Self-amplifying RNA constructs and uses thereof

Abstract

The present invention relates to a self-amplifying RNA construct that optimizes the expression of a target protein. Compared with conventional mRNA and enterovirus-based self-amplifying RNA, the enterovirus-based optimized self-amplifying RNA of the present invention increases the expression level of the target protein. Furthermore, the multi-antigen self-amplifying RNA simultaneously expresses one or more target proteins at a much higher expression rate than conventional mRNA. Therefore, the self-amplifying RNA construct of the present invention can be widely used in vaccines and the like.

Description

[Technical Field]

[0001] The present invention relates to self-amplifying RNA constructs and uses thereof. [Background technology]

[0002] Vaccines play an important role in health worldwide by preventing the infection and transmission of various diseases. According to the World Health Organization, vaccines are estimated to prevent 2 to 3 million deaths annually. Traditional vaccines, such as attenuated pathogens, inactivated pathogens, and subunit vaccines, have prevented a variety of deadly diseases. However, the rapid development and large-scale distribution of vaccines against various infectious pathogens that can evade the adaptive immune response remains challenging.

[0003] Meanwhile, nucleic acid therapeutics have been attracting attention as an alternative approach that can overcome the above-mentioned problems (Non-Patent Document 1). In particular, mRNA-based RNA vaccines, unlike some viral vaccines, are not integrated into the genome, making them safe and eliminating the risk of mutation. Furthermore, they can be produced using a cell-free method, enabling economical, rapid, and efficient vaccine production. Furthermore, single mRNA vaccines have the advantage of being able to encode multiple antigens, thereby enhancing the immune response against pathogens and avoiding the immune response against the vector in vivo.

[0004] Among these, self-amplifying RNA (saRNA) vaccines have constructs that encode structural proteins by replacing the structural proteins with an antigen protein (gene of interest, GOI) with RNA polymerase activity in nonstructural proteins. Therefore, self-amplifying RNA vaccines have the advantage of being able to sufficiently produce antibodies with just one vaccination, and much research has been conducted on these vaccine constructs. In fact, saRNA constructs can induce immune responses even when administered at doses approximately 1 / 10 to 1 / 100 of those used with mRNA, and it has been confirmed that antigen protein expression is maintained for up to 60 days when administered to mice.

[0005] Most of the self-amplifying RNA vaccines currently in use are based on the genome of alphaviruses (Non-Patent Document 2). In addition, there are viruses such as picornaviruses, flaviviruses, and coronaviruses that have self-amplifying RNA genomes containing RNA polymerase within non-structural proteins, but none of these, except for alphaviruses, have been commercialized yet, and further research is needed. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Vogel et al., Clin Microbiol Rev., 8(3):406-410 (1995) [Non-patent document 2] Bogers et al., J Infect Dis., 211(6):947-955(2015) Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the present inventors conducted research to develop a self-amplifying RNA vaccine construct that can be used rapidly and efficiently. As a result, a novel self-amplifying RNA construct was produced in which antigen protein expression was optimized by modifying the 5' untranslated region (UTR), protease cleavage site, stop codon, and poly(A) tail sequence of an enterovirus-based self-amplifying RNA construct. Furthermore, it was confirmed that the modified self-amplifying RNA construct can significantly increase antigen protein expression in cells compared to conventional mRNA and conventional enterovirus-based self-amplifying RNA constructs. Based on the above, the present inventors have completed the present invention. [Means for solving the problem]

[0008] To solve the above problem, in one aspect of the present invention, there is provided a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a nonstructural protein of a self-amplifying virus, a first protein of interest, and a protease cleavage site.

[0009] In another aspect of the present invention, there is provided a recombinant vector carrying the above polynucleotide.

[0010] In another embodiment of the present invention, there is provided a method for producing an RNA replicon, comprising producing the above-mentioned recombinant vector and synthesizing RNA from the recombinant vector.

[0011] In another aspect of the present invention, there is provided a vaccine composition comprising the above-mentioned polynucleotide or the above-mentioned recombinant vector as an active ingredient.

[0012] In another aspect of the present invention, there are provided 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 viral 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.

[0013] In another aspect of the present invention, there is provided a method for preventing or treating a disease, which comprises administering to a subject the above-described polynucleotide, the above-described recombinant vector, or a vaccine composition containing them. [Effects of the Invention]

[0014] Compared with conventional mRNA and enterovirus-based self-amplifying RNA constructs, the optimized enterovirus-based self-amplifying RNA constructs of the present invention increased the expression level of target proteins. Furthermore, the multi-antigen self-amplifying RNA constructs can simultaneously express more than one target protein, and the expression levels are much higher than those of conventional mRNA. Therefore, the self-amplifying RNA constructs of the present invention can be widely used in vaccines and other applications. [Brief explanation of the drawings]

[0015] [Figure 1a] Schematic representation of wild-type enterovirus RNA structure. [Figure 1b] FIG. 1 is a schematic diagram of the structure of an enterovirus-based self-amplifying RNA construct (saRNA). [Figure 2a]FIG. 1 shows the amino acid sequence of a 2A protease cleavage site contained in an enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention. [Figure 2b] 1 is a graph showing the results obtained by confirming the expression level of a target protein based on the amino acid sequence of the 2A protease cleavage site contained in an enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention using a luciferase system. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 3a] FIG. 1 is a schematic diagram of the structure of an enterovirus-based self-amplifying RNA construct with an inserted Kozak sequence according to one embodiment of the present invention. [Figure 3b] 1 is a graph showing the results obtained by confirming the expression level of a target protein by the sequence inserted into an enterovirus-based self-amplifying RNA construct with a Kozak sequence inserted therein according to one embodiment of the present invention using a luciferase system (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4a] FIG. 1 is a schematic diagram of the structure of an enterovirus-based self-amplifying RNA construct with an inserted poly(A) tail according to one embodiment of the present invention. [Figure 4b] This figure shows graphs illustrating the results obtained by confirming the expression level of a target protein by each sequence inserted into an enterovirus-based self-amplifying RNA construct with a poly(A) tail according to one embodiment of the present invention at the cellular level (bottom) and animal level (top) using a luciferase system. *p<0.05. [Figure 5] FIG. 1 is a schematic diagram comparing the structures of mRNA, an enterovirus-based self-amplifying RNA construct, and a self-amplifying RNA (modified saRNA) construct optimized by modifying an enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention. [Figure 6] Figure 1 shows a graph illustrating the results obtained by confirming the expression levels of a protein of interest by 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 at the cellular level using a luciferase system: **p<0.01, ***p<0.001, ****p<0.0001. [Figure 7] Figure 1 shows the results obtained by confirming the expression levels of a target protein by 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 at the animal level using a luciferase system. [Figure 8] This figure shows the results obtained by confirming the antigen protein expression levels at the cellular level by Western blotting for modified self-amplifying RNA constructs containing a severe fever with thrombocytopenia syndrome (SFTS) viral antigen as a target protein, as used in one embodiment of the present invention: Sa B: self-amplifying RNA construct containing the SFTS consensus B antigen; Sa ABDEF: self-amplifying RNA construct containing the SFTS consensus ABDEF antigen; mRNA B: mRNA containing the SFTS consensus B antigen. [Figure 9] This figure shows the results obtained by confirming the antigen protein expression levels at the cellular level by Western blotting for modified self-amplifying RNA constructs containing the spike protein antigen of coronavirus (SARS-CoV2) (top) or highly pathogenic avian influenza (HPAI) virus antigen (H5N8, bottom) as the target protein used in one embodiment of the present invention. [Figure 10] FIG. 1 is a schematic diagram of the structure of a modified self-amplifying RNA construct containing multiple antigens according to one embodiment of the present invention. [Figure 11]This figure shows a schematic diagram (top) of the modified self-amplifying RNA constructs containing two types of canine influenza virus antigens (H3N2 and H3N8) as target proteins used in one embodiment of the present invention, and the results (bottom) obtained by confirming the expression levels of the antigen proteins at the cellular level by Western blotting. 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. [Figure 12] FIG. 1 is a schematic diagram of a method for producing cell- and mouse-adapted Coxsackievirus B5. [Figure 13] Figure 1 shows a diagram (left) and graph (right) illustrating the results obtained by measuring and comparing the titers of wild-type Coxsackievirus B5 (WT-B5), cell-adapted Coxsackievirus B5 (Vp8-B5), and cell- and mouse-adapted Coxsackievirus B5 (Vp8Mp24-B5). ***p<0.001. [Figure 14] 1 is a schematic diagram of the RNA structure of Coxsackievirus B5 with increased proliferation rate according to one embodiment of the present invention. [Figure 15] FIG. 1 is a schematic diagram of the structure of an optimized enterovirus-based self-amplifying RNA construct according to one embodiment of the present invention. [Figure 16] FIG. 1 is a schematic diagram of the structure of a clone vector in the form of an enterovirus-based self-amplifying RNA construct used in the present invention. [Figure 17] FIG. 1 shows the schedule of animal experiments to confirm the antiviral effect against highly pathogenic influenza viruses of modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen as a target protein, which is used in one embodiment of the present invention. [Figure 18]FIG. 1 shows graphs illustrating the results obtained by administering modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen as a target protein, as used in one embodiment of the present invention, and then examining the production of neutralizing antibodies (top) and T cell responses (bottom) by removing the spleens of mice. [Figure 19] FIG. 1 is a graph showing the results obtained by examining the changes in body weight and survival rate of mice administered with modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen as a target protein, which is used in one embodiment of the present invention, and then infected with a highly pathogenic influenza virus. [Figure 20a] FIG. 1 is a graph showing the results obtained by measuring the virus titer remaining in each organ by extracting the organs of mice that had been administered modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen as a target protein, as used in one embodiment of the present invention, and then infecting them with a highly pathogenic influenza virus (on the third day). [Figure 20b] FIG. 1 is a graph showing the results obtained by measuring the virus titer remaining in each organ by extracting the organs of mice that had been administered modified self-amplifying RNA or mRNA containing a highly pathogenic influenza virus antigen as a target protein, as used in one embodiment of the present invention, and then infecting them with a highly pathogenic influenza virus (on the fifth day). [Figure 21a] FIG. 1 shows a schedule for animal experiments to confirm the neutralizing antibody production ability of modified self-amplifying RNA or mRNA containing severe fever with thrombocytopenia syndrome viral antigen (SFTS) as a target protein, which is used in one embodiment of the present invention. [Figure 21b]FIG. 1 shows the results obtained by administering modified self-amplifying RNA containing severe fever with thrombocytopenia syndrome virus antigens (A antigen, B antigen, DEF antigen, ABDEF antigen) used in one embodiment of the present invention as target proteins to mice (BALB / c mice), and then confirming by immunofluorescence staining whether neutralizing antibodies are produced in the blood of the mice. [Figure 21c] FIG. 1 is a graph showing the results obtained by administering modified self-amplifying RNA (B antigen, ABDEF antigen) or mRNA (B antigen) containing severe fever with thrombocytopenia syndrome viral antigen (SFTS) as a target protein, which is used in one embodiment of the present invention, to mice (C57BL6 mice), and then confirming whether neutralizing antibodies are produced in the blood of the mice. [Figure 22a] FIG. 1 is a schematic diagram showing the schedule for animal experiments (ferrets) to confirm the neutralizing antibody production ability of modified self-amplifying RNAs 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. [Figure 22b] FIG. 1 is a graph showing the results obtained by confirming whether neutralizing antibodies are produced in the blood of ferrets three weeks after the first administration 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. [Figure 22c] FIG. 1 is a graph showing the results obtained by confirming whether neutralizing antibodies are produced in the blood of ferrets three weeks after the second administration 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. [Figure 22d]FIG. 1 is a graph showing the results obtained by confirming whether neutralizing antibodies are produced in the blood of ferrets three weeks after the third administration 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. [Figure 23] FIG. 1 shows the results obtained by confirming the expression level of a modified self-amplifying 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 according to its concentration at the cellular level. [Figure 24] FIG. 1 shows a schedule for animal experiments to confirm the neutralizing antibody production 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. [Figure 25a] FIG. 1 is a graph showing the results obtained by analyzing the production of neutralizing antibodies in the blood of mice by confirming binding to inactivated canine influenza H3N2 virus 4 and 6 weeks (2 weeks after the second administration) after administering to mice 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. [Figure 25b] FIG. 1 is a graph showing the results obtained by analyzing the production of neutralizing antibodies in the blood of mice by confirming binding to inactivated canine influenza H3N8 virus 4 and 6 weeks (2 weeks after the second administration) after administering to the mice 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. [Figure 26]FIG. 1 shows the schedule for animal experiments in which mice are administered 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, as used in one embodiment of the present invention, and then infected with H3N2 virus or H3N8 virus at 4 and 6 weeks (2 weeks after the second administration), after which the antiviral activity against canine influenza is evaluated. [Figure 27a] FIG. 1 is a graph showing the results obtained by administering to mice 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, as used in one embodiment of the present invention, and then infecting the mice with canine influenza H3N2 virus after 4 weeks, and then observing changes in the body weight and survival rate of the mice. [Figure 27b] FIG. 1 is a graph showing the results obtained by administering to mice 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, as used in one embodiment of the present invention, and then infecting the mice with canine influenza H3N8 virus after 4 weeks, and then observing changes in the body weight and survival rate of the mice. [Figure 27c] FIG. 1 is a graph showing the results obtained by administering to mice 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, as used in one embodiment of the present invention, and then infecting the mice with canine influenza H3N2 virus 6 weeks later (2 weeks after the second administration), and observing changes in mouse weight and survival rate. [Figure 27d]FIG. 1 is a graph showing the results obtained by administering to mice 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, as used in one embodiment of the present invention, and then infecting the mice with canine influenza H3N8 virus 6 weeks later (2 weeks after the second administration), and then observing changes in mouse weight and survival rate. [Figure 28a] FIG. 1 is a graph showing the results obtained by administering to mice 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, as used in one embodiment of the present invention, and then infecting the mice with H3N2 virus or H3N8 virus, and measuring the titer of virus remaining in the lung tissue of the mice on day 3. [Figure 28b] FIG. 1 is a graph showing the results obtained by administering to mice 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, as used in one embodiment of the present invention, and then infecting the mice with H3N2 virus or H3N8 virus, and measuring the titer of virus remaining in the lung tissue of the mice on day 5. [Figure 29a] FIG. 1 is a schematic diagram of human papillomavirus (HPV) antigens (E6 / E7 domains, E6 / E7 sequential configuration, E6 / E7 crossover configuration, E6 / E7 odd and even configuration). [Figure 29b] Schematic diagram of engineered self-amplifying RNAs containing human papillomavirus (HPV) antigens (E6 / E7 domain, E6 / E7 sequential arrangement, E6 / E7 crossover arrangement, and E6 / E7 odd-even arrangement) as target proteins. G1: E6,E7 protein linker: E6 / E7 domain; G2: sequential: E6 / E7 sequential arrangement; G3: crossover: E6 / E7 crossover arrangement; G4: odd-even: E6 / E7 odd-even arrangement. [Figure 29c]This figure shows the results obtained by confirming the expression of modified self-amplifying RNAs containing human papillomavirus (HPV) antigens (E6 / E7 domain, E6 / E7 sequential arrangement, E6 / E7 crossover arrangement, E6 / E7 odd and even arrangement) as target proteins at the cellular level by Western blot. [Figure 30] FIG. 1 shows the schedule of animal experiments to confirm the cancer preventive activity of modified self-amplifying RNAs containing human papillomavirus (HPV) antigens (E6 / E7 domain, E6 / E7 sequential arrangement, E6 / E7 crossover arrangement, E6 / E7 odd and even arrangements) as target proteins. [Figure 31] FIG. 1 is a graph showing the results obtained by confirming T cell responses in the spleens of mice 4 weeks after administration (2 weeks after the second administration) of modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domain, E6 / E7 sequential arrangement, E6 / E7 crossover arrangement, E6 / E7 odd and even arrangement) as the target protein. [Figure 32] This figure shows a graph showing the results obtained by administering modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domains, E6 / E7 sequential arrangement, E6 / E7 crossover arrangement, E6 / E7 odd and even arrangement) as the target protein to mice, and then transplanting TC-1 tumor cells, which are cells expressing the E6 and E7 genes of human papillomavirus (type 16), into the mice 5 weeks later (3 weeks after the second administration), and measuring the tumor size. [Figure 33] FIG. 1 shows the schedule of animal experiments to confirm the tumor growth inhibitory activity of modified self-amplifying RNAs (four types) containing human papillomavirus (HPV) antigens (E6 / E7 domain, sequential E6 / E7 arrangement, crossover E6 / E7 arrangement, odd and even E6 / E7 arrangement) as target proteins. [Figure 34]This graph shows the results obtained by implanting TC-1 tumor cells, which express the E6 and E7 genes of human papillomavirus (type 16), into mice, and then measuring tumor size after administering four types of modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domain, E6 / E7 sequential configuration, E6 / E7 crossover configuration, and E6 / E7 odd and even configurations) as target proteins on day 3. PBS was used as a control. [Figure 35] This is a graph showing the results obtained by transplanting TC-1 tumor cells, which express the E6 and E7 genes of human papillomavirus (type 16), into mice, and then administering four types of modified self-amplifying RNA containing human papillomavirus (HPV) antigens (E6 / E7 domain, E6 / E7 sequential configuration, E6 / E7 crossover configuration, and E6 / E7 odd and even configurations) as the target protein on day 3, followed by measuring tumor size by treatment group. PBS was used as a control. [Figure 36a] This is a graph showing the results obtained by transplanting TC-1 tumor cells, which are cells expressing the E6 and E7 genes of human papillomavirus (type 16), into mice, and then administering a modified self-amplifying RNA (G1) containing a human papillomavirus (HPV) antigen (E6 / E7 domain) as a target protein or PBS on day 3, followed by measuring the tumor size in the mouse subject. G1: Protein GGGGS. [Figure 36b] This is a graph showing the results obtained by implanting TC-1 tumor cells, which are cells expressing the E6 and E7 genes of human papillomavirus (type 16), into mice, and then administering a modified self-amplifying RNA (G2) containing human papillomavirus (HPV) antigens (sequentially arranged E6 / E7) as the target protein and PBS on day 3, followed by measuring the tumor size in the mouse subject. G2: Continuous. [Figure 36c]This is a graph showing the results obtained by measuring tumor size in mouse subjects after implanting TC-1 tumor cells into mice and then administering a modified self-amplifying RNA (G3) containing a human papillomavirus (HPV) antigen (E6 / E7 crossover configuration) as the target protein or PBS on day 3. G3: crossover. [Figure 36d] This is a graph showing the results obtained by measuring tumor size in mouse subjects after implanting TC-1 tumor cells into mice and then administering modified self-amplifying RNA (G4) containing human papillomavirus (HPV) antigens (E6 / E7 odd and even configurations) as the target protein or PBS on day 3. G4: odd-even. [Figure 37] FIG. 1 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. [Figure 38] This figure shows the results obtained by confirming the expression levels of target proteins of the multiple antigen self-amplifying RNA constructs in Figure 37 by Western blot. NC: negative control, PC: positive control (wild-type H3N2 virus). DETAILED DESCRIPTION OF THE INVENTION

[0016] Polynucleotide Constructs Nonstructural proteins In one embodiment of the present invention, there is provided a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a nonstructural protein of a self-amplifying virus, a first protein of interest, and a protease cleavage site. In this case, the polynucleotide may be DNA or RNA. Furthermore, the self-amplifying virus may be a virus of the Picornaviridae family. Specifically, the self-amplifying virus may be a virus of the Enterovirus genus. More specifically, in the present invention, the nonstructural protein may be a nonstructural protein derived from an Enterovirus. Preferably, the nonstructural protein may be a nonstructural protein derived from a Coxsackievirus.

[0017] As used herein, the term "polynucleotide" refers to a single- or double-stranded sequence of nucleotides in which the 3' and 5' ends of each nucleotide are joined by a phosphodiester bond. Polynucleotides may be composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0018] As used herein, polynucleotides include DNA and RNA and may 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.

[0019] As used herein, the term "nucleic acid sequence" is used synonymously with "polynucleotide" and may be DNA or RNA. In this context, RNA may include mRNA. Furthermore, nucleic acid sequences may 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. Nucleic acids may also include synthetic nucleic acids (XNA), hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), locked nucleic acids (LNA), and peptide nucleic acids (PNA).

[0020] As used herein, the term "enterovirus" refers to a non-enveloped virus that belongs to the Enterovirus genus of the Picornaviridae family and is approximately 24 nm to 30 nm in size. Enteroviruses have an icosahedral shape and contain single-stranded positive-strand RNA approximately 7.2 kb to 7.5 kb in size as their genetic material. Enteroviruses include three serotypes of poliovirus (PV: 1-3), 23 serotypes of Coxsackievirus A (CVA: 1-22, 24), six serotypes of Coxsackievirus B (CVB: 1-6), 28 serotypes of Echovirus (ECV: 1-7, 9, 11-21, 24-27, 29-33), and other human enteroviruses (EV: 68-116).

[0021] Enteroviruses invade host cells, release RNA into the cytoplasm, and then act as mRNAs to initiate translation at the internal ribosome entry site (IRES) in the 5' untranslated region (UTR), thereby inducing the production of a large polyprotein. Enterovirus genetic material consists of a single open reading frame (ORF) and untranslated regions (UTRs), i.e., non-coding regions (NCRs) at the 5' and 3' ends that are not expressed as proteins. The ORF accounts for approximately 90% of the entire gene and is expressed as a single polyprotein. The polyprotein consists of approximately 2,185 amino acids and is cleaved into several different proteins by viral protease.

[0022] The polyprotein is divided into a P1 domain, a P2 domain, and a P3 domain, with the P1 domain being called a structural protein and the P2 and P3 domains being called non-structural proteins. The P1 domain of the polyprotein is the viral capsid protein, encoding components VP4, VP2, VP3, and VP1 from the N-terminus to the C-terminus, and the capsid is composed of a 32-mer capsomere. The P2 domain encodes, from the N-terminus to the C-terminus, the 2A protease (2A pro), 2B, and 2C, and the P3 domain encodes 3A, 3B (VPg proteins), and 3C proteases (3C pro ), and 3D polymerase (3D pol ) in this order. 2A protease and 3C protease are proteolytic enzymes that recognize and cleave the cleavage sites within the viral polyprotein, thereby processing the polyprotein into individual proteins. Meanwhile, 3D polymerase is an RNA-dependent RNA polymerase (RdRp) that synthesizes complementary RNA using RNA as a template during viral RNA self-replication.

[0023] In the present invention, the enterovirus may be poliovirus (PV; 1 to 3), rhinovirus (RV), enterovirus (A71, A76, A89 to A92, A120, B69, B73, B74, B75, B77, B78, B80 to B88, B93, B97, B98, B100, B101, B106, C99, C105, C109, C116, D68, D94, D111), coxsackievirus (A1 to A22, A24, B1 to B6, C96), echovirus (E2 to E7, E9, E11 to E21, E24 to E27, E29 to E33), or other human enterovirus (70, 79, 107, C104). Preferably, the enterovirus of the present invention may be Coxsackievirus B5.

[0024] In the present invention, the nonstructural protein derived from an enterovirus may contain the P2 domain and the P3 domain, in this case, the nonstructural protein may contain the P2 domain and the P3 domain in order from the N-terminus to the C-terminus.

[0025] 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 order 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, a polynucleotide may comprise or consist of, in order from the 5' end to the 3' end, a nucleic acid sequence encoding the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively. In one embodiment of the present invention, when the polynucleotide is DNA, it may comprise or consist of, in order from the 5' end to the 3' end, a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively. 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, in order from the 5' to the 3' end.

[0026] Furthermore, in the present invention, protease 2A, protease 2B, and protease 2C may be composed of amino acid sequences with one or several amino acid deletions, substitutions, and / or insertions, so long as they have the same biological function. Specifically, protease 2A, protease 2B, and protease 2C may comprise or consist of amino acid sequences that are 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 sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. In this case, the amino acid deletions, substitutions, and / or insertions may not cause changes in the secondary structure, activity, and other characteristics of the original proteins.

[0027] In the present invention, the 2A protease may further comprise a mutation.

[0028] As used herein, the term "mutation" refers to a form in which an amino acid or a portion of a polynucleotide encoding an amino acid is substituted compared to the wild type. In the present invention, a mutant may refer to a protein or nucleic acid (polynucleotide) containing a mutation.

[0029] That is, in the present invention, a 2A protease variant may have an amino acid sequence different from that of the wild-type 2A protease. However, the 2A protease variant may have activity equivalent to or similar to that of the wild-type 2A protease, or even improved activity. The 2A protease variant may have an increased protein expression level compared to the wild-type. Here, "2A protease activity" may mean specifically recognizing and cleaving a cleavage site within the amino acid sequence.

[0030] Specifically, the 2A protease variant may be a form in which some of the amino acids of the wild-type 2A protease have been substituted. A specific example of a 2A protease variant based on amino acid substitution is one in which the amino acid at position 87 in the amino acid sequence of SEQ ID NO: 11 has been substituted with another amino acid.

[0031] In this case, the "another amino acid" introduced by substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, excluding glycine.

[0032] In one embodiment of the present invention, the 2A protease variant may be one in which the 87th amino acid in the amino acid sequence of SEQ ID NO: 11 is substituted for glycine with serine (G87S). Preferably, the 2A protease variant may comprise the amino acid sequence of SEQ ID NO: 75. Thus, in the present invention, a polynucleotide may comprise or consist of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 75 as the 2A protease variant. Specifically, when the polynucleotide is DNA, the nucleic acid sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 73. When the polynucleotide is RNA, the nucleic acid sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 74.

[0033] Additionally, the 2A protease variant may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence of SEQ ID NO:75.

[0034] In the present invention, the nonstructural protein P3 domain may comprise 3A, 3B, and 3C proteases, and 3D polymerase. Specifically, the P3 domain of the present invention may comprise the 3A, 3B, and 3C proteases and 3D polymerase of Coxsackievirus B5. In this case, the P3 domain may comprise, in order from the N-terminus to the C-terminus, 3A, 3B, and 3C proteases, and 3D polymerase.

[0035] 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. Thus, in the present invention, a polynucleotide may sequentially comprise or consist of 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 a 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 sequentially 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 a nucleic acid sequence encoding the amino acid sequence of 2C; and when the polynucleotide is RNA, the nucleic acid sequence 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.

[0036] Furthermore, the 3A, 3B, and 3C proteases and 3D polymerases may be composed of amino acid sequences with one or several amino acid deletions, substitutions, and / or insertions, so long as they have the same biological function or the genetic location on the chromosome encoding the 3A, 3B, and 3C proteases and 3D polymerases is the same. Specifically, this may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequences of SEQ ID NOs: 14 to 17, respectively.

[0037] Target protein In the present invention, the polynucleotide may comprise a nucleic acid sequence encoding at least one or more proteins of interest, which may be linked to a protease cleavage site.

[0038] As used herein, the term "target protein" may refer to various proteins of medical or industrial interest and may include protein fragments, peptides, and variants. Industrially useful target proteins include hormones, hormone analogs, enzymes, enzyme inhibitors, cytokines, coagulation factors, transport proteins, receptors, receptor fragments, adhesion proteins, regulatory proteins, structural proteins, toxic proteins, transcription factors, antigens, antibodies, antibody fragments, monoclonal antibodies, etc. Preferably, the target protein may be an antigen, but is not limited thereto. The antigen may be any antigenic protein associated with an infectious disease or disease. Examples of antigens include tumor antigens, animal antigens, plant antigens, viral antigens, bacterial antigens, fungal antigens, protozoan antigens, autoimmune antigens, allergy antigens, etc. In this case, the antigen may be a surface antigen of tumor cells or a protein or peptide derived from the secreted form of a viral pathogen, a bacterial pathogen, a fungal pathogen, or a protozoan pathogen, respectively.

[0039] In the present invention, the viral pathogen may be isolated from or derived from, for example, filovirus, adenovirus, enterovirus, severe fever with thrombocytopenia syndrome virus (SFTSV; bunyaviridae), arbovirus, astrovirus, coronavirus, coxsackievirus, cytomegalovirus, dengue virus, Epstein-Barr virus, hepatitis virus, herpesvirus, 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, parovirus, poliovirus, rabies virus, respiratory syncytial virus, rhinovirus, rotavirus, rubella virus, smallpox virus, varicella-zoster virus, West Nile virus, Zika virus, etc.

[0040] In the present invention, the bacterial pathogen may be isolated from or derived from, for example, Campylobacter jejuni, Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitides, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, etc. In the present invention, the fungal pathogen may be, for example, one isolated from or derived from Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, Aspergillus species, etc.

[0041] In the present invention, the protozoan pathogen may be isolated from or derived from, for example, Plasmodium, Leishmania, Trypanosome, Cryptosporidium, Isospora, Naegleria fowleri, Acanthamoeba, Balamuthia mandrillaris, Toxoplasma gondii, Pneumocystis carinii, etc.

[0042] 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 tumors. Tumor antigens refer to tumor-specific antigens. Common tumor antigens include tumor-associated antigens that are more highly expressed in tumor cells than in normal cells. When cytotoxic T cells (Tc) recognize tumor-associated antigens, they can destroy tumor cells before they undergo rapid proliferation or metastasis.

[0043] In the present invention, the tumor antigen may be isolated from 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. Examples of tumor antigens include 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, CA15-3 / CA27-29, CA19-9, 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 (COTL1), collagen type XXIII, 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 lamini 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 / melan-A, MART-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-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-1, NY-ESO-B, OA1, OF A-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 / PARα, POTE, PRAME, PRDX5 / m, prostein, proteinar Ze-3 (PR3), PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, BAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAG E, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX_2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP, Sabah The protein may include 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.

[0044] As used herein, when a polynucleotide comprises one target protein, the target protein may be referred to indistinguishably as a "first target protein."

[0045] In the present invention, the first protein of interest may be located at the N-terminus of the nonstructural protein P2 domain. Specifically, the first protein of interest may 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 protein of interest may be located at the 5'-terminus of the nucleic acid sequence encoding the amino acid sequence of the 2A protease in a polynucleotide.

[0046] In one embodiment of the present invention, the first target protein may be a viral antigen, such as a severe fever with thrombocytopenia syndrome (SFTS) viral antigen, a SARS-CoV2 spike protein (COVID) antigen, a highly pathogenic avian influenza (HPAI) viral antigen, a canine influenza antigen, or a human papillomavirus (HPV) antigen.

[0047] As used herein, the term "severe fever with thrombocytopenia syndrome (SFTS) virus" refers to the virus that causes SFTS, also known as the Davie-Banda virus. The SFTS virus is currently classified into six genotypes (A, B, C, D, E, and F). SFTS is a disease characterized by high fever and thrombocytopenia as its main symptoms. It is transmitted by the tick Haemaphysalis longicornis, which belongs to the Ixodidae family and carries the SFTS virus. During blood feeding, the virus carried by the tick enters the body, multiplies, and causes the disease.

[0048] In the present invention, the severe fever with thrombocytopenia syndrome viral 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.

[0049] As used herein, the term "SARS-CoV2 (COVID) virus" refers to an enveloped, single-stranded, positive-sense RNA virus. The SARS-CoV2 (COVID) virus has a unique flame- or crown-like structure due to the presence of spike proteins, which are club-shaped protrusions, embedded in the envelope. The spike protein (S protein) is a spike-shaped protein used by coronaviruses to invade human cells and is composed of an S1 subunit and an S2 subunit. The virus infects the human body by binding to the angiotensin-converting enzyme 2 (ACE-2) receptor on human cells, penetrating the cells and delivering its genetic material (RNA) into the cells, where it self-replicates.

[0050] In the present invention, the SARS-CoV2 viral antigen may comprise the amino acid sequence of SEQ ID NO: 48.

[0051] 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, etc. This virus has various serotypes, such as 16 types of HA and 9 types of NA, depending on the HA and NA genes in the envelope, and it is known that there is no cross-protection between different serotypes. Of the various serotypes of AIV, all highly pathogenic avian influenza (HPAI) outbreaks to date are known to be caused by the H5 or H7 serotype. In particular, H5 HPAI viruses of the 2.3.4.4 and 2.3.2.1c lineages, which are H5 HPAI subtypes, have been brought into Japan by wild birds, and many cases of infection in wild birds have been reported.

[0052] In the present invention, the highly pathogenic avian influenza virus may comprise the amino acid sequence of SEQ ID NO:105.

[0053] As used herein, the term "canine influenza" refers to influenza that occurs in animals of the canine family. Canine influenza is believed to be caused by a mutant strain of influenza A virus that is identical to equine influenza virus H3N8. Influenza A virus is an enveloped, negative-sense, single-stranded RNA virus. Furthermore, a mutant avian influenza virus, the H3N2 virus, is known. Canine influenza virus is known to cause acute respiratory disease, exhibiting clinical symptoms such as severe coughing, fever, and runny nose.

[0054] In the present invention, the canine influenza may comprise the amino acid sequence of SEQ ID NO:53 or SEQ ID NO:56.

[0055] As used herein, the term "human papillomavirus (HPV)" refers to a DNA virus that infects humans and various animals through the skin or mucous membranes. To date, over 100 types of HPV have been discovered, and some types of HPV cause cancers such as cervical cancer and testicular cancer. In particular, human papillomavirus 16 (HPV 16) and human papillomavirus 18 (HPV 18) are found in 70% of cervical cancer patients worldwide and are classified as high-risk. HPVs consist of E1 to E7 genes required for viral replication, L1 and L2 genes expressing capsid proteins that constitute the virion, and an LCR that regulates viral replication and transcription. In particular, E6 and E7 are known to promote the degradation of p53 and RB proteins, respectively, thereby activating the cell cycle and inhibiting apoptosis, thereby inducing cancer development.

[0056] 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.

[0057] In one embodiment of the invention, the polynucleotide may comprise a nucleic acid sequence encoding the amino acid sequence of 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 / E7 domain antigen), SEQ ID NO:142 (HPV E6 / E7 sequential configuration antigen), SEQ ID NO:145 (HPV E6 / E7 cross-configuration antigen), or SEQ ID NO:148 (HPV E6 / E7 odd and even configuration antigen) at the 5' end of the nucleic acid sequence encoding the amino acid sequence of 2A protease.

[0058] In one embodiment of the invention, when the polynucleotide is DNA, the nucleic acid sequence may 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 may 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.

[0059] Furthermore, the SFTS antigen, COVID antigen, highly pathogenic avian influenza antigen, canine influenza antigen, or human papillomavirus antigen may consist of an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or inserted, so long as they have the same biological function or the gene encoding the SFTS antigen, COVID antigen, highly pathogenic avian influenza antigen, or canine influenza antigen on a chromosome is the same. Specifically, the antigen may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence of SEQ ID NO: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.

[0060] Protease cleavage site As used herein, the term "protease cleavage site" refers to a site that is specifically cleaved by a proteolytic enzyme.

[0061] In the present invention, the protease cleavage site linked to the first protein of interest may be cleaved by a protease derived from a nonstructural protein of a self-amplifying virus. In this case, the self-amplifying virus may be a virus of the genus Enterovirus. Preferably, this virus may be Coxsackievirus B5. In one embodiment of the present invention, the protease cleavage site may be cleaved by the 2A protease of an enterovirus. In this case, the protease cleavage site may be located between the first protein of interest and the 2A protease of the P2 domain of the nonstructural protein.

[0062] As used herein, the term "enterovirus 2A protease" refers to a proteolytic enzyme that first acts after enterovirus RNA is translated into a single polyprotein and cleaves the polyprotein into structural and nonstructural proteins. In this context, the 2A protease can cleave between any one amino acid selected from the group consisting of Tyr, Ala, Thr, Val, Phe, and Arg among the C-terminal amino acids of VP1 in the P1 domain and Gly, the N-terminal amino acid of the 2A protease in the P2 domain. In one embodiment, the 2A protease can cleave between Tyr and Gly. In one embodiment, the 2A protease can cleave between Ala and Gly. In one embodiment, the 2A protease can cleave between Thr and Gly. In one embodiment, the 2A protease can cleave between Val and Gly. In one embodiment, the 2A protease can cleave between Phe and Gly. In one embodiment, the 2A protease can cleave between Arg and Gly. Preferably, the 2A protease can cleave between Tyr among the C-terminal amino acids of VP1 in the P1 domain and Gly among the N-terminal amino acids of the 2A protease in the P2 domain.

[0063] Therefore, in the present invention, the 2A protease cleavage site is an amino acid sequence at the C-terminus of VP1, or the C-terminus of VP1, or the C-terminus of VP1 and the 2A protease (2A pro ) may comprise the N-terminus of

[0064] In this case, the amino acid sequence at the N-terminus of VP1 may contain 1 to 30 consecutive amino acids from the C-terminus of VP1. Specifically, the 2A protease cleavage site may contain 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 VP1. More 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, 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).

[0065] The N-terminal amino acid sequence may comprise 1 to 18 amino acids 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 from the N-terminus of the 2A protease. More specifically ... pro The amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive amino acids from the N-terminus of the amino acid sequence of the target protein.

[0066] 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 NOs: 18(T) to 23. Thus, the polynucleotide of the present invention may comprise a nucleic acid sequence encoding any one of the amino acid sequences selected from SEQ ID NOs: 18 to 23, between the 3' end of the nucleic acid sequence encoding the first protein of interest and the 5' end of the nucleic acid sequence encoding the 2A protease. Preferably, the polynucleotide of the present invention 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, the polynucleotide of the present invention may comprise or consist of any one of the nucleotide sequences selected from SEQ ID NOs: 24(ACC) to 29. Preferably, the polynucleotide of the present invention may comprise the nucleotide sequence of SEQ ID NO: 28. When the polynucleotide is RNA, the polynucleotide of the present invention may comprise or consist of any one of the nucleotide sequences selected from SEQ ID NOs: 24 and 30 to 34. Preferably, the polynucleotide of the present invention may comprise the nucleotide sequence of SEQ ID NO: 33.

[0067] Furthermore, the 2A protease cleavage site may be composed of an amino acid sequence in which one or more amino acids have been deleted, substituted, and / or inserted, so long as it has the same biological function or the genetic location on the chromosome encoding the 2A protease cleavage site is the same. Specifically, the 2A protease cleavage site may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to any one of the amino acid sequences selected from SEQ ID NOs: 18 to 23.

[0068] In the present invention, the polynucleotide may further comprise a nucleic acid sequence encoding a protein of interest. In the present specification, when a polynucleotide comprises a nucleic acid sequence encoding one or more proteins of interest, the proteins of interest can be described by dividing them into a first protein of interest and a second protein of interest. In this case, the second protein of interest may be located at the N-terminus of the first protein of interest. Therefore, in the present invention, the nucleic acid sequence encoding the second protein of interest may be located at the 5'-terminus of the nucleic acid sequence encoding the amino acid sequence of the first protein of interest in the polynucleotide. The proteins of interest and the first protein of interest are the same as those described above.

[0069] In one embodiment of the present invention, the first and second target proteins may be viral antigens. In one embodiment, the viral antigens may be canine influenza subtypes H3N2 and H3N8. In this case, the first target protein may be H3N2 or H3N8, and the second target protein may be H3N2 or H3N8. The first and second target proteins may be the same or different from each other. Thus, in one embodiment of the present invention, if the polynucleotide is DNA, the first target protein may consist of the nucleotide sequence of SEQ ID NO: 51 or SEQ ID NO: 54; if the polynucleotide is RNA, it may consist of the nucleotide sequence of SEQ ID NO: 52 or SEQ ID NO: 55. In one embodiment of the present invention, the first target protein may be H3N8 and may include the amino acid sequence of SEQ ID NO: 56, and the second target protein may be H3N2 and may include the amino acid sequence of SEQ ID NO: 53. Thus, the nucleic acid sequence encoding a first protein of interest of the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:54 or SEQ ID NO:55, and the nucleic acid sequence encoding a second protein of interest may comprise the nucleotide sequence of SEQ ID NO:51 or SEQ ID NO:52.

[0070] Between the first and second proteins of interest, there may be a 2A self-cleaving peptide sequence or a protease cleavage site.

[0071] As used herein, the term "2A self-cleaving peptide" refers to a peptide of 18 to 22 amino acids that can induce ribosome skipping during protein translation in cells. This peptide shares the core sequence motif of DxExNPGP and is found in a wide range of virus families. 2A self-cleaving peptides may be, for example, P2A, E2A, F2A, T2A, etc. In one embodiment, the 2A self-cleaving peptide may be P2A (porcine teschovirus 1 2A) and may comprise the amino acid sequence of SEQ ID NO: 187. Furthermore, when a 2A self-cleaving peptide is included, the 2A self-cleaving peptide may further comprise a linker (GSG, SEQ ID NO: 188) at the N-terminus of the peptide.

[0072] 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 Enterovirus genus. Preferably, the virus 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, the protease cleavage site 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 those described above.

[0073] In the present invention, the "enterovirus 3C protease" is a proteolytic enzyme that acts after the RNA of an enterovirus is translated into a single polyprotein. pro) is a precursor of the 3C protease and is a protein produced at an intermediate stage when the P3 domain is processed by the 3C protease into individual proteins. In this case, the 3CD protease also has protease activity. The 3C protease and 3CD protease play a role in cleaving the polyprotein, which has been cleaved by the 2A protease into structural proteins (P1 domain) and non-structural proteins (P2 domain and P3 domain), into individual proteins (VP0, VP3, VP1, 2A protease, 2B, 2C, 3A, 3B, 3C proteases, and 3D polymerase). In this case, "VP0" is an intermediate protein that is further processed into VP4 and VP2.

[0074] The 3C protease can cleave between any one amino acid selected from the group consisting of Gln, Glu, Ile, and Thr among the C-terminal amino acids of each of the individual proteins (VP3, VP1, 2A protease, 2B, 2C, 3A, 3B, and 3C proteases) and any one amino acid selected from the group consisting of Gly, Asn, Ser, Ala, Val, Cys, Trp, and Met among the N-terminal amino acids of each of the individual proteins (VP1, 2A protease, 2B, 2C, 3A, 3B, and 3C proteases, and 3D polymerase). In one embodiment, 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 is capable of cleaving between Gln and Ala. In one embodiment, the 3C protease is capable of cleaving between Gln and Val. In one embodiment, the 3C protease is capable of cleaving between Gln and Cys. In one embodiment, the 3C protease is capable of cleaving between Gln and Trp. In one embodiment, the 3C protease is capable of cleaving between Gln and Gly. In one embodiment, the 3C protease is capable of cleaving between Gln and Met. In one embodiment, the 3C protease is capable of cleaving between Glu and Gly. In one embodiment, the 3C protease is capable of cleaving between Glu and Asn. In one embodiment, the 3C protease is capable of cleaving between Glu and Ser. In one embodiment, the 3C protease is capable of cleaving between Glu and Ala. In one embodiment, the 3C protease is capable of cleaving between Glu and Val. In one embodiment, the 3C protease is capable of cleaving between Glu and Cys. In one embodiment, the 3C protease is capable of cleaving between Glu and Trp. In one embodiment, the 3C protease is capable of cleaving between Glu and Gly. In one embodiment, the 3C protease is capable of cleaving between Glu and Met.In one embodiment, the 3C protease is capable of cleaving between Ile and Gly. In one embodiment, the 3C protease is capable of cleaving between Ile and Asn. In one embodiment, the 3C protease is capable of cleaving between Ile and Ser. In one embodiment, the 3C protease is capable of cleaving between Ile and Ala. In one embodiment, the 3C protease is capable of cleaving between Ile and Val. In one embodiment, the 3C protease is capable of cleaving between Ile and Cys. In one embodiment, the 3C protease is capable of cleaving between Ile and Trp. In one embodiment, the 3C protease is capable of cleaving between Ile and Gly. In one embodiment, the 3C protease is capable of cleaving between Ile and Met. In one embodiment, the 3C protease is capable of cleaving between Thr and Gly. In one embodiment, the 3C protease is capable of cleaving between Thr and Asn. In one embodiment, the 3C protease is capable of cleaving between Thr and Ser. In one embodiment, the 3C protease is capable of cleaving between Thr and Ala. In one embodiment, the 3C protease is capable of cleaving between Thr and Val. In one embodiment, the 3C protease is capable of cleaving between Thr and Cys. In one embodiment, the 3C protease is capable of cleaving between Thr and Trp. In one embodiment, the 3C protease is capable of cleaving between Thr and Gly. In one embodiment, the 3C protease is capable of cleaving between Thr and Met. Preferably, the 3C protease is capable of cleaving between Gln among the C-terminal amino acids and Gly among the N-terminal amino acids of each individual protein.

[0075] Specifically, the 3C protease of the present invention can cleave between Gln, the C-terminal amino acid of VP3 of Coxsackievirus B5, and Gly, the N-terminal amino acid of VP1. The 3C protease can cleave between Gln, the C-terminal amino acid of 2A protease, and Gly, the N-terminal amino acid of 2B. The 3C protease can cleave between Gln, the C-terminal amino acid of 2B, and Gly, the N-terminal amino acid of 2C. The 3C protease can cleave between Gln, the C-terminal amino acid of 2C, and Gly, the N-terminal amino acid of 3A. The 3C protease can cleave between Gln, the C-terminal amino acid of 3A, and Gly, the N-terminal amino acid of 3B. The 3C protease can cleave between Gln, the C-terminal amino acid of 3B, and Gly, the N-terminal amino acid of 3C protease. The 3C protease can cleave between the C-terminal amino acid Gln of the 3C protease and the N-terminal amino acid Gly of the 3D polymerase.

[0076] 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 proteases.

[0077] Furthermore, in the present invention, the protease cleavage site may comprise any one amino acid sequence selected from the group consisting of the amino acid sequences of the C-terminus of VP3 and the N-terminus of VP1, the C-terminus of 2A protease and the N-terminus of 2B, the C-terminus of 2B and the N-terminus of 2C, the C-terminus of 2C and the N-terminus of 3A, the C-terminus of 3A and the N-terminus of 3B, the C-terminus of 3B and the N-terminus of 3C protease, and the C-terminus of 3C protease and the N-terminus of 3D polymerase.

[0078] Specifically, in the present invention, the 3C protease cleavage site may comprise 1 to 22 consecutive amino acids from the C-terminus of the amino acid sequence of any one protein selected from the group consisting of VP3, 2A protease, 2B, 2C, 3A, 3B, and 3C proteases.

[0079] More specifically, the 3C protease cleavage site may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive amino acids from the C-terminus of any one amino acid sequence selected from the group consisting of SEQ ID NO:80, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0080] Furthermore, in the present invention, the 3C protease cleavage site may comprise 1 to 11 consecutive amino acids from the C-terminus of the VP3 amino acid sequence and 1 to 11 consecutive amino acids from the N-terminus of the VP1 amino acid sequence. The 3C protease cleavage site may comprise 1 to 11 consecutive amino acids from the C-terminus of the 2A protease amino acid sequence and 1 to 11 consecutive amino acids from the N-terminus of the 2B amino acid sequence. The 3C protease cleavage site may comprise 1 to 11 consecutive amino acids from the C-terminus of the 2B amino acid sequence and 1 to 11 consecutive amino acids from the N-terminus of the 2C amino acid sequence. The 3C protease cleavage site may comprise 1 to 11 consecutive amino acids from the C-terminus of the 2C amino acid sequence and 1 to 11 consecutive amino acids from the N-terminus of the 3A amino acid sequence. The 3C protease cleavage site may comprise 1 to 11 consecutive amino acids from the C-terminus of the amino acid sequence of 3A and 1 to 11 consecutive amino acids from the N-terminus of the amino acid sequence of 3B. The 3C protease cleavage site may comprise 1 to 11 consecutive amino acids from the C-terminus of the amino acid sequence of 3B and 1 to 11 consecutive amino acids from the N-terminus of the amino acid sequence of the 3C protease. The 3C protease cleavage site may comprise 1 to 11 consecutive amino acids from the C-terminus of the amino acid sequence of the 3C protease and 1 to 11 consecutive amino acids from the N-terminus of the amino acid sequence of the 3D polymerase.

[0081] 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. 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: 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. 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: 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. 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: 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. 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: 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. 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: 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.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: 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.

[0082] The 3CD protease can cleave between VP0 and VP3. Specifically, the 3CD protease can cleave between any one amino acid selected from the group consisting of Gln, Lys, Tyr, and Met among the C-terminal amino acids of VP0 and any one amino acid selected from the group consisting of Gly, Trp, Tyr, and Asn among the N-terminal amino acids of VP3. In one embodiment of the present invention, the 3CD protease can cleave between Gln and Gly. In one embodiment, the 3CD protease can cleave between Gln and Trp. In one embodiment, the 3CD protease can cleave between Gln and Tyr. In one embodiment, the 3CD protease can cleave between Gln and Asn. In one embodiment, the 3CD protease can cleave between Lys and Gly. In one embodiment, the 3CD protease can cleave between Lys and Trp. In one embodiment, the 3CD protease is capable of cleaving between Lys and Tyr. In one embodiment, the 3CD protease is capable of cleaving between Lys and Asn. In one embodiment, the 3CD protease is capable of cleaving between Thr and Gly. In one embodiment, the 3CD protease is capable of cleaving between Thr and Trp. In one embodiment, the 3CD protease is capable of cleaving between Thr and Tyr. In one embodiment, the 3CD protease is capable of cleaving between Thr and Asn. In one embodiment, the 3CD protease is capable of cleaving between Met and Gly. In one embodiment, the 3CD protease is capable of cleaving between Met and Trp. In one embodiment, the 3CD protease is capable of cleaving between Met and Tyr. In one embodiment, the 3CD protease is capable of cleaving between Met and Asn. Preferably, the 3CD protease is capable of cleaving between the Gln among the C-terminal amino acids of VP0 and the Gly among the N-terminal amino acids of VP3.

[0083] Therefore, in the present invention, the 3CD protease cleavage site may comprise the C-terminal amino acid sequence of the amino acid sequence of VP2.

[0084] Additionally, the 3CD protease cleavage site may include the C-terminal amino acid sequence of VP2 and the N-terminal amino acid sequence of VP3.

[0085] Specifically, in the present invention, the 3CD protease cleavage site may comprise 1 to 40 consecutive amino acids from the C-terminus of VP2.

[0086] More specifically, the 3CD protease cleavage site may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids from the C-terminus of the amino acid sequence of SEQ ID NO:79.

[0087] Furthermore, the 3CD protease cleavage site may comprise a sequence of 1 to 20 consecutive amino acids from the C-terminus of VP2 and a sequence of 1 to 20 consecutive amino acids from the N-terminus of VP1.

[0088] More specifically, the 3CD protease cleavage site may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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.

[0089] In one embodiment of the present invention, the protease cleavage site may comprise 6 consecutive amino acids from the C-terminus of the amino acid sequence of 2C and 6 consecutive amino acids from the N-terminus of the amino acid sequence of 3A.

[0090] In one embodiment of the invention, the 3C protease cleavage site may comprise the amino acid sequence of SEQ ID NO: 50. Thus, in one embodiment of the 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.

[0091] Furthermore, the 3C protease cleavage site and the 3CD protease cleavage site may be composed of an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or inserted, so long as they have the same biological function or the genetic location on the chromosome encoding the 3C protease cleavage site is the same. Specifically, the cleavage site may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence of SEQ ID NO:50.

[0092] Thus, 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 nonstructural 2A protease protein, nucleic acid sequences encoding the first and second proteins of interest, and the amino acid sequences of SEQ ID NO:53, SEQ ID NO:50, SEQ ID NO:56, and SEQ ID NO:22, in 5' to 3' order. Thus, when the polynucleotide is DNA, the polynucleotide may comprise or consist, in 5' to 3' order, of the nucleotide sequences of SEQ ID NO:54, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:28. When the polynucleotide of the present invention is RNA, the nucleic acid sequence may comprise or consist, in 5' to 3' order, of the nucleotide sequences of SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:52, and SEQ ID NO:33.

[0093] Furthermore, in the present invention, when the second protein of interest comprises one or more proteins of interest, the second protein of interest may be composed of one or more identical or different proteins of interest, and a protease cleavage site may be located between each of the proteins of interest. In this case, the protease cleavage site may be a site cleaved by a protease derived from a nonstructural protein of a self-amplifying virus. Specifically, the protease cleavage site may be a site cleaved by a protease derived from a virus of the Picornaviridae family. More specifically, the protease cleavage site may be a site cleaved by the 2A protease, 3CD protease, or 3C protease of an enterovirus. Preferably, the protease cleavage site may be a site cleaved by the 2A protease, 3CD protease, or 3C protease of Coxsackievirus B5. The second protein of interest may be an antigen. Furthermore, 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 above.

[0094] stop codon 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 nonstructural protein of the self-amplifying virus.

[0095] As used herein, the term "stop codon" refers to a signal sequence that stops the translation of mRNA into amino acids, and generally refers to a stop codon or a sequence of a stop codon. There is no corresponding tRNA for a stop codon, but instead a protein called a "terminator" binds to it, and when the stop codon is reached during the translation process, the two units of the ribosome are separated, thereby terminating translation. Generally, the sequence of a DNA stop codon is TAG, TAA, or TGA, and the sequence of an RNA stop codon is UAG, UAA, or UGA.

[0096] Specifically, the polynucleotide of the present invention may contain one to three stop codons.

[0097] More specifically, the polynucleotide may contain one 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.

[0098] More specifically, the polynucleotide may contain two different stop codons arranged in succession.

[0099] When the polynucleotide of the present invention is DNA, in one embodiment, the stop codons may be arranged in the order of TAG and TAA from the 5' end to the 3' end. In one embodiment, the stop codons may be arranged in the order of TAG and TGA. In one embodiment, the stop codons may be arranged in the order of TAA and TGA. When the polynucleotide of the present invention is RNA, in one embodiment, the stop codons may be arranged in the order of UAG and UAA from the 5' end to the 3' end. In one embodiment, the stop codons may be arranged in the order of UAG and UGA. In one embodiment, the stop codons may be arranged in the order of UAA and UGA.

[0100] More specifically, the polynucleotide may contain three different stop codons arranged consecutively.

[0101] When the polynucleotide of the present invention is DNA, in one embodiment, the stop codons may be arranged in the order of TAG, TAA, and TGA from the 5' end to the 3' end. In one embodiment, the stop codons may be arranged in the order of TAG, TGA, and TAA. In one embodiment, the stop codons may be arranged in the order of TAA, TAG, and TGA. In one embodiment, the stop codons may be arranged in the order of TGA, TAG, and TAA. In one embodiment, the stop codons may be arranged in the order of TGA, TAG, and TAA. In one embodiment, the stop codons may be arranged in the order of TGA, TAA, and TAG. In one embodiment, the stop codons 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, the stop codons may be arranged in the order of UAG, UAA, and UGA from the 5' end to the 3' end. In one embodiment, the stop codons may be arranged in the order of UAG, UGA, and UAA. In one embodiment, the stop codons may be arranged in the order of UAA, UAG, and UGA. In one embodiment, the stop codons may be arranged in the order of UAA, UGA, and UAG. In one embodiment, the stop codons may be arranged in the order of UGA, UAG, and UAA. In one embodiment, the stop codons may be arranged in the order of UGA, UAA, and UAG. In one embodiment, the stop codons may comprise or consist of the nucleotide sequence of SEQ ID NO: 68 (UGAUAAUAG).

[0102] 5' untranslated region In the present invention, the polynucleotide may further comprise a 5' untranslated region (5'UTR) at the 5' end.

[0103] As used herein, the term "untranslated region" refers to a region in an RNA molecule that is transcribed but not translated into an amino acid sequence. The untranslated region may be located 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 functions to regulate transcription through various mechanisms in viruses, prokaryotes, and eukaryotes. Although the 5'UTR is commonly known as the untranslated region, it has been reported that it is translated into a protein product and regulates mRNA translation.

[0104] The elements of the 5' UTRs of eukaryotes and prokaryotes differ significantly. Prokaryotic 5' UTRs contain a ribosome binding site (RBS), also known as a Shine-Dalgarno sequence (SD sequence), which is typically located 3–10 base pairs upstream from the start codon. In contrast, eukaryotic 5' UTRs contain a Kozak consensus sequence (hereafter referred to as the "Kozak sequence") that encompasses the start codon.

[0105] The 5' untranslated region of the polynucleotide according to the invention may comprise an internal ribosome entry site (IRES).

[0106] As used herein, the term "internal ribosome entry site (IRES)" refers to 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 a translation initiation complex associated with the translation of a target protein binds, and the IRES is a cis-acting nucleotide sequence that induces the translation of the target protein by forming complex secondary and tertiary structures.

[0107] In the present invention, the IRES may comprise the nucleotide sequence of an IRES derived from a virus or a eukaryotic cell. In this case, the 5'UTR may have a 5'UTR structure comprising an IRES derived from a virus or a eukaryotic cell. In one embodiment of the present invention, the 5'UTR and IRES may be derived from the same self-amplifying virus as the nonstructural proteins. Specifically, the 5'UTR and IRES may be derived from a virus of the picornavirus genus. 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 or consist of the nucleic acid sequence of SEQ ID NO: 1 (DNA) or SEQ ID NO: 60 (RNA), and the IRES may comprise or consist of the nucleic acid sequence of SEQ ID NO: 58 (DNA) or SEQ ID NO: 59 (RNA).

[0108] The IRES may comprise a mutation, wherein the mutation is the same as described above.

[0109] Specifically, an IRES mutant may be one in which part of the nucleotide sequence of the wild-type IRES is replaced with a different nucleotide sequence.

[0110] More specifically, when the polynucleotide is DNA, the IRES mutant may be one in which any one nucleotide selected from the group consisting of nucleotides at positions 236, 386, and a combination thereof in the nucleotide sequence of SEQ ID NO: 58 is substituted with a nucleotide different from that in the nucleotide sequence of SEQ ID NO: 58. In one embodiment, the IRES mutant may be one in which the nucleotide at position 236 in the nucleotide sequence of SEQ ID NO: 58 is substituted with A, T, or G. In one embodiment, the IRES mutant may be one in which the nucleotide at position 386 in the nucleotide sequence of SEQ ID NO: 58 is substituted with A, T, or C. In one embodiment, the IRES mutant may be one in which the nucleotide at position 236 in the nucleotide sequence of SEQ ID NO: 58 is substituted with A, T, or G and the nucleotide at position 386 is substituted with A, T, or C. Preferably, the IRES mutant may be one in which the nucleotide at position 236, C, in the nucleotide sequence of SEQ ID NO: 58 is substituted with T (C236T). The mutant IRES may be one in which the 386th nucleotide, G, in the nucleotide sequence of SEQ ID NO: 58 is substituted with A (G386A). More preferably, the mutant IRES may be one in which the 236th nucleotide, C, is substituted with T (C236T) and the 386th nucleotide, G, is substituted with A (G386A) in the nucleotide sequence of SEQ ID NO: 58.

[0111] When the polynucleotide is RNA, the IRES mutant may be one in which any one nucleotide selected from the group consisting of nucleotides at positions 236, 386, and a combination thereof in the nucleotide sequence of SEQ ID NO: 59 is substituted with a nucleotide different from that in the nucleotide sequence of SEQ ID NO: 59. In one embodiment, the IRES mutant may be one in which the nucleotide at position 236 in the nucleotide sequence of SEQ ID NO: 59 is substituted with A, U, or G. In one embodiment, the IRES mutant may be one in which the nucleotide at position 386 in the nucleotide sequence of SEQ ID NO: 59 is substituted with A, U, or C. In one embodiment, the IRES mutant may be one in which the nucleotide at position 236 in the nucleotide sequence of SEQ ID NO: 59 is substituted with A, U, or G and the nucleotide at position 386 is substituted with A, U, or C. Preferably, the IRES mutant may be one in which the C at position 236 in the nucleotide sequence of SEQ ID NO: 59 is substituted with U (C236U). The mutant IRES may be one in which the 386th nucleotide, G, in the nucleotide sequence of SEQ ID NO: 59 is substituted with A (G386A). More preferably, the mutant IRES may be one in which the 236th nucleotide, C, is substituted with U (C236U) and the 386th nucleotide, G, is substituted with A (G386A) in the nucleotide sequence of SEQ ID NO: 59.

[0112] In one embodiment of the invention, the IRES variant may comprise or consist of the nucleotide sequence of SEQ ID NO:81 or SEQ ID NO:82.

[0113] Kozak sequence In the present invention, the polynucleotide may further comprise a Kozak sequence.

[0114] As used herein, the term "Kozak sequence" refers to a nucleotide sequence located upstream of the start codon at which eukaryotic mRNA begins translation into protein, which plays an important role in recognizing the start codon and initiating protein synthesis.

[0115] In the present invention, the Kozak sequence may be located at the 5' end of the nucleic acid sequence encoding the target protein (first target protein or second target protein). In the present invention, the Kozak sequence may comprise any one of the nucleotide sequences selected from SEQ ID NOs: 35 to 37. Preferably, the Kozak sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 35.

[0116] 3' untranslated region In the present invention, the polynucleotide may further comprise a 3' UTR. In this case, the 3' UTR may be located 3' from the termination codon. The 3' UTR, together with the 5' UTR, is operably linked to the nucleic acid sequence encoding the target protein and nonstructural protein, thereby improving the translation efficiency of the target protein and nonstructural protein or their transcription products. Furthermore, the 3' UTR plays an important role in ensuring that the transcription product, mRNA, is stably maintained in cells without being destroyed.

[0117] In the present invention, the 3'UTR can be derived from a virus or eukaryotic cell that is identical to the 5'UTR derived from a virus and / or eukaryotic cell having the above-mentioned IRES nucleotide sequence. In one embodiment of the present invention, the 3'UTR may be derived from the same self-amplifying virus as the nonstructural protein from which the 5'UTR is derived. Specifically, the 3'UTR may be derived from the same picornavirus virus as the 5'UTR. More specifically, the 3'UTR may be derived from the same enterovirus as the 5'UTR. Preferably, the 3'UTR may be derived from Coxsackievirus B5.

[0118] In one embodiment of the present invention, the 3'UTR may comprise or consist of the nucleotide sequence of SEQ ID NO: 10 (DNA) or SEQ ID NO: 69 (RNA).

[0119] Poly(A) tail In the present invention, the polynucleotide may further comprise a poly(A) tail sequence. The poly(A) tail sequence may be located at the 3' end of the nucleic acid sequence encoding the nonstructural protein of the self-amplifying virus. When the polynucleotide comprises a stop codon, the poly(A) tail sequence may be located at the 3' end of the stop codon. When the polynucleotide comprises a 3' UTR, the poly(A) tail sequence may be located at the 3' end of the nucleotide sequence of the 3' UTR. The poly(A) tail sequence can stabilize the transcribed nucleic acid molecule while further improving the translation efficiency of the target protein. The poly(A) tail sequence may comprise about 10 to about 500 adenosine (A) nucleotides. Specifically, the poly(A) tail sequence may be a nucleic acid sequence consisting of about 10 to about 500, about 50 to about 400, about 100 to about 300, or about 100 to about 200 adenosine nucleotides. Preferably, the poly(A) tail sequence may be consisting of about 120 adenosine nucleotides. In one embodiment of the present invention, the poly(A) tail sequence may comprise or consist of the nucleotide sequences of SEQ ID NOs: 38 to 40. Preferably, the poly(A) tail sequence may consist of the amino acid sequence of SEQ ID NO: 40.

[0120] Polynucleotide Structure The polynucleotide of the present invention has the following structural formula (I) in order from the 5' end to the 3' end: 5'-BC(1)-D-3' (I) (In structural formula (I), 5' and 3' are the 5' and 3' ends of the polynucleotide, respectively; B is a nucleic acid sequence encoding a first protein of interest, C(1) is a nucleic acid sequence encoding a protease cleavage site, and D is a nucleic acid sequence encoding a nonstructural protein of a self-amplifying virus).

[0121] More specifically, the polynucleotide has, in order from the 5' end to the 3' end, the following structural formula (II): 5'-U-[K] o -[B'-C(2)] n -BC(1)-DS-U'-P-3' (II) (In structural formula (II), 5' and 3' are the 5' and 3' ends of the polynucleotide, respectively; U is a nucleic acid sequence in the 5' untranslated region, K is the Kozak sequence, B' is a nucleic acid sequence encoding a second protein of interest, B is a nucleic acid sequence encoding a first protein of interest, D is a nucleic acid sequence encoding a nonstructural protein of the self-amplifying virus, S is a stop codon, U' is the nucleic acid sequence of the 3' untranslated region, P is a poly(A) tail, C(1) and C(2) are each independently a nucleic acid sequence encoding a protease cleavage site; n represents the number of [B'-C(2)] and is an integer of 0 to 10. When n is 2 to 10, each B' may be a nucleic acid sequence encoding the same or different target protein, and each C(2) may be a nucleic acid sequence encoding the same or different protease cleavage site. Furthermore, o is an integer of 0 or 1.

[0122] In this case, C(1) may be a 2A protease cleavage site, and C(2) may be a 2A protease cleavage site, a 3CD protease cleavage site, or a 3C protease cleavage site.

[0123] The 5' untranslated region, target protein, first target protein, second target protein, stop codon, 3' untranslated region, poly(A) tail, protease cleavage site, 2A protease cleavage site, 3CD protease cleavage site, and 3C protease cleavage site are the same as above.

[0124] The nonstructural protein may comprise the P2 and P3 domains of an enterovirus. Specifically, the nonstructural protein may comprise, from N-terminus to C-terminus, the P2 and P3 domains. More specifically, the nonstructural protein may comprise, from N-terminus to C-terminus, a 2A protease, 2B, 2C, 3A, 3B, and 3C proteases, and a 3D polymerase. In this case, the nonstructural protein, P2 domain, P3 domain, 2A protease, 2B, and 2C, 3A, 3B, and 3C proteases, and 3D polymerase are the same as those described above.

[0125] When the polynucleotide according to the present invention is RNA, the polynucleotide (hereinafter referred to interchangeably as "RNA construct") may be a self-amplifying RNA (saRNA). The RNA construct may be double-stranded or single-stranded, preferably single-stranded.

[0126] As used herein, the term "replicon" refers to a self-replicating nucleic acid sequence, and 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 generate one or more identical or substantially similar copies of the RNA replicon without a DNA intermediate. Hereinafter, in the present invention, saRNA virus vector, RNA replicon, saRNA, and saRNA construct may be referred to interchangeably.

[0127] Polynucleotide Embodiments In one embodiment, the polynucleotide may comprise the following structure from the 5' end to the C-terminus: [5'UTR-Kozak sequence-protein of interest-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 NOs: 111 to 115, 131, 134, 151 to 153, 155, 157, 162, or 111.

[0128] In one embodiment, the polynucleotide may comprise the following structure from the 5' to C-terminus: [5'UTR-Kozak sequence-second protein of interest-3C protease cleavage site-first protein of interest-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.

[0129] In one embodiment, the polynucleotide may comprise the following structure from the 5' to C-terminus: [5'UTR-Kozak sequence-second protein of interest-P2A self-cleaving peptide sequence-first protein of interest-2A protease cleavage site-nonstructural proteins (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.

[0130] Recombinant vector In another aspect of the present invention, there is provided a recombinant vector carrying the above-mentioned polynucleotide, wherein the polynucleotide is the same as above.

[0131] As used herein, the term "recombinant" means "made through genetic engineering" and means not occurring in nature.

[0132] As used herein, the term "vector" refers to a genetic construct that contains the necessary regulatory elements operably linked to express a gene insert, and is an expression vector (or recombinant vector) capable of expressing a protein of interest in a host cell. The term "operably linked" means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest are functionally linked to each other to perform their general functions. Operable linkage with a vector can be achieved using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes commonly known in the art. Specifically, recombinant vectors can be amplified in vitro by polymerase chain reaction (PCR), recombinantly produced by cloning, purified by cleavage and gel electroporation fractionation, or synthesized by chemical synthesis, but are not limited to these.

[0133] In the present invention, a recombinant vector can be used as a gene delivery vehicle to transport and express the polynucleotide of the present invention. Therefore, it is preferable to carry the polynucleotide in an appropriate expression vector. Suitable expression vectors that can be used in the present invention may contain, in addition to expression control elements such as a promoter, initiation codon, and termination codon, a signal sequence for membrane targeting or secretion. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding an immunogenic protein of interest, and must be functional in a subject when the gene construct is administered and must be in frame with the coding sequence.

[0134] As used herein, the term "promoter" refers to a nucleic acid sequence that controls the synthesis of a transcript, e.g., a transcript containing a coding sequence, by providing recognition and binding sites for RNA polymerase. Typical promoters may be constitutive or inducible. In the present invention, a promoter operably linked to a polynucleotide may be a promoter that can operate in animal cells, more preferably mammalian cells, to control the transcription of the polynucleotide. Promoters may include promoters derived from viruses, promoters derived from the genome of mammalian cells, or promoters derived from bacteriophages. For example, promoters may include, but are not limited to, the cytomegalovirus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, 94 promoter, T3 promoter, SM6 promoter, RSV promoter, EF1α promoter, metallothionein promoter, β-actin promoter, cancer cell-specific promoters (e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, and AFP promoter), and tissue-specific promoters (e.g., albumin promoter).

[0135] In one embodiment of the present invention, a polynucleotide can be used as a template and transcribed into an RNA nucleic acid molecule (saRNA construct, RNA replicon) through in vitro transcription (IVT) after being inserted into an appropriate expression vector. In this case, a promoter can be located at the 5' end of the polynucleotide to transcribe the linearized DNA into RNA.

[0136] "In vitro transcription" refers to the process by which RNA, particularly mRNA, is synthesized in a cell-free system in vitro. In this case, a cloning vector can be used to generate the transcription product. These cloning vectors are generally designated as transcription vectors and are included in the "recombinant vector" of the present invention. In the present invention, when the polynucleotide is RNA, the RNA may be in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription using an appropriate DNA template. In this case, the promoter regulating the transcription may be any promoter for RNA polymerase. Furthermore, the DNA template used for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA (a polynucleotide of the present invention), and introducing it into a vector suitable for in vitro transcription (a recombinant vector carrying a polynucleotide of the present invention). In the case where the nucleic acid (a polynucleotide of the present invention) is RNA, the cDNA can be obtained by reverse transcription.

[0137] Specifically, RNA can be synthesized using a DNA-dependent RNA polymerase such as T7 RNA polymerase, and in this case, the expression vector may contain a T7 promoter at the 5' end of the polynucleotide carried. When another RNA polymerase, such as SP6 RNA polymerase or T3 RNA polymerase, is used during IVT using an expression vector carrying a polynucleotide as a template, the vector may contain an SP6 promoter or a T3 promoter. In one embodiment of the present invention, the RNA polymerase may be T7 RNA polymerase, and the recombinant vector may contain a T7 promoter. Specifically, the recombinant vector may contain the nucleic acid sequence of SEQ ID NO: 94 upstream of the 5' end of the polynucleotide of the present invention, or may contain a promoter consisting of this sequence.

[0138] The vector that can be used as a template to synthesize the self-amplifying RNA construct may be, for example, a plasmid, cosmid, phage, viral vector, etc. In this case, the polynucleotide sequence may be a DNA sequence, preferably a plasmid DNA.

[0139] The term "plasmid" generally refers to a construct of extrachromosomal genetic material, i.e., a circular double-stranded DNA that can replicate independently of chromosomal DNA.

[0140] Furthermore, in the present invention, a recombinant vector can autonomously replicate in a host cell, in which case the vector can be introduced into the host cell, recombine, be inserted into the host cell genome, and be replicated together with the host cell genome.

[0141] In this case, the recombinant vector may further contain a promoter, regulator, or enhancer that controls the expression of the polynucleotide carried therein.

[0142] Additionally, the plasmid may contain a selection marker such as an antibiotic resistance gene, allowing host cells harboring the plasmid to be cultured under selective conditions.

[0143] Transformed host cells In another aspect of the present invention, there is provided a host cell transformed with a recombinant vector, the recombinant vector being the same as described above.

[0144] As used herein, the term "transformation" refers to the artificial production of a genetic change by introducing foreign DNA into a cell so that the DNA is replicable as a chromosomal element, or by introducing DNA into a host cell so that it is integrated into the chromosome.

[0145] The term "host cell" as used herein refers to a cell that feeds on other microorganisms or genes and is transformed into a host cell by a vector to produce various genetic or molecular effects in the host cell. Host cells are in a competent state to accept external DNA, and external DNA, such as a vector, can be inserted. When the vector is successfully introduced into the host cell, the genetic traits of the vector are imparted to the host cell. Host cells may include prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeast cells and insect cells), and may include mammalian cells, such as cells derived from humans, mice, hamsters, pigs, goats, or primates.

[0146] Transformation can be carried out by a variety of methods. Specifically, transformation methods that can be used include CaCl precipitation, the Hanahan method (CaCl precipitation with increased efficiency achieved by using a reducing agent such as dimethyl sulfoxide (DMSO)), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate-mediated transformation, lipofectamine-mediated transformation, and desiccation / inhibition-mediated transformation. Methods for transforming the plasmids of the present invention are not limited to the above examples, and any transformation method commonly used in the art can be used without limitation.

[0147] Method for producing RNA replicon In another aspect of the present invention, there is provided a method for producing an RNA replicon, comprising: i) producing a recombinant vector carrying a polynucleotide according to the present invention; and ii) synthesizing RNA from the recombinant vector, wherein the polynucleotide, recombinant vector, and RNA replicon are the same as those described above.

[0148] An RNA replicon can be obtained by in vitro transcription using the DNA of a recombinant vector as a template. Generally, the poly(A) tail sequence is encoded by a poly(dT) sequence on the DNA template. In the present invention, the poly(A) tail sequence can be added using an enzyme after transcription. Furthermore, in the present invention, the poly(A) tail sequence can be transcribed from the recombinant vector. Preferably, the poly(A) tail sequence can be transcribed from the recombinant vector.

[0149] Suitable methods for in vitro transcription are well known in the art and are known to those skilled in the art. For example, these methods are described in "Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) edited by C Nolan, Cold Spring Harbor Laboratory Press." In addition, various in vitro transcription kits are commercially available.

[0150] Vaccine Composition In another aspect of the present invention, there is provided a vaccine composition comprising as an active ingredient the polynucleotide or recombinant vector of the present invention, wherein the polynucleotide and recombinant vector are the same as those described above.

[0151] As used herein, the term "vaccine" refers to a drug used to stimulate the immune system using an organism for the purpose of preventing disease. Immune activation refers to the process of efficiently eliminating 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 related to the above is readily understood by those skilled in the art (Barrett, JT, Textbook of Immunology, 1983). In this specification, the terms "vaccine" and "vaccine composition" can be used interchangeably.

[0152] In the present invention, the vaccine composition may be for preventing or treating a protozoan, fungal, bacterial, or viral infection. In the present invention, the vaccine composition may be for preventing or treating a cancer.

[0153] In the vaccine composition, the polynucleotide or recombinant vector as an active ingredient may be contained in an amount sufficient to induce a typical immune response. In the vaccine composition, the active ingredient may be contained in an amount of about 0.0001 μg to about 1000 μg. Specifically, the active ingredient may be contained 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 to these.

[0154] In the vaccine composition of the present invention, the active ingredient may be contained in an amount sufficient to induce a typical immune response, i.e., any amount (effective amount) depending on the purpose, formulation, purpose of mixing, etc. Typically, the effective amount is determined within the range of 0.001% by weight to 20.0% by weight 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 ordinary skill of one skilled in the art.

[0155] Furthermore, the vaccine composition may contain a pharmaceutically acceptable carrier or diluent. Carriers used in the compositions of the present invention include pharmaceutically acceptable carriers, adjuvants, and vehicles, and are collectively referred to as "pharmaceutically acceptable carriers." Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond the extent that may be applicable to the subject of application (formulation). Pharmaceutically acceptable carriers that can be used in the compositions of the present invention include, but are not limited to, ion exchange carriers, alumina, 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, polyvinylpyrrolidone, cellulose-based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene blocked polymers, polyethylene glycol, and wool fat.

[0156] Meanwhile, the vaccine composition of the present invention can be administered in a "therapeutically effective amount," which can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health condition, sex, and sensitivity to the drug, the administration route, the administration method, the number of doses, the treatment period, and drugs used in combination or simultaneously.

[0157] As used herein, the term "administration" means introducing a given substance into a subject by an appropriate method, and as for the route of administration of a composition, the composition may be administered via any common route as long as it can reach the target tissue. For a vaccine to be effective in producing antibodies, the antigenic substance must be introduced into the body so that the antibody production mechanism of the vaccinated subject can be activated. Therefore, for an immune response, the polynucleotide or recombinant vector according to the present invention must first be introduced into the body.

[0158] When the polynucleotide or recombinant vector of the present invention is administered to a subject, it can be administered in the form of a liposome or vesicle by encapsulating it in a lipid nanoparticle (LNP) or coating it with a lipid. In this case, the lipid encapsulation can be in the form of a lipid aggregate or a micelle. LNP can be prepared using methods known in the art.

[0159] In the present invention, the antigen presented by the vaccine composition can be administered systemically via parenteral administration to stimulate a desired response. Parenteral administration can be performed in the form of, but is not limited to, intranasal administration, intranasal administration, oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, intracardiac administration, transdermal administration, subcutaneous administration, intradermal administration, intraperitoneal administration, enteral administration, topical administration, sublingual administration, or rectal administration. Preferably, it can be administered subcutaneously or intramuscularly.

[0160] Furthermore, in the present invention, the vaccine composition can be administered once or repeatedly multiple times.

[0161] Specifically, the vaccine composition can be administered repeatedly, but is not limited to, once, twice, three times, four times, or five times, as long as the vaccine is effective. The vaccine composition can enhance its preventive or therapeutic effect against a disease by repeated administration.

[0162] The subject to which the vaccine composition according to the present invention is administered may be a mammal, such as, for example, a human, cow, horse, pig, dog, sheep, goat, or cat, and preferably a human.

[0163] The preferred dosage of the vaccine composition may vary depending on factors such as the formulation method, administration method, age, body weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response susceptibility of the subject, and can be appropriately selected by those skilled in the art. The dosage appropriate for the subject 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 conventional vaccines. The dosage can be easily determined as needed through routine experimentation. A typical initial vaccine dose may be about 0.001 μg / kg to about 10 mg / kg, or about 0.01 μg / kg to about 1 mg / kg of antigen, with increased doses or multiple doses used as necessary to provide the desired level of protection. The vaccine composition may be administered once daily, daily, every other day, weekly, or every other week. Such dosages should in no way be construed as limiting the scope of the present invention.

[0164] The vaccine composition may be prepared, for example, as a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposomal suspension, polyplex, emulsion, lipid nanoparticles (LNPs) (with RNA on the surface or encapsulated within), or any other suitable form that can be administered to a human or mammal in need of treatment or vaccination.

[0165] When the vaccine composition is administered intranasally or parenterally, it can be administered, but is not limited to, in the form of a spray or aerosol, or by inhalation. Compositions for intranasal or intranasal administration are prepared according to techniques well known in the pharmaceutical art and may be prepared as a solution in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0166] For parenteral administration, the vaccine composition according to the present invention can be prepared as a sterile injectable preparation in the form of a sterile injectable aqueous or oleaginous suspension. Suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Acceptable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any fixed oil with low irritation properties can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injectable preparations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated forms.

[0167] The above-mentioned formulations are known in the art, and specific reference may be made to "Remington's Pharmaceutical Sciences" (19th edition, 1995), etc. The above-mentioned documents are incorporated herein by reference.

[0168] The vaccine composition of the present invention may further contain an adjuvant to enhance the immunogenicity of the vaccine. Auxiliary components such as adjuvants can be appropriately selected and used depending on the antigen type and the indication of the vaccine composition.

[0169] 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 those described above. The polynucleotide, recombinant vector, or vaccine composition can be administered once or multiple times. Specifically, it can be administered once, twice, three times, four times, or five times, and can be administered repeatedly indefinitely as long as its preventive or therapeutic effect can be fully exerted. As described above, repeated administration can enhance the preventive or therapeutic effect against the disease.

[0170] As used herein, the term "treatment" can be used to refer to both therapeutic and prophylactic treatment. In this context, the term "prevention" can be used to refer to alleviating or reducing a condition or disease in a subject. Treatment includes all applications or forms of drug therapy for treating disease in mammals, including humans. Furthermore, the term includes preventing or slowing the progression of a disease, partially or completely alleviating a disease by restoring or repairing damaged or defective function, or stimulating an inefficient process, or alleviating severe disease.

[0171] The disease may be an infectious disease caused by a protozoan, fungus, bacterium, or virus, or may be a cancerous disease.

[0172] The subject may be a mammal, preferably a human, suffering from or likely to suffer from a disease.

[0173] The dosage may vary depending on factors such as formulation method, administration method, age, weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response sensitivity.

[0174] Novel protease cleavage site sequences In another embodiment of the present invention, there is provided a nonstructural protein 2A protease cleavage site from enterovirus, comprising the amino acid sequence of SEQ ID NO: 22. The enterovirus, nonstructural protein, and 2A protease cleavage site are the same as above.

[0175] In another embodiment of the present invention, there is provided a nonstructural protein 3C protease cleavage site from enterovirus, comprising the amino acid sequence of SEQ ID NO: 50. The enterovirus, nonstructural protein, and 3C protease cleavage site are the same as above.

[0176] In another aspect of the invention there is provided an IRES mutant comprising the nucleic acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. The IRES mutant is as described above.

[0177] Novel 5' untranslated region variant sequences In another embodiment of the present invention, there is provided a 5' untranslated region variant comprising the nucleic acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87. The 5' untranslated region variant is the same as described above.

[0178] Novel Kozak sequence In another embodiment of the present invention, there is provided a Kozak sequence comprising the nucleic acid sequence of SEQ ID NO: 35. The Kozak sequence is the same as described above.

[0179] Novel 2A protease sequence In another embodiment of the present invention, there is provided a nonstructural protein 2A protease from enterovirus, comprising the amino acid sequence of SEQ ID NO: 75. The 2A protease is the same as described above.

[0180] Novel severe fever with thrombocytopenia syndrome (SFTS) virus antigen sequences In another aspect of the present invention, there is provided a severe fever with thrombocytopenia syndrome (SFTS) viral 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.

[0181] Novel human papillomavirus antigen sequences In another aspect of the present invention, 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 above. [Example]

[0182] MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples.

[0183] I. Optimized self-amplifying RNA production Preparation Example 1. Preparation of an enterovirus-based self-amplifying RNA-producing vector A clone vector was prepared to generate an enterovirus-based self-amplifying RNA construct with luciferase inserted as the target protein, which is used as a base scaffold in the production of the optimized self-amplifying RNA construct of the present invention.

[0184] Specifically, the cloning vector used in the present invention was prepared by inserting the Coxsackievirus B5 gene (SEQ ID NO: 99) into the pUC19-CMV / T7 (Addgene, Plasmid No. 50005) vector to produce an infectious clone, as described in "Development of a Universal Cloning System for Reverse Genetics of Human Enteroviruses" (W.S. Choi, et al., Microbiol. Spectr. 18; e0316722). In this clone, the P1 domain (structural protein, VP) was replaced with a nucleic acid encoding the target protein (luciferase, SEQ ID NO: 41) using the in-fusion cloning method (In-Fusion™ HD Cloning Kit, Takara Bio Inc.), producing a cloning vector in the form of a self-amplifying RNA construct (FIG. 16).

[0185] Example 1. Selection of 2A protease cleavage site sequence for self-amplifying RNA To prepare the modified self-amplifying RNA (saRNA) construct with optimized expression level of the target protein according to the present invention, we first confirmed the expression level of the target protein according to the amino acid sequence of the cleavage site of 2A protease, a nonstructural protein of enterovirus.

[0186] Example 1.1.2A Production of a Self-Amplifying RNA Construct with a Protease Cleavage Site Sequence Specifically, we constructed a library vector by inserting the nucleotide sequences of SEQ ID NOS: 24 to 29 into the 2A protease cleavage site of an enterovirus-based self-amplifying RNA construct containing luciferase (produced in-house by the method described in Preparation Example 1) using a known mutagenesis method, resulting in the insertion of one (VP1-1a.a, SEQ ID NOS: 18), five (VP1-5a.a, SEQ ID NOS: 19), ten (VP1-10a.a, SEQ ID NOS: 20), fifteen (VP1-15a.a, SEQ ID NOS: 21), twenty (VP1-20a.a, SEQ ID NOS: 22), or twenty-five (VP1-25a.a, SEQ ID NOS: 23) amino acid sequence into the vector DNA (Figure 2a). The vector DNA was linearized with BsmbI (NEB) restriction enzyme and incubated at 55°C for 1 to 6 hours, after which the restriction enzyme was inactivated at 80°C for 20 minutes. After confirming that the DNA was linear by electrophoresis on a 0.9% agarose gel, it was purified as follows.

[0187] In vitro transcription (IVT) was performed using the purified linearized DNA as a template using the Invitrogen™ MEGAscript™ T7 Transcription Kit for 3 hours at 37° C. After treatment with DNase to remove residual DNA template, the produced RNA was purified using lithium chloride (LiCl) precipitation (Table 1).

[0188] [Table 1]

[0189] Example 1.2.2A Comparison of expression levels of self-amplifying RNA constructs by protease cleavage site sequence The self-amplifying RNA constructs prepared by the method in Example 1.1 were transfected into 293T cells to confirm the expression level.

[0190] Specifically, 293T cells were cultured in a 6-well plate at 5 × 10 cells per well. 5 After seeding with 100 cells and culturing overnight, the medium was replaced with a medium containing no fetal bovine serum (FBS) 4 hours before transfection to prepare the cells.

[0191] Each purified RNA was transfected into cells at 5 μg per well using Lipofectamine™ 2000 transfection reagent (Invitrogen). 12 hours after transfection, the cells were harvested and centrifuged (12,000 rpm, 3 minutes) to precipitate the cells. Luciferase Cell Culture Lysis 1x Reagent (100 μl) was added and incubated at room temperature for 30 minutes. After the reaction was complete, the reaction solution was centrifuged again (12,000 rpm, 1 minute), and the luciferase activity of the supernatant (20 μl) was measured using the Luciferase Assay System reagent (Promega).

[0192] As a result, as shown in Figure 2b, the luciferase luminescence was highest in the group treated with a self-amplifying RNA construct (VP1-20a.a) into which 20 amino acids from the C-terminus of VP1 had been inserted.

[0193] Therefore, a 20 amino acid sequence from the C-terminus of VP1 (SEQ ID NO: 22) was used as the 2A protease cleavage site for the modified self-amplifying RNA construct with optimized expression levels of the present invention.

[0194] Example 2. Kozak sequence and stop codon selection Example 2.1. Production of a self-amplifying RNA construct with a Kozak sequence and a stop codon In order to prepare a modified self-amplifying RNA construct with an optimized expression level of a target protein in the present invention, the expression level of the target protein was confirmed by a Kozak sequence and a stop codon.

[0195] Self-amplifying RNA constructs were prepared in the same manner as in Example 1.1. The recombinant vectors contained Kozak sequences, Kozak 1 (SEQ ID NO: 35, CCACC), Kozak 2 (SEQ ID NO: 36, GCCACC), or Kozak 4 (SEQ ID NO: 37, AAG), inserted at the 3' end of the 5' UTR. The stop codon used was a sequence consisting of three different stop codons (3xSTOP) (SEQ ID NO: 9, TGATAATAG) at the 5' end of the 3' UTR (Table 2, Figure 3a).

[0196] [Table 2]

[0197] Example 2.2. Comparison of expression levels of self-amplifying RNA constructs with Kozak sequences and stop codons The expression of each self-amplifying RNA construct prepared by the method of Example 2.1 was measured in 293T cells using luciferase assay reagent (Promega) in the same manner as in Example 1.2, with a clone vector (original PC) prepared in-house from Coxsackievirus B5 used as a control.

[0198] As a result, as shown in Figure 3b, the self-amplifying RNA construct (Kozak 4) into which the Kozak sequence of Kozak 4 was inserted exhibited the highest luciferase activity. Furthermore, compared to the control, the self-amplifying RNA construct containing the stop codon of SEQ ID NO: 68 (SEQ ID NO: 9 for DNA) exhibited significantly increased luciferase activity.

[0199] Thus, the Kozak sequence of the modified self-amplifying RNA construct with optimized expression level of the present invention used was the sequence of SEQ ID NO: 42, and the stop codon used was the 3x stop codon of SEQ ID NO: 68 (SEQ ID NO: 9 for DNA).

[0200] Example 3. Selection of poly(A) tail sequence Example 3.1. Production of self-amplifying RNA constructs with poly(A) tail sequences To prepare the modified self-amplifying RNA construct of the present invention with an optimized expression level of a target protein, the expression level of the target protein was confirmed by using a poly(A) tail sequence.

[0201] Self-amplifying RNA constructs were prepared in the same manner as in Example 1.1. Recombinant vectors were prepared 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) residues into the 3' end of the 3' UTR (Table 3, Figure 4a).

[0202] [Table 3]

[0203] Example 3.2. Comparison of expression levels of self-amplifying RNA constructs with poly(A) tail sequences The expression of each self-amplifying RNA construct produced by the method of Example 3.1 was measured in 293T cells using luciferase assay reagent (Progmega) in the same manner as in Example 1.2.

[0204] Furthermore, the self-amplifying RNA constructs were encapsulated in NanoAssemblr™ Ignite (Precision NanoSystems) using a lipid nanoparticle (LNP) biodelivery system (ALC-0315 (MEC, HY-138170) / cholesterol (Sigma, C8667) / DMG-PEG 2000 (Avanti Polar Lipids, 880151P) / DOPE (Avanti Polar Lipids, 850725P)) and administered to mice to confirm expression levels at the animal level. Five- to six-week-old female BALB / c mice (n = 3) were used, and 1 μg of each self-amplifying RNA construct was intramuscularly administered to each mouse. Four hours after administration, luciferin was administered intravenously to the mice at a concentration of 150 mg / kg per mouse, and luciferase activity in the mice was monitored using an IVIS Spectrum (Perkin Elmer).

[0205] As a result, as shown in Figure 4b, the self-amplifying RNA construct with a 120x polyA tail inserted showed the highest luciferase activity at both the cellular and animal levels.

[0206] Therefore, the poly(A) tail of the modified self-amplifying RNA construct with optimized expression level of the present invention used was the sequence of SEQ ID NO:40.

[0207] II. Antigen expression using optimized self-amplifying RNA Example 4. Production of optimized self-amplifying RNA and confirmation of expression level Example 4.1. Optimized self-amplifying RNA production Similar to Example 1.1, optimized self-amplifying RNA constructs were prepared from recombinant vectors containing the nucleotide sequence of the 2A protease cleavage site (VP1-20a.a, SEQ ID NO: 28), Kozak sequence (Kozak 1, SEQ ID NO: 35), and poly(A) tail (120× poly(A), SEQ ID NO: 40) selected in Examples 1 to 3 (Table 4).

[0208] To compare the degree of improvement in expression level of the modified self-amplifying RNA construct, mRNA and a conventional enterovirus-based self-amplifying RNA construct (saRNA) were produced together and used as an experimental group (Figure 5). The target protein was cloned into a 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) construct using the 5'UTR and 3'UTR derived from human globulin, linearized, and then used as template DNA to prepare mRNA by reacting at 37°C for 6 hours using the MESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen). The mRNA was then purified in the same manner as the self-amplifying RNA construct (Table 4, Figure 5).

[0209] [Table 4]

[0210] Example 4.2. Confirmation of expression levels of optimized self-amplifying RNA 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. For confirmation at the cellular level, 293T cells were seeded into 12-well plates (2.5 × 10 cells per well) the day before transfection. 5 After incubation at 4°C for 1 hour, the cells were cultured overnight.

[0211] Transfection and luciferase activity measurement were carried out in the same manner as in Example 1.2. During transfection, RNA was added at concentrations of 0.01 μg, 0.1 μg, 0.5 μg, or 1 μg.

[0212] As a result, as shown in FIG. 6, the luciferase activity in the group treated with the modified self-amplifying RNA construct of the present invention was the highest across all RNA concentrations.

[0213] To confirm expression at the animal level, mRNA, self-amplifying RNA constructs, or modified self-amplifying RNA constructs were encapsulated in NanoAssemblr™ Ignite (Precision NanoSystems) using the LNP biodelivery system (ALC-0315 / cholesterol / PEG / DOPE) and then intramuscularly administered to mice, as described in Example 3.2. RNA was administered at concentrations of 0.01 μg, 0.1 μg, or 1 μg per mouse. Four hours after administration, luciferin was intravenously administered to the mice at a concentration of 150 mg / kg per mouse, and luciferase activity in the mice was monitored using an IVIS Spectrum (Perkin Elmer).

[0214] As a result, the highest luciferase activity was observed in the group administered with the modified self-amplifying RNA construct of the present invention, as shown in Figure 7. In particular, activity was clearly observed even in the group administered with 0.01 µg, while no luciferase activity was observed in the groups administered with the same concentrations of mRNA or conventional enterovirus-based self-amplifying RNA construct.

[0215] Example 5. Confirmation of antigen expression levels of optimized self-amplifying RNA constructs containing antigens Example 5.1. Production of an optimized self-amplifying RNA construct containing an antigen In the optimized modified self-amplifying RNA construct of Example 4.1, the target protein was replaced with a 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, and a recombinant vector was prepared. Optimized self-amplifying RNA constructs were prepared from the recombinant vector in the same manner as in Example 1.1 (Tables 5 to 7). The severe fever with thrombocytopenia syndrome virus antigens used were the nucleic acid sequence (SEQ ID NO: 43) encoding the SFTS B antigen (SEQ ID NO: 45) and the nucleic acid sequence (SEQ ID NO: 83) encoding the SFTS ABDEF antigen (SEQ ID NO: 85). The nucleic acid sequence (SEQ ID NO: 46) encoding the SARS-CoV2 spike protein antigen (SEQ ID NO: 48) was also used. The highly pathogenic avian influenza virus antigen used was the nucleic acid sequence (SEQ ID NO: 106) encoding the H5N8 antigen (SEQ ID NO: 105). To linearize the vector DNA, the vector DNA was reacted with BsmBI (NEB) restriction enzyme at 55°C for 6 hours and inactivated at 80°C for 10 minutes.

[0216] To compare the degree of improvement in expression level of the modified self-amplifying RNA constructs, mRNA was also produced and used as an experimental group. The target protein was cloned into the 5'UTR-target protein-3'UTR-poly(A) tail (50) using the 5'UTR and 3'UTR derived from human globulin, linearized, and then used as template DNA in a MESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen) at 37°C for 6 hours. The mRNA was then purified in the same manner as the self-amplifying RNA constructs (Tables 5 to 7).

[0217] [Table 5]

[0218] [Table 6]

[0219] [Table 7]

[0220] Example 5.2. Confirmation of expression levels of optimized self-amplifying RNA The expression level of the modified self-amplifying RNA construct prepared by the method of Example 5.1 was confirmed at the cellular level by Western blot. At this time, the efficiency of the cellular delivery system (LNP biodelivery system, Lipofectamine) was also confirmed using RNA containing the SFTS viral antigen (Figure 8).

[0221] First, to prepare the LNP-RNA experimental group, each of the above RNAs (including SFTS antigens) was encapsulated in NanoAssemblr™ Ignite (Precision NanoSystems) using the LNP biodelivery system (ALC-0315 / cholesterol / PEG / DOPE). The encapsulated RNA (LNP-RNA) was diluted 10-fold in 1x TE (Tris-EDTA) buffer containing 2% (vol / vol) Triton X-100 (Sigma-Aldrich). The fluorescence of the LNP-RNA was measured at wavelengths of 485 nm (excitation) and 528 nm (emission) using a microplate reader (BMG LABTECH, UK). The RNA concentration corresponding to the fluorescence value was also measured.

[0222] Transfection was performed by treating cells with LNP-RNA or each RNA prepared at various concentrations as described above. For each RNA, transfection was performed using Lipofectamine™ 2000 transfection reagent (Invitrogen) in the same manner as in Example 1.2. Furthermore, on the day before transfection, cells were transfected at 1 × 10 per well. 6After seeding the cells at a concentration of 1 / 3 cells into a 6-well plate, the medium was replaced with a medium without fetal bovine serum 4 hours before transfection, and the cells were cultured, prepared, and transfected.

[0223] The medium was replaced 4 hours after transfection and the cells were cultured for an additional 24 hours. After 24 hours, cells were harvested by centrifugation (12,000 rpm, 1 minute) and lysed by adding 1 ml of cell lysis buffer and vortexing. After incubation at room temperature for 10 minutes, protein concentration was measured. Protein samples were prepared by mixing 10 μg of protein with 2x sample buffer (SDS sample buffer with the same volume as the protein) and incubating at 95°C for 7 minutes. Each protein sample was electrophoresed on an SDS-PAGE gel. The gel was then transferred to a membrane, blocked with 5% lipoprotein solution, and incubated with primary antibodies for 16 hours at 4°C. Anti-Gc antibody (Novusbio, NBP2-41153, 1:2000) and anti-spike antibody (Sino Biological, 40591-MM42, 1:2000) were used as primary antibodies for each antigen.

[0224] After the primary antibody reaction, the membrane was washed five times with TBS-T buffer for 10 minutes each, and then the secondary antibody was added and incubated at room temperature for 1-2 hours. The secondary antibodies used were anti-rabbit HRP antibody (Abcam, ab205718, 1:2000) and anti-mouse HPR antibody (Invitrogen, 62-6520, 1:5000). After the secondary antibody reaction, each membrane was washed five times with TBS-T for 10 minutes each, and then detected using HRP reagent (Millipore).

[0225] As a result, as shown in Figure 8, RNA transfected using LNP showed higher expression levels than RNA transfected using Lipofectamine (top). Furthermore, it was confirmed that antigen expression was significantly higher in the optimized modified self-amplifying RNA constructs of the present invention containing the SFTS antigen (Figure 8) or SARS-CoV2 antigen (Figure 9) compared to mRNA.

[0226] III. Preparation of multi-antigen self-amplifying RNA Example 6. Preparation of multi-antigen self-amplifying RNA and confirmation of expression level Example 6.1. Preparation of multi-antigen self-amplifying RNA To prepare a vaccine capable of expressing two types of multiple antigens, the self-cleaving peptide P2A (porcine teschovirus-1 2A) or the sequence of the enterovirus 3C protease cleavage site was inserted into an enterovirus-based self-amplifying RNA construct, and a multi-antigen self-amplifying RNA construct was prepared in the same manner as in Example 1.1.

[0227] Luciferase was used as the first target protein, and a canine influenza H3N2 virus antigen was used as the second target protein. The multi-antigen self-amplifying RNA constructs were constructed in the following 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 (P2A-containing self-amplifying RNA construct: SEQ ID NO: 190, 3C protease-containing self-amplifying RNA construct: SEQ ID NO: 192). To optimize the expression of multiple antigens, we prepared self-amplifying RNA constructs by crossing the target protein positions, and compared the protein expression levels ([5'UTR-Kozak sequence-first target protein (luciferase)-P2A or 3C protease cleavage site-second target protein (H3N2 viral antigen)-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: 194, self-amplifying RNA construct containing 3C protease cleavage site: SEQ ID NO: 196). Additionally, we prepared and used a single-antigen (luciferase) self-amplifying RNA construct.

[0228] First, the expression level of the multi-antigen self-amplifying RNA construct was confirmed at the cellular level by luciferase and Western blot analysis. Luciferase activity was measured using Lipofectamine™ 2000 transfection reagent (Invitrogen) as described in Example 1.2, and Western blot analysis was performed as described in Example 5.2. Each cell was transfected with either the multi-antigen self-amplifying RNA construct or the single-antigen self-amplifying construct (1 μg or 5 μg, respectively).

[0229] Furthermore, the primary antibody used for Western blotting was an anti-H3N2 antibody produced in-house at a dilution of 1:2000, and the secondary antibody was an anti-mouse HRP antibody (Invitrogen, 62-6520, 1:20000). Anti-H3N2 antibodies were used by preparing acellular vaccines as follows: 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 in a 1:1 ratio and administered intramuscularly to mice. After the first administration, three additional doses were administered at 2-week intervals, and antibodies were generated by direct isolation from the blood.

[0230] As a result, as shown in Figure 37, it was confirmed that the luciferase luminescence was lower in the multi-antigen self-amplifying RNA construct compared to the single-antigen self-amplifying RNA construct, but high luciferase luminescence was observed in each self-amplifying RNA construct containing the P2A sequence and the 3C protease cleavage sequence.

[0231] Furthermore, as shown in Figure 38, it was confirmed that the construct containing the P2A sequence and the construct containing the 3C protease cleavage site exhibited similar levels of H3N2 expression.

[0232] The above results confirmed that the multi-antigen self-amplifying RNA construct was capable of simultaneously expressing two types of antigens.

[0233] Example 6.2. Preparation of multi-antigen self-amplifying RNA containing canine influenza antigens Using canine influenza subtypes H3N2 and H3N8, two antigens were inserted into an enterovirus-based self-amplifying RNA construct, and the amino acid sequence of a 3C protease cleavage site (SEQ ID NO: 50) was inserted between the antigens to prepare a multi-antigen self-amplifying RNA construct in the same manner as in Examples 1.1 and 4.1 above. Canine influenza subtypes H3N2 and H3N8 were used as antigens (Table 8, Figure 10, and top of Figure 11).

[0234] Furthermore, a self-amplifying RNA construct and mRNA containing only the H3N2 antigen were also prepared and used. In this case, the target protein was cloned into a 5'UTR-target protein-3'UTR-poly(A) tail (50) construct using the 5'UTR and 3'UTR derived from human globulin, as in Example 4.1. This construct was then linearized and used as template DNA to prepare mRNA using the MESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen).

[0235] [Table 8]

[0236] Example 6.3. Confirmation of antigen expression in multi-antigen self-amplified RNA The expression levels of the multi-antigen self-amplifying RNA constructs prepared by the method of Example 6.2 were confirmed at the cellular level by Western blot. Each RNA was transfected using Lipofectamine™ 2000 transfection reagent (Invitrogen) as described in Example 1.2. Each RNA was transfected at a concentration of 0.1 μg, 1 μg, or 5 μg per well. Four hours after transfection, the medium was replaced, and the cells were cultured for an additional 24 hours. PC WT virus was used as a control. The PC WT virus was canine influenza virus (A / dog / Korea / AS-01 / 2012). Western blot analysis was performed as described in Examples 5.2 and 6.1 above.

[0237] As shown in Figure 11, the group treated with the self-amplifying RNA construct showed significantly increased antigen expression compared to the group treated with mRNA (bottom left). The expression level was slightly lower in the group treated with the multi-antigen self-amplifying RNA construct compared to the group treated with the single-antigen self-amplifying RNA construct. Furthermore, in the group treated with the multi-antigen self-amplifying RNA construct, two types of antigens were observed in both fused and isolated forms (bottom right).

[0238] The above results confirmed that the multi-antigen self-amplifying RNA construct was capable of simultaneously expressing two types of antigens.

[0239] IV. Production of optimized self-amplifying RNA with improved amplification rate Example 7. Experiments on adapting viruses to cells and animals to increase virus growth rate Example 7.1. Production of cell-adapted Coxsackievirus B5 Cell-adapted virus was produced by infecting Vero cells with wild-type Coxsackievirus B5 (Vp8-B5 in Figure 12), which was isolated after passage from the cerebrospinal fluid of an infant with aseptic meningitis. Vero cells were cultured in DMEM (Gibco, Cat. No. 12430-054) containing 10% fetal bovine serum and 1 x 10 cells per well were cultured the day before infection. 6 Cells were prepared by seeding into 6-well plates at a concentration of 1000 x g.

[0240] On the day of infection, the virus was washed with saline and then diluted 1:100 in fetal bovine serum-free DMEM (Gibco, catalog number 12430-054) and used to treat the cells. After treatment, the cells were cultured for 2 hours, and then the culture medium was replaced with DMEM containing 2% fetal bovine serum (FBS). The cells were then cultured for an additional 2 to 4 days to check for cytopathic effects.

[0241] When morphological changes were observed in 80% to 90% of infected cells, the cells were harvested and centrifuged (1200 rpm, 3 min). 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.

[0242] As a result, it was confirmed that the passaged Coxsackievirus B5 showed an increased virus growth rate in infected cells compared to wild-type Coxsackievirus B5, and that cellular morphological changes appeared more rapidly.

[0243] Example 7.2. Production of mouse-adapted Coxsackievirus B5 The cell-adapted Coxsackievirus B5 obtained as described in Example 7.1 above was intraperitoneally administered to 3-4 week-old female BALB / c mice (SAMTACO) at a concentration of 5.5 TCID / 200 μl per mouse. Body weight changes were monitored for 7 days after administration to determine whether the virus caused pathogenicity in the mice. Weight loss was confirmed 3-4 days after infection. When weight loss was observed 4 days after infection, the mice were sacrificed by cervical dislocation, and the intestines were isolated. The isolated intestines were transferred to a tube and homogenized by adding 500 μl of culture medium. The homogenized intestinal tissue was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was collected. The virus concentration was measured using the supernatant, and other mice were infected with the virus in the same manner as described above. The above infection process was repeated 24 times in total (VP8Mp24-B5 in Figure 12).

[0244] Example 7.3. Evaluation of Growth Kinetics of Cell- and Mouse-Adapted Coxsackievirus B5 To confirm the changes in the growth rate of Coxsackievirus B5 obtained in the same manner as in Examples 7.1 and 7.2 above, the median tissue culture infectious dose (TCID 50 )Experiments were conducted.

[0245] Specifically, the viruses used were cell-adapted Coxsackievirus B5 (VP8-B5), cell- and mouse-adapted Coxsackievirus (VP8M24-B5), and wild-type Coxsackievirus B5 (WT-B5). Vero cells were infected with 1 × 10 cells per well the day before infection. 6 Each virus was prepared by inoculating a 96-well plate at a concentration of 1 / 10 cells. Each virus was serially diluted 1 / 10 from the stock solution for a total of seven times.

[0246] Vero cells prepared as described above were washed with saline and then treated with the prepared virus at various concentrations (50 μl per well). After 2 hours, the medium was replaced with DMEM containing 2% FBS, and the cells were further cultured. Three days after infection, when morphological changes were observed in 80%–90% of the cells, the cells were fixed with 10% formalin for 20 minutes. The cells were then stained with 1.25% crystal violet dye for 30 minutes, washed with distilled water, and the remaining cells in each well were examined. The results were quantified using the Reed-Münch method.

[0247] As a result, it was confirmed that the viral titer was significantly improved in the groups treated with VP8-B5 or VP8M24-B5 compared to WT-B5 (FIG. 13).

[0248] Example 7.4. Analysis of the Nucleic Acid Sequence of Mouse-Adapted Coxsackievirus B5 The nucleotide sequence of the mouse-adapted Coxsackievirus 5 obtained as in Example 7.2 above was analyzed.

[0249] Specifically, RNA was extracted from the virus to generate cDNA, and the entire viral genome was then amplified using universal primers (SEQ ID NO: 108, SEQ ID NO: 109). Sanger sequencing was performed using the PCR products obtained through the above process.

[0250] As a result, nucleotide sequence mutations were confirmed in the 5'UTR (two sites) and 2A protease (two sites) of the mouse-adapted Coxsackievirus B5 nucleic acid sequence compared to the wild-type Coxsackievirus B5 (Table 9, Figure 14). The mutated sequences are underlined in the table below.

[0251] [Table 9] TIFF2026508299000011.tif53170

[0252] Thus, an enterovirus-based optimized self-amplifying RNA construct of the present invention may comprise the components in Tables 10 and 11 below (Figure 15).

[0253] [Table 10]

[0254] [Table 11]

[0255] V. Evaluating the in vivo efficacy of self-amplifying RNA Example 8. Evaluation of vaccine efficacy of self-amplifying RNA containing highly pathogenic avian influenza virus antigens Example 8.1. Production of self-amplifying RNA and mRNA and animal experiments To evaluate the efficacy of the 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) (Table 7) containing highly pathogenic avian influenza virus prepared as in Example 5.1 was delivered to NanoAssemblr™ Ignite (Precision Immunoglobulin G1) 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) as in Example 5.2 above. The saRNA-LNP and mRNA-LNP were encapsulated in 50 μl of saRNA-LNP (Sinobiological, A / Broiler Duck / Korea / Buan2 / 2014) and administered intramuscularly to mice at concentrations of 10 μg, 1 μg, 0.1 μg, or 0.01 μg, respectively (first immunization). As a control, 5 μg of HA hemagglutinin purified protein (Sinobiological, A / Broiler Duck / Korea / Buan2 / 2014) (50 μl) and adjuvant (AddaVax, Invivogen) were mixed at a 1:1 ratio and administered intramuscularly to mice. PBS was administered as a control. Four weeks after the first immunization, mice were administered the self-amplifying RNA or mRNA in the same manner as above (second immunization). Blood was collected from the mice 4 weeks after the first administration and 2 weeks after the second administration (6 weeks after the first administration), and serum was obtained (Figure 17).

[0256] Example 8.2. Assessment of Neutralizing Antibody Formation For the experimental mice in Example 8.1 above, 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 was obtained to determine whether neutralizing antibodies were formed. In this case, the serum obtained as described above was inactivated by reacting it at 56°C for 1 hour before use.

[0257] Neutralizing antibody formation was assessed by hemagglutination assay using MDCK cells and confirmation of T cell responses.

[0258] Specifically, for the hemagglutination assay, 1 × 10 MDCK (Madin-Darby canine kidney) cells were used per well. 6 Cells were prepared by inoculating cells into a 96-well plate at a concentration of 100 cells / well and culturing overnight at 37°C. For the virus preparation, highly pathogenic avian influenza virus (A / Mallard / Korea / W452 / 2014 (H5N8)) was diluted at TCID100. Serum was diluted 10:1 with serum-free medium containing 1x trypsin (Thermo Fisher, 4370285) treated with TPCK (tosylsulfonylphenylalanyl chloromethyl ketone, Thermo Fisher Scientific, 20233). The virus dilution and trypsin dilution were then mixed at a 1:1 ratio and incubated at 37°C for 1 hour to prepare the virus dilution.

[0259] MDCK cells prepared as described above were washed twice with PBS and treated with 50 μl of the prepared virus dilution per well. The cells were then infected at 37°C. One hour after infection, the cell culture medium was replaced with serum-free medium containing TPCK (Thermo Fisher Scientific, 20233) containing trypsin (Thermo Fisher, 4370285) (1x) and further cultured. Forty-eight hours after infection, the cell culture medium was serially diluted 1:2 in PBS for a total of 11 times. Each dilution was then mixed 1:1 with a 0.5% turkey red blood cell dilution and incubated at room temperature for 30 minutes. If neutralizing antibodies were not formed, the virus would agglutinate with the red blood cells.

[0260] As a result, as shown in Figure 18 (top), in the serum obtained 4 weeks after the first vaccine administration, it was confirmed that the formation of neutralizing antibodies increased in the serum of the group administered the self-amplifying RNA vaccine (saRNA-2.3.4.4), depending on the vaccine concentration administered.

[0261] On the other hand, no neutralizing antibody formation was observed in the group administered the mRNA vaccine (mRNA-2.3.4.4). Furthermore, in serum samples obtained two weeks after the second vaccine administration (six weeks after the first vaccine administration), it was confirmed that the formation of neutralizing antibodies increased in the serum of the group administered the self-amplifying RNA vaccine (saRNA-2.3.4.4), depending on the vaccine concentration administered. In the groups administered the mRNA vaccine (mRNA-2.3.4.4), neutralizing antibody formation was confirmed only in the group administered the high dose (10 μg, 1 μg), but not in the group administered the low dose (0.1 μg, 0.01 μg).

[0262] T cell responses were confirmed using ELIspot analysis of antigen-specific IFN-γ-secreting cells using the BD™ ELISPOT Mouse IFNγ ELISPOT Set (BD, Cat. No. 551083) for each antibody required for the experiment.

[0263] Specifically, the capture antibody was diluted 1:200 in DPBS, and 100 μl of the diluted solution was added to each well of a 96-well plate. The plate was then incubated at 4°C for 18 hours to coat the plate. Each well was then washed with DPBS (Welgene) and blocked with RPMI 1640 medium containing 10% heat-treated bovine calf serum (BCS) and 1% penicillin / streptomycin (Gibco). Next, 1.0 × 10 spleen cells isolated from the experimental mice described in Example 8 were added per well. 6 After seeding each well with a concentration of 100 cells, the cells were stimulated by treating them with 20 μg / ml of H5N8 peptide (Peptron).

[0264] In this case, spleen cells from naive mice, mice administered with saRNA-B virus (10 μg), and mice administered with protein plus adjuvant (5 μg) were used as negative controls.

[0265] Each well was then washed three times with 200 μl of PBS-T. Then, 100 μl of primary antibody was added to each well, diluted 1:250 in DPBS containing 10% BCS, and the cells were then treated with secondary antibody. The number of spots generated by the HRP substrate reaction was counted.

[0266] As a result, as shown at the bottom of Figure 18, it was confirmed that the group administered self-amplifying RNA (saRNA-2.3.4.4) efficiently produced neutralizing antibodies even at low concentrations compared to the group administered mRNA (mRNA-2.3.4.4).

[0267] Furthermore, in both the self-amplifying RNA (saRNA-2.3.4.4) and mRNA (mRNA-2.3.4.4) groups, T cell activation was confirmed depending on the vaccine concentration administered. In particular, dose-dependent T cell responses were observed. In particular, the self-amplifying RNA (saRNA-2.3.4.4) group showed approximately two-fold higher T cell activity at 10 μg and approximately four-fold higher T cell activity at 1 μg compared to the mRNA (mRNA-2.3.4.4) group at the same concentration. These results confirmed that the CD8 T cell response in the self-amplifying RNA (saRNA-2.3.4.4) group was higher than that in the mRNA (mRNA-2.3.4.4) group.

[0268] Example 8.3. Measurement of changes in body weight and survival rate of mice In Example 8.1 above, two weeks after the second vaccination (six weeks after the first vaccination), laboratory mice were infected intranasally with 452 virus (10 MLD, A / mallard / Korea / W452 / 2014(H5N8)), and the weight and survival rate of the mice were monitored for 14 days.

[0269] As a result, as shown in Figure 19, the group administered with self-amplifying 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), and the groups administered with low concentrations (0.1 μg, 0.01 μg) showed a 0% survival rate after 9 days, similar to the group (PBS-administered, naive).

[0270] Example 8.4. Measurement of residual viral titers in mouse tissues Some of the experimental mice from Example 8.3 above were sacrificed on the third or fifth day after virus infection, and tissues from each organ were removed to measure the virus titer remaining in each organ.

[0271] As shown in Figures 20a and 20b, in the group administered with self-amplifying RNA (saRNA-2.3.4.4), no viral titer was detected in any of the groups administered with the self-amplifying RNA (saRNA-2.3.4.4), except in 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 detected in any tissue in the groups administered with high concentrations (10 μg, 1 μg), but viral titer was detected in all organs in the groups administered with low concentrations (0.1 μg, 0.01 μg). These results confirmed that self-amplifying RNA has superior activity compared to mRNA.

[0272] Example 9. Evaluation of neutralizing antibodies against severe fever with thrombocytopenia syndrome virus Example 9.1. Production of self-amplifying RNA and animal experiments To evaluate the efficacy of the self-amplifying RNA as a construct at the animal level, self-amplifying RNA containing a severe fever with thrombocytopenia syndrome virus (SFTS) antigen (self-amplifying RNA vaccine) was produced in the same manner as in Example 5.1 above (Tables 5 and 12). In this case, the severe fever with thrombocytopenia syndrome virus antigen used was 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).

[0273] [Table 12]

[0274] The self-amplifying RNA (self-amplifying RNA vaccine) or mRNA (mRNA vaccine) prepared as described above was delivered to NanoAssemblr™ Ignite (Precision Imaging) 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) in the same manner as in Example 8.1. The saRNA-LNPs and mRNA-LNPs were encapsulated in saRNA-LNPs (Nature NanoSystems) and administered to BALB / c mice (6-week-old, n=2) or C57BL / 6 mice (6-week-old, n=5) at a concentration of 5 μg / 100 μl / mouse (first immunization). Three weeks after the first immunization, a self-amplifying RNA vaccine at the same concentration was administered in the same manner (second immunization). Two weeks after the second immunization (5 weeks after the first immunization), blood was collected from the mice via retro-orbital bleeding, and serum was obtained (Figure 21a).

[0275] Example 9.2. Evaluation of neutralizing antibody production Example 9.2.1. Evaluation of Neutralizing Antibody Production in Mice The production of neutralizing antibodies was confirmed using immunofluorescence staining in Vero E6 cells.

[0276] Specifically, Vero E6 cells were cultured in DMEM (Gibco, Cat. No. 12430-054) containing 10% fetal bovine serum, and then plated in a 96-well plate at a density of 1.5 × 10 cells per well. 4 The cells were inoculated at a concentration of 100 TCID cells and cultured for 24 hours. Antibodies from mice were inactivated by incubation at 56°C for 30 minutes. The cells were incubated with 100 TCID antibodies against SFTS type D virus (NCCP No. 43265) in PBS. 50The virus was serially diluted 10 times with 1 / 2 of the original volume of PBS, mixed with the virus at a 1:1 ratio, and incubated at 37°C for 1 hour before treatment. One hour after treatment, the medium was replaced with DMEM containing 2% fetal bovine serum, and the cells were further cultured at 37°C for 120 hours.

[0277] After 120 hours of serum and virus treatment, cells were fixed with 80% acetone for 10 minutes at 4°C, dried for 5 minutes, and then washed with PBS. The fixed cells were treated with 3% BSA solution (100 μl) for 1.5 hours, blocked, and then washed six times with PBST. The cells were then treated with 50 μl of primary antibody (anti-SFTS antibody) per well and incubated for 3 hours at 37°C. The primary antibody was an in-house-produced antibody. We intramuscularly inoculated C57BL / 6 or BALB / c mice with our LNP vaccine against saRNA-A, saRNA-B, saRNA-DEF, or saRNA-ABDEF antigens at a concentration of 5 μg per mouse or 1 μg per mouse, three weeks apart. The antibodies were obtained two weeks after the second vaccination.

[0278] After the reaction was completed, each well was washed five times with PBST (100 μl) to remove unbound antibody, 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 this (50 μl per well), followed by reaction at 37° C. for 1.5 hours. Then, each well was washed three times with PBST, and the cells were treated with 200 μl of PBST per well, and the cells were observed using a fluorescence microscope.

[0279] As a result, as shown in Figure 21b, in the case of antibodies obtained from BALB / c mice, FITC fluorescence was observed in Vero E6 cells infected with the SFTS virus. On the other hand, no fluorescence was observed in Vero E6 cells not infected with the SFTS virus. Furthermore, as shown in Figure 21c, in the case of antibodies obtained from C57BL / 6 mice, it was confirmed that the self-amplifying RNA vaccine showed a maximum neutralizing antibody titer of 32 against the SFTS type D virus. In other words, it was confirmed that the self-amplifying RNA vaccine produced by our company produces antibodies against the SFTS virus.

[0280] Example 9.2.2. Evaluation of Neutralizing Antibody Production in Ferrets Four types of self-amplifying RNA (saRNA-LNP, self-amplifying RNA vaccine) prepared and encapsulated as in Example 9.1 were administered intramuscularly to each mouse at a concentration of 15 μg / 500 μl / mouse (initial immunization). Following the initial administration, a second and third administration were performed at 3-week intervals in the same manner as the initial administration (second and third immunizations). Three weeks after the initial, second, or third administration, blood was collected and serum was obtained by centrifugation (8500 rpm, 10 minutes) (FIG. 22a).

[0281] Cell experiments confirmed the production of neutralizing antibodies in ferret serum, which was inactivated by incubation at 56°C for 30 minutes before use.

[0282] Specifically, Vero E6 cells were plated in a 96-well plate at 1 × 10 cells per well. 4 The cells were inoculated at a concentration of 100 TCID and cultured for 24 hours. 50 The virus was prepared by mixing the SFTS type B virus (NCCP No. 43273), SFTS type C virus (NCCP No. 43332), SFTS type D virus (NCCP No. 43265), or SFTS type E virus (NCCP No. 43333) at a 1:1 ratio and incubating at 37°C for 1 hour.

[0283] The cells prepared as described above were treated with 50 μl of the virus and serum mixture per well and incubated for 1 hour. The medium was then replaced with DMEM containing 2% fetal bovine serum, and the cells were then cultured for 120 hours. 120 hours after infection, the cells were fixed with 80% acetone for 10 minutes at 4°C. The cells were dried for 5 minutes, washed with 150 μl of PBS, and blocked by treatment with 100 μl of 3% BSA solution for 1.5 hours. The cells were then washed three times with 100 μl of PBST and treated with 50 μl of in-house-produced primary antibody per well for 3 hours at 37°C. The cells were then washed five times with 100 μl of PBST and then treated with secondary antibody for 1.5 hours at 37°C. The secondary antibody used was a peroxidase-labeled anti-mouse IgG (gamma) antibody human serum (Seracare, 5220-0460) diluted 1:1000 in PBST and treated with 50 μl per well. After the secondary antibody reaction, the cells were stained using DAB staining and then observed to confirm the level of antibody production.

[0284] As a result, it was confirmed that three weeks after the first vaccination, the mean neutralizing antibody titer ranged from a minimum of 40 to a maximum of 320 (Figure 22b), and three weeks after the second vaccination, the mean neutralizing antibody titer ranged from a minimum of 160 to a maximum of 905 (Figure 22c). Furthermore, it was confirmed that three weeks after the third vaccination, the mean neutralizing antibody titer ranged from a minimum of 320 to a maximum of 5120 (Figure 22d).

[0285] Example 10. Evaluation of the efficacy of canine influenza virus vaccines Example 10.1. Production of self-amplifying RNA and confirmation of expression at the cellular level 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).

[0286] [Table 13]

[0287] Self-amplifying RNA (self-amplifying RNA vaccine) was encapsulated (saRNA-LNP) in NanoAssemblr™ Ignite (Precision NanoSystems) 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)). 293T cells were then treated with this at various concentrations to confirm the expression level of the self-amplifying RNA in the cells, as in Example 5.2. Anti-H3N2 and anti-H3N8 antibodies were used as primary antibodies, diluted at 1:2000 and 1:1000, respectively. The anti-H3N2 antibody was an antibody produced in-house, as in Example 6.2 above. Cells were plated at 1 × 10 per well the day before transfection. 6 The cells were seeded into a 6-well plate at a concentration of 1 / 4 cells per well, and the medium was replaced with a medium without fetal bovine serum 4 hours before transfection. The cells were then cultured and prepared.

[0288] The medium was replaced 4 hours after transfection and the cells were cultured for an additional 24 hours. After 24 hours, the cells were pelleted by centrifugation (12,000 rpm, 1 minute). The cells were then lysed with 1 ml of cell lysis buffer and vortexed. The lysate was incubated at room temperature for 10 minutes, after which the protein concentration was measured. Protein samples were prepared by mixing 10 μg of protein with 2x sample buffer (SDS sample buffer) at a 1:1 ratio and incubating at 95°C for 7 minutes. Each protein sample was electrophoresed on an SDS-PAGE gel. The membrane was then transferred to a membrane, blocked with a 5% lipoprotein solution, and then incubated with a primary antibody for 16 hours at 4°C. After incubation with the primary antibody, the membrane was washed five times with TBS-T buffer for 10 minutes each, followed by incubation with a secondary antibody for 1-2 hours at room temperature. After the secondary antibody reaction, each membrane was washed five times with TBS-T for 10 minutes each, and then detected using HRP reagent (Millipore).

[0289] The primary antibody (anti-H3N2 antibody) was manufactured in-house and used at a dilution of 1:2000. The secondary antibody was an anti-mouse HRP antibody (Invitrogen, 62-6520, 1:5000). The anti-H3N2 antibody was prepared as an LNP vaccine against our saRNA-H3N8 antigen and administered intramuscularly to mice at a concentration of 10 μg per mouse twice, four weeks apart. Serum was isolated two weeks after the second vaccination. Blood was collected in tubes without anticoagulant, left for 24 hours, and then centrifuged (8500 rpm, 15 minutes, repeated twice) to obtain serum.

[0290] The secondary antibody used was anti-mouse HPR antibody (1:5000, Invitrogen, 62-6520). After the secondary antibody reaction, each membrane was washed five times with TBS-T for 10 minutes each, and then detected using HRP reagent (Millipore).

[0291] As a result, as shown in FIG. 23, it was confirmed that the expression of each antigen increased in a concentration-dependent manner in response to treatment with self-amplifying RNA.

[0292] Example 10.2. Animal Experiments Self-amplifying RNA (self-amplifying RNA vaccine) encapsulated as in Example 10.1 above was administered to BALB / c mice at various concentrations. The self-amplifying RNAs used were 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 were administered intramuscularly at a concentration of 5 μg, 1 μg, or 0.1 μg per mouse (primary immunization).

[0293] Positive controls were prepared by mixing inactivated canine influenza virus (A / dog / Korea / AS-01 / 2012) and canine influenza H3N8 virus (A / dog / Massachusetts / 26810 / 2016) with aluminum hydroxide gel adjuvant (InvivoGen) at a 1:1 ratio. H3N2 or H3N8 canine influenza virus was inoculated into embryonated eggs and cultured at 37°C for 48 hours. The allantoic fluid was then collected and mixed with formalin (0.025% final concentration) and inactivated at 4°C for 72 hours to produce inactivated canine viruses. Virus inactivation was confirmed by inoculating the virus into other embryonated eggs and confirming no virus growth. As a positive control, inactivated H3N2 or H3N8 canine influenza virus was mixed and then cultured for 10 hours. 6 The mice were inoculated with a dose equal to or greater than HAU. As a negative control, PBS containing no antigen was used.

[0294] As a control, PBS was administered to a group without antigen (NC).

[0295] Furthermore, four weeks after the first administration, a second administration was performed in the same manner as the first administration (second immunization). Blood was collected from the mice two and four weeks after the first administration, and two weeks after the second administration (6 weeks after the first administration), and serum was obtained (Figure 24).

[0296] Example 10.3. Evaluation of neutralizing antibody production The production of neutralizing antibodies was evaluated using serum obtained from the experimental mice in Example 10.2 (4 and 6 weeks after the initial administration) by mixing the serum at a ratio of 1:3 (serum:RDE) using an RDE Receptor Destroying Enzyme kit (SEIKEN), incubating at 37°C for 18 hours, and then inactivating the RDE at 56°C for 30 minutes.

[0297] The formation of neutralizing antibodies was evaluated in two ways using hemagglutination inhibition tests and cell experiments. The hemagglutination inhibition tests and cell experiments were performed as described in Example 8.2 above. Blood cells were treated with canine influenza H3N2 virus (A / dog / Korea / AS-01 / 2012) at a level of 4-8 HAU (HA units) or diluted to 100 TCID, mixed with serum at a 1:1 ratio, and incubated at 37°C for 1 hour before use in the cell experiments.

[0298] For canine influenza H3N8 virus (A / dog / Massachusetts / 26810 / 2016), the virus was produced in-house and used. Blood cells were treated with the virus at the same level as above at HAU4-8 (HA units) or diluted to TCID100, mixed with serum at a 1:1 ratio, and incubated at 37°C for 1 hour before use in cell experiments.

[0299] In this case, the canine influenza H3N8 virus was produced by the following method.

[0300] The cells used were MDCK (Marbin-Darby canine kidney) cells and 293T cells, which were plated in a 6-well plate at a ratio of 1:2 (MDCK cells: 0.25 × 10 per well) on the day before transfection. 6 cells, 293T cells: 0.5 x 10 per well 6 The cells were co-cultured by inoculating them at a ratio of 1:1 (cells per 1000 cells).

[0301] Twenty-four hours after inoculation, the medium was replaced with medium lacking fetal bovine serum, and the cells were then used for experiments. Canine influenza viruses were prepared by incorporating polynucleotides (SEQ ID NOs: 169, 171, 173, 183, 185, 177, 179, 181) encoding the amino acid sequences 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 NO: 180), or M (SEQ ID NO: 182) into the PHW2000 vector (Hoffmann et al., PNAS, 97:6108-6113 (2000)) and then mixing equal amounts (1 μg) of each vector (plasmid DNA).

[0302] The prepared plasmid DNA and transfection reagent (TransIT-LT1, Mirus Bio) (18 μl) were mixed in an EP tube and incubated at room temperature for 3 minutes. After incubation, 194 μl of fetal bovine serum-free medium was added and incubated at room temperature for an additional 45 minutes. MDCK cells and 293T cells were transfected by treating them with the reaction solution and culturing them at 37°C for 24 hours. 24 hours after transfection, the cell culture medium was replaced with 1 ml of fetal bovine serum-free medium and cultured for an additional 24 hours. Then, fetal bovine serum-free medium containing 1 μg / ml TPCK was added and cultured for 48 hours. After 48 hours, the cell culture medium was harvested and used as the transfection sample.

[0303] To confirm virus production after transfection, 2.5 x 10 cells were cultured per well in a 6-well plate. 5 MDCK cells were prepared by inoculating them at a concentration of 1 / 3 cells and culturing them for 24 hours. After 24 hours, the cell culture medium was replaced with fetal bovine serum-free MEM medium (Corning, 10-010-CV). The cells were then treated with 1 ml of the transfection sample prepared as described above and incubated at 37°C for 1 hour. Medium containing 1 μg / ml TPCK without fetal bovine serum was added, and the cells were cultured for 48 hours. Virus production was confirmed by hemagglutination assay to confirm the production of canine influenza H3N8 virus before use.

[0304] In this experiment, PBS and virus were serially diluted 11 times in half in a 96-well round-bottom plate to prepare hemagglutination reactions. The reaction solution was treated with 0.5% diluted turkey red blood cells at a 1:1 ratio and incubated at room temperature for 30 minutes. If virus was produced, it was confirmed that the virus bound to blood cells.

[0305] As shown in Figures 25a and 25b, after the second administration, neutralizing antibodies were confirmed to be formed in all groups administered with self-amplifying RNA. The H3N2-containing self-amplifying RNA showed titers of 1024 MNT in the groups administered 1 μg and 5 μg, while the H3N8-containing self-amplifying RNA showed titers of 40960 MNT in the groups administered 1 μg and 5 μg. Furthermore, high neutralizing antibody production was confirmed even in the group administered 0.1 μg.

[0306] Example 10.4. Measurement of changes in body weight and survival rate of mice Self-amplifying RNA was administered to mice (first and second administrations) in the same manner as in Example 10.1 above. Four weeks after the first administration (28 days after vaccination, 28 dpv) and two weeks after the second administration (6 weeks after the first administration, 42 days after vaccination (42 dpv)), all administration groups except the control were infected intranasally with H3N2 canine influenza virus or H3N8 canine influenza virus (10 MLD), respectively, and the body weight and survival rate of the mice were monitored for 14 days. Furthermore, some mice were sacrificed on days 3 and 5 post-infection, and lung tissue was removed and the virus titer in the lung tissue was measured ( FIG. 26 ).

[0307] As a result, as shown in Figures 27a to 27d, it was confirmed that the group administered with multi-antigen self-amplifying RNA (SAH3N2+H3N8) had a higher survival rate and less weight loss than the groups administered with single-antigen self-amplifying RNA (SAH3N2 or SAH3H8).

[0308] Furthermore, as shown in Figures 28a and 28b, in mice infected with the virus 4 weeks after the first vaccination (28 dpv), the group administered the multi-antigen self-amplifying RNA (SAH3N2+H3N8) showed lower viral titers in lung tissue compared to the groups administered the single-antigen self-amplifying RNA (SAH3N2 or SAH3H8). In particular, examination of lung tissues excised 5 days post-infection (5 dpi) confirmed that H3N2 virus replication was significantly reduced in the lung tissue of mice infected with the H3N2 virus, while no viral replication was observed in the lung tissue of mice infected with the H3N8 virus (Figure 28a). Furthermore, in mice infected with the virus 2 weeks after the second vaccination (42 dpv), the group administered the self-amplifying RNA showed higher overall survival rates and less weight loss compared to the control group. Furthermore, it was confirmed that the virus did not grow in lung tissues excised 3 and 5 days after infection (Fig. 28b).

[0309] The above results confirmed that when H3N2 antigen and H3N8 antigen are administered in combination, the vaccine efficacy is superior compared to when H3N2 antigen or H3N8 antigen is administered alone, and that when administered in combination, sufficient virus inhibitory effect can be achieved with just the first administration.

[0310] Example 11. Evaluation of efficacy in preventing or treating cancer caused by human papillomavirus Example 11.1. Production of self-amplifying RNA containing human papillomavirus antigen and confirmation of its expression at the cellular level The following fusion proteins were used as human papillomavirus antigens (E6 / E7 domain, E6 / E7 sequential configuration, E6 / E7 crossover configuration (crossover), E6 / E7 odd and even configurations) (Figure 29a).

[0311] The E6 / E7 domain (SEQ ID NO: 139) is a fusion protein comprising, from the N-terminus to the C-terminus, the amino acid sequences of the E6 domain (SEQ ID NO: 159) of human papillomavirus, a linker (SEQ ID NO: 189, GGGGS), and the E7 domain (SEQ ID NO: 160).

[0312] The E6 / E7 sequential arrangement (sequential) (SEQ ID NO: 142) is a fusion protein in which the E6 domain (SEQ ID NO: 159) and the E7 domain are divided into eight and seven fragments, respectively, and contain the E6 and E7 domains sequentially from the N-terminus to the C-terminus.

[0313] Specifically, the E6 domain was divided into the following fragments: a fragment consisting of the amino acid sequence of positions 18 to 43 of the amino acid sequence of SEQ ID NO: 159 (E6-1); a fragment consisting of the amino acid sequence of positions 28 to 61 of the amino acid sequence of SEQ ID NO: 159 (E6-2); a fragment consisting of the amino acid sequence of positions 42 to 70 of the amino acid sequence of SEQ ID NO: 159 (E6-3); a fragment consisting of the amino acid sequence of positions 48 to 79 of the amino acid sequence of SEQ ID NO: 159 (E6-4); a fragment consisting of the amino acid sequence of positions 66 to 92 of the amino acid sequence of SEQ ID NO: 159 (E6-5); a fragment consisting of the amino acid sequence of positions 82 to 122 of the amino acid sequence of SEQ ID NO: 159 (E6-6); a fragment consisting of the amino acid sequence of positions 100 to 143 of the amino acid sequence of SEQ ID NO: 159 (E6-7); and a fragment consisting of the amino acid sequence of positions 126 to 151 of the amino acid sequence of SEQ ID NO: 159 (E6-8). The E7 domain was divided into the following fragments: a fragment consisting of the amino acid sequence of positions 5 to 21 of the amino acid sequence of SEQ ID NO: 160 (E7-1); a fragment consisting of the amino acid sequence of positions 9 to 33 of the amino acid sequence of SEQ ID NO: 160 (E7-2); a fragment consisting of the amino acid sequence of positions 24 to 45 of the amino acid sequence of SEQ ID NO: 160 (E7-3); a fragment consisting of the amino acid sequence of positions 36 to 64 of the amino acid sequence of SEQ ID NO: 160 (E7-4); a fragment consisting of the amino acid sequence of positions 47 to 69 of the amino acid sequence of SEQ ID NO: 160 (E7-5); a fragment consisting of the amino acid sequence of positions 65 to 88 of the amino acid sequence of SEQ ID NO: 160 (E7-6); and a fragment consisting of the amino acid sequence of positions 72 to 95 of the amino acid sequence of SEQ ID NO: 160 (E7-7). A sequential arrangement (continuous) of E6 / E7 is a fusion protein containing the above fragments in order from the N-terminus to the C-terminus 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.

[0314] The E6 / E7 crossover configuration is a fusion protein containing E6 domain fragments (8 types) and E7 domain fragments (7 types) sequentially from the N-terminus to the C-terminus 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.

[0315] The odd and even configurations of E6 / E7 are fusion proteins containing E6 domain fragments (8 types) and E7 domain fragments (7 types) sequentially from the N-terminus to the C-terminus 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.

[0316] Self-amplifying RNA constructs containing the above-mentioned human papillomavirus (type 16) E6 / E7 domain (SEQ ID NO: 139), sequential E6 / E7 arrangement (SEQ ID NO: 142), crossover E6 / E7 arrangement (SEQ ID NO: 145), or odd-even E6 / E7 arrangement (SEQ ID NO: 148) were prepared in the same manner as in Example 5.1, and the respective self-amplifying RNAs were designated G1, G2, G3, and G4 (also referred to as target proteins). The nucleotide sequences of each self-amplifying RNA are shown in Tables 14 and 15.

[0317] [Table 14]

[0318] [Table 15]

[0319] The expression of the self-amplifying RNA construct prepared as described above was confirmed at the cellular level in the same manner as in Example 5.2, and human papillomavirus antigens were produced.

[0320] Example 11.2. Evaluation of Cancer Prevention Activity Self-amplifying RNA prepared 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 mice (C57 / BL6, 6-week-old, female, n=8) at a concentration of 5 μg / 50 μl per mouse (first immunization). Two weeks after the first administration, a second administration was performed in the same manner as above (second immunization). Three weeks after the second administration, TC-1 tumor cell line was injected subcutaneously into the flank of the mice at a dose of 2 × 10 per mouse. 5 The mice were transplanted at a concentration of 100 cells / 100 μl, and the weight of the mice and tumor size were measured 15 times over 19 days. The control group was administered PBS, and the group administered G1 served as a positive control.

[0321] Additionally, tumor size was calculated by measuring width (W), length (L), and height (H) using the following formula I (https: / biopticon.com / resources / tumor-volume-measurements-by-calipers / ). <Formula I> Tumor size (V (t) )=6 / π×L×W×H10

[0322] Blood was collected from the mice two weeks after the first administration and two weeks after the second administration (four weeks after the first administration), and plasma was obtained. Two weeks after the second administration, some mice (n=3) were sacrificed and their spleens were removed (Figure 30).

[0323] Example 11.2.1. Confirmation of T Cell Responses Four weeks after the first vaccination, the second vaccination was administered. Two weeks later, the spleens were removed to obtain cells for the subsequent experiments. Mice (n=3) not injected with the TC-1 tumor cell line were used. A group administered with G1 was also used as a positive control.

[0324] Specifically, the capture antibody contained in the BD™ ELISPOT Mouse IFNγ ELISPOT Set (BD, 551083) was diluted 1:200 in DPBS, and 100 μl of the diluted solution was added to each well of a 96-well plate. The plate was then coated by incubation at 4°C for 18 hours. Each well was then washed with DPBS (Welgene) and blocked with RPMI 1640 medium containing 10% heat-treated bovine calf serum (BCS) and 1% penicillin / streptomycin (Gibco). Next, 1.0 × 10 spleen cells isolated from the experimental mice (n = 3 mice not injected with the TC-1 tumor cell line) used in Example 11.2 above were added per well. 6 After seeding each well with a concentration of 100 cells, the cells were stimulated by treatment with 20 μg / ml of E6 and E7 peptides (Miltenyibiotec, 130-095-997 and 130-095-999). Naive mouse spleen cells were used as a negative control. After washing each well three times with 200 μl of PBS-T, a primary antibody (BD™ ELISPOT Mouse IFNγ ELISPOT Set, BD, 551083) was diluted 1:250 in DPBS containing 10% BCS and added to each well (100 μl). The cells were then treated with a secondary antibody (BD™ ELISPOT Mouse IFNγ ELISPOT Set, BD, 551083), and the number of spots generated by the HRP substrate reaction was counted.

[0325] As a result, as shown in FIG. 31, 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.

[0326] Example 11.2.2. Cancer prevention effect of self-amplifying RNA After tumor implantation in the experimental mice of Example 11.2 above, mouse weights and tumor sizes were monitored for 19 days post-vaccination.

[0327] As a result, tumor formation and tumor growth were confirmed to be inhibited in all groups administered with G1 (positive control), G2, G3, and G4 compared to the negative control (PBS). Notably, tumor formation was observed only in the group (2 mice) administered with the positive control G1, and not in the other groups. These results confirmed that self-amplifying RNA can be effectively used as a cancer prevention vaccine.

[0328] Example 11.3. Evaluation of tumor growth inhibitory activity To confirm the tumor growth inhibitory effect, mice (C57 / BL6, 6-week-old, female) were inoculated with 1 × 10 cells / mouse into the flank. 5 TC-1 cells were implanted via subcutaneous injection at a concentration of cells / 100 μl. On days 3 and 7 after tumor cell implantation, self-amplifying RNA was administered (only the initial administration was performed) in the same manner as in Example 11.2 above, and the mouse weight and tumor size were measured over 22 days (22 times in total). The concentration of self-amplifying RNA administered to each mouse was 5 μg / 50 μl or 1 μg / 50 μl per mouse ( FIG. 34 ). PBS was administered as a negative control.

[0329] As a result, as shown in Figures 35 and 36a to 36d, tumor growth was confirmed to be inhibited in all groups administered 5 μg of self-amplifying RNA (vaccine) compared to the negative control (PBS). Furthermore, in the group administered G4, tumor growth was confirmed to be inhibited in all experimental mice, even in the group administered 1 μg.

[0330] The above results confirmed that tumor growth was inhibited or tumors were eliminated by just the initial administration of self-amplifying RNA.

Claims

1. a self-amplifying viral nonstructural protein; a first target protein; a protease cleavage site; A polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising:

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

3. 3. The polynucleotide of claim 2, wherein the nonstructural proteins comprise the P2 and P3 domains of an enterovirus.

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

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

13.

6. The polynucleotide of claim 4 , wherein the 2A protease comprises a mutation.

7. The polynucleotide of claim 6, wherein the mutation is a substitution of the 87th amino acid in the amino acid sequence of SEQ ID NO:

11.

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

75.

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

10. 3A comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14; 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 10. The polynucleotide of claim 9, wherein 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. 12. The polynucleotide of claim 11, wherein the protease cleavage site is cleaved by an enterovirus 2A protease.

13. 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 of claim 1 , wherein the polynucleotide further comprises a nucleic acid sequence encoding a second protein of interest.

15. 15. The polynucleotide of claim 14, wherein the protease cleavage site is located between the second protein of interest and the first protein of interest.

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

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

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

19. 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 of claim 1 or 14, wherein the first target protein and / or the second target protein is an antigen.

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

22. 22. The polynucleotide of 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. 21. 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. 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. 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. 21. The polynucleotide of claim 20, wherein the antigen is a human papillomavirus antigen.

30. 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 tumor antigens include 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, CA15-3 / CA27-29, CA19-9, 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 (COTL1), collagen type XXIII, 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, HP V-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamini 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, MAGEEL2, mammaglobin-A, MART-1 / melan-A, MART-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-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP , OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml / PARα, POTE, PRAME, PRDX5 / m, prostein, proteinase-3 (PR3), PS A, 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- 32. The polynucleotide of claim 31, wherein the polynucleotide is selected from the group consisting of 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. 2. The polynucleotide of claim 1, wherein the polynucleotide further comprises at least one stop codon at the 3' end of the nucleic acid sequence encoding a nonstructural protein of the self-amplifying virus.

34. 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, further comprising a 5' untranslated region (5'UTR) at the 5' end.

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

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

59.

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

39. The mutation is A substitution of any one nucleotide selected from the group consisting of positions 236, 386, and combinations thereof in the nucleotide sequence of SEQ ID NO: 58, or A substitution of any one nucleotide selected from the group consisting of positions 236, 386, and combinations thereof in the nucleotide sequence of SEQ ID NO: 59; 39. The polynucleotide of claim 38,

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

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

46. The polynucleotide of claim 1 , further comprising a poly(A) tail sequence.

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

48. The polynucleotide has the following structural formula (II) in order from the 5' end to the 3' end: 5'-U-[K] ｏ -[B'-C(2)] ｎ -B-C(1)-D-S-U'-P-3' (II) (In the structural formula (II), 5' and 3' are the 5' and 3' ends of the polynucleotide, respectively; U is a nucleic acid sequence of the 5' untranslated region, K is the Kozak sequence, B' is a nucleic acid sequence encoding a second protein of interest; B is a nucleic acid sequence encoding a first protein of interest, D is a nucleic acid sequence encoding a nonstructural protein of the self-amplifying virus, S is a stop codon, U' is the nucleic acid sequence of the 3' untranslated region, P is a 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, and when n is from 2 to 10, each B' is a nucleic acid sequence encoding the same or a different protein of interest, and each C(2) is a nucleic acid sequence encoding the same or a different protease cleavage site, and 48. The polynucleotide of claim 47, wherein o is an integer of 0 or 1.

49. 49. The polynucleotide of claim 47 or 48, wherein the nonstructural proteins comprise, in N-terminal to C-terminal order, the P2 and P3 domains of an enterovirus.

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

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

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

53. A recombinant vector comprising the polynucleotide of claim 1.

54. 1. A method for producing an RNA replicon, comprising: i) producing the recombinant vector of claim 53; ii) synthesizing RNA from the recombinant vector; A method comprising:

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

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

22.

57. A nonstructural protein 3C protease cleavage site 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) viral 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. 1. A method for preventing or treating a disease, comprising:

56. A method comprising administering to a subject the polynucleotide of claim 1, the recombinant vector of claim 53, or the vaccine composition of claim 55.

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