CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/113,107, filed November 12, 2020, the disclosure of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[0002] This application incorporates by reference a Sequence Listing submitted with this application as a text file, entitled 14671-001-228_ST25.txt, created on November 8, 2021, and is 880,739 bytes in size.

BACKGROUND

[0003] Coronaviruses are species of viruses belonging to the subfamily Coronavirinae in the family Coronaviridae, and are positive-sense RNA viruses that infect humans and animals and cause respiratory, gastrointestinal or neurologic disease. Human coronavrisues include 229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV-2. Common human coronaviruses include OC43, HKU1, 229E, and HCoV-NL63 that cause common colds and more severe lower respiratory tract infections in the very young and the elderly. Over the past two decades, three serious coronaviruses outbreaks have occurred, including SARS-CoV, MERS-CoV, and most recently, SARS-CoV-2 (also referred to as COVID-19), which resulted in a worldwide pandemic, infecting millions of people and causing substantial human death. There are currently no FDA-approved preventative or therapeutic vaccines for use against human coronaviruses and there is an urgent need for agents that can be used in methods for treating, preventing, or ameliorating coronaviruses, such as SARS-CoV-2, and diseases, disorders, and conditions related thereto.

[0004] Synthetic virus-like particles (SVLPs) have been utilized for vaccine development. Specifically, SVLP -based vaccine candidates using synthetic lipopeptide building blocks to which antigens are conjugated, which spontaneously self-assemble into immunogenic nanoparticles have been developed (see, WO2015/082501 and WO2020/127728). Additionally, certain SLVPs have been developed that include T-helper epitopes and coiled-coil domains (see, WO2018/229159 and W02008/068017). Despite these recent developments, there is still a need for SVLP -based vaccine candidates, most particularly for treating, preventing, or ameliorating coronaviruses, such as SARS-CoV-2, and diseases, disorders, and conditions related thereto.

SUMMARY

[0005] In a first aspect, disclosed herein is a lipoprotein comprising (a) a lipopeptide building block comprising: (i) a lipid moiety and (ii) an amphipathic domain; and (b) an antigen of a coronavirus. In certain embodiments, the lipoprotein is a nanomicelle. In certain embodiments, the lipid moiety is a Toll-like receptor (TLR) agonist. In certain embodiments, the TLR agonist is a TLR-2 agonist. In certain embodiments, the lipid moiety has a structure selected from the group consisting of LM1, LM2, LM3, LM4, LM5, LM6, LM7, and LM8. In certain embodiments, the lipid moiety is selected from the group consisting of Cys((R)- 2,3-di(palmitoyloxy)-2-propyl) (Pam2Cys), Cys((R)-2,3-di(palmitoyloxy)-2-propyl-N- palmitoyl) (Pam3Cys), unsaturated Pam3Cys, phosphatidylethanolamine, diphosphoryl hexaacyl lipid A, l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine, and 1,3-dipalmitoyl- glycero-2-phosphoethanolamine.

[0006] In certain embodiments, the amphipathic domain is a coiled-coil domain. In certain embodiments, the coiled-coil domain directs formation of helical bundles. In certain embodiments, the coiled-coil domain comprises a heptad motif, (abcdefg)n, wherein each a and d independently represents a hydrophobic residue and each b, c, e, f, and g independently represents a polar residue. In certain embodiments, the amphipathic domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-48. In certain embodiments, the amphipathic domain comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-48. In certain embodiments, the amphipathic domain consists of an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-48.

[0007] In certain embodiments, the lipoprotein further comprises a T-helper epitope. In certain embodiments, the lipopeptide building block further comprises a T- helper epitope. In certain embodiments, the T- helper epitope is selected from the group consisting of a flu, hepatitis B, tetanus toxin, diphtheria toxin, and measles T-helper epitope. In certain embodiments, the T-helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 49-77. In certain embodiments, the CD4+ T-helper cell epitope comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 49-77. [0008] In certain embodiments, the coronavirus is a human coronavirus. In certain embodiments, the coronavirus is an alpha coronavirus. In certain embodiments, the coronavirus is a beta coronavirus. In certain embodiments, the coronavirus is selected from the group consisting of 229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS- CoV-2. In certain embodiments, the coronavirus is SARS-CoV-2. In certain embodiments, the antigen comprises at least one of a B-cell epitope, CD4+ T-cell epitope, or CD8+ T-cell epitope. In certain embodiments, the antigen is a modified SARS-CoV-2 Spike protein epitope. In certain embodiments, the modified SARS-CoV-2 Spike protein epitope comprises at least one modification selected from the group consisting of a linker, a carboxamide, hairpin cap, a cysteine residue, and a furin cleavage site, a serin protease cleavage site, and an amino acid substitution.

[0009] In certain embodiments, the antigen comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 96-193. In certain embodiments, the antigen comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID Nos. 96-193. In certain embodiments, the antigen consists of an amino acid sequence selected from the group consisting of SEQ ID Nos. 96-193.

[0010] In certain embodiments, the lipoprotein further comprises one or more linkers. In certain embodiments, the antigen is coupled to the lipopeptide building block via the one or more linkers. In certain embodiments, the amphipathic domain is coupled to the lipid moiety via the one or more linkers. In certain embodiments, the one or more linkers is a peptide linker.

[0011] In certain embodiments, the one or more linkers comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 194-232.

[0012] In certain embodiments, the lipoprotein further comprises a purification tag coupled to the antigen. In certain embodiments, the purification tag is removable by treatment with a protease.

[0013] In certain embodiments, the purification tag comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 233-242.

[0014] In another aspect, disclosed herein is a lipoprotein comprising: (a) lipopeptide building block comprising: (i) a lipid moiety, (ii) an amphipathic domain, and (iii) a T-helper cell epitope; and (b) an antigen of SARS-CoV-2 coupled to the lipopeptide building block. [0015] In a further aspect, disclosed herein is an immunogenic composition comprising the lipoprotein of any one of the preceding embodiments. In certain embodiments, the immunogenic composition further comprises an adjuvant. In certain embodiments, the adjuvant is selected from the group consisting of aluminum, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), monophosphoryl lipid A (MPL), squalene, cytosine phosphoguanine, QS-21, microcrystalline tyrosine, MF59, mannide monooleate, GLA, SLA, E6020, resiquimod (R848), imidazoquinolines, imiquimod, 3M-052, 2',3'-cGAMP, 3', 3'- cGAMP, cGMP, cAMP, AMP, muramyl dipeptide, poly I:C, CpGand combinations thereof. [0016] In a further aspect, disclosed herein is a lipoprotein or an immunogenic composition of any one of the preceding embodiments, wherein the lipoprotein provides protective immunity to a subject against the coronavirus. In certain embodiments, the lipoprotein or immunogenic composition provides protective immunity to a subject against the coronavirus for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years, about 3 years, about 4 years, about 5 years, or about 10 years after administration to the subject. In certain embodiments, the lipoprotein induces an immune response in a subject sufficient to provide protective immunity to the subject against the coronavirus. In certain embodiments, the lipoprotein or the immunogenic composition induces the production of antibodies against at least a portion of the coronavirus spike protein. In certain embodiments, a single dose of the lipoprotein induces an immune response in a subject sufficient to provide protective immunity to the subject against the coronavirus.

[0017] In certain embodiments, two doses of the lipoprotein induce an immune response in a subject sufficient to provide protective immunity to the subject against the coronavirus. In certain embodiments, the lipoprotein does not induce antibody-dependent enhancement. In certain embodiments, the lipoprotein has an efficacy rate of greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, or greater than about 90%. In certain embodiments, the lipoprotein has an reactogenicity rate of less than about 20%, less than about 15%, less than about 10%, less than about 8%, less than about 5%, less than about 3%, less than about 2%, or less than about 1%.

[0018] In another aspect, disclosed herein is a method of immunizing a subject against a coronavirus comprising administering to the subject the lipoprotein or the immunogenic composition of any one of the preceding embodiments.

[0019] In yet another aspect, disclosed herein is a method of preventing or reducing risk of coronavirus infection in a subject comprising administering to the subject the lipoprotein or the immunogenic composition of any one of the preceding embodiments. [0020] In a further aspect, disclosed herein is a method of treating a coronavirus infection in a subject comprising administering to a subject the lipoprotein or the immunogenic composition of any one of the preceding embodiments.

[0021] In yet a further aspect, disclosed herein is a method of reducing symptoms of a coronavirus infection in a subject comprising administering to the subject the lipoprotein or the immunogenic composition of any one of the preceding embodiments. In certain embodiments, the subject is human. In certain embodiments, the subject is immunocompromised. In certain embodiments, the subject is 60 years of age or older. In certain embodiments, the subject is a pediatric subject.

[0022] In another aspect, disclosed herein is a method of immunizing a subject against a coronavirus comprising: (a) administering to the subject a first immunogenic composition; and (b) administering to the subject a second immunogenic composition, the second immunogenic composition comprising a lipoprotein, the lipoprotein comprising (1) a lipopeptide building block and (2) a first antigen of a coronavirus, wherein the lipopeptide building block comprises lipid moiety and an amphipathic domain, and wherein the second immunogenic composition is heterologous to the first immunogenic composition; and wherein the second immunogenic composition is administered to the subject before or after the first immunogenic composition. In certain embodiments, the second immunogenic composition is administered to the subject before the lipoprotein. In certain embodiments, the second immunogenic composition is administered to the subject after the lipoprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the structure of Construct No. 567 (in Table 6), specifically C- terminal structure of the peptide is depicted. SEQ ID NO:719.

[0024] FIG. 2 shows the structure of a lipoprotein construct having a Pam2Cys structure at the C-terminus.

[0025] FIG. 3 shows the structure of a lipoprotein construct having a Pam3Cys structure at the C-terminus.

[0026] FIG. 4 shows the structure of a lipoprotein construct having a Pam3Cys structure at the C-terminus.

[0027] FIG. 5 shows the structure of a lipoprotein construct having a Pam3Cys structure at the C-terminus. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present disclosure relates to a lipoprotein comprising (1) a lipopeptide building block (BB) (see, Section I herein) comprising a lipid moiety (LM) (see, Section I. A herein) and an amphipathic domain (AD) (see, Section I.B herein) and (2) an antigen of a coronavirus (A) (see Section II herein). In certain embodiments, the lipoprotein has the structure: BB-A, where BB represents the lipopeptide building block and A represents the antigen. In certain embodiments, the lipoprotein has the structure: LM-AD-A, where LM represents the lipid moiety, AD represents the amphipathic domain, and A represents the antigen. In certain embodiments, the lipopeptide building block and the antigen of a coronavirus are covalently linked, either directly or indirectly through a linker (L) (see, Section III herein). In certain embodiments, the lipoprotein has the structure: LM-L-AD-L-A. In certain embodiments, the lipopeptide building block further comprises a T-helper epitope (TH) (see, Section I.C herein), which can be linked, either directly or indirectly through a linker (L), to the amphipathic domain and the antigen. Accordingly, in certain embodiments, the lipoprotein has the structure LM-AD-TH-A, where wherein LM represents the lipid moiety, AD represents the amphipathic domain, TH represents the T-helper epitope, and A represents the antigen. In certain embodiments, each component of the lipoprotein can be either covalently linked to each other, either directly or indirectly through a linker (L). Accordingly, in certain embodiments, the lipoprotein has the structure LM-L-AD-L-TH-L-A, where each instance of linker (L) is optional. In certain embodiments, the lipoprotein construct can comprise the sequences set forth in Table 6 herein (see, Section IV herein). [0029] In certain embodiments, the lipoprotein is an immunogenic composition (see, Section V herein). In certain embodiments, disclosed herein are immunogenic compositions comprising the lipoprotein disclosed herein. In certain embodiments, the lipoprotein can be used to treat, prevent, or reduce the symptoms of a coronavirus infection (see, Section VI herein). In certain embodiments, the lipoprotein can be used as a coronavirus vaccine. In certain embodiments, the lipoprotein is a synthetic virus-like particle (SVLP). Without wishing to be bound by theory, it is thought that the disclosed lipoproteins which are SVLPs and thus exhibit a native virus-like shape can result in increased cross-reactivity and increased titers of functional antibodies, providing an advantage in utilizing the disclosed lipoproteins as vaccines. In certain embodiments, the lipoprotein is a nanomicelle. I. Lipopeptide Building Block

[0030] In certain embodiments, the lipoprotein comprises a lipopeptide building block. In certain embodiments, the lipopeptide building block comprises a lipid moiety as disclosed in Section I. A herein and an amphipathic domain as disclosed in Section I.B herein. In certain embodiments, the lipopeptide building block further comprises a T-helper epitope as disclosed in Section I.C herein.

A. Lipid Moiety

[0031] In certain embodiments, the lipid moiety is a Toll-like receptor (TLR) agonist, for example, a TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, or TLR- 10 agonist. In certain embodiments, the lipid moiety is a TLR-2 agonist.

[0032] In certain embodiments, the lipid moiety comprises hydrocarbyl chains. In certain embodiments, the lipid moiety comprises one or more hydrocarbyl chains. In certain embodiments, the lipid moiety comprises two hydrocarbyl chains. In certain embodiments, the lipid moiety comprises three hydrocarbyl chains. In certain embodiments, the lipid moiety is a phospholipid. The phospholipid can comprise either ester- or ether-linked extended alkyl or alkenyl side. In certain embodiments, the phospholipid comprises enantiomers of 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine. In certain embodiments, the phospholipid comprises achiral analogues, including, but not limited to, l,3-dipalmitoyl-glycero-2- phosphoethanolamine.

[0033] In certain embodiments, the lipid moiety is selected from one of Formula LM1 to LM8:

[0034] wherein R1 and R^ in formulas LM 1 and LM 2 are independently of each other hydrocarbyl or hydrocarbyl-C=0, and Y is H orCOOH; [0035] LM 3

[0036] wherein Rl, R4 and R in formula LM 3 are independently of each other hydrocarbyl or hydrocarbyl-C=0; or Rl and R^ are independently of each other hydrocarbyl or hydrocarbyl-C=0 , and R is H or acetyl or lower alkyl-C=0;

LM 4 LM 5

[0037] wherein Rl and R^ in formulas LM 4 and LM 5 are independently of each other hydrocarbyl or hydrocarbyl-C=0 , and n is 1, 2, 3 or 4; LM 6

[0038] wherein Rl and R^ in formula LM 6 are independently of each other a hydrocarbyl, X is

O or NH, and n is 1, 2, 3 or 4, or

[0039] wherein R1 and R^ in formulas LM 7 and LM 8 are independently of each other hydrocarbyl.

[0040] In certain embodiments, the lipid moiety is di-palmitoyl-S-glycerylcysteinyl of formula LM3, wherein R1 and R^ are palmitoyl, and R^ is H or acetyl.

[0041] In certain embodiments, the hydrocarbyl or hydrocarbyl chain is a straight or branched alkyl or alkenyl chain consisting of at least 7 carbon atoms, between 8 and 50 carbon atoms, between 8 and 25 carbon atoms, and optionally one, two or three double bonds. Alkenyl has one, two or three double bonds in the chain, each with E or Z geometry, as is customarily found in natural fatty acids and fatty alcohols. Also included in the definition of "hydrocarbyl" or "hydrocarbyl chain" is branched alkyl or alkenyl, for example alkyl bearing a methyl or ethyl substituent at the second or third carbon atom counted from the end of the chain, as e.g. as in 2-ethyl-hexyl. The term, "lower alkyl" means alkyl with 1 to 7 carbon atoms, including, for example, 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl or tert-butyl.

[0042] In certain embodiments, the lipid moiety is:

[0043] In certain embodiments, the lipid moiety is:

[0045] In certain embodiments, the lipid moiety is: wherein R ³ is -C(O)Cn-i5alky or -C(O)CisH3i.

[0047] In certain embodiments, the lipid moiety is: wherein R ³ is -C(O)Cn-i5alky or -C(O)Ci5H3i.

[0049] In certain embodiments, the lipid moiety is:

[0051] In certain embodiments, the lipid moiety is:

[0052] [0053] In certain embodiments, the lipid moiety is:

[0055] In certain embodiments, the lipid moiety is moiety is selected from the group consisting of Cys((R)-2,3-di(palmitoyloxy)-2-propyl) (also referred to as: Pam2Cys or dipalmitoyl-S-glyceryl cysteine), Cys((S)-2,3-di(palmitoyloxy)-2-propyl), Cys((R)-2,3- di(palmitoyloxy)-2-propyl-N-palmitoyl) (also referred to as: Pam3Cys or tripalmitoyl-S- glyceryl cysteine), unsaturated Pam3Cys, unsaturated Pam2Cys, N, N’ -dipalmitoyl. -2,3- diamino-propionamide (also referred to as: Pam2Dap), phosphatidylethanolamine, diphosphoryl hexaacyl lipid A, l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine, and 1,3- dipalmitoyl-glycero-2-phosphoethanolamine. In certain embodiments, the lipid moiety comprises Pam2Cys. In certain embodiments, the lipid moiety consists of Pam2Cys. In certain embodiments, the lipid moiety comprises Pam3Cys. In certain embodiments, the lipid moiety consists of Pam3Cys.

B. Amphipathic Domain

[0056] The amphipathic domain can be a peptide chain comprising alternating hydrophobic residues (X) and hydrophilic residues (Y) with a general pattern of XmZnXoZp wherein m = 1-5, n = 1-5, o = 1-5, p = 0-5. In certain embodiments, the amphipathic domain has an amphipathic alpha helical structure with opposing polar and nonpolar faces oriented along the long axis of the helix. In certain embodiments, the amphipathic domain has amphipathic beta strand structure in which hydrophobic side-chains point in one direction and polar side-chains in the other. In certain embodiments, the amphipathic domain associates into a helical bundle. In certain embodiments, the amphipathic domain directs formation a coiled-coil. In certain embodiments, the amphipathic domain directs formation of an amphipathic beta sheet with at least two parallel beta strands in which hydrophobic side-chains point in one direction and polar side-chains in the other.

[0057] In certain embodiments, the amphipathic domain is a peptide structure comprising one chain. In certain embodiments, the amphipathic domain is a peptide structure comprising at least two chains. In certain embodiments, the amphipathic domain comprises parallel beta strands. In certain embodiments, the amphipathic domain comprises at least two peptide chains are associated into a bundle. In certain embodiments, the at least two peptide chains each have an alpha helical secondary structure and are associated into a bundle. Accordingly, in certain embodiments, the amphipathic domain comprises, or consists of, a coiled-coil domain. In certain embodiments, the coiled-coil domain directs formation of helical bundles. Various amphipathic domains, including coiled-coil domains, that are suitable in certain embodiments disclosed herein are disclosed in WO 2008/068017, W02015/082501, WO 2018/229156, WO 2020/127728, WO 2009/109428, WO 2011/112999, US 9,044,514, US 9,994,646, US 7,811,577, US 8,728,785, each of which are herein incorporated by reference. [0058] In certain embodiments, the amphipathic domain comprises multiple repeat units consecutively linked to each other. The repeat units of the amphipathic domain segment may be identical or may be different, e.g. may contain at least one discontinuity, such as an insertion, deletion or exchange of at least one amino acid within the repeat unit. In certain embodiments, 1, 2, 3 or 4 amino acids within the repeat unit may be inserted, deleted, or exchanged.

[0059] In certain embodiments, the amphipathic domain comprises or consists of 2 to 10 repeat units including 2, 3, 4, 5, 6, 7, 8, 9 and 10 repeat units, 3 to 8 repeat units including 3, 4, 5, 6, 7, 8 repeat units, or 4 repeat units. Without wishing to be bound by theory, it is thought that an increase in the number of repeat units in the peptide moiety influences the stability of the domain. Accordingly, in certain embodiments, the amphipathic domain comprises at least two heptad repeats (e.g. two, three, four, five, six, seven, eight, nine, ten, or more, repeat units).

[0060] The amphipathic domain can be based on canonical repeat units and non- canonical repeat units. In certain embodiments, the amphipathic domain comprises canonical tandem heptad repeats that may form right-handed amphipathic alpha-helices, which then assemble to form helical bundles with left-handed coiled coils. In certain embodiments, the coiled-coil domain comprises non-canonical, non-heptad-based repeats that form coiled coils that are not necessarily left-handed or even regular supercoils.

[0061] Repeat units of amphipathic domains have a sequence with a certain number of amino acids, wherein the positions of the amino acids are labelled as lowercase letters. In a certain embodiments, the repeat unit of the coiled-coil domains consists of 7 to 15 amino acids or 7 to 11 amino acids. In certain embodiments, the coiled-coil domain comprises or consists of 7 amino acids, wherein the seven amino acid positions are designated with letters a, b, c, d, e, f and g. In a certain embodiments, the heptad motif includes amino acids having hydrophobic residues at positions a and d, and polar, helix- favoring residues at the other residues. In certain embodiments, each of a and d can independently be selected from Ala, He, Leu, Met, Vai, Phe, Trp and Tyr. In certain embodiments, residues a and d can each independently be Gin or Asn. In certain embodiments, residues a and d each do not comprise N or Q. In certain embodiments, residues b, c, e, f and g are selected from Ala, Glu, Lys and Gin.

[0062] In certain embodiments, the amphipathic domain comprises or consists of 2 to 10 repeat units or 3 to 8 repeat units. In certain embodiments, the amphipathic domain comprises or consists of 4 repeat units. In certain embodiments, the amphipathic domain comprises or consists of 15, 11 or 7 amino acids. In certain embodiments, the amphipathic domain consists of 7 amino acids (heptad motif).

[0063] In certain embodiments, the amphipathic domain comprises 3-8 tandemly linked heptad motifs, wherein positions a and d in each heptad motif (abcdefg) contain alpha-amino acids belonging to the Group 1 and/or to the Group 2 as defined herein below. In certain embodiments, not more than two of all the a and d positions may be occupied by any amino acid residue belonging to the Group 3, and not more than one of all the a and d positions may be occupied by any amino acid residue belonging to the Group 4 or Group 5 or by glycine. In certain embodiments, positions b, c, e, f and g, are each independently selected from alphaamino acids belonging to the Groups 3, 4 and 5. In certain embodiments, at least one amino acid in positions b, c, e, f and g are hydrophobic. In certain embodiments, at least two amino acids in positions b, c, e, f and g are hydrophobic. In certain embodiments, at least three amino acids in positions b, c, e, f and g are hydrophobic. In certain embodiments, positions b, c, e, f and g, are each independently selected from a hydrophobic amino acid. In certain embodiments, positions b, c, e, f and g are each independently selected from amino acids belonging to the Groups 1 and 2.

[0064] Group 1 comprises alpha-amino acid residues with small to medium sized hydrophobic side chains. A hydrophobic residue refers to an amino acid side chain that is uncharged at physiological pH and that is repelled by aqueous solution. These side chains generally do not contain hydrogen bond donor groups, such as primary and secondary amides, primary and secondary amines and the corresponding protonated salts thereof, thiols, alcohols, ureas or thioureas. However, they may contain hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides, or tertiary amines. Genetically encoded amino acids in this group include alanine, isoleucine, leucine, methionine and valine.

[0065] Group 2 comprises amino acid residues with aromatic or heteroaromatic side chains. An aromatic amino acid residue refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated aromatic pi-electron system. In addition, it may contain additional hydrophobic groups such as lower alkyl, aryl or halogen, hydrogen bond donor groups such as primary and secondary amines, and the corresponding protonated salts thereof, primary and secondary amides, alcohols, and hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides or tertiary amines. Genetically encoded aromatic amino acids include phenylalanine and tyrosine. A heteroaromatic amino acid residue refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated aromatic pi-system incorporating at least one heteroatom such as O, S and N . In addition such residues may contain hydrogen bond donor groups such as primary and secondary amides, primary and secondary amines and the corresponding protonated salts thereof, alcohols, and hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides or tertiary amines. Genetically encoded heteroaromatic amino acids include tryptophan and histidine.

[0066] Group 3 comprises amino acids containing side chains with polar non-charged residues. A polar non-charged residue refers to a hydrophilic side chain that is uncharged at physiological pH, but that is not repelled by aqueous solutions. Such side chains typically contain hydrogen bond donor groups such as primary and secondary amides, primary and secondary amines, thiols, and alcohols. These groups can form hydrogen bond networks with water molecules. In addition, they may also contain hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides, or tertiary amines. Genetically encoded polar noncharged amino acids include asparagine, cysteine, glutamine, serine and threonine. Non- proteinogenic polar amino acids include citrulline and homocysteine.

[0067] Group 4 comprises amino acids containing side chains with polar cationic residues and acylated derivatives thereof, such as acylamino-derived residues and urea-derived residues. Polar cationic side chains refer to a basic side chain, which is protonated at physiological pH. Genetically encoded polar cationic amino acids include arginine, lysine and histidine. Omitine is an example for a urea-derived amino acid residue.

[0068] Group 5 comprises amino acids containing side chains with polar anionic residues. Polar anionic refers to an acidic side chain, which is deprotonated at physiological pH. Genetically encoded polar anionic amino acids include aspartic acid and glutamic acid, for example, a polar anionic residue is -(CTLjaCOOH, wherein "a" is 1 to 4.

[0069] In certain embodiments, the amphipathic domain includes between 3 to 8 tandemly linked heptad motifs, wherein each heptad motif (abcdefg) may have any one of the following sequences: Ixxlxxx (referring respectively to the positions abcdefg); lxx2xxx (referring respectively to the positions abcdefg); 2xxlxxx (referring respectively to the positions abcdefg); or 2xx2xxx (referring respectively to the positions abcdefg); wherein 1 is a genetically encoded amino acid from Group 1; 2 is a genetically encoded amino acid from Group 2; and wherein x is a genetically encoded amino acid from Groups 1, 2, 3, 4 or 5 or glycine. The alpha-amino acid with a hydrophobic side chain is selected from alanine, isoleucine, leucine, methionine and valine; the alpha-amino acid with aromatic or heteroaromatic residue is selected from phenylalanine, tyrosine, tryptophan and histidine; the alpha-amino acid with polar non- charged residue is selected from asparagine, cysteine, glutamine, serine and threonine; the alpha-amino acids with polar cationic residue is selected from arginine, lysine and histidine; and the alpha-amino acid with polar anionic residue is selected from aspartic acid and glutamic acid.

[0070] In certain embodiments, the amphipathic domain comprises or consists of amino acid sequences identified in naturally occurring peptides and proteins, but excluding those of human origin, for example, coiled-coil domains identified in viral and bacterial proteins. [0071] In certain embodiments, the amphipathic domain can be (XiX2X3X4XsX6X7)n (SEQ ID NO: 722), wherein Xi is a hydrophobic amino acid residue or asparagine, X2, X3, and Xe are each any amino acid residue, X4 is a hydrophobic amino acid residue, X5 and X7 are each a charged amino acid residue, and n is from 1 to 10. In certain embodiments, the amphipathic domain can be (lEKKIEXi)n (SEQ ID NO:723), wherein Xi is selected from A, G, T, and S, and n is 2. In certain embodiments, the coiled-coil domain includes E/K salt bridges between the b and f positions IEEKIKA/G/T/S (SEQ ID NO:724)and IECKIAA (SEQ ID NO:725). In certain embodiments, the amphipathic domain includes a cysteine residue for conjugation.

[0072] Exemplary amino acid sequences that can be included in the amphipathic domain are listed in Table 1 below, wherein Xi is selected from Vai, He, Leu, and Phe, X2 is selected from Glu, Lys, Arg, His, Ser, Thr, Asn, Gin, and Cys, X3 is selected from Glu, Lys, Arg, His, Ser, Thr, Asn, Gin, X4 is selected from He, Leu, Phe, X5 is selected from Glu, Lys and Gly, and Xe is selected from Ala, Gly, Lys, and any other hydrophilic amino acid, and variants thereof in which one, two, or three amino acids are replaced by other amino acids or are deleted. [0073] Table 1. Amphipathic Domain Sequences.

[0074] In certain embodiments, the amphipathic domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-48

[0075] In certain embodiments, the amphipathic domain comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1-48.

[0076] In certain embodiments, the amphipathic domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-48.

C. T-helper Epitope

[0077] In certain embodiments, the lipopeptide building block further comprises a T- helper epitope. In certain embodiments, the T-helper epitope is selected from a flu, hepatitis B, tetanus toxin, diphtheria toxin, and measles T-helper epitope. In certain embodiments, the T-helper epitope is a flu T-helper epitope. In certain embodiments, the T-helper epitope is a tetanus toxin T-helper epitope. The T-helper epitopes that can be included in the lipopeptide building block are provided in Table 2 below, and variants thereof in which one, two, or three amino acids are replaced by other amino acids or are deleted.

[0078] Table 2. T-helper Epitope Sequences.

[0079] In certain embodiments, the T-helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-77.

[0080] In certain embodiments, the T-helper epitope comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 49-77.

[0081] In certain embodiments, the T-helper epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-77. D. Signal Sequence

[0082] In certain embodiments, the lipopeptide building block comprises a signal sequence for cell-culture based expression of the lipopeptide building block. In certain embodiments, the signal sequence is fused to the N-terminus of the lipopeptide building block and then cleaved upon processing of the lipopeptide. Exemplary signal sequences that can be included in the lipopeptide building block are provided in Table 3 below:

[0083] Table 3. Signal Sequences.

[0084] In certain embodiments, the signal peptide is cleaved from the lipopeptide for use with the methods of treatment disclosed herein. In certain embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 78-95.

[0085] In certain embodiments, the signal peptide comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 78-95.

[0086] In certain embodiments, the signal peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 78-95.

[0087]

II. Coronavirus Antigen

[0088] The lipoprotein can comprise a coronavirus antigen. In certain embodiments, the coronavirus antigen is conjugated directly or indirectly through a linker to a component of the lipopeptide building block. In certain embodiments, the coronavirus antigen is connected, either directly or indirectly through a linker, through its N- or C-terminus, to the N- or to the C-terminal of the amphipathic domain, or to an amino acid side chain of the amphipathic domain. In certain embodiments, the coronavirus antigen is connected, either directly or indirectly through a linker, through its N- or C-terminus, to the N- or to the C-terminal of the T-helper epitope, or to an amino acid side chain of the T-helper epitope. In certain embodiments, the coronavirus antigen is connected to a component of the lipopeptide building block through a side chain residue of the coronavirus antigen, for example, through a terminal or internal aspartic acid, glutamic acid, lysine, ornithine or cysteine side chain. [0089] Without wishing to be bound by theory, it is thought that the disclosed antigens provide targeted responses to both linear and conformational epitopes and exclude of diseaseenhancing epitopes. In certain embodiments, the coronavirus antigen is a human coronavirus antigen. In certain embodiments, the coronavirus antigen is an alpha coronavirus antigen. In certain embodiments, the coronavirus antigen is a beta coronavirus antigen. In certain embodiments, the coronavirus antigen is selected from the group consisting of a 229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV-2 antigen. In certain embodiments, the coronavirus antigen is a SARS-CoV-2 antigen. In certain embodiments, the antigen is selected from a B-cell epitope, CD4+ T-cell epitope, and CD8+ T-cell epitope. In certain embodiments, the antigen can comprise one or more of a B-cell epitope, CD4+ T-cell epitope, and CD8+ T-cell epitope. In certain embodiments, the antigen comprises a B-cell epitope. In certain embodiments, the antigen comprises a CD4+ T-cell epitope. In certain embodiments, the antigen comprises a CD8+ T-cell epitope. In certain embodiments, the antigen comprises a modified Spike protein epitope. In certain embodiments, the antigen comprises one or more epitopes from different coronaviruses. In certain embodiments, the antigen is a modified SARS-CoV-2 Spike protein epitope. In certain embodiments, the modified SARS-CoV-2 Spike protein epitope comprises at least one modification selected from the group consisting of a hairpin cap, a helix cap, a cysteine residue, a furin protease cleavage site, a serin protease cleavage site, a linker, a carboxamide, and an amino acid substitution. In certain embodiments, the amino acid substitution improves solubility. Accordingly, the amino acid substitution can be a hydrophilic amino acid. In certain embodiments, a hydrophobic amino acid may be substituted by a charged amino acid, such as Asp, Glu, Arg, Lys, ornitin (Orn), 1,3-diaminopropionic acid (Dap), or 1,4-diaminobutyric acid (Dab), 4-amino-proline (Amp). In certain embodiments, a hydrophobic amino acid may be substituted by a less hydrophobic amino acid such as Ala. In certain embodiments, the amino substitution improves the stability. For example, in certain embodiments, a small hydrophobic amino acid can be substituted by a larger hydrophobic amino acid to fill an empty cavity inside the lipoprotein or an L-amino acid may be substituted by a D-amino acid to increase the stability towards proteases. Certain coronavirus antigen sequences that can be included in the disclosed lipoprotein are provided in Table 4 below, and variants thereof in which one, two, or three amino acids are replaced by other amino acids or are deleted. SEQ ID NOs: 63-70 are wild-type receptor binding domain peptides of the Spike protein SI subunit from SARS-CoV-2, SARS-CoV-1, HuCoV-229E, HuCoV-OC43, HuCoV-NL63, HuCoV-HKUl/BRA/22/2007, HuCoV-HKUl or MERS, respectively; SEQ ID NOs: 71-73 are wild-type receptor binding motif peptides from SARS-CoV-2, SARS-CoV-1 or MERS; SEQ ID NOs: 74-81 are wild-type Spike protein SI subunit SD1 peptides from SARS-CoV- 2, SARS-CoV-1, HuCoV-229E, HuCoV-OC43, HuCoV-NL63, HuCoV-

HKUl /BRA/22/2007, HuCoV-HKUl or MERS; SEQ ID NOs: 82-89 are wt Spike SD2 peptides from SARS-CoV-2, SARS-CoV-1, HuCoV-229E, HuCoV-OC43, HuCoV-NL63, HuCoV-HKUl/BRA/22/2007, HuCoV-HKUl or MERS; SEQ ID NOs: 90-96 are wild-type Spike protein S2 subunit FP peptides from SARS-CoV-2, SARS-CoV-1, HuCoV-229E, HuCoV-OC43, HuCoV-NL63, HuCoV-HKUl/BRA/22/2007 or MERS; SEQ ID NOs: 97- 103 are wild-type Spike protein S2 subunit HR1 peptide peptides from SARS-CoV-2, SARS- CoV-1, HuCoV-229E, HuCoV-OC43, HuCoV-NL63, HuCoV-HKUl/BRA/22/2007 or MERS; SEQ ID NOs: 104-110 are wild-type Spike protein S2 subunit HR2 peptide peptides from SARS-CoV-2, SARS-CoV-1, HuCoV-229E, HuCoV-OC43, HuCoV-NL63, HuCoV- HKUl /BRA/22/2007, HuCoV-HKUl or MERS; SEQ ID NOs: 111-160 are modified Spike protein SI or S2 subunit peptides from SARS-CoV-2, SARS-CoV-1, HuCoV-229E, HuCoV- OC43, HuCoV-NL63, HuCoV-HKUl /BRA/22/2007, HuCoV-HKUl or MERS.

[0090] Table 4. Coronavirus Antigens

[0091] In certain embodiments, the coronavirus antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96-193.

[0092] In certain embodiments, the coronavirus antigen comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 96-193.

[0093] In certain embodiments, the coronavirus antigen consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 96-193.

[0094] In certain embodiments, the lipoprotein comprises a first coronavirus antigen and a second coronavirus antigen, wherein the first and second coronavirus antigens are connected via a linker, e.g., a peptide linker as disclosed herein. In certain embodiments, the N-terminus of the first coronavirus antigen is connected to the C-terminus of the second coronavirus antigen. In certain embodiments, the C-terminus of the first coronavirus antigen is connected to the N-terminus of the second coronavirus antigen.

III. Linkers

[0095] The components of the lipoprotein can be linked via various linkers. The linkers can include linkers having the structure LI to L16 below and/or short peptides of 2 to 20 amino acids (as discussed below). The amphipathic domain (AD) can be covalently linked to the lipid moiety (LM) at or near one terminus, i.e., the N-terminus or the C-terminus. In certain embodiments, the amphipathic domain is linked to the lipid moiety via the N-terminus of the amphipathic domain. In certain embodiments, the lipid moiety is directly linked to the amphipathic domain to form the structure: LM-AD. In certain embodiments, the lipid moiety is indirectly linked to the amphipathic domain via a linker to form the structure: LM-L-AD. Additionally, the coronavirus antigen can be linked to the lipopeptide building block (BB), for example, to the coiled-coil domain or to the T-helper epitope, via a linker, to form the structure BB-L-A.

[0096] If the components of the lipoprotein, for example, the amphipathic domain and the lipid moiety, or the coronavirus antigen and the BB, are directly linked, this can be accomplished, for example, through an amide bond between a lipid moiety carbonyl function and an amino function, e.g. the N-terminal amino function, of amphipathic domain. In certain embodiments, certain lipid moieties, e.g., LM1, LM2, and LM8 (as defined below), are connected through an amide bond between their amine function and a carboxy function, e.g., the C-terminal carboxy function, of the amphipathic domain. [0097] A person of ordinary skill in the art would know that a wide variety of suitable linkers and coupling strategies exist, including, but not limited to, linkers based on dicarboxylic acid derivatives, linkers containing one or multiple ethylene glycol units, amino acid residues (including a-, P-, y-, 6-amino acids), or sugar (carbohydrate) units, or containing heterocyclic rings. In certain embodiments, the linker can have the structure of LI to L16 herein, wherein n is between 1 and 45 and m is between 1 and 45, for example wherein n is between 1 and 20 and m is between 1 and 20, shown with the connecting functional group C=0 and/or X wherein X is O or NH:

[0098] In certain embodiments, linkers LI to L16 may be connected to the lipid moiety and amphipathic domain as follows.

[0099] In certain embodiments, a carbonyl function shown for LI to L16 may be connected to an amino function of a suitable lipid moiety and/or an amino function, e.g. the N-terminal amino function, of the amphipathic domain through an amide bond. In certain embodiments, a carbonyl function shown for LI to LI 6 may be connected to a lipid moiety by replacement of the corresponding carbonyl function in particular lipid moieties LM3 to LM7.

[00100] In certain embodiments, a functional group X shown for LI to LI 6 (with the meaning -NH or -O) may be connected to a carbonyl function of a suitable lipid moiety and/or a carboxy function, e.g. the C-terminal carboxy function, of the amphipathic domain through an amide bond (for X = NH) or through an ester bond (for X = O).

[00101] In certain embodiments, the terminal -CH2 group of L8 may be connected to an amino function of a suitable lipid moiety, an amino function, e.g. the N-terminal amino function, of the amphipathic domain, or a carbonyl function of a suitable lipid moiety. One of ordinary skill in the art would appreciate that the linker can be connected via a terminus of the amphipathic domain, or through a side chain residue of the amphipathic domain, for example, through such as a terminal or internal aspartic acid, glutamic acid, lysine, ornithine or cysteine side chain.

[00102] The coronavirus antigen can be conjugated directly or through a linker, either at the N- or C-terminus of the coronavirus antigen, and is connected either to the N- or to the C- terminal of the lipopeptide building block, or optionally to an amino acid side chain. Alternatively the coronavirus antigen can be conjugated to the lipopeptide building block through a side chain residue of the coronavirus antigen, such as a terminal or internal aspartic acid, glutamic acid, lysine, ornithine or cysteine side chain.

[00103] Suitable linkers for linking the coronavirus antigen to the lipopeptide building block include short peptides of 2 to 20 amino acids, hydroxyalkyl- or aminoalkyl-carboxylic acids, substituted or unsubstituted polyalkylenoxy glycols, e.g., containing one to twelve C2 and/or C3 alkylenoxy units, polyalkylenoxy glycol block co-polymers (e.g. pluronics), mono-, di-, tri- and oligosaccharides, which may comprise acetyl, glycerol-phosphate or other substituents at one or more positions, polysaccharides such as poly(sialylic acid) and derivatives (e.g. peptide conjugates) thereof, proteinogenic or non-proteinogenic amino acids, and Cl -C8 saturated or unsaturated hydrocarbons, and may comprise one or more of the following functional groups: a disulphide bond, amine, amide, acetal, ester, ether, thioether, hydrazone, hydrazide, imine, oxime, urea, thiourea, carbonate, iminocarbonate, amidine, amide, imide, an alkyl sue- cinimide, which may also be hydrolyzed to an amide, sulphonamide, sulfone, or a heterocyclic ring comprising one or more atoms selected from nitrogen and oxygen, for example a triazole, and combinations thereof. Any method used for conjugating peptides or other antigens to an antigen delivery system such as carrier protein, polymer, dendrimer, nanoparticle or virus-like particle, can be used to conjugate the coronavirus antigen to the lipopeptide building block component. Table 5 sets forth exemplary peptide linker sequences. In certain embodiments, the linker can have the sequence according to one of SEQ ID NOs: 194 to 230 herein, wherein z is any amino acid n is between 1 and 4 and m is between 1 and 4 and o is between 1 and 4 and x is between 1 and 20, and PEG is NH(CH2CH ₂ O)xCH2CH ₂ CO, with x = 1-20. Table 5. Exemplary Peptide Linkers. [00104] In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 194-232.

[00105] In certain embodiments, the peptide linker comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs 194-232.

[00106] In certain embodiments, the peptide linker consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 194-232.

IV. Lipoprotein Constructs

[00107] In certain embodiments, the lipoprotein can have the structure set forth in Table 6, wherein Xi is selected from Leu, Vai, and He, X ₂ is selected from Ala, Ser, Thr, Vai, and He, and X3 is selected from Gly, Ala, and Ser, and PEG is NH(CH ₂ CH ₂ O)xCH ₂ CE[ ₂ CO, with n = 1-20.

[00108] Table 6. Exemplary Lipoprotein Constructs

[00109] In certain embodiments, the lipoprotein comprises a structure selected from the group consisting of Constructs 1-573 as set forth in Table 6. In certain embodiments, the lipoprotein consists of a structure selected from the group consisting of Constructs 1-573 as set forth in Table 6. In certain embodiments, the lipoprotein comprises a structure as shown in Figures 1, 2, 3, 4, or 5. In certain embodiments, the lipoprotein comprises a structure as shown in Figure 1. In certain embodiments, the lipoprotein comprises a structure as shown in Figure 2. In certain embodiments, the lipoprotein comprises a structure as shown in Figure 3. In certain embodiments, the lipoprotein comprises a structure as shown in Figure 4. In certain embodiments, the lipoprotein comprises a structure as shown in Figure 5.

[00110] In certain embodiments, the lipoprotein constructs can comprise a purification tag coupled to the coronavirus antigen, for example, a hexa-His tag. In certain embodiments, the purification tag is directly coupled to the lipoprotein construct. In certain embodiments, the purification tag is indirectly coupled to the lipoprotein construct via a linker. In certain embodiments, the purification tag is removable by treatment with a protease. In certain embodiments, the purification tag comprises one of the amino acid sequences set forth in Table 7.

[00111] Table 7. Purification Tag Sequences

[00112] In certain embodiments, the purification tag comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 233-242.

[00113] In certain embodiments, the purification tag comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 231-240.

[00114] In certain embodiments, the purification tag consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 233-242.

V. Immunogenic Pharmaceutical Compositions

[00115] In certain embodiments, the lipoprotein is an immunogenic composition. In certain embodiments, the lipoprotein is a vaccine, for example, a coronavirus vaccine. In certain embodiments, the immunogenic composition is a vaccine. In certain embodiments, the immunogenic composition comprises the lipoprotein disclosed herein, either alone or in combination with a pharmaceutically acceptable carrier. In certain embodiments, the immunogenic composition comprises one or more adjuvants selected from, for example, a mineral salt (e.g. aluminium hydroxide, aluminium phosphate, aluminium sulfate, calcium phosphate), monophosphoryl lipid A (MPL), plant extracts containing saponins (e.g. QS-21), imidazo-quinolines (e.g. Imiquimod), muramyl dipeptides and tripeptides, lipopeptides, oilin- water emulsions (e.g. Montanide ISA 720), cytokines (e.g. IL-2 or GM-CSF), mycobacterial and bacterial derivatives (e.g. Freund's complete adjuvant), BCG, nucleic acid derivatives (e.g. polylC) and other adjuvants known to those skilled in the art. In certain embodiments, the adjuvant is selected from the group consisting of aluminum, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), monophosphoryl lipid A (MPL), squalene, cytosine phosphoguanine, QS-21, microcrystalline tyrosine, MF59, mannide monooleate, GLA, SLA, E6020, resiquimod (R848), imidazoquinolines, imiquimod, 3M-052, 2',3'-cGAMP, 3', 3'- cGAMP, cGMP, cAMP, AMP, muramyl dipeptide, poly I:C, CpG and combinations thereof. [00116] In certain embodiments, the immunogenic composition is encapsulated in or attached to bio-degradable polymers, which may for example be useful for controlled release, for example polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, poly orthoesters, poly acetals, poly dihydropyrans, poly cyanoacrylates, and crosslinked or amphipathic block copolymers of hydrogels, or may be formulated in liposomes.

[00117] The immunogenic composition can be prepared according to conventional means known to a person of ordinary skill in the art, such as, for example, dissolving and lyophilizing processes and/or may comprise excipients, for example preservatives, stabilizers, wetting agents, tonicity adjusting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffering substances to stabilize the pH. including but not limited to sodium phosphate, potassium phosphate, sodium chloride, polysorbate 80, mannitol, sucrose, trehalose, amino acids such as glycine, histidine or the monosodium salt of glutamic acid and proteins such as human serum albumin.

[00118] In certain embodiments, the immunogenic composition comprises from about 0.05% to about 99%, from about 0.05% to about 75%, from about 0.05% to about 50% , from about 0.05% to about 25%, from about 0.05% to about 10%, from about 0.1% to about 50%, from about 0.1% to about 25%, from about 0.1% to about 10%, from about 1% to about 90%, from about 1% to about 75%, from about 1% to about 50%, from about 1% to about 10%, from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, or from about 10% to about 25% of the active ingredient, i.e., the lipoprotein.

[00119] As would be appreciated by a person of ordinary skill in the art, the dosage of the active ingredient depends upon the intended recipient (e.g. species), its age, weight, and indi vidual condition, and the administration route. An optimal dosage for a particular active ingredient and a particular target population can be determined by standard studies involving observation of appropriate immune responses in subjects.

[00120] In certain embodiments, the lipoprotein or immunogenic composition provides protective immunity to a subject against a coronavirus. In certain embodiments, the lipoprotein or immunogenic composition decreases the severity of a coronavirus infection. In certain embodiments, the lipoprotein or immunogenic composition provides protective immunity to a subject against the coronavirus for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years, about 3 years, about 4 years, about 5 years, or about 10 years after administration to the subject. In certain embodiments, the lipoprotein or immunogenic composition induces the production of antibodies against at least a portion of a coronavirus spike protein.

[00121] In certain embodiments, a single dose of the lipoprotein or immunogenic composition induces an immune response in a subject sufficient to provide protective immunity to the subject against the coronavirus. In certain embodiments, a two doses of the lipoprotein or immunogenic composition induces an immune response in a subject sufficient to provide protective immunity to the subject against the coronavirus. In certain embodiments, the lipoprotein or immunogenic composition does not induce antibodydependent enhancement.

[00122] In certain embodiments, the lipoprotein or immunogenic composition has an efficacy rate of greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, or greater than about 90%. In certain embodiments, the lipoprotein or immunogenic composition has an reactogenicity rate of less than about 20%, less than about 15%, less than about 10%, less than about 8%, less than about 5%, less than about 3%, less than about 2%, or less than about 1%.

VI. Methods

[00123] In certain embodiments, contemplated herein is a method of immunizing a subject against a coronavirus comprising administering to the subject the lipoprotein or immunogenic composition disclosed herein. In certain embodiments, provided herein is a method of of eliciting an immune response in a subject comprising administering to the subject an immunogenically effective amount of a lipoprotein comprising a coronavirus antigen described herein.

[00124] In certain embodiments, provided herein is a method of preventing or reducing risk of a coronavirus infection in a subject comprising administering to the subject the lipopeptide or immunogenic composition disclosed herein. In certain embodiments, provided herein is a method of treating a coronavirus infection in a subject comprising administering to the subject the lipopeptide or immunogenic composition disclosed herein. In certain embodiments, provided herein is a method of reducing symptoms of a coronavirus infection in a subject comprising administering to the subject the lipopeptide or immunogenic composition disclosed herein.

[00125] In certain embodiments, the immunogenic composition is administered by any enteral or parenteral routes, for example, by intranasal, oral, sublingual, intramuscular, intradermal, transdermal, subcutaneous, or transcutaneous routes.

[00126] In certain embodiments, the lipoprotein or immunogenic composition is administered as a single dose. In certain embodiments, the lipoprotein or immunogenic composition is administered as two or more doses. In certain embodiments, the lipoprotein or immunogenic composition disclosed herein is administered as a booster vaccine, to boost preexisting immune responses in prior patients who have been previously infected with a coronavirus, or alternatively in heterologous prime-boost regimens with other coronavirus vaccines.

A. Heterologous Prime-Boost Regimens

[00127] In certain embodiments, the lipoprotein or immunogenic composition disclosed herein is administered in a heterologous prime-boost regimen in which a subject is administered a prime immunogenic composition and then later administered a booster immunogenic composition that is different from the first immunogenic composition. In certain embodiments, any aspect or component to the booster immunogenic composition can be different from the prime immunogenic composition, including, but not limited to, an antigen, adjuvant, excipient, lipopeptide, amphipathic domain, T-helper epitope, and the like. In certain embodiments, the booster immunogenic composition comprises an antigen that is heterologous to the prime immunogenic composition. In certain embodiments, the booster immunogenic composition comprises an adjuvant that is heterologous to the prime immunogenic composition. In certain embodiments, the booster immunogenic composition comprises an excipient that is heterologous to the prime immunogenic composition. In certain embodiments, the prime immunogenic composition comprises the lipoprotein disclosed herein and the boost immunogenic composition comprises a heterologous antigen. In certain embodiments, the boost immunogenic composition comprises the lipoprotein disclosed herein and the prime immunogenic composition comprises a heterologous antigen. The heterologous antigen can be another coronavirus antigen, such as a recombinant SARS-CoV-2 spike protein, messenger RNA- or DNA-encoding a corona virus antigen, which may be delivered by a non-replicating viral vector, virosome, liposome or a lipid nanoparticle, a killed inactivated coronavirus such as a P-propiolactone-treated coronavirus or a live attenuated coronavirus. In certain embodiments, a subject is administered the lipoprotein or immunogenic composition disclosed herein and subsequently administered a second immunogenic composition comprising a heterologous antigen. In certain embodiments, a subject is administered a first immunogenic composition comprising an antigen which is heterologous to antigens included in the lipoprotein or immunogenic composition disclosed herein, and subsequently the subject is administered the lipoprotein or immunogenic composition disclosed herein. In certain embodiments, the prime vaccine is selected from an inactivated coronavirus, a non-replicating viral vector coronavirus vaccine, a replicating viral vector coronavirus vaccine, a messenger RNA coronavirus vaccine, a DNA plasmid coronavirus vaccine, a coronavirus protein subunit vaccine, and a VLP coronavirus vaccine and the booster vaccine is a lipoprotein provided herein. In certain embodiments, the prime is a lipoprotein provided herein and the booster is selected from an inactivated coronavirus, a non-replicating viral vector coronavirus vaccine, a replicating viral vector coronavirus vaccine, a messenger RNA coronavirus vaccine, a DNA plasmid coronavirus vaccine, a coronavirus protein subunit vaccine, and a VLP coronavirus vaccine. The “booster” vaccine can be administered any time after the “prime” vaccine is administered, including, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, 4 years, or 5 years after administration of the “prime” vaccine.

[00128] In certain embodiments, the subject is an animal. In certain embodiments, the subject is a warm-blooded mammal. In certain embodiments, the subject is a non-human primate. In certain embodiments, the subject is a human. In certain embodiments, the subject is immunocompromised. In certain embodiments, the subject is 60 years of age or older. In certain embodiments, the subject is a pediatric subject. In certain embodiments, the subject is under the age of 10. In certain embodiments, the subject is under the age of 6.

[00129] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present application. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. [00130] The following examples further illustrate aspects of the present application.

However, they are in no way a limitation of the teachings or disclosure of the present application as set forth herein.

EXAMPLE

[00131] Example 1: Production of Lipoproteins

[00132] The lipoproteins disclosed herein are produced, depending on the composition, either by chemical synthesis using Fmoc solid phase peptide synthesis (SPPS) or by recombinant protein expression.

[00133] Chemical Synthesis. The peptide chain is assembled stepwise on a solid support using Fmoc protected amino acids and standard solid phase peptide synthesis (SPPS) methods. Following assembly of the peptide and removal of the N-terminal protecting groups, the lipid is coupled to the N-terminus using standard solid phase peptide synthesis (SPPS) methods. Following assembly, the lipoprotein is cleaved and the sidechain protecting groups are removed by treatment with acid (TFA). The crude lipopeptide is then purified by high performance liquid chromatography (HPLC) using a reversed phase column. Disulfide bonds are formed either prior to cleavage by on-resin oxidation or in solution using suitable oxidizing agents (Chan, Weng C., and Peter D. White. 2000. Fmoc solid phase peptide synthesis: a practical approach. New York: Oxford University Press.). For storage the lipoprotein is lyophilized or dissolved in a suitable storage buffer. The purity and identitiy are determined by HPLC and mass spectrometry (MS).

[00134] Expression: A signal peptide sequence containing a charged N-terminal region, a hydrophobic region and a C-terminal lipobox (consensus sequence X1X2X3C, where Xi is selected from Leu, Vai, He, X2 is selected from Ala, Ser, Thr, Vai, He, and X3 is selected from Ala, Ser, Gly) is fused to the N-terminus of the target lipoprotein. The resulting amino acid sequence is converted into a DNA sequence with codon optimization for expression in E. coli (or another suitable host). The resulting DNA sequence is synthesized chemically, cloned into an expression vector, transformed into host cells, and transformed cells are grown in LB-, TB- or another suitable medium. Protein expression is induced by inoculation with isopropylthio-B-D-galactoside (IPTG) (or another suitable reagent). Cells are harvested, disrupted, pelleted and the lipoproteins are extracted using a buffer containing 1% Triton-X- 100 (or another suitable detergent). The crude lipoproteins are purified by chromatography using immobilized metal ion affinity chromatography (IMAC), ion exchange (IEX), size exclusion (SEC) and/or reversed phase HPLC columns. The lipoprotein is folded in a refolding buffer. For storage the lipoprotein is lyophilized or dialyzed into in a storage buffer. Purity and identitiy are determined by SDS-PAGE, Western Blot or HPLC and MS.

[00135] Example 2: Immunization Studies

[00136] The lipoproteins disclosed herein are tested for immunogenicity. Test subjects are immunized one or more times with the lipoproteins disclosed herein with or without coadministration of an exogenous adjuvant. Blood sera are analyzed by ELISA to determine titers of antibodies binding to the antigens disclosed herein, competition assay to determine antibodies inhibiting SARS-CoV-2 S binding to ACE-2 or SARS-CoV-2 neutralization assays to determine neutralizing antibodies.

[00137] SARS-CoV-2 Antigen Antibody Assay. Blood is collected before the first immunization and after each immunization, and the sera are analyzed by ELISA for antibodies binding to the SARS-CoV-2 antigens disclosed herein. All immunized subjects exhibit high titers of target antigen-specific (e.g. SARS-CoV-2 antigen) antibodies compared to unimmunized control subjects.

[00138] SARS-CoV-2 Competition Assay: Blood is collected before the first immunization and after the final immunization, and the sera are analyzed by competition ELISA or SPR for antibodies reducing binding of a recombinant SARS-CoV-2 S protein fragments to ACE-2. All immunized subjects exhibit antibodies that reduce binding of the recombinant SARS-CoV-2 S protein fragment compared to unimmunized control subjects.

[00139] SARS-CoV-2 Pseudovirus Assay. Blood is collected before the first immunization and after the final immunization, and the sera are analyzed by SARS-CoV-2 spike pseudotyped lentivirus neutralization assay for neutralizing antibodies. All immunized subjects exhibit high neutralization titers compared to unimmunized control subjects.

[00140] SARS-CoV-2 Neutralization Assay. Blood is collected before the first immunization and after the final immunization, and the sera are analyzed by SARS-CoV-2 neutralizing antibody assay for neutralizing antibodies against SARS-CoV-2. All immunized subjects exhibit high neutralization titers compared to unimmunized control subjects.

[00141] SARS-CoV-2 Challenge and Viral Titrations. Immunized test subjects are exposed to an infectious dose of SARS-CoV-2. The lungs are analyzed for virus by virus titration or RT-PCR and pulmonary inflammation by histopathology or RT-PCR (based on several parameters of lung inflammation that are converted to a pathology score). All immunized subjects exhibit high low lung virus titers, and low lung pathology score compared to SARS-CoV-2 exposed control subjects. [00142] Example 3: Safety and Efficacy Studies

[00143] Subjects are immunized with any of the lipoproteins disclosed herein, including constructs comprising SARS-CoV-2 antigens.

[00144] SARS-CoV-2 Reinfection Rates. Immunized subjects who are naive to SARS- CoV-2 infection show lower infection rates as compared to control subjects. Immunized subjects who have previously been infected with SARS-CoV-2 show lower reinfection rates as compared to control subjects.

[00145] SARS-CoV-2 Safety and Efficacy. Immunized subjects show acceptable safety and efficacy against laboratory confirmed SARS-CoV-2 infection or reinfection.

[00146] Mild COVID-19 Efficacy and Reactogenicity Rates. Immunized subjects show efficacy rates of more than 20% against laboratory confirmed SARS-CoV-2 infection or reinfection with at least one of the following symptoms: difficulty of breathing, muscle or body aches, headache, loss of taste, loss of smell, sore throat, congestion, runny nose, nausea, vomiting, diarrhea. Immunized subjects show reactogenicity rates of less than 20%.

[00147] Severe COVID-19 Efficacy and Reactogenicity Rates. Immunized subjects show efficacy rates of more than 20% against laboratory confirmed severe SARS-CoV-2 infection or reinfection with any of the following: admission to the intensive care unit, respiratory failure, clinical signs of severe systemic illness, shock or death. Immunized subjects show reactogenicity rates of less than 20%.

[00148] Safety and Efficacy in Elderly, Immunocompromised, and Pediatric Populations. Subjects who are 60 years of age and older, pediatric subjects, and immunocompromised subjects are immunized. Immunized subjects show acceptable safety profiles and efficacy rates, are immunized with the lipoproteins disclosed herein, including Lipoproteins comprising SARS-CoV-2 antigens.

[00149] Example 4: Lipoprotein Constructs

[00150] Lipoprotein Construct 1

{Pam2Cys}GGEIAALKQEIAALKKENAALKFEIAALKQGPKYVKQNTLKLATKKKG G GGSTEIYQAGSTPCNGVEGFNCYFPLQSWTG{D-Ala}-NH2 (1) Lipoprotein construct 1 comprises the lipid Cys((R)-2,3-di(palmitoyloxy)-2-propyl) (Pam2Cys), the artificially designed coiled-coil: lAALKQEIAALKKENAALKFEIAAL (SEQ-ID No: 5), the SARS- CoV-2 RBD antigen STEIYQAGSTPCNGVEGFNCYFPLQSWTG{D-Ala} (SEQ-ID No: 150) and a C-terminal carboxamide. Lipoprotein construct 1 was synthesized by SPPS on ChemMatrix Rink amide resin (loading 0.4 mmol/g) loading 0.4 mmol/g), using DIC/C1- HOBt for activation and 20% piperidine in DMF for Fmoc removal. After each coupling step, any remaining free amino groups were capped with acetic anhydride. {Pam2Cys} was coupled to the N-terminus using Fmoc-Cys((R)-2,3-di(palmitoyloxy)propyl-OH. The two other cysteines were coupled using Fmoc-Cys(Acm)-OH. The disulfide bond was obtained by on-resin oxidation for 1 hour using 15 eq. of iodine in DMF followed by washing with 1 M ascorbate-DMF (lx), DMF (4x) and CH2CI2 (2x). For cleavage the resin was treated with TFA/TIPS/water 95:2.5:2.5 for 3 hours. The resin was filtered, the filtrate was precipitated and washed with cold ethyl ether, dried in vacuo, and the lipoprotein 1 was purified by reverse phase HPLC on a C4 column. Analytical HPLC (4.6x250 mm KLM PS/DVB column, 15-55% acetonitrile/0.1% TFA in water/0.1% TFA in 25 min.): tR = 14.15 min. Purity: 92.2%. ESI-MS: MW calc, for C42H684N104O118S3: 9186.95; MW found = 9187.6 Da (± 0.01%). The induction of SARS-CoV-2 inhibiting antibodies was tested by immunizing three New Zealand White rabbits by intramuscular injection with 150pg lipoprotein 1 in 0.2 ml PBS without adjuvant on days 0 and 21. As control, three New Zealand White rabbits were immunized with SARS-CoV-2 S RBM (residues 438-506, SEQ ID NO: 104) (LifeTein, Inc. NJ) in Imject alum adjuvant (Thermo Fisher Scientific Inc., MA). Blood sera collected on days 0 and 42 were analyzed for SARS-CoV-2 inhibiting antibodies using a commercial SARS-CoV-2 RBD/ACE-2 blocking ELISA kit (Cat. No L00847, GenScript USA, Inc., NJ). Inhibition rates were determined and calculated according to the manufacturer's protocol. Day 42 sera showed inhibition rates of 89%, 60% and 62%. Sera from confirmed COVID-19 patients showed inhibition rates of > 20%. No inhibition could be detected in the sera from animals immunized with SARS-CoV-2 S RBM or pre-immune sera, indicating that immunization with lipoprotein construct 1 elicits high titers of SARS-CoV-2 inhibiting antibodies.