Multi-epitope construct

Abstract

The present invention is positioned in the field of vaccination therapy. More specifically, the present invention relates to a multi-epitope construct comprising a nucleic acid sequence encoding a peptide derived from a coronavirus or a functional variant and fragment thereof. The present invention further relates to a combination, polypeptide, or pharmaceutical composition used for the treatment or prevention of coronavirus, particularly SARS-CoV-2 virus, in a subject.

Description

Technical Field

[0001] The present invention is positioned in the field of vaccination therapy. More specifically, the present invention relates to a multi-epitope construct comprising a nucleic acid sequence encoding a peptide derived from a coronavirus or a functional variant and fragment thereof. The present invention further relates to a combination, polypeptide, or pharmaceutical composition used for the treatment or prevention of coronavirus, particularly SARS-CoV-2 virus, in a subject.

Background Art

[0002] The occurrence of the COVID-19 pandemic is a threat to human populations worldwide due to the high infectivity of this infectious disease, which causes severe illness and large-scale deaths. To date, the global health burden and economy remain in a very critical situation. Currently, there are three main types of COVID-19 vaccines (mRNA vaccines, viral vector vaccines, and protein subunit vaccines), among which mRNA vaccines and vector vaccines have been successfully used in the global vaccination strategy. mRNA vaccines (Moderna; Patent Document 1 and Pfizer-BioNTech; Patent Document 2) use genetic information derived from SARS-CoV-2, the virus that causes COVID-19, to instruct the cells of the vaccinated person on how to produce a harmless protein specific to the virus. Viral vector vaccines (Johnson, AstraZeneca, Sputnik V, Convidecia) are those in which genetic information derived from SARS-CoV-2 is incorporated into a modified version of another virus. Protein subunit vaccines (Novavax) contain a harmless part (protein), not the whole SARS-CoV-2 virus.

[0003] mRNA vaccines are a promising alternative to conventional vaccination methods because they are easy to design, allow for rapid development, are expected to be manufactured at low cost, are safe to administer, induce both cellular and humoral immunity, and do not interact with genomic DNA. Recent improvements in mRNA vaccines act to increase translation into protein, modulate innate and acquired immunogenicity, and improve delivery. Some messenger RNA vaccines, such as the Pfizer-BioNTech COVID-19 vaccine, have the disadvantage of requiring storage at extremely low temperatures until supply, while other mRNA vaccines do not have such requirements.

[0004] SARS-CoV-2 is a member of the Betacoronavirus genus that causes pneumonia. SARS-CoV-2 is an enveloped virus with single-stranded RNA, belonging to the Coronaviridae family, and has the potential to cause infectious diseases in mammals, birds, and humans. The complete genome sequence of SARS-CoV-2 has been determined (Non-Patent Document 1) and is approximately 29.9 kb. The availability of the genome has created an opportunity to develop vaccines against this disease that causes such great harm. Regarding COVID-19 vaccines, all of the mRNA vaccines approved so far have used spike protein-based approaches (Moderna; Patent Document 1 and Pfizer-BioNTech; Patent Document 2). Spike protein-based vaccines block the binding of SARS-CoV-2 to host cell receptors and thus induce antibodies that neutralize viral infection, thereby providing highly effective protective immunity. A concerning feature of the spike protein of SARS-CoV-2 is how it changes over time as the virus evolves. Similar to many viruses, the viral genome of SARS-CoV-2 can mutate, and numerous such new variants have been described during the SARS-CoV-2 pandemic. These mutations change the biochemical properties of the spike protein, and thus there is a possibility that the neutralizing ability of neutralizing antibodies generated against previous variants is avoided or evaded as the virus evolves. Therefore, this creates an opportunity to explore improved vaccine designs that escape or reduce the impact of "variants of concern" (VOC).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Therefore, an object of the present invention is to design a vaccine based on a multi-epitope containing T cell epitopes based on structural proteins and non-structural proteins other than the spike protein derived from coronavirus, and to provide broad protective and persistent immunity (pan-coronavirus vaccine) against a large number of coronavirus variants and even strains. Optionally, this multi-epitope vaccine can be combined with one or more constructs encoding full-length spike variants that provide a neutralizing antibody response similar to that of the established COVID-19 vaccines described above.

Means for Solving the Problems

[0008] In a first aspect, the present invention relates to a multi-epitope construct comprising at least five nucleic acid sequences encoding a coronavirus-derived peptide or a functional variant and / or fragment thereof, wherein the peptide, variant and / or fragment thereof comprises an amino acid sequence having at least 95% sequence identity to a sequence selected from the list comprising SEQ ID NOs: 1 to 47.

[0009] In the following embodiments of the present invention, the nucleic acid sequence is optimized by codon optimization.

[0010] In certain embodiments, the present invention provides a multi-epitope construct as defined herein that further comprises one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant and / or fragment thereof, particularly the SARS-CoV-2 spike glycoprotein.

[0011] In certain embodiments, the present invention provides a combination comprising the above multi-epitope construct and a construct comprising one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant and fragment thereof, particularly the SARS-CoV-2 spike glycoprotein.

[0012] In yet another embodiment, the present invention provides the above multi-epitope construct, or the above combination, wherein the encoded peptide or a functional variant and fragment thereof is separated by at least one specific molecular linker selected from the list comprising a flexible linker, a rigid linker, and / or a cleavable linker.

[0013] In a further aspect, the present invention provides a multi-epitope construct of the present invention comprising at least five nucleic acid sequences, particularly RNA molecules, having at least 95% sequence identity to the sequences shown in SEQ ID NOs: 48 to 94.

[0014] In a further aspect, the present invention provides that the at least five nucleic acid sequences are from the following list: a. SEQ ID NO: 55, 89, 57, 53, 49, 92, 70, 71, 48, 74, 79, 77, 87, 65, 59, 88, 72, 60, 81; b. SEQ ID NO: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88, 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70; c. 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88; d. SEQ ID NO: 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70; e. SEQ ID NO: 60, 63, 54, 49, 84, 89, 71, 57, 65, 91, 48, 56, 66, 80, 79, 67, 78, 75, 59, 82; f. SEQ ID NO: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61, 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56; g. Any one of SEQ ID NOs: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61; Provided is a multi-epitope construct as defined herein, selected from any one of the foregoing.

[0015] In yet another aspect, the present invention provides a polypeptide encoded by the multi-epitope construct.

[0016] In certain embodiments, the present invention provides a pharmaceutical composition comprising the multi-epitope construct, the combination, or the polypeptide, and at least one pharmaceutically acceptable agent.

[0017] In another embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide, or the pharmaceutical composition formulated as liposomes or nanoparticles, such as lipid nanoparticles or polymer nanoparticles, particularly lipid nanoparticles.

[0018] In a further aspect, the present invention provides the multi-epitope construct, the combination, the polypeptide, or the pharmaceutical composition for use in a human or veterinary medicament.

[0019] In a further embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide, or the pharmaceutical composition for use in vaccination, particularly intramuscular vaccination.

[0020] In certain embodiments, the present invention provides the multi-epitope construct, the combination, the polypeptide, or the pharmaceutical composition for use in inducing an immune response against a coronavirus, particularly the SARS-CoV-2 virus, in a subject.

[0021] In yet another specific embodiment, the present invention provides the above multi-epitope construct, the above combination, the above polypeptide or the above pharmaceutical composition for use in treating or preventing coronavirus in a subject, particularly the SARS-CoV-2 virus.

[0022] In a further aspect, the present invention relates to a method of inducing an immune response against coronavirus, the method comprising administering to a subject a therapeutically effective amount of the above multi-epitope construct, the above combination, the above polypeptide or the above pharmaceutical composition.

[0023] Turning now to the drawings in particular, it is emphasized that the details shown are for the purpose of example only, and are an illustrative description of various embodiments of the present invention. These drawings are presented in order to provide the most useful and readily understood illustration of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention. This description will make apparent to those skilled in the art how some forms of the present invention may be actually embodied in conjunction with the drawings.

Brief Description of the Drawings

[0024]

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Mode for Carrying Out the Invention

[0025] The present invention will be further described below. In the following sections, various aspects of the present invention are defined in more detail. Each aspect so defined can, conversely, be combined with any single or plural aspects unless clearly indicated otherwise. In particular, any feature shown as preferred or advantageous can be combined with any other single or plural features shown as preferred or advantageous.

[0026] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or two or more compounds.

[0027] The terms "comprising", "comprises", and "comprised of", as used herein, are synonymous with "including", "includes", or "containing", "contains", and these terms are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. These terms also encompass "consisting of" and "consisting essentially of".

[0028] As used herein, the term "about" or "approximately", when referring to a measurable value such as a parameter, amount, duration, etc., means including variations of up to + / - 10%, preferably up to + / - 5%, more preferably up to + / - 1%, and still more preferably up to + / - 0.1% of the specified value, as long as such variations are appropriate for practicing the invention as disclosed. It is to be understood that the value modified by the term "about" or "approximately" is itself also specifically and preferably disclosed.

[0029] In the search for novel coronavirus vaccines, it was investigated whether nucleic acid vaccines based on the expression of proteins other than the spike glycoprotein derived from coronaviruses could induce broad, protective, and persistent immunity against multiple coronavirus strains. By using bioinformatics tools, a series of conserved peptide windows were selected for further epitope prediction. A series of conserved peptide windows were unexpectedly found to be potential antigenic determinants that elicit the production of specific T cells in the host and thus a strong antiviral immune response. As a result, the inventors designed a multi-epitope construct comprising nucleic acid sequences encoding at least five peptide windows or functional variants and fragments thereof derived from coronaviruses, which are used for the treatment or prevention of coronavirus infections, particularly SARS-CoV-2 virus infections, in a subject. The major advantage associated with multi-epitope vaccines is that they induce a broad immune response against different viral proteins, thereby reducing the risk of immune escape during viral evolution.

[0030] In a first aspect, the invention relates to a multi-epitope construct comprising at least five nucleic acid sequences encoding a peptide or a functional variant and / or fragment thereof derived from a coronavirus, wherein the peptide, variant, and / or fragment thereof comprises an amino acid sequence having at least 95% sequence identity to a sequence selected from the list comprising SEQ ID NOs: 1 to 47.

[0031] In the context of the present invention, the term "construct" refers to an artificially designed segment of nucleic acid that can be used to incorporate genetic material into a target tissue or cell. As used herein, a multi-epitope construct is delivered to the cytoplasm of a host cell as the transcript of interest, where it is expressed to yield a translated protein (s) that is located within the membrane, secreted, or located intracellularly. It should be understood that the translated protein (s) can be one or more immunogens derived from a coronavirus.

[0032] In some embodiments, the constructs defined in the present invention may be multi-epitope DNA constructs or multi-epitope RNA constructs, particularly multi-epitope messenger RNA (mRNA) constructs.

[0033] In the context of the present invention, the term "RNA" relates to a molecule containing ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. Specifically, this term refers to single-stranded RNA, but may also refer to double-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can include, for example, the addition of non-nucleotide material at the end(s) or within the RNA, e.g., at one or more nucleotides of the RNA. Nucleotides within an RNA molecule can also include non-standard nucleotides, e.g., nucleotides that do not occur naturally or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA.

[0034] According to the present invention, the term "RNA" includes "mRNA" which means "messenger RNA", preferably related thereto, can be produced using DNA as a template, and relates to a "transcript" encoding a peptide or protein. mRNA typically includes a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). RNA further includes a 3' poly(A) tail and / or a 5' cap analog. The half-life of mRNA in cells and in vitro is limited. Unless otherwise stated, in a representative RNA sequence with respect to the nucleic acid sequences described in this application, it may be described as "U", but when the sequence represents DNA, it will be understood by those skilled in the art that "U" is replaced by "T". Therefore, any RNA sequence disclosed herein and identified by a specific sequence identification number herein is also intended to disclose the corresponding DNA sequence complementary to the RNA, in which each "U" of the RNA sequence is replaced by "T".

[0035] For clarity, an mRNA molecule encompasses any coding RNA molecule that can be translated into a protein by a eukaryotic host.

[0036] In some embodiments, the RNA may be non-replicating RNA (NRM), which is also referred to as non-proliferating RNA. Non-proliferating mRNA has only one open reading frame encoding the antigen protein of interest. The total amount of mRNA used in the cell is equal to the amount of mRNA delivered by the vaccine, and thus the dosage strength is limited to the amount of RNA delivered.

[0037] In some embodiments, the RNA may be self-amplifying RNA (SAM). SAM has two open reading frames. The first open reading frame encodes the antigen protein of interest, similar to conventional mRNA. The second open reading frame encodes an RNA-dependent RNA polymerase (and its helper proteins) that self-replicates the mRNA construct in the cell and creates multiple self-copies.

[0038] In some embodiments, the RNA may be linear or circular RNA, preferably linear, more preferably linear non-proliferative RNA.

[0039] The term "modified mRNA molecule" means an mRNA molecule containing one or more modified nucleosides (referred to as "modified nucleic acids"), and the modified mRNA molecule has useful properties such as not substantially inducing the innate immune response of the cells into which the mRNA is introduced. These modified nucleic acids enhance the efficiency of protein production, the intracellular retention of nucleic acids, and the survival rate of the contacted cells, and have reduced immunogenicity.

[0040] In some embodiments, the modified nucleobases in the nucleic acid (e.g., RNA nucleic acid such as mRNA nucleic acid) include 1-methyl-pseudouridine (m1y), 1-ethyl-pseudouridine (e1y), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, the modified nucleobases in the nucleic acid (e.g., RNA nucleic acid such as mRNA nucleic acid) include 5-methoxymethyluridine, 5-methylthiouidine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, the polynucleotide includes at least two (e.g., two, three, four or more) combinations of any of the above modified nucleobases, including but not limited to chemical modifications.

[0041] mRNA is preferably produced by in vitro transcription using a DNA template. In one embodiment of the present invention, RNA is obtained by in vitro transcription. In vitro transcription method systems are known to those skilled in the art and may include a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system containing dithiothreitol (DTT) and magnesium ions, spermidine, and a suitable RNA polymerase, such as T7 RNA polymerase. The exact conditions used for the transcription reaction depend on the amount of RNA required for a particular application. Various in vitro transcription kits are commercially available.

[0042] In another embodiment, the present invention provides a multi-epitope DNA construct comprising at least five nucleic acid sequences encoding a coronavirus-derived peptide or a functional variant and fragment thereof, wherein the peptide, variant and / or fragment thereof comprises an amino acid sequence having at least 95% sequence identity to a sequence selected from the list comprising SEQ ID NOs: 1 to 47, and each U in the RNA sequence is replaced by T in the corresponding DNA sequence.

[0043] In the context of the present invention, the term "DNA construct" refers to a type of construct that functions by injecting a genetically engineered plasmid containing a DNA sequence encoding an antigen(s), which is then taken up, transcribed, and translated by a host cell into the gene of interest for which an immune response is desired, such that the cell directly produces the antigen, thereby eliciting a protective immunological response. This approach offers several potential advantages over conventional methods, including stimulating both B-cell and T-cell responses, improved vaccine stability, the absence of any infectious agents, and relatively easy large-scale production.

[0044] As will be understood by those skilled in the art, examples of DNA constructs include, but are not limited to, bacterial plasmids, bacteriophage vectors, artificial chromosomes, or fosmids.

[0045] In the context of the present invention, the term "epitope" refers to an antigenic determinant (e.g., a polypeptide of a coronavirus) capable of inducing an immune response, particularly a cellular or humoral immune response. The immune response should be understood as the ability of the immune system to recognize antigens presented by infected host cells, cytotoxic T cells and helper T cells, or to produce antibodies against the antigen. An epitope is a small site on an antigen that interacts with a specific antigen-binding site on an antigen-binding protein, e.g., the variable region of a molecular cell receptor. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes can be linear epitopes or conformational, i.e., epitopes composed of non-linear amino acids. A single antigen can have two or more epitopes. As used herein, the term "multi-epitope" refers to a series of potentially overlapping peptides that enable the induction of a broad immune response. The series of peptides can have T cell and B cell epitopes.

[0046] The term "epitope" also refers to the site on an antigen to which T cells and / or B cells respond. As used herein, the terms "epitope" or "antigen" should be understood to include immunogenic proteins and immunogenic fragments, unless otherwise specified. "Viral antigen" refers to an antigen encoded by a virus. Examples of viral antigens include, but are not limited to, antigens of coronaviruses such as COVID-19.

[0047] The multiepitope construct of the present invention comprises nucleic acid molecules encoding various sites of the coronavirus antigen. In the context of the present invention, the term "coronavirus antigen" should be understood as the arrangement of amino acids that constitutes part of the gene product encoded by a given coronavirus. The genomic organization of the coronavirus is as follows: 5'-leader-UTR-ORF1a-ORF1b-spike (S) gene-envelope (E) gene-membrane (M) gene-nucleocapsid (N) gene-3'-UTR-poly(A) tail. ORF1a and ORF1b encode replicase polyproteins that are themselves cleaved to form 16 non-structural proteins (nsp1-nsp16). ORF1a encodes nsp1-nsp10 and ORF1b encodes nsp11-nsp16. Accessory genes are distributed among the structural genes, and the number and functions of the accessory proteins are unique for a given coronavirus (e.g., for the SARS-CoV-2 virus, the following accessory genes are present: ORF3a, ORF6, ORF7a, ORF7b, ORF8a, ORF9b, and ORF10).

[0048] In the context of the present invention, the multiepitope construct can comprise at least two, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleic acid sequences encoding coronavirus peptides or functional variants and fragments thereof. In certain embodiments, the multiepitope construct comprises at least two nucleic acid sequences encoding coronavirus peptides or fragments thereof. In preferred embodiments, the multiepitope construct comprises at least five nucleic acid sequences encoding coronavirus peptides or fragments thereof.

[0049] In one embodiment, the multi-epitope construct of the present disclosure may comprise a nucleic acid sequence encoding a coronavirus peptide or a functional variant and fragment thereof selected from the list comprising the E gene, M gene, N gene, ORF3a, ORF6, ORF7a, ORF7b, ORF8a, ORF9b, and ORF10, ORF1a and / or ORF1b viral genome windows.

[0050] In certain embodiments, the ORF1a and ORF1b coronavirus peptides or functional variants and fragments can be selected from the list comprising NSP1, NSP2, NSP3, NSP4, NSP5, NSP6, NSP7, NSP8, NSP9, NSP10, NSP11, NSP12, NSP13, NSP14, NSP15, NSP16, or combinations thereof.

[0051] In a further embodiment, the multi-epitope construct of the present disclosure may comprise a nucleic acid sequence encoding a coronavirus peptide or a functional variant and fragment thereof selected from the list comprising E, M, N, NSP1, NSP3, NSP4, NSP5, NSP6, NSP8, NSP9, NSP12, NSP13, NSP14, NSP15, NSP16, ORF6, or combinations thereof.

[0052] In one embodiment, the sequence numbers of the amino acid sequences of the coronavirus antigens, and the sequence numbers of the nucleic acid sequences encoding them are listed in Tables 1 and 2, respectively.

[0053] It should be understood that the amino acid sequences described herein are not limiting and may include sequence variations (i.e., having a certain percentage of sequence identity to the described sequences).

[0054] In determining the degree of sequence identity between two amino acid sequences, one of ordinary skill in the art can take into account so-called "conservative" amino acid substitutions, which are generally described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of a similar chemical structure and has little or essentially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitutions preferably are substitutions in which one amino acid from one of the following groups (a)-(e) is replaced by another amino acid residue from the same group: (a) small, aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are as follows: substitution of Ala with Gly or Ser; substitution of Arg with Lys; substitution of Asn with Gln or His; substitution of Asp with Glu; substitution of Cys with Ser; substitution of Gln with Asn; substitution of Glu with Asp; substitution of Gly with Ala or Pro; substitution of His with Asn or Gln; substitution of Ile with Leu or Val; substitution of Leu with Ile or Val; substitution of Lys with Arg, Gln, or Glu; substitution of Met with Leu, Tyr, or Ile; substitution of Phe with Met, Leu, or Tyr; substitution of Ser with Thr; substitution of Thr with Ser; substitution of Trp with Tyr; substitution of Tyr with Trp; and / or substitution of Phe with Val, Ile, or Leu. Thus, in one embodiment, a sequence having a given percentage of sequence identity as set forth above herein is a sequence having one, two, three, or more conservative amino acid substitutions as compared to a reference sequence.

[0055] On the other hand, the variant antigens / polypeptides encoded by the nucleic acids of the present disclosure may contain amino acid changes that confer any of a number of desirable properties, such as enhancing their immunogenicity in a subject, enhancing their expression, and / or improving their stability or PK / PD properties. The variant antigens / polypeptides can be made using conventional mutagenesis techniques and assayed as necessary to determine whether their variants have the desired properties.

[0056] As will be understood by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are considered to fall within the scope of the coronavirus antigen of interest. For example, any protein fragment of the reference protein (meaning a polypeptide sequence of at least one amino acid residue that is shorter than the reference antigen sequence but identical in other respects) is presented herein, provided that the fragment is immunogenic against the coronavirus and confers a protective immune response thereto. In addition to variants that are identical to the reference protein but truncated, in some embodiments, the antigen contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences presented or referenced herein. The length of the antigen / antigenic polypeptide can range from about 4 amino acids, 6 amino acids, or 8 amino acids to the full-length protein.

[0057] Thus, in certain embodiments, the amino acid sequences described herein are at least and / or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence numbers listed in Table 1.

[0058] In one embodiment, the present invention is a multi-epitope construct comprising at least two, for example at least five, nucleic acid sequences encoding a peptide derived from a coronavirus or a functional variant and fragment thereof, wherein the peptide, variant and / or fragment thereof comprises an amino acid sequence selected from the list comprising SEQ ID NOs: 1 to 47, or an amino acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0059] In certain embodiments, the present invention relates to a multi-epitope construct comprising at least two, for example at least five, nucleic acid sequences encoding a peptide derived from a coronavirus or a functional variant and fragment thereof, wherein the peptide, variant and / or fragment thereof comprises an amino acid sequence selected from the list comprising SEQ ID NOs: 1, 2, 6, 8, 10, 12, 13, 18, 23-25, 27, 30, 32, 34, 40-42, 45, or an amino acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0060] In a further embodiment, the present invention relates to a multi-epitope construct comprising at least two, for example at least five, nucleic acid sequences encoding a peptide derived from a coronavirus or a functional variant and fragment thereof, wherein the peptide, variant and / or fragment thereof comprises an amino acid sequence selected from the list comprising SEQ ID NOs: 1, 2, 4, 5, 6, 8, 10-13, 18, 20, 21, 23-25, 27, 29, 32, 34, 36, 38, 40-42, 45, 47, or an amino acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0061] In another embodiment, the present invention relates to a multi-epitope construct comprising at least two, for example at least five, nucleic acid sequences encoding a peptide derived from a coronavirus or a functional variant and fragment thereof, wherein the peptide, variant and / or fragment thereof comprises an amino acid sequence selected from the list comprising SEQ ID NOs: 1, 2, 7, 9, 10, 12, 13, 16, 18-20, 24, 28, 31-33, 35, 37, 42, 44, or an amino acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0062] In certain embodiments, the present invention relates to a multi-epitope construct comprising at least two, for example at least five, nucleic acid sequences encoding a coronavirus-derived peptide or a functional variant and fragment thereof, comprising an amino acid sequence selected from the list comprising SEQ ID NO: 1, 2, 3, 7, 9, 10, 12-20, 22, 24, 26, 28, 31-33, 35, 37, 39, 42-44, 46, or an amino acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0063] In certain embodiments, the present invention relates to a multi-epitope construct comprising at least two, for example at least five, nucleic acid sequences encoding a coronavirus-derived peptide or a functional variant and fragment thereof, comprising an amino acid sequence selected from the list comprising SEQ ID NO: 1, 2, 10, 12, 13, 18, 20, 24, 32, 42, or an amino acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0064] Table 3 lists examples of multi-epitope constructs having corresponding nucleic acid sequences (SEQ ID NO: 95 to SEQ ID NO: 103) encoding the amino acid sequences of coronavirus antigens.

[0065] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.1) according to SEQ ID NO: 95, consisting of the nucleic acid sequences: SEQ ID NO: 55, 89, 57, 53, 49, 92, 70, 71, 48, 74, 79, 77, 87, 65, 59, 88, 72, 60, 81, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0066] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2) according to SEQ ID NO: 96, which is composed of the nucleic acid sequences: SEQ ID NO: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88, 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0067] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.2.1) according to SEQ ID NO: 97, which is composed of the nucleic acid sequences: SEQ ID NO: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0068] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.2.2) according to SEQ ID NO: 98, which is composed of the nucleic acid sequences: SEQ ID NO: 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0069] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.3) according to SEQ ID NO: 99, which is composed of the nucleic acid sequences: SEQ ID NO: 60, 63, 54, 49, 84, 89, 71, 57, 65, 91, 48, 56, 66, 80, 79, 67, 78, 75, 59, 82, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0070] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.4) according to SEQ ID NO: 100, which is composed of the nucleic acid sequences: SEQ ID NO: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61, 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0071] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.4.1) according to SEQ ID NO: 101, which is composed of the nucleic acid sequences: SEQ ID NO: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0072] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.4.2) according to SEQ ID NO: 102, which is composed of the nucleic acid sequences: SEQ ID NO: 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0073] In certain embodiments, the present invention relates to a multi-epitope construct (Construct 2.5) according to SEQ ID NO: 103, which is composed of the nucleic acid sequences: SEQ ID NO: 89, 59, 60, 49, 65, 79, 71, 67, 57, 48, or a multi-epitope construct having at least 90%, for example at least 95%, sequence identity thereto.

[0074] Table 4 lists examples of multi-epitope constructs having codon-optimized RNA sequences (three versions). Therefore, it is clear that the present invention also relates to multi-epitope constructs according to SEQ ID NOs: 104 to 130, or multi-epitope constructs having at least 90%, for example at least 95%, sequence identity thereto.

[0075] For clarity, a multi-epitope construct may refer to one nucleic acid (e.g., mRNA) molecule containing at least the above-mentioned five nucleic acid sequences encoding coronavirus epitopes, or may refer to two or more nucleic acid (e.g., mRNA) molecules, each mRNA molecule containing a specific set of at least five selected nucleic acid sequences encoding coronavirus epitopes. In other words, very long constructs (e.g., over 11 Kb) may not necessarily be fully expressed, and thus it may be preferable to encode the epitopes in two or more separate nucleic acid (e.g., mRNA) constructs (see also the examples).

[0076] In the context of the present invention, the term "coronavirus" includes, but is not limited to, all human coronaviruses (hCoV) such as HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, SARS-CoV-2, SARS-CoV, MERS-CoV, etc. The peptides or fragments thereof encoded by the multi-epitope constructs are common, at least, in sarbecoviruses including SARS-CoV-2, and thus it should be understood that they induce an immune response against all these types of coronaviruses. As used herein, the multi-epitope constructs can also be used in the context of a pan-coronavirus vaccine.

[0077] In certain embodiments, the present invention provides a combination comprising the above multi-epitope construct and a construct comprising one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant and fragment thereof, particularly the SARS-CoV-2 spike glycoprotein.

[0078] Alternatively, the present invention also provides a construct combining a sequence encoding a coronavirus glycoprotein and a sequence encoding a coronavirus-derived peptide or a functional variant and fragment thereof having an amino acid sequence selected from the list comprising SEQ ID NO: 1 to SEQ ID NO: 47, or having at least 90%, for example at least 95% sequence identity thereto.

[0079] In the context of the present invention, the term "combination" refers to at least two separate multi-epitope constructs, where one type of construct expresses a peptide or a fragment thereof as defined in Table 1, and at least one other type of multi-epitope construct expresses additional proteins and / or adjuvants. It should be understood that the combination may include a plurality of different multi-epitope designs.

[0080] Examples of additional proteins can be immunostimulatory proteins, co-stimulatory molecules, cytokines, chemokines, and / or innate pathway-inducing molecules. Adjuvants can be used to protect the vaccine from degradation or to enhance or extend vaccine immunogenicity.

[0081] In certain embodiments, the combination refers to at least two separate constructs, where one type of construct is a multi-epitope construct that expresses a peptide or a fragment thereof as defined in Table 1, and at least one other construct expresses a coronavirus spike glycoprotein.

[0082] In the context of the present invention, the term "spike glycoprotein (S)" means a special viral protein trimer that acts as a major mediator of attachment to host cell receptors and viral entry. In one embodiment, the combination is selected from a list comprising HCoV-OC43, HCoV-HKUI, HCoV-229E, HCoV-NL63, MERS-CoV, SARS-CoV-2, SARS-CoV, or combinations thereof, in particular nucleic acid sequences encoding the coronavirus glycoprotein of SARS-CoV-2 or functional variants and fragments thereof. It should be understood that the S-proteins of the Coronaviridae family have amino acid sequence similarity. For example, the S-protein of SARS-CoV and the S-protein of SARS-CoV2 share approximately 76% identity with respect to the amino acid sequence. Thus, the combinations described herein may include constructs comprising at least one nucleic acid molecule encoding a coronavirus glycoprotein (e.g., full-length S) or a functional variant or at least a fragment thereof of all types of coronaviruses, or at least one type of coronavirus.

[0083] In some embodiments, the construct encoding the coronavirus glycoprotein comprises a nucleic acid sequence encoding a coronavirus antigen variant. An antigen variant or other polypeptide variant refers to a molecule that has a different amino acid sequence from the wild-type sequence, native sequence, or reference sequence. Antigen / polypeptide variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Usually, the variant has at least 50% identity to the wild-type sequence, native sequence, or reference sequence. In some embodiments, the variant shares at least 80%, or at least 90% identity with the wild-type sequence, native sequence, or reference sequence.

[0084] Table 5 lists examples of codon-optimized RNA sequences of constructs encoding the SARS-CoV-2 spike glycoprotein (SEQ ID NOs: 131 to SEQ ID NOs: 134).

[0085] In another specific embodiment, the combination comprises the multi-epitope construct of the present invention and one or more nucleic acid sequences encoding a SARS-CoV-2 spike glycoprotein comprising the amino acid sequences shown in SEQ ID NOs: 131 to 134, or a construct having at least 90%, for example at least 95% sequence identity thereto.

[0086] In another specific embodiment, the combination refers to at least two separate constructs, one type of which is a multi-epitope construct expressing a peptide or a fragment thereof as defined in Table 1, and at least one other construct expressing at least two immunostimulatory proteins selected from the group comprising CD40L, CD70, and caTLR4. In a preferred embodiment, at least one other multi-epitope construct comprises nucleic acid molecules encoding CD40L and CD70 (i.e., "DiMix"). In a more preferred embodiment, at least one other construct may further comprise a nucleic acid molecule encoding caTLR4, resulting in a so-called "TriMix".

[0087] Throughout the present invention, the term "TriMix" represents a mixture of mRNA molecules encoding CD40L, CD70, and caTRLA4 immunostimulatory proteins.

[0088] In a more preferred embodiment, a specific combination of a coronavirus glycoprotein and TriMix is used to improve the immunostimulatory effect of a construct encoding a coronavirus epitope as defined in Table 1.

[0089] In a specific embodiment, the combination of the present invention comprises at least two separate constructs, one type of which is a multi-epitope construct expressing a peptide or a fragment thereof as defined in Table 1, and at least one other construct expressing a plurality of predefined additional proteins, particularly a coronavirus glycoprotein or an immunostimulatory protein, for example, TriMix.

[0090] In certain embodiments, the invention also provides constructs that express a peptide or fragment thereof as defined in Table 1 and that express one or more predefined additional proteins, particularly coronavirus glycoproteins or immunostimulatory proteins such as TriMix. It should be understood that in the context of the present invention, additional proteins and / or adjuvants may be encoded by the same additional construct or by a plurality of separate constructs.

[0091] In yet another embodiment, the invention provides the above multi-epitope construct, or the above combination, wherein the encoded peptide is separated by at least one specific molecular linker selected from the list comprising a flexible linker, a rigid linker, and / or a cleavable linker.

[0092] In yet another aspect, the invention provides a polypeptide encoded by the above multi-epitope construct.

[0093] In some embodiments, the multi-epitope construct encodes two or more polypeptides, referred to as a fusion protein. In some embodiments, the construct further encodes a linker located between at least one or each of the domains of the fusion protein. Linkers play a crucial role in splicing with the epitope and generating a wide range of conformations (flexibility), protein folding, and separation of functional domains, and thus make the protein structure more stable. Flexible linkers and rigid linkers covalently link functional domains to function as one molecule throughout the in vivo process and thus are not cleaved. In some embodiments, the flexible linker is selected from the group comprising Gly, Ser, Thr, Lys, Glu, Thr, Ala, or combinations thereof. Specific examples are a series of Gly residues and Ser residues («GS n」 linker), where n represents the length of this GS linker. Examples of the most widely used flexible linkers have the sequences of (Gly-Gly-Gly-Gly-Ser)n, GGS, GGSG, G. Other examples can be GS linkers containing additional amino acids to improve solubility and flexibility. Some other types of flexible linkers include KESGSVSSEQLAQFRSLD (SEQ ID NO: 135) and EGKSSGSGSESKST (SEQ ID NO: 136). In some embodiments, the rigid linker can be, for example, an alpha-helix-forming linker having a sequence of (EAAAK)n, which is frequently applied to construct many recombinant fusion proteins. Another type of rigid linker has a Pro-rich sequence, (XP) n and here X represents any amino acid, preferably Ala, Lys, or Glu. An example of a rigid linker can be a series of Glu residues and Pro residues (the "GP n " linker), where n represents the length of this GP linker, particularly GPG, GPPPG, GPGPG, GP8G, or PAPAP, PA.

[0094] On the other hand, the linker may be a linker cleavable in vivo in order to release the free functional domain in vivo. By using this type of linker, it is possible to reduce steric hindrance, improve biological activity, or achieve independent action / metabolism of the individual domains of the recombinant fusion protein after linker cleavage. For clarity, cleavable linkers known in the art can be used in connection with the present disclosure. Exemplary such linkers include F2A linker, T2A linker, P2A linker, E2A linker, and combinations thereof (see, for example, WO 2017 / 127750). It will be understood by those skilled in the art that other linkers recognized in the art may also be suitable for use in the constructs of the present disclosure (e.g., constructs encoded by the nucleic acids of the present disclosure). It will also be understood by those skilled in the art that other polycistronic constructs (mRNAs encoding two or more antigens / polypeptides separately within the same molecule) may also be suitable for the uses presented herein.

[0095] In some embodiments, the linker can be selected from the group consisting of GGGGS, GGS, GGGS, GGSG, G, GS, ankyrin repeat, EAAAK, GPG, GPPPG, GPGPG, GP8G, GTP, PAPAP, KK, AP, F2A, E2A, P2A, T2A, AAY, and / or AYY. In some embodiments, the fusion protein contains three domains separated by linkers and has the structure: domain-linker-domain-linker-domain.

[0096] In a further embodiment, the nucleic acid sequence encoding the coronavirus-derived peptide is part of a single nucleic acid molecule. This single nucleic acid molecule preferably enables the independent expression of several proteins. In a preferred embodiment, the nucleic acid sequences encoding the coronavirus-derived peptides are linked in a single nucleic acid molecule by an internal ribosome entry site (IRES) in the sequence, thereby enabling each of two or more nucleic acid sequences to be translated separately into an amino acid sequence. Alternatively, a sequence encoding a self-cleaving 2a peptide is incorporated between the coding sequences of different coronavirus antigens. Thus, two or more factors can be encoded by a single nucleic acid molecule only.

[0097] Accordingly, the present invention further provides a multi-epitope construct comprising nucleic acid sequences encoding two or more peptides derived from coronaviruses, wherein the two or more peptides derived from coronaviruses are translated separately from a single nucleic acid molecule by using an IRES between the two or more coding sequences. Alternatively, the present invention provides an mRNA molecule encoding two or more peptides derived from coronaviruses separated by a sequence encoding a self-cleaving 2a peptide that enables cleavage of two protein sequences after translation.

[0098] In the following embodiments of the present invention, the nucleic acid sequence is optimized by codon optimization.

[0099] Codon optimization methods are known in the art. For example, any one or more open reading frames (ORFs) of the sequences presented herein can be codon-optimized. In some embodiments, codon optimization is used to adapt codon frequencies in the target and host organisms to increase the GC content, to increase mRNA stability or reduce secondary structure, to minimize tandem repeat codons or base runs that may impair transcription or translation, to customize transcriptional and translational control regions, to insert or remove protein transport sequences, to add, remove or shuffle protein domains, to insert or delete restriction sites, to modify ribosome binding sites and mRNA degradation sites, to adjust the translation rate so that various domains of the protein fold properly, or to reduce or eliminate problematic secondary structure within the polynucleotide. Codon optimization tools, algorithms and services are known in the art. Codon-optimized mRNA sequences generated using different programs or approaches can vary widely. This is because different codon optimization strategies differ in how codon usage is quantified and how codon changes are implemented. Thus, mRNAs encoding the same polypeptide with different codon assignments can differ in the amount of protein expressed. In the context of the present invention, there may be an acceptable difference in the RNA sequence between the non-optimized and the optimized RNA sequences. In some embodiments, the open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0100] In some embodiments, the codon-optimized sequence shares less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65% sequence identity with the naturally occurring sequence ORF or wild-type sequence ORF (e.g., the naturally occurring mRNA sequence or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity with the naturally occurring sequence or wild-type sequence (e.g., the naturally occurring mRNA sequence or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon-optimized sequence shares 65% to 75% or about 80% sequence identity with the naturally occurring sequence or wild-type sequence (e.g., the naturally occurring mRNA sequence or wild-type mRNA sequence encoding a coronavirus antigen). In certain embodiments, the codon-optimized sequence shares 70% sequence identity with the naturally occurring sequence or wild-type sequence (e.g., the naturally occurring mRNA sequence or wild-type mRNA sequence encoding a coronavirus antigen).

[0101] In some embodiments, the codon-optimized sequence encodes an antigen that is as immunogenic as or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% higher) the coronavirus antigen encoded by the non-codon-optimized sequence.

[0102] In some embodiments, the codon-optimized RNA may have an enhanced G / C level. The G / C content of a nucleic acid molecule (e.g., mRNA) can affect the stability of the RNA. RNA having an increased amount of guanine (G) residues and / or cytosine (C) residues can be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO 02 / 098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modification of the translated region. Due to the degeneracy of the genetic code, this modification acts by replacing existing codons with codons that promote higher RNA stability without changing the resulting amino acid. This technique is not limited to the coding region of the RNA.

[0103] For the purpose of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered a difference at a single base (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using a known computer algorithm for sequence alignment, such as NCBI Blast v2.0, using standard settings.

[0104] For the purpose of comparing two or more amino acid sequences, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence is calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at corresponding positions within the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence and multiplying by [100%], where each deletion, insertion, substitution, or addition of an amino acid residue within the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as those described above for determining the degree of sequence identity of nucleotide sequences, and here too, using standard settings. In a particular method, the BLAST module of WU-BLAST-2 set to default parameters is utilized, and the overlap span and overlap fraction are set to 1 and 0.125, respectively.

[0105] In certain embodiments, the present invention provides a pharmaceutical composition comprising the above multi-epitope construct, the above combination, or the above polypeptide and at least one pharmaceutically acceptable agent.

[0106] As used herein, the term "composition" refers to any mixture of two or more articles or compounds (e.g., agents, modulators, regulators, etc.). The composition may be a solution, suspension, liquid or aqueous formulation, or any combination thereof.

[0107] In the context of the present invention, the term "pharmaceutical composition" refers to a composition having pharmaceutical properties. In other words, it refers to a composition having pharmacological and / or physiological effects. The pharmaceutical composition may include one or more pharmaceutically acceptable agents, such as excipients, carriers, diluents.

[0108] In some embodiments, pharmaceutically acceptable agents include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents for realizing a composition usable as a dosage form. Additional suitable pharmaceutical carriers and diluents, and the pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0109] As used herein and unless otherwise specified, the term "excipient" is for the purpose of long-term stabilization, e.g., preventing denaturation or aggregation over the expected shelf life, bulking up a liquid or solid formulation containing a small amount of an active compound (and thus often referred to as a "bulking agent", "filler", or "diluent"), or enhancing the active compound in the final dosage form, e.g., facilitating absorption, reducing viscosity, or enhancing solubility, and should be understood to be any substance formulated with the active compound that is included.

[0110] In another embodiment, the present invention provides the above multi-epitope construct, the above combination, the above polypeptide, or the above pharmaceutical composition formulated as liposomes or nanoparticles, such as lipid nanoparticles or polymer nanoparticles, particularly lipid nanoparticles.

[0111] The constructs, combinations, polypeptides, or pharmaceutical compositions defined herein can be formulated as lipid nanoparticles (LNPs) encapsulating the constructs to protect them from degradation and promote intracellular uptake.

[0112] In the context of the present invention, the term "lipid nanoparticle", or LNP for short, refers to nano-sized particles composed of one or more lipids, for example, a combination of different lipids. The possible lipids used in LNP can be, for example, but not limited to, at least one phospholipid, at least one modified lipid, such as PEG lipid, at least one ionizable lipid, and at least one sterol. The lipid nanoparticles and their compositions of the present disclosure are generally known in the art.

[0113] In the context of the present invention, the term "PEG lipid" or, alternatively, "PEGylated lipid" means any suitable lipid modified with a PEG (polyethylene glycol) group. For example, in the context of the present invention, the PEG lipid may be a C14-PEG lipid, such as DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000). The C14-PEG lipid contains a polyethylene glycol moiety that defines the molecular weight of the lipid and a fatty acid tail containing 14 carbon atoms. Alternatively, the PEG lipid of the present invention may be a C16 lipid or a C18 lipid.

[0114] In the context of the present invention, the term "ionizable" (or, alternatively, cationic) in the context of a compound or lipid means that there is any uncharged group in the above compound or lipid that emits an ion (usually an H+ ion) and dissociates, and thus the compound or lipid itself becomes positively charged. Alternatively, any uncharged group in the above compound or lipid can emit an electron and thus become negatively charged. As used herein, any type of ionizable lipid can be appropriately used. For example, a suitable ionizable lipid is an ionizable amino lipid containing two identical or different tails linked by an S-S bond.

[0115] In the context of the present invention, the term "phospholipid" means a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. These two components are most often joined by a glycerol molecule, and thus, for the phospholipids of the present invention, it is preferred that they be glycerol-phospholipids. Further, the phosphate group is often modified by a simple organic molecule, for example, choline (i.e., made into phosphocholine) or ethanolamine (i.e., made into phosphoethanolamine).

[0116] In the context of the present invention, the term "sterol" is also known as steroid alcohol and is a subgroup of steroids found naturally in plants, animals and fungi, or produced by some bacteria. In the context of the present invention, any suitable sterol such as those selected from the list including cholesterol, ergosterol, campesterol, oxysterol, androsterol, desmosterol, nikasterol, sitosterol and stigmasterol, preferably cholesterol, can be used.

[0117] In certain embodiments, the LNP contains from about 10 mol% to about 60 mol%, preferably from about 40 mol% to about 60 mol% of the ionizable lipid.

[0118] In yet another specific embodiment, the LNP contains from about 15 mol% to about 50 mol%, preferably from about 20 mol% to about 40 mol% of sterol.

[0119] In a further embodiment, the LNP contains from about 0.5 mol% to about 10 mol%, preferably from about 0.5 mol% to about 5 mol% of the PEG lipid.

[0120] In another specific embodiment, the LNP contains from about 5 mol% to about 40 mol%, preferably from about 5 mol% to about 15 mol% of the phospholipid.

[0121] Thus, in other specific embodiments, the LNP of the present invention comprises from about 10 mol% to about 70 mol% of the ionizable lipid, and / or from about 15 mol% to about 50 mol% of the sterol, and / or from about 0.5 mol% to about 10 mol% of the PEG lipid, and / or from about 5 mol% to about 40 mol% of the phospholipid.

[0122] In another specific embodiment, the LNP of the present invention comprises from about 40 mol% to about 60 mol% of the ionizable lipid, from about 20 mol% to about 40 mol% of the sterol, from about 0.5 mol% to about 5 mol% of the PEG lipid, and from about 5 mol% to about 15 mol% of the phospholipid.

[0123] In a specific embodiment, the LNP of the present invention comprises 50 mol% of the ionizable lipid, 10 mol% of the phospholipid, 1.5 mol% of the PEG lipid and 38.5 mol% of the sterol.

[0124] As used herein, the term "nanoparticle" refers to any particle having a diameter that makes the particle suitable for systemic administration, particularly intramuscular or intravenous administration, typically less than 1000 nanometers (nm), preferably less than 500 nm, even more preferably less than 200 nm, for example, having a diameter of 50 nm to 200 nm, preferably 70 nm to 160 nm.

[0125] In some embodiments, lipid nanoparticles are formed by a mixture of lipids. In some embodiments, the construct is formulated into lipid nanoparticles. In some embodiments, the lipid nanoparticles are first formed as empty lipid nanoparticles and combined with the vaccine construct immediately prior to administration (e.g., within a few minutes to 1 hour).

[0126] To avoid any misunderstanding, the LNPs of the present invention may contain a single multi-epitope construct, or a combination of multiple constructs, for example, one or more constructs encoding immunomodulatory proteins and / or one or more constructs encoding antigen-specific proteins.

[0127] In a very specific embodiment, the above construct encoding an immunomodulatory molecule can be combined with one or more constructs encoding peptides derived from coronaviruses. For example, the LNPs of the present invention may combine a construct encoding a peptide derived from a coronavirus with one or more constructs encoding immunostimulatory molecules CD40L, CD70, and / or caTLR4 (e.g., Dimix or Trimix), and further combine with one or more constructs encoding coronavirus glycoproteins.

[0128] Furthermore, it should be understood that the LNPs of the present invention may contain the above combination, the above polypeptide, or the above pharmaceutical composition according to the present invention.

[0129] In some embodiments, two or more different constructs (e.g., mRNA) encoding antigens may be formulated as the same lipid nanoparticles.

[0130] In a further aspect, the present invention provides the above multi-epitope construct, the above combination, the above polypeptide, or the above pharmaceutical composition for use in a human or animal pharmaceutical.

[0131] The pharmaceutical composition is particularly suitable as a vaccine.

[0132] In the context of the present invention, the term "vaccine", as used herein, means any preparation intended to confer acquired immunity (T cell response and antibodies) against a disease. For that purpose, the term "vaccine" as meant herein optionally formulated as an LNP, comprises at least one multi-epitope construct, at least one combination, at least one polypeptide or at least one pharmaceutical composition for which an acquired immune response is initiated.

[0133] In some embodiments, the vaccine may comprise a naked multi-epitope construct, a naked polypeptide suspended in a buffer solution.

[0134] The vaccine may be prophylactic (e.g., to prevent or ameliorate the effects of future infection by any natural or "wild-type" pathogen) or therapeutic (e.g., to actively treat or reduce the symptoms of a progressive disease). Administration of the vaccine is referred to as vaccination.

[0135] The present invention also provides vaccines for use in human or veterinary medicine. Use of the vaccine is also contemplated. Finally, the present invention provides a method of preventing and treating human and animal disorders by administering a vaccine to a subject in need thereof.

[0136] In certain embodiments, the present invention provides a vaccine for use in the treatment or prevention of coronavirus, particularly the SARS-CoV-2 virus, in a subject.

[0137] In the context of the present application, terms such as "treatment", "treating", "treat" etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or may be therapeutic in that it partially or completely stabilizes or cures a disease and / or the adverse effects resulting from the disease. "Treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing a disease or symptom from occurring in a subject who may be predisposed to the disease or symptom but who is not currently diagnosed as having it, (b) inhibiting the symptoms of a disease, i.e., preventing its occurrence, or (c) alleviating the symptoms of a disease, i.e., causing regression of the disease or symptom.

[0138] In another specific embodiment, the present invention provides a vaccine for use in inducing an immune response against a coronavirus, particularly the SARS-CoV-2 virus, in a subject.

[0139] The term "immune response" as used throughout this description is not intended to be limited to the types of immune responses that may be exemplified herein. Thus, this term encompasses all infectious agents for which vaccination is thought to be beneficial to the subject.

[0140] The vaccine of the present invention can be used to induce an immune response, particularly an immune response against a disease-related antigen or a cell expressing a disease-related antigen, for example, an immune response against a coronavirus antigen. The above immune response is preferably a T cell response. In one embodiment, the disease-related antigen is a coronavirus antigen. The antigen encoded by the construct contained in the nanoparticles described herein is preferably a disease-related antigen or one that elicits an immune response against a disease-related antigen or a cell expressing a disease-related antigen.

[0141] In a further aspect, the present invention relates to a method of inducing an immune response against a coronavirus, the method comprising administering to a subject a therapeutically effective amount of the vaccine.

[0142] In the context of the present invention, the vaccine can be administered prophylactically or therapeutically as part of an active immunization scheme to a healthy individual, or during the early stage of infection, the incubation period, or active infection after symptom onset.

[0143] In some embodiments, the vaccine can be administered as monotherapy or in combination therapy with other coronavirus vaccines.

[0144] In some embodiments, the vaccine can be administered as a single dose, two doses, three doses, four doses, or by repeated administration, if applicable.

[0145] In some embodiments, the administration period in monotherapy may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year, and may be repeated annually.

[0146] In another embodiment, the administration period between injections in combination therapy may be, but is not limited to, 1 minute to 30 minutes, 30 minutes to 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year.

[0147] In certain embodiments, the subject is injected with a dose of from about 10 micrograms (μg) to about 100 μg. The effective amount of the construct presented herein can be as low as 10 μg and can be administered, for example, as a single dose or as two doses of 5 μg. In some embodiments, the effective amount is from 10 μg to 100 μg as the total dose. For example, the effective amount can be 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg as the total dose. In some embodiments, the effective amount is 10 μg as the total dose. In some embodiments, the effective amount is 20 μg as the total dose. In some embodiments, the effective amount is 30 μg as the total dose. In some embodiments, the effective amount is 60 μg as the total dose. In some embodiments, the effective amount is 80 μg as the total dose. In some embodiments, the effective amount is 100 μg as the total dose.

[0148] In yet another embodiment, the present invention provides the above multi-epitope construct, the above combination, the above polypeptide or the above pharmaceutical composition for use in vaccination, particularly intramuscular vaccination.

[0149] In some embodiments, the vaccine can be administered intramuscularly, subcutaneously, intranasally, intradermally, or through the lymphatic system, in the same manner as the administration of inactivated vaccines known in the art.

[0150] The vaccine of the present invention is particularly intended for intramuscular administration, i.e., direct injection of a liquid substance into the muscle.

[0151] The present invention also provides a vaccine for intravenous administration, i.e., direct injection of a liquid substance into a vein.

[0152] Table 1: Amino acid sequences of peptides derived from coronaviruses

Table 1

[0153] Table 2: Nucleic acid sequences of peptides derived from coronaviruses

Table 2-1

Table 2-2

[0154] In the context of the present invention, the RNA sequences in the above table can also be replaced with the corresponding DNA sequences in which the "U" of the sequences defined herein is replaced with "T".

Examples

[0155] The present invention is illustrated by the following non-limiting examples.

[0156] Example 1: Prediction of conserved immunogenic T cell epitopes of SARS-CoV-2 SARS-CoV-2 conservation and epitope prediction multiplex epitope construction (MyNEO) (Table 2).

[0157] In silico predictions were made to determine which SARS-CoV-2 antigens are processed, transported to MHC molecules, stably bind to components of MHC molecules, and are likely to elicit an immune response. Predictions were made for each protein expressed by SARS-CoV-2. For each protein, each possible peptide of 9, 10, or 11 amino acid lengths was analyzed for its presentation potential on MHC class I alleles. MHC alleles for presentation prediction were selected based on their distribution in the human population. Furthermore, for each peptide predicted to be presented on MHC class I, a prediction was made regarding the likelihood that the presented peptide would elicit a T cell response.

[0158] Furthermore, for the prediction of peptide presentation and immunogenicity, sequence conservation analysis was performed to identify protein regions that are highly conserved among SARS-CoV-2 variants identified in individuals with SARS-CoV-2 infection. Prioritizing epitopes located within conserved protein regions has the obvious advantage of ensuring broad efficacy of the final vaccine against different SARS-CoV-2 strains that have already been identified. Also, due to the inherent relationship between conservation and functional importance, epitope selection based on evolutionary conservation minimizes the risk of epitope escape as the virus spreads through the human population and new mutations accumulate.

[0159] The level of conservation was calculated based on genomic sequence data sampled from individuals with SARS-CoV-2 infection. Conservation was calculated in a time-resolved and window-wise manner. For a specific window along the SARS-CoV-2 protein sequence, the average sequence conservation was calculated with higher weighting given to more recent variants to account for virus evolution.

[0160] Regarding the selection of coronavirus-derived peptides for vaccines, a composite scoring method was used to draw out more potent clone types and increase the likelihood of more effectively preventing T cell escape. Peptides were prioritized according to the overall level of conservation and the number and quality of the contained epitopes, with peptide regions containing multiple targetable antigens being preferred.

[0161] In the final construct, the selected peptides were concatenated in a chain into one long sequence. Neoepitopes, so-called junctional epitopes, may occur at the junctions between peptide sequences. Such junctional epitopes can interfere with the immune response to the relevant epitopes and non-related junctional epitopes may become advantageous. To reduce the potential negative impact of these epitopes, the windows were ordered so that the number of predicted junctional epitopes was minimized.

[0162] Example 2: Preparation of RNA constructs Preparation of RNA constructs To optimize processing and MHC class I and class II presentation, DNA sequences encoding coronavirus-derived peptides of different linear nucleic acid sequences defined in Table 2 were cloned in-frame into the LAMP1-derived signal peptide and DC-LAMP sequences. Since very long constructs (e.g., over 11 Kb) are not expressed sufficiently, it is also possible to encode the epitopes in two separate mRNA constructs. For example, construct 2.2 is 2 Kb and can also be generated as two separate constructs 2.2.1 (1 Kb) and construct 2.2.2 (1 Kb), or similarly, construct 2.4 (2 Kb) can be generated as two separate constructs 2.4.1 and construct 2.4.2. The resulting DNA sequences defined in Table 3 were prepared by modifying the wild-type or reference coding DNA sequences for stabilization and expression optimization. The sequences were introduced into a DNA vector containing a stabilizing 5'-UTR sequence and 3'-UTR sequence and further containing a run of at least 90 adenosines.

[0163] The obtained plasmid DNA construct was introduced into bacteria using a general protocol known in the art to transform and grow it. The finally purified and linearized plasmid DNA construct was then used for in vitro transcription of RNA. In vitro transcription using T7 RNA polymerase was carried out under appropriate buffer conditions in the presence of a nucleotide mixture containing N1-methyl-pseudouridine and a cap1 analog. N1-methyl-pseudouridine was incorporated during in vitro transcription to improve expression and reduce harmful innate immune activation by the mRNA construct itself. The obtained RNA construct was purified and used for in vitro and in vivo experiments.

[0164] Expression analysis of the linear T cell epitope RNA construct using Western blot To analyze epitope construct expression, HeLa cells were transfected with unformulated mRNA using Lipofectamine MessengerMAX as a transfection agent. HeLa cells were seeded in 6-well plates at a density of 320,000 cells per well. HeLa cells were transfected with 2 μg of unformulated mRNA using Lipofectamine MessengerMAX (Invitrogen). The mRNA constructs listed in Table 3, prepared according to Example 2, were used in the experiment, including a negative control (water for injection). 24 hours after transfection, HeLa cells were detached with trypsin, collected, and cell lysates were prepared. The cell lysates were subjected to SDS-PAGE followed by Western blot detection. Western blot analysis was performed using an anti-DC-LAMP protein antibody in combination with an appropriate secondary antibody.

[0165] Results of Example 2 The mRNA constructs used resulted in detectable expression that varied depending on sequence optimization (western blots not shown). The optimized RNA sequences of the preferred constructs are listed in Table 4, and the non-optimized RNA sequences of the constructs are listed in Table 3 and may include the following sequence numbers: 1. SARS-CoV-2 construct 2.1_DCL (SEQ ID NO: 95): Nucleic acid sequence: a multi-epitope construct containing SEQ ID NOs: 55, 89, 57, 53, 49, 92, 70, 71, 48, 74, 79, 77, 87, 65, 59, 88, 72, 60, 81 (see Figure 4). 2. SARS-CoV-2 construct 2.2_DCL (SEQ ID NO: 96): Nucleic acid sequence: a multi-epitope construct containing SEQ ID NOs: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88, 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70 (see Figure 5). 3. SARS-CoV-2 construct 2.2.1_DCL (SEQ ID NO: 97): Nucleic acid sequence: a multi-epitope construct containing SEQ ID NOs: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88 (see Figure 6). 4. SARS-CoV-2 construct 2.2.2_DCL (SEQ ID NO: 98): Nucleic acid sequence: a multi-epitope construct containing SEQ ID NOs: 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70 (see Figure 7). 5. SARS-CoV-2 construct 2.3_DCL (SEQ ID NO: 99): Nucleic acid sequence: a multi-epitope construct containing SEQ ID NOs: 60, 63, 54, 49, 84, 89, 71, 57, 65, 91, 48, 56, 66, 80, 79, 67, 78, 75, 59, 82 (see Figure 8). 6. SARS-CoV-2 construct 2.4_DCL (SEQ ID NO: 100): Nucleic acid sequence: a multi-epitope construct containing SEQ ID NOs: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61, 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56 (see Figure 9). 7. SARS-CoV-2 construct 2.4.1_DCL (SEQ ID NO: 101): Nucleic acid sequence: A multi-epitope construct containing SEQ ID NOs: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61 (see Figure 10). 8. SARS-CoV-2 construct 2.4.2_DCL (SEQ ID NO: 102): Nucleic acid sequence: A multi-epitope construct containing SEQ ID NOs: 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56 (see Figure 11). 9. SARS-CoV-2 construct 2.5_DCL (SEQ ID NO: 103): Nucleic acid sequence: A multi-epitope construct containing SEQ ID NOs: 89, 59, 60, 49, 65, 79, 71, 67, 57, 48 (see Figure 12).

[0166] Example 3: Vaccination of Mice with SARS-CoV-2 Epitopes Encoded by mRNA Preparation of RNA Constructs The SARS-CoV-2 epitope constructs were prepared as described in Example 1, formulated using LNP, and then used in in vivo vaccination experiments.

[0167] Vaccination of Mice and Flow Cytometry Female BALB / c mice (6 - 8 weeks old) are intramuscularly injected with the mRNA vaccine composition at a dose of 5 μg. As a negative control, one group of mice is vaccinated with buffer. All animals are vaccinated on day 0 and day 21.

[0168] Spleen cells from vaccinated mice are isolated according to standard protocols known in the art. Briefly, the isolated spleen is ground through a cell strainer, washed with PBS, and then the red blood cells are lysed. After extensive washing steps with PBS, the spleen cells are placed in RPMI and seeded into 96-well plates (2×10 cells per well) 6(cells). The cells are stimulated for 5 hours at 37°C in the presence of a protein transport inhibitor with a mixture of specific peptide epitopes (2 μg / ml of each peptide) that match the epitopes in the mRNA construct. After stimulation, the cells are washed and stained for intracellular cytokines using the Cytofix / Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: Thy1.2-Alexa700 (BioLegend), CD4-FITC (BioLegend), CD8-V450 (BD Biosciences), CD107a-BV711 (BD Biosciences), and IFNγ-PE (BD Biosciences). Zombie Aqua is used to distinguish live / dead cells (Invitrogen). Cells are acquired using an Attune flow cytometer (Thermo Fisher Scientific). Flow cytometry data are analyzed using the FlowJo software package (Tree Star, Inc.).

[0169] Results of Example 3 As shown in Figure 1, vaccination with mRNA encoding a linear epitope derived from a conserved region of the non-structural protein of SARS-CoV-2 induced a strong epitope-specific CD8 T cell response after prime / boost vaccination with mRNA-LNP.

[0170] Example 4: Vaccination of Mice with mRNA Construct 2.3 opt2 In this example, a mixed vaccination strategy was envisioned.

[0171] RNA constructs were prepared according to the details of Example 2, and vaccination and analysis of the results were performed according to the details of Example 3.

[0172] A humoral response is elicited by the full-length SARS-CoV-2 Spike mRNA, and a T cell response is induced by an mRNA (Construct 2.3 opt2 - SEQ ID NO: 117) encoding a chain protein containing epitopes encoded by the entire viral genome. Here, these two mRNA constructs are combined in vivo. A dose-response approach is selected, and the two components are tested at equimolar ratios from 0.2 μg to 10 μg each according to the following table:

[0173]

Table A

[0174] Results A very mild and transient weight loss (up to 5%) occurred after each vaccination event (prime or boost) (data not shown). This combination of antigens in the mRNA vaccine candidate does not increase signs of toxicity even at high doses (data not shown).

[0175] A clear induction of both IgG1 and IgG2a responses was observed after vaccination, and a specific boost effect by the second vaccination was recognized (Figure 2). The dose-range effect is more obvious for lower doses. A plateau effect of the vaccination dose was observed at doses higher than 1 μg.

[0176] Furthermore, a dose-response increase in multifunctional CD8+ T cell activation can be observed for all doses tested (Figure 3).

[0177] Conclusion With the mRNA combination, almost no visible toxicity is observed. Almost all cytokines tested, especially type II INF-related cytokines, increased after boosting, and a clear dose-response effect was observed.

[0178] High S-specific IgG1 titers are observed at both d21 and d35, and good induction from d21 to d35 is recognized. A clear plateau effect is observed for all doses above 1 μg.

[0179] Finally, a clear dose-range effect was observed for the CD8+ T cell response.

[0180] Therefore, these data demonstrate that the constructs disclosed herein have potential in vaccination strategies against COVID infection.

[0181] Table 3: Unoptimized RNA sequences of the multi-epitope construct

Table 3-1

Table 3-2

[0182] In the context of the present invention, the RNA sequences in the above table can also be replaced with the corresponding DNA sequences in which the "U" of the sequences defined herein is replaced by "T".

[0183] Table 4: Codon-optimized RNA sequences of multi-epitope constructs (_opt1; _opt2; _opt3 refer to three different codon-optimized sequences of the same construct).

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

[0184] Table 5: Codon-optimized RNA sequences of SARS-CoV-2 spike glycoprotein constructs (_opt1; _opt2 refer to two different codon-optimized sequences of the same construct).

Table 5-1

Table 5-2

Table 5-3

[0185] References Wu, F., Zhao, S., Yu, B. et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020).

Explanation of symbols

[0186] Drawing translation Figure 1 % of CD8+ T cells % of CD8+ T cells Construct Figure 2 S Delta-specific IgG1 S Delta-specific IgG1 Endpoint titers IgG1 Endpoint IgG1 titers empty S Delta-specific IgG2a S Delta-specific IgG2a Endpoint titers IgG2a Endpoint IgG2a titers Figure 3 CD8+ T cell response to restimulation CD8+ T cell response to restimulation % of CD8+ T cells % of CD8+ T cells Empty LNP Empty LNP

Claims

1. A multi-epitope nucleic acid construct encoding a peptide derived from a coronavirus or a functional variant and / or fragment thereof, wherein the peptide, variant and / or fragment thereof comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 1, 2, 7, 9, 10, 12, 13, 16, 18, 19, 20, 24, 28, 31, 32, 33, 35, 37, 42, and 44.

2. The multi-epitope construct according to claim 1, comprising a sequence selected from the list comprising SEQ ID NOs: 116, 117, or 118, particularly SEQ ID NO: 117, or a sequence having at least 95% sequence identity thereto.

3. The multi-epitope construct according to claim 1 or 2, further comprising one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant and / or fragment thereof, particularly the SARS-CoV-2 spike glycoprotein.

4. A combination comprising the multi-epitope construct according to any one of claims 1 to 3 and a construct comprising one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant and / or fragment thereof, particularly the SARS-CoV-2 spike glycoprotein. Multi multi!!

5. The multi-epitope construct according to any one of claims 1 to 3, or the combination according to claim 4, wherein the encoded peptide or a functional variant and / or fragment thereof is separated by at least one molecular linker selected from the list comprising a flexible linker, a rigid linker, and / or a cleavable linker. Multi multi!!

6. A polypeptide encoded by the multi-epitope construct according to any one of claims 1 to 3 or 5. Multi multi!!

7. A pharmaceutical composition comprising the multi-epitope construct according to any one of claims 1 to 3 or 5, the combination according to claim 4, or the polypeptide according to claim 6, and at least one pharmaceutically acceptable agent. Multi multi!!

8. The multi-epitope construct according to any one of claims 1 to 3 or 5, the combination according to claim 4, the polypeptide according to claim 6, or the pharmaceutical composition according to claim 7, formulated as a liposome or nanoparticle, such as a lipid nanoparticle or a polymer nanoparticle, particularly a lipid nanoparticle. Multi multi!!

9. The multi-epitope construct according to any one of claims 1 to 3 or 5, the combination according to claim 4, the polypeptide according to claim 6, or the pharmaceutical composition according to claim 8, which is used in a medicament for humans or animals. Multi multi!!

10. The multi-epitope construct according to any one of claims 1 to 3 or 5, the combination according to claim 4, the polypeptide according to claim 6, or the pharmaceutical composition according to claim 8, which is used for vaccination, particularly intramuscular vaccination. Multi multi!!

11. The multi-epitope construct according to any one of claims 1 to 3 or 5, the combination according to claim 4, the polypeptide according to claim 6, or the pharmaceutical composition according to claim 8, which is used for inducing an immune response against a coronavirus, particularly the SARS-CoV-2 virus, in a subject. Multi multi!!

12. The multi-epitope construct according to any one of claims 1 to 3 or 5, the combination according to claim 4, the polypeptide according to claim 6, or the pharmaceutical composition according to claim 8, which is used for treating or preventing a coronavirus infection, particularly an SARS-CoV-2 virus infection, in a subject. Multi multi!!

13. A method for inducing an immune response against a coronavirus, comprising administering to a subject a therapeutically effective amount of the multi-epitope construct according to any one of claims 1 to 3 or 5, the combination according to claim 4, the polypeptide according to claim 6, or the pharmaceutical composition according to claim 8. Multi multi!!

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