
  • the designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
  • Immunogenic peptide SEQ ID NO:5 QILPDPSKPSKRSC was derived from the surface of the S2 subunit, aa 804-816 C-terminal cysteine (C) is used for conjugation to KLFI.
  • the antibodies induced against the peptide should prevent access of the protease to the S2’ cleavage site, and thus prevent a conformational change to induce ‘fusion peptide’ interaction with the cellular membrane.
  • the peptide SEQ ID NO:6 CNQKLIANQFNSAIGKIQDSLSS was derived from the surface residues of the S2 subunit of the SARS-CoV-2 spike glycoprotein, amino acids 919-940. N-terminal cysteine (C) is used for conjugation to KLFI.
  • C N-terminal cysteine
  • the peptide drives production of antibodies against the S2 subunit of the S protein, that should inhibit its conformational changes and prevent fusion of the virus with the plasma membrane of a host cell.
  • the peptide will be used in combination with peptides derived from the S1 subunit.
  • EXAMPLE 7 THE IMMUNOGENIC PEPTIDE (7) SEQ ID NO:7
  • the immunogenic peptide SEQ ID NO: 7 CYTMSLGAENSVAYS was derived from the S2 subunit using the amino acid residues 695-708, which are on the surface of the protein. N-terminal cysteine (C) is used for conjugation to KLFI.
  • the vaccine containing the peptide conjugated to KLFI can induce antibodies against S2 subunit of S protein, that should inhibit its conformational changes and prevent fusion of virus with plasma membrane of host cell.
  • the peptide will be used in combination with peptides derived from the S1 subunit.
  • the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 13A, 13B).
  • the designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
  • EXAMPLE 8 Peptide vaccine preparation, immunization and analysis of humoral and cellular immune response.
  • the designed peptides SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 are conjugated to keyhole limpet hemocyanin (KLH) via a cysteine link or azide-phosphine link.
  • KLH keyhole limpet hemocyanin
  • tau peptides are synthetized as cysteinated peptides with an extra N-terminally located cysteine residue or N-terminal azide with the aim to obtain oriented attachment of the peptide on the surface of the KLH protein.
  • Peptides are coupled to the KLH carrier via bifunctional crosslinker N-[y-maleimidobutyryloxy]succinimide ester (GMBS) or NHS- Phosphine (Thermo Fisher Scientific).
  • conjugation buffer PBS with 0.9 M NaCI, 10 mM EDTA
  • conjugation buffer PBS with 0.9 M NaCI, 10 mM EDTA
  • 2 mg of active bi-functional cross-linker GMBS or NHS-Phosphine are dissolved in 50 pi of anhydrous dimethylformamide and mixed with 2 ml of KLH solution for 1 hour at room temperature.
  • un-reacted GMBS and NHS-Phosphine are removed on a 5 ml HiTrap Desalting column (GE Healthcare) equilibrated in conjugation buffer.
  • Conjugations are carried out at a 1 :1 ratio of peptide to activated KLH (w/w, 20 mg of peptide) for 2-16 h at room temperature (25°C).
  • the resulting conjugates are dialyzed against a 100-fold excess of PBS, with four dialysis buffer changes to remove unconjugated peptide. After dialysis, the conjugates are centrifuged at 21 ,000xg for 15 min at 2°C. Completeness of conjugation was confirmed by the absence of free peptide in the dialysis buffer, measured using LC-MS/MS.
  • the conjugates are aliquoted and stored at -20°C until used.
  • spleens After excision of spleens from the mice, single-cell suspension is prepared using teflon/glass homogenizer. Erythrocytes are precipitated in culture medium (DMEM containing 10% horse serum, 1 mM glutamine and 50 pg/ml gentamycin), bigger particles including pieces of spleen tissue and erythrocytes aggregates are let to settle down for 1 minute by gravity in 15 ml falcon tubes and supernatant is carefully removed.
  • culture medium DMEM containing 10% horse serum, 1 mM glutamine and 50 pg/ml gentamycin
  • Splenocytes are seeded at density 8x10 6 cells/well (in 24-well plates and 1 ml of culture medium) and stimulated in vitro with peptide (10 mM), peptide conjugated to KLH (200 pg/ml), S protein (20pg/ml) or concanavalin A (5 pg/ml) for 72 hours. Subsequently, protein transport inhibitor Brefeldin A (GolgiPlug, BD Biosciences) is added to cells according to manufacturer ' s instructions. Five hours later, cells are centrifuged for 10 min at 300g. Cell culture supernatants are collected, stored at -80°C for further cytokine’s detection and splenocytes are used for flow cytometry analysis.
  • peptide 10 mM
  • peptide conjugated to KLH 200 pg/ml
  • S protein 20pg/ml
  • concanavalin A 5 pg/ml
  • protein transport inhibitor Brefeldin A Gol
  • cells are washed with PBS and stained with antibodies against activated central memory T cell surface markers (CD4-Alexa Fluor 700, CD44- BV510, CD62L-PECy7, CCR7-APC and CD69-BV421) for 45 min at room temperature. Then, cells are washed, fixed in BD FACS Lysing Solution containing formaldehyde for 10 min, permeabilized in PBS containing 0,5% Triton X-100 for 20 min, split into two separate tubes and stained for intracellular cytokines (IL2-PE-CF594, IL10-PE and IL4- PE-CF594, IFNy-PE).
  • activated central memory T cell surface markers CD4-Alexa Fluor 700, CD44- BV510, CD62L-PECy7, CCR7-APC and CD69-BV4211
  • Activated B cells are measured using markers CD19 (for detection of B cells), CD138 (which is negative for all developmental stages of B cells except of plasmablasts and plasma cells), IgM and IgD (which are immunoglobulins produced by activated B cells) and CD69 and CD86 (markers of activation). Additionally, memory B cells are detected using additional marker CD62L, CD27 and immunoglobulins IgG, IgM (positive markers) and IgD (negative marker). Finally, cells are fixed, resuspended in PBS and analyzed immediately on LSRFortessaTM flow cytometer (BD Biosciences). For each analysis, 100 000 cells are acquired and cells expressing individual markers are gated according to fluorescence- minus- one (FMO) controls. Facs Diva software is used for data analysis.
  • FMO fluorescence- minus- one
  • EXAMPLE 9 In vitro tests of neutralizing activity of serum antibodies elicited against the SARS-CoV-2 spike protein peptide vaccines.
  • the recombinant receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S) (residues 319-591 , NCBI Reference Sequence: NC_045512.2) is fused with C-terminal HRV3C protease cleavage site, followed by an 8XhisTag (SEQ ID NO: 39) and a TwinStrepTag and.
  • S protein The entire ectodomain of S protein (residues 1-1208) is expressed with the mutations that stabilize the protein in its prefusion conformation: residues K986 and L987 are changed to prolines, the S1/S2 cleeavage site 683-RRAR- 686 (SEQ ID NO: 40) is replaced with the sequence GSAS (SEQ ID NO: 41).
  • the S protein is cloned with C-terminal T4 bacteriophage fibritin trimerization sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO. 206] , HRV3C protease cleavage site followed by a TwinStrepTag and an 8XhisTag (SEQ ID NO: 39).
  • Human ACE2 protein (residues 1-615, NCBI Reference Sequence: NP_001358344.1) is fused with a C- terminal HRV3C protease cleavage site followed by a TwinStrepTag and an 8XhisTag (SEQ ID NO: 39).
  • the proteins are cloned into mammalian expression vector under the control of the CMV promoter with the human Ig kappa light chain leader sequence (GenBank: MN197520.1 ) to drive their export into the media.
  • the plasmids amplified in E. coli are transfected into FreeStyle 293-FTM cells (Thermo Fisher Scientific) using the FreeStyleTM MAX Transfection Reagent (Thermo Fisher Scientific) according to manufacturers recomendations.
  • the transfected cells are incubated for 6-7 days, medium collected and the proteins are isolated using a stepwise chromatography steps.
  • the proteins are purifed from filtered cell culture supernatants (adjusted to 50 mM TRIS-HCI pH 8.0, 1 mM EDTA, 50 mM NaCI) by capturing them on the StrepTrapTM HP (GE Healthcare) column and eluting with desthiobiotin (SIGMA, 2.5 mM in 50 mM TRIS-HCI pH 8, 1 mM EDTA, 50 mM NaCI).
  • SIGMA desthiobiotin
  • the cell culture medium containing the S protein RBD-Fc fusion protein is adjusted to pH 8.0 with by titrating with 1 M Tris-HCI pH 8.0 and then the RBD-Fc fusion is purified using Protein A column (GE Healthcare).
  • the bound protein is eluted using 100 mM Glycine pH 2.7 and immediately neutralized with 1 M Tris-HCI pH 8.0.
  • the eluted proteins are further purified by size-exclusion chromatography on Superdex 200 10/300 Increase column (GE Healthcare) in 5 mM Tris pH 8.0, 200 mM NaCI.
  • Antibody isotype titers are compared based on the half-maximal effective concentration value of the dilution factor. The resulting signal was compared with that obtained for the non-vaccinated patients.
  • Affinity purification of vaccine induced antibodies from human sera Human serum is separated by centrifugation at 2000 xg for 10 minutes.
  • affinity purification of vaccine-specific antibodies the recombinant ectodomain of the S protein is coupled to superparamagnetic Dynabeads M280 Tosylactivated (Thermo Fisher Scientific) according to manufacturer’s instructions.
  • the prepared S-beads are incubated with human serum 6-fold diluted with PBS supplemented with Tween 20 and Complete® protease inhibitors (Roche) at +2 to +8°C for 16 hrs with head-over-tail rotation.
  • the bound antibodies are eluted with 0.2 M glycine pH 2.7 and immediately neutralized with 1 M Tris-HCI pH 8.0.
  • Control antibodies are isolated from a non-vaccinated human by using Dynabeads Protein G (GE Healthcare) and final concentration is determined by absorption spectroscopy. Quality of vaccine-induced antibodies isolated form human sera is analysed by SDS-polyacrylamide electrophoresis as follow.
  • Solution of the recombinant S protein ectodomain (100 pmol/L and 10 pmol/L) are incubated withserially diluted control human IgG antibodies (Merck) or serially diluted (10- fold) antibodies isolated from human sera at 37°C for 1 hr and then added to the ACE2 immobilized on the ELISA plates. The plates are incubated for 1 hr at room temperature. After washing, bound S protein ectodomain is detected with peroxidase-conjugated anti StrepTag antibody visualized with the chromogenic substrate TMB one (Kem-en-tec, Denmark). The reaction is stopped by adding 50 pi of 0.25M H2SO4 into each well.
  • the plate is read at 450 nm using Powerwave HT (Bio-Tek).
  • the neutralizing activity of the human serum antibodies are determined by calculated the inhibitory concentration of the antibody isolates that caused 50% drop in the S protein ectodomain binding activity (IC50) using the four-parameter logistic function.
  • EXAMPLE 10 THE IMMUNOGENIC PEPTIDE SEQ ID NO. 83 (PEPTIDE 8)
  • Immunogenic peptide SEQ ID NO: 83 was derived from the surface of the S1 subunit.
  • the designed peptide contains one B cell epitope located in the middle part of the peptide and two contact residues (Spike protein positions N439, Y449), which may be important for binding the virus to the ACE2 receptor.
  • the peptide-induced antibody may affect binding of virus to hACE2, thus inhibiting of entry into a host cell.
  • N-terminal cysteine (C) is added for conjugation to KLH (the carrier protein) through a maleimide linker.
  • the predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 16B).
  • the peptide exhibits suitable hydrophilicity profile, documented in FIG. 17.
  • EXAMPLE 11 THE IMMUNOGENIC PEPTIDE SEQ ID NO. 84 (PEPTIDE 9)
  • Immunogenic peptide SEQ ID NO: 84 was derived from the surface of the S1 subunit.
  • the designed peptide contains one B cell epitope located in middle part of the peptide.
  • the peptide carries several contact residues (Spike protein positions T500, N501 , G502, Y505), which may play important roles in binding the virus to its receptor (ACE2). Blocking these residues by peptide-induced antibodies may be therapeutically effective to prevent the S1 subunit - ACE2 interaction.
  • N-terminal cysteine (C) is used for conjugation to KLFI as a carrier protein using a maleimide linker
  • aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid.
  • the predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 18B).
  • the peptide exhibits suitable hydrophilicity profile, which is shown in FIG. 19.
  • EXAMPLE 12 THE IMMUNOGENIC PEPTIDE SEQ ID NO. 85 (PEPTIDE 10)
  • Immunogenic peptide SEQ ID NO: 85 was derived from the surface amino acids of the S1 subunit.
  • the peptide contains one B cell epitope located in middle region and two contact residues (Spike protein positions L455, F456), which may play important roles in binding the virus to receptor of host cell.
  • the peptide-induced antibodies may block binding of virus to hACE2 and entry of virus into the cell.
  • the N-terminal cysteine (C) is added for conjugation to KLH (as a carrier protein) through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid.
  • KLH as a carrier protein
  • D aspartic acid
  • the predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 20B).
  • the designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 21.
  • EXAMPLE 13 THE IMMUNOGENIC PEPTIDE SEQ ID NO. 86 (PEPTIDE 11)
  • Peptide SEQ ID NO: 86 was derived from surface S1 subunit.
  • the peptide contains one B cell epitope located in middle region.
  • the Alanine located at the position 10 (A475 of the Spike protein) of the peptide is one of the contact residues with ACE2, YQAGS residues (SEQ ID NO: 117) form a part of the contact surface of S1 with ACE2, which the virus may utilize to enter into the host cell.
  • the peptide-induced antibodies may have neutralizing activity and thus effectively block the virus from binding to the receptor of permissive cells.
  • the N-terminal cysteine (C) is added for conjugation to KLFI (as a carrier protein) through a maleimide linker.
  • KLFI as a carrier protein
  • the designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 23.
  • EXAMPLE 14 THE IMMUNOGENIC PEPTIDE SEQ ID NO. 87 (PEPTIDE 12)
  • Peptide SEQ ID NO: 87 was derived from the surface of S1 subunit.
  • the designed peptide contains one B cell epitope located in central part of peptide.
  • the Threonine located at position 5 (the Spike protein position T415) and lysine at position 7 (the Spike protein position K417) were identified as the contact residues, which the virus may exploit to enter the host cell. Binding of the peptide-induced antibodies to these strategic residues or near these residues may lead to the lowering number of virus - infected cells.
  • N-terminal cysteine (C) is added for conjugation to KLFI as a carrier protein through a maleimide linker.
  • Peptide SEQ ID NO: 88 was derived from the surface of the S1 subunit.
  • the designed peptide contains one B cell epitope located in medium part of peptide.
  • the residues R3, D5, E6, Q9, T 14 and K16 of the peptide were identified as part the S1 protein contact surface with ACE2, which the virus exploits to enter the host cell. Blocking of the positions important for binding to human ACE2 by peptide-induced antibodies may lead to the lower affinity between the viral RBD and host ACE2 in the initial viral attachment step. In the end this lowering of affinity may lead to lowering of virus efficiency.
  • the N- terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker.
  • EXAMPLE 16 THE IMMUNOGENIC PEPTIDE SEQ ID NO. 89 (PEPTIDE 14)
  • Peptide SEQ ID NO: 89 was derived from the surface of the S1 subunit.
  • the peptide contains one B cell epitope located in medium part of peptide and four residues which were identified as important contact sites which virus exploit for binding to host cell receptor.
  • the identified amino acids residues at the Spike protein positions F486 (phenylalanine), N487 (asparagine), Y489 (tyrosine) and Q493 (glutamine) enhance viral attachment to human ACE2. Occupation (blocking) of these strategic residues by peptide- induced antibodies may be therapeutically effective.
  • the N-terminal cysteine (C) is added for conjugation to KLFI as a carrier protein through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid.
  • the predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 28B).
  • the designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 29.

Abstract

The disclosures described herein relate to polypeptides, antibodies, and compositions for use in the treatment and diagnosis of severe acute respiratory syndrome (SARS) coronavirus-associated diseases or disorders. Provided are new immunogenic compositions related to SARS CoV2 S protein and method of preventing and/or treating severe acute respiratory syndrome and other SARS-CoV-related disease, including COVID-19. Also provided are diagnostic reagents for the detection of SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, and/or severe acute respiratory syndrome or other SARS-CoV-related disease.

Description

EPITOPE-BASED VACCINES FOR TREATMENT OF CORONAVIRUS
ASSOCIATED DISEASES
Reference to Sequence Listing Submitted Electronically
[0001] The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 24, 2020, is named SequenceListing.txt and is 60,034 bytes in size.
Field of the Disclosure
[0002] The disclosures described herein relate to polypeptides, antibodies, and compositions for use in the treatment and diagnosis of severe acute respiratory syndrome (SARS) coronavirus-associated diseases or disorders.
Background
[0003] Coronaviruses (CoVs) are large, enveloped, positive-strand ribonucleic acid (RNA) viruses, having the largest genomes among all RNA viruses. They are roughly spherical and moderately pleiomorphic and contain crown-shape peplomers that are 80- 160 nM in size (Sahin et al., 2020). Coronaviruses have a highly conserved genomic organization, with a large replicase gene (encoded by approximately two thirds of the genome) preceding structural and accessory genes; express many non-structural genes by ribosomal frameshifting; develop constant transcription errors and RNA-Dependent RNA Polymerase jumps; display several unique or unusual enzymatic activities encoded within the large replicase-transcriptase polyprotein; and expression of structural and accessory downstream genes by synthesis of 3' nested sub-genomic micro-ribonucleic acids (mRNAs) (Fehr and Perlman, 2015; Sahin et al., 2020). Coronaviruses have a high substitution rate, with an average of roughly 10-4 nucleotide substitutions per site per year (Su et al., 2016). They cause a variety of respiratory, enteric and neurologic diseases in birds and mammals. Infections are transmitted mainly via deposition of infected droplets or aerosols on the respiratory epithelium or via fecal-oral routes (Masters, 2006). [0004] There are four genera of coronaviruses: Alphacoronavirus (aCoV), Betacoronavirus (pCoV), Deltacoronavirus (bCoV) and Gammacoronavirus (yCoV). Bats and rodents are the sources of most aCoVs and bOo /s, while avian species are the sources of most bCoVs and yCoVs (Chan et al., 2013). Seven human coronaviruses have been identified, two of them belonging to the a-coronavirus subgroup, HCoV-229E, HCoV-NL63 and another five are part of the b-coronavirus subgroup, HCoV-OC43, HC0V-HKUI , SARS-CoV and Middle East respiratory syndrome (MERS) -CoV (Fehr and Perlman, 2015) and SARS-Cov2 (Dong et al., 2020, Lu et al., 2020). Coronaviruses can cause potentially fatal disease in humans such as MERS-Cov, SARS-Cov and the newly discovered SARS-Cov2. SARS first emerged in the human population in November 2002. The symptoms of SARS included fever, headache, body aches, and diarrhea. Most patients develop pneumonia. A total of 8,098 people in 29 countries became sick with SARS during the 2003 outbreak; of these 774 died (fatality rate of 9.6%) (http://www.who.int/csr/sars/country/ table2004_04_21). Recently, no circulation of SARS-CoV has been registered. MERS was first reported in Saudi Arabia in 2012. Since then, a total number of 2494 laboratory-confirmed cases of MERS-CoV and 858 associated deaths (fatality rate of 30%) in 27 countries were reported (https://www.who.int/emergencies/ mers-cov/en/). The clinical signs of MERS-CoV- infected patients are similar to SARS-CoV (Su et al., 2016). A continuous increase of human cases in the endemic areas has been reported (Jia et al., 2019). In December 2019, an outbreak of atypical pneumonia caused by a novel strain of coronavirus was emerged, which was subsequently named SARS-CoV-2. As the new coronavirus outbreak has spread to many other countries with more than 12 million infected cases and more than half a million deaths, the World Health Organization (WHO) declared the outbreak of SARS-CoV-2a pandemic on 11 March, 2020 (https://www.who.int/dg/speeches/ detail/who-director-general-s-opening-remarks-at- the-media-briefing-on-covid-19—11 -march-2020). [0005] The coronaviral genome encodes four canonical structural proteins, the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. N is a phosphoprotein which binds to the CoV RNA genome and makes up the nucleocapsid and participates in viral transcription. The M glycoprotein - the most abundant structural protein - coordinates virion assembly and release. The envelope protein is a short, integral membrane protein, which is abundantly expressed inside the infected cell, but only a small portion is incorporated into the virion envelope. Recent studies have expanded on its functions as an ion-channelling viroporin. The hydrophobic transmembrane domain contains at least one predicted amphipathic a-helix that oligomerizes to form an ion-conductive pore in membranes (Schoeman and Fielding, 2019).
[0006] The transmembrane S glycoprotein forms homotrimers and mediates receptor attachment and subsequent fusion between the viral and host cell membranes to facilitate viral entry into the host cell. The S protein assembles into trimers to form the distinctive surface spikes of coronaviruses (Song et al., 2004). The interaction between the S protein and receptor represents the key determinant of coronavirus host species range and tissue tropism (Masters, 2006). The S protein comprises two functional subunits: S1 is responsible for binding to the host cell receptor and S2 orchestrates the fusion of the viral and cellular membranes. In most coronaviruses, the S protein is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently bound in the prefusion conformation. In case of SARS-CoV-2, the S protein harbors a furin cleavage site at the S1/S2 boundary. The S protein is further cleaved by host proteases at the so-called S2 site located immediately upstream of the fusion peptide. This cleavage was shown to activate the protein for membrane fusion via extensive irreversible conformational changes (Walls et al., 2020). The S1 domain is the most divergent region of the molecule and can vary extensively across and within the coronavirus subgroups, while the S2 portion of the ectodomain, which is heavily glycosylated, is the most conserved part of the molecule across the coronaviruses (Masters, 2006). The SARS- CoV and Cov-2 spike glycoproteins showed large sequence difference (~55% identity) in the S1 domain, while the S2 domain region displays ~91% identity (Song et al., 2018; Vandakari and Wilce, 2020). In both, SARS-CoV and SARS-CoV2, the S protein binds to the angiotensin-converting enzyme 2 (ACE2) (Hoffmann et al., 2020), which is a component of the renin-angiotensin system. ACE2 is a monocarboxypeptidase that catalyses cleavage of the Angiotensin II - the major vasoactive peptide in renin- angiotensin system, to produce Angiotensin I (1 -7). It has been shown that ACE inhibiting drugs have an antihypertensive effect (Francis, 2000; Clarke and Turner, 2012).
[0007] Previous studies identified 14 positions on the S1 domain that are key for binding of SARS-CoV to human ACE2: T402, R426, Y436, Y440, Y442, L472, N473, Y475, N479, Y484, T486, T487, G488, and Y491 (Li et al., 2005). A predictive study of the SARS-CoV-2 showed that 8 out of these 14 positions were conserved in SARS-CoV- 2, whereas 6 positions were (semi)conservatively substituted: R426 SARS-CoV, N439 SARS-CoV-2; Y442 SARS-CoV, L455 SARS-CoV-2; L472 SARS-CoV , F486 SARS- CoV-2; N479 SARS-CoV, Q493 SARS-CoV-2; Y484 SARS-CoV Q498 SARS-CoV-2; and T487 SARS-CoV, N501 SARS-CoV-2 (Walls et al., 2020). In addition, another predictive study demonstrated thatR408, Q409, T445, V417, L461 , D467, S469, L491 , N492, D493, Y 494, T497, T150, Y504 residues on SARS-Cov2 S1 domain were crucial for interaction with CD26 receptors on the surface of the host cells (Vandakari and Wilce, 2020).
[0008] The receptor-mediated conformational change in S1 and the dissociation of S1 from S2 are thought to initiate a major rearrangement in the remaining S2 trimer. This rearrangement exposes a fusion peptide that interacts with the host cellular membrane, and it brings together the two heptad repeats in each monomer so as to form an antiparallel, six-helix “trimer-of-dimers” bundle. The trimer of dimers is extremely stable, forming a rod-like, protease-resistant complex (Bosch et al., 2004, 2005). Interestingly, the S trimer in SARS-CoV2 exists in multiple, distinct conformational states. S glycoprotein trimers are found to exist in partially opened states in SARS-CoV2, while they remain largely closed in less pathogenic human coronaviruses (Wrapp et al., 2020; Walls et al., 2020).
[0009] Multiple strategies have been proposed for SARS-CoV2 vaccines; most of them target the surface-exposed S glycoprotein. The strategy is based on the knowledge that antibodies targeting the S protein can interfere with its binding to the receptor ACE- 2, and thus may block the virus entry into the host cell (Amanat and Krammer, 2020). Several active vaccines are currently in development, and two of them are in clinical trials: a RNA-based vaccine (mRNA encapsulated in lipid nanoparticles) and a viral vector- based vaccine (Adenovirus type V vector). The majority of vaccines are still in a preclinical stage, namely RNA-based vaccines, DNA vaccines, recombinant protein-based vaccines, viral vector-based vaccines, live attenuated vaccines, inactivated virus vaccine, subunit vaccines and peptide- based vaccines (Dhama et al. , 2020).
[0010] Since older individuals are the most vulnerable to SARS-CoV2 driven pneumonia, it will be important to develop vaccines that protect this segment of the population. In general, older individuals respond less well to vaccination due to immune- senescence (Amanat and Krammer, 2020). Only a limited number of vaccines have been tested in the older population, so it is difficult to predict whether vaccines lacking an adjuvant would be successful in generating the effective antibody response in elderly people.
[0011] Another important aspect of the vaccine development is safety. Several vaccines against SARS-CoV such as recombinant spike protein-based vaccines, attenuated and whole inactivated vaccines as well as vectored vaccines were shown to protect animals from challenge with SARS-CoV. However, in some cases, vaccination with the live virus results in complications, including lung damage and infiltration of eosinophils in the mouse model (Bolles et al., 2011 ; Tseng et al., 2012).
[0012] RNA and DNA vaccines, recombinant protein-based vaccines, viral vector- based vaccines, live attenuated vaccines, inactivated virus vaccines and subunit vaccines against SARS-Cov2 virus will contain hundreds of antigenic epitopes - all of which are not necessary, and some may even be detrimental and induce allergenic or reactogenic responses. The development of peptide vaccines containing only epitopes capable of inducing a desirable T cell and B cell mediated immune response may prevent unwanted side effects of the abovementioned vaccines. The peptide vaccines are considered sufficient to activate the appropriate cellular and humoral responses, while eliminating allergenic or reactogenic responses. Taking into the consideration these findings, peptide vaccines represent an effective and safe therapeutic and preventive approach for the SARS-Cov2 virus.
Summary of the Disclosure
[0013] As described in detail below, the present disclosure is based, in part, on the development of peptides for immunotherapy of coronavirus-associated diseases or disorders. In one embodiment, the disease is Covid-19.
[0014] Embodiment 1. An isolated polypeptide, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of one or more of the following sequences:
DSKVGGNYNKLYRLF, SEQ ID NO:8;
DSKVGGNYNYLYR, SEQ ID NO:9;
DSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ, SEQ ID NO:10; LQSYGFQPTNGVGYQ, SEQ ID NO:11 ;
STEIYQAGSTPCNGVEGFNCYF, SEQ ID NO:12; VEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE, SEQ ID NO:13; QILPDPSKPSKRS, SEQ ID NO:14;
NQKLIANQFNSAIGKIQDSLSS, SEQ ID NO:15;
YTMSLGAENSVAYS, SEQ ID NO:16;
SNNLDSKVGGNYN, SEQ ID NO: 118;
GFQPTNGVGYQPYR, SEQ ID NO: 119;
RLFRKSNLKPFE, SEQ ID NO: 120;
RDISTEIYQAGSTP, SEQ ID NO: 121 ;
PGQTGKIADYNYKLPD, SEQ ID NO: 122;
RGDEVRQIAPGQTGK, SEQ ID NO: 123;
DNGVEGFNGYFPLQSYG, SEQ ID NO: 124;
SEQ ID NOs. 42 through 116; and SEQ ID Nos. 125 through 227; and a polypeptide, fragment, variant, or derivative thereof, comprising a contiguous sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of said sequences, wherein the polypeptide, fragment, variant, or derivative thereof is at least 7 amino and at most 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues-long; and wherein the polypeptide, or a fragment, variant, or derivative thereof is neither a fragment of SARS Cov2 Spike protein of NC_045512.2 nor a fragment of any other naturally-existing coronavirus Spike protein, optionally wherein the polypeptide, fragment, variant, or derivative thereof comprises at least one modification to any one of the peptides of SEQ ID NOs: 8 through 16, SEQ ID NOs:118 through 124, or SEQ ID NOs:140 through 175, wherein the modification is an addition, deletion, or mutation to the amino acid sequence and/or the addition of a moiety to either the N-terminus, C-terminus, and/or internally .
[0015] Embodiment 2. The isolated polypeptide of embodiment 1 , or a fragment, variant, or derivative thereof wherein the polypeptide is selected from:
Peptide (1): 442- CGSGDSKVGGNYNKLYRLFE -456, SEQ ID NO:1 ;
Peptide (2): 442- CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ -506, SEQ ID NO:2; Peptide (3): 469-SGSTEIYQAGSTPCNGVEGFNCYFAK-490, SEQ ID NO:3;
Peptide (4): 483-CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE-516, SEQ ID NO:4; Peptide (5): 804-QILPDPSKPSKRSC-816, SEQ ID NO:5;
Peptide (6): 919-CNQKLIANQFNSAIGKIQDSLSS-940, SEQ ID NO:6;
Peptide (7): 695-CYTMSLGAENSVAYS -708, SEQ ID NO:7;
Peptide (8): 438-CSNNLDSKVGGNYN-450, SEQ ID NO: 83;
Peptide (9): 496- CDGFQPTNGVGYQPYR-509, SEQ ID NO: 84; Peptide (10): 454-CDRLFRKSNLKPFE-465, SEQ ID NO: 85;
Peptide (11 ): 466-CRDISTEIYQAGSTP-479, SEQ ID NO: 86;
Peptide (12): 412-CSPGQTGKIADYNYKLPD-427, SEQ ID NO: 87;
Peptide (13): 403-CSRGDEVRQIAPGQTGK-417, SEQ ID NO: 88;
Peptide (14): 481 -CDNGVEGFNGYFPLQSYG-496, SEQ ID NO: 89;
Peptides (1 ) through (14) without the N-terminal or C-terminal Cys (SEQ ID NOs. 90 through 102) and/or with an extra Cys (SEQ ID NO. 103 through 106);
Peptide based on peptide (3) but wherein the two underlined Cys are any amino acids (SEQ ID NO: 115);
Peptides (1 ) through 14 without the spacer(s) and/or linker(s) (SED ID NOs.109 through 112);
Peptides derived from Peptides (1 ) through (14), wherein any one or more of the underlined amino acids is replaced by one or more different amino acids (SEQ ID NOS 107-108 and 1 14-116) or is absent;
Any other Peptide selected from those of SEQ ID NO: 1 18-140; and
A polypeptide, fragment, variant, or derivative thereof comprising a contiguous sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of said peptides, wherein the numbering of the amino acids in the Peptides is relative to the sequence of SARS CoV2 S protein, and the differences between the two are underlined; wherein the polypeptide is neither a fragment of SARS Cov2 S protein of NC_045512.2 nor a fragment of any other naturally-existing coronavirus S protein.
[0016] Embodiment 3. The isolated polypeptide of any one of embodiments 1 and 2, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of a continguos sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NO:1 through SEQ ID NO:16, SEQ ID NO: 83 through SEQ ID NO:116, or SEQ ID NO: 140 through SEQ ID NO:175, optionally wherein the polypeptide, fragment, variant, or derivative thereof comprises one or more conservative amino acid substitutions.
[0017] Embodiment 4. The isolated polypeptide of any one of embodiments 1 through 3, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof differs from the polypeptides of SEQ ID NO:1 through 16, SEQ ID NO: 83 through 116, or SEQ ID NO: 140 through 175 in that it has an addition, deletion, or insertion that comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,
18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,
41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,
64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,
87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 96, 97, 98, 99, or 100 amino acids.
[0018] Embodiment 5. The isolated polypeptide of any one of embodiments 1 through 4, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof is at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41 , at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51 , at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58 residues, at least 59, at least 60, at least 61 , at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 90, but fewer than 100 amino acid residues in length.
[0019] [0020] Embodiment 6. The isolated polypeptide of any one of embodiments 1 through 5, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof, is at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21 , at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31 , at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41 , at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51 , at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58 residues, at most 59, at most 60, at most 61 , at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71 , at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 90, at most 100 amino acid residues in length.
[0021] Embodiment 7. The isolated polypeptide of any one of embodiments 1 through 6, or a fragment, variant, or derivative thereof, wherein the polypeptide further comprises a moiety or a moiety and a linker, and/or a peptide corresponding to Peptides (1) through Peptide (14) without a N-terminal Cys.
[0022] Embodiment 8. The isolated polypeptide of any one of embodiments 1 through 7, or a fragment, variant, or derivative thereof, wherein the moiety is a carrier protein selected from any one or more of a Cys residue, an Asp residue, a Ser residue, Keyhole Limpet Hemocyanin (KLH) functional unit, Cys-KLH, a tetanus toxin heavy chain C fragment, a diphteria toxin, a diphtheria toxin variant CRM197, an H influenzae protein D, a Meningococcal outer membrane protein complex protein, an Outer-membrane lipoprotein carrier protein, or a Cholera toxin B subunit, a virus-like particle, biotin, avidin, streptavidin, neutravidin, serum albumin, an enzyme, a metallic nanomaterial, CRM197 and an outer membrane protein mixture from N. meningitidis (OMP), micro- and nano particles of biodegradable polymers including polylactic, polycaproic, polyglycolic, polymalic, polybutyric acids and their combination and modifications; hydrogels of polyethyleneglycol and its modifications; hyaluronic acid, dextran, chitosan, liposome- polymer hybrid carrier, and a fragment or derivative or combination thereof.
[0023] Embodiment 9. The isolated polypeptideof any one of embodiments 1 through 8, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of one or more of the polypeptides of SEQ ID NO:1 through SEQ ID NO:16, SEQ ID NO:118 through SEQ ID NO:124, SEQ ID NO:83 through SEQ ID NO: 89, and/or SEQ ID NO: 140 through 175 connected through a linker.
[0024] Embodiment 10. The isolated polypeptide of any one of embodiments 1 through 9, or a fragment, variant, or derivative thereof, wherein the linker is selected from GSG, GPAD (SEQ ID NO: 17), and SG.
[0025] Embodiment 11 . The isolated polypeptide of any one of embodiments 1 through 10, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of a peptide of SEQ ID Nos: 3, 5, 6, 7, 9, 11 , 12, 14, 15, 16, 83 through 89, or 118 through 124.
[0026] Embodiment 12. The isolated polypeptide of any one of embodiments 1 through 11 , or a fragment, variant, or derivative thereof, wherein the polypeptide further comprises one or more chemical modifications selected from an internal bridge, short- range cyclization, methylation, amidation, acetylation or substitution with other chemical groups cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, which can be N-terminal, C-terminal, or internal. [0027] [0028] Embodiment 13. The isolated polypeptide of any one of embodiments 1 through 12, or a fragment, variant, or derivative thereof, wherein the polypeptide is immunogenic.
[0029] Embodiment 14. A nucleic acid encoding one or more of the polypeptides, fragments, variants, or derivatives thereof of any one of embodiments 1 through 13.
[0030] Embodiment 15. The nucleic acid of embodiment 14, wherein the nucleic acid is DNA, RNA, or modified RNA.
[0031] Embodiment 16. A vector comprising one or more of the nucleic acids of any one of embodiments 14 and 15.
[0032] Embodiment 17. The vector of embodiment 16, wherein the vector is a retroviral vector, lentiviral vector, adenoviral vector, poxvirus vector, plasmid, or bacterial vector.
[0033] Embodiment 18. A recombinant cell comprising a polypeptide, or a fragment, variant, or derivative thereof, of any one of embodiments 1 through 13, a nucleic acid according to any one of embodiments 14 and 15, or a vector of any one of embodiments 16 and 17.
[0034] Embodiment 19. The recombinant cell of embodiment 18, wherein the cell is a prokaryotic cell (e.g. E. coli) or an eukaryotic cell (e.g. Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NS0, Sp2/0), a monkey kidney cell (COS-7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TRI cell, an MRC 5 cell, a FS4, or a MDCK cell. [0035] Embodiment 20. An immunogenic composition comprising one or more of the polypeptides of any one of embodiments 1 through 13, or a fragment, variant, or derivative thereof, nucleic acids of embodiments 14 and 15, vector of embodiments 16 and 17, or cell of embodiments 18 and 19.
[0036] Embodiment 21. The immunogenic composition of embodiment 20, comprising one or more of the polypeptides of any one of embodiments 1 through 13, or a fragment, variant, or derivative thereof, and an adjuvant.
[0037] Embodiment 22. The immunogenic composition of embodiment 21 , comprising a combination of any two of Peptides (1 ) through Peptide (14).
[0038] Embodiment 23. The immunogenic composition of embodiment 22, wherein the composition comprises a combination of Peptide (1) and (2), (1) and (3), (1) and (4), (1) and (5), 1 and (6), (1 ) and (7), (2) and (3), or any one of the 21 possible two-peptide combinations of these seven Peptides; or any one of the 21 possible two-peptide combinations of Peptides (8) through (14); or any one of the 91 two-peptide combinations, 364 three-peptide combinations, and 1001 four-peptide combinations of Peptides (1) through (14), or any one of the 1456 combinations of Peptides (1) through (14).
[0039] Embodiment 24. The immunogenic composition of embodiment 23, comprising a combination of any three of Peptides (1) through Peptide (14).
[0040] Embodiment 25. The immunogenic composition of embodiment 24, comprising a combination of Peptides (1), (2) and (3); (1), (2) and (4); (1), (3), and (4); and (2), (3), (4) or any one of the 35 possible three-peptide combinations of these seven Peptides; or any one of the 35 three-peptide combinations of Peptides (8) through (14); or any one of the 364 three-peptide combinations of Peptides (1) through (14); or any one of the 1001 four-peptide combinations of Peptides (1) through (14).
[0041] [0042] Embodiment 26. The immunogenic composition of any one of embodiments 19 through 25, wherein the immunogenic composition is a vaccine.
[0043] Embodiment 27. The immunogenic composition of any one of embodiments 19 through 26, wherein the adjuvant is aluminum hydroxide or one of its salts.
[0044] Embodiment 28. A method of preventing or treating severe acute respiratory syndrome or other SARS-CoV-related disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one of embodiments 20 through 27, or of a polypeptide of any one of embodiments 1 through 13.
[0045] Embodiment 29. The method of embodiment 28, wherein the efficacy is measured by measuring a reduced viral load or delays or prevention of a further increase in viral load.
[0046] Embodiment 30. The method of embodiment 29, wherein the efficacy is measured by elimination, reduction, or decrease in the intensity or frequency of one or more symptoms associated with SARS such as fever, cough, shortness of breath, organ failure (e.g. kidneys) and/or septic shock.
[0047] Embodiment 31. The method of any one of embodiments 28 through 30, wherein the subject has an underlying disorder such as diabetes, cancer, chronic lung disease, heart disease, or generally weakened immune system.
[0048] Embodiment 32. The method of any one of embodiments 28 through 31 , wherein the immunogenic composition induces a humoral response, such as an increased level of neutralizing antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine, or as compared to the same subject prior to administration of the immunogenic composition.
[0049] Embodiment 33. The method of any one of embodiments 28 through 32, wherein the immunogenic composition induces a cellular immune response, such as a CD8+T cell response, (including the production of cytokines such as interferon- gamma (TFN-g), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or any combinations thereof.
[0050] Embodiment 34. The method of any one of embodiments 28 through 33, wherein the therapeutically effective amount is prophylactic.
[0051] Embodiment 35. The method of any one of embodiments 28 through 34, wherein the immunogenic composition or polypeptide is administered by any route typically used for vaccination, including topical, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, inhalation, or combinations thereof. [0052] Embodiment 36. The method of any one of embodiments 28 through 35, wherein the immunogenic composition or polypeptide is administered in a single-dose vaccination schedule.
[0053] Embodiment 37. The method of any one of embodiments 28 through 36, further comprising the administration of another treatment for SARS.
[0054] Embodiment 38. The method of any one of embodiments 28 through 37, further comprising the administration of remdesivir, azithromycin, hydroxychloroquire, chloroquine, standard of care, or combinations thereof.
[0055] Embodiment 39. The method of any one of embodiments 28 through 38, further comprising the administration of hydroxychloroquine combination (200 mg X 3 per days for 10 days) with Azithromycin (500 mg on the 1st day then 250 mg per day for 5 more days); Anti-inflammatory therapy such as tocilizumab and favipiravir, tocilizumab, sarilumab, leronlimab, rintatolimod, BPI-002, REGN3048 and REGN 3051 , monoclonal antibody designed to bind SARS-CoV-2; Anti-Viral therapy such as remdesivir, lopinavir and ritonavir, danoprevir and ritonavir, favipiravir, darunavir and cobicistat, umifenovir, galidesivir, linebacker and equivir, compounds that inhibit the virus interaction with the receptor ACE2; or Immunotherapy such as RNA vaccines (e.g., targeting Spike protein), DNA vaccines (e.g., targeting Spike protein), recombinant protein vaccines (e.g., targeting Spike protein), viral-vector based vaccines (e.g., targeting Spike protein), live attenuated vaccines (e.g., targeting the whole virion), inactivated vaccines (e.g., targeting the whole virion), subunit vaccine (e.g., targeting the whole virion), and other peptide vaccines.
[0056] Embodiment 40. The method of any one of embodiments 28 through 39, wherein the disease is Covid-19.
[0057] Embodiment 41. A method of using a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, as a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
[0058] Embodiment 42. An immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19 for manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
[0059] [0060] Embodiment 43. Use of an immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, or a recombinant cell of any one of embodiments 18 and 19 for the manufacture of a medicament for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
[0061] Embodiment 44. An immunogenic composition of any one of embodiments
20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, or a recombinant cell of any one of embodiments 18 and 19 for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
[0062] Embodiment 45. Use of an immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19, or an antibody against a polypeptide of any one of embodiments 1 through 13, for the manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
[0063] Embodiment 46. An immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19 for manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
[0064] Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the present invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following embodiments/claims.
Brief Description of the Figures
[0065] FIG. 1 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:1. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.65 corresponds to sensitivity of 0.01969and specificity of 0.99272.
[0066] FIG. 2 shows the hydrophobicity prediction of the peptide SEQ ID NO:1 using the Kyte-Doolittle algorithm.
[0067] FIG. 3 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:2. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of two B-cell epitopes. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116.
[0068] FIG.4 shows the hydrophobicity prediction of the peptide SEQ ID NO:2 using the Kyte-Doolittle algorithm.
[0069] FIG. 5 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:3. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116.
[0070] FIG.6 shows the hydrophobicity prediction of the peptide SEQ ID NO:3 using the Kyte-Doolittle algorithm.
[0071] FIG. 7 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:4. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116.
[0072] FIG. 8 shows the hydrophobicity prediction of the peptide SEQ ID NO:4 using the Kyte-Doolittle algorithm.
[0073] FIG. 9 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:5. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.60 corresponds to sensitivity of 0.09559 and specificity of 0.95116.
[0074] FIG. 10 shows the hydrophobicity prediction of the peptide SEQ ID NO:5 using the Kyte-Doolittle algorithm.
[0075] FIG. 11 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:6. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
[0076] FIG. 12 shows the hydrophobicity prediction of the peptide SEQ ID NO:6 using the Kyte-Doolittle algorithm.
[0077] FIG. 13 shows the prediction of the B-cell epitope in the peptide SEQ ID NO:7. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.60 corresponds to sensitivity of 0.09559and specificity of 0.95116. [0078] FIG. 14 shows the hydrophobicity prediction of the peptide SEQ ID NO:7 using the Kyte-Doolittle algorithm.
[0079] FIG. 15: List of epitopes in immunogenic polypeptides (e.g.SEQ ID NO: 1 through 16). The figure discloses SEQ ID NOS 1 , 42-47, 2, 44, 48-56, 3, 57-67, 4, 68, 51 - 56, 69-72, 5, 73-74, 6, 75-80, 7, 81-83, 125, 42, 84, 69-71 , 85, 126, 86, 127, 87, 128-131 , 88, 132-135, 89 and 136-139, respectively, in order of appearance.
[0080] FIG. 16 shows the prediction of the B-cell epitope in the Peptide (8) SEQ ID NO: 83. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0. 60 corresponds to sensitivity of 0.09559 and specificity of 0.95116.
[0081] FIG. 17 shows the hydrophobicity prediction of the peptide SEQ ID NO: 83 using the Kyte-Doolittle algorithm.
[0082] FIG. 18 shows the prediction of the B-cell epitope in the Peptide (9) SEQ
ID NO: 84. The table (A) contains the score of each of the amino acid, the graph (A) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
[0083] FIG. 19 shows the hydrophobicity prediction of the peptide SEQ ID NO: 84 using the Kyte-Doolittle algorithm
[0084] FIG. 20 shows the prediction of the B-cell epitope in the Peptide (10) SEQ ID NO: 85. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.60 corresponds to sensitivity of 0.09559 and specificity of 0.95116.
[0085] FIG. 21 shows the hydrophobicity prediction of the peptide SEQ ID NO: 85 using the Kyte-Doolittle algorithm.
[0086] FIG. 22 shows the prediction of the B-cell epitope in the Peptide (11) SEQ ID NO: 86. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
[0087] FIG. 23 shows the hydrophobicity prediction of the peptide SEQ ID NO: 86 using the Kyte-Doolittle algorithm
[0088] FIG. 24 shows the prediction of the B-cell epitope in the Peptide (12) SEQ ID NO: 87. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
[0089] FIG. 25 shows the hydrophobicity prediction of the peptide SEQ ID NO: 87 using the Kyte-Doolittle algorithm.
[0090] FIG. 26 shows the prediction of the B-cell epitope in the Peptide (13) SEQ ID NO: 88. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
[0091] FIG. 27 shows the hydrophobicity prediction of the peptide SEQ ID NO: 88 using the Kyte-Doolittle algorithm
[0092] FIG. 28 shows the prediction of the B-cell epitope in the Peptide (14) SEQ ID NO: 89. The table (A) contains the score of each of the amino acid, the graph (B) visually shows the position of the B-cell epitope. The amino acid score value of 0.50 correlates to sensitivity of 0.58564 and specificity of 0.57158. Score value of 0.55 corresponds to sensitivity of 0.29159 and specificity of 0.81655.
[0093] FIG. 29 shows the hydrophobicity prediction of the peptide SEQ ID NO: 89 using the Kyte-Doolittle algorithm.
[0094] FIG. 30 shown predicted T cell epitopes (MFIC binding). Detailed Description
[0095] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the Specification.
[0096] Units, prefixes, and symbols used herein are provided using their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.
[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, Juo, “The Concise Dictionary of Biomedicine and Molecular Biology”, 2nd ed., (2001), CRC Press; “The Dictionary of Cell & Molecular Biology”, 5th ed., (2013), Academic Press; and “The Oxford Dictionary Of Biochemistry And Molecular Biology”, Cammack et al. eds., 2nd ed., (2006), Oxford University Press, provide those of skill in the art with a general dictionary for many of the terms used in this disclosure.
[0098] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. [0099] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and.”
[00100] The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include A and B; A or B; A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[00101] The terms “e.g.,” and “i.e.” as used herein, are used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification. [00102] The terms “or more”, “at least”, “more than”, and the like, e.g., “at least one” are understood to include but not be limited to at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more than the stated value. Also included is any greater number or fraction in between.
[00103] Conversely, the term “no more than” includes each value less than the stated value. For example, “no more than 100 nucleotides” includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73,
72, 71 , 70, 69, 68, 67, 66, 65, 64, 63, 62, 61 , 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 , 50,
49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27,
26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , and 0 nucleotides. Also included is any lesser number or fraction in between.
[00104] The terms “plurality”, “at least two”, “two or more”, “at least second”, and the like, are understood to include but not limited to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,
13, 14, 15, 16, 17, 18, 19 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36,
37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also included is any greater number or fraction in between. [00105] Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided. The term "consisting of" excludes any element, step, or ingredient not specified in the claim. In one embodiment, "consisting of" is defined as "closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. A claim which depends from a claim which "consists of" the recited elements or steps cannot add an element or step. The terms “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
[00106] Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The disclosure may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment of the disclosure" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the disclosure, without any restrictions regarding the scope of the disclosure and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non- optional features of the disclosure.
[00107] Unless specifically stated or evident from context, as used herein, the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of” may mean within one or more than one standard deviation per the practice in the art. “About” or “comprising essentially of” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms may mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “comprising essentially of” should be assumed to be within an acceptable error range for that particular value or composition.
[00108] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one- hundredth of an integer), unless otherwise indicated.
[00109] “Administering” refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Polypeptides, nucleic acids and host cells of the present description, and immunogenic compositions and vaccines thereof, may be administered to a subject in need thereof by routes known in the art, and may vary depending on the use. Routes of administration include, for example, local administration (e.g., to the lungs) and parenteral administration such as subcutaneous, intraperitoneal, intramuscular, intravenous, intraportal and intrahepatic. In a preferred embodiment, polypeptides, nucleic acids, composiitions, vaccines or host cells of the present disclosure, or immunogenic compositions thereof, are administered to a subject by local infusion, for example using an infusion pump and/or catheter system. Other exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the polypeptides, nucleic acids, composiitions, vaccines or host cells of the present disclosure, or immunogenic compositions thereof is administered via a non- parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal, or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering may also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
[00110] An "amino acid/s" or an "amino acid residue/s" may be a natural or non natural amino acid residue/s linked by peptide bonds or bonds different from peptide bonds. The amino acid residues may be in D- configuration or L -configuration (referred to herein as D- or L- enantiomers). An amino acid residue comprises an amino terminal part (NH3) and a carboxy terminal part (COOH) separated by a central part (R group) comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group. NH3 refers to the amino group present at the amino terminal end of an amino acid or peptide, and COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide. The generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature are listed in 37 C.F.R. 1.822(b)(2). Examples of non-natural amino acids are also listed in 37 C.F.R. 1.822(b)(4), other non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues. Naturally occurring amino acids may be further modified, e.g. amidation, hydroxyproline, y-carboxygiutamate, and O-phosphoserine. [00111] The term “antibody” (Ab) includes, without limitation, a glycoprotein immunoglobulin, which binds specifically to an antigen. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1 , CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprises one constant domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
[00112] Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-id) antibodies (including, e.g., anti-anti-ld antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), and antigen binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations.
[00113] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human lgG1 , lgG2, lgG3 and lgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG 1 ) that is encoded by the heavy chain constant region genes. The term “antibody” includes, by way of example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or nonhuman Abs; wholly synthetic Abs; and single chain Abs. A nonhuman Ab may be humanized by recombinant methods to reduce its immunogenicity in man. Where not expressly stated, and unless the context indicates otherwise, the term “antibody” also includes an antigen-binding fragment or an antigen binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab.
[00114] An “antigen binding molecule,” “antigen binding portion,” or “antibody fragment” refers to any molecule that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived. An antigen binding molecule may include the antigenic complementarity determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules. Peptibodies (i.e. , Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an epitope present in the surface of a viral particle. In some embodiments, the antigen binding molecule binds to an epitope in the SARS coronavirus S protein. In certain embodiments, the antigen binding molecule binds to any one of polypeptides of the disclosure. In further embodiments, the antigen binding molecule is an antibody fragment that specifically binds to the antigen, including one or more of the complementarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of avimers. [00115] As used herein, the term “variable region” or “variable domain” is used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called CDRs while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).
[00116] As used herein, an antigen binding molecule, an antibody, or an antigen binding molecule thereof, or a polypeptide of the dislosure “cross-competes” with a reference antibody or an antigen binding molecule thereof or polypeptide of the disclosure if the interaction between an antigen and the first binding molecule, an antibody, or an antigen binding molecule or polypeptide of the disclosure blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, an antigen binding molecule thereof, or polypeptide of the disclosure to interact with the antigen or with the coronavirus S protein. Cross competition may be complete, e.g., binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind the antigen, or it may be partial, e.g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind the antigen. In certain embodiments, an antigen binding molecule that cross- competes with a reference antigen binding molecule binds the same or an overlapping epitope as the reference antigen binding molecule. In other embodiments, the antigen binding molecule that cross-competes with a reference antigen binding molecule binds a different epitope as the reference antigen binding molecule.
[00117] Numerous types of competitive binding assays may be used to determine if one antigen binding molecule or polypeptide competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct labeled assay, solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1 -125 label (Morel et al., 1988, Molec. Immunol. 25:7-15), solid phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). These methods are also examples of methods for measuring the affinity of the peptides of the disclosure for the antibodies or coronaviral S protein of the disclosure. [00118] An “antigen” refers to any molecule that provokes an immune response or is capable of being bound by an antibody or an antigen binding molecule (e.g., a TCR). The immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, could serve as an antigen. An antigen may be endogenously expressed, i.e. expressed by genomic DNA, or may be recombinantly expressed. An antigen may be specific to a certain tissue, such as a cancer cell, or it may be broadly expressed. In addition, fragments of larger molecules may act as antigens. In one embodiment, the antigens are any one of the polypeptides of the disclosure.
[00119] A “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a therapeutic agent, e.g., polypeptide or antibody of the disclosure, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression may be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays. Dosages of the molecules of the present disclosure may vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated. A physician will ultimately determine appropriate dosages to be used.
[00120] A “patient” or ’’subject” as used herein includes any human or animal who is afflicted with coronavirus-related disease or disorder. The terms “subject” and “patient” are used interchangeably herein.
[00121] As used herein, the term “in vitro cell” refers to any cell, which is cultured ex vivo. In particular, an in vitro cell may include a T cell.
[00122] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
[00123] The terms “reducing” and “decreasing” are used interchangeably herein and indicate any change that is less than the original. “Reducing” and “decreasing” are relative terms, requiring a comparison between pre- and post- measurements. “Reducing” and “decreasing” include complete depletions.
[00124] “Treatment” or “treating” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity, or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, “treatment” or “treating” includes a partial remission. In another embodiment, “treatment” or “treating” includes a complete remission.
[00125] One of ordinary skill in the art is familiar with a variety of methods to calculate indentiy or homology of one polypeptide to the other. In one embodiment, to calculate percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that may be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span,” as determined by the algorithm.) In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm. In some embodiments, identity may be determined as percentage of identity using known computer algorithms such as the “PASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, I, et al. , Nucleic Acids Research 12(1) :387 (1984)), BLASTP, BLASTN, PASTA Atschul, S. F , et al, J Molec Biol 215:403 (1990); Guide to Huge Computers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology information database may be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG Gap” program (Madison Wis.)).
[00126] The term "similarity" and "similar" and grammatical variations thereof, as used herein, mean that an amino acid sequence contains a limited number of conservative amino acid substitutions compared to a peptide reference sequence, e.g. the variant peptide versus the parent peptide as defined herein. A variety of criteria can be used to indicate whether amino acids at a particular position in a peptide are similar. In making changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
[00127] Substitutions may be conservative or non-conservative amino acid substitutions. A "conservative substitution" is the replacement of one amino acid by a biologically, chemically or structurally similar residue. Biological similarity means that the substitution does not destroy a biological activity, e.g. T ceil reactivity or HLA coverage. Structural similarity means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size. Chemical similarity means that the residues have the same charge, or are both either hydrophilic or hydrophobic.
[00128] For example, a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain, for example amino acids with basic side chains (e.g., lysine, arginine, histidine) ; acidic side chains (e.g., aspartic acid, glutamic acid) ; uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine) ; nonpolar side chains
(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan ) ; beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, serine for threonine, and the like. Proline, which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., Leu, Val, He, and Ala). In certain circumstances, substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
[00129] ISOLATED AND PURIFIED POLYPEPTIDES
[00130] Immunogenic peptides to generate SARS-CoV-2 vaccine [00131] The S1 ectodomain subunit of SARS-CoV-2 spike (S) glycoprotein binds to the cellular receptor angiotensin-converting enzyme 2 (ACE2) present on permissive host cells. The membrane-proximal subunit S2 of SARS-CoV-2 is responsible for the fusion of the virus with the plasma membrane of host cell. Since the S protein is cleaved during egress of the virus from the host cells, the two subunits are held together only by non- covalent interactions. Binding of S1 to ACE2 destabilizes the prefusion S protein trimer, resulting in shedding of the S1 subunit and exposing the key S2’ protease cleavage site. Cleavage at this site initiates profound structural rearrangements of the S2 subunit, which exposes the membrane fusion peptide and facilitates membrane fusion.
[00132] Use of the attenuated virus or subunit vaccines (surface viral proteins) leads to the inclusion of unnecessary antigenic load that on one hand complicates the situation by inducing allergenic and/or reactogenic responses and on the other contributes little to the protective immune response by directing the immune response to immunodominant epitopes (Li et al., Vaccines 2014). These immunodominant epitopes might not be the correct target for the production of protective antibodies. On the contrary, the appropriately designed peptides will direct the protective antibody response to the desired epitopes that have essential functions in the entry of the virus to the host cells.
[00133] Structural and functional analyses have revealed key atomic-level interactions between the surface S glycoprotein receptor-binding domain (RBD) and its host receptor (ACE2) (Hoffmann et al., 2020; Walls et al., 2020; Wrapp et al., 2020). Based on the primary and 3D structures of the ectodomain of the spike S glycoprotein and the receptor ACE2 (PDB structure database 6M0J, DOI: 10.2210/pdb6M0J/pdb), suitable immunogenic peptides with potential for protective therapeutic function were derived from the S1 subunit (SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4) and from the S2 subunit (SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7). [00134] B-cell epitope probability prediction BepiPred-2.0 server (Jespersen et al., 2017) run at http://t00ls.iedb.0rg/bcell// was used to evaluate the physical and chemical properties of the designed peptides by Random Forest algorithm trained on epitopes and non-epitope amino acids determined from crystal structures.
[00135] Hydrophobicity of peptides (Kyte-Doolittle plots) were evaluated by ProtScale server at https://web.expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. Moreover, the accessibility of the selected target regions to antibodies on the native Spike glycoprotein on viral particles was ensured by designing peptides that do not contain or are not in a close proximity of glycosylation sites (Walls et al., 2020). The proposed peptides will be conjugated to a carrier, Keyhole limpet hemocyanin (KLH), to stimulate the immune system effectively. KLH is a highly immunogenic T-cell dependent antigen, xenogeneic to mammalian immune system, elicits strong immune response and promotes generation of antibodies against haptens. The peptides may also be conjugated to other molecules via other methods, including click-chemistry (e.g., Peptide (3)). The conjugation linker may be attached to the N-terminal or C-terminal end of the peptides.
[00136] The vaccines carrying the peptides derived from the S1 subunit conjugated to KLH will elicit antibodies that will bind and inhibit SARS-CoV-2 S1-Ace2 interaction and binding to host permissive cells. The vaccines containing the peptides derived from S2 subunit conjugated to KLH can induce antibodies preventing the conformational change and fusion of the virus with membrane of the host cell.
[00137] The KLH-conjugated peptides derived from S1 subunit (SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89 will be combined with KLH-conjugated peptides derived from S2 subunit (SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7). This approach allows to obtain a defined pool of vaccine induced antibodies with a combined mode of action, preventing the first step of the infection - viral binding to a host cell, and also preventing the second step - fusion of the virus with the plasma membrane of the host cell. All proposed peptides can also be produced with amide at its C-terminus. This might increase the binding capacity (crossreactivity) of vaccine induced antibodies with desired (therapeutic) epitopes on native surface S protein. The amino acid numbers are according to full length SARS-CoV-2 Spike protein of the virus isolate under the NCBI Reference Sequence NC_045512.2 (Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 , complete genome).
[00138] In one embodiment, the disclosure is directed to isolated or purified polypeptides encompassing at least one epitope from a coronavirus Spike protein, including the SARS CoV2 Spike protein.
[00139] The terms “isolated or purified” mean modified “by the hand of humans” from the natural state; in other words if an object exists in nature, it is said to be isolated or purified if it is modified or extracted from its natural environment or both. For example, a polynucleotide or a protein/peptide naturally present in a living organism is neither isolated nor purified; on the other hand, the same polynucleotide or protein/peptide separated from coexisting molecules in its natural environment, obtained by cloning, amplification and/or chemical synthesis is isolated for the purposes of the present disclosure. Furthermore, a polynucleotide or a protein/peptide which is introduced into an organism by transformation, genetic manipulation or by any other method, is “isolated” even if it is present in said organism. The term purified as used in the present disclosure means that the proteins/peptides according to the disclosure is essentially free from, contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. Flowever, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dinners or alternatively glycosylated or derivatized forms. By definition, isolated peptides are also non-naturally occurring, synthetic peptides. Methods for isolating or synthesizing peptides of interest with known amino acid sequences are well known in the artare essentially free of association with the other proteins or polypeptides, as is for example the product purified from the culture of recombinant host cells or the product purified from a nonrecombinant source.
[00140] In one embodiment, the SARS-CoV-2 spike (S) polypeptide is the peptide disclosed in NCBI access number NC_045512.2 (Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 , complete genome). The S polypeptide has 1273 amino acids. It contains a large N-terminal ectodomain consisting of two subunits S1 and S2, one transmembrane domain close to the C-terminus and a short intraviral Cys-rich domain. The S1 ectodomain subunit of SARS-CoV-2 spike (S) glycoprotein binds to the cellular receptor angiotensin-converting enzyme 2 (ACE2) present on permissive host cells. The membrane-proximal subunit S2 of SARS-CoV-2 is responsible for the fusion of the virus with the plasma membrane of the host cell. The peptides below are numbered relatively to the sequence of NCBI access number NC_045512.2.
[00141] In one embodiment, the SARS-CoV-2 polypeptide is a peptide selected from the following:
[00142] Peptide (1 ): 442- CGSGDSKVGGNYNKLYRLFE -456 (SEQ ID NO: 1 )
[00143] Peptide (2): 442- CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ -506 (SEQ ID NO: 2)
[00144] Peptide (3): 469-SGSTEIYQAGSTPCNGVEGFNCYFAK-490 (SEQ ID NO: 3) [00145] Peptide (4): 483-CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE-516 (SEQ ID NO: 4)
[00146] The differences between the peptides and the naturally existing SARS Cov2 S1 polypeptide are underlined. These peptides have SEQ ID Nos: 1 , 2, 3, 4.
[00147] In one embodiment, the SARS-CoV-2 polypeptide is a peptide selected from the following:
[00148] Peptide (5): 804-QILPDPSKPSKRSC-816 (SEQ ID NO: 5)
[00149] Peptide (6): 919-CNQKLIANQFNSAIGKIQDSLSS-940 (SEQ ID NO: 6)
[00150] Peptide (7): 695-CYTMSLGAENSVAYS -708 (SEQ ID NO: 7)
[00151] These peptides have SEQ ID Nos: 5, 6, 7.
[00152] In one embodiment, the SARS-CoV-2 peptide is a peptide selected from the following: [00153] Peptide (8): 438-CSNNLDSKVGGNYN-450, SEQ ID NO: 83
[00154] Peptide (9): 496- CDGFQPTNGVGYQPYR-509, SEQ ID NO: 84
[00155] Peptide (10): 454-CDRLFRKSNLKPFE-465, SEQ ID NO: 85
[00156] Peptide (11 ): 466-CRDISTEIYQAGSTP-479, SEQ ID NO: 86
[00157] Peptide (12): 412-CSPGQTGKIADYNYKLPD-427, SEQ ID NO: 87 [00158] Peptide (13): 403-CSRGDEVRQIAPGQTGK-417, SEQ ID NO: 88 [00159] Peptide (14): 481 -CDNGVEGFNGYFPLQSYG-496, SEQ ID NO: 89
[00160] In one embodiment, each of Peptides (1) through (14), if not already there, may have a N-terminal Cysteine to facilitate conjugation to KLH. In other words, the N- terminal (or C-terminal) Cys in all of the peptides is used for conjugation and may or may not be present. In one embodiment, the peptides may be conjugated to CRM197ln one embodiment, Peptide (1) is derived from the S1 subunit, modified by adding GSG linker sequence. In one embodiment, N-terminal C may be used for conjugation to KLH. In one embodiment, Peptide (1) may be combined with Peptide (3) and Peptides (5), (6), and/or (7) from the S2 subunit. In one embodiment, additional changes (K instead of Y451) and C-terminal E may be added to stabilize the peptide structure.
[00161] In one embodiment, Peptide (2) is derived from the S1 subunit and is generated by fusing two separated sequences: 442-454 and 492-506. The aim is to mimic a compact 3D structure similar to that present on native Spike protein, which makes contacts with ACE2 receptor on human epithelial cells. In one embodiment, the linker sequence (GPAD (SEQ ID NO: 17)) may form a beta turn was generated by using the Motivated proteins web site facility. In one embodiment, the N-terminal C may be used for conjugation to KLH. In one embodiment, the peptide may be produced with amide at its C-terminus.
[00162] In one embodiment, Peptide (3) is derived from the S1 subunit, and the last two amino acids Ala(A) and Lys(K) may be added in order to force the peptide to generate a compact structure by contacting the added K with E471. This may mimic the 3D structure of the native S protein contact site with ACE2. In one embodiment, the two internal cysteines (underlined) may be oxidized to form internal disulfide bond. In one embodiment, the N-terminus of the peptide is modified (e.g. by a chemoselective reactive group like azide which can bind selectively to alkyne or phosphine) and may be used for conjugation to KLH. In one embodiment, an SG may be used as a spacer. In one embodiment, the peptide may also be produced with amide at its C-terminus.
[00163] In one embodiment, Peptide (4) derived from S1 subunit, internal Cys (C488) is modified to Ala(A) to prevent dimerization, may change the 3D peptide structure. In one embodiment, N-terminal C may be used for conjugation to KLH. In one embodiment, the peptide may be produced with amide at its C-terminus.
[00164] In one embodiment, Peptide (5) will induce antibodies that prevent access of the protease to the S2’ cleavage site, and thus prevent a conformational change to induce ‘fusion peptide’ interaction with cellular membrane. In one embodiment, the C- terminal C may be used for conjugation to KLH. In one embodiment, the peptide may be produced with amide at its N-terminus. In one embodiment, Peptide (5) may be used in combination with peptides derived from the S1 subunit.
[00165] In one embodiment, Peptide (6) will induce antibodies against S2 subunit of S protein, that should inhibit its conformational changes and prevent fusion with cellular plasma membrane. In one embodiment, the N-terminal C may be used for conjugation to KLH. In one embodiment, the peptide may be produced with amide at its C-terminus. In one embodiment, Peptide (6) may be used in combination with peptides derived from the S1 subunit.
[00166] In one embodiment, Peptide (7) will induce antibodies against S2 subunit of S protein, that should inhibit its conformational changes and prevent fusion with cellular plasma membrane. In one embodiment, the N-terminal C may be used for conjugation to KLH. In one embodiment, the peptide may be produced with amide at its C-terminus. In one embodiment, Peptide (7) may be used in combination with peptides derived from the S1 subunit.
[00167] In one embodiment, Peptide (8) is derived from the surface of the S1 subunit. The designed peptide contains one B cell epitope located in the middle part of the peptide and two contact residues (Spike protein positions N439, Y449), which may be important for binding the virus to the ACE2 receptor. In one embodiment, the peptide- induced antibody may affect binding of virus to hACE2, thus inhibiting of entry into a host cell. N-terminal cysteine (C) is added for conjugation to KLH (the carrier protein) through a maleimide linker. In one embodiment, Peptide (8) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
[00168] In one embodiment, Peptide (9) is derived from the surface of the S1 subunit. The designed peptide contains one B cell epitope located in middle part of the peptide. In one embodiment, the peptide carries several contact residues (Spike protein positions T500, N501 , G502, Y505), which may play important roles in binding the virus to its receptor (ACE2). Blocking these residues by peptide-induced antibodies may be therapeutically effective to prevent the S1 subunit - ACE2 interaction. Two residues are added to the original SARS-CoV-2 spike protein sequence: N-terminal cysteine (C) is used for conjugation to KLH as a carrier protein using a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid. In one embodiment, Peptide (9) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
[00169] In one embodiment, Peptide (10) is derived from the surface amino acids of the S1 subunit. The peptide contains one B cell epitope located in middle region and two contact residues (Spike protein positions L455, F456), which may play important roles in binding the virus to receptor of host cell. In one embodiment, the peptide-induced antibodies may block binding of virus to hACE2 and entry of virus into the cell. The N- terminal cysteine (C) is added for conjugation to KLH (as a carrier protein) through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid. In one embodiment, Peptide (10) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
[00170] In one embodiment, Peptide (11) is derived from surface S1 subunit. The peptide contains one B cell epitope located in middle region. Alanine located at the position 10 (A475 of the Spike protein) of the peptide is one of the contact residues with ACE2, YQAGS residues (SEQ ID NO: 117) form a part of the contact surface of S1 with ACE2, which the virus can utilize to enter into the host cell. In one embodiment, the peptide-induced antibodies could have neutralizing activity and thus effectively block the virus from binding to the receptor of permissive cells. The N-terminal cysteine (C) is added for conjugation to KLH (as a carrier protein) through a maleimide linker. In one embodiment, Peptide (11 ) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
[00171] In one embodiment, Peptide (12) derived from the surface of S1 subunit. The designed peptide contains one B cell epitope located in central part of peptide. Threonine located at position 5 (the Spike protein position T415) and lysine at position 7 (the Spike protein position K417) were identified as the contact residues, which the virus may exploit to enter the host cell. In one embodiment, the binding of the peptide-induced antibodies to these strategic residues or near these residues may lead to the lowering number of virus -infected cells. N-terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker. For a better accessibility of predicted B- cell epitope to the immune system, serine was added to the N terminus. In one embodiment, Peptide (12) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
[00172] In one embodiment, Peptide (13) is derived from the surface of the S1 subunit. The designed peptide contains one B cell epitope located in medium part of peptide. The residues R3, D5, E6, Q9, T14 and K16 of the peptide were identified as part the S1 protein contact surface with ACE2, which the virus exploits to enter the host cell. In one embodiment, the blocking of the positions important for binding to human ACE2 by peptide-induced antibodies can lead to the lower affinity between the viral RBD and host ACE2 in the initial viral attachment step. In the end this lowering of affinity can lead to lowering of virus efficiency. The N-terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker. For a better accessibility of predicted B-cell epitope to the immune system, serine as a linker was added to the N terminus. In one embodiment, Peptide (13) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
[00173] In one embodiment, Peptide (14) is derived from the surface of the S1 subunit. The peptide contains one B cell epitope located in medium part of peptide and four residues which were identified as important contact sites which virus exploit for binding to host cell receptor. In one embodiment, the identified amino acids residues at the Spike protein positions F486 (phenylalanine), N487 (asparagine), Y489 (tyrosine) and Q493 (glutamine) enhance viral attachment to human ACE2. In one embodiment, the occupation (blocking) of these strategic residues by peptide-induced antibodies may be therapeutically effective. The N-terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid. In one embodiment, Peptide (14) may be used in combination with peptides derived from the S1 subunit, from the S2 subunit, or from both subunits.
[00174] In one embodiment, the polypeptide comprises one or more peptides of the S1 protein and/or the S2 protein. In one embodiment, the polypeptide comprises one or more epitopes from those disclosed on FIG. 15.
[00175] In one embodiment, the peptides may be linked directly as one sequence. In other embodiments, the peptides may be linked through a linker. In one embodiment, the polypeptide is Peptide (2).
[00176] Linkers may be used to link peptides and/or to conjugate the peptides to other molecules. Linkers may have one or more properties that may include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. In one embodiment, the linker is GSG. In another embodiment, the linker is GPAD (SEQ ID NO: 17). In one embodiment, the linker is selected from a GS-short linker GGGGSG (SEQ ID NO: 18), a GS-medium linker, and a GS-long linker GGGGSGGGGSGGGG (SEQ ID NO: 19). Linkers may be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, may be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage. In another embodiment, the linker is a cleavable linker, which may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition. The cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met-X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp-Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole. Protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used. The precise sequences are available in the art and the skilled person will have no difficulty in selecting a suitable cleavage site. By way of example, the protease cleavage region targeted by Factor Xa is I E G R (SEQ ID NO: 20). The protease cleavage region targeted by Enterokinase is D D D D (SEQ ID NO: 21). The protease cleavage region targeted by Thrombin is L V P R G (SEQ ID NO: 22). The cleavable linker region may be one that is targeted by endocellular proteases. Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage. Additional linkers include:
[00177] (GGGGS)n where n = 1 , 2, 3, or 4 (SEQ ID NO: 23)
[00178] (Gly)n where n = 6 or 8 (SEQ ID NO: 24)
[00179] (EAAAK)n where n = 1-3 (SEQ ID NO: 25)
[00180] A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 26)
[00181] PAPAP (SEQ ID NO: 27)
[00182] AEAAAKEAAAKA (SEQ ID NO: 28)
[00183] (Ala-Pro)n n - 10-34 aa (SEQ ID NO: 29)
[00184] VSQTSKLTR|AETVFPDV (SEQ ID NO: 30)
[00185] PLGlLWAc (SEQ ID NO: 31)
[00186] RVL|AEA (SEQ ID NO: 32)
[00187] EDVVCCISMSY (SEQ ID NO: 33)
[00188] GGIEGRIGS (SEQ ID NO: 34)
[00189] TRHRQPRIGWE (SEQ ID NO: 35)
[00190] AGNRVRRISVG (SEQ ID NO: 36) [00191] RRRRRRR|R|R (SEQ ID NO: 37)
[00192] GFLGI (SEQ ID NO: 38)
[00193] I = cleavable at this location
[00194] In one embodiment, the peptides may comprise and Aspartic acid (D) at the N-terminus (e.g., after Cys), which may be placed before, within, or after an SG spacer. Such peptides may be cleavable by hot formic acid after the D, which generates a peptide- specific fragment suitable for HPLC-UV or HPLC-MS/MS identification and/or quantification.
[00195] In one embodiment, Peptides (1) through (14) may be conjugated to other peptides, which may be, in some instances, B or T cell epitopes. In one embodiment, they are arranged as PADRE constructs.
[00196] The disclosure also provides for variants of the specific parent polypeptides identified above namely those of SEQ ID NOS 1 -16, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NOS 90-139, or those comprising one or more peptide epitopes disclosed in FIG. 15. A variant, which is also termed a "variant peptide" or "modified peptide" herein, is a peptide, that is derived from but not identical to a parent peptide as defined herein. A variant peptide may include a number of variations compared to the parent peptide as defined herein, for example to increase or decrease physical or chemical properties of the parent peptide as defined herein, for example to decrease its ability to resist oxidation, to improve or increase solubility in aqueous solution, to decrease aggregation, to decrease synthesis problems, etc. Furthermore, a variant peptide may comprise one or more of the same B or T cell epitopes as the parent peptide as defined herein. In one embodiment, this may be determined by the ability of the variant peptide to induce or stimulate in-vitro T cell proliferation using cultured PBMCs (peripheral blood monocytes) compared to the parent peptide as defined herein, optionally using same test conditions, or by the ability of the variant peptide to induce or stimulate production of cytokines, (e.g. cytokines, IL-5, IL-13 and/or IL-10) from T cells (obtained from cultured PBMC's) compared to the parent peptide as defined herein. [00197] In one embodiment, the epitopes of FIG. 15are predicted to be B cell epitopes. Various assays can be done to test for this activity, including the generation of neutralizing antibodies against SARS-Cov2 S protein upon injection into mice. Sera are collected and antibody titers are measured. Examples of such assays are given in the Examples section of this disclosure. It can also be tested whether sera from such mice induce antibody -dependent cellular cytotoxicity (ADCC) in vitro. Neutralizing antibodies reduce viral infectivity. In one embodiment, the epitopes of FIG. 15 may include B cell epitopes and the epitopes of FIG. 30 may include T cell epitopes. The latter were predicted using IEDB analysis resource Consensus tool in www.iedb.org; Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B. 2008. A systematic assessment of MFIC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol. 4(4):e1000048; Wang P, Sidney J, Kim Y, Sette A, Lund O, Nielsen M, Peters B. 2010. Peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinformatics. 11 :568.
[00198] As just mentioned, in one embodiment, the polypeptide is modified. In one embodiment, modification means that the polypeptide is not a fragment of the SARS Cov2 S protein and is different from the polypeptides whose sequence is specifically described in this disclosure by at least one amino acid residue either in sequence and/or in chemical modification. In one embodiment, an amide group is added at the 3' end of the peptide of the disclosure. In one embodiment, a Cys residue or a D residue, or an S residue is added at either the C terminus and/or N-terminus (or removed, in the case of Peptide (1) through Peptide (14)). In one embodiment the Cys residue is added through a linker. In one embodiment, the peptide can be modified by a chemoselective group, which allows coupling to a carrier ( e.g. azide binding to phosphine or alkyne). In one embodiment, the polypeptide may be modified by an internal bridge, short-range cyclization, methylation, amidation, acetylation or substitution with other chemical groups, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation. In one embodiment, a disulfide linkage may be present or absent in the peptides of the disclosure. In one embodiment, the polypeptide is modified by manosylation, glycosylation, amidation (specifically C-terminal amides), carboxylation or phosphorylation. In one embodiment, these modifications may preserve the biological activity of the original molecule. Functional derivatives of the peptides are also included in the present disclosure. Functional derivatives are meant to include peptides which differ in one or more amino acids in the overall sequence, which have deletions, substitutions, inversions or additions but preserve or improve the biological activity of the original peptide. In one embodiment, amino acid substitutions which may work not to essentially alter biological and immunological activities may be found. Amino acid replacements between related amino acids or replacements which may have occurred frequently in evolution include, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, lle/Val. Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence may provide advantageous physical, chemical, biochemical, and pharmacological properties, such as enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others. In one embodiment, the substitutions are located at the end of the amino acid chain. In one embodiment, the substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical nature, such as where leucine is replaced by isoleucine. Certain amino acid substitutions are more often tolerated than others, and these often show correlation with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement. Chemical and biological effects are not totally predictable and substitutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles. In some embodiments, the substitutions provide unexpected results.
[00199] In one embodiment, the polypeptide further comprises one or more moieties. In one embodiment, the moiety is a Cys residue. In one embodiment, the moiety is a carrier protein selected from any one of a Keyhole Limpet Hemocyanin (KLH) functional unit, a tetanus toxin heavy chain C fragment, a diphteria toxin, a diphtheria toxin variant CRM197, an H influenzae protein D, a Meningococcal outer membrane protein complex protein, an Outer-membrane lipoprotein carrier protein, or a Cholera toxin B subunit, a virus-like particle; biotin, avidin, streptavidin, neutravidin, serum albumin, keyhole limpet hemocyanin (KLH), an enzyme, a metallic nanomaterial, CRM197 and an outer membrane protein mixture from N. meningitidis (OMP), micro- and nano-particles of biodegradable polymers including polylactic, polycaproic, polyglycolic, polymalic, polybutyric acids and their combination and modifications; hydrogels of polyethyleneglycol and its modifications; hyaluronic acid, dextran, chitosan, liposome- polymer hybrid carrier, or a derivative or combination thereof. In one embodiment, the moiety is a Cys and a KLH. In one embodiment, the moiety is an azidopentanoyl moiety, which may or may not be conjugated to KLH. In one embodiment, the moiety or modification is an alkyne modification, a cyclooctyne derivative [e.g., BCN (Bicyclo[6.1 0]non-4- yne)-9-methanol; DBCO Dibenzocyclooctyne (11 ,12-didehydro-5, 6-dihydro-dibenz[b,f] azocine derivatives); DIFO 2-[(6,6-difluoro-4- cyclooctyn-1-yl)oxy] acetic acid; MOFO 2-(4-carboxybenzyl)- 2-fluorocyclooctyne.
[00200] In one embodiment, the variant may for instance include one or more deletions of amino acid residues from the N- and/or C- terminal end of the parent peptide as defined herein one or more additions of amino acid residues to the N- and/or C- terminal of the parent peptide as defined herein and/or one or more amino acid substitutions, additions or deletions within the amino acid sequence of the parent peptide as defined herein. One type of variant is a "derivative", where chemical modifications are introduced, for instance in the side-chains of one of more of the amino acid residues of the parent peptide's amino acid sequence (thus effectively resulting in a peptide that includes an amino acid residue substitution relative to the parent peptide as defined herein). A derivative can also include a chemical modification that involves the N-terminal amino group and/or the C-terminal COOH group. Derivatives are described in more detail herein. It is important to note that some derivatives of the parent peptides as defined herein, are those that could be obtained by substituting an amino acid residue with another naturally occurring amino acid residue, whereas other derivatives involve chemical modifications that result in the provision of peptides that could not be encoded by a nucleic acid sequence.
[00201] In one embodiment, a longer variant of the parent peptide as defined herein may be up to 60 amino acids in length, for example up to 55, 50, 45 amino acids in length. More typically, a longer variant peptide is up to about 100 amino acids in length, such as up to 70 amino acids in length. The longer variant may comprise the amino acid sequence of a parent peptide disclosed herein, or an amino acid sequence having at least 85% identity or similarity over the length of the amino acid sequence of the parent peptide as defined herein, or a fragment thereof, such as over at least 12 contiguous amino acids, for example over at least 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein. Typically, the longer variant comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity or similarity over the length of the amino acid sequence of the parent peptide as defined herein, or over at least 12 contiguous amino acids, for example over at least 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein. Therefore, in some embodiments, a variant of the parent peptide as defined herein is a longer peptide up to 50 amino acid residues in length that comprises one or more additional amino acid residues at the N- and/or C- terminal end of the parent peptide as defined herein, or comprises an amino acid sequence having at least 80%, such as at least 85%, 90% or 95% identity or similarity over at least 14 contiguous amino acids of the parent peptide as defined herein, such as over at least 15, 16, 17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein.
[00202] A variant of the parent peptide may also include a fragment of a parent peptide as defined herein disclosed herein. A fragment of the parent peptide can have one or more amino acids less than the parent peptide as defined herein, either comprising deletions from within the amino acid sequence of the parent peptide as defined herein and/or amino acid deletions from the N- and/or C- terminus of the parent peptide as defined herein. Typically, a fragment will have a length of at least 12 amino acids, for example at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 amino acids, and will have at least 65% identity or similarity over the length of the fragment, or over the length of at least 12 contiguous amino acids, when aligned with the parent peptide as defined herein. In some embodiments, the percentage identity or similarity is at least 70%, 75%, 80%, 85%, 90% or 95% over the length of the fragment, or over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide as defined herein. Therefore, in some embodiments, a variant thereof may be a shorter peptide comprising an amino acid sequence having at least 80%, such as at least 85%, 90% or 95% identity or similarity over at least 14 contiguous amino acids of the parent peptide as defined herein, such as over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide.
[00203] In some embodiments, the variant is a peptide consisting of 25- 45 amino acids and comprises an amino acid sequence having at least 80%, such as at least 85%, 90% or 95% identity or similarity over at least 25 or 30 contiguous amino acids of the parent sequence as defined here. In still other particular embodiments, the variant is a peptide consisting of 25-45 amino acids and comprises an amino acid sequence having at least 80% identity or similarity over at least 20-30 or 30-35 contiguous amino acids with the sequence of a parent peptide as defined herein. In still other particular embodiments, the variant is a peptide consisting of 15-25 amino acids and comprises an amino acid sequence having at least 80%, such as at least 85%, or at least 90%, or at least 95%, or at least 98% identity or similarity over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids of the parent peptide sequence as defined herein. In still other particular embodiments, the variant is a peptide consisting of 16-25 amino acids and comprises an amino acid sequence having at least 80% identity or similarity over at least 12, 13, 14,15, 16,17, 18, 19, 20, 25, 30, 40 contiguous amino acids with the sequence of a parent as defined herein further embodiments, the at least 80% identity or similarity is over at least 16, 17, 18 or 19 contiguous amino acids with the sequence of a parent peptide as defined herein and the percent identity or similarity may be at least is 80%, such as at least 85%, 90% or 95% over at least 16, 17, 18 or 19 contiguous amino acids with the sequence of a parent peptide . The term "identity" and "identical" and grammatical variations thereof, as used herein, mean that two or more referenced entities are the same (e.g., amino acid sequences). Thus, where two peptides are identical, they have the same amino acid sequence. The identity can be over a defined area, e.g. over at least 12, 13, 14, 15 or 16 contiguous amino acids with the sequence of a parent peptide as defined herein, optionally wherein the alignment is the best fit with gaps permitted. For example, to determine whether a variant peptide has at least 80% similarity or identity over at least 15 contiguous amino acid residues of the sequence of a parent peptide as defined herein , the variant peptide may be aligned with the parent peptide as defined herein and the percent identity calculated with respect to the identical amino acid residues found within the amino acid sequence of the variant peptide that overlaps with the 15 contiguous amino acids with the sequence of a parent peptide as defined herein.
[00204] In one embodiment, the disclosure provides an isolated polypeptide comprising one or more peptides that differ from the peptides of amino acid sequences of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 , SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NOS 90-140 by 1 , 2, 3, 4,
5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29,
30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acid residues, wherein the difference is an insertion, substitution, or deletion.
[00205] As mentioned, a variant of a parent peptide as defined herein may comprise additional amino acids or may consist of a fragment of the parent peptide as defined herein. In one embodiment, the polypeptide further comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,
11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33,
34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56,
57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79,
80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 96, 97, 98, 99, 100 or more additional amino acid residues at the N-terminus, in the middle of the sequence, and/or at the C-terminus, but the polypeptide is still not a fragment of SARS Cov2 S protein. [00206] In one embodiment, the polypeptide is at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21 , at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31 , at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41 , at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51 , at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58 residues, at most 59, at most 60, at most 61 , at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71 , at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 90, at most 100 amino acid residues in length.
[00207] In one embodiment, the polypeptide is between about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, and 90 to 100 amino acid residues in length.
[00208] In one embodiment, the polypeptide is at least, about, or no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or similar to any one of the polypeptides whose specific sequence is disclosed herein (SEQ ID NOs: 1 through 16), by at least one method, provided that the polypeptide is not a fragment of the SARS Cov2 S protein in that it comprises at least different amino acid residue in sequence and/or chemical modification.
[00209] In some embodiments of the disclosure, a variant of a parent peptide as defined herein comprises: a) one or more (e.g. 1 , 2, or 3) amino acid substitutions in the sequence of the parent peptide as defined herein, for example a glutamate residue at the N-terminus of the parent peptide as defined herein may be replaced with pyroglutamate and/or one or more cysteine residues in the parent peptide as defined herein may be replaced with serine or 2-aminobutyric acid; and/or b) one or more amino acid additions (e.g. 1 , 2, 3, 5, 4, 6, 7, 8) to the sequence of a parent peptide as defined herein , for example wherein the variant comprises one or more (e.g. 1 , 2, 3, or 4) lysine residue(s) and/or one or more (e.g. 1 , 2, 3, or 4) arginine residue(s) and/or one or more positively charged residues added at the N- and/or C-terminus of the parent peptide as defined herein or to a fragment of the parent peptide as defined herein consisting of at least 14 contiguous amino acids of the parent peptide as defined herein (such as at least 15 contiguous amino acids) ; and/or c) one or more amino acid deletions from the parent peptide as defined herein, for example wherein a hydrophobic residue up to three amino acids from the N- or C- terminus of the parent peptide as defined herein are deleted; and/or any two consecutive amino acids comprising the sequence Asp-Gly up to four amino acids from the N- or C- terminus of the parent peptide as defined herein are deleted. Furthermore, in some embodiments, a variant of a parent peptide as defined herein may comprise one, two, three or more lysine or arginine amino acid residue(s) added to the N- and/or C-terminus of the parent peptide as defined herein that have been extended with one or more, e.g. 1 , 2, 3, 4, or 5 amino acid residues.
[00210] A parent peptide as defined herein may be modified to contain "non-natural" modifications. Such peptides are also referred to as variants herein and more specifically they are referred to as derivative peptides or derivatives. The term derivative refers to a chemically modified form of a peptide disclosed herein. Typically, a derivative is formed by reacting a functional side group of an amino acid (e.g. amino, sulfhydryl or carboxy- group) with another molecule to form a covalent or non- covalent attachment of any type of molecule (naturally occurring or designed), such as a sugar moiety. Specific examples of derivatives of a peptide include glycosylation, acylation (e.g. acetylation), phosphorylation, amidation, formylation, ubiquitination and derivatization by protecting/blocking groups and any of numerous chemical modifications. Additional specific non- limiting examples are tagged peptides, fusion peptides, chimeric peptides including peptides having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide. Typically, a derivative comprises one or more modifications, for example selected from any of: (a) N-terminal acylation (e.g. acetylation or formylation); (b) C-terminal amidation (e.g. reaction with ammonia or an amine) ; (c) one or more hydrogens on the side chain amines of arginine and/or lysine replaced with a methylene group; (d) glycosylation and/or (e) phosphorylation. In a particular embodiment, the peptides are amidated at the C-terminal end.
[00211] Peptides are typically provided in the form of a salt, for example as a pharmaceutically acceptable and/or a physiologically acceptable salt. For example, the salt may be an acid addition salt with an inorganic acid, an acid addition salt with an organic acid, a salt with a basic inorganic acid, a salt with a basic organic acid, a salt with an acidic or basic amino acid or a mixture thereof. Typical examples of an acid addition salts with an inorganic acid are selected from any of the salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, or the like. An acid salt with an organic acid may be selected from any of the salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or the like. Salts with an inorganic base may be selected from a salt of an alkali metal salts such as sodium salts and potassium salts; alkali earth metal salts such as calcium salts and magnesium salts; and aluminum salts and ammonium salts. Salts with a basic organic base may be selected from any salt with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triet hanolamine, dicyclohexylamine, N,N-dibenzylethylenediamine, caffeine, piperidine, and pyridine. Salts with a basic amino acid may be selected from any salt with arginine, lysine, ornithine, or the like. Salts with an acidic amino acid may be selected from any salt with aspartic acid, glutamic acid, or the like. In one embodiment, the peptide is a TFA salt.
[00212] An isolated and purified peptide of the disclosure and fragments and variants thereof may be produced synthetically or recombinantly. A polypeptide that contains an epitope that induces an immune response against coronavirus may be synthesized by standard chemical methods, including synthesis by automated procedure. Alternatively, the polypeptide may be produced recombinantly. For example, the polypeptide may be expressed from a polynucleotide that is operably linked to an expression control sequence, such as a promoter, in a nucleic acid expression construct. The polypeptide may be expressed in mammalian cells, yeast, bacteria, insect or other cells under the control of appropriate expression control sequences. Cell-free translation systems may also be employed to produce such coronavirus proteins using nucleic acids, including RNAs, and expression constructs. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are routinely used by persons skilled in the art, and may include plasmids, cosmids, shuttle vectors, viral vectors, and vectors comprising a chromosomal origin of replication as disclosed therein. As will be appreciated by those of ordinary skill in the art, a nucleotide sequence encoding a coronavirus S polypeptide or variant thereof may differ from the sequences presented herein due to, for example, the degeneracy of the genetic code. A nucleotide sequence that encodes a coronavirus polypeptide variant includes a sequence that encodes a homologue or strain variant or other variant. Variants may result from natural polymorphisms or may be synthesized by recombinant methodology, for example to introduce an amino acid mutation, or chemical synthesis, and may differ from wild-type polypeptides by one or more amino acid substitutions, insertions, deletions, and the like. [00213] The current disclosure also provides nucleic acid molecules encoding any one or more of the polypeptides disclosed herein. In one embodiment, the nucleic acid is DNA. In another embodiment, the nucleic acid is RNA or modified RNA. In one embodiment, the nucleic acid sequence is codon optimized to improve expression of the polypeptide.
[00214] In another embodiment, the disclosure provides vectors comprising the nucleic acids of the disclosure. In one embodiment, the vector is an expression vector. In one embodiment, the vector is a retroviral vector, lentiviral vector, adenoviral vector, poxvirus vector, plasmid, bacterial vector. In one embodiment, the vector is a recombinant vaccinia virus.
[00215] In another embodiment, the disclosure provides recombinant host cells comprising a nucleic acid and/or vector of the disclosure. In one embodiment, the recombinant host cell is selected from a prokaryotic cell (e.g. E. coli) or an eukaryotic cell (e.g. Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NS0, Sp2/0), a monkey kidney cell (COS-7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TRI cell, an MRC 5 cell, a FS4, and MDCK cell.
[00216] IMMUNOGENIC COMPOSITIONS
[00217] In one embodiment, the disclosure provides immunogenic compositions that are useful in the prevention or treatment of severe acute respiratory syndrome (SARS) or other SARS-CoV-related disease, including Covid-19. In one embodiment, the immunogenic composition comprises one or more of the isolated or purified polypeptides disclosed in this disclosure, including, for example, Peptides (1) through (7), Peptides (8) through (14), fragments, variants, and/or derivatives of the same. The composition may comprise one or more of any other polypeptides described in this disclosure.
[00218] An “immunogenic composition,” as used in the present disclosure refers to a composition that comprises an immunogenic component capable of provoking an immune response in an individual, such as a human, or an animal (such as a mouse or a rat), optionally when suitably formulated with an adjuvant. Accordingly, in one embodiment the disclosure provides an immunogenic composition comprising an immunogenic SARS coronavirus S (spike) polypeptide, or a fragment or variant thereof, and an adjuvant. In another embodiment of the disclosure, the immunogenic composition of the disclosure is a vaccine, i.e. the immunogenic composition is capable of provoking a protective immune response against a SARS-CoV infection.
[00219] In one embodiment, the immunogenic composition of the present disclosure comprises one or more immunogenic SARS coronavirus S (spike) polypeptides, including fragments and variants thereof. The immunogenic S polypeptides may comprise any portion of an S protein that has an epitope capable of eliciting an immune response, for example an epitope capable of eliciting production of a neutralizing antibody and/or stimulating a cell-mediated immune response, against a SARS-CoV infection. In some embodiments, the immunogenic composition is a vaccine. A “vaccine” is an immunogenic composition capable of provoking a protective immune response against a SARS-CoV infection. As used in this disclosure, the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody.Exemplary epitopes identified in this disclosure can be found at FIG. 15 [00220] Induction of an immune response in a subject or host (human or non-human animal) by a SARS-CoV S polypeptide, fragment, or variant described herein, may be determined and characterized by methods described herein and routinely practiced in the art. These methods include in vivo assays, such as animal immunization studies, for example, using a rabbit, mouse, ferret, civet cat, African green monkey, or rhesus macaque model, and any one of a number of in vitro assays, such as immunochemistry methods for detection and analysis of antibodies, including Western immunoblot analysis, ELISA, immunoprecipitation, radioimmunoassay, and the like, and combinations thereof. Other methods and techniques that may be used to analyze and characterize an immune response include neutralization assays (such as a plaque reduction assay or an assay that measures cytopathic effect (CPE) or any other neutralization assay practiced by persons skilled in the art). These and other assays and methods known in the art may be used to identify and characterize S protein immunogens and variants thereof that have at least one epitope that elicits a protective humoral or cell-mediated immune response against SARS coronavirus. The statistical significance of the results obtained in the various assays may be calculated and understood according to methods routinely practiced by persons skilled in the relevant art.
[00221] In one embodiment, the SARS-CoV-2 polypeptide is a peptide selected from the following:
[00222] Peptide (1 ): 442- CGSGDSKVGGNYNKLYRLFE -456 (SEQ ID NO: 1 )
[00223] Peptide (2): 442- CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ -506 (SEQ ID NO: 2)
[00224] Peptide (3): 469-SGSTEIYQAGSTPCNGVEGFNCYFAK-490 (SEQ ID NO: 3) [00225] Peptide (4): 483-CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE-516 (SEQ ID NO: 4) [00226] The differences between the peptides and the naturally existing SARS Cov2 S1 polypeptide are underlined. These peptides have SEQ ID Nos: 1 , 2, 3, 4.
[00227] In one embodiment, the SARS-CoV-2 polypeptide is a peptide selected from the following:
[00228] Peptide (5): 804-QILPDPSKPSKRSC-816 (SEQ ID NO: 5)
[00229] Peptide (6): 919-CNQKLIANQFNSAIGKIQDSLSS-940 (SEQ ID NO: 6)
[00230] Peptide (7): 695-CYTMSLGAENSVAYS -708 (SEQ ID NO: 7):
[00231] Peptide (8): 438-CSNNLDSKVGGNYN-450, SEQ ID NO: 83;
[00232] Peptide (9): 496- CDGFQPTNGVGYQPYR-509, SEQ ID NO: 84;
[00233] Peptide (10): 454-CDRLFRKSNLKPFE-465, SEQ ID NO: 85;
[00234] Peptide (11 ): 466-CRDISTEIYQAGSTP-479, SEQ ID NO: 86;
[00235] Peptide (12): 412-CSPGQTGKIADYNYKLPD-427, SEQ ID NO: 87;
[00236] Peptide (13): 403-CSRGDEVRQIAPGQTGK-417, SEQ ID NO: 88;
[00237] Peptide (14): 481 -CDNGVEGFNGYFPLQSYG-496, SEQ ID NO: 89; Peptides
(1) through (14) without the N-terminal or C-terminal Cys (SEQ ID NOs. 90 through 102) and/or with an extra Cys (SEQ ID NO. 103 through 106);
[00238] Peptide based on peptide (3) but wherein the two underlined Cys are any amino acids (SEQ ID NO: 115);
[00239] Peptides (1) through 14 without the spacer(s) and/or linker(s) (SED ID NOs.109 through 112);
[00240] Peptides derived from Peptides (1 ) through (14), wherein any one or more of the underlined amino acids is replaced by one or more different amino acids (SEQ ID NOS 107-108 and 114-116) or is absent; and
[00241] Any other Peptide selected from those of SEQ ID NO: 118-140;.
[00242] In one embodiment, the peptide comprises, consists of, or consists essentialy of one or more of the following sequences:
[00243] Peptide (1A): 442- GSGDSKVGGNYNKLYRLFEC -456, SEQ ID NO: 90; [00244] Peptide (2A): 442- DSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQC- 506, SEQ ID NO: 91 ; [00245] Peptide (3A): 469-SGSTEIYQAGSTPCNGVEGFNCYFAKC-490, SEQ ID
NO: 92;
[00246] Peptide (4A): 483-VEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFEC-
516, SEQ ID NO: 93;
[00247] Peptide (5A): 804-QILPDPSKPSKRSC-815, SEQ ID NO: 106;
[00248] Peptide (6A): 919-NQKLIANQFNSAIGKIQDSLSSC-940, SEQ ID NO: 94; [00249] Peptide (7A): 695-YTMSLGAENSVAYSC -708, SEQ ID NO: 95;
[00250] Peptide (8A): 438-SNNLDSKVGGNYNC-450 [SEQ ID NO: 96;
[00251] Peptide (9A): 496- DGFQPTNGVGYQPYRC-509, SEQ ID NO: 97;
[00252] Peptide (1 OA): 454-DRLFRKSNLKPFEC-465, SEQ ID NO: 98;
[00253] Peptide (11 A): 466-RDISTEIYQAGSTPC-479, SEQ ID NO: 99;
[00254] Peptide (12A): 412-SPGQTGKIADYNYKLPDC-427, SEQ ID NO: 100;
[00255] Peptide (13A): 403-SRGDEVRQIAPGQTGKC-417, SEQ ID NO: 101 ;
[00256] Peptide (14A): 481 -DNGVEGFNGYFPLQSYGC-496, SEQ ID NO: 102;
[00257] Peptide (3B): 469-CSGSTEIYQAGSTPCNGVEGFNCYFAK-490, SEQ ID NO: 103;
[00258] Peptide (3C): 469-STEIYQAGSTPCNGVEGFNCYFAKC-490, SEQ ID NO: 104;
[00259] Peptide (3D) 469-STEIYQAGSTPCNGVEGFNCYFC-490, SEQ ID NO: 105;
[00260] Peptide (2D): CDSKVGGNYNYLYRXLQSYGFQPTNGVGYQ, SEQ ID NO:
107 (X CAN BE ANY ONE OR MORE AMINO ACIDS);
[00261] Peptide (2E): DSKVGGNYNYLYRXLQSYGFQPTNGVGYQC, SEQ ID NO:
108 (X CAN BE ANY ONE OR MORE AMINO ACIDS);
[00262] Peptide (8B): 438-NNLDSKVGGNYN-450, SEQ ID NO: 109 [00263] Peptide (9B): 496- GFQPTNGVGYQPYR-509, SEQ ID NO: 110;
[00264] Peptide (12B): 412-PGQTGKIADYNYKLPD-427, SEQ ID NO: 111 ;
[00265] Peptide (13B): 403-RGDEVRQIAPGQTGK-417, SEQ ID NO: 112;
[00266] Peptide (14B): 481 -NGVEGFNGYFPLQSYG-496, SEQ ID NO: 113;
[00267] Peptide (1 B): 442- DSKVGGNYNXLYRLFX -456, SEQ ID NO: 114; (X CAN BE ANY ONE OR MORE AMINO ACIDS)
[00268] Peptide (3B): 469-STEIYQAGSTPXNGVEGFNCXFX-490, SEQ ID NO: 115; (X CAN BE ANY ONE OR MORE AMINO ACIDS) ;
[00269] Peptide (4B): 483-VEGFNXYFPLQSYGFQPTNGVGYQPYRVVVLSFE-
516, SEQ ID 116 (X CAN BE ANY ONE OR MORE AMINO ACIDS);
[00270] DSKVGGNYNKLYRLFEGSGC (SEQ ID NO: 140)
[00271] DSKVGGNYNKLYRLFEDSGC (SEQ ID NO: 141 )
[00272] DSKVGGNYNKLYRLFEDGC (SEQ ID NO: 142)
[00273] DSKVGGNYNKLYRLFEDC (SEQ ID NO: 143)
[00274] STEIYQAGSTPCNGVEGFNCYFAKSGC (SEQ ID NO: 144)
[00275] STEIYQAGSTPCNGVEGFNCYFAKSDGC (SEQ ID NO: 145)
[00276] STEIYQAGSTPCNGVEGFNCYFAKGDC (SEQ ID NO: 146)
[00277] STEIYQAGSTPCNGVEGFNCYFAKDC (SEQ ID NO: 147)
[00278] SDSTEIYQAGSTPCNGVEGFNCYFAK (SEQ ID NO: 148)
[00279] SDSTEIYQAGSTPCNGVEGFNCYF (SEQ ID NO: 149)
[00280] SDGSTEIYQAGSTPCNGVEGFNCYFAK (SEQ ID NO: 150)
[00281] SDGSTEIYQAGSTPCNGVEGFNCYF (SEQ ID NO: 151 )
[00282] STEIYQAGSTPCNGVEGFNCYFAKC (SEQ ID NO: 104)
[00283] STEIYQAGSTPCNGVEGFNCYFAKDC (SEQ ID NO: 147)
[00284] STEIYQAGSTPCNGVEGFNCYFAKGDC (SEQ ID NO: 146)
[00285] STEIYQAGSTPCNGVEGFNCYFGC (SEQ ID NO: 152)
[00286] STEIYQAGSTPCNGVEGFNCYFGDC (SEQ ID NO: 153)
[00287] CSTEIYQAGSTPCNGVEGFNCYFAK (SEQ ID NO: 154)
[00288] CDSTEIYQAGSTPCNGVEGFNCYFAK (SEQ ID NO: 155)
[00289] CDSTEIYQAGSTPCNGVEGFNCYF (SEQ ID NO: 156)
[00290] CQILPDPSKPSKRS (SEQ ID NO: 106)
[00291] CSQILPDPSKPSKRS (SEQ ID NO: 157)
[00292] CGSQILPDPSKPSKRS (SEQ ID NO: 158)
[00293] QILPDPSKPSKRSDC (SEQ ID NO: 159)
[00294] NNLDSKVGGNYNSC (SEQ ID NO: 160) [00295] NNLDSKVGGNYNSDC (SEQ ID NO: 161 )
[00296] GFQPTNGVGYQPYRC (SEQ ID NO: 162)
[00297] GFQPTNGVGYQPYRDC (SEQ ID NO: 163)
[00298] GFQPTNGVGYQPYRGDC (SEQ ID NO: 164) [00299] RLFRKSNLKPFEDC (SEQ ID NO: 165)
[00300] RLFRKSNLKPFEGDC (SEQ ID NO: 166)
[00301] CDGRLFRKSNLKPFE (SEQ ID NO: 167)
[00302] PGQTGKIADYNYKLPDC (SEQ ID NO: 168)
[00303] PGQTGKIADYNYKLPDSC (SEQ ID NO: 169) [00304] CRGDEVRQIAPGQTGK (SEQ ID NO: 170)
[00305] RGDEVRQIAPGQTGKSC (SEQ ID NO: 171)
[00306] RGDEVRQIAPGQTGKSDC (SEQ ID NO: 172) [00307] NGVEGFNGYFPLQSYGDC (SEQ ID NO: 173) [00308] CSYTMSLGAENSVAYS (SEQ ID NO: 174)
[00309] CDSYTMSLGAENSVAYS (SEQ ID NO: 175)
[00310] RLFRRSNLKPFE (SEQ ID NO: 176)
[00311] RGDEVIQIAPGQTGK (SEQ ID NO: 177)
[00312] RGDEVREIAPGQTGK (SEQ ID NO: 178)
[00313] RGDEVIEIAPGQTGK (SEQ ID NO: 179)
[00314] RDISTEVYQAGSTP (SEQ ID NO: 180)
[00315] RDISTEIYQASSTP (SEQ ID NO: 181 )
[00316] RDISTEVYQASSTP (SEQ ID NO: 182)
[00317] STEIYQAGSTPCNGAEGFNCYF (SEQ ID NO: 183) [00318] STEIYQASSTPCNGVEGFNCYF (SEQ ID NO: 184) [00319] STEVYQAGSTPCNGVEGFNCYF (SEQ ID NO: 185) [00320] STEIYQASSTPCNGAEGFNCYF (SEQ ID NO: 186) [00321] STEVYQASSTPCNGVEGFNCYF (SEQ ID NO: 187) [00322] STEVYQASSTPCNGAEGFNCYF (SEQ ID NO: 188) [00323] NGAEGFNGYFPLQSYG (SEQ ID NO: 189)
[00324] GFQPTNGVGYQPHR (SEQ ID NO: 190) [00325] NGVEDFNGYFPLQSYG (SEQ ID NO: 191)
[00326] NGVEGVNGYFPLQSYG (SEQ ID NO: 192)
[00327] NGVKDVNGYFPLQSYG (SEQ ID NO: 193)
[00328] NGVEGFNGYPPLQSYG (SEQ ID NO: 194)
[00329] NGVEGFNGYFPLKSYG (SEQ ID NO: 195)
[00330] SNNLDSQVGGNYN (SEQ ID NO: 196)
[00331] SNNLDSKAGGNYN (SEQ ID NO: 197)
[00332] SNNLDSKVGGNYD (SEQ ID NO: 198)
[00333] DSQVGGNYNKLYRLF (SEQ ID NO: 199)
[00334] DSKAGGNYNKLYRLF (SEQ ID NO: 200)
[00335] DSKVGGNYDKLYRLF (SEQ ID NO: 201 )
[00336] DSKVGGNYNKLFRLF (SEQ ID NO: 202)
[00337] DSKVGGNYNKLYRFF (SEQ ID NO: 203)
[00338] PGQTGEIADYNYKLPD (SEQ ID NO: 204); and [00339] RGDEVRQIAPGQTGE (SEQ ID NO: 205).
[00340] In one embodiment, the composition comprises one or more fragments, variants, or derivatives of any one of Polypeptides (1 ) through (7). In one embodiment, the composition comprises one or more immunogenic SARS coronavirus S (spike) polypeptides consisting of Peptides (1) through (7), with or without an additional Cys residue at either the N- or C- terminus, and an adjuvant. In one embodiment, the composition comprises two of the listed polypeptides described herein. For example, any combination of peptide (1) and (2), (1) and (3), (1 ), and (5), 1 and (6), (1) and (7), (2) and (3), and any other one of the 21 possible two-peptide combinations of the seven listed peptides is within the scope of the disclosure.
[00341] In one embodiment, the composition comprises three polypeptides described herein. Exemplary combinations include that combination of peptides (1), (2) and (3); (1 ), (2) and (4); (1), (3), and (4); and (2), (3), (4)) and any other one of the 35 combination of three peptides that may be made with the seven specifically listed peptides in one embodiment, the composition comprises four of the peptides described herein in one embodiment, the composition comprises five, six, seven, eight, nine, ten, or any other combination of the peptides disclosed herein. Peptide combinations where one or more peptides have been replaced by modified forms of the same peptides (variants) are also included in this disclosure.
[00342] In one embodiment, the polypeptides are linked to one another, directly or via a linker. In one embodiment, the polypeptides exist in the composition as separate peptides.
[00343] In one embodiment, an adjuvant or an adjuvant component in the broadest sense is typically a (e.g. pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine. Conventionally the term refers in the context of the disclosure to a compound or composition that serves as a carrier or auxiliary substance for immunogens and/or other pharmaceutically active compounds. In this disclosure, the term is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or co-administered with an adjuvant in question. In the context of the present disclosure an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present disclosure. For example, an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent. Typically, "adjuvant" or "adjuvant component" has the same meaning and may be used mutually. Adjuvants may be divided, e.g., into immunopotentiators, antigenic delivery systems or even combinations thereof. In one embodiment, the adjuvant is selected from the group consisting of mineral substances, aluminum hydroxide, aluminum phosphate, bacterial extracts (e.g., bacterial liposaccharides, Freund's adjuvants, and/or MDP), oily emulsions, saponin, squalene, potassium aluminium sulfate, calcium phosphate hydroxide, a TLR agonist, CRM197, Montanide®, Freund's incomplete adjuvant, MF59, a CpG oligonucleotide, iscoms, iscom matrix, ISCOMATRIX™ adjuvant, Matrix M™ adjuvant, Matrix C™ adjuvant, Matrix Q™ adjuvant, AblSCQ®-100 adjuvant, AblSC()®-300 adjuvant, ISCOPREP™, an ISCOPREP™ derivative, adjuvant containing ISCOPREP™ or an ISCOPREP™ derivative, monophosphoryl lipid A ((3-0-desacyl-4'-monophosphoryl lipid A; MPL), AS01 (a liposome-based formulation of MPL and QS-21), AS04 (a liposome-based formulation of MPL and aluminium hydroxide), QS-21 , a QS-21 derivative, an adjuvant containing QS-21 or a QS21 derivative, and a combination of any of the foregoing. In one embodiment, the adjuvant is aluminum hydroxide.
[00344] In one embodiment, the immunogenic composition comprises in addition to the peptide combination, therapeutically inactive ingredients, such as a pharmaceutically acceptable or physiologically acceptable excipient and/or carrier, which are well-known to the person skilled in the art and may include, but are not limited to, solvents, emulsifiers, wetting agents, plasticizers, solubilizers (e.g. solubility enhancing agents) coloring substances, fillers, preservatives, anti-oxidants, anti-microbial agents, viscosity adjusting agents, buffering agents, pH adjusting agents, isotonicity adjusting agents, mucoadhesive substances, and the like. Examples of formulation strategies are well- known to the person skilled in the art).
[00345] In some embodiments, the immunogenic composition may be formulated for parenteral administration, such as formulated for injection, e.g. subcutaneous and/or intradermal injection. Therefore, in some embodiments, the immunogenic composition may be a liquid (i.e. formulated as a liquid), including a solution, a suspension, a dispersion, and a gelled liquid. For example, a liquid immunogenic composition may be formed by dissolving a powder, granulate or lyophilizate of a peptide combination described herein in a suitable solvent and then administering to a subject. Suitable solvents may be any solvent having physiologically acceptable properties and able to dissolve the peptide combination in desired concentrations. A desired concentration may depend on the aliquot to be administered (i.e. to be injected) and the desired single dose. In one embodiment, for the purposes of injection the aliquot is in the range of about 10 to 500 microliters, e.g. 50 to 300 microliters or less and a desired single dose is within range of 1 to 1000 nanomole. Therefore, a suitable solvent should be able to dissolve any peptide of the combination to achieve a final concentration of about 1 to 1000 mM for each of the peptides. Thus, in one embodiment, a liquid composition comprises each of the peptides of the combination in a concentration of 10 to 800 pM, for Example 20 to 500 pM or 20 to 300 pM. Typically, the concentration of each peptide is the same, such as in an equimolar concentration, but each peptide of the composition may be present in different concentrations. Typically, the solvent is an aqueous solution, optionally mixed with other solvents. Thus, a solvent may comprise at least 60% w/w of water, e.g. at least 65% w/w, 70% w/w, 75% w/w, 80% w/w , 85% w/w, 90% w/w or 95% w/w, 99% w/w of water, such as distilled water, such as sterile water. In some embodiments, the solvent is sterile distilled water, e.g. water for injection. An aqueous solution may comprise other solvents than water, for example DMSO (dimethylsulfoxide), glycerol, ethanol, acetonitrile, vegetable or synthetic oils. The pH of the aqueous phase of the solvent may be in a physiological acceptable range, typically in the range of 3 to 9, such as in the range of pH 3 to 8, such as in the range of pH 4 to 8, such as in the range of 5 to 8, such as in the range of 6 to 8. Thus, the liquid formulation may comprise a pH controlling agent or buffering agent
(e.g. citrate buffer, phosphate buffer, acetate buffer), optionally the pH may be adjusted with dilutions of strong base (e.g. sodium hydroxide or the like) and/or dilutions of strong acids (e.g. hydrochloric acid). Typically, the liquid formulation is isotonic, and optionally sterile. Therefore, in some embodiments, the formulation comprises saline, such as isotonic saline. The liquid may contain additional excipients, such as another solvent, a solubilizing enhancing agent (e.g. polyoxyethylene (20) sorbitan monolaurate (Tween® 20), ionic and non-ionic emulsifiers (e.g. poloxamers (Kolliphor®)), a dispersant, a thickener, a preservative, an anti-microbial agent, and/or an antioxidant. Non- limiting illustrative examples of solvents include water, saline, DMSO, glycerol, ethanol, acetonitrile, vegetable or synthetic oil.
[00346] Some peptides are known to be prone to oxidation or being unstable when exposed to water for a long period. Therefore, to achieve storage stable compositions, a immunogenic composition may be formulated to contain only a limited amount of water or aqueous solution, e.g. containing less than 10% w/w of water or aqueous solution, such as less than 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5% w/w of water or aqueous solution. Examples of immunogenic compositions with limited levels of water may include granulates, powders, for example lyophilizates, i.e. freeze-dried powders. Typically, the freeze-dried composition may be dissolved before use, for example dissolved in an aqueous, optionally sterile, solution, for example a solution having a pH in the range of 3-9, such as pH in the range of 3 to 8, such as pH in the range of 4 to 8.
[00347] A lyophilizate may contain additional ingredients, e.g. bulking agents and lyoprotectants e.g. sucrose, lactose, trehalose, mannose, mannitol, sorbitol, glucose, raffinose, glycine, histidine or mixtures thereof), buffering agents (e.g. sodium citrate, sodium phosphate, disodium phosphate, sodium hydroxide, Tris base, Tris acetate, Tris HCI or mixtures thereof), antioxidants, antimicrobial agents, solubilizers (e.g. polyoxyethylene (20) sorbitan monolaurate (Tween® 20))
[00348] Another aspect of the disclosure relates to a kit comprising a compartment and instructions, wherein the compartment comprises a immunogenic composition as described herein for single, sequential or simultaneous administration, and wherein the instructions are for use in treating allergy to grass, such as grass. A kit may further comprise packaging material comprising corrugated fiber, glass, plastic, foil, ampules, vials, blister pack, preloaded syringes or tubes, optionally that maintain sterility of the components. A kit may further comprise labels or inserts comprising printed matter or computer readable medium optionally including identifying components, dose amounts, clinical pharmacology and instructions for the clinician or for a subject using one or more of the kit components, prophylactic or therapeutic benefits, adverse side effects or manufacturer information.
[00349] In one embodiment, the kit additionally comprises a container comprising a solvent for dissolving the composition before use. Examples of suitable solvents are described supra. Optionally, the kit may also comprise a device for use in parenteral injection, e.g. for injecting the composition (e.g. dissolved composition) to a subcutaneous or intradermal tissue. A device may be any suitable device for that purpose, such as a needle or microneedle adapted for intradermal or subcutaneous delivery of the composition. For example, the device may be a microneedle or a device comprising a plurality of microneedles designed for intradermal delivery of liquids. In some embodiments, the kit comprises a means for delivery of the immunogenic composition via inhalation.
[00350] Another aspect of the disclosure relates to recombinant antibodies prepared with the immunogenic polypeptides of the disclosure. In one embodiment, the disclosure provides compositions comprising one of those antibodies and, optionally, an adjuvant. These antibodies may be used alone or in any of the other combinations described herein, including combination with a polypeptide. The combination may be in the same composition. The use of the combination may be separate administrations. In one embodiment, the antibodies are used as diagnostic agents. In one embodiment, the antibodies are used method of preventing, treating or ameliorating at least one symptom of SARS-Cov infection, or of decreasing the frequency or severity of at least one symptom of SARS-Cov infection, the method comprising administering an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to the S1 or S2 subunit of SARS-Cov2 S protein prepared using one or more of the polypeptides of the disclosure. The term “recombinant”, as used herein, refers to antibodies or antigen binding fragments thereof of the invention created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.
[00351] In one embodiment, the antibody or the polypeptide of the disclosure is used to detect a SARS-associated coronavirus infection. In one embodiment, the detection is carried out by ELISA. Accordingly, the disclosure also provides a SARS-Cov associated coronavirus detection kit comprising an antibody according to the disclosure.
[00352] The antibodies of the disclosure can be full-length (for example, an lgG1 or lgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab')2 or scFv fragment), and may be modified to affect functionality, e.g., to increase persistence in the host or to eliminate residual effector functions. In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to SARS-Cov and/or a SARS-Cov S protein, wherein the antibody or antigen-binding fragment thereof neutralizes SARS-Cov in vitro with an IC50 less than or equal to 10-9M and wherein the antibody or antigen-binding fragment thereof demonstrates a protective effect in vivo in a SARS-Cov infected animal.
[00353] In one embodiment, the antibodies of the disclosure are neutralizing antibodies. The term “neutralizing antibody”, as used herein (or an “antibody that neutralizes SARS-Cov2 activity” or “antagonist antibody”), is intended to refer to an antibody whose binding to SARS-Cov2 results in inhibition of at least one biological activity of SARS-Cov2. For example, an antibody of the invention may prevent or block SARS-Cov2 attachment to, fusion with, and/or entry into a host cell. In addition, a “neutralizing antibody” is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms “neutralizing antibody” and “an antibody that neutralizes” or “antibodies that neutralize” are used interchangeably herein. These antibodies can be used, alone or in combination, as prophylactic or therapeutic agents with other anti-viral agents upon appropriate formulation, or in association with active vaccination, or as a diagnostic tool. [00354] In one embodiment, the subject in need thereof may be administered at least one anti-SARS-Cov S protein antibody of the invention or an antigen-binding fragment thereof, or a pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof of the invention in combination with a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of an anti-viral drug, an anti-inflammatory drug (such as corticosteroids, and non-steroidal anti-inflammatory drugs, such as antibodies to TNF, a different antibody to SARS-Cov, a vaccine for SARS-Cov, and/or interferons (alpha/beta/or lambda).
[00355] METHODS OF USE
[00356] As described herein, S protein immunogens, fragments, and variants thereof described herein contain an epitope that elicits or induces an immune response, for instance a protective immune response, which may be a humoral response and/or a cell-mediated immune response. A protective immune response may be manifested by at least one of the following: preventing infection of a host by a coronavirus; modifying or limiting the infection; aiding, improving, enhancing, or stimulating recovery of the host from infection; and generating immunological memory that will prevent or limit a subsequent infection by a SARS coronavirus. In case of SARS CoV infection, the protective immune response may be assessed for instance by the viral load in lungs and upper respiratory tract, the score of pulmonary inflammation and lesions, the scores of viral antigen loads in lungs, the presence of neutralizing antibodies, the CD4+ T cell responses in PBMC, spleen and the cytokine secretion from spleen. A humoral response may include production of antibodies that neutralize infectivity, lyse the virus and/or infected cell, facilitate removal of the virus by host cells (for example, facilitate phagocytosis), and/or bind to and facilitate removal of viral antigenic material. A humoral response may also include a mucosal response, which comprises eliciting or inducing a specific mucosal IgA response. In one embodiment, the host is a human.
[00357] Accordingly, in one embodiment, the disclosure provides method of preventing or treating severe acute respiratory syndrome or other SARS-CoV-related disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one or more of the polypeptides of the disclosure. [00358] In one embodiment, the efficacy is measured by measuring a reduced viral load or delays or prevention of a further increase in viral load. In one embodiment, efficacy is measured by an elimination, reduction, or decrease in the intensity or frequency of one or more symptoms associated with SARS such as fever, cough, shortness of breath, organ failure (e.g. kidneys) and/or septic shock. In one embodiment, the subject has an underlying disorder such as diabetes, cancer, chronic lung disease, or generally weakened immune system. In one embodiment, the SARS is Covid-19. In one embodiment, the immunogenic composition induces a humoral response, such as an increased level of neutralizing antibodies associated with the subject administered the immunogenic composition as compared to a subject not administered the immunogenic composition. In one embodiment, the immunogenic composition induces a cellular immune response, such as a CD8+T cell response, (including the production of cytokines such as interferon- gamma (TFN-g), tumor necrosis factor alpha (TNF-alpha), interleukin- 2 (IL-2), or any combinations thereof. In one embodiment, the immunogenic composition is a vaccine.
[00359] In one embodiment, the therapeutically effective amount is prophylactic. In one embodiment, the immunogenic composition or polypeptide is administered by any route typically used for vaccination, including topical, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, inhalation, or combinations thereof. In one embodiment, the immunogenic composition or polypeptide is administered in a single-dose vaccination schedule.
[00360] The amount of the protein of the present disclosure present in each vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and the type and amount of adjuvant used. An optimal amount for a particular vaccine may be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Generally, it is expected that each dose will comprise 1-2000 or 1-1000pg of protein, for example 1- 200pg, or 10-100pg. This amount may relate to the peptide with or without any modification. An exemplary dose will contain 10-50pg, for example 15- 25 pg, suitably about 20pg of protein. In one embodiment, the dose may contain 40 pg or 160 pg of each peptide, conjugated to approximately 300 pg of KLFI (conjugated to KLH), or a total of 160 pg of peptide plus 1200 pg of KLFI, adding up to a total dose of 1360 pg of protein per dose, when the vaccine comprises a combination of four peptides. Alternatively, a "dose-sparing" approach may be used, for example in a pandemic situation. This is based on the finding that it is possible to provide the same protective effect using lower doses of antigen, due to the presence of an effective adjuvant. Accordingly, each human dose may contain a significantly lower quantity of polypeptide, for example from 0.1 to 10pg, or 0.5 to 5pg, or 1 to 3pg, suitably 2pg polypeptide per dose. By the term "human dose" is meant a dose which is in a volume suitable for human use. Generally, this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml. Following an initial vaccination, subjects typically receive a boost after a 2 to 4-week interval, for example a 3 week interval, optionally followed by repeated boosts for as long as a risk of infection exists. In a specific embodiment of the disclosure, a single-dose vaccination schedule is provided, whereby one dose of S protein polypeptides in combination with adjuvant is sufficient to provide protection against the SARS CoV2, without the need for any boost after the initial vaccination. In one embodiment, the vaccination schedule includes more than one dose, and each dose may be different. In one embodiment, the repeated doses are administered a week apart or less, between more than a week apart and a month apart or less; more than a month apart; more than 2 months apart, more than 3 months apart and other monthly regimens as suited.
[00361] In one embodiment, the method further comprises the administration of another treatment for SARS or other coronavirus-related disease or disorder. In one embodiment, the method further comprises the administration of remdesivir, azithromycin, hydroxychloroquire, chloroquine, or combinations thereof. In one embodiment, the method further comprises the administration of hydroxychloroquine combination (200 mg X 3 per days for 10 days) with Azithromycin (500 mg on the 1st day then 250 mg per day for 5 more days).
[00362] In one embodiment, the treatment with the polypeptides, nucleic acids, vectors, and immunogenic compositions of the disclosure is combined with one of the following:
[00363] Anti-inflammatory therapy such as tocilizumab and favipiravir, tocilizumab, sarilumab, leronlimab, rintatolimod, BPI-002, REGN3048 and REGN 3051 , monoclonal antibody designed to bind SARS-CoV-2;
[00364] Anti-Viral therapy such as remdesivir, lopinavir and ritonavir, danoprevir and ritonavir, favipiravir, darunavir and cobicistat, umifenovir, galidesivir, linebacker and equivir, compounds that inhibit the virus interaction with the receptor ACE2;
[00365] Immunotherapy such as RNA vaccines (e.g., targeting Spike protein), DNA vaccines (e.g., targeting Spike protein), recombinant protein vaccines (e.g., targeting Spike protein), viral-vector based vaccines (e.g., targeting Spike protein), live attenuated vaccines (e.g., targeting the whole virion), inactivated vaccines (e.g., targeting the whole virion), subunit vaccine (e.g., targeting the whole virion), and peptide vaccine. [00366] In one embodiment, the disclosure is directed to the use of the immunogenic compositions of the disclosure for the manufacture of a medicament for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19. In one embodiment, the disclosure is directed to an immunogenic composition of the disclosure for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
[00367] In one embodiment, the disclosure is related to methods of using the polypeptides and/or antibodies of the disclosure as diagnostic agents in diagnostic methods. In one embodiment, the polypeptides and/or antibodies of the disclosure are used in the detection of coronavirus-related diseases and viruses such as those in SARS and Covid-19. In one embodiment, the polypeptides are labeled for detection by immunohistochemisty, ELISA, and other methods known in the art for using polypeptides in diagnostic methods. In one embodiment, the polypeptides and/or antibodies of the disclosure are used to identify other immunogenic polypeptides and antibodies that bind to the same region of the S protein or to a complex of the S protein with the ACE2 protein at the surface of target cells. In one embodiment, the polypeptides and/or antibodies of the disclosure are used in competition assays.
EXAMPLES
[00368] The following Examples 1-9 and 10-16 describe selection and characterisation of exemplary therapeutic peptides for generation of a SARS-CoV-2 vaccine.
EXAMPLE 1. IMMUNOGENIC PEPTIDE (1) [SEQ ID NO:1]
[00369] Immunogenic peptide SEQ ID NO:1 (CGSGDSKVGGNYNKLYRLFE) is derived from the ACE2-contacting surface of the S1 subunit (SARS-CoV-2 Spike protein aa 442-456). The peptide contains the contact residues Y449, Y453, L455 and F456. For better accessibility of the predicted B-cell epitope to the immune system, GSG linker sequence was added to the N terminus. Additional changes as lysine (K) instead of tyrosine (Y) at position 451 and C-terminal glutamic acid (E) were added with the aim to stabilize the peptide structure in a conformation similar to that present in the native S protein. Moreover, N-terminal cysteine (C) was used for conjugation of the peptide to the KLH carrier protein. To determine the predicted antigenic index of the peptide and the position of the B-cell epitope the peptide sequence was analyzed by BepiPred-2.0 server (FIG.1A, 1 B). The designed peptide contains one B cell epitope located at the N-terminal part of the peptide.
[00370] Hydrophobicity of peptides were evaluated by ProtScale server at https://web.expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. The prediction is based on the method of Kyte and Doolittle, J Mol Biol, 1982. Designed peptide exhibits suitable hydrophobicity profile, which is shown in FIG. 2.
EXAMPLE 2: IMMUNOGENIC PEPTIDE (2) [SEQ ID NO:2]
[00371] The peptide SEQ ID NO 2
CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ (aa 442-506) of the Spike protein was derived from the parts of the ACE2-contacting surface of the S1 subunit. With the aim to mimic the complex 3D structure similar to that present on the native spike protein at the places making contacts with ACE2 receptor, the peptide was designed by fusing two peptides from of the S1 glycoprotein, aa 442-454 which include the contact residues Y449 and Y453, and aa 492-506 which include the contact residues Q493, T500, N501 , G502 and Y505. These two amino acid stretches appear in a close spatial vicinity forming a part of the S-ACE2 interaction domain The sequences were bridged using a short, beta turn forming linker sequence (GPAD (SEQ ID NO: 17)), which was designed by using the data from the Motivated proteins website.
[00372] To determine the predicted antigenic index of the peptide and the position of the B-cell epitope(s) the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 3A, 3B). The designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
[00373] The proposed linker located between two B cell epitopes of the selected peptides can lead to better accessibility of the predicted B-cell epitopes to the immune system. Moreover, N-terminal cysteine (C) was used for conjugation to KLFI as a carrier protein. [00374] Hydrophobicity of peptides were evaluated by ProtScale server at https://web.expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. The prediction is based on the method of Kyte and Doolittle, J Mol Biol, 1982. Designed peptide exhibits suitable hydrophobicity profile, which is shown in FIG. 4.
EXAMPLE 3: THE IMMUNOGENIC PEPTIDE (3) [SEQ ID NO:3]
[00375] The immunogenic peptide SEQ ID NO:3 (SGSTEIYQAGSTPCNGVEGFNCYFAK) was derived from the surface of the S1 subunit contacting ACE2 receptor, amino acids 469-490. Designed peptide spans the ACE2 contact site with the following contact residues: A475, F486, N487, Y489 and Q493. In order to mimic 3D structure of the native S protein contact site with receptor ACE2 on host cell, two amino acids (Alanine (A) and Lysine (K)) was added at the end of C terminus. This modification can generate a compact structure by contacting the added Lysine (K) with Glutamic acid (E) at position 471. In addition, the two internal cysteines (underlined) will be oxidized to form internal disulfide bond. Moreover, N-terminus of the peptide is modified (e.g. by a chemoselective reactive group like azide which can bind selectively to alkyne or phosphine) and is used for conjugation to KLH. Because of the better accessibility of the B-cell epitope to the immune system, the short SG sequence was used as a spacer. Alternatively, an N-terminal or C-terminal Cys may be added for conjugation to KLH, with or without the N-terminal spacer.
[00376] To determine the predicted antigenic index of the peptide and the position of the B-cell epitope(s) the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 5A, 5B). The designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
[00377] Hydrophobicity of peptides were evaluated by ProtScale server at https://web.expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. The prediction is based on the method of Kyte and Doolittle, J Mol Biol, 1982. Designed peptide exhibits suitable hydrophobicity profile, which is shown in FIG. 6. EXAMPLE 4: THE IMMUNOGENIC PEPTIDE (4) SEQ ID NO:4
[00378] The immunogenic peptide SEQ ID NO:4 CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE was derived from the ACE2- contacting surface of the S1 subunit, amino acids 483-516. The peptide contains the following ACE2 contact sites: F486, N487, Y489, Q493, T500, N501 , G502 and Y505. Internal Cysteine (C) at position 488 is modified to Alanine (A) to prevent dimerization, which will change the 3D peptide structure.
[00379] Moreover, N-terminal cysteine (C) is used for conjugation to KLFI.
[00380] To determine the predicted antigenic index of the peptide and the position of the B-cell epitope(s) the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 7A, 7B). The designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
[00381] Hydrophobicity of peptides were evaluated by ProtScale server at https:/web. expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. The prediction is based on the method of Kyte and Doolittle, J Mol Biol, 1982. Designed peptide exhibits suitable hydrophobicity profile, which is shown in FIG. 8.
EXAMPLE 5: THE IMMUNOGENIC PEPTIDE (5) SEQ ID NO:5
[00382] Immunogenic peptide SEQ ID NO:5 QILPDPSKPSKRSC was derived from the surface of the S2 subunit, aa 804-816 C-terminal cysteine (C) is used for conjugation to KLFI. The antibodies induced against the peptide should prevent access of the protease to the S2’ cleavage site, and thus prevent a conformational change to induce ‘fusion peptide’ interaction with the cellular membrane.
[00383] To determine the predicted antigenic index of the peptide and the position of the B-cell epitope(s) the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 9A, 9B). The designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
[00384] Hydrophobicity of peptides were evaluated by ProtScale server at https://web.expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. The prediction is based on the method of Kyte and Doolittle, J Mol Biol, 1982. Designed peptide exhibits suitable hydrophobicity profile, which is shown in FIG. 10.
EXAMPLE 6: THE IMMUNOGENIC PEPTIDE (6) SEQ ID NO:6
[00385] The peptide SEQ ID NO:6 CNQKLIANQFNSAIGKIQDSLSS was derived from the surface residues of the S2 subunit of the SARS-CoV-2 spike glycoprotein, amino acids 919-940. N-terminal cysteine (C) is used for conjugation to KLFI. The peptide drives production of antibodies against the S2 subunit of the S protein, that should inhibit its conformational changes and prevent fusion of the virus with the plasma membrane of a host cell. The peptide will be used in combination with peptides derived from the S1 subunit.
[00386] To determine the predicted antigenic index of the peptide and the position of the B-cell epitope(s) the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 11 A, 11 B). The designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
[00387] Hydrophobicity of peptides were evaluated by ProtScale server at https://web.expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. The prediction is based on the method of Kyte and Doolittle, J Mol Biol, 1982. Designed peptide exhibits suitable hydrophobicity profile, which is shown in FIG. 12.
EXAMPLE 7: THE IMMUNOGENIC PEPTIDE (7) SEQ ID NO:7
[00388] The immunogenic peptide SEQ ID NO: 7 CYTMSLGAENSVAYS was derived from the S2 subunit using the amino acid residues 695-708, which are on the surface of the protein. N-terminal cysteine (C) is used for conjugation to KLFI. The vaccine containing the peptide conjugated to KLFI can induce antibodies against S2 subunit of S protein, that should inhibit its conformational changes and prevent fusion of virus with plasma membrane of host cell. The peptide will be used in combination with peptides derived from the S1 subunit.
[00389] To determine the predicted antigenic index of the peptide and the position of the B-cell epitope(s) the peptide sequence was analyzed by BepiPred-2.0 server (FIG. 13A, 13B). The designed peptide contains two B cell epitopes located at the N-terminal part of the peptide.
[00390] Hydrophobicity of peptides were evaluated by ProtScale server at https://web.expasy.org/protscale/protscale-ref.html (Gasteiger et al., 2005) to determine their solubility in aqueous solutions. The prediction is based on the method of Kyte and Doolittle, J Mol Biol, 1982. Designed peptide exhibits suitable hydrophobicity profile, which is shown in FIG. 14.
EXAMPLE 8: Peptide vaccine preparation, immunization and analysis of humoral and cellular immune response.
[00391] The designed peptides SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 are conjugated to keyhole limpet hemocyanin (KLH) via a cysteine link or azide-phosphine link. To this end, tau peptides are synthetized as cysteinated peptides with an extra N-terminally located cysteine residue or N-terminal azide with the aim to obtain oriented attachment of the peptide on the surface of the KLH protein. Peptides are coupled to the KLH carrier via bifunctional crosslinker N-[y-maleimidobutyryloxy]succinimide ester (GMBS) or NHS- Phosphine (Thermo Fisher Scientific). To prepare the conjugation reaction, 20 mg of KLH (Calbiochem) are dissolved in conjugation buffer (PBS with 0.9 M NaCI, 10 mM EDTA) to a concentration of 10 mg/ml by gentle mixing for 10 minutes. For preparation of maleimide-activated or phosphine-activated KLH, 2 mg of active bi-functional cross-linker GMBS or NHS-Phosphine are dissolved in 50 pi of anhydrous dimethylformamide and mixed with 2 ml of KLH solution for 1 hour at room temperature. Subsequently, un-reacted GMBS and NHS-Phosphine are removed on a 5 ml HiTrap Desalting column (GE Healthcare) equilibrated in conjugation buffer. Conjugations are carried out at a 1 :1 ratio of peptide to activated KLH (w/w, 20 mg of peptide) for 2-16 h at room temperature (25°C). The resulting conjugates are dialyzed against a 100-fold excess of PBS, with four dialysis buffer changes to remove unconjugated peptide. After dialysis, the conjugates are centrifuged at 21 ,000xg for 15 min at 2°C. Completeness of conjugation was confirmed by the absence of free peptide in the dialysis buffer, measured using LC-MS/MS. The conjugates are aliquoted and stored at -20°C until used.
[00392] Vaccine preparation and administration. [00393] To prepare immunization doses with Alum adjuvant (Alhydogel, Brentag Biosector, Denmark), 200 pg of each respective peptide conjugate (dissolved in 150 pi of PBS) are emulsified at a 1 :1 (vol/vol) ratio with Alum adjuvant, in a final dose volume of 300 pi. Each suspension/emulsion is incubated with rotation at 4°C overnight to allow the peptide to adsorb onto the aluminium hydroxide particles. Prepared vaccine doses are subcutaneously injected into the C57BL mice (n=10/peptide/KLH) in three-weekly schedule, starting at 8 weeks of age. Mice are sacrificed 10 days after the last dose of the vaccine for the immunogenicity analysis.
[00394] Preparation and stimulation of splenocytes.
[00395] After excision of spleens from the mice, single-cell suspension is prepared using teflon/glass homogenizer. Erythrocytes are precipitated in culture medium (DMEM containing 10% horse serum, 1 mM glutamine and 50 pg/ml gentamycin), bigger particles including pieces of spleen tissue and erythrocytes aggregates are let to settle down for 1 minute by gravity in 15 ml falcon tubes and supernatant is carefully removed. Splenocytes are seeded at density 8x106 cells/well (in 24-well plates and 1 ml of culture medium) and stimulated in vitro with peptide (10 mM), peptide conjugated to KLH (200 pg/ml), S protein (20pg/ml) or concanavalin A (5 pg/ml) for 72 hours. Subsequently, protein transport inhibitor Brefeldin A (GolgiPlug, BD Biosciences) is added to cells according to manufacturer's instructions. Five hours later, cells are centrifuged for 10 min at 300g. Cell culture supernatants are collected, stored at -80°C for further cytokine’s detection and splenocytes are used for flow cytometry analysis.
[00396] Analysis of cellular immune response by flow cytometry.
[00397] After stimulation, cells are washed with PBS and stained with antibodies against activated central memory T cell surface markers (CD4-Alexa Fluor 700, CD44- BV510, CD62L-PECy7, CCR7-APC and CD69-BV421) for 45 min at room temperature. Then, cells are washed, fixed in BD FACS Lysing Solution containing formaldehyde for 10 min, permeabilized in PBS containing 0,5% Triton X-100 for 20 min, split into two separate tubes and stained for intracellular cytokines (IL2-PE-CF594, IL10-PE and IL4- PE-CF594, IFNy-PE). Activated B cells are measured using markers CD19 (for detection of B cells), CD138 (which is negative for all developmental stages of B cells except of plasmablasts and plasma cells), IgM and IgD (which are immunoglobulins produced by activated B cells) and CD69 and CD86 (markers of activation). Additionally, memory B cells are detected using additional marker CD62L, CD27 and immunoglobulins IgG, IgM (positive markers) and IgD (negative marker). Finally, cells are fixed, resuspended in PBS and analyzed immediately on LSRFortessa™ flow cytometer (BD Biosciences). For each analysis, 100 000 cells are acquired and cells expressing individual markers are gated according to fluorescence- minus- one (FMO) controls. Facs Diva software is used for data analysis.
[00398] Detection of secreted cytokines by ELISA.
[00399] Concentrations of IL-2, IL-4, IL-10 and IFN-gamma secreted to the cell culture media are measured by Mouse Quantikine ELISA kits (R&D Systems) according to the manufacturer’s protocol.
[00400] Determination of antibody response to the peptide vaccines.
[00401] 250 ng/well of peptide, peptide conjugated to BSA (2pg/ml), recombinant S protein (2, 5pg/ml) are separately coated onto 96-well plates (NUNC, Thermo Scientific, Denmark). After blocking, the plates are incubated with serially diluted sera (50 pl/well, 1 :100 to 1 :51 ,200 in PBS, in two-fold dilution steps) for 1 hour at 37°C. Bound serum antibodies are detected with peroxidase-conjugated secondary antibody (goat anti mouse immunoglobulin (Ig), Dako, Glostrup, Denmark) using TMB one (Kem-en-tec, Denmark) at room temperature for 20 minutes in the dark. The reaction is stopped by adding 50 mI of 0.25M H2S04 into each well. The plate is read at 450 nm using Powerwave HT (Bio-Tek).
[00402] Determination of isotypic profile of peptide vaccine-induced antibodies To determine the isotypes of the specific antibodies produced in response to vaccine, sera from immunized mice are serially diluted from 1 :100 to 1 :12,800 in twofold dilution steps and tested in duplicates by enzyme-linked immunosorbent assay (ELISA) against peptide and recombinant S protein. To detect mouse IgG 1 , lgG2b, lgG2c, lgG3 and IgM isotypes, anti-mouse subclass-specific horseradish peroxidase (HRP)-conjugated secondary antibodies are diluted 1 :5,000 in PBS (Pierce Biotechnology, Rockford, IL, USA). Antibody isotype levels are compared based on the half-maximal effective concentration value of the dilution factor.
EXAMPLE 9. In vitro tests of neutralizing activity of serum antibodies elicited against the SARS-CoV-2 spike protein peptide vaccines.
[00403] Recombinant SARS-CoV-2 spike protein and human ACE2 protein expression and purification.
[00404] The recombinant receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S) (residues 319-591 , NCBI Reference Sequence: NC_045512.2) is fused with C-terminal HRV3C protease cleavage site, followed by an 8XhisTag (SEQ ID NO: 39) and a TwinStrepTag and. The entire ectodomain of S protein (residues 1-1208) is expressed with the mutations that stabilize the protein in its prefusion conformation: residues K986 and L987 are changed to prolines, the S1/S2 cleeavage site 683-RRAR- 686 (SEQ ID NO: 40) is replaced with the sequence GSAS (SEQ ID NO: 41). The S protein is cloned with C-terminal T4 bacteriophage fibritin trimerization sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO. 206] , HRV3C protease cleavage site followed by a TwinStrepTag and an 8XhisTag (SEQ ID NO: 39). Human ACE2 protein (residues 1-615, NCBI Reference Sequence: NP_001358344.1) is fused with a C- terminal HRV3C protease cleavage site followed by a TwinStrepTag and an 8XhisTag (SEQ ID NO: 39).
[00405] The proteins are cloned into mammalian expression vector under the control of the CMV promoter with the human Ig kappa light chain leader sequence (GenBank: MN197520.1 ) to drive their export into the media. The plasmids amplified in E. coli are transfected into FreeStyle 293-F™ cells (Thermo Fisher Scientific) using the FreeStyle™ MAX Transfection Reagent (Thermo Fisher Scientific) according to manufacturers recomendations. The transfected cells are incubated for 6-7 days, medium collected and the proteins are isolated using a stepwise chromatography steps. The proteins (ACE2 and the S protein ectodomain) are purifed from filtered cell culture supernatants (adjusted to 50 mM TRIS-HCI pH 8.0, 1 mM EDTA, 50 mM NaCI) by capturing them on the StrepTrap™ HP (GE Healthcare) column and eluting with desthiobiotin (SIGMA, 2.5 mM in 50 mM TRIS-HCI pH 8, 1 mM EDTA, 50 mM NaCI). The cell culture medium containing the S protein RBD-Fc fusion protein is adjusted to pH 8.0 with by titrating with 1 M Tris-HCI pH 8.0 and then the RBD-Fc fusion is purified using Protein A column (GE Healthcare). The bound protein is eluted using 100 mM Glycine pH 2.7 and immediately neutralized with 1 M Tris-HCI pH 8.0. The eluted proteins are further purified by size-exclusion chromatography on Superdex 200 10/300 Increase column (GE Healthcare) in 5 mM Tris pH 8.0, 200 mM NaCI.
[00406] Determination of the titres of vaccine-induced antibodies in human by indirect ELISA. 250 ng/well of one of the SARS-CoV-2 S protein peptides or peptides conjugated to BSA (2pg/ml) or recombinant S protein ectodomain (2.5pg/ml) are separately coated onto 96-well plates (NUNC, Thermo Scientific, Denmark). After blocking with PBS-0.05% Tween 20 for 1 hr at 25°C, the plates are incubated overnight at 4°C with serially diluted human sera (50 pl/well, 1 :100 to 1 :51 ,200 in PBS, in two-fold dilution steps). After washing, bound serum antibodies are detected with peroxidase- conjugated secondary antibody (anti human immunoglobulins IgG/ HRP, Thermo Scientific). The amount of bound secondary antibodies is detected with the chromogenic substrate TMB one (Kem-en-tec, Denmark) incubated at room temperature for 20 minutes in the dark. The reaction is stopped by adding 50 mI of 0.25M H2S04 into each well. The plate is read at 450 nm using Powerwave HT (Bio-Tek). The titre of the antibodies in the serum as the highest dilution at which the absorbance at 450 nm was defined at least twice the absorbance of equally diluted non-immunized serum samples.
[00407] Determination of isotypic profile of vaccine-induced antibodies in human by indirect ELISA. To determine the isotypes of the specific human antibodies produced in response to vaccine, sera from immunized subjects are serially diluted from 1 :100 to 1 :12,800 in twofold dilution steps and tested in duplicates by enzyme-linked immunosorbent assay (ELISA) against peptide and recombinant S protein. To detect human IgG 1 , lgG2, lgG3, lgG4 and IgM isotypes, anti-human subclass-specific horseradish peroxidase (HRP)-conjugated secondary antibodies are diluted 1 :5,000 in PBS (Pierce Biotechnology, Rockford, IL, USA). Antibody isotype titers are compared based on the half-maximal effective concentration value of the dilution factor. The resulting signal was compared with that obtained for the non-vaccinated patients. [00408] Affinity purification of vaccine induced antibodies from human sera. Human serum is separated by centrifugation at 2000 xg for 10 minutes. For affinity purification of vaccine-specific antibodies, the recombinant ectodomain of the S protein is coupled to superparamagnetic Dynabeads M280 Tosylactivated (Thermo Fisher Scientific) according to manufacturer’s instructions. The prepared S-beads are incubated with human serum 6-fold diluted with PBS supplemented with Tween 20 and Complete® protease inhibitors (Roche) at +2 to +8°C for 16 hrs with head-over-tail rotation. The bound antibodies are eluted with 0.2 M glycine pH 2.7 and immediately neutralized with 1 M Tris-HCI pH 8.0. Control antibodies are isolated from a non-vaccinated human by using Dynabeads Protein G (GE Healthcare) and final concentration is determined by absorption spectroscopy. Quality of vaccine-induced antibodies isolated form human sera is analysed by SDS-polyacrylamide electrophoresis as follow. 1 pg of vaccine- induced, S protein ectodomain-specific antibodies and control sera are loaded onto 12% SDS polyacrylamide gel and electrophoresed in a Tris-glycine-SDS buffer system and stained with 0.05% Coomassie Blue R250 (Sigma Aldrich) in 10 % acetic acid, 40 % methanol in H20. The binding capacity of vaccine -induced antibodies to the S protein ectodomain is detected by indirect ELISA.
[00409] Analysis of the neutralizing activity of the serum antibodies raised against the S protein peptide vaccines. 250 ng/well of recombinant ACE2 protein (2.5 pg/ml) is immobilized onto 96-well plates (NUNC, Thermo Scientific, Denmark) for 2 hrs at room temperature and then the plates are blocked with PBS-0.05% Tween 20 for 1 hr at 25°C. Solution of the recombinant S protein ectodomain (100 pmol/L and 10 pmol/L) are incubated withserially diluted control human IgG antibodies (Merck) or serially diluted (10- fold) antibodies isolated from human sera at 37°C for 1 hr and then added to the ACE2 immobilized on the ELISA plates. The plates are incubated for 1 hr at room temperature. After washing, bound S protein ectodomain is detected with peroxidase-conjugated anti StrepTag antibody visualized with the chromogenic substrate TMB one (Kem-en-tec, Denmark). The reaction is stopped by adding 50 pi of 0.25M H2SO4 into each well. The plate is read at 450 nm using Powerwave HT (Bio-Tek). The neutralizing activity of the human serum antibodies are determined by calculated the inhibitory concentration of the antibody isolates that caused 50% drop in the S protein ectodomain binding activity (IC50) using the four-parameter logistic function.
EXAMPLE 10: THE IMMUNOGENIC PEPTIDE SEQ ID NO. 83 (PEPTIDE 8)
[00410] Immunogenic peptide SEQ ID NO: 83 was derived from the surface of the S1 subunit. The designed peptide contains one B cell epitope located in the middle part of the peptide and two contact residues (Spike protein positions N439, Y449), which may be important for binding the virus to the ACE2 receptor. The peptide-induced antibody may affect binding of virus to hACE2, thus inhibiting of entry into a host cell. N-terminal cysteine (C) is added for conjugation to KLH (the carrier protein) through a maleimide linker. The predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 16B). The peptide exhibits suitable hydrophilicity profile, documented in FIG. 17. EXAMPLE 11 : THE IMMUNOGENIC PEPTIDE SEQ ID NO. 84 (PEPTIDE 9)
[00411] Immunogenic peptide SEQ ID NO: 84 was derived from the surface of the S1 subunit. The designed peptide contains one B cell epitope located in middle part of the peptide. Moreover, the peptide carries several contact residues (Spike protein positions T500, N501 , G502, Y505), which may play important roles in binding the virus to its receptor (ACE2). Blocking these residues by peptide-induced antibodies may be therapeutically effective to prevent the S1 subunit - ACE2 interaction. Two residues are added to the original SARS-CoV-2 spike protein sequence: N-terminal cysteine (C) is used for conjugation to KLFI as a carrier protein using a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid. The predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 18B). The peptide exhibits suitable hydrophilicity profile, which is shown in FIG. 19.
EXAMPLE 12: THE IMMUNOGENIC PEPTIDE SEQ ID NO. 85 (PEPTIDE 10)
[00412] Immunogenic peptide SEQ ID NO: 85 was derived from the surface amino acids of the S1 subunit. The peptide contains one B cell epitope located in middle region and two contact residues (Spike protein positions L455, F456), which may play important roles in binding the virus to receptor of host cell. The peptide-induced antibodies may block binding of virus to hACE2 and entry of virus into the cell. The N-terminal cysteine (C) is added for conjugation to KLH (as a carrier protein) through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid. The predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 20B). The designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 21.
EXAMPLE 13: THE IMMUNOGENIC PEPTIDE SEQ ID NO. 86 (PEPTIDE 11)
[00413] Peptide SEQ ID NO: 86 was derived from surface S1 subunit. The peptide contains one B cell epitope located in middle region. The Alanine located at the position 10 (A475 of the Spike protein) of the peptide is one of the contact residues with ACE2, YQAGS residues (SEQ ID NO: 117) form a part of the contact surface of S1 with ACE2, which the virus may utilize to enter into the host cell. The peptide-induced antibodies may have neutralizing activity and thus effectively block the virus from binding to the receptor of permissive cells. The N-terminal cysteine (C) is added for conjugation to KLFI (as a carrier protein) through a maleimide linker. The predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 22B). The designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 23.
EXAMPLE 14: THE IMMUNOGENIC PEPTIDE SEQ ID NO. 87 (PEPTIDE 12)
[00414] Peptide SEQ ID NO: 87 was derived from the surface of S1 subunit. The designed peptide contains one B cell epitope located in central part of peptide. The Threonine located at position 5 (the Spike protein position T415) and lysine at position 7 (the Spike protein position K417) were identified as the contact residues, which the virus may exploit to enter the host cell. Binding of the peptide-induced antibodies to these strategic residues or near these residues may lead to the lowering number of virus - infected cells. N-terminal cysteine (C) is added for conjugation to KLFI as a carrier protein through a maleimide linker. For a better accessibility of predicted B-cell epitope to the immune system, serine was added to the N terminus. The predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 24B). The designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 25. EXAMPLE 15: THE IMMUNOGENIC PEPTIDE SEQ ID NO. 88 (PEPTIDE 13)
[00415] Peptide SEQ ID NO: 88 was derived from the surface of the S1 subunit. The designed peptide contains one B cell epitope located in medium part of peptide. The residues R3, D5, E6, Q9, T 14 and K16 of the peptide were identified as part the S1 protein contact surface with ACE2, which the virus exploits to enter the host cell. Blocking of the positions important for binding to human ACE2 by peptide-induced antibodies may lead to the lower affinity between the viral RBD and host ACE2 in the initial viral attachment step. In the end this lowering of affinity may lead to lowering of virus efficiency. The N- terminal cysteine (C) is added for conjugation to KLH as a carrier protein through a maleimide linker. For a better accessibility of predicted B-cell epitope to the immune system, serine as a linker was added to the N terminus. The predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 26B). Designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 27.
EXAMPLE 16: THE IMMUNOGENIC PEPTIDE SEQ ID NO. 89 (PEPTIDE 14)
[00416] Peptide SEQ ID NO: 89 was derived from the surface of the S1 subunit. The peptide contains one B cell epitope located in medium part of peptide and four residues which were identified as important contact sites which virus exploit for binding to host cell receptor. The identified amino acids residues at the Spike protein positions F486 (phenylalanine), N487 (asparagine), Y489 (tyrosine) and Q493 (glutamine) enhance viral attachment to human ACE2. Occupation (blocking) of these strategic residues by peptide- induced antibodies may be therapeutically effective. The N-terminal cysteine (C) is added for conjugation to KLFI as a carrier protein through a maleimide linker, aspartic acid (D) is added to facilitate quantification of the peptide in the conjugate by cleavage with formic acid. The predicted antigenic index of the peptide suggests its potential immunogenicity (FIG. 28B). The designed peptide exhibits suitable hydrophilicity profile, as shown in FIG. 29.
[00417] The subject matter described above is described by way of illustration only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the present disclosure, which is set forth in the following claims.
[00418] All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entireties as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.
[00419] LITERATURE
[00420] Amanat F, Krammer F. (2020) SARS-CoV-2 vaccines: status report. J Immun
[00421] Bolles M, Deming D, Long K, Agnihothram S, Whitmore A et al. (2011) A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge. J Virol 85:12201-12215.
[00422] Bosch BJ, de Flaan CAM, Smits SL, and Rottier PJM. (2005) Spike protein assembly into the coronavirion: Exploring the limits of its sequence requirements. Virology 334:306-318.
[00423] Bosch BJ, Martina BE, Van Der Zee R, Lepault J, Flaijema BJ, Versluis C et al. (2004). Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides. Proc. Natl. Acad. Sci. USA 101 :8455-8460.
[00424] Dhama K, Sharun K, Tiwari R, Dadar M, Malik YS, Singh KP, Chaicumpa W. (2020) COVID-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics. Flum Vaccin Immunother. 18:1-7.
[00425] Dong S, Sun J, Mao Z, Wang L, Lu YL, Li J. (2020) A guideline for homology modeling of the proteins from newly discovered betacoronavirus, 2019 novel coronavirus (2019-nCoV). J Med Virol. Mar 17. [00426] Francis GS. (2000) ACE inhibition in cardiovascular disease. N Engl J Med 342: 201-202.
[00427] Fehr AR, Perlman S. (2015) Coronaviruses: An Overview of Their Replication and Pathogenesis. Methods Mol Biol 1282:1-23.
[00428] Hoffmann M, Kleine-Weber H, Kruger N, Muller M, Drosten C, Pohlmann S. (2020) The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. bioRxiv, 10.1101/2020.01.31.929042.
[00429] Chan JF, To KK, Tse H, et al. (2013) Interspecies transmission and emergence of novel viruses: lessons from bats and birds. Trends Microbiol 21 (10):544- 555.
[00430] Jia W, Channappanavar R, Zhang C, Li M et al. (2019) Single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against MERS-CoV infection. Emerg Microbes Infect 8:760-772. [00431] Li F, Li W, Farzan M, Harrison SC. (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309:1864-1868. [00432] Li W, Joshi MD, Singhania S, Ramsey KH, Murthy AK (2014) Peptide Vaccine: Progress and Challenges. Vaccines 2:515-536.
[00433] Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W et al. (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395(10224):565-574.
[00434] Masters PS. (2006) The molecular biology of coronaviruses. Adv Virus Res. 66:193-292.
[00435] Sahin AR, Erdogan A, Mutlu Agaoglu P, Dineri Y, Cakirci AY, Senel ME, et al. (2020) 2019 Novel Coronavirus (COVID-19) Outbreak: A Review of the Current Literature. EJMO 4(1 ):1 -7.
[00436] Schoeman D, Fielding BC. (2019) Coronavirus envelope protein: current knowledge. Virology J 16:69. [00437] Song HC, Seo MY, Stadler K, Yoo BJ et al. (2004). Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein. J Virol 78:10328-10335.
[00438] Song WF, Gui M, Wang X et al. (2018) Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog 14(8):e1007236.
[00439] Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, Liu W, Bi Y, Gao GF. (2016) Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses. Trends Microbiol 24:490-502.
[00440] Tseng CT, Sbrana E, Iwata-Yoshikawa N, Newman PC, Garron T et al. (2012) Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus. PLoS One 7 e35421 .
[00441] Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 8674(20) :30262-2.
[00442] Vankadari N, Wilce JA (2020) Emerging WuHan (COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26. Emerg Microbes Infect. 17;9(1 ):601-604.
[00443] Wrapp D, Wang N, Corbett KS, Goldsmith JA et al. (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 10.1126/science. abb2507
[00444] http://www.who.int/csr/sars/country/table2004_04_21
[00445] https://www.who.int/emergencies/mers-cov/en/
[00446] https://www.who.int/dg/speeches/detail/who-director-general-s-opening- remarks-at-the-media-briefing-on-covid-19—11 -march-2020

Claims

What is claimed is:
1. An isolated polypeptide, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of one or more of the following sequences:
DSKVGGNYNKLYRLF, SEQ ID NO:8;
DSKVGGNYNYLYR, SEQ ID NO:9;
DSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ, SEQ ID NO:10; LQSYGFQPTNGVGYQ, SEQ ID NO:11 ;
STEIYQAGSTPCNGVEGFNCYF, SEQ ID NO:12; VEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE, SEQ ID NO:13; QILPDPSKPSKRS, SEQ ID NO:14;
NQKLIANQFNSAIGKIQDSLSS, SEQ ID NO:15;
YTMSLGAENSVAYS, SEQ ID NO:16;
SNNLDSKVGGNYN, SEQ ID NO: 118;
GFQPTNGVGYQPYR, SEQ ID NO: 119;
RLFRKSNLKPFE, SEQ ID NO: 120;
RDISTEIYQAGSTP, SEQ ID NO: 121 ;
PGQTGKIADYNYKLPD, SEQ ID NO: 122;
RGDEVRQIAPGQTGK, SEQ ID NO: 123;
DNGVEGFNGYFPLQSYG, SEQ ID NO: 124;
SEQ ID NOs. 42 through 116; and SEQ ID Nos. 125 through 227; and a polypeptide, fragment, variant, or derivative thereof, comprising a contiguous sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of said sequences, wherein the polypeptide, fragment, variant, or derivative thereof is at least 7 amino and at most 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues-long; and wherein the polypeptide, or a fragment, variant, or derivative thereof is neither a fragment of SARS Cov2 Spike protein of NC_045512.2 nor a fragment of any other naturally-existing coronavirus Spike protein, optionally wherein the polypeptide, fragment, variant, or derivative thereof comprises at least one modification to any one of the peptides of SEQ ID NOs: 8 through 16, SEQ ID NOs:118 through 124, or SEQ ID NOs:140 through 175, wherein the modification is an addition, deletion, or mutation to the amino acid sequence and/or the addition of a moiety to either the N-terminus, C-terminus, and/or internally .
2. The isolated polypeptide of claim 1 , or a fragment, variant, or derivative thereof wherein the polypeptide is selected from:
Peptide (1): 442- CGSGDSKVGGNYNKLYRLFE -456, SEQ ID NO:1 ;
Peptide (2): 442- CDSKVGGNYNYLYRGPADLQSYGFQPTNGVGYQ -506, SEQ ID NO:2; Peptide (3): 469-SGSTEIYQAGSTPCNGVEGFNCYFAK-490, SEQ ID NO:3;
Peptide (4): 483-CVEGFNAYFPLQSYGFQPTNGVGYQPYRVVVLSFE-516, SEQ ID NO:4; Peptide (5): 804-QILPDPSKPSKRSC-816, SEQ ID NO:5;
Peptide (6): 919-CNQKLIANQFNSAIGKIQDSLSS-940, SEQ ID NO:6;
Peptide (7): 695-CYTMSLGAENSVAYS -708, SEQ ID NO:7;
Peptide (8): 438-CSNNLDSKVGGNYN-450, SEQ ID NO: 83;
Peptide (9): 496- CDGFQPTNGVGYQPYR-509, SEQ ID NO: 84;
Peptide (10): 454-CDRLFRKSNLKPFE-465, SEQ ID NO: 85;
Peptide (11 ): 466-CRDISTEIYQAGSTP-479, SEQ ID NO: 86;
Peptide (12): 412-CSPGQTGKIADYNYKLPD-427, SEQ ID NO: 87;
Peptide (13): 403-CSRGDEVRQIAPGQTGK-417, SEQ ID NO: 88;
Peptide (14): 481 -CDNGVEGFNGYFPLQSYG-496, SEQ ID NO: 89; Peptides (1) through (14) without the N-terminal or C-terminal Cys (SEQ ID NOs. 90 through 102) and/or with an extra Cys (SEQ ID NO. 103 through 106);
Peptides based on peptide (3) but wherein the two underlined Cys are any amino acids (SEQ ID NO: 115);
Peptides (1) through 14 without the spacer(s) and/or linker(s) (SED ID NOs.109 through 112);
Peptides derived from Peptides (1 ) through (14), wherein any one or more of the underlined amino acids is replaced by one or more different amino acids (SEQ ID NOS 107-108 and 114-116) or is absent;
Any other Peptide selected from those of SEQ ID NO: 118-140; and
A polypeptide, fragment, variant, or derivative thereof comprising a contiguous sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of said peptides, wherein the numbering of the amino acids in the Peptides is relative to the sequence of SARS CoV2 S protein, and the differences between the two are underlined; wherein the polypeptide is neither a fragment of SARS Cov2 S protein of NC_045512.2 nor a fragment of any other naturally-existing coronavirus S protein.
3. The isolated polypeptide of any one of claims 1 and 2, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of a continguos sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NO:1 through SEQ ID NO:16, SEQ ID NO: 83 through SEQ ID NO:116, or SEQ ID NO: 140 through SEQ ID NO:175, optionally wherein the polypeptide, fragment, variant, or derivative thereof comprises one or more conservative amino acid substitutions.
4. The isolated polypeptide of any one of claims 1 through 3, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof differs from the polypeptides of SEQ ID NO:1 through 16, SEQ ID NO: 83 through 116, or SEQ ID NO: 140 through 175 in that it has an addition, deletion, or insertion that comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42,
43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65,
66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88,
89, 90, 91 , 92, 93, 94, 95, 96, 96, 97, 98, 99, or 100 amino acids.
5. The isolated polypeptide of any one of claims 1 through 4, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof is at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41 , at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51 , at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58 residues, at least 59, at least 60, at least 61 , at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71 , at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 90, but fewer than 100 amino acid residues in length.
6. The isolated polypeptide of any one of claims 1 through 5, or a fragment, variant, or derivative thereof, wherein the polypeptide, fragment, variant, or derivative thereof, is at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21 , at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31 , at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41 , at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51 , at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58 residues, at most 59, at most 60, at most 61 , at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71 , at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 90, at most 100 amino acid residues in length.
7. The isolated polypeptide of any one of claims 1 through 6, or a fragment, variant, or derivative thereof, wherein the polypeptide further comprises a moiety or a moiety and a linker, and/or a peptide corresponding to Peptides (1) through Peptide (14) without a N-terminal Cys.
8. The isolated polypeptide of any one of claims 1 through 7, or a fragment, variant, or derivative thereof, wherein the moiety is a carrier protein selected from any one or more of a Cys residue, an Asp residue, a Ser residue, Keyhole Limpet Hemocyanin (KLH) functional unit, Cys-KLH, a tetanus toxin heavy chain C fragment, a diphteria toxin, a diphtheria toxin variant CRM197, an H influenzae protein D, a Meningococcal outer membrane protein complex protein, an Outer-membrane lipoprotein carrier protein, or a Cholera toxin B subunit, a virus-like particle, biotin, avidin, streptavidin, neutravidin, serum albumin, an enzyme, a metallic nanomaterial, CRM197 and an outer membrane protein mixture from N. meningitidis (OMP), micro- and nano-particles of biodegradable polymers including polylactic, polycaproic, polyglycolic, polymalic, polybutyric acids and their combination and modifications; hydrogels of polyethyleneglycol and its modifications; hyaluronic acid, dextran, chitosan, liposome-polymer hybrid carrier, and a fragment or derivative or combination thereof.
9. The isolated polypeptideof any one of claims 1 through 8, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of one or more of the polypeptides of SEQ ID NO:1 through SEQ ID NO:16, SEQ ID NO:118 through SEQ ID NO:124, SEQ ID NO:83 through SEQ ID NO: 89, and/or SEQ ID NO: 140 through 175 connected through a linker.
10. The isolated polypeptide of any one of claims 1 through 9, or a fragment, variant, or derivative thereof, wherein the linker is selected from GSG, GPAD (SEQ ID NO: 17), and SG.
11 . The isolated polypeptide of any one of claims 1 through 10, or a fragment, variant, or derivative thereof, wherein the polypeptide comprises, consists, or consists essentially of a peptide of SEQ ID Nos: 3, 5, 6, 7, 9, 11 , 12, 14, 15, 16, 83 through 89, or 118 through 124.
12. The isolated polypeptide of any one of claims 1 through 11 , or a fragment, variant, or derivative thereof, wherein the polypeptide further comprises one or more chemical modifications selected from an internal bridge, short-range cyclization, methylation, amidation, acetylation or substitution with other chemical groups cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, which can be N-terminal, C- terminal, or internal.
13. The isolated polypeptide of any one of claims 1 through 12, or a fragment, variant, or derivative thereof, wherein the polypeptide is immunogenic.
14. A nucleic acid encoding one or more of the polypeptides, fragments, variants, or derivatives thereof of any one of claims 1 through 13.
15. The nucleic acid of claim 14, wherein the nucleic acid is DNA, RNA, or modified RNA.
16. A vector comprising one or more of the nucleic acids of any one of claims 14 and 15.
17. The vector of claim 16, wherein the vector is a retroviral vector, lentiviral vector, adenoviral vector, poxvirus vector, plasmid, or bacterial vector.
18. A recombinant cell comprising a polypeptide, or a fragment, variant, or derivative thereof, of any one of claims 1 through 13, a nucleic acid according to any one of claims 14 and 15, or a vector of any one of claims 16 and 17.
19. The recombinant cell of claim 18, wherein the cell is a prokaryotic cell (e.g. E. coli) or an eukaryotic cell (e.g. Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NSO, Sp2/0), a monkey kidney cell (COS-7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TRI cell, an MRC 5 cell, a FS4, or a MDCK cell.
20. An immunogenic composition comprising one or more of the polypeptides of any one of claims 1 through 13, or a fragment, variant, or derivative thereof, nucleic acids of claims 14 and 15, vector of claims 16 and 17, or cell of claims 18 and 19.
21. The immunogenic composition of claim 20, comprising one or more of the polypeptides of any one of claims 1 through 13, or a fragment, variant, or derivative thereof, and an adjuvant.
22. The immunogenic composition of claim 21 , comprising a combination of any two of Peptides (1) through Peptide (14).
23. The immunogenic composition of claim 22, wherein the composition comprises a combination of Peptide (1) and (2), (1) and (3), (1) and (4), (1) and (5), 1 and (6), (1 ) and (7), (2) and (3), or any one of the 21 possible two-peptide combinations of these seven Peptides; or any one of the 21 possible two-peptide combinations of Peptides (8) through (14); or any one of the 91 two-peptide combinations, 364 three- peptide combinations, and 1001 four-peptide combinations of Peptides (1) through (14), or any one of the 1456 combinations of Peptides (1 ) through (14).
24. The immunogenic composition of claim 23, comprising a combination of any three of Peptides (1) through Peptide (14).
25. The immunogenic composition of claim 24, comprising a combination of Peptides (1), (2) and (3); (1 ), (2) and (4); (1 ), (3), and (4); and (2), (3), (4) or any one of the 35 possible three-peptide combinations of these seven Peptides; or any one of the 35 three-peptide combinations of Peptides (8) through (14); or any one of the 364 three-peptide combinations of Peptides (1) through (14); or any one of the 1001 four- peptide combinations of Peptides (1) through (14).
26. The immunogenic composition of any one of claims 19 through 25, wherein the immunogenic composition is a vaccine.
27. The immunogenic composition of any one of claims 19 through 26, wherein the adjuvant is aluminum hydroxide or one of its salts.
28. A method of preventing or treating severe acute respiratory syndrome or other SARS-CoV-related disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one of claims 20 through 27, or of a polypeptide of any one of claims 1 through 13.
29. The method of claim 28, wherein the efficacy is measured by measuring a reduced viral load or delays or prevention of a further increase in viral load.
30. The method of claim 29, wherein the efficacy is measured by elimination, reduction, or decrease in the intensity or frequency of one or more symptoms associated with SARS such as fever, cough, shortness of breath, organ failure (e.g. kidneys) and/or septic shock.
31 . The method of any one of claims 28 through 30, wherein the subject has an underlying disorder such as diabetes, cancer, chronic lung disease, heart disease, or generally weakened immune system.
32. The method of any one of claims 28 through 31 , wherein the immunogenic composition induces a humoral response, such as an increased level of neutralizing antibodies associated with the subject administered the vaccine as compared to a subject not administered the vaccine, or as compared to the same subject prior to administration of the immunogenic composition.
33. The method of any one of claims 28 through 32, wherein the immunogenic composition induces a cellular immune response, such as a CD8+T cell response, (including the production of cytokines such as interferon- gamma (TFN-y), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or any combinations thereof.
34. The method of any one of claims 28 through 33, wherein the therapeutically effective amount is prophylactic.
35. The method of any one of claims 28 through 34, wherein the immunogenic composition or polypeptide is administered by any route typically used for vaccination, including topical, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, inhalation, or combinations thereof.
36. The method of any one of claims 28 through 35, wherein the immunogenic composition or polypeptide is administered in a single-dose vaccination schedule.
37. The method of any one of claims 28 through 36, further comprising the administration of another treatment for SARS.
38. The method of any one of claims 28 through 37, further comprising the administration of remdesivir, azithromycin, hydroxychloroquire, chloroquine, standard of care, or combinations thereof.
39. The method of any one of claims 28 through 38, further comprising the administration of hydroxychloroquine combination (200 mg X 3 per days for 10 days) with Azithromycin (500 mg on the 1st day then 250 mg per day for 5 more days); Anti inflammatory therapy such as tocilizumab and favipiravir, tocilizumab, sarilumab, leronlimab, rintatolimod, BPI-002, REGN3048 and REGN 3051 , monoclonal antibody designed to bind SARS-CoV-2; Anti-Viral therapy such as remdesivir, lopinavir and ritonavir, danoprevir and ritonavir, favipiravir, darunavir and cobicistat, umifenovir, galidesivir, linebacker and equivir, compounds that inhibit the virus interaction with the receptor ACE2; or Immunotherapy such as RNA vaccines (e.g., targeting Spike protein), DNA vaccines (e.g., targeting Spike protein), recombinant protein vaccines (e.g., targeting Spike protein), viral-vector based vaccines (e.g., targeting Spike protein), live attenuated vaccines (e.g., targeting the whole virion), inactivated vaccines (e.g., targeting the whole virion), subunit vaccine (e.g., targeting the whole virion), and other peptide vaccines.
40. The method of any one of claims 28 through 39, wherein the disease is Covid-19.
41 . A method of using a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, as a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV- related disease, preferably Covid-19.
42. An immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19 for manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
43. Use of an immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, or a recombinant cell of any one of embodiments 18 and 19 for the manufacture of a medicament for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
44. An immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, or a recombinant cell of any one of embodiments 18 and 19 for the prevention or treatment of severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
45. Use of an immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19, or an antibody against a polypeptide of any one of embodiments 1 through 13, for the manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV- related disease, preferably Covid-19.
46. An immunogenic composition of any one of embodiments 20 through 27, a polypeptide of any one of embodiments 1 through 13, or an antibody against a polypeptide of any one of embodiments 1 through 13, a nucleic acid of any one of embodiments 14 and 15, a vector of any one of embodiments 16 and 17, a recombinant cell of any one of embodiments 18 and 19 for manufacture of a diagnostic reagent to detect SARS virus, SARS Cov2 S protein, a complex of SARS Cov2 S protein and ACE2, or severe acute respiratory syndrome or other SARS-CoV-related disease, preferably Covid-19.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113527444A (en) * 2020-04-13 2021-10-22 中国科学院微生物研究所 Epitopes effective binding to antibodies of novel coronaviruses
WO2022013609A1 (en) * 2020-07-13 2022-01-20 Immunovaccine Technologies, Inc. Sars-cov-2 vaccine compositions and methods of preparation and use
GB2604692A (en) * 2020-11-04 2022-09-14 Beijing Shangwei Biotechnology Dev Co Ltd Method for preparing recombinant subunit vaccine against novel coronavirus
WO2023104154A1 (en) * 2021-12-08 2023-06-15 安达生物药物开发(深圳)有限公司 Antigenic polypeptide and use thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005018538A2 (en) * 2003-06-04 2005-03-03 Vaxim, Inc. Severe acute respiratory syndrome (sars) polypeptides, antibodies to sars polypeptides and the use thereof in diagnostic, vaccination and therapeutic applications
CN1609119A (en) * 2003-10-23 2005-04-27 中国医学科学院药物研究所 Peptide library, its synthesis process and active segment screened from the peptide library
EP1618127A2 (en) * 2003-04-10 2006-01-25 Chiron Corporation The severe acute respiratory syndrome coronavirus
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
CN112028978A (en) * 2020-09-07 2020-12-04 重庆医科大学 Novel coronavirus specific CD8+T cell epitope peptide and application thereof
RU2738081C1 (en) * 2020-10-14 2020-12-07 Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере зашиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) Peptide immunogens and a vaccine composition against coronavirus infection covid-19 using peptide immunogens
WO2021035325A1 (en) * 2019-08-27 2021-03-04 Fundação Oswaldo Cruz Protein receptacle, polynucleotide, vector, expression cassette, cell, method for producing the receptacle, method of identifying pathogens or diagnosing diseases, use of the receptacle and diagnostic kit

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1618127A2 (en) * 2003-04-10 2006-01-25 Chiron Corporation The severe acute respiratory syndrome coronavirus
WO2005018538A2 (en) * 2003-06-04 2005-03-03 Vaxim, Inc. Severe acute respiratory syndrome (sars) polypeptides, antibodies to sars polypeptides and the use thereof in diagnostic, vaccination and therapeutic applications
CN1609119A (en) * 2003-10-23 2005-04-27 中国医学科学院药物研究所 Peptide library, its synthesis process and active segment screened from the peptide library
WO2021035325A1 (en) * 2019-08-27 2021-03-04 Fundação Oswaldo Cruz Protein receptacle, polynucleotide, vector, expression cassette, cell, method for producing the receptacle, method of identifying pathogens or diagnosing diseases, use of the receptacle and diagnostic kit
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
CN112028978A (en) * 2020-09-07 2020-12-04 重庆医科大学 Novel coronavirus specific CD8+T cell epitope peptide and application thereof
RU2738081C1 (en) * 2020-10-14 2020-12-07 Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере зашиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) Peptide immunogens and a vaccine composition against coronavirus infection covid-19 using peptide immunogens

Non-Patent Citations (43)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. MN197520.1
"Guide to Huge Computers", 1994, ACADEMIC PRESS
"NCBI", Database accession no. NP_001358344.1
AMANAT FKRAMMER F.: "SARS-CoV-2 vaccines: status report", J IMMUN, 2020
ATSCHUL, S. F. ET AL., J MOLEC BIOL, vol. 215, 1990, pages 403
BOLLES MDEMING DLONG KAGNIHOTHRAM SWHITMORE A ET AL.: "A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge", J VIROL, vol. 85, 2011, pages 12201 - 12215
BOSCH BJDE HAAN CAMSMITS SLROTTIER PJM: "Spike protein assembly into the coronavirion: Exploring the limits of its sequence requirements", VIROLOGY, vol. 334, 2005, pages 306 - 318, XP004796061, DOI: 10.1016/j.virol.2005.02.001
BOSCH BJMARTINA BEVAN DER ZEE RLEPAULT JHAIJEMA BJVERSLUIS C ET AL.: "Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides", PROC. NATL. ACAD. SCI. USA, vol. 101, 2004, pages 8455 - 8460, XP002994902, DOI: 10.1073/pnas.0400576101
CARILLO ET AL., SIAM J APPLIED MATH, vol. 48, 1988, pages 1073
CHAN JFTO KKTSE H ET AL.: "Interspecies transmission and emergence of novel viruses: lessons from bats and birds", TRENDS MICROBIOL, vol. 21, no. 10, 2013, pages 544 - 555
CHEUNG ET AL., VIROLOGY, vol. 176, 1990, pages 546 - 552
DAYHOFF ET AL., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, vol. 5, 1978, pages 345 - 352
DEVEREUX ET AL., NUCL. ACID RES., vol. 12, 1984, pages 387
DEVEREUX, I ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. 1, 1984, pages 387
DHAMA KSHARUN KTIWARI RDADAR MMALIK YSSINGH KPCHAICUMPA W.: "COVID-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics", HUM VACCIN IMMUNOTHER, vol. 18, 2020, pages 1 - 7
DONG SSUN JMAO ZWANG LLU YLLI J: "A guideline for homology modeling of the proteins from newly discovered betacoronavirus, 2019 novel coronavirus (2019-nCoV", J MED VIROL, 2020
FEHR ARPERLMAN S.: "Coronaviruses: An Overview of Their Replication and Pathogenesis", METHODS MOL BIOL, vol. 1282, 2015, pages 1 - 23
FRANCIS GS: "ACE inhibition in cardiovascular disease", N ENGL J MED, vol. 342, 2000, pages 201 - 202
HENIKOFF ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 89, 1992, pages 10915 - 10919
HOFFMANN MKLEINE-WEBER HKRUGER NMULLER MDROSTEN CPOHLMANN S: "The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells", BIORXIV, 10.1101/2020.01.31.929042, 2020
JIA WCHANNAPPANAVAR RZHANG CLI M ET AL.: "Single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against MERS-CoV infection", EMERG MICROBES INFECT, vol. 8, 2019, pages 760 - 772
JUO: "The Concise Dictionary of Biomedicine and Molecular Biology", 2001, CRC PRESS
KIRKLAND ET AL., J. IMMUNOL., vol. 137, 1986, pages 3614 - 3619
KYTEDOOLITTLE, J MOL BIOL, 1982
LI FLI WFARZAN MHARRISON SC: "Structure of SARS coronavirus spike receptor-binding domain complexed with receptor", SCIENCE, vol. 309, 2005, pages 1864 - 1868
LI WJOSHI MDSINGHANIA SRAMSEY KHMURTHY AK: "Peptide Vaccine: Progress and Challenges", VACCINES, vol. 2, 2014, pages 515 - 536
LU RZHAO XLI JNIU PYANG BWU HWANG W ET AL.: "Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding", LANCET, vol. 395, no. 10224, 2020, pages 565 - 574
MASTERS PS: "The molecular biology of coronaviruses", ADV VIRUS RES, vol. 66, 2006, pages 193 - 292
MOLDENHAUER ET AL., SCAND. J. IMMUNOL., vol. 32, 1990, pages 77 - 82
MOREL ET AL., MOLEC. IMMUNOL., vol. 25, 1988, pages 7 - 15
PEARSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444
SAHIN ARERDOGAN AMUTLU AGAOGLU PDINERI YCAKIRCI AYSENEL ME ET AL.: "2019 Novel Coronavirus (COVID-19) Outbreak: A Review of the Current Literature", EJMO, vol. 4, no. 1, 2020, pages 1 - 7
SCHOEMAN DFIELDING BC: "Coronavirus envelope protein: current knowledge", VIROLOGY J, vol. 16, 2019, pages 69
SONG HCSEO MYSTADLER KYOO BJ ET AL.: "Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein", J VIROL, vol. 78, 2004, pages 10328 - 10335, XP002569185, DOI: 10.1128/JVI.78.19.10328-10335.2004
SONG WFGUI MWANG X ET AL.: "Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2", PLOS PATHOG, vol. 14, no. 8, 2018, pages e1007236
STAHLI ET AL., METHODS IN ENZYMOLOGY, vol. 9, pages 242 - 253
SU SWONG GSHI WLIU JLAI ACKZHOU JLIU WBI YGAO GF: "Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses", TRENDS MICROBIOL, vol. 24, 2016, pages 490 - 502, XP029538778, DOI: 10.1016/j.tim.2016.03.003
TSENG CTSBRANA EIWATA-YOSHIKAWA NNEWMAN PCGARRON T ET AL.: "Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus", PLOS ONE, vol. 7, 2012, pages e35421
VANKADARI NWILCE JA: "Emerging WuHan (COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26", EMERG MICROBES INFECT. 17, vol. 9, no. 1, 2020, pages 601 - 604
WALLS ACPARK YJTORTORICI MAWALL AMCGUIRE ATVEESLER D.: "Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein", CELL, vol. 8674, no. 20, 2020, pages 30262 - 2
WANG PSIDNEY JDOW CMOTHE BSETTE APETERS B.: "A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach", PLOS COMPUT BIOL., vol. 4, no. 4, 2008, pages e1000048, XP002598739, DOI: 10.1371/journal.pcbi.1000048
WANG PSIDNEY JKIM YSETTE ALUND ONIELSEN MPETERS B.: "Peptide binding predictions for HLA DR, DP and DQ molecules", BMC BIOINFORMATICS, vol. 11, 2010, pages 568, XP021085842, DOI: 10.1186/1471-2105-11-568
WRAPP DWANG NCORBETT KSGOLDSMITH JA ET AL.: "Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation", SCIENCE, 2020

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113527444A (en) * 2020-04-13 2021-10-22 中国科学院微生物研究所 Epitopes effective binding to antibodies of novel coronaviruses
WO2022013609A1 (en) * 2020-07-13 2022-01-20 Immunovaccine Technologies, Inc. Sars-cov-2 vaccine compositions and methods of preparation and use
GB2604692A (en) * 2020-11-04 2022-09-14 Beijing Shangwei Biotechnology Dev Co Ltd Method for preparing recombinant subunit vaccine against novel coronavirus
GB2604692B (en) * 2020-11-04 2023-12-06 Beijing Shangwei Biotechnology Dev Co Ltd Method for preparing recombinant subunit vaccine against novel coronavirus
WO2023104154A1 (en) * 2021-12-08 2023-06-15 安达生物药物开发(深圳)有限公司 Antigenic polypeptide and use thereof

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