Multi-epitope constructs

Abstract

The invention relates to 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 thereof and a fragment thereof. The invention also relates to combinations, polypeptides or pharmaceutical compositions for use in the treatment or prevention of coronavirus in a subject; in particular, SARS-CoV-2 virus.

Description

[Field of the Invention]

[0001] The present invention relates to 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 thereof and a fragment thereof. The present invention also relates to a combination, a polypeptide or a pharmaceutical composition for treating or preventing a coronavirus in a subject; in particular, the SARS-CoV-2 virus. [Background of the invention]

[0003] The emergence of the COVID-19 pandemic poses a threat to humanity worldwide, as the infection is highly contagious and causes severe illness and death. To date, the global health burden and economy remain very serious. Currently, COVD-19 vaccines are mainly divided into three types (mRNA vaccines, viral vector vaccines, and protein subunit vaccines), of which mRNA and vector vaccines have been successfully used in global vaccination strategies. mRNA vaccines (Moderna; WO2021154763A1 and Pfizer-BioNTech; WO2021188969A2) use genetic information from SARS-CoV-2, the virus that causes COVID-19, and instruct our cells how to make harmless proteins that are unique to the virus. In viral vector vaccines (Johnson, AstraZeneca, Sputnik V, Convidecia), genetic information from SARS-CoV-2 is placed in a modified version of a different virus. Protein subunit vaccines (Novavax) include harmless fragments (proteins) of SARS-CoV-2 instead of the entire virus.

[0004] mRNA vaccines represent a promising alternative to traditional vaccine approaches due to their ease of design, rapid development capabilities, potential for low-cost manufacturing, safe administration, induction of cellular and humoral immunity, and lack of interaction with genomic DNA. Recent improvements in mRNA vaccines increase protein translation, modulate innate and adaptive immunogenicity, and improve delivery. While a drawback of some messenger RNA vaccines (e.g., the Pfizer-BioNTech COVID-19 vaccine) is the need for ultra-cold storage prior to distribution, other mRNA vaccines have no such requirement.

[0005] SARS-CoV-2 is a member of the genus Betacoronavirus that causes pneumonia. SARS-CoV-2 is an enveloped virus with single-stranded RNA that belongs to the Coronaviridae family and can cause infections in mammals, birds, and humans. The entire genome of SARS-CoV-2 has been sequenced (Wu et al., 2020) and is approximately 29.9 kb. The availability of the genome provides an opportunity to develop vaccines against this devastating disease. For COVID-19 vaccines, all approved mRNA vaccines to date use a spike protein-based approach (Moderna; WO2021154763A1 and Pfizer-BioNTech; WO2021188969A2). Spike protein-based vaccines induce antibodies that block the binding of SARS-CoV-2 to host cell receptors and thus neutralize viral infection, resulting in effective protective immunity. A worrying feature of the SARS-CoV-2 spike protein is how it changes over time during viral evolution. As with many viruses, the viral genome of SARS-CoV-2 can mutate, and many such new variants have been described during the SARS-CoV-2 pandemic. These mutations can change the biochemical properties of the spike protein, thereby escaping or evading the neutralizing ability of neutralizing antibodies generated against previous variants as the virus evolves. This therefore provides an opportunity to find improved vaccine designs to avoid or reduce the impact of such new "variants of concern" (VOCs).

[0006] Therefore, the purpose of the present invention is to design a multi-epitope based vaccine containing T cell epitopes based on structural and non-structural proteins instead of the spike protein derived from the coronavirus to provide broad protection and lasting immunity against multiple coronavirus variants and even strains (pan-coronavirus vaccine). Optionally, this multi-epitope vaccine can be used in combination with one or more constructs encoding full-length spike mutants to provide neutralizing antibody responses similar to the above-mentioned established COVID-19 vaccines. [Summary of the invention]

[0007] In the first aspect, the present invention relates to a multi-epitope construct comprising at least five nucleic acid sequences encoding peptides derived from coronavirus or their functional variants and / or fragments, wherein the peptides, variants and / or fragments thereof comprise an amino acid sequence having at least 95% sequence identity with a sequence selected from SEQ ID NOs: 1 to 47.

[0008] In the following embodiments of the invention, the nucleic acid sequence is optimized by codon optimization.

[0009] In a specific embodiment, the present invention provides a multi-epitope construct as defined herein, which further comprises one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant thereof and fragments thereof, wherein the coronavirus glycoprotein is particularly the SARS-CoV-2 spike glycoprotein.

[0010] In a specific embodiment, the present invention provides a combination comprising the multi-epitope construct and a construct comprising one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant thereof and fragments thereof, wherein the coronavirus glycoprotein is particularly the SARS-CoV-2 spike glycoprotein.

[0011] In a further embodiment, the present invention provides the multi-epitope construct or the combination, wherein the encoded peptide or its functional variant and / or its fragment is separated from at least one specific molecular linker selected from the list, and the molecular linker includes: a flexible linker, a rigid linker, and / or a cleavable linker.

[0012] In a further aspect, the present invention provides a multi-epitope construct of the present invention, wherein the construct comprises at least five nucleic acid sequences having at least 95% sequence identity with the sequences specified in SEQ ID NO: 48 to 94; in particular an RNA molecule.

[0013] In a further aspect, the present invention provides a multi-epitope construct as defined herein, wherein the at least 5 nucleic acid sequences are selected from any one of the following list:

[0014] a.SEQ ID NO: 55, 89, 57, 53, 49, 92, 70, 71, 48, 74, 79, 77, 87, 65, 59, 88, 72, 60, 81;

[0015] 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;

[0016] c.SEQ ID NO: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88;

[0017] d.SEQ ID NO: 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70;

[0018] e.SEQ ID NO: 60, 63, 54, 49, 84, 89, 71, 57, 65, 91, 48, 56, 66, 80, 79, 67, 78, 75, 59, 82;

[0019] 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;

[0020] g. SEQ ID NO: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61.

[0021] In another aspect, the present invention provides a polypeptide encoded by the multi-epitope construct.

[0022] In a specific embodiment, the present invention provides a pharmaceutical composition comprising the multi-epitope construct, the combination, or the polypeptide, and at least one pharmaceutically acceptable agent.

[0023] In another embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition; which is formulated in a liposome or a nanoparticle, such as a lipid nanoparticle or a polymer nanoparticle; in particular a lipid nanoparticle.

[0024] In a further aspect, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition for use in human or veterinary medicine.

[0025] In a further embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition for use in vaccination, in particular intramuscular vaccination.

[0026] In a specific embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition for inducing an immune response against a coronavirus in a subject; in particular, the SARS-CoV-2 virus.

[0027] In another specific embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition for use in treating or preventing coronavirus in a subject; in particular, SARS-CoV-2 virus.

[0028] In a further aspect, the present invention relates to a method for inducing an immune response against a coronavirus, comprising: administering a therapeutically effective amount of the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition to a subject.

Brief Description of the Figures

[0030] With specific reference now to the drawings, emphasis is placed on the details shown by way of example and for illustrative discussion of various embodiments of the invention only. They are presented to provide what is believed to be the most useful and easiest description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show the structural details of the invention in greater detail, provided that a basic understanding of the invention is achieved. The description of the drawings makes it clear to those skilled in the art how several forms of the invention may be embodied in practice.

[0031] Figure 1 : Immunogenicity of mRNA constructs encoding SARS-CoV-2 epitopes in mice. Female BALB / c mice (6-8 weeks old) were injected intramuscularly (days 0 and 21) with mRNA vaccine compositions at a dose of 5 μg. As a negative control, one group of mice was vaccinated with buffer (TBS). Vaccination with mRNA encoding junctional epitopes derived from conserved regions of non-structural proteins of SARS-CoV-2 induced strong epitope-specific CD8 T cell responses after primary-prime administration of mRNA-LNP vaccines.

[0032] Figure 2 : Antigen (SARS-CoV-2 spike δ)-specific serum immunoglobulin (IgG1 and IgG2a) titers after intramuscular inoculation of mice with a dose-range amount of LNP-formulated mRNA vaccine at the time of initial administration (21 days) and follow-up administration (35 days). The indicated dose (0.2 to 10 μg) corresponds to the amount of spike-δ mRNA delivered to a single mouse in each injection event and is half the total amount of mRNA, as this is a combination containing the same amount of construct 2.3 mRNA. The obvious effect of the vaccine can be seen from the relatively high IgG titers, with a very obvious enhancement effect from d21 to d35, and a plateau effect with mRNA exceeding 1 mg. The included empty LNP group does not contain any mRNA in the formulation and is prepared according to the calculations of the other groups so as to include the same amount of lipid at higher doses.

[0033] Figure 3: CD8+ polyfunctional T cell responses after restimulation with a peptide pool corresponding to the vaccination antigen (SARS-CoV-2 spike, delta). Splenocytes were collected from the same group (animals vaccinated twice with an mRNA vaccine against SARS-CoV-2 formulated with different doses of LNPs consisting of equal amounts of spike delta mRNA and construct 2.3) on day 35 (terminal) and restimulated with a peptide cocktail that scanned the entire spike protein and analyzed by flow cytometry. We see a clear dose range response of CD8+ T cells expressing IFNg (and CD107a) after restimulation.

[0034] Figure 4 : The tandem sequence of SARS-CoV-2 construct 2.1_DCL (SEQ ID NO: 95).

[0035] Figure 5 : The tandem sequence of SARS-CoV-2 construct 2.2_DCL (SEQ ID NO: 96).

[0036] Figure 6 : The tandem sequence of SARS-CoV-2 construct 2.2.2_DCL (SEQ ID NO: 98).

[0037] Figure 7 : The tandem sequence of SARS-CoV-2 construct 2.3_DCL (SEQ ID NO: 99).

[0038] Figure 8 : The tandem sequence of SARS-CoV-2 construct 2.4_DCL (SEQ ID NO: 100).

[0039] Fig. 9 : The tandem sequence of SARS-CoV-2 construct 2.4.1_DCL (SEQ ID NO: 101).

[0040] Fig.10 : The tandem sequence of SARS-CoV-2 construct 2.4.2_DCL (SEQ ID NO: 102).

[0041] Fig.11 : The tandem sequence of SARS-CoV-2 construct 2.5_DCL (SEQ ID NO: 103). [Detailed description of the invention]

[0043] The invention will now be further described. In the following paragraphs, different aspects of the invention are defined in more detail. Unless expressly stated to the contrary, each aspect so defined may be combined with any other aspect or aspects. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or features indicated as preferred or advantageous.

[0044] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a compound" means one compound or more than one compound.

[0045] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. These terms also include "consisting of" and "consisting essentially of."

[0046] As used herein, the term "about" or "approximately" refers to a measurable value such as a parameter, amount, duration, etc., and is meant to include variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, still more preferably + / -0.1% or less, and more preferably + / -0.1% or less of the specified value, as long as such variations are suitable for implementation in the disclosed invention. It should be understood that the value referred to by the modifier "about" or "approximately" itself is also specifically and preferably disclosed.

[0047] In the search for a new pan-coronavirus vaccine, the inventors investigated whether nucleic acid vaccines based on the expression of other proteins besides the coronavirus-derived spike glycoprotein could be used to induce broad protection and lasting immunity against multiple coronavirus strains. By using bioinformatics tools, the inventors selected a set of conserved peptide windows for further epitope prediction. The inventors unexpectedly discovered a set of conserved peptide windows that are potential antigenic determinants that evoke the production of specific T cells, thereby generating a strong antiviral immune response in the host. As a result, the inventors designed a multi-epitope construct comprising a nucleic acid sequence encoding at least 5 peptide windows derived from coronaviruses or their functional variants and fragments thereof for the treatment or prevention of coronavirus infection in a subject; in particular, SARS-CoV-2 virus. The main advantage associated with multi-epitope vaccines is the induction of a broad immune response against different viral proteins, thereby reducing the risk of immune escape during viral evolution.

[0048] In a first aspect, the present invention relates to a multi-epitope construct comprising at least five nucleic acid sequences encoding peptides or functional variants thereof and / or fragments thereof derived from a coronavirus, wherein the amino acid sequence contained in the peptides, variants thereof and / or fragments thereof has at least 95% sequence identity with a sequence selected from the list comprising SEQ ID NOs: 1 to 47.

[0049] In the context of the present invention, the term "construct" refers to an artificially designed nucleic acid fragment that can be used to incorporate genetic material into target tissues or cells. As used herein, the multi-epitope construct is delivered to the host cell cytoplasm as a target transcript, where expression produces a translated protein located within the membrane, secreted, or intracellularly. It should be understood that the translated protein can be one or more immunogens from a coronavirus.

[0050] In some embodiments, the construct defined in the present invention may be a polyepitopic DNA construct or a polyepitopic RNA construct, in particular a polyepitopic messenger RNA (mRNA) construct.

[0051] In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribose group. Specifically, the term refers to single-stranded RNA, but may also refer to double-stranded RNA, isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA, which is different from naturally occurring RNA because it adds, deletes, replaces and / or changes one or more nucleotides. This change may include the addition of non-nucleotide substances, such as added to the end or interior of the RNA, such as at one or more nucleotides of the RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0052] According to the present invention, the term "RNA" includes and preferably relates to "mRNA", which means "messenger RNA", and to "transcripts", which can be produced using DNA as a template and encode peptides or proteins. mRNA generally includes a 5' untranslated region (5'-UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). RNA also includes a 3' poly (A) tail and / or a 5' cap analog. mRNA has a limited half-life in cells and in vitro. A skilled technician will understand that, unless otherwise stated, the nucleic acid sequences listed in the instant application may list "U" in the representative RNA sequence, but if the sequence represents DNA, "U" will be replaced by "T". Therefore, any RNA sequence disclosed herein and identified by a specific sequence identification number herein is also intended to disclose its corresponding DNA sequence complementary to the RNA, wherein each "U" of the RNA sequence is replaced by "T".

[0053] For the sake of clarity, mRNA molecules include any coding RNA molecule that can be translated into protein by a eukaryotic host.

[0054] In some embodiments, the RNA can be a non-replicating RNA (NRM), also known as a non-amplified RNA. Non-amplified mRNA has only one open reading frame, encoding the target antigen protein. The total amount of mRNA used by the cell is equal to the amount of mRNA delivered by the vaccine, so the dosage strength is limited to the amount of RNA delivered.

[0055] In some embodiments, the RNA can be a self-amplifying RNA (SAM). SAM has two open reading frames. The first open reading frame, like traditional mRNA, encodes the target antigen protein. The second open reading frame encodes RNA-dependent RNA polymerase (and its auxiliary protein), which self-replicate the mRNA construct in the cell and produce multiple self-copies.

[0056] In some embodiments, the RNA may be linear or circular RNA, preferably linear, more preferably linear non-amplified RNA.

[0057] The term "modified mRNA molecule" refers to mRNA molecules containing one or more modified nucleosides (referred to as "modified nucleic acids") that have useful properties, such as lack of substantial induction of an innate immune response to cells into which the mRNA is introduced. These modified nucleic acids increase the efficiency of protein production, intracellular retention of the nucleic acid and viability of contacting cells, and have reduced immunogenicity.

[0058] In some embodiments, the modified nucleobase in the nucleic acid (e.g., RNA nucleic acid, e.g., mRNA nucleic acid) includes 1-methyl pseudouracil (m1Ψ), 1-ethyl pseudouracil (e1Ψ), 5-methoxyuracil (mo5U), 5-methylcytidine (m5C) and / or pseudouracil (Ψ). In some embodiments, the modified nucleobase in the nucleic acid (e.g., RNA nucleic acid, e.g., mRNA nucleic acid) includes 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine and / or 5-methoxycytidine. In some embodiments, the polyribonucleotide includes at least two (e.g., 2, 3, 4 or more) combinations of any modified nucleobases described above, including but not limited to chemical modifications.

[0059] Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription. In vitro transcription methods are well known to technicians and may include a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system including dithiothreitol (DTT) and magnesium ions, spermidine and a suitable RNA polymerase, such as T7 RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA required for a specific application. There are multiple in vitro transcription kits on the market.

[0060] In another embodiment, the present invention also provides a multi-epitope DNA construct, which comprises at least five nucleic acid sequences encoding peptides or their functional variants and fragments derived from coronavirus, wherein the amino acid sequence contained in the peptides, their variants and / or their fragments has at least 95% sequence identity with the sequence selected from the list comprising SEQ ID NO: 1 to 47; wherein each U in the RNA sequence is replaced by T in the corresponding DNA sequence.

[0061] In the context of the present invention, the term "DNA construct" refers to a construct that works by injecting a genetically engineered plasmid containing a DNA sequence encoding an antigen, which is then taken up by the host cell and transcribed and translated into the target gene that encodes the target gene that is sought for the immune response, so that the cell directly produces the antigen, thereby eliciting a protective immune response. This approach has many potential advantages over traditional methods, including stimulation of B-cell and T-cell responses, improved vaccine stability, absence of any infectious pathogens, and relative ease of large-scale production.

[0062] As will be recognized by those skilled in the art, types of DNA constructs may include, but are not limited to, bacterial plasmids, bacteriophage vectors, artificial chromosomes, or fosmids.

[0063] In the context of the present invention, the term "epitope" refers to an antigenic determinant (e.g., a polypeptide of a coronavirus) that can induce an immune response, particularly a cellular or humoral immune response. An immune response should be understood as the ability of the immune system to produce cytotoxic and helper T cells that recognize antigens presented by infected host cells or antigen antibodies. An epitope is a small site on an antigen that interacts with a specific antigen binding site of an antigen-binding protein, such as a variable region of a molecular cell receptor. An epitope can be defined as structural or functional. A functional epitope is typically a subset of a structural epitope and has residues that directly cause an interaction affinity. An epitope can be linear or conformational, i.e., composed of nonlinear amino acids. A single antigen may have multiple epitopes. The term "polytope" used herein refers to a series of possibly overlapping peptides that allow a wide range of immune responses to be induced. This series of peptides may contain T cell and B cell epitopes.

[0064] The term "epitope" also refers to a site on an antigen to which T and / or B cells react. As used herein, it is understood that the term "epitope" or "antigen" includes immunogenic proteins and immunogenic fragments, unless otherwise indicated. "Viral antigens" refer to antigens encoded by a virus. They include, but are not limited to, antigens of coronaviruses, such as COVID-19.

[0065] The multi-epitope construct of the present invention comprises nucleic acid molecules encoding various sites of coronavirus antigens. In the context of the present invention, the term "coronavirus antigen" is understood to be the arrangement of amino acids constituting a part of the gene product encoded by a given coronavirus. The genome organization of 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, which themselves cleave to form 16 non-structural proteins (nsp1-nsp16). ORF1a encodes nsp1-nsp10, while ORF1b encodes nsp11-nsp16. Accessory genes are distributed between the number of structural genes and accessory proteins, and their functions are unique depending on the specific coronavirus (for example, for the SARS-CoV-2 virus, the following accessory genes are present: ORF3a, ORF6, ORF7a, ORF7b, ORF8a, ORF9b, and ORF10).

[0066] In the context of the present invention, the polyepitope construct can include at least two nucleic acid sequences encoding coronavirus peptides or their functional variants and fragments thereof, for example 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. In a specific embodiment, the polyepitope construct includes at least two nucleic acid sequences encoding coronavirus peptides or their fragments. In a preferred embodiment, the polyepitope construct includes at least five nucleic acid sequences encoding coronavirus peptides or their fragments.

[0067] In one embodiment, the multi-epitope construct of the present invention may include nucleic acid sequences encoding coronavirus peptides or their functional variants and fragments thereof, which are selected from: E gene, M gene, N gene, ORF3a, ORF6, ORF7a, ORF7b, ORF8a, ORF9b and ORF10, ORF1a and / or ORF1b viral genome windows.

[0068] In a specific embodiment, ORF1a and ORF1b coronavirus peptides or their functional variants and fragments thereof can be selected from the following list, which includes: NSP1, NSP2, NSP3, NSP4, NSP5, NSP6, NSP7, NSP8, NSP9, NSP10, NSP11, NSP12, NSP13, NSP14, NSP15, NSP16 or a combination thereof.

[0069] In a further embodiment, the multi-epitope construct of the present invention can include a nucleic acid sequence encoding a coronavirus peptide or a functional variant thereof and a fragment thereof can be selected from the following list, which includes: E, M, N, NSP1, NSP3, NSP4, NSP5, NSP6, NSP8, NSP9, NSP12, NSP13, NSP14, NSP15, NSP16, ORF6 or a combination thereof.

[0070] In one embodiment, the sequence numbers of the amino acid sequences used for coronavirus antigens, and the sequence numbers of the nucleic acid sequences encoding them are listed in Table 1 and Table 2, respectively.

[0071] It should be understood that the amino acid sequences described herein are not limiting and may encompass sequence variations (ie, having a percent sequence identity to the described sequence).

[0072] In determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic, non-polar 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 non-polar residues: Met, Leu, Ile, Val and Cys; (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu. Thus, in one embodiment, a sequence having a given percentage sequence identity as given above is a sequence having one, two, three or more conservative amino acid substitutions compared to a reference sequence.

[0073] On the other hand, 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, e.g., enhancing their immunogenicity, enhancing their expression, and / or improving their stability or PK / PD properties in a subject. Variant antigens / polypeptides can be prepared using conventional mutagenesis techniques and analyzed, as appropriate, to determine whether they have the desired properties.

[0074] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the coronavirus antigen of interest. For example, any protein fragment of a reference protein is provided herein (referring to a polypeptide sequence that is at least one amino acid residue shorter than the reference antigen sequence, but otherwise identical), provided that the fragment is immunogenic and confers a protective immune response to coronavirus. In addition to variants that are identical to the reference protein but truncated, in some embodiments, the antigen further comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, as shown in any sequence provided or cited herein. The length of the antigen / antigenic polypeptide ranges from about 4, 6 or 8 amino acids to the full-length protein.

[0075] 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 sequences listed in Table 1.

[0076] In one embodiment, the present invention relates to a multi-epitope construct comprising at least two, for example at least five nucleic acid sequences encoding peptides derived from coronaviruses or their functional variants and fragments thereof, wherein the peptides, their variants and / or their fragments comprise an amino acid sequence selected from the list comprising SEQ ID NOs: 1 to 47, or have at least 90% sequence identity thereto, for example at least 95% sequence identity thereto.

[0077] In a specific embodiment, the present invention relates to a multi-epitope construct comprising at least two, for example at least five nucleic acid sequences encoding peptides or functional variants thereof and fragments thereof derived from coronaviruses, wherein the peptides, variants thereof and / or fragments thereof comprise an amino acid sequence selected from the list comprising SEQ ID NO: 1, 2, 6, 8, 10, 12, 13, 18, 23-25, 27, 30, 32, 34, 40-42, 45, or have at least 90% sequence identity thereto, for example at least 95% sequence identity.

[0078] 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 peptides or functional variants thereof and fragments thereof derived from coronavirus, wherein the peptides, variants and / or fragments thereof comprise an amino acid sequence selected from the list comprising SEQ ID NO: 1, 2, 4, 5, 6, 8, 10-13, 18, 20, 21, 23-25, 27, 29, 32, 34, 36, 38, 40-42, 45, 47, or have at least 90% sequence identity thereto, for example at least 95% sequence identity thereto.

[0079] 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 peptides or functional variants thereof and fragments thereof derived from coronavirus, wherein the peptides, variants and / or fragments thereof comprise an amino acid sequence selected from the list comprising SEQ ID NO: 1, 2, 7, 9, 10, 12, 13, 16, 18 to 20, 24, 28, 31 to 33, 35, 37, 42, 44, or have at least 90% sequence identity thereto, for example at least 95% sequence identity.

[0080] In a specific embodiment, the present invention relates to a multi-epitope construct comprising at least two, for example at least five nucleic acid sequences encoding peptides or functional variants thereof and fragments thereof derived from coronaviruses, wherein the peptides, variants thereof and / or fragments thereof comprise 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 have at least 90% sequence identity thereto, for example at least 95% sequence identity.

[0081] In a specific embodiment, the present invention relates to a multi-epitope construct comprising at least two, for example at least five nucleic acid sequences encoding peptides derived from coronaviruses or their functional variants and fragments thereof, wherein the peptides, their variants and / or their fragments comprise an amino acid sequence selected from the list comprising SEQ ID NO: 1, 2, 10, 12, 13, 18, 20, 24, 32, 42, or have at least 90% sequence identity thereto, for example at least 95% sequence identity.

[0082] In Table 3, examples of multi-epitope constructs having corresponding nucleic acid sequences (SEQ ID NOs: 95 to 103) encoding amino acid sequences of coronavirus antigens are listed.

[0083] In a specific embodiment, the present invention relates to a multi-epitope construct according to SEQ ID NO: 95 (Construct 2.1), which construct comprises SEQ ID NO: 55, 89, 57, 53, 49, 92, 70, 71, 48, 74, 79, 77, 87, 65, 59, 88, 72, 60, 81 or a nucleic acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0084] In a specific embodiment, the present invention relates to a multi-epitope construct according to SEQ ID NO: 96 (Construct 2), which construct comprises 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 nucleic acid sequence having at least 90%, e.g., at least 95% sequence identity thereto.

[0085] In a specific embodiment, the present invention relates to a multi-epitope construct according to SEQ ID NO: 97 (Construct 2.2.1), which construct comprises SEQ ID NO: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88 or a nucleic acid sequence having at least 90%, such as at least 95% sequence identity thereto.

[0086] In a specific embodiment, the present invention relates to a multi-epitope construct according to SEQ ID NO: 98 (Construct 2.2.2), which construct comprises SEQ ID NO: 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70 or a nucleic acid sequence having at least 90%, such as at least 95% sequence identity thereto.

[0087] In a specific embodiment, the present invention relates to a multi-epitope construct composed according to SEQ ID NO: 99 (Construct 2.3), which construct comprises 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 nucleic acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0088] In a specific embodiment, the present invention relates to a multi-epitope construct composed according to SEQ ID NO: 100 (construct 2.4), which construct comprises SEQ ID NO: 91, 71, 78, 66, 86, 63, 60, 90, 90, 75, 73, 54, 67, 61, 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56 or a nucleic acid sequence having at least 90%, for example at least 95%, sequence identity thereto.

[0089] In a specific embodiment, the present invention relates to a multi-epitope construct according to SEQ ID NO: 101 (Construct 2.4.1), which construct comprises SEQ ID NO: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61 or a nucleic acid sequence having at least 90%, such as at least 95% sequence identity thereto.

[0090] In a specific embodiment, the present invention relates to a multi-epitope construct according to SEQ ID NO: 102 (Construct 2.4.2), which construct comprises SEQ ID NO: 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56 or a nucleic acid sequence having at least 90%, such as at least 95% sequence identity thereto.

[0091] In a specific embodiment, the present invention relates to a multi-epitope construct according to SEQ ID NO: 103 (Construct 2.5), which construct comprises SEQ ID NO: 89, 59, 60, 49, 65, 79, 71, 67, 57, 48 or a nucleic acid sequence having at least 90%, such as at least 95% sequence identity thereto.

[0092] Examples of multi-epitope constructs with codon-optimized RNA sequences (three versions) are listed in Table 4. Therefore, it is clear that the present invention also relates to multi-epitope constructs according to SEQ ID NO: 104-130, or having at least 90%, such as at least 95%, sequence identity thereto.

[0093] For the sake of clarity, a polyepitope construct may refer to a nucleic acid (e.g., mRNA) molecule comprising at least 5 nucleic acid sequences encoding coronavirus epitopes, or it may refer to two or more nucleic acid (e.g., mRNA) molecules, wherein each mRNA molecule comprises at least 5 selected nucleic acid sequences encoding a specific set of coronavirus epitopes. In other words, long constructs (e.g., >11 Kb) are not always well expressed, and therefore may be more preferably encoded epitopes on two or more separate nucleic acid (e.g., mRNA) constructs (see also Examples).

[0094] In the context of the present invention, the term "coronavirus" includes all human coronaviruses (hCoV), such as but not limited to HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, SARS-CoV-2, SARS-CoV, MERS-CoV. It should be understood that the peptides or fragments thereof encoded by the multi-epitope construct are at least common in Sarbeco viruses including SARS-CoV-2, and therefore, induce immune responses against all these types of coronaviruses. As described herein, the multi-epitope construct can also be used in the context of a pan-coronavirus vaccine.

[0095] In a specific embodiment, the present invention provides a combination comprising the multi-epitope construct and a construct comprising one or more nucleic acid sequences encoding a coronavirus glycoprotein or a functional variant thereof and fragments thereof, in particular a SARS-CoV-2 spike glycoprotein.

[0096] Optionally, the present invention also provides a construct in which a sequence encoding a coronavirus glycoprotein is combined with a sequence encoding a peptide derived from a coronavirus or a functional variant thereof and a fragment thereof (comprising an amino acid sequence selected from a list comprising SEQ ID NOs: 1 to 47 or having at least 90%, for example at least 95%, sequence identity therewith).

[0097] In the context of the present invention, the term "combination" refers to at least two separate multi-epitope constructs, wherein one type of construct expresses a peptide or fragment thereof as defined in Table 1, and at least one other type of multi-epitope construct expresses an additional protein and / or adjuvant. It should be understood that the combination may include a variety of different multi-epitope designs.

[0098] Examples of additional proteins may be immunostimulatory proteins, co-stimulatory molecules, cytokines, chemokines and / or innate pathway triggering molecules. Adjuvants may be used to protect vaccines from degradation or to enhance or prolong vaccine immunogenicity.

[0099] In a specific embodiment, the combination refers to at least two independent constructs, wherein one type of construct is a multi-epitope construct expressing a peptide or fragment thereof as defined in Table 1, and at least one other construct expressing a crown spike glycoprotein.

[0100] In the context of the present invention, the term "spike glycoprotein (S)" refers to a specialized viral protein trimer that acts as the primary mediator for binding to host cell receptors and viral entry. In one embodiment, the combination includes nucleic acid sequences encoding coronavirus glycoproteins or functional variants thereof and fragments thereof, which are selected from HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, MERS-CoV, SARS-CoV-2, SARS-CoV or a combination thereof, particularly SARS-CoV-2. It should be understood that the S proteins in the coronavirus family have amino acid sequence similarity. For example, the S proteins of SARS-CoV and SARS-CoV2 have approximately 76% identity in amino acid sequence. Therefore, the combination described herein may include a construct comprising at least one nucleic acid molecule encoding a coronavirus glycoprotein (e.g., full-length-S) or a functional variant thereof or at least a fragment thereof, and the construct covers all types of coronaviruses, or at least one type of coronavirus.

[0101] In some embodiments, the construct encoding the coronavirus glycoprotein comprises a nucleic acid sequence encoding a coronavirus antigen variant. Antigenic variants or other polypeptide variants refer to molecules that differ from wild-type, natural or reference sequences in amino acid sequence. Compared to natural or reference sequences, antigen / polypeptide variants may have substitutions, deletions and / or insertions at certain positions in the amino acid sequence. Typically, variants have at least 50% identity with wild-type, natural or reference sequences. In some embodiments, variants share at least 80% or at least 90% identity with wild-type, natural or reference sequences.

[0102] In Table 5, examples of codon-optimized RNA sequences encoding constructs of the SARS-CoV-2 spike glycoprotein (SEQ ID NOs: 131-134) are listed.

[0103] In another specific embodiment, the combination comprises a multi-epitope construct of the invention, combined with a construct comprising one or more nucleic acid sequences encoding a SARS-CoV-2 spike glycoprotein, wherein the spike glycoprotein comprises an amino acid sequence as specified in SEQ ID NOs: 131 to 134 or has at least 90% sequence identity thereto, such as at least 95% sequence identity.

[0104] In another specific embodiment, the combination refers to at least two independent constructs, one type of which is a multi-epitope construct expressing a peptide or fragment thereof as defined in Table 1, and at least one other construct expressing at least 2 immunostimulatory proteins selected from the group consisting of CD40L, CD70 and caTLR4. In a preferred embodiment, at least one other multi-epitope construct comprises a nucleic acid molecule encoding CD40L and CD70 (i.e., "DiMix"). In a more preferred embodiment, at least one other construct may additionally comprise a nucleic acid molecule encoding caTLR4, thereby generating a so-called "TriMix".

[0105] Throughout the invention, the term "TriMix" represents a mixture of mRNA molecules encoding CD40L, CD70 and caTRLA4 immunostimulatory proteins.

[0106] In a further preferred embodiment, a specific combination of coronavirus glycoproteins and TriMix is ​​used to enhance the immunostimulatory effect of constructs encoding coronavirus epitopes as defined in Table 1.

[0107] In a specific embodiment, the combination of the invention comprises at least two separate constructs, one type of which is a multi-epitope construct expressing a peptide or fragment thereof as defined in Table 1, and at least one other construct expressing a plurality of predefined additional proteins, in particular coronavirus glycoproteins or immunostimulatory proteins, such as TriMix.

[0108] In a specific embodiment, the present invention may also provide a construct expressing a peptide or a fragment thereof as defined in Table 1 and expressing one or more predefined additional proteins, in particular a coronavirus glycoprotein or an immunostimulatory protein, such as TriMix.

[0109] In the context of the present invention, it is to be understood that additional proteins and / or adjuvants may be encoded by the same additional construct; or by multiple separate constructs.

[0110] In a further embodiment, the present invention provides said multi-epitope construct or said combination, wherein the encoded peptide is separated from at least one specific molecular linker selected from a list comprising: a flexible linker, a rigid linker and / or a cleavable linker.

[0111] In another aspect, the present invention provides a polypeptide encoded by the multi-epitope construct.

[0112] In some embodiments, the multi-epitope construct encodes more than one polypeptide, called a fusion protein. In some embodiments, the construct also encodes a linker between at least one or each domain of the fusion protein. Linkers play a vital role in splicing epitopes together and producing an extended conformation (flexibility), protein folding, and separation of functional domains, thereby making the protein structure more stable. Flexible and rigid linkers covalently connect the functional domains together and function as a molecule throughout the in vivo process, so they will not be cut. In some embodiments, the flexible linker is selected from the group comprising Gly, Ser, Thr, Lys, Glu, Thr, Ala, or a combination thereof. Specifically, an example can be a Gly and Ser residue (GS n linker), where n represents the length of the GS linker. An example of the most widely used flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser)n, GGS, GGSG, G. Other examples may be GS linkers containing other amino acids to improve solubility and flexibility. Several other types of flexible linkers include KESGSVSSEQ LAQFRSLD (SEQ ID NO: 135) and EGKSSGSGSE SKST (SEQ ID NO: 136). In some embodiments, a rigid linker may be an alpha helix forming linker, for example, having the sequence (EAAAK)n, which is often used in the construction of many recombinant fusion proteins. Another type of rigid linker has a Pro-rich sequence (XP) n , wherein X represents any amino acid, preferably Ala, Lys or Glu. An example of a rigid linker may be a fragment of Glu and Pro residues "(GP n " linker), wherein n represents the length of the GP linker, in particular GPG, GPPPG, GPGPG, GP8G or PAPAP, PA.

[0113] On the other hand, the linker can be an in vivo cleavable linker to release free functional domains in vivo. This type of linker can reduce steric hindrance, improve biological activity, or realize the independent action / metabolism of each domain of the recombinant fusion protein after linker cleavage. For clarity, cleavable linkers known in the art can be used to connect with the present disclosure. Examples of such linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers and combinations thereof (e.g., see WO2017 / 127750). Skilled technicians will appreciate that linkers recognized in other fields may be suitable for use in constructs disclosed herein (e.g., encoded by nucleic acids disclosed herein). Skilled technicians will also appreciate that other polycistronic constructs (mRNAs encoding multiple antigens / polypeptides in the same molecule) may be suitable for use as provided herein.

[0114] In some embodiments, the linker can be selected from the group comprising GGGGS, GGS, GGGS, GGSG, G, GS, ankyrin repeats, EAAAK, GPG, GPPPG, GPGPG, GP8G, GTP, PAPAP, KK, AP, F2A, E2A, P2A, T2A, AAY and / or AYY. In some embodiments, the fusion protein comprises three domains with a linker in the middle, and its structure is: domain-linker-domain-linker-domain.

[0115] In a further embodiment, the nucleic acid sequence encoding the peptide from coronavirus is part of a single nucleic acid molecule. This single nucleic acid molecule is preferably capable of independently expressing multiple proteins. In a preferred embodiment, the nucleic acid sequence encoding the peptide from coronavirus is connected in a single nucleic acid molecule by an internal ribosome entry site (IRES), so that each of the two or more nucleic acid sequences can be translated into an amino acid sequence respectively. Alternatively, a self-cleaved 2a peptide coding sequence is incorporated between the coding sequences of different coronavirus antigens. In this way, two or more factors can be encoded by one nucleic acid molecule.

[0116] Therefore, the present invention further provides a multi-epitope construct comprising a nucleic acid sequence encoding two or more peptides from a coronavirus, wherein the two or more coronavirus-derived peptides are either individually translated 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 coronavirus-derived peptides separated by a self-cleaving 2a peptide coding sequence, capable of post-translationally cleaving two protein sequences.

[0117] In the following embodiments of the invention, the nucleic acid sequence is optimized by codon optimization.

[0118] Codon optimization methods are known in the art. For example, the ORF of any one or more sequences provided herein can be codon optimized. Codon optimization, in some embodiments, can be used to match the codon frequency in the target organism and the host organism to increase the GC content to increase the stability of the mRNA or reduce the secondary structure; minimize tandem repeat codons or base runs that may impair transcription or translation; customize transcription and translation control regions; insert or remove protein transport sequences; add, delete or randomly arrange protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust the translation rate so that the various domains of the protein fold correctly; or reduce or eliminate problematic secondary structures in polynucleotides. Codon optimization tools, algorithms and services are known in the art. Codon-optimized mRNA sequences generated using different programs or methods may vary greatly because different codon optimization strategies differ in how to quantify codon usage and implement codon changes. Therefore, mRNAs encoding the same polypeptide by different codon assignments may show changes in protein expression. In the context of the present invention, there may be acceptable RNA sequence changes between unoptimized RNA sequences and optimized RNA sequences. In some embodiments, open reading frame (ORF) sequences are optimized using an optimization algorithm.

[0119] In some embodiments, the codon optimized sequence has 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 a naturally occurring or wild-type sequence ORF (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon optimized sequence shares 65% to 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring 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 a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In a specific embodiment, the codon optimized sequence shares 70% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen).

[0120] In some embodiments, the immunogenicity of the antigen encoded by the codon-optimized sequence is as great or greater (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200%) as compared to the immunogenicity of the coronavirus antigen encoded by the non-codon-optimized sequence.

[0121] In some embodiments, codon optimized RNA can be RNA with enhanced G / C level. The G / C content of nucleic acid molecules (e.g., mRNA) may affect the stability of RNA. RNA with increased guanine (G) and / or cytosine (C) residues may be more functionally stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. For example, WO02 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by translation region sequence modification. Due to the degeneracy of the genetic code, the working principle of modification is to replace existing codons with those codons that promote higher RNA stability without changing the resulting amino acid. This method is limited to the coding region of RNA.

[0122] For comparison of 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 in the second nucleotide sequence at corresponding positions] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%]. Wherein, each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered as a difference of a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using a known sequence alignment computer algorithm (e.g., NCBI Blast v2.0) using standard settings.

[0123] For comparison of 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 in the corresponding positions of the second amino acid sequence] by [the number of amino acid residues in the total first amino acid sequence] and multiplying by [100%], wherein each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered to be a difference in 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 a known computer algorithm, such as the algorithm mentioned above for determining the degree of sequence identity of nucleotide sequences, also using standard settings. A specific method utilizes the BLAST module of WU-BLAST-2 set to default parameters, with the overlap span and overlap score set to 1 and 0.125, respectively.

[0124] In a specific embodiment, the present invention provides a pharmaceutical composition comprising the multi-epitope construct, the combination or the polypeptide, and at least one pharmaceutically acceptable agent.

[0125] As used herein, "composition" refers to any mixture of two or more products or components (eg, agents, regulators, modulators, etc.). It may be a solution, suspension, liquid or aqueous formulation or any combination thereof.

[0126] In the context of the present invention, by the term "pharmaceutical composition" is meant a composition having pharmaceutical properties. In other words, it refers to a composition that provides a pharmacological and / or physiological effect. A pharmaceutical composition may include one or more pharmaceutically acceptable agents, such as excipients, carriers, diluents.

[0127] In some embodiments, pharmaceutically acceptable agents include, but are not limited to, biocompatible carriers, adjuvants, additives, and diluents to achieve compositions that can be used as dosage forms. Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for using them, are described in Remington's Pharmaceutical Sciences.

[0128] As used herein, unless otherwise indicated, the term "excipient" is understood to include any substance formulated with the active compound for the purpose of long-term stability, such as preventing denaturation or aggregation during the expected shelf life, swelling a liquid or solid formulation containing a small amount of the effective active compound (thus often referred to as a "bulking agent", "filler" or "diluent"), or imparting an enhancing effect to the active compound in the final dosage form, such as promoting absorption, reducing viscosity or enhancing solubility.

[0129] In another embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition; formulated in a liposome or a nanoparticle, such as a lipid nanoparticle or a polymeric nanoparticle; in particular a lipid nanoparticle.

[0130] The constructs, combinations, polypeptides or pharmaceutical compositions defined herein may be formulated in lipid nanoparticles (LNPs) which encapsulate the constructs to protect them from degradation and to facilitate cellular uptake.

[0131] In the context of the present invention, by the term "lipid nanoparticle" or LNP, it is meant a nanoscale particle consisting of one or more lipids, such as a combination of different lipids. The possible lipids used in the LNP can be, for example, but not limited to, at least one phospholipid, at least one modified lipid such as a PEG lipid, at least one ionizable lipid, at least one sterol. Lipid nanoparticles of the present disclosure and compositions thereof are generally known in the art.

[0132] In the context of the present invention, the term "PEG lipid" or "PEGylated lipid" refers to any suitable lipid modified with a PEG (polyethylene glycol) group. For example, in the context of the present invention, the PEG lipid can be a C14-PEG lipid, such as DMG-PEG2000 (1,2-dimyristoyl-racemically-glycero-3-methoxypolyethylene glycol-2000). The C14-PEG lipid comprises a polyethylene glycol moiety that defines the molecular weight of the lipid, and a fatty acid tail comprising 14 C atoms. Alternatively, the PEG lipid of the present invention can be a C16- or C18-lipid.

[0133] In the context of the present invention, the term "ionizable" (or other cations) in the context of a compound or lipid refers to the presence of any uncharged group in the compound or lipid that is capable of dissociating by producing ions (usually H+ ions) and is therefore itself positively charged. Alternatively, any uncharged group in the compound or lipid may produce electrons and thus be negatively charged. As used herein, any type of ionizable lipid may be suitably used. For example, a suitable ionizable lipid is an ionizable amino lipid that consists of 2 identical or different tails connected by SS bonds.

[0134] In the context of the present invention, the term "phospholipid" refers to 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 linked together by a glycerol molecule, and therefore, glycerol-phospholipids are preferred in the phospholipids of the present invention. In addition, the phosphate group is usually modified with simple organic molecules, such as choline (i.e., phosphorylcholine) or ethanolamine (i.e., phosphorylethanolamine).

[0135] In the context of the present invention, the term "sterol", also known as steroid, is a subgroup of steroids naturally present in plants, animals and fungi, or can be produced by certain bacteria. In the context of the present invention, any suitable sterol can be used, for example selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, streptosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol.

[0136] In specific embodiments, the LNP comprises between about 10 mol% and 60 mol% of the ionizable lipid; preferably between 40 mol% and 60 mol%.

[0137] In yet another specific embodiment, the LNP comprises between about 15 mol% and 50 mol% sterol; preferably between 20 mol% and 40 mol%.

[0138] In a further embodiment, the LNP comprises between about 0.5 mol% and 10 mol% of the PEG lipid; preferably between 0.5 mol% and 5 mol%.

[0139] In another specific embodiment, said LNP comprises between about 5 mol% and 40 mol% of said phospholipid; preferably between 5 mol% and 15 mol%.

[0140] Thus, in more specific embodiments, the LNPs of the invention comprise between about 10 mol% and 70 mol% of said ionizable lipids; and / or between about 15 mol% and 50 mol% of said sterols; and / or between about 0.5 mol% and 10 mol% of said PEG lipids; and / or between about 5 mol% and 40 mol% of said phospholipids.

[0141] In another specific embodiment, the LNP of the present invention comprises between about 40 mol% and 60 mol% of said ionizable lipid; and between about 20 mol% and 40 mol% of said sterol; between about 0.5 mol% and 5 mol% of said PEG lipid; and between about 5 mol% and 15 mol% of said phospholipid.

[0142] In a specific embodiment, the LNP of the invention comprises 50 mol% ionizable lipids, 10 mol% phospholipids, 1.5 mol% PEG lipids, and 38.5 mol% sterols.

[0143] 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 having a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, even more preferably less than 200 nm, for example between 50 and 200 nm; preferably between 70 and 160 nm.

[0144] In some embodiments, the mixture of lipids forms lipid nanoparticles. In some embodiments, the construct is formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles are first formed as empty lipid nanoparticles and are combined with the construct of the vaccine immediately (e.g., within a few minutes to an hour) before administration.

[0145] To avoid any misunderstanding, the LNPs of the invention may comprise a single multi-epitope construct, or they may comprise multiple constructs, such as a combination of one or more constructs encoding immunomodulatory proteins and / or one or more constructs encoding antigen-specific proteins.

[0146] In very specific embodiments, the construct encoding the immunomodulatory molecule can be combined with one or more constructs encoding a peptide derived from a coronavirus. For example, the LNP of the present invention can include a construct encoding a peptide derived from a coronavirus; combined with one or more constructs encoding the immunostimulatory molecules CD40L, CD70 and / or caTLR4 (such as Dimix or Trimix); and further combined with one or more constructs encoding a coronavirus glycoprotein.

[0147] Furthermore, it should be understood that the LNP of the present invention may comprise the combination, the polypeptide or the pharmaceutical composition according to the present invention.

[0148] In some embodiments, two or more different constructs (eg, mRNAs) encoding antigens can be formulated in the same lipid nanoparticle.

[0149] In a further aspect, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition for use in human or veterinary medicine.

[0150] The pharmaceutical composition is particularly suitable as a vaccine.

[0151] In the context of the present invention, the term "vaccine" as used herein refers to any formulation intended to provide adaptive immunity (T cell response and antibodies) against a disease. For this purpose, the term "vaccine" as used herein includes at least one multi-epitope construct, at least one combination, at least one polypeptide or at least one pharmaceutical composition, optionally formulated as LNP, to enhance the adaptive immune response.

[0152] In some embodiments, the vaccine may include a naked multi-epitope construct, a naked polypeptide suspended in a buffer solution.

[0153] Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of future infection with any natural or "wild" pathogen) or therapeutic (e.g., to actively treat or alleviate the symptoms of an ongoing disease). The administration of a vaccine is called vaccination.

[0154] The invention also provides vaccines for use in human or veterinary medicine. It is also contemplated that the vaccines will be used. Finally, the invention provides a method for the prevention and treatment of human and veterinary diseases by administering the vaccine to a subject in need thereof.

[0155] In a specific embodiment, the present invention provides a vaccine for treating or preventing a coronavirus in a subject; in particular, the SARS-CoV-2 virus.

[0156] In the context of this application, the terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, i.e., completely or partially preventing a disease or its symptoms, and / or may be therapeutic, partially or completely stabilizing or curing a disease and / or an adverse reaction caused by the disease. "Treatment" includes any treatment of a disease in mammals, particularly humans, including: (a) preventing the disease or symptom from occurring in a subject who may be susceptible to the disease or symptom but has not yet been diagnosed with the disease or symptom; (b) inhibiting the symptoms of the disease, i.e., preventing its development; or (c) alleviating the symptoms of the disease, i.e., causing the disease or symptom to disappear.

[0157] In another specific embodiment, the present invention provides a vaccine for inducing an immune response against a coronavirus in a subject; in particular, the SARS-CoV-2 virus.

[0158] The term "immune response" as used throughout the description is not limited to the types of immune responses that may have been exemplified herein. Thus, the term includes all infectious agents against which vaccination would be beneficial to a subject.

[0159] The vaccine of the present invention can be used to induce an immune response, particularly an immune response to a disease-associated antigen or a cell expressing a disease-associated antigen, such as an immune response to a coronavirus antigen. Preferably, the immune response is a T cell response. In one embodiment, the disease-associated antigen is a coronavirus antigen. The antigen encoded by the construct contained in the nanoparticle described herein is preferably a disease-associated antigen or induces an immune response to a disease-associated antigen or a cell expressing a disease-associated antigen.

[0160] In a further aspect, the present invention relates to a method of inducing an immune response against a coronavirus, comprising: administering a therapeutically effective amount of the vaccine to a subject.

[0161] In the context of the present invention, the vaccine may be administered prophylactically or therapeutically as part of an active immunization program in healthy individuals, or during the early latent phase of infection or during active infection after the onset of symptoms.

[0162] In some embodiments, the vaccine can be administered as a monotherapy or as a combination therapy with other coronavirus vaccines.

[0163] In some embodiments, the vaccine may be administered as a single dose, two doses, three doses, four doses, or repeated doses as appropriate.

[0164] In some embodiments, the administration period in monotherapy can be, but is not limited to, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year, and can be repeated once a year.

[0165] In another embodiment, the administration time between injections in the combination therapy can 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.

[0166] In certain embodiments, a dose of about 10 to 100 μg (μg) is injected into the subject. As described herein, the effective amount of the construct can be as low as 10 μg, for example, administered in a single dose or two 5 μg doses. In some embodiments, the effective amount is a total dose of 10 μg to 100 μg. For example, the effective amount can be a total dose of 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg. In some embodiments, the effective amount is a total dose of 10 μg. In some embodiments, the effective amount is a total dose of 20 μg. In some embodiments, the effective amount is a total dose of 30 μg. In some embodiments, the effective amount is a total dose of 60 μg. In some embodiments, the effective amount is a total dose of 80 μg. In some embodiments, the effective amount is a total dose of 100 μg.

[0167] In yet another embodiment, the present invention provides the multi-epitope construct, the combination, the polypeptide or the pharmaceutical composition for use in vaccination, particularly intramuscular vaccination.

[0168] In some embodiments, the vaccine can be administered intramuscularly, subcutaneously, intranasally, intradermally, or via the lymphatic system, similar to the administration of inactivated vaccines known in the art.

[0169] The vaccines of the present invention are particularly useful for intramuscular administration, ie, injection of a liquid substance directly into a muscle.

[0170] The present invention also provides a vaccine; wherein the administration is intravenous, that is, the liquid substance is directly injected into the vein.

[0171] [Table 1: Amino acid sequences of coronavirus-derived peptides]

[0172]

[0173]

[0174] [Table 2: Nucleic acid sequences of coronavirus-derived peptides]

[0175]

[0176]

[0177]

[0178]

[0179] In the context of the present invention, the RNA sequences in the above table can also be replaced by corresponding DNA sequences, wherein "U" in the sequences defined herein is replaced by "T". [Example]

[0180] The invention is illustrated by the following non-limiting examples.

[0181] [Example 1: Prediction of conserved immunogenic T cell epitopes of SARS-COV-2]

[0182] SARS-CoV-2 conserved and epitope predicted multi-epitope construct (MyNEO) (Table 2).

[0183] Computer predictions were performed to determine which SARS-CoV-2 antigens are likely to be processed, transported to MHC molecules, stably associated with their components and 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 acids in length was analyzed for its likelihood of presentation on MHC class I alleles. The MHC alleles used for presentation predictions were selected based on their prevalence in the population. In addition, for each peptide predicted to be presented on MHC class I, the likelihood that the presented peptide would elicit a T cell response was predicted.

[0184] Additionally, for peptide presentation and immunogenicity prediction, sequence conservation analysis was performed to identify protein regions that are highly conserved among SARS-CoV-2 variants found in individuals infected with SARS-CoV-2. Prioritizing epitopes located in conserved protein regions has a distinct advantage as it ensures broad efficacy of the final vaccine against the different SARS-CoV-2 strains that have been identified. Furthermore, due to the intrinsic link 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 accumulates new mutations.

[0185] The conservation level is calculated based on genomic sequence data sampled from people infected with SARS-CoV-2. Conservation is calculated in a time-resolved and windowed manner. For a specific window on the SARS-CoV-2 protein sequence, the average sequence conservation is calculated and newer variants are weighted more heavily to account for viral evolution.

[0186] For the selection of coronavirus-derived peptides for the vaccine, a hybrid scoring approach was used to increase the chances of eliciting more potent clonotypes and more effectively preventing T cell escape. Peptides were prioritized based on their overall level of conservation and the number and quality of epitopes they contained, with preference given to peptide regions that encompass multiple targeted antigens.

[0187] In the final construct, the selected peptides are linked into one long sequence. It is possible that new epitopes may appear at the junctions between peptide sequences, so-called junction epitopes. Such junction epitopes may interfere with the immune response to related epitopes, thereby favoring unrelated junction epitopes. To reduce the potential negative effects of these epitopes, the windows are ordered in a way that minimizes the number of predicted junction epitopes.

[0188] [Example 2: Preparation of RNA constructs]

[0189] Preparation of RNA constructs

[0190] DNA sequences encoding different tandem nucleic acid sequences of coronavirus-derived peptides defined in Table 2 are cloned in frame into LAMP1-derived signal peptides and DC-LAMP sequences to optimize processing and presentation to MHC class I and class II. Since long constructs (e.g., >11Kb) are poorly expressed, epitopes can also be encoded on two separate mRNA constructs. For example, the 2Kb construct 2.2 can also generate two separate constructs 2.2.1 (1Kb) and construct 2.2.2 (1Kb); or similarly, construct 2.4 (2Kb) can also be generated in two separate constructs 2.4.1 and construct 2.4.2. The resulting DNA sequences defined in Table 3 are prepared by modifying wild-type or reference coding DNA sequences to achieve stability and expression optimization. The sequence is introduced into a DNA vector comprising stable 5'-UTR and 3'-UTR sequences, and also includes a fragment of at least 90 adenosines.

[0191] The obtained plasmid DNA constructs were transformed and propagated in bacteria using common protocols known in the art. Finally, the purified and linearized plasmid DNA constructs were used for subsequent RNA in vitro transcription. In vitro transcription was performed using T7 RNA polymerase under appropriate buffer conditions in the presence of a nucleotide mixture containing N1-methylpseudouridine and cap1 analogs. N1-methylpseudouridine was incorporated during in vitro transcription to improve expression and reduce harmful innate immune activation of the mRNA construct itself. The obtained RNA constructs were purified and used for in vitro and in vivo experiments. [Expression analysis of RNA constructs linked to T cell epitopes using western blot]

[0192] In order to analyze the expression of epitope constructs, HeLa cells were transfected with unformulated mRNA using Lipofectamine MessengerMAX as a transfection reagent. HeLa cells were seeded in 6-well plates at a density of 320,000 cells / well. HeLa cells were transfected with 2 μg of unformulated mRNA using Lipofectamine MessengerMAX (Invitrogen). The mRNA constructs prepared according to Example 2 were used in the experiment and are listed in Table 3, including negative controls (water for injection). 24 hours after transfection, HeLa cells were separated with trypsin, harvested and cell lysates were prepared. SDS-PAGE was performed on the cell lysates, and then western blot was performed. Western blot analysis was performed using anti-DC-LAMP protein antibodies in combination with suitable second antibodies.

[0193] [Results of Example 2]

[0194] 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, while the non-optimized RNA sequences of the constructs are listed in Table 3 and may include the following sequence numbers:

[0195] 1. SARS-CoV-2 construct 2.1_DCL (SEQ ID NO: 95): A multi-epitope construct comprising the following 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 (see Figure 4 ).

[0196] 2. SARS-CoV-2 construct 2.2_DCL (SEQ ID NO: 96): A multi-epitope construct comprising the following 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 (see Figure 5 ).

[0197] 3. SARS-CoV-2 construct 2.2.1_DCL (SEQ ID NO: 97): A multi-epitope construct comprising the following nucleic acid sequences: SEQ ID NO: 55, 65, 59, 76, 83, 60, 94, 71, 49, 74, 79, 87, 88 (see Figure 6 ).

[0198] 4. SARS-CoV-2 construct 2.2.2_DCL (SEQ ID NO: 98): A multi-epitope construct comprising the following nucleic acid sequences: SEQ ID NO: 89, 48, 58, 68, 92, 51, 52, 57, 85, 81, 53, 67, 72, 70 (see Figure 7 ).

[0199] 5. SARS-CoV-2 construct 2.3_DCL (SEQ ID NO: 99): A multi-epitope construct comprising the following 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 (see Figure 8 ).

[0200] 6. SARS-CoV-2 construct 2.4_DCL (SEQ ID NO: 100): a multi-epitope construct comprising the following 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 (see Fig. 9 ).

[0201] 7. SARS-CoV-2 construct 2.4.1_DCL (SEQ ID NO: 101): A multi-epitope construct comprising the following nucleic acid sequences: SEQ ID NO: 91, 71, 78, 66, 86, 63, 60, 80, 90, 75, 73, 54, 67, 61 (see Fig.10 ).

[0202] 8. SARS-CoV-2 construct 2.4.2_DCL (SEQ ID NO: 102): A multi-epitope construct comprising the following nucleic acid sequences: SEQ ID NO: 89, 48, 65, 79, 62, 57, 50, 93, 84, 49, 59, 64, 69, 82, 56 (see Fig.11 ).

[0203] 9. SARS-CoV-2 construct 2.5_DCL (SEQ ID NO: 103): A multi-epitope construct comprising the following nucleic acid sequences: SEQ ID NO: 89, 59, 60, 49, 65, 79, 71, 67, 57, 48 (see Figure 12). [Example 3: Vaccination of mice with mRNA-encoded SARS-COV-2 epitope vaccines]

[0204] Preparation of RNA constructs

[0205] SARS-CoV-2 epitope constructs were prepared as described in Example 1 and formulated with LNPs prior to use in in vivo vaccination experiments.

[0206] Mouse vaccination and flow cytometry

[0207] Female BALB / c mice (6-8 weeks old) were injected intramuscularly with the mRNA vaccine composition at a dose of 5 μg. As a negative control, one group of mice was immunized with buffer. All animals were immunized on days 0 and 21.

[0208] Splenocytes from vaccinated mice were isolated according to standard protocols known in the art. Briefly, isolated spleens were ground through a cell strainer and washed in PBS, followed by red blood cell lysis. After extensive washing steps with PBS, splenocytes in RPMI were seeded into 96-well plates (2 × 10 6 Cells were stimulated with a mixture of specific peptide epitopes (2 μg / ml for each peptide) that matched the epitopes in the mRNA constructs at 37°C in the presence of a protein transport inhibitor for 5 hours. After stimulation, cells were washed and intracellular cytokine staining was performed using Cytofix / Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were 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 was used to distinguish live cells / dead cells (Invitrogen). Cells were acquired using an Attune flow cytometer (Thermo Fisher Scientific). Flow cytometry data were analyzed using the FlowJo software package (Tree Star Inc.).

[0209] [Results of Example 3]

[0210] like Figure 1 As shown, vaccination with mRNA encoding junctional epitopes derived from conserved regions of SARS-CoV-2 nonstructural proteins induced strong epitope-specific CD8 T cell responses after primary / boosting vaccination with mRNA-LNP vaccination.

[0211] [Example 4: Vaccination of mice using mRNA construct 2.3opt2]

[0212] In this example, a combination vaccine strategy is envisioned.

[0213] RNA constructs were prepared according to the details of Example 2, and vaccination and results were analyzed according to the details of Example 3.

[0214] The humoral response was elicited by the full-length SARS-CoV-2 spike mRNA, and the T cell response was elicited by an mRNA encoding a tandem protein containing an epitope encoded by the full viral genome (construct 2.3opt2-SEQ ID NO: 117). Here, the two mRNA constructs were combined in vivo. A dose response approach was chosen, in which an equimolar ratio of the two components was tested, ranging from 0.2 μg to 10 μg of each component according to the following table:

[0215]

[0216]

result

[0217] Very mild transient weight loss (maximum 5%) occurred after each vaccination event (primary or booster) (data not shown). The antigen combination in the mRNA vaccine candidate did not cause increased signs of toxicity, even at higher doses (data not shown).

[0218] We observed a clear induction of IgG1 and IgG2a responses after vaccination, with a specific boosting effect of the second vaccination ( Figure 2 ). The dose range effect was more pronounced for lower doses. For doses above 1 mg, a plateau effect of vaccination dose was observed.

[0219] Furthermore, a dose-responsive increase in polyfunctional CD8+ T cell activation was seen at all doses tested ( Figure 3 ).

[0220]

in conclusion

[0221] There was little observable toxicity from the mRNA combinations. Almost all cytokines tested, especially INF type II-related cytokines, increased after chase administration, with a clear dose-response effect.

[0222] High S-specific IgG1 titers were observed on d21 and d35, with good induction from d21 to d35. A clear plateau effect was observed for all doses above 1 mg.

[0223] Finally, we observed a clear dose-range effect on CD8+ T cell responses.

[0224] Therefore, these data demonstrate that the constructs disclosed therein have potential in vaccination strategies against COVID infection.

[0225] [Table 3: Non-optimized RNA sequences for multi-epitope constructs]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] In the context of the present invention, the RNA sequences in the above table may also be replaced by corresponding DNA sequences, wherein "U" in the sequences as defined herein is replaced by "T".

[0233] [Table 4: Codon-optimized RNA sequences of multi-epitope constructs (_opt1; _opt2; _opt3 refer to three different codon-optimized sequences of the same construct)]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250] [Table 5: Codon-optimized RNA sequences of SARS-CoV-2 spike glycoprotein constructs (_opt1; _opt2 refers to two different codon-optimized sequences of the same construct)]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

References

[0259] Wu,F.,Zhao,S.,Yu,B.et al.,A new coronavirus associated with humanrespiratory disease in China.Nature 579,265-269(2020).

Claims

1. A multi-epitope nucleic acid construct encoding a peptide derived from a coronavirus or a functional variant thereof and / or a fragment thereof, wherein: The peptides, variants thereof and / or fragments thereof comprise 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, wherein The construct comprises a sequence selected from the group consisting of: SEQ ID NO: 116, 117 or 118; in particular SEQ ID NO: 117; or a sequence having at least 95% sequence identity thereto.

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

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 its functional variant and / or its fragment is separated from at least one molecular linker selected from the group consisting of a flexible linker, a rigid linker and / or a cleavable linker.

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

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

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 in liposomes or nanoparticles, such as lipid nanoparticles or polymer nanoparticles; in particular lipid nanoparticles.

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; for use in human or veterinary medicine.

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; for use in vaccination, in particular intramuscular vaccination.

11. A multi-epitope construct according to any one of claims 1 to 3 or 5, a combination according to claim 4, a polypeptide according to claim 6 or a pharmaceutical composition according to claim 8; for use in inducing an immune response against a coronavirus in a subject; in particular the SARS-CoV-2 virus.

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; for treating or preventing coronavirus infection in a subject; in particular SARS-CoV-2 virus.

13. A method for inducing an immune response against a coronavirus, comprising: 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 is administered to a subject.

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