Nucleic acid-based combination vaccine

Abstract

The present invention particularly relates to pharmaceutical compositions comprising at least one nucleic acid encoding at least one antigenic peptide or protein of a coronavirus, preferably a pandemic coronavirus, and at least one nucleic acid encoding at least one antigenic peptide or protein of an additional virus, such as influenza virus or RSV virus. The pharmaceutical compositions provided herein are suitable for use in the treatment or prevention of at least one coronavirus infection and at least one additional viral infection and may therefore be included in a combination vaccine. The nucleic acid sequences of the pharmaceutical compositions and combination vaccines are preferably associated with a polymeric carrier, a polycationic protein or peptide, or a lipid nanoparticle (LNP). The present invention also relates to primary, secondary, and additional medical uses of the pharmaceutical compositions and combination vaccines, as well as methods for treating or preventing coronavirus infection and additional viral infection.

Description

[Technical Field]

[0001] Introduction: The present invention relates in particular to a pharmaceutical composition comprising at least one nucleic acid encoding at least one antigenic peptide or protein of a coronavirus, preferably a pandemic coronavirus, and at least one nucleic acid encoding at least one antigenic peptide or protein of a further virus, such as influenza virus or RSV virus. The pharmaceutical compositions provided herein are suitable for use in the treatment or prevention of infections caused by at least one coronavirus and at least one further viral infection, and may therefore be included in a mixed vaccine. The nucleic acid sequences of the pharmaceutical compositions and mixed vaccines are preferably associated with a polymer carrier, a polycationic protein or peptide, or lipid nanoparticles (LNPs). The present invention also relates to first, second, and further medical uses of the pharmaceutical compositions and mixed vaccines, and to methods for treating or preventing coronavirus infections and further viral infections.

[0002] Coronaviruses are highly infectious, enveloped, positive-sounding single-stranded RNA viruses belonging to the Coronaviridae family. Coronaviruses (CoVs) are genetically very diverse, and individual virus species can infect multiple host species by crossing species barriers. Such transmission has led to human infections by SARS-related coronaviruses (SARS-CoV-1), Middle East Respiratory Syndrome coronaviruses (MERS-CoV), and SARS-CoV-2 (which causes COVID-19).

[0003] SARS-CoV-1, which causes Severe Acute Respiratory Syndrome (SARS), infected 8,422 people and killed 916 in 37 countries between 2002 and 2003. MERS-CoV was first identified in the Middle East in 2012. As of July 2016, there were reported to be 1,791 confirmed cases of MERS-CoV infection in 27 countries, including at least 640 deaths.

[0004] The coronavirus pandemic, which (presumably) began in Wuhan, China, at the turn of 2019 / 2020, is caused by an unknown coronavirus (SARS-CoV-2) that causes severe respiratory illness (COVID-19). By the end of May 2020, the number of confirmed cases of SARS-CoV-2 infection had reached approximately 6,000,000, spreading to almost every country in the world, and COVID-19-related deaths had exceeded 350,000.

[0005] These current coronavirus outbreaks represent a significant risk of a serious global pandemic caused by coronaviruses. Providing a vaccine that offers protection against (pandemic) coronaviruses is fundamentally important for global health. To prevent a recurrence of such coronavirus outbreaks, it would also be effective to provide a combination vaccine (or multi-pathogen vaccine) that protects against coronavirus infection and further infections caused by at least one additional viral infection, preferably a virus that also causes respiratory illness. Furthermore, since older populations are more susceptible to the effects of such viruses, it would be effective to provide a combination vaccine that is effective for older populations.

[0006] A suitable example of such a virus is the influenza virus. The influenza virus is an enveloped RNA virus. Three genera of this family, influenza viruses A, B, and C, cause influenza in humans. These differ in host range, surface glycoprotein variability, genomic composition, and morphology. Influenza viruses A and B are further classified based on the viral surface proteins hemagglutinin (HA) and neuraminidase (NA). Typically, two to three influenza strains circulate simultaneously during influenza epidemics. A typical influenza epidemic leads to an increased incidence of pneumonia and lower respiratory tract disease, resulting in increased hospitalization or mortality rates. The elderly or those with chronic underlying diseases are at very high risk of developing these complications, but young infants can also develop serious illnesses. The number of influenza cases worldwide exceeds one billion each year, resulting in an estimated 3 to 5 million severe cases and 300,000 to 500,000 deaths. Furthermore, for people who overcome mild illness symptoms in just a few days or weeks, there is a significant loss in both productivity and quality of life.

[0007] In addition to coronaviruses and influenza viruses, viruses of the Pneumoviridae family also cause severe respiratory illnesses. Examples of members of the Pneumoviridae family include respiratory syncytial virus (RSV) and metapneumovirus (hMPV). RSV is the most common cause of bronchiolitis and pneumonia in infants under one year of age. RSV also causes recurrent infections, including severe lower respiratory tract illnesses, which can occur at any age, particularly among the elderly or those with heart, lung, or immune system defects. hMPV is a significant cause of viral lower respiratory tract illnesses in infants. The seasonal epidemiology of hMPV appears similar to that of RSV, but the incidence of infection and illness seems much lower. However, hMPV can occur and be severe in the elderly, with almost the same frequency as influenza. hMPV is associated with more serious illnesses in adults with asthma and chronic obstructive pulmonary disease (COPD). Many hMPV outbreaks have been reported in long-term care facilities for children and adults, resulting in fatalities.

[0008] In addition to coronaviruses, influenza viruses, and pneumoviruses, viruses of the Paramyxoviridae family can cause severe respiratory illness. Members of the Paramyxoviridae family include parainfluenza virus (PIV) and henipavirus.

[0009] Parainfluenza viruses, such as PIV3 (PIV3), are the leading cause of widespread acute respiratory infections in infants and young children. Their incidence peaks between 4 and 12 months of age, accounting for 3-10% of hospitalizations, primarily due to bronchiolitis and pneumonia. PIV3 can be fatal and, in some cases, associated with neurological disorders such as febrile seizures. It can also lead to airway remodeling, a significant cause of morbidity. In developing regions worldwide, infants and young children are at the highest risk of death from primary PIV3 infection or secondary effects such as bacterial infections.

[0010] Henipaviruses, such as Hendra virus and Nipah virus, are emerging potential pandemic disease sources in recent years. Hendra virus was first identified in 1994 in Hendra, Queensland, Australia, following an outbreak of respiratory illness among 20 horses and 2 humans. In 1995, a second unrelated outbreak was confirmed in Mackay, Queensland, which occurred in August 1994, resulting in the deaths of 2 horses and 1 human. The fatality rate has been reported to be over 70% in horses and over 50% in humans. Nipah virus was first isolated in 1999 following an investigation of samples from outbreaks of encephalitis and respiratory illness among adult males in Malaysia and Singapore. The host of Nipah virus is still unknown, but fruit bats are thought to be its natural host. Human infection with Nipah virus is associated with encephalitis characterized by fever and drowsiness, as well as more serious central nervous system disorders such as coma, seizures, and respiratory failure. Some patients develop respiratory illnesses in the early stages of infection. During the 1998-1999 Nipah virus disease outbreak, approximately 40% of hospitalized patients with severe neurological complications died.

[0011] As outlined above, the object of the present invention is to provide a nucleic acid-based mixed vaccine that provides effective protection against at least one coronavirus, in particular at least one pandemic coronavirus, and further, at least one additional virus selected from, for example, at least one influenza virus, at least one Pneumoviridae virus, and / or at least one Paramyxoviridae virus.

[0012] Developing a combination vaccine is a major challenge because the various components of such vaccines are often incompatible with each other. Furthermore, different vaccine components may result in different effective vaccination intervals, which hinders the development of combination vaccines. In addition, it is important that all components of the combination vaccine induce an efficient immune response and that no single component exhibits immune dominance, or that the components do not show immune interference. Vaccines are particularly needed to protect the elderly population, which has a high mortality rate in the case of SARS-CoV-2.

[0013] Nucleic acid-based vaccination, including DNA or RNA, is a promising technology for providing novel and combination vaccines against emerging viruses. Nucleic acids can be genetically engineered and administered to human subjects. Transfected cells directly produce the encoded antigen (e.g., provided by DNA or RNA, particularly mRNA), resulting in a protective immune response.

[0014] The crucial role of virus-specific memory T cells in broad and long-term protection against SARS-CoV infection has been elucidated (see, for example, Non-Patent Document 1). Virus-specific CD8 T cells are necessary, for example, for pathogen clearance and mediating defense after viral attack. Effective combination vaccines, such as the SARS-CoV-2 vaccine, not only induce a robust functional humoral immune response against each pathogen, but also induce pathogen-specific CD8+ T cell and CD4+ T cell responses, such as SARS-CoV-2-specific CD8+ T cell and CD4+ T cell responses.

[0015] Therefore, the objective of the underlying invention is to provide a nucleic acid-based mixed vaccine or multi-pathogen vaccine that provides protection against coronavirus infection and protection against at least one further virus. [Prior art documents] [Non-patent literature]

[0016] [Non-Patent Document 1] 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

[0017] definition For clarity and ease of reading, the following definitions are given. The technical features referred to in these definitions can be read in all embodiments of the present invention. Additional definitions and explanations can be specifically provided in the context of these embodiments.

[0018] percentage In the context of numerical values, percentages are understood to be relative to the total number of each item. In other cases, unless otherwise stated in the context, percentages are understood to be weight percentages (wt.-%).

[0019] aboutThe term "approximately" is used when the determinants or values ​​do not need to be identical, i.e., 100% the same. Therefore, "approximately" means that the determinants or values ​​may differ by 0.1% to 20%, preferably 0.1% to 10%; in particular by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Those skilled in the art will recognize, for example, that a certain parameter or determinant may differ slightly depending on the method by which the parameter was determined. For example, if a determinant or value is defined herein as having a length of, for example, "about 1000 nucleotides," then its length may vary by 0.1% to 20%, preferably 0.1% to 10%, particularly by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Therefore, those skilled in the art will recognize that in certain examples, its length may vary by 1 to 200 nucleotides, preferably 1 to 200 nucleotides, particularly by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 nucleotides.

[0020] Adaptive immune response As used herein, the term “adaptive immune response” is intended to be recognized and understood by those skilled in the art, and to mean, for example, an antigen-specific response of the immune system (adaptive immune system). Antigen specificity allows for the generation of a response tailored to a particular pathogen or pathogen-infected cells. The ability to initiate these tailored responses is typically maintained in the body by “memory cells” (B cells). In the context of the present invention, the antigen is provided by a nucleic acid (e.g., RNA or DNA) encoding at least one antigenic peptide or protein derived from a coronavirus (component A) or a further virus (component B).

[0021] antigenAs used herein, the term “antigen” is intended to mean a substance that is recognized and understood by those skilled in the art, for example, by the immune system, preferably the adaptive immune system, and that can evoke an antigen-specific immune response, for example, by the production of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be, or may comprise, a peptide or protein that can be presented to T cells by MHC. Also, fragments, variants, and derivatives of peptides or proteins containing at least one epitope are understood as antigens in the context of the present invention. In the context of the present invention, an antigen may be a translation product of a provided nucleic acid, as identified herein.

[0022] Antigenic peptide or protein The terms “antigenic peptide or protein” or “immunogenic peptide or protein” are intended to mean peptides and proteins derived from proteins that stimulate the body’s adaptive immune system and produce an adaptive immune response, as recognized and understood by those skilled in the art. Therefore, an antigenic / immunogenic peptide or protein comprises at least one epitope (as defined herein) or antigen (as defined herein) of the protein from which it is derived (e.g., coronavirus (preferably SARS-CoV-2) spike protein (S), influenza virus HA, RSV virus F protein, etc.).

[0023] Cationic properties Unless it is clear from a specific context that it has a different meaning, the term "cationic" means that each structure has a positive charge, either permanently or in response to certain conditions such as pH. Therefore, the term "cationic" encompasses both "permanent cationicity" and "cationizable."

[0024] Cationic conversion possibleAs used herein, the term "cationicizable" means that a compound, group, or atom is positively charged at lower pH levels and uncharged at higher pH levels in its environment. Furthermore, in non-aqueous environments where the pH value cannot be determined, cationicizable compounds, groups, or atoms are positively charged at high hydrogen ion concentrations and uncharged at low hydrogen ion concentrations or activity. This depends on the individual properties of the cationicizable or polycationizable compound, particularly the pKa of each cationicizable group or atom, which determines whether it is charged or uncharged at its pH or hydrogen ion concentration. In a diluted aqueous environment, the proportion of positively charged cationicizable compounds, groups, or atoms can be estimated using the so-called Henderson-Hasselbalch formula, well known to those skilled in the art. For example, in some embodiments, if the compound or part is cationizable, it is preferable to be positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or even more preferably 6 to 8, more preferably 9 or less, 8 or less, or 7 or less, most preferably at a physiological pH, for example, about 7.3 to 7.4 (i.e., under physiological conditions), and particularly under physiological salt conditions of cells in vivo. In other embodiments, it is preferable that the cationizable compound or part is mainly neutral at a physiological pH, for example, about 7.0 to 7.4, but positively charged at lower pH values. In some embodiments, the preferred range of pKa of the cationizable compound or part is about 5 to about 7.

[0025] Code layout / code area As used herein, the terms “code sequence” or “code region” and the corresponding abbreviation “cds” are intended to mean a sequence of several nucleotide triplets that are recognized and understood by those skilled in the art and can be translated into peptides or proteins. In the context of the present invention, a code sequence may be a DNA sequence, preferably an RNA sequence, consisting of a number of nucleotides that are divisible by 3, beginning with a start codon and preferably ending with a stop codon.

[0026] originating fromAs used throughout this specification in the context of nucleic acids, the term “derived” means that a nucleic acid “derived” from another nucleic acid means that the nucleic acid is derived from another nucleic acid and shares, for example, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived. Those skilled in the art will recognize that sequence identity is typically calculated for nucleic acids of the same type, i.e., DNA sequences or RNA sequences. Therefore, when DNA “derived” from RNA or RNA “derived” from DNA, it is understood that in the first step, the RNA sequence is converted to the corresponding DNA sequence (particularly by substituting uracil (U) with thymidine (T) throughout the sequence), or conversely, the DNA sequence is converted to the corresponding RNA sequence (particularly by substituting T with U throughout the sequence). Subsequently, the sequence identity of the DNA sequence or the sequence identity of the RNA sequence is determined. Preferably, nucleic acid “derived” from nucleic acid also means a nucleic acid that has been modified compared to the nucleic acid from which it originates in order to further enhance the stability of RNA and / or to extend and / or increase protein production. In the context of amino acid sequences (e.g., antigenic peptides or proteins), the term “derived” means that an amino acid sequence derived from another amino acid sequence shares at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it originates.

[0027] EpitopeAs used herein, the term “epitope” (also referred to in the art as “antigenic determinant”) is intended to be recognized and understood by those skilled in the art, and to mean, for example, T cell epitopes and B cell epitopes. A T cell epitope or portion of an antigenic peptide or protein may include fragments having a length of about 6 to about 20 or more amino acids, for example, fragments processed and presented by an MHC class I molecule, preferably fragments having a length of about 8 to about 10 amino acids, for example, 8, 9, or 10 (and even 11 or 12) amino acids, or fragments processed and presented by an MHC class II molecule, preferably fragments having a length of about 13 to about 20 or more amino acids. These fragments are usually 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 native form. B cell epitopes are typically fragments located on the outer surface of a (native) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably 5 to 12 amino acids, and even more preferably 6 to 9 amino acids, and these can be recognized by antibodies, i.e., in their native form. Such epitopes of a protein or peptide may further be selected from any of the variants of such protein or peptide referred to herein. In this context, an epitope can be a conformation or discontinuous epitope consisting of segments of a protein or peptide as defined herein that are discontinuous in the amino acid sequence of the protein or peptide as defined herein but come together in a three-dimensional structure, or a continuous or linear epitope consisting of a single polypeptide chain.

[0028] pieceIn the context of nucleic acid sequences (e.g., RNA or DNA) or amino acid sequences, the term “fragment” as used herein is usually a shorter portion of a full-length sequence of, for example, a nucleic acid sequence or amino acid sequence. Therefore, a fragment typically consists of the same sequence as the corresponding stretch within the full-length sequence. Preferred fragments of sequences in the context of the present invention consist of a continuous sequence of entities, such as nucleotides or amino acids, corresponding to a continuous sequence of entities in the molecule from which the fragment originates, and represent at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the entire molecule (i.e., full length) from which the fragment originates (e.g., coronavirus spike protein (S), influenza HA / NA, etc.). In the context of proteins or peptides, the term “fragment” as used herein can typically include sequences of proteins or peptides as defined herein, which are truncated at the N-terminus and / or C-terminus with respect to their amino acid sequence compared to the amino acid sequence of the original protein. Therefore, such truncation can occur at either the amino acid level or the corresponding nucleic acid level. Therefore, sequence identity with respect to a fragment as defined herein may preferably mean the entire protein or peptide as defined herein, or the entire (coding) nucleic acid molecule of such protein or peptide. A protein or peptide fragment may contain at least one epitope of that protein or peptide.

[0029] Different species As used herein in the context of nucleic acid sequences or amino acid sequences, the terms “heterogeneous” or “heterogeneous sequence” are understood to mean a sequence (e.g., RNA, DNA, amino acids) that originates from another gene, another allele, or, for example, another species or virus. Two sequences are generally understood to be “heterogeneous” if they do not originate from the same gene or the same allele. That is, heterogeneous sequences may originate from the same organism or virus, but in practice they do not exist in the same nucleic acid or protein.

[0030] Humoral immune responseThe terms “humoral immunity” or “humoral immune response” are as recognized and understood by those skilled in the art and are intended to mean B cell-mediated antibody production and, optionally, the auxiliary processes associated with antibody production. Humoral immune responses can typically be characterized by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity may also mean the effector functions of antibodies, including the neutralization of pathogens and toxins, classical complement activation, phagocytosis and opsonin enhancement of pathogen elimination.

[0031] (Array) Identity The term “identity” as used herein in the context of nucleic acid sequences or amino acid sequences is intended to mean, for example, the percentage of two sequences that are identical, as will be recognized and understood by those skilled in the art. Two sequences, for example, nucleic acid sequences or amino acid (aa) sequences as defined herein, preferably the aa sequences encoded by the nucleic acid sequences as defined herein, or the aa sequences themselves, can be aligned for later comparison to determine the percentage of identity between them. Thus, for example, the position of a first sequence can be compared to the corresponding position of a second sequence. If there is a residue at the position in the first sequence that is identical to the residue at the position in the second sequence, then the two sequences are identical at that position. Otherwise, the sequences are different at that position. If an insertion occurs in the second sequence compared to the first sequence, a gap can be inserted in the first sequence to allow for further alignment. If a deletion occurs in the second sequence compared to the first sequence, a gap can be inserted in the second sequence to allow for further alignment. The percentage of identical arrays is a function obtained by dividing the number of identical positions by the total number of positions that are present in only one of the arrays. This percentage can be determined using an algorithm, such as the one built into the BLAST program.

[0032] immunogen, immunogenicityThe terms “immunogen” or “immunogenicity” are intended to be recognized and understood by those skilled in the art and to mean, for example, a compound that can 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 provided nucleic acid (components A and B) comprising at least one coding sequence encoding at least one antigenic peptide, for example, a protein derived from coronavirus spike protein (S) as defined herein (preferably from SARS-CoV-2). Typically, an immunogen induces an adaptive immune response.

[0033] Immune response The term "immune response" is as recognized and understood by those skilled in the art and is intended to mean, for example, a specific response of the adaptive immune system to a particular antigen (so-called specific or adaptive immune response) or a nonspecific response of the innate immune system (so-called nonspecific or innate immune response), or a combination thereof.

[0034] immune system The term “immune system” is intended to be recognized and understood by those skilled in the art and to mean, for example, the system of an organism capable of defending itself from infection. When a pathogen enters an organism through its physical barriers, the innate immune system immediately exhibits a nonspecific response. Even if the pathogen evades this innate response, vertebrates have an adaptive immune system, which is a second layer of defense. Here, the immune system adapts its response during infection to improve recognition of the pathogen. This improved response is then retained in the form of immunological memory even after the pathogen has been eliminated, allowing the adaptive immune system to launch a faster and more powerful attack each time the pathogen is encountered. According to this, the immune system includes the innate immune system and the adaptive immune system. Each of these two parts typically includes so-called humoral and cellular components.

[0035] innate immune systemThe term “innate immune system” (also known as non-specific or unspecific immune system) is intended to be recognized and understood by those skilled in the art and to mean, for example, a system that typically includes cells and mechanisms that defend a host from infection by other organisms in a non-specific manner. This means that cells of the innate system can recognize and respond to pathogens in a general manner, but unlike the adaptive immune system, they do not confer long-term immunity or protective immunity to the host. The innate immune system can be activated by ligands such as pattern recognition receptors, e.g., Toll-like receptors, NOD-like receptors, or RIG-I-like receptors.

[0036] Lipidoid compounds Lipidoid compounds, also simply called lipidoids, are lipid-like compounds, that is, amphiphilic compounds that possess lipid-like physical properties. In the context of this invention, the term lipid is considered to encompass lipidoid compounds.

[0037] nucleic acid, nucleic acid molecule In particular, in the context of components A and B, the terms “nucleic acid” or “nucleic acid molecule” as used herein will be recognized and understood by those skilled in the art. The terms “nucleic acid” or “nucleic acid molecule” preferably mean DNA(molecule) or RNA(molecule). This term is used synonymously with the term “polynucleotide.” Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently bonded to each other by phosphodiester bonds of a sugar / phosphate backbone. The terms “nucleic acid” or “nucleic acid molecule” also encompass modified nucleic acids(molecules), such as base-modified, sugar-modified, or backbone-modified DNA or RNA(molecules) as defined herein. Therefore, the nucleic acids of components A and B may be DNA or RNA.

[0038] nucleic acid sequence, DNA sequence, RNA sequence The terms “nucleic acid sequence,” “DNA sequence,” and “RNA sequence” are recognized and understood by those skilled in the art, and mean, for example, the specific and individual sequence of the nucleotide sequence.

[0039] Polyvalent vaccine or combination vaccineBoth terms are understood to be interconvertible herein. The multivalent or mixed vaccine of the present invention provides a multivalent (e.g., antigen) derived from multiple viruses (e.g., at least one coronavirus as defined herein and at least one further virus as defined herein). Nucleic acid-based mixed vaccines can also be thought of as “multipathogen vaccines”.

[0040] Persistent cationicity As used herein, the term “persistent cationicity” is recognized and understood by those skilled in the art and means, for example, that each compound, or group, or atom is positively charged at any pH value or the hydrogen ion activity of its environment. Typically, the positive charge is due to the presence of a quaternary nitrogen atom. When a compound has multiple such positive charges, it may be referred to as persistent polycationicity.

[0041] Stabilized RNA The term "stabilized RNA" refers to RNA that has been modified to be more stable than unmodified RNA against disintegration or degradation by environmental factors or enzymatic digestion such as exo- or endonuclease degradation. Preferably, in the context of the present invention, stabilized RNA is stabilized in cells such as prokaryotic or eukaryotic cells, preferably mammalian cells such as human cells. The stabilizing effect can also be exerted outside the cell, for example, in a storage buffer solution of a composition containing stabilized RNA.

[0042] T cell responseAs used herein, the terms “cellular immunity,” “cellular immune response,” or “cellular T cell response” are intended to be recognized and understood by those skilled in the art and to mean, 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. More generally, cellular immunity is based on the activation of cells in the immune system, rather than antibodies. Typically, a cellular immune response is characterized by the activation of antigen-specific cytotoxic T lymphocytes that can induce apoptosis in certain immune cells, such as dendritic cells or other cells, and present an epitope of a foreign antigen on their surface.

[0043] (Sequence) variants In the context of nucleic acid sequences, the term “variant” as used herein is intended to mean, for example, a variant of a nucleic acid sequence derived from another nucleic acid sequence, as recognized and understood by those skilled in the art. For example, a variant of a nucleic acid sequence may exhibit 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 at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence from which the variant is derived. The variant is a functional variant in the sense that it retains at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence from which it is derived. A "variant" of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% nucleotide identity across a sequence of at least 10, 20, 30, 50, 75, or 100 nucleotides of such nucleic acid sequence.

[0044] As used herein in the context of proteins or peptides, the term “variant” is intended to mean a protein or peptide variant having an amino acid sequence that differs from the original sequence in terms of one or more mutations / substitutions, such as one or more substitutions, insertions, and / or deletions of amino acids. Preferably, these fragments and / or variants have the same or equivalent specific antigenic properties (immunogenic variants, antigenic variants). Insertions and substitutions are possible, in particular, at sequence locations that do not alter the three-dimensional structure or affect binding regions. Changes to the three-dimensional structure due to insertions or deletions can be readily determined, for example, by using CD spectroscopy (circular dichroism spectroscopy). A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity over a sequence of at least 10, 20, 30, 50, 75, or 100 amino acids of such protein or peptide. Preferably, the protein variants include functional variants of the protein, which in the context of the present invention means that the variant exhibits essentially the same immunogenicity as the protein from which it is derived, or at least 40%, 50%, 60%, 70%, 80%, or 90% of that immunogenicity.

[0045] Brief Description of the Invention As further described in the claims and the underlying description, these objects of the present invention are addressed in particular by providing a pharmaceutical composition comprising or comprising: a nucleic acid (e.g., RNA or DNA) ("Component A") comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from a coronavirus; and a nucleic acid (e.g., RNA or DNA) ("Component B") comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from at least one further virus.

[0046] Preferably, the pharmaceutical composition or mixed vaccine of the present invention has at least some of the following advantageous features. • Translation of nucleic acids of component A and component B at the injection / vaccination site (e.g., muscle); Induction of an antigen-specific immune response to all encoded proteins (provided by components A and B), preferably at very low doses and administration regimens; • Suitability for vaccination of infants and / or neonates or the elderly, especially the elderly; • Suitability of compositions / vaccines for intramuscular administration; • Induction of a specific and functional humoral immune response against coronavirus, preferably SARS-CoV-2 (component A), and at least one further virus (component B); • Induction of a broad and functional cellular T cell response to coronavirus, preferably SARS-CoV-2 (component A), and at least one additional virus (component B); Induction of specific B-cell memory for coronavirus, preferably SARS-CoV-2 (component A), and at least one further virus (component B); • Induction of functional antibodies capable of effectively neutralizing coronaviruses such as SARS-CoV-2, and at least one additional virus; • Induction of functional antibodies capable of effectively neutralizing new variants of coronaviruses such as SARS-CoV-2; • Induction of mucosal IgA immunity through the induction of mucosal IgA antibodies; • Induction of a balanced B-cell and T-cell response; • Induction of protective immunity against coronavirus infection, such as SARS-CoV-2 or its new variants; • Rapid initiation of immune defense against coronavirus, preferably SARS-CoV-2 (component A), and at least one additional virus (component B); • The duration of the induced immune response to coronavirus, preferably SARS-CoV-2 (component A), and at least one further virus (component B); • No enhancement of viral infection (e.g., SARS-CoV-2 infection) due to vaccination or immunopathological action; • No antibody-dependent enhancement (ADE) caused by nucleic acid-based combination vaccines; • No excessive induction of systemic cytokine or chemokine responses after vaccine administration, which could lead to undesirable high reactiongenicity at the time of vaccination; • The combination vaccine is well-tolerated, free from side effects, and non-toxic; • Advantageous stability characteristics of nucleic acid-based combination vaccines; • Speed, adaptability, ease of use, and scalability of mixed vaccine production; • A favorable vaccination regimen that provides sufficient protection with only one or two doses; • Advantageous vaccination regimens that provide sufficient protection with only low-dose compositions / vaccines.

[0047] The present invention is based on the inventors' astonishing finding that a pharmaceutical composition comprising at least one nucleic acid encoding at least one peptide or protein derived from a coronavirus ("Component A") and at least one nucleic acid encoding at least one peptide or protein derived from a further virus ("Component B") can be efficiently co-expressed in human cells. Even more astonishingly and unexpectedly, administration of a pharmaceutical composition containing such components induces an antigen-specific immune response to the encoded coronavirus antigen and to at least one further viral antigen. These findings form the basis of the nucleic acid-based mixed vaccine of the present invention, which provides protection against at least one coronavirus (e.g., pandemic coronavirus) and at least one further virus (e.g., influenza virus and / or RSV virus) (see the Examples section).

[0048] In a first aspect, the present invention provides a pharmaceutical composition comprising: at least one component A comprising at least one nucleic acid comprising at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from at least one coronavirus, or an immunogenic fragment or immunogenic variant thereof; and at least one component B comprising at least one nucleic acid comprising at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from at least one further virus, or an immunogenic fragment or immunogenic variant thereof.

[0049] At least one coronavirus of component A may be suitably derived from or selected from pandemic coronaviruses such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV. At least one further virus of component B may be suitably derived from or selected from at least one further virus, such as at least one different coronavirus, at least one influenza virus, at least one Pneumoviridae virus (e.g., respiratory syncytial virus, and / or at least one metapneumovirus), and / or at least one Paramyxoviridae virus (e.g., parainfluenza virus, and / or at least one henipavirus).

[0050] In a second aspect, the present invention provides a mixed vaccine comprising the pharmaceutical composition (including component A and component B) as defined in the first aspect. Therefore, the second aspect relates to a multi-pathogen vaccine.

[0051] In a third aspect, the present invention provides a kit or kit of parts comprising at least one pharmaceutical composition (containing component A and component B) of the first aspect and / or at least one mixed vaccine of the second aspect.

[0052] Further aspects of the present invention relate to methods for treating or preventing multiple viral infections in a subject, as well as first and second medical uses of a pharmaceutical composition, a mixed vaccine, or a kit. Furthermore, methods for producing a pharmaceutical composition or a mixed vaccine are also provided.

[0053] Detailed description of the invention This application is submitted together with the electronic sequence listing, which is part of the specification (WIPO Standard ST.25). The entire information contained in the sequence listing is incorporated herein by reference. Wherever “sequence number” is referred herein, it refers to the corresponding nucleic acid sequence or amino acid (aa) sequence in the sequence listing that has the respective identifier. For many sequences, the sequence listing also provides additional details, such as specific structural features, sequence optimizations, GenBank (NCBI) or GISAID (epi) identifiers, or additional details regarding their coding capabilities. In particular, such information is related to numerical identifiers in the WIPO Standard ST.25 sequence listing. <223> It is given below. Therefore, the numerical identifier <223> The entirety of the information provided below is explicitly included herein and is understood to be an integral part of the specification of the underlying invention. Where “sequence numbers” of other published patent applications or patents are referenced, such sequences as amino acid sequences or nucleic acid sequences are expressly incorporated herein by reference. Thus, these sequences constitute an integral part of the underlying specification. With respect to “sequence numbers” included by reference, the numerical identifiers (of the respective sequence protocols) <223> The information provided below is also explicitly included herein in its entirety and is understood to be an integral part of the specification of the underlying invention. Accordingly, the “Sequence Numbers” listed in WO2014160463, WO2015024668, WO2017070626, WO2017070622, WO2017172890, WO2018081318, WO2018115527, WO2018170245, WO2018078053, WO2018115507, WO2019092153, and WO2019202035 are incorporated herein by reference and are therefore part of the specification.

[0054] Pharmaceutical composition: In a first aspect, the present invention relates to a pharmaceutical composition suitable for a mixed vaccine or a multi-pathogen vaccine.

[0055] The first aspect of the present invention, i.e., the specific features and embodiments described in the context of the pharmaceutical composition of the present invention, are equally applicable to a second aspect (the mixed vaccine of the present invention), a third aspect (the kit or kit of parts of the present invention), or further aspects including medical uses (first and second medical uses) and methods of treatment.

[0056] In a preferred embodiment, the pharmaceutical composition of the first embodiment is - Component A comprising at least one nucleic acid comprising at least one coding sequence that encodes at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one coronavirus, - Component B comprising at least one nucleic acid comprising at least one coding sequence that encodes at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one further virus, It includes or consists of.

[0057] The nucleic acids according to the present invention, for example, DNA or RNA, form the basis of nucleic acid-based pharmaceutical compositions or nucleic acid-based vaccines.

[0058] Such nucleic acid-based pharmaceutical compositions (first aspect) or nucleic acid-based vaccines (second aspect) described herein have advantages over classical vaccine approaches. Generally, protein-based vaccines or live attenuated vaccines are not ideal for use in developing countries due to their high production costs. Furthermore, protein-based vaccines, i.e., live attenuated 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. Moreover, using classical approaches, it remains difficult to provide a combination vaccine that is effective against coronaviruses and further viruses (e.g., issues with the compatibility of individual components).

[0059] In contrast, the nucleic acid-based pharmaceutical compositions and vaccines according to the present invention enable very rapid and cost-effective manufacturing. Therefore, compared to known vaccines, the nucleic acid-based vaccines of the present invention can be manufactured at a significantly lower cost and more quickly, making them particularly advantageous for use in developing countries and in the context of global pandemics.

[0060] One further advantage of the nucleic acid-based pharmaceutical composition or vaccine of components A and B is that each component is more stable to temperature compared to vaccines based on proteins or peptides. Furthermore, the inventors have found that each nucleic acid sequence encoding an antigen selected from or derived from coronavirus can be combined with nucleic acid sequences encoding antigens selected from or derived from further viruses (e.g., different coronaviruses, influenza viruses, RSV viruses, PIV, hMPV, Hendra, Nipah) without inhibiting the efficacy of the individual components (e.g., induction of cellular and humoral immune responses) (see Examples section).

[0061] Ingredient A: In various preferred embodiments, at least one component A of the pharmaceutical composition comprises at least one nucleic acid comprising at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one coronavirus.

[0062] Coronaviruses are classified into the genera alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, and unclassified coronaviruses. Coronaviruses are highly genetically mutable, and individual virus species can cross species barriers to infect multiple host species, potentially causing pandemics.

[0063] In a preferred embodiment, at least one coronavirus of component A is selected from or derived from at least one pandemic coronavirus.

[0064] In various embodiments, the at least one coronavirus of component A, or the at least one pandemic coronavirus of component A, is selected from at least one alpha coronavirus, at least one beta coronavirus, at least one gamma coronavirus, and / or at least one delta coronavirus, preferably pandemic alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus.

[0065] In various embodiments, at least one coronavirus of component A, or at least one pandemic coronavirus of component A, is a betacoronavirus. Preferably, the betacoronavirus is selected from at least one salvecovirus, at least one merubecovirus, at least one envecovirus, at least one novecovirus, and / or at least one hibecovirus.

[0066] In preferred embodiments, at least one coronavirus of component A, or at least one pandemic coronavirus of component A, is a betacoronavirus, preferably a salvecovirus. In the context of the present invention, preferred salvecoviruses may be selected from SARS-related coronaviruses. Preferred SARS-related coronaviruses may be selected from SARS-CoV-1 and / or SARS-CoV-2.

[0067] In a preferred embodiment, at least one coronavirus of component A, or at least one pandemic coronavirus of component A, is a betacoronavirus, preferably a Merbecovirus. In the context of the present invention, a preferred Merbecovirus may be selected from MERS-related coronaviruses. A preferred MERS-related coronavirus may be selected from MERS-CoV.

[0068] The term “coronavirus antigenic peptide or protein” refers to a peptide or protein selected from or derived from each coronavirus as defined herein, but also to fragments, variants, or derivatives thereof, preferably immunogenic fragments or immunogenic variants.

[0069] The terms "immunogenic fragment" or "immunogenic variant" are understood to refer to fragments / variants of the corresponding coronavirus antigen that can enhance the immune response in a target.

[0070] In the context of the present invention, any protein selected from or derived from coronaviruses, preferably pandemic coronaviruses, can be used in the context of the present invention and can be suitably encoded by the coding sequence or nucleic acid of component A. Furthermore, it is within the scope of the underlying invention that at least one antigenic peptide or protein may include or consist of a synthetically engineered or artificial coronavirus peptide or protein. The terms “synthetically engineered” coronavirus peptide or protein, or “artificial coronavirus peptide or protein,” refer to proteins that do not exist in nature. Therefore, “artificial coronavirus peptide or protein” or “synthetically engineered coronavirus peptide or protein” may differ from, for example, naturally occurring coronavirus peptide or protein by at least one amino acid, and / or may contain additional peptide or protein elements (e.g., heterogeneous elements), and / or may have an elongated or truncated N-terminus or C-terminus.

[0071] In a preferred embodiment, the nucleic acid of component A comprises at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from coronavirus, preferably pandemic coronavirus, wherein the at least one antigenic peptide or protein comprises at least one peptide or protein selected from or derived from a structural protein, an accessory protein, or a replicase protein, or any immunogenic fragment or immunogenic variant thereof.

[0072] Preferably, the structural protein is selected from or derived from coronavirus spike protein (S), coronavirus envelope protein (E), coronavirus membrane protein (M), or coronavirus nucleocapsid protein (N), or immunogenic fragments or variants of any of these.

[0073] In a particularly preferred embodiment, the encoded antigenic peptide or protein of component A comprises or consists of at least one peptide or protein selected from or derived from coronavirus spike proteins (S, S1, S2, or S1 and S2), or an immunogenic fragment or immunogenic variant thereof.

[0074] As used herein, the term spike protein (S) refers to the coronavirus protein. The spike protein (S) is a typical type I viral fusion protein present as a trimer on the viral surface, with each monomer consisting of a head (S1) and a stem (S2). Individual precursor S polypeptides form a homotrimer, undergoing glycosylation in the Golgi apparatus, processing that removes the signal peptide, and cleavage by cellular proteases to produce separate S1 and S2 polypeptide chains, which remain associated as S1 / S2 protomers within the homotrimer, thus forming a heterodimeric trimer. The S1 domain of the spike glycoprotein includes a receptor-binding domain (RBD) that (often) binds to the angiotensin-converting enzyme 2 receptor and mediates the fusion of the virus into host cells, an N-terminal domain that can first come into contact with target cells, and two subdomains, all of which are susceptible to neutralizing antibodies. The S2 domain consists of six helical bundle fusion cores involved in membrane fusion with the host endosomal membrane and is also a target of neutralization. The S2 subunit further includes two 7-repeat sequences (HR1 and HR2), a central helix typical of fusion glycoproteins, a transmembrane domain, and a cytoplasmic tail domain.

[0075] In a preferred embodiment, the spike protein (S) fragment is encoded by the nucleic acid of component A, and the fragment may be truncated at the N-terminus, for example, by deleting one to up to 100 N-terminal amino acids of the full-length coronavirus S protein. In some embodiments, the spike protein (S) fragment may be encoded by the nucleic acid of component A, and the fragment may be truncated at the C-terminus, for example, by deleting one to up to 200 C-terminal amino acids of the full-length coronavirus S protein. Such “spike protein (S) fragment” may contain amino acid substitutions and optionally at least one heterologous peptide or protein element (hereinafter described later for specific coronaviruses). In a preferred embodiment, the coronavirus spike protein (S) fragment may be truncated at the C-terminus, thereby deleting the C-terminal transmembrane domain.

[0076] In a preferred embodiment, at least one antigenic peptide or protein encoded by component A comprises or consists of a coronavirus spike protein (S), wherein the spike protein (S) lacks a transmembrane domain (TM or TMflex). While not wishing to be bound by theory, coronavirus spike proteins (S) lacking a transmembrane domain (TM or TMflex) as defined herein may be suitable for vaccines because such proteins are soluble and not immobilized on the cell membrane. Therefore, when administered to a subject or cells, a higher concentration of soluble protein may be produced (translated), potentially leading to an improved immune response.

[0077] While we do not wish to be bound by theory, the RBD and CND domains may be crucial for the immunogenicity of coronavirus spike protein (S). Both regions are located in the S1 fragment of the coronavirus spike protein. Therefore, in the context of the present invention, it may be preferable that the antigenic peptide or protein comprises or consists of the S1 fragment of the coronavirus spike protein, or its immunogenic fragment or immunogenic variant. Preferably, such S1 fragment may contain at least the RBD and / or CND domains as defined above.

[0078] In a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A comprises or consists of a coronavirus spike protein (S), wherein the spike protein (S) comprises or consists of a spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof.

[0079] In a preferred embodiment, the encoded antigenic peptide or protein comprises coronavirus spike protein fragment S1 and is deleting at least 70%, 80%, 90%, preferably 100%, of spike protein fragment S2. Such embodiments may be beneficial because the S1 fragment contains a neutralizing epitope, without the potential problems associated with full-length proteins containing S1 and S2.

[0080] While we do not wish to be bound by theory, it may be preferable that the antigenic peptide or protein of component A includes or consists of coronavirus spike protein fragment S1 and coronavirus spike protein fragment S2 (or at least a fragment thereof), since the formation of immunogenic coronavirus spike protein may be promoted.

[0081] Therefore, in a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A comprises or comprises coronavirus spike protein (S), wherein coronavirus spike protein (S) comprises or comprises coronavirus spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof, and coronavirus spike protein fragment S2, or an immunogenic fragment or immunogenic variant thereof.

[0082] In a particularly preferred embodiment, the encoded antigenic peptide or protein of component A comprises or consists of a full-length coronavirus spike protein, or an immunogenic fragment or immunogenic variant thereof. The term “full-length coronavirus spike protein” is understood to mean a coronavirus spike protein, preferably derived from a pandemic coronavirus, having an amino acid sequence that essentially corresponds to a complete spike protein.

[0083] In a particularly preferred embodiment, the coronavirus spike protein (S) provided by the nucleic acid of component A is designed or adapted to stabilize the S antigen in a pre-fusion conformation. The pre-fusion conformation is particularly advantageous in the context of efficient vaccines because the pre-fusion protein conformation contains several potential and accessible epitopes for neutralizing antibodies. Furthermore, the presence of the S protein in the pre-fusion conformation is intended to avoid immunopathological effects, such as disease exacerbation and / or antibody-dependent enhancement (ADE).

[0084] Therefore, in a preferred embodiment, the nucleic acid of component A comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from coronavirus, preferably pandemic coronavirus, wherein the at least one antigenic peptide or protein is selected from or derived from a spike protein (S), and the spike protein (S) is a pre-fusion stabilization spike protein (S_stab). Preferably, the pre-fusion stabilization spike protein comprises at least one pre-fusion stabilization mutation.

[0085] As used herein, the term “pre-fusion conformation” refers to the structural conformation adopted by the external domain of the coronavirus S protein after it has been processed into the mature coronavirus S protein in the secretory system and before the fusion event that leads to the transition to the post-fusion conformation of coronavirus S.

[0086] The “pre-fusion stabilization spike protein (S_stab)” described herein includes one or more amino acid substitutions, deletions, or insertions to a native coronavirus S sequence that enhance the retention of the pre-fusion conformation relative to the coronavirus S external domain trimer formed from the corresponding native coronavirus S sequence. The pre-fusion conformation resulting from one or more amino acid substitutions, deletions, or insertions may include, for example, energy stabilization (e.g., reducing the energy of the pre-fusion conformation relative to the open post-fusion conformation) and / or kinetic stabilization (e.g., reducing the transition rate from the pre-fusion conformation to the post-fusion conformation). Furthermore, the stabilization of the coronavirus S external domain trimer in the pre-fusion conformation may include increased resistance to denaturation compared to the corresponding native coronavirus S sequence.

[0087] Therefore, in a preferred embodiment, the coronavirus spike protein includes one or more amino acid substitutions that stabilize the S protein in the pre-fusion conformation, for example, substitutions that stabilize the membrane distal portion (including the N-terminal region) of the S protein in the pre-fusion conformation.

[0088] In some embodiments, the prefusion stabilization mutation involves an amino acid substitution at an amino acid position in the distal portion (including the N-terminal region) of the S protein, wherein the amino acid is substituted with one selected from A, I, L, M, F, V, G, or P (the amino acid position relating to the reference), preferably with P. Preferably, stabilization of the coronavirus spike protein is achieved by substituting two consecutive amino acids with an amino acid that stabilizes the spike protein in the prefusion conformation.

[0089] In preferred embodiments, at least one pre-fusion stabilization mutation includes a cavity-filling mutation that further stabilizes the pre-fusion state of the coronavirus S protein. The terms “cavity-filling mutation” or “cavity-filling amino acid substitution” refer to amino acid substitutions that fill cavities within the protein core of a protein, such as the S protein extradomain of the coronavirus. A cavity is essentially a cavity inside 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 present in the pre-fusion conformation of the coronavirus S extradomain core that collapse (e.g., decrease in volume) after transitioning to the post-fusion conformation.

[0090] In a preferred embodiment, at least one prefusion-stabilizing mutation includes a mutant protonation site that further stabilizes the prefusion state.

[0091] In preferred embodiments, at least one pre-fusion stabilization mutation includes an artificial intramolecular disulfide bond. Introducing such an artificial intramolecular disulfide bond can further stabilize the membrane distal portion (including the N-terminal region) of the S protein of the pre-fusion conformation, i.e., the conformation that specifically binds to one or more pre-fusion specification antibodies and / or the conformation that presents a preferred antigenic site present in the pre-fusion conformation but not present in the post-fusion conformation of the S protein. In some embodiments, at least one pre-fusion stabilization mutation includes 2, 3, 4, 5, 6, 7, or 8 different artificial intramolecular disulfide bonds.

[0092] In the context of the present invention, it is emphasized that the coronavirus S protein, preferably the pandemic coronavirus S protein, may be mutated as described above in order to stabilize the spike protein in the pre-fusion conformation. Therefore, the spike protein can be selected from coronaviruses, preferably alpha coronavirus, beta coronavirus, gamma coronavirus, delta coronavirus, and more preferably beta coronavirus.

[0093] According to various preferred embodiments, the nucleic acid of component A encodes at least one antigenic peptide or protein derived from a coronavirus as defined herein, preferably from a pandemic coronavirus, and furthermore, at least one heterologous peptide or protein element.

[0094] Preferably, at least one heterologous peptide or protein element can promote or improve the secretion of the encoded coronavirus antigenic peptide or protein (e.g., via a secretion signal sequence), promote or improve the fixation of the encoded antigenic peptide or protein on the plasma membrane (e.g., via a transmembrane element), promote or improve the formation of an antigen complex (e.g., via a multimerization domain or antigen clustering element), or promote or improve the formation of virus-like particles (VLP-forming sequences). Furthermore, the nucleic acid of component A can further encode a peptide linker element, a self-cleaving peptide, an immunoadjuvant sequence, or a dendritic cell target sequence.

[0095] A suitable polymerizing domain can be selected from the list of amino acid sequences relating to sequence numbers 1116-1167 of WO2017081082, or from fragments or variants of these sequences. A suitable transmembrane element can be selected from the list of amino acid sequences relating to sequence numbers 1228-1343 of WO2017081082, or from fragments or variants of these sequences. A suitable VLP-forming sequence can be selected from the list of amino acid sequences relating to sequence numbers 1168-1227 of patent application WO2017081082, or from fragments or variants of these sequences. A suitable peptide linker can be selected from the list of amino acid sequences relating to sequence numbers 1509-1565 of patent application WO2017081082, or from fragments or variants of these sequences. A suitable self-cleaving peptide can be selected from the list of amino acid sequences relating to sequence numbers 1434-1508 of patent application WO2017081082, or from fragments or variants of these sequences. Suitable immunological adjuvant sequences can be selected from the list of amino acid sequences relating to SEQ ID NOs. 1360-1421 of patent application WO2017081082, or from fragments or variants of these sequences. Suitable dendritic cell (DC) target sequences can be selected from the list of amino acid sequences relating to SEQ ID NOs. 1344-1359 of patent application WO2017081082, or from fragments or variants of these sequences. Suitable secretion signal peptides can be selected from the list of amino acid sequences relating to SEQ ID NOs. 1-1115 and SEQ ID NO. 1728 of published PCT patent application WO2017081082, or from fragments or variants of these sequences. In a preferred embodiment, at least one coding sequence further codes for one or more heterologous peptides or protein elements selected from signal peptides, linker peptides, helper epitopes, antigen clustering elements, trimerizing or multimerizing elements, transmembrane elements, or VLP-forming sequences.

[0096] In a preferred embodiment, the nucleic acid of component A encoding at least one antigenic protein derived from coronavirus further encodes at least one heterotrimerizing element, an antigen clustering element, or a VLP-forming sequence.

[0097] In preferred embodiments, the antigen clustering element can be selected from a ferritin element, a lumazine synthase element, a hepatitis B virus (HBsAg) surface antigen, or an encapsulin. When a stably clustered coronavirus spike protein is expressed, preferably in its pre-fusion conformation, the intensity and range of neutralizing activity against the encoded coronavirus antigen may be increased.

[0098] Lumazine synthase (lumazine, LS, LumSynth) is an enzyme present in a wide range of organisms that has particle-forming properties and is involved in riboflavin biosynthesis. In particularly preferred embodiments, lumazine synthase can be used to promote antigen clustering and can enhance or boost the immune response to the coronavirus antigen encoded in the present invention.

[0099] In a particularly preferred embodiment, the antigen clustering element (multimerized element) is obtained from lumazine synthase, and the amino acid sequence of the antigen clustering domain is preferably identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence of SEQ ID NO: 112, a fragment thereof, or a variant thereof.

[0100] Ferritin is a protein whose primary function is intracellular iron storage. Almost all organisms produce ferritin, which consists of 24 subunits, each composed of a 4-alpha-helix bundle, and this self-assembles into an octahedral symmetric quaternary structure. This property of self-assembling into nanoparticles makes it suitable for antigen transport and exposure.

[0101] In a particularly preferred embodiment, ferritin may be used to promote antigen clustering and facilitate an immune response to encoded coronavirus antigens, preferably spike proteins.

[0102] In a particularly preferred embodiment, the antigen clustering element (multimerized element) is preferably selected from or derived from ferritin in which the amino acid sequence of the antigen clustering domain is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence of SEQ ID NO: 113, a fragment thereof, or a variant thereof.

[0103] In some embodiments, the antigen clustering domain is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles. The addition of fragments of the surface antigen of the hepatitis B virus (HBsAg) sequence may be particularly effective in enhancing the immune response to nucleic acid-based vaccines against coronaviruses.

[0104] In a particularly preferred embodiment, HBsAg may be used to promote antigen clustering and facilitate the immune response to the encoded coronavirus antigen, preferably the spike protein as defined herein.

[0105] In some embodiments, the antigen clustering element is an encapsulin element. The addition of an encapsulin sequence may be particularly effective in enhancing the immune response to nucleic acid-based vaccines against coronaviruses. In a particularly preferred embodiment, the encapsulin may be used to promote antigen clustering and enhance the immune response to the encoded coronavirus antigen, preferably the coronavirus spike protein as defined herein.

[0106] Encapsrin is a protein isolated from the thermophilic Thermotoga maritima that can be used as an element to form antigenic (nano) particles by enabling the self-assembly of antigens. Encapsrin is assembled from 60 copies of identical 31 kDa monomers, each having a thin icosahedral T=1 symmetric cage structure with inner and outer diameters of 20 nm and 24 nm.

[0107] In embodiments in which the coding sequence of the nucleic acid of component A further codes for a heterologous antigen clustering element, it is particularly preferable to generate a fusion protein comprising the antigen clustering element and an antigenic peptide or protein derived from coronavirus. Preferably, the antigenic peptide or protein, preferably the spike protein, is deleting a C-terminal transmembrane domain (TM) or a portion of a C-terminal transmembrane domain (TM).

[0108] In other embodiments in which the coding sequence of the nucleic acid of component A further codes for the heterologous antigen clustering element defined above, it is particularly preferable to generate a fusion protein comprising the antigen clustering element and an antigenic peptide or protein derived from coronavirus spike protein fragment S1 (lacking S2, and / or TM, and / or TMflex). Furthermore, it may be preferable to use a linker element to separate the heterologous antigen clustering element from the antigenic peptide or protein (for example, the linker relating to SEQ ID NOs: 115, 13148, and 13152).

[0109] Further preferred polymerizing elements can be selected from the list of amino acid sequences relating to sequence numbers 1116-1167 of WO2017081082, or from fragments or variants of these sequences. Sequence numbers 1116-1167 of WO2017081082 are incorporated herein by reference.

[0110] In a preferred embodiment, the trimerizing element can be selected from Foldon elements. In a preferred embodiment, the Foldon element is a fibrin-foldon element. Expression of a stable trimer spike protein, preferably in its pre-fusion conformation, may increase the intensity and range of neutralizing activity against coronaviruses.

[0111] In a particularly preferred embodiment, the fibrin-foldon element may be used to promote antigen trimerization and thus enhance the immune response to the encoded coronavirus antigen, preferably the spike protein. Preferably, the foldon element is or is derived from a bacteriophage, preferably bacteriophage T4, most preferably bacteriophage T4 fibrin.

[0112] In a particularly preferred embodiment, the trimerizing element is selected from or derived from Foldon, and the amino acid sequence of the trimerizing element is preferably identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence of SEQ ID NO: 114, any fragment or variant thereof.

[0113] In embodiments in which the coding sequence of component A nucleic acid further codes for a heterologous trimer element, it is particularly preferable to generate a fusion protein comprising the trimer element and an antigenic peptide or protein derived from coronavirus. Preferably, the antigenic peptide or protein is a coronavirus-derived spike protein that lacks a C-terminal transmembrane domain or a portion of a C-terminal transmembrane domain (TMflex).

[0114] In other embodiments in which the coding sequence of the nucleic acid of component A further codes for the heterologous trimerizing element defined above, it is particularly preferable to generate a fusion protein comprising the trimerizing element and an antigenic peptide or protein derived from coronavirus spike protein fragment S1 (lacking S2, and / or TM, and / or TMflex). Furthermore, it may be preferable to use a linker element to separate the heterologous antigen clustering element from the antigenic peptide or protein (e.g., the linker relating to SEQ ID NOs: 115, 13148, and 13152).

[0115] Further preferred trimerizing elements can be selected from the list of amino acid sequences relating to sequence numbers 1116-1167 of WO2017081082, or from fragments or variants of these sequences. Sequence numbers 1116-1167 of WO2017081082 are incorporated herein by reference.

[0116] In preferred embodiments, VLP-forming sequences can be selected and fused to coronavirus antigens as defined herein. Stable clustering of spike proteins in the form of VLPs may increase the intensity and range of neutralizing activity against coronaviruses. VLPs structurally mimic infectious viruses and can induce potent cellular and humoral immune responses.

[0117] Suitable VLP-forming sequences can be selected from elements derived from hepatitis B virus core antigen, HIV-1Gag protein, or woodchuck hepatitis core antigen element (WhcAg).

[0118] In a particularly preferred embodiment, at least one VLP-forming sequence is a woodchuck hepatitis core antigen element (WhcAg). The WhcAg element can be used to facilitate VLP formation and promote an immune response to the encoded coronavirus antigen, preferably the spike protein.

[0119] In a particularly preferred embodiment, the VLP-forming sequence is selected from or derived from Foldon, and the amino acid sequence of the VLP-forming sequence is preferably identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence of SEQ ID NO: 13171, any fragment or variant thereof.

[0120] In embodiments in which the coding sequence of component A nucleic acid further codes for a heterologous VLP-forming sequence, it is particularly preferable to generate a fusion protein containing the VLP-forming sequence and an antigenic peptide or protein derived from a coronavirus. Preferably, the antigenic peptide or protein is a coronavirus-derived spike protein that lacks a C-terminal transmembrane domain or a portion of its C-terminal transmembrane domain.

[0121] In other embodiments in which the coding sequence of component A nucleic acid further codes for the heterologous VLP-forming sequence defined above, it is particularly preferable to generate a fusion protein comprising the VLP-forming sequence and an antigenic peptide or protein derived from coronavirus spike protein fragment S1 (lacking S2, and / or TM, and / or TMflex). Furthermore, it may be preferable to use a linker element to separate the heterologous antigen clustering element from the antigenic peptide or protein (e.g., the linker relating to SEQ ID NOs: 115, 13148, and 13152).

[0122] Further preferred VLP-forming sequences in the above context can be selected from the list of amino acid sequences relating to Sequence IDs 1168-1227 of patent application WO2017081082, or from fragments or variants of these sequences. Sequence IDs 1168-1227 of WO2017081082 are incorporated herein by reference.

[0123] In some embodiments, the antigenic peptide or protein includes a heterologous signal peptide. The heterologous signal peptide may be used to improve the secretion of the encoded coronavirus antigen.

[0124] Suitable secretory signal peptides can be selected from the list of amino acid sequences relating to SEQ ID NOs. 1-1115 and SEQ ID NO. 1728 of published PCT patent application WO2017081082, or from fragments or variants of these sequences. SEQ ID NOs. 1-1115 and SEQ ID NO. 1728 of WO2017081082 are incorporated herein by reference.

[0125] In embodiments in which the coding sequence of component A nucleic acid further encodes a heterologous secretion signal peptide, it is particularly preferable to generate a fusion protein comprising the heterologous secretion signal peptide and an antigenic peptide or protein derived from coronavirus. Preferably, the antigenic peptide or protein is a coronavirus-derived spike protein lacking the N-terminal endogenous secretion signal peptide (lacking aa1 to aa15).

[0126] In a preferred embodiment, the nucleic acid of component A comprises at least one coding sequence, or fragments and variants thereof, encoding at least one antigenic peptide or protein, selected from or derived from the coronaviruses defined herein. In a preferred embodiment, the nucleic acid of component A comprises at least one coding sequence, or fragments and variants thereof, encoding at least one antigenic peptide or protein, selected from or derived from the coronaviruses defined herein, wherein the at least one coronavirus is selected from at least one (pandemic) alpha coronavirus, at least one (pandemic) beta coronavirus, at least one (pandemic) gamma coronavirus, and / or at least one (pandemic) delta coronavirus.

[0127] In the context described herein, coding sequences, or fragments and variants thereof, that encode at least one antigenic protein of coronavirus as defined herein are understood to be suitable coding sequences and may be included in the nucleic acid of component A.

[0128] Preferred features and embodiments applicable to the nucleic acid of component A are described in the following paragraph, “Features and Embodiments of Nucleic Acids.”

[0129] Preferably, the nucleic acid of component A is compounded and / or complexed. Preferred features and embodiments applicable to the complexation of nucleic acids or the compounding of component A are described in the following "Compounding and Complexation" section.

[0130] In the context of this invention, the term "nucleic acid species" is understood to include not only a "single nucleic acid molecule" but also a collection of essentially identical nucleic acid molecules. Therefore, it can refer to multiple essentially identical nucleic acid molecules, such as DNA or RNA molecules.

[0131] In some embodiments, component A may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, such as DNA or RNA as defined herein, each encoding at least one antigenic peptide or protein, or a fragment or variant thereof, derived from the same coronavirus. In particular, the (genetically) identical coronavirus expresses an (essentially) identical repertoire of proteins or peptides, all of which have (essentially) identical amino acid sequences. In particular, the (genetically) identical coronavirus expresses essentially identical proteins, peptides, or polyproteins, preferably these proteins, peptides, or polyproteins that are identical in their amino acid sequences.

[0132] In some embodiments, component A comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, e.g., DNA or RNA as defined herein, each encoding at least one peptide or protein, or a fragment or variant thereof, derived from a genetically distinct coronavirus (e.g., different coronavirus isolates, different beta-coronaviruses, different pandemic coronaviruses). The terms “different” or “different coronaviruses” as used herein are understood as differences between at least two respective coronaviruses (e.g., different coronavirus isolates, different beta-coronaviruses, different pandemic coronaviruses, etc.), the differences manifesting on the genome of each different coronavirus. In particular, the (genetically) different coronaviruses may express at least one different protein, peptide, or polyprotein, and the at least one different protein, peptide, or polyprotein differs by at least one amino acid.

[0133] In a preferred embodiment, component A comprises at least one antigenic peptide or protein selected from or derived from at least one coronavirus, or at least one nucleic acid encoding an immunogenic fragment or immunogenic variant thereof, wherein component A is preferably administered intramuscularly or intradermally.

[0134] Preferably, intramuscular or intradermal administration of component A results in the expression of the encoded antigen construct in the subject. In embodiments where the nucleic acid is RNA, administration of component A results in the translation of RNA and the generation of the encoded coronavirus antigen in the subject. In embodiments where the nucleic acid is DNA (e.g., plasmid DNA, adenovirus DNA), administration of the composition results in the transcription of DNA to RNA in the subject, followed by the translation of the RNA into the encoded coronavirus antigen.

[0135] In some embodiments, administration of a pharmaceutical composition containing component A to a subject induces neutralizing antibodies, but not disease-enhancing antibodies. In particular, administration of a pharmaceutical composition containing component A, which encodes the pre-fusion stabilizing spike protein of coronavirus, to a subject does not induce immunopathological effects, such as disease enhancement and / or antibody-dependent enhancement (ADE).

[0136] Preferably, component A is suitable for vaccines, particularly for coronavirus vaccines, and preferably for the mixed vaccine of the present invention.

[0137] Embodiments relating to a specific coronavirus in the context of the present invention are shown below (component A-1, component A-2, component A-3).

[0138] Ingredient A-1: ​​SARS-CoV-2 In a particularly preferred embodiment, at least one coronavirus of component A is the SARS-CoV-2 virus (also referred to as component A-1).

[0139] Therefore, in a preferred embodiment of the first aspect, the nucleic acid of component A includes at least one antigenic peptide or protein selected from or derived from at least one SARS-CoV-2, or at least one coding sequence encoding an immunogenic fragment or immunogenic variant thereof.

[0140] It is understood that the general embodiments and features described in the paragraph for "Component A" may also be applicable to nucleic acids encoding SARS-CoV-2 antigenic peptides or proteins.

[0141] 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" used in this invention can be used interchangeably throughout this invention and relate to a new pandemic coronavirus that emerged in Wuhan, China at the turn of 2019 / 2020 and causes the disease known as COVID-19. According to the WHO (February 2020), the virus was officially named "SARS-CoV-2," and the associated disease was officially named "COVID-19."

[0142] SARS-CoV-2 belongs to the Coronaviridae family, specifically the orthocoronavirus genus, more specifically the betacoronavirus genus. Exemplary SARS-CoV-2 coronaviruses include, but are not limited to, the isolates given in Lists A and B below.

[0143] 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(BetaCoV_Wuhan_WIV05_2019_EPI_ISL_402128), EPI_ISL_402129, EPI_ISL_402130, EPI_ISL_402131, EPI_ISL_402132, EPI_IS L_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_4、 13809、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_4140 44、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_IS L_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_4 17569、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_417605、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_417628、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、EPI_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、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_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_IS L_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,EPI_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_ISL_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_418204、EPI_ISL_418231、EPI_ISL_418232、EPI_ISL_418233、EPI_ISL_418235、EPI_ISL_418236, EPI_ISL_418237, EPI_ISL_418238, EPI_ISL_418239, EPI_ISL_418240, EPI_ISL_418257, EPI_ISL_4 18260, EPI_ISL_418263, EPI_ISL_418264, or EPI_ISL_418265, or EPI_ISL_616802 (hCoV-19 / Denmark / DCGC-3024 / 2020). ,

[0144] Exemplary SARS-CoV-2 coronaviruses can also be defined or identified by genetic information provided by GenBank accession numbers, as shown in List B below.

[0145] List B: GenBank accession numbers for 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、Or MT192773.

[0146] The SARS-CoV-2 coronavirus has been assigned the NCBI taxonomic ID (NCBI:txid or taxID): 2697049.

[0147] In the context of the present invention, proteins selected from or derived from SARS-CoV-2 can be used in the context of the present invention and can be suitably encoded by a coding sequence or the nucleic acid of component A. Furthermore, it is within the scope of the underlying invention that at least one antigenic peptide or protein may include or consist of a synthetically engineered or artificial SARS-CoV-2 peptide or protein. The terms “synthetically engineered” SARS-CoV-2 peptide or protein, or “artificial SARS-CoV-2 peptide or protein,” refer to proteins that do not exist in nature. Therefore, “artificial SARS-CoV-2 peptide or protein” or “synthetically engineered SARS-CoV-2 peptide or protein” may differ from, for example, a naturally occurring SARS-CoV-2 peptide or protein by at least one amino acid, and / or may contain additional heterologous peptide or protein elements, and / or may have an elongated or truncated N-terminus or C-terminus.

[0148] In preferred embodiments, the nucleic acid of component A (particularly component A-1) comprises at least one antigenic peptide or protein selected from or derived from SARS-CoV-2, or at least one coding sequence encoding an immunogenic fragment or immunogenic variant thereof, wherein the at least one antigenic peptide or protein comprises at least one peptide or protein selected from or derived from a structural protein, an accessory protein, or a replicase protein, or any immunogenic fragment or immunogenic variant thereof.

[0149] Preferably, the structural protein is selected from or derived from a spike protein (S), an envelope protein (E), a membrane protein (M), or a nucleocapsid protein (N), or an immunogenic fragment or variant thereof.

[0150] In a particularly preferred embodiment of the pharmaceutical composition, the encoded antigenic peptide or protein of component A (particularly component A-1) comprises or consists of at least one peptide or protein selected from or derived from SARS-CoV-2 spike proteins (S, S1, S2, or S1 and S2), or an immunogenic fragment or immunogenic variant thereof.

[0151] Suitable antigenic peptide or protein sequences provided by the nucleic acid of component A are disclosed in Table 1, rows 1-41, columns A and B. In addition, further information regarding the aforementioned suitable antigenic peptide or protein sequences selected from or derived from SARS-CoV-2 is provided in the ST.25 sequence listing identifiers. <223> It is given below.

[0152] The following describes in detail preferred antigenic peptides or protein sequences selected from or derived from SARS-CoV-2, provided by the nucleic acid of component A (particularly component A-1).

[0153] When referring to amino acid (aa) residues and their positions in the SARS-CoV-2 spike protein (S), the numbering used herein, unless otherwise specified, refers to the position of each amino acid residue in the corresponding spike protein (S) of SARS-CoV-2 coronavirus isolate EPI_ISL_402128 (BetaCoV_Wuhan_WIV05_2019_EPI_ISL_402128) related to SEQ ID NO: 1. Each amino acid position is shown exemplarily for the spike protein (S) of SARS-CoV-2 coronavirus isolate EPI_ISL_402128 (SEQ ID NO: 1) when referring to the SARS-CoV-2 spike protein. Needless to say, those skilled in the art can adapt the teachings provided herein, exemplarily for SARS-CoV-2 EPI_ISL_402128 (SEQ ID NO: 1), to other antigenic peptides or proteins in other SARS-CoV-2 coronavirus isolates. Other SARS-CoV-2 coronavirus 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 (other SARS-CoV-2 isolates are listed in List A and / or List B).

[0154] Protein annotation of the SARS-CoV-2 spike protein (S) was performed using SEQ ID NO: 1 as the reference protein. The full-length SARS-CoV-2 reference protein S has 1273 amino acid residues and contains the following elements. - Secretory signal peptide: Amino acid positions aa1~aa15 (see SEQ ID NO: 28) - Spike protein fragment S1: Amino acid positions aa1~aa681 (see SEQ ID NO: 27) - Receptor-binding domain (RBD): Amino acid positions aa319~aa541 (see SEQ ID NO: 13243) - Important neutralizing domain (CND): Amino acid positions aa329~aa529 (see SEQ ID NO: 13310) - Spike protein fragment S2: Amino acid positions aa682~aa1273 (see SEQ ID NO: 30) - Transmembrane domain (TM): Amino acid positions aa1212~aa1273 (see SEQ ID NO: 49) - Transmembrane domain (TMflex): Amino acid positions aa1148~aa1273 (see SEQ ID NO: 13176)

[0155] Amino acid level variations can spontaneously occur between spike proteins derived from different SARS-CoV-2 isolates (exemplary SARS-CoV-2 isolates are shown in Lists A and B). In the context of the present invention, such amino acid variations can be applied to each antigenic peptide or protein derived from the SARS-CoV-2 spike protein described herein.

[0156] Therefore, each SARS-CoV-2 spike protein provided herein and considered suitable antigens in the context of the present invention may have one or more of the following amino acid mutations / substitutions (amino acid positions related to reference sequence number 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 / aa deletion; V70 or H70del / aa deletion; H69_V70 or H69del and H70del / aa 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 L 456F;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 L48 6F;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;H69 or H69del / aa deletion; V70 or H70del / aa deletion; H69_V70 or H69del and H70del / aa 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; L4 55F 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; G48 5S 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; Q506 K or K506Q; Q506H or H506Q; Y144 or Y144del / aadel; 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 / aa deletion; A243 or A243del / aa deletion; L244 or L244del / aa deletion; L242_A243_L244 or L242del and A243del and L244del / aa 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;and / or V1176F or F1176V.

[0157] The following amino acid mutations (amino acid positions related to reference sequence number 1) are particularly preferred: 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

[0158] In a preferred embodiment, the SARS-CoV-2 spike protein (S) contains the following amino acid mutations (amino acid positions corresponding to reference sequence number 1): L18F, D80A, D215G, delL242, delA243, delL244, R246I, K417N, E484K, N501Y, D614G, A701V.

[0159] Suitable SARS-CoV-2 spike protein (S) may be selected from or derived from novel SARS-CoV-2 variants as shown in the table below. (outside 1) TIFF0007848141000001.tif70170

[0160] In a particularly preferred embodiment, the SARS-CoV-2 spike protein (S) is selected from or derived from B.1.351.

[0161] In some embodiments, a fragment of the SARS-CoV-2 spike protein (S) may be encoded by the nucleic acid of component A, the fragment may be N-terminated, with 1 to 100 N-terminal amino acids deleted from the full-length SARS-CoV-2 coronavirus reference protein (SEQ ID NO: 1), and / or the fragment may be C-terminated, with 531 to 1273 C-terminal amino acids (aa) deleted from the full-length SARS-CoV-2 coronavirus reference protein (SEQ ID NO: 1). Such “spike protein (S) fragment” may further include amino acid substitutions (described below) and may further include at least one heterologous peptide or protein element (described below). In a preferred embodiment, the SARS-CoV-2 spike protein (S) fragment may have its C-terminus truncated, thereby deleting the C-terminal transmembrane domain (i.e., deletion of aa1212~aa1273 or deletion of aa1148~aa1273).

[0162] In some embodiments, the encoded antigenic peptide or protein of component A comprises or consists of a SARS-CoV-2 spike protein (S), wherein the spike protein (S) is deleting a transmembrane domain (TM) (amino acid positions aa1212-aa1273). In some embodiments, the encoded antigenic peptide or protein comprises or consists of a SARS-CoV-2 spike protein (S), wherein the spike protein (S) is deleting an extension of the transmembrane domain (TMflex) (amino acid positions aa1148-aa1273). While we do not wish to be bound by theory, SARS-CoV-2 spike protein (S) deleting the transmembrane domain (TM or TMflex) as defined herein may be suitable for SARS-CoV-2 vaccines because such proteins are soluble and not fixed to the cell membrane. Therefore, when administered to a subject, soluble proteins may be produced (translated) at higher concentrations, potentially leading to an improved immune response.

[0163] While we do not wish to be constrained by theory, the RBD(aa319~aa541) and CND(aa29~aa529) domains may be extremely important for the immunogenicity of the SARS-CoV-2 spike protein (S). Both regions are located in the S1 fragment of the spike protein. Therefore, in the context of the present invention, it may be preferable for the antigenic peptide or protein to contain or consist of the S1 fragment of the spike protein, or its immunogenic fragment or immunogenic variant.

[0164] Preferably, the S1 fragment of SARS-CoV-2 may contain at least the RBD and / or CND domains defined above.

[0165] In preferred embodiments, at least one encoded antigenic peptide or protein comprises or consists of receptor-binding domains (RBDs; aa319~aa541), the RBD comprising or consisting of spike protein fragments, or immunogenic fragments or immunogenic variants thereof.

[0166] In a more preferred embodiment, at least one encoded antigenic peptide or protein comprises or consists of a truncated receptor-binding domain (truncRBD; aa334~aa528), the RBD comprising or consisting of a spike protein fragment, or an immunogenic fragment or immunogenic variant thereof.

[0167] Such "spike protein (S) fragments" (RBD; aa319~aa541, or truncRBD, aa334~aa528) may further contain amino acid substitutions (described below) and may contain at least one heterologous peptide or protein element (described below).

[0168] In a particularly preferred embodiment, the at least one antigenic peptide or protein encoded by component A comprises or consists of a SARS-CoV-2 spike protein (S), wherein the SARS-CoV-2 spike protein (S) comprises or consists of a spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof.

[0169] In preferred embodiments, at least one encoded antigenic peptide or protein comprises the SARS-CoV-2 spike protein fragment S1 and is deleting at least 70%, 80%, 90%, preferably 100%, of the spike protein fragment S2 (aa682-aa1273). Such embodiments may be beneficial because the SARS-CoV-2 S1 fragment contains a neutralizing epitope, without the potential problems associated with full-length proteins containing S1 and S2.

[0170] While we do not wish to be bound by theory, it is preferable that the antigenic peptide or protein of component A contains or consists of SARS-CoV-2 spike protein fragment S1 and SARS-CoV-2 spike protein fragment S2 (or at least a fragment thereof), since the formation of immunogenic SARS-CoV-2 spike protein may be promoted.

[0171] Therefore, in a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A comprises or consists of a SARS-CoV-2 spike protein (S), the SARS-CoV-2 spike protein (S) comprising or consisting of a SARS-CoV-2 spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof, and a SARS-CoV-2 spike protein fragment S2, or an immunogenic fragment or immunogenic variant thereof.

[0172] In a particularly preferred embodiment, the encoded antigenic peptide or protein of component A comprises or consists of a full-length SARS-CoV-2 spike protein, or an immunogenic fragment or immunogenic variant thereof.

[0173] The term "full-length SARS-CoV-2 spike protein" is understood as a SARS-CoV-2 spike protein, preferably derived from the SARS-CoV-2 coronavirus, that has an amino acid sequence corresponding to an essentially complete spike protein. Therefore, the "full-length spike protein" may include aa1 to aa1273 (reference protein: SEQ ID NO: 1). Thus, the full-length SARS-CoV-2 spike protein may typically include the secretion signal peptide, spike protein fragment S1, spike protein fragment S2, receptor-binding domain (RBD), the important neutralization domain CND, and the transmembrane domain. Furthermore, variants containing specific amino acid substitutions (e.g., to enable pre-fusion stabilization of the S protein) or native amino acid deletions are also included in the term "full-length SARS-CoV-2 spike protein."

[0174] In a particularly preferred embodiment, the SARS-CoV-2 spike protein (S) provided by the nucleic acid of component A is designed or adapted to stabilize the antigen in a pre-fusion conformation. The pre-fusion conformation is particularly advantageous in the context of an efficient SARS-CoV-2 vaccine because the pre-fusion protein conformation contains several potential and accessible epitopes for neutralizing antibodies. Furthermore, the presence of protein in the pre-fusion conformation is intended to avoid immunopathological effects, such as disease exacerbation or antibody-dependent enhancement (ADE).

[0175] In preferred embodiments, administration of a nucleic acid (or composition or vaccine) encoding a pre-fusion stabilized spike protein to a subject induces spike protein neutralizing antibodies but not disease-enhancing antibodies. In particular, administration of a nucleic acid (or composition or vaccine) encoding a pre-fusion stabilized spike protein to a subject does not induce immunopathological effects such as disease enhancement and / or antibody-dependent enhancement (ADE).

[0176] Therefore, in a preferred embodiment, the nucleic acid of component A comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from the SARS-CoV-2 coronavirus, wherein the at least one antigenic peptide or protein is selected from or derived from a spike protein (S), and the spike protein (S) is a pre-fusion stabilization spike protein (S_stab). Preferably, the pre-fusion stabilization spike protein comprises at least one pre-fusion stabilization mutation.

[0177] Stabilization of the SARS-CoV-2 spike protein can be achieved by substituting at least one amino acid at position K986 and / or V987 with an amino acid that stabilizes the spike protein in the pre-fusion conformation (amino acid position related to Reference Sequence ID No. 1).

[0178] In some embodiments, the prefusion stabilization mutation of the SARS-CoV-2 spike protein includes an amino acid substitution at position K986, where amino acid K986 is substituted with one selected from A, I, L, M, F, V, G, or P (the amino acid position related to reference sequence number 1), preferably amino acid K986 is substituted with P. In some embodiments, the prefusion stabilization mutation includes an amino acid substitution at position V987, where amino acid V987 is substituted with one selected from A, I, L, M, F, V, G, or P (the amino acid position related to reference sequence number 1), preferably amino acid V987 is substituted with P.

[0179] Preferably, the stabilization of the SARS-CoV-2 spike protein can be achieved by substituting two consecutive amino acids at positions K986 and V987 with amino acids that stabilize the spike protein in the pre-fusion conformation (amino acid positions related to Reference Sequence ID No. 1).

[0180] In a preferred embodiment, the prefusion-stabilizing mutation of the SARS-CoV-2 spike protein includes amino acid substitutions at positions K986 and V987, where amino acids K986 and / or V987 are substituted with one selected from A, I, L, M, F, V, G, or P (amino acid positions related to reference sequence number 1).

[0181] Preferably, stabilization of the pre-fusion conformation is achieved by introducing two consecutive proline substitutions at residues K986 and V987 of the SARS-CoV-2 spike protein (amino acid positions related to reference sequence number 1).

[0182] Therefore, in a preferred embodiment, the SARS-CoV-2 pre-fusion stabilization spike protein (S_stab) comprises at least one pre-fusion stabilization mutation, the at least one pre-fusion stabilization mutation comprising the following amino acid substitutions: K986P and V987P (amino acid positions related to reference sequence number 1).

[0183] Therefore, proteins, fragments, or variants thereof selected from the NCBI protein accession numbers shown above, or from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, and 22929-22946, can be selected by those skilled in the art to introduce such amino acid changes into the SARS-CoV-2 spike protein, preferably amino acid substitutions: K986P and V987P (amino acid positions relating to Reference SEQ ID NO: 1).

[0184] In a preferred embodiment, at least one prefusion-stabilizing mutation of the SARS-CoV-2 spike protein includes a cavity-filling mutation that further stabilizes the prefusion state, and the mutation / amino acid substitution is selected from a list including T887W;A1020W;T887W and A1020W; or P1069F (amino acid position relating to Reference Sequence ID No. 1).

[0185] In some embodiments, at least one of the following amino acid substitutions, T887W;A1020W;T887W and A1020W; or P1069F, can be combined with the (K986P and V987P) substitution in the SARS-CoV-2 spike protein (amino acid positions related to Reference Sequence ID No. 1).

[0186] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes at least one of the following amino acid substitutions (amino acid positions related to Reference Sequence ID No. 1): T887W; K986P and V987P · A1020W; K986P and V987P T887W and A1020W; K986P and V987P P1069F; K986P and V987P

[0187] Therefore, the NCBI protein accession numbers of SARS-CoV-2 shown above, or proteins selected from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946, or their fragments or variants, are such amino acid changes, preferably T887W;A1020W;T An amino acid substitution selected from 887W and A1020W; or P1069F; or an amino acid substitution selected from (T887W; K986P and V987P); (A1020W; K986P and V987P); (T887W and A1020W; K986P and V987P); (P1069F; K986P and V987P) (amino acid position related to reference sequence number 1) can be selected by a person skilled in the art.

[0188] In some embodiments, at least one of the following amino acid substitutions F817P, A892P, A899P, and A942P can be combined with the (K986P and V987P) substitutions (amino acid positions related to reference sequence number 1).

[0189] In some embodiments, the SARS-CoV-2 coronavirus spike protein includes at least one of the following amino acid substitutions (amino acid positions related to Reference Sequence ID No. 1): F817P; K986P and V987P A892P; K986P and V987P A899P; K986P and V987P A942P; K986P and V987P

[0190] In a particularly preferred embodiment, the SARS-CoV-2 coronavirus spike protein includes the following amino acid substitutions (amino acid positions related to Reference Sequence ID No. 1): F817P, A892P, A899P, A942P, K986P and V987P (S_stab_PP_hex)

[0191] Therefore, proteins, or fragments or variants thereof, selected from the NCBI protein accession numbers shown above, or from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946, are selected from such amino acid changes, preferably F817P, A892P, A899P, and A942P. The amino acid substitutions to be introduced; or amino acid substitutions selected from (F817P;K986P and V987P);(A892P;K986P and V987P);(A899P;K986P and V987P);(A942P;K986P and V987P);(F817P, A892P, A899P, A942P, K986P and V987P) (amino acid positions related to reference sequence number 1) can be selected by those skilled in the art.

[0192] In a preferred embodiment, at least one pre-fusion stabilizing mutation of the SARS-CoV-2 spike protein comprises a mutational protonation that further stabilizes the pre-fusion state, wherein the mutation / amino acid substitution is selected from H1048Q and H1064N; H1083N and H1101N; or H1048Q, and H1064N, and H1083N, and H1101N (amino acid positions relating to Reference Sequence ID No. 1).

[0193] In some embodiments, the following amino acid substitutions H1048Q and H1064N; H1083N and H1101N; or at least one of H1048Q, H1064N, H1083N, and H1101N can be combined with (K986P and V987P) substitutions (amino acid positions related to reference sequence number 1) to form the SARS-CoV-2 spike protein.

[0194] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes at least one of the following amino acid substitutions (amino acid positions related to Reference Sequence ID No. 1): H1048Q and H1064N; K986P and V987P H1083N and H1101N; K986P and V987P H1048Q, H1064N, H1083N, and H1101N; K986P and V987P

[0195] Therefore, a protein selected from the NCBI protein accession numbers of SARS-CoV-2 shown above, or from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946, or a fragment or variant thereof, is a protein with such amino acid changes, preferably H1048Q and H1064N; H1083N and H1101N; or H1048Q, H10 An amino acid substitution selected from 64N, H1083N, and H1101N; or an amino acid substitution selected from (H1048Q and H1064N; K986P and V987P); (H1083N and H1101N; K986P and V987P); (H1048Q, H1064N, H1083N, and H1101N; K986P and V987P) (amino acid position related to reference sequence number 1) can be selected by those skilled in the art to introduce into the SARS-CoV-2 spike protein.

[0196] In a preferred embodiment, at least one pre-fusion stabilizing mutation of the SARS-CoV-2 spike protein includes an artificial intramolecular disulfide bond. Introducing such an artificial intramolecular disulfide bond can further stabilize the membrane distal portion (including the N-terminal region) of the SARS-CoV-2 S protein in the pre-fusion conformation, i.e., the conformation that specifically binds to one or more pre-fusion specification antibodies, and / or the conformation that presents a preferred antigen site present in the pre-fusion conformation but not in the post-fusion conformation of the SARS-CoV-2 S protein.

[0197] In a preferred embodiment, at least one pre-fusion stabilizing mutation of the SARS-CoV-2 spike protein comprises an artificial intramolecular disulfide bond, preferably at least one artificial intramolecular disulfide bond comprising at least two amino acid substitutions selected from the list including I712C, I714C, P715C, T874C, G889C, A890C, I909C, N914C, Q965C, F970C, A972C, R995C, G999C, S1003C, L1034C, V1040C, Y1047C, S1055C, P1069C, T1077C, Y1110C, or S1123C (amino acid position related to reference sequence number 1).

[0198] In a preferred embodiment, at least one pre-fusion stabilizing mutation of the SARS-CoV-2 spike protein comprises an artificial intramolecular disulfide bond, the at least one of which includes at least one of 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 (amino acid position relating to reference sequence number 1).

[0199] In some embodiments, at least one pre-fusion stabilization mutation of the SARS-CoV-2 spike protein comprises 2, 3, 4, 5, 6, 7, or 8 different artificial intramolecular disulfide bonds, each selected from 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; N914C and S1123C; T874C and S1055C; or N914C and S1123C (amino acid position related to reference sequence number 1).

[0200] In some embodiments, at least one, preferably two, three, four, five, or more, of 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 can be combined with the (K986P and V987P) substitution. For example, the pre-fusion stabilized SARS-CoV-2 S protein may contain two different artificial intramolecular disulfide bonds, e.g., I712C and T1077C; P715C and P1069C, and further, K986P and V987P substitutions (amino acid positions related to reference sequence number 1).

[0201] In a particularly preferred embodiment, the SARS-CoV-2 spike protein includes at least one of the following amino acid substitutions (amino acid positions related to reference sequence number 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

[0202] Therefore, the NCBI protein accession numbers of SARS-CoV-2 shown above, or proteins selected from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946, or fragments or variants thereof, are such amino acid changes, preferably I712C and Amino acid substitutions selected from 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;or (I712C;T1077C;K986P;V9 87P) or (I714C;Y1110C;K986P;V987P) or (P715C;P1069C;K986P;V987P) or (G889C;L1034C;K986P;V987P) or (I909C;Y1047C;K986P;V987P) or (Q965C;S1003C;K986P;V987P) or (F970C;G999C;K986P;V987P) or (A972C;R995C An amino acid substitution (amino acid position related to reference sequence number 1) selected from (;K986P;V987P) or (A890C and V1040C;K986P and V987P) or (T874C and S1055C;K986P and V987P) or (N914C and S1123C;K986P and V987P) can be selected by those skilled in the art to be introduced into the SARS-CoV-2 spike protein.

[0203] In the context of the present invention, it is noteworthy that the SARS-CoV-2 spike protein can be mutated or modified in order to stabilize the spike protein in the pre-fusion conformation, as described above (as illustrated for reference protein SEQ ID NO: 1).

[0204] According to various preferred embodiments, the nucleic acid of component A encodes at least one antigenic peptide or protein selected from or derived from SARS-CoV-2 as defined herein, and more preferably at least one heterologous peptide or protein element selected from or derived from signal peptides, linkers, helper epitopes, antigen clustering elements, trimerizing elements, transmembrane elements, and / or VLP-forming sequences.

[0205] Preferably, at least one heterologous peptide or protein element can promote or improve the secretion of the SARS-CoV-2 encoded antigenic peptide or protein (e.g., via a secretion signal sequence), promote or improve the fixation of the encoded antigenic peptide or protein of the present invention on the plasma membrane (e.g., via a transmembrane element), promote or improve the formation of an antigen complex (e.g., via a multimerization domain or antigen clustering element), or promote or improve virus-like particle (VLP-forming sequence). Furthermore, the nucleic acid of component A can further encode a peptide linker element, a self-cleaving peptide, an immunoadjuvant sequence, or a dendritic cell target sequence.

[0206] In a preferred embodiment, the nucleic acid of component A encoding at least one antigenic protein selected from or derived from SARS-CoV-2 as defined herein further encodes at least one heterotrimerizing element, an antigen clustering element, or a VLP-forming sequence.

[0207] In preferred embodiments, the antigen clustering element may be selected from a ferritin element, a lumazine synthase element, the surface antigen of hepatitis B virus (HBsAg), or encapsulin. Stably clustered SARS-CoV-2 spike proteins, preferably expressed in their pre-fusion conformation, may increase the intensity and range of neutralizing activity against the encoded SARS-CoV-2 peptide / protein.

[0208] In preferred embodiments, lumazine synthase is used to promote antigen clustering of SARS-CoV-2 proteins, which can promote or enhance the immune response to the encoded SARS-CoV-2 antigen, preferably the SARS-CoV-2 spike protein. In preferred embodiments, ferritin is used to promote antigen clustering of SARS-CoV-2 proteins, which can promote or enhance the immune response to the encoded SARS-CoV-2 antigen, preferably the SARS-CoV-2 spike protein. In preferred embodiments, HBsAg is used to promote antigen clustering of SARS-CoV-2 proteins, which can promote or enhance the immune response to the encoded SARS-CoV-2 antigen, preferably the SARS-CoV-2 spike protein. In preferred embodiments, encapsulin is used to promote antigen clustering of SARS-CoV-2 proteins, which can promote or enhance the immune response to the encoded SARS-CoV-2 antigen, preferably the SARS-CoV-2 spike protein.

[0209] In some embodiments in which the coding sequence of component A further codes for a heterologous antigen clustering element, it is particularly preferred to generate a fusion protein comprising the antigen clustering element and an antigenic peptide or protein derived from SARS-CoV-2. Preferably, the antigenic peptide or protein, preferably the spike protein, is deleting the C-terminal transmembrane domain (TM) (deleting aa1212~aa1273) or a portion of the C-terminal transmembrane domain (TMflex), for example, deleting aa1148~aa1273.

[0210] Therefore, an amino acid sequence that is identical to any of SEQ ID NOs. 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964, or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, can be modified to remove the endogenous transmembrane domain (TM) at the positions aa1212-aa1273, and thus, in the context of the present invention, can be used as a "C-terminus truncated" SARS-CoV-2 protein (amino acid position relating to Reference SEQ ID NO. 1). Furthermore, amino acid sequences that are identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964 can be modified to remove a portion of the endogenous transmembrane domain (TM) at the positions aa1212-aa1273, and therefore, in the context of the present invention, can be used as a "C-terminus truncated" SARS-CoV-2 protein (amino acid positions relating to Reference SEQ ID NO: 1). Suitable spike proteins lacking a C-terminal transmembrane domain (TM or TMflex) can be selected from SEQ ID NOs: 31-39, 1614-3623, and 13377-13510.

[0211] In other embodiments in which the coding sequence of component A further codes for the heterologous antigen clustering element defined above, it is particularly preferable to generate a fusion protein comprising the antigen clustering element and an antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein fragment S1 (lacking S2, and / or TM, and / or TMflex). Furthermore, it may be preferable to use a linker element (e.g., the linker relating to SEQ ID NOs: 115, 13148, and 13152) for separating the heterologous antigen clustering element from the antigenic peptide or protein.

[0212] In a preferred embodiment, the trimerizing element can be selected from Foldon elements. In a preferred embodiment, the Foldon element is a fibrin-foldon element. Expression of a stable trimer spike protein, preferably in its pre-fusion conformation, may increase the intensity and range of neutralizing activity against SARS-CoV-2.

[0213] In a particularly preferred embodiment, the fibrin-foldon element may be used to promote antigen trimerization and thus enhance the immune response to the encoded SARS-CoV-2 antigen, preferably the SARS-CoV-2 spike protein. Preferably, the foldon element is or is derived from a bacteriophage, preferably bacteriophage T4, most preferably bacteriophage T4 fibrin.

[0214] In some embodiments in which the coding sequence of the nucleic acid of component A further codes for a heterologous trimer element, it is particularly preferred to produce a fusion protein comprising the trimer element and an antigenic peptide or protein derived from SARS-CoV-2. Preferably, the antigenic peptide or protein, preferably the 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), for example, deleting aa1148~aa1273.

[0215] In other embodiments in which the coding sequence of the nucleic acid of component A further codes for the heterologous trimerizing element defined above, it is particularly preferable to generate a fusion protein comprising the trimerizing element and an antigenic peptide or protein derived from the SARS-CoV-2 spike protein fragment S1 (lacking S2, and / or TM, and / or TMflex). Furthermore, it may be preferable to use a linker element (e.g., the linker relating to SEQ ID NOs: 115, 13148, and 13152) to separate the heterologous antigen clustering element from the antigenic peptide or protein.

[0216] In preferred embodiments, VLP-forming sequences can be selected and fused to the SARS-CoV-2 antigen as defined herein. Stable clustering of SARS-CoV-2 spike proteins in the form of VLPs may increase the intensity and range of neutralizing activity against SARS-CoV-2. VLPs structurally mimic infectious viruses and can induce potent cellular and humoral immune responses.

[0217] Suitable VLP-forming sequences can be selected from elements derived from hepatitis B virus core antigen, HIV-1Gag protein, or woodchuck hepatitis core antigen element (WhcAg).

[0218] In a particularly preferred embodiment, at least one VLP-forming sequence is a woodchuck hepatitis core antigen element (WhcAg). The WhcAg element can be used to facilitate VLP formation and promote an immune response to the encoded coronavirus antigen, preferably the spike protein.

[0219] In some embodiments in which the coding sequence of the nucleic acid of component A further codes for a heterologous VLP-forming sequence, it is particularly preferable to generate a fusion protein comprising the VLP-forming sequence and an antigenic peptide or protein derived from SARS-CoV-2. Preferably, the antigenic peptide or protein, preferably the 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), for example, deleting aa1148~aa1273.

[0220] Therefore, amino acid sequences that are identical to any of SEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, 22737-22758, and 22929-22964, or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, can be modified to delete the endogenous transmembrane domain at the aa1212-aa1273 position, and thus, in the context of the present invention, can be used as a "C-terminus truncated" SARS-CoV-2 S protein. Furthermore, amino acid sequences that are identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, 22737-22758, or 22929-22964 can be modified to remove a portion of the endogenous transmembrane domain (TMflex) at the positions aa1148-aa1273, and therefore, in the context of the present invention, can be used as a "C-terminus truncated" SARS-CoV-2 S protein (amino acid positions relating to Reference SEQ ID NO: 1). Suitable SARS-CoV-2 spike proteins lacking a C-terminal transmembrane domain (TM or TMflex) can be selected from SEQ ID NOs: 31-39, 1614-3623, and 13377-13510.

[0221] In other embodiments in which the nucleic acid coding sequence further codes for the heterologous VLP-forming sequence defined above, it is particularly preferable to generate a fusion protein comprising the VLP-forming sequence and an antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein fragment S1 (lacking S2, and / or TM, and / or TMflex). Furthermore, it may be preferable to use a linker element (e.g., the linker relating to SEQ ID NOs: 115, 13148, and 13152) for separating the heterologous antigen clustering element from the antigenic peptide or protein.

[0222] In some embodiments, the antigenic peptide or protein includes the heterologous signal peptide defined above. The heterologous signal peptide can be used to enhance the secretion of the encoded SARS-CoV-2 antigen.

[0223] In some embodiments in which the coding sequence of the nucleic acid of component A further encodes a heterologous secretion signal peptide, it is particularly preferred and suitable to generate a fusion protein comprising the heterologous secretion signal peptide and an antigenic peptide or protein derived from SARS-CoV-2. Preferably, the antigenic peptide or protein, preferably the spike protein derived from SARS-CoV-2, is deleting the N-terminal endogenous secretion signal peptide (deleting aa1 to aa15). Therefore, amino acid sequences that are identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, or 22737-22758, or 22929-22964 can be modified to delete the endogenous secretory signal peptide at the positions aa1-aa15, and thus, in the context of the present invention, can be used as a "N-terminus truncated" SARS-CoV-2 protein.

[0224] List 1 below provides a more detailed description (e.g., naming, protein elements, etc.) of the preferred SARS-CoV-2 coronavirus antigenic peptides and proteins defined above.

[0225] List 1: Exemplary preferred SARS-CoV-2 antigen designs: • Full-length spike protein (S) containing aa1~aa1273; For example, see sequence numbers 1 and 274. • Stabilized S protein (S_stab_PP) containing aa1~aa1273 and K986P, V987P substitutions; For example, see sequence numbers 10 and 341. • Stabilized S protein (S_stab_PP) containing aa1~aa1273 and K986P, V987P substitutions; For example, see sequence number 22961. • Stabilized S protein (S_stab_PP) containing aa1~aa1273 and K986P, V987P substitutions; For example, see sequence number 22960. Stabilized S proteins containing aa1~aa1273 and K986P, V987P, F817P, A892P, A899P, and A942P proline substitutions; S_stab_PP_hex For example, see sequence number 22732. Stabilized S protein containing aa1~aa1273, K986P, V987P substitutions, and cavity-filling mutations (T887W, A1020W); S_stab_PP_cav For example, see sequence number 408. Stabilized S protein containing aa1~aa1273, K986P, V987P substitutions, and cavity-filling mutation (P1069F); S_stab_PP_cav For example, see sequence number 475. Stabilized S protein containing aa1~aa1273, K986P, V987P substitutions, and cavity-filling mutations (H1048Q, H1064N, H1083N, H1101N); S_stab_PP_prot For example, see Sequence ID 542. • Stabilizing S proteins containing aa1~aa1273 and artificial disulfide bonds (S_stab_disul) I712C, T1077C; For example, see sequence numbers 19, 609. • S(S_woTM) lacks a transmembrane domain including aa1~aa1211; For example, see sequence numbers 31 and 1614. • S(S_woTMflex) lacks the transmembrane domain flex, including aa1~aa1147; For example, see sequence number 2619. • S_woTM(S_stab_PP_woTM) including K986P and V987P substitutions; For example, see sequence numbers 40 and 1681. • S_woTMflex(S_stab_PP_woTMflex) includes K986P and V987P substitutions; For example, see sequence number 2686. • Spike protein fragments containing aa1~aa681(S1); For example, see sequence numbers 27 and 1279. • S_woTM containing lumazine synthase; For example, see sequence numbers 58 and 3624. • S_woTMflex containing lumazine synthase; For example, see sequence number 7644. • Contains lumazine synthase S_stab_PP_woTM; For example, see sequence numbers 85 and 3691. • Contains lumazine synthase S_stab_PP_woTMflex; For example, see sequence number 7711. • Contains ferritin element S_wo™; For example, see sequence numbers 67 and 4629. • Contains ferritin element S_woTMflex; For example, see sequence number 8649. • Contains ferritin element S_stab_PP_woTM; For example, see sequence numbers 94 and 4696. · S_stab_PP_woTMflex containing a ferritin element; For example, refer to SEQ ID NO: 8716. · S_woTM containing a folded element; For example, refer to SEQ ID NOs: 76, 5634. · S_woTMflex containing a folded element; For example, refer to SEQ ID NO: 9654. · S_stab_PP_woTM containing a folded element; For example, refer to SEQ ID NOs: 103, 5701. · S_stab_PP_woTMflex containing a folded element; For example, refer to SEQ ID NO: 9721. · S_woTM containing a VLP-sequence (WhcAg); For example, refer to SEQ ID NO: 6639. · S_woTMflex containing a VLP-sequence (WhcAg); For example, refer to SEQ ID NO: 10659. · S_stab_PP_woTM containing a VLP-sequence (WhcAg); For example, refer to SEQ ID NO: 6706. · S_stab_PP_woTMflex containing a VLP-sequence (WhcAg); For example, refer to SEQ ID NO: 10726. · truncRBD containing a folded element; For example, refer to SEQ ID NO: 22734. · truncRBD containing lumazine synthase (C-terminus); For example, refer to SEQ ID NO: 22735. · truncRBD containing lumazine synthase (N-terminus); For example, refer to SEQ ID NO: 22736. · truncRBD containing a ferritin element; For example, refer to SEQ ID NO: 22733.

[0226] The amino acid positions given in List 1 follow Reference SEQ ID NO: 1.

[0227] Preferred antigenic peptides or proteins selected from or derived from the SARS-CoV-2 coronavirus defined above are shown in Table 1 (rows 1 to 41). In the table, each of rows 1 to 41 corresponds to a suitable SARS-CoV-2 construct. Column A of Table 1 gives a brief description of the suitable SARS-CoV-2 antigen constructs. Column B of Table 1 lists the sequence numbers of the proteins (amino acids) of each SARS-CoV-2 antigen construct. Column C of Table 1 lists the sequence numbers of the corresponding wild-type or reference nucleic acid coding sequences. Column D of Table 1 lists the sequence numbers of the corresponding G / C optimized nucleic acid coding sequences (opt1, gc). Column E of Table 1 lists the sequence numbers of the corresponding human codon usage adapted nucleic acid coding sequences (opt3, human). Column F of Table 1 lists the sequence numbers of the corresponding G / C content modified nucleic acid coding sequences (opt10, gc mod).

[0228] Note that the description of the present invention explicitly includes the information given under the identifier <223> of the ST.25 sequence listing of this application. Preferred nucleic acid constructs containing the coding sequences of Table 1, for example, mRNA sequences containing the coding sequences of Table 1, are given in Tables 3A and B.

[0229] (Outer 2-1) TIFF0007848141000002.tif233170(Outer 2-2) TIFF0007848141000003.tif239170(Outer 2-3) TIFF0007848141000004.tif179170

[0230] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from SARS-CoV-2, encoded by at least one nucleic acid of component A (particularly component A-1), comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 1-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, or 26939, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the aforementioned amino acid sequences can be found in Table 1 (see rows 1-41 of columns A and B) and the ST.25 sequence listing identifiers for each sequence's sequence number. <223> It is also given under.

[0231] In a preferred embodiment, at least one antigenic peptide or protein (pre-fusion stabilizing spike protein (S_stab)) selected from or derived from SARS-CoV-2, encoded by at least one nucleic acid of component A (particularly component A-1), is 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, 133 This comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the following: 77-13510, 13521-14123, 22732, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, or 22947-22964, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the aforementioned amino acid sequences can be found in Table 1 (see rows 2-5, 12-15, 17-20, 22-25, 27-30, and 32-35 of columns A and B) and the ST.25 sequence listing identifiers for each sequence number. <223> It is also given under.

[0232] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from SARS-CoV-2 comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 10, 21, 22, 25, 27, 27, 27, 27, 34, 408, 475, 542, 74, 81

[0233] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from SARS-CoV-2 comprises at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs. 10-18, 341-407, or 22947-22964, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 1 (see row 2 of columns A and B) and the ST.25 sequence listing identifier for each sequence's SEQ ID NO. <223> It is also given under.

[0234] In a more preferred embodiment, the pre-fusion stabilizing spike protein (S_stab) comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 22960, 22961, or 22963, or an immunogenic fragment or immunogenic variant thereof.

[0235] In a particularly preferred embodiment, the pre-fusion stabilizing spike protein (S_stab) comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequences of sequence numbers 22961, or an immunogenic fragment or immunogenic variant thereof.

[0236] In a more preferred embodiment, at least one antigenic peptide or protein selected from or derived from SARS-CoV-2 comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 10 or 341, or an immunogenic fragment or immunogenic variant thereof.

[0237] In a preferred embodiment, the nucleic acid of component A (particularly component A-1) comprises at least one coding sequence encoding at least one antigenic peptide or protein, or fragments and variants thereof, derived from SARS-CoV-2 as defined above. In this context, coding sequences encoding at least one SARS-CoV-2 antigenic protein, or fragments and variants thereof, as defined herein, are understood as preferred coding sequences and may be included in the nucleic acid of component A.

[0238] In preferred embodiments, the nucleic acid of component A (particularly component A-1) comprises or comprises at least one antigenic peptide or protein selected from or derived from the SARS-CoV-2 coronavirus as defined herein, preferably at least one coding sequence, or a fragment or variant thereof, encoding one of SEQ ID NOs: 1-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, 26939. At the nucleic acid level, sequences (DNA or RNA sequences) encoding amino acid sequences, or fragments or variants thereof, that are identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 1-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, or 26939 can be selected, and are therefore understood to be preferred coding sequences of the present invention. Further information regarding the amino acid sequences can be found in Table 1 (see rows 1-41 of columns A and B), Tables 3A and B, and the ST.25 sequence listing identifiers for each sequence number. <223> It is also given under.

[0239] In a preferred embodiment, the nucleic acid of component A (particularly component A-1) is sequence numbers 116-132, 134-138, 140-143, 145-147, 148-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520, 1 4124~14177, 22759, 22764~22786, 22791~22813, 22818~22839, 22969~23184, 23189~23404, 23409~23624, 23629~23844, 23849~24064, 24069~24284, 24289~24504, 24509~2472 4. A coding sequence comprising at least one nucleic acid sequence encoding the SARS-CoV-2 antigen that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any nucleic acid sequence selected from 4, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, or 26709-26937, or a fragment or variant of any of these sequences. Further information regarding the nucleic acid sequences can be found in Table 1 (see columns C-F, rows 1-7, 9, 11-41), Tables 3A and 3B, and the ST.25 sequence listing identifiers for each sequence's sequence number. <223> It is also given under.

[0240] In a preferred embodiment, at least one coding sequence of component A nucleic acid is a codon-modified coding sequence as defined herein, and the amino acid sequence encoded by at least one codon-modified coding sequence, i.e., the SARS-CoV-2 peptide or protein, is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

[0241] The term "reference code sequence" refers to the code sequence that was the original sequence being modified and / or optimized.

[0242] In a particularly preferred embodiment, at least one coding sequence of nucleic acid component A is a codon-modified coding sequence, and the codon-modified coding sequence is a G / C optimized coding sequence, a human codon frequency-adapted coding sequence, or a G / C modified coding sequence.

[0243] In a preferred embodiment, the nucleic acid of component A (particularly component A-1) is sequence numbers 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, 24949~25164, 25 The present invention includes or comprises at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of 169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937, or a codon-modified nucleic acid sequence encoding the SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any fragment or variant of these sequences. Further information relating to each of these preferred nucleic acid sequences may be found in the sequence listing, in particular, the identifier <223> Further details can also be obtained from those given below. Preferred coding sequences of the first embodiment are provided in Table 1. Further information regarding the nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, 11-41, columns D-F), Tables 3A and 3B, and the ST.25 sequence listing identifiers for the sequence numbers of each sequence. <223> It is also given under.

[0244] In a particularly preferred embodiment, the nucleic acid of component A (particularly component A-1) is 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, 247 29~24800, 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, 26489~26560, 26597~26668, 26709~ The present invention includes or comprises at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of 26780, 26817-26888, and 26925-26937, or a G / C optimized coding sequence encoding the SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any fragment or variant of these sequences. Further information relating to each of these preferred nucleic acid sequences may be found in the sequence listing, particularly the identifier. <223> This can also be obtained from the details given below. A preferred code sequence of the first embodiment is provided in Table 1.Further information regarding the nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, 11-41 of column D), Tables 3A and 3B, and the ST.25 sequence listing identifiers for each sequence's sequence number. <223> It is also given under.

[0245] In a particularly preferred embodiment, the nucleic acid of component A (particularly component A-1) includes or comprises at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of SEQ ID NOs: 142, 143, 145, 150, 153, 157, 160, 164, 167, 171, 174, 11967-12033, 12473-12539, 12945-13011, or a human codon frequency-matched coding sequence encoding the SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any fragment or variant of these sequences. Further information regarding each of these preferred nucleic acid sequences may be found in the sequence listing, particularly the identifier <223> Further details can also be obtained from the information provided below. Preferred coding sequences of the first embodiment are provided in Table 1. Further information regarding the nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, 11-41 of column E), Tables 3A and 3B, and the ST.25 sequence listing identifiers for the sequence numbers of each sequence. <223> It is also given under.

[0246] In a particularly preferred embodiment, the nucleic acid of component A (particularly component A-1) is 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, 23149~2318 4, 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, 24909~24944, 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, 26561~26596, 26669~26704, 26781~26816, 2 The present invention comprises or includes at least one coding sequence comprising a codon-modified nucleic acid sequence selected from the group consisting of 6889 to 26924, or a G / C modified coding sequence encoding the SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any fragment or variant of these sequences. Further information relating to each of these preferred nucleic acid sequences may be found in the sequence listing, particularly the identifier. <223> Further details can also be obtained from the information provided below. Preferred coding sequences of the first embodiment are provided in Table 1. Further information regarding the nucleic acid sequences can be found in Table 1 (see rows 1-7, 9, 11-41 of column F), Tables 3A and 3B, and the ST.25 sequence listing identifiers for the sequence numbers of each sequence. <223> It is also given under.

[0247] In an even more preferred embodiment, the nucleic acid of component A (particularly, component A-1) is the codon-modified nucleic acid sequence selected from the group consisting of SEQ ID NOs: 136-138, 142, 143, 146, 147, 11731, 11798, 11804, 11805, 11808, 11810, 11811, 11812, 12035, 12049, 22759-22785, 22965-22982, 23077-23094, 23149, or a fragment or variant of any of these sequences, or is the G / C-modified coding sequence encoding a SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and includes or consists of at least one coding sequence. Further information regarding each of these preferred nucleic acid sequences can also be obtained from the details provided under the Sequence Listing, particularly identifier <223>. The preferred coding sequences of the first aspect are provided in Table 1. Further information regarding the nucleic acid sequences is also provided in Table 1 (refer to rows 1-7, 9, 11-41 of column F), Tables 3A and B, and under identifier <223> of the ST.25 Sequence Listing of the sequence numbers of each sequence.

[0248] In a particularly preferred embodiment, the nucleic acid of component A (particularly, component A-1) is the codon-modified nucleic acid sequence according to SEQ ID NO: 137, or a fragment or variant thereof, or is the G / C-modified coding sequence encoding a SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and includes or consists of at least one coding sequence.

[0249] In a particularly preferred embodiment, the nucleic acid of component A (particularly component A-1) includes or comprises at least one coding sequence comprising a G / C modified coding sequence encoding the SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the codon-modified nucleic acid sequence relating to sequence numbers 23090, 23091, 23093, 23094, or a fragment or variant thereof.

[0250] In a particularly preferred embodiment, the nucleic acid of component A (particularly component A-1) includes or comprises at least one coding sequence comprising a G / C modified coding sequence encoding the SARS-CoV-2 antigen that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the codon-modified nucleic acid sequence relating to sequence number 23091, or a fragment or variant thereof.

[0251] In further embodiments, the nucleic acid of component A (particularly component A-1) includes or comprises at least one coding sequence comprising a G / C modified coding sequence encoding the SARS-CoV-2 antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the codon-modified nucleic acid sequence relating to sequence numbers 23113, 23167, or a fragment or variant thereof.

[0252] Table 3A (columns C and D) shows preferred nucleic acid sequences of component A (particularly component A-1) containing particularly preferred mRNA sequences. In the table, each row represents a specific preferred SARS-CoV-2 construct of the present invention (compared to Table 1), a description of the SARS-CoV-2 construct is shown in column A of Table 3A, and the sequence number of the amino acid sequence of each SARS-CoV-2 construct is shown in column B. The corresponding sequence numbers of the coding sequences encoding each SARS-CoV-2 construct are shown in Table 1. Further information is provided by the sequence number identifiers of each sequence in the sequence listing. <223> It is given below.

[0253] Corresponding nucleic acids, preferably coding RNA sequences, in particular mRNA sequences containing preferred coding sequences, are shown in columns C and D, where column C shows nucleic acid sequences having the UTR combination "HSD17B4 / PSMB3" as defined herein, and column D shows nucleic acid sequences having the "alphaglobin" 3'UTR as defined herein.

[0254] (Outside 3-1) TIFF0007848141000005.tif244170(Outside 3-2) TIFF0007848141000006.tif250170(Outside 3-3) TIFF0007848141000007.tif57170

[0255] Table 3B provides more preferred nucleic acid sequences, preferably mRNA sequences, of the present invention. In the table, each column represents a specific suitable SARS-CoV-2 (nCoV-2019) construct of the present invention (compared to Tables 1 and 3B), column B represents "full-length spike protein; S" (row 1 in Tables 1 and 3A), and column C represents "stabilized spike protein; S_stab_PP" (row 2 in Tables 1 and 3A).

[0256] Row 1 shows the sequence numbers for the amino acid sequences of each SARS-CoV-2 construct. Table 1 shows the corresponding sequence numbers for the coding sequences that encode each SARS-CoV-2 construct. Further information can be found in the identifiers of each sequence number in the sequence listing. <223> Provide it below the identifier.

[0257] Corresponding nucleic acids, preferably coding RNA sequences, and in particular mRNA sequences containing preferred coding sequences, are shown in rows 2 to 16, with each row providing a combination of UTRs and a nucleic acid sequence having a preferred 3' end. (outside 4) TIFF0007848141000008.tif176170

[0258] In a preferred embodiment, the nucleic acid of component A (particularly component A-1), preferably RNA, is 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 Nucleic acid sequences selected from ~25824, 25829~26044, 26049~26264, 26269~26484, 26489~26704, 26709~26937, or nucleic acid sequences encoding the SARS-CoV-2 antigen that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any fragment or variant of these sequences. Further information regarding each nucleic acid sequence is provided by the identifier of each sequence number in the sequence listing. <223> The following is given below, as well as in Tables 3A (see columns C and D in particular) and 3B (see rows 2-16 in particular).

[0259] In a particularly preferred embodiment, component A (especially component A-1), preferably RNA, comprises or consists of a nucleic acid sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, a nucleic acid sequence encoding the SARS-CoV-2 antigen, selected from sequence numbers 162-175, 12676-13147, 14160-14177, 22813-22839, 23189-23404, or a fragment or variant of any of these sequences. Further information regarding each nucleic acid sequence is provided by the identifier of each sequence number in the sequence listing. <223> This is given below, as well as in Tables 3A (see column D in particular) and 3B (row 2).

[0260] In a particularly preferred embodiment, component A (especially component A-1), preferably RNA, comprises or consists of a nucleic acid sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, a nucleic acid sequence encoding the SARS-CoV-2 antigen, selected from sequence numbers 148-161, 12204-12675, 14142-14159, 22786-22812, 23409-23624, and 24729-24944, or a fragment or variant of any of these sequences. Further information regarding each nucleic acid sequence is provided by the identifier of each sequence number in the sequence listing. <223> The following is given below, as well as in Tables 3A (see column C in particular) and 3B (see rows 3 and 7).

[0261] In a particularly preferred embodiment, the nucleic acid of component A (particularly component A-1), preferably RNA, is 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, 2282 7, 22829, 22831, 22833, 22835, 22837, 22839, 23517-23624, 23297-23404, 24837-24944, or nucleic acid sequences encoding the SARS-CoV-2 antigen that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any fragment or variant of these sequences. Further information regarding each nucleic acid sequence is provided in the identifier of each sequence number in the sequence listing. <223> The following is given below, as well as in Table 3A (see in particular columns C and D, rows 2 and 6) and Table 3B (see column C).

[0262] In a more preferred embodiment, the nucleic acid of component A (particularly component A-1), preferably RNA, is sequence numbers 149, 156, 12338, 150, 157, 151, 158, 12541, 163, 170, 12810, 164, 171, 165, 172, 13013, 12342~12351, 12545~12554, 12814~12823, 13017~1302 6, comprising or consisting of nucleic acid sequences selected from 14133, or nucleic acid sequences encoding the SARS-CoV-2 antigen that are identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any fragment or variant of these sequences. Further information regarding each nucleic acid sequence is provided by the identifier of each sequence number in the sequence listing. <223> The following is given below, as well as in Tables 3A and 3B.

[0263] In a more preferred embodiment, component A (particularly component A-1) nucleic acid, preferably RNA, comprises or comprises a nucleic acid sequence encoding the SARS-CoV-2 antigen, which includes or comprises a nucleic acid sequence selected from SEQ ID NOs: 149, 150, 163, 164, 165, 24837, 23311, 23531, 24851, 23310, 23530, 24850, 23313, 23533, 24853, 23314, 23534, 24854, or a fragment or variant of any of these sequences. Further information regarding each nucleic acid sequence is provided by the identifier of each SEQ ID NO in the sequence listing. <223> The following is given below and in Table 3 (columns C and D, row 2).

[0264] In a particularly preferred embodiment, component A (especially component A-1), preferably RNA, comprises or consists of a nucleic acid sequence encoding the SARS-CoV-2 antigen selected from SEQ ID NO: 163 or a fragment or variant thereof.

[0265] In a particularly preferred embodiment, component A (especially component A-1), preferably RNA, comprises or consists of a nucleic acid sequence encoding the SARS-CoV-2 antigen selected from SEQ ID NO: 149 or a fragment or variant thereof.

[0266] In a particularly preferred embodiment, component A (especially component A-1), preferably RNA, comprises or consists of a nucleic acid sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the nucleic acid sequence of sequence number 24837, or a fragment or variant thereof.

[0267] In a particularly preferred embodiment, component A (especially component A-1), preferably RNA, comprises or consists of nucleic acid sequences that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the nucleic acid sequences of sequence numbers 23311, 23531, 24851, or fragments or variants thereof.

[0268] In a more preferred embodiment, the nucleic acid of component A (particularly component A-1), preferably RNA, comprises or consists of nucleic acid sequences that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the nucleic acid sequences of sequence numbers 23310, 23530, 24850, or fragments or variants thereof.

[0269] In a more preferred embodiment, component A (particularly component A-1), preferably RNA, comprises or consists of nucleic acid sequences that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the nucleic acid sequences of sequence numbers 23313, 23533, 24853, 23314, 23534, 24854, or fragments or variants of these sequences.

[0270] In a more preferred embodiment, component A (particularly component A-1), preferably RNA, comprises or consists of nucleic acid sequences that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the nucleic acid sequence of sequence number 26633 or 26907, or a fragment or variant of these sequences.

[0271] In a more preferred embodiment, the nucleic acid of component A (particularly component A-1), preferably RNA, is 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- A nucleic acid sequence selected from 25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, and 26709-26937, and a nucleic acid sequence encoding the SARS-CoV-2 antigen that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, wherein the RNA includes a cap1 structure as defined herein. Further information regarding each nucleic acid sequence is provided in the identifier of each sequence number in the sequence listing. <223> The following is given below, as well as in Tables 3A and 3B.

[0272] In further embodiments, the nucleic acid of component A (particularly component A-1), preferably RNA, is 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, 25829-26044, 26049-26264, 26269 A nucleic acid sequence selected from ~26484, 26489~26704, 26709~26937 comprises or consists of a nucleic acid sequence encoding the SARS-CoV-2 antigen that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA, wherein at least one, preferably all, uracil nucleotides in the RNA are replaced with pseudouridine (Ψ) nucleotides and / or N1-methylpseudridine (m1Ψ) nucleotides. Further information regarding each nucleic acid sequence is provided in the identifier of each sequence number in the sequence listing. <223> The following is given below, as well as in Tables 3A and 3B.

[0273] In further embodiments, the nucleic acid of component A (particularly component A-1), preferably RNA, is sequence numbers 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24 069~24284, 24289~24504, 24509~24724, 24729~24944, 24949~25164, 25169~25384, 25389~25604, 25609~25824, 25829~26044, 26049~26264, 26269~26484, 26489~ A nucleic acid sequence selected from 26704, 26709-26937, and a nucleic acid sequence encoding the SARS-CoV-2 antigen that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA, wherein the RNA comprises a cap1 structure as defined herein, and at least one, preferably all, uracil nucleotides in the RNA are replaced with pseudouridine (Ψ) nucleotides and / or N1-methylpseudridine (m1Ψ) nucleotides. Further information regarding each nucleic acid sequence is provided in the identifier of each sequence number in the sequence listing. <223> The following is given below, as well as in Tables 3A and 3B.

[0274] Preferred features and embodiments applicable to the nucleic acid of component A-1 are described in the following paragraph, “Features and Embodiments of Nucleic Acids.”

[0275] Preferably, the nucleic acid of component A-1 is compounded and / or complexed. Preferred features and embodiments applicable to the complexation of nucleic acids or the compounding of component A are described in the following "Compounding and Complexation" section.

[0276] In some embodiments, component A comprises multiple or at least one nucleic acid species, for example, DNA or RNA species as defined herein, each comprising at least one antigenic peptide or protein, or at least one coding sequence encoding an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one SARS-CoV-2. Preferably, component A as defined herein (in particular component A-1) comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acids, each as defined herein.

[0277] In some embodiments, component A may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, such as DNA or RNA as defined herein, each encoding at least one antigenic peptide or protein, or a fragment or variant thereof, derived from the same SARS-CoV-2. In particular, the (genetically) identical SARS-CoV-2 expresses (essentially) the same repertoire of proteins or peptides, and all proteins or peptides have (essentially) the same amino acid sequence. In particular, the (genetically) identical SARS-CoV-2 expresses essentially the same proteins, peptides, or polyproteins, and these proteins, peptides, or polyproteins preferably have no difference in their amino acid sequences.

[0278] In some embodiments, component A comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, e.g., DNA or RNA as defined herein, each encoding a genetically distinct SARS-CoV-2 (e.g., different SARS-CoV-2 isolates), or a fragment or variant thereof. The terms “different” or “different SARS-CoV-2” as used herein are understood as differences between at least two respective SARS-CoV-2 (e.g., different SARS-CoV-2 isolates), the differences manifesting on the genome of each different SARS-CoV-2. In particular, the (genetically) different SARS-CoV-2 may express at least one different protein, peptide, or polyprotein, the at least one different protein, peptide, or polyprotein differing by at least one amino acid.

[0279] In some embodiments, component A-1 comprises 2, 3, 4, or 5 nucleic acid species (e.g., DNA or RNA), preferably RNA species, wherein the nucleic acid species are sequence numbers 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 1 3519, 13520, 14124~14177, 22759, 22764~22786, 22791~22813, 22818~22839, 22969~23184, 23189~23404, 23409~23624, 23629~23844, 23849~24064, 24069~24284, 24289~24504, 24509~24724, 24729 A nucleic acid sequence selected from the group consisting of ~24944, 24949~25164, 25169~25384, 25389~25604, 25609~25824, 25829~26044, 26049~26264, 26269~26484, 26489~26704, 26709~26937 is identical to or at least 70%, 80%, 85%, 86%, 87%, 8 A nucleic acid sequence that is 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and optionally, at least one pharmaceutically acceptable carrier or excipient, wherein each of the 2, 3, 4, or 5 nucleic acid sequences encodes a different antigenic peptide or protein of the SARS-CoV-2 coronavirus.

[0280] Therefore, in some embodiments, component A-1 comprises two nucleic acid species (e.g., DNA or RNA), preferably RNA species, wherein the nucleic acid species are 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, and 25829. A nucleic acid sequence selected from the group consisting of ~26044, 26049~26264, 26269~26484, 26489~26704, and 26709~26937, is identical to or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence, and optionally comprises at least one pharmaceutically acceptable carrier or excipient, wherein each of the two nucleic acid sequences encodes a different antigenic peptide or protein of the SARS-CoV-2 coronavirus.

[0281] Therefore, in some embodiments, component A-1 comprises three nucleic acid species (e.g., DNA or RNA), preferably RNA species, wherein the nucleic acid species are 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, 25829-260 A nucleic acid sequence selected from the group consisting of 44, 26049-26264, 26269-26484, 26489-26704, and 26709-26937 is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence, and optionally at least one pharmaceutically acceptable carrier or excipient, wherein each of the 2, 3, 4, or 5 nucleic acid species encodes a different antigenic peptide or protein of the SARS-CoV-2 coronavirus.

[0282] Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species of component A-1 each encode a different pre-fusion stabilization spike protein (as defined in the first embodiment). Preferably, pre-fusion conformation stabilization is achieved by introducing two consecutive proline substitutions at residues K986 and V987 of the spike protein (amino acid positions related to reference sequence number 1). Thus, in preferred embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 pre-fusion stabilization spike proteins (S_stab) each contain at least one pre-fusion stabilization mutation, the at least one pre-fusion stabilization mutation containing the following amino acid substitutions: K986P and V987P (amino acid positions related to reference sequence number 1).

[0283] Preferably, the different spike proteins or pre-fusion stabilizing spike proteins are derived from at least B.1.1.7, B.1.351, P.1, or CAL.20C.

[0284] Preferably, different spike proteins or pre-fusion stabilization spike proteins have amino acid changes in the S protein, including the following: (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; and / or (v)S13I, W152C, L452R, and D614G.

[0285] Therefore, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different nucleic acid species of component A-1 each encode a different pre-fusion stabilization spike protein, and 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stabilization spike proteins are sequence numbers 10-26, 341-407, 609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22 The amino acid sequences selected from those that are identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the following: 746, 22748, 22750, 22752, 22754, 22756, 22758, 22947-22964, or immunogenic fragments or immunogenic variants thereof.

[0286] In a preferred embodiment, component A-1 comprises two, three, four, or five nucleic acid species, each containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of sequence numbers 10, 22961, 22960, 22963, 22941, or 22964, an amino acid sequence.

[0287] In a preferred embodiment, component A-1 comprises one nucleic acid species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences in Sequence ID No. 10, and the polyvalent composition further comprises at least two, three, or four additional RNA species selected from the following: i) A nucleic acid species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the amino acid sequences in Sequence ID No. 22961; and / or ii) A nucleic acid species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the amino acid sequences of sequence number 22960; and / or iii) A nucleic acid species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the amino acid sequences in Sequence ID No. 22963; and / or iv) A nucleic acid species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the amino acid sequences of sequence number 22941; and / or v) A nucleic acid species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of sequence numbers 22964.

[0288] Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different nucleic acid species of component A each contain nucleic acid coding sequences encoding different pre-fusion stabilization spike proteins, and the 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid coding sequences include SEQ ID NOs: 136-138, 140-143, 145-175, 11731-11813, 11815, 11817-12050, 12052, 12054-1 Nucleic acid sequences that are identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of 2203, 13514, 13515, 13519, 13520, 14124-14141, 22759, 22764-22785, or 22969-23184, or selected from fragments or variants of any of these.

[0289] In a preferred embodiment, component A-1 comprises one nucleic acid species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequences in Sequence ID No. 137, and the polyvalent composition further comprises at least two, three, or four additional RNA species selected from the following: i) A nucleic acid species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequence numbers 23091; and / or ii) A nucleic acid species comprising a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 23090; and / or iii) A nucleic acid species comprising a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 23093; and / or iv) A nucleic acid species comprising a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 22999; and / or v) A nucleic acid species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 23094.

[0290] Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different nucleic acid species of component A-1 each contain nucleic acid coding sequences encoding different pre-fusion stabilization spike proteins, and the 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid coding sequences include SEQ ID NOs: 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, 2282 RNA sequences that are identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the following: 9, 22831, 22833, 22835, 22837, 22839, 23523, 23525, 23527, 23529, 23530, 23589, 23737, 23957, 24397, 24837, 25057, 25277, 25717, or 26925-26937, or fragments or variants thereof, are selected from these.

[0291] In a preferred embodiment, component A-1 comprises or comprises one RNA species that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the RNA sequences in Sequence ID No. 163, and the polyvalent composition further comprises i) A single RNA species comprising or consisting of an RNA sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequence numbers 23311; and / or ii) A single RNA species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 23310; and / or iii) A single RNA species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 23313; and / or iv) A single RNA species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 23219; and / or v) comprising at least two, three, or four further RNA species selected from one RNA species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 23314, Preferably, each mRNA species contains a Cap1 structure, and optionally, each mRNA species does not contain modified nucleotides.

[0292] In a preferred embodiment, component A-1 comprises or comprises one RNA species that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 149 or 24837, and the polyvalent composition further comprises i) an RNA species comprising or consisting of an RNA sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 23531 or 24851; and / or ii) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NO: 23530 or 24850; and / or iii) A single RNA species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 23533 or 24853; and / or iv) A single RNA species containing a coding sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, either SEQ ID NO: 23439 or 24759; and / or v) comprising at least two, three, or four further RNA species selected from one RNA species containing a coding sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 23534 or 24854, Preferably, each mRNA species contains a Cap1 structure, and optionally, each mRNA species does not contain modified nucleotides.

[0293] In a more preferred embodiment, component A-1 includes at least two RNA sequences that are identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 149 or 24837, 23531 or 24851, 23530 or 24850, 23533 or 24853, 23439 or 24759, or 23534 or 24854.

[0294] In a preferred embodiment, component A-1 comprises at least one nucleic acid encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one SARS-CoV-2, wherein component A is preferably administered intramuscularly or intradermally.

[0295] Preferably, intramuscular or intradermal administration of component A-1 results in the expression of the encoded SARS-CoV-2 antigen construct in the subject. In embodiments where the nucleic acid is RNA, administration of component A results in the translation of RNA and the generation of the encoded SARS-CoV-2 antigen in the subject. In embodiments where the nucleic acid is DNA (e.g., plasmid DNA, adenovirus DNA), administration of the composition results in the transcription of DNA to RNA in the subject, followed by the translation of the RNA into the encoded SARS-CoV-2 antigen.

[0296] In some embodiments, administration of a pharmaceutical composition containing component A-1 to a subject induces neutralizing antibodies against SARS-CoV-2, but not disease-enhancing antibodies. In particular, administration of a pharmaceutical composition containing component A-1, which encodes the pre-fusion stabilizing spike protein of SARS-CoV-2, to a subject does not induce immunopathological effects such as disease enhancement and / or antibody-dependent enhancement (ADE).

[0297] In a preferred embodiment, administration of component A-1 induces an antigen-specific immune response, including a T-cell response and / or B-cell response to an encoded SARS-CoV-2 antigen, provided by at least one nucleic acid of component A-1.

[0298] Preferably, component A-1 is suitable for vaccines, particularly for SARS-CoV-2 vaccines, and preferably for the mixed vaccine of the present invention.

[0299] In some embodiments, the nucleic acid contained in component A-1 as defined herein is provided in amounts of about 100 ng to about 500 μg, about 1 μg to about 200 μg, about 1 μg to about 100 μg, about 5 μg to about 100 μg, preferably about 10 μg to about 50 μg, specifically about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg.

[0300] If component A-1 contains multiple or at least one nucleic acid species as defined herein, the amount of nucleic acid of each nucleic acid species is approximately 100 ng to approximately 500 μg, approximately 1 μg to approximately 200 μg, approximately 1 μg to approximately 100 μg, approximately 5 μg to approximately 100 μg, preferably approximately 10 μg to approximately 50 μg, specifically approximately 1 μg, 2 μg, and 3 μg. It is provided in amounts of 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12μg, 13μg, 14μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, or 100μg.

[0301] In some embodiments, the nucleic acid amounts of each nucleic acid species are essentially equal in mass. In other embodiments, the nucleic acid amounts of each nucleic acid species are selected to be equimolar.

[0302] Preferred embodiments of component A1 are provided below in the form of a list of items (see below).

[0303] Item 1: Component A-1 is, (a) At least one coding sequence encoding a SARS-CoV-2 spike protein (S), which is a pre-fusion stabilization spike protein (S_stab) containing at least one pre-fusion stabilization mutation, such as SEQ ID NOs: 10, 341, 22960, 22961, 22963, preferably at least 90% identical to SEQ ID NO: 10; (b) at least one heterogeneous untranslated region (UTR); and (c) at least one pharmaceutically acceptable carrier, A composition containing mRNA, which includes The mRNA is complexed or associated with lipid nanoparticles (LNPs), and the LNPs are (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analogue; and (iv) comprising at least one PEG-lipid, The molar ratios of (i) to (iv) relate to compositions in which approximately 20-60% cationic lipids, 5-25% neutral lipids, 25-55% sterols, and 0.5-15% PEG lipids are present. Item 2: Component A-1 according to Item 1, wherein the mRNA comprises at least one poly(A) sequence preferably containing 30 to 200 adenosine nucleotides and / or at least one poly(C) sequence preferably containing 10 to 40 cytosine nucleotides. Item 3: Component A-1 according to Item 1 or 2, wherein the mRNA comprises a 5' cap structure, preferably m7G, cap0, cap1, cap2, modified cap0, or modified cap1 structure. Item 4: Component A-1 as described in any of Items 1 to 3, wherein the S protein contains pre-fusion stabilization K986P and V987P mutations. Item 5: Component A-1 according to any one of Items 1 to 4, wherein the at least one code sequence is a codon-modified code sequence, and the at least one codon-modified code sequence is selected from a C-maximizing code sequence, a CAI-maximizing code sequence, a human codon frequency-adapted code sequence, a G / C content-modified code sequence, and a G / C-optimized code sequence, or any combination thereof. Item 6: Component A-1 according to any one of items 1 to 5, wherein the at least one coding sequence has a G / C content that is at least 10%, 20%, or 30% higher than the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence. Item 7: Component A-1 according to any one of items 1 to 6, wherein the at least one code sequence has a G / C content of at least about 50%, 55%, or 60%, preferably about 63.9%. Item 8: Component A-1 according to any one of items 1 to 7, wherein the mRNA contains a sequence that is at least 90% identical to sequence numbers 149, 163, 24837, 26633, 26907, preferably sequence number 163. Item 9: Component A-1 according to any one of items 1 to 8, wherein the at least one heterogeneous untranslated region is selected from at least one heterogeneous 5'-UTR and / or at least one heterogeneous 3'-UTR. Item 10: Component A-1 as described in Item 9, wherein the at least one heterologous 3'-UTR is derived from the 3'-UTR of a gene selected from PSMB3, ALB7, alphaglobin (referred to as "muag"), CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or comprises a nucleic acid sequence derived from an analog, fragment, or variant of any of these genes. Item 11: Component A-1 according to Item 9, comprising or comprising a nucleic acid sequence derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or derived from an analog, fragment, or variant of any of these genes. Item 12: Component A-1 according to any one of items 1 to 11, wherein the nucleic acid comprises at least one histone stem loop. Item 13: The mRNA comprises Component A-1 as described in any of Items 1 to 12, wherein the mRNA includes a nucleotide analog. Item 14: Component A-1 as described in any of items 1 to 13, wherein the mRNA does not contain a 1-methylpseudridine substitution. Item 15: Component A-1 according to any one of items 1 to 14, wherein the mRNA has RNA integrity of at least about 50%, preferably at least about 60%, more preferably at least about 70%, and most preferably at least about 80%. Item 16: Component A-1 as described in any of items 1 to 15, wherein the mRNA is purified mRNA obtained by RP-HPLC and / or TFF. Item 17: Component A-1 as described in any of items 1 to 16, wherein the mRNA is purified by RP-HPLC and / or TFF and contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts than RNA that has not been purified by RP-HPLC and / or TFF. Item 18: The LNP is given by Equation III: [ka] (In the formula, L 1 or L 2 These are, independently, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, or -NRaC(=O)O-, preferably L 1 Or L 2 is -O(C=O)- or -(C=O)O-; G 1 and G 2 These are, independently, unsaturated C1-C12 alkylenes or C1-C12 alkenylenes; G 3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R 1 and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R 3 is H, OR5, CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR5C(=O)R 4 ; R 4 is C1-C12 alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2.) Component A-1 according to any one of items 1 to 17, comprising a cationic lipid represented by the formula, or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof. Item 19: The LNP is of formula III-3:

Chemical formula

Chemical formula

[0304] Component A-2: SARS-related virus In a particularly preferred embodiment, at least one coronavirus of component A is a SARS-related virus, preferably a SARS-CoV-1 virus (also referred to as component A-2).

[0305] Accordingly, in the embodiment of the first aspect, the nucleic acid of component A includes at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from at least one SARS-related virus, preferably SARS-CoV-1, or an immunogenic fragment or immunogenic variant thereof.

[0306] It is understood that the general embodiments and features described in the paragraph for "Component A" may also be applicable to nucleic acids encoding SARS-CoV-1 antigenic peptides or proteins.

[0307] SARS-CoV-related viruses belong to the Coronaviridae family, particularly the orthocoronavirus, and more specifically the Betacoronavirus genus. SARS-CoV-1 (Severe Acute Respiratory Syndrome Coronavirus, SARS Coronavirus, SCV) causes Severe Respiratory Syndrome (SARS). An exemplary SARS-CoV-1 coronavirus can be identified by NCBI classification ID: 694009, NCBI reference: DQ182595.1.

[0308] Further preferred SARS-related viruses in the context of the present invention are SARS-CoV / Tor2, HCoV / OC43, HCoV / HKU1 / N5, HCoV / 229E / BN1 / GER / 2015, HCoV / NL63 / RPTEC / 2004, Bat SARS-like CoV / WIV1, BatCoV / HKU9-1 BF_005I, PDCoV / pig / thai / S5011 / 2015, PEDV / NPL-PEDv / 2013 / P10, PEDV / NPL-PEDv / 2013 / P10, or MHV / S.

[0309] In the context of the present invention, any protein selected from or derived from a SARS-related virus, preferably SARS-CoV-1, can be used in the context of the present invention and can be suitably encoded by the coding sequence or nucleic acid of component A (particularly component A-2). Furthermore, it is within the scope of the underlying invention that at least one antigenic peptide or protein may include or consist of a synthetically engineered or artificial SARS-CoV-1 peptide or protein. The terms “synthetically engineered” SARS-CoV-1 peptide or protein, or “artificial coronavirus peptide or protein,” refer to proteins that do not exist in nature. Therefore, “artificial coronavirus peptide or protein” or “synthetically engineered coronavirus peptide or protein” may differ from, for example, a naturally occurring SARS-CoV-1 peptide or protein by at least one amino acid, and / or may contain additional heterologous peptide or protein elements, and / or may have an elongated or truncated N-terminus or C-terminus.

[0310] In a preferred embodiment, the nucleic acid of component A comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from a SARS-related virus, preferably SARS-CoV-1, or an immunogenic fragment or immunogenic variant thereof, wherein the at least one antigenic peptide or protein comprises at least one peptide or protein selected from or derived from a structural protein, accessory protein, or replicase protein, or any immunogenic fragment or immunogenic variant thereof.

[0311] Preferably, the structural protein is selected from or derived from a spike protein (S), an envelope protein (E), a membrane protein (M), or a nucleocapsid protein (N), or an immunogenic fragment or variant thereof.

[0312] In a particularly preferred embodiment of the pharmaceutical composition, the encoded antigenic peptide or protein of component A (particularly component A-2) comprises or consists of at least one peptide or protein selected from or derived from SARS-related virus, preferably SARS-CoV-1 spike protein (S, S1, S2, or S1 and S2), or immunogenic fragments or immunogenic variants thereof.

[0313] Suitable antigenic peptide or protein sequences provided by the nucleic acid of component A (particularly component A-2) are disclosed in Table 4, rows 1-45, columns A and B. Further information regarding the aforementioned suitable antigenic peptide or protein sequences selected from or derived from SARS-related viruses, preferably SARS-CoV-1, is provided in the ST.25 sequence listing identifier. <223> It is given below.

[0314] The following describes in detail preferred antigenic peptides or protein sequences selected from or derived from SARS-CoV-1, which are provided by the nucleic acid of component A.

[0315] Amino acid level changes can spontaneously occur between spike proteins derived from different SARS isolates. In the context of the present invention, such amino acid changes can be applied to each antigenic peptide or protein derived from the SARS-related virus or SARS-CoV-1 spike protein described herein.

[0316] In some embodiments, a fragment of a SARS-related virus, preferably a SARS-CoV-1 spike protein (S), may be encoded by the nucleic acid of component A, the fragment may have its N-terminus truncated, and / or the fragment may have its C-terminus truncated. Such “spike protein (S) fragment” may further include amino acid substitutions (described below) and may further include at least one heterologous peptide or protein element (described below).

[0317] In some embodiments, the at least one antigenic peptide or protein encoded by component A comprises or consists of a SARS-related virus, preferably SARS-CoV-1 spike protein (S), wherein the spike protein (S) lacks a transmembrane domain. While we do not wish to be bound by theory, SARS-CoV-1 lacking the transmembrane domain (TM) as defined herein may be suitable for vaccines because such protein is soluble and not immobilized on the cell membrane. Therefore, when administered to a subject, a higher concentration of soluble protein may be produced (translated), potentially leading to an improved immune response.

[0318] While we do not wish to be bound by theory, the RBD and CND domains may be crucial for the immunogenicity of SARS-related viruses, preferably the SARS-CoV-1 spike protein (S). Both regions are located in the S1 fragment of the spike protein. Therefore, in the context of the present invention, it may be preferable that the antigenic peptide or protein contains or consists of the S1 fragment of the spike protein, or its immunogenic fragment or immunogenic variant.

[0319] Preferably, the S1 fragment of a SARS-related virus, preferably SARS-CoV-1, may contain at least the RBD and / or CND domains, as defined above.

[0320] In a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A (particularly component A-2) comprises or consists of a SARS-related virus, preferably SARS-CoV-1 spike protein (S), wherein the spike protein (S) comprises or consists of a spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof.

[0321] In a preferred embodiment, the encoded antigenic peptide or protein comprises a SARS-related virus, preferably a SARS-CoV-1 spike protein fragment S1, and is missing at least 70%, 80%, 90%, preferably 100%, of the spike protein fragment S2. Such embodiments may be beneficial because the SARS-CoV-1 S1 fragment contains a neutralizing epitope, without the potential problems associated with full-length proteins containing S1 and S2.

[0322] While we do not wish to be bound by theory, it may be preferable that the antigenic peptide or protein of component A (particularly component A-2) contains or consists of SARS-related viruses, preferably SARS-CoV-1 spike protein fragment S1 and SARS-CoV-1 spike protein fragment S2 (or at least fragments thereof), since the formation of immunogenic spike proteins may be promoted.

[0323] Therefore, in a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A (particularly component A-2) comprises or consists of a SARS-related virus, preferably a SARS-CoV-1 spike protein fragment (S), wherein the spike protein fragment (S) comprises or consists of a spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof, and a spike protein fragment S2, or an immunogenic fragment or immunogenic variant thereof.

[0324] In a particularly preferred embodiment, at least one antigenic peptide or protein encoded by component A (particularly component A-2) comprises or consists of a full-length SARS-related virus, preferably the SARS-CoV-1 spike protein, or an immunogenic fragment or immunogenic variant thereof.

[0325] In a particularly preferred embodiment, the SARS-related virus, preferably the SARS-CoV-1 spike protein fragment (S), provided by the nucleic acid of component A (component A-2), is designed or adapted to stabilize the antigen in a pre-fusion conformation. The pre-fusion conformation is particularly advantageous in the context of an efficient SARS-CoV-1 vaccine because the pre-fusion protein conformation contains several potential and accessible epitopes for neutralizing antibodies. Furthermore, the presence of protein in the pre-fusion conformation is intended to avoid immunopathological effects, such as disease exacerbation and / or antibody-dependent enhancement (ADE).

[0326] Therefore, in a preferred embodiment, the nucleic acid of component A (component A-2) comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from a SARS-related virus, preferably SARS-CoV-1, wherein the at least one antigenic peptide or protein is selected from or derived from a spike protein (S), and the spike protein (S) is a pre-fusion stabilization spike protein (S_stab). Preferably, the pre-fusion stabilization spike protein comprises at least one pre-fusion stabilization mutation.

[0327] Stabilization of the SARS-CoV-1 spike protein can be achieved by substituting at least one amino acid at position K968 and / or V969 with an amino acid that stabilizes the spike protein in the pre-fusion conformation (amino acid positions related to reference sequence number 14906).

[0328] In some embodiments, the prefusion stabilization mutation of the SARS-CoV-1 spike protein includes an amino acid substitution at position K968, where amino acid K968 is substituted with one selected from A, I, L, M, F, V, G, or P (the amino acid position related to reference sequence number 14906), preferably amino acid K968 is substituted with P. In some embodiments, the prefusion stabilization mutation includes an amino acid substitution at position V969, where amino acid V969 is substituted with one selected from A, I, L, M, F, V, G, or P (the amino acid position related to reference sequence number 14906), preferably amino acid V969 is substituted with P.

[0329] Therefore, in a preferred embodiment, the SARS-CoV-1 pre-fusion stabilization spike protein (S_stab) comprises at least one pre-fusion stabilization mutation, the at least one pre-fusion stabilization mutation comprising the following amino acid substitutions: K968P and V969P (amino acid positions corresponding to reference sequence number 14906).

[0330] A person skilled in the art can select SARS-related viral proteins or fragments or variants thereof to introduce such amino acid changes (e.g., such double proline mutations). For example, HCoV / OC43 spike protein (1-1353) (A1070P_L1071P), HCoV / OC43 / 1783A_10 spike protein (1-1362) (A1079P_L1080P), HCoV / HKU1 / N5 spike protein (1-1351) (N1067P_L1068P), HCoV / 229E / BN1 / GER / 2015 spike protein (1-1171) (I869P_I870P), HCoV / NL63 / RPTEC / 2004 spike protein (1-1356) (S1052P_I1053P), Bat Examples include SARS-like CoV / WIV1 spike protein (1-1256) (K969P_V970P), BatCoV / HKU9-1 BF_005I spike protein (1-1274) (G983P_L984P), PDCoV / pig / thai / S5011 / 2015 spike protein (1-1160) ((E855P_V856P), PEDV / NPL-PEDv / 2013 / P10 spike protein (1-1386) (I1076P_L1077P), and MHV / S spike protein (1-1361) (A1073P_L1074P).

[0331] In some embodiments, at least one pre-fusion stabilizing mutation of a SARS-related virus, preferably the SARS-CoV-1 spike protein, includes a cavity-filling mutation.

[0332] In some embodiments, at least one pre-fusion stabilizing mutation of a SARS-related virus, preferably the SARS-CoV-1 spike protein, includes a mutated protonation site.

[0333] In some embodiments, at least one pre-fusion stabilizing mutation of the SARS-related virus, preferably the SARS-CoV-1 spike protein, includes an artificial intramolecular disulfide bond. Introducing such an artificial intramolecular disulfide bond can further stabilize the membrane distal portion (including the N-terminal region) of the S protein of the pre-fusion conformation, i.e., the conformation that specifically binds to one or more pre-fusion specification antibodies, and / or the conformation that presents a preferred antigen site present in the pre-fusion conformation but not in the post-fusion conformation of the S protein.

[0334] A SARS-related virus, preferably a SARS-related virus, preferably a SARS-CoV-1 S protein or a fragment or variant thereof, can be selected by those skilled in the art to introduce at least one cavity-filling mutation, at least one mutant protonation site, and / or at least one artificial intramolecular disulfide bond.

[0335] According to various preferred embodiments, the nucleic acid of component A (particularly component A-2) encodes at least one antigenic peptide or protein selected from or derived from SARS-related viruses, preferably SARS-CoV-1 as defined herein, and further preferably at least one heterologous peptide or protein element selected from or derived from signal peptides, linkers, helper epitopes, antigen clustering elements, trimerizing elements, transmembrane elements, and / or VLP-forming sequences.

[0336] Preferably, at least one heterologous peptide or protein element can promote or improve the secretion (e.g., via a secretion signal sequence) of the SARS-related virus-encoded antigenic peptide or protein, promote or improve the fixation of the encoded antigenic peptide or protein on the plasma membrane (e.g., via a transmembrane element), promote or improve the formation of an antigen complex (e.g., via a multimerization domain or antigen clustering element), or promote or improve the formation of virus-like particles (VLP-forming sequences). Furthermore, the nucleic acid of component A (particularly component A-2) can further encode a peptide linker element, a self-cleaving peptide, an immunoadjuvant sequence, or a dendritic cell target sequence.

[0337] In a preferred embodiment, the nucleic acid of component A (particularly component A-2) encoding at least one antigenic protein selected from or derived from a SARS-related virus, preferably SARS-CoV-1 as defined herein, further encodes at least one heterotrimerizing element, an antigen clustering element as defined herein, or a VLP-forming sequence as defined herein.

[0338] The antigen clustering element can be selected from the ferritin element, the lumazine synthase element, the hepatitis B virus (HBsAg) surface antigen, or the capsule phosphorus as defined herein. In embodiments in which the coding sequence of component A (particularly component A-2) further codes for a heterologous antigen clustering element, it is particularly preferable to generate a fusion protein comprising the antigen clustering element and an antigenic peptide or protein derived from SARS-CoV-1. Preferably, the antigenic peptide or protein, preferably the SARS-CoV-1 spike protein, is deleting the C-terminal transmembrane domain (TM) or a portion of the TM. Furthermore, it may be preferable to use a defined linker element to separate the heterologous antigen clustering element from the antigenic peptide or protein.

[0339] The trimerizing element can be selected from the Foldon elements defined herein. In a preferred embodiment, the Foldon element is a fibrin-foldon element as defined herein. In embodiments in which the coding sequence of the nucleic acid of component A (particularly component A-2) further codes for a heterologous trimerizing element, it is particularly preferred and suitable to generate a fusion protein comprising the trimerizing element and an antigenic peptide or protein derived from a SARS-related virus, preferably SARS-CoV-1. Preferably, the antigenic peptide or protein is a spike protein derived from a SARS-related virus, preferably SARS-CoV-1 lacking a portion of the C-terminal transmembrane domain or TM. Furthermore, it may be preferable to use a defined linker element to separate the heterologous antigen clustering element from the antigenic peptide or protein.

[0340] A VLP-forming sequence can be selected and fused to a SARS-related virus, preferably a SARS-CoV-1 antigen, as defined herein. Suitable VLP-forming sequences can be selected from elements derived from the hepatitis B virus core antigen, the HIV-1 Gag protein, or the woodchuck hepatitis core antigen element (WhcAg) as defined herein. In embodiments in which the coding sequence of the nucleic acid of component A (particularly component A-2) further codes for a heterologous VLP-forming sequence, it is particularly preferable to generate a fusion protein comprising the VLP-forming sequence and an antigenic peptide or protein derived from SARS-CoV-1. Preferably, the antigenic peptide or protein is a SARS-CoV-1 spike protein lacking a C-terminal transmembrane domain (TM) or a portion of the TM. Furthermore, it may be preferable to use a linker element as defined herein to separate the heterologous antigen clustering element from the antigenic peptide or protein.

[0341] In some embodiments, the antigenic peptide or protein comprises the heterologous signal peptide defined above. The heterologous signal peptide can be used to improve the secretion of the encoded SARS-related virus, preferably SARS-CoV-1 antigen. In embodiments in which the coding sequence of the nucleic acid of component A (particularly component A-2) further encodes a heterologous secretion signal peptide, it is particularly preferable to produce a fusion protein comprising the heterologous secretion signal peptide and an antigenic peptide or protein derived from the SARS-related virus, preferably SARS-CoV-1. Preferably, the antigenic peptide or protein, preferably a spike protein derived from the SARS-related virus, preferably SARS-CoV-1, is deleting the N-terminal endogenous secretion signal peptide.

[0342] Table 4 (rows 1-45) shows preferred antigenic peptides or proteins selected from or derived from SARS-related viruses, preferably SARS-CoV-1 as defined above. In the table, each row from 1 to 45 corresponds to a preferred SARS-CoV-1 construct or SARS-related virus construct. Column A of Table 4 provides a brief description of the preferred antigen construct. Column B of Table 4 lists the sequence numbers of the proteins (amino acids) of each antigen construct. Column D of Table 4 lists the sequence numbers of the corresponding G / C optimized nucleic acid coding sequences (opt1, gc). Column E of Table 4 lists the sequence numbers of the corresponding human codon frequency-matched nucleic acid coding sequences (opt3, human).

[0343] The description of this invention refers to the identifiers in the ST.25 sequence listing of this application. <223> The information provided below is explicitly included. Preferred nucleic acid constructs containing the coding sequences of Table 4, for example, mRNA sequences containing the coding sequences of Table 4, are given in Table 5.

[0344] (outside 5) TIFF0007848141000012.tif230170

[0345] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from a SARS-related virus, preferably SARS-CoV-1, encoded by at least one nucleic acid of component A (particularly component A-2), comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 14906 to 14950, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 4 (see rows 1-45 of columns A and B) and the ST.25 sequence listing identifier for each sequence number. <223> It is also given under.

[0346] In further embodiments, at least one antigenic peptide or protein selected from or derived from SARS-CoV-1 encoded by at least one nucleic acid of component A (in particular component A-2) comprises or consists of at least one amino acid sequence that is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence numbers 29, 32, or 34 of published PCT patent application WO2017070626, or an immunogenic fragment or immunogenic variant thereof, the corresponding disclosure relating thereto is incorporated herein by reference.

[0347] In further embodiments, at least one antigenic peptide or protein selected from or derived from SARS-CoV-1, encoded by at least one nucleic acid of component A (particularly component A-2), comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 30 of published PCT patent application WO2018081318, or an immunogenic fragment or immunogenic variant thereof. SEQ ID NO: 7 or 30 of WO2018081318 and the corresponding disclosure relating thereto are incorporated herein by reference.

[0348] More suitable antigenic peptides or proteins selected from or derived from SARS-CoV-1 may be selected from or derived from Table 12 of WO2017070626. Accordingly, the entire contents of Table 12 of WO2017070626 are incorporated herein by reference.

[0349] In a preferred embodiment, the at least one antigenic peptide or protein (pre-fusion stabilizing spike protein (S_stab)) selected from or derived from a SARS-related virus, preferably SARS-CoV-1, encoded by at least one nucleic acid of component A (particularly component A-2), comprises or consists of at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 14907, 14910, 14914, 14916, 14920, 14924, 14928, 14932, 14936, 14940, 14944, or 14948, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the aforementioned amino acid sequences can be found in Table 4 and the ST.25 sequence listing identifiers for each sequence. <223> It is also given under.

[0350] In a preferred embodiment, the nucleic acid of component A (particularly component A-2) comprises at least one coding sequence encoding at least one antigenic peptide or protein, or fragments and variants thereof, derived from the SARS-related virus as defined above, preferably SARS-CoV-1. In this context, coding sequences encoding at least one SARS-related virus as defined herein, preferably SARS-CoV-1 antigenic protein, or fragments and variants thereof, are understood as preferred coding sequences and may be included in the nucleic acid of component A.

[0351] In preferred embodiments, the nucleic acid of component A (particularly component A-2) comprises or consists of at least one antigenic peptide or protein selected from or derived from SARS-related viruses as defined herein, preferably any of SEQ ID NOs: 14906-14950; SEQ ID NOs: 1-152, 1448-1548 of WO2018115527; SEQ ID NOs: 29, 32, or 34 of WO2017070626; SEQ ID NOs: 7 or 30 of WO2018081318, or at least one coding sequence encoding a fragment or variant thereof. At the nucleic acid level, sequences (DNA or RNA sequences) encoding amino acids that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of the following: SEQ ID NOs. 14906-14950; SEQ ID NOs. 1-152, 1448-1548 of WO2018115527; SEQ ID NOs. 29, 32, or 34 of WO2017070626; or SEQ ID NOs. 7 or 30 of WO2018081318, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or fragments or variants thereof, can be selected and thus understood to be preferred coding sequences of the present invention. Further information regarding the above amino acid sequences can be found in Table 4 (see rows 1-45 of columns A and B), Table 5, and the ST.25 sequence listing identifiers for each sequence's SEQ ID NO. <223> It is also given under.

[0352] In a preferred embodiment, the nucleic acid of component A (particularly component A-2) includes a coding sequence comprising at least one nucleic acid sequence encoding a SARS-related virus antigen, preferably a SARS-CoV-1 antigen, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any nucleic acid sequence selected from sequence numbers 14951 to 15220, or a fragment or variant of any of these sequences. Further information regarding the nucleic acid sequences can be found in Table 4 (see rows 1 to 45), Table 5, and the ST.25 sequence listing identifiers for each sequence's sequence number. <223> It is also given under.

[0353] In preferred embodiments, the nucleic acid of component A (particularly component A-2) is such that at least one coding sequence of component A (particularly component A-2) is a codon-modified coding sequence as defined herein, and the amino acid sequence encoded by at least one codon-modified coding sequence, i.e., the SARS-related peptide or protein, is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

[0354] In a particularly preferred embodiment, at least one coding sequence of nucleic acid component A (particularly component A-2) is a codon-modified coding sequence, and the codon-modified coding sequence is a G / C optimized coding sequence, a human codon frequency-adapted coding sequence, or a G / C modified coding sequence.

[0355] Table 5 (columns C and D) shows preferred nucleic acid sequences of component A (particularly component A-2) containing particularly preferred mRNA sequences. In the table, each row represents a specific preferred SARS-related virus construct of the present invention (compared to Table 4), a description of the SARS-related virus construct is shown in column A of Table 5, and the amino acid sequence number of each SARS-related virus construct is shown in column B. The corresponding sequence numbers of the coding sequences encoding each SARS-related virus construct are shown in Table 4. Further information is provided in the identifiers of each sequence number in the sequence listing. <223> It is given below.

[0356] (Outside 6-1) TIFF0007848141000013.tif248170(Outside 6-2) TIFF0007848141000014.tif11170

[0357] In a preferred embodiment, component A (particularly component A-2), preferably RNA, comprises or consists of a nucleic acid sequence selected from sequence numbers 15041 to 15220, or a nucleic acid sequence encoding a SARS-related virus antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any fragment or variant of these sequences. Further information regarding each nucleic acid sequence is provided by the sequence number identifier of each sequence in the sequence listing. <223> The following are also given below and in Table 5 (see columns C and D). Optionally, the nucleic acid sequence comprises a cap1 structure as defined herein, and / or at least one, preferably all, uracil nucleotides in the RNA sequence are replaced with pseudouridine (Ψ) nucleotides and / or N1-methylpseudridine (m1Ψ) nucleotides.

[0358] Preferred features and embodiments applicable to the nucleic acid of component A-2 are described in the following paragraph, “Features and Embodiments of Nucleic Acids.”

[0359] Preferably, the nucleic acid of component A-2 is compounded and / or complexed. Preferred features and embodiments applicable to the complexation of nucleic acids or the compounding of component A are described in the following "Compounding and Complexation" section.

[0360] In some embodiments, component A (particularly component A-2) comprises multiple or more than one nucleic acid species, e.g., DNA or RNA species as defined herein, each containing at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one SARS-related virus, preferably SARS-CoV-1. Preferably, component A (particularly component A-2) as defined herein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acids, each as defined herein.

[0361] In some embodiments, component A (in particular component A-2) may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, such as DNA or RNA as defined herein, each encoding at least one antigenic peptide or protein, or a fragment or variant thereof, derived from the same SARS-related virus. In particular, the (genetically) identical SARS-related viruses express an (essentially) identical repertoire of proteins or peptides, all of which have (essentially) identical amino acid sequences. In particular, the (genetically) identical SARS-related viruses express essentially identical proteins, peptides, or polyproteins, preferably these proteins, peptides, or polyproteins that are identical in their amino acid sequences.

[0362] In some embodiments, component A (in particular, component A-2) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, e.g., DNA or RNA as defined herein, each encoding at least one peptide or protein, or a fragment or variant thereof, derived from a genetically distinct SARS-related virus (e.g., different SARS-related virus isolates). The terms “different” or “different SARS-related viruses” as used herein are understood as differences between at least two respective SARS-related viruses (e.g., different SARS-related virus isolates), the differences manifesting on the genome of each different SARS-related virus. In particular, the (genetically) different SARS-related viruses may express at least one different protein, peptide, or polyprotein, the at least one different protein, peptide, or polyprotein differing by at least one amino acid.

[0363] In a preferred embodiment, component A-2 comprises at least one nucleic acid encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one SARS-related virus, preferably SARS-CoV-1, and component A-2 is preferably administered intramuscularly or intradermally.

[0364] Preferably, intramuscular or intradermal administration of component A-2 results in the expression of the encoded SARS-related virus antigen construct in the subject. In embodiments where the nucleic acid is RNA, administration of component A-2 results in the translation of RNA and the generation of the encoded SARS-related virus antigen in the subject. In embodiments where the nucleic acid is DNA (e.g., plasmid DNA, adenovirus DNA), administration of the composition results in the transcription of DNA to RNA in the subject, followed by the translation of the RNA into the encoded SARS-related virus antigen.

[0365] In some embodiments, administration of a pharmaceutical composition containing component A-2 to a subject induces neutralizing antibodies against SARS-related viruses, but not disease-enhancing antibodies. In particular, administration of a pharmaceutical composition containing component A-2, which encodes the pre-fusion stabilizing spike protein of SARS-related viruses, to a subject does not induce immunopathological effects such as disease enhancement and / or antibody-dependent enhancement (ADE).

[0366] In a preferred embodiment, administration of component A-2 induces an antigen-specific immune response, including a T-cell response and / or B-cell response to an encoded SARS-related viral antigen, provided by at least one nucleic acid of component A-2.

[0367] Preferably, component A-2 is suitable for vaccines, and in particular for SARS-related viruses, preferably SARS-CoV-1 vaccines, and preferably for the mixed vaccine of the present invention.

[0368] In some embodiments, the nucleic acid contained in component A-2 as defined herein is provided in amounts of about 100 ng to about 500 μg, about 1 μg to about 200 μg, about 1 μg to about 100 μg, about 5 μg to about 100 μg, preferably about 10 μg to about 50 μg, specifically about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg.

[0369] If component A-2 contains multiple or at least one nucleic acid species as defined herein, the amount of nucleic acid of each nucleic acid species is approximately 100 ng to approximately 500 μg, approximately 1 μg to approximately 200 μg, approximately 1 μg to approximately 100 μg, approximately 5 μg to approximately 100 μg, preferably approximately 10 μg to approximately 50 μg, specifically approximately 1 μg, 2 μg, and 3 μg. It is provided in amounts of 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12μg, 13μg, 14μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, or 100μg.

[0370] In some embodiments, the nucleic acid amounts of each nucleic acid species are essentially equal in mass. In other embodiments, the nucleic acid amounts of each nucleic acid species are selected to be equimolar.

[0371] Ingredient A-3: MERS-CoV In a particularly preferred embodiment, at least one coronavirus of component A is the MERS-CoV virus (also referred to as component A-3).

[0372] Accordingly, in some embodiments of the first aspect, the nucleic acid of component A includes at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one MERS-CoV.

[0373] It is understood that the general embodiments and characteristics described in the paragraph for "Component A" may also be applicable to nucleic acids encoding MERS-CoV antigenic peptides or proteins.

[0374] MERS-CoV belongs to the Coronaviridae family, specifically the orthocoronavirus genus, more specifically the Betacoronavirus genus. MERS-CoV (Middle East Respiratory Syndrome Coronavirus, MERS Coronavirus, EMC / 2012 (HCoV-EMC / 2012)) causes severe respiratory syndrome illness. An example of the MERS-CoV coronavirus can be identified by NCBI classification ID: 1335626, NCBI reference: NC_038294.1. Suitable MERS-CoV strains / isopropyls can be selected from MERS-CoV / MERS-CoV-Jeddah-Human-1, MERS-CoV / Ahasar_4_2013, MERS-CoV / Riyadh_14_2013, MERS-CoV / Riyadh_14_2013, MERS-CoV / Riyadh_14_2013 spike protein, MERS-CoV / England 1 spike protein, and MERS-CoV / England 1 spike protein (mutant).

[0375] In the context of the present invention, any protein selected from or derived from MERS-CoV can be used in the context of the present invention and may be suitably encoded by the coding sequence or nucleic acid of component A (particularly component A-3). Furthermore, it is within the scope of the underlying invention that at least one antigenic peptide or protein may include or consist of a synthetically engineered or artificial MERS-CoV peptide or protein. The terms “synthetically engineered” MERS-CoV peptide or protein, or “artificial coronavirus peptide or protein,” refer to proteins that do not exist in nature. Therefore, “artificial coronavirus peptide or protein” or “synthetically engineered coronavirus peptide or protein” may differ from, for example, a naturally occurring MERS-CoV peptide or protein by at least one amino acid, and / or contain additional heterologous peptide or protein elements, and / or have an elongated or truncated N-terminus or C-terminus.

[0376] In a preferred embodiment, the nucleic acid of component A comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from MERS-CoV, or an immunogenic fragment or immunogenic variant thereof, wherein the at least one antigenic peptide or protein comprises at least one peptide or protein selected from or derived from a structural protein, an accessory protein, a replicase protein, or an immunogenic fragment or immunogenic variant thereof.

[0377] Preferably, the structural protein is selected from or derived from a spike protein (S), an envelope protein (E), a membrane protein (M), or a nucleocapsid protein (N), or an immunogenic fragment or variant thereof.

[0378] In a particularly preferred embodiment of the pharmaceutical composition, the encoded antigenic peptide or protein of component A (particularly component A-3) comprises or consists of at least one peptide or protein selected from or derived from MERS-CoV spike proteins (S, S1, S2, or S1 and S2), or immunogenic fragments or immunogenic variants thereof.

[0379] Suitable antigenic peptide or protein sequences provided by the nucleic acid of component A (particularly component A-3) are disclosed in Table 6, rows 1-16, columns A and B. In addition, further information regarding the aforementioned suitable antigenic peptide or protein sequences selected from or derived from MERS-CoV is available in the ST.25 sequence listing identifiers. <223> It is given below.

[0380] The following describes in detail preferred antigenic peptides or protein sequences selected from or derived from MERS-CoV, provided by the nucleic acid of component A.

[0381] Amino acid level variations can spontaneously occur between spike proteins derived from different MERS-CoV isolates. In the context of the present invention, such amino acid variations can be applied to each antigenic peptide or protein derived from the MERS-CoV spike protein described herein.

[0382] In some embodiments, the MERS-CoV spike protein (S) fragment may be encoded by the nucleic acid of component A, the fragment may have its N-terminus truncated, and / or the fragment may have its C-terminus truncated. Such “spike protein (S) fragment” may further include amino acid substitutions (described below) and may further include at least one heterologous peptide or protein element (described below).

[0383] In some embodiments, component A, at least one encoded antigenic peptide or protein, comprises or consists of a MERS-CoV spike protein (S), wherein the spike protein (S) lacks a transmembrane domain (TM). While we do not wish to be bound by theory, the MERS-CoV spike protein (S) lacking a transmembrane domain (TM) as defined herein may be suitable for mixed vaccines because such protein is soluble and not immobilized on the cell membrane. Therefore, when administered to a subject, a higher concentration of soluble protein may be produced (translated), potentially leading to an improved immune response.

[0384] While we do not wish to be constrained by theory, the RBD and CND domains may be crucial for the immunogenicity of the MERS-CoV spike protein (S). Both regions are located on the S1 fragment of the spike protein. Therefore, in the context of this invention, it may be preferable for the antigenic peptide or protein to contain or consist of the S1 fragment of the spike protein, or its immunogenic fragment or immunogenic variant.

[0385] Preferably, the S1 fragment of MERS-CoV may contain at least the RBD and / or CND domains defined above.

[0386] In a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A (particularly component A-3) comprises or consists of a MERS-CoV spike protein (S), wherein the MERS-CoV spike protein (S) comprises or consists of a spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof.

[0387] In a preferred embodiment, the encoded antigenic peptide or protein comprises the MERS-CoV spike protein fragment S1 and is deleting at least 70%, 80%, 90%, preferably 100%, of the spike protein fragment S2. Such embodiments may be beneficial because the MERS-CoV S1 fragment contains a neutralizing epitope, without the potential problems associated with full-length proteins containing S1 and S2.

[0388] While we do not wish to be bound by theory, it may be preferable that the antigenic peptide or protein of component A (particularly component A-3) comprises or consists of MERS-CoV spike protein fragment S1 and MERS-CoV spike protein fragment S2 (or at least a fragment thereof), since the formation of immunogenic MERS-CoV spike protein may be promoted.

[0389] Therefore, in a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A (in particular component A-3) comprises or consists of a MERS-CoV spike protein (S), the MERS-CoV spike protein (S) comprising or consisting of a MERS-CoV spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof, and a MERS-CoV spike protein fragment S2, or an immunogenic fragment or immunogenic variant thereof.

[0390] In a particularly preferred embodiment, at least one encoded antigenic peptide or protein of component A (in particular, component A-3) comprises or consists of a full-length MERS-CoV spike protein, or an immunogenic fragment or immunogenic variant thereof.

[0391] In a particularly preferred embodiment, the MERS-CoV spike protein (S) provided by the nucleic acid of component A (particularly component A-3) is designed or adapted to stabilize the antigen in a pre-fusion conformation. The pre-fusion conformation is particularly advantageous in the context of an efficient MERS-CoV vaccine because the pre-fusion protein conformation contains several potential and accessible epitopes for neutralizing antibodies. Furthermore, the presence of protein in the pre-fusion conformation is intended to avoid immunopathological effects, such as disease exacerbation or antibody-dependent enhancement (ADE).

[0392] Therefore, in a preferred embodiment, the nucleic acid of component A (particularly component A-3) comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from MERS-CoV, wherein the at least one antigenic peptide or protein is selected from or derived from a spike protein (S), and the spike protein (S) is a pre-fusion stabilization spike protein (S_stab). Preferably, the pre-fusion stabilization spike protein comprises at least one pre-fusion stabilization mutation.

[0393] Stabilization of the MERS-CoV spike protein can be achieved by substituting at least one amino acid at position V1060 and / or L1061 with an amino acid that stabilizes the spike protein in the pre-fusion conformation (amino acid positions related to reference sequence number 14794).

[0394] In some embodiments, the prefusion stabilization mutation of the MERS-CoV spike protein includes an amino acid substitution at position V1060, where amino acid V1060 is replaced with one selected from A, I, L, M, F, V, G, or P (the amino acid position related to reference sequence number 14794), preferably with P. In some embodiments, the prefusion stabilization mutation includes an amino acid substitution at position L1061, where amino acid L1061 is replaced with one selected from A, I, L, M, F, V, G, or P (the amino acid position related to reference sequence number 14794), preferably with P.

[0395] Therefore, in a preferred embodiment, the MERS-CoV pre-fusion stabilization spike protein (S_stab) comprises at least one pre-fusion stabilization mutation, the at least one pre-fusion stabilization mutation comprising the following amino acid substitutions: V1060P and L1061P (amino acid positions related to reference sequence number 14794).

[0396] MERS-CoV spike protein or its fragments or variants can be selected by those skilled in the art to introduce such amino acid changes, preferably amino acid substitutions: V1060P and L1061P (amino acid positions corresponding to reference sequence number 14794).

[0397] MERS-CoV spike proteins or their fragments or mutants can be selected by those skilled in the art to introduce such amino acid changes (e.g., double proline mutations). Examples include MERS-CoV / MERS-CoV-Jeddah-Human-1 spike protein (1-1353)(V1060P_L1061P), MERS-CoV / Ahasar-4-2013 spike protein (1-1353)(V1060P_L1061P), MERS-CoV / Riyadh-14-2013 spike protein (1-1353)(V1060P_L1061P), and MERS-CoV / England-1 spike protein (1-1353)(V1060P_L1061P).

[0398] In some embodiments, at least one pre-fusion stabilization mutation of the MERS-CoV spike protein includes a cavity-filling mutation.

[0399] In some embodiments, cavity-filling substitutions to stabilize the pre-fusion conformation of the MERS-CoV S external domain may be selected from the following amino acid substitutions: N1072F and A1083I; N1072F and L1086F; N1072F and V1087I; N1072F and E1090I; T1076F and A1083I; T1076F and L1086F; T1076F and V1087I; T1076F and E10901; T1076I and A1083I; T1076I and L1086F; T1076I and V1087I; T1076I and E10901; A1018V; or A1018I.

[0400] In some embodiments, at least one pre-fusion stabilizing mutation in the MERS-CoV spike protein includes a mutated protonation site (R1020Q).

[0401] In some embodiments, at least one pre-fusion stabilizing mutation of the MERS-CoV spike protein includes a repacking substitution to stabilize the S extradomain, such as one of the following: E793M and K1102F; E793M, K1102F, and H1138F; D1068M and R1069W; A1083L; A1083L and V1087I; A1083L, V1087, and E1090L; A834L and Q1084M; Q1066M; S454F; R921W; S612F and G1052F; or P476V, T477A, ​​and R1057W.

[0402] In some embodiments, at least one pre-fusion stabilizing mutation of the MERS-CoV spike protein includes an artificial intramolecular disulfide bond. Introducing such an artificial intramolecular disulfide bond can further stabilize the membrane distal portion (including the N-terminal region) of the MERS-CoV spike protein in the pre-fusion conformation, i.e., the conformation that specifically binds to one or more pre-fusion specification antibodies, and / or the conformation that presents a preferred antigen site present in the pre-fusion conformation but not in the post-fusion conformation of the MERS-CoV spike protein.

[0403] In some embodiments, disulfide bond substitutions to stabilize the pre-fusion conformation of the MERS-CoV S extradomain can be selected from the following amino acid substitutions: T63C and V631C; T63C and Q638C; Q733C and D940C; S676C and D910C; V1087C (forming a disulfide bond with cysteine ​​present in the native sequence); A432C and L1058C; or A432C and D1059C to stabilize the S extradomain of the pre-fusion conformation.

[0404] MERS-CoV spike proteins or their fragments or variants can be selected by those skilled in the art to introduce at least one cavity-filling mutation, at least one mutant protonation site, and / or at least one artificial intramolecular disulfide bond.

[0405] According to various preferred embodiments, the nucleic acid of component A (particularly component A-3) encodes at least one antigenic peptide or protein selected from or derived from MERS-CoV as defined herein, and further preferably at least one heterologous peptide or protein element selected from or derived from signal peptides, linkers, helper epitopes, antigen clustering elements, trimerizing elements, transmembrane elements, and / or VLP-forming sequences.

[0406] Preferably, at least one heterologous peptide or protein element can promote or improve the secretion of the MERS-CoV-encoded antigenic peptide or protein (e.g., via a secretion signal sequence), promote or improve the fixation of the encoded antigenic peptide or protein in the plasma membrane (e.g., via a transmembrane element), promote or improve the formation of an antigen complex (e.g., via a multimerization domain or antigen clustering element), or promote or improve virus-like particle (VLP-forming sequence). Furthermore, the nucleic acid of component A (particularly component A-3) can further encode a peptide linker element, a self-cleaving peptide, an immunoadjuvant sequence, or a dendritic cell target sequence.

[0407] In a preferred embodiment, the nucleic acid of component A (in particular, component A-3), which encodes at least one antigenic protein selected from or derived from MERS-CoV as defined herein, further encodes at least one heterotrimerizing element as defined herein, an antigen clustering element as defined herein, or a VLP-forming sequence as defined herein.

[0408] The antigen clustering element may be selected from the ferritin element, the lumazine synthase element, the hepatitis B virus surface antigen (HBsAg), or the encapsulin as defined herein. In embodiments in which the coding sequence of component A (particularly component A-3) further codes for a heterologous antigen clustering element, it is particularly preferred to generate a fusion protein comprising the antigen clustering element and an antigenic peptide or protein derived from MERS-CoV. Preferably, the antigenic peptide or protein, preferably the MERS-CoV spike protein, is deleting its C-terminal transmembrane domain (TM) or a portion of its TM. Furthermore, it may be preferable to use a defined linker element to separate the heterologous antigen clustering element from the antigenic peptide or protein.

[0409] The trimerizing element can be selected from the Foldon elements defined herein. In a preferred embodiment, the Foldon element is a fibrin-foldon element as defined herein. In embodiments in which the coding sequence of the nucleic acid of component A (particularly component A-3) further codes for a heterologous trimerizing element, it is particularly preferred and suitable to generate a fusion protein comprising the trimerizing element and an antigenic peptide or protein derived from MERS-CoV. Preferably, the antigenic peptide or protein is a spike protein derived from MERS-CoV that is missing a portion of the C-terminal transmembrane domain or TM. Furthermore, it may be preferable to use a defined linker element to separate the heterologous antigen clustering element from the antigenic peptide or protein.

[0410] A VLP-forming sequence can be selected and fused to the MERS-CoV antigen as defined herein. Suitable VLP-forming sequences can be selected from elements derived from the hepatitis B virus core antigen, the HIV-1 Gag protein, or the woodchuck hepatitis core antigen element (WhcAg) as defined herein. In embodiments where the coding sequence of component A (particularly component A-3) of nucleic acid further codes for a heterologous VLP-forming sequence, it is particularly preferable to generate a fusion protein comprising the VLP-forming sequence and an antigenic peptide or protein derived from MERS-CoV. Preferably, the antigenic peptide or protein is a MERS-CoV-derived spike protein lacking a C-terminal transmembrane domain (TM) or a portion of the TM. Furthermore, it may be preferable to use a linker element as defined herein to separate the heterologous antigen clustering element from the antigenic peptide or protein.

[0411] In some embodiments, the antigenic peptide or protein includes the heterologous signal peptide defined above. The heterologous signal peptide can be used to improve the secretion of the encoded MERS-CoV antigen. In embodiments in which the coding sequence of the nucleic acid of component A (particularly component A-3) further encodes a heterologous secretion signal peptide, it is particularly preferable to generate a fusion protein comprising the heterologous secretion signal peptide and the antigenic peptide or protein derived from MERS-CoV. Preferably, the antigenic peptide or protein, preferably a spike protein derived from MERS-CoV, lacks an endogenous secretion signal peptide at its N-terminus.

[0412] Table 6 (rows 1-16) shows preferred antigenic peptides or proteins selected from or derived from the MERS-CoV defined above. In the table, rows 1-16 correspond to preferred MERS-CoV constructs. Column A of Table 6 provides a brief description of the preferred MERS-CoV antigen constructs. Column B of Table 6 lists the sequence numbers of the proteins (amino acids) of each MERS-CoV antigen construct. Column D of Table 6 lists the sequence numbers of the corresponding G / C optimized nucleic acid coding sequences (opt1, gc). Column E of Table 6 lists the sequence numbers of the corresponding human codon-frequency-matched nucleic acid coding sequences (opt3, human).

[0413] The description of this invention refers to the identifiers in the ST.25 sequence listing of this application. <223> The information provided below is explicitly included. Preferred nucleic acid constructs containing the coding sequences in Table 6, for example, mRNA sequences containing the coding sequences in Table 6, are given in Table 7.

[0414] (outside 7) TIFF0007848141000015.tif99170

[0415] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from MERS-CoV encoded by at least one nucleic acid of component A (particularly component A-3) comprises at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 14794-14809, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 6 (see rows 1-16 of columns A and B) and the ST.25 sequence listing identifier for each sequence number. <223> It is also given under.

[0416] In further embodiments, at least one antigenic peptide or protein selected from or derived from at least one MERS-CoV encoded by at least one nucleic acid of component A (particularly component A-3) comprises or consists of at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs. 1-152 or 1448-1548 of published PCT application WO2018115527, or an immunogenic fragment or immunogenic variant thereof. Accordingly, sequence numbers 1-152 or 1448-1548 of WO2018115527, and the corresponding disclosures relating thereto (e.g., the information in each sequence listing, Tables 1-4 and Table 7, column 1 or 2) are incorporated herein by reference.

[0417] In further embodiments, at least one antigenic peptide or protein selected from or derived from at least one MERS-CoV encoded by at least one nucleic acid of component A (particularly component A-3) comprises or consists of at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs. 2-4, 28-29 of published PCT application WO2018081318, or an immunogenic fragment or immunogenic variant thereof. Accordingly, SEQ ID NOs. 2-4, 28-29 of WO2018081318 and the corresponding disclosures relating thereto are incorporated herein by reference.

[0418] In further embodiments, at least one antigenic peptide or protein selected from or derived from at least one MERS-CoV encoded by at least one nucleic acid of component A (particularly component A-3) comprises or consists of at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 24-28 or 33 of published PCT application WO2017070626, or an immunogenic fragment or immunogenic variant thereof. Accordingly, sequence numbers 24-28 or 33 of WO2017070626 and the corresponding disclosures relating thereto are incorporated herein by reference.

[0419] Further preferred antigenic peptides or proteins selected from or derived from MERS-CoV may be selected from or derived from Table 12 of WO2017070626. Accordingly, the entire contents of Table 12 of WO2017070626 are incorporated herein by reference.

[0420] In a preferred embodiment, at least one antigenic peptide or protein (pre-fusion stabilizing spike protein (S_stab)) selected from or derived from MERS-CoV encoded by at least one nucleic acid of component A (particularly component A-3) comprises at least one amino acid sequence that is identical to or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 14795, 14797, 14799-14802, or 14804, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 6 (columns A and B) and the ST.25 sequence listing identifiers for each sequence. <223> It is also given under.

[0421] In a preferred embodiment, the nucleic acid of component A (particularly component A-3) comprises at least one coding sequence encoding at least one antigenic peptide or protein, or fragments and variants thereof, derived from MERS-CoV as defined above. In this context, coding sequences encoding at least one MERS-CoV antigenic protein, or fragments and variants thereof, as defined herein, are understood as preferred coding sequences and may be included in the nucleic acid of component A.

[0422] In preferred embodiments, the nucleic acid of component A (particularly component A-3) comprises or consists of at least one antigenic peptide or protein selected from or derived from MERS-CoV as defined herein, preferably any of SEQ ID NOs: 14794-14809; SEQ ID NOs: 1-152, 1448-1548 of WO2018115527; SEQ ID NOs: 2-4, 28-29 of WO2018081318; SEQ ID NOs: 24-28 or 33 of WO2017070626, or at least one coding sequence encoding a fragment or variant thereof. At the nucleic acid level, sequences (DNA or RNA sequences) encoding amino acid sequences, or fragments or variants thereof, can be selected that are identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any of sequence numbers 14794-14809; sequence numbers 1-152 and 1448-1548 of WO2018115527; sequence numbers 2-4 and 28-29 of WO2018081318; or sequence numbers 24-28 or 33 of WO2017070626. These sequences can therefore be understood to be suitable coding sequences for the present invention. Further information regarding the amino acid sequences can be found in Tables 6 and 7, and in the ST.25 sequence listing identifiers for each sequence. <223> It is also given under.

[0423] In a preferred embodiment, the nucleic acid of component A (particularly component A-3) includes a coding sequence comprising at least one nucleic acid sequence encoding the MERS-CoV antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences of sequence numbers 14810 to 14905, or a fragment or variant of any of these sequences. Further information regarding the nucleic acid sequences can be found in Tables 6 and 7, and the ST.25 sequence listing identifiers for each sequence's sequence number. <223> It is also given under.

[0424] In further embodiments, at least one coding sequence of at least one nucleic acid of component A (in particular, component A-3) comprises or comprises a MERS-CoV antigen, or an immunogenic fragment or immunogenic variant thereof, that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences of sequence numbers 153-304 or 1549-1649, 305-1368, 1650-2356, 2365, 2366, or 2373-2378 of published PCT application WO2018115527. Furthermore, sequence numbers 153-304 or 1549-1649, 305-1368, 1650-2356, 2365, 2366, 2373-2378 of WO2018115527, and the corresponding disclosures therein (e.g., each sequence listing, Tables 1-4 and Table 7, column 4 or 4) are incorporated herein by reference.

[0425] In further embodiments, at least one coding sequence of at least one nucleic acid of component A (in particular, component A-3) comprises or comprises a MERS-CoV antigen that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences of sequence numbers 20-23, 65-68 of published PCT application WO2017070626, or an immunogenic fragment or immunogenic variant thereof. Sequence numbers 20-23, 65-68 of WO2017070626 and the corresponding disclosures thereto (e.g., the information in each sequence listing, Tables 1-4 and 7) are incorporated herein by reference.

[0426] In preferred embodiments, at least one coding sequence of the nucleic acid of component A (particularly component A-3) is a codon-modified coding sequence as defined herein, and the amino acid sequence encoded by at least one codon-modified coding sequence, i.e., the MERS-CoV peptide or protein, is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

[0427] In a particularly preferred embodiment, at least one coding sequence of nucleic acid component A (particularly component A-3) is a codon-modified coding sequence, and the codon-modified coding sequence is a G / C optimized coding sequence, a human codon frequency-adapted coding sequence, or a G / C modified coding sequence.

[0428] Table 7 (columns C and D) shows preferred nucleic acid sequences of component A (particularly component A-3) containing particularly preferred mRNA sequences. In the table, each row represents a specific preferred MERS-CoV construct of the present invention (compared to Table 6), a description of the MERS-CoV construct is shown in column A of Table 7, and the sequence number of the amino acid sequence of each MERS-CoV construct is shown in column B. The corresponding sequence numbers of the coding sequences encoding each MERS-CoV construct are shown in Table 6. Further information is provided by the sequence number identifiers of each sequence in the sequence listing. <223> It is given below.

[0429] (outside 8) TIFF0007848141000016.tif99170

[0430] In a preferred embodiment, component A (particularly component A-3), preferably RNA, comprises or consists of a nucleic acid sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, a nucleic acid sequence encoding the MERS-CoV antigen, selected from sequence numbers 14842 to 14905, or a fragment or variant of any of these sequences. Further information regarding each nucleic acid sequence is provided by the sequence number identifier of each sequence in the sequence listing. <223> The following are given below and in Table 7 (see columns C and D in particular). Optionally, the nucleic acid sequence comprises a cap1 structure as defined herein, and / or at least one, preferably all, uracil nucleotides in the RNA sequence are replaced with pseudouridine (Ψ) nucleotides and / or N1-methylpseudridine (m1Ψ) nucleotides.

[0431] In further embodiments, component A (particularly component A-3), preferably RNA, comprises or consists of a nucleic acid sequence encoding the MERS-CoV antigen that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from sequence numbers 2373-2378 of WO2018115527 and sequence numbers 65-68 of WO2017070626, or a fragment or variant of any of these sequences. Further information regarding each nucleic acid sequence is provided by the identifier of each sequence number in the sequence listing of WO2018115527 or WO2017070626. <223> The following are given below and in Table 7 of WO2018115527. Optionally, the nucleic acid sequence comprises a cap1 structure as defined herein, and / or at least one, preferably all, uracil nucleotides in the RNA sequence are replaced with pseudouridine (Ψ) nucleotides and / or N1-methylpseudridine (m1Ψ) nucleotides.

[0432] Preferred features and embodiments applicable to the nucleic acid of component A-3 are described in the following paragraph, “Features and Embodiments of Nucleic Acids.”

[0433] Preferably, the nucleic acids of component A-3 are compounded and / or complexed. Preferred features and embodiments applicable to the complexation of nucleic acids or the compounding of component A are described in the following "Compounding and Complexation" section.

[0434] In some embodiments, component A (particularly component A-3) comprises multiple or more than one nucleic acid species, e.g., DNA or RNA species as defined herein, each containing at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one MERS-CoV. Preferably, component A (particularly component A-3) as defined herein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acids, each as defined herein.

[0435] In some embodiments, component A (in particular component A-3) may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, such as DNA or RNA as defined herein, each encoding at least one antigenic peptide or protein, or a fragment or variant thereof, derived from the same MERS-CoV. In particular, the (genetically) identical MERS-CoV expresses an (essentially) identical repertoire of proteins or peptides, all of which have (essentially) identical amino acid sequences. In particular, the (genetically) identical MERS-CoV expresses essentially identical proteins, peptides, or polyproteins, preferably these proteins, peptides, or polyproteins that are identical in their amino acid sequences.

[0436] In some embodiments, component A (in particular, component A-3) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, e.g., DNA or RNA as defined herein, each encoding at least one peptide or protein, or a fragment or variant thereof, derived from a genetically distinct MERS-CoV (e.g., different MERS-CoV isolates). The terms “different” or “different MERS-CoV” as used herein are understood as differences between at least two respective MERS-CoVs (e.g., different MERS-CoV isolates), the differences manifesting on the genome of each different MERS-CoV. In particular, the (genetically) different MERS-CoVs may express at least one different protein, peptide, or polyprotein, and the at least one different protein, peptide, or polyprotein differs by at least one amino acid.

[0437] In a preferred embodiment, component A-3 comprises at least one nucleic acid encoding at least one antigenic peptide or protein selected from or derived from at least one MERS-CoV, or an immunogenic fragment or immunogenic variant thereof, and component A-3 is preferably administered intramuscularly or intradermally.

[0438] Preferably, intramuscular or intradermal administration of component A-3 results in the expression of the encoded MERS-CoV antigen construct in the subject. In embodiments where the nucleic acid is RNA, administration of component A-3 results in the translation of RNA and the generation of the encoded MERS-CoV antigen in the subject. In embodiments where the nucleic acid is DNA (e.g., plasmid DNA, adenovirus DNA), administration of the composition results in the transcription of DNA to RNA in the subject, followed by the translation of the RNA into the encoded MERS-CoV antigen.

[0439] In some embodiments, administration of a pharmaceutical composition containing component A-3 to a subject induces neutralizing antibodies against MERS-CoV, but not disease-enhancing antibodies. In particular, administration of a pharmaceutical composition containing component A-3, which encodes the pre-fusion stabilizing spike protein of MERS-CoV, to a subject does not induce immunopathological effects such as disease enhancement and / or antibody-dependent enhancement (ADE).

[0440] In a preferred embodiment, administration of component A-3 induces an antigen-specific immune response, including a T-cell response and / or B-cell response to encoded MERS-CoV, provided by at least one nucleic acid of component A-3.

[0441] Preferably, component A-3 is suitable for vaccines, particularly for MERS-CoV, and preferably for the mixed vaccine of the present invention.

[0442] In some embodiments, the nucleic acid contained in component A-3 as defined herein is provided in amounts of about 100 ng to about 500 μg, about 1 μg to about 200 μg, about 1 μg to about 100 μg, about 5 μg to about 100 μg, preferably about 10 μg to about 50 μg, specifically about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg.

[0443] If component A-3 contains multiple or at least one nucleic acid species as defined herein, the amount of nucleic acid of each nucleic acid species is approximately 100 ng to approximately 500 μg, approximately 1 μg to approximately 200 μg, approximately 1 μg to approximately 100 μg, approximately 5 μg to approximately 100 μg, preferably approximately 10 μg to approximately 50 μg, specifically approximately 1 μg, 2 μg, and 3 μg. It is provided in amounts of 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12μg, 13μg, 14μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, or 100μg.

[0444] In some embodiments, the nucleic acid amounts of each nucleic acid species are essentially equal in mass. In other embodiments, the nucleic acid amounts of each nucleic acid species are selected to be equimolar.

[0445] Component B: In various preferred embodiments, at least one component B of the pharmaceutical composition comprises at least one nucleic acid comprising at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one further virus.

[0446] In the context of the present invention, the term "peptides or proteins selected from or derived from at least one further virus" in its broadest sense refers to peptides or proteins derived from further viruses, and the further virus is not selected as component A. Therefore, in embodiments in which SARS-CoV-2 is selected as at least one coronavirus of component A, the at least one further virus of component B may be a virus other than SARS-CoV-2.

[0447] In a preferred embodiment, at least one further virus of component B is selected from or derived from at least one different coronavirus, at least one influenza virus, at least one Pneumoviridae virus, and / or at least one Paramyxoviridae virus.

[0448] In a preferred embodiment, at least one Pneumoviridae virus is selected from at least one respiratory syncytial virus and / or at least one metapneumovirus.

[0449] In a preferred embodiment, at least one paramyxoviridae virus is selected from at least one parainfluenza virus and / or at least one henipavirus.

[0450] In a preferred embodiment, at least one paramyxoviridae virus is selected from at least one parainfluenza virus and / or at least one henipavirus.

[0451] According to some embodiments, the nucleic acid of component B comprises at least one coding sequence encoding at least one antigenic peptide or protein, or a fragment or variant thereof, selected from or derived from at least one further virus as defined herein. In preferred embodiments, the nucleic acid of component B comprises at least one coding sequence encoding at least one antigenic peptide or protein, or a fragment or variant thereof, selected from or derived from at least one further virus as defined herein, wherein the at least one further virus is selected from at least one different coronavirus, at least one influenza virus, at least one Pneumoviridae virus, and / or at least one Paramyxoviridae virus.

[0452] In this context, coding sequences encoding at least one antigenic protein of further viruses as defined herein, or fragments and variants thereof, are understood as preferred coding sequences and may therefore be included in the nucleic acid of component B.

[0453] Preferred features and embodiments applicable to the nucleic acid of component B are described in the following paragraph, “Features and Embodiments of Nucleic Acids.”

[0454] Preferably, the nucleic acid of component B is compounded and / or complexed. Preferred features and embodiments applicable to the complexation of nucleic acids or the compounding of component A are described in the following "Compounding and Complexation" section.

[0455] In some embodiments, component B comprises multiple or more than one nucleic acid species, e.g., DNA or RNA species as defined herein, each comprising at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one further virus. Preferably, component B as defined herein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acids, each as defined herein.

[0456] In some embodiments, component B may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, such as DNA or RNA as defined herein, each encoding at least one antigenic peptide or protein, or a fragment or variant thereof, derived from the same further virus. In particular, the (genetically) identical further virus expresses an (essentially) identical repertoire of proteins or peptides, all of which have (essentially) identical amino acid sequences. In particular, the (genetically) identical further virus expresses essentially identical proteins, peptides, or polyproteins, preferably these proteins, peptides, or polyproteins that are identical in their amino acid sequences.

[0457] In some embodiments, component B comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid species, e.g., DNA or RNA as defined herein, each encoding at least one peptide or protein, or a fragment or variant thereof, derived from a genetically distinct further virus (e.g., a different influenza virus, a different RSV virus, a different PIV). The terms “different” or “different further virus” as used herein are understood as differences between at least two of each further virus, the differences manifesting on the genome of each different further virus. In particular, the (genetically) different further virus may express at least one different protein, peptide, or polyprotein, and the at least one different protein, peptide, or polyprotein differs by at least one amino acid.

[0458] In a preferred embodiment, component B comprises at least one nucleic acid encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one further virus, wherein component B is preferably administered intramuscularly or intradermally.

[0459] Preferably, intramuscular or intradermal administration of component B results in the expression of further encoded viral antigen constructs in the subject. In embodiments where the nucleic acid is RNA, administration of component B results in the translation of RNA and the generation of further encoded viral antigens in the subject. In embodiments where the nucleic acid is DNA (e.g., plasmid DNA, adenovirus DNA), administration of the composition results in the transcription of DNA to RNA in the subject, followed by the translation of the RNA into further encoded viral antigens.

[0460] In a preferred embodiment, administration of component B induces an antigen-specific immune response, including a T-cell response and / or B-cell response to an encoded viral antigen, which is brought about by at least one nucleic acid of component B.

[0461] Preferably, component B is suitable for a vaccine, preferably a combination vaccine, or suitable for the combination vaccine of the present invention.

[0462] Embodiments relating to specific further viruses in the context of the present invention are shown below (components B-1, B-2, B-3, and B-4).

[0463] Component B-1: Different coronaviruses In a preferred embodiment, at least one further virus of component B is selected from at least one different coronavirus (also referred to as component B-1), wherein the at least one different coronavirus is not the coronavirus of component A.

[0464] It is understood that the general embodiments and characteristics described in the paragraph for "Component B" may also be applicable to nucleic acids encoding different coronavirus antigenic peptides or proteins.

[0465] Features and embodiments provided in the context of component A (including components A-1, A-2, and A-3) can similarly be applied to component B (in particular, component B-1). Therefore, any nucleic acid, such as DNA or RNA provided in the context of component A (including components A-1, A-2, and A-3), can similarly be selected for component B (in particular, component B-1).

[0466] In embodiments in which component A comprises at least one nucleic acid including at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one SARS-CoV-2 as defined herein, at least one different coronavirus of component B can be selected from any coronavirus, preferably any pandemic coronavirus, and at least one different coronavirus is not SARS-CoV-2. Thus, the nucleic acids provided in the context of component A (including components A-1, A-2, and A-3) can similarly be selected as component B (in particular, component B-1), and at least one nucleic acid of component B does not encode the SARS-CoV-2 antigen.

[0467] In embodiments in which component A comprises at least one nucleic acid including at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one SARS-CoV-1 as defined herein, at least one different coronavirus of component B can be selected from any coronavirus, preferably any pandemic coronavirus, and at least one different coronavirus is not SARS-CoV-1. Thus, the nucleic acids provided in the context of component A (including components A-1, A-2, and A-3) can similarly be selected as component B (in particular, component B-1), and at least one nucleic acid of component B does not encode the SARS-CoV-1 antigen.

[0468] In embodiments in which component A comprises at least one nucleic acid including at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one MERS-CoV as defined herein, at least one different coronavirus of component B can be selected from any coronavirus, preferably any pandemic coronavirus, and at least one different coronavirus is not MERS-CoV. Thus, the nucleic acids provided in the context of component A (including components A-1, A-2, and A-3) can similarly be selected as component B (in particular, component B-1), and at least one nucleic acid of component B does not encode the MERS-CoV antigen.

[0469] In a preferred embodiment, at least one antigenic peptide or protein, selected from or derived from at least one coronavirus, encoded by at least one nucleic acid of component B, is sequence numbers 1-111, 274-11663, 13176-13510, 13521-14123, 14794-14809, 14906-14950, 22732-22758, 22917, This comprises or consists of at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of 22923, 22929-22964, 26938, or 26939, or an immunogenic fragment or immunogenic variant thereof.

[0470] In a preferred embodiment, at least one antigenic peptide or protein, selected from or derived from at least one coronavirus, encoded by at least one nucleic acid of component B, is: SEQ ID NOs. 116-132, 134-138, 140-143, 145-147, 148-175, 11664-11813, 11815, 11817-12 050, 12052, 12054~13147, 13514, 13515, 13519, 13520, 14124~14177, 14810~14905, 14951~15220, 22759, 22764~22786, 22791~22813, 22818~22839, 22969~23184, 23189~23404, 23409~23624, 2 3629~23844, 23849~24064, 24069~24284, 24289~24504, 24509~24724, 24729~24944, 24949~25164, 25169~25384, 25389~25604, 25609~25824, 25829~26044, 26049~26264, 26269~26484, 26489~ The present invention comprises or consists of at least one nucleic acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences 26704, 26709-26937, or a fragment or variant of any of these sequences.

[0471] In a preferred embodiment, at least one nucleic acid of component B is sequence numbers 148-175, 12204-13147, 14142-14177, 14842-14905, 15041-15220, 22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604, 256 A nucleic acid sequence selected from 09-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704, 26709-26937, and a nucleic acid sequence encoding at least one different coronavirus antigen that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of these sequences, or comprising or comprising a fragment or variant of any of these sequences.

[0472] In some embodiments, component B (in particular, component B-1) may include multiple or at least one nucleic acid species, for example, DNA or RNA species as defined herein, each containing at least one coding sequence encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one different coronavirus. Preferably, component B as defined herein may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acids as defined herein.

[0473] In a preferred embodiment, component B-1 comprises at least one nucleic acid encoding at least one antigenic peptide or protein, or an immunogenic fragment or immunogenic variant thereof, selected from or derived from at least one different coronavirus, wherein component B-1 is preferably administered intramuscularly or intradermally.

[0474] Preferably, intramuscular or intradermal administration of component B-1 results in the expression of different encoded coronavirus antigen constructs in the subject. In embodiments where the nucleic acid is RNA, administration of component B-1 results in the translation of RNA and the generation of different encoded coronavirus antigens in the subject. In embodiments where the nucleic acid is DNA (e.g., plasmid DNA, adenovirus DNA), administration of the composition results in the transcription of DNA to RNA in the subject, followed by the translation of the RNA into different encoded coronavirus antigens.

[0475] In some embodiments, administration of a pharmaceutical composition containing component B-1 to a subject induces neutralizing antibodies against different coronaviruses, but not disease-enhancing antibodies. In particular, administration of a pharmaceutical composition containing component B-1, which encodes the pre-fusion stabilizing spike protein of coronaviruses, to a subject does not induce immunopathological effects such as disease enhancement and / or antibody-dependent enhancement (ADE).

[0476] In a preferred embodiment, administration of component B-1 induces an antigen-specific immune response, including a T-cell response and / or B-cell response to a different coronavirus antigen encoded by at least one nucleic acid of component B-1.

[0477] Preferably, component B-1 is suitable for vaccines, and in particular for different coronavirus vaccines, and preferably for the mixed vaccine of the present invention.

[0478] In some embodiments, the nucleic acid contained in component B-1 as defined herein is provided in amounts of about 100 ng to about 500 μg, about 1 μg to about 200 μg, about 1 μg to about 100 μg, about 5 μg to about 100 μg, preferably about 10 μg to about 50 μg, specifically about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg.

[0479] If component B-1 contains multiple or at least more than one of the nucleic acid species defined herein, the amount of nucleic acid of each nucleic acid species is approximately 100 ng to approximately 500 μg, approximately 1 μg to approximately 200 μg, approximately 1 μg to approximately 100 μg, approximately 5 μg to approximately 100 μg, preferably approximately 10 μg to approximately 50 μg, specifically approximately 1 μg, 2 μg, and 3 μg. It is provided in amounts of 4μg, 5μg, 6μg, 7μg, 8μg, 9μg, 10μg, 11μg, 12μg, 13μg, 14μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, 50μg, 55μg, 60μg, 65μg, 70μg, 75μg, 80μg, 85μg, 90μg, 95μg, or 100μg.

[0480] In some embodiments, the nucleic acid amounts of each nucleic acid species are essentially equal in mass. In other embodiments, the nucleic acid amounts of each nucleic acid species are selected to be equimolar.

[0481] Component B-2: Influenza virus In a preferred embodiment, at least one further virus of component B is selected from at least one influenza virus (also referred to as component B-2). Influenza viruses belong to the family Orthomyxoviridae (NCBI classification ID: 11308), which is further classified into alpha influenza viruses (the genus including influenza A virus), beta influenza viruses (the genus including influenza B virus), gamma influenza viruses (the genus including influenza C virus), and delta influenza viruses (the genus including influenza D virus).

[0482] It is understood that the general embodiments and features described in the paragraph for "Component B" may also be applicable to nucleic acids encoding influenza virus antigenic peptides or proteins.

[0483] In the context of the present invention, any influenza virus, regardless of a specific genotype, species, strain, isolate, or serotype, can be selected as "at least one additional virus" of component B.

[0484] Preferably, at least one influenza virus of component B (particularly component B-2) can be selected from at least one influenza A virus (NCBI classification ID: 11320), and / or at least one influenza B virus (NCBI classification ID: 11520), and / or at least one influenza C virus (NCBI classification ID: 11552), and / or at least one influenza D virus (NCBI classification ID: 1511084).

[0485] Therefore, in some embodiments, at least one influenza virus of component B (in particular, component B-2) is selected from at least one influenza A virus and / or at least one influenza B virus and / or at least one influenza C virus. In preferred embodiments, at least one influenza virus is selected from at least one influenza A virus and / or at least one influenza B virus.

[0486] At least one influenza A virus may be selected from influenza A viruses characterized by hemagglutinin (HA) selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. Preferably, the influenza A virus is selected from influenza viruses characterized by hemagglutinin (HA) selected from the group consisting of H1, H3, H5, H7, H9, or H10.

[0487] Furthermore, particularly preferred are influenza A viruses characterized by neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11. Most preferably, the influenza A viruses are selected from influenza viruses characterized by neuraminidase (NA) selected from the group consisting of N1, N2, and N8.

[0488] In some embodiments, the influenza virus is selected from at least one influenza A virus, preferably selected from at least one of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, and H10N8, and preferably from at least one of H1N1, H3N2, H5N1, and H5N8.

[0489] Suitable influenza virus A and B strains are FLUAV / H1N1 / A / California / 7 / 2009, FLUAV / H1N1 / A / Michigan / 45 / 2015, FLUAV / H1N1 / A / Netherlands / 602 / 2009, FLUAV / H3N2 / A / Hong Kong / 4801 / 2014, FLUAV / H0N0 / B / Brisbane / 60 / 2008, FLUAV / H0N0 / B / Phuket / 3073 / 2013, FLUAV / H1N1 / A / Brisbane / 02 / 2018, FLUAV / H1N1 / A / Brisbane / 02 / 2018, FLUAV / H1N1 / A / Guangdong-Maonan / SWL1536 / 2019, FLUAV / H1N1 / A / Guangdong-Maonan / SWL1536 / 2019, FLUAV / H3N2 / A / Singapore / INFIMH-16-0019 / 2016, FLUAV / H3N2 / A / Singapore / INFIMH-16-0019 / 2016 NYMC-X-307, FLUAV / H3N2 / A / Singapore / INFIMH-16-0019 / 2016-CDC-LV18A, FLUAV / H3N2 / A / Kansas / 14 / 2017, FLUAV / H3N2 / A / Kansas / 14 / 2017 CBER-22B You can choose from CDC19A, FLUAV / H3N2 / A / Switzerland / 8060 / 2017, FLUAV / H3N2 / A / Hong Kong / 2671 / 2019, FLUAV / H3N2 / A / Hong Kong / 45 / 2019, FLUAV / H3N2 / A / South Australia / 34 / 2019, FLUAV / H7N9 / A / Tri / Jiangxi Province / C483 / 2013 (H7N9), FLUAV / H10N8 / A / Jiangxi Province / IPB13 / 2013 (H10N8), and FLUAV / Vietnam / 1203 / 2004 (H5N1).

[0490] In some embodiments, the at least one antigenic peptide or protein encoded by at least one nucleic acid of component B (in particular, component B-2) comprises or consists of at least one influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acid protein (PA), polymerase basic proteins PB1, PB1-F2, and / or polymerase basic protein 2 (PB2), or fragments or variants thereof, or at least one peptide or protein selected from or derived from any synthetically engineered influenza virus peptide or protein.

[0491] In preferred embodiments, the at least one antigenic peptide or protein encoded by at least one nucleic acid of component B comprises or consists of at least one peptide or protein selected from or derived from at least one hemagglutinin (HA) or neuraminidase (NA), or an immunogenic fragment or immunogenic variant thereof.

[0492] In a particularly preferred embodiment, at least one coding region encodes at least one full-length protein of hemagglutinin (HA) and / or at least one full-length protein of neuraminidase (NA) of influenza virus or a variant thereof.

[0493] According to various embodiments, the nucleic acid of component B (in particular, component B-2) encodes at least one antigenic peptide or protein derived from the influenza virus as defined herein, and further, at least one heterologous peptide or protein element.

[0494] Preferably, at least one heterologous peptide or protein element can promote or improve the secretion of the encoded influenza antigenic peptide or protein (e.g., via a secretion signal sequence), promote or improve the fixation of the encoded antigenic peptide or protein of the present invention on the plasma membrane (e.g., via a transmembrane element), promote or improve the formation of an antigen complex (e.g., via a multimerization domain or antigen clustering element), or promote or improve virus-like particle (VLP-forming sequence). Furthermore, the nucleic acid of component B can further encode a peptide linker element, a self-cleaving peptide, an immunoadjuvant sequence, or a dendritic cell target sequence.

[0495] A suitable polymerizing domain can be selected from the list of amino acid sequences related to sequence numbers 1116-1167 of WO2017081082, or from fragments or variants of these sequences. A suitable transmembrane element can be selected from the list of amino acid sequences related to sequence numbers 1228-1343 of WO2017081082, or from fragments or variants of these sequences. A suitable VLP-forming sequence can be selected from the list of amino acid sequences related to sequence numbers 1168-1227 of WO2017081082, or from fragments or variants of these sequences. A suitable peptide linker can be selected from the list of amino acid sequences related to sequence numbers 1509-1565 of WO2017081082, or from fragments or variants of these sequences. A suitable self-cleaving peptide can be selected from the list of amino acid sequences related to sequence numbers 1434-1508 of WO2017081082, or from fragments or variants of these sequences. Suitable immunological adjuvant sequences can be selected from the list of amino acid sequences relating to sequence numbers 1360-1421 of WO2017081082, or from fragments or variants of these sequences. Suitable dendritic cell (DC) target sequences can be selected from the list of amino acid sequences relating to sequence numbers 1344-1359 of WO2017081082, or from fragments or variants of these sequences. Suitable secretory signaling peptides can be selected from the list of amino acid sequences relating to sequence numbers 1-1115 and sequence number 1728 of published PCT patent application WO2017081082, or from fragments or variants of these sequences.

[0496] In a preferred embodiment, at least one coding sequence further encodes one or more heterologous peptides or protein elements selected from signal peptides, linker peptides, helper epitopes, antigen clustering elements, trimerizing or multimerizing elements, transmembrane elements, or VLP-forming sequences.

[0497] Table 8 (rows 1-52) shows preferred antigenic peptides or proteins selected from or derived from the influenza viruses defined above. In the table, rows 1-52 correspond to preferred influenza constructs. Column A of Table 8 provides a brief description of the preferred influenza antigen constructs. Column B of Table 8 lists the sequence numbers of the proteins (amino acids) of each influenza antigen construct. Column D of Table 8 lists the sequence numbers of the corresponding G / C optimized nucleic acid coding sequences (opt1, gc). Column E of Table 8 lists the sequence numbers of the corresponding human codon frequency-matched nucleic acid coding sequences (opt3, human).

[0498] The description of this invention refers to the identifiers in the ST.25 sequence listing of this application. <223> The information provided below is explicitly included. Preferred nucleic acid constructs containing the coding sequences of Table 8, for example, mRNA sequences containing the coding sequences of Table 8, are given in Tables 9 and 10.

[0499] (Outside 9-1) TIFF0007848141000017.tif228170(Outside 9-2) TIFF0007848141000018.tif233170(Outside 9-3) TIFF0007848141000019.tif115170

[0500] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from influenza virus, encoded by at least one nucleic acid of component B (particularly component B-2), comprises or consists of at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 14178-14229, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 8 (see rows 1-52 of columns A and B) and the ST.25 sequence listing identifier for each sequence number. <223> It is also given under.

[0501] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from influenza A virus (HA), encoded by at least one nucleic acid of component B (particularly component B-2), comprises at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequence numbers 14178-14181 and 14184-14204 (HA of influenza A), or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 8 (columns A and B) and the ST.25 sequence listing identifiers for each sequence number. <223> It is also given under.

[0502] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from influenza A virus (HA), encoded by at least one nucleic acid of component B (particularly component B-2), comprises at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequence numbers 14210-14213 and 14216-14227 (NAs of influenza A), or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 8 (columns A and B) and the ST.25 sequence listing identifiers for each sequence number. <223> It is also given under.

[0503] In a preferred embodiment, at least one antigenic peptide or protein selected from or derived from influenza B virus (HA and NA), encoded by at least one nucleic acid of component B (particularly component B-2), comprises at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of sequence numbers 14182-14183, 14205-14209, 14214-14215, or 14228-14229, or an immunogenic fragment or immunogenic variant thereof. Further information regarding the amino acid sequences can be found in Table 8 (columns A and B) and the ST.25 sequence listing identifiers for each sequence number. <223> It is also given under.

[0504] In further embodiments, at least one antigenic peptide or protein, selected from or derived from at least one influenza virus and encoded by at least one nucleic acid of component B (particularly component B-2), comprises or consists of at least one amino acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs. 1-444, 458, 460, 462-479, or 543-565 of published PCT application W...

Claims

1. A pharmaceutical composition comprising or comprising at least one component A and at least one component B, Component A comprises at least one nucleic acid containing at least one coding sequence encoding a SARS-CoV-2 spike protein, selected from a protein whose amino acid sequence is sequence number 10 or an immunogenic variant thereof having at least 90% sequence identity with sequence number 10, wherein the encoded SARS-CoV-2 spike protein contains pre-fusion stabilization K986P and V987P mutations. The aforementioned component B contains multiple mRNAs, At least three of the plurality of mRNAs are independently selected from hemagglutinin (HA) of influenza A virus or influenza B virus represented by the amino acid sequence of SEQ ID NO: 14178, 14181, 14182, or 14183, or each contains at least one coding sequence of an antigenic peptide or protein having at least 90% amino acid sequence identity with respect to the HA. At least one of the plurality of mRNAs comprises at least one antigenic peptide or protein coding sequence that is selected from neuraminidase (NA) of influenza A virus or influenza B virus, represented by the amino acid sequence of SEQ ID NO: 14210, 14213, or 14214, or has at least 90% amino acid sequence identity with respect to the NA. A pharmaceutical composition characterized in that at least one nucleic acid of component A and / or a plurality of mRNAs of component B complex with or associate with one or more lipids to form lipid nanoparticles (LNPs).

2. The pharmaceutical composition according to claim 1, wherein the SARS-CoV-2 spike protein comprises the amino acid substitution D614G.

3. The pharmaceutical composition according to claim 1 or 2, wherein the SARS-CoV-2 spike protein comprises the amino acid substitution E484K.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the SARS-CoV-2 spike protein comprises amino acid substitutions K417N, E484K, N501Y, and D614G.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the SARS-CoV-2 spike protein further comprises an important neutralizing domain (CND).

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 90% sequence identity with amino acid positions 329 to 529 of SEQ ID NO:

10.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the transmembrane domain of the amino acid sequence of Sequence ID No. 10 is modified.

8. The pharmaceutical composition according to any one of claims 1 to 7, further comprising a peptide or protein encoded by at least one nucleic acid of at least one of the components A, wherein the peptide or protein comprises or consists of at least one amino acid sequence identical to any of SEQ ID NOs: 1-9, 11-111, 274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, or 26939.

9. The pharmaceutical composition according to claim 1 or 8, wherein at least one influenza virus is selected from H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, and H10N8.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein at least one mRNA among the plurality of mRNAs of component B comprises a coding sequence of an antigenic peptide or protein selected from hemagglutinin (HA) of influenza A virus or having at least 90% amino acid sequence identity with respect to the HA, and at least one other mRNA among the plurality of mRNAs comprises a coding sequence of an antigenic peptide or protein selected from hemagglutinin (HA) of influenza B virus or having at least 90% amino acid sequence identity with respect to the HA.

11. At least one amino acid sequence of a peptide or protein encoded by a plurality of mRNAs of component B is identical to any of SEQ ID NOs. 14178 to 14229, or comprises at least one amino acid sequence having at least 90% sequence identity. and / or, At least one coding sequence of the plurality of mRNAs of component B is identical to, or has at least 90% sequence identity with, any nucleic acid sequence selected from sequence numbers 14230-14333, 14334-14541, 26946, and 26947-26955. A pharmaceutical composition according to any one of claims 1 to 10.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein component B comprises mRNA sequences encoding at least seven influenza virus antigens.

13. The component B comprises a plurality of mRNA sequences that encode influenza virus antigens, and the influenza virus is FLUAV / H1N1 / A / California / 7 / 2009 FLUAV / H1N1 / A / Michigan / 45 / 2015 FLUAV / H1N1 / A / Netherlands / 602 / 2009 FLUAV / H3N2 / A / Hong Kong / 4801 / 2014, FLUBV / H0N0 / B / Brisbane / 60 / 2008, and Select from FLUBV / H0N0 / B / Phuket / 3073 / 2013 Or, Component B comprises at least four mRNA sequences encoding at least four different influenza virus antigens, wherein the influenza virus antigens are selected from the HA protein of FLUAV / H3N2 / A / Hong Kong / 4801 / 2014, the HA protein of FLUAV / H1N1 / A / California / 7 / 2009, the HA protein of FLUBV / H0N0 / B / Phuket / 3073 / 2013, the HA protein of FLUBV / H0N0 / B / Brisbane / 60 / 2008, the NA protein of FLUAV / H3N2 / A / Hong Kong / 4801 / 2014, the NA protein of FLUAV / H1N1 / A / California / 7 / 2009, and the NA protein of FLUBV / H0N0 / B / Brisbane / 60 / 2008. A pharmaceutical composition according to any one of claims 1 to 12.

14. The component B comprises at least seven nucleic acid sequences encoding at least seven influenza virus antigens as the sequences of the plurality of mRNAs, The seven influenza virus antigens mentioned above are: HA protein of FLUAV / H3N2 / A / Hong Kong / 4801 / 2014 HA protein of FLUAV / H1N1 / A / California / 7 / 2009 HA protein of FLUBV / H0N0 / B / Phuket / 3073 / 2013 HA protein of FLUBV / H0N0 / B / Brisbane / 60 / 2008, NA protein of FLUAV / H3N2 / A / Hong Kong / 4801 / 2014 The NA protein of FLUAV / H1N1 / A / California / 7 / 2009, and The NA protein of FLUBV / H0N0 / B / Brisbane / 60 / 2008, A pharmaceutical composition according to any one of claims 1 to 13.

15. The component B is (1) to (7): (1) mRNA encoding an influenza virus antigen whose amino acid sequence is identical to or at least 90% identical to sequence number 14178, (2) mRNA encoding an influenza virus antigen whose amino acid sequence is identical to or at least 90% identical to sequence number 14181, (3) mRNA encoding an influenza virus antigen whose amino acid sequence is identical to or at least 90% identical to sequence number 14182, (4) mRNA encoding an influenza virus antigen whose amino acid sequence is identical to or at least 90% identical to sequence number 14183, (5) mRNA encoding an influenza virus antigen whose amino acid sequence is identical to or at least 90% identical to sequence number 14210, (6) mRNA encoding an influenza virus antigen whose amino acid sequence is identical to or at least 90% identical to sequence number 14213, and (7) mRNA encoding an influenza virus antigen whose amino acid sequence is identical to or at least 90% identical to sequence number 14214, Includes seven mRNAs, each selected as at least one from the following: A pharmaceutical composition according to any one of claims 1 to 13.

16. At least one coding sequence of the nucleic acid of component A and / or at least one coding sequence of the plurality of mRNAs of component B is a codon-modified coding sequence, At least one of the codon-modified coding sequences is selected from C-maximizing coding sequences, CAI-maximizing coding sequences, human codon frequency-adapted coding sequences, G / C content-modified coding sequences, and G / C-optimized coding sequences, or any combination thereof. A pharmaceutical composition according to any one of claims 1 to 15.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein at least one coding sequence of component A and / or a plurality of mRNAs of component B have a G / C content of at least 50%.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein at least one nucleic acid of component A and / or a plurality of mRNAs of component B include at least one heterologous untranslated region selected from at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR.

19. The at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence derived from PSMB3, ALB7, alphaglobin, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or an analog, fragment, or variant of any of these genes. and / or, The at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence derived from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or an analog, fragment, or variant of any of these genes. The pharmaceutical composition according to claim 18.

20. At least one nucleic acid of component A and / or a plurality of mRNAs of component B are - Alphaglobin 3'-UTR; - HSD17B4 5'-UTR and PSMB3 3'-UTR (HSD17B4 / PSMB3); - SLC7A3 5'-UTR and PSMB3 3'-UTR (SLC7A3 / PSMB3); or - RPL31 5'-UTR and RPS9 3'-UTR (RPL31 / RPS9) Includes at least one code array that is operably linked to, and / or, At least one nucleic acid of component A and / or a plurality of mRNAs of component B include at least one coding sequence operably linked to HSD17B4 5'-UTR and PSMB3 3'-UTR (HSD17B4 / PSMB3), A pharmaceutical composition according to any one of claims 1 to 19.

21. At least one nucleic acid of component A and / or a plurality of mRNAs of component B each contain at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides. and / or, At least one nucleic acid of component A and / or multiple mRNAs of component B include at least one histone stem-loop or histone stem-loop structure. and / or, The pharmaceutical composition according to any one of claims 1 to 20, wherein at least one nucleic acid of component A and / or a plurality of mRNAs of component B include a 5' cap structure selected from m7G, cap0, cap1, cap2, modified cap0, or modified cap1 structures.

22. At least one nucleic acid of component A and / or a plurality of mRNAs of component B are purified mRNAs. and / or, At least one nucleic acid of component A and / or multiple mRNAs of component B are mRNAs, and the mRNAs have at least 50% RNA integrity. A pharmaceutical composition according to any one of claims 1 to 21.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein at least one nucleic acid of component A and / or a plurality of mRNAs of component B comprises at least one modified nucleotide selected from pseudouridine (Ψ) and N1-methylpseudridine (m1Ψ).

24. The pharmaceutical composition according to any one of claims 1 to 23, comprising at least one pharmaceutically acceptable carrier.

25. At least one nucleic acid of component A and / or a plurality of mRNAs of component B are incorporated into separate lipid nanoparticles (LNPs). and / or, At least one nucleic acid of component A and / or multiple mRNAs of component B are co-combined in lipid nanoparticles (LNPs). A pharmaceutical composition according to any one of claims 1 to 24.

26. The pharmaceutical composition is a freeze-dried composition, a spray-dried composition, or a spray freeze-dried composition, The lyophilized composition or the spray-dried composition according to any one of claims 1 to 25, wherein the lyophilized composition or the spray-dried composition has a water content of less than 10%.

27. ​​When the pharmaceutical composition is administered to a subject, at least one nucleic acid of component A and / or at least one mRNA of component B are translated into a peptide or protein suitable for inducing an immune response. and / or, When the pharmaceutical composition is administered to a subject, an antigen-specific humoral immune response to the peptide or protein encoded by component A and an antigen-specific humoral immune response to the peptide or protein encoded by component B are induced in the subject. and / or, When the pharmaceutical composition is administered to a subject, an antigen-specific T cell response to the peptide or protein encoded by component A and an antigen-specific T cell response to the peptide or protein encoded by component B are induced in the subject. A pharmaceutical composition according to any one of claims 1 to 26.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein when the pharmaceutical composition is administered to a subject, antigen-specific B cell memory for a peptide or protein encoded by component A and antigen-specific B cell memory for a peptide or protein encoded by component B are established in the subject.

29. A mixed vaccine comprising the pharmaceutical composition according to any one of claims 1 to 28, The aforementioned combination vaccine is characterized by being a vaccine against at least one SARS-CoV-2 virus and at least one influenza virus.

30. A pharmaceutical composition according to any one of claims 1 to 28, or a mixed vaccine according to claim 29, and At least one liquid vehicle for solubilization, and A kit or kit of parts characterized by including a technical manual providing information on the dosage and usage of each kit component.

31. For use as a medicine, For use in the treatment or prevention of infections caused by SARS-CoV-2 and at least one influenza virus, or disorders associated with such infections, Or, For use in the treatment or prevention of disorders caused by infectious diseases, and the subject is a mammal. Or, For use in the treatment or prevention of disorders caused by infections with SARS-CoV-2 and at least one influenza virus, the drug prevents moderate and severe COVID-19 disease and at least one further disease associated with coronavirus infection and influenza virus infection in at least 80% of subjects treated for 2 weeks to 3 months after administration. A pharmaceutical composition according to any one of claims 1 to 28, a mixed vaccine according to claim 29, or a kit or kit of parts according to claim 30.

32. For use in the treatment or prevention of disorders caused by infectious diseases, The subjects will be administered a single dose containing 5 μg to 50 μg of RNA. Or, The subjects will be administered two doses containing 5 μg to 50 μg of RNA. A pharmaceutical composition according to any one of claims 1 to 28, a mixed vaccine according to claim 29, or a kit or kit of parts according to claim 30.

33. A method for stabilizing a pharmaceutical composition according to any one of claims 1 to 28, or a mixed vaccine according to claim 29, A method characterized by comprising freeze-drying the pharmaceutical composition or the mixed vaccine to produce a stabilized pharmaceutical composition or the mixed vaccine having a water content of less than 10%.

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