Coronavirus vaccine

Abstract

The present invention provides nucleic acids, compositions, polypeptides, and vaccines suitable for use in the treatment or prevention of infection with coronavirus (preferably coronavirus SARS-CoV-2) or disorders associated with such infection (preferably COVID-19). [Solution] Preferably, the composition and vaccine contain one or more nucleic acid sequences. Preferably, the nucleic acid sequences are associated with lipid nanoparticles (LNPs). The present invention is also directed toward first and second pharmaceutical uses of the above-mentioned nucleic acids, compositions, polypeptides, combinations, vaccines and kits, as well as methods for treating or preventing coronavirus infection (preferably coronavirus infection).

Description

Detailed description of the invention

[0001] [Words] The present invention is particularly directed to nucleic acids suitable for use in the treatment or prevention of infection with coronavirus (preferably coronavirus SARS-CoV-2) or disorders associated with such infection (preferably COVID-19). The present invention is also directed to compositions, polypeptides and vaccines. Preferably, the compositions and vaccines comprise one or more of the above-described nucleic acid sequences. Preferably, the nucleic acid sequences are associated with polymeric carriers, polycationic proteins or peptides, or lipid nanoparticles (LNPs). The present invention is also directed to first and second pharmaceutical uses of the above-described nucleic acids, compositions, polypeptides, vaccines and kits, as well as methods for the treatment or prevention of coronavirus infection (preferably infection with SARS-CoV-2).

[0002] Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses belonging to the Coronaviridae family.

[0003] Typical coronaviruses cause a variety of diseases in various vertebrates, including mammals, birds, and fish.

[0004] Coronaviruses are genetically highly variable. Furthermore, individual virus species can cross species barriers and infect multiple species of hosts. This host crossover has resulted in the emergence of human-infectious coronaviruses such as SARS-related coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV). The coronavirus outbreak that began in Wuhan, China, at the end of 2019 and the beginning of 2020 was initially attributed to an unknown coronavirus. This virus was initially called nCoV-2019 or Wuhan human coronavirus (WHCV), and was later given the formal name SARS-CoV-2.

[0005] Typical symptoms of SARS-CoV-2 viral infection (also known as COVID-19 disease (Coronavirus disease 2019)) include fever, cough, difficulty breathing, pneumonia, and gastrointestinal symptoms (such as diarrhea). In severe cases, respiratory failure can occur, requiring mechanical ventilation and treatment in an intensive care unit. On January 30, 2020, the World Health Organization (WHO) declared a global health emergency in response to this new coronavirus outbreak. On March 11, the WHO declared COVID-19 a pandemic. It also noted that there were more than 118,000 cases of this coronavirus disease in more than 110 countries and territories worldwide, and that there was a persistent risk of further global spread. By the end of March 2020, SARS-CoV-2 infection had reached more than 800,000 confirmed cases, spreading to almost every country in the world and causing more than 400,000 COVID-19-related deaths.

[0006] Currently, there is no vaccine or specific treatment for infection with SARS-CoV-2 and / or COVID-19 disease.

[0007] Patients diagnosed with SARS-CoV-2 infection receive only supplemental treatment based on their individual symptoms and clinical condition. Given the significant risk of a major global pandemic, there is an urgent need for safe and effective treatment or prevention of SARS-CoV-2 infection. Vaccines are particularly necessary to protect the elderly, who are at high mortality rates.

[0008] Nucleoside (DNA or RNA, etc.) vaccines are a promising technology for novel vaccines against emerging viruses. Genetically modified nucleic acids can be administered to human subjects. Transfected cells then directly produce the encoded antigen (e.g., antigens given by DNA or RNA, especially those given by mRNA). This results in a protective immune response.

[0009] The central role of virus-specific memory T cells in broad and long-term protection against SARS-CoV infection has been elucidated (see, for example, Channappanavar, Rudragouda, et al. "Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection." Journal of virology 88.19 (2014): 11034-11044). For example, virus-specific CD8 T cells are necessary for mediating pathogen clearance and defense after viral attack. Therefore, an effective SARS-CoV-2 vaccine must not only induce a robust and functional humoral immune response, but also induce a SARS-CoV-2-specific CD8+ T cell and CD4+ T cell response.

[0010] Therefore, the inherent problem of the present invention is to provide a nucleic acid-based vaccine against coronavirus infection (particularly infection with SARS-CoV-2). A further problem of the present invention is to provide an effective coronavirus vaccine that can be stored and transported in conditions other than frozen, and that enables rapid and large-scale production of the coronavirus vaccine.

[0011] As further defined in the claims and as inherent herein, the above-mentioned problems are solved, in particular, by nucleic acids (such as RNA or DNA) provided by the present invention. These nucleic acids have one or more coding sequences encoding one or more antigenic peptides or antigenic proteins derived from the coronavirus SARS-CoV-2.

[0012] Furthermore, preferably, the nucleic acids described above, or compositions / vaccines containing such nucleic acids, satisfy at least some of the following advantageous characteristics. • Nucleic acids are translated at the injection / vaccination site (such as the muscle). • Low doses and regimens highly effectively induce an antigen-specific immune response against the encoded SARS-CoV-2 protein. It is suitable for vaccination of infants and / or newborns and the elderly (especially the elderly). • Suitable for intramuscular administration of the composition / vaccine. It induces a specific and functional humoral immune response to coronaviruses (such as SARS-CoV-2). • Induces a broad and functional T-cell response to coronaviruses (such as SARS-CoV-2). It induces B-cell memory specific to coronaviruses (such as SARS-CoV-2). • Induce functional antibodies that can effectively neutralize viruses (such as SARS-CoV-2). • Induce functional antibodies that can effectively neutralize emerging SARS-CoV-2 variants. • Induces IgA antibodies in the mucosa, thereby inducing mucosal immunity mediated by IgA. • Induces balanced B-cell and T-cell responses. • Induces protective immunity against coronavirus infection (such as SARS-CoV-2 or its emerging variants). • Rapidly initiates immune defense against coronaviruses (such as SARS-CoV-2). • The immune response induced against coronaviruses (such as SARS-CoV-2) is long-lasting. • Does not promote infection with SARS-CoV-2 due to vaccination or immunopathological effects. • Nucleoside-based SARS-CoV-2 vaccines do not induce antibody-dependent enhancement (ADE). • The vaccine should not cause excessive systemic cytokine or chemokine reactions after administration, which could lead to undesirable hyperreactiveness in the vaccine. • The vaccine is well-tolerated, has no side effects, and is non-toxic. • Nucleic acid-based vaccines have the advantage of characteristic stability. • Coronavirus vaccine production is rapid, applicable, simple, and scalable. • There are advantages to vaccination regimens that provide sufficient protection with one or two doses. • The vaccine regimen has the advantage of providing sufficient protection with only a small dose of the composition / vaccine.

[0013] [Definition] For clarity and readability, the following definitions are provided. Any technical features mentioned in these definitions can be read and applied to each and all embodiments of the present invention. Further definitions and descriptions may be specifically provided in connection with these embodiments.

[0014] Percentages related to numbers should be understood as relative values ​​to the total number of individual items. In other examples, unless the context clearly indicates otherwise, percentages should be understood as percentages of weight (weight %).

[0015] Approximately: The term "approximately" is used when the determinant or value does not need to be identical (100%). Therefore, "approximately" means that there is a variation of 0.1% to 20% in the determinant or value. This variation is preferably 0.1% to 10%, and particularly 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. As those skilled in the art will understand, for example, a parameter or determinant may vary slightly depending on the method by which that parameter is determined. For example, when a determinant or value such as length is defined herein as "approximately 1000 nucleotides," there may be a variation of 0.1% to 20% in that length. This displacement is preferably 0.1% to 10%, and particularly 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Thus, as a person skilled in the art will understand from this specific example, the length displacement is 1 to 200 nucleotides. This displacement is preferably 1 to 200 nucleotides, and particularly 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 nucleotides.

[0016] Adaptive Immune Response: In this specification, the term “adaptive immune response” will be recognized and understood by those skilled in the art. This term is intended to represent, for example, an antigen-specific response of the immune system (adaptive immune system). Antigen specificity enables a controlled response that is specific to a pathogen or cells infected with a pathogen. Typically, it is memory cells (B cells) that enable such controlled responses in the body. In relation to the present invention, the antigen is given by one or more antigenic peptides or nucleic acids (such as RNA or DNA) encoding antigenic proteins derived from coronavirus. Preferably, the coronavirus is SARS-CoV-2 (nCoV-2019).

[0017] Antigen: In this specification, the term “antigen” will be recognized and understood by those skilled in the art. The term is intended to represent, for example, a substance recognized by the immune system (preferably the adaptive immune system). This substance can induce an antigen-specific immune response. An example of an antigen-specific immune response is the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be a peptide or protein presented to T cells by MHC, or a part thereof. Also, a peptide or protein fragment, variant, and derivative having one or more epitopes are understood to be an antigen in relation to the present invention. An example of a peptide or protein is one derived from the spike protein (S) of a coronavirus (preferably SARS-CoV-2 (nCoV-2019)). In relation to the present invention, an antigen may also be a translation product of a given nucleic acid identified herein.

[0018] Antigenic peptides or antigenic proteins: The terms “antigenic peptides or antigenic proteins” or “immunogenic peptides or immunogenic proteins” are recognized and understood by those skilled in the art. These terms refer to peptides or proteins derived from, for example, (antigenic or immunogenic) proteins. These peptides or proteins stimulate the body’s adaptive immune system, resulting in an adaptive immune response. Therefore, an antigenic / immunogenic peptide or protein has one or more epitopes (as defined in this section) or an antigen (as defined in this section) of the protein (as defined in this section) from which it is derived. An example of such a protein is the spike protein (S) of the coronavirus (preferably SARS-CoV-2 (nCoV-2019)).

[0019] Cationicity: Unless it is clear from a specific context that it has a different meaning, the term "cationic" means that the respective structure is positively charged. This positive charge may be permanent or temporary. If temporary, it becomes positively charged in response to specific conditions such as pH. Therefore, the term "cationic" includes both "permanently cationic" and "cationizable."

[0020] Cationizable: In this specification, the term “cationizable” means that a compound, group, or atom is positively charged in a low pH environment and not positively charged in a high pH environment. Furthermore, in a non-aqueous environment where pH cannot be determined, a cationizable compound, group, or atom is positively charged when the hydrogen ion concentration is high and not positively charged when the hydrogen ion concentration or activity is low. The pH or hydrogen ion concentration at which the presence or absence of charge changes varies depending on the individual properties of the cationizable or polycationizable compound (in particular, depending on the pKa of the cationizable group or atom). In a diluted aqueous environment, the proportion of positively charged cationizable compounds, groups, or atoms can be estimated by the so-called Henderson-Hasselbalch formula, which is well known to those skilled in the art. For example, in some embodiments, if a compound or part is cationizable, it is positively charged at pH approximately 1–9. The pH at which it is positively charged is preferably 4–9, 5–8, or 6–8, more preferably less than 9, less than 8, or less than 7, and most preferably the physiological pH. Physiological pH is, for example, about 7.3 to 7.4, and refers to physiological conditions, particularly the physiological salt conditions of cells in vivo. In other embodiments, preferably, the cationizable compound or moiety is mainly neutral at physiological pH (e.g., about 7.0 to 7.4) but positively charged at low pH. In some embodiments, the pKa of the cationizable compound or moiety is preferably about 5 to about 7.

[0021] Coding Sequence / Coding Region: In this specification, the terms “coding sequence” or “coding region” and their abbreviation “cds” will be recognized and understood by those skilled in the art. The terms are intended to represent, for example, a sequence of triplets of multiple nucleotides that can be translated into a peptide or protein. In relation to the present invention, the coding sequence may be a DNA sequence consisting of a number of nucleotides divisible by 3, and is preferably an RNA sequence. The coding sequence begins with a start codon and preferably ends with a stop codon.

[0022] Derived from: In this specification, when the term “derived from” is used in relation to nucleic acids (i.e., when referring to nucleic acids derived from (another) nucleic acid), the term means nucleic acids derived from (another) nucleic acid. The sequence identity between this nucleic acid and the nucleic acid from which it is derived is, for example, 60% or more, 70% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. As those skilled in the art will understand, sequence identity is usually calculated between nucleic acids of the same type (DNA sequences or RNA sequences). Therefore, as would be understood, if "DNA is derived from RNA" or "RNA is derived from DNA," the first step is to convert the RNA sequence to the corresponding DNA sequence (in particular, by substituting uracil (U) with thymidine (T) in the sequence). Or, conversely, convert the DNA sequence to the corresponding RNA sequence (in particular, by substituting T with U in the sequence). Next, the sequence identity of the DNA sequence or RNA sequence is determined. Preferably, "nucleic acid derived from nucleic acid" also refers to a nucleic acid that has been modified compared to the nucleic acid from which it originates. Examples of this include modifications that increase RNA stability or modifications that prolong and / or increase protein production. In relation to amino acid sequences (such as antigenic peptides or antigenic proteins), the term "derived from" means an amino acid sequence derived from another amino acid sequence. The sequence identity between this amino acid sequence and the amino acid sequence from which it is derived is, for example, 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0023] Epitope: In this specification, the term “epitope” (also referred to in the art as “antigen determinant”) is as recognized and understood by those skilled in the art. The term is intended to represent, for example, T cell epitopes and B cell epitopes. T cell epitopes (or portions of antigenic peptides or antigenic proteins) preferably have fragments about 6 to about 20 amino acids or more in length. For example, the length of fragments modified and presented by MHC class I molecules is preferably about 8 to about 10 amino acids (e.g., 8, 9, or 10 amino acids; or even 11 or 12 amino acids). The length of fragments modified and presented by MHC class II molecules is preferably about 13 to about 20 amino acids or more. Typically, these fragments are recognized by T cells in the form of a complex consisting of the peptide fragment and the MHC molecule. That is, the fragments are not usually recognized in their original form. B cell epitopes are typically fragments located on the outer surface of a protein or peptide antigen (in its original form). The length of this fragment is preferably 5 to 15 amino acids, more preferably 5 to 12 amino acids, and even more preferably 6 to 9 amino acids. This fragment can be recognized by an antibody in its original form. Furthermore, such an epitope of a protein or peptide may be selected from any of the variants of the protein or peptide described herein. In this regard, the epitope may be structural or discontinuous. Such an epitope consists of multiple segments of the protein or peptide described herein. These segments are discontinuous in the amino acid sequence of the protein or peptide described herein, but together form a three-dimensional structure. Alternatively, the epitope may be a continuous or linear epitope consisting of a single polypeptide chain.

[0024] Fragment: In this specification, when used in relation to nucleic acid sequences (such as RNA or DNA) or amino acid sequences, the term "fragment" can usually refer to a shortened portion of a full-length sequence, such as a nucleic acid sequence or amino acid sequence. Therefore, a fragment typically consists of the same type of sequence as the corresponding full-length sequence. Preferably, in relation to the present invention, a sequence fragment consists of a contiguous portion of the subject (such as a nucleotide or amino acid). A sequence fragment corresponds to a contiguous portion of the subject in the molecule from which the fragment originates. The contiguous portion represents 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the entire molecule (full-length molecule) from which the fragment originates. An example of a molecule from which a fragment originates is the coronavirus spike protein (S), preferably derived from SARS-CoV-2 (nCoV-2019). In this specification, when used in relation to proteins or peptides, the term "fragment" can usually refer to sequences of proteins or peptides described herein. In other words, the amino acid sequence is shortened at the N-terminus and / or C-terminus compared to the amino acid sequence of the original protein. Thus, such shortening may occur at the amino acid level or at the corresponding nucleic acid level. Sequence identity to such fragments described herein may preferably refer to the entire protein or peptide described herein (or to the entire nucleic acid molecule encoding the protein or peptide). A protein or peptide fragment may have one or more epitopes of the protein or peptide.

[0025] Heterogeneous: In this specification, when used in relation to nucleic acid sequences or amino acid sequences, the terms “heterogeneous” or “heterogeneous sequence” refer to sequences (RNA, DNA, amino acids, etc.) that should be understood to originate from another gene, another allele, another species, or a virus. If two sequences do not originate from the same gene or allele, they are generally understood to be heterogeneous. That is, even if heterogeneous sequences originate from the same organism or virus, they do not exist in nature in the same nucleic acid or protein.

[0026] Humoral Immune Response: The terms “humoral immunity” or “humoral immune response” are recognized and understood by those skilled in the art. These terms are intended to represent, for example, antibody production mediated by B cells, and in any configuration, also the incidental processes associated with antibody production. Typically, the humoral immune response can be characterized by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation, etc. Humoral immunity also refers to the effector function of antibodies. Examples of effector function include neutralization of pathogens and toxins, classical complement activation, and promotion of phagocytosis and pathogen elimination by opsonins.

[0027] (Sequence) Identity: When used herein in relation to nucleic acid sequences or amino acid sequences, the term “identity” will be recognized and understood by those skilled in the art. The term is intended to represent, for example, the proportion of two sequences that are identical. To determine the proportion of two sequences that are identical (for example, nucleic acid sequences or amino acid sequences (aa sequences) described herein, preferably the aa sequences encoded by the nucleic acid sequences described herein or the aa sequences themselves), the sequences are aligned and then compared with each other. For example, a position in the first sequence may be compared with a corresponding position in the second sequence. If a position in the first sequence and a corresponding position in the second sequence are the same residue, then the two sequences are identical at that position. If the residues are different, then the two sequences are different at that position. If the second sequence has an insertion relative to the first sequence, a gap may be inserted into the first sequence before further alignment. If the second sequence has a deletion relative to the first sequence, a gap may be inserted into the second sequence before further alignment. The ratio of identical positions between two arrays is a function obtained by dividing the number of identical positions by the total number of positions. The total number of positions includes positions that exist in only one of the arrays. The ratio of identical positions between two arrays may also be determined by an algorithm. An example of such an algorithm is the one built into the BLAST program.

[0028] Immunogen, Immunogenicity: The terms “immunogen” or “immunogenicity” will be recognized and understood by those skilled in the art. These terms are intended to represent, for example, compounds that stimulate / induce an immune response. Preferably, an immunogen is a peptide, polypeptide, or protein. In the sense of the present invention, an immunogen is a translation product of a given nucleic acid. This nucleic acid has one or more coding sequences encoding one or more antigenic peptides / proteins derived from the spike protein (S) of SARS-CoV-2 (nCoV-2019) as described herein. Typically, an immunogen induces an adaptive immune response.

[0029] Immune response: The term “immune response” is recognized and understood by those skilled in the art. This term is intended to represent, for example, a specific response of the adaptive immune system to a particular antigen (so-called specific response or adaptive immune response), a nonspecific response of the innate immune system (so-called nonspecific response or innate immune response), or a combination of these.

[0030] Immune System: The term "immune system" is recognized and understood by those skilled in the art. This term is intended to represent, for example, the biological system that protects an organism from infection. When a pathogen penetrates an organism's physical barriers and invades it, the innate immune system produces a rapid and nonspecific response. If the pathogen evades this innate response, vertebrates have a second layer of defense: the adaptive immune system. The immune system adapts its response to infection, improving its recognition of the pathogen. This improved response is maintained as immunological memory even after the pathogen has been eliminated. Because of this immunological memory, the adaptive immune system can launch a rapid and powerful attack each time it encounters the same pathogen. Therefore, the immune system includes both the innate and adaptive immune systems. Both of these immune systems typically have so-called humoral and cellular components.

[0031] Innate Immune System: The term "innate immune system (also known as nonspecific immune system)" is recognized and understood by those skilled in the art. This term refers to a system typically containing cells and mechanisms that protect the host in a nonspecific manner, for example, from infection by other organisms. In other words, cells in this innate system recognize and respond to pathogens in a general manner, but do not confer long-term or protective immunity to the host, as the adaptive immune system does. The innate immune system can be activated by pattern recognition receptor ligands. Examples of pattern recognition receptors include Toll-like receptors, NOD-like receptors, or RIG-I-like receptors.

[0032] Lipidoid compounds: Lipidoid compounds (also simply called lipidoids) are lipid-like compounds. That is, they are amphiphilic compounds that have physical properties similar to lipids. In relation to this invention, the term "lipid" is considered to include lipidoid compounds.

[0033] Permanently Cationic: In this specification, the term “permanently cationic” will be recognized and understood by those skilled in the art. This term means that a compound, group, or atom is positively charged, for example, in any pH or hydrogen ion-active environment. Typically, the positive charge arises as a result of the presence of a quaternary nitrogen atom. If a compound has multiple such positive charges, it may be referred to as “permanently polycationic.”

[0034] Stabilized RNA: The term "stabilized RNA" refers to RNA that has been modified to be more resistant to degradation compared to unmodified RNA. Examples of degradation include environmental factors or enzymatic digestion (such as degradation by exonucleases or endonucleases). Preferably, in relation to the present invention, the stabilized RNA is stabilized in cells. Examples of cells include prokaryotic cells or eukaryotic cells, and preferably mammalian cells (such as human cells). For example, to preserve a composition containing stabilized RNA, the stabilization effect may be exerted extracellularly (e.g., in a buffer solution).

[0035] T-cell response: In this specification, the terms “cellular immunity,” “cellular immune response,” or “cellular T-cell response” will be recognized and understood by those skilled in the art. These terms are intended to represent, for example, the activation of macrophages, natural killer cells (NKs), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. In more general terms, cellular immunity is based on the activation of cells in the immune system, not on antibodies. Typically, a cellular immune response may be characterized by the activation of specific immune cells or other cells, such as antigen-specific cytotoxic T lymphocytes that can induce apoptosis in cells, or dendritic cells that present epitopes of foreign antigens on their surface.

[0036] Variant (of a sequence): In this specification, when used in relation to nucleic acid sequences, the term “variant” will be recognized and understood by those skilled in the art. The term is intended to represent, for example, a variant of a nucleic acid sequence derived from another nucleic acid sequence. For example, a variant of a nucleic acid sequence may have one or more nucleotide deletions, insertions, additions and / or substitutions compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more identical to the nucleic acid sequence from which the variant is derived. A variant is a functional variant if it possesses 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the functions of the derived sequence. The nucleotide identity of a nucleic acid sequence variant may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more over 10 or more nucleotides of the nucleic acid sequence, 20 or more, 30 or more, 50 or more, 75 or more, or 100 or more.

[0037] In this specification, when used in relation to proteins or peptides, the term “variant” is intended to represent a variant of a protein or peptide having an amino acid sequence that differs from the original sequence in, for example, one or more mutations / substitutions (substitutions, insertions, and / or deletions of one or more amino acids). Preferably, the fragment and / or variant has the same or equivalent specific antigenic properties (immunogenic variant, antigenic variant). Insertions and substitutions may occur, among other things, at positions in the sequence that do not change the three-dimensional structure or affect the binding region. Changes in the three-dimensional structure due to insertions or deletions can be easily determined, for example, using a CD spectrum (circular dichroism spectrum). The amino acid identity of a protein or peptide variant may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more over 10, 20 or more, 30 or more, 50 or more, 75 or more, or 100 or more amino acids of the protein or peptide. Preferably, the protein variant includes a functional variant of the protein. In relation to the present invention, a functional protein variant is a variant that exhibits substantially the same immunogenicity as the derived protein, or exhibits 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of immunogenicity.

[0038] [Summary of the Invention] This invention is based on the inventors' remarkable finding that one or more peptides or proteins derived from the coronavirus SARS-CoV-2 (formerly known as nCoV-2019), obtained from the coding sequences of nucleic acids (such as RNA), can be effectively expressed in human cells (Examples 2a, 2b, and 2c). Even more remarkably, administration of compositions containing such nucleic acids (such as RNA) induces an antigen-specific immune response against coronaviruses (particularly SARS-CoV-2) (see Examples).

[0039] Furthermore, unexpectedly, the inventors demonstrate that the coding RNA of the present invention induces high levels of functional antibodies. These antibodies exhibit high viral neutralizing antibody titers (VNTs) and T cell responses. The vaccine induces bipositive CD4+ T cells and CD8+ T cells (see, e.g., Examples 7 and 10). The vaccine also protects hamsters and non-human primates from SARS-CoV-2 attack infection (see Examples 9 and 15). Thus, these findings indicate that the coding RNA or composition / vaccine of the present invention is suitable for use as a vaccine (such as a vaccine in human subjects).

[0040] These findings form the basis for the supply of nucleic acid-based coronavirus vaccines.

[0041] In a first aspect, the present invention provides nucleic acids for coronavirus vaccines (preferably SARS-CoV-2 coronavirus vaccines). The nucleic acids have one or more coding sequences. The coding sequences encode one or more antigenic peptides or antigenic proteins of the SARS-CoV-2 coronavirus. Alternatively, the coding sequences encode an immunogenic fragment or immunogenic variant thereof.

[0042] In a second embodiment, the present invention provides a composition (preferably an immunogenic composition). The composition comprises one or more nucleic acids according to the first embodiment. Preferably, the composition comprises one or more nucleic acids (e.g., one or more coding RNAs) that are complexed, encapsulated, or associated with one or more lipids, thereby forming lipid nanoparticles.

[0043] In a third aspect, the present invention provides an antigenic polypeptide for a coronavirus vaccine. Preferably, the antigenic polypeptide is for a SARS-CoV-2 composition or vaccine.

[0044] In a fourth embodiment, the present invention provides a coronavirus vaccine (preferably a SARS-CoV-2 vaccine). The vaccine comprises one or more nucleic acids of the first embodiment, a composition of the second embodiment, or one or more polypeptides of the third embodiment.

[0045] In a fifth aspect, the present invention provides a kit or kit of parts, comprising one or more nucleic acids of the first aspect, one or more compositions of the second aspect, one or more polypeptides of the third aspect, and one or more vaccines of the fourth aspect.

[0046] In a sixth embodiment, the present invention provides a combination comprising two or more isolated elements, one or more of which are selected from two nucleic acids of the first embodiment, two or more compositions of the second embodiment, two or more polypeptides of the third embodiment, and two or more vaccines of the fourth embodiment.

[0047] Further aspects of the present invention relate to methods for treating or preventing coronavirus infection (preferably infection with SARS-CoV-2) in subjects. The invention also relates to first and second pharmaceutical uses of nucleic acids, compositions, and vaccines. Furthermore, it relates to methods for producing the above-mentioned nucleic acids, compositions, or vaccines.

[0048] [Detailed description of the invention] This application also submits an electronic sequence listing (WIPO Standard ST.25). This sequence listing constitutes part of this specification. The information contained in the sequence listing is incorporated in its entirety by reference. Where a sequence number is referred to in this specification, it refers to the corresponding nucleic acid sequence or amino acid sequence (aa sequence) that has the same identifier in the sequence listing. For many sequences, the sequence listing contains additional details. For example, it contains additional information on specific structural features, sequence optimization, GenBank (NCBI) or GISAID (epi) identifiers, and coding capabilities. In particular, such information is included in the WIPO Standard ST.25 sequence listing. <223> Write it in the column. Therefore, <223> The information contained in the section is explicitly included in this specification. Furthermore, this information should be understood as an integral part of this specification, forming the basis of the invention.

[0049] [Nucleic acid for coronavirus vaccine] In a first embodiment, the present invention relates to nucleic acids suitable for coronavirus vaccines.

[0050] It should be noted that certain features and embodiments described in relation to the first aspect of the present invention (nucleic acids of the present invention) are also applicable to the second aspect (compositions of the present invention), the third aspect (polypeptides of the present invention), the fourth aspect (vaccines of the present invention), the fifth aspect (kits or kits of parts of the present invention), or further aspects (such as medical uses and therapeutic methods).

[0051] Coronaviruses are classified into the genera Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus, and unclassified coronaviruses. Coronaviruses are genetically highly variable. Furthermore, individual virus species can cross species barriers and infect hosts of multiple species. Examples of human coronaviruses include SARS-related coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), and coronavirus SARS-CoV-2 (formerly known as "Wuhan human coronavirus" or "nCoV-2019"). Therefore, nucleic acids are suitable for vaccines against coronaviruses. Preferably, they are suitable for vaccines against coronaviruses that are pathogens to humans. Most preferably, they are suitable for vaccines against the novel coronavirus SARS-CoV-2 (nCoV-2019).

[0052] The terms “nucleic acid” or “nucleic acid molecule” will be recognized and understood by those skilled in the art. Preferably, in this specification, the terms “nucleic acid” or “nucleic acid molecule” refer to DNA(molecule) or RNA(molecule). Preferably, this term is used synonymously with the term “polynucleotide.” Preferably, a nucleic acid or nucleic acid molecule is a polymer containing or consisting of nucleotide monomers. In this polymer, the nucleotide monomers are covalently bonded to each other by phosphodiester bonds of a sugar / phosphate backbone. The term “nucleic acid molecule” also includes modified nucleic acid molecules. Examples of modified nucleic acid molecules include base-modified DNA or RNA molecules, sugar-modified DNA or RNA molecules, and backbone-modified DNA or RNA molecules (as defined herein).

[0053] The nucleic acid (such as DNA or RNA) of the first embodiment can form the basis of a nucleic acid composition or vaccine. Generally, protein-based vaccines or attenuated live vaccines are expensive to manufacture and are not the best option for use in developing countries. Furthermore, protein-based vaccines or attenuated live vaccines require long development periods and are not suitable for rapid responses to pandemic virus outbreaks (such as the 2019 / 2020 coronavirus SARS-CoV-2 outbreak). In contrast, the nucleic acid vaccine according to the present invention can be manufactured rapidly and at low cost. Therefore, compared to conventional vaccines, the nucleic acid vaccine of the present invention can be manufactured at a significantly lower cost and more quickly. This is particularly advantageous for use in developing countries. A further advantage of the nucleic acid vaccine of the present invention is its higher temperature stability compared to protein-based vaccines or peptide-based vaccines. However, polypeptide-based vaccines are also inherent in the scope of the present invention (see, for example, the third embodiment).

[0054] The terms “nucleic acid sequence,” “DNA sequence,” and “RNA sequence” are recognized and understood by those skilled in the art. These terms, for example, refer to a specific and unique sequence of nucleotides.

[0055] In a preferred embodiment of the first aspect, the nucleic acid has one or more coding sequences. The coding sequences encode one or more antigenic peptides or antigenic proteins derived from the SARS-CoV-2 (nCoV-2019) coronavirus. Alternatively, the coding sequences encode an immunogenic fragment or immunogenic variant thereof.

[0056] The term "antigenic peptide or antigenic protein derived from SARS-CoV-2 coronavirus" should be understood as follows: (i) an antigen from SARS-CoV-2 coronavirus in which the amino acid sequence of the antigenic peptide or antigenic protein (or fragment thereof) is identical to that of the SARS-CoV-2 coronavirus protein (or fragment thereof). (ii) an antigen derived from SARS-CoV-2 coronavirus in which the amino acid sequence of the antigenic peptide or antigenic protein (or fragment thereof) is not identical to that of the corresponding SARS-CoV-2 coronavirus protein (or fragment thereof).

[0057] Therefore, in a preferred embodiment of the first aspect, the nucleic acid has one or more coding sequences. The coding sequences encode one or more antigenic peptides or antigenic proteins that are of or derived from the SARS-CoV-2 (nCoV-2019) coronavirus. Alternatively, the coding sequences encode an immunogenic fragment or immunogenic variant thereof.

[0058] The term “antigenic peptide or antigenic protein of or derived from SARS-CoV-2 (nCoV-2019) coronavirus” should be understood as follows: (i) an antigen from SARS-CoV-2 coronavirus whose amino acid sequence of antigenic peptide or antigenic protein (or fragment thereof) is identical to the sequence of the SARS-CoV-2 coronavirus protein (or fragment thereof).

[0059] (ii) Antigens derived from SARS-CoV-2 coronavirus, which are antigenic peptides or The amino acid sequence of the antigenic protein (or fragment thereof) is not identical to the sequence of the corresponding SARS-CoV-2 coronavirus protein (or fragment thereof).

[0060] In a preferred embodiment, the nucleic acid has one or more coding sequences. The coding sequences encode one or more antigenic peptides or antigenic proteins that are of or derived from the SARS-CoV-2 (nCoV-2019) coronavirus. Alternatively, the coding sequences encode an immunogenic fragment or immunogenic variant thereof. Here, the nucleic acid has one or more heterologous untranslated regions (UTRs).

[0061] The terms “untranslated region,” “UTR,” or “UTR element” are recognized and understood by those skilled in the art. These terms are intended to represent, for example, a portion of a nucleic acid molecule typically located at the 5' or 3' of the coding sequence. UTRs are not translated into proteins. UTRs may be portions of nucleic acids (such as DNA or RNA). UTRs may contain elements that regulate gene expression (also called regulatory elements). Regulatory elements may include ribosome binding sites, miRNA binding sites, and so on.

[0062] In this specification, the terms “Human Coronavirus 2019,” “Wuhan Human Coronavirus (WHCV),” “nCoV-2019 Coronavirus,” “nCoV-2019,” “Wuhan Seafood Market Pneumonia Virus,” “Wuhan Coronavirus,” “WHCV Coronavirus,” “HCoV-19,” “SARS2,” “COVID-19 Virus,” “hCov-19,” “SARS-CoV-2,” or “Coronavirus SARS-CoV-2” may be used interchangeably in this invention. These terms relate to the coronavirus that caused a novel pandemic. This coronavirus appeared in the city of Wuhan, People's Republic of China, at the end of 2019 to the beginning of 2020 and caused the disease COVID-19. According to the WHO, this virus was formally named “SARS-CoV-2,” and the disease associated with it was formally named “COVID-19” (February 2020).

[0063] The SARS-CoV-2 virus belongs to the Coronaviridae family, specifically to the Orthocoronavirus family, and more specifically to the Betacoronavirus genus. Examples of SARS-CoV-2 coronaviruses include (but are not limited to) the isolates listed in Lists A and B below.

[0064] List A: Exemplary SARS-CoV-2 coronavirus isolates (EPI / GISAID) EPI_ISL_402119、EPI_ISL_402120、EPI_ISL_402121、EPI_ISL_402123、EPI_ISL_402124 (hCoV-19 / Wuhan / WIV04 / 2019)、EPI_ISL_402125、EPI_ISL_402127、EPI_ISL_402128 (hCoV-19 / Wuhan / WIV05 / 2019; WIV05; SARS-CoV-2 / Wuhan / WIV05 / 2019__EPI_ISL_402128), EPI_ISL_402129, EPI_ISL_402130, EPI_ISL_402131, EPI_ISL_402132, EPI_ISL_403928, EPI_ISL_403929, EPI_ISL_403930, EPI_ISL_403931, EPI_ISL_403932, EPI_ISL_403933, EPI_ISL _403934、EPI_ISL_403935、EPI_ISL_403936、EPI_ISL_403937、EPI_ISL_403962、EPI_ISL_403963、EPI_ISL_404227、EPI_ISL_404228、EPI_ISL_404253、EPI_ISL_404895、EPI_ISL_405839、EPI_ISL_406030、EPI_ISL_406031、EPI_ISL_406034、 EPI_ISL_406036、EPI_ISL_406223、EPI_ISL_406531、EPI_ISL_406533、EPI_ISL_406534、EPI_ISL_406535、EPI_ISL_406536、EPI_ISL_406538、EPI_ISL_406592、EPI_ISL_406593、EPI_ISL_406594、EPI_ISL_406595、EPI_ISL_406596、EPI_ISL _406597、EPI_ISL_406798、EPI_ISL_406800、EPI_ISL_406801、EPI_ISL_406844、EPI_ISL_406862、EPI_ISL_406716、EPI_ISL_406717、EPI_ISL_406970、EPI_ISL_406973、EPI_ISL_407071、EPI_ISL_407073、EPI_ISL_407079、EPI_ISL_407084、EPI_ISL_407193、EPI_ISL_407214、EPI_ISL_407215、EPI_ISL_407313、EPI_ISL_407893、EPI_ISL_407894、EPI_ISL_407896、EPI_ISL_407976、EPI_ISL_407987、EPI_ISL_407988、EPI_ISL_408008、EPI_ISL_408009、EPI_ISL_408010、EPI_ISL_408430、EPI_ISL_408431、EPI_ISL_408478、EPI_ISL _408479、EPI_ISL_408480、EPI_ISL_408481、EPI_ISL_408482、EPI_ISL_408484、EPI_ISL_408486、EPI_ISL_408488、EPI_ISL_408489、EPI_ISL_408514、EPI_ISL_408515、EPI_ISL_408665、EPI_ISL_408666、EPI_ISL_408667、EPI_ISL_408668、EPI_ISL_408669、EPI_ISL_408670、EPI_ISL_408976、 EPI_ISL_408977、EPI_ISL_409067、EPI_ISL_410044、EPI_ISL_410045、EPI_ISL_410218、EPI_ISL_410301、EPI_ISL_410486、EPI_ISL_410531、EPI_ISL_410532、EPI_ISL_410535、EPI_ISL_410536、EPI_ISL_410537、EPI_ISL_410538、EPI_ISL_410539、EPI_ISL_410540、EPI_ISL_410541、EPI_ISL _410542、EPI_ISL_410713、EPI_ISL_410714、EPI_ISL_410715、EPI_ISL_410716、EPI_ISL_410717、EPI_ISL_410718、EPI_ISL_410719、EPI_ISL_410720、EPI_ISL_410984、EPI_ISL_411060、EPI_ISL_411066、EPI_ISL_411218、EPI_ISL_411219、EPI_ISL_411220、EPI_ISL_411902、EPI_ISL_411915、EPI_ISL_411926、EPI_ISL_411927、EPI_ISL_411929、EPI_ISL_411950、EPI_ISL_411951、EPI_ISL_411952、EPI_ISL_411953、EPI_ISL_411954、EPI_ISL_411955、EPI_ISL_411956、EPI_ISL_411957、EPI_ISL_412026、EPI_ISL_412028、EPI_ISL_412029、EPI_ISL_412030、EPI_ISL_412459、EPI_ISL _412862、EPI_ISL_412869、EPI_ISL_412870、EPI_ISL_412871、EPI_ISL_412872、EPI_ISL_412873、EPI_ISL_412898、EPI_ISL_412899、EPI_ISL_412912、EPI_ISL_412966、EPI_ISL_412967、EPI_ISL_412968、EPI_ISL_412969、EPI_ISL_412970、EPI_ISL_412972、EPI_ISL_412973、EPI_ISL_412974、 EPI_ISL_412975、EPI_ISL_412978、EPI_ISL_412979、EPI_ISL_412980、EPI_ISL_412981、EPI_ISL_412982、EPI_ISL_412983、EPI_ISL_413014、EPI_ISL_413015、EPI_ISL_413016、EPI_ISL_413017、EPI_ISL_413018、EPI_ISL_413021、EPI_ISL_413022、EPI_ISL_413023、EPI_ISL_413024、EPI_ISL _413213、EPI_ISL_413214、EPI_ISL_413455、EPI_ISL_413456、EPI_ISL_413457、EPI_ISL_413458、EPI_ISL_413459、EPI_ISL_413485、EPI_ISL_413486、EPI_ISL_413488、EPI_ISL_413489、EPI_ISL_413490、EPI_ISL_413513、EPI_ISL_413514、EPI_ISL_413515、EPI_ISL_413516、EPI_ISL_413518、EPI_ISL_413519、EPI_ISL_413520、EPI_ISL_413521、EPI_ISL_413522、EPI_ISL_413523、EPI_ISL_413555、EPI_ISL_413557、EPI_ISL_413558、EPI_ISL_413559、EPI_ISL_413560、EPI_ISL_413562、EPI_ISL_413563、EPI_ISL_413566、EPI_ISL_413572、EPI_ISL_413573、EPI_ISL_413577、EPI_ISL _413579、EPI_ISL_413580、EPI_ISL_413581、EPI_ISL_413582、EPI_ISL_413583、EPI_ISL_413584、EPI_ISL_413587、EPI_ISL_413589、EPI_ISL_413590、EPI_ISL_413591、EPI_ISL_413592、EPI_ISL_413593、EPI_ISL_413594、EPI_ISL_413595、EPI_ISL_413596、EPI_ISL_413597、EPI_ISL_413598、 EPI_ISL_413599、EPI_ISL_413600、EPI_ISL_413602、EPI_ISL_413603、EPI_ISL_413604、EPI_ISL_413606、EPI_ISL_413607、EPI_ISL_413608、EPI_ISL_413609、EPI_ISL_413610、EPI_ISL_413611、EPI_ISL_413612、EPI_ISL_413613、EPI_ISL_413614、EPI_ISL_413615、EPI_ISL_413616、EPI_ISL _413617、EPI_ISL_413618、EPI_ISL_413619、EPI_ISL_413620、EPI_ISL_413621、EPI_ISL_413622、EPI_ISL_413647、EPI_ISL_413648、EPI_ISL_413691、EPI_ISL_413692、EPI_ISL_413693、EPI_ISL_413694、EPI_ISL_413697、EPI_ISL_413711、EPI_ISL_413729、EPI_ISL_413746、EPI_ISL_413748、EPI_ISL_413749、EPI_ISL_413750、EPI_ISL_413751、EPI_ISL_413761、EPI_ISL_413791、EPI_ISL_413809、EPI_ISL_413852、EPI_ISL_413853、EPI_ISL_413854、EPI_ISL_413856、EPI_ISL_413857、EPI_ISL_413858、EPI_ISL_413860、EPI_ISL_413861、EPI_ISL_413862、EPI_ISL_413863、EPI_ISL _413928、EPI_ISL_413931、EPI_ISL_413996、EPI_ISL_413997、EPI_ISL_413999、EPI_ISL_414005、EPI_ISL_414006、EPI_ISL_414007、EPI_ISL_414008、EPI_ISL_414009、EPI_ISL_414011、EPI_ISL_414012、EPI_ISL_414019、EPI_ISL_414020、EPI_ISL_414021、EPI_ISL_414022、EPI_ISL_414023、 EPI_ISL_414027、EPI_ISL_414040、EPI_ISL_414041、EPI_ISL_414042、EPI_ISL_414043、EPI_ISL_414044、EPI_ISL_414045、EPI_ISL_414363、EPI_ISL_414366、EPI_ISL_414367、EPI_ISL_414368、EPI_ISL_414369、EPI_ISL_414414、EPI_ISL_414423、EPI_ISL_414428、EPI_ISL_414429、EPI_ISL _414433、EPI_ISL_414435、EPI_ISL_414439、EPI_ISL_414443、EPI_ISL_414445、EPI_ISL_414446、EPI_ISL_414451、EPI_ISL_414457、EPI_ISL_414468、EPI_ISL_414470、EPI_ISL_414476、EPI_ISL_414477、EPI_ISL_414479、EPI_ISL_414480、EPI_ISL_414481、EPI_ISL_414482、EPI_ISL_414483、EPI_ISL_414484、EPI_ISL_414485、EPI_ISL_414487、EPI_ISL_414500、EPI_ISL_414505、EPI_ISL_414509、EPI_ISL_414510、EPI_ISL_414511、EPI_ISL_414517、EPI_ISL_414519、EPI_ISL_414520、EPI_ISL_414521、EPI_ISL_414522、EPI_ISL_414523、EPI_ISL_414524、EPI_ISL_414525、EPI_ISL _414526、EPI_ISL_414527、EPI_ISL_414528、EPI_ISL_414529、EPI_ISL_414530、EPI_ISL_414531、EPI_ISL_414532、EPI_ISL_414534、EPI_ISL_414535、EPI_ISL_414545、EPI_ISL_414546、EPI_ISL_414547、EPI_ISL_414548、EPI_ISL_414549、EPI_ISL_414552、EPI_ISL_414554、EPI_ISL_414555、 EPI_ISL_414556、EPI_ISL_414557、EPI_ISL_414558、EPI_ISL_414559、EPI_ISL_414560、EPI_ISL_414561、EPI_ISL_414562、EPI_ISL_414564、EPI_ISL_414565、EPI_ISL_414566、EPI_ISL_414569、EPI_ISL_414571、EPI_ISL_414574、EPI_ISL_414577、EPI_ISL_414578、EPI_ISL_414579、EPI_ISL _414580、EPI_ISL_414586、EPI_ISL_414587、EPI_ISL_414588、EPI_ISL_414589、EPI_ISL_414590、EPI_ISL_414591、EPI_ISL_414592、EPI_ISL_414593、EPI_ISL_414594、EPI_ISL_414595、EPI_ISL_414596、EPI_ISL_414597、EPI_ISL_414600、EPI_ISL_414601、EPI_ISL_414616、EPI_ISL_414617、EPI_ISL_414618、EPI_ISL_414619、EPI_ISL_414620、EPI_ISL_414621、EPI_ISL_414622、EPI_ISL_414623、EPI_ISL_414624、EPI_ISL_414625、EPI_ISL_414626、EPI_ISL_414627、EPI_ISL_414628、EPI_ISL_414629、EPI_ISL_414630、EPI_ISL_414631、EPI_ISL_414632、EPI_ISL_414633、EPI_ISL _414635、EPI_ISL_414637、EPI_ISL_414638、EPI_ISL_414641、EPI_ISL_414642、EPI_ISL_414643、EPI_ISL_414646、EPI_ISL_414648、EPI_ISL_414663、EPI_ISL_414684、EPI_ISL_414685、EPI_ISL_414686、EPI_ISL_414687、EPI_ISL_414688、EPI_ISL_414689、EPI_ISL_414690、EPI_ISL_414691、 EPI_ISL_414692、EPI_ISL_414936、EPI_ISL_414937、EPI_ISL_414938、EPI_ISL_414940、EPI_ISL_414941、EPI_ISL_415105、EPI_ISL_415128、EPI_ISL_415129、EPI_ISL_415136、EPI_ISL_415141、EPI_ISL_415142、EPI_ISL_415147、EPI_ISL_415150、EPI_ISL_415151、EPI_ISL_415152、EPI_ISL _415153、EPI_ISL_415154、EPI_ISL_415155、EPI_ISL_415156、EPI_ISL_415157、EPI_ISL_415158、EPI_ISL_415159、EPI_ISL_415710、EPI_ISL_416426、EPI_ISL_416457、EPI_ISL_416481、EPI_ISL_416489、EPI_ISL_416491、EPI_ISL_416492、EPI_ISL_416514、EPI_ISL_416515、EPI_ISL_416516、EPI_ISL_416517、EPI_ISL_416518、EPI_ISL_416538、EPI_ISL_416539、EPI_ISL_416683、EPI_ISL_416685、EPI_ISL_416704、EPI_ISL_416711、EPI_ISL_416713、EPI_ISL_416715、EPI_ISL_416717、EPI_ISL_416744、EPI_ISL_416830、EPI_ISL_416831、EPI_ISL_416832、EPI_ISL_417020、EPI_ISL _417021、EPI_ISL_417022、EPI_ISL_417023、EPI_ISL_417024、EPI_ISL_417025、EPI_ISL_417026、EPI_ISL_417027、EPI_ISL_417028、EPI_ISL_417034、EPI_ISL_417200、EPI_ISL_417201、EPI_ISL_417202、EPI_ISL_417203、EPI_ISL_417204、EPI_ISL_417374、EPI_ISL_417375、EPI_ISL_417376、 EPI_ISL_417377、EPI_ISL_417379、EPI_ISL_417382、EPI_ISL_417408、EPI_ISL_417409、EPI_ISL_417410、EPI_ISL_417411、EPI_ISL_417412、EPI_ISL_417413、EPI_ISL_417420、EPI_ISL_417435、EPI_ISL_417436、EPI_ISL_417437、EPI_ISL_417438、EPI_ISL_417439、EPI_ISL_417440、EPI_ISL _417441、EPI_ISL_417442、EPI_ISL_417467、EPI_ISL_417468、EPI_ISL_417504、EPI_ISL_417505、EPI_ISL_417506、EPI_ISL_417507、EPI_ISL_417508、EPI_ISL_417509、EPI_ISL_417510、EPI_ISL_417512、EPI_ISL_417513、EPI_ISL_417514、EPI_ISL_417515、EPI_ISL_417516、EPI_ISL_417517、EPI_ISL_417526、EPI_ISL_417527、EPI_ISL_417528、EPI_ISL_417529、EPI_ISL_417530、EPI_ISL_417531、EPI_ISL_417532、EPI_ISL_417533、EPI_ISL_417534、EPI_ISL_417536、EPI_ISL_417537、EPI_ISL_417538、EPI_ISL_417539、EPI_ISL_417540、EPI_ISL_417541、EPI_ISL_417542、EPI_ISL _417543、EPI_ISL_417544、EPI_ISL_417545、EPI_ISL_417546、EPI_ISL_417547、EPI_ISL_417548、EPI_ISL_417550、EPI_ISL_417551、EPI_ISL_417552、EPI_ISL_417553、EPI_ISL_417554、EPI_ISL_417555、EPI_ISL_417556、EPI_ISL_417557、EPI_ISL_417558、EPI_ISL_417559、EPI_ISL_417560、 EPI_ISL_417561、EPI_ISL_417562、EPI_ISL_417563、EPI_ISL_417564、EPI_ISL_417565、EPI_ISL_417566、EPI_ISL_417567、EPI_ISL_417568、EPI_ISL_417569、EPI_ISL_417570、EPI_ISL_417571、EPI_ISL_417572、EPI_ISL_417573、EPI_ISL_417574、EPI_ISL_417575、EPI_ISL_417576、EPI_ISL _417577、EPI_ISL_417578、EPI_ISL_417579、EPI_ISL_417580、EPI_ISL_417581、EPI_ISL_417582、EPI_ISL_417583、EPI_ISL_417584、EPI_ISL_417585、EPI_ISL_417586、EPI_ISL_417587、EPI_ISL_417588、EPI_ISL_417589、EPI_ISL_417590、EPI_ISL_417591、EPI_ISL_417592、EPI_ISL_417593、EPI_ISL_417594、EPI_ISL_417595、EPI_ISL_417596、EPI_ISL_417597、EPI_ISL_417598、EPI_ISL_417599、EPI_ISL_417600、EPI_ISL_417601、EPI_ISL_417602、EPI_ISL_417603、EPI_ISL_417604、EPI_ISL_4 17605、EPI_ISL_417606、EPI_ISL_417607、EPI_ISL_417608、EPI_ISL_417609、EPI_ISL_417610、EPI_ISL_417611、EPI_ISL_417612、EPI_ISL_417613、EPI_ISL_417614、EPI_ISL_417615、EPI_ISL_417616、EPI_ ISL_417617、EPI_ISL_417618、EPI_ISL_417619、EPI_ISL_417620、EPI_ISL_417621、EPI_ISL_417622、EPI_ISL_417623、EPI_ISL_417624、EPI_ISL_417625、EPI_ISL_417626、EPI_ISL_417627、EPI_ISL_41762 8、EPI_ISL_417629、EPI_ISL_417630、EPI_ISL_417631、EPI_ISL_417632、EPI_ISL_417633、EPI_ISL_417634、EPI_ISL_417635、EPI_ISL_417636、EPI_ISL_417637、EPI_ISL_417638、EPI_ISL_417639、EPI_ISL、 _417640、EPI_ISL_417641、EPI_ISL_417642、EPI_ISL_417643、EPI_ISL_417644、EPI_ISL_417645、EPI_ISL_417646、EPI_ISL_417647、EPI_ISL_417648、EPI_ISL_417649、EPI_ISL_417650、EPI_ISL_417651、EPI_ISL_417652、EPI_ISL_417653、EPI_ISL_417654、EPI_ISL_417666、EPI_ISL_417667、E PI_ISL_417668、EPI_ISL_417669、EPI_ISL_417670、EPI_ISL_417671、EPI_ISL_417672、EPI_ISL_417676、EPI_ISL_417678、EPI_ISL_417680、EPI_ISL_417685、EPI_ISL_417699、EPI_ISL_417700、EPI_ISL_417703、EPI_ISL_417706、EPI_ISL_417709、EPI_ISL_417712、EPI_ISL_417716、EPI_ISL_417717 717、EPI_ISL_417724、EPI_ISL_417733、EPI_ISL_417737、EPI_ISL_417740、EPI_ISL_417742、EPI_ISL_417743、EPI_ISL_417746、EPI_ISL_417750、EPI_ISL_417752、EPI_ISL_417753、EPI_ISL_417754、EPI_ISL_417762、EPI_ISL_417763、EPI_ISL_417764、EPI_ISL_417766、EPI_ISL_417774、EPI_ISL_417766 SL_417808、EPI_ISL_417809、EPI_ISL_417813、EPI_ISL_417814、EPI_ISL_417815、EPI_ISL_417816、EPI_ISL_417818、EPI_ISL_417819、EPI_ISL_417820、EPI_ISL_417821、EPI_ISL_417822、EPI_ISL_417823、EPI_ISL_417824、EPI_ISL_417825、EPI_ISL_417826、EPI_ISL_417827、EPI_ISL_417829、EPI_ISL_417830、EPI_ISL_417831、EPI_ISL_417832、EPI_ISL_417833、EPI_ISL_417834、EPI_ISL_417835、EPI_ISL_417836、EPI_ISL_417837、EPI_ISL_417838、EPI_ISL_417839、EPI_ISL_417864、EPI_ISL_417917、EPI_ISL_417918、EPI_ISL_417920、EPI_ISL_417925、EPI_ISL_417926、EPI_ISL _417931、EPI_ISL_417932、EPI_ISL_417933、EPI_ISL_417935、EPI_ISL_417936、EPI_ISL_417937、EPI_ISL_417938、EPI_ISL_417939、EPI_ISL_417940、EPI_ISL_417941、EPI_ISL_417942、EPI_ISL_417943、EPI_ISL_417944、EPI_ISL_417945、EPI_ISL_417946、EPI_ISL_417947、EPI_ISL_417948、 EPI_ISL_417949、EPI_ISL_417950、EPI_ISL_417951、EPI_ISL_417953、EPI_ISL_417955、EPI_ISL_417958、EPI_ISL_417959、EPI_ISL_417960、EPI_ISL_417962、EPI_ISL_417964、EPI_ISL_417965、EPI_ISL_417966、EPI_ISL_417968、EPI_ISL_417970、EPI_ISL_417971、EPI_ISL_417973、EPI_ISL _417974、EPI_ISL_417976、EPI_ISL_417977、EPI_ISL_417982、EPI_ISL_417983、EPI_ISL_417984、EPI_ISL_417985、EPI_ISL_418009、EPI_ISL_418017、EPI_ISL_418018、EPI_ISL_418019、EPI_ISL_418020、EPI_ISL_418021、EPI_ISL_418022、EPI_ISL_418023、EPI_ISL_418024、EPI_ISL_418025、EPI_ISL_418026、EPI_ISL_418027、EPI_ISL_418029、EPI_ISL_418030、EPI_ISL_418031、EPI_ISL_418032、EPI_ISL_418033、EPI_ISL_418034、EPI_ISL_418037、EPI_ISL_418038、EPI_ISL_418040、EPI_ISL_418046、EPI_ISL_418047、EPI_ISL_418048、EPI_ISL_418050、EPI_ISL_418052、EPI_ISL _418053、EPI_ISL_418054、EPI_ISL_418063、EPI_ISL_418064、EPI_ISL_418067、EPI_ISL_418071、EPI_ISL_418072、EPI_ISL_418073、EPI_ISL_418074、EPI_ISL_418075、EPI_ISL_418076、EPI_ISL_418077、EPI_ISL_418078、EPI_ISL_418079、EPI_ISL_418080、EPI_ISL_418081、EPI_ISL_418082、 EPI_ISL_418101、EPI_ISL_418102、EPI_ISL_418103、EPI_ISL_418104、EPI_ISL_418105、EPI_ISL_418126、EPI_ISL_418127、EPI_ISL_418128、EPI_ISL_418129、EPI_ISL_418130、EPI_ISL_418131、EPI_ISL_418132、EPI_ISL_418133、EPI_ISL_418134、EPI_ISL_418135、EPI_ISL_418136、EPI_ISL _418137、EPI_ISL_418138、EPI_ISL_418139、EPI_ISL_418140、EPI_ISL_418148、EPI_ISL_418149、EPI_ISL_418150、EPI_ISL_418151、EPI_ISL_418152、EPI_ISL_418153、EPI_ISL_418154、EPI_ISL_418155、EPI_ISL_418156、EPI_ISL_418157、EPI_ISL_418158、EPI_ISL_418159、EPI_ISL_418160、EPI_ISL_418161, EPI_ISL_418162, EPI_ISL_418163, EPI_ISL_418164, EPI_ISL_418165, EP I_ISL_418183, EPI_ISL_418184, EPI_ISL_418185, EPI_ISL_418186, EPI_ISL_418187, EPI_ ISL_418188, EPI_ISL_418189, EPI_ISL_418190, EPI_ISL_418191, EPI_ISL_418192, EPI_IS L_418193, EPI_ISL_418194, EPI_ISL_418195, EPI_ISL_418197, EPI_ISL_418198, EPI_ISL_ 418199, EPI_ISL_418200, EPI_ISL_418201, EPI_ISL_418202, EPI_ISL_418203, EPI_ISL_41 8204, EPI_ISL_418231, EPI_ISL_418232, EPI_ISL_418233, EPI_ISL_418235, EPI_ISL_4182 36, EPI_ISL_418237, EPI_ISL_418238, EPI_ISL_418239, EPI_ISL_418240, EPI_ISL_418257 , EPI_ISL_418260, EPI_ISL_418263, EPI_ISL_418264, EPI_ISL_418265 or EPI_ISL_616802 (hCoV-19 / Denmark / DCGC-3024 / 2020), Exemplary SARS-CoV-2 coronaviruses can also be defined and identified by their genetic information, which is provided by GenBank accession numbers. The accession numbers are shown in List B below.

[0065] List B: GenBank accession numbers of various SARS-CoV-2 isolates NC_045512、LC528232、LC528233、LC529905、MN908947、MN938384、MN938385、MN938386、MN938387、MN938388、MN938389、MN938390、MN970003、MN970004、MN975262、MN975263、MN975264、MN975265、MN975266、MN975267、MN975268、MN985325、MN988668、MN988669、MN994467、MN994468、MN996527、MN996528、MN996529、MN996530、MN996531、MN997409、MT007544、MT012098、MT019529、MT019530、MT019531、MT019532、MT019533、MT020880、MT020881、MT027062、MT027063、MT027064、MT039873、MT039887、MT039888、MT039890、MT044257、MT044258、MT049951、MT050493、MT066156、MT066175、MT066176、MT072688、MT093571、MT093631、MT106052、MT106053、MT106054、MT118835、MT121215、MT123290、MT123291、MT123292、MT123293、MT126808、MT135041、MT135042、MT135043、MT135044、MT152824、MT159705、MT159706、MT159707、MT159708、MT159709、MT159710、MT159711、MT159712、MT159713、MT159714、MT159715、MT159716、MT159717、MT159718、MT159719、MT159720、MT159721、MT159722、MT163716、MT163717、MT163718、MT163719、MT163720、MT163721、MT184907、MT184908、MT184909、MT184910、MT184911、MT184912、MT184913、MT188339、MT188340、MT188341、MT192759、MT192765、MT192772 orMT192773 The NCBI Taxonomy ID (NCBI:txid or taxID) for the SARS-CoV-2 coronavirus is 2697049.

[0066] The terms “antigenic peptide or antigenic protein of nCoV-2019 coronavirus” or “antigenic peptide or antigenic protein of SARS-CoV-2 coronavirus” refer to any peptide or protein of or derived from the SARS-CoV-2 (nCoV-2019) coronavirus as defined above. They also refer to fragments, variants, or derivatives of such peptides or proteins (preferably immunogenic fragments or immunogenic variants).

[0067] The terms “immunogenic fragment” or “immunogenic variant” should be understood as any fragment / variant that corresponds to an antigen of the SARS-CoV-2 (nCoV-2019) coronavirus capable of eliciting an immune response in a subject. Preferably, the encoded SARS-CoV-2 antigen (peptide or protein) becomes expressible in the subject as a result of intramuscular or intradermal administration of the nucleic acid of the first embodiment.

[0068] In this specification, the term “expression” refers to the production of a peptide or protein of the SARS-CoV-2 coronavirus. This SARS-CoV-2 coronavirus peptide or protein is provided from the coding sequence of the nucleic acid of the first embodiment. For example, “RNA expression” refers to the production of a protein by translating the RNA into a polypeptide (e.g., production after administering the RNA to a cell or subject). The protein produced is, for example, a peptide or protein of or derived from the SARS-CoV-2 coronavirus. “DNA expression” refers to the production of a protein by transcribing DNA into RNA and subsequently translating it into a polypeptide (e.g., production after administering the DNA to a cell or subject). The protein produced is, for example, a peptide or protein of or derived from the SARS-CoV-2 coronavirus. In this specification, the terms “expression” and “production” may be used interchangeably. Preferably, the term “expression” refers to the production of a specific peptide or protein when a nucleic acid is administered to a cell or organism. In the embodiment, the nucleic acid is suitable for a vaccine, and more preferably for a coronavirus vaccine. In a preferred embodiment, the nucleic acid is suitable for a SARS-CoV-2 coronavirus vaccine.

[0069] In connection with the present invention, any protein of or derived from the SARS-CoV-2 coronavirus may be utilized, or may be appropriately encoded by a coding sequence or nucleic acid of the first embodiment. Within the scope of the basis of the present invention, one or more antigenic peptides or antigenic proteins may include or consist of synthetically modified or artificial coronavirus peptides or proteins. The terms “synthetically modified coronavirus peptides or proteins” or “artificial coronavirus peptides or proteins” refer to proteins that do not exist in nature. Therefore, artificial coronavirus peptides or proteins or synthetically modified coronavirus peptides or proteins may differ from natural coronavirus peptides or proteins in, for example, by one or more amino acids, have additional heterologous peptide or protein elements, or have elongated or shortened N-terminuses or C-terminuses.

[0070] In a preferred embodiment, the nucleic acid has one or more coding sequences encoding one or more antigenic peptides or antigenic proteins of the SARS-CoV-2 coronavirus or their immunogenic fragments or immunogenic variants. The one or more antigenic peptides or antigenic proteins include one or more peptides or proteins that are structural proteins, accessory proteins, replicase proteins, or immunogenic fragments or immunogenic variants of any of these, or derived therefrom.

[0071] In a preferred embodiment, the nucleic acid has one or more coding sequences encoding one or more antigenic peptides or antigenic proteins of the SARS-CoV-2 coronavirus. The one or more antigenic peptides or antigenic proteins are structural proteins or comprise one or more peptides or proteins derived therefrom. The structural proteins are selected from spike proteins (S), envelope proteins (E), membrane proteins (M), nucleocapsid proteins (N), or immunogenic fragments or variants of any of these.

[0072] In a particularly preferred embodiment, one or more encoded antigenic peptides or antigenic proteins include or consist of a spike protein (S) or an immunogenic fragment or immunogenic variant thereof.

[0073] The spike protein is a typical type I viral fusion protein. It exists as a trimer on the surface of the virus. Each monomer consists of a head (S1) and a stem (S2). Individual precursor S polypeptides form a homotrimer, which is glycosylated in the Golgi apparatus and has its signal peptide removed. It is then cleaved by intracellular proteases to separate S1 and S2 polypeptide chains. In the homotrimer, S1 and S2 associate as an S1 / S2 protomer, forming a heterodimeric trimer. The S1 domain of the spike glycoprotein contains a receptor-binding domain (RBD). The RBD binds to the angiotensin-converting enzyme 2 receptor (or is considered likely to do so), mediating fusion between the virus and the host cell. The N-terminal domain first contacts the target cell, followed by two subdomains. Both of these domains are sensitive to neutralizing antibodies. The S2 domain, consisting of a fusion core of six helical bundles, is involved in membrane fusion with the host endosomal membrane. The S2 domain is also a target for neutralization. The S2 subunit typically has two 7-repetition sequences (HR1 and HR2) fused with glycoproteins, as well as a central helix, a transmembrane domain, and an intracellular tail domain.

[0074] The sequences of suitable antigenic peptides or antigenic proteins provided by the nucleic acids of the present invention are shown in rows 1 to 41 of columns A and B in Table 1. Additional information regarding the sequences of these suitable antigenic peptides or antigenic proteins can be found in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0075] The following describes in detail suitable sequences of antigenic peptides or antigenic proteins provided by the nucleic acids of the present invention.

[0076] In a preferred embodiment, one or more encoded antigenic peptides or antigenic proteins are identical to any one of SEQ ID NOs: 1-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, 26939, or consist of one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, one or more encoded antigenic peptides or antigenic proteins are any of these immunogenic fragments or immunogenic variants. For additional information regarding the above amino acid sequences, see Table 1 (rows 1-41 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0077] It should be noted that when referring to amino acid residues (aa residues) and their positions in the spike protein (S), any number used refers to the positions of amino acid residues in the corresponding spike protein (S) of the SARS-CoV-2 (nCoV-2019) coronavirus isolate EPI_ISL_402128 (BetaCoV_Wuhan_WIV05_2019_EPI_ISL_402128) related to SEQ ID NO: 1 (unless otherwise stated). In this disclosure, the positions of individual amino acids are illustrative examples of their positions in the spike protein (S) of the SARS-CoV-2 coronavirus isolate EPI_ISL_402128 (SEQ ID NO: 1). Those skilled in the art will naturally be able to apply the disclosures of this specification, as exemplified with respect to SARS-CoV-2 EPI_ISL_402128 (SEQ ID NO: 1), to other antigenic peptides or antigenic proteins in other SARS-CoV-2 coronavirus isolates. Examples of such isolates include (but are not limited to) EPI_ISL_404227, EPI_ISL_403963, EPI_ISL_403962, EPI_ISL_403931, EPI_ISL_403930, EPI_ISL_403929, EPI_ISL_402130, EPI_ISL_402129, EPI_ISL_402128, EPI_ISL_402126, EPI_ISL_402125, EPI_ISL_402124, EPI_ISL_402123, EPI_ISL_402120, and EPI_ISL_402119. For further isolates of SARS-CoV-2, see List A, List B, and / or Table 25.

[0078] Protein annotation is performed using Sequence ID No. 1 as the reference protein. The full-length spike protein (S) of the SARS-CoV-2 coronavirus reference protein has a size of 1273 amino acid residues and contains the following elements. • Secretory signal peptides: aa1~aa15 (see SEQ ID NO: 28) • Spike protein fragment S1: aa1~681 (see SEQ ID NO: 27) • Receptor-binding domain (RBD): aa319~aa541 (see Sequence ID No. 13243) • Neutralizing critical domain (CND): aa329~aa529 (see SEQ ID NO: 13310) • Spike protein fragment S2: aa682~aa1273 (see SEQ ID NO: 30) • Transmembrane domain (TM): aa1212~aa1273 (see Sequence ID No. 49) • Transmembrane domain (TMflex): aa1148~aa1273 (see Sequence ID No. 13176) It should be noted that spike proteins derived from different SARS-CoV-2 isolates have naturally occurring amino acid-level mutations (see Lists A and B for exemplary SARS-CoV-2 isolates). In connection with the present invention, such amino acid mutations may be applied to antigenic peptides or antigenic proteins derived from the spike proteins described herein.

[0079] Accordingly, in relation to the present invention, the spike proteins described herein as suitable antigens may have one or more of the following amino acid mutations (amino acid positions are relative to SEQ ID NO: 1). • D614G or G614D H49Y or Y49H • V367F or F367V • P1263L or L1263P • V483A or A483V • S939F or F939S • S943P or P943S L5F or F5L L8V or V8L · S940F or F940S • C1254F or F1254C • Q239K or K239Q • M153T or T153M · V1040F or F1040V • A845S or S845A • Y145H or H145Y A831V or V831A • M1229I or I1229M • H69 or H69del (amino acid deletion) • V70 or H70del (amino acid deletion) • H69_V70 or H69del and H70del (amino acid deletion) • A222V or V222A • Y453F or F453Y • S477N or N477S I692V or V692I • R403K or K403R • K417N or N417K • N437S or S437N • N439K or K439N • V445A or A445V V445I or I445V • V445F or F445V G446V or V446G G446S or S446G G446A or A446G • L455F or F455L • F456L or L456F • K458N or N458K • A475V or V475A G476S or S476G G476A or A476G • S477I or I477S • S477R or R477S • S477G or G477S S477T or T477S • T478I or I478T • T478K or K478T • T478R or R478T • T478A or A478T • E484Q or Q484E • E484K or K484E • E484A or A484E • E484D or D484E G485R or R485G G485S or S485G F486L or L486F • N487I or I487N • Y489H or H489Y · F490S or S490F · F490L or L490F • Q493L or L493Q • Q493K or K493Q • S494P or P494S • S494L or L494S P499L or L499P • T500I or I500T • N501Y or Y501N • N501T or T501N • N501S or S501N • V503F or F503V • V503I or I503V G504D or D504G • Y505W or W505Y • Q506K or K506Q • Q506H or H506Q • Y144 or Y144del (amino acid deletion) A570D or D570A • P681H or H681P • T716I or I716T • S982A or A982S • D1118H or H1118D • L18F or F18L · D80A or A80D • D215G or G215D • L242 or L242del (amino acid deletion) • A243 or A243del (amino acid deletion) • L244 or L244del (amino acid deletion) • L242_A243_L244 or L242del, A243del, and L244del (amino acid deletion) • R246I or I246R · A701V or V701A ·T20N or N20T P26S or S26P ·D138Y or Y138D · R190S or S190R H655Y or Y655H • T1027I or I1027T • S13I or I13S W152C or C152W L452R or R452L • R346T or T346R • P384L or L384P • L452M or M452L • F456A or A456F F456K or K456F F456V or V456F • E484P or P484E • K417T or T417K G447V or V447G L452Q or Q452L • A475S or S475A • F486I or I486F · F490Y or Y490F • Q493R or R493Q S494A or A494S <P499H or H499P P499S or S499P G502V or V502G • T748K or K748T • A522S or S522A • V1176F or F1176V The following amino acid mutations are particularly preferred (amino acid positions are based on SEQ ID NO: 1). H69del, V70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, E484K, N501Y, D614G and A701V ·K417N, E484K, N501Y, and D614G • E484K and D614G L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T1027I · S13I, W152C, L452R, and D614G • delH69, delV70, Y453F, D614G, I692V and M1229I • E484K, E484P, or E484Q G446V G485R In some embodiments, a fragment of the spike protein (S) may be encoded by the nucleic acid of the present invention. This fragment may have a shortened N-terminus, deleting N-terminal amino acids from aa1 to aa100 compared to the full-length reference protein of SARS-CoV-2 coronavirus (SEQ ID NO: 1). Alternatively, this fragment may have a shortened C-terminus, deleting C-terminal amino acids from aa531 to aa1273 compared to the full-length reference protein of SARS-CoV-2 coronavirus (SEQ ID NO: 1). Such a fragment of the spike protein (S) may further have amino acid substitutions (described below) or one or more heterologous peptide or protein elements (described below). In a preferred embodiment, the fragment of the spike protein (S) has a shortened C-terminus and lacks a C-terminal transmembrane domain (i.e., deleting aa1212~aa1273 or aa1148~aa1273).

[0080] In other embodiments, one or more encoded antigenic peptides or antigenic proteins include or consist of a spike protein (S), which is derived from SARS-CoV-2 coronavirus that lacks a transmembrane domain (TM; amino acid positions: aa1212-aa1273). In embodiments, one or more encoded antigenic peptides or antigenic proteins include or consist of a spike protein (S), which is derived from SARS-CoV-2 coronavirus that lacks an extension site of the transmembrane domain (TMflex; amino acid positions: aa1148-aa1273). While not intended to be theoretically bound, the spike proteins (S) lacking a transmembrane domain (TM or TMflex) described herein are suitable for coronavirus vaccines because the protein is considered soluble and not immobilized on the cell membrane. If the protein is soluble, it can be administered to the subject at high concentrations (when translated), resulting in an improved immune response.

[0081] While not intended to be theoretically bound, the RBD domains (aa319-aa541) and CND domains (aa29-aa529) are important for immunogenicity. Both of these regions are located in the S1 fragment of the spike protein. Therefore, preferred in relation to the present invention, the antigenic peptide or antigenic protein contains or consists of the S1 fragment of the spike protein. Alternatively, the antigenic peptide or antigenic protein is its immunogenic fragment or immunogenic variant. Preferably, the S1 fragment may contain at least the RBD domains and / or CND domains described above.

[0082] In a preferred embodiment, one or more encoded antigenic peptides or antigenic proteins include or consist of receptor-binding domains (RBDs; aa319~aa541). These RBDs include or consist of spike protein fragments. Alternatively, the RBDs are immunogenic fragments or immunogenic variants thereof.

[0083] In a more preferred embodiment, one or more encoded antigenic peptides or antigenic proteins include or consist of a truncated receptor-binding domain (truncRBD; aa334~aa528). This RBD includes or consists of a spike protein fragment. Alternatively, the RBD is an immunogenic fragment or immunogenic variant thereof.

[0084] Such a fragment of spike protein (S) (RBD; aa319~aa541 or truncRBD; aa334~aa528) may further have amino acid substitutions (described below) or one or more heterologous peptide or protein elements (described below).

[0085] In a particularly preferred embodiment, one or more encoded antigenic peptides or antigenic proteins include or consist of a spike protein (S). The spike protein (S) includes or consists of a spike protein fragment S1. Alternatively, the spike protein (S) is an immunogenic fragment or immunogenic variant thereof.

[0086] Therefore, in a preferred embodiment, one or more antigenic peptides or antigenic proteins (containing or consisting of spike protein fragment S1) that are encoded are SEQ ID NOs: 1-27, 29, 31-48, 58-111, 274-1345, 1480-1546, 1614-11663, 13377-13510, 13521-14123, 22732, 22737-22758, It contains, or consists of, one or more amino acid sequences that are identical to any one of 22929-22964, or are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, one or more antigenic peptides or antigenic proteins are immunogenic fragments or immunogenic variants of any of these. Additional information regarding these amino acid sequences can be found in Table 1 (see rows 1-6, 9, and 11-41 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0087] In a preferred embodiment, one or more encoded antigenic peptides or antigenic proteins contain spike protein fragment S1 and do not contain 70%, 80%, 90%, preferably 100%, of the spike protein fragment S2 (aa682 to aa1273). The advantage of this embodiment is that the neutralizing epitope of the S1 fragment is not constrained by the potential problems associated with full-length proteins containing S1 and S2.

[0088] Therefore, in a preferred embodiment, one or more encoded antigenic peptides or antigenic proteins (substantially consisting of spike protein fragment S1) are identical to any one of SEQ ID NOs: 27, 1279-1345, 29, 1480-1546, 13243-13309, 22733-22736, 26938, 26939, or consist of one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, one or more antigenic peptides or antigenic proteins are any immunogenic fragment or immunogenic variant thereof. Additional information regarding the above amino acid sequences can be found in Table 1 (rows 6 and 9 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0089] While not intended to be theoretically bound, it may be preferable for the antigenic peptide or antigenic protein to contain, or consist of, spike protein fragment S1 and spike protein fragment S2 (at least a fragment of S2). This is because the formation of immunogenic spike proteins may be promoted.

[0090] Therefore, in a particularly preferred embodiment, one or more encoded antigenic peptides or antigenic proteins include or consist of a spike protein (S). This spike protein (S) includes or consists of a spike protein fragment S1 or its immunogenic fragment or immunogenic variant, and a spike protein fragment S2 or its immunogenic fragment or immunogenic variant.

[0091] In a preferred embodiment, one or more encoded antigenic peptides or antigenic proteins include or consist of spike protein fragment S1 and spike protein fragment S2. Furthermore, one or more antigenic peptides or antigenic proteins are identical to any one of SEQ ID NOs: 1-26, 31-48, 58-111, 274-1278, 1614-11663, 13377-13510, 13521-14177, 22732, 22737-22758, or 22929-22964, or contain one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, one or more antigenic peptides or antigenic proteins are immunogenic fragments or immunogenic variants of any of these. For additional information regarding the above amino acid sequences, please refer to Table 1 (rows 1, 5, 11-35, and 38 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0092] In a particularly preferred embodiment, one or more encoded antigenic peptides or antigenic proteins include or consist of a full-length spike protein. Alternatively, one or more antigenic peptides or antigenic proteins are immunogenic fragments or immunogenic variants thereof.

[0093] The term "full-length spike protein" should be understood as a spike protein (preferably derived from the SARS-CoV-2 coronavirus) that has an amino acid sequence that substantially corresponds to a complete spike protein. Therefore, a full-length spike protein may include aa1-aa1273 (reference protein: SEQ ID NO: 1). Thus, a full-length spike protein may typically include a secreted signal peptide, spike protein fragment S1, spike protein fragment S2, receptor-binding domain (RBD), neutralizing critical domain (CND), and transmembrane domain. Notably, variants with specific amino acid substitutions (e.g., those that stabilize the S protein in a pre-fusion structure) and variants with native amino acid deletions are also included in the category of "full-length spike protein."

[0094] Therefore, in a preferred embodiment, one or more encoded antigenic peptides or antigenic proteins are full-length S proteins. The full-length S protein is identical to or consists of one or more amino acid sequences that are 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the full-length S protein is one of these immunogenic fragments or immunogenic variants. For additional information regarding the above amino acid sequences, see Table 1 (see the first row of columns A and B) and the sequence number of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0095] In a particularly preferred embodiment, the spike protein (S) provided by the nucleic acid of the first embodiment is, by design or adaptation, a stable antigen in a pre-fusion conformation. The pre-fusion conformation is particularly advantageous when used in connection with coronavirus vaccines, because some of the potential epitopes for neutralizing antibodies can only be utilized in the protein's pre-fusion conformation. Maintaining the protein in a pre-fusion conformation also serves to avoid immunopathological effects such as disease exacerbation and / or antibody-dependent enhancement (ADE).

[0096] In preferred embodiments, administering a nucleic acid (or composition or vaccine) encoding a stable spike protein in a pre-fusion structure to a subject induces neutralizing antibodies against the spike protein, but not antibodies that exacerbate the disease. In particular, administering a nucleic acid (or composition or vaccine) encoding a stable spike protein in a pre-fusion structure to a subject does not induce disease exacerbation and / or immunopathological effects such as antibody-dependent enhancement (ADE).

[0097] Therefore, in a preferred embodiment, the nucleic acid of the present invention has one or more coding sequences encoding one or more antigenic peptides or antigenic proteins of or derived from the SARS-CoV-2 coronavirus. The one or more antigenic peptides or antigenic proteins are or derived from a spike protein (S). The spike protein (S) is a spike protein (S_stab) that is stable in the pre-fusion structure. Preferably, the spike protein that is stable in the pre-fusion structure has one or more pre-fusion structural stabilization mutations.

[0098] In this specification, the term "pre-fusion conformation" refers to the structural conformation introduced into the extracellular domain of the coronavirus S protein. This conformation is the conformation after modification in the secretory system to become the mature coronavirus S protein, and is the conformation before the fusion-inducing event occurs and the coronavirus S changes to the post-fusion conformation.

[0099] The “pre-fusion stable spike protein (S_stab)” described herein has one or more amino acid substitutions, deletions, or insertions compared to the original form of the coronavirus S sequence. This increases the time that the extracellular domain trimer of coronavirus S is maintained in the pre-fusion conformation compared to the extracellular domain trimer of coronavirus S formed by the original form of the coronavirus S sequence. The stabilization to the pre-fusion conformation by one or more amino acid substitutions, deletions, or insertions may be, for example, energetic stabilization (e.g., making the energy of the pre-fusion conformation lower than that of the open post-fusion conformation) or kinetic stabilization (e.g., reducing the probability of transition from the pre-fusion conformation to the post-fusion conformation). Furthermore, stabilizing the extracellular domain trimer of coronavirus S in the pre-fusion conformation can increase its resistance to denaturation compared to the original form of the coronavirus S sequence.

[0100] Therefore, in a preferred embodiment, the spike protein has one or more amino acid substitutions that stabilize the S protein in a pre-fusion conformation. Examples of such substitutions include those that stabilize the distal membrane region (such as the N-terminal region) of the S protein in a pre-fusion conformation.

[0101] The SARS-CoV-2 coronavirus spike protein may be stabilized by substituting one or more amino acids at positions K986 and / or V987 with amino acids that stabilize the spike protein in a pre-fusion conformation (amino acid positions are relative to SEQ ID NO: 1).

[0102] In a particularly preferred embodiment, the amino acid at the K986 position is substituted in the pre-fusion structural stabilization mutation (the amino acid position is based on SEQ ID NO: 1). The amino acid substitution at K986 is selected from A, I, L, M, F, V, G, or P. Preferably, the amino acid at K986 is substituted with P. In a further preferred embodiment, the amino acid at the K986 position is substituted in the pre-fusion structural stabilization mutation (the amino acid position is based on SEQ ID NO: 1). The amino acid substitution at V987 is selected from A, I, L, M, F, V, G, or P. Preferably, the amino acid at V987 is substituted with P.

[0103] Preferably, the spike protein of the SARS-CoV-2 coronavirus may be stabilized by substituting two amino acids at positions K986 and V987 with amino acids that stabilize the spike protein in a pre-fusion conformation (amino acid positions are based on SEQ ID NO: 1).

[0104] In preferred embodiments, the pre-fusion structural stabilization mutation involves substitutions at the K986 and V987 amino acids (amino acid positions relative to SEQ ID NO: 1). The amino acid substitutions at K986 and / or V987 are selected from A, I, L, M, F, V, G, or P.

[0105] Preferably, the K986 and V987 residues of the spike protein are stabilized in a pre-fusion conformation by substituting them with two proline molecules (amino acid positions are based on SEQ ID NO: 1).

[0106] Therefore, in a preferred embodiment, a spike protein (S_stab) stable in the pre-fusion structure has one or more pre-fusion structural stabilization mutations. In one or more pre-fusion structural stabilization mutations, amino acids are substituted at K986P and V987P (amino acid positions are relative to SEQ ID NO: 1).

[0107] Therefore, a person skilled in the art may select any protein, fragment thereof, or variant selected from any of the above-mentioned NCBI protein accession numbers or SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, and 22929-22946 and introduce amino acid mutations. Preferably, the amino acid substitutions are K986P and V987P (amino acid positions are relative to SEQ ID NO: 1).

[0108] In a preferred embodiment, one or more pre-fusion structural stabilizing mutations have cavity-filling mutations that further stabilize the pre-fusion state. These mutations / amino acid substitutions are selected from the group including T887WN;A1020W;T887WN and A1020W;P1069F (amino acid positions are relative to SEQ ID NO: 1).

[0109] The term "cavity-filling mutation" or "cavity-filling amino acid substitution" refers to amino acid substitutions that fill cavities in the protein core of a protein (such as the extracellular domain of the coronavirus S protein). A cavity is a substantial empty space in a folded protein where no amino acids or amino acid side chains are present. In some embodiments, cavity-filling amino acid substitutions are introduced to fill cavities in the pre-fusion conformation of the extracellular domain core of coronavirus S. These cavities are lost (e.g., due to a decrease in volume) after the transition to the post-fusion conformation.

[0110] In some embodiments, one or more of the amino acid substitutions F817P;A892P;A899P and A942P may be combined with the substitutions K986P and V987P (amino acid positions are relative to SEQ ID NO: 1).

[0111] In a preferred embodiment, the spike protein of the SARS-CoV-2 coronavirus has one or more of the following amino acid substitutions (amino acid positions are relative to SEQ ID NO: 1): F817P; K986P and V987P A892P; K986P and V987P A899P; K986P and V987P A942P; K986P and V987P In a particularly preferred embodiment, the spike protein of the SARS-CoV-2 coronavirus has the following amino acid substitutions (amino acid positions relative to SEQ ID NO: 1): ·F817P, A892P, A899P, A942P, K986P and V987P(S_stab_PP_hex) Therefore, a person skilled in the art may select any protein, fragment thereof, or variant selected from any of the above-mentioned NCBI protein accession numbers or SEQ ID NOs. 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 and introduce an amino acid mutation. Preferably, the amino acid substitution is selected from: F817P, A892P, A899P, A942P. Alternatively, the amino acid substitution is selected from the following: F817P, K986P and V987P; A892P, K986P and V987P; A899P, K986P and V987P; A942P, K986P and V987P; F817P, A892P, A899P, A942P, K986P and V987P. The amino acid position is based on SEQ ID NO: 1.

[0112] In a particularly preferred embodiment, one or more of the amino acid substitutions T887W;A1020W;T887W and A1020W;P1069F may be combined with the K986P and V987P substitutions (amino acid positions are based on SEQ ID NO: 1).

[0113] In other particularly preferred embodiments, the SARS-CoV-2 coronavirus spike protein has one or more amino acid substitutions (amino acid positions are relative to SEQ ID NO: 1): T887W; K986P and V987P · A1020W; K986P and V987P T887W and A1020W; K986P and V987P P1069F; K986P and V987P Therefore, a person skilled in the art may select any protein, fragment thereof, or variant selected from any of the above-mentioned NCBI protein accession numbers or SEQ ID NOs. 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 and introduce amino acid mutations. Preferably, the amino acid substitutions are selected from: T887W; A1020W; T887W and A1020W; P1069F. Alternatively, amino acid substitutions are selected from the following: T887W, K986P and V987P; A1020W, K986P and V987P; T887W, A1020W, K986P and V987P; P1069F, K986P and V987P. The amino acid positions are based on SEQ ID NO: 1.

[0114] In a preferred embodiment, one or more pre-fusion structural stabilizing mutations have a mutant protonation site that further stabilizes the pre-fusion state. These mutations / amino acid substitutions are selected from the following: H1048Q and H1064N; H1083N and H1101N; H1048Q, H1064N, H1083N and H1101N (amino acid positions are relative to SEQ ID NO: 1).

[0115] In some embodiments, one or more of the amino acid substitutions of H1048Q and H1064N; H1083N and H1101N; H1048Q, H1064N, H1083N and H1101N may be combined with the substitutions of K986P and V987P (amino acid positions are based on SEQ ID NO: 1).

[0116] In a particularly preferred embodiment, the spike protein of the SARS-CoV-2 coronavirus has one or more of the following amino acid substitutions (amino acid positions are relative to SEQ ID NO: 1): H1048Q and H1064N; K986P and V987P H1083N and H1101N; K986P and V987P H1048Q, H1064N, H1083N and H1101N; K986P and V987P Therefore, a person skilled in the art may select any protein, fragment thereof, or variant selected from any of the above-mentioned NCBI protein accession numbers or SEQ ID NOs. 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 and introduce amino acid mutations. Preferably, the amino acid substitutions are selected from: H1048Q and H1064N; H1083N and H1101N; H1048Q, H1064N, H1083N and H1101N. Alternatively, amino acid substitutions are selected from the following: H1048Q, H1064N, K986P and V987P; H1083N, H1101N, K986P and V987P; H1048Q, H1064N, H1083N, H1101N, K986P and V987P. The amino acid positions are based on SEQ ID NO: 1.

[0117] In a preferred embodiment, one or more pre-fusion structural stabilization mutations have an artificial intramolecular disulfide bond. By introducing an artificial intramolecular disulfide bond, the distal membrane region (such as the N-terminal region) of the S protein can be further stabilized to the pre-fusion conformation. That is, it can be further stabilized to a conformation that specifically binds to one or more pre-fusion specific antibodies, or it can exhibit preferred antigenicity that is present in the pre-fusion conformation but not in the post-fusion conformation of the S protein.

[0118] In preferred embodiments, one or more pre-fusion structural stabilization mutations form artificial intramolecular disulfide bonds. One or more artificial intramolecular disulfide bonds are formed by the substitution of two or more amino acids selected from the group including: I712C, I714C, P715C, T874C, G889C, A890C, I909C, N914C, Q965C, F970C, A972C, R995C, G999C, S1003C, L1034C, V1040C, Y1047C, S1055C, P1069C, T1077C, Y1110C, S1123C (amino acid positions are relative to SEQ ID NO: 1).

[0119] In preferred embodiments, one or more pre-fusion structural stabilization mutations form artificial intramolecular disulfide bonds. These artificial intramolecular disulfide bonds are formed by the substitution of one or more amino acids from the following: I712C and T1077C; I714C and Y1110C; P715C and P1069C; G889C and L1034C; I909C and Y1047C; Q965C and S1003C; F970C and G999C; A972C and R995C; A890C and V1040C; T874C and S1055C; N914C and S1123C (amino acid positions are relative to SEQ ID NO: 1).

[0120] In further embodiments, one or more pre-fusion structural stabilization mutations include two, three, four, five, six, seven, or eight different artificial intramolecular disulfide bonds. Each of these artificial intramolecular disulfide bonds may be an amino acid substitution selected from the following: I712C and T1077C; I714C and Y1110C; P715C and P1069C; G889C and L1034C; I909C and Y1047C; Q965C and S1003C; F970C and G999C; A972C and R995C; A890C and V1040C; T874C and S1055C; N914C and S1123C (amino acid positions relative to SEQ ID NO: 1).

[0121] In further embodiments, one or more (preferably two, three, four, five or more) amino acid substitutions from I712C and T1077C; I714C and Y1110C; P715C and P1069C; G889C and L1034C; I909C and Y1047C; Q965C and S1003C; F970C and G999C; A972C and R995C; A890C and V1040C; T874C and S1055C; N914C and S1123C may be combined with substitutions of K986P and V987P. For example, a pre-fusion-stabilized S protein may have two different artificial intramolecular disulfide bonds (such as I712C and T1077C; P715C and P1069C), as well as additional K986P and V987P substitutions. The amino acid positions are based on SEQ ID NO: 1.

[0122] In a particularly preferred embodiment, the spike protein of the SARS-CoV-2 coronavirus has one or more of the following amino acid substitutions (amino acid positions are relative to SEQ ID NO: 1). I712C and T1077C; K986P and V987P I714C and Y1110C; K986P and V987P P715C and P1069C; K986P and V987P G889C and L1034C; K986P and V987P I909C and Y1047C; K986P and V987P • Q965C and S1003C; K986P and V987P F970C and G999C; K986P and V987P A972C and R995C; K986P and V987P A890C and V1040C; K986P and V987P T874C and S1055C; K986P and V987P N914C and S1123C; K986P and V987P Therefore, a person skilled in the art may select any protein, fragment thereof, or variant selected from any of the above-mentioned NCBI protein accession numbers or SEQ ID NOs. 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 and introduce amino acid mutations into it. Preferably, amino acid substitutions are selected from the following: I712C and T1077C; I714C and Y1110C; P715C and P1069C; G889C and L1034C; I909C and Y1047C; Q965C and S1003C; F970C and G999C; A972C and R995C; A890C and V1040C; T874C and S1055C; N914C and S1123C. Alternatively, the amino acid substitution may be selected from the following: I712C, T1077C, K986P and V987P; I714C, Y1110C, K986P and V987P; P715C, P1069C, K986P and V987P; G889C, L1034C, K986P and V987P; I909C, Y1047C, K986P and V987P Q965C, S1003C, K986P and V987P; F970C, G999C, K986P and V987P; A972C, R995C, K986P and V987P; A890C, V1040C, K986P and V987P; T874C, S1055C, K986P and V987P; N914C, S1123C, K986P and V987P. Amino acid positions are based on Sequence ID No. 1.

[0123] It should be emphasized that, in relation to the present invention, any SARS-CoV-2 coronavirus spike protein may be stabilized in a pre-fusion conformation by having the above-mentioned mutation (exemplified with reference protein SEQ ID NO: 1).

[0124] Therefore, in a preferred embodiment, the spike protein (S_stab) stable in the pre-fusion structure is SEQ ID NOs: 10-26, 40-48, 85-111, 341-1278, 1681-2618, 2686-3623, 3691-4628, 4696-5633, 5701-6638, 6706-7643, 7711-8648, 8716-9653, 9721-10658, 10726-11663, 13377-13510, 13521-14123, 22732, 22738, 22740, 22 It contains, or consists of, one or more amino acid sequences that are identical to any one of 742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22947~22964, or are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, a spike protein (S_stab) that is stable in a pre-fusion structure is one of these immunogenic fragments or immunogenic variants. For additional information regarding the above amino acid sequences, please refer to Table 1 (rows 2-5, 12-15, 17-20, 22-25, 27-30, 32-35, and 38 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0125] In a particularly preferred embodiment, the spike protein (S_stab) stable in the pre-fusion structure is found in SEQ ID NOs: 10-26, 341-407, 609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22947-22964. It contains, or consists of, one or more amino acid sequences that are identical to any one of the above, or are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, a spike protein (S_stab) that is stable in a pre-fusion structure is one of these immunogenic fragments or immunogenic variants. For additional information regarding the above amino acid sequences, see Table 1 (see rows 2 and 5 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0126] In a more preferred embodiment, the pre-fusion stable spike protein (S_stab) is identical to any one of SEQ ID NOs. 10-18, 341-407, or 22947-22964, or contains or consists of one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the pre-fusion stable spike protein (S_stab) is any of these immunogenic fragments or immunogenic variants. Additional information regarding the above amino acid sequences can be found in Table 1 (see the second row of columns A and B) and the SEQ ID NOs of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0127] In a more preferred embodiment, the pre-fusion stable spike protein (S_stab) is identical to any one of SEQ ID NOs. 22960, 22961, or 22963, or contains or consists of one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the pre-fusion stable spike protein (S_stab) is any of these immunogenic fragments or immunogenic variants.

[0128] In a more preferred embodiment, the pre-fusion stable spike protein (S_stab) is identical to either SEQ ID NOs. 10 or 341, or contains or consists of one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the pre-fusion stable spike protein (S_stab) is any of these immunogenic fragments or immunogenic variants.

[0129] According to various preferred embodiments, the nucleic acids of the present invention encode one or more antigenic peptides or antigenic proteins of the SARS-CoV-2 coronavirus described herein, in addition to one or more heterologous peptide or protein elements.

[0130] Preferably, one or more heterologous peptide or protein elements may promote or enhance the secretion of the encoded antigenic peptide or antigenic protein of the present invention (e.g., by a secretion signal sequence). Alternatively, they may promote or enhance the adhesion of the encoded antigenic peptide or antigenic protein of the present invention to the plasma membrane (e.g., by a transmembrane element). Alternatively, they may promote or enhance the formation of an antigen complex (e.g., by a multimerizing domain or antigen clustering element). Alternatively, they may promote or enhance the formation of a virus-like particle (VLP-forming sequence). Furthermore, the nucleic acid of the first embodiment may further encode a peptide linker element, a self-cleaving peptide, an immunoadjuvant sequence, or a dendritic cell targeting sequence.

[0131] A suitable polymerizing domain may be selected from among amino acid sequences of sequence numbers 1116-1167 of WO2017 / 081082 or fragments or variants of these sequences. A suitable transmembrane element may be selected from among amino acid sequences of sequence numbers 1228-1343 of WO2017 / 081082 or fragments or variants of these sequences. A suitable VLP-forming sequence may be selected from among amino acid sequences of sequence numbers 1168-1227 of WO2017 / 081082 or fragments or variants of these sequences. A suitable peptide linker may be selected from among amino acid sequences of sequence numbers 1509-1565 of WO2017 / 081082 or fragments or variants of these sequences. A suitable self-cleaving peptide may be selected from among amino acid sequences of sequence numbers 1434-1508 of WO2017 / 081082 or fragments or variants of these sequences. A suitable immunoadjuvant sequence may be selected from among the amino acid sequences of sequence numbers 1360-1421 of WO2017 / 081082 or fragments or variants of these sequences. A suitable dendritic cell (DC) targeting sequence may be selected from among the amino acid sequences of sequence numbers 1344-1359 of WO2017 / 081082 or fragments or variants of these sequences. A suitable secreted signal peptide may be selected from among the amino acid sequences of sequence numbers 1-1115, 1728 of WO2017 / 081082 or fragments or variants of these sequences.

[0132] In preferred embodiments, one or more coding sequences further encode one or more heterologous peptide or protein elements selected from signal peptides, linker peptides, helper epitopes, antigen clustering elements, trimerizing or multimerizing elements, transmembrane elements, or VLP-forming sequences.

[0133] In a preferred embodiment, the nucleic acid of the present invention encodes one or more antigenic proteins derived from the SARS-CoV-2 coronavirus, in addition to one or more heterologous trimer elements, antigen clustering elements, or VLP-forming sequences.

[0134] (Antigen clustering element or multimerizing element) In preferred embodiments, the antigen clustering element may be selected from ferritin elements, lumazine synthase elements, hepatitis B virus surface antigen (HBsAg), or encapsrin. By expressing a stably clustered spike protein (preferably in a pre-fusion structure), the neutralizing activity against SARS-CoV-2 can be greatly broadened.

[0135] Lumazine synthase (LS, LumSynth) is an enzyme with particle-forming properties. This enzyme is present in various organisms and is involved in riboflavin biosynthesis.

[0136] In particularly preferred embodiments, lumazine synthase is used to promote antigen clustering. This may promote or enhance the immune response to the encoded coronavirus antigen of the present invention.

[0137] In particularly preferred embodiments, the antigen clustering element (multimerizing element) is or derived from rumazine synthase. Preferably, the amino acid sequence of the antigen clustering domain is identical to the amino acid sequence of SEQ ID NO: 112, a fragment thereof, or a variant thereof, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0138] Ferritin is a protein whose primary function is iron storage within cells. Almost all living organisms produce ferritin. Ferritin is composed of 24 subunits, each subunit consisting of a bundle of four α-helics, and it self-assembles into an octahedral symmetric quaternary structure. Its ability to self-assemble into nanoparticles makes it suitable for carrying and exposing antigens.

[0139] In a particularly preferred embodiment, ferritin is used to promote antigen clustering. This can enhance the immune response to the encoded coronavirus antigen (preferably a spike protein).

[0140] In particularly preferred embodiments, the antigen clustering element (multimerizing element) is or derived from ferritin. Preferably, the amino acid sequence of the antigen clustering domain is identical to one of the amino acid sequences, fragments, or variants thereof of SEQ ID NO: 113, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0141] In some embodiments, the antigen clustering domain is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles. The addition of sequence fragments of hepatitis B virus surface antigen (HBsAg) may be particularly effective in improving the immune response to nucleic acid-based vaccines against coronaviruses.

[0142] In particularly preferred embodiments, HBsAg is used to promote antigen clustering. This can enhance the immune response to the encoded coronavirus antigen (preferably the spike protein described herein).

[0143] In some embodiments, the antigen clustering element is an encapsulin element. The addition of an encapsulin sequence may be particularly effective in improving the immune response to nucleic acid-based vaccines against coronaviruses. In particularly preferred embodiments, encapsulin is used to promote antigen clustering, thereby enhancing the immune response to the encoded coronavirus antigen (preferably the spike protein described herein).

[0144] Encapsrin is a protein isolated from the thermophilic bacterium Thermotoga maritima. Encapsrin elements can be used as components for self-assembling antigens and forming antigen (nano) particles. Encapsrin is formed from 60 copies of identical monomers, each 31 kDa in size. Each monomer is a thin icosahedral T=1 symmetric cage structure with an inner diameter of 20 nm and an outer diameter of 24 nm.

[0145] In some embodiments, the coding sequence of the nucleic acid further codes for heterologous antigen clustering elements. Particularly preferably, the heterologous antigen clustering elements produce a fusion protein. This fusion protein contains the antigen clustering elements and an antigenic peptide or antigenic protein derived from SARS-CoV-2. Preferably, the antigenic peptide or antigenic protein (preferably the spike protein) is deleting its C-terminal transmembrane domain (TM) (deleting aa1212~aa1273), or a portion of its C-terminal transmembrane domain (TMflex) (e.g., deleting aa1148~aa1273).

[0146] Therefore, any amino acid sequence that is identical to any one of SEQ ID NOs. 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964, or identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, may be modified to remove the intrinsic transmembrane domain (TM) located at aa1212-aa1273 (amino acid positions are based on SEQ ID NO. 1). Thus, it can be used as a C-terminal shortened protein in relation to the present invention. Furthermore, any amino acid sequence that is identical to any one of SEQ ID NOs. 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964, or identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, may be modified to remove a portion of the intrinsic transmembrane domain (TMflex) located at aa1148-aa1273 (amino acid positions are based on SEQ ID NO. 1). Thus, it can be used as a C-terminal shortened protein in relation to the present invention. A suitable spike protein lacking a C-terminal transmembrane domain (TM or TMflex) may be selected from SEQ ID NOs: 31-39, 1614-3623, and 13377-13510.

[0147] In other embodiments, when the coding sequence of the nucleic acid further codes for the heterologous antigen clustering elements described above, a fusion protein is produced, particularly preferably. This fusion protein contains the antigen clustering elements and an antigenic peptide or antigenic protein (deleting S2, TM, and / or TMflex) derived from the SARS-CoV-2 spike protein fragment S1. Furthermore, preferably, a linker element (e.g., the linker of SEQ ID NOs: 115, 13148, 13152) is used to separate the heterologous antigen clustering elements from the antigenic peptide or antigenic protein.

[0148] In a preferred embodiment, one or more antigenic peptides or antigenic proteins containing heterologous antigen clustering elements contain or consist of one or more amino acid sequences. The heterologous antigen clustering elements are one or more amino acid sequences that are identical to any one of SEQ ID NOs. 58-75, 85-102, 3624-5633, 7644-9653, 13588-13721, 13856-13989, 22733, 22735, or 22736, or are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, heterogeneous antigen clustering elements are either of these immunogenic fragments or immunogenic variants. For additional information regarding the above amino acid sequences, see Table 1 (rows 16 and 25 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0149] Further preferred polymerizing elements may be selected from the amino acid sequences described in Sequence IDs 1116-1167 of WO2017 / 081082, or from fragments or variants of these sequences. Sequence IDs 1116-1167 of WO2017 / 081082 are incorporated herein by reference.

[0150] (trimerization element) In a preferred embodiment, the trimerizing element may be selected from foldon elements. In a preferred embodiment, the foldon element is a fibrin-foldon element. By expressing a stable trimer spike protein (preferably in a pre-fusion structure), the neutralizing activity against SARS-CoV-2 can be greatly broadened.

[0151] In particularly preferred embodiments, fibrin-foldon elements are used to promote antigen trimerization. This can enhance the immune response to the encoded coronavirus antigen (preferably the spike protein). Preferably, the foldon element is of or derived from a bacteriophage. Preferably, the foldon element is of or derived from bacteriophage T4. Most preferably, the foldon element is fibrin of bacteriophage T4 or derived from it.

[0152] In a particularly preferred embodiment, the trimer is foldon or derived therefrom. Preferably, the amino acid sequence of the trimer is identical to any one of the amino acid sequences, fragments, or variants of SEQ ID NO: 114, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0153] In other embodiments, when the coding sequence of a nucleic acid further codes for a different trimer, a fusion protein is produced, particularly preferably. This fusion protein comprises the trimer and an antigenic peptide or antigenic protein derived from SARS-CoV-2. Preferably, the antigenic peptide or antigenic protein (preferably a spike protein derived from SARS-CoV-2) is deleting its C-terminal transmembrane domain (deleting aa1212~aa1273), or a portion of its C-terminal transmembrane domain (TMflex) (e.g., deleting aa1148~aa1273).

[0154] Therefore, any amino acid sequence that is identical to any one of SEQ ID NOs. 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22947-22964, or identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, may be modified to delete the intrinsic transmembrane element located at aa1212-aa1273. Thus, it can be used as a C-terminal shortened protein in relation to the present invention. Furthermore, any amino acid sequence that is identical to any one of SEQ ID NOs. 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22947-22964, or identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, may be modified to remove a portion of the intrinsic transmembrane domain (TMflex) located at aa1148-aa1273 (amino acid positions are based on SEQ ID NO. 1). Thus, it can be used as a C-terminal shortened protein in relation to the present invention. A suitable spike protein lacking a C-terminal transmembrane domain (TM or TMflex) may be selected from SEQ ID NOs: 31-39, 1614-3623, and 13377-13510.

[0155] In other embodiments, when the coding sequence of the nucleic acid further codes for the heterologous trimer elements described above, a fusion protein is produced, particularly preferably. This fusion protein contains the trimer elements and an antigenic peptide or antigenic protein (lacking S2, TM, and / or TMflex) derived from the SARS-CoV-2 spike protein fragment S1. Furthermore, a linker element (e.g., the linker described in SEQ ID NOs: 115, 13148, and 13152) may be used to separate the heterologous antigen clustering elements from the antigenic peptide or antigenic protein.

[0156] In a preferred embodiment, one or more antigenic peptides or antigenic proteins containing heterologous trimer elements contain or consist of one or more amino acid sequences. The amino acid sequences are identical to any one of SEQ ID NOs: 76-84, 103-111, 5634-6638, 9654-10658, 13722-13788, 13990-14056, and 22734, or one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the amino acid sequences are any of these immunogenic fragments or immunogenic variants. Additional information regarding the above amino acid sequences can be found in Table 1 (rows 26, 30, and 41 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0157] Further preferred trimerizing elements may be selected from the amino acid sequences of sequence numbers 1116-1167 of WO2017 / 081082, or from fragments or variants of these sequences. Sequence numbers 1116-1167 of WO2017 / 081082 are incorporated herein by reference.

[0158] (VLP forming element) In preferred embodiments, a VLP-forming sequence may be selected and fused with the coronavirus antigen described herein. Expressing clustered spike proteins that are stable in VLP form significantly broadens the neutralizing activity against SARS-CoV-2. VLPs structurally mimic infectious viruses and can induce potent cellular and humoral immune responses.

[0159] Suitable VLP-forming sequences may be selected from elements derived from hepatitis B virus core antigen, HIV-1 Gag protein, or woodchuck hepatitis core antigen element (WhcAg).

[0160] In a particularly preferred embodiment, one or more VLP-forming sequences are woodchuck hepatitis core antigen elements (WhcAg). The WhcAg elements are used to promote VLP formation, thereby stimulating the immune response to the encoded coronavirus antigen (preferably a spike protein).

[0161] In a particularly preferred embodiment, the VLP-forming sequence is foldon or derived therefrom. Preferably, the amino acid sequence of the VLP-forming sequence is identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0162] In a further embodiment, when the coding sequence of the nucleic acid further codes for a different VLP-forming sequence, a fusion protein is produced, particularly preferably. The fusion protein comprises the VLP-forming sequence and an antigenic peptide or antigenic protein derived from SARS-CoV-2 (preferably a spike protein derived from SARS-CoV-2). The antigenic peptide or antigenic protein is deleting its C-terminal transmembrane domain (deleting aa1212~aa1273), or a portion of its C-terminal transmembrane domain (TMflex) (e.g., deleting aa1148~aa1273).

[0163] Therefore, any amino acid sequence that is identical to any one of sequence numbers 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964, or identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, may be modified to delete the intrinsic transmembrane element located at aa1212-aa1273. Thus, it can be used as a C-terminal shortened protein in relation to the present invention. Furthermore, any amino acid sequence that is identical to any one of SEQ ID NOs. 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964, or identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, may be modified to remove a portion of the intrinsic transmembrane domain (TMflex) located at aa1148-aa1273 (amino acid positions are based on SEQ ID NO. 1). Thus, it can be used as a C-terminal shortened protein in relation to the present invention. A suitable spike protein lacking a C-terminal transmembrane domain (TM or TMflex) may be selected from SEQ ID NOs: 31-39, 1614-3623, and 13377-13510.

[0164] In other embodiments, when the nucleic acid coding sequence further codes for the heterologous VLP-forming sequence described above, a fusion protein is produced, particularly preferably. The fusion protein comprises the VLP-forming sequence and an antigenic peptide or antigenic protein (deleting S2, TM, and / or TMflex) derived from the SARS-CoV-2 spike protein fragment S1. Furthermore, preferably, a linker element (e.g., the linker of SEQ ID NOs: 115, 13148, 13152) is used to separate the heterologous antigen clustering elements from the antigenic peptide or antigenic protein.

[0165] In a preferred embodiment, one or more antigenic peptides or antigenic proteins containing heterogeneous VLP-forming sequences contain or consist of one or more amino acid sequences. The amino acid sequences are identical to any one of SEQ ID NOs: 6639-7643, 10659-11663, 13789-13855, 14057-14123, or one or more amino acid sequences that are identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the amino acid sequences are any of these immunogenic fragments or immunogenic variants. Additional information regarding the above amino acid sequences can be found in Table 1 (rows 31 and 35 of columns A and B) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0166] In this regard, further preferred VLP-forming sequences may be selected from the amino acid sequences of sequence numbers 1168-1227 of WO2017 / 081082, or from fragments or variants of these sequences. Sequence numbers 1168-1227 of WO2017 / 081082 are incorporated herein by reference.

[0167] (Heterogeneous secreted signal peptides) In some embodiments, the antigenic peptide or antigenic protein contains heterologous signal peptides. Heterologous signal peptides can be used to enhance the secretion of the encoded coronavirus antigen.

[0168] Suitable secreted signal peptides may be selected from the amino acid sequences of SEQ ID NOs. 1-1115 and 1728 of WO2017 / 081082, or from fragments or variants of these sequences. SEQ ID NOs. 1-1115 and 1728 of WO2017 / 081082 are incorporated herein by reference.

[0169] In embodiments in which the nucleic acid coding sequence further codes for a heterologous secreted signal peptide, a fusion protein is produced, particularly preferably. The fusion protein contains the heterologous secreted signal peptide and an antigenic peptide or antigenic protein derived from SARS-CoV-2. Preferably, the antigenic peptide or antigenic protein (preferably a spike protein derived from SARS-CoV-2) is deleting the N-terminal endogenous secreted signal peptide (deleting aa1 to aa15). Therefore, any amino acid sequence that is identical to any one of SEQ ID NOs. 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964, or identical by 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, may be modified to delete the endogenous secretory signal peptide located at aa1-aa15. Thus, it can be used as an N-terminal truncated protein in relation to the present invention.

[0170] List 1 below specifies in detail (regarding nomenclature, protein elements, etc.) the preferred SARS-CoV-2 coronavirus antigenic peptides and proteins mentioned above.

[0171] List 1: Exemplary preferred protein design of the present invention • Full-length spike protein (S) containing aa1~aa1273 →See Sequence IDs 1, 274, etc. Stabilized S protein (S_stab_PP) containing aa1~aa1273 and having K986P and V987P substitutions. →See Sequence IDs 10, 341, etc. Stabilized S protein (S_stab_PP) containing aa1~aa1273 and having K986P and V987P substitutions. →See Sequence ID 22961, etc. Stabilized S protein (S_stab_PP) containing aa1~aa1273 and having K986P and V987P substitutions. →See Sequence ID 22960, etc. • A stabilized S protein (S_stab_PP_hex) containing aa1~aa1273 and having proline substitutions K986P, V987P, F817P, A892P, A899P and A942P, which includes aa1~aa1273 and has proline substitutions of K986P, V987P, F817P, A892P, A899P and A942P. →See Sequence ID 22732, etc. A stabilized S protein (S_stab_PP_cav) containing aa1~aa1273, with K986P and V987P substitutions and cavity-filling mutations (T887W, A1020W). →See Sequence ID 408, etc. • A stabilized S protein (S_stab_PP_cav) containing aa1~aa1273, with K986P and V987P substitutions and a cavity-filling mutation (P1069F). →See Sequence ID 475, etc. A stabilized S protein (S_stab_PP_prot) containing aa1~aa1273, with K986P and V987P substitutions and cavity-filling mutations (H1048Q, H1064N, H1083N, H1101N). →See Sequence ID 542, etc. • A stabilized S protein (S_stab_disul) containing aa1~aa1273 and possessing artificial disulfide bonds I712C and T1077C. →See Sequence IDs 19, 609, etc. • Contains aa1~aa1211 and does not have a transmembrane domain (S(S_woTM)) →See Sequence IDs 31, 1614, etc. • Contains aa1~aa1147 and does not have a transmembrane domain bending portion. →See Sequence ID 2619, etc. S_woTM(S_stab_PP_woTM) has substitutions for K986P and V987P. →See Sequence IDs 40, 1681, etc. S_woTMflex(S_stab_PP_woTMflex) has replacements for K986P and V987P. →See Sequence ID 2686, etc. • Spike protein fragment S1 (S1) containing aa1~aa681 →See Sequence IDs 27, 1279, etc. S_woTM possesses lumazine synthase. →See Sequence IDs 58, 3624, etc. • S_woTMflex possesses lumazine synthase →See Sequence ID 7644, etc. • Contains lumazine synthase S_stab_PP_woTM →See Sequence IDs 85, 3691, etc. • Contains lumazine synthase S_stab_PP_woTMflex →See Sequence ID 7711, etc. S_woTM contains ferritin elements. →See Sequence IDs 67, 4629, etc. • S_woTMflex contains ferritin elements →See Sequence ID 8649, etc. • Contains ferritin elements S_stab_PP_woTM →See Sequence IDs 94, 4696, etc. • Contains ferritin elements S_stab_PP_woTMflex →See Sequence ID 8716, etc. • S_woTM has Foldon elements →See Sequence IDs 76, 5634, etc. • S_woTMflex has a Foldon element →See Sequence ID 9654, etc. • S_stab_PP_woTM has a Foldon element →See Sequence IDs 103, 5701, etc. • Has a folded element S_stab_PP_woTMflex →Refer to SEQ ID NO: 9721 etc. ·S_woTM having a VLP sequence (WhcAg) →Refer to SEQ ID NO: 6639 etc. ·S_woTMflex having a VLP sequence (WhcAg) Refer to SEQ ID NO: 10659 etc. S_stab_PP_woTM having a VLP sequence (WhcAg) →Refer to SEQ ID NO: 6706 etc. ·S_stab_PP_woTMflex constituting a VLP sequence (WhcAg) →Refer to SEQ ID NO: 10726 etc. truncRBD having a foldon element →Refer to SEQ ID NO: 22734 etc. ·truncRBD having lumazine synthase (C-terminal) →Refer to SEQ ID NO: 22735 etc. ·truncRBD having lumazine synthase (N-terminal) →Refer to SEQ ID NO: 22736 etc. ·truncRBD having a ferritin element: →Refer to, for example, SEQ ID NO: 22733 ※The amino acid positions in List 1 are based on SEQ ID NO: 1.

[0172] In a particularly preferred embodiment of the first aspect, one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The amino acid sequences are identical to any one of SEQ ID NOs: 10, 21, 22, 25, 27, 274, 341, 408, 475, 542, 743, 810, 1011, 1145, 1212, 1279, 8716, 10726, 22732-22758, 22929-22942, 22947-22964, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the amino acid sequence may be one of these immunogenic fragments or immunogenic variants.

[0173] Table 1 shows preferred antigenic peptides or antigenic proteins derived from the SARS-CoV-2 coronavirus described above (rows 1 to 41). In the table, each row from 1 to 41 corresponds to a preferred SARS-CoV-2 coronavirus construct. Column A of Table 1 contains a brief description of the preferred antigen construct. Column B of Table 1 contains the sequence numbers of the proteins (amino acids) of each antigen construct. Column C of Table 1 contains the sequence numbers of the corresponding wild-type nucleic acid coding sequences. Column D of Table 1 contains the sequence numbers of the corresponding nucleic acid coding sequences (opt1,gc) with optimized G / C ratios. Column E of Table 1 contains the sequence numbers of the corresponding nucleic acid coding sequences (opt3,human) adapted to human codon usage frequency. Column F of Table 1 contains the sequence numbers of the corresponding nucleic acid coding sequences (opt10,gc mod) with modified G / C content. For a detailed description of the coding sequences, see the paragraph (Preferred Coding Sequences).

[0174] In particular, the detailed description of the present invention refers to the ST25 sequence listing of this application. <223> The information described in the column is explicitly included. Preferred nucleic acid constructs containing the coding sequences in Table 1 (e.g., mRNA sequences having the coding sequences in Table 1) are shown in Tables 3a and 3b.

[0175] [Table 1]

[0176] JPEG2026048878000002.jpg121169

[0177] JPEG2026048878000003.jpg125169

[0178] (Preferred coding sequence) According to a preferred embodiment, the nucleic acid of the present invention has one or more coding sequences encoding one or more antigenic peptides or antigenic proteins (preferably those described above) or fragments and variants thereof derived from the SARS-CoV-2 (nCoV-2019) coronavirus. In this regard, any coding sequences or fragments and variants thereof encoding one or more antigenic proteins as described herein can be understood as preferred coding sequences. Therefore, they can be included in the nucleic acid of the present invention.

[0179] In a preferred embodiment, the nucleic acid of the first embodiment may have, or consist of, one or more coding sequences encoding one or more antigenic peptides or antigenic proteins of the SARS-CoV-2 coronavirus described herein. Preferably, the coding sequences encode one of sequence numbers 1-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, 26939, or a fragment or variant thereof. It should be understood that, at the nucleic acid level, any sequence (DNA or RNA sequence) can be selected that codes for an amino acid sequence that is identical to or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Therefore, these sequences can be understood as preferred coding sequences of the present invention. For additional information regarding the above amino acid sequences, please refer to Table 1 (see rows 1-41 of columns A and B), Table 3a, Table 3b, and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0180] In a preferred embodiment, the nucleic acid of the first embodiment has a coding sequence. The coding sequence includes one or more nucleic acid sequences. The nucleic acid sequences are sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520, 14124-14177, 2 2759, 22764~22786, 22791~22813, 22818~22839, 22969~23184, 23189~23404, 23409~23624, 23629~23844, 23849~24064, 24069~24284, 24289~24504, 245 The sequences are identical to those described in 09-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, 26709-26937, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequences are fragments or variants of any of these sequences. For additional information regarding the nucleic acid sequences mentioned above, please refer to Table 1 (rows 1-7, 9, and 11-41 of columns C-F), Table 3a, Table 3b, and the sequence numbers of each sequence in the ST25 sequence list. <223> Also, write it in the section marked "...".

[0181] Alternatively, the nucleic acid of the first embodiment has a coding sequence. The coding sequence includes one or more nucleic acid sequences. The nucleic acid sequences are sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520, 14124-14177, 22759, and 22764. ~22786, 22791~22813, 22818~22839, 22969~23184, 23189~23404, 23409~23624, 23629~23844, 23849~24064, 24069~24284, 24289~24504, 24509~24724, 24729~2494 4. The sequence is identical to the sequences described in 24949~25164, 25169~25384, 25389~25604, 25609~25824, 25829~26044, 26049~26264, 26269~26484, 26489~26704, 26709~26937 (all uracil (U) in each sequence is substituted with thymidine (T)), or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant of any of these sequences. For additional information regarding the nucleic acid sequences mentioned above, please refer to Table 1 (rows 1-7, 9, and 11-41 of columns C-F), Table 3a, Table 3b, and the sequence numbers of each sequence in the ST25 sequence list. <223> Also, write it in the section marked "...".

[0182] In a preferred embodiment, the nucleic acid of the first embodiment has a coding sequence. The coding sequence comprises one or more nucleic acid sequences. The nucleic acid sequence is identical to the sequences described in SEQ ID NOs: 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, or identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, a nucleic acid sequence is a fragment or variant of any of these sequences. For additional information regarding the nucleic acid sequences mentioned above, see Table 1 (rows 1-7, 9, and 11-41 of columns C-F), Table 3a, Table 3b, and the sequence number of each sequence in the ST25 sequence list. <223> Also, write it in the section marked "...".

[0183] Alternatively, the nucleic acid of the first embodiment has a coding sequence. The coding sequence contains one or more nucleic acid sequences. The nucleic acid sequences are sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184 (each sequence contains The sequence is identical to the sequence described above (where all uracil (U) is substituted with thymidine (T)), or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant of any of these sequences. For additional information regarding the above nucleic acid sequences, see Table 1 (rows 1-7, 9, and 11-41 of columns C-F), Table 3a, Table 3b, and the sequence number of each sequence in the ST25 sequence list. <223> Also, write it in the section marked "...".

[0184] In a preferred embodiment, the nucleic acid of the first embodiment is an artificial nucleic acid (artificial DNA, artificial RNA, etc.).

[0185] In this specification, the term “artificial nucleic acid” is intended to refer to nucleic acids that do not exist in nature. In other words, an artificial nucleic acid can be understood as a non-natural nucleic acid molecule. These nucleic acid molecules may be non-natural due to their own sequence (e.g., coding sequences with altered G / C content, UTRs) or due to other modifications (e.g., modifications to the nucleotide structure). Typically, artificial nucleic acids are designed and fabricated by genetic engineering to produce nucleotides of the corresponding desired artificial sequence. In this context, artificial nucleic acids may be sequences that do not exist in nature (wild-type sequences / sequences that differ from naturally occurring sequences by one or more nucleotides). The term “artificial nucleic acid” is understood to mean not only a single molecule but also a collection of substantially identical nucleic acid molecules. Therefore, an artificial nucleic acid may refer to multiple substantially identical nucleic acid molecules. In this specification, the term “artificial nucleic acid” may refer to artificial DNA (or preferably artificial RNA).

[0186] In a preferred embodiment, the nucleic acid of the first embodiment (preferably DNA or RNA) is a modified and / or stabilized nucleic acid. Preferably, the nucleic acid of the first embodiment is a modified and / or stabilized artificial nucleic acid.

[0187] Accordingly, according to preferred embodiments, the nucleic acids of the present invention may be provided as "stabilized artificial nucleic acids" or "stabilized coding nucleic acids." These may be nucleic acids with improved degradation resistance in vivo, improved stability in vivo, or improved translationability in vivo. Specific preferred modifications / fittings related to the above are described below. These are modifications / fittings suitable for stabilizing nucleic acids. Preferably, the nucleic acids of the present invention may be provided as "stabilized RNA," "stabilized coding RNA," "stabilized DNA," or "stabilized coding DNA."

[0188] Such stabilization can be achieved by the dried nucleic acids (such as dried DNA or dried RNA) and / or purified nucleic acids (such as purified DNA or purified RNA) described herein. Alternatively or in addition, such stabilization can also be achieved, for example, by modifying the backbone of the nucleic acids of the present invention with phosphoric acid. In connection with the present invention, the modification of the backbone refers to the modification in which the phosphate backbone of the nucleotides contained in the nucleic acid is chemically modified. In this connection, nucleotides that can be preferably used have, for example, a phosphorothioate-modified phosphate backbone. Preferably, one or more of the phosphoric acid oxygens contained in the phosphate backbone are replaced with sulfur atoms. Examples of the stabilized nucleic acids (preferably stabilized RNA) include non-ionic phosphate analogs. Examples of non-ionic phosphate analogs include alkylphosphonates and arylphosphonates in which the charged phosphoric acid oxygen is replaced with an alkyl or aryl group, and phosphodiesters and alkylphosphotriesters in which the charged oxygen residue remains in an alkylated state. Such backbone modifications typically include modifications from the group consisting of methylphosphonate, phosphoramidate, and phosphorothioate (but are not limited thereto). An example thereof is cytidine-5'-O-(1-thiophosphate).

[0189] Suitable modifications for stabilizing the nucleic acids of the present invention will be described below.

[0190] In a preferred embodiment, the nucleic acid (such as RNA or DNA) has one or more coding sequences with modified codons.

[0191] In a preferred embodiment, one or more of the coding sequences of the nucleic acid are coding sequences with modified codons. Preferably, the amino acid sequence encoded by one or more of the coding sequences with modified codons does not vary from the amino acid sequence encoded by the corresponding wild-type coding sequence or reference coding sequence.

[0192] The term "codon-modified coding sequence" refers to a coding sequence in which one or more codons (triplets of nucleotides encoding a single amino acid) differ from the corresponding wild-type coding sequence or reference coding sequence. Preferably, in relation to the present invention, a codon-modified coding sequence may have improved degradation resistance in vivo, improved stability in vivo, or improved translatability in vivo. In its broadest sense, codon modification utilizes the degeneracy of the genetic code. That is, when multiple codons encode the same amino acid and they can be used interchangeably (see Table 2), the coding sequence is optimized / modified and applied in vivo as described above.

[0193] The term "reference coding array" refers to a coding array. This coding array is the original array before modification and / or optimization.

[0194] In a preferred embodiment, one or more coding sequences of the nucleic acid are codon-modified coding sequences. The codon-modified coding sequences are selected from C-maximized coding sequences, CAI-maximized coding sequences, coding sequences adapted to codon use in humans, coding sequences with modified G / C content, G / C optimized coding sequences, or any combination thereof.

[0195] When transfected into mammalian host cells, nucleic acids containing the modified codon coding sequence remain stable for 12–18 hours or longer (e.g., 12, 24, 36, 48, 60, 72 hours or longer) and are expressed by mammalian host cells (such as muscle cells).

[0196] When transfected into mammalian host cells, nucleic acids having a coding sequence with modified codons are translated into proteins. The amount of protein translated is equal to or greater than the amount of naturally occurring protein or the amount of protein obtained when mammalian host cells are transfected with a wild-type coding sequence or a reference coding sequence. Preferably, the amount of protein translated is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 100% or more, or 200% or more greater.

[0197] In preferred embodiments, the nucleic acids of the present invention may be modified to increase (preferably maximize) the C content of one or more coding sequences compared to the C content of the corresponding wild-type coding sequence or reference coding sequence. Such sequences are referred to herein as "C-maximized coding sequences." Preferably, the amino acid sequence encoded by the C-maximized coding sequence of the nucleic acid is unchanged compared to the amino acid sequence encoded by the wild-type coding sequence or reference coding sequence. C-maximized nucleic acid sequences can preferably be produced by the modification method described in WO2015 / 062738. In this regard, the disclosure of WO2015 / 062738 is incorporated herein by reference.

[0198] In preferred embodiments, nucleic acids may be modified to optimize the G / C content of one or more coding sequences compared to the G / C content of the corresponding wild-type coding sequence or reference coding sequence. Such sequences are referred to herein as “G / C-optimized coding sequences.” In this context, “optimized” preferably means coding sequences with substantially maximized G / C content. Preferably, the amino acid sequence encoded by the G / C-optimized coding sequence of nucleic acid remains unchanged compared to the amino acid sequence encoded by the wild-type coding sequence or reference coding sequence. G / C-optimized nucleic acid sequences (RNA or DNA) can be prepared by the method described in WO2002 / 098443. In this context, the disclosures of WO2002 / 098443 are incorporated in their entirety into the present invention. Throughout this specification (sequence listings) <223> The G / C optimized coding sequence (including the column) is indicated by the abbreviation "opt1" or "gc".

[0199] In a preferred embodiment, the nucleic acid may be modified to adapt the codons in one or more coding sequences to the frequency of codon use in humans. Such sequences are referred to herein as "coding sequences adapted to human codon use." Even codons encoding the same amino acid have different frequencies of occurrence in humans. Therefore, it is preferable to modify the nucleic acid coding sequence to adapt the frequency of codons encoding the same amino acid to the frequency of codons that occur naturally according to the frequency of codon use in humans. For example, for the amino acid Ala, it is preferable to adapt the wild-type coding sequence or reference coding sequence such that the frequency of codon "GCC" is 0.40, the frequency of codon "GCT" is 0.28, the frequency of codon "GCA" is 0.22, and the frequency of codon "GCG" is 0.10 (see Table 2). Thus, the procedure exemplified for Ala is applied to each amino acid encoded by the nucleic acid coding sequence to obtain sequences adapted to human codon use. Throughout this specification (sequence listings) <223> (Including the column marked with an asterisk), coding sequences adapted for codon use in humans are indicated by the abbreviations "opt3" or "human".

[0200] [Table 2]

[0201] In embodiments, nucleic acids of the present invention may be modified such that the G / C content of one or more coding sequences is altered compared to the G / C content of the corresponding wild-type coding sequence or reference coding sequence. Such sequences are referred to herein as “G / C-modified coding sequences.” In this context, the terms “G / C optimized” or “G / C content modified” refer to nucleic acids in which the number of guanosine nucleotides and / or cytosine nucleotides is modified (preferably increased) compared to the corresponding wild-type coding sequence or reference coding sequence. Such an increase in number can be achieved by substituting codons containing adenosine nucleotides or thymidine nucleotides with codons containing guanosine nucleotides or cytosine nucleotides. Advantageously, nucleic acid sequences with improved G / C content are more stable or expressible than sequences with improved A / U content. Preferably, the amino acid sequence encoded by the G / C-modified coding sequence of the nucleic acid is unchanged compared to the amino acid sequence encoded by the wild-type sequence or reference sequence. Preferably, the G / C content of the nucleic acid coding sequence is increased by 10%, 20%, 30%, and preferably 40% or more compared to the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence. In this specification, a coding sequence with a modified G / C content is referred to as "opt10" or "gc mod".

[0202] In embodiments, nucleic acids may be modified to increase (preferably maximize) the codon compatibility index (CAI) of one or more coding sequences. Such sequences are referred to herein as "coding sequences with maximized CAI." Preferably, all codons that are relatively rare in humans, etc., contained in the wild-type nucleic acid sequence or reference nucleic acid sequence are replaced with codons that are common in humans, etc. In this case, the high-frequency codons and the relatively rare codons code for the same amino acid. Preferably, the most frequent codon is used for each amino acid of the coded protein (see Table 2; high-frequency human codons are marked with an asterisk). Preferably, the nucleic acid has one or more coding sequences, and the codon compatibility index (CAI) of one or more coding sequences is 0.5 or higher, 0.8 or higher, 0.9 or higher, or 0.95 or higher. Most preferably, the codon compatibility index (CAI) of one or more coding sequences is 1 (CAI=1). For example, in the case of the amino acid Ala, the wild-type coding sequence or a reference coding sequence may be adapted so that the most frequent codon in humans, "GCC," is always used for Ala. By applying the procedure exemplified for Ala to each amino acid encoded by the nucleic acid coding sequence in this way, a coding sequence with maximized CAI can be obtained.

[0203] In a particularly preferred embodiment, one or more coding sequences of the nucleic acid are coding sequences with modified codons. The coding sequences with modified codons are selected from coding sequences with optimized G / C ratios, coding sequences adapted to codon usage in humans, or coding sequences with modified G / C ratios.

[0204] In a preferred embodiment, the nucleic acid of the first embodiment has one or more coding sequences, which include or consist of modified nucleic acid sequences with altered codons. Nucleic acid sequences with modified codons include: SEQ ID NOs: 136-138, 140-143, 145-175, 11731-11813, 11815, 11817-12050, 12052, 12054-13147, 14142-14177, 22759, 22764-22786, 22791-22813, 22818-22839, 22969-23184, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, The sequence is identical to, or 70% or more identical to, a nucleic acid sequence with modified codons selected from the group consisting of 24949~25164, 25169~25384, 25389~25604, 25609~25824, 25829~26044, 26049~26264, 26269~26484, 26489~26704, and 26709~26937, or identical to, or 70% or more identical to, 80% or more identical to, 85% or more identical to, 86% or more identical to, 87% or more identical to, 88% or more identical to, 89% or more identical to, 90% or more identical to, 91% or more identical to, 92% or more identical to, 93% or more identical to, 94% or more identical to, 95% or more identical to, 96% or more identical to, 97% or more identical to, 98% or more identical to, or 99% or more identical to, a nucleic acid sequence with modified codons, or Additional information regarding what the above-mentioned preferred nucleic acid sequences encode is also included in the sequence listing. In particular, <223> Details are provided in the column. Preferred coding sequences for the first embodiment are shown in Table 1. Additional information regarding the above nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, and 11-41 of columns D-F), Table 3a, Table 3b, and the sequence numbers of each sequence in the ST25 sequence list. <223> Also, write it in the section marked "...".

[0205] Alternatively, the nucleic acid of the first embodiment has one or more coding sequences. One or more coding sequences include or consist of nucleic acid sequences with modified codons. The nucleic acid sequences with modified codons are sequence numbers 136-138, 140-143, 145-175, 11731-11813, 11815, 11817-12050, 12052, 12054-13147, 14142-14177, 22759, 22764-22786, and 22791-22813. , 22818~22839, 22969~23184, 23189~23404, 23409~23624, 23629~23844, 23849~24064, 24069~24284, 24289~24504, 24509~24724, 24729~24944, 24949~2516 4. The sequences are identical to, or 70% or more identical to, 80% or more identical to, 85% or more identical to, 86% or more identical to, 87% or more identical to, 88% or more identical to, 94% or more identical to, 95% or more identical to, 96% or more identical to, 97% or more identical to, 98% or more identical to, 98% or more identical to, 99% or more identical to, 25169~25384, 25389~25604, 25609~25824, 25829~26044, 26049~26264, 26269~26484, 26489~26704, 26709~26937 (all uracil (U) in each sequence are replaced by thymidine (T)). Alternatively, the codon-modified nucleic acid sequences are fragments or variants of any of these sequences. For additional information regarding the nucleic acid sequences mentioned above, please refer to Table 1 (rows 1-7, 9, and 11-41 of columns D-F), Table 3a, Table 3b, and the sequence numbers of each sequence in the ST25 sequence list. <223> Also, write it in the section marked "...".

[0206] In a particularly preferred embodiment, the nucleic acid of the first embodiment has one or more coding sequences. The one or more coding sequences include or consist of G / C optimized coding sequences. The G / C optimized coding sequences are sequence numbers 136-138, 140, 141, 148, 149, 152, 155, 156, 159, 162, 163, 166, 169, 170, 173, 11731-11813, 11815, 11817-11966, 12271-12472, 12743-12944, 13514, 13515, 14124-14132, 14142-14150, 14160-14168, 22759, 22764-227 86, 22791~22813, 22818~22839, 22969~23040, 23077~23148, 23189~23260, 23297~23368, 23409~23480, 23517~23588, 23629~23700, 23737~23808, 23849~23920, 23957~24028, 24069~24140, 24177~24248, 24289~24360, 24397~24468, 24509~2458 0, 24617~24688, 24729~24800, 24837~24908, 24949~25020, 25057~25128, 25169~25240, 25277~25348, 25389~25460, 25497~25568, 25609~25680, 25717~25788, 25829~25900, 25937~26008, 26049~26120, 26157~26228, 26269~26340, 26377~26448 , is identical to a nucleic acid sequence with modified codons selected from the group consisting of 26489-26560, 26597-26668, 26709-26780, 26817-26888, and 26925-26937, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, a G / C optimized coding sequence is a fragment or variant of any of these sequences.Additional information regarding what the above-mentioned preferred nucleic acid sequences encode is also included in the sequence listing. In particular, <223> Details are provided in the column. Preferred coding sequences for the first embodiment are shown in Table 1. Additional information regarding the above nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, and 11-41 of column D), Table 3a, Table 3b, and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0207] In a particularly preferred embodiment, the nucleic acid of the first embodiment has one or more coding sequences. The one or more coding sequences include or consist of coding sequences adapted for codon use in humans. The coding sequences adapted for codon use in humans are identical to, or are identical to, nucleic acid sequences with modified codons selected from the group consisting of SEQ ID NOs: 142, 143, 145, 150, 153, 157, 160, 164, 167, 171, 174, 11967-12033, 12473-12539, and 12945-13011 by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, coding sequences adapted for codon use in humans are fragments or variants of any of these sequences. Additional information regarding what the preferred nucleic acid sequences described above encode is also included in the sequence listings. In particular, <223> Details are provided in the column. Preferred coding sequences for the first embodiment are shown in Table 1. Additional information regarding the above nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, and 11-41 of column E), Table 3a, and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0208] In a particularly preferred embodiment, the nucleic acid of the first embodiment has one or more coding sequences. One or more coding sequences include or consist of G / C modified coding sequences. The G / C modified coding sequences are sequence numbers 146, 147, 151, 154, 158, 161, 165, 168, 172, 175, 12034~12050, 12052, 12054~12203, 12540~12675, 13012~13147, 13519, 13520, 14133~14141, 14151~14159, 14169~14177, 23041~23076, 23149~231 84, 23261~23296, 23369~23404, 23481~23516, 23589~23624, 23701~23736, 23809~23844, 23921~23956, 24029~24064, 24141~24176, 24249~24284, 24361~24396, 24469~24504, 24581~24616, 24689~24724, 24801~24836, 24 909~24944, 25021~25056, 25129~25164, 25241~25276, 25349~25384, 25461~25496, 25569~25604, 25681~25716, 25789~25824, 25901~25936, 26009~26044, 26121~26156, 26229~26264, 26341~26376, 26449~26484, 26561~2 The nucleic acid sequence is identical to, or is identical to, a nucleic acid sequence with a modified codon selected from the group consisting of 6596, 26669-26704, 26781-26816, and 26889-26924 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Alternatively, the G / C modified coding sequence is a fragment or variant of any of these sequences. Additional information regarding what the above preferred nucleic acid sequences encode is also included in the sequence listing. In particular, <223> Details are provided in the section. Table 1 shows preferred coding sequences for the first embodiment.For additional information regarding the nucleic acid sequences mentioned above, please refer to Table 1 (rows 1-7, 9, and 11-41 of column F) and the sequence numbers of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0209] In a more preferred embodiment, the nucleic acid of the first embodiment has one or more coding sequences. The one or more coding sequences include or consist of G / C modified coding sequences. The G / C modified coding sequences are sequence numbers 136-138, 142, 143, 146, 147, 11731, 11798-11801, 11804, 11805, 11808, 11810-11812, 11923, 11953, 12035, 12049, 22759-22785, 22965-22982, 23077-2309 4. A nucleic acid sequence with a modified codon selected from the group consisting of 23,149, or identical to such a sequence by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Alternatively, a coding sequence with modified G / C, or a fragment or variant of any of these sequences. Additional information regarding what the above preferred nucleic acid sequences encode is also provided in the sequence listing. In particular, <223> Details are provided in the column. Table 1 shows preferred coding sequences for the first embodiment. Additional information regarding the above nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, and 11-41 of column F) and the sequence number of each sequence in the ST25 sequence listing. <223> Also, write it in the section marked "...".

[0210] In a particularly preferred embodiment, the nucleic acid of the first embodiment has one or more coding sequences. The one or more coding sequences encode the SARS-CoV-2 antigen and include or consist of a G / C modified coding sequence. The G / C modified coding sequence is identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to, the G / C modified coding sequence is a fragment or variant thereof.

[0211] In a particularly preferred embodiment, the nucleic acid of the first embodiment has one or more coding sequences. The one or more coding sequences encode the SARS-CoV-2 antigen and include or consist of a G / C modified coding sequence. The G / C modified coding sequence is identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the G / C modified coding sequence is a fragment or variant thereof.

[0212] In further embodiments, the nucleic acid of the first embodiment has one or more coding sequences. The one or more coding sequences include or consist of coding sequences encoding the SARS-CoV-2 antigen. The coding sequences encoding the SARS-CoV-2 antigen are identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the coding sequences encoding the SARS-CoV-2 antigen are fragments or variants thereof.

[0213] (UTR) In preferred embodiments, the nucleic acids of the present invention have a protein-coding region (coding sequence or cds) and a 5'UTR and / or 3'UTR. In particular, the UTR may contain regulatory sequence elements. Regulatory sequence elements determine the metabolism, stability, and localization of the nucleic acid (e.g., RNA). Furthermore, the UTR may have sequence elements that facilitate translation. In the medical applications of nucleic acid sequences (e.g., DNA, RNA), translating the nucleic acid into one or more peptides or proteins is crucial for therapeutic effects. Depending on the specific combination of 3'UTR and / or 5'UTR, the expression of the peptide or protein of the present invention, encoded by the coding sequence to which they are operably linked, can be facilitated. After administration (preferably intramuscular) of a nucleic acid molecule having the UTR combination to a subject, the antigenic peptide or antigenic protein is advantageously expressed rapidly and transiently. Therefore, nucleic acids having the specific combinations of 3'UTR and / or 5'UTR described herein are particularly suitable for administration as vaccines. They are especially suitable for intramuscular, intradermal, or intraepidermal administration to subjects.

[0214] Preferably, the nucleic acid of the present invention has one or more heterologous 5'UTRs and / or one or more heterologous 3'UTRs. The heterologous 5'UTRs or heterologous 3'UTRs may be derived from naturally occurring genes or may be synthetically modified. In a preferred embodiment, the nucleic acid (preferably RNA) has one or more coding sequences described herein that are operably linked to one or more (heterologous) 3'UTRs and / or one or more (heterologous) 5'UTRs. In a preferred embodiment, the nucleic acid (such as RNA or DNA) has one or more heterogeneous 3'UTRs.

[0215] The terms “3' untranslated region,” “3'UTR,” or “3'UTR element” are recognized and understood by those skilled in the art. These terms refer to, for example, a portion of a nucleic acid molecule located on the 3' side (i.e., downstream) of a coding sequence that is not translated into a protein. The 3'UTR may be a portion of a nucleic acid (such as RNA or DNA) located between the coding sequence and a terminal poly(A) sequence (of any configuration). The 3'UTR may contain elements (also called regulatory elements) that control gene expression. Examples of such regulatory elements include ribosome binding sites and miRNA binding sites.

[0216] Preferably, the nucleic acid has a 3'UTR. The 3'UTR can be derived from genes related to the extension of the RNA half-life (genes that provide stable RNA).

[0217] In some embodiments, the 3'UTR has one or more polyadenylation signals, protein binding sites that affect the intracellular positional stability of nucleic acids, or one or more miRNAs or miRNA binding sites.

[0218] MicroRNAs (or miRNAs) are non-coding RNAs with a length of 19 to 25 nucleotides. MicroRNAs bind to the 3'UTR of nucleic acid molecules and downregulate gene expression. This downregulation is achieved by reducing the stability of the nucleic acid molecule or by inhibiting translation. For example, microRNAs are known to regulate RNA, thereby regulating protein expression. Protein expression regulation occurs, for example, in the liver (miR-122), heart (miR-ld, miR-149), endothelial cells (miR-17-92, miR-126), adipose tissue (let-7, miR-30c), kidney (miR-192, miR-194, miR-204), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), muscle (miR-133, miR-206, miR-208), and lung epithelial cells (let-7, miR-133, miR-126). The RNA may have one or more microRNA target sequences, microRNA sequences, or microRNA species. Such sequences correspond to, for example, any high level of microRNA (as disclosed in US2005 / 0261218 and US2005 / 0059005).

[0219] Therefore, by removing or introducing the aforementioned miRNA or miRNA-binding site from the 3'UTR, the expression of nucleic acids (such as RNA) in a desired cell type or tissue (such as muscle cells) can be regulated.

[0220] In a preferred embodiment, the nucleic acid has one or more heterologous 3'UTRs. One or more heterologous 3'UTRs contain nucleic acid sequences derived from the 3'UTRs of genes selected from PSMB3, ALB7, α-globin (also represented as muag), CASP1, COX6B1, GNAS, NDUFA1, and RPS9. Alternatively, one or more heterologous 3'UTRs contain nucleic acid sequences derived from a homolog, fragment, or variant of any one of these genes. Preferably, the nucleic acid sequence is identical to the nucleic acid sequences described in SEQ ID NOs. 253-268, 22902-22905, 22892-22895, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence may be a fragment or variant of any of these. Particularly preferred nucleic acid sequences in this regard may be derived from WO2019 / 077001A1 (in particular, claim 9 of WO2019 / 077001A1). The 3'UTR sequences corresponding to claim 9 of WO2019 / 077001A1 (such as sequence numbers 23-34 of WO2019 / 077001A1 or fragments or mutations thereof) are incorporated herein by reference.

[0221] In a particularly preferred embodiment, the nucleic acid has a 3'UTR derived from the α-globin gene. The 3'UTR derived from the α-globin gene (muag) may contain or consist of a nucleic acid sequence. The nucleic acid sequence is identical to sequence numbers 267, 268, 22896-22901, 22906-22911, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0222] In a further embodiment, the nucleic acid has a 3'UTR derived from the RPS9 gene. The 3'UTR derived from the RPS9 gene may contain or consist of a nucleic acid sequence. The nucleic acid sequence is identical to SEQ ID NOs. 263, 264, 22894, 22895, 22904, 22905, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0223] In a preferred embodiment, the nucleic acid has a 3'UTR derived from the PSMB3 gene. The 3'UTR derived from the PSMB3 gene may contain or consist of a nucleic acid sequence. The nucleic acid sequence is identical to SEQ ID NOs. 253, 254, 22892, 22893, 22902, 22903, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0224] In other embodiments, the nucleic acid has a 3'UTR, which contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to sequence numbers 22876-22891, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0225] In other embodiments, the nucleic acid has a 3'UTR as described in WO2016 / 107877. The disclosure of WO2016 / 107877 relating to 3'UTR sequences is incorporated herein by reference. Preferred 3'UTRs are sequence numbers 1-24, 49-318 of WO2016 / 107877, or fragments or variants of these sequences. In other embodiments, the nucleic acid has a 3'UTR as described in WO2017 / 036580. The disclosure of WO2017 / 036580 relating to 3'UTR sequences is incorporated herein by reference. Preferred 3'UTRs are sequence numbers 152-204 of WO2017 / 036580, or fragments or variants of these sequences. In other embodiments, the nucleic acid has a 3'UTR as described in WO2016 / 022914. The disclosures of WO2016 / 022914 relating to 3'UTR sequences are incorporated herein by reference. Particularly preferred 3'UTRs are the nucleic acid sequences described in Sequence IDs 20-36 of WO2016 / 022914, or fragments or variants thereof.

[0226] In a preferred embodiment, the nucleic acid (such as RNA or DNA) has one or more heterogeneous 5'UTRs.

[0227] The terms “5' untranslated region,” “5'UTR,” or “5'UTR element” are recognized and understood by those skilled in the art. These terms refer to, for example, a portion of a nucleic acid molecule located on the 5' side (i.e., upstream) of a coding sequence that is not translated into a protein. The 5'UTR may also be a portion of nucleic acid located on the 5' side of a coding sequence. Typically, the 5'UTR begins at the transcription start site and ends before the start codon of the coding sequence. The 5'UTR may contain elements (also called regulatory elements) that control gene expression. Examples of such regulatory elements include ribosome binding sites and miRNA binding sites. The 5'UTR may be post-transcriptionally modified. Post-transcriptional modification may occur, for example, enzymatically or by adding a 5' cap structure (such as mRNA, as described later).

[0228] Preferably, the nucleic acid has a 5'UTR. The 5'UTR can be derived from genes related to the extension of the RNA half-life (genes that provide stable RNA).

[0229] In some embodiments, the 5'UTR has one or more protein binding sites that affect the position or stability of RNA within the cell, or one or more miRNAs or miRNA binding sites (as described above).

[0230] Therefore, by removing or introducing the aforementioned miRNA or miRNA-binding site into the 5'UTR, the expression of nucleic acids (such as RNA) in a desired cell type or tissue (such as muscle cells) can be regulated.

[0231] In a preferred embodiment, the nucleic acid has one or more heterologous 5'UTRs. One or more heterologous 5'UTRs contain nucleic acid sequences derived from the 5'UTRs of genes selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2. Alternatively, one or more heterologous 5'UTRs are homologs, fragments, or variants of any one of these genes. The nucleic acid sequence is identical to sequence numbers 231-252, 22870-22875, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence may be a fragment or variant of any of these. In this regard, particularly preferred nucleic acid sequences may be selected from WO2019 / 077001A1 (especially claim 9 of WO2019 / 077001A1). The 5'UTR sequences corresponding to claim 9 of WO2019 / 077001A1 (such as sequence numbers 1-20 or fragments or variants thereof of WO2019 / 077001A1) are incorporated herein by reference.

[0232] In a preferred embodiment, the nucleic acid has a 5'UTR derived from the RPL31 gene. The 5'UTR derived from the RPL31 gene contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to or identical to sequence numbers 243, 244, 22872, 22873 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0233] In other embodiments, the nucleic acid has a 5'UTR derived from the SLC7A3 gene. The 5'UTR derived from the SLC7A3 gene contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to SEQ ID NOs. 245, 246, 22874, 22875, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0234] In a particularly preferred embodiment, the nucleic acid has a 5'UTR derived from the HSD17B4 gene. The 5'UTR derived from the HSD17B4 gene contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to SEQ ID NOs. 231, 232, 22870, 22871, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0235] In other embodiments, the nucleic acid has a 5'UTR, which contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to sequence numbers 22848-22869, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0236] In other embodiments, the nucleic acid has a 5'UTR as described in WO2013 / 143700. The disclosures of WO2013 / 143700 relating to 5'UTR sequences are incorporated herein by reference. Particularly preferred 5'UTRs are nucleic acid sequences derived from SEQ ID NOs. 1-1363, 1395, 1421, and 1422 of WO2013 / 143700, or fragments or variants thereof. In other embodiments, the nucleic acid has a 5'UTR as described in WO2016 / 107877. The disclosures of WO2016 / 107877 relating to 5'UTR sequences are incorporated herein by reference. Particularly preferred 5'UTRs are nucleic acid sequences derived from SEQ ID NOs. 25-30 and 319-382 of WO2016 / 107877, or fragments or variants thereof. In other embodiments, the nucleic acid has a 5'UTR as described in WO2017 / 036580. The disclosure of WO2017 / 036580 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are the nucleic acid sequences described in SEQ ID NOs. 1 to 151 of WO2017 / 036580, or fragments or variants thereof. In other embodiments, the nucleic acid has a 5'UTR as described in WO2016 / 022914. The disclosure of WO2016 / 022914 relating to 5'UTR sequences is incorporated herein by reference. Particularly preferred 5'UTRs are the nucleic acid sequences described in SEQ ID NOs. 3 to 19 of WO2016 / 022914, or fragments or variants thereof.

[0237] Preferably, in a preferred embodiment, the nucleic acid has one or more coding sequences as described herein that encode one or more antigenic proteins as described herein (preferably derived from SARS-CoV-2 (nCoV-2019) coronavirus). The coding sequences are operably linked to a 3'UTR and / or 5'UTR. The 3'UTR and / or 5'UTR are selected from the following 5'UTR / 3'UTR combinations (also referred to as “UTR designs”). a-1(HSD17B4 / PSMB3) a-2 (NDUFA4 / PSMB3) a-3 (SLC7A3 / PSMB3) a-4 (NOSIP / PSMB3) a-5 (MP68 / PSMB3) b-1(UBQLN2 / RPS9) b-2(ASAH1 / RPS9) b-3 (HSD17B4 / RPS9) b-4 (HSD17B4 / CASP1) b-5 (NOSIP / COX6B1) c-1(NDUFA4 / RPS9) c-2(NOSIP / NDUFA1) c-3(NDUFA4 / COX6B1) c-4 (NDUFA4 / NDUFA1) c-5 (ATP5A1 / PSMB3) d-1(Rpl31 / PSMB3) d-2 (ATP5A1 / CASP1) d-3 (SLC7A3 / GNAS) d-4(HSD17B4 / NDUFA1) d-5(Slc7a3 / Ndufa1) e-1 (TUBB4B / RPS9) e-2 (RPL31 / RPS9) e-3 (MP68 / RPS9) e-4 (NOSIP / RPS9) e-5 (ATP5A1 / RPS9) e-6(ATP5A1 / COX6B1) f-1(ATP5A1 / GNAS) f-2(ATP5A1 / NDUFA1) f-3 (HSD17B4 / COX6B1) f-4 (HSD17B4 / GNAS) f-5 (MP68 / COX6B1) g-1 (MP68 / NDUFA1) g-2 (NDUFA4 / CASP1) g-3 (NDUFA4 / GNAS) g-4(NOSIP / CASP1) g-5 (RPL31 / CASP1) h-1(RPL31 / COX6B1) h-2 (RPL31 / GNAS) h-3(RPL31 / NDUFA1) h-4(Slc7a3 / CASP1) h-5(SLC7A3 / COX6B1) i-1 (SLC7A3 / RPS9) i-2 (RPL32 / ALB7) i-2 (RPL32 / ALB7) i-3 (alpha-globin gene) In a particularly preferred embodiment, the nucleic acid has one or more coding sequences described herein. The coding sequence codes for one or more antigenic proteins (preferably derived from SARS-CoV-2 (nCoV-2019) coronavirus). The coding sequence is operably ligated to the 5'UTR of HSD17B4 and the 3'UTR of PSMB3 (HSD17B4 / PSMB3, UTR design: a-1).

[0238] As the inventors have shown, this embodiment is particularly beneficial for inducing an immune response to SARS-CoV-2. In this regard, it has been shown that a sufficient virus-neutralizing antibody titer can be obtained with just one vaccination.

[0239] In a more preferred embodiment, the nucleic acid has one or more coding sequences described herein. The one or more coding sequences encode one or more antigenic proteins described herein (preferably derived from SARS-CoV-2 (nCoV-2019) coronavirus). The coding sequences are operably ligated to the 5'UTR of SLC7A3 and the 3'UTR of PSMB3 (SLC7A3 / PSMB3, UTR design: a-3).

[0240] In a more preferred embodiment, the nucleic acid has one or more coding sequences described herein. The one or more coding sequences encode one or more antigenic proteins described herein (preferably derived from SARS-CoV-2 (nCoV-2019) coronavirus). The coding sequences are operably ligated to the 5'UTR of RPL31 and the 3'UTR of RPS9 (RPL31 / RPS9, UTR design: e-2).

[0241] In a particularly preferred embodiment, the nucleic acid has one or more coding sequences described herein. The one or more coding sequences encode one or more antigenic proteins described herein (preferably derived from SARS-CoV-2 (nCoV-2019) coronavirus). The coding sequences are operably ligated to the 3'UTR (- / muag) of alpha-globin (muag) (UTR design: i-3).

[0242] In some embodiments, nucleic acids (such as DNA or RNA) can be monocistron, bicistron, or multicistron.

[0243] The term "monocistrone" will be recognized and understood by those skilled in the art. This term is intended to refer to, for example, a nucleic acid having only one coding sequence. In this specification, the terms "biocistrone" or "multicistronone" will be recognized and understood by those skilled in the art. These terms are intended to refer to, for example, a nucleic acid having two coding sequences (biocistrone) or a nucleic acid having more than two coding sequences (multicistronone).

[0244] In a preferred embodiment, the nucleic acid of the first embodiment is a monocistrone.

[0245] In other embodiments, the nucleic acid is a monocistrone. The coding sequence of the nucleic acid encodes two or more different antigenic peptides or antigenic proteins derived from the SARS-CoV-2 coronavirus. Thus, the coding sequence may encode two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, and more antigenic peptides or antigenic proteins derived from the SARS-CoV-2 coronavirus. The antigenic peptides or antigenic proteins may or may not be linked by an amino acid linker sequence. The linker sequence may include a rigid linker, a flexible linker, a cleaving linker, or a combination thereof. In this specification, such a construct is referred to as a “multi-antigen construct”.

[0246] In further embodiments, the nucleic acid may be a bicistron or a multicistron. The nucleic acid has two or more coding sequences. The two or more coding sequences encode two or more different antigenic peptides or antigenic proteins derived from the SARS-CoV-2 coronavirus. Therefore, coding sequences contained in a bicistron or multicistron nucleic acid are suitable for encoding the distinguishable antigenic proteins or antigenic peptides, their immunogenic fragments or immunogenic variants described herein. Preferably, the coding sequences contained in a bicistron or multicistron construct are fragmented by one or more IRES sequences (internal ribosome entry site sequences). Therefore, the expression "encodes two or more antigenic peptides or antigenic proteins" may mean, for example, a bicistron or multicistron nucleic acid encoding two, three or more, four or more, five or more, six or more, or more (preferably different) antigenic peptides or antigenic proteins from different isolates of the SARS-CoV-2 coronavirus. Alternatively, a bicistronic nucleic acid or multicistronic nucleic acid may encode, for example, two or more (preferably different) antigenic peptides or antigenic proteins derived from the same SARS-CoV-2 coronavirus, such as two, three or more, four or more, five or more, six or more, or more. In this regard, suitable IRES sequences may be selected from the group of nucleic acid sequences described in Sequence IDs 1566-1662 of WO2017 / 081082, or from fragments or variants of these sequences. In this regard, the disclosures of WO2017 / 081082 relating to IRES sequences are incorporated herein by reference.

[0247] In relation to the present invention, it should be understood that specific combinations of coding sequences can be achieved by any combination of monocistrone, bicistrone, and multicistrone DNA constructs, RNA constructs, and / or multiantigen constructs. This results in a group of nucleic acids encoding multiple antigenic peptides or antigenic proteins as described herein.

[0248] In a preferred embodiment, the A / U(A / T) content around the ribosome binding site of the nucleic acid may be increased compared to the A / U(A / T) content around the ribosome binding site of the wild-type nucleic acid or a reference nucleic acid. This modification (increasing the A / U(A / T) content around the ribosome binding site) improves, for example, the efficiency of ribosome binding to nucleic acids (such as RNA). Improved efficiency of ribosome binding to the ribosome binding site allows for efficient translation of nucleic acids.

[0249] Therefore, in a particularly preferred embodiment, the nucleic acid has a ribosome-binding site also referred to as a Kozak sequence. The ribosome-binding site is identical to any one of sequence numbers 180, 181, 22845-22847, or is identical by 80% or more, 85% or more, 90% or more, or 95% or more. Alternatively, the ribosome-binding site is a fragment or variant thereof.

[0250] In a preferred embodiment, the nucleic acid of the present invention has one or more poly(N) sequences. Examples of one or more poly(N) sequences include one or more poly(A) sequences, one or more poly(U) sequences, one or more poly(C) sequences, or a combination thereof.

[0251] In a preferred embodiment, the nucleic acid (preferably RNA) of the present invention has one or more poly(A) sequences.

[0252] In this specification, the terms “poly(A) sequence,” “poly(A) tail,” or “3' poly(A) tail” will be recognized and understood by those skilled in the art. These terms refer to a sequence of adenosine nucleotides, typically located at the 3' end of linear (or circular) RNA, up to approximately 1000 nucleotides. Preferably, the poly(A) sequence is substantially homopolymer. For example, the length of a poly(A) sequence of 100 adenosine nucleotides is substantially 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by one or more nucleotides different from adenosine nucleotides. For example, the length of a poly(A) sequence of 100 adenosine nucleotides may be greater than 100 nucleotides. In this case, in addition to the 100 adenosine nucleotides, it includes one or more nucleotides (or a series of nucleotides) different from adenosine nucleotides. It should be understood that while the “poly(A) sequence” as used herein typically relates to RNA, in the context of this invention, the term also relates to the corresponding sequence contained within a DNA molecule (such as the poly(T) sequence).

[0253] The poly(A) sequence may contain approximately 10 to 500, 10 to 200, 40 to 200, or 40 to 150 adenosine nucleotides. Preferably, the length of the poly(A) sequence may be approximately 10 or more, 50 or more, 64 or more, 75 or more, 100 or more, 200 or more, 300 or more, 400 or more, or 500 or more adenosine nucleotides.

[0254] In a preferred embodiment, the nucleic acid has one or more poly(A) sequences. One or more poly(A) sequences contain about 30 to about 200 adenosine nucleotides. In a particularly preferred embodiment, the poly(A) sequence contains about 64 adenosine nucleotides (A64). In another particularly preferred embodiment, the poly(A) sequence contains about 100 adenosine nucleotides (A100). In another embodiment, the poly(A) sequence contains about 150 adenosine nucleotides.

[0255] In a further embodiment, the nucleic acid has one or more poly(A) sequences. The one or more poly(A) sequences contain about 100 adenosine nucleotides. The poly(A) sequences are interrupted by nucleotides other than adenosine. Preferably, they are interrupted by 10 nucleotides other than adenosine (A30-N10-A70).

[0256] The poly(A) sequences described herein may be located directly at the 3' end of a nucleic acid. Preferably, they may be located directly at the 3' end of RNA.

[0257] In a preferred embodiment, the 3'-terminal nucleotide (i.e., the last nucleotide at the 3' end of a polynucleotide chain) is the 3'-terminal A nucleotide of one or more poly(A) sequences. The term "directly located at the 3' end" must be understood to mean precisely located at the 3' end. In other words, the 3' end of the nucleic acid consists of a poly(A) sequence that terminates with an A nucleotide.

[0258] As the inventors have shown, this embodiment is particularly beneficial for inducing an immune response to SARS-CoV-2. In this regard, it has been shown that a sufficient virus-neutralizing antibody titer can be obtained with just one vaccination.

[0259] In a particularly preferred embodiment, the nucleic acid sequence (preferably RNA) has a poly(A) sequence. The poly(A) sequence contains 70 or more adenosine nucleotides. The nucleotide at the 3' end is an adenosine nucleotide.

[0260] In this regard, it has been shown that IFN-α induction by RNA vaccines is reduced by using adenosine nucleotides at the end of the molecule. This fact is particularly important because IFN-α induction is considered to be the main cause of fever in vaccinated subjects, and this fever should obviously be avoided.

[0261] In embodiments where the nucleic acid is RNA, the poly(A) sequence of the nucleic acid is preferably obtained from a DNA template in RNA transcription in vitro. In other embodiments, the poly(A) sequence does not necessarily need to be transcribed from a DNA template but is obtained in vitro by common chemical synthesis methods. In other embodiments, the poly(A) sequence is produced by enzymatic polyadenylation of RNA (after RNA transcription in vitro). In this case, commercially available polyadenylation kits and corresponding protocols well known in the art are used. Alternatively, the poly(A) sequence is produced using immobilized poly(A) polymerase. In this case, for example, the method and means described in WO2016 / 174271 are used.

[0262] Nucleic acids may have poly(A) sequences obtained by enzymatic polyadenylation. Most nucleic acid molecules contain about 100 (+ / -20) to about 500 (+ / -50) adenosine nucleotides. Preferably, they contain about 250 (+ / -20) adenosine nucleotides.

[0263] In other embodiments, the nucleic acid may have a poly(A) sequence derived from template DNA. The nucleic acid may further contain one or more additional poly(A) sequences resulting from enzymatic polyadenylation. See, for example, WO2016 / 091391.

[0264] In a further embodiment, the nucleic acid has one or more polyadenylation signals.

[0265] In other embodiments, the nucleic acid may have one or more poly(C) sequences.

[0266] In this specification, the term "poly(C) sequence" refers to a sequence of up to about 200 cytosine nucleotides. In preferred embodiments, the poly(C) sequence contains about 10 to about 200, about 10 to about 100, about 20 to about 70, about 20 to about 60, or about 10 to about 40 cytosine nucleotides. In a particularly preferred embodiment, the poly(C) sequence contains about 30 cytosine nucleotides.

[0267] In a preferred embodiment, the nucleic acid has one or more histone stem-loops (hSLs).

[0268] The term "histone stem-loop" (abbreviated as hSL in sequence listings, etc.) refers to a nucleic acid sequence that is primarily found in histone mRNA and forms a stem-loop secondary structure.

[0269] The histone stem-loop sequence / structure may preferably be selected from the histone stem-loop sequences disclosed in WO2012 / 019780. The disclosures relating to histone stem-loop sequences / structures in that document are incorporated herein by reference. The histone stem-loop sequences that can be used in the present invention may preferably be derived from formula (I) or (II) of WO2012 / 019780. In a more preferred embodiment, the nucleic acid may have one or more histone stem-loop sequences derived from one or more specific formulas (Ia) or (IIa) described in WO2012 / 019780.

[0270] In a preferred embodiment, the nucleic acid of the present invention has one or more histone stem loops. The histone stem loop (hSL) includes or consists of a nucleic acid sequence. The nucleic acid sequence is identical to SEQ ID NO: 178 or 179, or is identical by 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0271] In other embodiments, the RNA of the first embodiment does not have the histone stem loop described herein.

[0272] In some embodiments (particularly in embodiments relating to RNA), the nucleic acid has a 3' terminal sequence element. The 3' terminal sequence element comprises a poly(A) sequence and, optionally, a histone stem-loop sequence. Thus, the nucleic acid of the present invention has one or more 3' terminal sequence elements. One or more 3' terminal sequence elements contain or consist of a nucleic acid sequence. The nucleic acid sequence is identical to SEQ ID NOs. 182-230, 22912, 22913, or is 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0273] In preferred embodiments (particularly in embodiments relating to RNA), the nucleic acid has a 3' terminal sequence element. The 3' terminal sequence element comprises a poly(A) sequence and, optionally, a histone stem-loop sequence. Thus, the nucleic acid of the present invention has one or more 3' terminal sequence elements. The 3' terminal sequence element comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to SEQ ID NOs. 182, 187, 189, 192, 199, 207, or is identical by 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant thereof.

[0274] In various embodiments (particularly in embodiments relating to RNA), the nucleic acid has a 5' terminal sequence element, or a fragment or variant thereof, as described in SEQ ID NOs: 176, 177, 22840-22844. Such a 5' terminal sequence element includes, for example, a binding site for T7 RNA polymerase. Furthermore, the first nucleotide of the 5' terminal start sequence is preferably 2'-O-methylated (e.g., 2'-O-methylated guanosine or 2'-O-methylated adenosine).

[0275] Preferably, the nucleic acid (such as RNA or DNA) of the first embodiment contains about 50 to about 20,000 nucleotides, about 500 to about 10,000 nucleotides, or about 1,000 to about 10,000 nucleotides. Preferably, it is about 1,000 to about 5,000 nucleotides. More preferably, it is about 2,000 to about 5,000 nucleotides.

[0276] In some embodiments, nucleic acids are DNA or RNA.

[0277] In various embodiments, the DNA is a construct of plasmid DNA or linear coding DNA. The DNA contains or consists of the nucleic acid elements described herein (coding sequence, UTR, poly(A / T), polyadenylation signal, promoter, etc.).

[0278] In a preferred embodiment, the nucleic acid is a DNA expression vector. Such a DNA expression vector may be selected from the group consisting of bacterial plasmids, adenoviruses, poxviruses, parapoxviruses (Orff virus), vaccinia viruses, fowlpox viruses, herpesviruses, adeno-associated viruses (AAVs), alphaviruses, lentiviruses, lambda phages, lymphocytic choriomeningitis viruses, Listeria sp., and Salmonella sp.

[0279] Preferably, the DNA may further have a promoter. The promoter is operably ligated to the SARS-CoV-2 antigen coding sequence. Examples of promoters operably ligated to an antigen coding sequence include the Simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) promoter (e.g., the bovine immunodeficiency virus (BIV) long-term repeat (LTR) promoter), the Moloney virus promoter, the avian leukemia virus (ALV) promoter, the cytomegalovirus (CMV) promoter (e.g., the CMV pre-early promoter), the Epstein-Barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter. The promoter may also be derived from a human gene (e.g., human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein). The promoter may also be a tissue-specific promoter (e.g., a (natural or synthetic) muscle-specific promoter, a skin-specific promoter). Examples of such promoters are disclosed in US20040175727. In preferred embodiments, the vector is pVAX, pcDNA3.0, or provax. Alternatively, the vector is any other expression vector that expresses DNA encoding a coronavirus antigen, causing cells to translate the sequence to produce an antigen recognized by the immune system.

[0280] Further suitable plasmid DNA may be prepared to efficiently produce the SARS-CoV-2 antigen encoded by the cell line. For example, in insect cell lines, vectors as defined in claims 1 to 33 of WO2009 / 150222A2 can be used. Claims 1 to 33 of WO2009 / 150222A2 are incorporated herein by reference.

[0281] In other embodiments, the nucleic acid of the first embodiment is an adenovirus vector. Such an adenovirus vector may have one or more coding sequences encoding one or more SARS-CoV-2 antigenic peptides or antigenic proteins described herein.

[0282] In connection with the present invention, any suitable adenovirus vector can be used (such as those described in WO2005 / 071093 or WO2006 / 048215). Preferably, the adenovirus vector used is a monkey adenovirus. This avoids the attenuation of the immune response after vaccination due to common existing antibodies against humans (such as AdHu5). Examples of suitable monkey adenovirus vectors include AdCh63 (WO2005 / 071093) or AdCh68 (Cohen et al J. Gen Virol 2002 83: 151). However, other vectors may be used. Preferably, adenoviruses. The vector lacks the E1 region, which causes it to lose replication ability in human cells. Other regions of the adenovirus (such as E3 and E4) may also be deleted.

[0283] In further embodiments, the nucleic acid of the first embodiment is an Orff virus vector. Such an adenovirus vector may have one or more coding sequences encoding one or more SARS-CoV-2 antigenic peptides or antigenic proteins described herein.

[0284] In a particularly preferred embodiment of the present invention, the nucleic acid of the present invention is RNA.

[0285] Preferably, the number of nucleotides in the RNA is typically about 50 to 20,000, about 500 to 10,000, or about 1,000 to 10,000. Preferably, it is about 1,000 to 5,000. More preferably, it is about 2,000 to 5,000.

[0286] According to a preferred embodiment, the nucleic acid is RNA (preferably coding RNA).

[0287] In a preferred mechanism, the coding RNA may be selected from mRNA, (coding) self-replicating RNA, (coding) circular RNA, (coding) viral RNA, or (coding) replicon RNA.

[0288] In other embodiments, the coding RNA is circular RNA. In this specification, “circular RNA” or “circRNA” should be understood as a circular polynucleotide construct encoding one or more antigenic peptides or antigenic proteins as described herein. Preferably, such circRNA is a single-stranded RNA molecule. In preferred embodiments, the circRNA has one or more coding sequences. The one or more coding sequences encode one or more antigenic proteins, immunogenic fragments or immunogenic variants thereof, derived from the SARS-CoV-2 coronavirus.

[0289] In a further embodiment, the coding RNA is a replicon RNA. The term “replicon RNA” will be recognized and understood by those skilled in the art. The term refers, for example, to optimized self-replicating RNA. Examples of such constructs include replicase elements derived from alphaviruses (such as SFV, SIN, VEE, or RRV), and substitutions of structural viral proteins with a given nucleic acid (a coding sequence encoding an antigenic peptide or antigenic protein of the SARS-CoV-2 coronavirus). Alternatively, the replicase may be provided as a construct of independent coding RNA or coding DNA. Downstream of the replicase, there may be a subgenome promoter that controls the replication of the replicon RNA.

[0290] In a particularly preferred embodiment, one or more nucleic acids are not replicon RNA or self-replicating RNA.

[0291] In a particularly preferred embodiment, the nucleic acid of the present invention is mRNA.

[0292] Preferably, the mRNA does not contain replicase elements (such as nucleic acids that encode replicases).

[0293] The terms "RNA" and "mRNA" are recognized and understood by those skilled in the art. These terms refer to, for example, ribonucleic acid molecules (polymers composed of nucleotides). These nucleotides are typically adenosine monophosphate monomers, uridine monophosphate monomers, guanosine monophosphate monomers, and cytidine monophosphate monomers, linked together along a so-called backbone. This backbone is formed by phosphodiester bonds between the sugar (ribose) of an adjacent first monomer and the phosphate group of a second monomer. The specific sequence of monomers is called an RNA sequence. The nucleotide coding sequence provided by mRNA (messenger RNA) can be translated into the amino acid sequence of a specific peptide or protein.

[0294] In connection with the present invention, the coding RNA (preferably mRNA) may provide one or more coding sequences encoding the antigenic protein of SARS-CoV-2. One or more coding sequences are translated into (functional) antigens after administration (for example, after administration to a subject such as a human subject).

[0295] Therefore, coding RNA (preferably mRNA) is suitable for vaccines (preferably SARS-CoV-2 vaccines).

[0296] Preferably, the coding RNA may be modified by the addition of a 5' cap structure. Preferably, the 5' cap structure stabilizes the coding RNA (after administration to the subject), promotes the expression of the encoded antigen, or reduces stimulation of the innate immune system. The 5' cap structure is particularly important in embodiments where the nucleic acid is linear coding RNA (such as linear mRNA) or linear coding replicon RNA.

[0297] Therefore, in a preferred embodiment, the RNA (particularly coding RNA) has a 5' cap structure. Preferably, the RNA has a cap0 structure, a cap1 structure, a cap2 structure, a modified cap0 structure, or a modified cap1 structure.

[0298] In this specification, the term “5' cap structure” will be recognized and understood by those skilled in the art. This term is intended to refer to, for example, a 5' modified nucleotide (particularly a guanine nucleotide) located at the 5' end of RNA (such as mRNA). Preferably, the 5' cap structure is linked to the RNA via a 5'-5' triphosphate bond.

[0299] Examples of suitable 5' cap structures in relation to the present invention include cap0 (methylation of a first nucleic acid base such as m7GpppN), cap1 (additional methylation of ribose of a nucleotide adjacent to m7GpppN), cap2 (additional methylation of ribose of a second nucleotide downstream of m7GpppN), cap3 (additional methylation of ribose of a third nucleotide downstream of m7GpppN), cap4 (additional methylation of ribose of a fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (phosphothioate modified ARCA, etc.), inosine, N1-methyl-guanosine, 2'-fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, and 2-azidoguanosine.

[0300] The 5' cap (cap0 or cap1) structure may be formed during chemical RNA synthesis or during in vitro RNA transcription using a cap analog (co-transcription capping).

[0301] In this specification, the term “cap analog” will be recognized and understood by those skilled in the art. The term is intended to represent, for example, a non-polymerizable dinucleotide or trinucleotide having a cap function. The cap function is the function of facilitating translation or localization or preventing degradation of the nucleic acid molecule (especially RNA molecules) when incorporated into the 5' end of a nucleic acid molecule. Non-polymerizable means that the cap analog is incorporated only into the 5' end. This is because, lacking a 5' triphosphate, it cannot be extended in the 3' direction by template-dependent polymerases (especially template-dependent RNA polymerases). Examples of capped analogs include (but are not limited to) chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; non-methylated capped analogs (such as GpppG); dimethylated capped analogs (such as m2,7GpppG); trimethylated capped analogs (such as m2,2,7GpppG); dimethylated symmetrical capped analogs (such as m7Gpppm7G); or anti-reverse capped analogs (ARCA; such as m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives). Further capping analogs have also been made public (WO2008 / 016473, WO2008 / 157688, WO2009 / 149253, WOWO2011 / 015347, and WO2013 / 059475). In connection therewith, further preferred capping analogs are disclosed in WO2017 / 066793, WO2017 / 066781, WO2017 / 066791, WO2017 / 066789, WO2017 / 053297, WO2017 / 066782, WO2018 / 075827, and WO2017 / 066797. Disclosures referring to capping analogs are incorporated herein by reference.

[0302] In some embodiments, the modified cap1 structure is prepared using the trinucleotide cap analogs disclosed in WO2017 / 053297, WO2017 / 066793, WO2017 / 066781, WO2017 / 066791, WO2017 / 066789, WO2017 / 066782, WO2018 / 075827, and WO2017 / 066797. In particular, the modified cap1 structure can be prepared co-transferred using any cap structure derived from the structures disclosed in claims 1 to 5 of WO2017 / 053297 as appropriate. Furthermore, the modified cap1 structure can be prepared co-transferred using any cap structure derived from the structure described in claim 1 or 21 of WO2018 / 075827 as appropriate.

[0303] In a preferred embodiment, the (coding) RNA (particularly mRNA) has a cap1 structure.

[0304] In preferred embodiments, a 5' cap structure can be suitably co-transcribed into the RNA transcription reaction in vitro described herein using the trinucleotide cap analog described herein.

[0305] In a preferred embodiment, the cap1 structure of the coding RNA of the present invention is formed by co-transcription capping using the trinucleotide capping analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. A preferred cap1 analog in this regard is m7G(5')ppp(5')(2'OMeA)pG.

[0306] In another preferred embodiment, the cap1 structure of the RNA of the present invention is formed by co-transcriptional capping using the trinucleotide capping analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0307] In another embodiment, the cap0 structure of the RNA of the present invention is formed by co-transcription capping using the cap analog 3'OMe-m7G(5')ppp(5')G.

[0308] In other embodiments, the 5' cap structure is formed by enzymatic capping using a capping enzyme, resulting in a cap0, cap1, or cap2 structure. Examples of capping enzymes include vaccinia virus capping enzyme and / or cap-dependent 2'-O methyltransferase. The 5' cap structure (cap0 or cap1) may be added using an immobilized capping enzyme and / or cap-dependent 2'-O methyltransferase, utilizing the methods and means disclosed in WO2016 / 193226.

[0309] In a preferred embodiment, when measured by a capping assay, approximately 70%, 75%, 80%, 85%, 90%, and 95% of the RNA (species) have a cap1 structure. In a preferred embodiment, when measured by a capping assay, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% of the RNA (species) do not have a cap1 structure. In another preferred embodiment, when measured by a capping assay, approximately 70%, 75%, 80%, 85%, 90%, and 95% of the RNA (species) have a cap0 structure. In a preferred embodiment, when measured by a capping assay, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, and 1% of the RNA (species) do not have a cap0 structure.

[0310] The term "RNA species" is understood to include not only a single molecule but also a collection of substantially identical RNA molecules. Therefore, the present invention may relate to multiple substantially identical (coding) RNA molecules.

[0311] To determine the presence or absence of a cap0 or cap1 structure, the capping assays disclosed in WO2015 / 101416 (in particular, the capping assays described in claims 27-46 of WO2015 / 101416) can be used. Other capping assays available for determining the presence or absence of a cap0 or cap1 structure in RNA are disclosed in PCT / EP2018 / 08667, WO2014 / 152673, and WO2014 / 152659.

[0312] In a preferred embodiment, the RNA has an m7G(5')ppp(5')(2'OMeA) cap structure. In this embodiment, the coding RNA has a 5'-terminal m7G cap and additional methylation of the ribose of the nucleotide adjacent to m7GpppN (in this case, 2'-O-methylated adenosine). Preferably, when measured by a capping assay, about 70%, 75%, 80%, 85%, 90%, and 95% of the RNA (species) have the cap1 structure.

[0313] In another preferred embodiment, the RNA has an m7G(5')ppp(5')(2'OMeG) cap structure. In this embodiment, the coding RNA has a 5'-terminal m7G cap and additional methylation of the ribose of the adjacent nucleotide (in this case, 2'-O-methylated guanosine). Preferably, when measured by a capping assay, about 70%, 75%, 80%, 85%, 90%, and 95% of the coding RNA (species) have the cap1 structure.

[0314] Therefore, the first nucleotide of the RNA sequence or mRNA sequence (the downstream nucleotide of the m7G(5')ppp structure) may be 2'-O-methylated guanosine or 2'-O-methylated adenosine.

[0315] According to some embodiments, RNA is modified RNA. Modifications include not only sugar or base modifications but also skeletal modifications.

[0316] Modified RNA may have nucleotide analogs / modifications (such as modifications to the backbone, sugars, or bases). In relation to the present invention, backbone modification refers to a modification in which the phosphate of the nucleotide backbone of RNA is chemically altered. In relation to the present invention, sugar modification refers to a chemical modification of the sugar of the RNA nucleotide. In relation to the present invention, base modification refers to a chemical modification of the base portion of the RNA nucleotide. In this regard, the nucleotide analog or modification is preferably selected from nucleotide analogs available for transcription and / or translation.

[0317] In particularly preferred embodiments, the nucleotide analogs / modifications that may be included in the modified RNA described herein are preferably selected from the following: 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate Acid, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate Phosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate or promycin-5'-triphosphate, xanthosine-5'-triphosphate. Particularly preferred, the nucleotide used for base modification is selected from the group consisting of: 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate,Pseudouridine-5'-triphosphate, pyridine-4-onyribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, 5-taurinomethyl-2-thiouridine, 1-tau Linomethyl-4-thiouridine, 5-methyluridine, 1-methylpsuduridine, 4-thio-1-methylpsuduridine, 2-thio-1-methylpsuduridine, 1-methyl-1-deazapsuduridine, 2-thio-1-methyl-1-deazapsuduridine, dihydrouridine, dihydropsuduridine, 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypsuduridine, 4-methoxy-2-thiopsuduridine Douridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine , Zebralin, 5-Aza-Zebralin, 5-Methyl-Zebralin, 5-Aza-2-Thio-Zebralin, 2-Thio-Zebralin, 2-Methoxy-Cytidine, 2-Methoxy-5-Methyl-Cytidine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, 2-Aminopurine, 2,6-Diaminopurine, 7-Deaza-Adenine, 7-Deaza-8-Aza-Adenine, 7-Deaza-2-Aminopurine, 7-Deaza-8-Aza-2-Aminopurine, 7-Deaza-2,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 1-Methyladenosine, N6-Methyladenosine, N6-Isopentenyladenosine, N6-(cis-Hydroxyisopentenyl)adenosine, 2-Methylthio-N6-(cis-Hydroxyisopentenyl)adenosine, N6-Glycinylcarbamoyladenosine, N6-Threonylcarbamoyladenosine, 2-Methylthio-N6-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-Methyladenine, 2-Methylthio-Adenine, 2-Methoxy-Adenine, Inosine, 1-Methyl-Ino Syn, iosin, wibutosin, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio O-guanosine, N2,N2-dimethyl-6-thio-guanosine, 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseudruridine, 6-aza-cytidine, 2-thiocytidine, α-thiocytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iod-uridine, N1-methyl-pseudruridine, 5,6-dihydrouridine, α-thiouridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0318] In some embodiments, one or more modified nucleotides are selected from the following: pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0319] In some embodiments, 100% of the uracil contained in the coding sequence described herein is chemically modified. Preferably, the 5-position of the uracil is chemically modified.

[0320] Particularly preferred in connection with the present invention are pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine.

[0321] However, in some embodiments, the polynucleotide molecule of the embodiment lacks the position substituted with N1-methylpseudridine (m1ψ). In further embodiments, the polynucleotide molecule of the embodiment lacks the positions substituted with pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. In further embodiments, the polynucleotide molecule of the embodiment has a coding sequence consisting only of G nucleotides, C nucleotides, A nucleotides, and U nucleotides.

[0322] Incorporating modified nucleotides such as pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and / or 5-methoxyuridine into the coding sequence of RNA can be advantageous. This is because it can modulate or (in some cases) reduce undesirable innate immune responses (when coding RNA or vaccines are administered).

[0323] In some embodiments, the RNA has one or more coding sequences encoding the SARS-CoV-2 antigenic protein described herein. The coding sequences contain one or more modified nucleotides selected from pseudouridine (ψ) and N1-methylpseudridine (m1ψ). Preferably, all uracil nucleotides are substituted with pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides.

[0324] In a preferred embodiment, the RNA does not have a position substituted with N1-methylpseudridine (m1ψ). In a further embodiment, the RNA does not have a position substituted with pseudouridine (ψ), N1-methylpseudridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine.

[0325] In preferred embodiments, the RNA consists only of G nucleotides, C nucleotides, A nucleotides, and U nucleotides. Therefore, the RNA does not contain modified nucleotides (except for the cap analog).

[0326] (Nucleic acid (preferably mRNA) construct suitable for coronavirus vaccines) In various embodiments, the nucleic acid (preferably mRNA) preferably has the following elements in the 5' to 3' direction. (A) 5' cap structure (preferably as described herein) (B) 5' terminal initiation element (preferably one of those specified herein) (C) 5'UTR of any configuration (preferably as described herein) (D) Ribosome binding site (preferably as described herein) (E) One or more coding sequences (preferably those described herein) (F)3'UTR (preferably as described herein) (G) Poly(A) sequences of any configuration (preferably those described herein) (H) Poly(C) sequences of any configuration (preferably those described herein) (I) Histone stem loop of any configuration (preferably as described herein) (J) 3' terminal sequence elements of any configuration (preferably those described herein) In a preferred embodiment, the nucleic acid (preferably mRNA) preferably has the following elements in the 5' to 3' direction. (A) A 5' cap structure selected from m7G(5'), m7G(5')ppp(5')(2'OMeA) or m7G(5')ppp(5')(2'OMeG). (B) A 5'-terminated start element selected from sequence numbers 176, 177, or their fragments or variants. (C) Any configuration, 5'UTR derived from the HSD17B4 gene (D) Ribosome binding sites selected from sequence numbers 180, 181, 22845-22847, or their fragments or variants. (E) Sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, and one or more coding sequences selected from their fragments or variants. (F) 3'UTR derived from the PSMB3 gene or the α-globin gene (muag) (G) Poly(A) sequences containing approximately 30 to 500 adenosine molecules in any configuration. (H) A poly(C) sequence with an arbitrary configuration containing approximately 10 to 100 cytosines. (I) Any configuration, a histone stem loop selected from sequence number 178 or 179. (J) Any configuration, with 3' terminal sequence elements selected from sequence numbers 182-230. In a particularly preferred embodiment, the nucleic acid (preferably mRNA) has the following elements in the 5' to 3' direction. (A) Cap1 structure as described herein (B) Coding sequences selected from sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, or their fragments or variants. (C) 3'UTR derived from the 3'UTR of the muag gene described herein (preferably those described in SEQ ID NOs. 267, 268, 22896-22901, 22906-22911) (D) A poly(A) sequence containing approximately 64 A nucleotides (E) Poly(C) sequence containing approximately 10 to 100 cytosines (F) Histone stem loop selected from sequence number 178 or 179 In a preferred embodiment, the nucleic acid (preferably mRNA) has the following elements in the 5' to 3' direction. (A) Cap1 structure as described herein (B) 5'UTR derived from the HSD17B4 gene as described herein (preferably the one described in SEQ ID NO: 231 or 232) (C) Sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, and coding sequences selected from their fragments or variants. (D) A 3'UTR derived from the 3'UTR of the PSMB3 gene as described herein (preferably the one described in SEQ ID NO: 253 or 254) (E) A poly(A) sequence containing approximately 64 A nucleotides (F) A poly(C) sequence with an arbitrary configuration containing approximately 10 to 100 cytosines. (G) Histone stem loop selected from sequence number 178 or 179 (H) Any configuration, 3' end sequence elements of sequence numbers 182-230 In a particularly preferred embodiment, the nucleic acid (preferably mRNA) has the following elements in the 5' to 3' direction. (A) Cap1 structure as described herein (B) 5'UTR derived from the HSD17B4 gene as described herein (preferably as described in SEQ ID NO: 231 or 232) (C) Sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, and coding sequences selected from their fragments or variants. (D) A 3'UTR derived from the 3'UTR of the PSMB3 gene described herein (preferably SEQ ID NO: 253 or 254) (E) Histone stem loop selected from sequence number 178 or 179 (F) A poly(A) sequence containing approximately 100 A nucleotides (preferably corresponding to the 3' end) In a more preferred embodiment, the nucleic acid (preferably mRNA) has the following elements in the 5' to 3' direction. (A) Cap1 structure as described herein (B) 5'UTR derived from the HSD17B4 gene as described herein (preferably the one described in SEQ ID NO: 231 or 232) (C) Sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, and coding sequences selected from their fragments or variants. (D) A 3'UTR derived from the 3'UTR of the PSMB3 gene as described herein (preferably the one described in SEQ ID NO: 253 or 254) (F) A poly(A) sequence containing approximately 100 A nucleotides (preferably corresponding to the 3' end) In a more preferred embodiment, the nucleic acid (preferably mRNA) has the following elements in the 5' to 3' direction. (A) Cap1 structure as described herein (B) 5'UTR derived from the SLC7A3 gene as described herein (preferably the one described in SEQ ID NO: 245 or 246) (C) Sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, and coding sequences selected from their fragments or variants. (D) A 3'UTR derived from the 3'UTR of the PSMB3 gene as described herein (preferably the one described in SEQ ID NO: 253 or 254) (E) Any configuration, a histone stem loop selected from sequence number 178 or 179. (F) A poly(A) sequence containing approximately 100 A nucleotides (preferably corresponding to the 3' end) In a more preferred embodiment, the nucleic acid (preferably mRNA) has the following elements in the 5' to 3' direction. (A) Cap1 structure as described herein (B) 5'UTR derived from the RPL31 gene as described herein (preferably the one described in SEQ ID NO 243 or 243) (C) Sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184, and coding sequences selected from their fragments or variants. (D) A 3'UTR derived from the 3'UTR of the RPS9 gene as described herein (preferably the one described in SEQ ID NO: 263 or 264) (E) Any configuration, a histone stem loop selected from sequence number 178 or 179. (F) A poly(A) sequence containing approximately 100 A nucleotides (preferably corresponding to the 3' end) Table 3a shows preferred nucleic acid sequences (preferably mRNA sequences) of the present invention. In Table 3a, each row represents a particularly preferred SARS-CoV-2 (nCoV-2019) construct of the present invention (corresponding to Table 1). Column A of Table 3a contains a description of the SARS-CoV-2 construct. Column B contains the sequence number of the amino acid sequence for each SARS-CoV-2 construct. The sequence numbers corresponding to the coding sequences encoding each SARS-CoV-2 construct are listed in Table 1. Additional information is available for each sequence number in the sequence listing. <223> It is written in the section.

[0327] Columns C and D describe the corresponding nucleic acids (preferably coding RNA sequences, and especially preferably mRNA sequences having a coding sequence). Column C describes nucleic acid sequences having the UTR combination "HSD17B4 / PSMB3" as described herein. Column D describes nucleic acid sequences having the 3' UTR of α-globin as described herein.

[0328] [Table 3a]

[0329] JPEG2026048878000006.jpg110169

[0330] JPEG2026048878000007.jpg105169

[0331] Table 3b shows further preferred nucleic acid sequences (preferably the mRNA sequences of the present invention). In Table 3b, each column represents a particularly preferred SARS-CoV-2 (nCoV-2019) construct of the present invention (corresponding to Tables 1 and 3a). Column B lists the full-length spike protein (S) from the first row of Tables 1 and 3a. Column C lists the stabilized spike protein (S_stab_PP) compared to the second row of Tables 1 and 3a.

[0332] The first row lists the sequence numbers of the amino acid sequences of each SARS-CoV-2 construct. The sequence numbers corresponding to the coding sequences encoding each SARS-CoV-2 construct are listed in Table 1. Additional information is provided for each sequence number in the sequence listing. <223> It is written in the section.

[0333] Rows 2 through 16 describe the corresponding nucleic acids (preferably coding RNA sequences, and especially preferably mRNA sequences having a coding sequence). Each row describes the combination of UTRs and nucleic acid sequences having a preferred 3' end.

[0334] [Table 3b]

[0335] In a preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequences are: SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824 , is identical to a nucleic acid sequence selected from the group consisting of 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant of any of these sequences. Additional information about each nucleic acid sequence is provided for each sequence number in the sequence listing. <223> This information is also included in the section, Table 3a (see columns C and D in particular), and Table 3b (see rows 2 through 16 in particular).

[0336] In a particularly preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to, or 99% or more identical to, a nucleic acid sequence of any of these sequences. Additional information regarding each nucleic acid sequence is provided for each sequence number in the sequence listing. <223> This information is also included in the column, Table 3a (see column D in particular), and Table 3b (row 2).

[0337] In a particularly preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to, or 70% or more identical to, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 148-161, 12204-12675, 14142-14159, 22786-22812, 23409-23624, and 24729-24944, or identical to, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant of any of these sequences. Additional information regarding each nucleic acid sequence is provided for each SEQ ID NO in the sequence listing. <223> This information is provided in the relevant sections, Table 3a (see column C in particular), and Table 3b (see rows 3 and 7).

[0338] In a particularly preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequences are sequence numbers 149-154, 156-161, 163-168, 170-175, 12338, 12352, 12541, 12555, 12810, 12824, 13013, 13027, 22786, 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 22813, 22819, 22821, 22823, 22825, 22827, and 22829. , 22831, 22833, 22835, 22837, 22839, 23517~23624, 23297~23404, 24837~24944 are identical to, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant of any of these sequences. Additional information about each nucleic acid sequence is provided for each sequence number in the sequence listing. <223> This information is provided in the relevant sections, Table 3a (see columns C and D, rows 2 and 6) and Table 3b (see column C).

[0339] In a more preferred embodiment, the nucleic acid (preferably RNA) contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 149, 156, 12338, 150, 157, 151, 158, 12541, 163, 170, 12810, 164, 171, 165, 172, 13013, 12342-12351, 12545-12554, 12814-12823, 13017-13026, and 14133, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, the nucleic acid sequence is a fragment or variant of any of these sequences. Additional information about each nucleic acid sequence is provided for each sequence number in the sequence listing. <223> This is described in the column, Table 3a, and Table 3b.

[0340] In a more preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 149, 150, 151, 163, 164, and 165. Alternatively, the nucleic acid sequence is a fragment or variant of any of these sequences. Additional information regarding each nucleic acid sequence is provided for each SEQ ID NO in the sequence listing. <223> This information is provided in the relevant section and in Table 3 (see columns C and D, row 2).

[0341] In a particularly preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of Sequence ID No. 163, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0342] In further particularly preferred embodiments, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of Sequence ID No. 149, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0343] In further particularly preferred embodiments, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of Sequence ID No. 24837, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0344] In a more preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequences of SEQ ID NOs. 23311, 23531, and 24851, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0345] In a more preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of SEQ ID NOs. 23310, 23530, and 24850, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0346] In a more preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequences of SEQ ID NOs. 23313, 23533, 24853, 23314, 23534, and 24854, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0347] In further embodiments, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of Sequence ID No. 26633, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0348] In further embodiments, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of Sequence ID No. 26907, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0349] In a more preferred embodiment, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequences are: SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824 , is identical to a nucleic acid sequence selected from the group consisting of 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The RNA sequence has the Cap1 structure described herein. Additional information regarding each nucleic acid sequence is provided for each sequence number in the sequence listing. <223> This is described in the column, Table 3a, and Table 3b.

[0350] In further embodiments, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequences are: SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824 , is identical to a nucleic acid sequence selected from the group consisting of 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In the RNA sequence, one or more uracil nucleotides (preferably all uracil nucleotides) are substituted with pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides. Additional information regarding each nucleic acid sequence is provided for each sequence number in the sequence listing. <223> This is described in the column, Table 3a, and Table 3b.

[0351] In further embodiments, the nucleic acid (preferably RNA) comprises or consists of a nucleic acid sequence. The nucleic acid sequences are: SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824 , is identical to a nucleic acid sequence selected from the group consisting of 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The RNA sequence has the cap1 structure described herein. In the RNA sequence, one or more uracil nucleotides (preferably all uracil nucleotides) are substituted with pseudouridine (ψ) nucleotides and / or N1-methylpseudridine (m1ψ) nucleotides. Additional information regarding each nucleic acid sequence is provided for each sequence number in the sequence listing. <223> This is described in the column, Table 3a, and Table 3b.

[0352] As described throughout the specification, additional information regarding preferred amino acid sequences or nucleic acid sequences (coding sequences, DNA sequences, RNA sequences) may be derived from the sequence listing. In particular, as described below. <223> Details are provided in the section marked "".

[0353] In certain embodiments, the nucleic acid of the present invention is RNA. RNA can be produced by methods well known in the art. Examples of such methods include chemical synthesis (such as solid-phase RNA synthesis) and in vitro methods (such as RNA transcription reactions in vitro). Therefore, in preferred embodiments, RNA is obtained by RNA transcription in vitro.

[0354] Therefore, in a preferred embodiment, the nucleic acid of the present invention is preferably RNA transcribed in vitro.

[0355] The term "in vitro RNA transcription" or "in vitro transcription" refers to the process of synthesizing RNA in a cell-free system (in vitro). RNA may be obtained by DNA-dependent transcription of a suitable DNA template in vitro. The DNA template according to the present invention is a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA transcription in vitro may be any promoter for any DNA-dependent RNA polymerase. Examples of DNA-dependent RNA polymerases include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or Syn5 RNA polymerase. In a preferred embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme, and then RNA is transcribed in vitro.

[0356] The reagents used for RNA transcription in vitro typically include the following: • A DNA template (linear plasmid DNA or PCR product) that has a promoter sequence with high binding affinity to RNA polymerases such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5). • Ribonucleotide triphosphates (NTPs) for four types of bases (adenine, cytosine, guanine, and uracil) • Capacitor analogs as specified herein, in any configuration • Modified nucleotides as described herein, in any configuration. • DNA-dependent RNA polymerases (such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or Syn5 RNA polymerase) capable of binding to promoter sequences contained in the DNA template. • Ribonuclease inhibitors for inactivating any potentially contaminating ribonuclease (RNase) in any configuration. • A pyrophosphatase with any configuration for degrading pyrophosphates (which can inhibit RNA transcription in vitro). • Mg, a cofactor of polymerase 2+ MgCl2 supplies ions • A buffer (TRIS or HEPES) for maintaining a suitable pH value, which may contain an optimal concentration of antioxidant (such as DTT) and / or polyamine (such as spermidine) (e.g., a buffer system containing TRIS-citrate disclosed in WO2017 / 109161). In a preferred embodiment, the cap1 structure of the RNA of the present invention is formed by co-transcription capping using the trinucleotide capping analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. A preferred cap1 analog that can be suitably used in the preparation of the coding RNA of the present invention is m7G(5')ppp(5')(2'OMeA)pG.

[0357] In a particularly preferred embodiment, the RNA cap 1 structure of the present invention is formed by co-transcription capping using the trinucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0358] In another embodiment, the RNA cap 0 structure of the present invention is formed by co-transcription capping using the cap analog 3'OMe-m7G(5')ppp(5')G.

[0359] In further embodiments, the nucleotide mixture used in the transcription of RNA in vitro may further contain the modified nucleotides described herein. In this regard, preferred modified nucleotides may be selected from pseudouridine (Ψ), N1-methylpseudridine (m1Ψ), 5-methylcytosine, and 5-methoxyuridine. In certain embodiments, the uracil nucleotides in the nucleotide mixture are (partially or completely) substituted with pseudouridine (Ψ) and / or N1-methylpseudridine (m1Ψ) to obtain the modified RNA.

[0360] In preferred embodiments, the nucleotide mixture used in in vitro RNA transcription does not contain the modified nucleotides described herein. In preferred embodiments, the nucleotide mixture used in in vitro RNA transcription comprises only G nucleotides, C nucleotides, A nucleotides, and U nucleotides, and, in an optional configuration, the capping analogs described herein.

[0361] In preferred embodiments, the nucleotide mixture used in the in vitro RNA transcription reaction (i.e., the fraction of each nucleotide in the mixture) may preferably be optimized for a given RNA sequence (see WO2015 / 188933).

[0362] In this regard, in vitro transcription is carried out in the presence of a sequence-optimized nucleotide mixture and a capping analog in an optional configuration. Preferably, the sequence-optimized nucleotide mixture does not contain chemically modified nucleotides.

[0363] In this regard, a sequence-optimized nucleoside triphosphate (NTP) mixture is a mixture of nucleoside triphosphates (NTPs) for transcribing RNA molecules of a given sequence in vitro, and contains four types of nucleoside triphosphates: GTP, ATP, CTP, and UTP. Each fraction of the four types of nucleoside triphosphates in the sequence-optimized nucleoside triphosphate mixture corresponds to the fraction of each nucleotide contained in the RNA molecule. If ribonucleotides are not present in the RNA molecule, the corresponding nucleoside triphosphate is not present in the sequence-optimized nucleoside triphosphate (NTP) mixture.

[0364] In embodiments of this specification that require the preparation of two or more different RNAs (for example, embodiments that require the preparation of two, three, four, five, six, seven, eight, nine, ten or more different RNAs (see Second Embodiment)), the procedure described in WO2017 / 109134 can be suitably used.

[0365] In connection with the production of nucleic acid vaccines, it may be necessary to provide GMP-grade nucleic acids (such as GMP-grade RNA or DNA). GMP-grade RNA or DNA can be produced by manufacturing processes approved by regulatory authorities. Therefore, in a particularly preferred embodiment, RNA is produced based on current Good Manufacturing Practice (GMP) standards, preferably by implementing various quality control steps at the DNA and RNA levels in accordance with WO2016 / 180430. In a preferred embodiment, the RNA of the present invention is GMP-grade RNA (particularly GMP-grade mRNA). Therefore, RNA for vaccines is preferably GMP-grade RNA.

[0366] Preferably, the obtained RNA product is purified. Purification is performed using PureMessenger (R) (RP-HPLC according to CureVac, Tuebingen, Germany; WO2008 / 077592), tangential flow filtration (as described in WO2016 / 193206), and / or oligo-d(T) purification (see WO2016 / 180430).

[0367] Preferably, the RNA according to the present invention is purified by reverse-phase high-pressure liquid chromatography (RP-HPLC). Preferably, a microporous styrene / divinylbenzene column (e.g., particle size: 30 μm, pore size: 4000 Å) is used for RP-HPLC. Further purification is performed using a filter cassette equipped with a cellulose-based membrane with a molecular weight cutoff of approximately 100 kDa.

[0368] In relation to this, RNA purified by RP-HPLC and / or TFF produces approximately 5%, 10%, or 20% less double-stranded RNA byproducts than RNA that is not purified by RP-HPLC and / or TFF.

[0369] Alternatively, purified RNA obtained by RP-HPLC and / or TFF contains approximately 5%, 10%, or 20% fewer double-stranded RNA byproducts compared to RNA obtained by oligo-dT purification, precipitation, filtration, and / or anion exchange chromatography.

[0370] In a more preferred embodiment, the nucleic acid (preferably RNA) is freeze-dried (e.g., according to WO2016 / 165831 or WO2011 / 069586). This yields a temperature-stable dry nucleic acid (powder), such as RNA or DNA, as described herein. The nucleic acid (particularly RNA) of the present invention may also be dried by spray drying or spray freeze-drying (e.g., according to WO2016 / 184575 or WO2016 / 184576) to obtain a temperature-stable RNA (powder) as described herein. Accordingly, in relation to the production and purification of nucleic acids (especially RNA), the disclosures of WO2017 / 109161, WO2015 / 188933, WO2016 / 180430, WO2008 / 077592, WO2016 / 193206, WO2016 / 165831, WO2011 / 069586, WO2016 / 184575, and WO2016 / 184576 are incorporated herein by reference.

[0371] Therefore, in a preferred embodiment, the nucleic acid is a dried nucleic acid (particularly dried RNA).

[0372] In this specification, the term "dried RNA" should be understood as RNA that has been freeze-dried, spray-dried, or spray-freeze-dried in order to obtain temperature-stable dried RNA (powder), as described above.

[0373] In a preferred embodiment, the nucleic acid of the present invention is purified nucleic acid (particularly purified RNA).

[0374] In this specification, the term "purified nucleic acid" should be understood as nucleic acid that has a higher purity than the starting material after a particular purification process. Typical impurities that are substantially absent in purified nucleic acid include peptides or proteins, spermidine, BSA, incomplete nucleic acid sequences, nucleic acid fragments, free nucleotides, bacterial impurities, or impurities derived from the purification method. In this regard, it is desirable that the purity of the nucleic acid be as close to 100% as possible. It is also desirable that the amount of full-length nucleic acid be as close to 100% as possible. Therefore, in this specification, the purity of purified nucleic acid is greater than 75%, greater than 80%, greater than 85%. In particular, the purity of purified nucleic acid is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%. Most preferably, the purity of purified nucleic acid is 99% or higher. For example, purity may be determined by HPLC analysis. The above ratio corresponds to the ratio of the peak area of ​​the target nucleic acid to the total area of ​​all peaks representing by-products. Alternatively, purity may be determined by, for example, agarose gel electrophoresis or capillary gel electrophoresis.

[0375] In a preferred embodiment, the nucleic acid of the present invention is purified RNA.

[0376] In this specification, the terms "purified RNA" or "purified mRNA" should be understood as RNA that is purer than the starting material (such as RNA transcribed in vitro) after a specific purification process (such as HPLC, TFF, oligo-d(T) purification, or precipitation). Typical impurities substantially absent from purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent in vitro transcription of RNA, such as RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, and DNase), spermidine, BSA, incomplete RNA sequences, RNA fragments (short double-stranded RNA fragments, incomplete sequences, etc.), free nucleotides (modified nucleotides, conventional NTPs, capping analogs), template DNA fragments, and buffer components (HEPES, TRIS, MgCl2). Other potential impurities that may originate from fermentation methods include bacterial impurities (bioburden, bacterial DNA) or impurities from the purification process (such as organic solvents). Therefore, in this regard, it is desirable that the purity of RNA be as close to 100% as possible. Furthermore, it is desirable that the RNA purity be as close to 100% as possible in terms of the amount of full-length RNA transcript. Therefore, in this specification, the purity of purified RNA is greater than 75%, greater than 80%, and greater than 85%. In particular, the purity of purified RNA is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, and greater than 98%. Most preferably, the purity of purified RNA is 99% or higher. For example, purity may be determined by HPLC analysis. The above percentages correspond to the ratio of the peak area of ​​the target RNA to the total area of ​​all peaks representing byproducts. Alternatively, purity may be determined by agarose gel electrophoresis or capillary gel electrophoresis, for example.

[0377] In a particularly preferred embodiment where the nucleic acid is RNA, the RNA is purified by RP-HPLC and / or TFF. Double-stranded RNA, uncapped RNA, and / or RNA fragments are removed from the purified RNA.

[0378] When double-stranded RNA is formed as a byproduct during RNA transcription in vitro, it can induce an innate immune response (particularly IFN-α). IFN-α is a major cause of fever in vaccinated subjects. This is, of course, an undesirable side effect. Current techniques for immunoblotting of dsRNA (using dot blot, serologically specific electron microscopy (SSEM), or ELISA, etc.) are used to detect and size dsRNA species from a mixture of nucleic acids.

[0379] Preferably, the RNA of the present invention is purified by RP-HPLC and / or TFF as described herein. This reduces the amount of dsRNA.

[0380] In preferred embodiments, the RNA of the present invention has approximately 5%, 10%, or 20% fewer double-stranded RNA byproducts compared to RNA that has not been purified by RP-HPLC and / or TFF.

[0381] In preferred embodiments, RNA purified by RP-HPLC and / or TFF contains approximately 5%, 10%, or 20% fewer double-stranded RNA byproducts compared to RNA purified by oligo-dT purification, precipitation, filtration, and / or AEX.

[0382] It should be understood that the “dried RNA,” “purified RNA,” or “GMP-grade RNA” described herein are highly stable (in vitro, in vivo) and may have improved efficacy (e.g., improved mRNA translationability in vivo). Therefore, they are particularly suitable for medical applications (such as vaccines).

[0383] The degree of capping of RNA obtained after co-transcription capping as described herein (and after purification as described herein) can be determined using the capping assay described in WO2015 / 101416 (in particular, claims 27-46 of WO2015 / 101416). Alternatively, the capping assay described in PCT / EP2018 / 08667 can be used.

[0384] In embodiments, the nucleic acids of the present invention may be prepared and purified using an automated apparatus for transcribing RNA in vitro. Such apparatus can also be used for the production of compositions or vaccines (see embodiments 2 and 3). Preferably, the apparatus described in WO2020002598 (in particular claims 1 to 59 and / or 68 to 76 (b) and Figures 1 to 18 of WO2020002598) can be suitably used.

[0385] Preferably, the methods described herein may be applied to methods for producing RNA compositions or vaccines as detailed below.

[0386] [Compositions, pharmaceutical compositions] The second aspect relates to a composition comprising one or more nucleic acids of the first aspect.

[0387] Notably, embodiments relating to the composition of the second embodiment may also be read and understood as preferred embodiments of the vaccine of the fourth embodiment. Similarly, embodiments relating to the vaccine of the fourth embodiment may also be read and understood as preferred embodiments of the composition of the second embodiment (containing the nucleic acid of the first embodiment). Furthermore, features and embodiments described in relation to the first embodiment (the nucleic acid of the present invention) should be read and understood as preferred embodiments of the composition of the second embodiment.

[0388] In a preferred embodiment, the composition comprises one or more nucleic acids according to the first embodiment. The nucleic acid encodes one or more antigenic peptides or antigenic proteins that are SARS-CoV-2 (formerly nCoV-2019) coronavirus, or its immunogenic fragment or immunogenic variant, or derived therefrom.

[0389] In a preferred embodiment, the composition comprises one or more nucleic acids according to the first embodiment. The one or more nucleic acids encode one or more antigenic peptides or antigenic proteins that are SARS-CoV-2 coronavirus, its immunogenic fragment or immunogenic variant, or derived therefrom. Preferably, the composition is administered intramuscularly or intradermally.

[0390] Preferably, the composition is administered intramuscularly or intradermally, causing the encoded SARS-CoV-2 antigen construct to be expressed in the subject. In embodiments where the nucleic acid is RNA, administration of the composition causes the RNA to be translated in the subject, producing the encoded SARS-CoV-2 antigen. In embodiments where the nucleic acid is DNA (plasmid DNA, adenovirus DNA, etc.), administration of the composition causes the DNA to be transcribed into RNA in the subject, and then the RNA to be translated into the encoded SARS-CoV-2 coronavirus antigen.

[0391] Preferably, the composition of the second embodiment is suitable for vaccines. In particular, the composition of the second embodiment is suitable for coronavirus vaccines (preferably SARS-CoV-2 (nCoV-2019) vaccines).

[0392] In relation to the present invention, “composition” refers to any type of composition. A composition may contain certain components. Examples of certain components include nucleic acids (such as RNA or DNA) encoding one or more antigenic peptides or antigenic proteins of or derived from the SARS-CoV-2 coronavirus. The nucleic acids are associated with polymeric carriers or LNPs, etc. In any configuration, a composition may also be associated with further components (usually with one or more pharmaceutically acceptable carriers or excipients). A composition may be a dry composition (such as a powder or granules) or a solid unit (such as a lyophilized form). Alternatively, a composition may be in liquid form, with each component independently dissolved or dispersed (such as suspended or emulsified).

[0393] In a preferred embodiment of the second aspect, the composition comprises one or more nucleic acids (such as DNA or RNA) of the first aspect, preferably RNA. The composition comprises, in any configuration, one or more pharmaceutically acceptable carriers or excipients.

[0394] In embodiments of the second aspect, the composition comprises one or more nucleic acids of the first aspect, preferably plasmid DNA and adenovirus DNA. The composition optionally comprises one or more pharmaceutically acceptable carriers or excipients.

[0395] In a preferred embodiment of the second aspect, the composition comprises one or more nucleic acids (such as DNA or RNA), preferably RNA. The nucleic acid comprises or consists of a nucleic acid sequence. Sequence IDs 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520, 14124-14177, 22759, 22 764~22786, 22791~22813, 22818~22839, 22969~23184, 23189~23404, 23409~23624, 23629~23844, 23849~24064, 24069~24284, 24289~24504, 24509~24724 , are identical to nucleic acid sequences selected from the group consisting of 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The composition comprises one or more pharmaceutically acceptable carriers or excipients in any configuration.

[0396] In a particularly preferred embodiment of the second aspect, the composition comprises one or more nucleic acids (such as DNA or RNA), preferably RNA. The nucleic acid comprises or consists of a nucleic acid sequence. The nucleic acid sequences are: SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824 The nucleic acid sequence is identical to, or is identical to, a nucleic acid sequence selected from the group consisting of 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. The composition comprises one or more pharmaceutically acceptable carriers or excipients in any configuration.

[0397] Most preferably, the composition contains one or more nucleic acids (such as DNA or RNA), and preferably RNA. The nucleic acid contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of Sequence ID No. 163, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0398] Most preferably, the composition contains one or more nucleic acids (such as DNA or RNA), and preferably RNA. The nucleic acid contains or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequence of Sequence ID No. 149, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0399] In a particularly preferred embodiment, the composition comprises one or more nucleic acids (such as DNA or RNA), preferably RNA. The nucleic acid comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequences of SEQ ID NOs. 24837, 23311, 23531, 23310, 23530, 23313, and 23533, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0400] In further embodiments, the composition comprises one or more nucleic acids (such as DNA or RNA), preferably RNA. The nucleic acid comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to the nucleic acid sequences of SEQ ID NOs. 26633 and 26907, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0401] In a particularly preferred embodiment of the second aspect, the composition comprises one or more nucleic acids (such as DNA or RNA), preferably RNA. The nucleic acid comprises or consists of a nucleic acid sequence. The nucleic acid sequence is identical to, or is identical to, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 149-151, 163-165, 24837, 23311, 23531, 24851, 23310, 23530, 23313, and 23533, by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The composition comprises, in any configuration, one or more pharmaceutically acceptable carriers or excipients.

[0402] In this specification, the terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” preferably include the liquid or non-liquid base of the composition for administration. When the composition is provided in liquid form, the carrier may be water (water free of pyrogens), isotonic saline, or a buffer (water-soluble) solution (such as phosphate buffer or citrate buffer). Water (or preferably a buffer, more preferably an aqueous buffer) may be used. This water contains a sodium salt (preferably 50 mM or more of a sodium salt), a calcium salt (preferably 0.01 mM or more of a calcium salt), and optionally a potassium salt (preferably 3 mM or more of a potassium salt). According to a preferred embodiment, the sodium salt, calcium salt, and optionally a potassium salt may exist in the form of their halides (such as chloride, iodide, or bromide), hydroxides, carbonates, bicarbonates, or sulfates. Examples of sodium salts include NaCl, NaI, NaBr, Na2CO3, NaHCO3, and Na2SO4. Examples of potassium salts with arbitrary compositions include KCl, KI, KBr, K2CO3, KHCO3, and K2SO4. Examples of calcium salts include CaCl2, CaI2, CaBr2, CaCO3, CaSO4, and Ca(OH)2.

[0403] Furthermore, the organic anions of the aforementioned cations may be present in the buffer. Therefore, in embodiments, the nucleic acid composition may contain pharmaceutically acceptable carriers or excipients. One or more pharmaceutically acceptable carriers or excipients may be used, for example, to improve stability, facilitate transfection into cells, enable persistence or extension, promote in vivo translation of the encoded coronavirus protein, or alter the in vivo release profile of the encoded coronavirus protein. In addition to conventional excipients (any and all solvents, dispersions, diluents or other liquid vehicles, dispersants or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, etc.), excipients of the present invention include (but are not limited to) lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, polynucleotide-transfected cells, hyaluronidases, nanoparticle mimetic compounds, and combinations thereof. Similarly, in embodiments, one or more compatible solid or liquid fillers, diluents, or capsule compounds suitable for administration to a subject may be used. In this specification, the term “compatible” means that the components of the composition can be mixed with one or more nucleic acids (or any number of nucleic acids). This mixing is carried out in a manner that does not cause interactions that substantially reduce the bioactivity or pharmaceutically effective effect of the composition under typical use conditions (such as intramuscular or intradermal administration). To make the composition suitable for administration to the subject being treated, the pharmaceutically acceptable carrier or excipient must be sufficiently pure and sufficiently low in toxicity.Examples of compounds that can be used as pharmaceutically acceptable carriers or excipients include sugars (such as lactose, glucose, trehalose, mannose, and sucrose); starches (such as corn starch or potato starch); dextrose; cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; animal fat; solid lubricants (such as stearic acid and magnesium stearate); calcium sulfate; vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oils derived from the cocoa genus); polyols (such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol); and alginic acid.

[0404] One or more pharmaceutically acceptable carriers or excipients may be selected, preferably, to be suitable for intramuscular or intradermal delivery / administration of the composition. Therefore, the composition is preferably a pharmaceutical composition, and preferably a composition for intramuscular administration.

[0405] Subjects to whom the composition (preferably a pharmaceutical composition) is intended to be administered include, but are not limited to, humans and / or other primates; mammals (including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and / or rats); and birds (including, but not limited to, poultry, chickens, ducks, geese and / or turkeys and other commercially relevant birds).

[0406] The pharmaceutical composition of the present invention may preferably be sterilized and may not contain pyrogens.

[0407] (Polyvalent composition of the present invention) In embodiments, the compositions described herein (such as polyvalent compositions) may contain more than one or more nucleic acid species (for example, RNA species described in relation to the first aspect of the present invention). Preferably, the compositions described herein may contain two, three, four, five, six, seven, eight, nine, or ten different nucleic acids described in relation to the first aspect.

[0408] In embodiments, the composition (such as a polyvalent composition) may contain two or more different nucleic acid species described in relation to the first embodiment. Each nucleic acid species encodes one or more antigenic peptides or antigenic proteins, or fragments or variants thereof, derived from the same coronavirus. In particular, the (genetically) identical coronaviruses described above express (substantially) the same repertoire of proteins or peptides, and all proteins or peptides have (substantially) the same amino acid sequence. In particular, the (genetically) identical coronaviruses described above express substantially the same proteins, peptides or polyproteins, and preferably these proteins, peptides or polyproteins have the same amino acid sequence.

[0409] In embodiments, the composition (such as a polyvalent composition) may contain two or more different nucleic acid species described in relation to the first embodiment. Each nucleic acid species encodes one or more peptides or proteins, or fragments or variants thereof, derived from genetically distinct coronaviruses (such as different coronavirus isolates). In this specification, the terms “different” or “different coronaviruses” should be understood as differences between two or more coronaviruses (such as different coronavirus isolates). These differences appear in the genomes of the different coronaviruses. In particular, the aforementioned (genetically) different coronaviruses may express one or more different proteins, peptides or polyproteins, and one or more different proteins, peptides or polyproteins may differ in one or more amino acids.

[0410] In a preferred embodiment, multiple or more nucleic acid sequences of the polyvalent composition encode different spike proteins (preferably pre-fusion structure-stabilizing spike proteins).

[0411] In this regard, it is particularly preferable that the different spike proteins or pre-fusion structure-stabilizing spike proteins originate from different SARS-CoV-2 virus variants / isolated strains. The spike proteins are particularly preferable to originate from B.1.1.7, B.1.351, P.1, or CAL.20C.

[0412] In this regard, it is even more preferable that different spike proteins or pre-fusion structure-stabilized spike proteins have one or more amino acid mutations in the spike protein from (i) to (v) below. (i) delH69, delV70, Y453F, D614G, I692V and M1229I (ii) delH69, delV70, delY144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H (iii) L18F, D80A, D215G, delL242, delA243, delL244, R246I, K417N, E484K, N501Y, D614G and A701V (iv) L18F, T20N, P26S, D138Y, R190S, K417T, E484K , N501Y, D614G, H655Y and T1027I (v)S13I, W152C, L452R, and D614G In embodiments, the composition (such as a polyvalent composition) contains two, three, four, or five nucleic acid species (such as DNA or RNA), preferably containing RNA species. The nucleic acid species contains or consists of nucleic acid sequences. Nucleic acid sequences include SEQ ID NOs: 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520, 14124-14177, and 2275. 9, 22764~22786, 22791~22813, 22818~22839, 22969~23184, 23189~23404, 23409~23624, 23629~23844, 23849~24064, 24069~24284, 24289~24504, 24509~24 The nucleic acid sequence is identical to, or 99% or more identical to, selected from the group consisting of 724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937. Each of the two, three, four, or five nucleic acid species encodes a different antigenic peptide or antigenic protein of the SARS-CoV-2 coronavirus.

[0413] Therefore, in the embodiment, the composition (such as a polyvalent composition) contains two nucleic acid species (such as DNA or RNA), preferably an RNA species. The nucleic acid species contains or consists of a nucleic acid sequence. The nucleic acid sequences are: SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824 The nucleic acid sequences are identical to, or are identical to, a nucleic acid sequence selected from the group consisting of 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. The composition is of any configuration and contains one or more pharmaceutically acceptable carriers or excipients. Each of the two nucleic acid species encodes a different antigenic peptide or antigenic protein of the SARS-CoV-2 coronavirus.

[0414] In the embodiment, the composition (such as a polyvalent composition) contains three nucleic acid species (such as DNA or RNA), preferably containing an RNA species. The nucleic acid contains or consists of a nucleic acid sequence. The nucleic acid sequences are: SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824 The nucleic acid sequences are identical to, or are identical to, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, nucleic acid sequences selected from the group consisting of 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937. The compositions are of any configuration and contain one or more pharmaceutically acceptable carriers or excipients. Each of the two, three, four, or five nucleic acid species encodes a different antigenic peptide or antigenic protein of the SARS-CoV-2 coronavirus.

[0415] Particularly preferred embodiments of the polyvalent composition are shown below.

[0416] Preferably, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or even more different nucleic acid species contained in the polyvalent composition each encode a different pre-fusion structure-stabilizing spike protein (as described in the first embodiment). Preferably, the spike protein is stabilized in the pre-fusion conformation by substituting the K986 and V987 residues with two proline molecules (amino acid positions are based on SEQ ID NO: 1). Therefore, in a preferred embodiment, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more pre-fusion structure-stabilizing spike proteins (S_stab) each have one or more pre-fusion structure-stabilizing mutations. In one or more pre-fusion structure-stabilizing mutations, amino acid substitutions of K986P and V987P occur (amino acid positions are based on SEQ ID NO: 1).

[0417] Therefore, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or even more different nucleic acid species contained in the polyvalent composition each encode a different pre-fusion structure-stabilized spike protein. Two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or even more stabilized spike proteins include SEQ ID NOs: 10-26, 341-407, 609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, The amino acid sequences are selected from those that are identical to any one of 22756, 22758, 22947-22964, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more or more, stabilized spike proteins are any of these immunogenic fragments or immunogenic variants.

[0418] In a preferred embodiment, the polyvalent composition comprises one nucleic acid species having a coding sequence. The coding sequence codes for an amino acid sequence. The amino acid sequence is identical to any one of sequence numbers 10, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The polyvalent composition further comprises two or more, three or more, or four or more further RNA species selected from the following: (i) A single nucleic acid species having a coding sequence. The coding sequence codes for an amino acid sequence. The amino acid sequence is identical to any one of sequence numbers 22961, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (ii) A single nucleic acid species having a coding sequence. The coding sequence codes for an amino acid sequence. The amino acid sequence is identical to any one of sequence numbers 22960, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iii) A single nucleic acid species having a coding sequence. The coding sequence codes for an amino acid sequence. The amino acid sequence is identical to any one of sequence numbers 22963, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iv) A single nucleic acid species having a coding sequence has a coding sequence that codes for an amino acid sequence. The amino acid sequence is identical to any one of sequence numbers 22941, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (v) A single nucleic acid species having a coding sequence. The coding sequence codes for an amino acid sequence. The amino acid sequence is identical to any one of sequence numbers 22964, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0419] In a preferred embodiment, the polyvalent composition comprises two or more nucleic acid species having coding sequences. The coding sequences encode amino acid sequences. The amino acid sequences are identical to any one of SEQ ID NOs: 10, 22961, 22960, 22963, 22941, and 22964, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0420] Preferably, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more or more different nucleic acid species contained in the polyvalent composition have nucleic acid coding sequences. Each nucleic acid coding sequence codes for a different pre-fusion structure stabilizing spike protein. Two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more or more or more nucleic acid coding sequences include SEQ ID NOs: 136-138, 140-143, 145-175, 11731-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520, 1 Either identical to any one of 4124-14141, 22759, 22764-22785, or 22969-23184, or identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more or more nucleic acid coding sequences are fragments or variants of any of these.

[0421] In a preferred embodiment, the polyvalent composition comprises one nucleic acid species having a coding sequence. The coding sequence is identical to any one of sequence numbers 137, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The polyvalent composition further comprises two or more, three or more, or four or more additional RNA species selected from the following: (i) A single nucleic acid species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23091, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (ii) A single nucleic acid species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23090, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iii) A single nucleic acid species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23093, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iv) A single nucleic acid species having a coding sequence. The coding sequence is identical to any one of sequence numbers 22999, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (v) A single nucleic acid species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23094, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0422] Preferably, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or even more different nucleic acid species contained in the polyvalent composition have nucleic acid coding sequences. Each nucleic acid coding sequence codes for a different pre-fusion structure stabilizing spike protein. Nucleic acid coding sequences with 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more or even more are sequence numbers 149-151, 163-165, 12338, 12541, 12810-12813, 12901, 12931, 13013, 22792, 22794, 22796, 22798, 22802, 22804, 22806, 22810, 22813, 22819, 22821, 22823, 22825, 22827, 22829, 22831, 22833, 22835, 22837, 22839 , 23297~23314, 23369, 23517~23520, 23523~23525, 23527, 23529, 23530, 23589, 23737, 23957, 24397, 24837, 25057, 25277, 25717, 26925~26937 are identical to any one of these, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Alternatively, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or even more nucleic acid coding sequences are any of these fragments or variants.

[0423] In a preferred embodiment, the polyvalent composition contains or comprises RNA species. The RNA sequences are identical to any one of sequence numbers 163, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The polyvalent composition further contains two or more, three or more, or four or more additional RNA species selected from the following: (i) A single RNA species that contains or consists of an RNA sequence. The RNA sequence is identical to any one of sequence numbers 23311, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (ii) A single RNA species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23310, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iii) A single RNA species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23313, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iv) A single RNA species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23219, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (v) A single RNA species having a coding sequence. The coding sequence is identical to any one of sequence numbers 23314, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0424] In the above embodiment, preferably, each mRNA species has a Cap1 structure. In any configuration, each mRNA species does not contain modified nucleotides.

[0425] In a preferred embodiment, the polyvalent composition comprises an RNA species, which comprises or consists of an RNA sequence, which is identical to either SEQ ID NO: 149 or 24837, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The polyvalent composition further contains two or more, three or more, or four or more additional RNA species selected from the following: (i) A single RNA species that contains or consists of an RNA sequence. The RNA sequence is identical to either SEQ ID NO: 23531 or 24851, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (ii) A single RNA species having a coding sequence. The coding sequence is identical to either SEQ ID NO: 23530 or 24850, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iii) A single RNA species having a coding sequence. The coding sequence is identical to either SEQ ID NO: 23533 or 24853, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (iv) A single RNA species having a coding sequence. The coding sequence is identical to either SEQ ID NO: 23439 or 24759, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. (v) A single RNA species having a coding sequence. The coding sequence is identical to either SEQ ID NO: 23534 or 24854, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0426] In the above embodiment, preferably, each mRNA species has a Cap1 structure. In any configuration, each mRNA species does not contain modified nucleotides.

[0427] In a more preferred embodiment, the polyvalent composition comprises two or more RNA species. The two or more RNA species are identical to any one of SEQ ID NOs: 149 or 24837, 23531 or 24851, 23530 or 24850, 23533 or 24853, 23439 or 24759 or 23534 or 24854, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0428] In embodiments, nucleic acids such as DNA or RNA (preferably RNA species) contained in the polyvalent composition may be formulated separately (formulations specified below). In preferred embodiments, nucleic acids such as DNA or RNA (preferably RNA species) contained in the polyvalent composition may be co-formulated separately (formulations specified below).

[0429] (compound) In a preferred embodiment of the second aspect, one or more nucleic acids (preferably one or more RNAs), such as DNA or RNA, are complexed or associated with a further compound. This results in a formulated composition. In connection therewith, the formulation may function as a transfection reagent. In connection therewith, the formulation may have the function of inhibiting the degradation of nucleic acids.

[0430] In a preferred embodiment of the second aspect, one or more nucleic acids (preferably one or more RNAs), such as DNA or RNA, and one or more additional nucleic acids of an optional composition are complexed or associated with, or at least partially complexed or associated with, one or more cationic compounds or polycationic compounds. Preferably, the cationic compound or polycationic compound is a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combination thereof.

[0431] In this specification, the terms “cationic compound or polycationic compound” will be recognized and understood by those skilled in the art. The terms are intended to represent, for example, a positively charged molecule. This molecule is positively charged when the pH is about 1–9, about 3–8, about 4–8, or about 5–8. More preferably, the molecule is positively charged when the pH is about 6–8. Even more preferably, the molecule is positively charged when the pH is about 7–8. Most preferably, the molecule is positively charged at physiological pH (e.g., about 7.2–7.5). Therefore, cationic components (such as cationic peptides, cationic proteins, cationic polymers, cationic polysaccharides, cationic lipids, etc.) can be any positively charged compound or polymer that is positively charged under physiological conditions. “Cationic or polycationic peptides or proteins” may contain one or more positively charged amino acids or two or more positively charged amino acids. Such amino acids are selected from, for example, Arg, His, Lys, or Orn. ​​Therefore, "polycationic" components also fall within the range of those that have two or more positive charges under given conditions.

[0432] In this regard, particularly preferred cationic or polycationic compounds may be selected from the following group of cationic or polycationic peptides, proteins or fragments thereof: protamine, nucleolin, spermine or spermidine, or other cationic peptides or cationic proteins. Examples of other cationic peptides or cationic proteins include poly-L-lysine (PLL), polyarginine, basic polypeptides, cell membrane permeable peptides (CPPs) (HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22-derived peptides or VP22 analog peptides, HSV) Examples include VP22 (herpes simplex), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptide, arginine-rich peptide, lysine-rich peptide, MPG peptide, Pep-1, L-oligomer, calcitonin peptide, Antennapedia-derived peptide, pAntep, pIsl, FGF, lactoferrin, transportan, buphorin 2, Bac715-24, Syn(B), SynB(1), pVEC, hCT-derived peptide, SAP, and histones. More preferably, nucleic acids such as DNA or RNA (e.g., coding RNA, preferably mRNA) are complexed with one or more polycations. Preferably, one or more polycations are protamines or oligofectamines. Most preferably, one or more polycations are complexed with protamines.

[0433] In a preferred embodiment, one or more nucleic acids (preferably one or more RNAs), such as DNA or RNA, are complexed with protamine.

[0434] Further preferred examples of cationic or polycationic compounds that can be used as transfection reagents or compounding reagents include cationic polysaccharides (such as chitosan and polybrene); cationic lipids (such as DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DOTAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9, oligofectamine, etc.); and cationic or polycationic polymers. Examples of cationic or polycationic polymers include modified polyamino acids (β-amino acid polymers, reverse polyamides, etc.); modified polyethylene (PVP, etc.); modified acrylates (pDMAEMA, etc.); modified amidoamines (pAMAM, etc.); modified polyβ-aminoesters (PBAEs) (diamine-terminated 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers, etc.); dendrimers (polypropylamine dendrimers, pAMAM-based dendrimers, etc.); polyimines (PEI, poly(propyleneimine), etc.); polyallylamines; sugar-backed polymers (cyclodextran-based polymers, dextran-based polymers, etc.); silane-backed polymers (PMOXA-PDMS copolymers, etc.); and block polymers consisting of a combination of one or more cationic blocks (selected from the cationic polymers mentioned above) and one or more hydrophilic or hydrophobic blocks (polyethylene glycol, etc.).

[0435] In this regard, one or more nucleic acids (preferably one or more RNAs), such as DNA or RNA, are complexed or at least partially complexed with a cationic compound or polycationic compound and / or polymeric carrier. Preferably, one or more nucleic acids are complexed or at least partially complexed with a cationic protein or cationic peptide. In this regard, the disclosures of WO2010 / 037539 and WO2012 / 113513 are incorporated herein by reference. "Partially" means that only a portion of the nucleic acid is complexed with the cationic compound, and the remaining nucleic acid is in an uncomplexed form (free form).

[0436] In embodiments, the composition comprises one or more nucleic acids (preferably one or more RNAs), such as DNA or RNA. The one or more nucleic acids are complexed with one or more cationic compounds or polycationic compounds (preferably protamines). The composition further comprises one or more free nucleic acids (uncomplexed nucleic acids).

[0437] In this regard, particularly preferably, one or more nucleic acids (preferably one or more RNAs), such as DNA or RNA, are complexed with protamine, or at least partially complexed. Preferably, the molar ratio of nucleic acids (particularly RNA of RNA complexed with protamine) to free RNA may be selected from about 0.001:1 to about 1:0.001 (this range also includes about 1:1). Preferably, complexed RNA is obtained by adding the RNA sample to a protamine-trehalose solution to complex it with protamine. In this case, the weight ratio (w / w) of RNA:protamine is 2:1.

[0438] A more preferable cationic or polycationic protein or peptide that can be used for complexation is given by formula (Arg) of WO2009 / 030481 or WO2011 / 026641 l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa)x This may be derived from WO2009 / 030481 or WO2011 / 026641 relating thereto, which are incorporated herein by reference.

[0439] In a preferred embodiment, one or more nucleic acids (preferably one or more RNAs), such as DNA or RNA, are complexed, or at least partially complexed, with one or more cationic or polycationic proteins or peptides (preferably selected from SEQ ID NOs. 269-273), or any combination thereof.

[0440] According to various embodiments, the composition of the present invention comprises one or more nucleic acids such as DNA or RNA (preferably one or more RNAs as described in the context of the first embodiment) and a polymeric carrier.

[0441] In this specification, the term “polymeric carrier” will be recognized and understood by those skilled in the art. The term is intended to represent a compound that facilitates the transport and / or complexation of other compounds (such as cargo nucleic acids). Polymeric carriers are typically carriers formed from polymers. Polymeric carriers may be associated with cargo (such as DNA or RNA) by covalent or non-covalent interactions. Polymers may have different subunits, such as copolymers.

[0442] In this regard, suitable polymerizable carriers include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextrans, albumins, gelatins, alginates, collagens, chitosans, cyclodextrins, protamines, PEGylated protamines, PEGylated PLLs, and polyethyleneimines (PEIs), dithiobis(succinimidylpropionate) (DSPs), and dimethyl-3,3'-dithiobispropionimide. Poly(DTBP), Poly(ethyleneimine)biscarbamate (PEIC), Poly(L-lysine) (PLL), Histidine-modified PLL, Poly(N-vinylpyrrolidone) (PVP), Poly(propyleneimine) (PPI), Poly(amidoamine) (PAMAM), Poly(amidoethyleneimine) (SS-PAEI), Triethylenetetramine (TETA), Poly(β-aminoester), Poly(4-hydroxy-L-proline ester) (PHP), Poly(allylamine), Poly(α-[4-aminobutyl]-L-glycolic acid) (PAGA), Poly(D (L-lactic acid-glycolic acid copolymer) (PLGA), poly(N-ethyl-4-vinylpyridinium bromide), poly(phosphazene) (PPZ), poly(phosphoester) (PPE), poly(phosphoramide) (PPA), poly(N-2-hydroxypropyl methacrylamide) (pHPMA), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2-aminoethylpropylene phosphate) (PPE_EA), galactosylated chitosan, N-dodecylated chitosan, histone, collagen, dextran Permine is an example. In one embodiment, the polymer may be an inert polymer (such as PEG, but not limited to these). In one embodiment, the polymer may be a cationic polymer (such as PEI, PLL, TETA, poly(allylamine), poly(N-ethyl-4-vinylpyridinium bromide), pHPMA, pDMAEMA, but not limited to these). In one embodiment, the polymer may be biodegradable PEI (such as DSP, DTBP, PEIC, but not limited to these).In one embodiment, the polymer may be biodegradable (such as, but not limited to, histone-modified PLL, SS-PAEI, poly(β-amino ester), PHP, PAGA, PLGA, PPZ, PPE, PPA, and PPE-EA).

[0443] A suitable polymeric carrier may be a polymeric carrier formed by a cationic compound crosslinked by a disulfide bond. The cationic compounds crosslinked by a disulfide bond may be the same or different from each other. The polymeric carrier may further contain additional components. The polymeric carrier used according to the present invention may be a mixture of a ...

Claims

1. A nucleic acid having one or more coding sequences encoding one or more antigenic peptides or antigenic proteins, or immunogenic fragments or immunogenic variants thereof, The one or more antigenic peptides or antigenic proteins mentioned above are of or derived from the SARS-CoV-2 coronavirus. A nucleic acid having one or more heterologous untranslated regions (UTRs).

2. The nucleic acid according to claim 1, which is suitable for use as a vaccine.

3. The above-mentioned one or more antigenic peptides or antigenic proteins contain one or more peptides or proteins, or consist of such peptides or proteins. One or more of the above peptides or proteins are structural proteins, accessory proteins, replicase proteins, or immunogenic fragments or variants of any of these, or are derived therefrom. The nucleic acid according to claim 1 or 2.

4. The nucleic acid according to claim 3, wherein the above-mentioned structural protein is a spike protein (S), an envelope protein (E), a membrane protein (M), a nucleocapsid protein (N), or an immunogenic fragment or immunogenic variant of any of these, or derived therefrom.

5. The nucleic acid according to any one of claims 1 to 4, wherein the one or more antigenic peptides or antigenic proteins are spike protein (S), an immunogenic fragment or immunogenic variant thereof, or derived therefrom.

6. The above-mentioned one or more antigenic peptides or antigenic proteins contain one or more amino acid sequences, or consist of such amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 5: (i) It is identical to any one of sequence numbers 1-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, 26939, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

7. The nucleic acid according to any one of claims 4 to 6, wherein the spike protein (S) comprises or consists of a spike protein fragment S1 or an immunogenic fragment or immunogenic variant thereof.

8. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 6: (i) It is identical to any one of sequence numbers 1-27, 29, 31-48, 58-111, 274-1345, 1480-1546, 1614-11663, 13377-13510, 13521-14123, 22732, 22737-22758, 22929-22964, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii) It is an immunogenic fragment or immunogenic variant of any of (i).

9. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 8: (i) It is identical to any one of sequence numbers 27, 1279-1345, 29, 1480-1546, 13243-13309, 22733-22736, 26938, 26939, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

10. The spike protein (S) mentioned above is, Spike protein fragment S1 or its immunogenic fragment or immunogenic variant, Spike protein fragment S2 or its immunogenic fragment or immunogenic variant, The nucleic acid according to any one of claims 4 to 9, comprising or consisting of the following.

11. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 10: (i) It is identical to any one of sequence numbers 1-26, 31-48, 58-111, 274-1278, 1614-11663, 13377-13510, 13521-14177, 22732, 22737-22758, 22929-22964, or it is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

12. The nucleic acid according to any one of claims 4 to 11, wherein the spike protein (S) is a spike protein (S_stab) that is stable in a pre-fusion structure and has one or more pre-fusion structure stabilization mutations.

13. The nucleic acid according to claim 12, wherein one or more of the above-mentioned pre-fusion structure-stabilizing mutations include the following amino acid substitutions: K986P; and, V987P.

14. The nucleic acid according to claim 12 or 13, wherein the one or more pre-fusion structure-stabilizing mutations include a cavity-filling mutation.

15. The nucleic acid according to claim 14, wherein one or more cavity-filling mutations are selected from the group including the following: T887W; A1020W; T887W and A1020W; or, P1069F.

16. The nucleic acid according to any one of claims 12 to 15, wherein one or more of the above-mentioned pre-fusion structure stabilization mutations include a mutant protonation site.

17. The nucleic acid according to claim 16, wherein one or more mutant protonation sites are selected from the group including the following: H1048Q and H1064N; H1083N and H1101N; or, H1048Q, H1064N, H1083N, and H1101N.

18. The nucleic acid according to any one of claims 12 to 17, wherein one or more artificial intramolecular disulfide bonds are formed by the one or more pre-fusion structure stabilization mutations described above.

19. The nucleic acid according to claim 18, wherein one or more of the above-mentioned artificial intramolecular disulfide bonds are formed by the following amino acid substitutions: I712C and T1077C; I714C and Y1110C; P715C and P1069C; G889C and L1034C; I909C and Y1047C; Q965C and S1003C; F970C and G999C; A972C and R995C; A890C and V1040C; T874C and S1055C; or, N914C and S1123C.

20. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 19: (i) Sequence numbers 10-26, 40-48, 85-111, 341-1278, 1681-2618, 2686-3623, 3691-4628, 4696-5633, 5701-6638, 6706-7643, 7711-8648, 8716-9653, 9721-10658, 10726-11663, 13377-13510, 13521-14123, 22732, 22738, 22740, 22742, 2 It is identical to any one of 2744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22947-22964, or it is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

21. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 20: (i) It is identical to any one of sequence numbers 10-26, 341-407, 609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22947-22964, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

22. The above one or more coding sequences further encode one or more heterogeneous peptide or protein elements. The above heterogeneous peptide or protein elements are selected from signal peptides, linkers, helper epitopes, antigen clustering elements, trimer elements, transmembrane elements and / or VLP-forming sequences. The nucleic acid according to any one of claims 1 to 21.

23. The nucleic acid according to claim 22, wherein the one or more heterologous peptide elements or protein elements are heterologous antigen clustering elements, heterologous trimerizing elements and / or VLP-forming sequences.

24. The nucleic acid according to claim 22 or 23, wherein the one or more heterogeneous antigen clustering elements are selected from ferritin elements, lumazine synthase elements, hepatitis B virus surface antigen (HBsAg), or encapsulin.

25. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 24: (i) It is identical to any one of sequence numbers 58-75, 85-102, 3624-5633, 7644-9653, 13588-13721, 13856-13989, 22733, 22735, or 22736, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

26. The above one or more heterologous trimer elements are Foldon elements, Preferably, the element is fibrinfoldon. The nucleic acid according to claim 22 or 23.

27. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 26: (i) It is identical to any one of sequence numbers 76-84, 103-111, 5634-6638, 9654-10658, 13722-13788, 13990-14056, 22734, 26938, or 26939, or it is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

28. The nucleic acid according to claim 22 or 23, wherein one or more of the above-mentioned VLP-forming sequences are woodchuck hepatitis core antigen elements (WhcAG).

29. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 28: (i) It is identical to any one of sequence numbers 6639-7643, 10659-11663, 13789-13855, or 14057-14123, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

30. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 29: (i) It is identical to any one of sequence numbers 1, 10, 21, 22, 25, 27, 274, 341, 408, 475, 542, 743, 810, 1011, 1145, 1212, 1279, 8716, 10726, 22732-22758, 22929-22942, 22947-22964, or it is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

31. The above one or more antigenic peptides or antigenic proteins contain or consist of one or more amino acid sequences. The above amino acid sequence satisfies either (i) or (ii) below, the nucleic acid according to any one of claims 1 to 30: (i) It is identical to any one of sequence numbers 10, 22960, 22961, or 22963, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

32. The above one or more coding sequences contain or consist of one or more nucleic acid sequences. The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 31: (i) Sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520, 14124-14177, 2275 9, 22764-22786, 22791-22813, 22818-22839, 22969-23184, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509- It is identical to any one of 24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, 26709-26937, or it is identical to 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is a fragment or variant of any of the sequences.

33. One or more antigenic peptides or antigenic proteins contain an S protein. The above S protein is It possesses the K986P and V987P mutations, which are pre-fusion structural stabilizing mutations, and It contains or consists of an amino acid sequence identical to sequence number 10, or is an immunogenic fragment or immunogenic variant of either of these. The nucleic acid according to any one of claims 1 to 32.

34. The above one or more coding sequences are coding sequences with modified codons. Preferably, the amino acid sequence encoded by one or more of the modified codon coding sequences is unchanged compared to the amino acid sequence encoded by the corresponding wild-type coding sequence or reference coding sequence. The nucleic acid according to any one of claims 1 to 33.

35. The nucleic acid according to claim 34, wherein one or more of the coding sequences with modified codons are selected from a coding sequence with maximized C, a coding sequence with maximized CAI, a coding sequence adapted for codon use in humans, a coding sequence with modified G / C content, a coding sequence with optimized G / C, or any combination thereof.

36. The nucleic acid according to claim 34 or 35, wherein one or more of the coding sequences with modified codons are coding sequences with optimized G / C ratio, coding sequences adapted to codon use in humans, or coding sequences with modified G / C content.

37. The one or more coding sequences described above have, or consist of, a coding sequence optimized for G / C that includes a nucleic acid sequence. The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 36: (i) Sequence numbers 136-138, 140, 141, 148, 149, 152, 155, 156, 159, 162, 163, 166, 169, 170, 173, 11731-11813, 11815, 11817-11966, 12271-12472, 12743-12944, 13514, 13515, 14124-14132, 14142-14150, 14160-14168, 22759, 22764-22786, 22791- 22813, 22818-22839, 22969-23040, 23077-23148, 23189-23260, 23297-23368, 23409-23480, 23517-23588, 23629-23700, 23737-23808, 23849-23920, 23957-24028, 24069-24140, 24177-24248, 24289-24360, 24397-24468, 24509-24580 , 24617-24688, 24729-24800, 24837-24908, 24949-25020, 25057-25128, 25169-25240, 25277-25348, 25389-25460, 25497-25568, 25609-25680, 25717-25788, 25829-25900, 25937-26008, 26049-26120, 26157-26228, 26269-26340, 2637 It is identical to any one of the following: 7-26448, 26489-26560, 26597-26668, 26709-26780, 26817-26888, 26925-26937, or it is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is a fragment or variant of any of the sequences.

38. The one or more coding sequences described above have, or consist of, coding sequences adapted for codon use in humans, which include nucleic acid sequences. The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 37: (i) It is identical to any one of sequence numbers 142, 143, 145, 150, 153, 157, 160, 164, 167, 171, 174, 11967-12033, 12473-12539, 12945-13011, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is a fragment or variant of any of the sequences.

39. The above one or more coding sequences have, or consist of, coding sequences with a modified G / C content that include nucleic acid sequences. The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 38: (i) Sequence numbers 146, 147, 151, 154, 158, 161, 165, 168, 172, 175, 12034-12050, 12052, 12054-12203, 12540-12675, 13012-13147, 13519, 13520, 14133-14141, 14151-14159, 14169-14177, 23041-23076, 23149-23184, 2326 1-23296, 23369-23404, 23481-23516, 23589-23624, 23701-23736, 23809-23844, 23921-23956, 24029-24064, 24141-24176, 24249-24284, 24361-24396, 24469-24504, 24581-24616, 24689-24724, 24801-24836, 24 909-24944, 25021-25056, 25129-25164, 25241-25276, 25349-25384, 25461-25496, 25569-25604, 25681-25716, 25789-25824, 25901-25936, 26009-26044, 26121-26156, 26229-26264, 26341-26376, 26449-26484 , is identical to any one of 26561-26596, 26669-26704, 26781-26816, or 26889-26924, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) A fragment or variant of any of these sequences.

40. The G / C content of one or more of the above coding sequences is approximately 50% or more, approximately 55% or more, or approximately 60% or more. Preferably, it is about 63.9%. The nucleic acid according to any one of claims 1 to 39.

41. The above one or more coding sequences encode the S protein, The above S protein has the K986P and V987P mutations, which are pre-fusion structural stabilization mutations. The above coding sequence has or consists of a coding sequence that optimizes G / C, which includes a nucleic acid sequence. The above nucleic acid sequences include sequence numbers 137, 23090, 23091, 23093, 23094, or fragments or variants thereof. The nucleic acid according to any one of claims 1 to 40.

42. The nucleic acid according to any one of claims 1 to 41, wherein the one or more heterogeneous untranslated regions are selected from one or more heterogeneous 5'UTRs and / or one or more heterogeneous 3'UTRs.

43. The above one or more heterogeneous 3'UTRs contain or consist of nucleic acid sequences. The nucleic acid according to claim 42, wherein the above nucleic acid sequence satisfies either (i) or (ii) below: (i) Derived from the 3'UTR of a gene selected from PSMB3, ALB7, α-globin, CASP1, COX6B1, GNAS, NDUFA1, and RPS9; (ii) Derived from any one homolog, fragment, or variant of the gene in (i).

44. The above one or more different 5'UTRs contain or consist of nucleic acid sequences. The nucleic acid according to claim 42, wherein the above nucleic acid sequence satisfies either (i) or (ii) below: (i) Derived from the 5'UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2; (ii) Derived from any one homolog, fragment, or variant of the gene in (i).

45. The UTR designs for one or more heterogeneous 5'UTRs and one or more heterogeneous 3'UTRs are selected from a-1 (HSD17B4 / PSMB3), a-3 (SLC7A3 / PSMB3), e-2 (RPL31 / RPS9), and i-3 (- / muag). Particularly preferred are the UTR designs a-1 (HSD17B4 / PSMB3) and i-3 (- / muag). The nucleic acid according to claim 42.

46. The above nucleic acid has one or more poly(A) sequences and / or one or more poly(C) sequences, Preferably, the poly(A) sequence contains 30 to 200 adenosine nucleotides. Preferably, the poly(C) sequence contains 10 to 40 cytosine nucleotides. The nucleic acid according to any one of claims 1 to 45.

47. The nucleic acid according to any one of claims 1 to 46, wherein the nucleic acid has one or more histone stem loops.

48. The nucleic acid according to any one of claims 1 to 47, wherein the nucleic acid is DNA or RNA.

49. The nucleic acid according to any one of claims 1 to 48, wherein the nucleic acid is coding RNA.

50. The nucleic acid according to claim 49, wherein the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA.

51. The nucleic acid according to any one of claims 1 to 50, wherein the nucleic acid (preferably coding RNA) is mRNA.

52. The nucleic acid according to claim 51, wherein the mRNA is not replicon RNA or self-replicating RNA.

53. The above mRNA is, It has one or more poly(A) sequences containing 30 to 200 adenosine nucleotides, and The nucleotide at the 3' end is adenosine. The nucleic acid according to claim 51.

54. The above RNA (preferably coding RNA) has a 5' cap structure, Preferably, the 5' cap structure is The nucleic acid according to any one of claims 48 to 51. It is an m7G structure, a cap0 structure, a cap1 structure, a cap2 structure, a modified cap0 structure, or a modified cap1 structure. Preferably, it has a 5'cap1 structure. The nucleic acid according to any one of claims 48 to 51.

55. The nucleic acid (preferably mRNA) described above comprises the following elements in the 5' to 3' direction, according to any one of claims 48 to 54: (A) 5'cap1 structure; (B) The coding sequence of sequence number 137, or a fragment or variant thereof; (C) A 3'UTR derived from the 3'UTR of the α-globin gene (preferably the 3'UTR of SEQ ID NO: 267 or 268); (D) A poly(A) sequence containing approximately 64 A nucleotides; (E) A poly(C) sequence containing approximately 30 C nucleotides; (F) Histone stem loop of sequence number 178 or 179.

56. The nucleic acid (preferably mRNA) described above comprises the following elements in the 5' to 3' direction, according to any one of claims 48 to 54: (A) 5'cap1 structure (B) A 5'UTR derived from the 5'UTR of the HSD17B4 gene (preferably the 5'UTR of SEQ ID NO: 231 or 232); (C) The coding sequence of sequence number 137, or a fragment or variant thereof; (D) A 3'UTR derived from the 3'UTR of the PSMB3 gene (preferably the 3'UTR of SEQ ID NO: 253 or 254); (E) Histone stem loop selected from sequence number 178 or 179; (F) A poly(A) sequence containing approximately 100 A nucleotides.

57. The nucleic acid according to claim 56, wherein the nucleotide at the 3' end is adenosine.

58. The nucleic acid (preferably mRNA) described above comprises the following elements in the 5' to 3' direction, according to any one of claims 48 to 54: (A) 5'cap1 structure; (B) The coding sequence of sequence number 23090 or 23091, or a fragment or variant thereof. (C) 3'UTR derived from the 3'UTR of the α-globin gene (preferably the 3'UTR of SEQ ID NO: 267 or 268) (D) A poly(A) sequence containing approximately 64 A nucleotides; (E) A poly(C) sequence containing approximately 30 C nucleotides; (F) Histone stem loop of sequence number 178 or 179.

59. The nucleic acid (preferably mRNA) described above comprises the following elements in the 5' to 3' direction, according to any one of claims 48 to 54: (A) 5'cap1 structure; (B) A 5'UTR derived from the 5'UTR of the HSD17B4 gene (preferably the 5'UTR of SEQ ID NO: 231 or 232); (C) The coding sequence of sequence number 23090 or 23091, or a fragment or variant thereof. (D) A 3'UTR derived from the 3'UTR of the PSMB3 gene (preferably the 3'UTR of SEQ ID NO: 253 or 254) (E) Histone stem loop selected from sequence number 178 or 179; (F) A poly(A) sequence containing approximately 100 A nucleotides.

60. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 59: Sequence numbers 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824, 258 The nucleic acid sequence is identical to, or is identical to, a nucleic acid sequence selected from the group consisting of 29-26044, 26049-26264, 26269-26484, 26489-26704, or 26709-26937148 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more; (ii)(i) is a fragment or variant of any of the sequences.

61. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The above nucleic acid sequence satisfies either (i) or (ii) below, (i) A nucleic acid sequence selected from the group consisting of SEQ ID NOs: 149, 156, 12338, 150, 157, 151, 158, 12541, 163, 170, 12810, 164, 171, 165, 172, 13013, 12342-12351, 12545-12554, 12814-12823, 13017-13026, 14133 is identical to, or identical to, 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is a fragment or variant of any of the sequences; Preferably, the nucleic acid sequence satisfies (iii) or (iv) below, according to any one of claims 1 to 60: (iii) Select from sequence numbers 149, 150, 151, 163, 164, and 165; (iv)(iii) A fragment or variant of any of the sequences is selected.

62. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 61: (i) The nucleic acid sequence is identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii) is a fragment or variant of (i).

63. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 62: (i) The nucleic acid sequence is identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii) is a fragment or variant of (i).

64. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 63: (i) The nucleic acid sequence is identical to, or 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii) is a fragment or variant of (i).

65. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The nucleic acid described above is the nucleic acid according to any one of claims 1 to 64, satisfying either (i) or (ii) below: (i) identical to, or identical to, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 23311, 23531, and 24851 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; (ii) a fragment or variant of (i).

66. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 65: (i) identical to, or identical to, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 23310, 23530, and 24850 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; (ii) a fragment or variant of (i).

67. The above nucleic acid contains or consists of a nucleic acid sequence (preferably an RNA sequence). The nucleic acid sequence described above satisfies either (i) or (ii) below, according to any one of claims 1 to 66: (i) The nucleic acid sequence is identical to, or is identical to, a nucleic acid sequence selected from the group consisting of sequence numbers 23313, 23533, 24853, 23314, 23534, and 24854 by 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; (ii) is a fragment or variant of (i).

68. The nucleic acid according to any one of claims 1 to 67, wherein the nucleic acid is RNA that does not have a 1-methylpseudridine substitution.

69. The nucleic acid according to any one of claims 1 to 68, wherein the nucleic acid is RNA that does not contain chemically modified nucleotides.

70. The above nucleic acid is RNA transcribed in vitro. RNA transcription in vitro is performed in the presence of a sequence-optimized nucleotide mixture and a capping analog. Preferably, the sequence-optimized nucleotide mixture does not contain chemically modified nucleotides. The nucleic acid according to any one of claims 1 to 69.

71. The nucleic acid mentioned above is purified RNA. Preferably, the RNA is purified by RP-HPLC and / or TFF. The nucleic acid according to any one of claims 1 to 70.

72. The nucleic acid mentioned above is purified RNA. The above purified RNA is purified by RP-HPLC and / or TFF. The purified RNA described above contains approximately 5%, 10%, or 20% fewer double-stranded RNA byproducts compared to RNA that has not been purified by RP-HPLC and / or TFF. The nucleic acid according to any one of claims 1 to 71.

73. The nucleic acid mentioned above is purified RNA. The above purified RNA is purified by RP-HPLC and / or TFF. The purified RNA described above contains approximately 5%, 10%, or 20% fewer double-stranded RNA byproducts compared to RNA purified by oligo-dT purification, precipitation, filtration, and / or anion exchange chromatography. The nucleic acid according to any one of claims 1 to 72.

74. A composition comprising one or more nucleic acids according to any one of claims 1 to 73, In any configuration, the above composition comprises one or more pharmaceutically acceptable carriers. composition.

75. The composition according to claim 74, comprising mRNA described in SEQ ID NOs: 149, 163, 24837, 23311, 23531, 23310, 23530, 23313, or 23533, or a fragment or variant of any of these sequences.

76. The composition according to claim 74, wherein the composition is a polyvalent composition containing a plurality or two or more nucleic acids as described in any one of claims 1 to 73.

77. The multiple or more nucleic acid sequences of the above polyvalent composition each encode a different spike protein. Preferably, the spike protein is a pre-fusion structure-stabilizing spike protein. The composition according to claim 76.

78. The composition according to claim 77, wherein the above-mentioned different spike proteins or pre-fusion structure-stabilizing spike proteins are derived from different variants / isolated strains of the SARS-CoV-2 virus.

79. The composition according to claim 78, wherein the above-mentioned different spike proteins or pre-fusion structure-stabilizing spike proteins are derived from at least B. 1.1.7, B. 1.351, P. 1, or CAL. 20C.

80. The composition according to claim 78, wherein the above-mentioned different spike proteins or pre-fusion structure-stabilizing spike proteins have one or more amino acid mutations in the S protein from (i) to (v) below: (i) delH69, delV70, Y453F, D614G, I692V and M1229I; (ii) delH69, delV70, delY144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H; (iii) L18F, D80A, D215G, delL242, delA243, delL244, R246I, K417N, E484K, N501Y, D614G and A701V; (iv) L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y and T1027I; (v) S13I, W152C, L452R and D614G.

81. The above polyvalent composition contains two or more nucleic acid species, The above nucleic acid species has a coding sequence that codes for an amino acid sequence that is identical to any one of SEQ ID NOs: 10, 22961, 22960, 22963, 22941, or 22964, or is identical to 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The composition according to any one of claims 76 to 78.

82. The above polyvalent composition contains two or more RNA species, The above RNA species are identical to any one of sequence numbers 149 or 24837, 23531 or 24851, 23530 or 24850, 23533 or 24853, 23439 or 24759, or 23534 or 24854, or are identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The composition according to any one of claims 76 to 78.

83. The composition according to any one of claims 74 to 80, comprising mRNA having an RNA integrity of 70% or more.

84. The above composition contains mRNA with a capping rate of 70% or more. Preferably, the capping rate is 70% or more, 80% or more, or 90% or more, and the mRNA species contains a Cap1 structure. The composition according to any one of claims 74 to 83.

85. The above one or more nucleic acids are complexed or associated with one or more cationic compounds or polycationic compounds, or are at least partially complexed or associated with them. Preferably, the cationic compound or polycationic compound is a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combination thereof. The composition according to any one of claims 74 to 84.

86. The above one or more nucleic acids are complexed or associated with one or more lipids or lipid carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes. Preferably, the above one or more nucleic acids are encapsulated, The composition according to claim 85.

87. The composition according to claim 85 or 86, wherein one or more nucleic acids are complexed with one or more lipids, thereby forming lipid nanoparticles.

88. The composition according to claim 86 or 87, wherein the above-mentioned LNP contains a cationic lipid relating to formula III-3. 【Chemistry 1】

89. The composition according to any one of claims 86 to 88, wherein the above LNP comprises a PEG lipid of formula (IVa): 【Chemistry 2】 During the ceremony, The average value of n is between 30 and 60. Preferably, the average value of n is approximately 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54. Most preferably, the average value of n is 49 or 45.

90. The composition according to any one of claims 86 to 88, wherein the above LNP comprises a PEG lipid of formula (IVa): 【Transformation 3】 In the formula, n is an integer selected such that the average molecular weight of the PEG lipid is approximately 2500 g / mol.

91. The composition according to any one of claims 86 to 90, wherein the above-mentioned LNP comprises one or more neutral lipids and / or one or more steroids or steroid analogs.

92. The above neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Preferably, the molar ratio of the cationic lipid to the DSPC is (approximately 2:1) to (approximately 8:1). The composition according to claim 91.

93. The steroid mentioned above is cholesterol. Preferably, the molar ratio of the cationic lipid to cholesterol is (approximately 2:1) to (approximately 1:1). The composition according to claim 91.

94. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids, preferably lipids of formula (III), more preferably lipid III-3; (ii) One or more neutral lipids, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) One or more steroids or steroid analogs, preferably cholesterol; (iv) One or more polymer-compounded lipids, preferably PEG lipids derived from formula (IVa) with n=49; The composition according to any one of claims 86 to 93, wherein the molar ratio of (i) to (iv) above is approximately 20 to 60% cationic lipids, 5 to 25% neutral lipids, 25 to 55% sterols, and 0.5 to 15% PEG lipids.

95. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids, preferably lipids of formula (III), more preferably lipid III-3; (ii) One or more neutral lipids, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) One or more steroids or steroid analogs, preferably cholesterol; (iv) One or more polymer-compounded lipids, preferably PEG lipids derived from formula (IVa) with n=45; The composition according to any one of claims 86 to 93, wherein the molar ratio of (i) to (iv) above is approximately 20 to 60% cationic lipids, 5 to 25% neutral lipids, 25 to 55% sterols, and 0.5 to 15% PEG lipids.

96. The molar ratio of (i) to (iv) above is approximately 50:10:38.5:1.

5. Preferably, the ratio is 47.5:10:40.8:1.

7. More preferably, the ratio is 47.4:10:40.9:1.

7. The composition according to claim 94 or 95.

97. The nucleic acid mentioned above is RNA, The amount of free RNA (uncomplexed or unencapsulated RNA) contained in the above composition is less than approximately 20%. Preferably, the amount of free RNA is less than approximately 15%. More preferably, the amount of free RNA is less than approximately 10%. The composition according to any one of claims 87 to 96.

98. The weight ratio of lipids to nucleic acids is approximately 10:1 to 60:

1. Preferably, the ratio is (approximately 20:1) to (approximately 30:1), For example, it is approximately 25:

1. The composition according to any one of claims 87 to 97.

99. The N / P ratio of the LNPs encapsulating the above nucleic acids is approximately 1 to approximately 10. Preferably, it is about 5 to about 7. More preferably, it is about 6. The composition according to any one of claims 87 to 98.

100. The polydispersion index (PDI) of the above composition is less than approximately 0.

4. Preferably, it is less than about 0.

3. More preferably, it is less than about 0.

2. Most preferably, it is less than about 0.

1. The composition according to any one of claims 87 to 99.

101. The Z-average particle size of the above LNP is approximately 60 to 120 nm. Preferably, it is less than about 120 nm. More preferably, less than approximately 100 nm. Most preferably less than about 80 nm, The composition according to any one of claims 86 to 100.

102. The composition according to any one of claims 86 to 101, wherein, of the above LNPs, the amount of LNPs having a particle size greater than approximately 500 nm is less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, and less than approximately 1%.

103. The composition according to any one of claims 86 to 102, wherein, of the above LNPs, the amount of LNPs having a particle size of less than about 20 nm is less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, and less than 1%.

104. Of the lipid carriers, those that are spherical account for over 80%, over 85%, over 90%, and over 95%. Preferably, it has a solid or partially solid core. The composition according to any one of claims 86 to 103.

105. The turbidity of the above composition is approximately 150 to approximately 0.0 FNU. Preferably, it is about 50 FNU or less. More preferably, it is about 25 FNU or less. The composition according to any one of claims 86 to 104.

106. It also contains sugars at a concentration of approximately 50 to 300 mM. Preferably, it contains sucrose at a concentration of about 150 mM. The composition according to any one of claims 74 to 105.

107. It also contains salts with a concentration of approximately 10 to 200 mM. Preferably, it contains NaCl at a concentration of about 75 mM. The composition according to any one of claims 74 to 106.

108. It also contains a buffer with a concentration of 1 to approximately 100 mM. Preferably, Na at a concentration of about 10 mM. ３ PO ４ Includes, The composition according to any one of claims 74 to 107.

109. The pH of the above composition is approximately 7.0 to 8.

0. Preferably, it is about 7.

4. The composition according to any one of claims 74 to 108.

110. It contains lipid nanoparticles that encapsulate RNA, The above RNA encodes the S protein of SARS-CoV-2, which has the pre-fusion structural stabilization mutations K986P and V987P. The above LNP includes (i) to (iv) below: (i) Cationic lipid of formula III-3; (ii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) Cholesterol; (iv) PEG lipids of formula IVa (n=49); The molar ratios of (i) to (iv) above are approximately 47.4% cationic lipids, 10% DSPC, 40.9% cholesterol, and 1.7% PEG lipids. The above RNA is identical to the nucleic acid sequence of Sequence ID No. 163 or 149, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The above RNA is unmodified. The above RNA has a 5'cap1 structure, The integrity of the above RNA is approximately 70% or higher. The N / P ratio of the LNP encapsulating the above RNA is approximately 6. The Z-average particle size of the LNPs encapsulating the above RNA is approximately 60 to 120 nm. The amount of free RNA (uncomplexed or unencapsulated RNA) contained in the above composition is less than approximately 20%. In any configuration, the above composition consists of sucrose at a concentration of approximately 150 mM, NaCl at a concentration of approximately 75 mM, and Na at a concentration of approximately 10 mM. ３ PO ４ It also includes, In any configuration, the pH of the above composition is approximately 7.

4. The composition according to any one of claims 86 to 109.

111. It contains lipid nanoparticles that encapsulate RNA, The above RNA encodes the S protein of SARS-CoV-2, which has the pre-fusion structural stabilization mutations K986P and V987P. The above LNP includes (i) to (iv) below: (i) Cationic lipid of formula III-3; (ii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) Cholesterol; (iv) PEG lipids of formula IVa (n=45); The molar ratios of (i) to (iv) above are approximately 47.4% cationic lipids, 10% DSPC, 40.9% cholesterol, and 1.7% PEG lipids. The above RNA is identical to the nucleic acid sequence of Sequence ID No. 163 or 149, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The above RNA is unmodified. The above RNA has a 5'cap1 structure, The integrity of the above RNA is approximately 70% or higher. The N / P ratio of the LNP encapsulating the above RNA is approximately 6. The Z-average particle size of the LNPs encapsulating the above RNA is approximately 60 to 120 nm. The amount of free RNA (uncomplexed RNA) contained in the above composition is less than approximately 20%. In any configuration, the above composition consists of sucrose at a concentration of approximately 150 mM, NaCl at a concentration of approximately 75 mM, and Na at a concentration of approximately 10 mM. ３ PO ４ It includes, In any configuration, the pH of the above composition is approximately 7.

4. The composition according to any one of claims 86 to 109.

112. It contains unmodified RNA, The above RNA is identical to the nucleic acid sequence of Sequence ID No. 163, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The above RNA is formulated in lipid nanoparticles (LNPs). In the above LNP, the molar ratio of cationic lipid III-3, DSPC, cholesterol, and PEG lipid of formula (IVa) is approximately 50:10:38.5:1.5 (mol%). Preferably, the ratio is 47.5:10:40.8:1.7 (mol%). More preferably, the ratio is 47.4:10:40.9:1.7 (mol%). The composition according to any one of claims 86 to 110.

113. It contains unmodified RNA, The above RNA is identical to the nucleic acid sequence of Sequence ID No. 149, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The above RNA is formulated in lipid nanoparticles (LNPs). In the above LNP, the molar ratio of cationic lipid III-3, DSPC, cholesterol, and PEG lipid of formula (IVa) is approximately 50:10:38.5:1.5 (mol%). Preferably, the ratio is 47.5:10:40.8:1.7 (mol%). More preferably, the ratio is 47.4:10:40.9:1.7 (mol%). The composition according to any one of claims 86 to 110.

114. The above composition contains mRNA encoding the SARS-CoV-2 spike protein (S), The above mRNA is a spike protein (S_stab) that is stable in a pre-fusion structure and has one or more pre-fusion structural stabilization mutations. The composition according to any one of claims 74 to 112.

115. The composition according to claim 114, wherein the mRNA encoding the spike protein of SARS-CoV-2 is 95% or more identical to SEQ ID NO: 163, or encodes the same coronavirus spike protein as SEQ ID NO:

163.

116. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids; (ii) one or more neutral lipids; (iii) One or more steroids or steroid analogs; (iv) One or more PEG lipids; The composition according to claim 114, wherein the molar ratio of (i) to (iv) above is approximately 20 to 60% cationic lipids, 5 to 25% neutral lipids, 25 to 55% sterols, and 0.5 to 15% PEG lipids.

117. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The composition according to claim 114, wherein the molar ratio of (i) to (iv) above is approximately 20 to 60% cationic lipids, 5 to 25% neutral lipids, 25 to 55% sterols, and 0.5 to 15% PEG lipids.

118. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratio of (i) to (iv) above is approximately 47.5:10:40.8:1.

7. The composition according to claim 114.

119. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratios of (i) to (iv) above are 47.4:10:40.9:1.

7. The composition according to claim 114.

120. The composition according to any one of claims 107 to 118, wherein the ratio of mRNA to total lipids is approximately 0.03 to 0.04 (w / w).

121. The above mRNA is complexed with one or more lipids, thereby forming lipid nanoparticles (LNPs). The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratios of (i) to (iv) above are approximately 47.5:10:40.8:1.

7. The ratio of mRNA to total lipids is approximately 0.03 to 0.04 (w / w). The composition according to claim 120.

122. The above mRNA is complexed with one or more lipids, thereby forming lipid nanoparticles (LNPs). The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratios of (i) to (iv) above are 47.4:10:40.9:1.

7. The ratio of mRNA to total lipids is approximately 0.03 to 0.04 (w / w). The composition according to claim 120.

123. The composition described above is a freeze-dried composition, as described in any one of claims 74 to 99, 110 to 122.

124. The composition according to claim 123, wherein the water content of the freeze-dried composition is less than about 10%.

125. The composition according to claim 124, wherein the water content of the freeze-dried composition is about 0.5% to 5%.

126. The nucleic acid mentioned above is RNA, The above composition is stable after being stored as a liquid at approximately 5°C for approximately two weeks or more. The composition according to any one of claims 86 to 122.

127. The nucleic acid mentioned above is RNA, The above composition is stable after being stored as a liquid at approximately 5°C for more than one month. The composition according to claim 126.

128. The nucleic acid mentioned above is RNA, The composition according to claim 126, wherein the composition is stable after being stored as a liquid at approximately 5°C for approximately 2 weeks to approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, or approximately 1 year.

129. The nucleic acid mentioned above is RNA, After being stored as a liquid at approximately 5°C for approximately two weeks or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the above RNA remains undamaged. The composition according to claim 126.

130. The nucleic acid mentioned above is RNA, After being stored as a liquid at approximately 5°C for more than one month, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the above RNA remains undamaged. The composition according to claim 129.

131. The nucleic acid mentioned above is RNA, The composition according to claim 126, wherein, after being stored as a liquid at approximately 5°C for approximately 2 weeks to approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, or approximately 1 year, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the RNA remains undamaged.

132. The nucleic acid mentioned above is RNA, After being stored as a liquid at approximately 5°C for about two weeks, more than 80% of the above RNA remained undamaged. The composition according to claim 126.

133. The composition according to any one of claims 86 to 132, comprising a low-cohesive lipid.

134. The nucleic acid mentioned above is RNA, The concentration of the above RNA is approximately 10 μg / mL to approximately 10 mg / mL. Preferably, the concentration is about 100 μg / mL to about 1 mg / mL. The composition according to any one of claims 86 to 133.

135. The nucleic acid mentioned above is RNA, The concentration of the above RNA is 100 μg / mL or higher. More preferably, 200 μg / mL or more. Most preferably, it is 500 μg / mL or more. The composition according to any one of claims 86 to 133.

136. The nucleic acid mentioned above is RNA, The RNA integrity of the above RNA is approximately 50% or higher. Preferably, it is about 60% or more. More preferably, it is about 70% or more. Most preferably, it is about 80% or more. The composition according to any one of claims 86 to 135.

137. The nucleic acid mentioned above is RNA, The free RNA contained in the above composition is less than approximately 20%. Preferably, it is less than about 15%. More preferably, less than approximately 10%. The composition according to any one of claims 86 to 136.

138. The nucleic acid mentioned above is RNA, The divalent cation contained in the above composition is less than approximately 100 nM per gram of RNA. Preferably, the concentration is less than approximately 50 nM per gram of RNA. More preferably, the amount is less than approximately 10 nM per gram of RNA. The composition according to any one of claims 86 to 137.

139. The above divalent cation is Mg ２＋ and / or Ca ２＋ A composition according to claim 138, selected from the above.

140. The concentration of the above lipids is approximately 250 μg / mL to approximately 250 mg / mL. Preferably, the concentration is about 2.5 mg / mL to about 25 mg / mL. The composition according to any one of claims 86 to 139.

141. The concentration of the above lipids is approximately 2.5 mg / mL or higher. Preferably, it is 5 mg / mL or more. More preferably, 12.5 mg / mL or more. The composition according to any one of claims 86 to 140.

142. The concentration of the above-mentioned low-aggregation lipids is approximately 17.5 μg / mL to approximately 17.5 mg / mL. Preferably, the concentration is about 175 μg / mL to about 1.75 mg / mL. The composition according to any one of claims 133 to 142.

143. The concentration of the above-mentioned low-aggregating lipids is approximately 175 μg / mL or higher. Preferably, it is about 350 μg / mL or more. More preferably, 875 μg / mL or more. The composition according to any one of claims 133 to 142.

144. The nucleic acid mentioned above is RNA, The weight ratio of the above lipids to the above RNA is approximately 10:1 to 60:

1. Preferably, the ratio is (approximately 20:1) to (approximately 30:1), More preferably, it is about 25:

1. The composition according to any one of claims 86 to 143.

145. The nucleic acid mentioned above is RNA, The N / P ratio of the above lipid carrier to the above RNA is approximately 1 to approximately 10. Preferably, it is about 5 to about 7. More preferably, it will be about 6. The composition according to any one of claims 86 to 144.

146. The nucleic acid mentioned above is RNA, The molar ratio of low-aggregating lipids contained in the lipid carrier encapsulating the above RNA is approximately 0.5% to 15%. Preferably, it is about 1.0% to about 2.5%. More preferably, about 1.7% The composition according to any one of claims 86 to 143.

147. The composition according to any one of claims 133 to 143, wherein the low-aggregating lipid is a polymer-compounded lipid (for example, a PEG-compounded lipid).

148. The nucleic acid mentioned above is RNA, The above RNA and the lipid carrier encapsulating the RNA are purified by one or more of the following (i) to (iii), in the composition according to any one of claims 86 to 147: (i) One or more purification steps (preferably one or more TFF steps); (ii) One or more clarification steps; (iii) One or more filtration steps.

149. The ethanol contained in the above composition is less than approximately 500 ppm. Preferably, it is less than about 50 ppm. More preferably, less than approximately 5 ppm. The composition according to any one of claims 86 to 148.

150. The osmolality of the above composition is approximately 250 mOsmol / kg to approximately 450 mOsmol / kg. Preferably, it is about 335 mOsmol / kg. The composition according to any one of claims 86 to 154.

151. The above composition is stable for more than one week after being stored as a liquid at approximately 25°C. Preferably, it is stable for two weeks or more. More preferably, stable for more than 3 weeks. Most preferably, it is stable for four weeks or more. The composition according to any one of claims 86 to 150.

152. The above composition is stable for more than one day after being stored as a liquid at approximately 40°C. Preferably, it is stable for two days or more. More preferably, it is stable for more than 3 days. Most preferably, it is stable for 4 days or more. The composition according to any one of claims 86 to 151.

153. When stored in liquid form, the loss of integrity of the above RNA is less than approximately 30%. Preferably, it is less than about 20%. More preferably, less than approximately 10%. The composition according to any one of claims 86 to 152.

154. When stored as a liquid, the increase in free RNA is less than 10%. Preferably, it is 5% or less. The composition according to any one of claims 86 to 153.

155. The nucleic acid mentioned above is RNA, When stored as a liquid, the increase in PDI of the lipid carrier encapsulating the RNA is approximately 0.2 or less. Preferably, it is about 0.1 or less. The composition according to any one of claims 86 to 154.

156. The nucleic acid mentioned above is RNA, When stored as a liquid, the increase in the Z-average particle size of the lipid carrier encapsulating the above RNA is 20% or less. Preferably, it is 10% or less. The composition according to any one of claims 86 to 155.

157. When stored as a liquid, the increase in turbidity of the above composition is 20% or less. Preferably, it is 10% or less. The composition according to any one of claims 86 to 156.

158. When stored as a liquid, the increase or decrease in pH and / or osmolality is 20% or less. Preferably, it is 10% or less. The composition according to any one of claims 86 to 157.

159. When stored as a liquid, the decrease in potency of the above composition is less than approximately 30%. Preferably, it is less than about 20%. More preferably, less than approximately 10%. The composition according to any one of claims 86 to 158.

160. The nucleic acid mentioned above is RNA, The above RNA is purified RNA. Preferably, the RNA is purified by RP-HPLC and / or tangential flow filtration (TFF). The composition according to any one of claims 86 to 133.

161. It further contains one or more antagonists of one or more RNA sensing pattern recognition receptors, Preferably, the system comprises one or more antagonists of the TLR7 receptor and / or the TLR8 receptor. The composition according to any one of claims 74 to 160.

162. The one or more antagonists of the TLR7 receptor and / or TLR8 receptor are single-stranded oligonucleotides. Preferably, it is p5'-GAG CGmG CCA-3', The composition according to claim 161.

163. A polypeptide for vaccines comprising one or more antigenic peptides or antigenic proteins, The above antigenic peptide or antigenic protein is either the coronavirus SARS-CoV-2 or its immunogenic fragment or immunogenic variant, or is derived therefrom. Preferably, the amino acid sequence of the antigenic peptide or antigenic protein is a polypeptide that satisfies either (i) or (ii) below: (i) It is identical to any one of the amino acid sequences of SEQ ID NOs: 1-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, 26939, or is identical by 70% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more; (ii)(i) is an immunogenic fragment or immunogenic variant.

164. A vaccine containing one or more of the following (i) to (iii): (i) The nucleic acid according to any one of claims 1 to 73; (ii) The composition according to any one of claims 74 to 162; (iii) The polypeptide according to claim 163.

165. The above vaccines induce an adaptive immune response, Preferably, the above adaptive immune response is a protective adaptive immune response against coronavirus. Preferably, the coronavirus is SARS-CoV-2, according to claim 164.

166. The vaccine according to claim 164 or 165, wherein the vaccine is a polyvalent vaccine comprising (i) or (ii) below: (i) A plurality or two or more nucleic acids as described in any one of claims 1 to 73; (ii) A plurality or two or more compositions from any one of claims 74 to 162.

167. A kit or kit of parts comprising one or more of the following (i) to (iv): (i) the nucleic acid according to any one of claims 1 to 73; (ii) The composition according to any one of claims 74 to 162; (iii) The polypeptide according to claim 163; (iv) The vaccine according to any one of claims 164 to 166; It has an arbitrary configuration and is equipped with a liquid medium for solubilization. A kit or kit of parts, with optional configurations and equipped with technical instructions providing information on the administration and dosage of the components.

168. For use as a medicine, any of the following (i) to (v): (i) The nucleic acid according to any one of claims 1 to 73; (ii) The composition according to any one of claims 74 to 162; (iii) The polypeptide according to claim 163; (iv) The vaccine according to any one of claims 164 to 166; (v) A kit or kit of parts according to claim 167.

169. Any of the following (i) to (v) for the treatment or prevention of infection with coronavirus (preferably SARS-CoV-2 coronavirus) or disorders associated with such infection (preferably COVID-19): (i) The nucleic acid according to any one of claims 1 to 73; (ii) The composition according to any one of claims 74 to 162; (iii) The polypeptide according to claim 163; (iv) The vaccine according to any one of claims 164 to 166; (v) A kit or kit of parts according to claim 167.

170. A method for treating or preventing a disability, The above method includes the step of applying or administering one or more of the following (i) to (v) to a subject that requires it: (i) The nucleic acid according to any one of claims 1 to 73; (ii) The composition according to any one of claims 74 to 162; (iii) The polypeptide according to claim 163; (iv) The vaccine according to any one of claims 164 to 166; (v) A kit or kit of parts according to claim 167.

171. A method for treating or preventing the disorder according to claim 170, wherein the disorder is an infection with a coronavirus (preferably SARS-CoV-2 coronavirus) or a disorder associated with said infection (preferably COVID-19).

172. The above subjects are mammalian subjects, Preferably, the subject is a human subject. A method for treating or preventing the disorder described in claim 170 or 171.

173. The above human subjects were elderly human subjects. Preferably, they are 50 years of age or older, 60 years of age or older, 65 years of age or older, or 70 years of age or older. A method for treating or preventing the disorder described in any one of claims 170 to 172.

174. The method for treating or preventing the disorder described in claim 173, wherein the human subject is 61 years of age or older.

175. The method for treating or preventing the disorder described in any one of claims 170 to 172, wherein the human subject is between 18 and 60 years of age.

176. The subject described above is pregnant, and the method for treating or preventing the disorder described in any one of claims 170 to 172.

177. The method according to any one of claims 170 to 175, satisfying either (i) or (ii) below: (i) After the first administration of the above composition, less than 25% of subjects experience a Grade 3 systemic adverse event; (ii) Less than 30% of subjects experience a local adverse event of grade 2 or higher after the first administration of the above composition.

178. The method according to any one of claims 170 to 175, wherein 40% or less of subjects experience a Grade 3 systemic adverse event after a second dose of the composition.

179. The human subjects described above were newborns or infants. Preferably, the age is 3 years or younger, 2 years or younger, 1.5 years or younger, 1 year or younger (12 months or younger), 9 months or younger, 6 months or younger, or 3 months or younger, or 6 months to 2 years. A method for treating or preventing the disorder described in any one of claims 170 to 172.

180. The method according to claim 170, further defined as a method for reducing the disease burden on the above-mentioned subject.

181. The method according to claim 180, wherein the severity of one or more symptoms of COVID-19 disease is reduced by the method described above.

182. The method according to claim 181, wherein the probability that the subject will require hospitalization, admission to an intensive care unit, oxygen therapy, and / or mechanical ventilation is reduced.

183. The method according to claim 181, wherein the probability of the subject developing severe or moderate COVID-19 disease is reduced by the method described above.

184. The method according to claim 181, wherein the subject is protected from severe COVID-19 disease for approximately six months or more by the method described above.

185. By the above method, the subject was protected from severe COVID-19 disease upon exposure to the SARS-CoV-2 variant. The S protein of the above SARS-CoV-2 mutant strain has one or more amino acid mutations compared to SEQ ID NO:

1. The method according to claim 184.

186. The method according to claim 185, wherein the S protein of the above SARS-CoV-2 mutant strain has one or more amino acid mutations from (i) to (v) below: (i) delH69, delV70, Y453F, D614G, I692V and M1229I; (ii) delH69, delV70, delY144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H; (iii) L18F, D80A, D215G, delL242, delA243, delL244, R246I, K417N, E484K, N501Y, D614G and A701V; (iv) L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y and T1027I; (v) S13I, W152C, L452R and D614G.

187. The above method reduces the probability that the subject will develop fever, difficulty breathing, loss of smell, and / or loss of taste. The method according to claim 181.

188. The above method reduces the probability that the subject will develop fever, difficulty breathing, loss of smell, and / or loss of taste. The method according to claim 181.

189. The method according to claim 170, wherein the subject is suffering from a disease or is in an immunocompromised state.

190. The method according to claim 189, wherein the subject has liver disease, kidney disease, diabetes, hypertension, heart disease, lung disease, cancer, or is HIV positive.

191. The method according to claim 170, wherein the subject described above has not received immunosuppressant treatment for more than 14 days in the past six months.

192. The method according to claim 170, wherein the subject described above satisfies (i) and / or (ii) below: (i) Not having received a live vaccine for 28 days or more prior to the above administration; (ii) The person has not received an inactivated vaccine for 14 days or more prior to the above administration.

193. A method for promoting an immune response in a subject, The above method includes at least the step of administering the first composition to a subject, The above-mentioned first composition comprises one or more of the following (i) to (v): (i) nucleic acids (preferably mRNA) according to any one of claims 1 to 73; (ii) The composition according to any one of claims 74 to 162; (iii) The polypeptide according to claim 163; (iv) The vaccine according to any one of claims 164 to 166; (v) A kit or kit of parts according to claim 167.

194. The method according to claim 193, wherein the subject has a history of being infected with SARS-CoV-2.

195. The method according to claim 193, wherein the subject has previously been treated with at least one SARS-CoV-2 vaccine composition.

196. The method according to claim 195, wherein the first SARS-CoV-2 vaccine composition described above was an mRNA vaccine.

197. The method according to claim 196, wherein the first SARS-CoV-2 vaccine composition was BNT162 or mRNA-1273.

198. The method according to claim 195, wherein the first SARS-CoV-2 vaccine composition described above was a protein subunit vaccine.

199. The method according to claim 198, wherein the first SARS-CoV-2 vaccine composition was NVX-CoV2373 or COVAX.

200. The method according to claim 195, wherein the first SARS-CoV-2 vaccine composition described above was an adenovirus vector vaccine.

201. The method according to claim 200, wherein the first SARS-CoV-2 vaccine composition was ADZ1222 or Ad26.CoV-2.S.

202. The method according to any one of claims 193 to 201, wherein antibodies that bind to SARS-CoV-2 can be detected from the above-mentioned subject.

203. The method according to claim 202, wherein an antibody that binds to the SARS-CoV-2 S protein can be detected in the above-mentioned subject.

204. The method according to claim 202, wherein an antibody that binds to the N protein of SARS-CoV-2 can be detected from the above-mentioned subject.

205. The method according to any one of claims 195-201, wherein the patient was administered the above-mentioned first SARS-CoV-2 vaccine composition approximately three months or more ago, approximately six months or more ago, approximately nine months or more ago, approximately one year or more ago, approximately 1.5 years or more ago, approximately two years or more ago, or three years or more ago.

206. The method according to any one of claims 195 to 201, wherein the patient was administered the above-mentioned first SARS-CoV-2 vaccine composition approximately three months to two years ago or approximately six months to two years ago.

207. The method according to any one of claims 193 to 206, wherein 80% or more, 85% or more, 90% or more, or 95% or more of the treated subjects are prevented from developing moderate and severe COVID-19 disease.

208. By the above method, 80% or more, 85% or more, 90% or more, or 95% or more of the treated subjects were prevented from developing moderate and severe COVID-19 disease. That period lasts from approximately two weeks to approximately one year after the above administration. The method according to claim 207.

209. By the above method, 80% or more, 85% or more, 90% or more, or 95% or more of the treated subjects were prevented from developing moderate and severe COVID-19 disease. The period of administration may range from approximately two weeks to three months, six months, nine months, one year, 1.5 years, two years, or three years from the date of administration. The method according to claim 207.

210. The method according to any one of claims 193 to 209, wherein 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the subjects treated by the above method satisfy (i) and / or (ii) below: (i) The above subjects are prevented from becoming infected with SARS-CoV-2; (ii) Transmission of SARS-CoV-2 from the above-mentioned subjects is prevented.

211. The method according to claim 210, wherein 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the treated subjects satisfy (i) and / or (ii) below: (i) For approximately two weeks to one year following the administration of the above-mentioned drug, the subject will be protected from infection with SARS-CoV-2; (ii) Transmission of SARS-CoV-2 from the subject is prevented for approximately two weeks to one year following the administration of the above-mentioned substance.

212. The method according to claim 211, wherein 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the subjects treated by the above method satisfy the following (i) and / or (ii): (i) The subject is prevented from being infected with SARS-CoV-2, for a period of approximately 2 weeks to 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, or 3 years from the date of administration; (ii) Transmission of SARS-CoV-2 from the above subjects is prevented, for a period of approximately 2 weeks to approximately 3 months, approximately 6 months, approximately 9 months, approximately 1 year, approximately 1.5 years, approximately 2 years, or approximately 3 years from the time of administration.

213. The process further includes administering at least the second composition to the subject, The method according to any one of claims 193 to 212, wherein the second composition described above comprises one or more of the following (i) to (v): (i) nucleic acids (preferably mRNA) according to any one of claims 1 to 61; (ii) The composition according to any one of claims 74 to 128; (iii) The polypeptide according to claim 163; (iv) The vaccine according to any one of claims 164 to 166; (v) A kit or kit of parts according to claim 167.

214. The second composition is administered after the first composition. That period is approximately 7 days or more later. The method according to claim 213.

215. The second composition is administered after the first composition. The period is approximately 10 days or more, approximately 14 days or more, approximately 21 days or more, approximately 28 days or more, approximately 35 days or more, approximately 42 days or more, approximately 49 days or more, or approximately 56 days or more. The method according to claim 214.

216. The second composition is administered after the first composition. That period is approximately 7 days to 56 days later. The method according to claim 213.

217. The second composition is administered after the first composition. The timeframe is approximately 14 days to 56 days later, approximately 21 days to 56 days later, or approximately 28 days to 56 days later. The method according to claim 216.

218. The process further includes administering at least the third composition to the subject, The method according to any one of claims 193 to 212, wherein the third composition described above comprises one or more of the following (i) to (v): (i) The nucleic acid according to any one of claims 1 to 61; (ii) The composition according to any one of claims 74 to 162; (iii) The polypeptide according to claim 163; (iv) The vaccine according to any one of claims 164 to 166; (v) A kit or kit of parts according to claim 167.

219. The method according to any one of claims 213 to 218, wherein 80% or more, 85% or more, 90% or more, or 95% or more of the treated subjects are prevented from developing moderate and severe COVID-19 disease.

220. By the above method, more than 80%, more than 85%, more than 90%, or more than 95% of the treated subjects were prevented from developing moderate and severe COVID-19 disease. The period is approximately 2 weeks to 1 year from the administration of the second composition or any subsequent compositions. The method according to claim 219.

221. By the above method, 80% or more, 85% or more, 90% or more, or 95% or more of the treated subjects were prevented from developing moderate and severe COVID-19 disease. The period is approximately 2 weeks to 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, or 3 years from the administration of the second composition or any subsequent composition. The method according to claim 219.

222. The method according to any one of claims 193 to 221, further defined as a method for promoting an antibody, CD4+ T cell response, or CD8+ T cell response in the above-mentioned subject.

223. The method according to any one of claims 193 to 221, further defined as a method for promoting a neutralizing antibody reaction in the above-mentioned subject.

224. The above method accelerates the antibody reaction. The production of antibodies that bind to the coronavirus spike protein in the above-mentioned subject due to the above antibody reaction is approximately 10 to 500 for every 1 coronavirus neutralizing antibody. The method according to any one of claims 193 to 221.

225. The above method accelerates the antibody reaction. The production of antibodies that bind to the spike protein through the above antibody reaction is approximately 200 or less for every 1 coronavirus neutralizing antibody. The method according to claim 224.

226. The above method accelerates the antibody reaction. The production of antibodies that bind to the coronavirus spike protein through the above antibody reaction is approximately 10 to 300, 20 to 300, 20 to 200, or 30 to 100 for every 1 unit of coronavirus neutralizing antibody. The method according to claim 224.

227. The above method accelerates the antibody reaction. The production of antibodies that bind to the coronavirus spike protein through the above antibody reaction is approximately 30 to 80 times the amount of coronavirus neutralizing antibody. The method according to claim 226.

228. The above method accelerates the antibody reaction. The production of antibodies that bind to the receptor-binding domain (RBD) of the coronavirus spike protein in the above-mentioned subject due to the above-mentioned antibody reaction is approximately 1 to 500 for every 1 coronavirus neutralizing antibody. The method according to claim 223.

229. The above method accelerates the antibody reaction. The production of antibodies that bind to the RBD of the spike protein through the above antibody reaction is approximately 50 or less in proportion to 1 coronavirus neutralizing antibody. The method according to claim 228.

230. The above method accelerates the antibody reaction. The production of antibodies that bind to the RBD of the spike protein through the above antibody reaction is approximately 1 to 200, 2 to 100, 3 to 200, 5 to 100, or 5 to 50 for every 1 unit of coronavirus neutralizing antibody. The method according to claim 228.

231. The above method accelerates the antibody reaction. The production of antibodies that bind to the RBD of the coronavirus spike protein through the above antibody reaction is approximately 5 to 20 times the amount of coronavirus neutralizing antibody. The method according to claim 230.

232. The method according to claim 222, wherein the subject has a history of being infected with SARS-CoV-2.

233. The method according to claim 222, further defined as a method for promoting a protective immune response in the above-mentioned subject.

234. The method according to any one of claims 193 to 233, wherein the subject is a human subject.

235. The method according to claim 234, wherein the age of the subject is 6 months to 100 years, 6 months to 80 years, 1 year to 80 years, 1 year to 70 years, 2 years to 80 years, or 2 years to 60 years.

236. The above subjects are newborns or infants. The ages of the subjects mentioned above are 3 years or younger, 2 years or younger, 1.5 years or younger, 1 year or younger (12 months or younger), 9 months or younger, 6 months or younger, or 3 months or younger, or 6 months to 2 years. The method according to claim 234.

237. The above subject was an elderly subject, The method according to claim 234, wherein the age of the subject is 50 years or older, 60 years or older, 65 years or older, or 70 years or older.

238. The above subject was an elderly subject, The above subjects are 60 years of age or older. The method according to claim 237.

239. The method according to any one of claims 234 to 238, wherein the subject has a Native American, African, Asian, or European genetic background.

240. The above subjects have a genetic background of Native American, African, Asian, or European descent. The genetic background is approximately 10% or more, approximately 25% or more, or approximately 50% or more. The method according to claim 238.

241. The method according to claim 238, wherein the subject has a Native American genetic background.

242. The above subjects have a Native American genetic background. The genetic background is approximately 10% or more, approximately 25% or more, or approximately 50% or more. The method according to claim 238.

243. The above method does not substantially induce an increase in Th2 cytokines in the above subjects. Preferably, the Th2 cytokine is IL-4, IL-13, TNF and / or IL-1β. The method according to any one of claims 193 to 242.

244. The method according to any one of claims 193 to 242, further defined as a method for inducing a Th1-directed immune response in the above-mentioned subject.

245. The method according to any one of claims 193 to 244, wherein the subject described above is receiving anticoagulation therapy.

246. The method according to any one of claims 193 to 245, wherein the above composition is administered by intramuscular injection.

247. The above composition contains mRNA encoding the coronavirus spike protein (S), The spike protein (S) described above is a spike protein (S_stab) that is stable in a pre-fusion structure and possesses one or more pre-fusion structural stabilization mutations. The method according to any one of claims 193 to 246.

248. The above mRNA encodes the coronavirus spike protein, which is more than 95% identical to sequence number 163. The method according to claim 247.

249. The method according to claim 248, wherein the above mRNA encodes the coronavirus spike protein which is identical to sequence number 163.

250. The method according to claim 247, wherein the mRNA above codes for a coronavirus spike protein that is 95% or more identical to sequence number 149.

251. The method according to claim 248, wherein the mRNA described above encodes the coronavirus spike protein which is identical to sequence number 149.

252. A single dose of the above composition provides a sufficient immune response to protect the subject from severe COVID-19 disease. That period lasted for more than six months. The method according to claim 250 or 251.

253. A single dose of the above composition provides a sufficient immune response to protect the subject from severe COVID-19 disease. The duration can range from approximately 6 months to approximately 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, or 5 years. The method according to claim 252.

254. The method according to any one of claims 247 to 249, wherein the mRNA is complexed with one or more lipids, thereby forming an LNP.

255. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids; (ii) one or more neutral lipids; (iii) One or more steroids or steroid analogs; (iv) One or more PEG lipids; The method according to claim 254, wherein the molar ratio of (i) to (iv) above is approximately 20-60% cationic lipids, 5-25% neutral lipids, 25-55% sterols, and 0.5-15% PEG lipids.

256. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The method according to claim 255, wherein the molar ratio of (i) to (iv) above is approximately 20 to 60% cationic lipids, 5 to 25% neutral lipids, 25 to 55% sterols, and 0.5 to 15% PEG lipids.

257. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratio of (i) to (iv) above is approximately 47.5:10:40.8:1.

7. The method according to claim 256.

258. The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratios of (i) to (iv) above are 47.4:10:40.9:1.

7. The method according to claim 256.

259. The method according to any one of claims 254 to 258, wherein the ratio of mRNA to total lipids is approximately 0.03 to 0.04 (w / w).

260. The above mRNA is complexed with one or more lipids, thereby forming lipid nanoparticles (LNPs). The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratios of (i) to (iv) above are approximately 47.5:10:40.8:1.

7. The ratio of mRNA to total lipids is approximately 0.03 to 0.04 (w / w). The method according to claim 249.

261. The above mRNA is complexed with one or more lipids, thereby forming lipid nanoparticles (LNPs). The above LNP includes (i) to (iv) below: (i) One or more cationic lipids relating to formula III-3; (ii) DSPC (iii) Cholesterol; (iv) PEG lipids related to formula IVa; The molar ratios of (i) to (iv) above are 47.4:10:40.9:1.

7. The ratio of mRNA to total lipids is approximately 0.03 to 0.04 (w / w). The method according to claim 249.

262. The method according to any one of claims 247 to 261, wherein the composition administered to the subject contains about 2 μg to about 50 μg of mRNA.

263. The method according to claim 262, wherein the composition administered to the subject contains about 10 μg to about 50 μg of mRNA.

264. The method according to claim 263, wherein the composition administered to the subject contains about 10 μg to about 30 μg of mRNA.

265. The method according to claim 264, wherein the composition administered to the subject contains approximately 12 μg of mRNA.

266. The method according to claim 264 or 265, wherein seroconversion occurs in 100% of subjects administered the composition as a result of the above administration.

267. The method according to any one of claims 193 to 266, wherein the human subject is 61 years of age or older.

268. The method according to any one of claims 193 to 266, wherein the human subject is 18 to 60 years old.

269. The method according to any one of claims 193 to 268, wherein the human subject described above has a history of vaccine allergies.

270. The method according to any one of claims 193 to 269, wherein an anti-PEG antibody can be detected from the above-mentioned subject.

271. The method according to any one of claims 193 to 270, including (i) to (iii) below: (i) A step of obtaining a composition according to any one of claims 74 to 162, wherein the composition is a freeze-dried composition; (ii) A step of preparing a liquid composition by solubilizing the above freeze-dried composition in a pharmaceutically acceptable liquid carrier; (iii) A step of administering an effective amount of the liquid composition to the subject.

272. A method for stabilizing the composition according to any one of claims 74 to 162, The process includes a step of freeze-drying the above composition to prepare a stabilized composition. method.

273. The method according to claim 272, wherein the water content of the stabilizing composition is less than about 10%.

274. The method according to claim 273, wherein the water content of the stabilizing composition is about 0.5% to 5.0%.

275. A stabilized freeze-dried composition prepared by the method described in any one of claims 272 to 274.

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