KR20240141041A - Canine influenza virus vaccine composition using self amplifying rna construct - Google Patents

Abstract

The present invention relates to a self-amplifying saRNA construct in which expression of a target protein is optimized, and a canine influenza virus vaccine produced based thereon. The optimized self-amplifying RNA based on enterovirus according to the present invention improved the expression amount of the target protein compared to existing mRNA and enterovirus-based saRNA. In addition, it effectively expressed canine influenza virus H3N2, and the expression rate was also significantly higher than that of existing mRNA. Therefore, the self-amplifying RNA construct according to the present invention can be applied to a canine influenza virus vaccine.

Description

{CANINE INFLUENZA VIRUS VACCINE COMPOSITION USING SELF AMPLIFYING RNA CONSTRUCT}

The present invention relates to a canine influenza virus vaccine composition using a novel self-amplifying RNA construct and its use.

Influenza viruses have various subtypes, and depending on the type of virus, they can cause respiratory diseases accompanied by chills, fever, and muscle pain. This is due to the diversity of hemagglutinin (HA) and neuraminidase (NA), which are proteins that determine the immunogenicity of the virus. For example, there are 18 types of HA and 11 types of NA for influenza A, and mutations frequently occur. Therefore, in order to develop a vaccine, there is the burden of having to predict the prevalent strains in advance and produce the vaccine, and there are various problems, such as the fact that vaccine efficacy is significantly reduced due to the occurrence of new variants despite the prediction of prevalent strains. In addition, vaccines such as universal influenza virus vaccines are being developed to overcome this diversity. However, due to issues with vaccine efficacy, the development of various vaccines is necessary.

Accordingly, the inventors of the present invention studied to develop a saRNA vaccine construct that can be used rapidly and efficiently. As a result, a novel saRNA construct optimized for expression of an antigen protein was produced by modifying the 5' untranslated region (UTR), protease cleavage site, stop codon, and polyadenosine tail sequence of an enterovirus-based saRNA construct. In addition, the modified saRNA construct was confirmed to significantly increase the expression of an antigen protein in cells compared to existing mRNA and existing enterovirus-based saRNA constructs, thereby completing the present invention. In addition, when this was applied to a canine influenza virus vaccine, it was confirmed that antibodies were effectively produced, thereby completing the present invention.

To achieve the above object, one aspect of the present invention provides a polynucleotide comprising a nucleic acid sequence encoding a non-structural protein of a self-amplifying virus; a canine influenza virus H3N2 variant protein; and a fusion protein comprising a protease cleavage site.

Another aspect of the present invention provides a recombinant vector loaded with the polynucleotide.

Another aspect of the present invention provides a vaccine composition comprising the polynucleotide or recombinant vector as an active ingredient.

The optimized self-amplifying RNA construct based on enterovirus according to the present invention improved the expression amount of the target protein compared to existing mRNA and enterovirus-based saRNA constructs. In addition, the multi-antigen saRNA construct could simultaneously express more than one target protein, and the expression rate was also significantly higher compared to existing mRNA. Therefore, a vaccine comprising a nucleic acid sequence encoding a canine influenza virus H3N2 variant protein using the technology of the present invention can be widely used as a preventive vaccine for companion dogs.

Figure 1 is a schematic diagram of the structures of wild type enterovirus (Enterovirus, left) and enterovirus-based self-amplifying RNA construct (saRNA, self-amplifying RNA, right).
Figure 2 is a graph showing the amino acid sequence of the 2A protease cleavage site included in the enterovirus-based saRNA construct (top) and the results of confirming the expression level of the target protein according to each sequence using the luciferase system (bottom). *p<0.05, **p<0.01, ***p<0.001, ns:not significant
Figure 3 is a graph showing the results of inserting a Kozak sequence into an enterovirus-based saRNA construct (top) and confirming the expression level of the target protein according to each inserted sequence using a luciferase system (bottom). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001
Figure 4 is a graph showing the results of inserting a poly(A) tail into an enterovirus-based saRNA construct (top) and confirming the expression level of the target protein according to each inserted sequence at the cell (bottom right) and animal (bottom left) levels using a luciferase system (bottom right). *p<0.05
Figure 5 is a schematic diagram comparing the structures of mRNA, an enterovirus-based saRNA construct, and an optimized saRNA (modified saRNA) construct obtained by modifying the enterovirus-based saRNA construct.
Figure 6 is a graph showing the results of confirming the expression level of the target protein by mRNA, enterovirus-based saRNA (sa luci) construct, and modified saRNA construct at the cellular level using the luciferase system. **p<0.01, ***p<0.001, ****p<0.0001
Figure 7 is a diagram showing the results of confirming the expression level of the target protein by mRNA, enterovirus-based saRNA (sa luci) construct, and modified saRNA construct at the animal level using a luciferase system.
Figure 8 is a drawing showing the results of Western blot analysis of the antigen protein expression rate at the cellular level of a modified saRNA construct containing a severe fever with thrombocytopenia syndrome (SFTS) virus antigen as a target protein used in one specific example of the present invention. Sa B: saRNA construct containing SFTS consensus B antigen, RNA containing the antigen, Sa ABDEF: saRNA construct containing SFTS consensus ABDEF antigen, mRNA B: mRNA containing SFTS consensus B antigen
FIG. 9 is a drawing showing the results of confirming the antigen protein expression rate at the cellular level of a modified saRNA construct containing the spike protein antigen of coronavirus (SARS-CoV2) (left) or highly pathogenic avian influenza (HPAI) virus antigen (H5N8, right) as a target protein used in one specific example of the present invention, by Western blot.
Figure 10 is a schematic diagram of the structure of a modified saRNA construct containing multiple antigens.
Figure 11 is a schematic diagram (top) of a modified saRNA construct containing two types of canine influenza virus antigens (H3N2 and H3N8) as target proteins in one specific example of the present invention, and a drawing showing the results of confirming the level of antigen protein expression at the cellular level by Western blot. Ma saRNA: modified saRNA construct containing H3N2 and H3N8, single saRNA: modified saRNA construct containing H3N2, PC WT virus: wild-type canine influenza virus
Figure 12 is a schematic diagram illustrating a method for producing cell- and mouse-adapted coxsackievirus B5.
Figure 13 is a drawing (left) and graph (right) comparing the titers of wild-type (WT-B5), cell-adapted (Vp8-B5), and cell and mouse-adapted (Vp8Mp24-B5) Coxsackievirus B5. ***p<0.001
Figure 14 is a schematic diagram showing a Coxsackievirus B5 with an increased proliferation rate according to one specific example of the present invention.
FIG. 15 is a schematic diagram showing an optimized enterovirus-based saRNA construct according to one specific example of the present invention.
Figure 16 is a schematic diagram of the structure of a clone vector in the form of an enterovirus-based saRNA construct used in the present invention.
Figure 17 is a schematic diagram designing a gene for expression of H3N2 canine influenza HA (hemagglutinin).
Figure 18 is a schematic diagram for increasing the antigenicity of H3N2 canine influenza HA.
Figure 19a is a schematic diagram of the structure of a wild type enterovirus.
Figure 19b is a schematic diagram of the structure of an enterovirus-based self-amplifying RNA construct (saRNA, self-amplifying RNA, right).
Figure 20 shows the expression level of the saRNA construct confirmed at the cellular level through Western blot.
Figure 21 is a schematic diagram of an experiment in which a dog was inoculated with a canine influenza self-amplifying RNAP and the antigen expression of the canine influenza self-amplifying RNA-LNP was confirmed in vitro.
Figures 22a to 22c show that neutralizing antibodies were produced after the second vaccination was completed.

Polynucleotide construct

nonstructural protein

One aspect of the present invention provides a polynucleotide comprising a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus; a first target protein; and a fusion protein comprising a protease cleavage site. At this time, the polynucleotide may be DNA or RNA. In addition, the self-amplifying virus may be a virus of the family Picornaviridae. Specifically, the self-amplifying virus may be a virus of the genus Enterovirus. More specifically, in the present invention, the nonstructural protein may be a nonstructural protein derived from Enterovirus. Preferably, it may be a nonstructural protein derived from Coxsackievirus.

At this time, the first target protein may be an antigen protein derived from a canine influenza virus. Specifically, the canine virus may be influenza virus A, B or C. In addition, the antigen protein may be H3N2 or a variant thereof. In addition, the H3N2 may be a form in which the Arg-Gly sequence is mutated into a Val-Gly sequence. Specifically, the H3N2 variant may have an amino acid sequence of SEQ ID NO: 109.

As used herein, the term "polynucleotide" means a single or double stranded covalent sequence of nucleotides in which the 3' and 5' termini of each nucleotide are linked by phosphodiester bonds. A polynucleotide may be composed of deoxyribonucleotide bases (DNA) or ribonucleotide bases (RNA).

The polynucleotide herein includes DNA and RNA, and may be produced synthetically in vitro or isolated from natural sources. The size of the polynucleotide is typically expressed in the number of base pairs (bp) for double-stranded polynucleotides, or in the number of nucleotides (nt) for single-stranded polynucleotides. 1,000 bp or nt is equal to one kilobase (kb). Polynucleotides less than about 40 nucleotides in length are typically referred to as 'oligonucleotides'.

In this specification, the nucleic acid sequence is used in the same sense as polynucleotide, and may be DNA or RNA. In this case, RNA may include mRNA. In addition, the nucleic acid sequence may further include modified DNA or RNA, for example, methylated DNA or RNA, or RNA processed by post-translational modification, 3' processing such as cleavage and polyadenylation, and splicing. The nucleic acid may also include synthetic nucleic acid (XNA), hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA).

Homology with respect to the polynucleotides, nucleic acid sequences or base sequences described herein is not limited to mere 100% sequence identity. In this regard, two sequences are said to be "substantially similar" if the sequences have at least about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more or about 100% similarity. Likewise, with respect to two sequences, sequences are said to be "substantially complementary" if the sequences are completely complementary base by base, or at least about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more or about 99% or more of the bases. That is, mismatches may occur between bases of the sequences to be hybridized, and these may occur between bases in at least about 1% or less, about 5% or less, about 10% or less, about 20% or less, or at most about 30% or less.

The term "enterovirus" as used herein refers to a virus that belongs to the genus Enterovirus in the family Picornavirus and does not have an envelope, measuring 24 to 30 nm. It has an icosahedral shape and contains a single-stranded positive sense RNA measuring about 7.2 to 7.5 kb as its genetic material. Enteroviruses include three serotypes of Poliovirus (PV: 1 to 3); 23 serotypes of Coxsackievirus group A (CVA: 1 to 22, 24); and six serotypes of Coxsackievirus group B (CVB: 1 to 6); It consists of 28 serotypes of echovirus (ECV: 1–7, 9, 11–21, 24–27, 29–33) and other human enteroviruses (EV: 68–116).

The above enterovirus, after entering a host cell, releases RNA into the cytoplasm, and then functions as mRNA by itself to initiate translation at the IRES (internal ribosomal entry site) of the 5' untranslated region (UTR), thereby inducing the production of a large polyprotein. The genetic material of the above enterovirus consists of one ORF (open reading frame) and an untranslated region (UTR; or NCR, non-coding region) at the 5' and 3' ends that is not expressed as a protein. The ORF accounts for approximately 90% of the entire gene and is expressed as one polyprotein. The polyprotein consists of approximately 2,185 amino acids and is divided into several different proteins by the protease of the virus.

The above polyprotein is divided into a P1 domain, a P2 domain, and a P3 domain, and the P1 domain is called a structural protein, and the P2 domain and the P3 domain are called nonstructural proteins. The P1 domain of the above polyprotein encodes components VP4, VP2, VP3, and VP1 in order from the N-terminus to the C-terminus as a capsid protein of the virus, and the capsid is composed of a 32-mer capsomer. The P2 domain encodes 2A protease (2A ^pro ), 2B, and 2C in order from the N-terminus to the C-terminus, and the P3 domain encodes 3A, 3B (VPg protein), 3C protease (3C ^pro ), and 3D polymerase (3D ^pol ) in order. The above 2A protease and 3C protease is a proteolytic enzyme that recognizes and cleaves the cleavage site within the viral polyprotein, thereby processing it into individual proteins. Meanwhile, 3D The polymerase is an RNA-dependent RNA polymerase (RdRp) that synthesizes complementary RNA using RNA as a template during viral RNA self-replication.

In the present invention, the enterovirus is poliovirus (PV; 1-3), rhinovirus (RV), enterovirus (A71, A76, A89-A92, A120, B69, B73, B74, B75, B77, B78, B80-B88, B93, B97, B98, B100, B101, B106, C99, C105, C109, C116, D68, D94, D111), Coxsackievirus (A1-A22, A24, B1-B6, C96), echovirus (E2-E7, E9, E11-E21, E24-E27, E29-E33), and other human enteroviruses. 70, 79, 107, C104). Preferably, the enterovirus of the present invention may be Coxsackievirus B5.

In the present invention, the nonstructural protein derived from the enterovirus may include a P2 domain and a P3 domain. At this time, the nonstructural protein may include a P2 domain and a P3 domain in order from the N terminus to the C terminus.

In the present invention, the P2 domain may include 2A protease, 2B and 2C. Specifically, the P2 domain of the present invention may include 2A protease, 2B and 2C of Coxsackievirus B5. At this time, the P2 domain may include 2A protease, 2B and 2C in order from the N terminus to the C terminus. In one embodiment, the 2A protease, 2B and 2C may include the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively. Accordingly, the polynucleotide in the present invention may include or consist of a nucleic acid sequence encoding the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, in order from the 5' end to the 3' end. In one embodiment of the present invention, if the polynucleotide is DNA, it may include or consist of the base sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 in order from the 5' end to the 3' end. If the polynucleotide is RNA, it may include or consist of the base sequences of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63 in order from the 5' end to the 3' end.

In addition, in the present invention, the 2A protease, 2B and 2C may be composed of an amino acid sequence in which one or more amino acids are added, deleted, substituted and/or inserted, as long as they have the same biological function or the gene positions encoding the 2A protease, 2B and 2C on the chromosome are the same. Specifically, the 2A protease, 2B and 2C may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively. At this time, the secondary structure and hydropathic nature of the polypeptide of the original amino acid may not change due to addition, deletion, substitution and/or insertion of the amino acid.

In the present invention, the 2A protease may additionally comprise a mutation.

The term "mutant" as used herein means a form in which an amino acid or a part of a polynucleotide encoding an amino acid is substituted compared to the wild type. In the present invention, a protein or nucleic acid (polynucleotide) containing the mutation may be described as a mutant.

That is, in the present invention, the 2A protease variant may have a different amino acid sequence from the wild-type 2A protease. However, the 2A protease variant may have an activity equivalent to or similar to the wild-type 2A protease or an activity further improved. The 2A protease variant may have an increased protein expression rate compared to the wild-type. Here, "2A protease activity" may mean specifically recognizing and cleaving a cleavage site within the amino acid sequence.

Specifically, the 2A protease variant may be one in which some of the amino acids of the wild-type 2A protease are substituted. One specific example of a 2A protease variant resulting from an amino acid substitution may be one in which the 87th amino acid in the amino acid sequence of SEQ ID NO: 11 is substituted.

At this time, the "other amino acid" introduced by the substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, excluding glycine.

In one embodiment of the present invention, the 2A protease variant may be one in which glycine, the 87th amino acid in the amino acid sequence of SEQ ID NO: 11, is substituted with serine (G87S). Preferably, it may include the amino acid of SEQ ID NO: 75. Therefore, in the present invention, the polynucleotide may include or consist of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 75 as the 2A protease variant. Specifically, when the polynucleotide is DNA, the nucleic acid sequence may include or consist of the base sequence of SEQ ID NO: 73. When the polynucleotide is RNA, the nucleic acid sequence may include or consist of the base sequence of SEQ ID NO: 74.

Additionally, the 2A protease variant may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to the amino acid sequence of SEQ ID NO: 75.

In the present invention, the nonstructural protein P3 domain may include 3A, 3B, 3C protease and 3D polymerase. Specifically, the P3 domain of the present invention may include 3A, 3B, 3C protease and 3D polymerase of Coxsackievirus B5. At this time, the P3 domain may include 3A, 3B, 3C protease and 3D polymerase in order from the N terminus to the C terminus.

In one embodiment, the 3A, 3B, 3C proteases and 3D polymerases may each comprise amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17. Accordingly, in the present invention, the polynucleotide may comprise or consist of nucleic acid sequences sequentially encoding amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 from the 3' end of the nucleic acid sequence encoding the amino acid sequence of 2C. In one embodiment of the present invention, if the polynucleotide is DNA, the nucleic acid sequence may sequentially include or consist of the base sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 from the 3' end of the nucleic acid sequence encoding the amino acid sequence of 2C, and if the nucleic acid sequence is RNA, the nucleic acid sequence may sequentially include or consist of the base sequences of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

In addition, the 3A, 3B, 3C proteases and 3D polymerases may be composed of amino acid sequences in which one or more amino acids are added, deleted, substituted and/or inserted, as long as they have the same biological function or the gene positions encoding the 3A, 3B, 3C proteases and 3D polymerases on the chromosome are the same. Specifically, the amino acid sequences may include or consist of amino acid sequences having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with the amino acid sequences of SEQ ID NOs: 14 to 17, respectively.

Target protein

In the present invention, the polynucleotide may include a nucleic acid sequence encoding at least one or more target proteins. In this case, the target protein may be linked to a protease cleavage site.

The term "target protein" as used herein may be various proteins for medical or industrial purposes, and may include protein fragments, peptides, and variants. Industrially useful target proteins include hormones, hormone analogs, enzymes, enzyme inhibitors, cytokines, coagulation factors, transport proteins, receptors, receptor fragments, attachment proteins, regulatory proteins, structural proteins, toxic proteins, transcription factors, antigens, antibodies, antibody fragments, monoclonal antibodies, and the like. Preferably, it may be an antigen, but is not limited thereto. The antigen may be any antigenic protein associated with infection or disease. Examples of the antigen may include tumor antigens, animal antigens, plant antigens, viral antigens, bacterial antigens, fungal antigens, and protozoan antigens, autoimmune antigens, or allergy antigens. In this case, the antigen may be a surface antigen of a tumor cell, and a protein and peptide derived from a secreted form of a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen, respectively.

In the present invention, the viral pathogens include, for example, Filovirus, Adenovirus, Enterovirus, Severe fever with thrombocytopenia syndrome virus (SFTSV; Bunyavirus), Arbovirus, Astrovirus, Coronavirus, Coxsackie virus, Cytomegalovirus, Dengue virus, Epstein-Barr virus, Hepatitis virus, Herpesvirus, Human immunodeficiency virus, Human papilloma virus, Human T-lymphotropic virus, Influenza virus, Canine influenza virus virus), highly pathogenic avian influenza virus, JC virus, Lymphocytic choriomeningitis virus, Measles virus, Molluscum contagiosum virus, Mumps virus, Norovirus, Parovirus, Poliovirus, Rabies virus, Respiratory syncytial virus, Rhinovirus, Rotavirus, Rubella virus, Smallpox virus, Varicella zoster virus, West nile virus, Zika virus, etc.

In the present invention, the bacterial pathogen may be isolated from or derived from, for example, Campylobacter jejuni, Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitides, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, etc. In the present invention, the fungal pathogen may be isolated from or derived from, for example, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, Aspergillus species, etc.

In the present invention, the protozoan pathogen may be isolated from or derived from, for example, Plasmodium, Leishmania, Trypanosome, Cryptosporidiums, Isospora, Naegleria fowleri, Acanthamoeba, Balamuthia mandrillaris, Toxoplasma gondii, Pneumocystis carinii, etc.

In the present invention, the tumor antigen may be isolated from or derived from cystitis, breast cancer, colon and rectal cancer, endometrial cancer, renal cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, etc. For example, 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, α-5-β-1-integrin, α-5-β-6-integrin, α-actinin-4/m, α-methylacyl-coA racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, β-catenin/m, BING-4, CA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein (COTL1), collagen type XXIII, COX-2, CT_9/BRD6, Cten, cycline B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, h TERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin Receptor, kallikrein-2(KLK2), kallikrein-4(LKL4), Ki67, KIAA0205, KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, 1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin-A, MART-1/melan-A, MART-2, MART_2/m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3 (PR3), PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, BAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1, SSX_2/HOM-MEL-40, , STAMP-1, STEAP, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFβ, TGFβRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, These may include VEGF, VEGFR-2/FLK-1, and WT1.

In the present specification, when the polynucleotide comprises one target protein, the target protein may be described interchangeably with the “first target protein”.

In the present invention, the first target protein may be located at the N-terminus of the nonstructural protein P2 domain. Specifically, it may be located at the N-terminus of the 2A protease of the nonstructural protein P2 domain. Accordingly, in the present invention, the nucleic acid sequence encoding the first target protein may be located at the 5'-terminus of the nucleic acid sequence encoding the amino acid sequence of the 2A protease in the polynucleotide.

In one specific example of the present invention, the first target protein may be a viral antigen. In one embodiment, the viral antigen may be a severe fever with thrombocytopenia syndrome (SFTS) virus antigen, a spike protein of SARS-CoV2 (COVID) antigen, a highly pathogenic avian influenza (HPAI) virus antigen, or a canine influenza antigen. Accordingly, in the present invention, the polynucleotide may include a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 85, SEQ ID NO: 48, SEQ ID NO: 105, SEQ ID NO: 53, or SEQ ID NO: 56 at the 5' end of a nucleic acid sequence encoding an amino acid sequence of 2A protease. In one embodiment of the present invention, when the polynucleotide is DNA, the nucleic acid sequence may include or consist of a base sequence of SEQ ID NO: 43, SEQ ID NO: 83, SEQ ID NO: 46, SEQ ID NO: 106, SEQ ID NO: 51, or SEQ ID NO: 54. When the polynucleotide is RNA, the nucleic acid sequence may include or consist of a base sequence of SEQ ID NO: 44, SEQ ID NO: 84, SEQ ID NO: 47, SEQ ID NO: 107, SEQ ID NO: 52, or SEQ ID NO: 55.

Preferably, the first target protein may be an H3N2 protein of a canine influenza virus. Additionally, a variant of the H3N2 protein may be used as an antigen, which may include the amino acid sequence of SEQ ID NO: 109.

In addition, the SFTS antigen, COVID antigen, highly pathogenic avian influenza or canine influenza antigen may be composed of an amino acid sequence having the same biological function or having the same gene location encoding the SFTS antigen, COVID antigen, highly pathogenic avian influenza or canine influenza antigen on the chromosome, or having one or more amino acids added, deleted, substituted and/or inserted. Specifically, the SFTS antigen may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 85, SEQ ID NO: 48, SEQ ID NO: 105, SEQ ID NO: 53 or SEQ ID NO: 56.

The term "protease cleavage site" used in the present invention means a site specifically cleaved by a proteolytic enzyme. In the present invention, the protease cleavage site linked to the first target protein may be a site cleaved by a protease derived from a nonstructural protein of the self-amplifying virus. At this time, the self-amplifying virus may be a virus of the genus Enterovirus. Preferably, it may be Coxsackievirus B5. In one specific example of the present invention, the protease cleavage site may be a site cleaved by a 2A protease of the Enterovirus. At this time, the protease cleavage site may be located between the first target protein and the 2A protease of the nonstructural protein P2 domain.

As used herein, the term "2A protease of enterovirus" is a protein decomposition enzyme that operates first after enterovirus RNA is translated into a single polyprotein, and plays a role in cleaving the polyprotein into structural proteins and nonstructural proteins. At this time, the 2A protease can cleave between any one amino acid selected from the group consisting of Tyr, Ala, Thr, Val, Phe, and Arg among the C-terminal amino acids of VP1 of the P1 domain and Gly, which is the N-terminal amino acid of the 2A protease of the P2 domain. In one specific example of the present invention, the 2A protease can cleave between Tyr and Gly. In one specific example, it can cleave between Ala and Gly. In one specific example, it can cleave between Thr and Gly. In one specific example, it can cleave between Val and Gly. In one specific example, it can cleave between Phe and Gly. In one specific example, it can cleave between Arg and Gly. Preferably, it can cleave between Tyr among the C-terminal amino acids of VP1 in the P1 domain and Gly among the N-terminal amino acids of 2A protease in the P2 domain.

Therefore, in the present invention, the 2A protease cleavage site may include the C-terminal amino acid sequence of VP1.

Additionally, the 2A protease cleavage site may comprise the C-terminal amino acid sequence of VP1 and the N-terminal amino acid sequence of 2A protease.

Specifically, in the present invention, the 2A protease cleavage site may include 1 to 26 consecutive amino acids from the C-terminus of VP1.

More specifically, the 2A protease cleavage site may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 70.

Additionally, the 2A protease cleavage site may comprise 1 to 13 consecutive amino acid sequences from the C-terminus of VP1 and 1 to 13 consecutive amino acid sequences from the N-terminus of VP1.

More specifically, it may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 70 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 11.

In one embodiment of the present invention, the 2A protease cleavage site may include any one amino acid sequence selected from SEQ ID NO: 18(T) to SEQ ID NO: 23. Accordingly, the polynucleotide of the present invention may include a nucleic acid sequence encoding any one amino acid sequence selected from SEQ ID NO: 18 to SEQ ID NO: 23 between the 3' end of the nucleic acid sequence encoding the first target protein and the 5' end of the nucleic acid sequence encoding the 2A protease. Preferably, it may include a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22. In one embodiment of the present invention, when the polynucleotide is DNA, it may include or consist of any one base sequence selected from SEQ ID NO: 24(ACC) to SEQ ID NO: 29. Preferably, it may include the base sequence of SEQ ID NO: 28. When the polynucleotide is RNA, it may include or consist of any one base sequence selected from SEQ ID NO: 24 and SEQ ID NO: 30 to SEQ ID NO: 34. Preferably, it may contain a base sequence of sequence number 33.

In addition, the 2A protease cleavage site may be composed of an amino acid sequence having one or more amino acids added, deleted, substituted and/or inserted, as long as the location of the gene encoding the 2A protease cleavage site on the chromosome is the same or has the same biological function. Specifically, the 2A protease cleavage site may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with any one amino acid sequence selected from SEQ ID NO: 18 to SEQ ID NO: 23.

In the present invention, the polynucleotide may additionally include a nucleic acid sequence encoding a target protein. In the present specification, when the polynucleotide includes a nucleic acid sequence encoding one or more target proteins, the target proteins may be described as a first target protein and a second target protein. At this time, the second target protein may be located at the N-terminus of the first target protein. Accordingly, in the present invention, the nucleic acid sequence encoding the second target protein may be located at the 5'-terminus of the nucleic acid sequence encoding the amino acid sequence of the first target protein in the polynucleotide. The target protein and the first target protein are the same as described above.

In one specific embodiment of the present invention, the first target protein and the second target protein may be viral antigens. In one embodiment, the viral antigens may be H3N2 and H3N8, which are subtypes of canine influenza. In this case, the first target protein may be H3N2 or H3N8, and the second target protein may be H3N2 or H3N8. The first and second target proteins may be the same or different from each other. Accordingly, in one embodiment of the present invention, when the polynucleotide is DNA, the first target protein may be composed of a base sequence of SEQ ID NO: 51 or SEQ ID NO: 54, and when the polynucleotide is RNA, the first target protein may be composed of a base sequence of SEQ ID NO: 52 or SEQ ID NO: 55. In one embodiment of the present invention, the first target protein may be H3N8 and include an amino acid sequence of SEQ ID NO: 56, and the second target protein may be H3N2 and include an amino acid sequence of SEQ ID NO: 53. Therefore, the nucleic acid sequence encoding the first target protein of the polynucleotide may include the base sequence of SEQ ID NO: 54 or SEQ ID NO: 55, and the nucleic acid sequence encoding the second target protein may include the base sequence of SEQ ID NO: 51 or SEQ ID NO: 52.

A protease cleavage site may exist between the first target protein and the second target protein. At this time, the protease cleavage site may be a site cleaved by a protease derived from a nonstructural protein of the self-amplifying virus. The self-amplifying virus may be a virus of the family Picornavirus, and specifically, a virus of the genus Enterovirus. Preferably, it may be Coxsackievirus B5. In one specific example of the present invention, the protease cleavage site may be a site cleaved by 2A protease, 3CD protease, or 3C protease of Enterovirus. More specifically, it may be a site cleaved by 2A protease, 3CD protease, or 3C protease of Coxsackievirus B5. Here, the protease cleavage site, the 2A protease, and the 2A protease cleavage site are the same as described above.

In the present invention, "3C protease of enterovirus" is a protein decomposition enzyme that operates second after enterovirus RNA is translated into one polyprotein. The "3CD protease (3CD ^pro )" is a precursor of 3C protease and is a protein produced in an intermediate step in which the P3 domain is processed into each individual protein by the 3C protease. At this time, the 3CD protease also has protease activity. The 3C protease and 3CD protease play a role in cleaving the polyprotein, which is cleaved into a structural protein (P1 domain) and a nonstructural protein (P2 domain and P3 domain) by the 2A protease, into each individual protein (VP0, VP3, VP1, 2A protease, 2B, 2C, 3A, 3B, 3C protease and 3D polymerase). At this time, "VPO" is an intermediate protein that is additionally processed into VP4 and VP2.

3C protease can cleave between any one amino acid selected from the group consisting of Gln, Glu, Ile, and Thr among the C-terminal amino acids of each individual protein (VP3, VP1, 2A protease, 2B, 2C, 3A, 3B, and 3C protease) and any one amino acid selected from the group consisting of Gly, Asn, Ser, Ala, Val, Cys, Trp, and Met among the N-terminal amino acids of each individual protein (VP1, 2A protease, 2B, 2C, 3A, 3B, 3C protease, and 3D polymerase). In one specific embodiment of the present invention, 3C protease can cleave between Gln and Gly. In one specific embodiment, 3C protease can cleave between Gln and Asn. In one specific embodiment, 3C protease can cleave between Gln and Ser. In one embodiment, the 3C protease can cleave between Gln and Ala. In one embodiment, the 3C protease can cleave between Gln and Val. In one embodiment, the 3C protease can cleave between Gln and Cys. In one embodiment, the 3C protease can cleave between Gln and Trp. In one embodiment, the 3C protease can cleave between Gln and Gly. In one embodiment, the 3C protease can cleave between Gln and Met. In one embodiment, the 3C protease can cleave between Glu and Gly. In one embodiment, the 3C protease can cleave between Glu and Asn. In one embodiment, the 3C protease can cleave between Glu and Ser. In one embodiment, the 3C protease can cleave between Glu and Ala. In one embodiment, the 3C protease can cleave between Glu and Val. In one embodiment, the 3C protease can cleave between Glu and Cys. In one embodiment, the 3C protease can cleave between Glu and Trp. In one embodiment, the 3C protease can cleave between Glu and Gly. In one embodiment, the 3C protease can cleave between Glu and Met. In one embodiment, the 3C protease can cleave between Ile and Gly. In one embodiment, the 3C protease can cleave between Ile and Asn. In one embodiment, the 3C protease can cleave between Ile and Ser. In one embodiment, the 3C protease can cleave between Ile and Ala. In one embodiment, the 3C protease can cleave between Ile and Val. In one embodiment, the 3C protease can cleave between Ile and Cys. In one embodiment, the 3C protease can cleave between Ile and Trp. In one embodiment, the 3C protease can cleave between Ile and Gly. In one embodiment, the 3C protease can cleave between Ile and Met. In one embodiment, the 3C protease can cleave between Thr and Gly. In one embodiment, the 3C protease can cleave between Thr and Asn. In one embodiment, the 3C protease can cleave between Thr and Ser. In one embodiment, the 3C protease can cleave between Thr and Ala. In one embodiment, the 3C protease can cleave between Thr and Val. In one embodiment, the 3C protease can cleave between Thr and Cys. In one specific embodiment, the 3C protease can cleave between Thr and Trp. In one specific embodiment, the 3C protease can cleave between Thr and Gly. In one specific embodiment, the 3C protease can cleave between Thr and Met. Preferably, the cleavage can be between Gln among the C-terminal amino acids and Gly among the N-terminal amino acids of each individual protein.

Specifically, the 3C protease according to the present invention can cleave between Gln, the C-terminal amino acid of VP3 of Coxsackievirus B5, and Gly, the N-terminal amino acid of VP1. It can cleave between Gln, the C-terminal amino acid of 2A protease, and Gly, the N-terminal amino acid of 2B. It can cleave between Gln, the C-terminal amino acid of 2B, and Gly, the N-terminal amino acid of 2C. It can cleave between Gln, the C-terminal amino acid of 2C, and Gly, the N-terminal amino acid of 3A. It can cleave between Gln, the C-terminal amino acid of 3A, and Gly, the N-terminal amino acid of 3B. It can cleave between Gln, the C-terminal amino acid of 3B, and Gly, the N-terminal amino acid of 3C protease. It can cleave between Gln, the C-terminal amino acid of 3C protease, and Gly, the N-terminal amino acid of 3D polymerase.

Therefore, in the present invention, the 3C protease cleavage site may include the C-terminal amino acid sequence of any one protein selected from the group consisting of VP3, 2A protease, 2B, 2C, 3A, 3B and 3C protease.

In addition, in the present invention, the protease cleavage site may include any one amino acid sequence selected from the group consisting of the amino acid sequences of the C-terminus of VP3 and the N-terminus of VP1, the C-terminus of 2A protease and the N-terminus of 2B, the C-terminus of 2B and the N-terminus of 2C, the C-terminus of 2C and the N-terminus of 3A, the C-terminus of 3A and the N-terminus of 3B, the C-terminus of 3B and the N-terminus of 3C protease, the C-terminus of 3C protease and the N-terminus of 3D polymerase.

Specifically, in the present invention, the 3C protease cleavage site may include 1 to 22 consecutive amino acids from the C-terminus of the amino acid sequence of any one protein selected from the group consisting of VP3, 2A protease, 2B, 2C, 3A, 3B and 3C protease.

More specifically, the 3C protease cleavage site may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive amino acids from the C-terminus of any one amino acid sequence selected from the group consisting of SEQ ID NO: 80, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

In addition, in the present invention, the 3C protease cleavage site may include 1 to 11 consecutive amino acids from the C-terminal amino acid sequence of VP3 and 1 to 11 consecutive amino acids from the N-terminal amino acid sequence of VP1. It may include 1 to 11 consecutive amino acids from the C-terminal amino acid sequence of 2A protease and 1 to 11 consecutive amino acids from the N-terminal amino acid sequence of 2B. It may include 1 to 11 consecutive amino acids from the C-terminal amino acid sequence of 2B and 1 to 11 consecutive amino acids from the N-terminal amino acid sequence of 2C. It may include 1 to 11 consecutive amino acids from the C-terminal amino acid sequence of 2C and 1 to 11 consecutive amino acids from the N-terminal amino acid sequence of 3A. It may include 1 to 11 consecutive amino acids from the C-terminal amino acid sequence of 3A and 1 to 11 consecutive amino acids from the N-terminal amino acid sequence of 3B. It may include 1 to 11 consecutive amino acids from the C-terminal amino acid sequence of 3B and 1 to 11 consecutive amino acids from the N-terminal amino acid sequence of 3C protease. It may include 1 to 11 consecutive amino acids from the C-terminal amino acid sequence of 3C protease and 1 to 11 consecutive amino acids from the amino acid sequence of the N-terminal amino acid sequence of 3D polymerase.

More specifically, the 3C protease cleavage site may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 80 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 70. It may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 11 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 12. It may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 12 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 13. It may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 13 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 14. It may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 14 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 15. It may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 15 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 16. It may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 16 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 17.

3CD protease can cleave between VP0 and VP3. Specifically, it can cleave between any one amino acid selected from the group consisting of Gln, Lys, Tyr, and Met among the C-terminal amino acids of VP0 and any one amino acid selected from the group consisting of Gly, Trp, Tyr, and Asn among the N-terminal amino acids of VP3. In one specific example of the present invention, 3CD protease can cleave between Gln and Gly. In one specific example, it can cleave between Gln and Trp. In one specific example, it can cleave between Gln and Tyr. In one specific example, it can cleave between Gln and Asn. In one specific example, it can cleave between Lys and Gly. In one specific example, it can cleave between Lys and Trp. In one specific example, it can cleave between Lys and Tyr. In one specific example, it can cleave between Lys and Asn. In one specific example, it can cleave between Thr and Gly. In one specific example, it can cleave between Thr and Trp. In one specific example, it can cleave between Thr and Tyr. In one specific example, it can cleave between Thr and Asn. In one specific example, it can cleave between Met and Gly. In one specific example, it can cleave between Met and Trp. In one specific example, it can cleave between Met and Tyr. In one specific example, it can cleave between Met and Asn. Preferably, it can cleave between Gln among the C-terminal amino acids of VP0 and Gly among the N-terminal amino acids of VP3.

Therefore, in the present invention, the 3CD protease cleavage site may include the C-terminal amino acid sequence of the amino acid sequence of VP2.

Additionally, the 3CD protease cleavage site may include the C-terminal amino acid sequence of VP2 and the N-terminal amino acid sequence of VP3.

Specifically, in the present invention, the 3CD protease cleavage site may include 1 to 40 consecutive amino acids from the C-terminus of VP2.

More specifically, the 3CD protease cleavage site may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 79.

Additionally, the 3CD protease cleavage site may comprise 1 to 20 consecutive amino acid sequences from the C-terminus of VP2 and 1 to 20 consecutive amino acid sequences from the N-terminus of VP1.

More specifically, it may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 79 and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 80.

In one specific embodiment of the present invention, the protease cleavage site may include a sequence of 6 amino acids continuous from the C-terminal amino acid sequence of the amino acid sequence of 2C and a sequence of 6 amino acids continuous from the N-terminal amino acid sequence of 3A.

In one embodiment of the present invention, the 3C protease cleavage site may comprise an amino acid sequence of SEQ ID NO: 50. Accordingly, in one embodiment of the present invention, the nucleic acid sequence encoding the 3C protease cleavage site may comprise or consist of a base sequence of SEQ ID NO: 49 or SEQ ID NO: 57, respectively.

In addition, the 3C protease cleavage site and the 3CD protease cleavage site may be composed of an amino acid sequence in which one or more amino acids are added, deleted, substituted and/or inserted, as long as they have the same biological function or the same genetic location encoding the 3C protease cleavage site on the chromosome. Specifically, the amino acid sequence may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to the amino acid sequence of SEQ ID NO: 50.

Therefore, in one embodiment of the present invention, the polynucleotide of the present invention may include a nucleic acid sequence encoding the first and second target proteins, in the order of the 5' to 3' end, a nucleic acid sequence encoding the amino acid sequences of SEQ ID NO: 53, SEQ ID NO: 50, SEQ ID NO: 56 and SEQ ID NO: 22, at the 5' end of the nucleic acid sequence encoding the 2A protease protein of the nonstructural protein. Accordingly, when the polynucleotide is DNA, it may include or consist of the base sequences of SEQ ID NO: 54, SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 28, in the order of the 5' to the 3' end. When the polynucleotide of the present invention is RNA, the nucleic acid sequence may include or consist of the base sequences of SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 52 and SEQ ID NO: 33, in the order of the 5' to the 3' end.

In addition, when the second target protein in the present invention comprises one or more target proteins, the second target protein may be composed of one or more identical or different target proteins, and a protease cleavage site may be located between each target protein. At this time, the protease cleavage site may be a site cleaved by a protease derived from a nonstructural protein of a self-amplifying virus. Specifically, it may be a site cleaved by a protease derived from a picornavirus. More specifically, it may be a site cleaved by 2A protease, 3CD protease, or 3C protease of enterovirus. Preferably, it may be a site cleaved by 2A protease, 3CD protease, or 3C protease of coxsackievirus B5. The second target protein may be an antigen. Additionally, the protease cleavage site, 2A protease, 3CD protease, 3C protease, 2A protease cleavage site, 3CD protease cleavage site and 3C protease cleavage site are the same as described above.

Termination codon

In the present invention, the polynucleotide may additionally include at least one stop codon at the 3' end of the nucleic acid sequence encoding the nonstructural protein of the self-amplifying virus.

The term "termination codon" as used herein is a signal sequence for stopping translation of mRNA into amino acids, and generally refers to the sequence of a termination codon or stop codon. There is no corresponding tRNA at a termination codon, but instead a protein called a "termination factor" binds, and when the termination codon is reached during the translation process, the two units of the ribosome separate, thereby terminating translation. Generally, the sequence of the termination codon of DNA is TAG, TAA, or TGA, and the sequence of the termination codon of RNA is UAG, UAA, or UGA.

Specifically, the polynucleotide of the present invention may include one to three stop codons.

More specifically, the polynucleotide may comprise one stop codon. In one specific embodiment, when the polynucleotide is DNA, the stop codon may be any one selected from the group consisting of TAG, TAA, and TGA. When the polynucleotide is RNA, the stop codon may be any one selected from the group consisting of UAG, UAA, and UGA.

More specifically, the polynucleotide may comprise two different stop codons located consecutively.

If the polynucleotide of the present invention is DNA, in one specific example, if the polynucleotide is DNA, the stop codon may be positioned in the order of TAG and TAA from the 5' end to the 3' end. In one specific example, it may be positioned in the order of TAG and TGA. In one specific example, it may be positioned in the order of TAA and TGA. If the polynucleotide of the present invention is RNA, in one specific example, it may be positioned in the order of UAG and UAA from the 5' end to the 3' end. In one specific example, it may be positioned in the order of UAG and UGA. In one specific example, it may be positioned in the order of UAA and UGA.

More specifically, the polynucleotide may comprise three different stop codons located sequentially.

When the polynucleotide of the present invention is DNA, in one specific example, the stop codon may be positioned in the order of TAG, TAA, and TGA from the 5' end to the 3' end. In one specific example, the stop codon may be positioned in the order of TAG, TGA, and TAA. In one specific example, the stop codon may be positioned in the order of TAA, TAG, and TGA. In one specific example, the stop codon may be positioned in the order of TAA, TGA, and TAG. In one specific example, the stop codon may be positioned in the order of TGA, TAG, and TAA. In one specific example, the stop codon may be positioned in the order of TGA, TAA, and TAG. In one embodiment of the present invention, the stop codon may include or consist of the base sequence of SEQ ID NO: 9 (TGATAATAG). When the polynucleotide of the present invention is RNA, in one specific example, the stop codon may be positioned in the order of UAG, UAA, and UGA from the 5' end to the 3' end. In one specific example, it may be positioned in the order of UAG, UGA, and UAA. In one specific example, it may be positioned in the order of UAA, UAG, and UGA. In one specific example, it may be positioned in the order of UAA, UGA, and UAG. In one specific example, it may be positioned in the order of UGA, UAG, and UAA. In one specific example, it may be positioned in the order of UGA, UAA, and UAG. In one embodiment of the present invention, the stop codon may include or consist of the base sequence of SEQ ID NO: 68 (UGAUAAUAG).

5' untranslated area

In the present invention, the polynucleotide may additionally include a 5' untranslated region (5' UTR) at the 5' end.

As used herein, the term "untranslated region" refers to a region within an RNA molecule that is transcribed but not translated into an amino acid sequence. The untranslated region may be upstream of the start codon (5' UTR) and downstream of the stop codon (3' UTR). The 5' UTR is a region that regulates the translation of transcription and acts to regulate transcription through different mechanisms in viruses, prokaryotes, and eukaryotes. Although generally known as an untranslated region, it is sometimes known to be translated into a protein product and to regulate the translation of mRNA.

The elements of the 5' UTRs of eukaryotes and prokaryotes differ significantly. Prokaryotic 5' UTRs contain a ribosome binding site (RBS), also known as the Shine-Dalgarno sequence (SD sequence), which is typically located 3-10 base pairs upstream of the start codon. In contrast, eukaryotic 5' UTRs contain a Kozak consensus sequence (hereinafter referred to as the "Kozak sequence") that contains the start codon.

The 5' untranslated region of the polynucleotide according to the present invention may include an internal ribosomal entry site (IRES).

The term "internal ribosome entry site (IRES)" as used herein is a base sequence that is generally located in the 5' UTR of an RNA virus and can induce translation of RNA in a cap-independent manner. In the present invention, the 5' UTR is a region where a translational initiation complex associated with translation of a target protein binds, and the IRES is a cis-acting base sequence that induces translation of a target protein by forming complex secondary and tertiary structures.

In the present invention, the IRES may include a base sequence of an IRES derived from a virus (viral) or a eukaryotic cell (eukaryotic). At this time, the 5' UTR may have a 5' UTR structure including a viral or eukaryotic IRES derived from the IRES. In one specific example of the present invention, the 5' UTR and the IRES may be derived from the same self-amplifying virus as the nonstructural protein. They may be derived from a virus of the genus Picornavirus. More specifically, the 5' UTR and the IRES may be derived from an enterovirus. In one embodiment of the present invention, the 5' UTR may include or consist of a nucleic acid sequence of SEQ ID NO: 1 (DNA) or SEQ ID NO: 60 (RNA), and at this time, the IRES may include or consist of a nucleic acid sequence of SEQ ID NO: 58 (DNA) or SEQ ID NO: 59 (RNA).

The above IRES may contain a mutation, wherein the mutation is the same as described above.

Specifically, the mutant of the IRES may have a portion of the base sequence of the wild-type IRES replaced with a different base sequence.

More specifically, when the polynucleotide is DNA, any one nucleotide selected from the group consisting of the 236th, 386th, and a combination thereof in the base sequence of SEQ ID NO: 58 may be substituted with a nucleotide different from the base sequence of SEQ ID NO: 58. As a specific example, the 236th nucleotide in the base sequence of SEQ ID NO: 58 may be substituted with A, T, or G. As a specific example, the 386th nucleotide in the base sequence of SEQ ID NO: 58 may be substituted with A, T, or C. As a specific example, the 236th nucleotide in the base sequence of SEQ ID NO: 58 may be substituted with A, T, or G, and the 386th nucleotide may be substituted with A, T, or C. Preferably, the 236th nucleotide in the base sequence of SEQ ID NO: 58 may be substituted with T (C236T). In the base sequence of sequence number 58, G at the 386th nucleotide may be substituted with A (G386A). More preferably, in the base sequence of sequence number 58, C at the 236th nucleotide may be substituted with T (C236T), and G at the 386th nucleotide may be substituted with A (G386A).

When the above polynucleotide is RNA, any one nucleotide selected from the group consisting of the 236th, 386th, and a combination thereof in the base sequence of SEQ ID NO: 59 may be substituted with a nucleotide different from the base sequence of SEQ ID NO: 59. As a specific example, the 236th nucleotide in the base sequence of SEQ ID NO: 59 may be substituted with A, U, or G. As a specific example, the 386th nucleotide in the base sequence of SEQ ID NO: 59 may be substituted with A, U, or C. As a specific example, the 236th nucleotide in the base sequence of SEQ ID NO: 59 may be substituted with A, U, or G, and the 386th nucleotide may be substituted with A, U, or C. Preferably, C, which is the 236th nucleotide in the base sequence of SEQ ID NO: 59, may be substituted with U (C236U). In the base sequence of sequence number 59, G at the 386th nucleotide may be substituted with A (G386A). More preferably, in the base sequence of sequence number 59, C at the 236th nucleotide may be substituted with U (C236U), and G at the 386th nucleotide may be substituted with A (G386A).

In one embodiment of the present invention, the variant of the IRES may comprise or consist of the base sequence of SEQ ID NO: 81 or SEQ ID NO: 82.

Cossack sequence

In the present invention, the polynucleotide may additionally include a Kozak sequence.

The term "Kozak sequence" as used herein refers to a base sequence located upstream of the initiation codon at which translation of eukaryotic mRNA into protein begins, and plays an important role in recognizing the initiation codon and initiating protein synthesis.

In the present invention, the Kozak sequence may be located at the 5' end of a nucleic acid sequence encoding a target protein (a first target protein or a second target protein). In the present invention, the Kozak sequence may include any one base sequence selected from SEQ ID NO: 35 to SEQ ID NO: 37. Preferably, it may include the base sequence of SEQ ID NO: 35 or consist of the base sequence.

3' untranslated area

In the present invention, the polynucleotide may additionally include a 3' UTR. At this time, the 3' UTR may be located at the 3' end of the stop codon. The 3' UTR is functionally linked to a nucleic acid sequence encoding a target protein and a non-structural protein together with the 5' UTR, thereby improving the translation efficiency of the target protein and the non-structural protein, or their transcripts. In addition, it plays an important role in ensuring that the transcript, mRNA, is not destroyed within the cell and is stably maintained.

In the present invention, the 3' UTR may use a 3' UTR derived from a virus or eukaryotic cell that is identical to the 5' UTR derived from a virus and/or eukaryotic cell having the IRES base sequence described above. In one specific example of the present invention, the 3' UTR may be derived from the same self-amplifying virus as the nonstructural protein derived from the 5' UTR. Specifically, it may be derived from a virus of the genus Picornavirus identical to the 5' UTR. More specifically, it may be derived from the same enterovirus as the 5' UTR. Preferably, it may be derived from Coxsackievirus B5.

In one embodiment of the present invention, the 3' UTR may include or consist of the base sequence of SEQ ID NO: 10 (DNA) or SEQ ID NO: 69 (RNA).

In the present invention, the polynucleotide may additionally include a poly(A) tail sequence. The poly(A) tail sequence may be located at the 3' end of a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus. When the polynucleotide includes a stop codon, the poly(A) tail sequence may be located at the 3' end of the stop codon. When the polynucleotide includes a 3' UTR, the poly(A) tail sequence may be located at the 3' end of the base sequence of the 3' UTR. The poly(A) tail sequence may stabilize a transcribed nucleic acid molecule and further improve the translation efficiency of the target protein. The poly(A) tail sequence may include about 10 to about 500 adenosine (A) nucleotides. Specifically, it may be a nucleic acid sequence consisting of about 10 to about 500, about 50 to about 400, about 100 to about 300, or about 100 to about 200 adenosine nucleotides. Preferably, it may consist of about 120 adenosine nucleotides. In one embodiment of the present invention, the poly adenosine tail sequence may include or consist of a base sequence of SEQ ID NO: 38 to SEQ ID NO: 40. Preferably, it may consist of a base sequence of SEQ ID NO: 40.

Structure of polynucleotide

The polynucleotide of the present invention may be composed of the following structural formula (I) in order from the 5' end to the 3' end:

5'-B-C(1)-D-3' (I)

5' and 3' are the 5' and 3' ends of the polynucleotide, respectively,

B is a nucleic acid sequence encoding the first target protein,

C(1) is a nucleic acid sequence encoding a protease cleavage site,

D is a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus.

More specifically, the polynucleotide may be composed of the following structural formula (II) in order from the 5' end to the 3' end:

5'-U-K-[B'-C(2)]n-B-C(1)-D-S-U'-P-3' (II)

5' and 3' are the 5' and 3' ends of the polynucleotide, respectively,

U is the nucleic acid sequence of the 5' untranslated region,

K is the Cozak sequence,

B' is a nucleic acid sequence encoding a second target protein,

B is a nucleic acid sequence encoding the first target protein,

D is a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus,

S is the stop codon,

U' is the nucleic acid sequence of the 3' untranslated region,

P is a polyadenosine tail,

C(1) and C(2) are each independently a nucleic acid sequence encoding a protease cleavage site,

n represents the number of [B'-C(2)] and is an integer from 0 to 10.

When n is 2 to 10, B' is a nucleic acid sequence encoding the same or different target protein, and C(2) is also a nucleic acid sequence encoding the same or different protease cleavage site.

At this time, C(1) is a 2A protease cleavage site, and C(2) can be a 2A protease, 3CD protease or 3C protease cleavage site.

The 5' untranslated region, target protein, first target protein, second target protein, nonstructural protein, stop codon, 3' untranslated region, polyadenosine tail, protease cleavage site, 2A, 3CD and 3C protease cleavage sites are the same as described above.

When the polynucleotide according to the present invention is RNA, the polynucleotide (hereinafter, interchangeably used with "RNA construct") may be self-amplifying RNA (saRNA). The RNA construct may be double-stranded or single-stranded, and preferably single-stranded.

The term "replicon" as used herein refers to a self-replicating nucleic acid sequence, and in the present invention, the RNA replicon refers to an RNA molecule that can be replicated by an RNA-dependent RNA polymerase. The RNA replicon can produce one or more identical or substantially similar copies of an RNA replicon without a DNA intermediate. Hereinafter, the saRNA virus vector, RNA replicon, saRNA, and saRNA construct may be described interchangeably in the present invention.

Recombinant vector

Another aspect of the present invention provides a recombinant vector loaded with the polynucleotide, wherein the polynucleotide is the same as described above.

The term "recombinant" as used herein means "created through genetic manipulation," meaning something that does not occur naturally.

As used herein, the term "vector" refers to a genetic construct that is an expression vector (or recombinant vector) capable of expressing a target protein in a host cell, and includes essential regulatory elements that are operably linked so that a gene insert is expressed. The term "operably linked" refers to a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein being functionally linked so as to perform a general function. The operably linked vector can be performed using a genetic recombination technique well known in the art to which the present invention pertains, and the site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art to which the present invention pertains. Specifically, the recombinant vector can be amplified in vitro by polymerase chain reaction (PCR), recombinantly produced by cloning, purified by cleavage and gel-electroporation fractionation, or synthesized by a chemical synthesis method, but is not limited thereto.

In the present invention, the recombinant vector can be used as a gene delivery vehicle for transporting and expressing the polynucleotide according to the present invention. Therefore, it is preferable that the polynucleotide is loaded into a suitable expression vector. A suitable expression vector that can be used in the present invention can include a signal sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, an initiation codon, and a termination codon. The initiation codon and the termination codon are generally considered to be part of a nucleotide sequence encoding an immunogenic target protein, and must necessarily exhibit function in a subject when the genetic construct is administered, and must be in frame with the coding sequence.

The term "promoter" as used herein refers to a nucleic acid sequence that controls the synthesis of a transcript, for example a transcript comprising a coding sequence, by providing a recognition and binding site for RNA polymerase. A typical promoter may be constitutive or inducible. In the present invention, the promoter operably linked to the polynucleotide is preferably one that can operate in an animal cell, more preferably a mammalian cell, to control transcription of the polynucleotide. The promoter may include a promoter derived from a virus, a promoter derived from the genome of a mammalian cell, or a promoter derived from a bacteriophage. For example, it can include, but is not limited to, the cytomegalovirus (CMV) promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the tk promoter of HSV, the 94 promoter, the T3 promoter, the SM6 promoter, the RSV promoter, the EF1α promoter, the metallothionein promoter, the β-actin promoter, cancer cell-specific promoters (e.g., the TERT promoter, the PSA promoter, the PSMA promoter, the CEA promoter, the E2F promoter, and the AFP promoter), and tissue-specific promoters (e.g., the albumin promoter).

In one embodiment of the present invention, the polynucleotide may be loaded into a suitable expression vector and then used as a template for transcription into a nucleic acid molecule in the form of RNA (saRNA construct, RNA replicon) through in vitro transcription (IVT). At this time, a promoter may be located at the 5' end of the polynucleotide for transcription from linearized DNA into RNA.

The above "in vitro transcription" refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. At this time, a cloning vector can be applied to the production of a transcript. These cloning vectors are generally designed as transcription vectors and are included in the "recombinant vector" in the present invention. In the present invention, when the polynucleotide is RNA, the RNA may be in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription using an appropriate DNA template. At this time, the promoter for regulating transcription may be any promoter of RNA polymerase. In addition, the DNA template used for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA (a polynucleotide of the present invention), and introducing it into an appropriate vector for in vitro transcription (a recombinant vector loaded with the polynucleotide of the present invention). When the nucleic acid (the polynucleotide of the present invention) is RNA, cDNA can be obtained by reverse transcription.

Specifically, the RNA can be synthesized using a DNA-dependent RNA polymerase such as T7 RNA polymerase, and at this time, the expression vector can include a T7 promoter at the 5' end of the loaded polynucleotide. When another RNA polymerase, for example, SP6 or T3 RNA polymerase, is used in IVT using the expression vector loaded with the polynucleotide as a template, the vector can include an SP6 or T3 promoter. In one embodiment of the present invention, the RNA polymerase can be T7 RNA polymerase, and the recombinant vector can include a T7 promoter. Specifically, the recombinant vector can include a promoter including or consisting of the nucleic acid sequence of SEQ ID NO: 94 at the 5' end upstream of the polynucleotide according to the present invention.

A vector that can be used as a template for synthesizing the above saRNA construct may be, for example, a plasmid, a cosmid, a phage, a viral vector, etc. In this case, the polynucleotide sequence may be a DNA sequence, and preferably, may be plasmid DNA.

The above "plasmid" generally refers to a structure of extrachromosomal genetic material, usually a circular DNA duplex that can replicate independently of chromosomal DNA.

In addition, in the present invention, the recombinant vector can replicate spontaneously within a host cell. At this time, the vector can be introduced into a host cell, recombined and inserted into the host cell genome, and replicated together with the host cell genome.

At this time, the recombinant vector may additionally include a promoter, regulator, or enhancer for controlling the expression of the loaded polynucleotide.

Additionally, the plasmid may contain a selection marker, such as an antibiotic resistance gene, and the host cell harboring the plasmid may be cultured under selective conditions.

Transformed host cells

Another aspect of the present invention provides a host cell transformed with the recombinant vector. The recombinant vector is the same as described above.

The term "transformation" as used herein means the phenomenon of artificially causing genetic changes by introducing external DNA into a cell, such that the DNA becomes replicable as a chromosomal element or by completion of chromosomal integration by introducing the DNA into the host cell.

The above "host cell" refers to a cell that supplies nutrition by parasitizing other microorganisms or genes, and a cell in which a vector exerts various genetic or molecular effects within the host cell by transforming the host cell. The host cell is in a state of competence that can accept external DNA, and external DNA such as a vector can be inserted, and when the vector is successfully introduced into the host cell, the genetic characteristics of the vector are provided to the host cell. The host cell may include a prokaryotic cell (e.g., E. coli) or a eukaryotic cell (e.g., yeast cell and insect cell), and may include a mammalian cell such as a cell derived from a human, mouse, hamster, pig, goat, or primate.

The above transformation can be performed by various methods. Specifically, the transformation method may be a CaCl ₂ precipitation method, a Hanahan method that increases efficiency by using a reducing substance called DMSO (dimethyl sulfoxide) in the CaCl ₂ precipitation method, electroporation, a calcium phosphate precipitation method, a protoplast fusion method, a stirring method using silicon carbide fibers, an Agrobacterium-mediated transformation method, a transformation method using PEG, dextran sulfate, lipofectamine, and a drying/inhibition-mediated transformation method. The method for transforming the plasmid of the present invention is not limited to the above examples, and a transformation method commonly used in the art may be used without limitation.

The transformed host cell of the present invention can be used in a method for producing the target protein of the present invention. The method can include a process of culturing the host cell and recovering the produced target protein. The recovered target protein or peptide can be purified from the culture supernatant.

Method for producing RNA replicons

Another aspect of the present invention provides a method for producing an RNA replicon, comprising the steps of: i) producing a recombinant vector loaded with a polynucleotide according to the present invention; and ii) synthesizing RNA from the recombinant vector. The polynucleotide, the recombinant vector, and the RNA replicon are the same as those described above.

The above RNA replicon can be obtained by in vitro transcription using the DNA of the recombinant vector as a template. Generally, the poly adenosine tail sequence is encoded by a poly-(dT) sequence on the DNA template. In the present invention, the poly adenosine tail sequence can be added using an enzyme after transcription. In addition, in the present invention, the poly adenosine tail sequence can be transcribed from a recombinant vector. Preferably, it can be transcribed from a recombinant vector.

Suitable methods for in vitro transcription are well known in the art and are known to those skilled in the art. For example, see [Molecular Cloning, A Laboratory Manual, 2nd edition. (1989) editor C Nolan, Cold Spring Harbor Laboratory Press]. In addition, a variety of in vitro transcription kits are commercially available.

Vaccine composition

Another aspect of the present invention provides a vaccine composition comprising the polynucleotide or recombinant vector of the present invention as an active ingredient. Here, the polynucleotide and the recombinant vector are the same as described above.

The term "vaccine" as used herein refers to a drug that uses a living organism to stimulate the immune system for the purpose of preventing disease. Immune activation refers to the process of efficiently removing antigens by producing antibodies, stimulating T cells, or stimulating other immune cells (e.g., macrophages, natural killer cells) in the body. A detailed overview of immunology on the above is readily understood by those skilled in the art (Barrett, J.T., Textbook of Immunology, 1983). The terms "vaccine" and "vaccine composition" may be used interchangeably herein.

In the present invention, the vaccine composition may be for treating protozoan, fungal, bacterial or viral infections. In the present invention, the vaccine composition may be for treating cancer.

In the above vaccine composition, the active ingredient, a polynucleotide or recombinant vector, may be included in an amount sufficient to induce a typical immune response. In the above vaccine composition, the active ingredient may be included in an amount of about 0.0001 ㎍ to about 1000 ㎍. Specifically, it may be included in an amount of about 0.0001 ㎍ to about 1000 ㎍, about 0.001 ㎍ to about 100 ㎍, or about 0.01 ㎍ to about 10 ㎍, but is not limited thereto.

In the vaccine composition of the present invention, the effective ingredient may be included in any amount (effective amount) depending on the amount, use, formulation, compounding purpose, etc. sufficient to induce a typical immune response. Typically, the effective amount may be determined within the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. Here, the "effective amount" refers to the amount of the recombinant microorganism, which is an effective ingredient, sufficient for the vaccine composition to induce a typical immune response of the vaccine. This effective amount can be experimentally determined within the normal ability range of a person skilled in the art.

In addition, the vaccine composition may include a pharmaceutically acceptable carrier or diluent. The carrier used in the composition of the present invention includes pharmaceutically acceptable carriers, adjuvants, and vehicles, and is collectively referred to as a "pharmaceutically acceptable carrier." Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity that is more than the target of application (prescription) can adapt to. Pharmaceutically acceptable carriers that may be used in the compositions of the present invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffering substances (e.g., various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based matrices, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-blocked polymers, polyethylene glycol, and wool fat.

Meanwhile, the vaccine composition of the present invention can be administered in a "therapeutically effective amount." The "therapeutically effective amount" can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, the route of administration, the method of administration, the number of administrations, the treatment period, and drugs used in combination or simultaneously.

The term "administration" as used herein means introducing a given substance into a subject in an appropriate manner, and the route of administration of the composition may be administered through any common route as long as it can reach the target tissue. In order for a vaccine to be effective in producing antibodies, the antigenic substance must be introduced into the body so that the antibody-producing mechanism of the subject treated with the vaccine can be realized. Therefore, in order to induce an immune response, the polynucleotide or recombinant vector according to the present invention must first be introduced into the body.

When administering the polynucleotide or recombinant vector according to the present invention to a subject, it may be encapsulated as a lipid nanoparticle (LNP) or coated with lipid and administered in the form of a liposome or vesicle. At this time, the lipid-encapsulated product may exist in the form of a lipid aggregate or micelle. The LNP may be manufactured using a method known in the art.

In order to stimulate a desirable response by the antigen presented by the vaccine composition of the present invention, it can be administered systemically through parenteral administration. Parenteral administration can be administered in the form of intranasal, intranasal, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intradermal, intraperitoneal, enteral, topical, sublingual or rectal administration, but is not limited thereto. Preferably, it can be administered subcutaneously or intramuscularly.

The subject of administration of the vaccine composition according to the present invention may be a mammal such as a human, cow, horse, pig, dog, sheep, goat, or cat, and preferably a human.

The preferred dosage of the above vaccine composition can be prescribed in various ways depending on factors such as the formulation method of the subject of administration, the method of administration, the age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the subject of administration, and can be appropriately selected by a person skilled in the art. The dosage suitable for the subject of administration varies depending on the antigenicity of the gene product and is not particularly limited as long as it is an amount sufficient to induce a typical immune response of the existing vaccine. The dosage can be easily determined through routine experimental procedures. A typical initial vaccine dose can be about 0.001 μg/kg to about 10 mg/kg or about 0.01 μg/kg to about 1 mg/kg of antigen, and the amount can be increased or multiple doses can be used as needed to provide a desired level of protection. The vaccine composition can be administered once a day, daily, every other day, weekly, or every other week. Such dosage should not be construed as limiting the scope of the present invention in any way.

The above vaccine composition may be prepared in the form of, for example, a powder, a tablet, a capsule, a liquid, an ointment, a cream, a gel, a hydrogel, an aerosol, a spray, a micellar solution, a transdermal patch, a liposomal suspension, a polyplex, an emulsion, a lipid nanoparticle (LNP) (having RNA on its surface or encapsulated therein) or any other suitable form which can be administered to a human or mammal in need of treatment or vaccination.

When the above vaccine composition is administered intranasally or parenterally, it may be administered in the form of a spray or aerosol, or by inhalation, but is not limited thereto. The composition for intranasal or intranasal administration is prepared according to techniques well known in the pharmaceutical field, and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption promoters for enhancing bioavailability, fluorocarbons, and/or other solubilizers or dispersants known in the art.

The vaccine composition according to the present invention, when administered parenterally, may be prepared in the form of a sterile injectable preparation as a sterile aqueous or oily suspension for injection. The suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic, parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Acceptable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any fixed oil having little irritation, including synthetic mono- or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in injectable preparations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated forms.

With regard to the above formulation, it is known in the art, and specifically, reference can be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)], etc. The above literature is considered to be a part of this specification.

The vaccine composition of the present invention may additionally include an adjuvant to enhance the immunogenicity of the vaccine. Auxiliary components such as an adjuvant may be appropriately selected and used depending on the type of antigen and the indication of the vaccine composition.

Another aspect of the present invention provides a method for preventing or treating a disease, comprising administering to a subject the polynucleotide, recombinant vector or vaccine composition. In this case, the vaccine composition and administration are the same as described above.

The term "treatment" as used herein can be used to mean both therapeutic treatment and preventive treatment. In this case, "prevention" can be used to mean alleviating or reducing a pathological condition or disease of an individual. The treatment includes all applications or any form of medication for treating a disease in mammals, including humans. In addition, the term includes inhibiting or slowing the progression of a disease; restoring or repairing damaged or defective functions, partially or completely alleviating a disease, or stimulating an ineffective process, or alleviating a serious disease.

The above disease may be an infectious disease caused by protozoa, fungi, bacteria or viruses, or may be a cancer disease.

The subject may be a mammal suffering from or capable of suffering from the disease, preferably a human.

The above dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity.

Another aspect of the present invention provides a nonstructural protein 2A protease cleavage site derived from enterovirus comprising the amino acid sequence of SEQ ID NO: 22. The enterovirus, the nonstructural protein and the 2A protease cleavage site are the same as described above.

Another aspect of the present invention provides a nonstructural protein 3C protease cleavage site derived from enterovirus comprising an amino acid sequence of SEQ ID NO: 50. The enterovirus, the nonstructural protein and the 3C protease cleavage site are the same as described above.

Another aspect of the present invention provides an IRES variant comprising a nucleic acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. The IRES variant is as described above.

Another aspect of the present invention provides a 5' untranslated region variant comprising the nucleic acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87. The 5' untranslated region variant is the same as described above.

Another aspect of the present invention provides a Kozak sequence comprising a nucleic acid sequence of SEQ ID NO: 35. The Kozak sequence is identical to that described above.

Another aspect of the present invention provides a nonstructural protein 2A protease derived from enterovirus comprising an amino acid sequence of SEQ ID NO: 75. The 2A protease is the same as described above.

Another aspect of the present invention provides a severe fever with thrombocytopenia syndrome (SFTS) virus antigen comprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 85.

The term "severe fever with thrombocytopenia syndrome" used in the present invention refers to a disease with high fever and thrombocytopenia as its main symptoms, and is caused by infection with the SFTS virus. SFTS is transmitted by the Haemaphysalis longicornis tick of the Ixodidae family of the Ixodiformes order, which vectors the SFTS virus, and the virus carried by the tick enters the body during blood sucking, multiplies, and causes the disease.

Another aspect of the present invention provides a fusion protein comprising a target protein and a protease cleavage site. The target protein in the fusion protein may be a first target protein, a second target protein, or may include both the first and second target proteins. When the target protein is the first target protein, the protease cleavage site may be a site cleaved by a 2A protease derived from a nonstructural protein of an enterovirus. In this case, the 2A protease may be located at the C-terminus of the first target protein. When the target protein is the second target protein, the protease cleavage site may be a site cleaved by a 2A, 3CD protease or 3C protease derived from a nonstructural protein of an enterovirus. In this case, the 2A, 3CD or 3C protease cleavage site may be located at the C-terminus of the second target protein. When the above target protein includes both the first and second target proteins, the second target protein may be located at the N-terminus of the first target protein, and the two target proteins may be linked via a 2A protease, 3CD protease or 3C protease cleavage site derived from a nonstructural protein of an enterovirus. At this time, the 2A protease may be located at the C-terminus of the first target protein. In addition, the second target protein may be composed of one or more identical or different target proteins, and at this time, the proteins may be linked via a protease cleavage site selected from the group consisting of 2A, 3CD and 3CD proteases. The protease cleavage sites may be identical or different.

Here, the target protein, the first target protein, the second target protein, the enterovirus nonstructural protein, and the 2A, 3CD and 3C protease cleavage sites are the same as described above.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

I. Production of optimized self-amplifying RNA

Manufacturing Example 1. Production of a vector containing enterovirus-based saRNA

In the present invention, a clone vector in the form of an enterovirus-based saRNA construct with luciferase inserted as a target protein, which is used as a basic backbone in the production of an optimized self-amplifying RNA construct, was produced.

The clone vector used in the present invention was constructed by loading the Coxsackievirus B5 gene (SEQ ID NO: 99) into the pUC19-CMV/T7 (Addgene, plasmid #50005) vector using the same method described in "Development of a Universal Cloning System for Reverse Genetics of Human Enteroviruses" (WS Choi, et al., Microbiol Spectr. 18; e0316722) to produce an infectious clone. Using the In-fusion cloning (In-Fusion®HD Cloning Kit, Takara) method, the P1 domain (structural protein, VP) of the clone was replaced with the target protein (luciferase, SEQ ID NO: 41) to produce a clone vector in the form of a saRNA construct (Fig. 16).

Example 1. Selection of 2A protease cleavage site sequences of self-amplifying RNA

In order to produce a modified self-amplifying RNA (saRNA) construct with an optimized expression rate of the target protein according to the present invention, the expression level of the target protein was first confirmed according to the amino acid sequence of the cleavage site of 2A protease, a nonstructural protein of enterovirus.

Example 1.1. Production of saRNA constructs by 2A protease cleavage site sequence type

Specifically, a library vector was produced by inserting the base sequences of SEQ ID NOs: 24 to 29 into the cleavage site of the 2A protease of an enterovirus-based saRNA construct clone vector having a luciferase produced in-house by the method of Manufacturing Example 1 inserted as a target protein using a known mutagenesis method so that 1 (VP1-1a.a, SEQ ID NO: 18), 5 (VP1-5a.a, SEQ ID NO: 19), 10 (VP1-10a.a, SEQ ID NO: 20), 15 (VP1-15a.a, SEQ ID NO: 21), 20 (VP1-20a.a, SEQ ID NO: 22), or 25 (VP1-25a.a, SEQ ID NO: 23) amino acid sequences were included. BsmbI (NEB) restriction enzyme was added to the above vector DNA, and linearized by reaction at 55°C for 1 to 6 hours, and then the restriction enzyme was inactivated by reaction at 80°C for 20 minutes. The DNA was confirmed to be linear DNA by electrophoresis on a 0.9% agarose gel, and then purified as follows.

In vitro transcription (IVT) was performed using the purified linearized DNA as a template using the Invitrogen™MEGAscript™T7 Transcription Kit at 37°C for 3 hours. After treatment with DNase to remove residual DNA template, the produced RNA was purified using lithium chloride (LiCl) precipitation (Table 1).

VP1-1a.a saRNA VP1-5a.a saRNA VP1-10a.a saRNA VP1-15a.a saRNA VP1-20a.a saRNA VP1-25a.a saRNA DNA vector saRNA DNA vector saRNA DNA vector saRNA DNA vector saRNA DNA vector saRNA DNA vector saRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Target protein Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 2A protease cleavage site Sequence number 24 Sequence number 24 Sequence number 25 Sequence number 30 Sequence number 26 Sequence number 31 Sequence number 27 Sequence number 32 Sequence number 28 Sequence number 33 Sequence number 29 Sequence number 34 2A protease Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 3’ UTR Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 poly(A) tail Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38 Sequence number 38

Example 1.2. Comparison of expression rates of saRNA constructs according to 2A protease cleavage site sequences

The saRNA construct produced by the method of Example 1.1 was transfected into 293T cells and the expression level was confirmed.

Specifically, 293T cells were seeded at 5 × 10 ⁵ cells/well in a 6-well plate, cultured overnight, and then the cell medium was replaced with medium without fetal bovine serum (FBS) 4 hours before transfection to prepare the cells.

Each of the purified RNAs above was transfected into cells at 5 μg per well using Lipofectamine®2000 Transfection Reagent (Invitrogen). 12 hours after transfection, cells were harvested, centrifuged (12,000 rpm, 3 minutes) to precipitate the cells, and Luciferase Cell Culture Lysis 1× Reagent was added (100 μl) and reacted at room temperature for 30 minutes. After the reaction was complete, the reaction solution was centrifuged again (12,000 rpm, 1 minute), and the luciferase activity of the supernatant (20 μl) was measured using Luciferase Assay System reagent (Promega).

As a result, as shown in Fig. 2, the luminescence of luciferase was found to be the highest in the group treated with the saRNA construct (VP1-20a.a) into which 20 amino acids at the C-terminus of VP1 were inserted.

Therefore, the 20 amino acid sequence (SEQ ID NO: 22) at the C-terminus of VP1 was used as the 2A protease cleavage site of the modified saRNA construct with optimized expression rate of the present invention.

Example 2. Selection of Kozak sequence and stop codon

Example 2.1. Production of saRNA constructs by Kozak sequence and stop codon type

In order to produce a modified saRNA construct with an optimized expression rate of the target protein in the present invention, the expression level of the target protein according to the Kozak sequence and stop codon was confirmed.

The saRNA construct was produced in the same manner as in Example 1.1, and at this time, the recombinant vector had a Kozak sequence of Kozak 1 (SEQ ID NO: 35, CCACC), Kozak 2 (SEQ ID NO: 36, GCCACC), or Kozak 4 (SEQ ID NO: 37, AAG) inserted at the 3' end of the 5' UTR. The stop codon used was a sequence consisting of three different stop codons in succession at the 5' end of the 3' UTR (3× stop) (SEQ ID NO: 9, TGATAATAG) (Table 2).

Kozak1 saRNA Kozak2 saRNA Kozak4 saRNA 3X stop saRNA DNA vector saRNA DNA vector saRNA DNA vector saRNA DNA vector saRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Cossack sequence Sequence number 35 Sequence number 35 Sequence number 36 Sequence number 36 Sequence number 37 Sequence number 37 - - Target protein Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 2A protease cleavage site Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 2A protease Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence - - - - - - Sequence number 9
(TGATAATAG) Sequence number 68
(UGAUAAUAG) 3’ UTR Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40

Example 2.2. Comparison of saRNA construct expression rates according to Kozak sequence and stop codon

The expression of each saRNA construct produced by the method of Example 2.1 was measured in 293T cells using Luciferase assay reagent (Progmega) in the same manner as in Example 1.2. At this time, a clone vector (original P.C) produced in-house from Coxsackievirus B5 was used as a control.

As a result, as shown in Fig. 3, the luciferase activity was highest in the saRNA construct (Kozak 4) into which the Kozak sequence of Kozak 4 was inserted. In addition, compared to the control group, the luciferase activity significantly increased in the saRNA construct into which the stop codon of sequence number 68 (sequence number 9 for DNA) was inserted.

Therefore, the Kozak sequence of the modified saRNA construct with optimized expression rate of the present invention used the sequence of SEQ ID NO: 42, and the stop codon used the 3× stop of SEQ ID NO: 68 (SEQ ID NO: 9 in the case of DNA).

Example 3. Selection of polyadenosine tail sequences

Example 3.1. Production of saRNA constructs by polyadenosine tail sequence type

In order to produce a modified saRNA construct with an optimized expression rate of the target protein of the present invention, the expression level of the target protein according to the poly adenosine tail (poly(A) tail) sequence was confirmed.

The saRNA construct was produced in the same manner as in Example 1.1, and at this time, the recombinant vector was produced by inserting 50 (50× poly(A), SEQ ID NO: 38), 100 (100× poly(A), SEQ ID NO: 39), or 120 (120× poly(A), SEQ ID NO: 40) poly(A) units into the 3' end of the 3' UTR (Table 3).

50X poly(A) saRNA 100X poly(A) saRNA 120X poly(A) saRNA DNA vector saRNA DNA vector saRNA DNA vector saRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Cossack sequence Sequence number 35 Sequence number 35 Sequence number 35 Sequence number 35 Sequence number 35 Sequence number 35 Target protein Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 Sequence number 41 Sequence number 42 2A protease cleavage site Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 2A protease Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence Sequence number 9 Sequence number 68 Sequence number 9 Sequence number 68 Sequence number 9 Sequence number 68 3’ UTR Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 poly(A) tail Sequence number 38 Sequence number 38 Sequence number 39 Sequence number 39 Sequence number 40 Sequence number 40

Example 3.2. Comparison of saRNA construct expression rates according to polyadenosine tail sequences

The expression of each saRNA construct produced by the method of Example 3.1 was measured in 293T cells using Luciferase assay reagent (Progmega) in the same manner as in Example 1.2.

In addition, the saRNA construct was encapsulated using a lipid nanoparticle (LNP) biodelivery system (ALC-0315 (MEC, HY-138170)/cholesterol (Sigma, C8667)/DMG-PEG 2000 (Avanti Polar Lipids, 880151P)/DOPE (Avanti Polar Lipids, 850725P) in NanoAssemblr®Ignite (Precision NanoSystems) and administered to mice to confirm the expression level at the animal level. At this time, female BLAB/c mice of 5 to 6 weeks of age were used (n=3), and 1 ㎍ of each saRNA construct was administered intramuscularly to each mouse. Four hours after administration, luciferin was administered intravenously to the mice at a concentration of 150 mg/kg per mouse, and the luciferase activity in the mice was measured using IVIS Spectrum (Perkin Observed through Elmer).

As a result, as shown in Fig. 4, the luciferase activity was highest in the saRNA construct with a 120× poly(A) tail inserted at both the cellular and animal levels.

Therefore, the poly adenosine tail of the modified saRNA construct with optimized expression rate of the present invention used the sequence of SEQ ID NO: 40.

II. Antigen expression using optimized self-amplifying RNA

Example 4. Production of optimized self-amplifying RNA

An optimized saRNA construct was produced from a recombinant vector containing the base sequence of the 2A protease cleavage site (VP1-20a.a, SEQ ID NO: 28), the Kozak sequence (Kozak1, SEQ ID NO: 35), and the poly adenosine tail (120Х poly(A), SEQ ID NO: 40) selected in Examples 1 to 3 using the same method as in Example 1.1 (Table 4).

In order to compare the degree of improvement in the expression rate of the modified saRNA construct, mRNA and existing enterovirus-based saRNA constructs (saRNA) were produced together and used as an experimental group (Fig. 5). At this time, mRNA was cloned using the 5' and 3' UTR of the target protein derived from human globulin into the structure of 5' UTR (SEQ ID NO: 95) - target protein (SEQ ID NO: 41) - 3' UTR (SEQ ID NO: 97) - poly adenosine tail (50X poly(A), SEQ ID NO: 38), linearized, and then used as template DNA to react at 37°C for 6 hours using the MESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen), and then purified in the same way as the saRNA construct (Table 4).

Modified saRNA mRNA DNA vector saRNA DNA vector mRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 95 Sequence number 96 Cossack sequence Sequence number 35 Sequence number 35 - - Target protein Sequence number 41
(Luciferase) Sequence number 42
(Luciferase) Sequence number 41
(Luciferase) Sequence number 42
(Luciferase) 2A protease recognition site Sequence number 28 Sequence number 33 - - 2A protease Sequence number 2 Sequence number 61 - - 2B Sequence number 3 Sequence number 62 - - 2C Sequence number 4 Sequence number 63 - - 3A Sequence number 5 Sequence number 64 - - 3B Sequence number 6 Sequence number 65 - - 3C Protease Sequence number 7 Sequence number 66 - - 3D Sequence number 8 Sequence number 67 - - Termination sequence Sequence number 9 Sequence number 68 - - 3’ UTR Sequence number 10 Sequence number 69 Sequence number 97 Sequence number 98 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 38 Sequence number 38

Example 4.2. Confirmation of expression rate of optimized self-amplifying RNA

The expression level of the modified saRNA construct produced by the method of Example 4.1 was confirmed at the cellular and animal levels. For confirmation at the cellular level, 293T cells were used and seeded in 12-well plates (2.5 × 10 ⁵ cells/well) the day before transfection and then cultured overnight.

Transfection and luciferase activity measurement were performed in the same manner as in Example 1.2. During transfection, RNA was treated at a concentration of 0.01 μg, 0.1 μg, 0.5 μg, or 1 μg.

As a result, as shown in Fig. 6, the luciferase activity of the modified saRNA construct treatment group of the present invention was the highest at all concentrations of RNA.

To confirm expression at the animal level, the mRNA, saRNA construct or modified saRNA construct was encapsulated in NanoAssemblr®Ignite (Precision NanoSystems) using an LNP biodelivery system (ALC-0315/cholesterol/PEG/DOPE) in the same manner as in Example 3.2 and then administered intramuscularly to mice. At this time, RNA was administered at a concentration of 0.01 ㎍, 0.1 ㎍ or 1 ㎍ per mouse, respectively. Four hours after administration, luciferin was administered intravenously to the mice at a concentration of 150 mg/kg per mouse, and the luciferase activity in the mice was observed using IVIS Spectrum (Perkin Elmer).

As a result, as shown in Fig. 7, the highest luciferase activity was observed in the modified saRNA construct administration group of the present invention. In particular, the activity was clearly observed even in the 0.01 ㎍ administration group, and no luciferase activity was observed in the mRNA and enterovirus-based existing saRNA construct administration groups at the same concentration.

Example 5.1. Production of optimized self-amplifying RNA constructs containing antigens

In the optimized modified saRNA construct of Example 4.1, instead of luciferase, the target protein was replaced with a severe fever with thrombocytopenia syndrome (SFTS) virus antigen, a spike protein antigen of SARS-CoV2, a highly pathogenic avian influenza (HPAI) virus antigen, or a canine influenza virus antigen, thereby producing a recombinant vector, and an optimized saRNA construct was produced from the recombinant vector in the same manner as in Example 1.1 (Tables 5 to 7). At this time, the severe fever with thrombocytopenia syndrome virus antigen used was a nucleic acid sequence (SEQ ID NO: 43) encoding SFTS B antigen (SEQ ID NO: 45) and a nucleic acid sequence (SEQ ID NO: 83) encoding SFTS ABDEF antigen (SEQ ID NO: 85). The nucleic acid sequence (SEQ ID NO: 46) encoding the spike protein antigen (SEQ ID NO: 48) of SARS-CoV2 was used, respectively. The nucleic acid sequence (SEQ ID NO: 106) encoding the H5N8 antigen (SEQ ID NO: 105) was used as the highly pathogenic avian influenza virus antigen. For linearization of vector DNA, BsmBI (NEB) restriction enzyme was reacted at 55°C for 6 hours, and inactivated at 80°C for 10 minutes.

In order to compare the degree of improvement in the expression rate of the modified saRNA construct, mRNA was also produced and used as an experimental group. At this time, mRNA was linearized by cloning the target protein into the structure of 5' UTR-target protein-3' UTR-poly adenosine tail (50) using 5' and 3' UTR derived from human globulin, and then using this as template DNA, it was reacted at 37℃ for 6 hours using MESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen), and then purified in the same way as the saRNA construct (Tables 5 to 7).

Modified SFTS B antigen saRNA Modified SFTS ABDEF antigen saRNA SFTS B antigen mRNA DNA vector saRNA DNA vector saRNA DNA vector mRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 95 Sequence number 96 Cossack sequence Sequence number 35 Sequence number 35 Sequence number 35 Sequence number 35 - - Target protein Sequence number 43 Sequence number 44 Sequence number 83 Sequence number 84 Sequence number 43 Sequence number 44 2A protease recognition site Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 - - 2A protease Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence Sequence number 9 Sequence number 68 Sequence number 9 Sequence number 68 - - 3’ UTR Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 97 Sequence number 98 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 38 Sequence number 38

Modified SARS antigen saRNA SARS antigen mRNA DNA vector saRNA DNA vector mRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 95 Sequence number 96 Cossack sequence Sequence number 35 Sequence number 35 - - Target protein Sequence number 46 Sequence number 47 Sequence number 46 Sequence number 47 2A protease recognition site Sequence number 28 Sequence number 33 - - 2A protease Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence Sequence number 9 Sequence number 68 - - 3’ UTR Sequence number 10 Sequence number 69 Sequence number 97 Sequence number 98 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 38 Sequence number 38

Modified H5N8 antigen saRNA H5N8 antigen mRNA DNA vector saRNA DNA vector mRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 95 Sequence number 96 Cossack sequence Sequence number 35 Sequence number 35 - - Target protein Sequence number 106 Sequence number 107 Sequence number 106 Sequence number 107 2A protease recognition site Sequence number 28 Sequence number 33 - - 2A protease Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence Sequence number 9 Sequence number 68 - - 3’ UTR Sequence number 10 Sequence number 69 Sequence number 97 Sequence number 98 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 38 Sequence number 38

Example 5.2. Confirmation of expression rate of optimized self-amplifying RNA

The expression level of the modified saRNA construct produced by the method of Example 5.1 was confirmed at the cellular level through Western blot. At this time, the efficiency for the cell delivery system (LNP biodelivery system, lipofectamine) using RNA containing the SFTS virus antigen was also confirmed (Fig. 8).

First, to prepare the LNP-RNA experimental group, each of the above RNAs (RNA containing SFTS antigen) was encapsulated using the LNP biodelivery system (ALC-0315/Cholesterol application/PEG/DOPE) in NanoAssemblr®Ignite (Precision NanoSystems). The encapsulated RNA (LNP-RNA) was diluted 10-fold in 1× TE (Tris-EDTA) buffer containing 2% (v/v) Triton X-100 (Sigma Aldrich), and the fluorescence of LNP-RNA was measured at wavelengths of 485 nm (excitation) and 528 nm (emission) using a microplate reader (BMG LABTECH, UK). At this time, the RNA concentration corresponding to the fluorescence value was also measured.

As prepared above, LNP-RNA or each RNA was treated and transfected into cells at different concentrations. At this time, each RNA was transfected in the same manner as Example 1.2 using Lipofectamine®2000 Transfection Reagent (Invitrogen). In addition, the cells were seeded in a 6-well plate at a concentration of 1 × 10 ⁶ cells/well the day before transfection, and then cultured by replacing the medium with one that did not contain fetal bovine serum 4 hours before transfection, and then transfection was performed.

The medium was replaced 4 hours after transfection, and the culture was continued for an additional 24 hours. After 24 hours, the cells were harvested by centrifugation (12,000 rpm, 1 minute), Cell Lysis buffer was added (1 mL), and the cells were lysed by vortexing. After incubation at room temperature for 10 minutes, the protein concentration was measured, and 10 μg of protein was mixed with 2× sample buffer (SDS sample buffer having the same volume as the protein) and incubated at 95°C for 7 minutes to prepare protein samples. Each protein sample was electrophoresed on SDS-PAGE, transferred to a membrane, blocked with a 5% lipoprotein solution, and then primary antibodies were added and reacted at 4°C for 16 hours. Anti-Gc antibody (Novusbio, NBP2-41153, 1:2000) and anti-Spike antibody (Sino Biological, 40591-MM42, 1:2000) were used for each antigen as primary antibodies, respectively.

After the primary antibody reaction, the membrane was washed five times for 10 minutes each with TBS-T buffer, and then the secondary antibody was added and reacted at room temperature for 1 to 2 hours. The secondary antibodies used were anti-rabbit-HRP antibody (abcam, ab205718, 1:2000) and anti-mouse-HPR antibody (Invitrogen, 62-6520, 1:5000). After the secondary antibody reaction, each membrane was washed five times for 10 minutes each with TBS-T and then detected using HRP reagent (Millipore).

As a result, as shown in Fig. 8, RNA using LNP showed a higher expression rate than RNA transfected using lipofectamine (top). In addition, it was confirmed that antigen expression was significantly higher in the optimized modified saRNA construct of the present invention including SFTS (Fig. 8) antigen or SARS-CoV2 (Fig. 9) antigen compared to mRNA.

III. Production of multi-antigen self-amplifying RNA

Example 6.1. Production of multi-antigen self-amplifying RNA

To produce a vaccine capable of expressing two antigens at a low dose, two antigens were inserted into an enterovirus-based saRNA construct, and a 3C protease cleavage site amino acid sequence (SEQ ID NO: 50) was inserted between the antigens to produce a multi-antigen saRNA construct using the same method as in Examples 1.1 and 4.1. At this time, H3N2 and H3N8, subtypes of canine influenza, were used as antigens (Table 8, Figures 10 and 11, top).

In addition, saRNA constructs and mRNA containing only H3N2 antigens were also produced and used. At this time, mRNA was linearized by cloning the target protein into the structure of 5' UTR-target protein-3' UTR-polyadenosine tail (50) using 5' and 3' UTR derived from human globulin, and then using this as template DNA, it was produced in the same manner as Example 4.1 using the MESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen).

H3N2 antigen saRNA H3N2/H3N8 antigen saRNA H3N2 antigen mRNA DNA vector saRNA DNA vector saRNA DNA vector mRNA 5’ UTR Sequence number 1 Sequence number 60 Sequence number 1 Sequence number 60 Sequence number 95 Sequence number 96 Cossack sequence Sequence number 35 Sequence number 35 Sequence number 35 Sequence number 35 - - Target protein 2 - - Sequence number 51 Sequence number 52 - - 3C protease cleavage site - - Sequence number 49 Sequence number 57 - - Target protein 1 Sequence number 51 Sequence number 52 Sequence number 54 Sequence number 55 Sequence number 51 Sequence number 52 2A protease cleavage site Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 - - 2A protease Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 Sequence number 2 Sequence number 61 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence Sequence number 9 Sequence number 68 Sequence number 9 Sequence number 68 - - 3’ UTR Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 Sequence number 97 Sequence number 98 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 38 Sequence number 38

Example 6.2. Confirmation of antigen expression of multi-antigen self-amplified RNA

The expression level of the multi-antigen saRNA construct produced by the method of Example 6.1 was confirmed at the cellular level through Western blot. Each RNA was transfected in the same manner as Example 1.2 using Lipofectamine®2000 Transfection Reagent (Invitrogen). At this time, each RNA was treated at a concentration of 0.1 ㎍, 1 ㎍, or 5 ㎍ per well. The medium was replaced 4 hours after transfection and cultured for an additional 24 hours. At this time, P.C WT virus was used as a control. At this time, P.C WT virus is canine influenza virus (A/canine/Korea/AS-06/2012).

The above transfected cells were sampled using the same method as in Example 5.2, and the expression of the antigen was confirmed through Western blotting. At this time, the primary antibody, anti-H3N2 antibody, was used by diluting a self-produced antibody at 1:2000, and the secondary antibody was anti-mouse-HRP antibody (Invitrogen, 62-6520, 1:20000). At this time, the anti-H3N2 antibody was used by producing an acellular vaccine using the following method. First, 50 ㎕ of purified H3N2 hemagglutinin protein (Mybiosource, 32-5658) at a concentration of 0.1 ㎍/㎕ and adjuvant (AddaVax, Invivogen) were mixed at a 1:1 ratio and administered intramuscularly to mice. After the first administration, three additional vaccinations were performed at two-week intervals, and then antibodies were produced by directly separating from the blood.

As a result, as shown in Fig. 11, it was confirmed that antigen expression was significantly increased in the saRNA construct treatment group compared to the mRNA treatment group (bottom left). It was observed that the multi-antigen saRNA construct treatment group had a somewhat lower expression rate compared to the saRNA construct containing a single antigen. In addition, in the multi-antigen saRNA construct treatment group, two types of antigens were observed in a fused form and a separated form (bottom right).

Through the above results, it was confirmed that the multi-antigen saRNA construct can simultaneously express two antigens.

IV. Production of optimized self-amplifying RNA with improved proliferation rate

Example 7. Virus cell and animal adaptation experiments for increasing virus proliferation rate

Example 7.1. Production of cell-adapted coxsackievirus B5

A cell-adapted virus was produced by infecting Vero cells with wild-type Coxsackievirus B5 (Vp8-B5 in Fig. 12). At this time, Coxsackievirus B5 was isolated after passage from the cerebrospinal fluid of an infant showing aseptic meningitis. Vero cells were cultured in DMEM (Gibco, cat. No. 12430-054) containing 10% fetal bovine serum, and were seeded in 6-well plates at a concentration of 1 × 10 ⁶ cells/well the day before infection. Prepared by vaccination.

On the day of infection, the virus was washed with saline, diluted 1:100 in DMEM (Gibco, cat. No. 12430-054) containing no fetal bovine serum, and treated with the cells. After treatment, the culture medium was replaced with DMEM containing 2% fetal bovine serum (FBS), and the medium was cultured for 2 to 4 days, and cell lesions were checked.

When morphological deformation was observed in 80-90% of the infected cells, the cells were collected and centrifuged (1200 rpm, 3 minutes), and the cell pellet was frozen and thawed three times to extract the virus from the cells. The extracted virus was stored at -80℃ until the next infection. The above process was repeated eight times.

As a result, compared to wild-type Coxsackievirus B5, it was confirmed that the subcultured Coxsackievirus B5 showed an increased rate of virus replication within infected cells, resulting in faster cell shape deformation.

Example 7.2. Production of mouse-adapted Coxsackievirus B5

The cell-adapted Coxsackievirus B5 obtained by the same method as in Example 7.1 was administered intraperitoneally to 3-4 week-old female BALB/c mice (SAMTACO) at a concentration of 5.5 TCID/200 μl per mouse. The body weight change was observed for 7 days after administration to determine whether the virus caused pathogenicity in the mice. As a result, it was confirmed that the body weight decreased 3-4 days after infection. Four days after infection when the body weight loss was observed, the mice were sacrificed by dislocating the cervical vertebrae, and the intestines were separated. The separated intestines were transferred to a tube, and 500 μl of culture medium was added to homogenize them. The homogenized intestinal tissues were centrifuged at 12,000 rpm for 3 minutes, and the supernatant was collected. The supernatant was used to measure the virus concentration, and other mice were infected using the same method as above. The above infection process was performed a total of 24 times (VP8Mp24-B5-B5 in Figure 12).

Example 7.3. Evaluation of growth rate of cell- and mouse-adapted coxsackievirus B5

In order to confirm the change in the proliferation rate of Coxsackievirus B5 obtained through the same method as in Examples 7.1 and 7.2 above, a medium tissue culture infectious dose (TCID 50) experiment was conducted using Vero cells.

Specifically, the viruses used were cell-adapted Coxsackievirus B5 (VP8-B5), cell- and mouse-adapted Coxsackievirus (VP8M24-B5), and wild-type Coxsackievirus B5 (WT-B5). Vero cells were prepared by inoculating 96-well plates at a concentration of 1 × 10 ⁶ cells/well the day before infection. Each virus was prepared by serially diluting 7 times, 1/10 at a time, from the original solution.

The Vero cells prepared as above were washed with saline, and then treated with the prepared concentrations of viruses (50 ㎕/well) and, after 2 hours, replaced with DMEM medium containing 2% FBS and further cultured. Three days after infection, when 80-90% cell shape deformation was observed, each cell was treated with 10% formalin and reacted for 20 minutes to fix the cells. Then, the cells were stained with 1.25% crystal violet staining reagent for 30 minutes, washed with distilled water, and the cells remaining in each well were observed. In addition, the results were quantified using the Reed-Muench method.

As a result, it was confirmed that the virus titer was significantly improved in the VP8-B5 or VP8M24-B5 treatment groups compared to WT-B5 (Fig. 13).

Example 7.4. Nucleic acid sequence analysis of mouse-adapted coxsackievirus B5

The base sequence of mouse-adapted coxsackievirus 5 obtained by the same method as in Example 7.2 above was analyzed.

Specifically, RNA was extracted from the virus to produce cDNA, and then the entire viral genome was amplified using universal primers (SEQ ID NO: 108, SEQ ID NO: 109). Sanger sequencing was performed using the PCR product obtained through the above process.

As a result, we were able to confirm mutations in the base sequences of the 5' UTR (2 sites) and 2A protease (2 sites) of the nucleic acid sequence of the mouse-adapted coxsackievirus B5 compared to the wild-type coxsackievirus B5 (Table 9 and Figure 14). The mutated sequences are underlined in the table below.

domain order Sequence number 5' UTR DNA TTAAAACAGCCTGTGGGTTGTACCCACCCACAGGGCCCACTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGTGCCTGTTTTATATCCCCTTTCCCCGATTGTAACTTAGAAGATGTACACACTGATCAATAGCGGGCATAGCACACCAGCTATGTCTTGATCAAGCACTCCTGTATCCCCGGACCAAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAACCGGCTAACTACTTCGA GAAACCCAGTAGTATCATGAAAGTTGCGAGGCGTTTCGTTCAGCACACCCCCAGTGTAGATCGGGTCGATGAGTCACCGCATCCCCCACGGGCGACCGTGGCGGTGGCTGCGCTGGCGGCCTGCCTACGG A GCAACCCGTAGGACGCTTCAATACAGACATGGTGCGAAGAGTCGATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATGCCCTCAACCCAGGGGGTATTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCCTTATACTGGCTGCTTATGGTTGACAATTGAAAGATTGTTGCCATATAGCTATTGGATTGGGCCATCCA GTGTCTAATAGAGCTGTTATTTACCTCTTTGTTGGATTTATACCACTCAACCTAAAAGAAGTTAAAACACTACAGCTCATTTTACAACTGAATACAGCAAA 86 RNA UUAAAACAGCCUGUGGGUUGUACCCACCCACAGGGCCCACUGGGCCUAGACUCUGGUAUCACGGUACCUUUGUGUGCCUGUUUUAUAUCCCCUUUCCCCGAUUGUAACUUAGAAGAUGUACACACUGAUCAAUAGCGGGCAUAGCACACCAGCUAUGUCUUGAUCAAGCACUCCUGUAUCCCCGGACCAAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAACGUUCGU U ACCCGGCUA ACUACUUCGAGAAACCCAGUAGUAUCAUGAAAGUUGCGAGGCGUUUCGUUCAGCACACCCCCAGUGUAGAUCGGGUCGAUGAGUCACCGCAUCCCCCACGGGCGACCGUGGCGGUGGCUGCGCUGGCGGCCUGCCUACGG A GCAACCCGUAGGACGCUUCAAUACAGACAUGGUGCGAAGAGUCGAUUGAGCUAGUUAGUAGUCCUCCGGCCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACAUGCCCUCAACCCAGGGGGUAUUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUGUCCGUGUUUCCUUUUAUCCUUAUACUGGCUUGCUUAUGGUGACAAUUGAAAGAUUGUGCCAUAUAG CUAUUGGAUUGGCCAUCCAGUGUCUAAAUAGAGCUGUUAUUUACCUCUUUGUUGGAUUUAUACCACUCAACCUAAAAGAAGUUAAAACACUACAGCUCAUUUUACAACUGAAUACAGCAAA 87 2A ^pro DNA GGTTCATGTGGGCAACAATCAGGAGCTGTGTACGTGGGCAATTACAGGATTGTAAACAGACACCTGGCAACGAGCCACGATTGGCAAAATTGCGTGTGGGAGGACTACAACAGGGATCTCCTGGTCAGCACAACAACTGCTCATGGTTGTGATACTATAGCACGGTGCCAATGTACTGCTGGCGTGTACTTTTGTGCATCTAGAAACAAGCACTACCCAGTGAGCTTCGAGGGTCCAGGTCTAGTGAAAGTTCAGGAG A GCGAGTACTACCCCGAGAG G TACCAGTCGCACGTACTCCTTGCAGTTGGATTTTCAGAACCGGGTGATTGTGGAGGTATTTTGAGGTGTGAACATGGAGTCATTGGGCTCGTTACCATGGGTGGTGAGGGTGTAGTAGGCTTCGCTGACGTGCGGGATCTTTTGTGGTTGGAAGACGACGCCATGGAGCAG 73 RNA GGUUCAUGUGGGCAACAAUCAGGAGCUGUGUAACGGUGGGCAAUUACAGGAUUGUAAACAGACACCUGGCAACGAGCCACGAUUGGCAAAAUUGCGUGUGGGAGGACUACAACAGGGAUCUCCUGGUCAGCACAACAACUGCUCAUGGUUGUGAUACUAUAGCACGGUGCCAAUGUACUGCUGGCGUGUACUUUUGUGCAUCUAGAAACAAGCACUACCCAGUGAGCUUCGAGGGUCCAGGUCUAGUGAAA GUUCAGG A GAGCGAGUACUACCCCGAGAG G UACCAGUCGCACGUACUCCUUGCAGUUGGAUUUUCAGAACCGGGUGAUUGUGGAGGUAUUUUGAGGUGUGAACAUGGAGUCAUUGGCUCGUUACCAUGGGUGGUGAGGGUGUAGUAGGCUUCGCUGACGUGCGGGAUCUUUUGUGGUUGGAAGACGACGCCAUGGAGCAG 74 AA GSCGQQSGAVYVGNYRIVNRHLATSHDWQNCVWEDYNRDLLVSTTTAHGCDTIARCQCTAGVYFCASRNKHYPVSFEGPGLVKVQE S EYYPERYQSHVLLAVGFSEPGDCGGILRCEHGVIGLVTMGGEGVVGFADVRDLLWLEDDAMEQ 75

Therefore, the optimized self-amplifying RNA construct based on the enterovirus of the present invention may include the components of Tables 10 and 11 below (Fig. 15).

Single antigen saRNA Wild type Mutant DNA vector saRNA DNA vector saRNA 5’ UTR Sequence number 1 Sequence number 61 Sequence number 86 Sequence number 87 Cossack sequence Sequence number 35 Sequence number 35 Sequence number 35 Sequence number 35 Target protein GOI GOI GOI GOI 2A protease recognition site Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 2A protease Sequence number 2 Sequence number 61 Sequence number 73 Sequence number 74 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence Sequence number 9 Sequence number 68 Sequence number 9 Sequence number 68 3’ UTR Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40

Multi-antigen saRNA Wild type Variant DNA vector saRNA DNA vector saRNA 5’ UTR Sequence number 1 Sequence number 61 Sequence number 86 Sequence number 87 Cossack sequence Sequence number 35 Sequence number 35 Sequence number 35 Sequence number 35 Target protein 2 GOI GOI GOI GOI 3C protease cleavage site Sequence number 49 Sequence number 57 Sequence number 49 Sequence number 57 Target protein 1 GOI GOI GOI GOI 2A protease cleavage site Sequence number 28 Sequence number 33 Sequence number 28 Sequence number 33 2A protease Sequence number 2 Sequence number 61 Sequence number 73 Sequence number 74 2B Sequence number 3 Sequence number 62 Sequence number 3 Sequence number 62 2C Sequence number 4 Sequence number 63 Sequence number 4 Sequence number 63 3A Sequence number 5 Sequence number 64 Sequence number 5 Sequence number 64 3B Sequence number 6 Sequence number 65 Sequence number 6 Sequence number 65 3C Protease Sequence number 7 Sequence number 66 Sequence number 7 Sequence number 66 3D Sequence number 8 Sequence number 67 Sequence number 8 Sequence number 67 Termination sequence Sequence number 9 Sequence number 68 Sequence number 9 Sequence number 68 3’ UTR Sequence number 10 Sequence number 69 Sequence number 10 Sequence number 69 poly(A) tail Sequence number 40 Sequence number 40 Sequence number 40 Sequence number 40

V. Production of a Canine Influenza Virus Vaccine

Example 8. Design of H3N2 canine influenza antigen expression gene

Figure 17 is a schematic diagram designing a gene for expression of H3N2 canine influenza HA (hemagglutinin). The canine influenza virus vaccine strain currently commercially available in the United States is A/Canine/Illinois/12191/2015. In the present invention, the HA antigen gene of the H3N2 virus was analyzed from 2006 to 2021.

Since 2015, mutations have been continuously occurring in the HA antigen gene of H3N2 viruses. By analyzing the HA antigen gene of H3N2 viruses from 2015 to the present, we created a gene that is most homologous to the HA of the latest circulating H3N2 virus, including mutations.

Figure 18 is a schematic diagram for increasing the antigenicity of H3N2 canine influenza HA. In the HA protein of the influenza virus, gene cleavage occurs between Arg in the H1 unit and Gly in the H2 unit within the host cell, and gene cleavage was suppressed by replacing Arg in the H3N2 homologous gene produced in the present invention with Val. Suppression of gene cleavage induces complete protein expression of HA, and codon optimization was performed to increase the expressibility in the target animal, the dog.

Example 9. Schematic diagram of the H3N2 canine influenza self-amplifying RNA structure.

FIG. 19a is a schematic diagram of the structure of a wild type Enterovirus. In addition, FIG. 19b is a schematic diagram of the structure of an Enterovirus-based self-amplifying RNA construct (saRNA, self-amplifying RNA). The clone vector used in the present invention is a pUC19-CMV/T7 (Addgene, plasmid #50005) vector that contains the Coxsackievirus B5 gene (SEQ ID NO: 99) as described in "Development of a Universal Cloning System for Reverse Genetics of Human Enteroviruses" (WS Choi, et al., Microbiol Spectr. 18;e0316722) was loaded in the same manner as described in the previous publication to produce an infectious clone. Using the In-fusion cloning (In-Fusion®HD Cloning Kit, Takara) method, the P1 domain (structural protein, VP) of the clone was replaced with the target protein, canine influenza virus antigen (H3N2), to produce a clone vector in the form of a saRNA construct.

Example 10. Confirmation of in vitro antigen expression of H3N2 canine influenza self-amplifying RNA

The expression level of the saRNA construct was confirmed at the cellular level through Western blot (Fig. 20). At this time, the efficiency of the cell delivery system (LNP biodelivery system, lipofectamine) using RNA containing the H3N2 canine influenza virus antigen was also confirmed.

First, to prepare the LNP-RNA experimental group, RNA (RNA containing H3N2 antigen) was encapsulated using the LNP biodelivery system (ALC-0315/Cholesterol application/PEG/DOPE) in NanoAssemblr®Ignite (Precision NanoSystems). The encapsulated RNA (LNP-RNA) was diluted 10-fold in 1ХTE (Tris-EDTA) buffer containing 2% (v/v) Triton X-100 (Sigma Aldrich), and the fluorescence of LNP-RNA was measured at wavelengths of 485 nm (excitation) and 528 nm (emission) using a microplate reader (BMG LABTECH, UK). At this time, the RNA concentration corresponding to the fluorescence value was also measured.

As prepared above, LNP-RNA or each RNA was treated and transfected into cells at various concentrations. At this time, each RNA was transfected into 293T cells with the saRNA construct produced using Lipofectamine®2000 Transfection Reagent (Invitrogen) to confirm the expression level. Specifically, 293T cells were seeded at 1Х10 ⁶ cells/well in a 6-well plate, cultured overnight, and then 4 hours before transfection, the cell medium was replaced with a medium not supplemented with fetal bovine serum (FBS) to prepare the cells.

The medium was replaced 4 hours after transfection, and the culture was continued for an additional 24 hours. After 24 hours, the cells were harvested by centrifugation (12,000 rpm, 1 minute), and Cell Lysis buffer was added (1 mL) and vortexed to lyse the cells. After incubation at room temperature for 10 minutes, the protein concentration was measured, and 10 ㎍ of protein was mixed with 2Х sample buffer (SDS sample buffer having the same volume as the protein) and incubated at 95°C for 7 minutes to prepare protein samples. Each protein sample was electrophoresed on SDS-PAGE, transferred to a membrane, blocked with 5% lipoprotein solution, and then primary antibodies were added and reacted at 4°C for 16 hours. The primary antibody, anti-H3N2 antibody, was a self-produced antibody diluted 1:2,000 and used.

At this time, anti-H3N2 antibodies were produced using an acellular vaccine by the following method. First, 50 ㎕ of purified H3N2 hemagglutinin protein (Mybiosource, 32-5658) at a concentration of 0.1 ㎍/㎕ and adjuvant (AddaVax, Invivogen) were mixed in a 1:1 ratio and administered intramuscularly to mice. After the first administration, three additional vaccinations were administered at two-week intervals, and antibodies were produced by directly separating from the blood.

After the primary antibody reaction, the membrane was washed five times for 10 minutes each with TBS-T buffer, and then the secondary antibody was added and reacted at room temperature for 1 to 2 hours. Secondary anti-mouse-HRP antibody (Invitrogen, 62-6520, 1:5000) was used. After the secondary antibody reaction, each membrane was washed five times for 10 minutes each with TBS-T, and then detected using HRP reagent (Millipore).

As a result, as shown in Fig. 20, RNA transfected using LNP showed a higher expression rate than RNA transfected using lipofectamine.

Example 11. H3N2 canine influenza self-amplifying RNA-LNP target animal inoculation experiment

The H3N2 canine influenza self-amplifying RNA-LNP produced by the method described in Example 10 was vaccinated into puppies. Two puppies were vaccinated with 25 ug of each concentration, three puppies with 10 ug, and four puppies with 5 ug by intramuscular administration. As a control group, two puppies were vaccinated with a vaccine commercially available from the P.C. Central Vaccine Research Institute (Fluvax H3N2, Central Vaccine Research Institute), and one pup was vaccinated with PBS containing no N.C antigen (Fig. 21).

Serum was obtained by directly isolating from the blood of dogs in the 2nd and 4th weeks after the 1st vaccination, and in the 4th week, the second vaccination was performed after producing self-amplified RNP-LNP using the method described in Example 10 above. Serum was obtained by directly isolating from the blood of dogs in the 2nd week (total of 6 weeks) after the 2nd vaccination, and then neutralizing antibody formation was evaluated.

Example 12. Evaluation of neutralizing antibody formation in animals using H3N2 canine influenza self-amplifying RNA-LNP

As described in Example 11 above, the sera from weeks 2, 4, and 6 were inactivated by reacting at 56°C for 1 hour, and then neutralizing antibody formation was evaluated. The neutralizing antibody formation evaluation was conducted using two test methods: a blood cell agglutination inhibition reaction and an experiment using cells.

For the hemagglutination inhibition reaction test, the sera separated from a 96-well round-bottom plate were serially diluted 11 times, each 1/10, in a medium not containing fetal bovine serum (FBS), and the canine influenza virus (A/canine/Korea/AS-06/2012) was subjected to a hemagglutination reaction to adjust the HAU to 4-8 (HA unit), and the virus was reacted for 30 minutes at room temperature. After the reaction, 0.5% turkey red blood cell dilution was added in a 1:1 ratio and the reaction was performed for 30 minutes at room temperature to confirm the neutralizing antibody value. If the target animal has formed neutralizing antibodies, the antibodies bind to the virus and inhibit the binding of the virus to the red blood cells. The method using cells (serum neutralization test) is a method in which the sera are diluted stepwise in the same way as above, reacted with the virus, and then infected with MDCK cells to confirm.

Specifically, MDCK (Madin-Darby canine kidney cells) were seeded at 1Х10 ⁶ cells/well in a 96-well plate, cultured overnight at 37°C, and washed twice with PBS before use. The virus was diluted to TCID 100 using canine influenza virus (A/canine/Korea/AS-06/2012), added 1:1 with diluted serum, reacted at 37°C for 1 hour, and infected with MDCK cells prepared the previous day at 37°C for 1 hour. Culture medium without fetal bovine serum (FBS) and TPCK treated trypsin were diluted 1X and prepared, and replaced with the prepared culture medium 1 hour after infection. The cytopathic effect was confirmed 48 hours after infection, and a hemagglutination reaction test was performed to confirm the concentration of neutralized antibodies. Red blood cells were isolated from Turkish blood and diluted to a concentration of 0.5% in PBS for use in the experiment.

As a result, it was confirmed that neutralizing antibodies were formed in all experimental groups that completed the second vaccination (25ug, 10ug, 5ug), and it was confirmed that neutralizing antibodies were formed with only the first vaccine. In addition, a higher neutralizing antibody formation value was confirmed than that of the central vaccine protein vaccine, a control group sold on the market (Fig. 22a to Fig. 22c).

Attach electronic file of sequence list

Claims (24)

Non-structural protein of self-amplifying virus;
Canine influenza virus H3N2 variant protein; and
A polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a protease cleavage site. In the first paragraph,
A polynucleotide having the amino acid sequence of sequence number 109, wherein the above-mentioned influenza virus H3N2 variant protein. In the first paragraph,
The above nonstructural protein is a polynucleotide derived from a nonstructural protein of a Picornaviridae virus. In the third paragraph,
A polynucleotide wherein the nonstructural protein comprises a P2 domain and a P3 domain of Enterovirus. In paragraph 4,
A polynucleotide wherein the above P2 domain comprises 2A protease, 2B and 2C. In paragraph 5,
The above 2A protease comprises a base sequence encoding an amino acid sequence of sequence number 11,
2B contains a base sequence encoding the amino acid sequence of sequence number 12,
2C is a polynucleotide comprising a base sequence encoding an amino acid sequence of sequence number 13. In paragraph 5,
A polynucleotide comprising the above 2A protease mutation. In Article 7,
The above mutation is a polynucleotide in which the amino acid at position 87 in the amino acid sequence of sequence number 11 is substituted. In the third paragraph,
A polynucleotide wherein the above P3 domain comprises 3A, 3B, 3C protease and 3D polymerase. In Article 9,
The above 3A contains a base sequence encoding an amino acid sequence of sequence number 14,
3B contains a base sequence encoding the amino acid sequence of sequence number 15,
3C protease comprises a base sequence encoding the amino acid sequence of sequence number 16,
A polynucleotide comprising a base sequence encoding an amino acid sequence of SEQ ID NO: 17. In the first paragraph,
A polynucleotide wherein the protease cleavage site is cleaved by a protease derived from a nonstructural protein of a self-amplifying virus. In Article 11,
A polynucleotide wherein the protease cleavage site is cleaved by 2A protease of enterovirus. In Article 12.
A polynucleotide wherein the protease cleavage site comprises a base sequence encoding an amino acid sequence of SEQ ID NO: 22. In the first paragraph,
A polynucleotide, wherein the polynucleotide further comprises at least one stop codon at the 3' end of a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus. In Article 14,
A polynucleotide wherein the termination codon comprises a base sequence of SEQ ID NO: 9 or SEQ ID NO: 68. In the first paragraph,
A polynucleotide, wherein the polynucleotide additionally comprises a 5' untranslated region (5' UTR) at the 5' end. In Article 16,
A polynucleotide wherein the above 5' untranslated region comprises an internal ribosomal entry site (IRES). In Article 17,
A polynucleotide wherein the above IRES comprises a base sequence of SEQ ID NO: 58 or SEQ ID NO: 59. In the first paragraph,
A polynucleotide further comprising a non-translated region (3' UTR) at the 3' end of the polynucleotide. In the first paragraph,
A polynucleotide, wherein the polynucleotide further comprises a poly(A) tail sequence. In the first paragraph,
The above polynucleotide is a polynucleotide having the following structural formula (I) in order from the 5' end to the 3' end:
5'-BC(1)-D-3' (I)
5' and 3' are the 5' and 3' ends of the polynucleotide, respectively,
B is a nucleic acid sequence encoding a canine influenza virus H3N2 variant protein,
C(1) is a nucleic acid sequence encoding a protease cleavage site,
D is a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus. In the first paragraph,
A polynucleotide, wherein the polynucleotide is DNA or RNA. A recombinant vector comprising the polynucleotide of claim 1. A vaccine composition comprising the polynucleotide of claim 1 or the recombinant vector of claim 23 as an active ingredient.