WO2022015662A1

WO2022015662A1 - Coronavirus immunogenic t cell epitope compositions and uses thereof

Info

Publication number: WO2022015662A1
Application number: PCT/US2021/041313
Authority: WO
Inventors: Bertrand Georges
Original assignee: Altimmune, Inc; ANDERSON, Koren
Priority date: 2020-07-12
Filing date: 2021-07-12
Publication date: 2022-01-20

Abstract

Provided in the present disclosure are immunogenic compounds, pharmaceutical formulations thereof and their use for inducing a protective immune response against 2019 novel coronavirus (SARS-CoV-2) infection in a mammal.

Description

CORONAVIRUS IMMUNOGENIC T CELL EPITOPE COMPOSITIONS AND USES

THEREOF

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Ser. No. 63/050,852 filed on 12 July 2020, which is hereby incorporated into this application in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created on 09 July 2021, is named ALT2030PCT_ST25.txt and is 313,448 bytes in size.

FIELD OF THE DISCLOSURE

[0003] This application pertains generally to an immunogenic coronavirus peptide pharmaceutical formulation for administration to a mammalian subject that induces an immune response in the subject and optionally provides protection against novel 2019 Coronavirus (SARS- CoV-2).

BACKGROUND INFORMATION

[0004] The coronaviruses are a diverse group of large enveloped, positive-stranded RNA (ss RNA) viruses that cause respiratory and enteric diseases in humans and other animals. For example, Human coronaviruses 229E (HCoV-229E), OC43 (HCoV-OC43), NL63, and HKU 1 are endemic in the human population and cause up to 30% of common colds. Coronaviruses of animals (e.g., porcine transmissible gastroenteritis virus (TGEV), murine hepatitis virus (MHV) and avian infectious bronchitis virus (IBV)) cause respiratory, gastrointestinal, neurological, or hepatic disease in their respective hosts.

[0005] Coronavirus has a positive-sense, non-segmented, single-stranded RNA genome, which encodes at least 18 viral proteins (such as non- structural proteins (NSP) 1-13, structural proteins E, M, N, S, and an RNA-dependent RNA polymerase). Coronavirus has three major surface glycoproteins (designated S, E, and M), and some coronaviruses have another surface glycoprotein referred to as hemagglutinin esterase (HE), in addition, the N (nucleocapsid) protein is a basic phosphoprotein, which is generally associated with the genome and has been reported to be antigenic (Holmes and Lai, Fields Virology, Chapter 34, 1996). The S (spike) protein, a major antigen of coronavirus, has two domains: SI, which is believed to be involved in receptor binding and S2, believed to mediate membrane fusion between the virus and target cell (Holmes and Lai, 1996, supra).

[0006] The S (spike) protein may form non-covalently linked homotrimers (oligomers), which may mediate receptor binding and virus infectivity. Homotrimers of S proteins are likely necessary for presenting the correct native conformation of receptor binding domains and for eliciting a neutralizing antibody response. In addition, intracellular processing of S protein is associated with significant posttranslation oligosaccharide modification. The posttranslation oligosaccharide modification (glycosylation) expected by N-glycan motif analysis indicates that the S protein has as many as 23 sites for such modification. In addition, C-terminal cysteine residues may also participate in protein folding and preserving the native (functional) S protein conformation. The S protein of some coronaviruses can be proteolytically processed near the center of the S protein by a trypsin-like protease in the Golgi apparatus or by extracellularly localized enzymes into to a linked polypeptide, containing an N-terminal SI and a C-terminal S2 polypeptide. Presently, the coronaviruses are subdivided into a-genus, b-genus (e.g., MERS, SARS, SARS-CoV-2), and g- genus.

[0007] Coronavirus infection is achieved through fusion of the lipid bilayer of the viral envelope with host cell membranes. Membrane fusion is mediated by the viral spike (S) glycoprotein on the viral envelope. The S-gly coprotein is synthesized as a precursor of about 180 kDa that oligomerizes in the endoplasmic reticulum and is incorporated into budding virions in a pre-Golgi compartment. S 1 contains the receptor-binding site and thus contributes to defining the host range of the virus. S2 is the transmembrane subunit which contributes to mediating fusion between viral and cellular membranes. S2 contains two 4,3-hydrophobic repeat domains (HR) that are predicted to form coiled-coil structures. These regions are denoted HR-1 and HR-2, and are separated by an intervening stretch of amino acid residues called the interhelical domain. These coiled-coil regions may play an important role in defining the oligomeric structure of the spike protein in its native state and its fusogenic ability. [0008] The novel coronovirus SARS-CoV-2 (initally reported as 2019-nCoV and officially named SARS-CoV-2 by the Coronavirus Study Group (a working group of the International Committee on Taxonomy of Viruses) based on phylogeny, taxonomy and estabilished practice (BioRxiv; doi.org/10.1101/2020.02.07.937862)) is a new strain that has not been previously identified in humans and was first reported in Wuhan, Hubei Province, China. SARS-CoV-2 is the cause of the ongoing 2019-20 Wuhan coronavirus outbreak, a global health emergency. Genomic sequencing has shown that it is a positive-sense, single-stranded RNA coronavirus (GenBank Accession No. MN908947.3; RefSeq NC_045512; “Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1”). Coronaviruses are primarily spread through close contact, in particular through respiratory droplets from coughs and sneezes within a range of about 6 feet (1.8 m). Common signs of infection include respiratory symptoms, fever, cough, shortness of breath and breathing difficulties. In more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, kidney failure and even death. There is an urgent need for immunogenic peptides and proteins, as well as compositions comprising the same (e.g., vaccines) that can be used to induce and/or enhance an immune response against SARS-CoV-2. This disclosure provides solutions to these problems.

SUMMARY OF THE DISCLOSURE

[0007] In some embodiments, this disclosure provides reagents, compositions, and methods for inducing and/or improving (e.g., enhancing) an immune response against coronavirus, in particular novel 2019 coronalvirus SARS-CoV-2. For instance, in some embodiments, this disclosure provides peptide constructs comprising (e.g., representing, corresponding to, and/or being derived from) at least one coronavirus T cell epitope (preferably at least one SARS-CoV-2 antigen(s)). In preferred embodiments, the peptide constructs comprise at least one such peptide conjugated to a flurocarbon vector (e.g., as a fluoropeptide construct). As discussed herein, such peptide constructs (and/or immunogenic compositions comprising the same) can be used to induce immune responses against coronavirus (preferably SARS-CoV-2). In some embodiments, this disclosure provides methods comprising the administration of such peptide constructs to animals and/or human beings to induce and/or enhance an immune response (e.g., the production of antibodies and/or CD8⁺ T cells (and/or other T cells)) having specificity for coronavirus (preferably SARS-CoV-2) T cell and/or B cell epitope(s). In some embodiments, such immune response is protective against coronavirus, preferably SARS-CoV-2, and/or effective in ameliorating the symptoms caused by and/or infection by such coronavirus, and in preferred embodiments can be protective against coronavirus, preferably SARS-CoV-2, challenge.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and examples sections, serve to explain the principles and implementations of the disclosure.

[0010] Figure 1A-J. Exemplary SARS-CoV-2 complete genome (Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome; GenBank: MN908947.3).

[0011] Figure 2A-E. SARS-CoV-2 Polyprotein (GenBank: QHD43415.1; SEQ ID NO: 2).

[0012] Figure 3. SARS-CoV-2 surface glycoprotein (e.g. Spike protein) including the native leader sequence (GenBank: QHD43416.1; SEQ ID NO: 3).

[0013] Figure 4. SARS-CoV-2 ORF3A protein (GenBank: QHD43417.1; SEQ ID NO: 4).

[0014] Figure 5. SARS-CoV-2 envelope protein (GenBank: QHD43418.1; SEQ ID NO: 5).

[0015] Figure 6. SARS-CoV-2 membrane glycoprotein (GenBank: QHD43419.1; SEQ ID NO: 6)·

[0016] Figure 7. SARS-CoV-2 ORF6 protein (GenBank: QHD43420.1; SEQ ID NO: 7).

[0017] Figure 8. SARS-CoV-2 ORF7a protein (GenBank: QHD43421.1; SEQ ID NO: 8).

[0018] Figure 9. SARS-CoV-2 ORF8 protein (GenBank: QHD43422.1; SEQ ID NO: 9).

[0019] Figure 10. SARS-CoV-2 nucleocapsid phosphoprotein (GenBank: QHD43423.2; SEQ ID NO: 10).

[0020] Figure 11. SARS-CoV-2 ORF10 protein (GenBank: QHD43423.2; SEQ ID NO: 11).

[0021] Figures 12. Profiles of high affinity HLA class I and HLA class II binding motifs across the entire SARS-CoV-2 proteome. [0022] Figure 13. Profiles of high and moderate affinity HLA class I and HLA class II binding motifs across the entire SARS-CoV-2 proteome.

[0023] Figure 14. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 328. [0024] Figure 15. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 329. [0025] Figure 16. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 330. [0026] Figures 17. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 331. [0027] Figure 18. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 332.

[0028] Figure 19. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 333.

[0029] Figure 20. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 334. [0030] Figure 21. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 335. [0031] Figure 22. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 336. [0032] Figure 23. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 337.

[0033] Figure 24. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 338.

[0034] Figure 25. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 339. [0035] Figure 26. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 340. [0036] Figures 27. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 341. [0037] Figure 28. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 342.

[0038] Figure 29. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 343.

[0039] Figure 30. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 344. [0040] Figure 31. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 345. [0041] Figure 32. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 346. [0042] Figure 33. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 347.

[0043] Figure 34. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 348.

[0044] Figure 35. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 349. [0045] Figure 36. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 350.

[0046] Figure 37. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 351.

[0047] Figure 38. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 352.

[0048] Figure 39. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 353.

[0049] Figure 40. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 354.

[0050] Figure 41. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 355.

[0051] Figure 42. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 356.

[0052] Figure 43. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 357.

[0053] Figure 44. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 358.

[0054] Figure 45. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 359.

[0055] Figure 46. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 360.

[0056] Figure 47. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 361.

[0057] Figure 48. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 362.

[0058] Figure 49. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 363.

[0059] Figure 50. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 364.

[0060] Figure 51. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 365.

[0061] Figure 52. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 366.

[0062] Figure 53. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 367.

[0063] Figure 54. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 368.

[0064] Figure 55. Map of HLA class I and HLA class II binding motifs for SEQ ID NO: 369.

[0065] Figures 56A through 56E. SARS-CoV-2 concatenated Polyprotein SEQ ID NO: 410; Derived from SEQ ID NO: 1 (GenBank: MN908947.3). DETAILED DESCRIPTION

[0066] SARS-CoV-2, initially reported as 2019-nCoV and subsequently officially named COVID- 19 by the World Health Organization (WHO), is a new and highly pathogenic virus, only emerging in December 2019. The present disclosure relates to an immunogenic composition (e.g., vaccine) comprising a peptide vector comprising (e.g., representing, corresponding to, and/or being derived from) at least one 2019 novel coronavirus SARS-CoV-2 antigen(s), compositions comprising the same (preferably immunogenic compositions), and the use thereof for inducing a protective immune response against SARS-CoV-2. In preferred embodiments, the immunogenic compositions induce and/or enhance an immune response against the SARS-CoV-2 antigen as the antigen occurs in naturally circulating coronavirus (e.g., SARS-CoV-2). Accordingly, the immunogenic compositions disclosed herein provide for treatments for SARS-CoV-2 virus.

[0067] Immunogenic Compositions and Vaccines

[0068] In some embodiments, this disclosure provides peptide constructs and compositions comprising the same (e.g., immunogenic compositions). In embodiments, the peptide constructs comprise a coronavirus peptide comprising one or more T-cell epitopes and a vector configured for intracellular delivery of the peptide. In embodiments, the peptide constructs are delivered to antigen presenting cells, wherein the peptides are processed and presented inducing an immune response. In certain embodiments, the peptide constructs comprise an amphiphilic fluorocarbon construct conjugated to a “peptide” (R of Formula I) comprising at least one coronavirus (preferably SARS-CoV-2) antigen (e.g., epitope). Conjugation of a fluorocarbon vector to the immunogenic peptide increases intracellular delivery to antigen presenting cells (APC) (see, e.g., U.S. Pat. No. 9,119,811 B2 (Bradley, et al. Issued Sept. 1, 2015 and/or U.S. Pat. No. 10,300,132 B2 (Georges, et al. Issued May 29, 2019) both of which are hereby incorporated in their entireties into this disclosure). Such peptide constructs are referred to herein collectively as “anti- coronavirus peptide constructs” (or “anti-SARS-CoV-2 peptide constructs” wherein the peptide vectors are specific for anti-SARS-CoV-2). As discussed herein, such anti-coronavirus (e.g., anti- SARS-CoV-2) peptide constructs (and/or immunogenic compositions comprising the same) can preferably be used to induce mucosal and/or systemic cell-mediated and/or antibody immune responses against a coronavirus (in preferred embodiments SARS-CoV-2 (e.g., against protective SARS-CoV-2 epitopes such as spike (S) protein receptor binding domain (RBD)). In some embodiments, such anti-coronavirus (in preferred embodiments anti-SARS-CoV-2) peptide constructs (and/or immunogenic compositions comprising the same) stimulate an immune response if used prophylactically or as a treatment (e.g., interfering directly) with coronavirus (in preferred embodiments SARS-CoV-2) infection if administered during the pre-exposure period (few days before infection) or during the post-exposure period. In some embodiments, this disclosure describes the administration of such vectors to animals and/or human beings to induce and/or enhance an immune response (e.g., the production of antibodies and/or CD8⁺ T cells (and/or other T cells)) having specificity for coronavirus (in preferred embodiments SARS-CoV-2 T cell epitope(s) (e.g., a dominant epitopes). In some embodiments, such immune response is protective against coronavirus (in preferred embodiments SARS-CoV-2) and/or effective in ameliorating the symptoms and/or infection by coronavirus (in preferred embodiments SARS-CoV-2), and in some embodiments can be protective against a coronavirus (in preferred embodiments SARS-CoV-2) challenge. Thus, in some embodiments, this disclosure describes the use of an immunogenic composition(s) comprising one or more anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs to provide solutions to relating to coronavirus, and in preferred embodiments SARS- CoV-2, transmission and infection.

[0069] In some embodiments, this disclosure provides methods for preventing and/or treating coronavirus (in preferred embodiments SARS-CoV-2) infection (i.e., COVID) in a human being, the method comprising administering to a human being an anti-coronavirus (preferably anti- SARS-CoV-2) peptide construct comprising at least one peptide of from about 15 to about 60 amino acids, and comprising at least one CD8⁺ T-cell epitope, and at least one CD4⁺ T-cell epitope, the epitope(s) being derived from or corresponding to coronavirus (in preferred embodiments SARS-CoV-2) antigens (i.e., comprising amino acid sequences included in such antigens, and/or immunogenic derivatives and/or fragments thereof); the peptide being covalently attached to a vector configured for intracellular delivery comprising a chain of 3 to 30 carbon atoms, at least one of which is substituted with fluorine, chlorine, bromine or iodine, optionally wherein the vector is a fluorocarbon vector. In some embodiments, the peptide construct can have the formula C_mZ_n- C_yH_x(Sp)-R (Formula I), wherein m=3 to 30; Z is fluorine, chlorine, bromine or iodine; n < 2m+l; y=0 to 15; x < 2y; (m+y)=3-30; Sp is an optional spacer moiety; and R is the “peptide” which comprises at least one coronavirus (preferably SARS-CoV-2) antigen and/or epitope thereof (preferably about eight to about 60 amino acid residues, more preferably about eight to about 35 amino acid residues, and more preferably about eight to about 25 amino acid residues); wherein Sp is optionally derived from a lysine residue or has the formula -CONH-(CH2)4-CO-. In preferred embodiments of Formula I, Z is fluorine (F). In some embodiments, then, this disclosure provides such anti-coronavirus (preferably anti-SARS-CoV-2) peptide construct and methods for making the same. In some embodiments, the methods disclosed herein can comprise administering a composition one or more of such anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs, preferably as an anti-coronavirus or anti-SARS-CoV-2 composition. In some embodiments, each anti-coronavirus peptide construct (preferably an anti-SARS-CoV-2 peptide construct), or a collection of such constructs, elicits an anti-coronavirus (preferably anti-SARS- CoV-2) response in peripheral blood mononuclear cells (PBMC) from at least one coronavirus (in preferred embodiments SARS-CoV-2)-infected individual in an in vitro assay. In some embodiments, the composition can further comprise one or more other antigens and/or one or more adjuvants.

[0070] Intracellular Delivery Vectors

[0071] In preferred embodiments of Formula I, Z is fluorine (F) such that the peptide is part of a fluorocarbon vector (e.g., as a “anti-coronavirus fluoropeptide construct”). The fluorocarbon portion of the anti-coronavirus fluoropeptide construct can comprise one or more chains derived from perfluorocarbon or mixed fluorocarbon/hydrocarbon radicals, and may be saturated or unsaturated, each chain having from 3 to 30 carbon atoms. Thus, the chains in the fluorocarbon attachment are typically saturated or unsaturated, preferably saturated. The chains in the fluorocarbon attachment may be linear or branched, but preferably are linear. Each chain typically has from 3 to 30 carbon atoms, from 5 to 25 carbon atoms, or from 8 to 20 carbon atoms. In order to covalently link the fluorocarbon vector to the peptide, a reactive group, or ligand, for example -CO-, -H-, S, O or any other suitable group is included in the vector. The use of such ligands for achieving covalent linkages is well known in the art. The reactive group may be located at any position on the fluorocarbon vector. Coupling of the fluorocarbon vector to the peptide may be achieved through functional groups such as -OH, -SH, -COOH and - ¾, naturally present or introduced onto any site of the peptide. Examples of such linkages include amide, hydrazone, disulphide, thioether and oxime bonds. Optionally, a spacer element (peptidic or non-peptidic) can be incorporated to permit cleavage of the peptide from the fluorocarbon element for processing within an antigen-presenting cell and to optimize steric presentation of the peptide. The spacer can also be incorporated to assist in the synthesis of the molecule and to improve its stability and/or solubility. Examples of spacers include polyethylene glycol (PEG) or amino acids such as lysine or arginine that may be cleaved by proteolytic enzymes. Preferably the C_mFn-C_yH_x moiety of Formula I is linear. It is preferred that m is from 5 to 15, more preferably from 8 to 12. It is also preferred that y is from 0 to 8, more preferably from 0 to 6 or 0 to 4. It is preferred that the CmFn- C_yH_x moiety is saturated (i.e., n=2m+l and x=2y) and linear, and that m=8 to 12 and y=0 to 6 or 0 to 4. In a particular example, the fluorocarbon vector is derived from 2H, 2H, 3H, 3H- perfluoroundecanoic acid of the following formula:

Thus, a preferred fluorocarbon attachment is the linear saturated moiety C8Fn(CH2)2 which is derived from C8Fn(CH2)2COOH. Further examples of fluorocarbon attachments have the following formulae: C6Fi3(CH2)2-, CvFi5(CH2)2-, C9Fi9(CH2)2-, CioF2i(CH2)2-, C5Fn(CH2)3-, C6Fi3(CH2)3-, CVFI5(CH2)3-, C8Fn(CH2)3-, and C9Fi9(CH2)3-, which are derived from C₆Fi₃(CH₂)₂COOH, C_VF_I5(CH₂)₂ COOH, C₉F_I9(CH₂)₂COOH, C_IOF_2I(CH₂)₂COOH, C₅F_H(CH₂)3COOH, C₆Fi3(CH₂)3 COOH, C₇F_I5(CH₂)3COOH, CsF^aE^COOH, and C₉Fi9(CH₂)3COOH, respectively. Preferred examples of suitable structures for the fluorocarbon vector-antigen constructs have the formula:

in which Sp and R are as defined above. In certain embodiments Sp is derived from a lysine residue and has the formula -CONH-(CH2)4-CH(NH2)-CO-. In the context of this disclosure, the fluorocarbon attachment may be modified such that the resulting compound is still capable of delivering the peptide to antigen presenting cells. Thus, for example, a number of the fluorine atoms may be replaced with other halogen atoms such as chlorine, bromine or iodine. In addition, it is possible to replace a number of the fluorine atoms with methyl groups and still retain the properties of the molecule described herein. The peptides may be linked to the fluorocarbon vector via a spacer moiety. The spacer moiety is preferably a lysine residue. This spacer residue may be present in addition to any terminal lysine residues as described above, so that the peptide may, for example, have a total of four N-terminal lysine residues. Accordingly, the preferred formulation of the invention may comprise fluorocarbon-linked peptides in which the peptides have a C- terminal or N-terminal lysine residue, preferably an N-terminal lysine residue. The terminal lysine in the peptides is preferably linked to a fluorocarbon having the formula CsFn (CTh^COOH. The fluorocarbon is preferably coupled to the epsilon chain of the N-terminal lysine residue. It is contemplated that the pharmaceutical compositions described herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more immunogenic peptides optionally each covalently linked to its own fluorocarbon vector. In preferred embodiments, the anti-coronavirus fluoropeptide construct comprises a fluorocarbon having the formula C_mF_n-C_yH_x, where m=3 to 30, n < 2m+l, y=0 to 15, x < 2y, (m+y)=3-30, that in some embodiments may be derived from:

[0072] In some embodiments, the composition(s) may comprise at least one peptide (R in Formula I) comprising at least about eight (8) amino acids of one of the sequences disclosed herein (e.g., about 8 to about 60, or about 15 to about 60, or at least about 8 to about 20, contiguous amino acids corresponding to a coronavirus polypeptide, preferably a SARS-CoV-2 polypeptide). In preferred embodiments, each peptide comprises at least one CD8⁺ T-cell epitope and at least one CD4⁺ T-cell epitope. In some embodiments, one or more of the peptides may comprise one or more amino acid(s) at the N-terminus and/or C-terminus to increase the net positive charge and/or to reduce hydrophobicity of the peptide. Some aspects of certain peptide constructs, including fluoropeptide constructs, and the like have been previously described in U.S. Pat. No. 9,119,811 B2 (Bradley, et al. Issued Sept. 1, 2015 and/or U.S. Pat. No. 10,300,132 B2 (Georges, et al. Issued May 29, 2019) for Hepatitis B virus (HBV), that may be applicable to the anti-coronavirus (in preferred embodiments anti-SARS-CoV-2) peptide constructs disclosed herein, which are hereby incorporated in their entireties into this disclosure. For instance, some of the methods for synthesizing the HBV peptide constructs of that patent may be applicable to the anti-coronavirus (in preferred embodiments SARS-CoV-2) peptide constructs disclosed herein. In some embodiments, a peptide can be synthesized by FMOC (fluorenylmethyloxycarbonyl chloride) solid-phase synthesis. A fluorocarbon chain (e.g., CsF^CFh^COOH) can then be incorporated on the epsilon-chain of an additional N-terminal lysine of each peptide to derive the fluorocarbon- linked peptide. Purified fluorocarbon-linked peptides or unmodified peptides can be obtained through cleavage in the presence of trifluoroacetic acid (TFA) and a final purification by reverse phase-high performance liquid chromatography (RP-HPLC). In preferred embodiments, the preparations exhibit a fluorocarbon purity of 90% or greater. Other embodiments are also contemplated herein as would be understood by those of ordinary skill in the art.

[0073] T-Cell Epitope Peptides

[0074] In some embodiments, the coronavirus antigen(s) of the anti-coronavirus peptide construct can be a peptide (i.e., R of Formula I) corresponding to or derived from an antigen of a coronavirus (preferably SARS-CoV-2) virus. In some embodiments, the one or more anti-coronavirus (preferably anti-SARS-CoV-2) peptide construct(s) includes a peptide (R of Formula I) that represents, corresponds to, is derived from a coronavirus (preferably SARS-CoV-2) antigen, and preferably induces and/or enhances an immune response against a coronavirus (preferably SARS- CoV-2). In some embodiments, the peptide comprises at least (e.g., about 8 to about 60, or about 15 to about 60, or at least about 8 to about 20, contiguous amino acids corresponding to a coronavirus polypeptide, preferably a SARS-CoV-2 polypeptide, such as but not limited to the peptide sequences disclosed herein. In preferred embodiments, the peptide comprising one or more immunogenic domains of a coronavirus (preferably SARS-CoV-2) antigen comprising at least one B cell and/or T cell epitope, preferably at least one CD4⁺ T cell epitope and at least one CD8⁺ T cell epitope. Thus, in some embodiments, the anti-coronavirus (preferably SARS-CoV- 2) peptide construct(s) can comprise one or more peptide sequences of from 8 to 60 amino acids in length (and concatenated if more than one peptide sequence is included) (as R of Formula I), wherein each peptide sequence comprise at least one CD8⁺ T cell epitope and/or at least one CD4⁺ T cell epitope, and/or at least one B cell epitope. In some embodiments, a concatenated peptide of this disclosure can include T cell epitopes separated by a spacer sequence containing amino acids such as glycine, serine or alanine residues, among others, and/or ubiquitin or a signal peptide. [0075] This disclosure describes certain epitopes but one of skill in the art understands how to identify additional epitopes within a larger sequence using bioinformatic methodologies such as publicly available tools accessible at the immune epitope database (IEDB), in vitro assay based on PBMCs from infection -positive subjects combined with short linear peptides scanning the antigen sequence or in vitro assay based on serum using short linear or conformational peptides scanning the antigen sequence. For instance, the identification of conserved regions with clusters of T cell epitopes (at least one CD8⁺ T-cell epitope and at least one CD4⁺ T-cell epitope) has been previously applied to other pathogens including mutating viruses. For example, Heiny et al (Evolutionarily conserved protein sequences of influenza A viruses, avian and human, as vaccine targets. PLoS One. 2007 Nov 21;2(11)) described a process for the identification of influenza A vaccine targets using a bioinformatics approach addressing antigen conservation and presence of HLA class I & HLA class II binding motifs. Similarly, Khan et al. (Conservation and variability of dengue virus proteins: implications for vaccine design. PLoS Negl Trop Dis. 2008 Aug 13;2(8)) used a combination of bioinformatics and experimental approaches for the identification of immunologically relevant peptides from Dengue viruses that can be considered a framework for large-scale and systematic analysis of other pathogens as presented by the authors. And Zhang et al. (Hotspot Hunter: a computational system for large-scale screening and selection of candidate immunological hotspots in pathogen proteomes. BMC Bioinformatics. 2008;9 Suppl 1 : S 19) have developed a computational methodology to identify T cell epitopes clusters from large size pathogen proteomes, including of variant strains that may be exploited to facilitate the development of epitope-based vaccines. In addition, HLA class I binding motifs and HLA class II binding motifs can be performed using the ANN prediction tool available at https://www.iedb.org/ using, for instance, a high binding affinity threshold of 50nM (see, e.g., http://tools.iedb.org/mhci/help/; see also Propredll (http://crdd.osdd.net/raghava/propred/)). Bioinformatics models such as these exist, that can be used to identify conserved antigen regions containing multiple HLA class I and/or HLA class II binding motifs within the most conserved SARS-CoV-2 domains to confer the highest population coverage. Population coverage refers to the ability of the vaccine or immuno-therapeutics to induce an T cell immune response in a percentage of the population. Population coverage is proportional to the number and the allelic frequencies of HLA molecules by which the peptide(s) will be presented to the immune system at the population level. For a peptide-based vaccine, the induction of T cells responses in the vast majority of the population will be thus dependent upon the number and specificity of HLA- restricted epitopes contained within a peptide sequence. In addition, achieving a broad population coverage will also be depending upon the total number and specificity of HLA-restricted epitopes contained in the vaccine that can be achieved by combining multiple peptides in the same formulation. In some embodiments, peptides can be further selected based on their ability to stimulate T cell responses in coronavirus (preferably SARS-CoV-2) patients, including but not limited to actively infected or recovered patients, using an in vitro PBMC assay. In some embodiments, such techniques can comprise selection of antigen regions for screening (e.g., identification of conserved regions within/between coronavirus genotypes and HLA class I and HLA class II binding motifs therein), in vitro screening of PBMCs (e.g., delineation of large immunodominant regions (e.g., 35-64 amino acid residues) using, e.g., peptide pools, pools of pools, and species of pools), bioinformatics (e.g., sequence length reduction of immunodominant regions to, e.g., 31-40 amino acid residues, while maximizing the presence of HLA class I and HLA class II binding motifs and conservation), in vitro screening of PBMCs to delineate restricted immunodominant regions (e.g., for different viral phenotypes), bioinformatics and peptide design for selection of peptides (e.g., 8 to 20 amino acid residues), in vitro screening of PBMCs, determination of chemical stability, formulation studies, immunogenicity in mice, and further assessment in PBMC in vitro assays, HLA binding assays, and further in vivo testing in mice, followed by, in preferred embodiments, by human clinical studies. Using such exemplary techniques, one of ordinary skill in the art could identify those peptides using a combination of bioinformatics for identification of HLA class I and HLA class II binding motifs within conserved regions of SARS-CoV-2 proteins and in vitro screening assay using PBMCs from HBV patients with the aim to progressively identify and delimit immunodominant regions of the HBV proteome suitable for selection. These and other techniques can be used to identify epitopes for use as disclosed herein as would be understood by those of ordinary skill in the art. Thus, one of ordinary skill in the art would understand that this disclosure contemplates the use of additional epitopes and peptides that may be otherwise available in the art.

[0076] In some embodiments, the immunogenic compositions of this disclosure can be monovalent or multivalent (i.e. including one, or more than one antigen epitope) by including one anti-coronavirus (preferably anti-SARS-CoV-2) peptide construct representing multiple coronavirus (preferably SARS-CoV-2) antigens (e.g., concatenated) and/or multiple anti- coronavirus (preferably anti-SARS-CoV-2) peptide constructs each representing one or more different coronavirus (preferably SARS-CoV-2) antigens. In some embodiments, the immunogenic composition comprises one or more peptide constructs, each comprising one or more coronavirus (preferably SARS-CoV-2) antigen(s). In some embodiments, this disclosure provides compositions and methods for inducing an immune response against coronavirus (preferably SARS-CoV-2) in a mammalian subjects, including human subjects. In certain embodiments provided herein is an immunogenic composition comprising an anti-coronavirus (preferably SARS-CoV-2) peptide construct comprising (e.g., representing, corresponding to, derived from) at least one coronavirus (preferably SARS-CoV-2) antigen or at least one immunogenic fragment thereof. An immunogenic composition as used herein refers to any one or more peptide constructs capable of priming, potentiating, activating, eliciting, stimulating, augmenting, boosting, amplifying, or enhancing an adaptive (specific) immune response, which may be cellular (T cell), humoral (B cell) and/or mucosal, or a combination thereof. In some embodiments, the cellular response is preferably driven by CD8⁺ T cells and/or CD4⁺ T cells with an antiviral phenotype (e.g., production interferon-gamma (IFN-g)), and in some embodiments can be a peripheral T cell response or a resident T cell response in the nasal mucosa or respiratory tract. Preferably, the immune response is protective (i.e., as a vaccine), which may include neutralization of a virus (decreasing or eliminating virus infectivity) and/or reduction in symptoms or viral shedding.

[0077] The coronavirus (preferably SARS-CoV-2) antigens, immunogens (i.e., antigens that induce an anti-coronavirus (preferably SARS-CoV-2) immune response in a mammal), fragments, and variants thereof that can be included as peptide(s) (i.e., R in Formula I) can contain one or more epitopes that can elicit, induce, and/or enhance an immune response against coronavirus (preferably SARS-CoV-2), preferably a protective immune response, which may be an antibody (i.e., humoral) response and/or a cell-mediated immune response. An anti-coronavirus (preferably anti-SARS-CoV-2) protective immune response may be manifested by at least one of the following exemplary measures: preventing infection of a host by coronavirus (preferably SARS-CoV-2); modifying or limiting the infection by coronavirus (preferably SARS-CoV-2); aiding, improving, enhancing, or stimulating recovery of the host from infection by coronavirus (preferably SARS- CoV-2); and generating immunological memory that will prevent or limit a subsequent infection by coronavirus (preferably SARS-CoV-2). A humoral response may include production of antibodies that neutralize infectivity, lyse the coronavirus (preferably SARS-CoV-2) virus and/or infected cell, facilitate removal of the coronavirus (preferably SARS-CoV-2)virus by host cells (for example, facilitate phagocytosis), and/or bind to and facilitate removal of coronavirus (preferably SARS-CoV-2) viral antigenic material. An antibody response may also include a serum/plasma and/or mucosal immune response, which can comprise eliciting or inducing an anti- coronavirus (preferably SARS-CoV-2)-specific antibody response. In certain embodiments is provided a method for inducing a combined mucosal, humoral and/or cell-mediated protective immune response in a human subject against coronavirus (preferably SARS-CoV-2) infection.

[0078] Provided herein are pharmaceutically acceptable compositions (which may also be referred to as formulations), preferably immunogenic compositions, suitable and/or configured for administration to a mammalian subject and configured to induce an immune response against one or more coronavirus (preferably SARS-CoV-2) antigen(s) (e.g., an immunogen(s)), and optionally induce a protective immune response against coronavirus (preferably SARS-CoV-2) (i.e., as a vaccine)). In preferred embodiments, the pharmaceutical formulation is an immunogenic composition that upon administration induces an immune response against at least one coronavirus (preferably SARS-CoV-2) antigen(s), and preferably the coronavirus (preferably SARS-CoV-2) virus, in a mammalian subject. In some embodiments, the pharmaceutical formulation is a vaccine and/or therapeutic composition configured to induce a protective immune response in a mammalian subject, which is protective against coronavirus (preferably SARS-CoV-2), and in preferred embodiments induces, stimulates, and/or enhances an immune response (e.g., preferably a protective immune response) against coronavirus (preferably SARS-CoV-2) infection.

[0079] In some embodiments, the coronavirus (preferably SARS-CoV-2) antigen(s) (e.g., at least one epitope of which is included in “peptide” R of Formula I) can be from (i.e., represent, correspond to, be derived from, induce an immune response to) any coronavirus (preferably SARS- CoV-2) component. In some embodiments, the coronavirus (preferably SARS-CoV-2) component can be a coronavirus (preferably SARS-CoV-2) spike (S) protein, an immunogenic fragment thereof, or a consensus spike (S) antigen derived from the sequences of spike antigens from multiple strains of coronavirus (preferably SARS-CoV-2, but in some embodiments including closely related SARS isolates) identified during the 2019/2020 outbreak (initially sequenced and provided in GenBank MN908497; NCBI Reference Sequence: NC_045512.2 “Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1”). In some embodiments, the antigen can be encoded by SEQ ID NO: 1, and/or correspond to and/or be derived and/or comprise at least one T cell and/or B cell epitope of SEQ ID NO: 2, and/or exhibit about 80% or more to the same. In some embodiments, the antigen can be from at least one of the SI and/or S2 domains of spike protein, or immunogenic fragments thereof. In some embodiments, the antigen can be from the receptor binding domain (RBD) and/or N-terminal domain (NTD) of SI. In some embodiments, the antigen can be from SEQ ID NO: 3, or at least one domain sequence (e.g., T cell or B cell epitope) of SEQ ID NO: 3.

[0080] In some embodiments, the peptide comprises one or more immunogenic domains of SEQ ID NO: 1 (Fig. 1). In some embodiments, the peptide comprises one or more immunogenic domains (e.g., comprise at least one T cell and/or B cell epitope thereof) of any of SEQ ID NOS: 2 to 11 (Figs. 2-11), or exhibit about 80% or greater homology thereto. In some embodiments, the peptide comprises at least one epitope of a coronavirus (preferably SARS-CoV-2-)-spike (S) protein receptor binding domain (RBD), or at least one immunogenic fragment thereof, wherein the composition is configured to induce neutralizing antibody to the spike protein RBD, in a mammalian subject. Putative studies indicate the spike protein via its receptor binding domain of SI binds to the angiotensin-converting enzyme 2 (ACE2) receptor (Y. Wan et ah; receptor recognition by novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SAR.S; J. Virol doi: 10.1128/JVI.00127-20; posted online 29 January 2020). Generating an immune response against at least the RBD of spike protein is an attractive target for inducing neutralization antibodies, wherein spike protein mediates coronavirus entry into host cells by first binding to a host receptor (e.g., ACE2) and then fusing viral and host membranes. In preferred embodiments, the spike protein for SARS-CoV-2 is SEQ ID NO: 3 (GenBank: QHD43416.1) (Fig. 3), and the anti -coronavirus (preferably anti-SARS-CoV-2) peptide constructs can comprise any one or more antigens thereof.

[0081] In some embodiments, the peptide is a sequence, or immunogenic fragment thereof, of SEQ ID NOS: 2-11, or one encoded by SEQ ID NO: 1, or a sequence having at least 80% homology to the same. In certain embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 3. In certain embodiments, the peptide comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 4. In preferred embodiments, the peptide comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 5. In preferred embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 6. In preferred embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 7. In preferred embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 8. In preferred embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 9. In preferred embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 10. In preferred embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to SEQ ID NO: 11

[0082] In some embodiments, the peptide can comprise an antigen of the SI domain of the coronavirus (preferably SARS-CoV-2) spike protein (e.g., SEQ ID NO 3), or an immunogenic fragment thereof. In certain embodiments, the peptide comprises at least amino acid resides 331 to 527 of SEQ ID NO: 3, wherein the amino acid position numbering is based on the full-length spike protein sequence. In certain embodiments, the peptide comprises a sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to amino acid resides 331 to 527 of SEQ ID NO: 3; wherein the amino acid position numbering is based on the full length spike protein sequence.

[0083] In embodiments, the present immunogenic composition is a multivalent composition. In certain embodiments, the immunogenic composition can comprise one or more anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs comprises a peptide representing, corresponding to, and/or being derived from one or more coronavirus (preferably SARS-CoV-2) structural proteins (envelope (E), membrane (M), and nucleocapsid (N)) or non- structural proteins from derived from ORFlab, ORF3a, ORF6, ORF7a and/or ORF8. Each of those structural proteins is presented herein as SEQ ID NO: 5; SEQ ID NO: 6; and, SEQ ID NO: 10, respectively. (Figs. 5, 6 and 10). The complete SARS-CoV-2 proteome, represented as a concatenated polypeptide, is provided as SEQ ID NO: 410 (Fig. 56). In some embodiments, those structural proteins may be encoded from different anti-coronavirus (preferably SARS-CoV-2) peptide constructs and provided as a multivalent formulation with one or more anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs representing, corresponding to, and/or being derived from at least one epitope of a coronavirus (preferably SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and/or at least one immunogenic fragment thereof; and/or anti-coronavirus (preferably anti-SARS-CoV-2) agents.

[0084] In some embodiments, the peptide of the anti-SARS-CoV-2 peptide constructs can comprise one or more antigens derived from and/or corresponding to one or more S protein antigens. (Ahmed, et al. Preliminary Identification of Potential Vaccine Targets for the COVID- 19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses, 12: 254 (2020)). In some embodiments, the anti-coronavirus peptide constructs can comprise one or more SARS-CoV-2 T cell epitopes, for instance, any one or more of the peptides shown in Table 1 (or one having about 80% or greater homology therewith):

Table 1

[0085] In some preferred embodiments, the anti-coronavirus peptide constructs can encode multiple epitopes, separately or as part of a single polypeptide (e.g., concatenated, optionally separated by a linker amino acid sequence of two to ten amino acids). In some embodiments, the anti-coronavirus peptide constructs multiple epitopes as in the exemplary groups shown in Table 2:

Table 2

[0086] Other combinations of epitopes are also contemplated herein as would be understood by those of ordinary skill in the art.

[0087] In some embodiments, the anti-coronavirus peptide constructs can comprise one or more of the following epitopes that can be B cell epitopes: DVVNQNAQALNTLVKQL (SEQ ID NO: 283), FF GMSRIGME VTP S GT W (SEQ ID NO: 284), EAEVQIDRLITGRLQSL (SEQ ID NO: 285), GLPNNT AS WFT ALTQHGK (SEQ ID NO: 286),

EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 287), GTTLPK (SEQ ID NO: 288), EVAKNLNESLIDLQELG (SEQ ID NO: 289), IRQGTDYKHWPQIAQFA (SEQ ID NO: 290), GAALQIPFAMQMAYRFN (SEQ ID NO: 291), KHIDAYKTFPPTEPKKDKKK (SEQ ID NO: 292), GAGICASY (SEQ ID NO: 293), KHWPQIAQFAPSASAFF (SEQ ID NO: 294), AISSVLNDILSRLDKVE (SEQ ID NO: 295), YNVTQ AF GRRGPEQTQGNF (SEQ ID NO: 296), GSFCTQLN (SEQ ID NO: 297), KTFPPTEPKKDKKKK (SEQ ID NO: 298), ILSRLDKVEAEVQIDRL (SEQ ID NO: 299), LLPAAD (SEQ ID NO: 300), KGIYQTSN (SEQ ID NO: 301), LNKHIDAYKTFPPTEPK (SEQ ID NO: 302), AMQMAYRF (SEQ ID NO: 303), LPQGTTLPKG (SEQ ID NO: 304), KNHTSPDVDLGDISGIN (SEQ ID NO: 305), LPQRQKKQ (SEQ ID NO: 306), M A YRFN GIG VT QNVL YE (SEQ ID NO: 307), PKGFYAEGSRGGSQASSR (SEQ ID NO: 308), AATKMSECVLGQSKRVD (SEQ ID NO: 309), QFAPSAS FFGMSRIGM (SEQ ID NO: 310), PF AMQM A YRFN GIGVT Q (SEQ ID NO: 311), QGTDYKHW (SEQ ID NO: 312), Q ALNTL VKQL S SNF GAI (SEQ ID NO: 313), QLPQGTTLPKGFYAE (SEQ ID NO: 314), QLIRAAEIRASANLAAT (SEQ ID NO: 315), QLPQGTTLPKGFYAEGSR (SEQ ID NO: 316), QQFGRD (SEQ ID NO: 317), QLPQGTTLPKGFYAEGSRGGSQ (SEQ ID NO: 318), RASANLAATKMSECVLG (SEQ ID NO: 319), TFPPTEPK (SEQ ID NO: 320), RLITGRLQSLQTYVTQQ (SEQ ID NO: 321), RRPQGLPNNTASWFT (SEQ ID NO: 322), EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 323), SQASSRSS (SEQ ID NO: 324), SLQTYVTQQLIRAAEIR (SEQ ID NO: 325), SRGGSQ ASSRS S SRSR (SEQ ID NO: 326), and DLGDISGINASVVNIQK (SEQ ID NO: 327); and/or combinations of the same. In some preferred embodiments, the vectors can encode multiple of such epitopes, separately or as part of a single polypeptide (e.g., in some embodiments concatenated, optionally separated by a linker amino acid sequence of two to ten amino acids).

[0088] In some embodiments, the coronavirus (preferably SARS-CoV-2) peptide sequences included in the anti-coronavirus peptide constructs can be selected based on the ability to stimulate CD4⁺ and/or CD8⁺ T cell responses, and can in some embodiments be selected based on the prediction of proteome regions containing the highest number of HLA class I and HLA class II binding motifs across a range of selected HLA alleles. In some embodiments, analysis of HLA class II binding motifs across the SARS-CoV-2 sequences can be performed using NetMHCpan EL 4.0 available at IEDB (http://tools.iedb.org/mhci/; Jurtz, et al. NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data. J Immunol. 2017;199(9):3360-3368). In some embodiments, the NetMHCpan EL 4.0 can be used to identify binding motifs having a length varying from 9 to 11 amino acids to HLA class I molecules and assigned a percentage rank (% Rank) that can be included in the anti- coronavirus peptide constructs. In some embodiments, high affinity binding peptides can be identified as those exhibiting a %-Rank <0.1 while moderate affinity binding peptides can be considered to have a %-rank comprised between >0.1 and <0.5. In preferred embodiments, the NetMHCpan EL 4.0 prediction can be performed with a set of 18 HLA-A alleles, 32 HLA-B alleles and 20 HLA-C alleles shown here: HLA-A*01 :01, HLA-A*02:01, HLA-A*02:06, HLA-A*03:01, HLA-A* 11:01, HLA-A*23:0, HLA-A*24:02, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:03, HLA-A*68:01, HLA-A*68:02, HLA-A*74:01, HLA-B*07:02, HLA-B*08:01, HLA-B* 13:01, HLA-B* 13:02, HLA-B* 14:02, HLA-B*15:01, HLA-B*15:02, HLA-B*15:25, HLA-B*18:01, HLA-B*27:02, HLA-B*27:05, HLA-B*35:01, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*39:01, HLA-B*40:01, HLA-B*40:02, HLA-B*44:02, HLA-B*44:03, HLA-B*46:01, HLA-B*48:01, HLA-B*49:01, HLA-B*50:01, HLA-B*51:01, HLA-B*52:01, HLA-B*53:01, HLA-B*55:01, HLA-B*56:01, HLA-B*57:01, HLA-B*58:01, HLA-B*58:02, HLA-C*01:02, HLA-C*02:02, HLA-C*02:09, HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*04:01, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C* 12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, HLA-C*16:01 and HLA-C* 17:01. Other HLA class I alleles may also be suitable as would be understood by those of ordinary skill in the art.

[0089] In some embodiments, HLA class II binding motifs within the SARS-CoV-2 polypeptide sequences can be performed using NetMHCII 2.3 (http://www.cbs.dtu.dk/services/NetMHCII/; Jensen et al. Improved methods for predicting peptide binding affinity to MHC class II molecules. Immunology. 2018 Jul;154(3):394-406.) which is based on ensembles of artificial neural networks trained on quantitative peptide binding affinity data from the Immune Epitope Database (IEDB). In some embodiments, NetMHCII 2.3 can be used to identify peptides that can presented by HLA class II molecules by determining, e.g., the percentage rank (%-Rank) (related to the affinity of the peptides for the HLA molecules) and the core nine amino acid binding motif. In some embodiments, high affinity HLA class II binding peptides can be identified as those exhibiting a %-Rank <2 while moderate affinity binding peptides can be considered to have a %-Rank > 2 and 10. In preferred embodiments, the NetMHCII 2.3 system can be based on a set of 20 HLA-DR alleles, 20 HLA-DQ alleles and 9 HLA-DP alleles shown here: DRal*0101-DRp 1*0101, DRal*0101-DRpl*0301, DRal*0101-DRpl*0401, DRal*0101-DRp 1*0701, DRal*0101- DRp 1*0801, DRal*0101-DRp 1*0802, DRal*0101-DRp 1*0901, DRal*0101-DRp 1*1001, DRal*0101-DRpl*1101, DRal*0101-DRpl*1201, DRal*0101-DRpl*1301, DRal*0101- DRpl*1302, DRal*0101-DRpl*150, DRal*0101-DRp 1*1602, DRal*0101-DRp3*0101,

DRal*0101-DRp3*0202, DRal*0101-DRp3*0301, DRal*0101-DRp4*0101, DRal*0101- DRp4*0103, DRal*0101-DRp5*0101, DPal*0103-DPpl*0301, DPal*0103-DPpl*0401,

DPal *0103-ϋRb 1*0402, DPal*0103-DPpl*0601, DPal*0201-DPp 1*0101, DPal*0201- ϋRb1*0501, DPal*0201-DPpi*1401, DPal*0301-DPpl*0402, DPal*0103-DPpl*0201, DQal*0101-DQpl*0501, DQal*0102-DQpl*0501, DQal*0102-DQpl*0502, DQal*0102- DQP1*0602, DQal *0103-DQP 1*0603, DQal*0104-DQpl*0503, DQal*0201-DQpl*0202, DQal *020 l-DQP 1*0301, DQal*0201-DQpl*0303, DQal*0201-DQpl*0402, DQal*0301- DQP1*0301, DQal*0301-DQP 1*0302, DQal*0303-DQp 1*0402, DQal*0401-DQpl*0402, DQal *050 l-DQP 1*0201, DQal*0501-DQpl*0301, DQal*0501-DQpl*0302, DQal*0501- DQpl*0303, DQal*0501-DQpl*0402 and DQal*0601-DQpl*0402. Other HLA class II alleles may also be suitable as would be understood by those of ordinary skill in the art.

[0090] The number of HLA class I binding motifs across the selected 70 HLA class I alleles and the number of HLA class II binding motifs across the selected 49 HLA class II alleles having a moderate, high or high + moderate affinity were respectively calculated for each 41 amino-acid long window scanning the SARS-CoV-2 sequences and presented in Figs. 12-55. For this analysis, predicted transmembrane domains were deselected due to their high hydrophobicity. Based on this analysis, forty-two (42) long peptide sequences with a length varying from 34 to 124 amino- acids as presented in Table 4 were selected based on the highest content in HLA class I and/or HLA class II motifs across the SRAS-CoV-2 proteome. In some embodiments, one or more peptide sequences having an appropriate length (and antigenicity / immunogenicity) for inclusion in the anti-coronavirus peptide constructs can be selected from the peptides shown in Table 3.

Table 3

Selected SARS-CoV-2 long peptide sequences containing high density HLA class I and/or HLA class II binding motifs (SEQ ID NO: 328 to 369)

[0091] For each selected long sequences SEQ ID NO: 328 to SEQ ID NO: 369, the number of HLA class I and HLA class II alleles for which high or moderate binding was predicted are presented in Table 4.

Table 4

Number of HLA class I and HLA class II alleles for which high or moderate binding was predicted within the selected SARS-CoV-2 long peptide sequences containing high density HLA class I and/or HLA class II binding motifs (SEQ ID NO: 328 to 369)

[0092] In addition to the long peptide sequences SEQ ID NO: 328 to SEQ ID NO: 369, thirty-nine (39) shorter sequences with a length varying from 31 to 47 amino acid residues were also identified. The thirty-nine shorter peptide sequence correspond to portions of sequence within SEQ ID NO: 328 to SEQ ID NO: 369 with the highest number of HLA class I and class II binding motifs are shown in Table 5 (SEQ ID NOS: 370 to 408).

Table 5

Selected SARS-CoV-2 shorter peptide sequences containing high density HLA class I and/or HLA class II binding motifs (SEQ ID NO: 370 to 408)

For each of SEQ ID NO: 370-408 (Table 5), the number of HLA class I and HLA class II alleles for which high or moderate binding was predicted are presented in Table 6. Table 6

Number of HLA class I and HLA class II alleles for which high or moderate binding was predicted within the selected SARS-CoV-2 shorter peptide sequences containing high density HLA class I and/or HLA class II binding motifs (SEQ ID NOS: 370-408)

[0093] For each selected sequence from SEQ ID NO: 370 to SEQ ID NO: 408, a map of HLA class I and HLA class II binding motif are presented in Figs. 14 to 55 respectively. The N-terminal amino acid (i.e., amino acid #1) for each HLA class I and II motif therein is identified by an X or an O, wherein X further indicates a high affinity motif and O indicates moderate affinity for the HLA binding motif. Exemplary HLA binding motifs can be deduced from Figs. 14 to 55 by including the X or O amino acid residue and including the subsequent eight to ten amino acids in the motif such that each motif includes nine to eleven amino acid residues (i.e., each motif is a 9- 11 amino acid peptide) with reference to the SEQ ID NO. indicated therein. Any such binding motifs can be used as immunogens, alone and/or in combination, in the vectors disclosed herein. Other binding motifs of any of SEQ ID NOS. 370-408 may also be suitable for inclusion in the SARS-CoV-2 immunization vectors herein as would be understood by those of ordinary skill in the art.

[0094] Other epitopes and/or peptides, and combinations thereof, are also contemplated herein as would be understood by those of ordinary skill in the art.

[0095] Receptor Binding Antagonists

[0096] In some preferred embodiments, the anti-SARS-CoV-2 peptide constructs can include multiple of such epitopes, separately (e.g., multiple anti-SARS-CoV-2 peptide constructs in an immunogenic composition) or as part of a single anti-SARS-CoV-2 peptide constructs (e.g., in some embodiments concatenated, optionally separated by a linker amino acid sequence of two to ten amino acids). Other peptides, and combinations thereof, are also contemplated herein as would be understood by those of ordinary skill in the art.

[0097] In some embodiments, the anti-SARS-CoV-2 peptide construct compositions (i.e., those comprising one or more exogenous antigens) can comprise one or more antigens to which the induced and/or enhanced immune response acts to block SARS-CoV-2 entry into or attachment to a cell (e.g., antibodies that act as receptor binding antagonists). For instance, coronaviruses such as SARS-CoV-2 are known to use homotrimers of the spike (S) protein for host cell attachment, fusion and entry into the host cell, and can involve sialic acids and/or ACE2 (a cell membrane C- terminal anchored protein that catalyzes the cleavage of angiotensin 1 into angiotensin 1-9, and of angiotensin II into the vasodilator angiotensin 1-7, thus playing a key role in systemic blood pressure regulation (Alifano, et al. Renin-angiotensin system at the heart of COVID-19 pandemic. Biochimie, 174: 30-33 (2020)). Host cell proteases are known to process coronavirus S protein to generate two subunits (SI and S2), which remain non-covalently bound in the pre-fusion conformation of the virus (see, e.g., Tortorici, et al. Structural basis for human coronavirus attachment to sialic acid receptors. Nat. Struc. Mol. Biol. 26: 481-489 (2019)). The SI subunit comprises four domains NTD, RBD, SD1 and SD1 with NTD and RBD separated by a linker sequence. In some embodiments, the anti-SARS-CoV-2 peptide constructs can induce and/or enhance an immune response against at least one or both of the SI domains (NTD and/or RBD) or at least part of the full-length S protein (with or without transmembrane domain), that, in some preferred embodiments, interferes with the binding of the S protein (e.g., an antibody against the SI RDB and/or NTD domain(s)) to the above-mentioned sialic acids and/or ACE2, thereby interfering with entry of SARS-CoV-2 into a host cell and/or its effect on ACE2. In some embodiments, the anti- coronavirus (preferably anti-SARS-CoV-2) peptide construct can also or alternatively comprise induce and/or enhance an immune response that interferes with the binding of the S protein to its host cells receptors such as ACE2, preferably without interfering with the normal physiological function of ACE2 (i.e., other than its ability to serve as a receptor for SARS- CoV-2). In preferred embodiments, the anti-coronavirus (preferably anti-SARS-CoV-2) peptide construct can induce an immune response that interferes with the interaction of RBD and ACE2 at one or more of the 15 residues from ACE2 (24(Q), 27(T), 30(D), 31(K), 34(H), 35(E), 37(E), 38(D), 41(Y), and 42(Q) in al; one residue (residue 82 M) in a2; residues 353(K), 354(G), 355(D), and 357(R) at the linker between b3 and b4) that are currently understood to interact with RBD (Han, et al. Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2. ACS Nano 2020, Publication Date: April 14, 2020 (https://doi.org/10.1021/acsnano.0c02857); Yan, et al. Structural Basis for the Recognition of the SARS-CoV-2 by Full-Length Human ACE2. Science 367: 1444-1448 (2020)); preferably without affecting the normal physiological function (i.e., other than its ability to serve as a receptor for SARS-CoV-2) of the ACE2 protein.

[0098] Formulations

[0099] In some embodiments, this disclosure provides anti-coronavirus (preferably anti-SARS- CoV-2) peptide constructs as compositions (preferably immunogenic compositions) comprising the same, that are in some embodiments vaccine formulations, suitable and/or configured for administration to a mammalian subject for the prevention and/or treatment of coronavirus infection (preferably SARS-CoV-2) infection (“anti-coronavirus pharmaceutical formulations”), preferably wherein the coronavirus of the anti-coronavirus pharmaceutical formulations is SARS-CoV-2 (i.e., an “anti-SARS-CoV-2 pharmaceutical formulation”). In some embodiments, the anti-coronavirus peptide constructs disclosed herein can comprise any coronavirus antigen known to one of skill in art, and prepared for administration to a mammal, preferably wherein the coronavirus antigen is a SARS-CoV-2 antigen.

[00100] In some embodiments, the immunogenic composition (e.g., vaccine) comprises one or more anti-coronavirus (anti-SARS-CoV-2) peptide constructs including one or more coronavirus antigen(s) (preferably SARS-CoV-2 antigens). In some embodiments, the one or more additional anti- coronavirus (preferably anti-SARS-CoV-2) agent(s) can induce an immune response to the same or different coronavirus (preferably SARS-CoV-2) as the anti-coronavirus (anti-SARS-CoV-2) peptide constructs). In preferred embodiments, the coronavirus is SARS- CoV-2, and preferably the Wuhan 2019 isolate (see, e.g., SEQ ID NO: 1 and other SARS-CoV-2 sequences or strains available to those of ordinary skill in the art). The other components may be included to induce a humoral response with antibodies to a different epitope than that presented in the instant adenoviral vector containing spike protein antigen. In other embodiments, the other component(s) may be included to induce a different arm of the immune system, such as cell- mediated or mucosal immune response to a coronavirus antigen.

[00101] In certain embodiments, the effective amount induces a protective immune response configured to provide seroprotection, or cellular protection (e.g., based on a cellular immune response such as T cells) to the human subject for at least 1 month (e.g., 28 days or 4 weeks), at least 2 months, at least 3 months, at least 6 months, at least 8 months, at least 12 months, at least 13 months, or at least 14 months against SARS-CoV-2 infection. In certain embodiments, the protective immune response comprises a combined mucosal, humoral and/or T cell response.

[00102] With respect to dosages, routes of administration, formulations, adjuvants, and uses for recombinant viruses and expression products therefrom, compositions of the invention may be used for parenteral, topical, or mucosal administration, preferably by intradermal, subcutaneous, intranasal or intramuscular routes. When mucosal administration is used, it is possible to use oral, ocular or nasal routes. In exemplary embodiments, the present immunogenic compositions (e.g., vaccine) are administered intranasally. In exemplary embodiments, the present immunogenic compositions (e.g., vaccine) are administered intranasally to the mammalian subject.

[00103] The immunogenic compositions (e.g., formulations, pharmaceutical compositions comprising one or more anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs) can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary art. In some embodiments, the immunogenic compositions can be prepared by solubilizing at least one peptide (R in Formula I), such as a fluorocarbon -linked peptide (e.g., one or more lyophilized peptide constructs). Suitable, exemplary approaches for solubilizing fluorocarbon vector-peptide conjugates are described in U.S. Pat. No. 9,119,811 B2 (Bradley, et al. Issued Sept. 1, 2015 and/or U.S. Pat. No. 10,300,132 B2 (Georges, et al. Issued May 29, 2019), which are hereby incorporated in their entireties into this disclosure. In some embodiments, the fluorocarbon-linked peptide(s) can be solubilized in acetic acid (e.g., about 80% acetic acid) or other solvents (e.g., phosphate buffered saline (PBS), propan-2-ol, tert-butanol, acetone) to disperse one or more of the fluorocarbon-linked peptides in the blend as a first step in formulating the composition (e.g., as a pharmaceutical product for administration to a mammalian subject) . Where sterile filtration is to be used, it is preferred that the solubilization produces micelles comprising the fluorocarbon-linked peptide(s) with a diameter of less than 0.22 pm (e.g., self-assemblies such as nanometric spherical micelles (>220nm)). The peptide or fluorocarbon- linked peptide used as a starting material is typically desiccated. Peptides and fluorocarbon-linked peptides that comprise peptides shorter than 20 amino acids and/or that have fewer than 50% hydrophobic residues can be solubilized in a solvent other than acetic acid (e.g., phosphate buffered saline (PBS), propan-2-ol, tert-butanol, acetone). Acetic acid can be used where the peptide has more than 20 amino acids and/or has more than 50% hydrophobic residues. The concentration of fluorocarbon-linked peptide in the solution typically is from about 0.1 mM to about 10 mM, such as about 0.5 mM, 1 mM, 2 mM, 2.5 mM or 5 mM (e.g., about 10 mg/mL). The input components may be blended homogenously together to the desired ratios with any aggregates dispersed, rendered sterile and presented in a suitable format for administration. Such examples could include the introduction of a vortexing and/or sonication post-blending or post dilution stage to facilitate solubilization. Other permutations of the manufacturing process flow could include sterile filtration being performed at an earlier stage of the process or the omission of lyophilization to permit a liquid final presentation. Where the different peptides or fluorocarbon- linked peptides are solubilized separately, for example in different solvents or in different concentrations of acetic acid, the solubilized peptides or fluorocarbon-linked peptides are blended to create a mixture of peptides or fluorocarbon-linked peptides. The optional adjuvant and/or one or more pharmaceutically acceptable excipients can also be added to the solubilized peptide/fluorocarbon-linked peptide or mixture of peptides/fluorocarbon-linked peptides. Typically, the solubilized fluorocarbon-linked peptides are mixed with the excipient and/or adjuvant. After solubilization and blending the solution of fluorocarbon-linked peptide(s) may be diluted. For example, the blend may be diluted in water.

[00104] The solution containing the peptides or fluorocarbon-linked peptides is preferably sterilized. Sterilization is particularly preferred where the formulation is intended for systemic use. Any suitable means of sterilization may be used, such as heat, ionizing radiation, ultraviolet (UV) sterilization, or filter sterilization (e.g., produced aseptically according to a process involving membrane sterile filtration). Preferably, filter sterilization is used. Sterile filtration may include a 0.45 pm filter followed by a 0.22 pm sterilizing grade filter train. In some embodiments, sterile filtration requires the use of a 0.22pm filter and the individual peptide constructs in a mixture of multiple peptides be rendered soluble or forming supramolecular structure having a size smaller than the pore size of the filter, if they are not, all or most of the insoluble peptide will be unable to pass through the filter. Accordingly, sterile filtration implies a prior solubilization of the peptide constructs according to a pharmaceutically acceptable process, such as described above where acetic acid is used to solubilize fluorocarbon-linked peptides that are insoluble in aqueous solution (e.g., to produce micelles of less than 0.22 pm diameter (e.g., self-assemblies such as nanometric spherical micelles (>220nm)) and sufficiently stable in solution to avoid filter blockage and/or losses). Sterilization may be carried out before or after addition of any excipients and/or adjuvants.

[00105] In some embodiments, the composition may be in dried, such as lyophilized, form. The composition of the invention may be an aqueous solution, for example an aqueous solution formed by dissolving a lyophilizate or other dried formulation in an aqueous medium. The aqueous solution is typically pH neutral. Drying the formulation facilitates long-term storage. Any suitable drying method may be used. Lyophilization is preferred but other suitable drying methods may be used, such as vacuum drying, spray-drying, spray freeze-drying or fluid bed drying. The drying procedure can result in the formation of an amorphous cake within which the peptides or fluorocarbon-linked peptides are incorporated. For long-term storage, the sterile composition may be lyophilized. Lyophilization can be achieved by freeze-drying. Freeze-drying typically includes freezing and then drying. For example, the fluorocarbon-linked peptide mixture may be frozen for 2 hours at -80°C. and freeze-dried in a freeze-drying machine for 24 hours. [00106] In some embodiments, one or more fluorocarbon peptide constructs is/are solubilizing in a acetic acid solvent (e.g., about 80% acetic acid), sterile filtered, and dried, wherein the dried mixture is reconstituted in an aqueous solution to form a pharmaceutically acceptable homogenous mixture. Other mixtures are also contemplated herein as would be understood by those of ordinary skill in the art.

[00107] Pharmaceutically acceptable compositions may be solid compositions. The fluorocarbon-linked peptide composition may be obtained in a dry powder form. A cake resulting from lyophilization can be milled into powder form. A solid composition according to the invention thus may take the form of free-flowing particles. The solid composition typically is provided as a powder in a sealed vial, ampoule or syringe. If for inhalation, the powder can be provided in a dry powder inhaler. The solid matrix can alternatively be provided as a patch. A powder may be compressed into tablet form. The dried, for example, lyophilized, peptide or fluorocarbon-linked peptide composition may be reconstituted prior to administration. As used herein, the term "reconstitution" is understood to mean dissolution of the dried vaccine product prior to use. Following drying, such as lyophilization, the immunogenic peptide, for example, the fluorocarbon-linked peptide product, preferably is reconstituted to form an isotonic, pH neutral, homogeneous suspension. The formulation is typically reconstituted in the aqueous phase, for example by adding Water for Injection, histidine buffer solution (such as 28 mM L-histidine buffer), sodium bicarbonate, Tris-HCl or phosphate buffered saline (PBS). The reconstituted formulation is typically dispensed into sterile containers, such as vials, syringes or any other suitable format for storage or administration. The composition may be stored in a container, such as a sterile vial or syringe, prior to use.

[00108] The compositions / formulations can be administered in dosages and by techniques well known to those skilled in the clinical arts taking into consideration such factors as the age, sex, weight, and the route of administration. The formulations can be administered alone (i.e., as the sole active agent(s)) or can be co-administered or sequentially administered with compositions, e.g., with "other" immunogenic compositions or therapeutic compositions thereby providing multivalent or "cocktail" or combination compositions of the invention and methods employing them. In some embodiments, the formulations may comprise sucrose as a cryoprotectant and polysorbate-80 as a non-ionic surfactant. In certain embodiments, the formulations further comprise free-radical oxidation inhibitors ethanol and histidine, the metal-ion chelator ethylenediaminetetraacetic acid (EDTA), or other agents with comparable activity (e.g., block or prevent metal-ion catalyzed free-radical oxidation).

[00109] The compositions (e.g., formulations) may be present in a liquid preparation for mucosal administration, e.g., oral, nasal, ocular, etc., formulations such as suspensions and, preparations for parenteral, subcutaneous, intradermal, intramuscular, intravenous (e.g., injectable administration) such as sterile suspensions or emulsions. In such formulations the adenoviral vector may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, viscosity enhancing excipients or the like. Certain specialized formulations for mucosal administration can be used, including mucoadhesives, mucosal penetrants and mucosal disruptants. The formulations can also be lyophilized or frozen. The formulations can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, preservatives, and the like, depending upon the route of administration and the preparation desired. The formulations can contain at least one adjuvant compound. In exemplary embodiments, the present immunogenic compositions (e.g., vaccines) are non-adjuvanted. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[00110] An “effective amount” of an anti-coronavirus (preferably SARS-CoV-2) and/or immunogenic composition comprising one is one administered to a host in a form, dose, and/or administration regimen sufficient to induce an anti-coronavirus (anti-SARS-CoV-2) immune response (e.g., humoral, mucosal and/or cell-mediated immune response) that in some embodiments can be protective from coronavirus (preferably SARS-CoV-2) infection (and/or CoV disease progression). In some embodiments, such as described in the examples herein, a host to which the effective amount was administered can exhibit an induction of (e.g., the appearance of) and/or an increase in the number and/or function of anti-coronavirus (anti-SARS-CoV-2) antibody-producing cells (e.g., B cells, plasma cells) that produce antibodies that bind to CoV and/or antigens (or immunogens) thereof, such as an anti-coronavirus (anti-SARS-CoV-2) specific immunoglobulin G (IgG) response. In some embodiments, such as described in the examples herein, a host to which the effective amount was administered can exhibit an induction of (e.g., the appearance of) and/or an increase in the number and/or function of cells forming an anti- coronavirus (anti-SARS-CoV-2) cell-mediated response (e.g., T cells, granulocytes, natural killer (NK) cells, and the like). In some embodiments, a host to which the effective amount was administered can exhibit an induction of (e.g., the appearance of) and/or an increase in the number and/or function of anti-coronavirus (anti-SARS-CoV-2) antibody-producing cells and cells forming an anti-coronavirus (anti-SARS-CoV-2) cell-mediated response.

[00111] In order to determine whether a host to which the effective amount was administered exhibits exhibit an induction of (e.g., the appearance of) and/or an increase in the number and/or function of anti-coronavirus (preferably anti-SARS-CoV-2) antibody-producing cells, in some embodiments, a coronavirus (preferably SARS-CoV-2)-specific enzyme-linked immunosorbent assay (ELISA) can be used. As shown in the examples using a murine model, following administration of an immunogenic composition (e.g., 21 days after administration), mice can bled to provide samples for determining the presence of a systemic antibody response using a coronavirus (preferably SARS-CoV-2)-specific ELISA (e.g., to determine coronavirus (SARS- CoV-2)-specific IgG response has occurred). Briefly, ELISA can be performed by coating polystyrene 96-well plates overnight at 4°C with 1 pg/ml of coronavirus (preferably SARS-CoV- 2) S protein in sodium carbonate buffer (pH 9.3). Plates can be washed (e.g., three times in PBS with 0.02% Tween 20) and blocked (e.g., with non-fat dried milk) for a suitable amount of time and temperature (e.g., one hour at 37°C with PBS, 2% BSA, and 0.02% Tween 20). Serum from coronavirus (preferably SARS-CoV-2)-vaccinated mice can be serially diluted (e.g., in PBS) and incubated at an appropriate temperature and time (e.g., 37°C), washed (e.g., four times with PBS with 0.02% Tween 20) and then incubated with a labeled secondary antibody (e.g., biotin-labeled goat anti-mouse secondary antibody) for an appropriate amount of time (e.g., one hour). The samples can then be washed and incubated with an appropriate reagent (e.g., HRP-conjugated streptavidin), and developed using an appropriate agent (e.g., tetramethylbenzidine substrate), the reaction being stopped with the addition of an appropriate reagent (e.g., 2 N H2SO4), and emission (450 nm) read using an microplate reader. In some embodiments, administration to a host of an effective amount of an immunogenic composition comprising one or more anti-coronavirus, preferably anti-SARS-CoV-2, peptide constructs comprising one or more coronavirus (preferably SARS-CoV-2) antigens (e.g., Spike protein) can result in the expression of coronavirus (preferably SARS-CoV-2)-specific (e.g., S protein-specific) antibodies of a particular type (e.g., IgA, IgM, IgG) and/or amount (e.g., a particular reciprocal mean endpoint indicative of a response (e.g., as compared to naive hosts). Other assay systems can also be used to determine whether an effective amount has been administered such as, for instance but without limitation, neutralizing antibody assays.

[00112] In order to determine whether a host to which the effective amount was administered exhibits exhibit an induction of (e.g., the appearance of) and/or an increase in the number and/or function of cells forming a coronavirus (preferably anti-SARS-CoV-2) cell- mediated response, cell types and/or numbers and/or cytokine expression and/or functional assays can be used. For instance, in some embodiments, T cells of a host to which (or whom) an immunogenic composition was administered can be isolated and studied (e.g., physically isolated from other cells and/or as present within a biological sample such as blood). In some embodiments, an intracellular cytokine staining assay can be performed to determine the type and/or number of cells expressing a particular cytokine, and/or the level of such cytokine being expressed therein. Briefly, a biological sample (e.g., blood, spleen) of a host to which an immunogenic composition has been administered can be isolated at a particular point following administration (e.g., eight to 21 days post-administration). Cells (e.g., approximately 10⁶ cells in cell culture media (e.g., RPMI with 10% FBS and HEPES)) isolated from said biological sample(s) can then be plated in a culture plate(s) (e.g., round bottom 96 well plate), stimulated for an appropriate amount of time, temperature, etc. (e.g., 6 hours at 37°C, 5% CO2) in the presence of stimulator(s) (e.g., 10 pg/ml brefeldin A and either a-CD3 (2C11 clone) or 10 pg of CoV peptide (e.g., the spike antigen SARS-CoV-2 peptide (SEQ ID NO 3) in 90% DMSO). Following coronavirus (preferably SARS-CoV-2) peptide stimulation, cells can be washed (e.g., once with phosphate-buffered saline (PBS)) and stained for the following cell surface markers indicating cell type (e.g., a-CD8-PerCP-Cy 5.5 (clone 53-6.7), a-CD3-AF700 (clone 500A2), and a-CD19- BV605 (clone 1D3)). Cells can then be fixed (e.g., using formalin), permeabilized, stained for intracellular cytokine markers (e.g., a-IRN-g-APC (clone B27)), and analyzed by flow cytometry (e.g., using an Attune-NXT). In some embodiments, an effective amount can be an amount of immunogenic composition that raises the number of cells expressing the cytokine (e.g., IFN-g) and/or the amount expressed by such cells.

[00113] In some embodiments, the anti-coronavirus (anti-SARS-CoV-2) immune response is protective, meaning that it can protect a host from experiencing one or more of the symptoms of coronavirus (preferably SARS-CoV-2) infection. In some embodiments, a protective immune response prevents coronavirus (preferably SARS-CoV-2) infection, which can be demonstrated by challenge of a host to which (or whom) the effective amount was administered. In some embodiments, an immunogenic composition, and/or effective amount thereof, that is protective is a vaccine. To determine if an immunogenic composition is protective, a pre-clinical animal model can be used. For instance, in some embodiments, a coronavirus (preferably SARS-CoV-2) immunogenic composition can be administered to mice susceptible to infection and disease and the mice can be challenged by live coronavirus (preferably SARS-CoV-2) at a subsequent time (e.g., 7-21 days following administration) and monitored for survival and/or symptoms in comparison to the control group. Symptoms of coronavirus (preferably SARS-CoV-2) infection can also be monitored, including clinical signs of disease (e.g., upper and lower respiratory symptoms). Thus, in some embodiments, in order to determine whether an coronavirus (preferably SARS-CoV-2) immunogenic composition is protective (i.e., is a vaccine), one of ordinary skill in the art can conduct an animal challenge study.

[00114] Methods of Use

[00115] In some embodiments, this disclosure provides methods for inducing an immune response against coronavirus, preferably against SARS-CoV-2, the method comprising administering an effective amount of the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition (i.e., comprising an anti-coronavirus (preferably anti-SARS-CoV) peptide construct) to a mammalian subject. In certain embodiments, is provided a method for administration of a pharmaceutical dose of a present therapeutic/immunogenic composition (e.g., vaccine) configured to induce an immune response (e.g., a protective immune response as a vaccine) via any suitable route of administration (e.g., injection, intramuscular injection, transdermal, intranasal). In preferred embodiments, the mammalian subject is a human being and the coronavirus antigen is from SARS-CoV-2. In some embodiments, the mammalian subject is a human being infected by a coronavirus, preferably SARS-CoV-2, (e.g., a hospitalized human being). In some embodiments, the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition can be used to treat coronavirus (preferably SARS-CoV-2) infection (e.g., in such an infected and/or hospitalized human being).

[00116] Dosage of the immunogenic anti-coronavirus (preferably anti-SARS-CoV-2) composition, when used with or without an adjuvant, can may range from about 1 to about 1000 pg of anti-coronavirus (preferably anti-SARS-CoV-2) peptide, such as about any of 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 pg, as a single dose or distributed among more than one dose (e.g., administered simultaneously or within a particular time period such as an hour, day, or week). Other doses may also be suitable as would be understood by those of ordinary skill in the art.

[00117] One of skill in the art understands that an effective dose in a mouse may be scaled for larger animals such as a human, dogs, pigs, etc. In that way, through allometric scaling (also referred to as biological scaling) a dose in a larger animal may be extrapolated from a dose in a mouse to obtain an equivalent dose based on body weight or body surface area of the animal.

[00118] In certain embodiments, non-invasive administration of the immunogenic anti- coronavirus (preferably anti-SARS-CoV-2) composition, includes, but is not limited to, topical application to the skin, and/or intranasal and/or mucosal and/or perlingual and/or buccal and/or oral and/or oral cavity and/or intramuscular administration. Dosage forms for the application of the immunogenic anti-coronavirus (preferably anti-SARS-CoV-2) composition may include liquids, ointments, powders and sprays. The active component may be admixed under sterile conditions with a physiologically acceptable carrier and any preservative, buffers, propellants, or absorption enhancers as may be needed.

[00119] In embodiments, the present SARS-CoV-2 pharmaceutical formulation is used to provide protection against seasonal coronavirus. In certain other embodiments, the present SARS- CoV-2 pharmaceutical formulation is used to provide protection against pandemic SARS-CoV-2. In certain other embodiments, the present SARS-CoV-2 pharmaceutical formulation is used to provide protection against SARS-CoV-2. In embodiments, the seroprotection lasts at least about 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 12 month or at least about 13 months.

[00120] In some embodiments, is provided a method for inducing an immune response against coronavirus, the method comprising administering a single dose of a present immunogenic composition/formulation/dosage to a mammalian subject (e.g., a human being). In certain embodiments, the method comprises administration of an effective amount of the immunogenic composition to the mammalian subject, wherein the immune response provides protection against challenge with SARS-CoV-2. In certain embodiments, is provided a method of inducing a combined mucosal, humoral and/or T cell protective immune response in a human subject against coronavirus comprising administering to a human subject a single dose of the anti-coronavirus (preferably anti-SARS-CoV-2) pharmaceutical formulation (immunogenic composition), or a pharmaceutical dosage thereof, wherein the administration induces serum antibodies, mucosal antibodies and T cells against coronavirus. In embodiments, the human subject is seroprotected at least about 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 12 month or at least about 13 months. In certain embodiments, the human subject is seroprotected for at least about 9 months. In some embodiments, the methods comprise administering at least a prime and boost dose of a present immunogenic composition/formulation/dosage. In certain embodiments, the boost dose is administered about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks or 52 weeks, 2 years, 3 years, 4 years, 5 years, or more after administration of the prime dose; or after the boost dose (e.g., a second, third or more boost).

[00121] In some embodiments, this disclosure comprises immunogenic compositions comprising anti-coronavirus (preferably anti-SARS-CoV-2) peptide vectors and the use of such immunogenic compositions to prevent and/or treat coronavirus infection, preferably wherein the coronavirus is SARS-CoV-2, and methods for doing so. In some embodiments, such anti- coronavirus (preferably anti-SARS-CoV-2) peptide vectors can be co-administered with one or more other anti-coronavirus (preferably anti-SARS-CoV-2) peptide agents. In some embodiments, such co-administration can refer to administration of a single immunogenic composition comprising one or more anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs and the one or more coronavirus (preferably anti-SARS-CoV-2) agents (e.g., rdAd anti- SARS-CoV-2 vectors), and/or essentially simultaneous, and/or sequential administration of the same (e.g., at different sites, as part of different compositions). Such anti-coronavirus (preferably anti-SARS-CoV-2) peptide vectors can also be administered as part of a prime-boost protocol, in which an immunogenic composition comprising one or more anti- coronavirus (preferably anti- SARS-CoV-2) peptide constructs and/or other anti-SARS-CoV-2 agents is administered before or after (e.g., 7-21 days before and/or after) administration of an immunogenic composition comprising the same or different one or more other types of anti-SARS-CoV-2 agent(s), or vice versa (or in an alternating administration protocol). In some embodiments, this disclosure provides methods for inducing (and/or enhancing) an immune response against coronavirus (preferably SARS-CoV-2) in a mammalian subject in need thereof by administering an effective amount of such composition(s). In some embodiments, the immune response against coronavirus (preferably SARS-CoV-2) induced or enhanced by administration of such immunogenic compositions preferably begins within about twenty-four hours of administration and preferably lasts for at least about 21 days.

[00122] In some embodiments, the anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs can be combined with a replication deficient adenoviral vector (“rdAd”) in the prevention and/or treatment of coronavirus infection. In some embodiments, the rdAd can be one that does not express an exogenous antigen (exogenous as to the adenovirus from the adenoviral vector is derived), such vectors being referred to herein as “AdE” vectors. In some embodiments, the replication-defective adenoviral vector for use in treating and/or preventing coronavirus infection can be one that does not express one or more coronavirus antigens, but expresses one or more antigens of a different type of infectious agent (e.g., influenza virus) (referred to herein as “AdD”). In certain embodiments is provided an immunogenic composition comprising a rdAd vector comprising an expression cassette comprising a coding sequence encoding at least one coronavirus (preferably SARS-CoV-2) antigen, referred to herein as hAd5-SARS-CoV-2 vectors. In some embodiments, the immunogenic compositions of this disclosure can comprise a different type of such vectors (e.g., AdE, or AdD), alone or in combination with hAd5-SARS-CoV-2 vectors). In some embodiments, these types of vectors can be collectively, or a subset of at least two such vectors, referred to as “rdAd anti-SARS-CoV-2 vectors”. A SARS-CoV-2 immunogenic composition (e.g., vaccine) is a pharmaceutical formulation comprising one or more such anti- SARS-CoV-2 peptide vectors. Such rdAd vectors are described in U.S. Ser. Nos. 62/977,078; 62/992,553; 63/005,923; and, 63/016,902, which are hereby incorporated in its entirety into this disclosure.

[00123] In some preferred embodiments, such methods can comprise intranasal administration of one or more rdAd anti-SARS-CoV-2 vector(s) as an immunogenic composition in an effective amount of (e.g., at least about 10⁷ ifu of an rdAd anti-SARS-CoV-2 vectors) as disclosed in U.S. Ser. Nos. 62/977,078; 62/992,553; 63/005,923 and 63/016,902, which are hereby incorporated in its entirety into this disclosure. In some embodiments, the administering of the anti-coronavirus (preferably SARS-CoV-2) peptides and/or other rdAd anti-coronavirus (preferably SARS-CoV-2) agents in multiple doses can be about any of 7, 10, 14, 21, 28, 35, 42, 49, or 56 days apart. The anti-coronavirus (preferably SARS-CoV-2) peptide constructs (and/or the one or more other anti-coronavirus (preferably anti-SARS-CoV-2) agents may be administered by any suitable route, preferably by an invasive administration methods, such as intravenous, intramuscular, or subcutaneous administration (e.g., injection). Preferably, immunogenic compositions comprising rdAd anti-coronavirus (preferably SARS-CoV-2) vector(s) can be administered intranasally. In some embodiments, the host is an animal, such as an adult or child human being, optionally wherein the host is immunocompromised. In preferred embodiments, the immune response against the coronavirus (preferably SARS-CoV-2) lasts for at least about 40-50 days, and can be re-initiated by re-administration of the one or more anti-coronavirus (preferably SARS-CoV-2) peptide constructs and/or additional anti-coronavirus (preferably anti-SARS-CoV- 2) agents. Other embodiments of such immunogenic compositions, and/or methods are also contemplated herein as would be understood by those of ordinary skill in the art.

[00124] In some embodiments, the methods can comprise administration with one or more anti-cytokine reagents. In some embodiments, then, to prevent and/or treat coronavirus (preferably SARS-CoV-2) infection, an anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition can be administered to a mammal, such as a human being, with one or more anti cytokine reagent(s) (i.e., co-administered). Such co-administration can be carried out as single mixture (e.g., one or more anti-cytokine reagents can be included in the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition), or as separate compositions administered essentially simultaneously and/or at or near the same anatomical site, or at different anatomical sites by an appropriate route (e.g., intranasal administration of the anti-coronavirus (preferably anti-SARS-CoV-2)immunogenic composition and intradermal or intravenous administration of the one or more anti-cytokine reagent(s)), and in an effective amount that can vary for each type of anti-cytokine reagent (and as is known in the art). In some embodiments, the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition can be administered as a single dose, as can the one or more anti-cytokine reagents. In some embodiments, the one or more anti-cytokine reagents can be administered multiple times (e.g., any of about 7, 14, 21 days, or any of about one, two or three months) after administration of the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition including, in some embodiments, an initial administration with the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition. Exemplary anti-cytokine reagents for administration to a mammal to prevent and/or treat anti-coronavirus (preferably anti-SARS-CoV-2)can include, but are not limited to, one or more anti-IL-la reagent(s), one or more anti-IL5 reagent(s), one or more anti-IL-6 reagent(s), one or more anti-IL-12 reagent(s), one or more anti-IL-17 reagent(s), one or more anti-MCP-1 reagent(s), one or more anti-TNF-a reagent(s), one or more anti-GM-CSF reagent(s), and/or one or more anti-RANTES reagent(s). In some embodiments, the one or more anti -cytokine reagents would not include one or more anti-MIPa reagent(s) and/or one or more anti-RANTES reagent(s). Exemplary anti-cytokine reagents that can be used as described herein can include, for example, any of those shown in Table 7.

Table 7

In some embodiments, one or more additional anti-coronavirus (preferably anti-SARS-CoV-2) agents can also be administered to the subject(s) before, essentially simultaneously, or after administration of anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition such as, for instance, chloroquine (e.g., pharmaceutical salt and/or derivative thereof; e.g., hydroxychloroquine 400mg per day for 5 days or 200 mg three times per day for 10 days) and/or azithromycin (e.g., 500 mg on first day followed by four daily 250 mg doses) and/or remdesivir (e.g., 200 mg initial followed by lOOmg daily doses) and/or any other suitable reagent. Other administration / dosing schemes, anti-cytokine reagents, combinations thereof, and combinations with other anti-coronavirus (preferably anti-SARS-CoV-2) agents as are available to those of ordinary skill in the art can be suitable for use as disclosed herein, as would be understood by those of ordinary skill in the art.

[00125] In some embodiments, a subject (e.g., human being) can be tested for coronavirus (preferably SARS-CoV-2) infection by a suitable technique (e.g., polymerase chain reaction (PCR), nasal swab to detect viral particles). An immunogenic composition comprising one or more rdAd anti-SARS-CoV-2 vectors (e.g., as viral particles; SARS-CoV-2 immunogenic composition) can then be administered to individuals that test positive for coronavirus infection. Preferably, such administration can be completed within seven to ten days after initial exposure to the coronavirus. In some embodiments, an anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition can be administered to individuals at high risk for infection and/or symptoms (e.g., respiratory symptoms, death) such as immunocompromised individuals and/or suffering from another disease condition (e.g., kidney failure), and/or persons in high risk situations (e.g., travelers to pandemic areas, enclosed spaces such as cruise ships), whether or not such individuals have tested positive for coronavirus infection.

[00126] In some embodiments, the rdAd compositions administered with the anti- coronavirus (preferably anti-SARS-CoV-2) peptide constructs can be administered to a host comprising nostrils, wherein such nostrils are tilted upwards (i.e., the dorsal position), to generate a strong immunogenic response via intranasal administration. Other administration and dosing strategies are also contemplated herein as would be understood by those of ordinary skill in the art.

[00127] In some embodiments, this disclosure provides an immunogenic composition comprising a peptide construct comprising a peptide (R in Formula I) including at least one coronavirus (preferably SARS-CoV-2) antigen, optionally wherein said antigen comprises a coronavirus (preferably SARS-CoV-2) spike (S) protein receptor binding domain (RBD) wherein said immunogenic composition is configured to induce neutralizing antibody and/or cellular immune response against coronavirus (preferably SARS-CoV-2) in a mammalian subject to which said immunogenic composition is administered. In some embodiments, the peptide includes at least one coronavirus (preferably SARS-CoV-2) antigen of coronavirus (preferably SARS-CoV- 2) polypeptide of SEQ ID NOS. 2-11, encoded by SEQ ID NO: 1, or an antigen having about 80% homology therewith, such as spike (S) protein or SI domain of the spike protein; a sequence presented in SEQ ID NO: 3, a sequence having at least 80% homology to SEQ ID NO: 3; amino acids 331 to 527 of SEQ ID NO: 3; a spike protein RBD sequence comprises one or more of the following residues: L455, F486, Q493, S494 and/or N501, preferably in some embodiments Q493 and N501, preferably in some embodiments a residue selected from Y455, F455 or S455, preferably in some embodiments a residue selected from L486 or P486, preferably in some embodiments a residue selected from N493, R493 or K493, preferably in some embodiments a residue selected from D494 or G494, preferably in some embodiments a residue selected from T501 or S501; comprises at least one antigen of one or more of the coronavirus (preferably SARS- CoV-2) structural proteins envelope (E), membrane (M) or nucleocapsid (N). In some embodiments, the peptide includes one or more CD8⁺ T cell epitopes, and/or one or more CD4⁺ T cell epitopes (preferably, e.g., as in Tables 1-6) and/or at least one or more B cell epitopes (preferably, e.g., SEQ ID NOS. 283-327). In some embodiments, the immunogenic composition induces the production of neutralizing antibodies seroprotective against coronavirus (preferably SARS-CoV-2) infection in a mammalian subject, optionally wherein the mammalian subject is a human being.

[00128] In some embodiments, this disclosure provides pharmaceutical formulations comprising an effective amount of such immunogenic composition (i.e., a composition comprising one or more anti-coronavirus (preferably anti-SARS-CoV-2) peptide constructs) and, a pharmaceutically acceptable diluent or carrier, optionally wherein the diluent is phosphate- buffered saline. In some embodiments, the pharmaceutical formulation is configured for non- invasive administration, and/or for intranasal administration to the mammalian subject. In some embodiments, administration of the pharmaceutical formulation to the mammalian subject induces a protective immune response in the mammalian subject, optionally a combined mucosal, humoral and T cell protective immune response. In some embodiments, the pharmaceutically acceptable carrier is in a spray or aerosol form. In some embodiments, the effective amount is at least 10⁷ viral particles (vp), at least 10⁸ viral particles (vp), or at least 10⁹ viral particles (vp) (of the rdAd anti-SARS-CoV-2 vector(s)). In some embodiments, the pharmaceutical formulation is configured as a single dose, or as two or more doses. In some embodiments, this disclosure provides anti-coronavirus pharmaceutical formulations suitable for an administration to a human subject, comprising: an effective amount of at least about one (1) to about 1000 pg of the anti- coronavirus (preferably anti-SARS-CoV-2) peptide construct (within an immunogenic composition) comprising at least one peptide including an antigen of a coronavirus (preferably SARS-CoV-2) spike (S) protein receptor binding domain (RBD), or at least one immunogenic fragment thereof, wherein the effective amount induces a combined mucosal, humoral and/or T cell protective immune response; and, a pharmaceutically acceptable diluent or carrier. In some embodiments, the formulation is configured to provide seroprotection to the human subject for at least 9 months against coronavirus (preferably SARS-CoV-2).

[00129] In some embodiments, this disclosure provides methods for inducing an immune response against coronavirus, preferably SARS-CoV-2, the method comprising administering an effective amount of an immunogenic composition (or formulation or dosage form) disclosed herein to a mammalian subject, preferably wherein the immune response is protective against SARS- CoV-2. In preferred embodiments, the method comprises administration of an effective amount of an immunogenic composition disclosed herein to a mammalian subject, wherein the immune response provides protection against challenge with SARS-CoV-2. In some embodiments, this disclosure provides methods for inducing a combined mucosal, humoral and/or T cell protective immune response in a human subject against coronavirus comprising: administering intranasally to a human subject a single dose of a coronavirus, preferably SARS-CoV-2, pharmaceutical formulation or dosage disclosed herein, wherein the administration induces serum antibodies, mucosal antibodies and T cells against SARS-CoV-2, optionally whereby the human subject is seroprotected for at least about 9 months. Preferably, the seroprotection lasts for at least 12 months, at least 13 months or at least 14 months.

[00130] In some embodiments, this disclosure provides methods such as those above, further comprising administering one or more anti-cytokine reagents (see, e.g., Table 7) to the human being to prevent and/or treat coronavirus (preferably SARS-CoV-2) infection, optionally wherein the one or more anti -cytokine reagents include one or more anti -IL- la reagent(s), one or more anti-IL5 reagent(s), one or more anti-IL-6 reagent(s), one or more anti-IL-12 reagent(s), one or more anti-IL-17 reagent(s), one or more anti-MCP-1 reagent(s), one or more anti-TNF-a reagent(s), one or more anti-GM-CSF reagent(s), and/or one or more anti-RANTES reagent(s). In some embodiments, the one or more anti-cytokine reagents does not include one or more anti- MIRa reagent(s) and/or one or more anti-RANTES reagent(s). In some embodiments, the one or more anti-cytokine reagent(s) are co-administered substantially with the effective amount of the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition. In some embodiments, the one or more anti-cytokine reagent(s) are not administered substantially with the effective amount of the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition. In some embodiments, the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition is administered to the mammal once and the one or more anti -cytokine reagent(s) are administered multiple times. In some embodiments, the anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic composition is co-administered to the mammal with the one or more anti-cytokine reagent(s), and the one or more anti-cytokine reagent(s) are subsequently administered to the mammal. In some embodiments, this disclosure provides methods for treating and/or inhibiting (e.g., ameliorating) the symptoms of a respiratory viral infection in a mammal, said respiratory viral infection causing elevated expression of interleukin-6 (IL-6), interleukin- 1- alpha (IL-la) and/or interleukin- 12 (IL-12) in the lung of said mammal which can cause deleterious effects in a host. In some embodiments, such methods comprise administering an effective amount of anti-coronavirus (preferably anti-SARS-CoV-2) immunogenic compositionto the subject, whereby expression of IL-6, IL-la, and/or IL-12 in the lung is reduced thereby alleviating said symptoms for up to about 28 days following administration of the vector. In some embodiments, such methods cause the expression of monocyte chemoattractant protein 1 (MCP- 1), tumor necrosis factor alpha (TNF-a), granulocyte macrophage colony stimulating factor (GM- CSF), RANTES, and/or IL-17 are reduced in the lung following administration of the vector. In some embodiments, the expression of macrophage inflammatory protein 1 alpha (MIP-la) and/or RANTES are not reduced following administration of the vector. In some embodiments, this disclosure provides methods for inducing an anti -viral immune response in a mammalian subject in need thereof with, or at risk of, a respiratory viral infection, the method comprising: administering an effective amount an anti-coronavirus (preferably anti-SARS-CoV- 2) immunogenic composition to the subject, wherein the anti-coronavirus (preferably anti-SARS- CoV-2) immune response generates increased expression of monocyte chemoattractant protein 1 (MCP-1) and/or interferon alpha (IFN-g) following the administration step. In some embodiments of such methods, the mammalian subject (e.g., human being) is infected by coronavirus (preferably SARS-CoV-2) (e.g., in the hospital being treated for SARS-CoV-2 infection) prior to the administering of the pharmaceutical formulation thereto. In some embodiments, one or more additional anti-coronavirus (preferably SARS-CoV-2) agents can be administered to the subject(s) before, essentially simultaneously, or after administration of the anti-coronavirus (preferably anti- SARS-CoV-2) immunogenic composition such as, for instance, chloroquine (e.g., pharmaceutical salt and/or derivative thereof; e.g., hydroxychloroquine 400 mg per day for 5 days or 200 mg three times per day for 10 days) and/or azithromycin (e.g., 500 mg on first day followed by four daily 250 mg doses) and/or remdesivir (e.g., 200 mg initial followed by lOOmg daily doses) and/or any other suitable reagent.

[00131] In some embodiments, this disclosure provides anti-coronavirus (preferably anti- SARS-CoV-2) immunogenic compositions comprising one or more peptide constructs comprising one or more coronavirus antigens (preferably one or more peptides) comprising one or more T cell epitopes of Table 1, one or more groups of T cell epitopes of Table 2, one or more groups of T cell epitopes of Table 3; one or more groups of T cell epitopes of Table 4; one or more groups of T cell epitopes of Table 5; one or more groups of T cell epitopes of Table 6; and/or one or more B cell epitopes of SEQ ID NOS. 283-327; optionally, wherein the peptides are concatenated, and optionally separated by a linker amino acid sequence of two to ten amino acids.

[00132] Definitions

[00133] As used herein, the terms "a" or "an" are used, as is common in patent documents, to include one or more than one, independent of any other instances or usages of "at least one" or "one or more."

[00134] As used herein, the term "or" is used to refer to a nonexclusive or, such that "A or B" includes "A but not B," "B but not A," and "A and B," unless otherwise indicated.

[00135] As used herein, the term "about" is used to refer to an amount that is approximately, nearly, almost, or in the vicinity of being equal to or is equal to a stated amount, e.g., the state amount plus/minus about 5%, about 4%, about 3%, about 2% or about 1%. [00136] The compositions, formulations and methods of the present invention may comprise, consist essentially of, or consist of the components and ingredients of the present invention as well as other ingredients described herein. As used herein, "consisting essentially of means that the compositions, formulations and methods may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed compositions, formulations and methods.

[00137] It should also be noted that, as used in this specification and the appended claims, the term "configured" describes a system, apparatus, or other structure that is constructed or configured to perform a particular task or adopt a particular configuration. The term "configured" can be used interchangeably with other similar phrases such as arranged and configured, constructed and arranged, adapted and configured, adapted, constructed, manufactured and arranged, and the like.

[00138] As used herein, an “adjuvant” refers to a substance that enhances the body’s immune response to an antigen. In embodiments, the present monovalent influenza pharmaceutical formulation is a non-adjuvanted vaccine composition.

[00139] By “administration” is meant introducing a vaccine composition of the present disclosure into a subject; it may also refer to the act of providing a composition of the present disclosure to a subject (e.g., by prescribing).

[00140] As used herein, the term “ambient temperature” is the air temperature for storing the present monovalent influenza pharmaceutical formulation. In embodiments, the ambient temperature is a room temperature, such as selected from any temperature within the range from about 15 to 30°C, preferably from about 20 to 25°C.

[00141] The term “therapeutically effective amount” as used herein refers to that amount of the compound being administered which will induce a combined, mucosal, humoral and cell mediated immune response. The term also refers to an amount of the present compositions that will relieve or prevent to some extent one or more of the symptoms of the condition to be treated. In reference to conditions/diseases that can be directly treated with a composition of the disclosure, a therapeutically effective amount refers to that amount which has the effect of preventing the condition/disease from occurring in a mammal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the condition/disease (prophylactic treatment), alleviation of symptoms of the condition/disease, diminishment of extent of the condition/disease, stabilization (e.g., not worsening) of the condition/disease, preventing the spread of condition/disease, delaying or slowing of the condition/disease progression, amelioration or palliation of the condition/disease state, and combinations thereof. The term “effective amount” refers to that amount of the compound being administered which will produce a reaction that is distinct from a reaction that would occur in the absence of the compound.

[00142] As used herein, the term "percent (%) homology" and grammatical variations thereof in the context of two sequences (e.g., protein sequences), refers to two or more sequences or subsequences (i.e., fragment thereof) that have at least about 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%, and/or 100% nucleotide or amino acid residue identity (homology), when compared and aligned for maximum correspondence, as measured using one of the well-known sequence comparison algorithms or by visual inspection.

[00143] As used herein, an “immunogenic composition” refers to a composition, typically comprising at least one type of peptide construct as disclosed herein and at least one pharmaceutically acceptable carrier, that when administered to a host induces, stimulates, and/or enhances an immune response against at least one coronavirus (preferably SARS-CoV-2) antigen(s). A “vaccine” refers to such an immunogenic composition that when administered induces, stimulates, and/or enhances a protective immune response against coronavirus (preferably SARS-CoV-2) virus (e.g., protects the host against challenge with coronavirus (preferably SARS- CoV-2virus). In certain embodiments, an immunogenic composition (e.g., vaccine) can comprise one or more peptide constructs comprising at least one coronavirus antigen antigen(s), preferably coronavirus (preferably SARS-CoV-2) antigen(s), along with other components of an immunogenic composition (e.g., vaccine) suitable for administration to a mammalian host, including for example one or more adjuvants, slow release compounds, solvents, buffers, additional anti-coronavirus (preferably anti-SARS-CoV-2) agents, etc. In certain embodiments, an immunogenic composition and/or vaccine can comprise a protein and/or carbohydrate and/or lipid and/or other antigen, including but not limited to one or more killed antigen(s) (e.g., a killed or completely inactive virus) or a live attenuated antigen (e.g., an attenuated virus). In some embodiments, the immunogenic composition(s) and/or vaccine(s) improve immune responses to any antigen regardless of the antigen source or its function. [00144] As used herein, a “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to the human subject and does not abrogate the biological activity and properties of the administered vaccine compositions.

[00145] As used here, the term “seroconversion” is defined as a four-fold or greater increase in serum neutralization antibody titers (e.g., anti-Sl/S2 antibody or anti-RBD of SI antibody) after vaccination (e.g., administration of a present immunogenic composition).

[00146] As used herein, the term “seropositive” means a measurable (e.g., detectable in an in vitro assay) in serum neutralization antibody after vaccination (e.g., administration of a present immunogenic composition).

[00147] As used herein, the term “seroprotected” means a subject post vaccination that is protected from infection via generation of serum neutralization antibodies. In a population, this is referred to as a percentage (%) of seroprotected individuals (e.g., 50%). In embodiments, the present immunogenic compositions and methods of use provide seroprotection to the mammalian subject, such as a human subject, against SARS-CoV-2 infection.

[00148] The terms “treat”, “treating”, and “treatment” are an approach for obtaining beneficial or desired clinical results. Specifically, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (e.g., not worsening) of disease, delaying or slowing of disease progression, substantially preventing spread of disease, amelioration or palliation of the disease state, and remission (partial or total) whether detectable or undetectable. In addition, “treat”, “treating”, and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. As used herein, the terms “prophylactically treat” or “prophylactically treating” refers completely, substantially, or partially preventing a disease/condition or one or more symptoms thereof in a host. Similarly, “delaying the onset of a condition” can also be included in “prophylactically treating” and refers to the act of increasing the time before the actual onset of a condition in a patient that is predisposed to the condition.

[00149] As used herein, a “vaccine” refers to a composition comprising an anti-coronavirus (preferably SARS-CoV-2) peptide construct, along with other components of a vaccine formulation, including for example adjuvants, slow release compounds, solvents, etc. In embodiments of the invention, vaccines improve immune responses to any antigen regardless of the antigen source or its function.

[00150] As referred to herein an "antigen" means a substance that induces and/or enhances a specific immune response against the antigen, and/or an infectious agent expressing such antigen, in a subject, including humans and/or animals. The antigen may comprise an epitope, a hapten, and/or any combination thereof.

[00151] Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed.

[00152] Other embodiments are also contemplated herein as would be understood by those of ordinary skill in the art.

EXAMPLES

[00153] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to use the embodiments provided herein and are not intended to limit the scope of the disclosure nor are they intended to represent that the Examples below are all of the experiments or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by volume, and temperature is in degrees Centigrade. It should be understood that variations in the methods as described can be made without changing the fundamental aspects that the Examples are meant to illustrate.

[00154] Example 1: Materials and Methods

[00155] A peptide (R of Formula I) including at least one SARS-CoV-2 antigen (or epitope) is synthesized by FMOC (fluorenylmethyloxycarbonyl chloride) solid-phase synthesis. A fluorocarbon chain (e.g., CsFnlCFhjiCOOH) is then incorporated on the epsilon-chain of an additional N-terminal lysine of the peptide to derive the fluorocarbon-linked peptide. Purified fluorocarbon-linked peptides or unmodified peptides can be obtained through cleavage in the presence of trifluoroacetic acid (TFA) and a final purification by reverse phase-high performance liquid chromatography (RP-HPLC). In preferred embodiments, the preparations exhibit a fluorocarbon-linked peptide (i.e., anti-coronavirus peptide construct) purity of 90% or greater.

[00156] Example 2: Superiority of the Fluorocarbon-conjugated Peptides Compared to Unconjugated Peptides in their Ability to Promote T Cell Responses In Vivo

[00157] In this example, the immunogenicity in mice of a fluorocarbon-conjugated anti- coronavirus peptide is compared to an equivalent unconjugated peptides. Female BALB/c mice (n=7/group) are immunized intramuscularly with the fluorocarbon-conjugated anti-coronavirus peptide at a dose of 50 pg per peptide in a volume of 50 pL, or with an equimolar dose of unconjugated peptide in a volume of 50 pL. Mice are immunized on day 0 and sacrificed on day 14. Splenocytes of the sacrificed mice are stimulated in vitro with 5 pg/mL/peptide for 18 hours in an ELISpot assay. The plates are washed with PBS, incubated with an IFN-g detection peroxidase-labelled antibody, followed by a substrate, according to the manufacturer's instructions. The developed spots were counted using an automated plate counting system (CTL Europe) to quantify the number of åFN-y⁺ SFCS. Significantly higher magnitude T cell responses are observed in mice immunized with the mixture of fluorocarbon-conjugated peptides compared to the equivalent mixture of unconjugated peptides. The conjugation of a fluorocarbon vector to the coronavirus-derived peptide sequences are thereby shown to promote higher and broader T cell responses compared to the equivalent unconjugated peptides.

[00158] Example 3: Fluorocarbon-conjugated Peptides Promote a CTL/CD8+ T Cell Response

[00159] The quality of the immune response induced by a fluorocarbon-conjugated anti- coronavirus peptide is evaluated in mice. Female BALB/c mice (n=7/group) are immunized intramuscularly with the fluorocarbon-conjugated anti-coronavirus peptide at a dose of 25 pg per peptide in a volume of 50 pL. Mice are immunized on day 0 and sacrificed on day 14. Splenocytes derived from the sacrificed mice were stimulated in vitro with either a CTL epitope derived from coronavirus at concentrations ranging from 10¹ to 10⁹ pg/ml for 18 hours in an ELISpot assay. The plates are washed with PBS, incubated with an IFN-g detection peroxidase-labelled antibody, followed by a substrate, according to the manufacturer's instructions. The developed spots were counted using an automated plate counting system (CTL Europe) to quantify the number of IFN- g⁺ SFCs. The fluorocarbon-conjugated anti-coronavirus peptide is shown to promote T cell responses against CTL epitopes after a single immunization.

[00160] Example 4: Synergy Between Fluorocarbon-peptides Contained in the Same Formulation

[00161] The immunogenicity of a fluorocarbon-conjugated anti-coronavirus peptide administered in mice alone or as part of a co-formulation with other fluorocarbon-conjugated anti- coronavirus peptides is evaluated in mice. Female BALB/c mice (n=7/group) are immunized intramuscularly with the fluorocarbon-conjugated anti-coronavirus peptide at a dose of 25 pg per peptide in a volume of 50 pL. Mice are immunized on day 0 and sacrificed on day 14. Splenocytes derived from the sacrificed mice were stimulated in vitro with either a CTL epitope derived from coronavirus at concentrations ranging from 10¹ to 10⁹ pg/ml for 18 hours in an ELISpot assay. The plates are washed with PBS, incubated with an IFN-g detection peroxidase-labelled antibody, followed by a substrate, according to the manufacturer's instructions. The developed spots were counted using an automated plate counting system (CTL Europe) to quantify the number of IFN- g⁺ SFCs. A higher magnitude of coronavirus-specific T cell responses is observed in mice immunized with the mixture of fluorocarbon-conjugated peptides than is observed following administration of the single peptide.

[00162] While certain embodiments have been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the following claims.

Claims

CLAIMS What is claimed is:

1. A peptide construct comprising at least one coronavirus peptide having from about 15 to about 60 amino acids, at least one coronavirus CD8⁺ T-cell epitope, at least one coronavirus CD4⁺ T-cell epitope, and/or at least one coronavirus B cell epitope; the coronavirus peptide being covalently attached to a vector configured for intracellular delivery of the coronavirus peptide.

2. The peptide construct of claim 1, wherein the vector comprises a chain of 3 to 30 carbon atoms, at least one of which is substituted with fluorine, chlorine, bromine or iodine.

3. The peptide construct of claim 1 or 2, wherein the vector comprises a chain of 3 to 30 carbon atoms, at least one of which is substituted with fluorine.

4. The peptide construct of any preceding claim wherein the peptide binds two or more HLA class I alleles.

5. The peptide construct of any preceding claim wherein the peptide construct has the formula CmFn-C_yH_x(Sp)-R, where m=3 to 30, n < 2m+l, y=0 to 15, x < 2y, (m+y)=3-30, Sp is an optional spacer moiety, and R is the coronavirus peptide; wherein Sp is optionally derived from a lysine residue or has the formula -CONH-(CH₂)₄-CO-.

6. The method of any preceding claim wherein the vector comprises:

wherein Sp is an optional spacer moiety and R is the coronavirus peptide, and Sp is optionally derived from a lysine residue or has the formula -CONH-(CH2)4-CO-.

7. The peptide construct of any preceding claim wherein the vector is derived from 2H, 2H, 3H, 3H-perflouroundecanoic acid having the formula:

8. The peptide construct of any preceding claim wherein the coronavirus is SARS-CoV-2.

9. The peptide construct of any preceding claim wherein the coronavirus peptide has at least 80% identity to an epitope of any one of SEQ ID NOS. 1-11 or 27-408.

10. A composition comprising a peptide construct of any preceding claim, the composition optionally comprising at least one pharmaceutically acceptable excipient.

11. The composition of claim 10 which is a lyophilized mixture of one or more peptide constructs, mannitol, L-histidine buffer, and saline.

12. A method for treating and/or preventing coronavirus infection, the method comprising administering the peptide construct or composition of any preceding claim to a human being.

13. The method of claim 12, further comprising administering an adjuvant substantially with the fluorocarbon peptide, optionally wherein the adjuvant is IC31®.

14. The method of any preceding claim wherein a lyophilized mixture of at least one peptide construct, mannitol, L-histidine buffer, and saline is reconstituted prior to administration to the human being.

15. The method of any one of claims 12-14 wherein the coronavirus is SARS-CoV-2.

16. The method of any of claims 12-15 wherein the peptide construct is administered to the human being in at least four, five or six doses, wherein each dose is separated by at least about 28 days.

17. The method of any of claims 12-16 preceding claim wherein the peptide construct is administered intramuscularly, subcutaneously, sublingually, intranasally, orally, via a transdermal route or via a transmucosal route.