Immunogenic composition and a method for preparing thereof

Abstract

The present invention relates to an immunogenic composition and a method for preparing the same.

Description

Technology Field

[0001] The present invention relates to an immunogenic composition and a method for preparing the same. Background Technology

[0003] RNA 5'-capping is the first step of the pre-mRNA processing in eukaryotic cells, in which a cap is placed on the 5' end of transcribed RNA. Naturally occurring cap structures contain a ribo-guanosine residue that is methylated at position N7 of the guanine base. In living eukaryotes, the formation of a 5' cap (cap-0) at the 5' end of the mRNA molecule, in which 7-methylguanosine (m7G) is linked via a 5' triphosphate bond, is mediated by the enzyme methyltransferase. Additionally, eukaryotic mRNA may contain further modifications, including the methylation of the 2' hydroxyl group of the first ribose sugar at the 5' end (cap-1) and the methylation of the 2' hydroxyl group of the second ribose sugar (cap-2). Through this 5' capping process of mRNA, the mRNA acquires resistance to degrading enzymes such as 5' exonucleases when it exits the nucleus into the cytoplasm.

[0004] A specific gene can be artificially expressed by transfecting capped mRNA encoding that gene into eukaryotes or by microinjecting it into cells or embryos. However, if uncapped RNA is used in this process, the RNA not only degrades rapidly but also exhibits immunogenicity, leading to a sharp decrease in protein translation efficiency. Therefore, it is crucial that 5' capping is properly performed to synthesize mRNA in vitro.

[0005] Since capped RNA transcripts can be applied to therapeutic and / or preventive fields requiring protein synthesis, such as nucleic acid therapeutics and vaccines, it is necessary to develop cap structures that can maintain mRNA stability in vivo, improve translation efficiency, or reduce side effects such as immunogenicity when applied as RNA therapeutics or vaccines. Prior art literature

[0007] (Patent Document 0001) KR 10-2022-0046693 A The problem to be solved

[0008] The object of the present invention is to provide an immunogenic composition comprising a novel Cap structure and a method for preparing the same. means of solving the problem

[0010] One embodiment of the present invention is an immunogenic composition.

[0011] In one embodiment, the immunogenic composition comprises a polynucleotide having a 5'Cap structure of any one of the following formulas 1 to 4:

[0012] [Chemical Formula 1]

[0013]

[0014] [Chemical Formula 2]

[0015]

[0016] [Chemical Formula 3]

[0017]

[0018] [Chemical Formula 4]

[0019]

[0020] As an immunogenic composition according to the prior embodiment, the polynucleotide comprises a 5'Cap structure represented by Chemical Formula 3 or 4.

[0021] As an immunogenic composition according to any one of the prior embodiments, the immunogenic composition comprises a polynucleotide having a 5'Cap structure represented by Formula 3; and a polynucleotide having a 5'Cap structure represented by Formula 4.

[0022] As an immunogenic composition according to any one of the preceding embodiments, the polynucleotide comprises a 5'UTR (untranslated region) sequence.

[0023] As an immunogenic composition according to any one of the aforementioned embodiments, the 5'UTR (untranslated region) sequence is derived from HBA2 (Hemoglobin subunit alpha 2).

[0024] As an immunogenic composition according to any one of the preceding embodiments, the 5'UTR sequence comprises SEQ ID NO. 3 or a sequence having 80% or more homology or identity with it.

[0025] As an immunogenic composition according to any one of the preceding embodiments, the polynucleotide comprises a 3'UTR (untranslated region) sequence.

[0026] As an immunogenic composition according to any one of the aforementioned embodiments, the 3'UTR sequence is derived from HBB (Hemoglobin subunit beta).

[0027] As an immunogenic composition according to any one of the preceding embodiments, the 3'UTR sequence comprises SEQ ID NO. 5 or a sequence having 80% or more homology or identity with it.

[0028] An immunogenic composition according to any one of the preceding embodiments, wherein the polynucleotide comprises SEQ ID NO. 3 or a 5'UTR sequence having 80% or more homology or identity therewith; and SEQ ID NO. 5 or a 3'UTR sequence having 80% or more homology or identity therewith.

[0029] As an immunogenic composition according to any one of the prior embodiments, the polynucleotide comprises a sequence encoding a peptide or protein derived from a coronavirus or influenza virus that exhibits immunogenicity.

[0030] An immunogenic composition according to any one of the aforementioned embodiments, wherein the polynucleotide comprises a sequence encoding the S protein of a coronavirus or influenza virus or a fragment thereof.

[0031] As an immunogenic composition according to any one of the preceding embodiments, the coronavirus is SARS-CoV-2.

[0032] As an immunogenic composition according to any one of the preceding embodiments, the S protein of the coronavirus comprises an A701V variant.

[0033] As an immunogenic composition according to any one of the preceding embodiments, the polynucleotide comprises CDS of SEQ ID NO. 4.

[0034] As an immunogenic composition according to any one of the aforementioned embodiments, the polynucleotide comprises a poly A tail.

[0035] As an immunogenic composition according to any one of the aforementioned embodiments, the immunogenic composition may be formulated.

[0036] As an immunogenic composition according to any one of the aforementioned embodiments, the polynucleotide may be contained within lipid nanoparticles.

[0037] As an immunogenic composition according to any one of the aforementioned embodiments, the immunogenic composition may induce immunity against coronavirus or influenza virus.

[0038] Another embodiment for implementing the present invention is a method for preparing an immunogenic composition.

[0039] In one embodiment, the manufacturing method comprises the step of performing mRNA synthesis by mixing a composition comprising a DNA template with one or more compounds of any one of the following chemical formulas 5 to 8 or a salt thereof:

[0040] [Chemical Formula 5]

[0041]

[0042] [Chemical Formula 6]

[0043]

[0044] [Chemical Formula 7]

[0045]

[0046] [Chemical Formula 8]

[0047]

[0049] A manufacturing method according to any one of the preceding embodiments, wherein the manufacturing method comprises the step of performing mRNA synthesis by including a composition comprising one or more compounds of Chemical Formula 7 or 8 or a salt thereof and a composition comprising a DNA template.

[0050] A method of manufacturing according to any one of the aforementioned embodiments, wherein the DNA template comprises a sequence complementary to the mRNA polynucleotide.

[0051] A manufacturing method according to any one of the aforementioned embodiments, wherein the DNA template comprises a sequence encoding a peptide or protein derived from a coronavirus or influenza virus that exhibits immunogenicity.

[0052] A manufacturing method according to any one of the preceding embodiments, wherein the manufacturing method comprises the step of performing poly A tailing of mRNA.

[0053] As a manufacturing method according to any one of the aforementioned embodiments, the mRNA synthesis is performed in vitro.

[0054] A manufacturing method according to any one of the aforementioned embodiments, wherein the manufacturing method includes a step of formulation of mRNA.

[0055] A manufacturing method according to any one of the preceding embodiments, wherein the manufacturing method comprises the step of manufacturing mRNA in a form contained in lipid nanoparticles.

[0056] Another embodiment for implementing the present invention is a composition for preparing an immunogenic composition.

[0057] In one embodiment, the composition for preparing the immunogenic composition comprises one or more compounds of Formulas 5 to 8 and a DNA template.

[0058] As a composition according to any one of the aforementioned embodiments, the composition for preparing the immunogenic composition comprises components necessary for RNA synthesis.

[0059] As a composition according to any one of the aforementioned embodiments, the composition for preparing the immunogenic composition comprises a polymerase.

[0060] Another embodiment for implementing the present invention is a method for preventing a viral infectious disease, comprising the step of administering the immunogenic composition to an individual.

[0061] In one embodiment, the virus may be selected from coronavirus and influenza virus.

[0063] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below. Additionally, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the level of the art to which the present invention pertains and the content of the present invention.

[0064] Furthermore, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present invention described in this invention using only ordinary experiments. In addition, such equivalents are intended to be included in the present invention.

[0065] One aspect of the present invention provides an immunogenic composition.

[0066] The above immunogenic composition may include a polynucleotide comprising a 5'Cap structure of any one of the following chemical formulas 1 to 4:

[0067] [Chemical Formula 1]

[0068]

[0069] [Chemical Formula 2]

[0070]

[0071] [Chemical Formula 3]

[0072]

[0073] [Chemical Formula 4]

[0074]

[0076] For example, the polynucleotide may include a 5'Cap structure represented by the chemical formula 3 or 4.

[0077] For example, the immunogenic composition may include a polynucleotide having a 5'Cap structure represented by Formula 3; and a polynucleotide having a 5'Cap structure represented by Formula 4.

[0078] In the present invention, the term "5' cap" refers to a modified entity that caps the end of mRNA. The 5' cap can generally be formed by a modification, for example, a guanine derivative. Examples of such guanine nucleotide derivatives are included in Chemical Formulas 1 to 4 above.

[0079] For example, the 5' cap can be attached to the 5' end via a 5'->5' triphosphate bond. For example, the 5' cap can be methylated. For example, the polynucleotide of the present invention can be 5'-capped in vitro. Methods known in the art can be used for capping, that is, for forming a cap structure. For example, if a cap analogue is added during in vitro transcription, capping can be performed along with transcription. This is also referred to as co-transcriptional capping. In the case of the co-transcriptional method, the cap is incorporated into the RNA molecule simultaneously with transcription during the in vitro transcription process. As another example, capping can be performed via an enzymatic reaction after in vitro transcription. Enzymes such as triphosphatase and guanyltransferase may be involved in such enzymatic reactions.

[0081] In the present invention, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, and refers to a DNA or RNA strand of a certain length or longer.

[0082] A polynucleotide containing a specific 5'-Cap structure that may be included in the immunogenic composition disclosed in the present invention may be RNA. More specifically, it may be mRNA. In the present invention, mRNA refers to messenger RNA. mRNA generally contains a sequence encoding a protein or peptide and is involved in the transcription of RNA.

[0083] The polynucleotide of the present invention may include, without limitation, a desired RNA sequence that can be used as a desired immunogenic composition, and may include, without limitation, a sequence in which the induction of an immune response is enhanced due to the Cap structure of the present invention.

[0084] The polynucleotide included in the immunogenic composition of the present invention may include a sequence encoding a virus-derived peptide or protein that exhibits immunogenicity. The sequence encoding the peptide or protein may also be referred to as "CDS".

[0085] The CDS region included in the mRNA of the present invention may undergo various modifications to the coding region within a range that does not alter the amino acid sequence, taking into account the degeneracy of codons or codons preferred by the organism intended to express the variant of the present application.

[0086] The virus of the present invention may be a pathogenic virus that causes disease in humans and / or animals, and examples of such viruses may include viruses of the genus Orthomyxoviridae, viruses of the genus Picornaviridae, viruses of the genus Retroviridae, viruses of the genus Herpes, viruses of the genus Filoviridae, viruses of the genus Coronaviridae, viruses of the genus Hepadnaviridae, viruses of the genus Flaviviridae, viruses of the genus Bunyaviridae, etc. Specifically, the virus of the present invention may be a coronavirus or an influenza virus.

[0087] In the present invention, the term "coronavirus" refers to a positive-sense single-stranded RNA virus (ssRNA) of about 80-220 nm in size that belongs to the Coronaviridae family and has a spherical outer membrane. As an example, the coronavirus may be SARS-CoV-2.

[0088] In the present invention, the term "SARS-CoV-2" refers to the virus that causes coronavirus disease 2019 (COVID-19), a respiratory disease. SARS-CoV-2 is a positive single-stranded RNA virus. Taxonomically, SARS-CoV-2 is a strain of SARS-CoV, has zoonotic origins, and is considered to have close genetic similarity to bat coronaviruses. The said SARS-CoV-2 is transmitted primarily through close contact between people and / or through droplets produced during coughing or sneezing, and is known to enter human cells primarily by the spike (S) protein on the outer shell of the virus binding to the angiotensin converting enzyme 2 (ACE2) receptor present on the surface of human cells.

[0089] In the present invention, the term "influenza virus" refers to a virus belonging to the family Orthomyxoviridae. The influenza virus is a single-stranded, helical RNA virus that can be classified into types A, B, and C based on the composition of its nucleic acid. The form that causes infection in humans is known to be primarily type A influenza virus. The subtype of the type A influenza virus is determined by the surface antigens hemagglutinin (HA) and neuraminidase (NA). This HA plays a role in the virus attaching to somatic cells and has 16 subtypes (H1 to H16). NA plays an important role in the virus being released from infected cells and spreading to new respiratory cells, and it has 9 subtypes (N1 to N9).

[0090] The above-mentioned influenza virus may be influenza virus A, influenza virus B, or influenza virus C, and specifically, may be H1N1 influenza virus A, H2N2 influenza virus A, H3N2 influenza virus A, H2N3 influenza virus A, H5N1 influenza virus A, H7N9 influenza virus A, or influenza virus B. In addition, the above-mentioned influenza virus may cause influenza, the common cold, pharyngitis, bronchitis, and pneumonia, and may cause avian influenza, swine influenza, or goat influenza in animals other than humans.

[0091] The "coronavirus" and "influenza virus" of the present invention include variants thereof. For example, SARS-CoV-2 includes, without limitation, all variant forms that can be classified as SARS-CoV-2, and may include, for example, variants referred to as alpha (B.1.1.7), beta (B.1.351, B.1.351.2, B.1.351.3), delta (B.1.617.2, AY.1, AY.2, AY.3), gamma (P.1, P.1.1, P.1.2), and omecro (B.1.1.529, BA.1, BA.1.1, BA.2, BA.3) variants. Variant SARS-CoV-2 may include specific substitutions or combinations of substitutions in the spike protein. Specifically, the S protein of SARS-CoV-2 of the present invention may include an A701V substitution. However, it is not limited thereto. Additionally, the SARS-CoV-2 may include L452R, E484K, K417N / T, E484K, N501Y, or combinations of substitutions thereof.

[0093] The polynucleotide included in the immunogenic composition of the present invention may include a sequence encoding the S protein of a coronavirus or influenza virus or a fragment thereof.

[0094] For example, the CDS sequence included in the mRNA of the present invention may have or include the sequence of SEQ ID NO. 4, or be composed of said sequence, or essentially be composed of said sequence. For example, it may have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with SEQ ID NO. 4, or be composed of or essentially composed of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with the sequence of SEQ ID NO. 4, but is not limited thereto.

[0095] For example, the mRNA of the present invention may have or include the nucleotide sequence of SEQ ID NO. 2, or be composed of or essentially composed of the sequence of SEQ ID NO. 2. For example, the mRNA of the present invention may have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with the sequence of SEQ ID NO. 2, or be composed of or essentially composed of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with the sequence of SEQ ID NO. 2, but is not limited thereto.

[0097] The polynucleotide included in the immunogenic composition of the present invention may include a 5'UTR sequence.

[0098] In the present invention, the term "5'UTR sequence" refers to an untranslated region located upstream of the start codon. The 5'UTR may be involved in regulating the translation of RNA and / or stabilizing mRNA.

[0099] The 5' UTR sequence of the present invention can be modified or optimized to improve mRNA stability and / or translation accuracy. For example, avoiding gene sequences similar to the beginning of the ORF in the 5' UTR can effectively prevent incorrect frame initiation during mRNA translation. Additionally, certain specific sequences can be added to the 5' UTR to improve mRNA stability and translation accuracy. For example, a Kozak sequence can be inserted to configure the translation process to start more accurately.

[0100] For example, the 5'UTR (untranslated region) sequence of the present invention may be derived from HBA2 (Hemoglobin subunit alpha 2). For example, the 5'UTR sequence may have or include the sequence of SEQ ID NO. 3, or be composed of or essentially constitute the sequence. For example, it may have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with SEQ ID NO. 3, or may be composed of or necessarily composed of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with the sequence of SEQ ID NO. 3, but is not limited thereto.

[0102] The polynucleotide included in the immunogenic composition of the present invention may include a 3'UTR (untranslated region) sequence.

[0103] In the present invention, the "3'UTR sequence" refers to an untranslated region located downstream of the coding region of mRNA.

[0104] The 3' UTR sequence of the present invention can be modified or optimized to improve mRNA stability and / or translation accuracy. For example, by using an appropriate sequence for the 3' UTR, mRNA stability can be increased and the half-life can be increased.

[0105] For example, the above 3'UTR sequence may be derived from HBB (Hemoglobin subunit beta). For example, the above 3'UTR sequence may have or include the sequence of SEQ ID NO. 5, or be composed of the above sequence, or essentially be composed of. For example, it may have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with SEQ ID NO. 5, or may be composed of or necessarily composed of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with the sequence of SEQ ID NO. 5, but is not limited thereto.

[0106] For example, a polynucleotide included in the immunogenic composition of the present invention may include SEQ ID NO. 3 or a 5'UTR sequence having 80% or more homology or identity therewith, and SEQ ID NO. 5 or a 3'UTR sequence having 80% or more homology or identity therewith.

[0108] The polynucleotides of the present invention may include modified nucleotides. In the present invention, "modified nucleotide" means any atypical nucleoside, nucleotide, or its corresponding phosphorylated form. Modified nucleotides may have one or more backbone or base modifications. Examples of modified nucleotides include dI, dU, 8-oxo-dG, dX, and THF, and may include other natural or non-natural nucleotides known in the art.

[0109] In the present invention, the term “non-natural” means a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature. Such polynucleotides, polypeptides, carbohydrates, lipids, or compositions may differ from natural polynucleotides, polypeptides, carbohydrates, lipids, or compositions in one or more aspects. For example, a polymer (e.g., polynucleotide, polypeptide, or carbohydrate) may differ in terms of the type and arrangement of constituent building blocks (e.g., nucleotide sequence, amino acid sequence, or sugar molecule). A polymer may differ from a natural polymer in terms of the molecule(s) linking them.

[0111] The polynucleotide included in the immunogenic composition of the present invention may additionally include a poly A tail.

[0112] In the present invention, the term "poly A tail" refers to a continuous or discontinuous sequence of adenylate residues typically located at the 3' end of an RNA molecule. The poly A tail may follow the mRNA 3'UTR of the present invention. Such a poly A tail may consist of or contain 20 or more, 25 or more, 40 or more, 60 or more, 80 or more, or 100 or more adenyl (A) nucleotides. Based on the number of nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are A nucleotides, but the remaining nucleotides may be nucleotides other than A nucleotides, e.g., U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidilic acid). For example, a poly A tail may include modifications that delay the degradation of mRNA.

[0114] In the present invention, the term "immunogenicity" refers to a property that induces an immune response when introduced into the body. Infection in an individual can be prevented by administering the immunogenic composition of the present invention to an individual. Therefore, the immunogenic composition of the present invention may be a vaccine composition.

[0115] The above-mentioned immunogenic composition may induce immunity against coronavirus or influenza virus. The virus is as described above.

[0116] The above-mentioned immunogenic composition may be in a formulation. Such formulation may be in a solid or liquid form, or a combination thereof. For example, the polynucleotide of the present invention may be formulated in a form suitable for infusion or intravenous administration.

[0117] For example, the polynucleotide of the present invention may be formulated in the form of particles. The particles may be lipid nanoparticles. For example, they may be LNP (Lipid nanoparticle) or LPX (Lipoplex). LNP particles include an ionizable cationic lipid as one component and may also include other components such as helper lipids and stabilizers that encapsulate mRNA or help with delivery efficiency and stabilization. For example, LNP may include cationic lipids and neutral lipids (e.g., phospholipids), sterols or steroids (e.g., cholesterol), PEG-conjugated lipids, etc.At this time, specific examples of the above cationic lipids include ALC-0315 used in the BNT162b2 vaccine, SM-102 used in the mRNA-1273 vaccine, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA). DLin-KC2-DMA, (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLinMC3-DMA, CAS 1224606-06-7), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), etc. may be used, but are not specifically limited thereto.

[0118] In one embodiment, the lipid nanoparticles comprise cationic lipids of the aforementioned types (in the range of 35 to 60 mol% of total lipids), phospholipids such as DSPC (in the range of 5 to 15 mol% of total lipids), cholesterol (in the range of 30 to 50 mol% of total lipids), DMG-PEG 2000 It may contain PEG-conjugated lipids such as (in the range of 0.5 to 2.5 mol% of total lipids), but is not particularly limited thereto.

[0120] In one embodiment, the lipid nanoparticles may include the BNT162b2 or mRNA-1273 vaccine shown below, or more specifically, particles composed of ALC-0315:DSPC:Chol:ALC-0159 (47.5:10:40.8:1.7 mol%), but are not particularly limited thereto.

[0121]

[0123] In one embodiment, lipid compounds disclosed in WO2014 / 172045A1, WO2020 / 252589A1, and WO2021 / 000041A1 may be used as the lipids of the lipid nanoparticles without special limitation, and PNI123 / DSPC / Cholesterol / PEG-DMG (50:10:38.5 or 37.5:1.5 or 2.5 mol%) and PNI123 / DOPE / Cholesterol / PEG-DMG may be used, but are not particularly limited thereto.

[0124] LPX particles can be manufactured by mixing polynucleotides with liposomes.

[0125] For example, the polynucleotide of the present invention may be formulated in a colloidal form. For example, the polynucleotide of the present invention may be formulated in a particle form to form a dispersed phase of the colloid. However, it is not limited thereto.

[0126] The above immunogenic composition may further comprise a pharmaceutically acceptable carrier, excipient, binder, carrier, preservative, buffer, isotonic agent, emulsifying agent, or wetting agent.

[0127] The above immunogenic composition can be used to generate antibodies in mammals.

[0128] The immunogenic composition of the present invention may be provided in a single dose or a multidose dose. “Multidose dose” or “multidose dose” refers to a unit of formulation containing a dose that can be administered to one subject or more than one subject more than once (e.g., two or more times).

[0129] The immunogenic composition of the present invention may comprise an immunologically effective amount of polynucleotide. The term "immunologically effective amount" is an amount effective in inducing an antibody response to an antigen when administered to an individual. The immunologically effective amount may vary depending on the health and physical condition of the individual to be treated, the age of the individual, the capacity of the individual's immune system to synthesize antibodies, the level of protection required, the formulation of the composition, the treating physician's assessment of the medical situation, and other relevant factors.

[0130] The immunogenic composition provided in the present invention may include an adjuvant. In the present invention, "adjuvant" refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention. The adjuvant is often provided to enhance an immune response.

[0132] Another aspect of the present invention provides a method for preparing the immunogenic composition.

[0133] The method for preparing the above immunogenic composition may include the step of performing mRNA synthesis by mixing a composition comprising a DNA template with one or more compounds of Chemical Formulas 5 to 8 or a salt thereof:

[0134] [Chemical Formula 5]

[0135]

[0136] [Chemical Formula 6]

[0137]

[0138] [Chemical Formula 7]

[0139]

[0140] [Chemical Formula 8]

[0141]

[0143] Examples of the above salts include sodium salts, potassium salts, Tris, ammonium salts, magnesium salts, etc., but are not necessarily limited thereto.

[0144] For example, the above salt may be a sodium salt. Specifically, it may be a trisodium salt. However, it is not limited thereto.

[0146] Specifically, the above manufacturing method may include the step of performing mRNA synthesis by including a composition comprising one or more compounds of chemical formula 7 or 8 or a salt thereof and a composition comprising a DNA template.

[0147] For example, the DNA template may have or include the nucleotide sequence of SEQ ID NO. 1, or be composed of or essentially composed of the sequence of SEQ ID NO. 1. As an example, the DNA template may have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with the sequence of SEQ ID NO. 1, or be composed of or essentially composed of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% homology or identity with the sequence of SEQ ID NO. 1, but is not limited thereto.

[0148] The DNA template may include a sequence complementary to the mRNA polynucleotide. The DNA template may include any promoter for any RNA polymerase to initiate transcription. For example, the DNA template may be obtained by cloning a nucleic acid, such as cDNA, and introducing it into a suitable expression cassette or vector for transcription. The DNA template may include a sequence encoding a peptide or protein derived from a coronavirus or influenza virus that exhibits immunogenicity. This is as described above.

[0149] The above mRNA synthesis can be performed in vitro. Such a method for mRNA synthesis is known in the art.

[0150] For example, the above DNA template may include a dA nucleotide corresponding to a poly A tail of mRNA. Nucleotides such as dA, dG, dT, and dC may be included within the dA nucleotide sequence corresponding to the poly A tail.

[0151] The above manufacturing method may include a step of performing poly A tailing of mRNA. Poly A tailing may be performed by transcribing a poly A tail encoded by template DNA by a dependent RNA polymerase, or by attaching a poly A sequence to synthesized mRNA.

[0152] The above manufacturing method may include a step of formulating mRNA. When formulating, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used. For example, the above manufacturing method may include a step of preparing mRNA in a form contained within lipid nanoparticles.

[0154] Another aspect of the present invention provides a composition for preparing the immunogenic composition.

[0155] A composition for preparing the above immunogenic composition may comprise one or more compounds of the aforementioned chemical formulas 5 to 8 or a salt thereof; and a DNA template. The compounds of the above chemical formulas and the DNA template are as described above.

[0156] The composition for preparing the above-mentioned immunogenic composition may include components necessary for synthesizing RNA. Specifically, it may include components for in vitro transcription (IVT). For example, the composition may include RNA polymerase. For example, the composition may include ATP, GTP, UTP, CTP, or analogues thereof. For example, the composition may include a buffer solution.

[0158] Another aspect of the present invention provides a method for preventing a viral infectious disease, comprising the step of administering the immunogenic composition to an individual.

[0159] The immunogenic composition of the present invention can be used to prevent diseases caused by viral infection. The term "prevention" in the present invention refers to any act that suppresses or delays the onset of a disease.

[0160] The above-mentioned entities include, without limitation, animals capable of developing viral infectious diseases. Specifically, they include mammals such as cattle, pigs, sheep, chickens, dogs, and humans, birds, etc., and include, without limitation, entities in which viral infectious diseases are treated by the administration of the above-mentioned composition of the present invention.

[0161] The above immunogenic composition and virus are as described above. Effects of the invention

[0163] mRNA containing a specific 5'Cap structure disclosed in the present invention has high in vitro transcription efficiency and excellent immunogenicity of the composition containing it, so it can be usefully used in vaccine manufacturing. Specific details for implementing the invention

[0165] The present invention will be explained in more detail below through examples and experimental examples. However, these examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0167] <Manufacturing Example> Cap Reagent Manufacturing

[0168] Compounds of Preparation Examples 20-22 corresponding to three Cap Reagents were prepared.

[0170] Explanation of abbreviations

[0171] The abbreviations used below are as follows.

[0172] TBSCl: Tertiary-butyldimethylsilyl chloride

[0173] DMAP: 4-Dimethylaminopyridine

[0174] DMSO: Dimethyl sulfoxide

[0175] TFA: Trifluoroacetic acid

[0176] DCM: Dichloromethane

[0177] TEP: Triethylphosphate

[0178] DPS: 2,2'-Dipyridyl Disulfide

[0179] PPh3: Triphenylphosphine

[0180] DMF: Dimethylformamide

[0181] DMS: Dimethyl sulfate

[0182] PW: Distilled water

[0183] EDTA: Ethylenediaminetetraacetic acid

[0184] THF: Tetrahydrofuran

[0185] ACN: Acetonitrile

[0186] TEA: Triethylamine

[0187] TCA: Trichloroacetic acid

[0189] Scheme 1

[0190]

[0191] Preparation Example 1: Preparation of 5'-O-tert-butyldimethylsilyl-guanosine (1)

[0192] 20 g (70.6 mmol) of guanosine was dissolved in 200 ml of dimethyl sulfoxide, and then 21.6 g (155.4 mmol) of triethylamine, 21.2 g (141.2 mmol) of tert-butyldimethylsilyl chloride, and 860 mg (7 mmol) of dimethylaminopyridine were added under nitrogen. After stirring at room temperature for 24 hours, the mixture was slowly added dropwise to distilled water at 0 to 5°C, and the resulting precipitate was filtered. The filtered precipitate was purified using column chromatography to obtain 24.4 g (87%) of the target compound (1). 1 H NMR (400 MHz, CD3OD, 25℃): δ= 7.96 (s, 1H), 5.89 (d, 1H), 4.47 (t, 1H), 4.33 (t, 1H), 4.08 (m, 1H), 3.94 ~ 3.87 (m, 2H), 0.94 (m, 9H), 0.12 (m, 6H).

[0194] Preparation Example 2: Preparation of 2',3'-pivaloyl-5'-O-tert-butyldimethylsilyl-guanosine (2)

[0195] 10 g (25.2 mmol) of 5'-O-tert-butyldimethylsilyl-guanosine (1) was dissolved in 100 ml of anhydrous pyridine, and 23.4 g (125.6 mmol) of pivalic anhydrous was added and stirred at room temperature. After the reaction was complete, 500 ml of ethyl acetate was added, and the mixture was washed with 400 ml of saturated sodium bicarbonate aqueous solution and 400 ml of 20% citric acid, dried with magnesium sulfate, and subjected to vacuum distillation. 10.26 g (72%) of the target compound (2) was obtained by column chromatography. 1 ¹H NMR (400 MHz, DMSO- d6, 25℃): δ= 10.70 (s, 1H), 7.83 (d, 1H), 6.51 (br, 2H), 5.93 (d, 1H), 5.69 (m, 1H), 5.40 (m, 1H), 4.21 (m, 1H), 3.86 (m, 2H), 1.20 (s, 9H), 1.04 (s, 9H), 0.88 (s, 9H), 0.08 (s, 6H).

[0197] Preparation Example 3: Preparation of 2',3'-pivaloyl-guanosine (3)

[0198] 10 g (17.6 mmol) of 2',3'-pivaloyl-5'-O-tert-butyldimethylsilyl-guanosine (2) was dissolved in 300 mL of dichloromethane, and 8 mL of distilled water and 70 mL of trifluoroacetic acid were added sequentially. After stirring for 30 minutes, the mixture was neutralized with 2N sodium hydroxide. The organic layer and the aqueous layer were separated, the aqueous layer was extracted with 300 mL of dichloromethane, the organic layer was collected, washed with a saturated aqueous sodium bicarbonate solution and brine, dried with magnesium sulfate, and subjected to vacuum distillation. Using column chromatography, 5.16 g (64.7%) of the target compound (3) was obtained. 1 ¹H NMR (400 MHz, DMSO- d6 , 25℃): δ= 10.69 (s, 1H), 7.97 (d, 1H), 6.49 (br, 2H), 5.92 (d, 1H), 5.72 (dd, 1H), 5.45 (t, 1H), 5.40 (dd, 1H), 4.16 (m, 1H), 3.67 (m, 2H), 1.21 (m, 9H), 1.05 (m, 9H).

[0200] Example 4: Preparation of 2',3'-pivaloyl-guanosine 5'-monophosphate (pG(2',3' Pivaloyl))(4)

[0201] 4.51 g (10 mmol) of 2',3'-pivaloyl-guanosine (3) was added to 40 mL of triethyl phosphate and dissolved at 60-70 °C. The reaction mixture was cooled to 0 °C, 2.8 mL (30 mmol) of phosphorus oxychloride was added, and the temperature was raised to room temperature and stirred under nitrogen for 3 hours. 100 mL of 1 M triethylammonium bicarbonate buffer (pH 8.5) was slowly added dropwise and stirred for 8 hours, after which 1 L of distilled water was added. The reaction mixture was separated using a DEAE Sephadex column (140 x 210 mm) and azeotropically mixed with 50 mL of methanol four times to obtain 3.79 g (yield 60.0%) of the triethylammonium salt of the target compound (4). 1 ¹H NMR (400 MHz, DMSO- d6 , 25℃): δ= 10.72 (s, 1H), 8.03 (s, 1H), 6.68 (br, 2H), 5.94 (d, 1H), 5.83 (m, 1H), 5.46 (m, 1H), 4.28 (m, 1H), 4.00 (m, 2H), 1.21 (m, TEA overlapped, 9H), 1.03 (m, 9H). LC-MS (ESI, m / z) = 532.17 [M + H + ]

[0203] Preparation Example 5: Preparation of 2',3'-Pivaloyl-Guanosine 5'-Diphosphate Diphosphate (ppG(2',3' Pivaloyl)) (5)

[0204] 3.16 g (5.0 mmol) of pG(2',3' Pivaloyl) triethylammonium salt (4) was added to 250 mL of dimethylformamide, followed by the addition of 3.40 g (50 mmol) of imidazole, 6.56 g (25 mmol) of triphenylphosphine, 5.51 g (25 mmol) of 2,2'-dipyridyl disulfide, and 0.51 g (5.0 mmol) of triethylamine, and the mixture was stirred for 5 hours. The reaction mixture was added to a solution of 2.45 g (20 mmol) of sodium perchlorate dissolved in 430 mL of acetone, cooled to 4 °C, and then the resulting crystals were filtered, washed with cold acetone, and vacuum dried.

[0205] Dried im-pG (2',3' Pivaloyl) was added to 30 mL of dimethylformamide, followed by the addition of 1.36 g (10 mmol) of zinc chloride and 5.98 g (30 mmol) of triethylammonium phosphate, and the mixture was stirred at room temperature for 3 to 16 hours. After the reaction was complete, a mixture of 870 mL of distilled water and 9.79 g (33.5 mmol) of ethylenediaminetetraacetic acid was added to the reaction mixture and stirred for 20 minutes. The reaction mixture was neutralized to a pH of 6 to 7 using a 1 M aqueous sodium bicarbonate solution, and the reaction mixture was separated using a DEAE Sephadex column (140 x 210 mm). The mixture was then azeotropically mixed with 35 mL of methanol four times to obtain 2.44 g (yield 60.0%) of the triethylammonium salt of the target compound (5). 1 H NMR (400 MHz, DO, 25℃): δ= 7.99 (s, 1H), 6.00 (d, 1H), 5.71 (m, 1H), 5.52 (m, 1H), 4.49 (m, 1H), 4.16 (m, 2H), 1.11 (m, TEA overlapped, 9H), 0.95 (m, 9H). 31 P NMR (162 MHz, DO, 25°C): δ= -8.38 (d, 1P), -10.69 (d, 1P).

[0207] Preparation Example 6: Preparation of 7-methyl-2',3'-pivaloyl-guanosine 5'-diphosphate (pp7mG(2',3' Pivaloyl)) (6)

[0208] 2.44 g (3.0 mmol) of ppG(2',3' Pivaloyl) triethylammonium salt (5) was dissolved in 50 mL of distilled water, and the pH of the reaction solution was adjusted to 4.0 using glacial acetic acid. 10 mL (105.45 mmol) of dimethyl sulfate was slowly added to the reaction solution over 30 minutes, and the mixture was stirred for 4 hours. At this time, the pH of the reaction solution was maintained at 4.0 ± 0.5 using 0.1 N sodium hydroxide. The reaction solution was extracted three times with 150 mL of dichloromethane to remove unreacted dimethyl sulfate, the aqueous layer was adjusted to pH 5.5, and 500 mL of distilled water was added. The reaction mixture was separated using a DEAE Sephadex column (140 x 210 mm) to obtain 1.74 g of triethylammonium salt (80% yield) by distillation and vacuum drying. 1 H NMR (400 MHz, DO, 25°C): δ= 9.35 (s, 1H), 6.30 (d, 1H), 5.71 (m, 1H), 5.58 (m, 1H), 4.69 (m, 1H), 4.32 ~ 4.15 (m, 2H), 4.11 (s, 3H), 1.25(m, 9H), 1.19 (m, 9H). LC-MS (ESI, m / z) = 624.14 [M - H] -

[0210] Preparation Example 7: 7-methyl-2',3'-pivaloyl-guanosine 5'-diphosphate imidazolide (im-pp7mG(2',3'Pivaloyl) (7) preparation

[0211] 1.26 g (1.73 mmol) of pp7 mG (2',3' Pivaloyl) triethylammonium salt (6) was dissolved in 25 mL of dimethylformamide, and 941.63 mg (13.83 mmol) of imidazole, 1.14 g (5.19 mmol) of 2,2'-dipyridyl disulfide, 0.482 mL (3.46 mmol) of triethylamine, and 1.36 g (5.19 mmol) of triphenylphosphine were added and stirred for 6 to 8 hours. The reaction mixture was added to a solution of 847 mg (6.92 mmol) of sodium perchlorate dissolved in 175 mL of acetone, cooled to 4 °C, and then the resulting crystals were filtered, washed with cold acetone, and vacuum dried to obtain 967 mg of the target compound (yield 80.0%) (7).

[0212] Scheme 2

[0213]

[0214] Preparation Example 8: Preparation of 2',2'-difluoro-guanosine 5'-monophosphate (pG(2'FF)) (8)

[0215] 2',2'-difluoroguanosine was commercially available and used as a starting material without further processing. 3.03 g (10 mmol) of 2',2'-difluoroguanosine was added to 40 mL of triethyl phosphate and dissolved at 60–70 °C. The reaction mixture was cooled to 0 °C, 2.8 mL (30 mmol) of phosphorus oxychloride was added, and the temperature was raised to room temperature and stirred under nitrogen for 3 hours. 100 mL of 1 M triethylammonium bicarbonate buffer (pH 8.5) was slowly added dropwise and stirred for 8 hours, after which 1 L of distilled water was added. The reaction mixture was separated using a DEAE Sephadex column (140 x 210 mm) and azeotropically mixed with 50 mL of methanol four times to obtain 2.9 g (yield 60%) of the triethylammonium salt of the target compound (8). 1 H NMR (400 MHz, DO, 25°C): δ= 8.41 (s, 1H), 6.50 (t, 1H), 4.60 (m, 1H), 4.24 ~ 4.14 (m, overlapped, 3H);19 F NMR (377 MHz, DO, 25°C): δ= -119.81 (s, 2F); 31 P NMR (162 MHz, DO, 25°C): δ = 2.04 (s, 1P).

[0217] Preparation Example 9: Preparation of 2',2'-difluoro-guanosine 5'-diphosphate diphosphate (ppG(2'FF)) (9)

[0218] 2.42 g (5.0 mmol) of pG(2'FF) triethylammonium salt (8) was added to 250 mL of dimethylformamide, followed by the addition of 3.40 g (50 mmol) of imidazole, 6.56 g (25 mmol) of triphenylphosphine, 5.51 g (25 mmol) of 2,2'-dipyridyl disulfide, and 0.51 g (5.0 mmol) of triethylamine, and the mixture was stirred for 5 hours. The reaction mixture was added to a solution of 2.45 g (20 mmol) of sodium perchlorate dissolved in 430 mL of acetone, cooled to 4°C, and then the resulting crystals were filtered, washed with cold acetone, and vacuum dried. Dried im-pG(2'-FF) was added to 30 mL of dimethylformamide, followed by the addition of 1.36 g (10 mmol) of zinc chloride and 5.98 g (30 mmol) of triethylammonium phosphate, and the mixture was stirred at room temperature for 3 to 16 hours. After the reaction was complete, a mixture of 870 mL of distilled water and 9.79 g (33.5 mmol) of ethylenediaminetetraacetic acid was added to the reaction mixture and stirred for 20 minutes. The reaction mixture was neutralized to a pH of 6 to 7 using a 1 M aqueous sodium bicarbonate solution, and the reaction mixture was separated using a DEAE Sephadex column (140 x 210 mm) and azeotropically mixed with 35 mL of methanol four times to obtain 2.0 g (yield 60.0%) of the triethylammonium salt of the target compound (9). 1 H NMR (400 MHz, DO, 25°C): δ= 8.38 (s, 1H), 6.50 (t, 1H), 4.65 (m, 1H), 4.32 ~ 4.20 (m, overlapped, 3H); 19 F NMR (377 MHz, DO, 25°C): δ=-119.87 (s, 2F);31 P NMR (162 MHz, DO, 25°C): δ= -8.71 (d, 1P), -10.55 (d, 1P).

[0220] Preparation Example 10: Preparation of 7-methyl-2',2'-difluoro-guanosine 5'-diphosphate (pp7mG(2'FF)) (10)

[0221] 2.0 g (3.0 mmol) of ppG(2'FF) triethylammonium salt (9) was dissolved in 50 mL of distilled water, and the pH of the reaction mixture was adjusted to 4.0 using glacial acetic acid. 10 mL (105.45 mmol) of dimethyl sulfate was slowly added to the reaction mixture over 30 minutes, and the mixture was stirred for 4 hours. During this time, the pH of the reaction mixture was maintained at 4.0 ± 0.5 using 0.1 N sodium hydroxide. The reaction mixture was extracted three times with 150 mL of dichloromethane to remove unreacted dimethyl sulfate, the aqueous layer was adjusted to pH 5.5, and 500 mL of distilled water was added. The reaction mixture was separated for pp7 mG(2'-FF) using a DEAE Sephadex column (140 x 210 mm), distilled, and vacuum dried to obtain 1.41 g (yield 80.0%) of the triethylammonium salt of the target compound (10). 1 H NMR (400 MHz, DO, 25°C): δ = 9.34 (s, 1H), 6.62 (d, 1H), 4.75 (m, 1H), 4.40 (m, 1H), 4.30 (m, 2H), 3.82 (s, 3H). 19 F NMR (377 MHz, DO, 25°C): δ= -121.12 (s, 1F), -121.23 (s, 1F). LC-MS (ESI, m / z) = 476.01 [M - H] -

[0223] Preparation Example 11: 7-methyl-2',2'-difluoro-guanosine 5'-diphosphate imidazolide (Im-pp7mG(2'FF)) (11) Preparation

[0224] 1.0 g (1.73 mmol) of pp7 mG (2'FF) triethylammonium salt (10) was dissolved in 25 mL of dimethylformamide, and 941.63 mg (13.83 mmol) of imidazole and 1.14 g (5.19 mmol) of 2,2'-dipyridyl disulfide were added. 0.482 mL (3.46 mmol) of trimethylamine and 1.36 g (5.19 mmol) of triphenylphosphine were added to the reaction mixture and stirred for 6-8 hours. The reaction mixture was added to a solution of 847 mg (6.92 mmol) of sodium perchlorate dissolved in 175 mL of acetone, cooled to 4°C, and then the resulting crystals were filtered, washed with cold acetone, and vacuum dried to obtain 761 mg (yield 80.0%) of the target compound (11).

[0226] Scheme 3

[0227]

[0229] Preparation Example 12: Preparation of 3'-Pivaloyl-Guanosine Monophosphate (pG(3' Pivaloyl)) (12)

[0230] Commercially available guanosine monophosphate was used as a starting material without further treatment. 5 g (11.8 mmol) of guanosine monophosphate was dissolved in 50 ml of distilled water and the pH was adjusted to 9.5 using 1 M NaOH. 9.56 g (51.3 mmol) of pivalic anhydrate was added at room temperature and stirred at room temperature. The reaction was carried out for 4 to 12 hours at room temperature while maintaining a pH of 9.5 to 9.6 using 1 M NaOH. After the reaction was complete, 50% acetic acid was added to adjust the pH to 7.0, and the mixture was filtered. The filtrate was separated using a DEAE Sephadex column (140 x 210 mm) to separate the target compound, and the solution was distilled and dried under nitrogen to obtain 4.06 g (yield 53.0%) of the triethylammonium salt of the target compound (12). 1H NMR (400 MHz, D2O, 25℃):δ= 8.10 (m, 1H), 8.07 (m, 0.45H), 6.03 (m, 0.45H), 5.92 (m, 1H), 5.50 (m, 0.45H), 5.36 (m, 1H), 4.98 (t, 1H), 4.67 (t, 0.45H), 4.41 (m, 1H), 4.30 (m, 0.45H), 4.00 (m, overlapped, 4.35H), 1.22 (s, 9H), 1.13 (s, 4.5H); 31 P NMR (162 MHz, D2O, 25℃): δ= 2.46 (s, 0.56P), 2.25 (s, 1P).

[0232] Preparation Example 13: Preparation of 3'-Pivaloyl-Guanosine 5'-Diphosphate Diphosphate (ppG(3' Pivaloyl)) (13)

[0233] 4.09 g (6.29 mmol) of pG(3' Pivaloyl) triethylammonium salt (12) was added to 70 mL of dimethylformamide, followed by the addition of 2.55 g (37.5 mmol) of imidazole, 4.91 g (18.7 mmol) of triphenylphosphine, 4.09 g (18.6 mmol) of 2,2'-dipyridyl disulfide, and 0.64 g (6.3 mmol) of triethylamine, and the mixture was stirred for 1 to 12 hours. After the reaction was complete, the reaction mixture was added to a solution of 3.08 g (25.16 mmol) of sodium perchlorate dissolved in 556 mL of acetone, cooled to 4°C, and then the resulting crystals were filtered, washed with cold acetone, and vacuum dried. Dried im-pG (3' Pivaloyl) was added to 90 mL of dimethylformamide, followed by the addition of 2.57 g (18.87 mmol) of zinc chloride and 5.98 g (37.74 mmol) of triethylammonium phosphate, and the mixture was stirred at room temperature for 3 to 16 hours. After the reaction was complete, a mixture of 1.1 L of distilled water and 11 g (37.74 mmol) of ethylenediaminetetraacetic acid was added to the reaction mixture and stirred for 20 minutes. The reaction mixture was neutralized to a pH of 6 to 7 using a 1 M aqueous sodium bicarbonate solution, and the reaction mixture was separated using a DEAE Sephadex column (140 x 210 mm) and azeotropically mixed with 35 mL of methanol four times to obtain 3.66 g (yield 70.0%) of the triethylammonium salt of the target compound (13). 1 H NMR (400 MHz, DO, 25°C): δ= 8.11 (s, 1H), 8.05 (s, 0.51H), 6.04 (d, 0.52H), 5.94 (d, 1H), 5.55 (t, 0.52H), 5.43 (dd, 1H), 5.06 (dd, 1H), 4.46 (m, 1H), 4.33 (m, 0.53H), 4.29 ~ 4.17 (m, overlapped, 4H); 31 P NMR (162 MHz, DO, 25°C): δ= -9.62 (m, 1.5P), -10.74 (m, 1.5P).

[0235] Preparation Example 14: Preparation of 7-methyl-3'-pivaloyl-guanosine 5'-diphosphate (pp7mG(3' Pivaloyl)) (14)

[0236] 3.25 g (3.91 mmol) of ppG (3' Pivaloyl) triethylammonium salt (13) was dissolved in 66 mL of distilled water, and the pH of the reaction mixture was adjusted to 4.0 using glacial acetic acid. 19.7 g (156 mmol) of dimethyl sulfate was slowly added to the reaction mixture over 30 minutes and stirred for 4 hours. During this time, the pH of the reaction mixture was maintained at 4.0 ± 0.5 using 1 M sodium hydroxide. The reaction mixture was extracted three times with 195 mL of dichloromethane to remove unreacted dimethyl sulfate, and the aqueous layer was titrated to pH 5.5. The reaction mixture was separated using a DEAE Sephadex column (140 x 210 mm) to obtain 2.33 g (yield 80%) of triethylammonium salt (14). 1H NMR (400 MHz, DO, 25℃): δ= 9.29 (s, 0.69H), 9.24 (s, 0.53H), 6.18 (d, 0.85H), 6.10 (d, 1H), 5.56 (dd, 0.85H), 5.36 (dd, 1H), 4.94 (m, 1H), 4.73 (m, 0.85H), 4.57 (m, 1H), 4.41 (m, 0.85H), 4.35 (m, 0.85H), 4.25 ~ 4.13 (m, overlapped, 2.85H), 4.10 (s, 3H), 4.09 (s, 0.85H), 1.22 (s, 9H), 1.19 (s, 7.6H). LC-MS (ESI, m / z) = 542.10 [M + H+]

[0238] Preparation Example 15: Preparation of 7-methyl-3'-pivaloyl-guanosine 5'-diphosphate imidazolide (im-pp7mG(3'Pivaloyl) (15)

[0239] 1.29 g (1.73 mmol) of pp7 mG (3' Pivaloyl) triethylammonium salt (14) was dissolved in 22 mL of dimethylformamide, and 707 mg (10.38 mmol) of imidazole and 1.14 g (5.19 mmol) of 2,2'-dipyridyl disulfide were added. 175 mg (1.73 mmol) of triethylamine and 1.36 g (5.19 mmol) of triphenylphosphine were added to the reaction mixture and stirred for 1 to 12 hours. After the reaction was complete, the reaction mixture was added to a solution of 847 mg (6.92 mmol) of sodium perchlorate dissolved in 175 mL of acetone, cooled to 4 °C, and then the resulting crystals were filtered, washed with cold acetone, and vacuum dried to obtain 796 mg of the target compound (yield 75.0%) (15).

[0241] Scheme 4

[0242]

[0244] Preparation Example 16: Preparation of N2-isobutyryl-2',3'-diacetoxy-guanosine (16)

[0245] 2.4 g (3.66 mmol) of N2-isobutyryl-5'-O-DMT guanosine was dissolved in 12 ml of dichloromethane, followed by the addition of 1.47 ml (18.3 mmol) of pyridine and 1.78 ml (18.3 mmol) of acetic anhydride, and the mixture was stirred at room temperature for 5 hours. 24 ml of ethyl acetate was added to the reaction mixture for extraction, after which it was washed with 14.4 ml of saturated aqueous sodium bicarbonate, 14.4 ml of 20% aqueous citric acid solution, and 14.4 ml of distilled water. The organic layer was dried with sodium sulfate, and the residue obtained from vacuum distillation was nitrogen-dried for one day. 36 ml of 3% aqueous trichloroacetic acid solution was added to the dried material and reacted at room temperature for 3 hours; subsequently, 24 ml of methanol was added and the mixture was stirred for an additional 2 hours and 30 minutes. The reaction mixture was subjected to vacuum distillation, 36 ml of dichloromethane and 18 ml of distilled water were added, and then a saturated aqueous sodium bicarbonate solution was added to neutralize it. The organic layer was separated, dried with sodium sulfate, and then vacuum distilled. The residue from vacuum distillation was dissolved in 12 ml of ethyl acetate and then slowly added to 108 ml of hexene at room temperature to induce crystallization. The resulting crystals were filtered, washed three times with 12 ml of hexene, and vacuum dried to obtain 1.43 g (yield 89.6%) of the target compound (16). LC-MS (ESI, m / z) = 438.16 [M + H + ]

[0247] Preparation Example 17: Preparation of (N2-isobutyryl-2',3'-diacetoxy-guanocinyl)-N6-benzoyl-2'-methoxy-adenocinyl cyanoethyl phosphate ester (17)

[0248] 5'-O-DMT-N6-benzoyl-2'-methoxy-adenosine amidite was commercially available and used as a starting material without further processing. 1.43 g (3.28 mmol) of N2-isobutyryl-2',3'-diacetoxy-guanosine (16) and 3.78 g (4.26 mmol) of 5'-O-DMT-N6-benzoyl-2'-methoxy-adenosine amidite were dissolved in 28.4 mL of 1H-tetrazole (0.45 M acetonitrile solution, 12.79 mmol) and stirred at room temperature for 1 hour. 544 mg (2.15 mmol) of iodine was dissolved in 56.7 ml of a tetrahydrofuran:distilled water:pyridine mixture (v:v:v, 7:2:1), and the solution was added to the reaction mixture and stirred for 45 minutes. After adding 7.2 ml of a 10% sodium thiosulfate aqueous solution to the reaction mixture, 43 ml of dichloromethane and 14.3 ml of distilled water were added, and the organic layer was separated. The separated organic layer was dried with sodium sulfate, subjected to vacuum distillation, and then nitrogen dried for one day. 21.5 ml of a 3% trichloroacetic acid aqueous solution was added to the dried material and reacted at room temperature for 3 hours; then, 14.3 ml of methanol was added and stirred for an additional 2 hours and 30 minutes. The reaction mixture was vacuum distilled, 21.5 ml of dichloromethane and 10.7 ml of distilled water were added, and a saturated aqueous sodium bicarbonate solution was added to neutralize the mixture. The separated organic layer was washed with distilled water, dried with sodium sulfate, and subjected to vacuum distillation. The residue from vacuum distillation was dissolved in 28.6 ml of dichloromethane and then slowly added to 86 ml of methyl tert-butyl ether at room temperature to induce crystallization. The resulting crystals were filtered and vacuum dried for one day to obtain 2.7 g (yield 87.8%) of the target compound (17). LC-MS (ESI, m / z) = 938.28 [M + H + ]

[0250] Preparation Example 18: p(OCE) 2 A bz (2'OMe)p(OCE)G ib (2',3'OAc) (18) manufacturing

[0251] 2 g (2.13 mmol) of (N2-isobutyryl-2',3'-diacetoxy-guanocinyl)-N6-benzoyl-2'-methoxy-adenocinyl cyanoethyl phosphate ester (17) and 1.11 ml (4.26 mmol) of bis(2-cyanoethyl)-N,N-diisopropylphosphomidite were dissolved in 9.47 mL of 1H-tetrazole (0.45 M acetonitrile solution, 4.26 mmol) and stirred at room temperature for 30 minutes. 811 mg (3.2 mmol) of iodine was dissolved in 30 ml of a tetrahydrofuran:distilled water:pyridine mixture (v:v:v, 7:2:1), the solution was added to the reaction mixture, and the mixture was stirred for 30 minutes. After adding 20 ml of a 10% aqueous sodium thiosulfate solution to the reaction mixture, 100 ml of dichloromethane and 40 ml of distilled water were added, and the organic layer was separated. The separated organic layer was dried with sodium sulfate and subjected to vacuum distillation. The vacuum distilled concentrate was dissolved in 27 ml of dichloromethane and then slowly added to 133 ml of methyl tert-butyl ether at room temperature to induce crystallization. The resulting crystals were filtered and vacuum dried for one day to obtain 2.2 g of the target compound (18) (yield 92.2%). LC-MS (ESI, m / z) = 1124.30 [M + H + ]

[0253] Preparation Example 19: Preparation of pA(2'OMe)pG (19)

[0254] p(OCE)2A bz (2'OMe)p(OCE)G ib2 g (1.78 mmol) of (2',3'OAc) (18) was added to a mixture of 44 mL of methanol and 44 mL of concentrated ammonia and stirred at 50 to 55°C for 24 hours. When the reaction was complete, the solvent was distilled under reduced pressure, and 20 mL of methanol was added to the concentrate and distilled three times. The concentrate was dissolved in distilled water and purified using a DEAE Sepharose column and reverse-phase chromatography to obtain 1.13 g (yield 70%) of the triethylammonium salt (19) of the target compound. 1H NMR (400 MHz, DO, 25℃): δ= 8.44 (s, 1H), 8.12 (s, 1H), 7.90 (s, 1H), 6.07 (m, 1H), 5.80 (m, 1H), 4.46 ~ 4.01 (m, 8H), 3.45 (m, 3H) LC-MS (ESI, m / z) = 707.13 [M + H+]

[0256] Scheme 5

[0257]

[0259] Preparation Example 20: Preparation of 7mG(2',3'Pivaloyl)pppA(2'OMe)pG (20)

[0260] 0.55 g of magnesium chloride was added and dissolved in 27.5 mL of dimethylformamide. 0.69 g (0.99 mmol) of Im-pp7mG(2',3'Pivaloyl) (7) and 0.5 g (0.55 mmol) of pA(2'OMe)pG (19) were added to the reaction mixture and stirred at room temperature for 24 hours. After the reaction was complete, 275 mL of 25 mM aqueous ethylenediaminetetraacetic acid solution was added dropwise to terminate the reaction, and after cooling to room temperature, the mixture was neutralized with 1 M aqueous sodium bicarbonate solution. The reaction mixture was purified using a DEAE Sepharose column (140 x 210 mm), distilled, and vacuum dried. After dissolving the dried solid in 2.5 ml of distilled water, the solution was added to 15 ml of acetone in which 299 mg (2.44 mmol) of sodium perchlorate was dissolved, and the solution was cooled to 4 ℃. Then, the resulting crystals were filtered, washed with cold acetone, and vacuum dried to obtain 0.46 g (yield 60.0%) of the sodium salt (20) of the target compound. 1 H NMR (400 MHz, DO, 25℃): δ= 8.20 (s, 1H), 7.91 (s, 1H), 7.75 (s, 1H), 5.75 ~ 5.72 (m, 2H), 5.65 (m, 1H), 5.33 (m, 1H), 5.24 (m, 1H), 4.60 (m, 1H), 4.29 ~ 3.96 (m, 12H), 3.91 (m, 3H), 3.26 (m, 3H), 1.02 (s, 9H), 0.90 (s, 9H)); 31 P NMR (162 MHz, DO, 25°C): δ= -0.32 (s, 1P), -11.01 to -11.22 (dd, 2P), -22.26 (t, 1P). LC-MS (ESI, m / z) = 1314.27 [M + H + ].

[0262] Scheme 6

[0263]

[0265] Preparation Example 21: Preparation of 7mG(2'FF)pppA(2'OMe)pG (21)

[0266] The compound of Preparation Example 21 was prepared in the same manner as Preparation Example 20, but using Im-pp7mG(2'FF) (11) of Preparation Example 11 instead of Im-pp7mG(2',3'Pivaloyl) (7) of Preparation Example 20. 1 H NMR (400 MHz, DO, 25°C): δ= 8.32 (s, 1H), 8.03 (s, 1H), 7.87 (s, 1H), 6.45 (d, 1H), 5.89 (d, 1H), 5.76 (d, 1H), 4.87 ~ 4.83 (m, 2H), 4.55 (m, 1H), 4.45 (m, 3H), 4.32 to 4.14 (m, 8H), 3.68 (s, 3H), 3.36 (s, 3H); 19 F NMR (377 MHz, DO, 25°C): δ= -120.73 (s, 1F), -120.95 (s, 1F); 31 P NMR (162 MHz, DO, 25°C): δ= -0.32 (s, 1P), -10.82 to -11.14 (dd, 2P), -22.45 (t, 1P). LC-MS (ESI, m / z) = 1164.16 [M - H] -

[0268] Scheme 7

[0269]

[0270] Preparation Example 22: Preparation of 7mG(3'Pivaloyl)pppA(2'OMe)pG (22)

[0271] The same method as in Preparation Example 20 was used, but instead of Im-pp7mG(2',3'Pivaloyl) (7) of Preparation Example 20, Im-pp7mG(3'Pivaloyl) (15) of Preparation Example 15 was used to prepare the compound of Preparation Example 22 (positional isomers of 2'O and 3'O: molar ratio of 0.65:1). The structure of the compound of Preparation Example 22 of Scheme 7 corresponds to 3'Pivaloyl. 1H NMR (400 MHz, DO, 25°C): δ= 8.17 (d, 1.65H), 7.93 (d, 1.65H), 7.76 (d, 1.65H), 5.79 ~ 5.64 (m, 4.95H), 5.26 (m, 0.65H), 5.11 (m, 1H), 3.88 (d, 4.95H), 3.26 (d, 4.95H), 1.06 (s, 9H), 1.02 (s, 5.85H); 31 P NMR (162 MHz, DO, 25°C): δ= -0.35 (s, 1.65P), -10.85 (dd, 3.3P), -22.31 (t, 1.65P). LC-MS (ESI, m / z) = 1230.22 [M + H + ].

[0273] Example 1. Verification of mRNA synthesis and IVT yield according to the type of Cap Reagent

[0274] In this example, we aimed to verify the IVT yield of the mRNA synthesis process according to the type of Cap Reagent.

[0276] Example 1-1. mRNA Synthesis Process and Materials

[0277] To synthesize the mRNA used in the above vaccine, a substance required for the mRNA synthesis process (In vitro transcription, IVT), including a linear plasmid DNA (Sequence No. 1) template stock solution (L-DNA), was added to a reaction vessel (RNase-free tube), and mRNA synthesis was carried out for 2 to 3 hours in a heating block (37 ℃) according to the conditions in Table 1.

[0278] 10X Transcription buffer was added to the IVT reaction volume to achieve a concentration of 1X, and the composition of the 10X Transcription buffer was equal to the volume ratio of Tris-Cl buffer : Spermidine : Triton X = 4 : 2 : 1.

[0279] The mRNA synthesis process conditions above are as shown in Table 1 below, and the Cap Reagent may be Cap Reagent 1 to 3 (Table 2) prepared in the aforementioned preparation examples or Trilink Clean Cap Reagent AG (3'OMe) (Catalog No: N-7413).

[0280] To terminate the mRNA synthesis process, DNase1 was added at a concentration of 2 KU / mL and reacted in a heating block (37 ℃) for 15 to 30 minutes. Afterward, mRNA concentration analysis was repeated three times on the mRNA samples after synthesis was completed using a fluorometer, and the average result was calculated to determine the mRNA concentration. The mRNA content was then calculated by multiplying the concentration by the reaction volume.

[0282] No. substance name density 1 Linear plasmid DNA template 0.05 mg / mL 2 ATP solution 10 mM 3 CTP solution 10 mM 4 N1-Methylpseudo-UTP solution 10 mM 5 GTP solution 2 mM 6 Cap Reagent 8 mM 7 DTT solution 10 mM 8 MgCl2solution 50 mM 9 RNase inhibitor 2 KU / mL 10 Pyrophosphatase 20 unit / mL 11 T7 RNA Polymerase 300 KU / mL

[0283]

[0284]

[0285] Example 1-2. Derivation of IVT Process Yield

[0286] To determine the IVT process yield, the IVT yield according to the Cap Reagent was quantified by dividing the mRNA content synthesized using the four Cap Reagents above by the L-DNA content added. Specifically, an IVT yield of 100 means that 100 μg of mRNA was synthesized using 1 μg of L-DNA. Subsequently, the IVT yield from Trilink’s Clean Cap Reagent AG (3’OMe), a representative Cap Reagent manufacturer, was set to 100%. The IVT yield from Hanmi Fine Chemical’s Cap Reagents 1 to 3 was divided by the IVT yield from Trilink’s Clean Cap Reagent to calculate the IVT process yield (%) of Hanmi Fine Chemical relative to Trilink, and the results are shown in Table 3. Specifically, when the IVT yield from Trilink’s Clean Cap is 50 and the IVT yield from Hanmi Fine Chemical’s Cap Reagent is 60, the IVT yield (%) from Hanmi Fine Chemical’s Cap Reagent is 120%.

[0288] [Mathematical Formula 1]

[0289] IVT Process Yield = (Amount of synthesized mRNA) / (Amount of added L-DNA)

[0291] [Mathematical Formula 2]

[0292] IVT Process Yield (%) = {(IVT Process Yield by Hanmi Fine Chemical Cap Reagent 1 ~ 3) / (IVT Process Yield by Trilink Clean Cap Reagent)} * 100 (%)

[0294] Candidates IVT Yield (%) Comparison group (Trilink) Cap 1 101.8 % 100.0 % Cap 2 71.5 % 100.0 % Cap 3 105.3 % 100.0 %

[0295] As a result of the experiment, the IVT efficiency was in the order of Cap 3, Cap 1, and Cap 2, with the best efficiency confirmed in Cap 3 and showing higher efficiency compared to the control group.

[0296] Examples 1-3. Poly A tailing process

[0297] In this example, a Poly(A) tailing process was performed on mRNA prepared using Caps 1, 2, and 3 to attach a 3' poly(A) tail of 50 to 200 nt in length to the 3' end of the IVT mRNA. Specifically, after adding the materials required for the Poly(A) tailing process, including IVT mRNA in which the mRNA synthesis reaction was terminated by adding DNase 1, to a reaction vessel (RNase-free tube), the Poly(A) tailing process was performed for 30 minutes to 1 hour in a heating block (37 ℃) according to the conditions in Table 4. Subsequently, to terminate the Poly(A) tailing process, the reaction vessel was left at 4 ℃.

[0299] No. substance name Volume ratio 1 IVT mRNA 20 2 10X Poly(A) Polymerase Reaction Buffer 10 3 10 mM ATP solution 10 4 Poly (A) Polymerase enzyme (4 KU / mL) 2 5 Nulcease-free water 58

[0301] Example 2. LNP encapsulation after mRNA preparation

[0302] In this example, mRNA stock solution with the Poly(A) tailing process completed and a lipid mixture were formulated using Caps 1, 2, and 3 of Example 1 to produce mRNA-LNP (Lipid nanoparticle) in which mRNA is encapsulated within the lipid.

[0303] Specifically, the mRNA stock solution, after the Poly (A) tailing reaction and purification were completed, was diluted to 0.17 mg / mL with Formulation Buffer, and the commercial lipid mixture GenVoy-ILM (PRECISION NANOSYSTEMS) (PNI Catalog Number NWW0041, specification 2mL) was diluted 1 / 2 with ethanol and filled into syringes. Subsequently, a microtube cartridge was mounted on an Ignite™ instrument, and the syringes containing the mRNA dilution and the lipid mixture dilution were loaded. The reaction was carried out at a volume ratio of 3:1, and the formulation process was performed under conditions of an N / P ratio of 4. The formulation reaction solution recovered in the tube was buffer-exchanged and concentrated with more than 20 times the volume of PBS using a Centrifugal Ultrafiltration Unit MWCO 50 kDa.

[0304] The recovered mRNA-LNP stock solution was stored at 4°C, and after measuring the mRNA content and encapsulation ratio, it was used for administration in animal experiments.

[0306] Example 3. Derivation of encapsulation efficiency of mRNA-LNP finished drug product

[0307] In this example, the Total-RNA content and Free-RNA content of the mRNA-LNP finished drug product prepared in Example 2 were measured to calculate the ratio of mRNA incorporated into the LNP.

[0308] The mRNA-LNP finished drug product was lysed using a surfactant such as Triton X to enable the release of encapsulated mRNA, and the total RNA concentration was measured. The free RNA concentration not incorporated into the mRNA-LNP was measured through a TE buffer reaction. Encapsulation efficiency can be calculated based on the percentage concentration of the total RNA content minus the free RNA content relative to the total RNA content.

[0309] Specifically, a 2% Triton X solution was prepared, and Total-RNA was prepared by mixing mRNA-LNP samples with 2% Triton X in a 1:1 volume ratio in a microplate, and Free-RNA was prepared by mixing mRNA-LNP samples with TE Buffer in a 1:1 volume ratio, followed by a reaction at 37°C for 10 minutes using a shaker. After specifically staining RNA using the Ribogreen assay method, RNA concentration was calculated using fluorescence values ​​measured at fluorescence excitation (485 nm) and emission (528 nm) wavelengths using a microplate reader.

[0311] [Mathematical Formula 3]

[0312] RNA encapsulation efficiency = [{(Total-RNA concentration) - (Free-RNA concentration)} / (Total RNA concentration)] * 100 (%)

[0313] <<Encapsulation efficiency% 결과> > Group Cap material used Formulation Encapsulation efficiency % 1 Cap 1 GenVoy LNP 91.2 2 Cap 2 GenVoy LNP 89.7 3 Cap 3 GenVoy LNP 90.2 4 CleanCap (TriLink Clean Cap AG (3'OMe)) GenVoy LNP 86.4

[0315] Example 4. Evaluation of Immunogenicity of Vaccine Formulation Using Animal Model

[0316] In this example, we intended to investigate the effect of mRNA prepared using Caps 1, 2, and 3 of Example 1 on the immunogenicity of the vaccine. To this end, mRNA stock solutions prepared using Caps 1, 2, and 3 as disclosed in Example 2 (experimental group) and mRNA stock solutions prepared using CleanCap (Trilink) (control group) were formulated with a Lipid Mixture, and then the prepared mRNA-LNP stock solutions were inoculated into an animal model (mouse) at a dose of 10 µg.

[0317] Using serum from the inoculated animal model as a sample, the antibody titer of IgG generated by the vaccine formulation was measured using ELISA (Enzyme-Linked Immunosorbent Assay). Specifically, the surface of a 96-well plate was coated with 1 μg / mL of antigen and incubated at 2–8°C for 18 ± 2 hours. After washing the incubated plate, it was blocked with BSA solution for 1 hour at room temperature. Subsequently, the plate was washed, and a serum dilution prepared through serial dilution was dispensed onto the plate. The secondary antibody, Goat Anti-Mouse IgG-Alkaline phosphatase conjugates, was diluted and added to the plate, followed by an incubation at room temperature for 2 hours. Afterward, the plate was washed, 1 mg / mL of p-nitrophenylamine buffer was added, and the reaction was carried out again at room temperature for 2 hours. Afterward, 3 M NaOH was added to each well to stop the reaction, and the absorbance at 405 nm and 690 nm was measured to calculate the Optical Density (OD) value of the IgG antibody titer.

[0318] Table 6 shows the results of quantifying the OD values ​​of IgG antibodies by vaccine formulation inoculation for each Cap substance candidate group.

[0319] Candidates Experimental group (OD) Comparison group (OD) OD difference Cap 1 3867.6 77053.9 -73186.3 Cap 2 34.5 77053.9 -77019.4 Cap 3 89528.4 77053.9 12474.5

[0320] As a result, as shown in Table 6 above, it was confirmed that when Cap 3 among the Cap candidate substances according to one embodiment is used, the induction of an immune response by vaccination is enhanced compared to the control group.

[0322] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 Immunogenic composition comprising a polynucleotide having a 5'-Cap structure of any one of the following chemical formulas 1, 3, and 4: [Chemical Formula 1] [Chemical Formula 3] [Chemical Formula 4] Claim 2 A composition according to claim 1, wherein the polynucleotide comprises a 5'Cap structure represented by Chemical Formula 3 or 4. Claim 3 The immunogenic composition of claim 1 comprises: a polynucleotide having a 5'-Cap structure represented by Chemical Formula 3; and a polynucleotide having a 5'-Cap structure represented by Chemical Formula 4. Claim 4 A composition according to claim 1, wherein the polynucleotide comprises a 5'UTR (untranslated region) sequence. Claim 5 A composition according to claim 4, wherein the 5'UTR sequence is derived from HBA2 (Hemoglobin subunit alpha 2). Claim 6 A composition according to claim 4, wherein the 5'UTR sequence comprises SEQ ID NO. 3 or a sequence having 80% or more homology or identity therewith. Claim 7 A composition according to claim 1, wherein the polynucleotide comprises a 3'UTR (untranslated region) sequence. Claim 8 A composition according to claim 7, wherein the 3'UTR sequence is derived from HBB (Hemoglobin subunit beta). Claim 9 A composition according to claim 7, wherein the 3'UTR sequence comprises SEQ ID NO. 5 or a sequence having 80% or more homology or identity therewith. Claim 10 A composition according to claim 1, wherein the polynucleotide comprises: a 5'UTR sequence having at least 80% homology or identity with SEQ ID NO. 3 or a 3'UTR sequence having at least 80% homology or identity with SEQ ID NO.

5. Claim 11 A composition according to claim 1, wherein the polynucleotide comprises a sequence encoding a peptide or protein derived from a coronavirus or influenza virus that exhibits immunogenicity. Claim 12 In claim 11, the composition wherein the polynucleotide comprises CDS of SEQ ID NO.

4. Claim 13 A composition according to claim 1, wherein the polynucleotide comprises a poly A tail. Claim 14 A composition according to claim 1, wherein the polynucleotide is contained within lipid nanoparticles. Claim 15 A composition according to any one of claims 1 to 14, wherein the immunogenic composition induces immunity against coronavirus or influenza virus. Claim 16 A method for preparing an immunogenic composition comprising the step of performing mRNA synthesis by mixing a composition comprising a DNA template with one or more compounds of any one of the following chemical formulas 5, 7, and 8 or a salt thereof: [Chemical Formula 5] [Chemical Formula 7] [Chemical Formula 8] Claim 17 A method of preparation according to claim 16, wherein the DNA template comprises a sequence encoding a peptide or protein derived from a coronavirus or influenza virus that exhibits immunogenicity. Claim 18 In claim 16, the manufacturing method comprises the step of performing poly A tailing of mRNA. Claim 19 In claim 16, the above manufacturing method comprises the step of manufacturing mRNA in a form contained in lipid nanoparticles.

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