Self-amplifying RNA constructs and uses thereof

Abstract

The present invention relates to a self-amplifying RNA construct by which the expression of a target protein is optimized. Compared with traditional mRNA and self-amplification RNA based on the enterovirus, the optimized self-amplification RNA based on the enterovirus has the advantage that the expression level of the target protein is improved. In addition, the multi-antigen self-amplification RNA can simultaneously express one or more target proteins, and the expression rate of the multi-antigen self-amplification RNA is far higher than that of traditional mRNA. Therefore, the self-amplifying RNA constructs according to the present invention can be widely used in vaccines and the like.

Description

Technical Field

[0001] The present invention relates to a self-amplifying RNA construct and use thereof. Background Art

[0002] Vaccines play a vital role in health worldwide by preventing infection and transmission of various diseases. The World Health Organization estimates that vaccines prevent 2 to 3 million deaths annually. Traditional vaccines, such as attenuated pathogens, inactivated pathogens, and subunit vaccines, have prevented a wide range of fatal diseases. However, the rapid development and large-scale deployment of vaccines against various infectious pathogens that evade the adaptive immune response remain challenging.

[0003] On the other hand, nucleic acid therapy has attracted attention as an alternative that can overcome the above problems (Vogel et al., Clin Microbiol Rev., 8(3):406-410 (1995). In particular, mRNA-based RNA vaccines are safe because they are not integrated into the genome like some viral vaccines, so there is no problem of mutation, and they can be manufactured in a cell-free manner, allowing for economical, rapid and efficient production of vaccines. In addition, the advantage of a single mRNA vaccine is that it can encode multiple antigens, thereby enhancing the immune response to pathogens, and can evade the body's immune response to the carrier.

[0004] Among them, the construct of self-amplifying RNA (saRNA) vaccine has RNA polymerase activity in the non-structural protein and encodes the structural protein by replacing the structural protein with the antigen protein (target gene, GOI). Therefore, the advantage of self-amplifying RNA vaccine is that only one vaccination is needed to fully produce antibodies, and it is currently being studied as a vaccine construct. In fact, even if it is administered at a dose of about 1 / 100 to about 1 / 10 of the mRNA dose, the saRNA construct can induce an immune response, and it has been demonstrated that the expression of the antigen protein can be maintained for up to 60 days after administration to mice.

[0005] Most self-amplifying RNA vaccines currently in use are based on alphavirus genomes (Bogers et al., J Infect Dis., 211(6):947-955 (2015)). In addition, some self-amplifying RNA viruses contain RNA polymerases within the nonstructural proteins of their genomes, such as picornaviruses, flaviviruses, and coronaviruses. However, except for alphaviruses, these other viruses have not yet been commercialized and further research is needed. Summary of the Invention

[0006] Technical issues

[0007] Therefore, the present inventor is committed to developing the self-amplification RNA vaccine construct that can be used quickly and efficiently.The result is, by modifying 5 ' untranslated region (UTR), protease cleavage site, stop codon and poly (A) tail sequence of the self-amplification RNA construct based on enterovirus, construct the new self-amplification RNA construct that is optimized for antigen protein expression.In addition, it is confirmed that compared with conventional mRNA and conventional self-amplification RNA construct based on enterovirus, the modified self-amplification RNA construct can significantly improve the expression of antigen protein in cell.Based on the above, the present inventor has completed the present invention.

[0008] Solutions to technical problems

[0009] To achieve the above objectives, in one aspect of the present invention, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein is provided, wherein the polynucleotide comprises: a non-structural protein of a self-amplifying virus; a first target protein; and a protease cleavage site.

[0010] In another aspect of the present invention, a recombinant vector carrying the polynucleotide is provided.

[0011] In another aspect of the present invention, a method for producing an RNA replicon is provided, comprising: producing the recombinant vector; and synthesizing RNA from the recombinant vector.

[0012] In another aspect of the present invention, a vaccine composition is provided, which comprises the polynucleotide or the recombinant vector as an active ingredient.

[0013] In another aspect of the present invention, a 2A protease cleavage site comprising the amino acid sequence of SEQ ID NO:22; a 3C protease cleavage site comprising the amino acid sequence of SEQ ID NO:50; an IRES comprising the nucleic acid sequence of SEQ ID NO:81 or SEQ ID NO:82; a 5' untranslated region comprising the nucleic acid sequence of SEQ ID NO:86 or SEQ ID NO:87; a Kozak sequence comprising the nucleic acid sequence of SEQ ID NO:35; a 2A protease comprising the amino acid sequence of SEQ ID NO:75; a severe fever with thrombocytopenia syndrome virus antigen comprising the amino acid sequence of SEQ ID NO:45, SEQ ID NO:85, SEQ ID NO:116 or SEQ ID NO:119; and a human papillomavirus (HPV) antigen comprising the amino acid sequence of SEQ ID NO:139, SEQ ID NO:142, SEQ ID NO:145 or SEQ ID NO:148.

[0014] In another aspect of the present invention, a method for preventing or treating a disease is provided, comprising administering the polynucleotide, the recombinant vector, or a vaccine composition comprising the same to a subject.

[0015] Effects of the Invention

[0016] Compared with traditional mRNA and enterovirus-based self-amplification RNA constructs, the optimized self-amplification RNA construct based on enterovirus according to the present invention improves the expression level of target protein. In addition, this multi-antigen self-amplification RNA construct can express one or more target proteins simultaneously, and its expression level is much higher than the expression level of traditional mRNA. Therefore, the self-amplification RNA construct according to the present invention can be widely used in vaccines, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1a Schematic diagram of the RNA structure of wild-type enterovirus.

[0018] Figure 1b Schematic diagram of the structure of an enterovirus-based self-amplifying RNA construct (saRNA).

[0019] Figure 2a is a diagram showing the amino acid sequence of a 2A protease cleavage site included in an enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention.

[0020] Figure 2b This graph shows the results of confirming the expression level of a target protein using a luciferase system based on the amino acid sequence of the 2A protease cleavage site included in an enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significantly different.

[0021] Figure 3a is a schematic diagram of the structure of an enterovirus-based self-amplifying RNA construct into which a Kozak sequence is inserted according to one embodiment of the present invention.

[0022] Figure 3b This is a graph showing the results obtained by confirming the expression level of a target protein using a luciferase system according to an inserted sequence in an enterovirus-based self-amplifying RNA construct into which a Kozak sequence is inserted according to one embodiment of the present invention. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0023] Figure 4ais a schematic diagram of the structure of an enterovirus-based self-amplifying RNA construct into which a polyadenylic acid tail (poly(A) tail) is inserted according to one embodiment of the present invention.

[0024] Figure 4b Graphs showing the results obtained by confirming the expression level of a target protein at the cellular (bottom) and animal (top) levels using a luciferase system according to each inserted sequence in an enterovirus-based self-amplifying RNA construct into which a polyadenylic acid tail (poly(A) tail) is inserted according to one embodiment of the present invention. *p<0.05.

[0025] Figure 5 Schematic diagram comparing the structures of an mRNA, an enterovirus-based self-amplifying RNA construct, and a self-amplifying RNA (modified saRNA) construct optimized by modifying the enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention.

[0026] Figure 6 This is a graph showing the results obtained by confirming the expression levels of target proteins at the cellular level using a luciferase system using mRNA, an enterovirus-based self-amplifying RNA (sa luci) construct, and a modified self-amplifying RNA construct according to one embodiment of the present invention. **p<0.01, ***p<0.001, ****p<0.0001.

[0027] Figure 7 is a graph showing the results obtained by confirming the expression level of a target protein at the animal level by using a luciferase system by mRNA, enterovirus-based self-amplifying RNA (sa luci) construct, and modified self-amplifying RNA construct according to one embodiment of the present invention.

[0028] Figure 8 This figure shows the results of Western blotting, confirming the cellular expression levels of antigenic proteins from a modified, self-amplifying RNA construct containing a severe fever with thrombocytopenia syndrome (SFTS) virus antigen as a target protein, used in one embodiment of the present invention. SaB: Self-amplifying RNA construct containing the SFTS consensus B antigen; SaABDEF: Self-amplifying RNA construct containing the SFTS consensus ABDEF antigen; mRNAB: mRNA containing the SFTS consensus B antigen.

[0029] Figure 9The figure shows the results obtained by Western blotting to confirm the antigen protein expression level at the cellular level of a modified self-amplifying RNA construct containing a coronavirus (SARS-CoV2) spike protein antigen (top) or a highly pathogenic avian influenza (HPAI) virus antigen (H5N8, bottom) as the target protein used in one embodiment of the present invention.

[0030] Figure 10 is a schematic diagram of the structure of a modified self-amplifying RNA construct comprising multiple antigens according to one embodiment of the present invention.

[0031] Figure 11 A schematic diagram showing a modified self-amplifying RNA construct containing two types of canine influenza virus antigens (H3N2 and H3N8) as target proteins used in one embodiment of the present invention (top); and Western blotting results confirming the expression levels of the antigen proteins in cells (bottom). Ma saRNA: modified self-amplifying RNA construct containing H3N2 and H3N8, single saRNA: modified self-amplifying RNA construct containing H3N2, PC WT virus: wild-type canine influenza virus.

[0032] Figure 12 Schematic diagram of the method for generating cell- and mouse-adapted coxsackievirus B5.

[0033] Figure 13 Figures (left) and (right) show the results obtained by measuring and comparing the titers of wild-type (WT-B5), cell-adapted (Vp8-B5), and cell- and mouse-adapted (Vp8Mp24-B5) coxsackievirus B5. ***p<0.001.

[0034] Figure 14 is a schematic diagram of the RNA structure of Coxsackievirus B5 with increased proliferation rate according to one embodiment of the present invention.

[0035] Figure 15 is a schematic diagram of the structure of an optimized enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention.

[0036] Figure 16 is a schematic diagram of the structure of the enterovirus-based self-amplifying RNA construct cloning vector used in the present invention.

[0037] Figure 17 This is a diagram showing an animal experiment schedule for confirming the antiviral effect of a modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen as a target protein, used in one embodiment of the present invention, against highly pathogenic influenza viruses.

[0038] Figure 18 The graph shows the results obtained by extracting the spleen of mice after administering modified self-amplified RNA or mRNA containing a highly pathogenic influenza virus antigen target protein used in one embodiment of the present invention and confirming the production of neutralizing antibodies (top) and T cell responses (bottom).

[0039] Figure 19 This is a graph showing the results obtained by confirming changes in mouse body weight and survival rate after mice were infected with highly pathogenic influenza virus, and the mice were administered modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen target protein used in one embodiment of the present invention.

[0040] Figure 20a This is a graph showing the results obtained by extracting organs from mice after infection with highly pathogenic influenza virus (day 3) and measuring the virus titer remaining in each organ. The mice were administered modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen target protein, which is used in one embodiment of the present invention.

[0041] Figure 20b This is a graph showing the results obtained by extracting organs from mice after infection with highly pathogenic influenza virus (day 5) and measuring the virus titer remaining in each organ. The mice were administered modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen target protein, which is used in one embodiment of the present invention.

[0042] Figure 21a This is a diagram showing an animal experiment schedule for confirming the neutralizing antibody-producing ability of a modified self-amplifying RNA or mRNA containing severe fever with thrombocytopenia syndrome virus antigen (SFTS) as a target protein used in one embodiment of the present invention.

[0043] Figure 21b This figure shows the results of immunofluorescence staining to confirm whether neutralizing antibodies are produced in the blood of mice (BALB / c mice) after administering modified self-amplifying RNA containing severe fever with thrombocytopenia syndrome virus antigens (A antigen, B antigen, DEF antigen, ABDEF antigen) as target proteins used in one embodiment of the present invention.

[0044] Figure 21cThis is a graph showing the results obtained by confirming whether neutralizing antibodies are produced in the blood of mice (C57BL6 mice) after administering to mice a modified self-amplifying RNA (B antigen, ABDEF antigen) or mRNA (B antigen) containing the severe fever with thrombocytopenia syndrome virus antigen (SFTS) as the target protein used in one embodiment of the present invention.

[0045] Figure 22a It is a schematic diagram showing the timeline of animal experiments (ferrets) used to confirm the neutralizing antibody-producing ability of modified self-amplifying RNA containing severe fever with thrombocytopenia syndrome virus antigens (A antigen, B antigen, DEF antigen, ABDEF antigen) as target proteins used in one embodiment of the present invention.

[0046] Figure 22b This is a graph showing the results obtained by confirming whether neutralizing antibodies are produced in the blood of ferrets 3 weeks after the first administration of modified self-amplified RNA containing severe fever with thrombocytopenia syndrome virus antigens (A antigen, B antigen, DEF antigen, ABDEF antigen) as target proteins used in one embodiment of the present invention.

[0047] Figure 22c The graph shows the results obtained by confirming whether neutralizing antibodies are produced in the blood of ferrets 3 weeks after the second administration of modified self-amplified RNA containing severe fever with thrombocytopenia syndrome virus antigens (A antigen, B antigen, DEF antigen, ABDEF antigen) as target proteins used in one embodiment of the present invention.

[0048] Figure 22d The graph shows the results obtained by confirming whether neutralizing antibodies are produced in the blood of ferrets 3 weeks after the third administration of modified self-amplified RNA containing severe fever with thrombocytopenia syndrome virus antigens (A antigen, B antigen, DEF antigen, ABDEF antigen) as target proteins used in one embodiment of the present invention.

[0049] Figure 23 This figure shows the results of confirming the expression level of the modified self-amplified RNA containing a canine influenza virus antigen (H3N2 or H3N8) as a target protein used in one embodiment of the present invention by Western blotting, based on the concentration at the cellular level.

[0050] Figure 24This is a diagram showing an animal experiment schedule for confirming the neutralizing antibody-producing ability of a modified single-antigen self-amplifying RNA (H3N2 or H3N8) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein used in one embodiment of the present invention.

[0051] Figure 25a This is a graph showing the results of analyzing the production of neutralizing antibodies in mouse blood 4 weeks and 6 weeks (2 weeks after the second administration) after administration of modified single-antigen self-amplified RNA (H3N2 or H3N8) or multi-antigen self-amplified RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein used in one embodiment of the present invention to mice, by confirming binding to inactivated canine influenza virus H3N2.

[0052] Figure 25b This is a graph showing the results of analyzing the production of neutralizing antibodies in the blood of mice 4 weeks and 6 weeks (2 weeks after the second administration) after administration of modified single-antigen self-amplified RNA (H3N2 or H3N8) or multi-antigen self-amplified RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein used in one embodiment of the present invention to mice, by confirming binding to inactivated canine influenza virus H3N8.

[0053] Figure 26 This is a diagram showing an animal experiment schedule for evaluating antiviral activity against canine influenza after administering modified single-antigen self-amplifying RNA (H3N2 or H3N8) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein used in one embodiment of the present invention to mice, and then infecting the mice with H3N2 or H3N8 virus at 4 weeks and 6 weeks (2 weeks after the second administration).

[0054] Figure 27a This is a graph showing the results obtained by administering modified single-antigen self-amplifying RNA (H3N2) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein, used in one embodiment of the present invention, to mice, and then infecting the mice with canine influenza virus H3N2 at 4 weeks, and then examining changes in mouse body weight and survival rate.

[0055] Figure 27b This is a graph showing the results obtained by administering to mice a modified single-antigen self-amplifying RNA (H3N8) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein used in one embodiment of the present invention, and then infecting the mice with canine influenza virus H3N8 at 4 weeks, and then examining changes in mouse body weight and survival rate.

[0056] Figure 27c This is a graph showing the results obtained by administering to mice a modified single-antigen self-amplifying RNA (H3N2) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein used in one embodiment of the present invention, and then confirming changes in mouse body weight and survival rate after infecting the mice with canine influenza virus H3N2 6 weeks (2 weeks after the second administration).

[0057] Figure 27d This is a graph showing the results obtained by administering to mice a modified single-antigen self-amplifying RNA (H3N8) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein used in one embodiment of the present invention, and then infecting the mice with canine influenza virus H3N8 6 weeks later (2 weeks after the second administration), and then confirming changes in mouse body weight and survival rate.

[0058] Figure 28a This is a graph showing the results obtained by administering to mice a modified single-antigen self-amplifying RNA (H3N2) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein, used in one embodiment of the present invention, and then infecting the mice with the H3N2 or H3N8 virus, and measuring the virus titer remaining in the lung tissue of the mice on day 3.

[0059] Figure 28b This is a graph showing the results obtained by administering to mice a modified single-antigen self-amplifying RNA (H3N2) or multi-antigen self-amplifying RNA (H3N2 and H3N8) containing a canine influenza virus antigen as a target protein, used in one embodiment of the present invention, and then measuring the virus titer remaining in the lung tissue of the mice on the 5th day after infecting the mice with the H3N2 or H3N8 virus.

[0060] Figure 29a It is a schematic diagram of human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, alternating arrangement of E6 / E7, and even-odd arrangement of E6 / E7).

[0061] Figure 29b Schematic diagram of a modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, cross arrangement of E6 / E7, and even-odd arrangement of E6 / E7) as target proteins. G1 E6, E7 protein linker: E6 / E7 domains, G2 continuous: sequential arrangement of E6 / E7, G3 cross: cross arrangement of E6 / E7, G4 odd-even: odd-even arrangement of E6 / E7.

[0062] Figure 29c The figure shows the results obtained by confirming the expression of modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, alternating arrangement of E6 / E7, and odd-even arrangement of E6 / E7) as target proteins at the cellular level by Western blotting.

[0063] Figure 30 This is a diagram showing an animal experiment schedule for confirming the cancer prevention activity of modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, alternating arrangement of E6 / E7, and even-odd arrangement of E6 / E7) as target proteins.

[0064] Figure 31 This is a graph showing the results obtained by confirming T cell responses in mouse spleens 4 weeks after administration of modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, cross arrangement of E6 / E7, and even-odd arrangement of E6 / E7) as target proteins (2 weeks after the second administration).

[0065] Figure 32 The present invention is a graph showing the results obtained by measuring the tumor size 5 weeks after administering modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, cross arrangement of E6 / E7, and odd-even arrangement of E6 / E7) as target proteins to mice, and then transplanting TC-1 tumor cells, which express the E6 and E7 genes of human papillomavirus (type 16), into the mice (3 weeks after the second administration).

[0066] Figure 33 It is a diagram showing the animal experiment schedule for confirming the tumor growth inhibitory activity of modified self-amplifying RNA (4 types) containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, cross arrangement of E6 / E7, and odd-even arrangement of E6 / E7) as target proteins.

[0067] Figure 34 This graph shows the results of measuring tumor size after TC-1 tumor cells expressing the E6 and E7 genes of human papillomavirus (type 16) were transplanted into mice and then administered modified self-amplifying RNA (four types) containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, alternating arrangement of E6 / E7, and even-odd arrangement of E6 / E7) as target proteins on day 3. PBS served as a control group.

[0068] Figure 35 This graph shows the results of measuring tumor size according to the administration group after TC-1 tumor cells expressing the E6 and E7 genes of human papillomavirus (type 16) were transplanted into mice and then administered modified self-amplifying RNA (four types) containing human papillomavirus (HPV) antigens (E6 / E7 domains, sequential arrangement of E6 / E7, alternating arrangement of E6 / E7, and even-odd arrangement of E6 / E7) as target proteins on day 3. PBS served as a control group.

[0069] Figure 36a This graph shows the results of measuring tumor size in mice transplanted with TC-1 tumor cells expressing the E6 and E7 genes of human papillomavirus (HPV) type 16 (E6 / E7 domains) and then administered with modified self-amplifying RNA (G1) containing the target protein (E6 / E7 domains) and PBS on day 3. G1: Protein GGGGS.

[0070] Figure 36b This graph shows the results of measuring tumor size in mice transplanted with TC-1 tumor cells expressing the E6 and E7 genes of human papillomavirus (HPV) type 16 (E6 / E7) genes, followed by administration of modified self-amplifying RNA (G2) containing target proteins and PBS on day 3. G2: Continuous.

[0071] Figure 36c This graph shows the results of measuring tumor size in mice transplanted with TC-1 tumor cells and then administered with a modified self-amplifying RNA (G3) containing a human papillomavirus (HPV) antigen (cross-array of E6 / E7) as a target protein and PBS on day 3. G3: Cross-array.

[0072] Figure 36d This graph shows the results of measuring tumor size in mice transplanted with TC-1 tumor cells and then administered with a modified self-amplifying RNA (G4) containing human papillomavirus (HPV) antigens (E6 / E7 odd-even arrangement) as target proteins and PBS on day 3. G4: odd-even.

[0073] Figure 37It is a graph showing the results obtained by confirming the target protein expression level of a multi-antigen self-amplifying RNA construct (first target protein: luciferase, second target protein: canine influenza H3N2 virus antigen) or a single-antigen self-amplifying RNA construct (target protein: luciferase) according to one embodiment of the present invention using a luciferase system.

[0074] Figure 38 was shown by Western blotting, confirming Figure 37 Graph showing the results obtained from the expression levels of target proteins of the multi-antigen self-amplified RNA constructs. NC: negative control, PC: positive control (wild-type H3N2 virus) DETAILED DESCRIPTION

[0075] Best Mode for Carrying Out the Invention

[0076] Polynucleotide constructs

[0077] Nonstructural proteins

[0078] In one aspect of the present invention, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein is provided, comprising: a non-structural protein of a self-amplifying virus; a first target protein; and a protease cleavage site. In this case, the polynucleotide may be DNA or RNA. In addition, the self-amplifying virus may be a virus of the Picornaviridae family. Specifically, the self-amplifying virus may be a virus of the genus Enterovirus. More specifically, in the present invention, the non-structural protein may be a non-structural protein derived from an enterovirus. Preferably, it may be a non-structural protein derived from a Coxsackievirus.

[0079] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleotide sequence in which the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. A polynucleotide can be composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0080] In this specification, polynucleotides include DNA and RNA and can be produced synthetically in vitro or isolated from natural sources. The size of a polynucleotide is typically expressed in base pairs (bp) for double-stranded polynucleotides or in nucleotides (nt) for single-stranded polynucleotides.

[0081] In the present specification, a nucleic acid sequence is used in the same sense as a polynucleotide, and can be DNA or RNA. At this time, the RNA can include mRNA. Furthermore, the nucleic acid sequence can also include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has undergone post-translational modification, 3' processing such as cleavage and polyadenylation, and splicing. The nucleic acid can also include synthetic nucleic acids (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).

[0082] As used herein, the term "enterovirus" refers to a virus belonging to the Enterovirus genus of the Picornaviridae family, and has a size of about 24 nm to about 30 nm, without an envelope. It has an icosahedral shape, and contains a single-stranded positive-sense RNA of about 7.2 kb to about 7.5 kb in size as its genetic material. The enterovirus consists of three serotypes of poliovirus (PV: 1-3), 23 serotypes of coxsackievirus A group (CVA: 1-22, 24), six serotypes of coxsackievirus B group (CVB: 1-6), 28 serotypes of echovirus group (ECV: 1-7, 9, 11-21, 24-27, 29-33), and other human enteroviruses (EV: 68-116).

[0083] The enterovirus enters a host cell, releases the RNA into the cytoplasm, and then induces the production of a large polyprotein by initiating translation as mRNA itself at the IRES (internal ribosome entry site) of the 5' untranslated region (UTR). The genetic material of the enterovirus consists of one open reading frame (ORF) and untranslated regions (UTRs) or non-coding regions (NCRs) that are not expressed as proteins at the 5' and 3' ends. The ORF accounts for about 90% of the entire gene, and is expressed as one polyprotein. The polyprotein consists of about 2185 amino acids, and is divided into several different proteins by a viral protease.

[0084] The above-described one polyprotein is divided into a P1 domain, a P2 domain, and a P3 domain, and the P1 domain is referred to as a structural protein, and the P2 and P3 domains are referred to as non-structural proteins. The P1 domain of the above-described polyprotein is a capsid protein of the virus, and encodes components VP4, VP2, VP3, and VP1 in order from the N-terminus to the C-terminus, and the capsid is composed of a 32-mer capsid body. 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). 2A protease and 3C protease are proteolytic enzymes that recognize and cleave cleavage sites within the viral polyprotein, processing it into individual proteins. Meanwhile, 3D polymerase is an RNA-dependent RNA polymerase (RdRp) that uses RNA as a template to synthesize complementary RNA during viral RNA self-replication.

[0085] In the present invention, the enterovirus may be 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.

[0086] In the present invention, the nonstructural protein derived from enterovirus may include a P2 domain and a P3 domain. In this case, the nonstructural protein may include the P2 domain and the P3 domain in order from the N-terminus to the C-terminus.

[0087] In the present invention, the P2 domain may comprise 2A protease, 2B, and 2C. Specifically, the P2 domain of the present invention may comprise 2A protease, 2B, and 2C of Coxsackievirus B5. In this case, the P2 domain may comprise 2A protease, 2B, and 2C in sequence from the N-terminus to the C-terminus. In one embodiment, 2A protease, 2B, and 2C may comprise the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. Therefore, in the present invention, the polynucleotide may comprise, or consist of, nucleic acid sequences encoding the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, from the 5' end to the 3' end. In one embodiment of the present invention, when the polynucleotide is DNA, it may comprise or consist of the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, from the 5' end to the 3' end. When the polynucleotide is RNA, it may comprise or consist of the nucleotide sequences of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively, from the 5' end to the 3' end.

[0088] In addition, in the present invention, 2A protease, 2B and 2C can be composed of an amino acid sequence in which one or more amino acids are deleted, substituted and / or inserted, as long as they have the same biological function. Specifically, 2A protease, 2B and 2C can respectively 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 sequence of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. In this case, the deletion, substitution and / or insertion of amino acids may not cause changes in the properties of the original protein, such as secondary structure and activity.

[0089] In the present invention, 2A protease may further include mutations.

[0090] As used herein, the term "variant" refers to a form in which an amino acid or a portion of a polynucleotide encoding an amino acid is substituted compared to a wild type. In the present invention, a variant may refer to a protein or nucleic acid (polynucleotide) comprising the variation.

[0091] That is, in the present invention, a 2A protease variant may have an amino acid sequence that differs from that of the wild-type 2A protease. However, the 2A protease variant may have activity comparable to or similar to that of the wild-type 2A protease, or may have further enhanced activity. Compared to the wild-type, the 2A protease variant may have increased protein expression levels. Here, "2A protease activity" may refer to the ability to specifically recognize and cleave a cleavage site within the amino acid sequence.

[0092] Specifically, the 2A protease variant may be a form in which a portion of the amino acids of the wild-type 2A protease are substituted. A specific example of a 2A protease variant obtained by amino acid substitution may be a variant in which the amino acid at position 87 in the amino acid sequence of SEQ ID NO: 11 is substituted with another amino acid.

[0093] At this time, the "another amino acid" introduced by substitution can 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.

[0094] In one embodiment of the present invention, the 2A protease variant may be a variant in which the 87th amino acid glycine in the amino acid sequence of SEQ ID NO: 11 is substituted by serine (G87S). Preferably, it may comprise the amino acid sequence of SEQ ID NO: 75. Therefore, in the present invention, the polynucleotide may comprise the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 75 as a 2A protease variant or consist of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 75 as a 2A protease variant. Specifically, when the polynucleotide is DNA, the nucleic acid sequence may comprise the nucleotide sequence of SEQ ID NO: 73 or consist of the nucleotide sequence of SEQ ID NO: 73. When the polynucleotide is RNA, the nucleic acid sequence may comprise the nucleotide sequence of SEQ ID NO: 74 or consist of the nucleotide sequence of SEQ ID NO: 74.

[0095] In addition, the 2A protease variant may comprise or consist of an amino acid sequence that is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the amino acid sequence of SEQ ID NO:75.

[0096] In the present invention, the nonstructural protein P3 domain may comprise 3A, 3B, 3C proteases, and 3D polymerase. Specifically, the P3 domain of the present invention may comprise 3A, 3B, 3C proteases, and 3D polymerase of Coxsackievirus B5. In this case, the P3 domain may comprise 3A, 3B, 3C proteases, and 3D polymerase in order from the N-terminus to the C-terminus.

[0097] In one embodiment, the 3A, 3B, 3C proteases, and 3D polymerase may comprise the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. Therefore, in the present invention, the polynucleotide may comprise or consist of the nucleic acid sequences encoding the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, at the 3' end of the nucleic acid sequence encoding the amino acid sequence of 2C. In one embodiment of the present invention, when the polynucleotide is DNA, the nucleic acid sequence may comprise or consist of the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, at the 3' end of the nucleic acid sequence encoding the amino acid sequence of 2C, and when the polynucleotide is RNA, it may comprise or consist of the nucleotide sequences of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67, respectively.

[0098] 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 deleted, substituted and / or inserted, as long as they have the same biological function or the genes encoding the 3A, 3B, 3C proteases and 3D polymerases are located in the same position on the chromosome. Specifically, they 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: 14 to SEQ ID NO: 17, respectively.

[0099] Target protein

[0100] In the present invention, the polynucleotide may comprise 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.

[0101] As used herein, the term "target protein" can be various proteins used for medical or industrial purposes, and can include protein fragments, peptides and variants. Industrially useful target proteins include hormones, hormone analogs, enzymes, enzyme inhibitors, cytokines, coagulation factors, transporters, receptors, receptor fragments, attachment proteins, regulatory proteins, structural proteins, toxic proteins, transcription factors, antigens, antibodies, antibody fragments, monoclonal antibodies, etc. Preferably, it can be an antigen, but is not limited thereto. The antigen can be any antigenic protein associated with an infection or disease. Examples of antigens can include tumor antigens, animal antigens, plant antigens, viral antigens, bacterial antigens, fungal antigens, protozoan antigens, autoimmune antigens or allergic antigens, etc. In this case, the antigen can be a surface antigen of a tumor cell, or a secreted form of a protein and peptide derived from a viral pathogen, a bacterial pathogen, a fungal pathogen or a protozoan pathogen, respectively.

[0102] In the present invention, the viral pathogen can be isolated from or derived from, for example, filovirus, adenovirus, enterovirus, severe fever with thrombocytopenia syndrome (SFTSV; bunyavirus), arbovirus, astrovirus, coronavirus, coxsackievirus, cytomegalovirus, dengue virus, Epstein-Barr virus, hepatitis virus, herpes virus, human immunodeficiency virus, human papillomavirus (HPV), human T-lymphotropic virus, influenza virus, canine influenza virus, highly pathogenic avian influenza (HPAI) virus, JC virus, lymphocytic choriomeningitis virus, measles virus, molluscum contagiosum virus, mumps virus, norovirus, parvovirus, poliovirus, rabies virus, respiratory syncytial virus, rhinovirus, rotavirus, rubella virus, smallpox virus, varicella-zoster virus, West Nile virus, Zika virus, etc.

[0103] In the present invention, bacterial pathogens can be isolated from or derived from, for example, Campylobacter jejuni, Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, etc. In the present invention, fungal pathogens can be isolated from or derived from, for example, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida spp., Aspergillus spp., etc.

[0104] In the present invention, the protozoan pathogen can be isolated from or derived from, for example, Plasmodium, Leishmania, Trypanosome, Cryptosporidiums, Isospora, Naegleria fowleri, Acanthamoeba, Balamuthia mandrillaris, Toxoplasma gondii, Pneumocystis carinii, etc.

[0105] As used herein, the term "tumor antigen" refers to an antigen presented by the class I major histocompatibility complex on the surface of tumor cells. Tumor-specific antigens presented only by tumor cells are generally generated by mutations in the tumor. Tumor antigens refer to tumor-specific antigens. Common tumor antigens include tumor-associated antigens that are expressed more in tumor cells than in normal cells. When cytotoxic T cells (Tc) recognize tumor-associated antigens, they can destroy tumor cells before rapid proliferation or metastasis occurs.

[0106] In the present invention, the tumor antigen can be isolated or derived from cystitis, breast cancer, colorectal cancer, endometrial cancer, renal cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, etc. For example, it can be 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, BRCA1 / 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, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, coactosin-like protein protein, COTL1), type XXIII collagen, COX-2, CT_9 / BRD6, Cten, cyclin B1, cyclin D1, CypB, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / 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, hTERT, 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, M AGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, Mammaglobin-A, MART-1 / Melanin-A, MA RT-2, MART_2 / m, matrix protein 22, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, MN / CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class I / m, NA88-A, N-acetylglucosamine transferase-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, OG T / 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, prostate protein, 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, SSX-4, 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, VEGF, VEGFR-2 / FLK-1 and WT1, etc.

[0107] In this specification, when a polynucleotide contains a target protein, the target protein can be described interchangeably with the "first target protein".

[0108] In the present invention, the first target protein can be located at the N-terminus of the nonstructural protein P2 domain. Specifically, it can be located at the N-terminus of the 2A protease in the nonstructural protein P2 domain. Therefore, in the present invention, the nucleic acid sequence encoding the first target protein can be located at the 5' end of the nucleic acid sequence encoding the amino acid sequence of the 2A protease in the polynucleotide.

[0109] In one embodiment of the present invention, the first target protein can be a viral antigen. In one embodiment, the viral antigen can be a severe fever with thrombocytopenia syndrome (SFTS) viral antigen, a SARS-CoV2 spike protein (coronavirus pneumonia (COVID)) antigen, a highly pathogenic avian influenza (HPAI) viral antigen, a canine influenza antigen, or a human papillomavirus (HPV) antigen.

[0110] As used herein, the term "severe fever with thrombocytopenia syndrome (SFTS) virus" is also known as Dabie bandavirus, a virus that causes SFTS. The SFTS virus is currently divided into six genotypes (A, B, C, D, E, and F). SFTS is a disease with high fever and thrombocytopenia as its main symptoms, transmitted by the longicorn tick, a member of the Ixodidae family of the suborder Opisthomyma, which carries the SFTS virus. During the blood-sucking process, the virus carried by the tick enters the body, proliferates, and causes the disease.

[0111] In the present invention, the severe fever with thrombocytopenia syndrome virus antigen may comprise the amino acid sequence of SEQ ID NO:45, SEQ ID NO:85, SEQ ID NO:116 or SEQ ID NO:119.

[0112] As used herein, the term "SARS-CoV2 (COVID) virus" refers to an enveloped, single-stranded, positive-sense RNA virus. It has a distinctive flame- or crown-shaped structure due to the rod-shaped protrusions of its spike protein embedded in the envelope. The spike protein (S protein) is a spike-shaped protein used by the coronavirus to invade human cells and is composed of S1 and S2 subunits. It infects the human body by binding to the angiotensin-converting enzyme 2 (ACE-2) receptor on human cells, penetrating the cells, and pushing genetic material (RNA) into them for self-replication.

[0113] In the present invention, the SARS-CoV2 viral antigen may comprise the amino acid sequence of SEQ ID NO:48.

[0114] As used herein, the term "highly pathogenic avian influenza (HPAI) virus" refers to a virus that can cause avian influenza in chickens, ducks, wild birds, and the like. The virus has various serotypes, such as 16 types of HA and 9 types of NA, depending on the genes of HA and NA in the envelope, and it is known that there is no cross-protection between different serotypes. Among the various serotypes of AIV, all highly pathogenic avian influenza (HPAI) that have occurred to date are known to be caused by H5 or H7 serotypes. In particular, H5 HPAI viruses of the 2.3.4.4 and 2.3.2.1c lineages, i.e., H5 HPAI subtypes, have been introduced into the country through wild birds, and many cases of infection in wild birds have been reported to date.

[0115] In the present invention, the highly pathogenic avian influenza virus may comprise the amino acid sequence of SEQ ID NO: 105.

[0116] As used herein, the term "canine influenza" refers to influenza that occurs in canines. It is hypothesized that canine influenza is caused by a variant of the same influenza A virus as the equine influenza virus H3N8. Influenza A is an enveloped, negative-sense, single-stranded RNA virus. Furthermore, the H3N2 virus is known to be a mutant avian influenza virus. Canine influenza viruses are known to cause acute respiratory illness, with clinical symptoms such as severe coughing, fever, and runny nose.

[0117] In the present invention, the canine influenza virus may comprise the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 56.

[0118] As used herein, the term "human papillomavirus (HPV)", also known as human papillomavirus, is a DNA virus that infects humans and various animals through the skin or mucous membranes. To date, more than 100 types of human papillomavirus have been discovered, and some types of human papillomavirus can cause cancer, such as cervical cancer and testicular cancer. In particular, human papillomavirus type 16 (HPV 16) and human papillomavirus type 18 (HPV 18) are found in 70% of cervical cancer patients worldwide and are classified as high-risk groups. Human papillomavirus contains: E1 to E7, which are genes necessary for viral replication; L1 and L2, which are genes expressing capsid proteins that constitute the virion; and LCR, which regulates viral replication and transcription. In particular, E6 and E7 are known to induce cancer development by promoting the degradation of p53 and RB proteins, respectively, thereby activating the cell cycle and inhibiting apoptosis.

[0119] In the present invention, the human papillomavirus antigen may comprise the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 142, SEQ ID NO: 145 or SEQ ID NO: 148.

[0120] In an embodiment of the application, the polynucleotide can comprise, at the 5' end of the nucleic acid sequence encoding the amino acid sequence of the 2A protease, a nucleic acid sequence encoding the following amino acid sequence: SEQ ID NO: 45 (SFTS B antigen), SEQ ID NO: 85 (SFTS ABDEF antigen), SEQ ID NO: 116 (SFTS A antigen), SEQ ID NO: 119 (SFTS DEF antigen), SEQ ID NO: 48 (SARS antigen), SEQ ID NO: 105 (H5N8 antigen), SEQ ID NO: 53 (H3N2 antigen), SEQ ID NO: 56 (H3N8 antigen), SEQ ID NO: 139 (HPV E6), SEQ ID NO: 142 (HPV E6 / E7 sequential arrangement antigen), SEQ ID NO: 145 (HPV E6 / E7 cross arrangement antigen), or SEQ ID NO: 148 (HPV E6 / E7 odd-even arrangement antigen).

[0121] In an embodiment of the application, when the polynucleotide is DNA, the nucleic acid sequence can comprise or consist of the nucleotide 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 can comprise or consist of the nucleotide sequence of SEQ ID NO: 44, SEQ ID NO: 84, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 47, SEQ ID NO: 107, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 140, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 149.

[0122] In addition, the SFTS antigen, COVID antigen, highly pathogenic avian influenza, canine influenza antigen or human papillomavirus antigen can be composed of an amino acid sequence in which one or more amino acids are deleted, substituted and / or inserted, as long as they have the same biological function or the gene encoding the SFTS antigen, COVID antigen, highly pathogenic avian influenza or canine influenza antigen is located at the same position on the chromosome. Specifically, it can 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 sequence of SEQ ID NO: 45, SEQ ID NO: 85, SEQ ID NO: 116, SEQ ID NO: 119, SEQ ID NO: 48, SEQ ID NO: 105, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 139, SEQ ID NO: 142, SEQ ID NO: 145 or SEQ ID NO: 148.

[0123] Protease cleavage site

[0124] As used herein, the term "protease cleavage site" refers to a site that is specifically cleaved by a proteolytic enzyme.

[0125] In the present invention, the protease cleavage site attached to the first target protein can be a site cleaved by a protease derived from a nonstructural protein of a self-amplifying virus. In this case, the self-amplifying virus can be a virus of the genus Enterovirus. Preferably, it can be Coxsackievirus B5. In one embodiment of the present invention, the protease cleavage site can be a site cleaved by the 2A protease of an enterovirus. In this case, the protease cleavage site can be located between the first target protein and the 2A protease of the nonstructural protein P2 domain.

[0126] As used herein, the term "2A protease of enterovirus" is a proteolytic enzyme that first acts after the RNA of enterovirus is translated into a single polyprotein, and acts in cleaving the polyprotein into structural proteins and non-structural 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 in the C-terminal amino acid of VP1 of the P1 domain and the N-terminal amino acid Gly of the 2A protease of the P2 domain. In one embodiment of the present application, the 2A protease can cleave between Tyr and Gly. In one embodiment, it can cleave between Ala and Gly. In one embodiment, it can cleave between Thr and Gly. In one embodiment, it can cleave between Val and Gly. In one embodiment, it can cleave between Phe and Gly. In one embodiment, it can cleave between Arg and Gly. Preferably, it can cleave between Tyr in the C-terminal amino acid of VP1 of the P1 domain and Gly in the N-terminal amino acid of the 2A protease of the P2 domain.

[0127] Accordingly, in the present application, the 2A protease cleavage site can comprise the amino acid sequence of the C-terminus of VP1 or the C-terminus of VP1, or the C-terminus of VP1 and the N-terminus of the 2A protease (2A pro ) of enterovirus.

[0128] At this time, the amino acid sequence of the N-terminus of VP1 can comprise 1 to 30 consecutive amino acids from the C-terminus of VP1. Specifically, the 2A protease cleavage site can 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, 26, 27, 28, 29, or 30 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO: 70 (VP1 protein). More specifically, it can 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, 26, 27, 28, 29, or 30 consecutive amino acid sequences from the C-terminus of the amino acid sequence of SEQ ID NO: 70 (VP1 protein).

[0129] The amino acid sequence at the N-terminus may comprise a sequence of 1 to 18 amino acids starting from the N-terminus of the 2A protease. Specifically, the 2A protease cleavage site may comprise a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 consecutive amino acids starting from the N-terminus of the 2A protease. More specifically, it may comprise the amino acid sequence (2A) of SEQ ID NO: 11. pro A sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 consecutive amino acids starting from the N-terminus of a protein.

[0130] In one embodiment of the present invention, the 2A protease cleavage site may comprise any one of the amino acid sequences selected from SEQ ID NO: 18 (T) to SEQ ID NO: 23. Therefore, the polynucleotide of the present invention may comprise a nucleic acid sequence encoding any one of the amino acid sequences 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 comprise 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 comprise any one of the nucleotide sequences selected from SEQ ID NO: 24 (ACC) to SEQ ID NO: 29 or consist of any one of the nucleotide sequences selected from SEQ ID NO: 24 (ACC) to SEQ ID NO: 29. Preferably, it may comprise the nucleotide sequence of SEQ ID NO: 28. When the polynucleotide is RNA, it may comprise any one of the nucleotide sequences selected from SEQ ID NO: 24 and SEQ ID NO: 30 to SEQ ID NO: 34 or consist of any one of the nucleotide sequences selected from SEQ ID NO: 24 and SEQ ID NO: 30 to SEQ ID NO: 34. Preferably, it may comprise the nucleotide sequence of SEQ ID NO:33.

[0131] In addition, the 2A protease cleavage site may consist of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or inserted, as long as it has the same biological function or the gene encoding the 2A protease cleavage site is located at the same position on the chromosome. Specifically, it may comprise or consist of an amino acid sequence that is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to any one of the amino acid sequences selected from SEQ ID NO: 18 to SEQ ID NO: 23.

[0132] In the present application, the polynucleotide can further comprise a nucleic acid sequence encoding a target protein. In the present specification, when the polynucleotide comprises a nucleic acid sequence encoding one or more target proteins, the target protein can be described by dividing the target protein into a first target protein and a second target protein. At this time, the second target protein can be located at the N-terminus of the first target protein. Accordingly, in the present application, the nucleic acid sequence encoding the second target protein can be located at the 5' end 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.

[0133] In an embodiment of the present application, the first target protein and the second target protein can be viral antigens. In an embodiment, the viral antigens can be H3N2 and H3N8, which are subtypes of canine influenza. At this time, the first target protein can be H3N2 or H3N8, and the second target protein can be H3N2 or H3N8. The first target protein and the second target protein can be the same as or different from each other. Accordingly, in an embodiment of the present application, when the polynucleotide is DNA, the first target protein can consist of the nucleotide sequence of SEQ ID NO: 51 or SEQ ID NO: 54, and when the polynucleotide is RNA, it can consist of the nucleotide sequence of SEQ ID NO: 52 or SEQ ID NO: 55. In an embodiment of the present application, the first target protein can be H3N8 and can comprise the amino acid sequence of SEQ ID NO: 56, and the second target protein can be H3N2 and can comprise the amino acid sequence of SEQ ID NO: 53. Accordingly, the nucleic acid sequence encoding the first target protein of the polynucleotide can comprise the nucleotide sequence of SEQ ID NO: 54 or SEQ ID NO: 55, and the nucleic acid sequence encoding the second target protein can comprise the nucleotide sequence of SEQ ID NO: 51 or SEQ ID NO: 52.

[0134] A 2A self-cleaving peptide sequence or a protease cleavage site can exist between the first target protein and the second target protein.

[0135] As used herein, the term "2A self-cleaving peptide" refers to a peptide consisting of 18 to 22 amino acids, which can induce ribosomal skipping during protein translation in a cell. The peptide shares a core sequence motif of DxExNPGP and is found in a wide range of viral families. The 2A self-cleaving peptide can be, for example, P2A, E2A, F2A, T2A, etc. In an embodiment, the 2A self-cleaving peptide can be P2A (porcine enterovirus-1 2A) and can comprise the amino acid sequence of SEQ ID NO: 187. Furthermore, when the 2A self-cleaving peptide is included, it can further comprise a linker (GSG, SEQ ID NO: 188) at the N-terminus of the peptide.

[0136] The protease cleavage site may be a site cleaved by a protease derived from a nonstructural protein of a self-amplifying virus. The self-amplifying virus may be a virus of the Picornaviridae family, specifically, a virus of the genus Enterovirus. Preferably, it may be Coxsackievirus B5. In one embodiment of the present invention, the protease cleavage site may be a site cleaved by the 2A protease, 3CD protease, or 3C protease of an enterovirus. More specifically, it may be a site cleaved by the 2A protease, 3CD protease, or 3C protease of Coxsackievirus B5. Here, the protease cleavage site, 2A protease, and 2A protease cleavage site are the same as described above.

[0137] In the present invention, "enteroviral 3C protease" is the second proteolytic enzyme to act after the enteroviral RNA is translated into a single polyprotein. pro )" is a precursor of 3C protease and is a protein produced in the intermediate step of processing the P3 domain into each individual protein by 3C protease. At this time, 3CD protease also has protease activity. 3C protease and 3CD protease play a role in cleaving the polyprotein into individual proteins (VP0, VP3, VP1, 2A protease, 2B, 2C, 3A, 3B, 3C protease and 3D polymerase), and the polyprotein is cleaved into structural proteins (P1 domain) and non-structural proteins (P2 domain and P3 domain) by 2A protease. At this time, "VPO" is an intermediate protein that is further processed into VP4 and VP2.

[0138] The 3C protease can cleave between any one of the amino acids selected from the group consisting of Gln, Glu, Ile, and Thr at the C-terminal amino acid of each individual protein (VP3, VP1, 2A protease, 2B, 2C, 3A, 3B, and 3C protease) and any one of the amino acids selected from the group consisting of Gly, Asn, Ser, Ala, Val, Cys, Trp, and Met at the N-terminal amino acid of each individual protein (VP1, 2A protease, 2B, 2C, 3A, 3B, 3C protease, and 3D polymerase). In one embodiment of the present invention, the 3C protease can cleave between Gln and Gly. In one embodiment, the 3C protease can cleave between Gln and Asn. In one embodiment, the 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 may cleave between Gln and Trp. In one embodiment, the 3C protease may cleave between Gln and Gly. In one embodiment, the 3C protease may cleave between Gln and Met. In one embodiment, the 3C protease may cleave between Glu and Gly. In one embodiment, the 3C protease may cleave between Glu and Asn. In one embodiment, the 3C protease may cleave between Glu and Ser. In one embodiment, the 3C protease may cleave between Glu and Ala. In one embodiment, the 3C protease may cleave between Glu and Val. In one embodiment, the 3C protease may cleave between Glu and Cys. In one embodiment, the 3C protease may cleave between Glu and Trp. In one embodiment, the 3C protease may cleave between Glu and Gly. In one embodiment, the 3C protease may cleave between Glu and Met. In one embodiment, the 3C protease may cleave between Ile and Gly. In one embodiment, the 3C protease may cleave between Ile and Asn. In one embodiment, the 3C protease may cleave between Ile and Ser. In one embodiment, the 3C protease may cleave between Ile and Ala. In one embodiment, the 3C protease can cut between Ile and Val. In one embodiment, the 3C protease can cut between Ile and Cys. In one embodiment, the 3C protease can cut between Ile and Trp. In one embodiment, the 3C protease can cut between Ile and Gly. In one embodiment, the 3C protease can cut between Ile and Met.In one embodiment, the 3C protease can cut between Thr and Gly. In one embodiment, the 3C protease can cut between Thr and Asn. In one embodiment, the 3C protease can cut between Thr and Ser. In one embodiment, the 3C protease can cut between Thr and Ala. In one embodiment, the 3C protease can cut between Thr and Val. In one embodiment, the 3C protease can cut between Thr and Cys. In one embodiment, the 3C protease can cut between Thr and Trp. In one embodiment, the 3C protease can cut between Thr and Gly. In one embodiment, the 3C protease can cut between Thr and Met. Preferably, it can cut between the Gln in the C-terminal amino acid and the Gly in the N-terminal amino acid of each individual protein.

[0139] Specifically, the 3C protease according to the present invention can cleave between the C-terminal amino acid Gln of VP3 and the N-terminal amino acid Gly of VP1 of Coxsackievirus B5. It can cleave between the C-terminal amino acid Gln of the 2A protease and the N-terminal amino acid Gly of 2B. It can cleave between the C-terminal amino acid Gln of 2B and the N-terminal amino acid Gly of 2C. It can cleave between the C-terminal amino acid Gln of 2C and the N-terminal amino acid Gly of 3A. It can cleave between the C-terminal amino acid Gln of 3A and the N-terminal amino acid Gly of 3B. It can cleave between the C-terminal amino acid Gln of 3B and the N-terminal amino acid Gly of 3C protease. It can cleave between the C-terminal amino acid Gln of 3C protease and the N-terminal amino acid Gly of 3D polymerase.

[0140] Therefore, in the present invention, the 3C protease cleavage site may comprise 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.

[0141] In addition, in the present invention, the protease cleavage site may comprise any one amino acid sequence selected from the group consisting 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.

[0142] Specifically, in the present invention, the 3C protease cleavage site may comprise 1 to 22 consecutive amino acids starting 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.

[0143] More specifically, the 3C protease cleavage site can 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 starting from the C-terminus of any one of the amino acid sequences 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.

[0144] In addition, in the present invention, the 3C protease cleavage site may comprise 1 to 11 consecutive amino acids starting from the C-terminus of the amino acid sequence of VP3 and 1 to 11 consecutive amino acids starting from the N-terminus of the amino acid sequence of VP1. It may comprise 1 to 11 consecutive amino acids starting from the C-terminus of the amino acid sequence of 2A protease and 1 to 11 consecutive amino acids starting from the N-terminus of the amino acid sequence of 2B. It may comprise 1 to 11 consecutive amino acids starting from the C-terminus of the amino acid sequence of 2B and 1 to 11 consecutive amino acids starting from the N-terminus of the amino acid sequence of 2C. It may comprise 1 to 11 consecutive amino acids starting from the C-terminus of the amino acid sequence of 2C and 1 to 11 consecutive amino acids starting from the N-terminus of the amino acid sequence of 3A. It may comprise 1 to 11 consecutive amino acids starting from the C-terminus of the amino acid sequence of 3A and 1 to 11 consecutive amino acids starting from the N-terminus of the amino acid sequence of 3B. It may comprise 1 to 11 consecutive amino acids starting from the C-terminus of the amino acid sequence of 3B and 1 to 11 consecutive amino acids starting from the N-terminus of the amino acid sequence of 3C protease. It may comprise 1 to 11 consecutive amino acids starting from the C-terminus of the amino acid sequence of 3C protease and 1 to 11 consecutive amino acids starting from the N-terminus of the amino acid sequence of 3D polymerase.

[0145] 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.

[0146] 3CD protease can cleave between VP0 and VP3. Specifically, it can cleave between any one of the amino acids selected from the group consisting of Gln, Lys, Tyr, and Met at the C-terminal amino acid of VP0 and any one of the amino acids selected from the group consisting of Gly, Trp, Tyr, and Asn at the N-terminal amino acid of VP3. In one embodiment of the present invention, 3CD protease can cleave between Gln and Gly. In one embodiment, it can cleave between Gln and Trp. In one embodiment, it can cleave between Gln and Tyr. In one embodiment, it can cleave between Gln and Asn. In one embodiment, it can cleave between Lys and Gly. In one embodiment, it can cleave between Lys and Trp. In one embodiment, it can cleave between Lys and Tyr. In one embodiment, it can cleave between Lys and Asn. In one embodiment, it can cleave between Thr and Gly. In one embodiment, it can cleave between Thr and Trp. In one embodiment, it can cleave between Thr and Tyr. In one embodiment, it may cleave between Thr and Asn. In one embodiment, it may cleave between Met and Gly. In one embodiment, it may cleave between Met and Trp. In one embodiment, it may cleave between Met and Tyr. In one embodiment, it may cleave between Met and Asn. Preferably, it may cleave between the Gln in the C-terminal amino acid of VP0 and the Gly in the N-terminal amino acid of VP3.

[0147] Therefore, in the present invention, the 3CD protease cleavage site may comprise the C-terminal amino acid sequence of the VP2 amino acid sequence.

[0148] Furthermore, the 3CD protease cleavage site may comprise the C-terminal amino acid sequence of VP2 and the N-terminal amino acid sequence of VP3.

[0149] Specifically, in the present invention, the 3CD protease cleavage site may comprise 1 to 40 consecutive amino acids starting from the C-terminus of VP2.

[0150] More specifically, the 3CD protease cleavage site can 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 starting from the C-terminus of the amino acid sequence of SEQ ID NO: 79.

[0151] Furthermore, the 3CD protease cleavage site may comprise a 1 to 20 consecutive amino acid sequence starting from the C-terminus of VP2 and a 1 to 20 consecutive amino acid sequence starting from the N-terminus of VP1.

[0152] More specifically, it may comprise 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.

[0153] In one embodiment of the present invention, the protease cleavage site may comprise 6 consecutive amino acids starting from the C-terminus of the amino acid sequence of 2C and 6 consecutive amino acids starting from the N-terminus of the amino acid sequence of 3A.

[0154] In one embodiment of the present invention, the 3C protease cleavage site may comprise the amino acid sequence of SEQ ID NO: 50. Therefore, in one embodiment of the present invention, the nucleic acid sequence encoding the 3C protease cleavage site may comprise or consist of the nucleotide sequence of SEQ ID NO: 49 or SEQ ID NO: 57, respectively.

[0155] In addition, the 3C protease cleavage site and the 3CD protease cleavage site can be composed of an amino acid sequence in which one or more amino acids are deleted, substituted and / or inserted, as long as they have the same biological function or the gene encoding the 3C protease cleavage site is located at the same position on the chromosome. Specifically, it can comprise or consist of an amino acid sequence that has 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 sequence of SEQ ID NO: 50.

[0156] Therefore, in one embodiment of the present invention, the polynucleotide of the present invention may comprise, at the 5' end of the nucleic acid sequence encoding the 2A protease protein of the non-structural protein, the following nucleic acid sequences encoding the first target protein and the second target protein, and nucleic acid sequences encoding the amino acid sequences of SEQ ID NO: 53, SEQ ID NO: 50, SEQ ID NO: 56, and SEQ ID NO: 22. Therefore, when the polynucleotide is DNA, it may comprise, or consist of, the nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 28 from the 5' end to the 3' end. When the polynucleotide of the present invention is RNA, the nucleic acid sequence may comprise or consist of the nucleotide sequences of SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 52 and SEQ ID NO: 33 from the 5' end to the 3' end.

[0157] In addition, in the present invention, when the second target protein comprises one or more target proteins, the second target protein can be composed of one or more identical or different target proteins, and the protease cleavage site can be located between each target protein. In this case, the protease cleavage site can be a site that is cleaved by a protease derived from a non-structural protein of a self-amplifying virus. Specifically, it can be a site that is cleaved by a protease derived from a Picornaviridae virus. More specifically, it can be a site that is cleaved by the 2A protease, 3CD protease, or 3C protease of an enterovirus. Preferably, it can be a site that is cleaved by the 2A protease, 3CD protease, or 3C protease of Coxsackievirus B5. The second target protein can be an antigen. In addition, 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.

[0158] stop codon

[0159] In the present invention, the polynucleotide may further comprise at least one stop codon at the 3' end of the nucleic acid sequence encoding the self-amplifying viral non-structural protein.

[0160] As used herein, the term "stop codon" is a signal sequence for terminating mRNA translation into amino acids, and generally refers to a stop codon or a sequence of a stop codon. There is no corresponding tRNA in the stop codon, but it is bound by a protein called a "terminator", and when the stop codon is reached during the translation process, the two units of the ribosome are separated, thereby terminating translation. Generally speaking, the sequence of the stop codon of DNA is TAG, TAA or TGA, and the sequence of the stop codon of RNA is UAG, UAA or UGA.

[0161] In particular, the polynucleotides of the present invention may comprise one to three stop codons.

[0162] More specifically, the polynucleotide may comprise a stop codon. In one 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.

[0163] More specifically, the polynucleotide may comprise two different consecutively positioned stop codons.

[0164] When the polynucleotide of the present invention is DNA, in one embodiment, when the polynucleotide is DNA, the stop codon can be positioned in the order of TAG and TAA from the 5' end to the 3' end. In one embodiment, it can be positioned in the order of TAG and TGA. In one embodiment, it can be positioned in the order of TAA and TGA. When the polynucleotide of the present invention is RNA, in one embodiment, it can be positioned in the order of UAG and UAA from the 5' end to the 3' end. In one embodiment, it can be positioned in the order of UAG and UGA. In one embodiment, it can be positioned in the order of UAA and UGA.

[0165] More specifically, the polynucleotide may comprise three different consecutively positioned stop codons.

[0166] When the polynucleotide of the present invention is DNA, in one embodiment, the stop codon may be positioned in the order of TAG, TAA, and TGA from the 5' end to the 3' end. In one embodiment, it may be positioned in the order of TAG, TGA, and TAA. In one embodiment, it may be positioned in the order of TAA, TAG, and TGA. In one embodiment, it may be positioned in the order of TAA, TGA, and TAG. In one embodiment, it may be positioned in the order of TGA, TAG, and TAA. In one embodiment, it may be positioned in the order of TGA, TAA, and TAG. In one embodiment, the stop codon may comprise or consist of the nucleotide sequence of SEQ ID NO:9 (TGATAATAG). When the polynucleotide of the present invention is RNA, in one embodiment, it may be positioned in the order of UAG, UAA, and UGA from the 5' end to the 3' end. In one embodiment, it may be positioned in the order of UAG, UGA, and UAA. In one embodiment, it may be positioned in the order of UAA, UAG, and UGA. In one embodiment, it can be positioned in the order of UAA, UGA and UAG. In one embodiment, it can be positioned in the order of UGA, UAG and UAA. In one embodiment, it can be positioned in the order of UGA, UAA and UAG. In one embodiment of the invention, the stop codon can comprise the nucleotide sequence of SEQ ID NO:68 (UGAUAAUAG) or consist of the nucleotide sequence of SEQ ID NO:68 (UGAUAAUAG).

[0167] 5' untranslated region

[0168] In the present invention, the polynucleotide may further comprise a 5' untranslated region (5'UTR) at the 5' end.

[0169] 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 can be present upstream of the start codon (5'UTR) and downstream of the stop codon (3'UTR). The 5'UTR is a region that regulates translation and transcription, and regulates transcription by different mechanisms in viruses, prokaryotes, and eukaryotes. Although it is generally considered to be an untranslated region, it has been reported that it is translated into a protein product and regulates the translation of mRNA.

[0170] The elements of the 5'UTR of eukaryotes and prokaryotes are very different. The 5'UTR of prokaryotes contains a ribosome binding site (RBS), also known as a Shine-Dalgarno sequence (SD sequence), which is typically located 3-10 base pairs upstream of the start codon. In contrast, the 5'UTR of eukaryotes contains a Kozak consensus sequence (hereinafter referred to as a "Kozak sequence") containing the start codon.

[0171] The 5' untranslated region of the polynucleotide according to the present invention may comprise an internal ribosome entry site (IRES).

[0172] As used herein, the term "internal ribosome entry site (IRES)" is a nucleotide sequence that is typically located in the 5'UTR of an RNA virus and can induce RNA translation in a cap-independent manner. In the present invention, the 5'UTR is a region to which the translation initiation complex associated with the translation of the target protein binds, and the IRES is a cis-acting nucleotide sequence that induces the translation of the target protein by forming a complex secondary structure and a tertiary structure.

[0173] In the present invention, the IRES may comprise a nucleotide sequence from an IRES of a virus or eukaryotic cell. In this case, the 5'UTR may have a 5'UTR structure comprising an IRES from a virus or eukaryotic cell. In one embodiment of the present invention, the 5'UTR and IRES may be derived from a self-amplifying virus identical to the non-structural protein. Specifically, they may be derived from a virus of the genus Picornavirus. More specifically, the 5'UTR and IRES may be derived from an enterovirus. In one embodiment of the present invention, the 5'UTR may comprise a nucleic acid sequence of SEQ ID NO: 1 (DNA) or SEQ ID NO: 60 (RNA) or consist of a nucleic acid sequence of SEQ ID NO: 1 (DNA) or SEQ ID NO: 60 (RNA), and in this case, the IRES may comprise a nucleic acid sequence of SEQ ID NO: 58 (DNA) or SEQ ID NO: 59 (RNA) or consist of a nucleic acid sequence of SEQ ID NO: 58 (DNA) or SEQ ID NO: 59 (RNA).

[0174] The IRES may contain a mutation. Here, the mutation is the same as described above.

[0175] Specifically, the variant of IRES may be a variant in which a portion of the nucleotide sequence of a wild-type IRES is replaced by a different nucleotide sequence.

[0176] More specifically, when the polynucleotide is DNA, it can be a polynucleotide in which any one of the nucleotides selected from the group consisting of position 236, 386, and a combination thereof in the nucleotide sequence of SEQ ID NO: 58 is substituted with a nucleotide different from the nucleotide sequence of SEQ ID NO: 58. In one embodiment, it can be a nucleotide in which the 236th nucleotide in the nucleotide sequence of SEQ ID NO: 58 is substituted with A, T, or G. In one embodiment, it can be a nucleotide in which the 386th nucleotide in the nucleotide sequence of SEQ ID NO: 58 is substituted with A, T, or C. In one embodiment, it can be a nucleotide in which the 236th nucleotide in the nucleotide sequence of SEQ ID NO: 58 is substituted with A, T, or G, and the 386th nucleotide is substituted with A, T, or C. Preferably, it can be a nucleotide in which the 236th nucleotide C in the nucleotide sequence of SEQ ID NO: 58 is substituted with T (C236T). It can be a nucleotide in which the 386th nucleotide G in the nucleotide sequence of SEQ ID NO: 58 is substituted with A (G386A). More preferably, it can be a nucleotide in which the 236th nucleotide C in the nucleotide sequence of SEQ ID NO: 58 is substituted with T (C236T), and the 386th nucleotide G is substituted with A (G386A).

[0177] When the polynucleotide is RNA, it can be a polynucleotide in which any one of the nucleotides selected from the group consisting of position 236, 386, and a combination thereof in the nucleotide sequence of SEQ ID NO: 59 is substituted with a nucleotide different from the nucleotide sequence of SEQ ID NO: 59. In one embodiment, it can be a nucleotide in which the 236th nucleotide in the nucleotide sequence of SEQ ID NO: 59 is substituted with A, U, or G. In one embodiment, it can be a nucleotide in which the 386th nucleotide in the nucleotide sequence of SEQ ID NO: 59 is substituted with A, U, or C. In one embodiment, it can be a nucleotide in which the 236th nucleotide in the nucleotide sequence of SEQ ID NO: 59 is substituted with A, U, or G, and the 386th nucleotide is substituted with A, U, or C. Preferably, it can be a nucleotide in which the 236th nucleotide C in the nucleotide sequence of SEQ ID NO: 59 is substituted with U (C236U). It can be a nucleotide in which the 386th nucleotide G in the nucleotide sequence of SEQ ID NO: 59 is substituted with A (G386A). More preferably, it can be a nucleotide in which the 236th nucleotide C in the nucleotide sequence of SEQ ID NO: 59 is substituted with U (C236U), and the 386th nucleotide G is substituted with A (G386A).

[0178] In one embodiment of the present application, the variant of the IRES can comprise or consist of the nucleotide sequence of SEQ ID NO: 81 or SEQ ID NO: 82.

[0179] Kozak sequence

[0180] In the present application, the polynucleotide can further comprise a Kozak sequence.

[0181] As used herein, the term "Kozak sequence" refers to a nucleotide sequence located upstream of the start codon, where the eukaryotic mRNA begins to be translated into a protein, and plays an important role in recognizing the start codon and initiating protein synthesis.

[0182] In the present application, the Kozak sequence can be located at the 5' end of the nucleic acid sequence encoding the target protein (the first target protein or the second target protein). In the present application, the Kozak sequence can comprise any one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 37. Preferably, it can comprise or consist of the nucleotide sequence of SEQ ID NO: 35.

[0183] 3' untranslated region

[0184] In the present application, the polynucleotide can further comprise a 3' UTR. At this time, the 3' UTR can be located at the 3' end of the stop codon. The 3' UTR is functionally linked to the nucleic acid sequence encoding the target protein and the non-structural protein together with the 5' UTR, thereby improving the translation efficiency of the target protein and the non-structural protein or its transcript. In addition, it plays an important role in stably maintaining mRNA (transcript) from being destroyed by cells.

[0185] In the present application, the 3' UTR can use the 3' UTR of the virus or eukaryotic cell origin having the above-mentioned IRES nucleotide sequence, which is the same as the 5' UTR. In one embodiment of the present application, the 3' UTR can be derived from the same self-replicating virus as the non-structural protein derived from the 5' UTR. Specifically, it can be derived from the same virus of the genus Picornavirus as the 5' UTR. More specifically, it can be derived from the same enterovirus as the 5' UTR. Preferably, it can be derived from Coxsackievirus B5.

[0186] In one embodiment of the present application, the 3' UTR can comprise or consist of the nucleotide sequence of SEQ ID NO: 10 (DNA) or SEQ ID NO: 69 (RNA).

[0187] Poly(A) tail

[0188] In the present invention, the polynucleotide may also include a polyadenylic acid tail (poly (A) tail) sequence. The poly (A) tail sequence may be located at the 3' end of the nucleic acid sequence encoding the self-amplification virus non-structural protein. 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 3'UTR nucleotide sequence. The poly (A) tail sequence may also improve the translation efficiency of the target protein while stabilizing the transcribed nucleic acid molecule. The poly (A) tail sequence may include approximately 10 to approximately 500 adenosine nucleotides. Specifically, it may be a nucleic acid sequence consisting of approximately 10 to approximately 500, approximately 50 to approximately 400, approximately 100 to approximately 300, or approximately 100 to approximately 200 adenosine nucleotides. Preferably, it may be composed of approximately 120 adenosine nucleotides. In one embodiment of the present invention, the poly(A) tail sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 38 to SEQ ID NO: 40. Preferably, it may consist of the nucleotide sequence of SEQ ID NO: 40.

[0189] The structure of polynucleotides

[0190] 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:

[0191] 5'-BC(1)-D-3'(I)

[0192] In structural formula (I),

[0193] 5' and 3' are the 5' and 3' ends of the polynucleotide, respectively,

[0194] B is a nucleic acid sequence encoding the first target protein,

[0195] C(1) is a nucleic acid sequence encoding a protease cleavage site, and

[0196] D is a nucleic acid sequence encoding a nonstructural protein of the self-amplifying virus.

[0197] More specifically, the polynucleotide may be composed of the following structural formula (II) in order from the 5' end to the 3' end:

[0198] 5'-U-[K]o-[B'-C(2)]nBC(1)-DS-U'-P-3'(II)

[0199] In structural formula (II),

[0200] 5' and 3' are the 5' and 3' ends of the polynucleotide, respectively,

[0201] U is the nucleic acid sequence of the 5' untranslated region,

[0202] K is the Kozak sequence,

[0203] B' is a nucleic acid sequence encoding a second target protein,

[0204] B is a nucleic acid sequence encoding the first target protein,

[0205] D is a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus,

[0206] S is the stop codon,

[0207] U' is the nucleic acid sequence of the 3' untranslated region,

[0208] P is the poly(A) tail,

[0209] C(1) and C(2) are each independently a nucleic acid sequence encoding a protease cleavage site,

[0210] n represents the number of [B'-C(2)] and is an integer from 0 to 10, and

[0211] When n is 2-10, each B' is a nucleic acid sequence encoding the same or different target proteins, and each C(2) is a nucleic acid sequence encoding the same or different protease cleavage sites. In addition, o is an integer of 0 or 1.

[0212] In this case, C(1) may be a 2A protease cleavage site, and C(2) may be a 2A protease, 3CD protease, or 3C protease cleavage site.

[0213] The 5' untranslated region, target protein, first target protein, second target protein, stop codon, 3' untranslated region, poly(A) tail, protease cleavage site, 2A, 3CD and 3C protease cleavage sites are the same as above.

[0214] The nonstructural protein may comprise the P2 domain and P3 domain of an enterovirus. Specifically, it may comprise the P2 domain and the P3 domain in order from the N-terminus to the C-terminus. More specifically, it may comprise the 2A protease, 2B, 2C, 3A, 3B, 3C protease, and 3D polymerase in order from the N-terminus to the C-terminus. In this case, the nonstructural protein, the P2 domain, the P3 domain, the 2A protease, the 2B and 2C, 3A, 3B, 3C protease, and the 3D polymerase are the same as described above.

[0215] When the polynucleotide according to the present invention is RNA, the polynucleotide (hereinafter, used interchangeably with "RNA construct") can be a self-amplifying RNA (saRNA). The RNA construct can be double-stranded or single-stranded, and preferably can be single-stranded.

[0216] As used herein, the term "replicon" refers to a self-replicating nucleic acid sequence. In the present invention, an RNA replicon refers to an RNA molecule that can be replicated by an RNA-dependent RNA polymerase. An RNA replicon can produce one or more identical or substantially similar RNA replicon copies without a DNA intermediate. Hereinafter, in the present invention, saRNA viral vectors, RNA replicons, saRNA, and saRNA constructs can be described interchangeably.

[0217] Embodiments of polynucleotides

[0218] In one embodiment, the polynucleotide may comprise the structure of [5'UTR-Kozak sequence-target protein-2A protease cleavage site-nonstructural protein (2A protease-2B-2C-3A-3B-3V-C-3D)-stop codon-3'UTR-poly (A) tail] from the 5' end to the C-terminus. In one embodiment, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 111 to SEQ ID NO: 115, SEQ ID NO: 131, SEQ ID NO: 134, SEQ ID NO: 151 to SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 162, or SEQ ID NO: 111.

[0219] In one embodiment, the polynucleotide may comprise, from the 5' end to the C-terminus, a structure of [5'UTR-Kozak sequence-second target protein-3C protease cleavage site-first target protein-2A protease cleavage site-nonstructural protein (2A protease-2B-2C-3A-3B-3V-C-3D)-stop codon-3'UTR-poly (A) tail]. In one embodiment, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 192, or SEQ ID NO: 193.

[0220] In one embodiment, the polynucleotide may comprise, from the 5' end to the C-terminus, the structure of [5'UTR-Kozak sequence-second target protein-P2A self-cleaving peptide sequence-first target protein-2A protease cleavage site-nonstructural protein (2A protease-2B-2C-3A-3B-3V-C-3D)-stop codon-3'UTR-poly(A) tail]. In one embodiment, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 190 or SEQ ID NO: 191.

[0221] Recombinant vector

[0222] In another aspect of the present invention, a recombinant vector loaded with a polynucleotide is provided. Here, the polynucleotide is the same as described above.

[0223] As used herein, the term "recombinant" means "made through genetic manipulation" and means something that does not occur in nature.

[0224] As used herein, the term "vector" refers to a gene construct comprising essential regulatory elements operably connected to express a gene insert, which is an expression vector (or recombinant vector) that can express a target protein in a host cell. The term "operably connected" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the target protein are functionally connected to perform a general function. Gene recombination techniques well-known in the art to which the present invention belongs can be used to be operably connected to the vector, and enzymes generally known in the art to which the present invention belongs, etc. can be used to easily perform site-specific DNA cutting and connection. Specifically, the recombinant vector can be amplified in vitro by polymerase chain reaction (PCR), produced by cloning and recombination, purified by cutting and gel electroporation fractionation, or synthesized by chemical synthesis, but is not limited thereto.

[0225] In the present invention, recombinant vector can be used as gene delivery vehicle, for transporting and expressing polynucleotide according to the present invention.Therefore, preferably polynucleotide is loaded into suitable expression vehicle.Except expression control elements such as promoter, start codon and terminator codon, suitable expression vehicle that can be used for the present invention can comprise the signal sequence for membrane targeting or secretion.Start codon and terminator codon are generally considered to be a part of the nucleotide sequence of encoding immunogenic target protein, and it must show function in object when using genetic construct, and must be in the reading frame of coding sequence.

[0226] As used herein, the term "promoter" refers to a nucleic acid sequence that controls the synthesis of a transcript, e.g., a transcript comprising a coding sequence, by providing a recognition and binding site for RNA polymerase. A universal promoter can be constitutive or inducible. In the present invention, the promoter operably linked to the polynucleotide can be a promoter operable to control the transcription of the polynucleotide, preferably in an animal cell, more preferably in a mammalian cell. The promoter can comprise a promoter derived from a virus, a promoter derived from a mammalian cell genome, or a promoter derived from a bacteriophage. For example, it can comprise, but is not limited to, a cytomegalovirus (CMV) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, a 94 promoter, a T3 promoter, an SM6 promoter, an RSV promoter, an EF1a promoter, a metallothionein promoter, a beta-actin promoter, a cancer cell-specific promoter (e.g., a TERT promoter, a PSA promoter, a PSMA promoter, a CEA promoter, an E2F promoter, and an AFP promoter), and a tissue-specific promoter (e.g., an albumin promoter).

[0227] In one embodiment of the present invention, the polynucleotide can be used as a template for transcription into a nucleic acid molecule in the form of RNA (saRNA construct, RNA replicon) by in vitro transcription (IVT) after being loaded into a suitable expression vehicle. At this time, the promoter can be located at the 5' end of the polynucleotide for transcription into RNA from linearized DNA.

[0228] "In vitro transcription" refers to a process of synthesizing RNA, particularly mRNA, in vitro in a cell-free system. At this time, a cloning vehicle can be applied to the production of a transcript. These cloning vehicles are generally designed as transcription vehicles, and are included in the "recombinant vehicle" of the present invention. In the present invention, when the polynucleotide is RNA, the RNA can be in vitro transcribed RNA (IVT-RNA), and can be obtained by in vitro transcription using a suitable DNA template. At this time, the promoter for regulating transcription can be any promoter of RNA polymerase. In addition, the DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA (polynucleotide of the present invention), and introducing it into a suitable vehicle for in vitro transcription (recombinant vehicle loaded with the polynucleotide of the present invention). When the nucleic acid (polynucleotide of the present invention) is RNA, cDNA can be obtained by reverse transcription.

[0229] Specifically, RNA can be synthesized using a DNA-dependent RNA polymerase, such as T7 RNA polymerase. In this case, the expression vector can contain a T7 promoter at the 5' end of the loaded polynucleotide. When another RNA polymerase, such as SP6 or T3 RNA polymerase, is used during IVT using a loaded polynucleotide expression vector as a template, the vector can contain 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 contain a T7 promoter. Specifically, the recombinant vector can contain the nucleic acid sequence of SEQ ID NO: 94 at the 5' end upstream of the polynucleotide according to the present invention, or can contain a promoter consisting of this sequence.

[0230] The vector that can be used as a template for synthesizing a self-amplified RNA construct can be, for example, a plasmid, a cosmid, a phage, a viral vector, etc. In this case, the polynucleotide sequence can be a DNA sequence, and preferably a plasmid DNA.

[0231] "Plasmid" generally refers to a structure of extrachromosomal genetic material, that is, a circular DNA duplex that can replicate independently of chromosomal DNA.

[0232] In addition, in the present invention, the recombinant vector can replicate spontaneously in the host cell. At this time, the vector can be introduced into the host cell, recombined and inserted into the host cell genome, and replicated together with the host cell genome.

[0233] In this case, the recombinant vector may further comprise a promoter or regulator, or enhancer for controlling the expression of the loaded polynucleotide.

[0234] Additionally, the plasmid may contain a selectable marker, such as an antibiotic resistance gene, and host cells maintaining the plasmid may be cultured under selective conditions.

[0235] Transformed host cells

[0236] In another aspect of the present invention, a host cell transformed with a recombinant vector is provided. The recombinant vector is the same as described above.

[0237] As used herein, the term "transformation" refers to the phenomenon of artificially causing genetic changes by introducing foreign DNA into cells so that the DNA becomes replicable as a chromosomal element, or by introducing DNA into host cells to complete chromosomal integration.

[0238] Above-mentioned " host cell " refers to the cell that makes other microorganisms or gene parasitize and provides nutrition, and refers to wherein carrier is transformed into host cell and causes the cell of various hereditary or molecular effects in host cell.Host cell is in the competent state that can accept external DNA, and external DNA such as carrier can be inserted, and if carrier is successfully introduced into host cell, then the hereditary character of carrier is provided to host cell.Host cell can comprise prokaryotic cell (for example, Escherichia coli) or eukaryotic cell (for example, yeast cell and insect cell), and can comprise mammalian cell, such as the cell that is derived from people, mouse, hamster, pig, goat or primate.

[0239] Transformation can be carried out by various methods. Specifically, for transformation methods, CaCl precipitation, the Hanahan method (its efficiency has been increased by using a reducing agent such as dimethyl sulfoxide (DMSO) in the CaCl precipitation), electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, transformation using PEG, dextran sulfate, lipofectamine or desiccation / inhibition-mediated transformation, etc. can be used. The method for transforming the plasmid of the present invention is not limited to the above examples, and transformation methods commonly used in the art can be used without limitation.

[0240] Methods for producing RNA replicons

[0241] In another aspect of the present invention, a method for producing an RNA replicon is provided, comprising: i) producing a recombinant vector loaded with a polynucleotide according to the present invention; and ii) synthesizing RNA from the recombinant vector. The polynucleotide, recombinant vector, and RNA replicon are the same as described above.

[0242] RNA replicon can be obtained by in vitro transcription using the DNA of a recombinant vector as a template. Generally speaking, the poly (A) tail sequence is encoded by the poly-(dT) sequence on the DNA template. In the present invention, the poly (A) tail sequence can be added with an enzyme after transcription. In addition, in the present invention, the poly (A) tail sequence can be transcribed from a recombinant vector. Preferably, it can be transcribed from a recombinant vector.

[0243] Suitable methods for in vitro transcription are well known in the art and are known to those skilled in the art. For example, they are described in [Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) ed. C Nolan, Cold Spring Harbor Laboratory Press]. In addition, various in vitro transcription kits are commercially available.

[0244] Vaccine composition

[0245] In another aspect of the present invention, a vaccine composition comprising the polynucleotide or recombinant vector of the present invention as an active ingredient is provided. Here, the polynucleotide and recombinant vector are the same as described above.

[0246] As used herein, the term "vaccine" refers to a drug that uses a living organism to stimulate the immune system in order to prevent disease. Immune activation refers to the process of effectively removing antigens by producing antibodies, stimulating T cells, or stimulating other immune cells in the body (e.g., macrophages, natural killer cells). A detailed overview of immunology regarding the above content is readily understood by those of ordinary skill in the art (Barrett, JT, "Textbook of Immunology," 1983). In this specification, "vaccine" or "vaccine composition" can be used interchangeably.

[0247] In the present invention, the vaccine composition can be used to prevent or treat protozoan, fungal, bacterial or viral infections. In the present invention, the vaccine composition can be used to prevent or treat cancer.

[0248] The vaccine composition may include a polynucleotide or recombinant vector as an active ingredient in an amount sufficient to induce a typical immune response. The vaccine composition may include an active ingredient in an amount of about 0.0001 μg to about 1000 μg. Specifically, the active ingredient may be included in an amount of about 0.0001 μg to about 1000 μg, about 0.001 μg to about 100 μg, or about 0.01 μg to about 10 μg, but is not limited thereto.

[0249] The vaccine composition of the present invention may contain an active ingredient in an amount sufficient to induce a typical immune response, and any amount (effective amount) may be used depending on the purpose, formulation, mixing purpose, etc. Typically, the effective amount will be determined within the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. Here, the term "effective amount" refers to the amount of the recombinant microorganism as the active ingredient that is sufficient for the vaccine composition to induce a typical immune response of the vaccine. This effective amount can be determined experimentally within the normal capabilities of those skilled in the art.

[0250] In addition, the vaccine composition may include a pharmaceutically acceptable carrier or diluent. The carrier used for the composition of the present invention includes a pharmaceutically acceptable carrier, an adjuvant and a solvent, 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 can be adapted beyond the application (prescription) target. Pharmaceutically acceptable carriers that can be used for the composition of the present invention include but are not limited to ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer 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 dihydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrates, polyethylene glycol, sodium carboxymethyl cellulose, polyarylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin, etc.

[0251] 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 one of ordinary skill in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health condition, gender, the patient's sensitivity to drugs, the route of administration, the method of administration, the number of administrations, the treatment cycle, the drugs to be used in combination or concurrently, and the like.

[0252] As used herein, the term "administer" means introducing a given substance into a subject by an appropriate method, and with respect to the route of administration of the composition, the composition can be administered by any general route as long as it can reach the target tissue. In order for the vaccine to effectively produce antibodies, the antigenic substance must be introduced into the body so that the antibody production mechanism of the immunized subject is achieved. Therefore, the polynucleotide or recombinant vector according to the present invention must first be introduced into the body for an immune response.

[0253] When polynucleotides according to the present invention or recombinant vectors are used to a subject, they can be encapsulated in lipid nanoparticles (LNPs) or coated with lipids and used in the form of liposomes or vesicles. In this case, the lipid-encapsulated product can be in the form of lipid aggregates or micelles. LNPs can be prepared using methods known in the art.

[0254] In the present invention, in order to stimulate the desired response by the antigen presented by the vaccine composition, systemic administration can be performed by parenteral administration. Parenteral administration can be performed in the form of intranasal, intranasal, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardial, transdermal, subcutaneous, intradermal, intraperitoneal, enteral, topical, sublingual or rectal administration, but is not limited thereto. Preferably, it can be administered subcutaneously or intramuscularly.

[0255] Further, in the present application, the vaccine composition can be administered once or repeatedly several times.

[0256] Specifically, it can be administered once, twice, three times, four times, or five times, but is not limited thereto, and can be administered repeatedly as long as the vaccine is effective. As described above, by repeated administration, the vaccine composition can increase the prophylactic or therapeutic effect on the disease.

[0257] The subject to which the vaccine composition according to the present application is administered can be, for example, a mammal such as a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat, and preferably can be a human.

[0258] The preferred amount of use of the vaccine composition can be prescribed in various ways depending on factors such as the formulation method of the subject to be administered, the administration method, the age, body weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the subject to be administered, and can be appropriately selected by one of ordinary skill in the art. The amount of use suitable for the subject to be administered 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 a conventional vaccine. The amount of use can be easily determined as a desired amount through a routine experimental process. A typical initial vaccine dose can be from about 0.001 µg / kg to about 10 mg / kg or from about 0.01 µg / kg to about 1 mg / kg of antigen, with increased or multiple doses as needed to provide the desired level of protection. The vaccine composition can be administered once a day, and can be administered daily, every other day, weekly, or every other week. Such an amount of use should not be construed as limiting the scope of the present application in any way.

[0259] The vaccine composition can 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 liposome suspension, a polymer, an emulsion, a lipid nanoparticle (LNP) (having RNA on the surface or encapsulated therein), or any other suitable form that can be administered to a human or mammal in need of treatment or vaccination.

[0260] When the vaccine composition is administered intranasally or parenterally, it can be administered in the form of a spray or an 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 art, and can be prepared into a saline solution using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons, and / or other solubilizers or dispersants known in the art.

[0261] When administered parenterally, the vaccine composition according to the present invention can be prepared into the form of a sterile injectable aqueous or oily suspension as a sterile injectable preparation. Suspensions can be prepared using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. Sterile injectable preparations can also be sterile injectable solutions or suspensions (e.g., solutions in 1,3-butanediol) in non-toxic, parenterally acceptable diluents or solvents. Acceptable solvents and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are also commonly used as solvents or suspension media. For this purpose, any fixed oil with low irritation can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives can be used in injectable preparations, as can pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated forms.

[0262] The above preparations are known in the art, and specific references can be found in [Remington's Pharmaceutical Sciences (19th edition, 1995)] etc. The above references are considered to be part of this specification.

[0263] The vaccine composition of the present invention may further comprise an adjuvant to enhance the immunogenicity of the vaccine. Depending on the type of antigen and the indication of the vaccine composition, auxiliary components such as adjuvants may be appropriately selected and used.

[0264] In another aspect of the present invention, a method for preventing or treating a disease is provided, comprising administering a polynucleotide, a recombinant vector, or a vaccine composition to a subject. In this case, the vaccine composition and administration are the same as described above. The polynucleotide, recombinant vector, or vaccine composition can be administered once or repeatedly several times. Specifically, it can be administered once, twice, three times, four times, or five times, and can be repeated indefinitely as long as its preventive or therapeutic effect can be fully demonstrated. As described above, repeated administration can enhance the preventive or therapeutic effect of the disease.

[0265] As used herein, the term "treat" can be used to refer to both therapeutic and prophylactic treatments. In this context, the term "prevent" can be used to refer to alleviating or reducing a pathological condition or disease in a subject. Treatment encompasses all applications or forms of medication used to treat disease in mammals, including humans. Furthermore, the term encompasses inhibiting or slowing the progression of a disease; restoring or repairing an impaired or defective function, thereby partially or completely alleviating the disease; or stimulating an inefficient process; or alleviating a severe disease.

[0266] The disease may be an infectious disease caused by protozoa, fungi, bacteria or viruses, and may also be cancer.

[0267] The subject may be a mammal that has or may have the disease, and preferably may be a human.

[0268] The dosage can be prescribed in various ways depending on factors such as preparation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity.

[0269] Novel protease cleavage site sequence

[0270] In another aspect of the present invention, a 2A protease cleavage site of a nonstructural protein derived from enterovirus is provided, comprising the amino acid sequence of SEQ ID NO: 22. The enterovirus, nonstructural protein and 2A protease cleavage site are the same as described above.

[0271] In another aspect of the present invention, a 3C protease cleavage site of a nonstructural protein derived from enterovirus is provided, comprising the amino acid sequence of SEQ ID NO: 50. The enterovirus, nonstructural protein and 3C protease cleavage site are the same as described above.

[0272] In another aspect of the present invention, an IRES variant is provided that comprises the nucleic acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. The IRES variant is the same as described above.

[0273] New 5' untranslated region variant sequence

[0274] In another aspect of the present invention, a 5' untranslated region variant is provided 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.

[0275] New Kozak sequence

[0276] In another aspect of the present invention, a Kozak sequence is provided comprising the nucleic acid sequence of SEQ ID NO: 35. The Kozak sequence is the same as described above.

[0277] New 2A protease sequence

[0278] In another aspect of the present invention, a non-structural protein 2A protease derived from enterovirus is provided, comprising the amino acid sequence of SEQ ID NO: 75. The 2A protease is the same as described above.

[0279] New severe febrile thrombocytopenia syndrome (SFTS) virus antigen sequences

[0280] In another aspect of the present application, there is provided a severe fever with thrombocytopenia syndrome (SFTS) virus antigen comprising the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 85, SEQ ID NO: 116, or SEQ ID NO: 119. The severe fever with thrombocytopenia syndrome virus is the same as described above.

[0281] New human papillomavirus antigen sequences

[0282] In another aspect of the present application, there is provided a human papillomavirus antigen comprising the amino acid sequence of SEQ ID NO: 138, SEQ ID NO: 141, SEQ ID NO: 144, or SEQ ID NO: 147. The human papillomavirus is the same as described above.

[0283] Modes of carrying out the present application

[0284] Hereinafter, the present application will be described in greater detail by the following examples. However, the following examples are for illustration of the present application only, and the scope of the present application is not limited only to these examples.

[0285] I. Production of optimized self-amplifying RNA

[0286] Preparation Example 1. Production of a vector for producing an enterovirus-based self-amplifying RNA

[0287] A cloning vector for producing an enterovirus-based self-amplifying RNA construct in which luciferase, which is used as a basic backbone for producing an optimized self-amplifying RNA construct in the present application, is inserted as a target protein was produced.

[0288] Specifically, the cloning vector used in the present application was produced by loading the coxsackievirus B5 gene (SEQ ID NO: 99) into a pUC19-CMV / T7 (Addgene, plasmid #50005) vector in the same manner as described in "Development of a universal cloning system for human enterovirus reverse genetics" (WS Choi et al., Microbiol Spectr. 18; e0316722), thereby producing an infectious clone. In the cloning, the P1 domain (structural protein, VP) was replaced with a nucleic acid encoding a target protein (luciferase, SEQ ID NO: 41) using an In-fusion cloning (HD Cloning Kit, Takara) method, to produce a cloning vector in the form of a self-amplifying RNA construct ( ). Figure 16

[0289] ​Example 1. Selection of 2A protease cleavage site sequence for self-amplified RNA

[0290] According to the present invention, in order to generate a modified self-amplifying RNA (saRNA) construct with optimized target protein expression level, the target protein expression level is first confirmed based on the amino acid sequence of the 2A protease (a non-structural protein of enterovirus) cleavage site.

[0291] Example 1.1. Generation of self-amplifying RNA constructs based on the 2A protease cleavage site sequence

[0292] Specifically, by using a known mutagenesis method, the nucleotide sequences of SEQ ID NO: 24 to SEQ ID NO: 29 were inserted into the cleavage site of the 2A protease of a cloning vector in the form of an enterovirus-based self-amplifying RNA construct in which luciferase was inserted as the target protein, which was produced in-house by the method of Preparation Example 1, thereby generating a library vector, which resulted in a sequence of 1 (VP1-1 aa, SEQ ID NO: 18), 5 (VP1-5 aa, SEQ ID NO: 19), 10 (VP1-10 aa, SEQ ID NO: 20), 15 (VP1-15 aa, SEQ ID NO: 21), 20 (VP1-20 aa, SEQ ID NO: 22) or 25 (VP1-25 aa, SEQ ID NO: 23) amino acids being included ( Figure 2a BsmbI (NEB) restriction enzyme was added to the above-mentioned carrier DNA and linearized by reacting at 55°C for 1 to 6 hours, and then inactivated by reacting at 80°C for 20 minutes. The DNA was confirmed to be linear by electrophoresis on a 0.9% agarose gel and then purified as follows.

[0293] Purified linearized DNA was used as template and the DNA was purified by Invitrogen TM MEGAscript TM In vitro transcription (IVT) was performed using a T7 transcription kit at 37°C for 3 hours. After treatment with DNase to remove residual DNA template, the resulting RNA was purified by lithium chloride (LiCl) precipitation (Table 1).

[0294] [Table 1]

[0295]

[0296]

[0297] Example 1.2. Comparison of expression levels of self-amplifying RNA constructs based on 2A protease cleavage site sequences

[0298] The self-amplifying RNA construct produced by the method of Example 1.1 was transfected into 293T cells to confirm the expression level.

[0299] Specifically, 293T cells were seeded at 5 x 105cells / well in a 6-well plate, cultured overnight, and then the medium was replaced with a medium without fetal bovine serum (FBS) 4 hours before transfection to prepare the cells. 5

[0300] Each of the purified RNAs was transfected into the cells at 5 μg per well using Lipofectamine 2000 (Invitrogen). Twelve hours after the transfection, the cells were harvested, centrifuged (12,000 rpm, 3 minutes) to precipitate the cells, Luciferase Cell Culture Lysis 1x Reagent (100 μl) was added and reacted at room temperature for 30 minutes. After the completion of the reaction, the reaction solution was centrifuged again (12,000 rpm, 1 minute), and the luciferase activity of the supernatant (20 μl) was measured using the Luciferase Assay System Reagent (Promega).

[0301] As shown in Table 1, the luminescence of luciferase was the highest in the group treated with the self-amplifying RNA construct (VP1-20 a.a) in which 20 amino acids from the C-terminus of VP1 were inserted. Figure 2b

[0302] Accordingly, the 20-amino acid sequence (SEQ ID NO: 22) from the C-terminus of VP1 was used as a 2A protease cleavage site of the modified self-amplifying RNA construct having the optimized expression level according to the present application.

[0303] Example 2. Selection of Kozak sequence and stop codon

[0304] Example 2.1. Production of self-amplifying RNA construct according to Kozak sequence and stop codon

[0305] In order to produce the modified self-amplifying RNA construct having the optimized expression level of the target protein in the present application, the expression level of the target protein according to the Kozak sequence and the stop codon was confirmed.

[0306] ​​​The self-amplified RNA construct was generated in the same manner as in Example 1.1. In this case, the recombinant vector was a vector in which the Kozak sequence of Kozak1 (SEQ ID NO: 35, CCACC), Kozak2 (SEQ ID NO: 36, GCCACC) or Kozak4 (SEQ ID NO: 37, AAG) was inserted into the 3' end of the 5'UTR. The stop codon used was a sequence consisting of three different stop codons located consecutively at the 5' end of the 3'UTR (3×Stop) (SEQ ID NO: 9, TGATAATAG) (Table 2, Figure 3a ).

[0307] [Table 2]

[0308]

[0309] Example 2.2. Comparison of expression levels of self-amplifying RNA constructs based on Kozak sequence and stop codon

[0310] In the same manner as in Example 1.2, the expression of each self-amplifying RNA construct produced by the method of Example 2.1 was measured in 293T cells using a luciferase assay reagent (Promega). At this time, a cloning vector (original PC) produced in-house by coxsackievirus B5 was used as a control group.

[0311] Results, such as Figure 3b As shown, the luciferase activity was highest in the self-amplifying RNA construct (Kozak4) into which the Kozak sequence of Kozak 4 was inserted. In addition, the luciferase activity was significantly increased in the self-amplifying RNA construct containing the stop codon of SEQ ID NO: 68 (SEQ ID NO: 9 in the case of DNA) compared to the control group.

[0312] Thus, the Kozak sequence used for the modified self-amplifying RNA construct of the present invention with optimized expression levels is the sequence of SEQ ID NO: 42, and the stop codon used is 3x stop of SEQ ID NO: 68 (SEQ ID NO: 9 in the case of DNA).

[0313] Example 3. Selection of poly(A) tail sequence

[0314] Example 3.1. Generation of self-amplifying RNA constructs based on poly(A) tail sequences

[0315] In order to generate a modified self-amplifying RNA construct having an optimized expression level of the target protein of the present invention, the target protein expression level according to the polyadenylic acid tail (poly(A) tail) sequence was confirmed.

[0316] Self-amplifying RNA constructs were generated in the same manner as in Example 1.1. This time, recombinant vectors were generated by inserting 50 (50× poly(A), SEQ ID NO: 38), 70 (70× poly(A), SEQ ID NO: 39), or 120 (120× poly(A), SEQ ID NO: 40) poly(A) at the 3′ end of the 3′ UTR (Table 3, Figure 4a ).

[0317] [Table 3]

[0318]

[0319] Example 3.2. Comparison of expression levels of self-amplifying RNA constructs based on poly(A) tail sequences

[0320] In the same manner as in Example 1.2, the expression of each self-amplifying RNA construct produced by the method of Example 3.1 was measured in 293T cells using a luciferase assay reagent (Progmega).

[0321] In addition, the self-amplifying RNA construct was encapsulated in 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). Ignite (PrecisionNanoSystems), and applied to mice to confirm expression levels at the animal level. At this time, female BALB / c mice (n=3) aged 5-6 weeks were used, and 1 μg of each self-amplification RNA construct was administered intramuscularly to each mouse. 4 hours after administration, luciferin was administered intravenously to mice at a concentration of 150 mg / kg per mouse, and the luciferase activity in the mice was observed by IVIS spectrum (Perkin Elmer).

[0322] Results, such as Figure 4b As shown, at the cellular and animal levels, luciferase activity was highest in the self-amplifying RNA construct inserted with a 120× poly(A) tail.

[0323] Therefore, the poly(A) tail of the modified self-amplifying RNA construct of the present invention with optimized expression level used is the sequence of SEQ ID NO: 40.

[0324] II. Antigen Expression Using Optimized Self-Amplified RNA

[0325] Example 4. Production of optimized self-amplifying RNA and confirmation of expression level

[0326] Example 4.1. Production of optimized self-amplifying RNA

[0327] The optimized self-amplifying RNA construct was produced from the recombinant vector containing the nucleotide sequence of the 2A protease cleavage site (VP1-20 a.a., SEQ ID NO: 28), Kozak sequence (Kozak1, SEQ ID NO: 35), and poly(A) tail (120x poly(A), SEQ ID NO: 40) selected in Examples 1 to 3 in the same manner as in Example 1.1 (Table 4).

[0328] In order to compare the degree of increase in expression level of the modified self-amplifying RNA construct, mRNA and a regular enterovirus-based self-amplifying RNA construct (saRNA) were produced together and used as an experimental group (Example 4.2). Figure 5 At this time, the target protein was cloned into the structure of 5'UTR (SEQ ID NO: 95)-target protein (SEQ ID NO: 41)-3'UTR (SEQ ID NO: 97)-poly(A) tail (50x poly(A), SEQ ID NO: 38) by using 5' and 3'UTR derived from human globulin, and linearized, and then using MESSAGE mMACHINE TM T7 transcription kit (Invitrogen) was reacted for 6 hours at 37℃ using it as a template DNA, and then purified in the same manner as the self-amplifying RNA construct, thereby preparing mRNA (Table 4, Figure 5 ).

[0329] [Table 4]

[0330]

[0331] Example 4.2. Confirmation of expression level of optimized self-amplifying RNA

[0332] The expression level of the modified self-amplifying RNA construct produced by the method of Example 4.1 was confirmed at the cellular and animal levels. In order to confirm at the cellular level, 293T cells were used, seeded into a 12-well plate (2.5x 105cells / well) one day before transfection, and then cultured overnight. 5

[0333] 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.​

[0334] Results, such as Figure 6 As shown, the luciferase activity in the group treated with the modified self-amplifying RNA construct of the present invention was the highest at all RNA concentrations.

[0335] To confirm the expression at the animal level, mRNA, self-amplifying RNA constructs or modified self-amplifying RNA constructs were encapsulated in LNP biodelivery system (ALC-0315 / cholesterol / PEG / DOPE) in the same manner as in Example 3.2. Ignite (Precision NanoSystems) was then administered intramuscularly to mice. RNA was administered at concentrations of 0.01 μg, 0.1 μg, or 1 μg per mouse. Four hours after administration, luciferin was administered intravenously to mice at a concentration of 150 mg / kg per mouse, and luciferase activity was observed in the mice using an IVIS spectrometer (Perkin Elmer).

[0336] Results, such as Figure 7 As shown, the highest luciferase activity was observed in the group administered with the modified self-amplifying RNA construct of the present invention. In particular, activity was clearly observed even in the group administered with 0.01 μg, and no luciferase activity was observed in the group administered with the same concentration of mRNA and a conventional enterovirus-based self-amplifying RNA construct.

[0337] Example 5. Confirmation of Antigen Expression Levels of Optimized Self-Amplifying RNA Constructs Containing Antigens

[0338] Example 5.1. Generation of optimized self-amplifying RNA constructs containing antigens

[0339] In the optimized modified self-amplifying RNA construct of Example 4.1, the target protein was replaced with severe fever with thrombocytopenia syndrome (SFTS) virus antigen, SARS-CoV2 spike protein antigen, highly pathogenic avian influenza (HPAI) virus antigen or canine influenza virus antigen (canine influenza) instead of luciferase to produce a recombinant vector, and the optimized self-amplifying RNA 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 SARS-CoV2 spike protein antigen (SEQ ID NO:48) was used respectively. The highly pathogenic avian influenza virus antigen used was a nucleic acid sequence (SEQ ID NO:106) encoding H5N8 antigen (SEQ ID NO:105). For linearization of the vector DNA, it was reacted with BsmBI (NEB) restriction enzyme at 55°C for 6 hours and inactivated at 80°C for 10 minutes.

[0340] In order to compare the degree of improvement in expression level of the modified self-amplifying RNA construct, mRNA was also produced and used as an experimental group. In this case, the target protein was cloned into a structure of 5'UTR-target protein-3'UTR-poly (A) tail (50) by using 5' and 3'UTRs derived from human globin, and it was linearized and then used as a template DNA using MESSAGE mMACHINE TM The reaction was allowed to proceed at 37°C for 6 hours using a T7 transcription kit (Invitrogen), and then purified in the same manner as for the self-amplified RNA construct to prepare mRNA (Tables 5 to 7).

[0341] [Table 5]

[0342]

[0343]

[0344] [Table 6]

[0345]

[0346] [Table 7]

[0347] Example 5.2. Confirmation of expression levels of optimized self-amplified RNA

[0348] The expression level of the modified self-amplifying RNA construct produced by the method of Example 5.1 was confirmed at the cellular level by Western blotting. At this time, the efficiency of the cellular delivery system (LNP biodelivery system, lipofectamine) was also confirmed using RNA containing SFTS virus antigens ( Figure 8 ).

[0349] First, to prepare the LNP-RNA experimental group, each of the above RNAs (including RNA of SFTS antigen) was encapsulated in a LNP biodelivery system (ALC-0315 / cholesterol / PEG / DOPE). The encapsulated RNA (LNP-RNA) was diluted 10-fold in 1× TE (Tris-EDTA) buffer containing 2% (v / v) Triton X-100 (Sigma Aldrich) in an Ignite (Precision NanoSystems). The fluorescence of the LNP-RNA was then measured using a microplate reader (BMGLABTECH, UK) at 485 nm (excitation) and 528 nm (emission). The RNA concentration corresponding to the fluorescence value was also measured.

[0350] Transfection was performed by treating cells with different concentrations of LNP-RNA or each RNA prepared as described above. At this time, for each RNA, the same method as in Example 1.2 was used. 2000 transfection reagent (Invitrogen) was used for transfection. In addition, cells were plated at 1×10 6 The cells were seeded in a 6-well plate at a concentration of 10 cells / well, and then the medium was replaced with a medium without fetal bovine serum 4 hours before transfection, and the cells were cultured and prepared, and transfection was performed.

[0351] The culture medium was replaced 4 hours after transfection and cultured for another 24 hours. After 24 hours, the cells were harvested by centrifugation (12,000 rpm, 1 minute) and lysed by adding cell lysis buffer (1 ml) and vortexing. After reacting 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 with an equal volume of protein) and reacted at 95°C for 7 minutes to prepare protein samples. Each protein sample was electrophoresed in SDS-PAGE. Thereafter, it was transferred to a membrane, blocked with 5% lipoprotein solution, then a primary antibody was 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 as primary antibodies for their respective antigens.

[0352] After the primary antibody reaction, the membrane was washed 5 times with TBS-T buffer for 10 minutes each time, and then the secondary antibody was added and reacted at room temperature for 1 to 2 hours. As the secondary antibody, anti-rabbit-HRP antibody (abcam, ab205718, 1: 2000) and anti-mouse-HPR antibody (Invitrogen, 62-6520, 1: 5000) were used. After the secondary antibody reaction, each membrane was washed 5 times with TBS-T for 10 minutes each time, and then detected with HRP reagent (Millipore).

[0353] Results, such as Figure 8 As shown, RNA transfected with LNP showed a higher expression level than RNA transfected with lipofectamine (top). In addition, compared with mRNA, it was confirmed that the expression of SFTS antigen ( Figure 8 ) or SARS-CoV2 antigen ( Figure 9 ) of the present invention, antigen expression was significantly higher.

[0354] III. Generation of Multi-Antigen Self-Amplifying RNA

[0355] Example 6. Generation of multi-antigen self-amplifying RNA and confirmation of expression levels

[0356] Example 6.1. Generation of multi-antigen self-amplifying RNA

[0357] In order to produce a vaccine capable of expressing two multiple antigens, the enterovirus self-cleavage peptide P2A (porcine teschovirus-1 2A) or 3C protease cleavage site sequence was inserted into the enterovirus-based self-amplifying RNA construct, and the multi-antigen self-amplifying RNA construct was produced in the same manner as in Example 1.1.

[0358] In this case, luciferase was used as the first target protein, and canine influenza virus antigen (H3N2) was used as the second target protein. The multi-antigen self-amplifying RNA construct was constructed in the order [5'UTR-Kozak sequence-second target protein (H3N2 virus antigen)-P2A self-cleaving peptide or 3C protease cleavage site-first target protein (luciferase)-2A protease cleavage site-2A protease-2B-2C-3C protease-3D-termination sequence-3'UTR-poly(A) tail] (Self-amplifying RNA construct containing P2A: SEQ ID NO: 190, self-amplifying RNA construct containing 3C protease cleavage site: SEQ ID NO: 192). At this time, in order to optimize the expression of multiple antigens, the positions of the target proteins were crossed to prepare self-amplifying RNA constructs, and the expression levels of the proteins were compared (5'UTR-Kozak sequence-first target protein (luciferase)-P2A or 3C protease cleavage site-second target protein (H3N2 virus antigen)-2A protease cleavage site-2A protease-2B-2C-3C protease-3D-terminator sequence-3'UTR-poly (A) tail) (self-amplifying RNA construct comprising P2A: SEQ ID NO: 194, self-amplifying RNA construct comprising 3C protease cleavage site: SEQ ID NO: 196). In addition, a single antigen (luciferase) self-amplifying RNA construct was also prepared and used.

[0359] First, the expression level of the multi-antigen self-amplifying RNA construct was confirmed at the cellular level by luciferase and Western blotting. Luciferase activity was measured using 2000 transfection reagent (Invitrogen), and Western blotting was performed in the same manner as in Example 5.2. At this time, the multi-antigen self-amplifying RNA construct or the single-antigen self-amplifying construct (1 μg or 5 μg, respectively) was transfected into each cell.

[0360] In addition, the primary antibody used in the Western blotting method was an anti-H3N2 antibody produced in-house and diluted to 1:2000, and the secondary antibody was an anti-mouse HRP antibody (Invitrogen, 62-6520, 1:20000). The anti-H3N2 antibody was used by producing an acellular vaccine by the following method. First, 50 μl of purified H3N2 hemagglutinin protein (Mybiosource, 32-5658) at a concentration of 0.1 μg / μl and adjuvant (AddaVax, Invivogen) were mixed at a ratio of 1:1 and administered intramuscularly to mice. After the first administration, three additional administrations were performed at two-week intervals, and the antibody was produced by direct isolation from the blood.

[0361] Results, such as Figure 37 As shown, it was confirmed that the luminescence of luciferase in the multi-antigen self-amplifying RNA construct was lower than that in the single-antigen self-amplifying RNA construct, but the luminescence of luciferase in each self-amplifying RNA construct containing the P2A sequence and the 3C protease cleavage sequence was higher.

[0362] In addition, if Figure 38 As shown, it was confirmed that the construct comprising the P2A sequence and the construct comprising the 3C protease cleavage site showed similar levels of H3N2 expression.

[0363] The above results confirmed that the multi-antigen self-amplifying RNA construct can express two antigens simultaneously.

[0364] Example 6.2. Generation of multi-antigen self-amplifying RNA containing canine influenza antigens

[0365] Using canine influenza subtypes H3N2 and H3N8, two antigens were inserted into an enterovirus-based self-amplifying RNA construct, and a 3C protease cleavage site amino acid sequence (SEQ ID NO: 50) was inserted between the antigens to generate a multi-antigen self-amplifying RNA construct in the same manner as in Examples 1.1 and 4.1 above. In this case, H3N2 and H3N8, which are canine influenza subtypes, were used as antigens (Table 8, Figure 10 and Figure 11 (top of the ).

[0366] In addition, a self-amplifying RNA construct and mRNA containing only H3N2 antigens were also generated and used. In this case, the target protein was cloned into a structure of 5'UTR-target protein-3'UTR-polyadenylic acid tail (50) by using 5' and 3'UTRs derived from human globin, and it was linearized and then used as template DNA using MESSAGE mMACHINE TM The reaction was performed using T7 transcription kit (Invitrogen) in the same manner as in Example 4.1 to prepare mRNA.

[0367] [Table 8]

[0368]

[0369]

[0370] Example 6.3. Confirmation of Antigen Expression by Multi-Antigen Self-Amplified RNA

[0371] The expression level of the multi-antigen self-amplifying RNA construct produced by the method of Example 6.2 was confirmed at the cellular level by Western blotting. 2000 transfection reagent (Invitrogen) was transfected in the same manner as in Example 1.2. At this time, each RNA was treated with a concentration of 0.1 μg, 1 μg or 5 μg per well. The culture medium was replaced 4 hours after transfection, and the cells were cultured for another 24 hours. At this time, PC WT virus was used as a control group. At this time, P.CWT virus was canine influenza virus (A / canine / Korea / AS-01 / 2012). Western blotting was performed in the same manner as in Examples 5.2 and 6.1 above.

[0372] Results, such as Figure 11 Shown, confirmed that compared with the group processed with mRNA, antigen expression significantly increased (lower left) in the group processed with self-amplification RNA construct.Observe, compared with the group processed with the self-amplification RNA construct comprising single antigen, the expression level in the group processed with multi-antigen self-amplification RNA construct is slightly low.In addition, in the group processed with multi-antigen self-amplification RNA construct, observe two types of antigens (lower right) of fusion form and separation form.

[0373] The above results confirmed that the multi-antigen self-amplifying RNA construct can express two antigens simultaneously.

[0374] IV. Generation of Optimized Self-Amplifying RNA with Enhanced Proliferation Rates

[0375] Example 7. Virus cell and animal adaptation experiments with enhanced virus proliferation rates

[0376] Example 7.1. Production of Cell-Adapted Coxsackievirus B5

[0377] By using wild-type coxsackievirus B5 ( Figure 12 Vero cells were infected with Coxsackievirus B5 (Vp8-B5) to produce cell-adapted virus. 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 infected with 1×10 6 The cells were seeded into 6-well plates at a concentration of 10 cells / well.

[0378] On the day of infection, the virus was washed with physiological saline and then diluted 1:100 in DMEM (Gibco, Cat. No. 12430-054) without fetal bovine serum and treated with it. After treatment, the cells were cultured for 2 hours, and then the culture medium was replaced with DMEM containing 2% fetal bovine serum (FBS), and the cells were cultured for another 2-4 days to examine cytopathic effects.

[0379] When morphological changes were observed in 80% to 90% of infected cells, the cells were harvested and centrifuged (1,200 rpm, 3 minutes). The cell pellet was then frozen and thawed three times to extract the virus from the cells. The extracted virus was stored at -80°C until the next infection. The above process was repeated eight times.

[0380] As a result, it was confirmed that subcultured coxsackievirus B5 showed an increased virus proliferation rate in infected cells, and morphological changes of cells occurred more rapidly, compared to wild-type coxsackievirus B5.

[0381] Example 7.2. Generation of Mouse-Adapted Coxsackievirus B5

[0382] Cell-adapted coxsackievirus B5 obtained in the same manner as in Example 7.1 above was intraperitoneally administered to female BALB / c mice (SAMTACO) aged 3-4 weeks at a concentration of 5.5TCID / 200 μl per mouse. Body weight changes were observed for 7 days after administration to determine whether the virus causes pathogenicity in mice. As a result, weight loss was confirmed 3-4 days after infection. 4 days after infection, when weight loss was observed in mice, the mice were killed by cervical dislocation and then the intestines were separated. The separated intestines were transferred to a test tube and then 500 μl of culture medium was added for homogenization. The homogenized intestinal tissue was centrifuged at 12,000 rpm for 3 minutes, and then the supernatant was harvested. The supernatant was used to measure the virus concentration and was used to infect other mice in the same manner as above. The above-mentioned infection process was carried out a total of 24 times ( Figure 12 VP8Mp24-B5 in ).

[0383] Example 7.3. Evaluation of the proliferation rate of cell- and mouse-adapted coxsackievirus B5

[0384] In order to confirm the changes in the proliferation rate of Coxsackievirus B5 obtained in the same manner as in Examples 7.1 and 7.2 above, a medium tissue culture infectious dose (TCID 50) experiment was performed using Vero cells.

[0385] 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). 6 Vero cells were seeded in 96-well plates at a concentration of 10 cells / well. Each virus was prepared by serially diluting the stock solution 1 / 10 for a total of 7 times.

[0386] Vero cells prepared as described above are washed with normal saline, then processed with the virus concentration (50 μ l / well) of each preparation. Two hours later, replace the culture medium with the DMEM culture medium containing 2% FBS, and further culture the cells. After infection for three days, when observing morphological changes in 80% to 90% of the cells, process each cell with 10% formalin and react for 20 minutes with fixed cells. Then, with 1.25% crystal violet dye, the cells were dyed for 30 minutes, then washed with distilled water, and observed the remaining cells in each well. In addition, the Reed-Muench method is used to quantify the result.

[0387] As a result, it was confirmed that the virus titer was significantly increased in the groups treated with VP8-B5 or VP8M24-B5 compared to WT-B5 ( Figure 13 ).

[0388] Example 7.4. Analysis of the nucleic acid sequence of mouse-adapted coxsackievirus B5

[0389] The nucleotide sequence of mouse-adapted coxsackievirus 5 obtained in the same manner as in Example 7.2 above was analyzed.

[0390] Specifically, RNA was extracted from the virus to generate cDNA, and then the entire viral gene was amplified using universal primers (SEQ ID NO: 108, SEQ ID NO: 109). The PCR product obtained by the above process was used for Sanger sequencing.

[0391] As a result, nucleotide sequence mutations were confirmed in the 5'UTR (2 sites) and 2A protease (2 sites) of the nucleic acid sequence of mouse-adapted coxsackievirus B5 compared to wild-type coxsackievirus B5 (Tables 9 and Figure 14 ). The mutated sequences are underlined in the table below.

[0392] [Table 9]

[0393]

[0394]

[0395] Therefore, the optimized self-amplifying RNA construct based on enterovirus of the present invention may comprise the components of Table 10 and Table 11 below ( Figure 15 ).

[0396] [Table 10]

[0397]

[0398]

[0399] [Table 11]

[0400]

[0401] V. Evaluation of the in vivo efficacy of self-amplifying RNA

[0402] Example 8. Evaluation of vaccine efficacy of self-amplifying RNA containing highly pathogenic avian influenza virus antigens

[0403] Example 8.1. Production of self-amplifying RNA and mRNA and animal experiments

[0404] To evaluate the efficacy of self-amplifying RNA as a construct at the animal level, the self-amplifying RNA (SEQ ID NO: 111) or mRNA (mRNA vaccine, SEQ ID NO: 167) containing highly pathogenic avian influenza produced in the same manner as in Example 5.1 above (Table 7) was encapsulated (saRNA-LNP, mRNA-LNP) in the same manner as in Example 5.2 above using a lipid nanoparticle biodelivery system (ALC-0315 (MEC, HY-138170) / cholesterol (Sigma, C8667) / DMG-PEG 2000 (Avanti Polar Lipids, 880151P) / DOPE (Avanti Polar Lipids, 850725P). Ignite (Precision NanoSystems), and the mice were intramuscularly administered at a concentration of 10 μg, 1 μg, 0.1 μg or 0.01 μg, respectively (first immunization). As a control group, 5 μg of HA hemagglutinin purified protein (Sinobiological, A / broiler duck / Korea / Buan2 / 2014) hemagglutinin (50 μl) and adjuvant (AddaVax, Invivogen) were mixed at a ratio of 1: 1 and administered intramuscularly to the mice. At this time, PBS was administered to the control group. Four weeks after the first administration (immunization), self-amplified RNA or mRNA was administered to the mice in the same manner as above (second immunization). Blood was collected from the mice 4 weeks after the first administration of the vaccine and 2 weeks after the second administration (6 weeks after the first administration), and serum ( Figure 17 ).

[0405] Example 8.2. Evaluation of Neutralizing Antibody Formation

[0406] In the laboratory mice of the above-described Example 8.1, blood was collected from the mice 4 weeks after the first administration of the vaccine and 2 weeks after the second administration (6 weeks after the first administration), serum was obtained, and whether neutralizing antibodies were formed was determined. At this time, the serum obtained as described above was inactivated by reacting at 56°C for 1 hour, and then used.

[0407] The formation of neutralizing antibodies was evaluated by using MDCK cells to confirm hemagglutination test and T cell response.

[0408] Specifically, for the hemagglutination test, MDCK cells (Madin-Darby canine kidney cells) were seeded into a 96-well plate at a concentration of 1 × 10 6 cells / well, and then incubated at 37°C overnight to prepare cells. For the virus, highly pathogenic avian influenza virus (A / mallard / Korea / W452 / 2014 (H5N8)) was diluted in TCID 100, and serum was diluted with serum-free medium containing TPCK (toluene-p- sulfonyl-phenylalanyl chloromethyl ketone, Thermo Fisher Scientific, 20233)-treated trypsin (Thermo Fisher, 4370285) (1 ×) at a ratio of 10:1. Then, the virus dilution and trypsin dilution were mixed at a ratio of 1:1, and reacted at 37°C for 1 hour to prepare a virus dilution.

[0409] The MDCK cells prepared as described above were washed twice with PBS, treated with 50 μl of the prepared virus dilution per well, and reacted at 37°C to infect the cells. One hour after the infection, the cell culture solution was replaced with serum-free medium containing trypsin (Thermo Fisher, 4370285) (1 ×) containing TPCK (Thermo Fisher scientific, 20233), and further incubated. Forty-eight hours after the infection, the cell culture solution was serially diluted 1 / 2 in PBS a total of 11 times, and then each dilution was mixed with a turkey red blood cell dilution at a concentration of 0.5% at a ratio of 1:1, and reacted at room temperature for 30 minutes. At this time, if no neutralizing antibodies were formed, the virus and the red blood cells were aggregated.

[0410] As a result, as shown in Figure 18 (top), for the serum obtained 4 weeks after the first vaccine administration, the formation of neutralizing antibodies was confirmed to increase depending on the concentration of the administered vaccine in the serum of the group administered with the self-replicating RNA vaccine (saRNA-2.3.4.4).

[0411] On the other hand, the formation of neutralizing antibodies was not confirmed in the group administered with the mRNA vaccine (mRNA-2.3.4.4). In addition, for the serum obtained 2 weeks after the second vaccine administration (6 weeks after the first vaccine administration), it was confirmed that the formation of neutralizing antibodies increased according to the vaccine concentration administered in the serum of the group administered with the self-amplified RNA vaccine (saRNA-2.3.4.4). In the group administered with the mRNA vaccine (mRNA-2.3.4.4), the formation of neutralizing antibodies was confirmed only in the group administered with a high dose (10 μg, 1 μg), but not in the group administered with a low dose (0.1 μg, 0.01 μg).

[0412] T cell responses were confirmed using ELIspot analysis of cells secreting antigen-specific IFN-γ. TM ELISPOT Mouse IFNγ ELISPOT Set (BD, Cat. 551083) was used.

[0413] Specifically, the capture antibody was diluted in DPBS at a ratio of 1:200, and then 100 μl was added to each well of each 96-well plate, and then coated by reacting at 4°C for 18 hours. Thereafter, each well was washed with DPBS (Welgene) and blocked with RPMI1640 medium containing 10% heat-treated BCS (bovine calf serum, Welgene) and 1% penicillin / streptomycin (Gibco). Then, the spleen cells isolated from the experimental mice of Example 8 above were incubated at 1.0×10 6 Cells were seeded into each well at a concentration of 10 cells / well, and then the cells were stimulated by treating them with 20 μg / ml of H5N8 peptide (Peptron).

[0414] At this time, for the negative control group, Splenocytes from mice treated with saRNA-B virus (10 μg) and protein + adjuvant (5 μg).

[0415] After that, each well was washed three times with PBS-T (200 μl), and then the primary antibody was diluted in DPBS containing 10% BCS at a ratio of 1:250 and added to each well at 100 μl for reaction. After that, the cells were treated with secondary antibody, and the number of spots generated by HRP substrate reaction was counted.

[0416] Results, such as Figure 18 As shown in the bottom of the figure, it was confirmed that the group administered with self-amplified RNA (saRNA-2.3.4.4) effectively produced neutralizing antibodies even at a low concentration compared to the group administered with mRNA (mRNA-2.3.4.4).

[0417] In addition, it was confirmed that in the group administered with self-amplifying RNA (saRNA-2.3.4.4) and the group administered with mRNA (mRNA-2.3.4.4), T cells were activated depending on the vaccine concentration administered. In particular, it was confirmed that a dose-dependent T cell response was shown. In particular, compared with the group administered with the same concentration of mRNA (mRNA-2.3.4.4), the group administered with 10 μg of self-amplifying RNA (saRNA-2.3.4.4) showed about twice the T cell activity, and the group administered with 1 μg showed about four times the T cell activity. Based on the above results, it was confirmed that the CD8 T cell response in the group administered with self-amplifying RNA (saRNA-2.3.4.4) was higher than that in the group administered with mRNA (mRNA-2.3.4.4).

[0418] Example 8.3. Measurement of changes in mouse body weight and survival rate

[0419] In Example 8.1 above, laboratory mice were infected with 452 virus (10 MLD, A / mallard / Korea / W452 / 2014 (H5N8)) via the nasal cavity 2 weeks after the second vaccine administration (6 weeks after the first vaccine administration), and the body weight and survival rate of the mice were monitored for 14 days.

[0420] Results, such as Figure 19 As shown, the group administered with self-amplified RNA (saRNA-2.3.4.4) showed a 100% survival rate. On the other hand, the group administered with mRNA (mRNA-2.3.4.4) showed a 100% survival rate only at high concentrations (10 μg, 1 μg), while the group administered with low concentrations (0.1 μg, 0.01 μg) showed a 0% survival rate after 9 days, similar to the control group (PBS-administered group, no treatment) ).

[0421] Example 8.4. Measurement of Residual Virus Titers in Mouse Tissues

[0422] Some of the experimental mice in Example 8.3 were sacrificed on the 3rd or 5th day after virus infection, and tissues of each organ were extracted to measure the residual virus titer in each organ.

[0423] Results, such as Figure 20a and Figure 20bAs shown, in the group administered with self-amplifying RNA (saRNA-2.3.4.4), no viral titer was confirmed in any of the groups except the lung tissue of the group administered with 0.01 μg. On the other hand, in the group administered with mRNA (mRNA-2.3.4.4), no viral titer was confirmed in any tissue in the groups administered with high concentrations (10 μg, 1 μg). However, the above results confirmed that self-amplifying RNA has better activity than mRNA in the groups administered with mRNA.

[0424] Example 9. Evaluation of Neutralizing Antibodies Against Severe Febrile Disease with Thrombocytopenia Syndrome Virus

[0425] Example 9.1. Production of self-amplifying RNA and animal experiments

[0426] In order to evaluate the efficacy of self-amplifying RNA as a construct at the animal level, self-amplifying RNA (self-amplifying RNA vaccine) containing severe fever with thrombocytopenia syndrome virus (SFTS) antigen was produced in the same manner as in Example 5.1 above (Tables 5 and 12). At this time, type A (SEQ ID NO: 116), type B (SEQ ID NO: 45), type DEF (SEQ ID NO: 119), or type ABDEF (SEQ ID NO: 85) was used as a severe fever with thrombocytopenia syndrome virus antigen.

[0427] [Table 12]

[0428]

[0429] The self-amplifying RNA (self-amplifying RNA vaccine) or mRNA (mRNA vaccine) produced as described above was encapsulated (saRNA-LNP, mRNA-LNP) in the same manner as in Example 8.1 using a lipid nanoparticle biodelivery system (ALC-0315 (MEC, HY-138170) / cholesterol (Sigma, C8667) / DMG-PEG 2000 (Avanti Polar Lipids, 880151P) / DOPE (Avanti Polar Lipids, 850725P). Ignite (Precision NanoSystems) was added and administered to BALB / c mice (6 weeks old, n=2) or C57BL / 6 mice (6 weeks old, n=5) at a concentration of 5 μg / 100 μl / mouse per mouse (first immunization). Three weeks after the first administration (immunization), the same concentration of the self-amplifying RNA vaccine was administered in the same manner (second immunization). Two weeks after the second administration (5 weeks after the first administration), blood was collected from the mice by orbital bleeding, and serum was obtained ( Figure 21a ).

[0430] Example 9.2. Evaluation of Neutralizing Antibody Production

[0431] Example 9.2.1. Evaluation of Neutralizing Antibody Production in Mice

[0432] The production of neutralizing antibodies was confirmed using immunofluorescence staining in Vero E6 cells.

[0433] Specifically, Vero E6 cells were cultured in DMEM (Gibco, catalog number 12430-054) containing 10% fetal bovine serum and then cultured at 1.5 × 10 4 The cells were seeded into 96-well plates at a concentration of 10 cells / well and cultured for 24 hours. The antibodies obtained from mice were inactivated by reaction at 56°C for 30 minutes. Antibodies against SFTSD virus (NCCP No. 43265) (100 TCID 50 ) were serially diluted 1 / 2 in PBS a total of 10 times, mixed with virus at a 1:1 ratio, and incubated at 37°C for 1 hour before cell incubation. One hour after incubation, the culture medium was replaced with DMEM supplemented with 2% fetal bovine serum, and incubation was continued at 37°C for 120 hours.

[0434] 120 hours after serum and virus treatment, the cells were fixed with 80% acetone at 4 ° C for 10 minutes, dried for 5 minutes, and then washed with PBS. The fixed cells were treated with 3% BSA solution (100 μl), blocked for 1.5 hours, and then washed 6 times with PBST. Thereafter, the cells were treated with 50 μl of primary antibody (anti-SFTS antibody) per well and then reacted at 37 ° C for 3 hours. At this time, the primary antibody was an internally produced antibody and was obtained two weeks after the second vaccination by using an LNP vaccine for antigens against saRNA-A, saRNA-B, saRRNA-DEF or saRNA-ABDEF constructed by our company, and intramuscularly inoculated C57BL / 6 or BALB / c mice twice at a three-week interval at a concentration of 5 μg / mouse or 1 μg / mouse.

[0435] After the reaction was completed, each well was washed 5 times with PBST (100 μl) to remove unbound antibodies, and then the secondary antibody (Alexa Fluor 488 goat anti-mouse IgG antibody, Invitrogen, A11029) was diluted in PBST at a ratio of 1:500 and treated with the cells (50 μl / well), followed by reaction at 37° C. for 1.5 hours. Then, each well was washed three times with PBST, treated with 200 μl PBST per well, and observed with a fluorescence microscope.

[0436] Results, such as Figure 21bAs shown in FIG, in the case of antibodies obtained from BALB / c mice, FITC fluorescence values ​​were observed in Vero E6 cells infected with SFTS virus. On the other hand, no fluorescence values ​​were observed in Vero E6 cells not infected with SFTS virus. Figure 21c As shown, when antibodies were obtained from C57BL / 6 mice, it was confirmed that the self-amplifying RNA vaccine had a maximum neutralizing antibody value of 32 against the SFTSD virus. In other words, it was confirmed that antibodies against the SFTSD virus were formed using the self-amplifying RNA vaccine produced by our company.

[0437] Example 9.2.2. Evaluation of Neutralizing Antibody Production in Ferrets

[0438] Self-amplifying RNA (4 types) (saRNA-LNP, self-amplifying RNA vaccine) produced and encapsulated in the same manner as in Example 9.1 was administered intramuscularly to each mouse at a concentration of 15 μg / 500 μl / mouse (first immunization). After the first administration, the second and third administrations were performed at intervals of 3 weeks in the same manner as the first administration (secondary immunization and tertiary immunization). Blood was collected 3 weeks after the first, second, or third administration, and serum was obtained by centrifugation (8,500 rpm, 10 minutes). Figure 22a ).

[0439] The production of neutralizing antibodies in ferret serum was confirmed by cell experiments. At this time, the serum was inactivated by reacting at 56°C for 30 minutes before use.

[0440] Specifically, Vero E6 cells were cultured at a rate of 1 × 10 4 Cells were seeded at a concentration of 100 cells / well in a 96-well plate and cultured for 24 hours. Viruses were prepared by mixing serum with 100 TCID50 of SFTS type B (NCCP No. 43273), SFTS type C (NCCP No. 43332), SFTSD (NCCP No. 43265), or SFTS type E (NCCP No. 43333) virus at a ratio of 1:1 and reacting at 37°C for 1 hour.

[0441] The cells prepared as described above were treated with a mixture of 50 μl of virus and serum per well, reacted for 1 hour, and then the culture medium was replaced with a DMEM culture medium containing 2% fetal bovine serum, and the cells were then cultured for 120 hours. After infection for 120 hours, the cells were fixed with 80% acetone at 4°C for 10 minutes. The cells were dried for 5 minutes, washed with PBS (150 μl), and then blocked by treating with 3% BSA solution (100 μl) for 1.5 hours. Then, the cells were washed three times with PBST (100 μl), then treated with a primary antibody produced in-house with 50 μl per well, and the reaction was carried out at 37°C for 3 hours. Thereafter, the cells were washed five times with PBST (100 μl), then treated with a secondary antibody, and the reaction was carried out at 37°C for 1.5 hours. At this time, an anti-mouse IgG (γ) antibody (Seracare, 5220-0460) labeled with human serum adsorbed and peroxidase as a secondary antibody was diluted in PBST at a ratio of 1: 1000, and the cells were treated with 50 μl / well. After the secondary antibody reaction, the cells were stained using DAB staining and then observed to confirm the extent of antibody production.

[0442] The results confirmed that 3 weeks after the first vaccination, the average neutralizing antibody value increased from a minimum of 40 to a maximum of 320 ( Figure 22b ), and 3 weeks after the second vaccination, the average neutralizing antibody value increased from a minimum of 160 to a maximum of 905 ( Figure 22c In addition, it was confirmed that the average neutralizing antibody value increased from a minimum of 320 to a maximum of 5120 (3 weeks after the third vaccination). Figure 22d ).

[0443] Example 10. Evaluation of the efficacy of canine influenza virus vaccines

[0444] Example 10.1. Generation of self-amplified RNA and confirmation of expression at the cellular level

[0445] Single-antigen self-amplifying RNA or multi-antigen self-amplifying RNA containing canine influenza virus antigens (H3N2; SEQ ID NO: 53, H3N8; SEQ ID NO: 56) was produced in the same manner as in Example 6.2 above (Tables 8 and 13).

[0446] [Table 13]

[0447]

[0448] Self-amplifying RNA (self-amplifying RNA vaccine) was encapsulated (saRNA-LNP) in a lipid nanoparticle biodelivery system (ALC-0315 (MEC, HY-138170) / cholesterol (Sigma, C8667) / DMG-PEG 2000 (Avanti Polar Lipids, 880151P) / DOPE (Avanti Polar Lipids, 850725P)). Ignite (Precision NanoSystems), and then treated 293T cells at different concentrations to confirm the expression level of self-amplified RNA in the cells in the same manner as in Example 5.2. Anti-H3N2 antibody and anti-H3N8 antibody were used as primary antibodies after dilution at a ratio of 1:2000 or 1:1000, respectively. In this case, the anti-H3N2 antibody was produced in-house in the same manner as in Example 6.2 above. The day before transfection, cells were plated at 1×10 6 The cells were seeded in a 6-well plate at a concentration of 100 cells / well, and then the medium was replaced with a medium without fetal bovine serum 4 hours before transfection, and the cells were cultured and prepared.

[0449] The culture medium was replaced 4 hours after transfection and cultured for another 24 hours. After 24 hours, the cell pellet was obtained by centrifugation (12,000 rpm, 1 minute), and the cells were then lysed by treating with cell lysis buffer (1 ml) and vortexing. The lysate was reacted at room temperature for 10 minutes, and then the protein concentration was measured. 10 μg of protein was mixed with 2× sample buffer (SDS sample buffer) in a ratio of 1:1, and then reacted at 95°C for 7 minutes to prepare protein samples. Each protein sample was electrophoresed in SDS-PAGE. Thereafter, it was transferred to a membrane, blocked with a 5% lipoprotein solution, and then a primary antibody was added and reacted at 4°C for 16 hours. After the primary antibody reaction, the membrane was washed 5 times with TBS-T buffer for 10 minutes each time, and then the secondary antibody was added and reacted at room temperature for 1 to 2 hours. After the secondary antibody reaction, each membrane was washed 5 times with TBS-T for 10 minutes each time, and then detected with HRP reagent (Millipore).

[0450] The primary antibody (anti-H3N2 antibody) was produced in-house and used at a dilution of 1:2000, and the secondary antibody was an anti-mouse-HRP antibody (Invitrogen, 62-6520, 1:5000). At this time, the anti-H3N2 antibody was prepared as an LNP vaccine of the saRNA-H3N8 antigen constructed by our company, and was intramuscularly administered to mice twice at a concentration of 10 μg / mouse at an interval of 4 weeks, and then the serum was separated and used 2 weeks after the second vaccination. Serum was obtained by collecting blood in a test tube without anticoagulant, leaving it for 24 hours, and then centrifuging (8,500 rpm, 15 minutes, repeated 2 times).

[0451] The secondary antibody used was anti-mouse HPR antibody (1:5000, Invitrogen, 62-6520). After the secondary antibody reaction, each membrane was washed 5 times with TBS-T, each time for 10 minutes, and then detected with HRP reagent (Millipore).

[0452] Results, such as Figure 23 As shown, it was confirmed that the expression of each antigen increased in a concentration-dependent manner depending on the treatment with the self-amplified RNA.

[0453] Example 10.2. Animal experiments

[0454] Self-amplifying RNA (self-amplifying RNA vaccine) encapsulated in the same manner as in Example 10.1 above was administered to BALB / c mice at various concentrations. At this time, the self-amplifying RNA used included H3N2 single-antigen self-amplifying RNA (SAH3N2), H3N8 single-antigen self-amplifying RNA (SAH3N8), and H3N2 and H3N8 multi-antigen self-amplifying RNA (SAH3N2+H3N8), and was administered intramuscularly (first immunization) at concentrations of 5 μg, 1 μg, or 0.1 μg per mouse.

[0455] At this time, a positive control group was used by mixing inactivated canine influenza virus (A / canine / Korea / AS-01 / 2012) and canine influenza H3N8 virus (A / canine / Massachusetts / 26810 / 2016) with aluminum hydroxide gel adjuvant (InvivoGen) at a ratio of 1:1. The inactivated canine virus was prepared by inoculating H3N2 or H3N8 canine influenza virus into hatching eggs, incubating them at 37°C for 48 hours, obtaining allantoic fluid, mixing it with formalin (containing formalin at a final concentration of 0.025%), and inactivating it at 4°C for 72 hours. Whether the virus was inactivated was determined by confirming that the virus did not proliferate after inoculating another hatching egg with the virus. As a positive control group, the inactivated H3N2 or H3N8 canine influenza virus was mixed and then inoculated with 10 6The negative control group was vaccinated with PBS without antigen.

[0456] PBS was administered to the group containing no antigen (NC) as a control group.

[0457] In addition, the second administration was performed 4 weeks after the first administration in the same manner as the first administration (second immunization). Blood was collected from mice 2 weeks and 4 weeks after the first administration and 2 weeks after the second administration (6 weeks after the first administration), and serum was obtained ( Figure 24 ).

[0458] Example 10.3. Evaluation of Neutralizing Antibody Production

[0459] The production of neutralizing antibodies was evaluated using serum obtained from the laboratory mice of Example 10.2 above (4 and 6 weeks after the first administration). At this time, the serum was prepared by mixing at a ratio of 1:3 (serum:RDE) using an RDE receptor destroying enzyme kit (SEIKEN), reacting at 37°C for 18 hours, and then inactivating RDE at 56°C for 30 minutes.

[0460] Neutralizing antibody formation was assessed using both hemagglutination inhibition and cell-based assays. Hemagglutination inhibition and cell-based assays were performed in the same manner as in Example 8.2. Blood cells were treated with canine influenza H3N2 virus (A / canine / Korea / AS-01 / 2012) at a HAU 4-8 (HA unit) level or diluted to TCID100, mixed with serum at a 1:1 ratio, and incubated at 37°C for 1 hour before use in the cell-based assay.

[0461] For canine influenza H3N8 virus (A / canine / Massachusetts / 26810 / 2016), the virus was produced in-house and used, and blood cells were treated at the same HAU 4-8 (HA unit) level as above, or diluted to TCID100, mixed with serum at a ratio of 1:1, reacted at 37°C for 1 hour, and then used in cell experiments.

[0462] At this time, canine influenza H3N8 virus was generated by the following method.

[0463] The cells used were MDCK (Marbin-Darby canine kidney) cells and 293T cells, and they were isolated one day before transfection by mixing them at a ratio of 1:2 (MDCK cells: 0.25×10 6 cells / well, 293T cells: 0.5×10 6 cells / well) were seeded in 6-well plates for co-culture.

[0464] 24 hours after seeding, the culture medium was replaced with culture medium without fetal bovine serum, and then the cells were used for experiments. Canine influenza virus was prepared by loading polynucleotides (SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181) encoding the amino acid sequence of PB2 (SEQ ID NO: 170), PB1 (SEQ ID NO: 172), PA (SEQ ID NO: 174), H3N2-HA (SEQ ID NO: 184), H3N8-HA (SEQ ID NO: 186), NP (SEQ ID NO: 178), NA (SEQ ID N: 180), or M (SEQ ID NO: 182) into a PHW2000 vector (Hoffmann et al., PNAS, 97: 6108-6113 (2000)), and then mixing equal amounts (1 μg) of each vector (plasmid DNA).

[0465] The plasmid DNA prepared as described above and transfection reagent (TransIT-LT1, Mirus Bio) (18 μ l) were mixed in an EP tube and reacted at room temperature for 3 minutes. After the reaction, a culture medium (194 μ l) was added that did not contain fetal bovine serum, and the reaction was continued for another 45 minutes at room temperature. The MDCK cells and 293T cells prepared were treated with the reaction solution and transfected by culturing for 24 hours at 37°C. 24 hours after transfection, the cell culture fluid was replaced with a culture medium (1 ml) that did not contain fetal bovine serum, and the culture was continued for another 24 hours. The culture medium containing 1 μ g / ml TPCK was then added that did not contain fetal bovine serum, and the culture was continued for 48 hours. After 48 hours, the cell culture fluid was obtained and used as a transfection sample.

[0466] To confirm the production of virus after transfection, 2.5 × 10 5 MDCK cells were prepared by seeding the cells at a concentration of 10 cells / well in a 6-well plate and culturing for 24 hours. After 24 hours, the cell culture medium was replaced with MEM medium (Corning, 10-010-CV) without fetal bovine serum, and then the cells were treated with the transfection sample (1 ml) prepared as described above and reacted at 37°C for 1 hour. A medium without fetal bovine serum containing 1 μg / ml TPCK was added, and the cells were cultured for 48 hours. Virus production was then confirmed by hemagglutination assay, and the production of canine influenza H3N8 virus was confirmed before use.

[0467] At this point, a hemagglutination reaction was prepared by serially diluting PBS and virus 11 times in half in a 96-well round-bottom plate. The reaction solution was treated with a 0.5% turkey red blood cell dilution at a 1:1 ratio and allowed to react at room temperature for 30 minutes. If virus was produced at this point, viral binding to blood cells was confirmed.

[0468] Results, such as Figure 25a and Figure 25b As shown, neutralizing antibodies were confirmed to be formed in all groups after the second administration of self-amplified RNA. At this time, the self-amplified RNA containing H3N2 showed a titer of 1024 MNT in the group administered 1 μg and the group administered 5 μg, while the self-amplified RNA containing H3N8 showed a titer of 40960 MNT in the group administered 1 μg and the group administered 5 μg. In addition, high-level neutralizing antibody production was confirmed even in the group administered 0.1 μg.

[0469] Example 10.4. Measurement of changes in mouse body weight and survival rate

[0470] In the same manner as in Example 10.1 above, mice were administered self-amplified RNA (first administration and second administration). At this time, 4 weeks after the first administration (28 days after vaccination, 28 dpv) and 2 weeks after the second administration (6 weeks after the first administration, 42 days after vaccination (42 dpv)), all administration groups except the control group were infected with H3N2 or H3N8 canine influenza virus (10 MLD) via the nasal cavity, and the body weight and survival rate of the mice were monitored for 14 days. In addition, some mice were sacrificed on the 3rd and 5th days after infection, lung tissue was extracted, and the virus titer in the lung tissue was measured ( Figure 26 ).

[0471] Results, such as Figures 27a to 27d As shown, it was confirmed that the group administered with multi-antigen self-amplifying RNA (SAH3N2+H3N8) had a high survival rate and a low weight loss compared with the group administered with single-antigen self-amplifying RNA (SAH3N2 or SAH3H8).

[0472] In addition, if Figure 28a and Figure 28bAs shown in the figure, in the case of mice infected with the virus 4 weeks after the first vaccine administration (28 dpv), it was confirmed that the virus titer in the lung tissue of the group administered with the multi-antigen self-amplifying RNA (SAH3N2+H3N8) was reduced compared with the group administered with the single antigen self-amplifying RNA (SAH3N2 or SAH3H8). In particular, when the lung tissues extracted 5 days after the virus infection (5 dpi) were examined, it was confirmed that the proliferation of the H3N2 virus in the lung tissues of the mice infected with the H3N2 virus was significantly reduced, and no viral proliferation was observed in the lung tissues of the mice infected with the H3N8 virus ( Figure 28a In addition, in the case of mice infected with the virus 2 weeks after the second vaccine administration (42 dpv), it was confirmed that all mice in the group administered with self-amplified RNA had a higher survival rate and less weight loss than the control group. In addition, it was confirmed that there was no virus proliferation in the lung tissues extracted 3 days and 5 days after infection ( Figure 28b ).

[0473] The above results confirmed that the vaccine efficacy was better when H3N2 antigen and H3N8 antigen were administered in combination than when H3N2 antigen or H3N8 antigen was administered alone, and that a sufficient virus suppression effect could be obtained with only the first administration in the case of combined administration.

[0474] Example 11. Evaluation of the efficacy of preventing or treating cancer caused by human papillomavirus

[0475] Example 11.1. Generation of self-amplified RNA containing human papillomavirus antigens and confirmation of expression at the cellular level

[0476] The following fusion proteins were used as human papillomavirus antigens (E6 / E7 domains, sequential arrangement of E6 / E7, cross arrangement (swap) of E6 / E7, odd-even arrangement of E6 / E7) ( Figure 29a ).

[0477] The E6 / E7 domain (SEQ ID NO: 139) is a fusion protein that comprises the amino acid sequence of the human papillomavirus E6 domain (SEQ ID NO: 159), linker (SEQ ID NO: 189, GGGGS), and E7 domain (SEQ ID NO: 160) in order from N-terminus to C-terminus.

[0478] The continuous arrangement of E6 / E7 (SEQ ID NO: 142) is a fusion protein that divides the E6 domain (SEQ ID NO: 159) and the E7 domain into 8 segments and 7 segments, respectively, and contains the E6 domain and the E7 domain in sequence from the N-terminus to the C-terminus.

[0479] Specifically, the E6 domain is divided into a fragment consisting of amino acids 18 to 43 of the amino acid sequence of SEQ ID NO: 159 (E6-1), a fragment consisting of amino acids 28 to 61 of the amino acid sequence of SEQ ID NO: 159 (E6-2), a fragment consisting of amino acids 42 to 70 of the amino acid sequence of SEQ ID NO: 159 (E6-3), a fragment consisting of amino acids 48 to 79 of the amino acid sequence of SEQ ID NO: 159 (E6-4), a fragment consisting of amino acids 66 to 92 of the amino acid sequence of SEQ ID NO: 159 (E6-5), a fragment consisting of amino acids 82 to 122 of the amino acid sequence of SEQ ID NO: 159 (E6-6), a fragment consisting of amino acids 100 to 143 of the amino acid sequence of SEQ ID NO: 159 (E6-7), and a fragment consisting of amino acids 100 to 143 of the amino acid sequence of SEQ ID NO: 159 (E6-8). The E7 domain is divided into a fragment consisting of amino acids 126 to 151 of the amino acid sequence of SEQ ID NO: 159 (E6-8). The E7 domain is divided into a fragment consisting of amino acids 5 to 21 of the amino acid sequence of SEQ ID NO: 160 (E7-1), a fragment consisting of amino acids 9 to 33 of the amino acid sequence of SEQ ID NO: 160 (E7-2), a fragment consisting of amino acids 24 to 45 of the amino acid sequence of SEQ ID NO: 160 (E7-3), a fragment consisting of amino acids 36 to 64 of the amino acid sequence of SEQ ID NO: 160 (E7-4), a fragment consisting of amino acids 47 to 69 of the amino acid sequence of SEQ ID NO: 160 (E7-5), a fragment consisting of amino acids 65 to 88 of the amino acid sequence of SEQ ID NO: 160 (E7-6), and a fragment consisting of amino acids 72 to 95 of the amino acid sequence of SEQ ID NO: 160 (E7-7). The sequential arrangement (continuous) of E6 / E7 is a fusion protein that contains the above-mentioned fragments in order from N-terminus to C-terminus, such as E6-1, E6-2, E6-3, E6-4, E6-5, E6-6, E6-7, E6-8, E7-1, E7-2, E7-3, E7-4, E7-5, E7-6 and E7-7.

[0480] The exchange arrangement of E6 / E7 is a fusion protein that contains E6 domain fragments (8 types) and E7 domain fragments (7 types) in order from N-terminus to C-terminus, such as E6-1, E7-1, E6-2, E7-2, E6-3, E7-3, E6-4, E7-4, E6-5, E7-5, E6-6, E7-6, E6-7, E7-7 and E6-8.

[0481] The odd-even arrangement of E6 / E7 is a fusion protein that contains E6 domain fragments (8 types) and E7 domain fragments (7 types) in order from N-terminus to C-terminus, such as E6-1, E6-3, E6-5, E6-7, E6-2, E6-4, E6-6, E6-8, E7-1, E7-3, E7-5, E7-7, E7-2, E7-4 and E7-6.

[0482] Self-amplifying RNA constructs containing the E6 / E7 domain of human papillomavirus (type 16) (SEQ ID NO: 139), a sequential arrangement of E6 / E7 (sequential) (SEQ ID NO: 142), an exchange arrangement of E6 / E7 (exchange) (SEQ ID NO: 145), or an even-odd arrangement of E6 / E7 (odd) (SEQ ID NO: 148) were generated in the same manner as in Example 5.1, and each self-amplifying RNA was designated G1, G2, G3, and G4 (also referred to as target protein). The nucleotide sequence of each self-amplifying RNA is shown in Tables 14 and 15.

[0483] [Table 14]

[0484]

[0485] [Table 15]

[0486]

[0487] The expression of the self-amplifying RNA construct produced as described above was confirmed at the cellular level in the same manner as in Example 5.2, and human papillomavirus antigens were produced.

[0488] Example 11.2. Evaluation of Cancer Preventive Activity

[0489] The self-amplified RNA produced in the same manner as in Example 11.1 above was encapsulated (RNA-LNP) in the same manner as in Example 10.1 above and administered intramuscularly to each mouse (C57 / BL6, 6-week-old, female, n=8) at a concentration of 5 μg / 50 μl / mouse (first immunization). Two weeks after the first administration, a second administration was performed in the same manner as described above (second immunization). Three weeks after the second administration, the TC-1 tumor cell line was inoculated at 2×10 5 The cells were transplanted into the flank of the mice by subcutaneous injection at a concentration of 10 cells / 100 μl / mouse, and the body weight and tumor size of the mice were measured 15 times for 19 consecutive days. At this time, PBS was administered to the control group, and the group administered with G1 was used as a positive control group.

[0490] In addition, tumor size was calculated by measuring width (W), length (L), and height (H) using the following mathematical formula I (https: / / biopticon.com / resources / tumor-volume-measurements-by-calipers / ).

[0491] <Mathematical Formula I>

[0492] Tumor size (V (t) )=6 / π×L×W×H10

[0493] Blood was collected from mice 2 weeks after the first administration and 2 weeks after the second administration (4 weeks after the first administration), and plasma was obtained, and some mice (n=3) were sacrificed 2 weeks after the second administration, and the spleens ( Figure 30 ).

[0494] Example 11.2.1. Confirmation of T cell responses

[0495] Four weeks after the first inoculation, a second inoculation was performed. Two weeks later, spleens were extracted to obtain cells, and then the experiment was performed. At this time, mice not injected with the TC-1 tumor cell line (n=3) were used. In addition, the group administered with G1 served as a positive control group.

[0496] Specifically, BD TMThe capture antibody included in the ELISPOT mouse IFNγ ELISPOT kit (BD, 551083) was diluted in DPBS at a ratio of 1:200, and then 100 μl was added to each well of each 96-well plate, and then coated by reacting at 4 ° C for 18 hours. Thereafter, each well was washed with DPBS (Welgene) and blocked with RPMI 1640 medium containing 10% heat-treated BCS (calf serum, Welgene) and 1% penicillin / streptomycin (Gibco). Then, splenocytes isolated from the laboratory mice of Example 11.2 above (mice not injected with TC-1 tumor cell line, n=3) were incubated at 1.0×10 6 The cells were seeded into each well at a concentration of 10 cells / well and then stimulated by treating them with 20 μg / ml of E6 and E7 peptides (Miltenyibiotec, 130-095-997 and 130-095-999). Mouse spleen cells were used as a negative control group. Afterwards, each well was washed three times with PBS-T (200 μl), and then the primary antibody (BD Biosciences TM ELISPOT mouse IFNγ ELISPOT set (BD, 551083) was diluted in DPBS containing 10% BCS at a ratio of 1:250 and added to each well (100 μl) for reaction. TM Cells were treated with ELISPOT Mouse IFNγ ELISPOT Set (BD, 551083), and the number of spots generated by the HRP substrate reaction was counted.

[0497] Results, such as Figure 31 As shown, the lowest T cell response was observed in the group administered with G1, and high T cell responses were observed in the groups administered with G2 to G4.

[0498] Example 11.2.2. Anti-cancer effect of self-amplifying RNA

[0499] After tumors were transplanted into the laboratory mice described in Example 11.2 above, the body weight and tumor size of the mice were monitored for 19 days after vaccination.

[0500] As a result, it was confirmed that tumor formation and growth were suppressed in all groups administered with G1 (positive control group), G2, G3, and G4, compared to the negative control group (PBS). In particular, tumor formation was observed only in the group administered with G1, which served as the positive control group (two mice), while no tumor formation was observed in the other administration groups. These results confirm that self-amplifying RNA is sufficiently useful as a cancer prevention vaccine.

[0501] Example 11.3. Evaluation of tumor growth inhibitory activity

[0502] To confirm the tumor growth inhibitory effect, TC-1 cells were cultured at 1 × 10 5 The concentration of cells / 100 μl was transplanted into the flank of mice (C57 / BL6, 6 weeks old, female) by subcutaneous injection. On the 3rd and 7th days after tumor cell transplantation, the self-amplifying RNA was administered in the same manner as in Example 11.2 above (only the first administration was performed), and the body weight and tumor size of the mice were measured over 22 days (22 times in total). At this time, the concentration of the self-amplifying RNA administered to each mouse was 5 μg / 50 μl / mouse or 1 μg / 50 μl / mouse ( Figure 34 ). At this time, PBS was administered to the negative control group.

[0503] Results, such as Figure 35 and Figures 36a to 36d As shown, it was confirmed that tumor growth was suppressed in all groups administered with 5 μg of self-amplifying RNA (vaccine) compared to the negative control group (PBS). In addition, in the case of the group administered with G4, it was confirmed that tumor growth was suppressed in all experimental mice even in the group administered with 1 μg.

[0504] From the above results, it was confirmed that tumor growth was suppressed or tumors were eliminated only by the first administration of self-amplifying RNA.

Claims

1. A polynucleotide comprising a nucleic acid sequence encoding a fusion protein, comprising: Nonstructural proteins of self-amplifying viruses; a first target protein; and Protease cleavage site.

2. The polynucleotide according to claim 1, wherein the nonstructural protein is derived from a nonstructural protein of a Picornaviridae virus.

3. The polynucleotide of claim 2, wherein the nonstructural protein comprises the P2 domain and the P3 domain of an enterovirus.

4. The polynucleotide of claim 3, wherein the P2 domain comprises 2A protease, 2B, and 2C.

5. The polynucleotide according to claim 4, wherein The 2A protease comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11, The 2B comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12, and The 2C comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

13. The polynucleotide of claim 4 , wherein the 2A protease comprises a mutation. 7 . The polynucleotide according to claim 6 , wherein the mutation is a substitution of amino acid 87 in the amino acid sequence of SEQ ID NO:

11.

8. The polynucleotide of claim 7, wherein the mutant comprises the amino acid sequence of SEQ ID NO:

75.

9. The polynucleotide of claim 3, wherein the P3 domain comprises 3A, 3B, 3C proteases and 3D polymerase.

10. The polynucleotide according to claim 9, wherein Said 3A comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14, Said 3B comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15, The 3C protease comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16, and The 3D polymerase comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

17.

11. The polynucleotide of claim 1, wherein the protease cleavage site is cleaved by a protease derived from a nonstructural protein of a self-amplifying virus.

12. The polynucleotide of claim 11, wherein the protease cleavage site is cleaved by enterovirus 2A protease.

13. The polynucleotide of claim 12, wherein the protease cleavage site comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

22.

14. The polynucleotide according to claim 1, wherein the polynucleotide further comprises a nucleic acid sequence encoding a second target protein. The polynucleotide according to claim 14 , wherein the protease cleavage site is located between the second target protein and the first target protein.

16. The polynucleotide of claim 15, wherein the protease cleavage site is derived from a nonstructural protein of an autoamplifying virus.

17. The polynucleotide of claim 16, wherein the protease cleavage site is cleaved by a protease derived from a Picornaviridae virus.

18. The polynucleotide of claim 17, wherein the protease cleavage site is cleaved by 2A protease, 3CD protease or 3C protease of enterovirus.

19. The polynucleotide of claim 18, wherein the protease cleavage site comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO:

50.

20. The polynucleotide according to claim 1 or 14, wherein the first target protein and / or the second target protein is an antigen.

21. The polynucleotide of claim 20, wherein the antigen is a severe fever with thrombocytopenia syndrome virus antigen.

22. The polynucleotide according to claim 21, wherein the antigen comprises any one amino acid sequence selected from the group consisting of SEQ ID NO:45, SEQ ID NO:85, SEQ ID NO:116 and SEQ ID NO:

119.

23. The polynucleotide of claim 20, wherein the antigen is a SARS-CoV2 viral antigen.

24. The polynucleotide of claim 23, wherein the antigen comprises the amino acid sequence of SEQ ID NO:

48.

25. The polynucleotide of claim 20, wherein the antigen is a highly pathogenic avian influenza virus antigen.

26. The polynucleotide of claim 25, wherein the antigen comprises the amino acid sequence of SEQ ID NO:

105.

27. The polynucleotide of claim 20, wherein the antigen is a canine influenza virus antigen.

28. The polynucleotide of claim 27, wherein the antigen comprises the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO:

56.

29. The polynucleotide of claim 20, wherein the antigen is a human papillomavirus antigen.

30. The polynucleotide of claim 29, wherein the antigen comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 139, SEQ ID NO: 142, SEQ ID NO: 145 and SEQ ID NO:

148.

31. The polynucleotide of claim 20, wherein the antigen is a tumor antigen.

32. The polynucleotide of claim 31, wherein the tumor antigen is selected from the group consisting of 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, BRCA1 / 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, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, hairy-like protein (COTL1), type XXIII collagen, COX-2, CT_9 / BRD6, Cten, cyclin B1, cyclin D1, CypB, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / 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, hTERT, 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, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, Mammaglobin-A, MART-1 / Melanin-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-acetylglucosamine transferase-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, prostate protein, 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, SSX-4, 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, VEGF, VEGFR-2 / FLK-1 and WT1. 、 33. The polynucleotide of claim 1, wherein the polynucleotide further comprises at least one stop codon located at the 3' end of the nucleic acid sequence encoding the nonstructural protein of the self-amplifying virus.

34. The polynucleotide of claim 33, wherein the stop codon comprises the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO:

68.

35. The polynucleotide of claim 1, wherein the polynucleotide further comprises a 5' untranslated region (5'UTR) at the 5' end.

36. The polynucleotide of claim 35, wherein the 5' untranslated region comprises an IRES (internal ribosome entry site).

37. The polynucleotide of claim 36, wherein the IRES comprises the nucleotide sequence of SEQ ID NO: 58 or SEQ ID NO:

59.

38. The polynucleotide of claim 37, wherein the IRES comprises a mutation.

39. The polynucleotide of claim 38, wherein the mutation is: a substitution of any one nucleotide selected from the group consisting of position 236, position 386, and a combination thereof in the nucleotide sequence of SEQ ID NO: 58; or Substitution of any one nucleotide selected from the group consisting of position 236, position 386, and a combination thereof in the nucleotide sequence of SEQ ID NO:

59.

40. The polynucleotide of claim 39, wherein the mutation comprises the nucleotide sequence of SEQ ID NO: 81 or SEQ ID NO:

82.

41. The polynucleotide of claim 1, wherein the polynucleotide further comprises a Kozak sequence.

42. The polynucleotide of claim 41, wherein the Kozak sequence comprises the nucleotide sequence of SEQ ID NO:

35.

43. The polynucleotide of claim 1, wherein the polynucleotide further comprises an untranslated region (3'UTR) at the 3' end of the polynucleotide.

44. The polynucleotide of claim 35 or 43, wherein the untranslated region is derived from a self-amplifying virus.

45. The polynucleotide of claim 44, wherein the untranslated region is derived from a virus of the Picornaviridae family.

46. ​​The polynucleotide of claim 1, wherein the polynucleotide further comprises a polyadenylic acid tail (poly(A) tail) sequence.

47. The polynucleotide according to claim 1, wherein the polynucleotide consists of the following structural formula (I) in order from the 5' end to the 3' end: 5'-BC(1)-D-3'(I) In the structural formula (I), 5' and 3' are the 5' end and 3' end of the polynucleotide, respectively, B is a nucleic acid sequence encoding the first target protein, C(1) is a nucleic acid sequence encoding the protease cleavage site, and D is a nucleic acid sequence encoding a nonstructural protein of the self-amplifying virus.

48. The polynucleotide according to claim 47, wherein the polynucleotide is composed of the following structural formula (II) from the 5' end to the 3' end: 5'-U-[K]o-[B'-C(2)]nBC(1)-DS-U'-P-3'(II) In the structural formula (II), 5' and 3' are the 5' end and 3' end of the polynucleotide, respectively, U is the nucleic acid sequence of the 5' untranslated region, K is the Kozak 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 the poly(A) tail, C(1) and C(2) are each independently a nucleic acid sequence encoding a protease cleavage site, n is an integer from 0 to 10. When n is from 2 to 10, each of B' is a nucleic acid sequence encoding the same or different target proteins, each of C(2) is also a nucleic acid sequence encoding the same or different protease cleavage sites, and o is an integer of 0 or 1.

49. The polynucleotide according to claim 47 or 48, wherein the non-structural protein comprises the P2 domain and the P3 domain of enterovirus in order from N-terminus to C-terminus.

50. The polynucleotide of claim 49, wherein the P2 domain comprises 2A protease, 2B, and 2C in order from N-terminus to C-terminus.

51. The polynucleotide of claim 49, wherein the P3 domain comprises 3A, 3B, 3C, and 3D in order from N-terminus to C-terminus.

52. The polynucleotide of claim 1, wherein the polynucleotide is DNA or RNA.

53. A recombinant vector comprising the polynucleotide according to claim 1.

54. A method for producing an RNA replicon, comprising: i) producing the recombinant vector according to claim 53; and ii) Synthesis of RNA from the recombinant vector.

55. A vaccine composition comprising the polynucleotide according to claim 1 or the recombinant vector according to claim 53 as an active ingredient.

56. A nonstructural protein 2A protease cleavage site derived from enterovirus, comprising the amino acid sequence of SEQ ID NO:

22.

57. A non-structural protein 3C protease cleavage site derived from enterovirus, comprising the amino acid sequence of SEQ ID NO:

50.

58. An IRES comprising the nucleic acid sequence of SEQ ID NO: 81 or SEQ ID NO:

82.

59. A 5' untranslated region comprising the nucleic acid sequence of SEQ ID NO: 86 or SEQ ID NO:

87.

60. A Kozak sequence comprising the nucleic acid sequence of SEQ ID NO:

35.

61. A nonstructural protein 2A protease derived from enterovirus, comprising the amino acid sequence of SEQ ID NO:

75.

62. A severe fever with thrombocytopenia syndrome (SFTS) virus antigen comprising the amino acid sequence of SEQ ID NO:45, SEQ ID NO:85, SEQ ID NO:116 or SEQ ID NO:

119.

63. A human papillomavirus (HPV) antigen comprising the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 142, SEQ ID NO: 145 or SEQ ID NO:

148.

64. A method for preventing or treating a disease, comprising: The polynucleotide of claim 1, the recombinant vector of claim 53, or the vaccine composition of claim 55 is administered to a subject.

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