TW202449157A - 5'-utr containing mrna constructs with improved translation efficiency and vaccine compositions comprising same - Google Patents

Abstract

The present invention relates to an mRNA construct containing 5'-UTR with improved translation efficiency and a vaccine composition comprising same, and more particularly to an mRNA construct containing 5'-UTR with improved translation efficiency that have been engineered to include specific motifs, a codon-optimized signal sequence and an antigen encoding sequence, and a vaccine composition comprising the same. The mRNA construct according to the present invention contains a 5'-UTR with improved translation efficiency, which can effectively induce the expression of an antigenic polypeptide and is useful for vaccine development as it can be expected to increase immunogenicity as a vaccine.

Description

mRNA constructs comprising 5'-UTR with improved translation efficiency and vaccine compositions comprising the same

The present invention relates to an mRNA construct comprising a 5'-UTR with improved translation efficiency and a vaccine composition comprising the construct, and more particularly, to an mRNA construct comprising a 5'-UTR with improved translation efficiency that has been engineered to include a specific motif, a codon-optimized signal sequence and an antigen coding sequence, and a vaccine composition comprising the construct.

People have used vaccines since the 16th century. The first clear report of their use was in 1798 in an article about a smallpox vaccine, and since then many different types of vaccines have been developed to treat a variety of diseases and to prevent them (Pollard, A.J et al., Nat Rev Immunol Vol. 21, pp. 83-100, 2021).

Vaccines can be divided into live vaccines (live, attenuated), inactivated vaccines (dead), toxoid vaccines, subunit vaccines, VLP vaccines, other membrane-based vaccines, protein-polysaccharide conjugate vaccines, viral vector-based vaccines and gene vaccines.

Live vaccines were first used for smallpox, toxoid vaccines were first used for diphtheria, subunit vaccines were first used for anthrax, VLP vaccines were first used for hepatitis B, other film vaccines were first used for serogroup B meningococcal, protein-polysaccharide conjugate vaccines were first used for influenza B, viral vector vaccines were first used for Ebola, and mRNA vaccines were first used for SARS-CoV-2 (Pollard, A.J et al, Nat Rev Immunol Vol. 21, pp. 83-100, 2021).

Influenza virus (IV) is a single-stranded, negative-sense RNA virus composed of eight RNA segments of a single negative strand, which expresses 10 to 13 different proteins and belongs to the Orthomyxoviridae family. It is divided into type A (A), type B (B), and type C (C) based on the antigenicity of nucleoprotein (NP) and matrix (M) proteins, which are structural proteins attached to the surface of the virus (Lamb RA et al., Fields Virology, pp. 1487-1531, 2001), and mainly type A and type B are known to be pathogenic in humans. Type A can infect not only humans but also pigs and birds. The only host of type B is humans.

Influenza viruses consist of two surface glycoproteins (surface antigens), hemagglutinin and neuraminidase. Influenza viruses are divided into multiple subtypes based on the type and combination of hemagglutinin (an antigenic protrusion that binds to host cell receptors (sialic acid)) and neuraminidase (which cleaves sialic acid to release the sialic acid-bound virus into the cell). These subtypes are mainly classified under influenza A. There are currently 18 different hemagglutinins, namely H1 to H18, and 11 different neuraminidase, namely N1 to N11, which can theoretically combine to form 198 different subtypes of influenza A. Influenza B viruses are divided into two strains based on antigenic type, namely Victoria and Yamagata.

Influenza is characterized by antigenic variation, which is typical of RNA viruses and varies slightly or significantly almost every year. Antigenic drift occurs almost every year, especially for influenza A and B, resulting in seasonal epidemics. Therefore, the World Health Organization (WHO) integrates information on viral epidemics and publishes vaccine recommendations for the current season around February each year. Antigenic drift is a point mutation within the same subtype that produces a new hemagglutinin or neuraminase with slightly altered antigenicity through a small antigenic variation. Antigenic shift is a subtype change, such as H3N2 → H2N2, which produces a new virus. In addition to these two modifications, culture in embryonic eggs can induce mutations that can lead to differences in antigenicity that are not found in cell culture, so vaccines using viruses grown in mammalian cells are preferred.

Due to antigenic drift and an expiration date of less than one year, influenza vaccines should be given annually. In the northern hemisphere, the recommended time for vaccination is October to December each year due to the length of the influenza season (December to April) and the vaccine's expiration date (average 6 months).

Current influenza vaccines include killed vaccines and live attenuated influenza vaccines (LAIV). Killed vaccines are produced by killing viruses grown in fertilized eggs (hatching eggs) with formalin or by killing viruses grown in cell culture with formalin, harvesting surface antigens, and injecting them intramuscularly (i.m.) as vaccine antigens. Live attenuated vaccines are administered by nasal spray.

Killed vaccines include whole virus vaccines, which use whole viruses; split vaccines (subvirions), which are prepared by removing the viral envelope with Triton X-100; and subunit vaccines, which are prepared by purifying the hemagglutinin and neuraminidase components. Hemagglutinin and neuraminidase are antigens that directly elicit neutralizing antibody responses, and hemagglutinin is the main neutralizing antigen. Whole virus vaccines are not widely used in Korea and worldwide and are only available in a few countries or regions because they can cause side effects in children. On the other hand, component vaccines (such as split vaccines or subunit vaccines) are very safe and effective and are the most commonly used.

In addition, vaccines containing immune adjuvants (such as MF-59) or virosome vaccines (which form virus-like vesicles) have been developed to enhance immune responses and are being used in some countries or regions. In order to provide an adequate level of immune protection against influenza virus infection, especially for preventive or therapeutic purposes, it is necessary to incorporate effective and safe adjuvants into vaccines.

Antibodies against a specific subtype or influenza virus acquired through natural infection or vaccination generally do not generate protective antibodies against other types or subtypes of influenza virus and may not be sufficiently immunogenic against new variants within a single antigen.

Annual vaccination is a challenge because influenza viruses undergo both major and minor mutations each year, causing the prevalence of the disease to vary from year to year, making it difficult to rely on previous year's vaccine to provide protection.

Current seasonal influenza vaccines only provide protective immunity against the virus strains used in the vaccine, so there is a need to develop an affordable and effective influenza vaccine that can provide a sufficient degree of cross-protective immunity across various subtypes. In order to develop a universal vaccine with cross-protective immunity, antigens with minimal antigenic variation or methods to stimulate mucosal immunity are needed. One such method is to use one or more HA2 domains of HA with low antigenic variation as vaccine antigens (Korean Patent No. 10-1637955).

On the other hand, it has been reported that when DNA or RNA encoding a target gene is directly injected into an animal, the target gene will be expressed in the living animal and this expression will confer immunity, thus starting the development of gene vaccines (Wolff JA et al. Science, 247: 1465-8, 1990).

DNA and mRNA can be used as active pharmaceutical ingredient (API) nucleic acid molecules in genetic vaccines, and DNA is known to be relatively stable and easy to handle compared to mRNA. However, in the case of DNA, there is a potential risk that the DNA fragment administered to the patient's genome may be inserted into an unwanted location, causing genetic damage. In addition, unwanted anti-DNA antibodies may appear. Another problem is that the expression level of peptides or proteins expressed by DNA administration and subsequent transcription/translation is limited. The presence of specific transcription factors that regulate DNA transcription has a significant impact on the expression level of the administered DNA, and in the absence of specific transcription factors, DNA transcription cannot produce sufficient amounts of mRNA, and therefore, the extent of translated peptides or proteins is limited.

On the other hand, when mRNA is used as an API for gene delivery, mRNA does not require transcription and is able to synthesize proteins directly in the cytoplasm without entering the cell nucleus like DNA, thus eliminating the risk of getting stuck in cell chromosomes and causing unwanted genetic damage. In addition, compared with DNA, it has a shorter half-life and therefore does not cause long-term gene modification (Sayour EJ, et al., J Immunother Cancer Vol. 3, 13, 2015). When delivered to cells, typical mRNA vaccines are activated only for a short time to express target proteins and are destroyed by enzymatic reactions within a few days, retaining specific immune responses to the expressed target antigens (proteins).

In addition, when using mRNA as a gene delivery tool, it does not need to cross the nuclear membrane to work, but only needs to cross the cell membrane, so it can express the same amount of target protein as DNA with a smaller amount than DNA. In addition, mRNA itself has immunostimulatory properties, so a smaller amount of mRNA is needed to produce the same immune effect as DNA. By using mRNA instead of DNA for gene vaccination, the risk of unwanted genome integration and anti-DNA antibody generation is minimized or avoided. However, mRNA is considered to be a rather unstable molecule that can be easily degraded by ubiquitous ribonucleases.

Although many advances have been made over the years, challenges remain in the field of effective methods for mRNA vaccination that can elicit an adaptive immune response, such as inefficient translation of mRNA due to premature degradation of the antigen or inefficient release of mRNA from cells. In addition, there is an urgent need to reduce the dose of mRNA vaccines to reduce potential safety issues and make the vaccine more affordable for the third world.

There are many challenges in delivering nucleic acids to elicit a desired response in biological systems. While nucleic acid-based therapeutic approaches, such as vaccines, hold great promise, more efficient delivery of nucleic acids to appropriate sites within cells or organisms is needed to realize this potential.

However, the use of nucleic acids for therapeutic and preventive purposes currently faces two challenges. First, naked mRNA is easily digested by nucleases in plasma. Second, naked mRNA has a limited ability to enter the intracellular compartment where relevant translation occurs. To address these issues, lipid nanoparticles (LNPs) composed of various lipid components (such as neutral lipids, cholesterol, PEG, PEGylated lipids or oligonucleotides) and cationic lipids have been tried to block the degradation of mRNA in plasma and promote the cellular uptake of nucleic acids.

In addition, an mRNA vaccine containing several lipid nanoparticles related to influenza vaccines has been reported (Korean Patent Publication No. 10-2018-0096591).

Therefore, the inventors of the present invention have made sincere efforts to solve the above-mentioned problems and developed mRNA constructs containing 5'-UTRs with improved translation efficiency, and confirmed that by selecting artificial nucleic acid molecules that do not generate secondary structures, have low uridine content, and do not contain sequences that reduce stability in the combination of artificial nucleic acid molecules with a length of 30 bp, 5'-UTRs with improved translation efficiency can be obtained, and mRNA constructs including them have better performance as vaccines, thereby completing the present invention.

One object of the present invention is to provide mRNA constructs encoding antigenic polypeptides or immunogenic fragments thereof.

Another object of the present invention is to provide a vaccine composition comprising an mRNA construct.

In order to achieve the above-mentioned purpose, the present invention provides an mRNA construct encoding an antigenic polypeptide or an immunogenic fragment thereof, wherein the mRNA construct comprises: In order from 5' to 3', a) a 5'-cap structure; b) a 5'-untranslated region (5'-UTR) polynucleotide represented by a nucleic acid sequence according to the following formula (I) or (II): Formula (I): AG[N22]GCCACC, Formula (II): AGGA[N19]RGCCACC, wherein R represents A, G or U; c) a polynucleotide encoding a signal peptide; d) a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof; e) a 3'-untranslated region (3'-UTR); and f) a poly(A) tail or a poly(A) tail-like sequence consisting of 10 to 1000 adenines (A).

The present invention further provides influenza vaccine compositions comprising mRNA constructs.

The present invention further provides use of the vaccine composition for preventing influenza.

The present invention further provides a method for preventing influenza, comprising the step of administering the vaccine composition.

The present invention further provides the use of the vaccine composition for preparing an agent for preventing influenza.

Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those of ordinary skill in the art. Generally, the nomenclature and experimental procedures described herein are those known and commonly used in the art.

In the present invention, attempts were made to determine that mRNA constructs comprising 5'-UTRs with improved translation efficiency can be used to express influenza antigenic proteins at higher efficiency than wild-type mRNA.

That is, in one embodiment of the present invention, it was found that the use of an mRNA construct including a 5'-UTR polynucleotide with improved translation efficiency (FIG. 1) significantly improved the expression efficiency of influenza antigen protein (FIGS. 3 to 6).

Therefore, from one perspective, the present invention relates to,

An mRNA construct encoding an antigenic polypeptide or an immunogenic fragment thereof, comprising: In order from 5' to 3' a) a 5'-cap structure; b) a 5'-untranslated region (5'-UTR) polynucleotide represented by a nucleic acid sequence according to the following formula (I) or (II): Formula (I): AG[N22]GCCACC, Formula (II): AGGA[N19]RGCCACC, wherein R represents A, G or U; c) a polynucleotide encoding a signal peptide; d) a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof; e) a 3'-untranslated region (3'-UTR); and f) a poly(A) tail or a poly(A) tail-like sequence consisting of 10 to 1000 adenines (A).

The 5'-cap (CAP) of natural mRNA accompanies nuclear export, increases mRNA stability, and binds to mRNA cap binding protein (CBP), forming mature circular mRNA species through the combination of poly(A) binding protein and CBP, leading to mRNA stability at the cellular and translational levels. During the mRNA splicing process, the cap further helps remove introns near the 5'.

In the present invention, the 5'-cap is usually a modified nucleotide (cap analog) added to the 5' end of the mRNA molecule, especially a guanine nucleotide. Preferably, the 5'-cap is added using a 5'-5'-triphosphate bond (also known as m7GpppN). Other examples of 5'-cap structures include glyceryl, inverted deoxybasic (abasic) residue (part), 4',5'-methylene nucleotide, 1-(β-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexanol nucleotide, L-nucleotide, α-nucleotide, modified alkali nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, 4'-thionucleotide, 1,5-anhydrohexanol nucleotide, 1,5-thio ... nucleotide), acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide portion, a 3'-3'-inverted non-alkaline portion, a 3'-2'-inverted nucleotide portion, a 3'-2'-inverted non-alkaline portion, 1,4-butanediol phosphate, 3'-amidophosphonate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate or a bridged or non-bridged methylphosphonate portion.

These modified 5'-cap structures can be used to modify the mRNA sequence of the synthetic nucleic acid molecules of the present invention.

Other modified 5'-cap structures that can be used in the present invention include CAP1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), CAP2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), CAP3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), CAP4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine.

In the present invention, the 5'-cap structure can be formed by chemical RNA synthesis or by in vitro transcription of RNA using a cCAP analog (co-transcriptional capping), or the cap structure can be formed in vitro using a capping enzyme (eg, a commercially available capping kit).

As used herein, a cap analog refers to a non-polymeric dinucleotide having a cap function that promotes translation or localization and/or prevents degradation of an RNA molecule when the cap is introduced to the 5' end of the RNA molecule. Non-polymeric means that the cap analog has no 5' triphosphate group and therefore only binds at the 5' end and cannot be extended in the 3' direction by a template-dependent RNA polymerase.

The cap analog can include a chemical structure selected from but not limited to the group consisting of: m7GpppA, m7GpppAmpG, a dimethylated cap analog, a trimethylated cap analog (e.g., m2,2,7GpppA), a dimethylated symmetric cap analog (e.g., m7Gpppm7A) or an anti-reverse cap analog (e.g., ARCA; m7,2'OmeGpppA, m7,2'dGpppA, m7,3'OmeGpppA, m7,3'dGpppA, and tetraphosphate derivatives thereof).

Other cap analogs have been described previously (US 7,074,596, WO 2008/016473, WO 2008/157688, WO 2009/149253, WO 2011/015347 and WO 2013/059475).

In the present invention, the 5'-cap structure may be selected from but not limited to the group consisting of m7GpppAmpG, m7,3'OmeApppG and m7GpppA.

In the present invention, the 5'-UTR may be any one of the sequences represented by SEQ ID NOs: 1 to 34 in Table 1 below, more preferably any one selected from the group consisting of SEQ ID NOs: 1, 2, 15, 28 and 30, but is not limited thereto.

[Table 1] SEQ ID NO. Name sequence 1 GC00 AGGAGGACUGGACUGGACUGGACUGCCACC 2 GC01 AGGAGAAGAGAGAAAAGUAAAAUAGCCACC 3 GC02 AGGAGUAAAAGUAAAAUAGUAAAAGCCACC 4 GC03 AGGAGAAAGAAAGAGUAAAAUAGGCCACC 5 GC04 AGGAUUAAAAAUAGAAAGAAUAAGGCCACC 6 GC05 AGGACAACAAACAAAAACAAACUAGCCACC 7 GC06 AGGACAGAGCACUAGAACAAAACAGCCACC 8 GC07 AGGACCACUCUCAACCAAAACCUAGCCACC 9 GC08 AGGAGACACUAACACCCCGACCGAGCCACC 10 GC09 AGGACCGGAACACAAAAACUAGCAGCCACC 11 GC10 AGGAACUACACUAGCCACUAAACAGCCACC 12 GC11 AGGACAACUAGACAGAGACACCGAGCCACC 13 GC12 AGGACACACACCCAGACCACCGAGCCACC 14 GC13 AGGACACAACUACCGAACUACUCAGCCACC 15 GC14 AGGAGAACUAGUACCCACCCGACAGCCACC 16 GC15 AGGAGAGAAAAAAACAACUACACAGCCACC 17 GC16 AGGAGAAACAACUAGGACCACAGAGCCACC 18 GC17 AGGACCUAAAACACCUCGACAGCAGCCACC 19 GC18 AGGAAAAAACUCACCUCUCAGACAGCCACC 20 GC19 AGGAGAGAAACUACACAGACUAGAGCCACC twenty one GC20 AGGAACAACAGACUCACUCACUCAGCCACC twenty two GC21 AGGAAAACUAAACAGAACCUAACAGCCACC twenty three GC22 AGGAACAGAGAACACUAGAGAGCAGCCACC twenty four GC23 AGGACCACACAGACCCACCUACUAGCCACC 25 GC24 AGGAAACCGACUAAAAACCCUCUAGCCACC 26 GC25 AGGAACAACACCAGCAGAACUCGAGCCACC 27 GC26 AGGAACAACAGUCGUAACCCUCUAGCCACC 28 GC27 AGGACCCUAACCAAACCCACUCUAGCCACC 29 GC28 AGGACACAACUCUACCCGACUACAGCCACC 30 GC29 AGGAACACAGUCAGCACCAACACAGCCACC 31 GC30 AGGACACUCAGACAACCCACACUAGCCACC 32 GC31 AGGACCUCAAAAAAAAACUCUAGCCACC 33 GC32 AGGACAACAGCUCACACCCCCCUAGCCACC 34 GC33 AGGACCCAGAACCCACCUAACAGAGCCACC

As used herein, the term "UTR" refers to a "non-translated region" located upstream (5') and/or downstream (3') of the coding region of the nucleic acid molecules described herein, and thus is typically located on one side of the coding region. Thus, the term "UTR" typically includes a 3' non-translated region ("3'-UTR") and a 5'-non-translated region ("5'-UTR"). UTRs typically include or can consist of nucleic acid sequences that are not translated into proteins. Typically, UTRs include "regulatory elements".

The term "regulatory element" refers to a nucleic acid sequence that has gene regulatory activity, expression of a transcribable nucleic acid sequence that is operably linked (cis or trans), and particularly the ability to affect transcription or translation. The term includes promoters, enhancers, internal ribosome entry sites (IRES), introns, leader sequences, transcription termination signals (such as polyadenylation signals and poly-U sequences), and other regulatory elements of expression. Regulatory elements may act constitutively or in a time-specific and/or cell-specific manner. Optionally, regulatory elements may act through the interaction (e.g., mobilization and binding) of regulatory proteins, wherein the regulatory proteins may regulate (induce, enhance, reduce, eliminate or prevent) expression, particularly transcription of a gene.

The UTR is preferably "operably linked" to the coding region, i.e., arranged in a functional relationship in such a way as to control (i.e., mediate or regulate, preferably enhance) the expression of the coding sequence.

In the present invention, the term "5'-UTR" refers to a portion of a nucleic acid molecule that is located 5' (i.e., "upstream") of an open reading frame that is not translated into protein. In the context of the present invention, a 5'-UTR starts at the transcription start site and ends one nucleotide before the start codon of the open reading frame.

The 5'-UTR may comprise elements that regulate gene expression, so-called "regulatory elements". Such a regulatory element may be, for example, a ribosome binding site. The 5'-UTR may be modified by post-transcriptional modification, for example by adding a 5'-cap. Thus, the 5'-UTR may preferably correspond to a nucleic acid located between the 5'-cap and the start codon, in particular the sequence of a mature mRNA, more specifically the nucleotides at the 3' end of the 5'-cap, preferably the nucleotides at the 5' end of the start codon (transcription start site) of a protein coding sequence among the nucleotides directly following the 3' end of the 5'-cap, preferably the nucleotides directly before the 5' end of the start codon (transcription start site) of a protein coding sequence.

The nucleotides directly at the 3' end of the 5'-cap of the mature mRNA usually correspond to the transcription start site. The length of the 5'UTR is usually less than 500, 400, 300, 250 or less than 200 nucleotides. In some examples, its length can be 10, 20, 30 or 40 or more nucleotides, preferably 10 or 50 or less nucleotides.

In the present invention, the signal peptide may be derived from an antigenic polypeptide, immunoglobulin E (IgE) or tissue plasminogen activator (tPA), but is not limited thereto.

In the present invention, the signal peptide of the antigenic polypeptide may be represented by the following amino acid sequence: SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 73 (MKXIIALSXILCLVFA, wherein X is T, A, Y or N) or SEQ ID NO: 77 (MKAILVVXLYTFTTANA, wherein X is L or M). More preferably, it may be represented by the following amino acid sequence, but not limited to: SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 74 (MKTIIALSYILCLVFA), SEQ ID NO: 75 (MKTIIALSNILCLVFA), SEQ ID NO: 76 (MKAIIALSNILCLVFA), SEQ ID NO: 78 (MKAILVVMLYTFTTANA) or SEQ ID NO: 79 (MKAILVVLLYTFTTANA).

In the present invention, the signal peptide of IgE may be represented by the amino acid sequence of SEQ ID NO: 80, but is not limited thereto.

In the present invention, the signal peptide of tPA can be represented by the amino acid sequence of SEQ ID NO: 81, but is not limited thereto.

[Table 2] Signal peptide sequences SEQ ID NO. Name sequence 72 B_Ya_Vic_HA_WT_AA MKAIIVLLMVVTSNA 73 A_H3N2_HA_WT_AA_Norm MKXIIALSXILCLVFA 74 A_H3N2_HA_WT_AA_P MKTIIALSYILCLVFA 75 A_H3N2_HA_WT_AA_22 MKTIIALSNILCLVFA 76 A_H3N2_HA_WT_AA_25 MKAIIALSNILCLVFA 77 A_H1N1_HA_WT_AA_Norm MKAILVVXLYTFTTANA 78 A_H1N1_HA_WT_AA_P MKAILVVMLYTFTTANA 79 A_H1N1_HA_WT_AA_20 MKAILVVLLYTFTTANA 80 IgE_WT_AA MDWTWILFLVAAATRVHS 81 tPA_WT_AA MDAMKRGLCCVLLLCGAVFVSA

In the present invention, the polynucleotide encoding the signal peptide may be codon-optimized, and is preferably any one of the sequences represented by SEQ ID NOs: 39 to 50, but is not limited thereto.

In the present invention, the polynucleotide encoding the 5'-UTR and the signal peptide can be any one selected from but not limited to the group consisting of: (i) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 39; (ii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 42; (iii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 45; (iv) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 48; (v) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 28 and the signal peptide of SEQ ID NO: 39; (vi) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 28 and the signal peptide of SEQ ID NO: 42; (vii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 28 and the signal peptide of SEQ ID NO:45; (viii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:48; (ix) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:41; (x) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:44; (xi) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:47; and (xii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:50.

In the present invention, according to one embodiment of the present invention, the term "GC00-WT-HA" is an abbreviation of 5'-UTR signal peptide-ORF, meaning that different strains have different 5'-UTR, signal peptide and ORF sequence compositions despite using the same expression, as described in Tables 4 to 7 of the present invention.

In the present invention, the antigenic polypeptide may be one or more selected from but not limited to the group consisting of: tumor antigen, pathogenic antigen, self-antigen, alloantigen and allergenic antigen.

As used herein, the term "tumor antigen" refers to an antigenic (poly)peptide or protein derived from or associated with a tumor (preferably a malignant tumor) or cancer. The terms "cancer" and "tumor" used herein are used interchangeably and refer to tumors characterized by uncontrolled and usually rapid cell proliferation that attempts to invade surrounding tissues and metastasize to distant parts of the body. These terms include benign and malignant tumors. Malignant tumors are typically characterized by degeneration (anaplasia), invasiveness, and metastasis; benign tumors generally do not have these characteristics. The terms "cancer" and "tumor" refer specifically to cancers of the blood and lymphatic systems and tumors characterized by tumor growth. "Tumor antigens" generally originate from tumor/cancer cells, preferably mammalian tumor/cancer cells, and may be located in or on tumor cells originating from a mammal (preferably a mammal, preferably a human) or in or on the interior or surface of a tumor, such as a systemic tumor or a solid tumor. "Tumor antigens" generally include tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are generally caused by tumor-specific mutations and are specifically expressed by tumor cells. TAAs are more common and are generally present in tumors and "normal" (healthy, non-tumor) cells.

In the present invention, the tumor antigen can be a protein or nucleic acid sequence associated with the tumor, wherein each nucleic acid sequence encodes a different peptide or protein; and wherein at least one nucleic acid sequence can encode 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, α-5-β-1-integrin, α-5-β-6-integrin, α-coactin-4/m, α-methylacyl-coenzyme A racemase, AT-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, β-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 1 5-3/CA 27-29, CA 19-9, CA72-4, CA125, calcitonin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collagen XXIII (collagen XXIII), COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, 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, HERVK-MEL, HLA-A*0201 - R1 7I, HLA-A1 1/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES- 85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, i67, 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, MAG E-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B1 6. MAGE-B1 7. MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGED2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H I, MAGEL2, Mammaglobulin A, MART-1/Melan-A, MART-2, MART-2/m, matrix protein 22, MC1 R, M-CSF, ME 1/m, mesothelin, MG50/PXDN, MMP1 1, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin type l/m, NA88-A, N-acetylglucosamine transferase-V, neo-PAP, neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESOB, OA1, OFA-iLRP, OGT, OGT/m, OS-9, OS- 9/m, bone calcification, osteopontin, pi 5, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 -kinase, Pin-1, Pml/PARα, POTE, PRAME, PRDX5/m, prostate-specific protein (Prostein), proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD1 68, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp1 7. SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, Survivin, Survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF-β, TGF-βRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, WT1, and lymphocyte immunoglobulin genotype or lymphocyte T cell receptor genotype, or homologs, fragments, variants or derivatives of the above tumor antigens.

In the present invention, the tumor antigen is selected from but not limited to the group consisting of: NY-ESO-1, HER-2/neu, MAGE-1, tyrosinase, MUC1, CEA, Mam-A, hTERT, Syalyl-Tn, WT1, α-fetoprotein, CA-125, gp-100, p53, Ras, Src, EGFRvIII, PSMA, GD2, Bcr-abl, survivin, PSA, EphA2, PAP, AFP, EpCAM, ALK, mesothelin, PSCA, MART-1, Melan-A, SCP-1, SPAG9, AKAP4 and OY-TES-1.

In the present invention, the pathogenic antigen may be selected from the group consisting of bacterial, viral, fungal and protozoan antigens.

In the present invention, the pathogenic antigen can be derived from influenza virus, respiratory syncytial virus (RSV), coronavirus, herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), malaria, Staphylococcus aureus, dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus and yellow fever virus, or any homologs, homologues, fragments, variants or derivatives of the above proteins.

In the present invention, the antigenic polypeptide or immunogenic protein thereof is an influenza virus antigenic polypeptide, which is at least one selected from but not limited to the group consisting of: a defined antigenic subdomain of hemagglutinin (HA), also known as HA1, HA2 or a combination of HA1 and HA2, and neuraminase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1) and nonstructural protein 2 (NS2).

In the present invention, the influenza virus antigenic polypeptide may be, but is not limited to, influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), or an immunogenic fragment of HA1 or HA2.

In the present invention, the influenza antigenic polypeptide or immunogenic protein thereof is derived from an influenza virus strain selected from the group consisting of influenza B Yamagata virus, influenza B Victoria virus, influenza A H3N2 virus and influenza A H1N1 virus, and more preferably may be HA protein derived from each virus strain, but is not limited thereto.

In the present invention, the polynucleotide encoding the influenza antigen polypeptide or its immunogenic protein may be codon-optimized, preferably any one of the sequences represented by SEQ ID No. 55 to 66, but not limited thereto.

In the present invention, the polynucleotide encoding the 5'-UTR and the signal peptide; and the polynucleotide encoding the antigenic polypeptide or its immunogenic protein are selected from but not limited to any one of the following groups: (i) polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 39; and polynucleotide encoding the antigenic polypeptide or its immunogenic protein of SEQ ID NO: 55; (ii) polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 42; and polynucleotide encoding the antigenic polypeptide or its immunogenic protein of SEQ ID NO: 58; (iii) polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 45; and polynucleotide encoding the antigenic polypeptide or its immunogenic protein of SEQ ID NO: 61; (iv) polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 48; and polynucleotide encoding the 5'-UTR of SEQ ID NO: 49; NO:64 or an immunogenic protein thereof; (v) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:39; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:55; (vi) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:42; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:58; (vii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:45; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:61; (viii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:48; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:64; (ix) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:48; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:64; NO: 1 and the signal peptide of SEQ ID NO: 41; and a polynucleotide encoding an antigenic polypeptide of SEQ ID NO: 57 or an immunogenic protein thereof; (x) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 44; and a polynucleotide encoding an antigenic polypeptide of SEQ ID NO: 60 or an immunogenic protein thereof; (xi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 47; and a polynucleotide encoding an antigenic polypeptide of SEQ ID NO: 63 or an immunogenic protein thereof; and (xii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 50; and a polynucleotide encoding an antigenic polypeptide of SEQ ID NO: 66 or an immunogenic protein thereof.

In the present invention, the 3'-UTR is selected from but not limited to the group consisting of: human α-globulin 3'-UTR; β-globulin 3'-UTR; CYBA 3'-UTR; albumin 3'-UTR; growth hormone (GH) 3'-UTR; VEEV 3'-UTR; hepatitis B virus (HBV) 3'-UTR; α-globulin 3'-UTR; DEN 3'-UTR; PAV barley yellow dwarf virus (BYDV-PAV) 3'-UTR; elongation factor 1 α1 (EEF1A1) 3'-UTR; manganese superoxide dismutase (MnSOD) 3'-UTR; mitochondrial H(+)-ATP synthase β subunit (β-mRNA) 3'-UTR; GLUT1 3'-UTR; MEF2A 3'-UTR; β-F1-ATPase 3'-UTR; its functional fragments and their combinations.

In the present invention, the 3'-UTR can be represented by the sequence of SEQ ID NO:71.

As used herein, the term "3'-UTR" generally refers to a portion of an mRNA located between the protein coding region (i.e., open reading frame, coding region) and the poly (A) sequence of an mRNA. The 3'-UTR of an mRNA is not translated into an amino acid sequence. 3'-UTR sequences are generally encoded by genes that are transcribed into their respective mRNAs during gene expression. This genomic sequence is first transcribed into an immature mRNA, which contains optional introns. The immature mRNA is then further processed into a mature mRNA during the maturation process. This maturation process includes steps such as 5'-capping, shearing of the immature mRNA to remove optional introns, and modification of the 3' end (such as polyadenylation of the 3' end of the immature mRNA and selective endo- or exonuclease cleavage).

In the present invention, the 3'-UTR corresponds to the sequence at the 3' end of the stop codon in the protein coding region in the mature mRNA, preferably corresponds to the sequence at the 3' place of the stop codon in the protein coding region and extends to the 5' of the poly (A) sequence, preferably the nucleotides 5' of the poly (A) sequence. The term "corresponding" means that the 3'-UTR sequence can be an RNA sequence, such as an mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence.

In the present invention, the poly(A) tail or poly(A) tail-like sequence further comprises a poly(A) tail. In a further embodiment, a terminal group on the poly(A) tail can be introduced for stabilization. In another embodiment, the poly(A) tail comprises a des-3' hydroxyl tail.

During RNA processing, a long chain of adenine nucleotides (poly(A) tail) can be added to a polynucleotide (such as an mRNA molecule) to increase stability. Immediately after transcription, the 3' end of the transcript can be cleaved to release the 3' hydroxyl group. Poly-A polymerase then adds the chain of adenine nucleotides to the RNA. The process known as polyadenylation adds the poly(A) tail, which can be, for example, about 80 to about 250 residues in length (including about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues in length). The poly(A) tail can also be added after the product has exited the nucleus.

According to the present invention, terminal groups on the poly(A) tail can be introduced for stabilization. The polynucleotides of the present invention can include a des-3' hydroxyl tail. They can further include structural moieties or 2'-O-methyl modifications as taught by Junjie Li et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the contents of which are incorporated herein by reference in their entirety).

The unique poly (A) tail length provides certain advantages for the polynucleotides of the present invention. Typically, if a poly (A) tail is present, its length is greater than 30 nucleotides. In other embodiments, the length of the poly (A) tail is greater than 35 nucleotides (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and more than 3,000 nucleotides).

In some embodiments, a polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 1 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2,000 to 3,000, 2,000 to 2,500, and 2,500 to 3,000).

In some embodiments, the poly (A) tail is designed according to the length of the entire polynucleotide or the length of a specific region of the polynucleotide. This design can be based on the length of the coding region, the length of a specific feature or region, or the length of the final product expressed by the polynucleotide.

In this regard, the length of the poly(A) tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% longer than the polynucleotide or its feature. The poly(A) tail can also be designed as a part of the polynucleotide to which it belongs. In this regard, the poly(A) tail can be the total length of the fragment, a region of the fragment, or the total length of the fragment - at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the length of the poly(A) tail. In addition, engineered binding sites and conjugation of polynucleotides with poly-A binding proteins can enhance expression.

In addition, multiple different polynucleotides can be 3'-linked together via poly-A binding protein (PABP) using modified nucleotides at the 3' end of the poly(A) tail. Transfection experiments can be performed in appropriate cell lines and protein production can be tested by ELISA at 12 hours, 24 hours, 48 hours, 72 hours and 7 days after transfection.

In some embodiments, the polynucleotides of the present invention are designed to include a poly A-G quadruplex region. The G-quadruplex is a cyclic hydrogen-bonded secondary structure of four guanine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this experiment, the G-quadruplex was incorporated into the end of the poly (A) tail. The resulting polynucleotides were tested for stability, protein yield, and other parameters including half-life at different time points. It was found that the poly-A-G quadruplex resulted in a protein yield from mRNA equivalent to at least 75% of the protein yield using a 120-nucleotide poly (A) tail alone.

In the present invention, the poly (A) tail-like sequence may be any nucleic acid sequence capable of performing the function of a poly (A) tail, and may preferably be characterized by one or more nucleotides selected from the group consisting of uracil (U), cytosine (C) and guanine (G) other than adenine inserted between a plurality of adenines or at the end of the poly (A) tail, but is not limited thereto.

In the present invention, the mRNA construct may include one or more backbone-modified, sugar-modified or base-modified nucleic acids, but is not limited thereto.

Sugar decoration:

The modified nucleosides or nucleotides that can be incorporated into the modified mRNA compounds comprising the mRNA sequences described herein can be modified in the sugar portion. For example, the 2'hydroxyl group (OH) can be modified or substituted with many different "oxy" or "deoxy" substituents. Examples of "oxy-containing"2'hydroxyl group modifications can include, but are not limited to, alkoxy or allyloxy groups (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, _{aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH2CH2O ) nCH2CH2OR ;} "lock" nucleic acids (LNA), in which the _2'hydroxyl group is linked to the 4' carbon of the same ribose through, for example, a methylene crosslink; and amino groups (-O-amine, in which the amino group, e.g., NRR can be alkylamine, dialkylamine, heterocyclo, arylamine, diarylamine, heteroarylamine, or diheteroarylamine, ethylenediamine, polyamine) or aminoalkoxy groups.

The "deoxy" modification may include an amine group (e.g., _NH2 ; an alkylamine, a dialkylamine, a heterocyclic group, an arylamine, a diarylamine, a heteroarylamine, a diheteroarylamine, or an amino acid); or the amine group may be attached to the sugar via a linker, wherein the linker includes at least one of atoms C, N, and O.

The sugar group may further comprise one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, the modified mRNA may include nucleotides containing a sugar such as arabinose.

Frame modification:

The phosphate backbone can be further modified in modified nucleosides and nucleotides, which can be incorporated into modified mRNA compounds comprising mRNA sequences described herein. The phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with other substituents. In addition, the modified nucleosides or nucleotides can include completely replacing the unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, alkyl phosphate or aryl phosphonate and phosphotriester. Both unattached oxygens of dithiophosphates are replaced by sulfur.

Phosphate linkers can also be modified by replacing the attached oxygen with nitrogen (cross-linked phosphamides), sulfur (cross-linked phosphorothioates), or carbon (cross-linked methylenephosphonates).

Alkaline Modification:

The modified nucleosides or nucleotides that can be incorporated into the modified mRNA compounds comprising the mRNA sequences described herein can be modified in the nucleobase portion. Examples of nucleobases found in mRNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides or nucleotides described herein can be chemically modified at the major groove surface. In some embodiments, the major groove chemical modification can include an amine group, a thiol group, an alkyl group, or a halogen group.

In a particularly preferred embodiment of the present invention, the nucleotide analog/modification is preferably selected from: 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-aminopurine-nucleoside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate. Phosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine- 5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine nucleoside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate , 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate.

Particularly preferred is a nucleotide for base modification selected from the group consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate.

In some embodiments, the modified nucleosides include 5-pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-methoxyuridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.

In some embodiments, the modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-methylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-methylisocytidine, pyrrolyl-cytidine, pyrrolyl-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio -1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-methylisocytidine and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, glycoside, N6-isopentenyl adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycoamidomethyladenosine, N6-threonamidomethyladenosine, 2-methylthio-N6-threonamidomethyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine and 2-methoxy-adenine.

In other embodiments, the modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl- Guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine and N2,N2-dimethyl-6-thioguanosine.

In some embodiments, the nucleotide may be modified at the major groove and may include a methyl or halogen group replacing the hydrogen at C-5 of uracil. In certain embodiments, the modified nucleoside is 5'-O-(1-phosphorothioate)-adenosine, 5'-O-(1-phosphorothioate)-cytidine, 5'-O-(1-phosphorothioate)-guanosine, 5'-O-(1-phosphorothioate)-uridine or 5'-O-(1-phosphorothioate)-pseudouridine.

In other specific embodiments, the modified mRNA may include a nucleoside modification selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5- Methyl-uridine, pyrrolyl-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-oxo-adenosine, and 7-deaza-adenosine.

In another aspect, the invention relates to influenza vaccine compositions comprising mRNA constructs.

As used in the present invention, "vaccine" is generally understood as a preventive or therapeutic substance that provides at least one antigen, preferably an antigenic peptide or protein. "Providing at least one antigen" means, for example, that the vaccine comprises an antigen or that the vaccine comprises, for example, a molecule encoding an antigen. Thus, it is particularly envisioned that the vaccines of the present invention comprise at least one synthetic nucleic acid (RNA) molecule encoding at least one antigenic peptide (polypeptide) or protein as defined herein, which may be derived from, for example, a tumor antigen, a bacterial, viral, fungal or protozoan antigen, a self-antigen, an allergen or an alloantigen, and when expressed and presented to the immune system, preferably induces an immune response against the corresponding antigen. However, synthetic nucleic acid (RNA) molecules encoding non-antigenic peptides (polypeptides) or proteins of interest may also be used in the vaccines of the present invention.

In the present invention, the mRNA construct of the vaccine composition can be complexed with one or more lipids to form lipid nanoparticles or liposomes.

Lipid nanoparticles may include, but are not limited to, cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids.

In the present invention, the mRNA construct can be provided in a complexed form, i.e. in a complexed or conjugated form with one or more (poly) cationic compounds, preferably (poly) cationic polymers, (poly) cationic peptides or proteins, such as protamine, (poly) cationic polysaccharides and/or (poly) cationic lipids. In this regard, the term "complexed" or "conjugated" refers to an intrinsically stable combination of at least one synthetic nucleic acid (RNA) molecule with one or more of the above compounds in a larger complex or element, without covalent bonding.

Lipids

According to a preferred embodiment, the mRNA construct of the present invention is complexed or conjugated with lipids (particularly cationic and/or neutral lipids) to form one or more lipid nanoparticles or liposomes. Therefore, in some embodiments, the synthetic nucleic acid (RNA) molecules of the present invention can be provided in the form of lipid-based preparations (particularly liposomes comprising synthetic nucleic acid (RNA) molecules and/or lipid nanoparticles).

Lipid Nanoparticles

According to some preferred embodiments, the mRNA construct of the present invention is complexed or conjugated with lipids (particularly cationic and/or neutral lipids) to form one or more lipid nanoparticles.

Preferably, lipid nanoparticles (LNPs) may include: (a) at least one mRNA complex of the present invention, (b) a cationic lipid, (c) an aggregation reducing agent (e.g., a polyethylene glycol (PEG) lipid or a PEG-modified lipid), (d) optionally a non-cationic lipid (e.g., a neutral lipid), and (e) optionally, a sterol.

In some embodiments, in addition to at least one mRNA construct of the present invention, the LNP further comprises (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and a PEG-lipid, which may be included in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% polyethylene glycol lipid.

In some embodiments, the mRNA constructs of the present invention can be formulated as amino alcohol lipids. The amino alcohol lipids useful in the present invention can be prepared by the method described in U.S. Patent No. 8,450,298, the entire contents of which are incorporated herein by reference.

Liposome

In some embodiments, the mRNA constructs of the present invention are formulated into liposomes. Liposomes based on cationic lipids can form complexes with negatively charged nucleic acids (e.g., RNA) through electrostatic interactions, thereby producing complexes that provide biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; in cells, liposomes are processed through the phagocytic pathway and the nucleic acid is subsequently released from the endosome/vessel into the cytoplasm. Given that liposomes are essentially analogs of biological membranes and can be prepared from natural and synthetic phospholipids, liposomes have long been considered as drug delivery vehicles due to their excellent biocompatibility.

Liposomes are usually composed of a lipid bilayer, which can be composed of cationic, anionic, or neutral (phospholipids) and cholesterol, surrounding an aqueous core. Both the lipid bilayer and the aqueous space can contain hydrophobic or hydrophilic compounds, respectively. Liposomes can have one or more lipid membranes. Liposomes can be single-layered (called unilamellar) or multi-layered (called multilamellar).

In vivo, the properties and behavior of liposomes can be altered by adding hydrophilic polymer coatings such as polyethylene glycol (PEG) to the liposome surface to provide steric stability. In addition, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the ends of attached PEG chains.

Liposomes typically exist in the form of spherical vesicles that can range in size from 20 nm to several microns. Liposomes can vary in size, including but not limited to multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can range in diameter from 50 to 500 nm. Liposome designs can include but are not limited to opsonins or ligands to improve the attachment of liposomes to unhealthy tissues or to initiate events such as but not limited to endocytosis. Liposomes can include low or high pH to enhance the delivery of drug formulations.

In the present invention, the vaccine composition may further include one or more adjuvants or activators.

In the broadest sense, an "adjuvant" or "adjuvant ingredient" is generally a pharmacological and/or immunological agent that can modify (e.g., enhance) the efficacy of another active agent (e.g., a therapeutic agent or a vaccine). In this regard, an "adjuvant" can be understood as any compound suitable for supporting the administration and delivery of the vaccine composition of the present invention. In particular, an adjuvant can preferably enhance the immunostimulatory properties of the vaccine to which it is added. In addition, such an adjuvant can elicit or increase an immune response of the innate immune system, i.e., a non-specific immune response, without binding thereto.

"Adjuvants" generally do not elicit an adaptive immune response. To date, "adjuvants" have not been able to act as antigens. That is, when administered, the vaccines of the present invention generally elicit an adaptive immune response due to the antigenic peptide or protein encoded by at least one coding sequence of the synthetic nucleic acid (RNA) molecule contained in the vaccine.

Suitable adjuvants may be selected from any adjuvant known to those of ordinary skill in the art and suitable for use in the context of the present invention, i.e., drugs that help induce an immune response in mammals, such as TDM, MDP, muramyl dipeptide, pluronic, vitiligo solution, aluminum hydroxide, ADJUMER™ (polyphosphazene); aluminum phosphate gel; dextran from algae; algamulin; aluminum hydroxide gel (vitiligo); high protein adsorbing aluminum hydroxide gel; low viscosity aluminum hydroxide gel; low viscosity aluminum hydroxide gel; AF or SPT (squalane emulsion (5%), Tween 80 (0.2%), fluoride L121 (1.25%), phosphate-buffered saline (pH 7.4); AVRIDINETM (propylenediamine); a group of substances corresponding to pathogen-associated molecular patterns (PAMPs) that react with pattern recognition receptors (PRRs); CpG DNA; lipoproteins; flagella; poly I:C; saponin; squalene; capric glyceride; 3D-MPL; or detoxified lipooligosaccharides (dLOS).

In another aspect, the present invention relates to use of the vaccine composition for preventing influenza.

As used herein, "influenza" refers to the infectious disease caused by influenza viruses and is used interchangeably with pandemic cold, circulating cold, or influenza (FLU).

As used herein, "prevention" means any action to inhibit or delay the development of influenza by administering the vaccine composition of the present invention.

In another aspect, the present invention relates to a method for preventing influenza, comprising the step of administering a vaccine composition.

In another aspect, the present invention relates to the use of the vaccine composition in the preparation of an agent for preventing influenza.

Embodiment

The present invention is described in more detail with reference to the following embodiments. These embodiments are intended only to illustrate the present invention, and it is obvious to those having ordinary knowledge in the art that the scope of the present invention should not be interpreted as being limited by these embodiments.

Embodiment 1. mRNA Construct preparation

1-1 Preparation of mRNA sequences

33 5'-UTRs were screened by the method of PCT/KR2022/019491, and another 5'-UTR of SEQ ID NO: 1 was selected as the 5'-UTR sequence for HA protein expression (Table 3).

In addition, the signal sequence and ORF sequence of each strain were prepared by codon optimization based on the wild-type Yamagata, Victoria, H3N2 and H1N1 signal sequences (SEQ ID NOs: 51, 52, 53 and 54, respectively) and ORF sequences (SEQ ID NOs: 67, 68, 69 and 70, respectively) listed in Table 3. In addition, the signal sequence of the wild-type strain was replaced with codon-optimized IgE or tPA.

[Table 3] Serial ID NO. Name sequence 1 GC00 AGGAGGACUGGACUGGACUGGACUGCCACC 2 GC01 AGGAGAAGAGAGAAAAGUAAAAUAGCCACC 15 GC14 AGGAGAACUAGUACCCACCCGACAGCCACC 28 GC27 AGGACCCUAACCAAACCCACUCUAGCCACC 30 GC29 AGGAACACAGUCAGCACCAACACAGCCACC 35 B_Ya_UTR_WT AGCAGAAGCAGAGCAUUUUCUAAUAUCCACAAA 36 B_Vic_UTR_WT AGCAGAAGCAGAGCAUUUUCUAAUAUCCACAAA 37 A_H3N2_UTR_WT AGCAAAAGCAGGGGAUAAUUCUAUUAACC 38 A_H1N1_UTR_WT AGCAAAAGCAGGGGAAAAUAAAAGCAAAAAAA 39 B_Ya_Sig_WT AUGAAGGCCAUAAUCGUCCUCCUGAUGGUAGUCACUUCGAAUGCC 40 B_Ya_Sig_IgE AUGGACUGGACCUGGAUUCUUUUCCUGGUAGCCGCGGCGACGAGAGUGCAUUCU 41 B_Ya_Sig_tPA AUGGACGCAAUGAAAAGGGGAUUGUGCUGCGUGCUGCUCCUGUGCGGAGCCGUGUUUGUAUCCGCA 42 B_Vic_Sig_WT AUGAAGGCAAUCAUUGUUCUGCUCAUGGUGGUCACGUCGAACGCU 43 B_Vic_Sig_IgE AUGGACUGGACGUGGAUCUUAUUCCUCGUGGCAGCUGCCACCCGCGUUCACUCG 44 B_Vic_Sig_tPA AUGGACGCUAUGAAGCGGGGGCUAUGUUGCGUGCUCUUGUUGUGCGGGCCGGUGUUCGUGUCGGCA 45 A_H3N2_Sig_WT AUGAAGACCAUUAUUGCCCUCUCCUACAUCUUGUGCCUGGUAUUCGCU 46 A_H3N2_Sig_IgE AUGGAUUGGACAUGGAUACUGUUCCUAGUGGCAGCCGCAACUCGCGUUCACUCC 47 A_H3N2_Sig_tPA AUGGACGCCAUGAAAAGGGGCCUGUGCUGCGUCUUGUUGUUGUGCGGUGCUGUUUUCGUCAGUGCC 48 A_H1N1_Sig_WT AUGAAAGCUAUCCUCGUAGUUAUGCUGUACACGUUCACCACGGCCAAUGCA 49 A_H1N1_Sig_IgE AUGGAUUGGACAUGGAUUCUCUUUCUCGUAGCAGCUGCUACACGUGUUCACUCA 50 A_H1N1_Sig_tPA AUGGAUGCGAUGAAGCGCGGAUUGUGCUGCGUACUGCUGCUCUGCGGAGCCGUUUUUGUUAGCCGG 51 B_Ya_Sig_WT_C AUGAAGGCAAUAAUUGUAACUACUCAUGGUAGUAACAUCCAACGCA 52 B_Vic_Sig_WT_C AUGAAGGCAAUAAUUGUAACUACUCAUGGUAGUAACAUCCAAUGCA 53 A_H3N2_Sig_WT_C AUGAAGACUAUCAUUGCUUUGAGCUACAUUCUAUGUCUUGUUUUCGCU 54 A_H1N1_Sig_WT_C AUGAAGGCAAUACUAGUAGUUAUGCUGUAUACAUUUACAACCGCAAAUGCA 55 B_Ya_HA_WT GACAGAAUCUGCACUGGGAUCACGUCGAGCAACUCUCCACAUGUGGUUAAGACGGCCACUCAGGGUGAGGUGAACGUCACGGGAGAUCCCCCUCACCACCCCCACCAAGAGCUAUUUUGCCAACUUGAAGGGCACACGCACCAGGGGUAAGCUGUGCCCGGACUGCCUGAAUUGUACAGAUCUGGACGUGGCGCUGGGACGUCCCAUG UGCGUGGGCACGACUCCUAGCGCCAAGGCGUCCAUAUUGCAUGAGGUAAGACCUGUUACUAGCGGGUGCUUCCCAAUCAUGCAUGAUCGUACAAAGAUCAGGCAGCUCCCCAAUCUGCUUAGGGGCUACGAGAAGAUCCGGCUGUCGACGCAGAAUGUGAUUGAUGCCGAGAAAGCUCCUGGUGGCCCCUACAGGUUGGGGACUUCAGGGAGCU GCCCCAAUGCUACUAGCAAGAUUGGGUUUUUCGCCACUAUGGCCUGGGCCGUGCCUAAGGAUAAUUAUAAGAACGCGACCAAUCCACUAACGGUGGAGGUGCCUUAUAUUUGUACCGAGGGCGAAGACCAAAUCACGGUGUGGGGGUUUCAUUCUGACGACAAGACGCAGAUGAAAAGCCUGUAUGGGGACAGCAACCCCCAAAAGUUUACCAG CUCUGCGAACGGUGUGACCACACACUACGUUUCGCAGAUUGGUGACUUUCCCUGACCAGACGGAGGACGGCGGCCUGCCGCAGAGCGGCAGGAUCGUUGUGGAUUACAUGAUGCAAAAGCCGGGUAAGACCGGCACCAUCGUGUAUCAGCGGGGGGUUUUGCUGCCCCAAAAGGUAUGGUGCGCCAGUGGGAGGAGUAAGGUCAUUAAGGGCAGC UUGCCCUUGAUUGGCGAGGCCGAUUGCCUGCAUGAGGAGUACGGUGGCUUGAAUAAGAGCAAGCCGUAUUACACUGGUAGCACGCGAAGGCCAUCGGCAAUUGCCCAAUAUGGGUAAAGACCCCUCUCAAGCUGGCGAACGGUACCAAGUACAGGCCCCCCGCCAAGCUCCUAAAGGAGCGGGGCUUCUUUGGCGCGAUAGCUGGCUUCCUG GAGGGUGGAUGGGAGGGGAUGAUCGCUGGCUGGCACGGCUAUACCAGUCAUGGGGCCCAUGGCGUGGCCGUUGCCGCCGACCUGAAAAGCACCCAAGAGGCUAUUAACAAGAUUACGAAGAAUCUUAAUAGCCUCUCAGAGCUGGAGGUUAAGAACCUCCAGCGUUUGUCUGGGGCUAUGGACGAGUUGCACAACGAGAUCCUGGAGCUCGACG AAAAGGUCGACGAUUUGAGAGCUGAUACAAUCAGCUCUCAGAUCGAGCUUGCGGUUCUGUUGAGCAAUGAGGGAAUUAUCAAUAGCGAGGACGAGCACUUGCUCGCCUUAGAGCGCAAGCUGAAGAAGAUGCUGGGGCCCUCAGCGGUCGACAUCGGUAAGGGUGCUUUGAGACCAAGCUAAGUGCAACCAGACUUGUCUGGAUAGGAUCGC GGCCGGCACUUUUCAAUGCCGGCGAGUUCAGUCUGCCGACUUUCGACAGUCUGAACAUCACGGCCGCGUCGUUGAACGACGAUGGCUUGGACAACCACACGAUCCUGCUGUACUACAGUACAGCUGCUAGCAGUCUGGCAGUUACUCUCAUGCUCGCCAUAUUCAUCGUGUAUAUGGUGAGCAGAGAUAACGUCAGCUGCAGUAUAUGCCUUUAA 56 B_Ya_HA_IgE GACAGGAUAUGUACCGGCAUCACUUCGUCGAACUCGCCCCAUGUGGUGAAAACCGCCACACAGGGCGAGGUGAACGUGACAGGGGUCAUACCCCUGACCACUACGCCGACGAAGUCUUAUUUCGCGAACCUCAAAGGUACGCGGACCCGGGGCAAGCUGUGCCCCGACUGCCUGAAUUGUACGGAUCUUGACGUGGCGCUGGGAAGGCCUAUG UGCGUGGGGACCCCCAGUGCAAAGGCCAGCAUCUUACACGAGGUCCGCCCCGUGACCUCGGGGUGCUUUCCCAUCAUGCACGAUCGUACGAAAAUCAGGCAGUUGCCGAAUCUGUUGCGUGGCUACGAGAAGAUCAGGCUGUCUACGCAGAACGUGAUAGACGCAGAGAAGGCCCCGGGCGGUCCGUACCGCUUGGGGACUUCUGGUUCAU GUCCCAAUGCCACCAGUAAAAUUGGCUUCUUCGCCACUAUGGCGUGGGCUGUCCCGAAGGACAACUACAAGAAUGCCACAAACCCCCUUACGGUGGAGGUGCCGUACAUCUGCACCGAAGGGGAGGACCAGAUCACGGUGUGGGGCUUCCAUAGCGACGAUAAGACGCAGAUGAAAAGCCUGUACGGCGAUAGCAACCCCCAAAAGUUUACCAG CUCUGCCAAUGGUGUGACAACACACUAUGUGAGCCAGAUUGGAGACUUCCCCGAUCAGACGGAGGAUGGCGGCCUGCCACAGAGUGGCAGGAUCGUCGUUGACUACAUGAUGCAGAAGCCGGGUAAGACCGGUACCAUCGUGUAUCAACGGGGGGUUCUGUUGCCUCAGAAAGUUUGGUGUGCCAGCGGGAGGAGCAAGGUCAUAAAGGGAAGU CUCCCUUUAAUUGGCGAGGCCGAUUGCCUGCAUGAGGAGUACGGCGGACUUAAUAAGUCUAAGCCGUACUACACUGGUAAGCACGCGAAGGCCAUUGGCAAUUGCCCAAUAUGGGUAAAGACCCCUCUUAAGCUGGCGAACGGAACUAAGUACAGGCCUCCUGCCAAGCUGCUGAAGGAGCGUGGCUUCUUCGGUGCCAUCGCCGGCUUCCUU GAGGGAGGCUGGGAAGGCAUGAUUGCCGGUUGGCACGGUUAUACCAGCCACGGUGCGCAUGGUGUGGCCGUGGCGGCCGACCUGAAAUCGACUCAGGAAGCAAUCAACAAGAUCACCAAGAACUUGAACAGCUUGUCCGAGCUGGAGGUUAAGAAUCUCCAGCGUCUUUCGGGCGCUAUGGACGAGCUCCACAAUGAGAUUCUGGAGCUUGAUG AGAAGGUGGACGACUUAAGGGCUGACACGAUCAGCAGCCAGAUAGAGCUGGCUCCUGCUGAGCAACGAGGGCAUCAUCAACAGCGAGGACGAGCACUUGCUGGCUUUGGAACGCAAGUUAAAAAAGAUGCUGGGGCCCUCGGCCGUAGACAUUGGUAACGGGUGUUUUGAGACCAAGCAUAAGUGUAAUCAGACGUGCCUGGAUAGGAUAGC CGCCGGUACGUUCAACGCCGGCGAGUUUUCUCUACCAACGUUUGAUUCACUUAAUAUCACUGCCGCCUCGCUGAAUGAUGAUGGCCUGGAUAACCAUACUAUCCUGCUGUAUUACAGCACCGCUGCCAGCAGUCUGGCGGUCACCCUUAUGUUGGCCAUCUUCAUCGUCUACAUGGUGAGCAGGGACAACGUGAGCUGCUCUAUCUGUCUGUGA 57 B_Ya_HA_tPA GACCGGAUUUGCACAGGCAUUACGUCGAGCAACAGUCCCCACGUGGUGAAAACCGCCACCCAGGGCGAGGUGAACGUGACCGGCGUGAUCCCGCUGACUACGACUCCUACGAAGUCGUACUUCGCGAAUUUGAAAGGCACGCGGACUCGAGGCAAGCUGUGUCCCGAUUGCCUGAACUGCACUGAUCUGGACGUGGCUCUGGGCCGGCCUAUG UGCGUUGGGACUACUCCCAGCGCCAAGGCCAGUAUACUUCACGAGGUGCGGCCCGUGACCAGCGGCUGCUUCCCGAUUAUGCACGACAGGACAAAGAUUCGGCAGCUGCCGAAUCUGCUGAGGGGCUACGAGAAGAUCCGCCUGUCAACGCAAAACGUGAUAGACGCGGAGAAGGCCCCCGGCGGUCCUUACCGUCUAGGGACGAGCGGCAGCU GCCCGAAUGCGACCUCUAAGAUCGGCUUCUUCGCCACUAUGGCGUGGGCUGUCCCCAAGGACAACUACAAGAACGCCACUAAUCCUCUGACGGUUGAGGUGCCUUACAUUUGCACGGAAGGCGAGGACCAGAUCACAGUGUGGGGCUUCCAUAGCGAUGACAAGACUCAGAUGAAAUCUCUGUACGGCGACAGCAAUCCCCAGAAGUUCACCAG UUCUGCUAACGGUGACGACGCACUACGUUAGCCAGAUUGGGGACUUCCCGGAUCAGACCGAAGACGGCGGCCUGCCGCAGAGCGGCAGGAUCGUUGUUGACUAUAUGAUGCAAAAGCCCGGUAAAACCGGGACCAUCGUGUAUCAACGGGGGGUGCUGUUGCCUCAGAAGGUAUGGUGUGCCAGCGGGCGGAGUAAAGUGAUCAAGGGCAGC UUGCCCUUGAUCGGCGAGGCCGAUUGCCUUCACGAGGAGUAUGGCGGGCUUAAUAAGUCCAAGCCAUACUACACUGGGAAGCACGCAAAGGCCAUCGGCAAUUGCCCAAUCUGGGUAAAGACUCCCCUCAAGCUGGCGAACGGUACCAAGUACAGGCCCCCCGCGAAGCUGCUGAAGGAGCGGGGUUUCUUCGGGGCUAUCGCUGGCUUCCUG GAAGGAGGCUGGGAGGGCAUGAUUGCCGGCUGGCAUGGGUAUACCAGCCACGGCGCCCACGGCGUAGCCGUGGCUGCCGACCUGAAGUCAACUCAGGAGGCGAUCAACAAGAUUACAAAAAAUCUGAAUAGUCUUAGCGAGCUGGAGGUUAAAAACCUUCAGCGGCUGUCGGGCGCUAUGGACGAGCUCCACAAUGAGAUACUGGAGCUUGACG AGAAGGUGGACGAUUUGCGAGCUGAUACAAUCAGCUCGCAGAUUGAGCUCGCUGUGUCUUGCUGUCAAAUGAGGGUAUUCAUCAACUCAGAGGAUGAGCAUCUUCUGGCCCUGGAAAGGAAGUUAAAGAAGAUGCUCGGUCCUAGUGCCGUCGACAUCGGGAACGGCUGCUUCGAGACCAAGCAUAAAGUGCAAUCAGACUUGCUUAGACCGGAUCGC GGCCGGCACGUUCAACGCCGGCGAGUUUUCACUGCCGACGUUCGACAGCUUGAACAUCACAGCGGCCAGCCUAAACGACGAUGGUCUGGACAACCACACCAUCCUGCUGUAUACAGCACGGCGGCCAGUAGCCUGGCCGUUACGCUUAUGCUGGCCAUCUUCAUUGUGUAUAUGGUGAGCAGAGACAAUGUCUCUUGCUCAAUAUGUCUCUAA 58 B_Vic_HA_WT GACCGCAUUUGCACAGGUAUCACCUCCAGCAAUUCGCCCCAUGUGGUAAAGACUGCCACACAGGGCGAAGUAAACGUGACCGGCGUGAUCCCCCUUACCACCACGCCCACCAAGAGCCACUUCGCAAAUCUGAAGGGCACCGAGACCCGGGGCAAGCUGUGCCCCAAAUGCUUGAACUGCACUGAUCUGGAUGUCGCCUUGGGCAGACCUAAG UGCACCGGCAAAAUCCCGUCUGCACGUGUGAGCAUCCUGCAUGAAGUGAGGCCCGUUACAAGCGGUUGCUUCCCUAUUAUGCACGAUCGCACCAAGAUUCGGCAGCUGCCCAACUUGCUGAGGGGCUAUGAGCACGUUAGGUUGUCUACUCAUAACGUGAUCAAUGCCGAGGACGCUCCCGGCAGGCCCUAUAAGAUCGGCACGUCGGGCAGC UGCCCGAAUAUCACCAACGGCAAUGGGUUUUUUGCCACGAUGGCAUGGGCGGUUCCGAAAAAAUAAAACGGCGACUAAUCCACUGACCAUCGAGGUGCCUUAUAUUUGCACCGAGGGCGAGGACCAGAUAACGGUGUGGGGCUUCCACUCUGAUAGCGAGACCCAGAUGGCUAAGCUGUACGGGGAUUCGAAGCCCCAGAAGUUCACCAGCAGC GCCAACGGCGUGACUACUCACUAUGUGUCUCAGAUUGGUGGGUUUCCCAACCAAACUGAAGACGGCGGCCUGCCGCAAAGUGGCAGGAUCGUCGUCGACUAUAUGGUGCAGAAGUCUGGCAAGACGGGUACCAUAACCUAUCAGAGGGGCAUUUUGCUUCCUCAGAAGGUAUGGUGCGCGUCUGGCCGGUCUAAGGUGAUAAAGGGGUCUUUA CCCCUGAUCGGAGAGGCCGAUUGCUUGCACGAGAAGUACGGCGGACUUAACAAGUCUAAGCCGUACUACACGGGGUGAGCACGCGAAGGCGAUCGGCAACUGUCCGAUUUGGGUGAAGACCCCUUUAAAGCUGGCAAAUGGCACGAAGUAUCGUCCUCCGGCGAAACUUCUCAAAGAACGAGGGUUCUUUGGUGCCAUUGCCGGCUUCUUGGAG GGUGGGUGGGAGGGGAUGAUCGCGGGCUGGCACGGUUACACCUCGCACGGCGCGCAUGGCGUAGCCGUGGCGGCGGAUCUGAAGUCUACUCAGGAGGCGAUCAAUAAGAUCACCAAGAACCUGAAUAGCCUCUCGGAGCUGGAGGUGAAAAACCUGCAGAGGCUGUCGGGAGCGAUGGACGAGCUACACAACGAGAUACUCGAAUUGGACGAG AAGGUGGACGAUCUUCGUGCCGACACCAUCUCGAGCCAAAUCGAGCUCGCUGUUCUAUUGAGCAACGAGGGCAUCAUCAACAGUGAGGACGAGCACUUGCUGGCCCUGGAACGUAAGUUGAAGAAGAUGCUGGGUCCGAGCGCCGUUGAGAUUGGGAACGGCUGUUUCGAAACGAAGCAUAAGUGCAAUCAGACGUGCCUGGACCGUAUAGCG GCAGGUACGUUUGAUGCAGGUGAGUUCAGCCUGCCGACGUUCGACAGCCUGAACAUCACCGCCGCUUCCUUGAAUGACGACGGCUUGGACAACCAUACCAUCCUUCUCUACUACUCCACGGCCGCAAGUAGCUUGGCCGUCACGUUGAUAGCCAUCUUCGUUGUGUAUAUGGUGAGCAGGGACAAUGUGUCUUGCAGCAUUUGCUUGUAA 59 B_Vic_HA_IgE GAUCGGAUAUGUACGGGCAUCACUUCGAGCAACAGCCCCCAUGUGGUCAAAACUGCUACCCAGGGCGAGGUCAAUGUGACGGGGGUAAUCCCCCUCACAACGACUCCCACAAAGAGUCACUUUGCGAACCUGAAGGGUACGGAGACCCGUGGGAAGCUGUGUCCGAAGUGCCUGAACUGUACCGACCUGGACGUGGCGCUGGGACGUCCUAAG UGCACCGGCAAGAUUCCCAGCGCCCGGGUGAGCAUACUGCACGAGGUGAGACCUGUGACUAGCGGUUGCUUCCCGAUCAUGCAUGAUCGCACCAAGAUUCGGCAGUUACCCAAUCUCCUUCGCGGCUAUGAGCAUGUGAGGUUGUCGACCCAUAACGUUAUAAAUGCUGAAGAUGCGCCUGGCCGUCCCUAUAAAAUAGGGACCAGCGGUAGC UGCCCUAAUAUCACCAACGGUAACGGCUUUUUUGCGACUAUGGCUUGGGCGGGCCCAAGAAUAAGACCGCCACGAAUCCGCUCACGAUUGAGGUUCCUUAUAUUUGUACUGAGGGGGAAGACCAGAUAACCGUCUGGGGCUUCCACUCUGACUCCGAAACGCAGAUGGCGAAGCUGUACGGCGACAGUAAACCCCAGAAGUUUACGUCGUCU GCUAACGGUGUCACCACUCAUUACGUGAGUCAGAUUGGAGGCUUCCCCAAUCAGACGGAAGAUGGGGGCCUGCCUCAAUCGGGGGCGGAUUGUGGUGGACUAUAUGGUGCAGAAGUCCGGUAAGACCGGCACCAUAACCUAUCAGAGGGGCAUCUUGCUUCCUCAGAAGGUGUGGUGCGCUUCGGGGCGAAGCAAGGUGAUAAAGGGCAGCCUA CCGCUGAUUGGCGAGGCGGACUGCCUGCAUGAGAAGUACGGUGGGCUUAACAAGUCUAAGCCGUACUACACGGGCGAGCACGCGAAGGCGAUUGGUAACUGCCCGAUCUGGGUGAAAACCCCCCUUAAGCUGGCGAACGGCACGAAGUAUCGCCCCCCGGCGAAACUUCUGAAGGAGCGAGGCUUCUUCGGUGCCAUCGCCGGCUUCUUGGAG GGGGGUUGGGAGGGCAUGAUUGCUGGGUGGCAUGGCUACACUAGCCAUGGAGCCCAUGGCGUAGCCGUUGCCGCGGACCUGAAGUCCACGCAGGAGGCCAUCAACAAGAUUACUAAGAACCUCAACAGCUUAAGCGAGCUGGAGGUUAAGAAUCUUCAGAGGCUGUCUGGCGCUAUGGAUGAGUUACAUAACGAAAUUCUGGAGCUGGACGAG AAGGUCGACGACCUGAGGGCCGACACUAUCAGCUCCCAGAUAGAGCUCGCCGUAUUGUUGAGUAAUGAGGGCAUUAUCAACAGUGAGGACGAGCAUCUACUGGCCUUGGAGAGGAAGCUGAAGAAGAUGUUGGGGCCUUCGGCGGUCGAAAUCGGCAACGGCUGCUUCGAGACUAAGCAUAAAUGCAACCAGACGUGCUUGGAUCGAAUAGCC GCCGGUACCUUCGACGCCGGUGAGUUCAGCCUGCCGACGUUCGACAGCCUGAACAUCACGGCCGCCAGUCUCAAUGAUGAUGGCCUGGAUAACCAUACCAUCCUGCUGUACUACAGCACAGCCCGUCCAGUUUAGCCGUGACACUAAUGAUCGCAAUAUUCGUUGUGUACAUGGUUUCUAGGGAUAAUGUUAGUUGUAGCAUCUGUCUCUGA 60 B_Vic_HA_tPA GACCGAAUAUGCACUGGUAUAACGAGCAGCAAUAGCCCCCACGUGGUGAAGACCGCCACCCAGGGUGAGGUGAAUGUUACCGGCGUUAUCCCUCACCACUACCCCCACGAAGUCGCACUUUGCGAACCUCAAAGGCACCGAAACCCGGGGAAAGCUGUGUCCGAAAUGUCUCAACUGCACUGAUCUGGAUGUGGCCCUCGGACGCCCGAAG UGCACUGGGAAAAUUCCCAGUGCACGGGUGUCCAUACUGCACGAAGUGCGGCCUGUGACGAGCGGCUGCUUUCCGAUCAUGCAUGACCGUACGAAGAUACGGCAACUUCCCAAUUUGUUAAGGGGUUACGAGCAUGUCCGGUUGUCGACACAUAAUGUGAUCAACGCCGAGGACGCGCCUGGUAGGCCCUACAAAAUCGGCACAAGCGGCAGC UGCCCUAAUAUCACGAACGGCAAUGGCUUUUUUGCAACCAUGGCUUGGGCCGUGCCCAAGAACAAGACAGCCACGAAUCCGCUCACGAUUGAGGUUCCGUAUAUCUGUACUGAAGGGGAAGACCAGAUCACGGUCUGGGGCUUCCAUUCCGACAGCGAGACCCAGAUGGCUAAGCUGUACGGCGAUUCGAAGCCCCAGAAGUUCACGUCUAGU GCGAAUGGUGUCACCACGCACUACGUGAGCCAGAUCGGUGGUUUUCCCAAUCAGACAGAAGAUGGCGGAUUACCUCAGUCGGGGCGGAUCGGUUGACUACAUGGUGCAAAAGAGCGGUAAGACGGGUACCAUAACCUAUCAGAGAGGCAUCUUGCUUCCUCAGAAGGUUUGGUGCGCGUCUGGCCGUUCGAAGGUUAUUAAGGGCAGCCUG CCGUUGAUCGGCGAAGCCGAUUGCCUUCACGAGAAGUACGGCGGGCUGAACAAGAGUAAGCCGUACUACACGGGAGAGCAUGCCAAGGCGAUUGGCAACUGUCCGAUAUGGGUGAAGACCCCGCUGAAGUUGGCCAACGGUACCAAGUACCGUCCUCCCGGCCAAACUUCUGAAAGAGCGGGGCUUCUUUGGUGCCAUUGCAGGCUUCCUGGAG GGUGGGUGGGAGGGCAUGAUCGCCGGGGCACGGCUACACGAGCCAUGGAGCCCAUGGCGUGGCCGUGGCCGCCGACCUCAAGUCAACACAGGAGGCGAUUAACAAAAUUACCAAGAAUCUUAAUUCUUUGUCCGAACUAGAGGUGAAGAAUCUGCAGAGGUUAAGUGGCGCGAUGGAUGAGCUGCACAACGAGAUUCUCGAACUUGACGAG AAGGUGGAUGAUCUCCGCGCUGACACGAUAAGCAGUCAGAUCGAGCUGGCUGUCUUGUUGAGCAACGAGGGGAUCAUCAACUCGGAGGACGAGCACCUUCUCGCACUUGAGCGAAAGCUGAAGAAGAUGCUGGGUCCCAGCGCUGUCGAGAUCGGGAAUGGGUGCUUCGAGACCAAGCAUAAAGUGCAAUCAGACGUGUCUAGACCGGAUUGCG GCCGGCACCUUCGAUGCCGGCGAGUUCAGUUUACCCACGUUUGACUCAUUGAACAUCACCGCCGCUAGCCUCAAUGAUGACGGUCUUGACAACCAUACCAUAUUGCUAUAUUAUAGUACGGCCGCCAGUAGCCUGGCGGUUACUUUAAUGAUAGCGAUCUUUGUGGUGUACAUGGUUAGUCGGGACAACGUGUCAUGCAGCAUUUGCCUUUGA 61 A_H3N2_HA_WT CAGAAGAUCCCCGGUAACGACAACUCGACCGCUACUCUUUGUCUGGGCCAUCAUGCUGUGCCGAACGGCACGAUCGUGAAAACGAUCACCAACGAUCGCAUUGAGGUUACUAAUGCGACCGAAUUGGUUCAGAAUAGCAGCAUCGGUGAGAUUUGUGAUUCCCCCCACCAGAUCCUGGAUGGGGGCAAUUGCACUCUUAUUGAUGC UCUGUUGGGGGAUCCCCAGUGUGAUGGCUUCCAGAAUAAAGAGUGGGAUCUUUUCGUGGAGCGGAGCAGGGCUAAUAGCAACUGCUACCCCUACGACGUGCCCGACUACGCCAGCUUAAGGAGCUUGGUGGCUAGUUCGGGCACGUUGGAGUUCAAAAAUGAAUCCUUCAACUGGACUGGCGUGAAACAGAAUGGCACCAGUUCGGC CUGCAUAAGGGGCAGCAGUAGCUCGUUCUUUAGCAGGCUGAAUUGGUUGACGCAUCUGAAUUAUAAGUAUCCGGCGUUGAAUGUGACGAUGCCCAAUAAUGAGCAGUUUGAUAAGCUGUACAUAUGGGGCGUCCAUCACCCACGCACGGAUAAGGAUCAGAUCAGUCUGUUCGCCCAGCCCAGUGGGCGAAUAACCGUGUCCACUA AGAGGUCUCAGCAAGCCGUUAUUCCGAAUAUCGGCUCUAGGCCUCGAAUUAGGGACAUCCCGAGUAGAAUAUCUAUUUAUUGGACGAUCGUGAAGCCCGGUGACAUACUGUUGAUAAACAGCACCGGGAAUCUGAUCGCUCCGAGGGGGUACUUCAAGAUUAGGAGUGGCAAGAGCUCGAUUAUGAGGUCUGACGCCCCGAUAGGCA AGUGCAAGUCCGAGUGCAUUACUCCCAACGGCAGUAUCCCGAACGAUAAGCCCUUCCAGAAUGUGAACCGGAUAACUUAUGGCGCCUGCCCCCGGUAUGUUAAGCAGAGUACCCUCAAGCUGGCGACCGGCAUGAGGAACGUGCCUGAGAAACAGACCCGGGGCAUUUUCGGCGCCAUAGCCGGAUUCAUAGAGAAUGGUUGGGAG GGCAUGGUGGACGGCUGGUACGGCUUCAGGCACCAGAACUCGGAGGGCCGUGGGCAGGCGGCCGAUCUGAAGUCUACCCAGGCUGCCAUCGACCAGAUAAAUGGUAAGCUGAACCGUCUGAUCGGUAAGACUAACGAGAAGUUUCACCAGAUCGAGAAGGAGUUCAGCGAGGUGGAAGGCCGGGUGCAGGACCUGGAGAAGUACGUC GAAGACACCAAGAUCGAUUUGUGGAGCUACAAUGCCGAGCUGCUGGUCGCUCCGAAAACCAGCAUACCAUUGAUCUGACUGAUUCGGAGAUGAAUAAGCUGUUCGAAGACCAAGAAGCAGCUCAGGGAGAAUGCCGAGGCAUGGGCAACGGUUGUUUCAAGAUCUACCACAAGUGUGACAACGCUUGUAUCGGUAGUAUCCGA AACGAAACGUAUGAUCACAACGUAUACAGGGACGAGGCCCUGAAUAAUCGUUUUCAGAUCAAGGGCGUGGAGCUGAAAAGCGGCUACAAAGACUGGAUCCUGUGGAUCAGCUUUGCUAUGAGUUGCUUUUUGCUCUGCAUCGCCCUGUUAGGCUUUAUAAUGUGGGCCUGCCAGAAGGGCAAUAUUAGGUGCAACAUUUGCAUCUGA 62 A_H3N2_HA_IgE CAGAAGAUCCCUGGGAAUGAUAACAGCACCGCCACACUAUGUCUGGGCCAUCACGCUGUGCCGAACGGCACAAUUGUGAAGACGAUCACCAACGACAGGAUCGAAGUGACCAAUGCCACCGAGCUGGUGCAGAACAGCAGCAUAGGCGAUCUGCGAUUCCCCCCACCAGAUCCUGGAUGGGGGGAAUUGUACUUUGAUCGAUGC CUUGCUUGGUGACCCCCAGUGUGAUGGCUUCCAAAACAAGGAGUGGGACUUGUUCGUGGAGCGUAGCAGAGCUAAUAGUAAUUGCUACCCUUACGACGUGCCGGACUACGCUUCACUCAGGUCCUUGGUGGCGUCCUCGGGGACCCUCGAGUUCAAGAACGAGUCGUUCAAUUGGACCGGAGUCAAGCAGAACGGCACGAGCAGCGC UUGCAUCAGGGGCUCUAGUUCAUCGUUCUUCAGCCGGUUGAACUGGCUGACGCACCUGAAACUACAAGUAUCCCGCGUUGAACGUUACGAUGCCCAACGAACAGUUCGACAAGCUGUACAUAUGGGGGGUGCACCAUCCGAGGACCGACAAAGACCAGAUCUCGCUGUUCGCCCAGCCGAGUGGCAGGAUCACUGUGUCGACCA AGCGUAGCCAGCAGGCUGUGAUCCCAAAUAUUGGUUCUCGACCGCGGAUAAGGGAUAUCCCGUCGAGGAUUAGUAUUUAUUGGACCAUCGUGAAGCCCGGUGAUAUUCUGUUAAUAAACAGCACCGGGAAUCUGAUAGCCCCGCGGGGCUAUUUUAAGAUUCGCUCUGGUAAGAGUAGUAUCAUGAGGUCCGAUGCCCCAAUAGGCA AGUGUAAGAGCGAGUGCAUCACUCCCAAUGGUUCUAUUCCCAACGAUAAGCCCUUCCAGAACGUUAAUAGGAUCACCUAUGGUGCCUGUCCGCGCUACGUGAAGCAGAGUACUCUCAAGCUGGCAACUGGCAUGAGGAAUGUCCCCGAGAAGCAAACUCGGGGCAUCUUCGGUGCCAUUGCCGGCUUUAUCGAGAACGGCUGGGAG GGCAUGGUCGAUGGGUGGUACGGCUUCAGGCACCAGAACUCGGAAGGCCGCGGUCAAGCGGCCGACCUGAAGUCCACCCAAGCGGCCAUUGAUCAACAAUGGUAAGCUGAACCGCUUGAUUGGUAAGACUAACGAAAAGUUUCACCAAAUCGAGAAGGAGUUCAGCGAGGUGGAGGGCCGGGUUCAGGACCUGGAGAAGUACGUA GAAGAUACCAAGAUCGAUCUGUGGAGUUACAAUGCGGAGUUGUUGGUUGCACUGGAGAACCAGCAUACCAUUGAUCUGACCGACAGUGAGAUGAACAAGCUGUUUGAGAAAACUAAAAAGCAGCUGCGUGAGAACGCCGAAGACAUGGGCAACGGCUGCUUUAAGAUUUAUCAUAAGUGCGAUAAUGCAUGCAUAGGCAGCAUACGU AAUGAGACCUACGAUCAUAACGUGUACAGGGACGAGGCCCUGAACAAUAGGUUCCAAAUCAAAGGUGUGGAGCUUAAGAGCGGCUACAAAGACUGGAUUCUGUGGAUCAGUUUUGCGAUGAGCUGUUUUCUGCUAUGCAUUGCCUUCUGGGGUUCAUUAUGUGGGCCUGCCAGAAGGGCAAUAUAAGAUGUAAUAUCUGCAUUUGA 63 A_H3N2_HA_tPA CAGAAAAUUCCGGGCAACGACAAUAGCACCGCGACAUUGUGCCUGGGCCACCACGCGGUGCCCAACGGCACAAUCGUGAAAACCAUCACGAACGACAGGAUCGAGGUGACUAACGCCACUGAGCUGGUACAGAAUAGCAGCAUAGGAGAGAUCUGUGACAGCCCCCAUCAGAUUCUCGACGGGGGGAAUUGCACCCUGAUCGACGC GCUGCUGGGGGACCCCCAAUGCGACGGCUUCCAGAACAAGGAGUGGGAUCUGUUCGUGGAGCGUAGCAGGGCUAACAGCAACUGCUACCCCUACGACGUGCCGGAUUACGCUUCUCUGAGGAGCCUUGUUGCCAGUUCUGGUACGCUGGAGUUCAAGAAUGAGUCAUUCAAUUGGACUGGCGUGAAGCAGAACGGCACCAGCAGUGC UUGCAUCAGGGGGCAGUUCUUCCUCUUUCUUUAGCCGGUUGAACUGGCUGACGCAUGAAUUACAAGUACCCCGCGUUGAAUGUGACGAUGCCUAACAAUGAACAGUUUGACAAGCUGUACAUUUGGGGCGUUCACCAUCCACGCACCGAUAAGGAUCAGAUUAGCCUGUUCGCUCAGCCGAGCGGGCGGAUUACGGUUUCGACCA AGCGGUCGCAACAAGCCGUGAUCCCGAAUAUUGGCAGUCGUCCUAGGAUUCGCGAUAUCCCGAGUCGGAUAUCGAUUUACUGGACUAUCGUGAAACCUGGGGACAUACUGCUGAUCAAUUCGACGGGCAAUCUGAUCGCUCCUCGGGGGUACUUUAAGAUCAGGUCGGGCAAGAGUAGCAUCAUGAGGUCCGAUGCGCCGAUUGGCA AAUGCAAAAGCGAGUGCAUUACGCCGAAUGGCUCGAUCCCCAACGACAAGCCCUUCCAGAAUGUUAAUCGCAUUACCUACGGCGCGUGUCCUAGGUAUGUGAAACAAUCGACACUGAAGCUGGCGACUGGCAUGAGGAAUGUCCCGGAGAAACAGACUCGGGGCAUCUUCGGUGCCAUCGCCGGCUUCAUCGAGAACGGCUGGGAG GGCAUGGUGGAUGGCUGGUACGGCUUCAGGCAUCAGAACUCCGAGGGGAGGGGCCAGGCCGCUGACCUGAAGUCUACACAGGCCGCCAUCGACCAGAUCAAUGGCAAGCUGAACCGUCUUAUUGGUAAGACUAAUGAGAAGUUUCACCAGAUUGAGAAGGAGUUCUCAGAGGUGGAGGGUCGGGUCCAGGAUCUGGAGAAGUACGUU GAGGAUACGAAGAUCGACCUGUGGAGCUACAAUGCCGAGCUGCUCGUGGCUCUUGAAAACCAGCAUACUAUUGAUCUUACUGACAGUGAGAUGAAUAAGCUGUUCGAGAAGACCAAGAAGCAGCUCAGAGAGAACGCUGAGGAUAUGGGCAACGGCUGUUUCAAGAUCUAUCACAAGUGUGACAACGCUUGUAUCGGCAGCAUAAGG AACGAGACCUACGAUCACAACGUGUAUAGGGACGAAGCUCUGAACAAUAGGUUCCAGAUCAAGGGUGUGGAGCUUAAGAGCGGUUAUAAGGAUUGGAUCCUGGAUCUCGUUUGCUAUGAGCUGCUUUUUGUUGUGCAUCGCCCUUCUUGGCUUCAUAAUGUGGGCCUGCCAGAAGGGUAACAUAAGAUGCAACAUAUGUAUCUGA 64 A_H1N1_HA_WT GAUACACUCUGCAUUGGCUAUCACGCCAACAAUUCUACCGACACGGUUGACACCGUGUUGGAGAAGAACGUGACGGUGACGCACAGCGUGAACCUACUAGAGGAUAAGCACAAUGGCAAGCUGUGCAAACUGCGGGGCGUGGCCCCACUACACCUGGGCAAGUGCAAUAUCGCUGGCUGGAUUCUGGGUAACCCGAAUGUGAGU CACUGAGCACCGCUCGGUCCUGGAGUUACAUCGUAGAAACCAGCAACAGCGAUAAUGGCACCUGCUACCCCGGCGACUUCAUAAAUUAUGAGGAGCUGAGGGAGCAGUUGUCCAGCGUGAGUAGCUUUGAGAGAUUUGAGAUCUUUCCAAAGACCAGCAGCUGGCCCAACCAUGACAGCGAUAACGGCGUGACGGCUGCAUGCCCC CACGCCGGCGCCAAGUCCUUUUUAAAGAAUCUUAUUUGGUUGGUCAAAAAGGGUAAGUCUUACCCUAAGAUCAAUCAGACCUAUAUCAAUGAUAAGGGUAAGGAGGUGCUGGUGCUGGGGCAUUCACCAUCCGCCGACUAUCGCUGACCAGCAGUCAUUGUACCAGAACGAGGACGCGUACGUUUUCGUGGGCACUAGCCGGUA UUCGAAGAAGUUCAAGCCGGAGAUUGCGACGCGGCCAAAGGUCCGCGAUCGCGAGGGUCGGAUGAACUACUAUUGGACGCUGGUUGAGCCCGGUGAUAAGAUCACUUUUGAGGCCACCGGGAACCUGGUGGCCCCCAGGUAUGCUUUUACCAUGGAGCGGGACGCUGGGUCCGGUAUUAUCAUUUCGGAUACACCGGUGCACGACU GUAACACUACAUGUCAGACACCUGAGGGGGCAAUUAAUACUAGCUUGCCCUUUCAGAACGUGCACCCGAUCACGAUUGGCAAGUGCCCUAAAUACGUGAAGUCGACAAAAUUGAGGCUGGCUACUGGCCUGAGGAACGUUCCCUCGAUCCAGUCGAGGGGCCUCUUUGGAGCCAUAGCCGGCUUCAUCGAAGGCGGGUGGACCGGG AUGGUGGAUGGCUGGUACGGCUACCACCACCAUCAGAACGAGCAGGGUAGUGGCUACGCUGCCGAUCUGAAGUCCACCCAGAAUGCCAUCGAUAAGAUCACCAACAAGGUGAACUCGGUGAUUGAAAAGAUGAACACCCAGUUCACCGCUGUAGGUAAGGAGUUCAAUCACCUGGAGAAGAGGAUCGAAAACCUGAAUAAGAAAGUCGA CGACGGCUUCUUAGACAUUUGGACCUACAAUGCUGAGCUGCUGGUGCUGCUGGAGAACGAGCGAACGCUGGAUUAUCAUGAUUCUAAUGUGAAGAACCUUUACGAGAAGGUGCGAAAUCAGCUUAAAAACAACGCCAAGGAGAUCGGUAAUGGGUGCUUUGAGUUCUACCAUAAGUGUGACAAUACUUGCAUGGAGAGCGUCAAGA ACGGCACCUAUGACUAUCCCAAGUACAGCGAGGAAGCCAAGUUGAACAGGGAGAAGAUCGACGGCGUUAAGCUGGAUUCCACCAGGAUCUACCAGAUCCUGGCCAUUUACAGCACCGUGGCUUCGUCAUUGGGCUGGUGGUGAGCCUCGGCGCGAUCAGCUUCUGGAUGUGCCUCGAAUGGUAGUCUGCAGUGCAGAAUUUGCAUU 65 A_H1N1_HA_IgE GACACCUUGUGUAUAGGAUACCACGCCAACAAUAGCACCGACACUGUAGACACGGUGUUGGAGAAGAACGUAACCGUUACACACAGUGUGAAUUUGCUCGAGGACAAACACAACGGCAAAUUAUGUAAGCUGAGGGGGGUGGCACCCCUUCACCUUGGGAAAUGUAAUAUUGCCGGCUGGAUUUUAGGGAAUCCAGAGUGUGAGUC CUUGAGCACCGCUCGUAGCUGGUCUUAUAUUGUUGAGACCAGCACAGCGAUAACGGUACGUGCUAUCCUGGCGAUUUUAUAAACUAUGAGGAGCUGAGAGAGCAGUUGUCGUCGGUGUCGAGUUUCGAACGUUUCGAAAUUUUUCCAAAGACCAGCAGCUGGCCAAAUCAUGAUUCAGACAACGGCGUGACCGCUGCCUGCCCC ACGCCGGGGCCAAGUCGUUCUACAAGAACUUGAUAUGGCUGGUGAAGAAGGGUAAGAGUUACCCUAAGAUUAACCAGACUUAUAUCAACGAUAAAGGGAAGGAGGUGCUGGUGCUGUGGGGCAUCCACCAUCCUCCUACUAUCGCGGAUCAGCAGUCUCUGUACCAGAAUGAGGAUGCCUACGUAUUUGUGGGCACUUCUCGGUAC AGUAAGAAGUUCAAACCUGAGAUCGCCACUAGGCCGAAAGUGCGCGAUCGGGAGGGCAGGAAUGAAUUAUAUUGGACGCUGGUGGAGCCCGGCGACAAGAUUACCUUUGAGGCCACCGGUAACCUGGGGCCCCGAGGUACGCUUUCACCAUGGAACGCGACGCCGGGAGUGGCAUCAUCAUUAGCGACACGCCGGUGCAUGACUGC AACACCACCUGUCAGACACCUGAGGGUGCCAUUAAUACAUCCCUGCCCUUCCAGAAUGUGCAUCCGAUAACGAUUGGGAAGUGUCCUAAAUACGUUAAGAGCACGAAGCUGAGACUGGCCACCGGCUUGAGGAAUGUUCCGAGCAUUCAGUCGAGGGGGCUGUUUUGGUGCCAUCGCCGGCUUUAUCGAGGGCGGCUGGACUGGCAU GGUGGAUGGAUGGUACGGCUACCACCACCAGAACGAACAAGGCAGCGGUUAUGCCGCUGAUCUGAAGUCCACUCAGAACGCGAUCGAUAGAUCACGAACAAAGUGAACAGCGUGAUUGAGAAGAUGAAUACCCAGUUCACUGCGGUGGGCAAGGAGUUCAAUCAUCUCGAGAAGAGGAUCGAGAAUCUUAACAAGAAAGUCGACG ACGGCUUUUUGGAUAUCUGGACCUACAACGCCGAGCUGCUGGUCUUGUUGGAGAAUGAGAGAACUCUCGACUACCAUGACAGCAACGUGAAGAACCUCUACGAGAAGGUGAGAAACCAGCUGAAAAAUAACGCCAAGGAGAUCGGUAACGGCUGUUUCGAGUUCUACCACAAGUGUGAUAAUACUUGCAUGGAGAGCGUGAAGAAU GGCACGUAUGAUUAUCCUAAGUAUAGCGAAGAGGCUAAACUUAAUCGAGAGAAAAUCGAUGGCGUUAAGUUGGACAGCACCAGGAUCUACCAGAUCUUGGCCAUCUAUAGCACGGUGGCUAGCAGCCUUGUGCUGGUGGUGAGCCUUGGGGCAAUAUCUUUCUGGAUGUGUUCCAACGGCUCGCUGCAAUGUAGGAUCUGCAUAUAA 66 A_H1N1_HA_tPA GACACUCUGUGUAUUCGGCUACCAUGCCAACAAUAGCACCGACACCGUGGACACCGUCUUGGAGAAGAACGUCACCGUGACGCACUCGGUAAACCUUGGAGGACAAACACAAUGGGAAGCUGUGCAAGUUGCGCGGCGUUGCCCCGCUGCACCUGGGCAAGUGCAAUAUCGCUGGCUGGAUCCUGGGAAAUCCCGAGUGUGAGAG CUUGUCUACGGCUCGGUCGUGGUCUUACAUUGUGGAGACCAGCAACAGCGAUAACGGUACUUGCUACCCGGGGACUUUAUAAAUUAUGAGGAGCUGCGGGAGCAACUUUCCAGUGUGUCGUCCUUCGAGAGGUUCGAGAUCUUCCCAAAGACGUCGAGUUGGCCCAACCAUGACUCCGACAACGGUGUGACCGCAGCUUGUCCUC AUGCCGGCGCGAAGAGCUUCUACAAGAACCUCAUUUGGCUGGUAAAGAAGGGCAAGUCCUACCCCAAGAUUAAUCAGACAUAUAUUAAUGACAAGGGGAAGGAGGUGCUGGUGCUGUGGGGCAUUCACCACCCGCCUACCAUAGCCGACCAGCAGUCUCUGUACCAGAAUGAGGAUGCCUACGUGUUCGUGGGCACCUCACGGUAC AGCAAGAAAUUCAAGCCCGAGAUAGCCACGAGGCCCAAGGUGAGGGACAGGGAGGGCAGAAUGAACUAUUACUGGACAUUGGUGGAGCCCGGGGACAAGAUUACGUUCGAGGCCACCGGCAACCUGGGGCCCCGCGUUACGCCUUCACGAUGGAGCGUGAUGCGGGCAGCGGCAUUAUUAUAAGUGAUACGCCGGUGCACGACUGU AAUACCACUUGUCAGACACCUGAAGGUGCCAUCAAUACCAGCCUGCCCUUCCAGAAUGUCCACCCCAUCACCAUAGGCAAGUGCCCUAAGUAUGUGAAGAGUACAAAGCUGAGGCUGGCAACCGGCCUGAGGAAUGUGCCUUCCAUACAGAGUCGCGGCCUGUUUGGGCCCAUUGCUGGCUUUAUCGAGGGCGGCUGGACCGGCAU GGUGGAUGGGUGGUACGGCUACCAUCAUCAAAAUGAGCAGGGCAGCGGCUAUGCAGCCGAUCUGAAGAGCACCCAGAACGCGAUAGACAAGAUCACCAAUAAGGUGAACUGUCAUCGAGAAGAUGAACACCCAGUUCACCGCCGUGGGUAAAGAGUUCAAUCAUCUAGAGAAGCGCAUUGAAAACCUUAACAAAAAGGUCGAUG ACGGCUUCCUGGAUAUCUGGACCUACAAUGCCGAACUGCUGGUACUGUUGGAGAACGAACGUACUCUGGAUUACCACGAUAGUAACGUGAAGAAUCUCUACGAGAAGGUGCGAAACCAGCUUAAGAAUAACGCCAAGGAAAUCGGUAUGGUUGUUUCGAGUUCUACCAUAAGUGCGACAACACGUGCAUGGAGAGCGUGAAGAAC GGCACAUACGAUUAUCCGAAGUAUAGCGAAGAGGCUAAGCUGAAUCGGGAGAAGAUCGAUGGCGUAAAGCUGGAUUCGACCAGGAUCUAUCAGAUCCUGGCCAUCUACUGGCAGUGGCUAGCAGUUUGGUGUUGGUGGUGAGCCUGGGUGCCAUCUCUUUCUGGAUGUGCUCUAACGGGUCCUUGCAGUGCAGGAUCUGUAUCUAG 67 B_Ya_HA_WT_C GAUCGAAUCUGCACUGGGAUAACAUCUUCAAACUCACCUCAUGUGGUCAAAACAGCUACUCAAGGGGAGGUCAAUGUGACUGGCGUGAUACCACUGACAACAACACCAACAAAAUCUUAUUUUGCAAAUCUCAAAGGAACAAGGACCAGAGGGAAACUAUGCCCGGACUGUCUCAACUGUACAGAUCUGGAUGUGGCCUUGGGCAGGCCAAUG UGUGUGGGGACCACACCUUCUGCUAAAGCUUCAAUACUCCAUGAGGUCAGACCUGUUACAUCCGGGUGCUUUCCUAUAAUGCACGACAGAACAAAAAUCAGGCAACUACCCAAUCUUCUCAGAGGAUAUGAAAAGAUCAGGUUAUCAACCCAAAACGUUAUCGAUGCAGAAAAAGCACCAGGAGGACCCUACAGACUUGGAACCUCAGGAUCUU GCCCUAACGCUACCAGUAAAAUCGGAUUUUUUGCAACAAUGGCUUGGGCUGUCCCAAAGGACAACUACAAAAAUGCAACGAACCCACUAACAGUGGAAGUACCAUACAUUUGUACAGAAGGGGAAGACCAAAUUACUGUUUGGGGGUUCCAUUCGGAUGACAAAACCCAAAUGAAGAGCCUCUAUGGAGACUCAAAUCCUCAAAAGUUCACCUC AUCUGCUAAUGGAGUAACCACGCAUUAUGUUUCUCAGAUUGGCGACUUCCCAGAUCAAACAGAAGACGGAGGACUACCACAAAGCGGCAGAAUUGUUGAUUACAUGAUGCAAAAACCUGGGAAAACAGGAACAAUUGUCUAUCAAAGGGGUGUUUUGUUGCCUCAAAAGGUGUGGUGCGCGAGUGGCAGGAGCAAAGUAAUAAAAGGGUCA UUGCCUUUAAUUGGUGAAGCAGAUUGCCUUCAUGAAGAAUACGGUGGAUUAAACAAAAGCAAGCCUUACUACACAGGAAAACAUGCAAAAGCCAUAGGAAAUUGCCCAAUAUGGGUAAAAACACCUUUGAAGCUUGCCAAUGGAACCAAAUAUAGACCUCCUGCAAAACUAUUGAAGGAAAGGGGUUUCUUCGGAGCUAUUGCUGGUUUCCUA GAAGGAGGAUGGGAAGGAAUGAUUGCAGGUUGGCACGGAUACACAUCUCACGGAGCACAUGGAGUGGCAGUGGCGGCAGACCUUAAGAGUACACAAGAAGCUAUAAAUAAGAUAACAAAAAAUCUCAAUUCUUUGAGUGAACUAGAAGUAAAGAACCUUCAAAGACUAAGUGGUGCCAUGGAUGAACUCCACAACGAAAUACUCGAGCUGGAUG AAAAGUGGAUGAUCUCAGAGCUGACACUAUAAGCUCACAAAUAGAACUUGCAGUCUUGCUUUCCAACGAAGGAAUAAUAAACAGUGAAGACGAGCAUCUAUUGGCACUUGAGAGAAAACUAAAGAAAAUGCUGGGUCCCUCUGCUGUAGACAUAGGAAACGGAUGCUUCGAAACCAAACACAAGCAACCAGACCUGCUUAGACAGGAUAGC UGCUGGCACCUUUAAUGCAGGAGAAUUUUCUCCCCACUUUUGAUUCAUUGAACAUUACUGCUGCAUCUUUUAAAUGAUGAUGGAUUGGAUAACCAUACUAUACUGCUCUAUUACUCAACUGCUGCUUCUAGUUUGGCUGUAACAUUAAUGCUAGCUAUUUUUAUUGUUUAUAUGGUCUCCAGAGACAACGUUUCAUGCUCCAUCUGUCUAUAA 68 B_Vic_HA_WT_C GAUCGAAUCUGCACUGGGAUAACAUCGUCAAACUCACCACAUGUCGUCAAAACUGCUACUCAAGGGGAGGUCAACGUGACCGGUGUAAUACCACUGACAACAACACCCACCAAAUCUCAUUUUGCAAAUCUCAAAGGAACAGAAACCAGGGGGAAACUAUGCCCAAAAUGCCUCAACUGCACAGAUCUGGAUGUAGCCUUGGGCAGACCAAAA UGCACAGGGAAAAUACCCUCUGCAAGGGUUUCAAUACUCCAUGAAGUCAGACCUGUUACAUCUGGGUGCUUUCCUAUAAUGCACGAUAGAACAAAAAUUAGACAGCUGCCUAACCUUCUCCGAGGAUACGAACAUGUCAGGUUAUCAACUCACAACGUUAUCAAUGCAGAAGAUGCACCAGGAAGACCCUACAAAAUUGGAACCUCAGGGUCU UGCCCUAACAUUACCAAUGGAAACGGAUUCUUCGCAACAAUGGCUUGGGCCGUCCCAAAAAACAAAACAGCAACAAAUCCAUUAACAAUAGAAGUACCAUACAUUUGUACAGAAGGAGAAGACCAAAUUACCGUUUGGGGGUUCCACUGACAGCGAGACCCAAAUGGCAAAGCUCUAUGGGGACUCAAAGCCCCAGAAGUUCACCUCAUCU GCCAACGGAGUGACCACACAUUACGUUUCACAGAUUGGUGGCUUCCCAAAUCAAACAGAAGACGGAGGACUACCACAAAGUGGCAGAAUUGUUGUUGAUUACAUGGUGCAGAAAUCUGGAAAAACAGGAACAAUUACCUAUCAAAGAGGUAUUUUAUUGCCUCAAAAGGUGUGGUGCGCAAGUGGCAGGAGCAAGGUAAUAAAAGGAUCCUUG CCCUUAAUUGGAGAAGCAGAUUGCCUCCAUGAAAAAAUACGGUGGAUUAAACAAAAGCAAGCCUUACUACACAGGGGAACAUGCAAAGGCCAUAGGAAAUUGCCCAAUAUGGGUGAAAACACCCUUGAAGCUGGCCAAUGGAACCAAAUAUAGACCCCCUGCAAAACUAUUAAAGGAAAGAGGUUUCUUCGGAGCCAUUGCUGGUUUCUUAGAG GGAGGAUGGGAAGGAAUGAUUGCAGGUUGGCACGGAUACACAUCCCAUGGGGCACAUGGAGUAGCGGUGGCAGCUGACCUUAAGAGCACUCAAGAGGCCAUAAACAAGAUAACAAAAAAUCUCAACUCUUUGAGUGAGCUGGAAGUAAAGAAUCUUCAAAGACUAAGCGGUGCCAUGGAUGAACUCCACAACGAAAUACUAGAACUAGAUGAG AAAGUGGAUGAUCUCAGAGCUGAUACAAUAAGCUCACAAAUAGAACUCGCAGUCCUGCUUUCCAAUGAAGGAAUAAUAAACAGUGAAGAUGAACAUCUUGGCGCUUGAAAGAAAGCUGAAGAAAAUGCUGGGCCCCUCUGCUGUAGAGAUAGGGAAUGGAUGCUUUGAAACCAAACACAAGUGCAACCAGACCUGUCUCGACAGAAUAGCU GCUGGUACCUUUGAUGCAGGAGAGAAUUUUCUCCCCACCUUUGAUUCACUGAAUAUUACUGCUGCAUCUUUAAAUGACGACGGAUUGGACAAUCAUACUAUACUGCUUUACUACUCAACUGCUGCCUCCAGUUUGGCUGUAACACUGAUGAUAGCUAUCUUUGUUGUUUAUAUGGUCUCCAGAGACAAUGUUUCUUGCUCCAUUUGUCUAUGA 69 A_H3N2_HA_WT_C CAAAAAAUCCCUGGAAAUGACAAUAGCACGGCAACGCUGUGCCUUGGGCACCAUGCAGUACCAAACGGAACGAUAGUGAAAACAAUCACAAAUGACCGAAUUGAAGUUACUAAUGCUACUGAGUUGGUUCAGAAUUCCUCAAUAGGUGAAAUAUGCGACAGUCCUCAUCAGAUCCUUGAUGGAGGGAACUGCACACUAAUAGAUGC UCUAUUGGGGGACCCUCAGUGUGACGGCUUUCAAAAUAAGGAAUGGGACCUUUUGUUGAACGAAGCAGAGCCAACAGCAACUGUUACCCUUAUGAUGUGCCGGAUUAUGCCUCCCUUAGGUCACUAGUUGCCUCAUCCGGCACACUGGAGUUUAAAAAUGAAAGCUUCAAUUGGACUGGAGUCAAACAAAACGGAACAAGUUCUGC GUGCAUAAGGGGAUCUAGUAGUAGUUUUUUUAGUAGAUUAAAUUGGUUGACCCACUUAAACUACAAAUAUCCAGCACUGAACGUGACUAUGCCAAACAAUGAACAAUUUGACAAAUUGUACAUUUGGGGGGUUCACCACCCGAGAACGGACAAGGACCAAAUCUCCCUGUUUGCUCAACCAUCAGGAAGAAUCACAGUAUCUACCA AAAGAAGCCAACAAGCUGUAAUCCCAAAUAUAGGAUCUAGACCCAGAAUAAGGGAUAUCCCUAGCAGAAUAAGCAUCUAUUGGACAAUAGUAAAACCGGGAGACAUACUUUUGAUUAACAGCACAGGGAAUCUAAUUGCUCCUAGGGGUUACUUCAAAAUACGAAGUGGGAAAAGCUCAAUAAUGAGAUCAGAUGCACCCAUUGGCA AAUGCAAGUCUGAAUGCAUCACUCCAAAUGGAAGCAUUCCCAAUGACAAACCGUUCCAAAAUGUAAACAGGAUCACAUACGGGGCCUGUCCCAGAUAUGUCAAGCAAAGCACCCUGAAAUUGGCAACAGGAAUGCGAAAUGUACCAGAGAAACAAACCAGAGGCAUAUUUGGCGCAAUAGCGGGUUUCAUAGAAAAUGGAUGGGAG GGAAUGGUGGAUGGUUGGUACGGUUUCAGGCAUCAAAAUUCUGAGGGAAGAGGACAAGCAGCAGAUCUCAAAAGCACUCAAGCAGCAAUCGAUCAAAUCAAUGGGAAGCUGAAUCGAUUGAUCGGAAAAACCAACGAGAAAUUCCAUCAGAUUGAAAAAGAAUUCUCAGAAGUAGAAGGAAGAGUUCAAGACCUUGAGAAAUAUGUU GAGGACACUAAAAUAGAUCUCUGGUCAUACAACGCUGAGCUUCUUGUUGCCCUGGAGAACCAACAUACAAUUGACCUAACUGACUCAGAAAUGAACAAACUGUUUGAAAAAACAAAGAAGCAACUGAGGGAAAAUGCUGAGGAUAUGGGAAAUGGUUGUUUCAAAAUAUACCACAAAUGUGACAAUGCCUGCAUAGGAUCAAUAAGA AAUGAAACUUAUGACCACAAUGUGUACAGGGAUGAAGCAUUAAACAACCGGUUCCAGAUCAAGGGAGUUGAGCUGAAGUCAGGGUACAAAGAUUGGAUCCUAUGGAUUUCCUUUGCCAUGUCAUGUUUUUGCUUUGUAUUGCUUUGUUGGGGUUCAUCAUGUGGGCCUGCCAAAAGGGCAACAUUAGAUGCAACAUUUGCAUUUGA 70 A_H1N1_HA_WT_C GACACAUUAUGUAUAGGUUAUCAUGCGAACAAUUCAACAGACACUGUGGACACAGUACUAGAAAAGAAUGUAACAGUAACACACUGUCAAUCUUGGAAGACAAGCAUAACGGAAAACUAUGCAAACUAAGAGGGGUAGCCCCAUUGCAUUUGGGUAAAUGUAACAUUGCUGGCUGGAUCCUGGGAAAUCCAGAGUGUGAAUC ACUCUCCACAGCAAGAUCAUGGUCCUACAUUGUGGAAACAUCUAAUUCAGACAAUGGAACGUGUUACCCAGGAGAUUUCAUCAAUUAUGAGGAGCUAAGAGAGCAAUUGAGCUCAGUGUCAUCAUUUGAAAGGUUUGAAAUAUUCCCCAAGACAAGUUCAUGGCCUAAUCAUGACUCGGACAAUGGUGUAACGGCAGCAUGUCCUC ACGCUGGAGCAAAAAGCUUCUACAAAAACUUGAUAUGGCCGGUUAAAAAAGGAAAAUCAUACCCAAAGAUCAACCAAACCUACAUUAAUGAUAAAGGGAAAGAAGUCCUCGUGCUGGGGGCAUUCACCAUCCACCUACUAUUGCCGACCAACAAAGUCUCUAUCAGAAUGAAGAUGCAUAUGUUUUUGUGGGGACAUCAAGAUAC AGCAAGAAGUUCAAGCCGGAAAUAGCAAGACCCAAAGUGAGGGAUCGAGAAGGGAGAAUGAACUAUUACUGGACACUAGUAGAACCGGGAGACAAAAUAACAUUCGAAGCAACUGGUAAUCUAGUGGCACCGAGAUAUGCAUUCACAAUGGAAAGAGAUGCUGGAUCUGGUAUUAUCAUUUCAGAUACACCAGUCCACGAUUGC AAUACAACUUGUCAGACACCCGAGGGUGCUAUAAACACCAGCCUCCCAUUUCAGAAUGUACAUCCGAUCACAAUUGGGAAAUGUCCAAAGUAUGUAAAAAGCACAAAAUUGAGACUGGCCACAGGAUUGAGGAAUGUCCCGUCUAUUCAAUCUAGAGGCCUAUUCGGGGCCAUUGCUGGCUUCAUCGAAGGGGGGUGGACAGGGAU GGUAGAUGGAUGGUACGGUUAUCACCAUCAAAAUGAGCAGGGGUCAGGAUAUGCAGCCGAUCUGAAGAGCACACAAAAUGCCAUUGAUAAGAUUACUAACAAAGUAAAUUCUGUUAUUGAAAAGAUGAAUACACAGUUCACAGCAGUUGGUAAAGAGUUCAACCACCUUGAAAAAAGAAUAGAGAAUCUAAAUAAAAAGGUUGAUG AUGGUUUCCUGGACAUUUGGACUUACAAUGCCGAACUGUUGGUUCUACUGGAAAACGAAAGAACUUUGGACUAUCACGAUUCAAAUGUGAAGAACUUGUAUGAAAAAGUAAGAAAACCAGUUAAAAAACAAUGCCAAGGAAAUUGGAAACGGCUGCUUUGAAUUUUACCACAAAUGCGACAACACAUGCAUGGAAAGUGUCAAGAAU GGGACUUAUGACUACCCAAAAUACUCAGAGGAAGCAAAAUUAAACAGAGAAAAAAUAGAUGGAGUAAAGCUGGACUCAACAAGGAUCUACCAGAUUUUGGCGAUCUAUUCAACUGUUGCCAGUUCAUUGGUACUGGUAGUCUCCCUGGGGGCAAUCAGCUUCUGGAUGUGCUCUAAUGGGUCUCUACAGUGUAGAAUAUGUAUUUAA 71 3'-UTR UAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

Information on the mRNA generated for the in vivo protein expression assays is shown in Tables 4 to 7 below.

[Table 4] Influenza B Yamagata strain Name 5'-UTR Sequence ID NO. Signal peptide sequence ID NO. ORF sequence ID NO. 3'-UTR Sequence ID NO. Poly(A) tail GC00_WT_HA 1 39 55 71 120 bp GC01_WT_HA 2 39 55 71 120 bp GC14_WT_HA 15 39 55 71 120 bp GC27_WT_HA 28 39 55 71 120 bp GC29_WT_HA 30 39 55 71 120 bp GC00_IgE_HA 1 40 56 71 120 bp GC01_IgE_HA 2 40 56 71 120 bp GC14_IgE_HA 15 40 56 71 120 bp GC27_IgE_HA 28 40 56 71 120 bp GC29_IgE_HA 30 40 56 71 120 bp GC00_tPA_HA 1 41 57 71 120 bp GC01_tPA_HA 2 41 57 71 120 bp GC14_tPA_HA 15 41 57 71 120 bp GC27_tPA_HA 28 41 57 71 120 bp GC29_tPA_HA 30 41 57 71 120 bp Control group 35 51 67 71 120 bp

[Table 5] Type B influenza strain Victoria Name 5'-UTR Sequence ID NO. Signal peptide sequence ID NO. ORF sequence ID NO. 3'-UTR Sequence ID NO. Poly(A) tail GC00_WT_HA 1 42 58 71 120 bp GC01_WT_HA 2 42 58 71 120 bp GC14_WT_HA 15 42 58 71 120 bp GC27_WT_HA 28 42 58 71 120 bp GC29_WT_HA 30 42 58 71 120 bp GC00_IgE_HA 1 43 59 71 120 bp GC01_IgE_HA 2 43 59 71 120 bp GC14_IgE_HA 15 43 59 71 120 bp GC27_IgE_HA 28 43 59 71 120 bp GC29_IgE_HA 30 43 59 71 120 bp GC00_tPA_HA 1 44 60 71 120 bp GC14_tPA_HA 15 44 60 71 120 bp GC27_tPA_HA 28 44 60 71 120 bp Control group 36 52 68 71 120 bp

[Table 6] Influenza A (H3N2) strains Name 5'-UTR Sequence ID NO. Signal peptide sequence ID NO. ORF sequence ID NO. 3'-UTR Sequence ID NO. Poly(A) tail GC00_WT_HA 1 45 61 71 120 bp GC01_WT_HA 2 45 61 71 120 bp GC14_WT_HA 15 45 61 71 120 bp GC27_WT_HA 28 45 61 71 120 bp GC29_WT_HA 30 45 61 71 120 bp GC00_IgE_HA 1 46 62 71 120 bp GC14_IgE_HA 15 46 62 71 120 bp GC27_IgE_HA 28 46 62 71 120 bp GC29_IgE_HA 30 46 62 71 120 bp GC00_tPA_HA 1 47 63 71 120 bp GC14_tPA_HA 15 47 63 71 120 bp GC27_tPA_HA 28 47 63 71 120 bp Control group 37 53 69 71 120 bp

[Table 7] Influenza A (H1N1) strains Name 5'-UTR Sequence ID NO. Signal peptide sequence ID NO. ORF sequence ID NO. 3'-UTR Sequence ID NO. Poly(A) tail GC00_WT_HA 1 48 64 71 120 bp GC01_WT_HA 2 48 64 71 120 bp GC14_WT_HA 15 48 64 71 120 bp GC27_WT_HA 28 48 64 71 120 bp GC29_WT_HA 30 48 64 71 120 bp GC01_IgE_HA 2 49 65 71 120 bp GC14_IgE_HA 15 49 65 71 120 bp GC29_IgE_HA 30 49 65 71 120 bp GC00_tPA_HA 1 50 66 71 120 bp GC01_tPA_HA 2 50 66 71 120 bp GC14_tPA_HA 15 50 66 71 120 bp GC27_tPA_HA 28 50 66 71 120 bp GC29_tPA_HA 30 50 66 71 120 bp Control group 38 54 70 71 120 bp

1-2. mRNA Synthesis and purification

The mRNA constructs of Tables 4 to 7 prepared in Example 1.1 according to the method described in PCT/KR2022/019491 were purified by in vitro transcription.

Specifically, IVT was performed under the conditions in Table 8, and then after the reaction was completed, the template DNA was removed by treating 1 U of DNase I per 1 ug of DNA and reacting at 37°C for 15-30 min, and the IVT product was purified using Invitrogen's MEGAclear™ kit (Austin, TX) according to the manufacturer's method.

[Table 8] Element Volume ATP, CTP, GTP, N1-methyl pseudoUTP (final 10 mm) 20μL each RNase inhibitors 5μL Pyrophosphatase, inorganic (yeast) 0.5μL 100mm Clean Cap AG (3' OMe) 6μL Template DNA 6 μg 10X T7 polymerase reaction buffer 20μL T7 RNA polymerase 11.8μL Nuclease-free water Up to 200μL Total 200μL

Embodiment 2. mRNA-LNP Preparation of the complex

Ionic lipids, phospholipids, cholesterol, and PEG-lipid conjugates were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 and mixed with citrate buffer (pH 4, 50 mm) in which mRNA was dissolved at a volume ratio of 1:3. MC3 (MedChemExpress, USA) was used as ionic lipids, DSPC (Avanti Polar Lipids, USA) was used as phospholipids, cholesterol (Sigma Aldrich, USA) was used as cholesterol, and 1-2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) (Avanti Polar Lipids, USA) was used as PEG-lipid conjugates.

To generate lipid nanoparticles (LNPs), ionic lipids and mRNA were mixed using NanoAssemblr IgniteTM (Precision Nanosystems, Inc., Canada) at a total flow rate (TFR) of 12 mL/min to achieve a nitrogen-to-phosphorus ratio (N/P ratio) of 4 between ionic lipids and mRNA. The prepared LNPs were prepared by ethanol removal, buffer exchange and concentration using an Amicon ultracentrifugation screening program with MWCO 10 kDa (Millipore, USA), and dilution, concentration and exchange using 1X DPBS (Thermo Scientific, USA). For cryopreservation, a final 300 mM sucrose solution was added as a cryoprotectant and stored frozen (-80°C).

Embodiment 3. Animal experiments mRNA Confirmation of structure performance

3-1. will mRNA The constructs were injected into animals and the tissues were harvested for HA Ways of expression

Balb/c female mice aged 6-7 weeks were purchased (OrientBio, Korea) and maintained under specific pathogen-free conditions. The hair of the right leg of the mice was shaved one day before injection, and 50 μL of the mRNA-LNP complex prepared in Example 2 was injected intramuscularly into the right tibia using an insulin syringe. Six hours after the injection, the injected right tibia was harvested, collected in a 1.7 mL test tube, and stored in an ultra-low temperature freezer at -70°C until the expression of HA protein in the muscle lysate was evaluated (Figure 2).

3-2. Method for obtaining muscle tissue lysate

Preparation of muscle lysate to detect HA protein in injected muscle. To obtain muscle lysate, the harvested muscle tissue was placed in a tube containing tissue lysis buffer (tissue lysis buffer composition: 1x cell lysis buffer (Cell signaling technology, Cat No. #9803), protease and phosphatase inhibitor micro tablets, EDTA-free 1 tablet/10 mL, ThermoFisher scientific, Cat No. A32961) and stainless steel beads, and then each tissue was lysed using a tissue lyser.

The tube containing the lysed tissue was centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a new 1.7 mL tube and used for HA protein expression.

3-3. Muscle lysate HA Protein measurement

Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate HA protein expression in muscle. ELISA was performed using an ELISA kit, and capture antibodies from Sinobiological (Yamagata: Cat No. 11053-MM09, Victoria: Cat No. 11053-MM06, H3N2: 11056-RP01, H1N1: Cat No. SEK001) were diluted in PBS (phosphate buffered saline, Lonza, Cat No. 17-516Q) and added to the ELISA plate for protein coating at a final concentration of 1 to 2 μg/mL. After incubation in a 4°C refrigerator for 16 to 20 hours, the ELISA plate was washed three times (wash buffer composition: 0.05% Tween-20 in PBS, Tween-20 (Sigma-Aldrich, Cat No. P1379-500 mL)) and saturated with phosphate-buffered saline (PBS) solution containing 2% bovine serum albumin (BSA: Sigma-Aldrich, Cat No. A3803-100G) for 1 hour at room temperature.

The amount of protein in the previously obtained muscle hydrolysate was quantified by bicinchoninic acid (BCA) quantification (ThermoFisher Scientific, Cat No. 23225), and an equal amount of protein was used to measure HA protein in muscle.

After blocking, the ELISA plate was washed three times with the same washing buffer, and the prepared muscle lysate was added to the ELISA plate and incubated at room temperature for 2 hours, and then washed three times. HA detection antibodies conjugated with horseradish peroxidase (HRP) or biotin from Sinobiological (Yamagata & Victoria: Cat No. SEK-11053, H3N2: Cat No. 11056-R014B, H1N1: Cat No. SEK001) were diluted to 0.5 to 1 μg/mL in PBS containing 0.5% bovine serum albumin, and 100 μL of the antibody solution was added and incubated at room temperature for 1 hour.

For H3N2, the ELISA plate was washed three times with the same washing buffer, and streptavidin-HRP (Sigma-Aldrich, Cat No. S2438-250UG) was diluted 1:5,000 in PBS containing 0.5% bovine serum albumin, and 100 μL of the diluted solution was added and further incubated at room temperature for 20 minutes. The ELISA plate was then washed three times and observed with TMB (3,3',5,5'-tetramethylbenzidine) solution (ThermoFisher Scientific, Cat No. 34028) at room temperature for 10 minutes. Afterwards, stop solution (SeraCare, Cat No. 5150-0021) was added to terminate the color development reaction, and the absorbance was measured at a wavelength of 450 nm using a microplate analyzer (VersaMax) from Moleculardevices to compare the expression of HA protein in muscle.

3-4. Injection into animals mRNA Constructs and serum collection to evaluate HA Methods of specific immune response

Animal experiments were performed to evaluate the immune response of the mRNA constructs. Balb/c female mice aged 6-7 weeks were purchased (OrientBio, Korea) and raised under specific pathogen-free conditions. The mice were immunized intramuscularly with 0.1 μg of each mRNA-LNP complex in 50 μL in the right tibia using an insulin syringe (Figure 7). Blood samples were collected 4 weeks after immunization to obtain serum.

The blood samples were placed at room temperature for 30 minutes to allow the blood to clot and then centrifuged at 10,000 rpm for 10 minutes at 4°C. After centrifugation, the supernatant was transferred to a new 1.7 mL tube and stored in a -20°C freezer until the humoral immune response was tested.

3-5.HA Antigenic specificity IgG Measurement of antibody measurement values

Enzyme-linked immunosorbent assay (ELISA) was performed to measure the HA antigen-specific IgG titer in the serum. The influenza A/H1N1 virus split vaccine was diluted in PBS and added to the ELISA plate for protein coating. After incubation in a 4°C refrigerator for at least 12 hours, the plate was washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 and saturated with PBS containing 2% bovine serum albumin (BSA Sigma, Cat No. A3803) at room temperature for 1 hour. After blocking, the ELISA plate was washed three times, and the serum sample diluted 2,000 times in PBS containing 2% BSA was added to the plate and incubated for 2 hours at room temperature. The plate was then washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Southern Biotech, Cat No. 1031-05) at room temperature for 1 hour. The ELISA plate was then washed three times and observed with TMB (3,3',5,5'-tetramethylbenzidine, ThermoFisher Scientific, Cat No. 34028) for 0 minutes. After observation, stop solution (SeraCare, Cat No. 5150-0021) was added to terminate the color development reaction, and the optical density was measured by spectroscopy at a wavelength of 450 nm using a microplate analyzer (VersaMax) from Moleculardevices.

Embodiment 4. Based on 5'UTR Comparison of signal sequence variation with influenza B virus /Yamagata Plant HA Expression

To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence generated in Example 1 with the sequence of the influenza virus itself.

As a result, it was found that the mRNA sequence produced in Example 1 showed higher in vivo protein expression compared with the HA sequence of influenza B virus/Yamagata strain itself, as shown in Figure 3 (Figure 3, control group vs. each lane).

Embodiment 5. Based on 5'UTR Comparison of signal sequence variation with influenza B virus /Victoria Plant HA Expression

To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence generated in Example 1 with the sequence of the influenza virus itself.

The results confirmed that the mRNA sequence produced in Example 1 showed higher in vivo protein expression compared with the HA sequence of influenza B virus/Victoria strain itself, as shown in Figure 4 (Figure 4, control group vs. each lane).

Embodiment 6. Based on 5'UTR Comparison of influenza A virus with signal sequence variation /H3N2 Plant HA Expression

To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence generated in Example 1 with the sequence of the influenza virus itself.

As a result, it was found that the mRNA sequence produced in Example 1 showed higher in vivo protein expression compared with the HA sequence of influenza A virus/H3N2 strain itself, as shown in Figure 5 (Figure 5, control group vs. each lane).

Embodiment 7. Based on 5'UTR Comparison of influenza A virus with signal sequence variation /H1N1 Plant HA Expression

To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence generated in Example 1 with the sequence of the influenza virus itself.

As a result, it was found that the mRNA sequence produced in Example 1 showed higher in vivo protein expression compared with the HA sequence of influenza A virus/H1N1 strain itself, as shown in Figure 6 (Figure 6, control group vs. each lane).

Embodiment 8. Based on 5'UTR Comparison of signal sequence changes with influenza A virus /H1N1 Immune response in strains

To enhance the immunogenicity of the mRNA construct, an anti-influenza specific IgG ELISA assay was performed using the method of Example 3 to compare the mRNA sequence generated in Example 1 with the sequence of the influenza virus itself.

The results confirmed that the mRNA sequence produced in Example 1 showed higher immunogenicity compared with the antigen of influenza A virus/H1N1 strain itself, as shown in Figure 8 (Figure 8, control group vs. each lane).

Embodiment 9. Screening for high expression and immunogenicity 5'UTR and signal sequence combination

Based on the results of Examples 4 to 8, 5'UTR and signal sequence combinations with high HA expression and high immunogenicity in vivo were screened. The criteria were determined as: the combination had an in vivo HA expression at least 1.5 times higher than the control group and an immunogenicity at least 3 times higher than the control group.

First, the in vivo HA expression of Examples 4 to 7 was summarized as the fold difference relative to the control group, as shown in Table 9 below. In Table 9, GC00-HA is equivalent to GC00_WT_HA, GC00-IgE is equivalent to GC00_IgE_HA, and GC00-tPA is equivalent to GC00_tPA_HA. The remaining terms can be used interchangeably.

[Table 9] Project H1N1 H3N2 Yamagata Victoria Control group 1.0 1.0 1.0 1.0 GC00-HA 1.6 2.2 4.3 5.5 GC01-HA 1.3 1.2 3.2 2.0 GC14-HA 1.3 1.6 3.5 4.7 GC27-HA 1.6 1.7 3.1 4.0 GC29-HA 1.9 2.6 5.3 4.5 GC14-IgE 1.1 1.2 2.2 3.5 GC29-IgE 1.1 1.2 2.8 4.0 GC00-tPA 3.6 1.7 3.8 2.8 GC14-tPA 2.6 1.5 3.2 2.1 GC27-tPA 2.0 1.2 4.3 2.1

As shown in Table 9, the combinations found to have at least 1.5-fold increase in expression in all strains were GC00-HA, GC27-HA, GC29-HA, GC00-tPA, and GC14-tPA.

The immunogenicity of the H1N1 strain of Example 8 is then summarized in Table 10 below as the fold difference compared to the control group.

[Table 10] Project H1N1 Control group 1.0 GC00-HA 3.6 GC01-HA 3.5 GC14-HA 5.4 GC27-HA 4.7 GC29-HA 2.5 GC01-IgE 1.7 GC14-IgE 1.9 GC29-IgE 1.8 GC00-tPA 4.0 GC01-tPA 4.4 GC14-tPA 2.8 GC27-tPA 3.0 GC29-tPA 3.2

As shown in Table 10, the combinations found to have at least 3-fold immunogenicity were GC00-HA, GC01-HA, GC14-HA, GC27-HA, GC00-tPA, GC01-tPA, GC27-tPA and GC29-tPA, and among them, the combinations having at least 1.5-fold HA expression were GC00-HA (GC00_WT_HA), GC27-HA (GC27_WT_HA) and GC00-tPA (GC00_tPA_HA), which were selected as the final combinations.

Industrial Practicality

The mRNA construct according to the present invention comprises a 5'-UTR polynucleotide with improved translation efficiency, which can effectively induce the expression of antigenic polypeptides, which is useful for vaccine development because it can be expected to increase the immunogenicity of the vaccine.

Although certain aspects of the present invention have been described in detail above, it is obvious to those skilled in the art that these specific descriptions are only preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the essential scope of the present invention is defined by the appended patent application and its equivalents.

without

Figure 1 is a schematic diagram of the components of an mRNA construct according to one embodiment of the present invention.

FIG. 2 is a schematic diagram showing an animal testing process according to an embodiment of the present invention.

FIG3 shows the protein expression results of influenza B/Yamagata HA mRNA constructs prepared according to one embodiment of the present invention in mouse muscle.

FIG. 4 shows the protein expression results of influenza B/Victoria HA mRNA constructs prepared according to one embodiment of the present invention in mouse muscle lysate.

FIG5 shows the protein expression results of influenza A/H3N2 HA mRNA constructs prepared according to one embodiment of the present invention in mouse muscle lysate.

FIG6 shows the protein expression results of influenza A/H1N1 HA mRNA constructs prepared according to one embodiment of the present invention in mouse muscle lysate.

FIG. 7 is a schematic diagram showing an in vivo immunogenicity testing process according to an embodiment of the present invention.

FIG. 8 shows the anti-HA specific IgG titer after immunization with influenza A/H1N1 HA mRNA constructs prepared according to one embodiment of the present invention.

TW202449157A_113120279_SEQL.xml

without.

Claims (26)

An mRNA construct encoding an antigenic polypeptide or an immunogenic fragment thereof, comprising: in order from 5' to 3', a) a 5'-cap structure; b) a 5'-untranslated region (5'-UTR) polynucleotide represented by a nucleic acid sequence according to the following formula (I) or (II): Formula (I): AG[N ₂₂ ]GCCACC, Formula (II): AGGA[N ₁₉ ]RGCCACC, wherein R represents A, G or U; c) a polynucleotide encoding a signal peptide; d) a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof; e) a 3'-untranslated region (3'-UTR); and f) a poly(A) tail or a poly(A) tail-like sequence consisting of 10 to 1000 adenines (A). An mRNA construct as described in claim 1, wherein the 5'-cap structure is selected from the group consisting of m7GpppAmpG, m7GpppApG and m7,3'OmeApppG. The mRNA construct according to claim 1, wherein the 5'-UTR is any one of the sequences represented by SEQ ID NOs: 1 to 34. The mRNA construct of claim 3, wherein the 5'-UTR is any one selected from the group consisting of SEQ ID NO: 1, 2, 15, 28 and 30. The mRNA construct of claim 1, wherein the signal peptide is derived from an antigenic polypeptide, immunoglobulin E (IgE) or tissue plasminogen activator (tPA). The mRNA construct of claim 5, wherein the signal peptide is an amino group represented by SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 73 (MKXIIALSXILCLVFA, wherein X is T, A, Y or N), SEQ ID NO: 77 (MKAILVVXLYTFTTANA, wherein X is L or M), SEQ ID NO: 80 (MDWTWILFLVAAATRVHS) or SEQ ID NO: 81 (MDAMKRGLCCVLLLCGAVFVSA). The mRNA construct of claim 1, wherein the polynucleotide encoding the signal peptide is codon-optimized. The mRNA construct according to claim 7, wherein the polynucleotide encoding the signal peptide is any one of the sequences represented by SEQ ID NOs: 39 to 50. The mRNA construct of claim 1, wherein the polynucleotide encoding the 5'-UTR and the signal peptide is selected from any one of the following groups: (i) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 39; (ii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 42; (iii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 45; (iv) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 48; (v) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 28 and the signal peptide of SEQ ID NO: 39; (vi) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 28 and the signal peptide of SEQ ID NO: 42; (vii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 28 and the signal peptide of SEQ ID NO:45; (viii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:48; (ix) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:41; (x) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:44; (xi) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:47; and (xii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:50. The mRNA construct according to claim 1, wherein the antigenic polypeptide is at least one selected from the group consisting of tumor antigens, pathogenic antigens, self-antigens, alloantigens and allergenic antigens. An mRNA construct as described in claim 10, wherein the tumor antigen is selected from the group consisting of: NY-ESO-1, HER-2/neu, MAGE-1, tyrosinase, MUC1, CEA, Mam-A, hTERT, Syalyl-Tn, WT1, α-fetoprotein, CA-125, gp-100, p53, Ras, Src, EGFRvIII, PSMA, GD2, Bcr-abl, survivin, PSA, EphA2, PAP, AFP, EpCAM, ALK, mesothelin, PSCA, MART-1, Melan-A, SCP-1, SPAG9, AKAP4 and OY-TES-1. The mRNA construct of claim 10, wherein the pathogenic antigen is selected from the group consisting of bacterial, viral, fungal and protozoan antigens. The mRNA construct of claim 12, wherein the virus is an influenza virus. An mRNA construct as described in claim 10, wherein the antigenic polypeptide is influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), or an immunogenic fragment of HA1 or HA2. The mRNA construct of claim 14, wherein the antigenic polypeptide is derived from an influenza virus strain selected from the group consisting of influenza B Yamagata virus, influenza B Victoria virus, influenza A H3N2 virus and influenza A H1N1 virus. An mRNA construct as described in claim 1, wherein the polynucleotide encoding the antigenic polypeptide or its immunogenic protein is codon optimized. The mRNA construct according to claim 16, wherein the polynucleotide encoding the antigenic polypeptide or the immunogenic protein thereof is any one of the sequences represented by SEQ ID NOs: 55 to 66. The mRNA construct of claim 1, wherein the polynucleotide encoding the 5'-UTR and the signal peptide; and the polynucleotide encoding the antigenic polypeptide or the immunogenic protein thereof are any one selected from the group consisting of: (i) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 39; and a polynucleotide encoding the antigenic polypeptide or the immunogenic protein thereof of SEQ ID NO: 55; (ii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 42; and a polynucleotide encoding the antigenic polypeptide or the immunogenic protein thereof of SEQ ID NO: 58; (iii) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: 45; and a polynucleotide encoding the antigenic polypeptide or the immunogenic protein thereof of SEQ ID NO: 61; (iv) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide of SEQ ID NO: NO:48; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:64; (v) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:39; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:55; (vi) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:42; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:58; (vii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:45; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:61; (viii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:28 and the signal peptide of SEQ ID NO:48; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:55; NO:64 or an immunogenic protein thereof; (ix) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:41; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:57; (x) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:44; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:60; (xi) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:47; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:63; and (xii) a polynucleotide encoding the 5'-UTR of SEQ ID NO:1 and the signal peptide of SEQ ID NO:50; and a polynucleotide encoding the antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO:66. The mRNA construct of claim 1, wherein the 3'-UTR is selected from the group consisting of: human α-globulin 3'-UTR; β-globulin 3'-UTR; CYBA 3'-UTR; albumin 3'-UTR; growth hormone (GH) 3'-UTR; VEEV 3'-UTR; hepatitis B virus (HBV) 3'-UTR; α-globulin 3'-UTR; DEN 3'-UTR; PAV barley yellow dwarf virus (BYDV-PAV) 3'-UTR; elongation factor 1 alpha 1 (EEF1A1) 3'-UTR; manganese superoxide dismutase (MnSOD) 3'-UTR; mitochondrial H(+)-ATP synthase beta unit (β-mRNA) 3'-UTR; GLUT1 3'-UTR; MEF2A 3'-UTR; and β-F1-ATPase 3'-UTR. The mRNA construct of claim 18, wherein the 3'-UTR is a sequence represented by SEQ ID NO: 71. An mRNA construct as described in claim 1, wherein, in the poly (A) tail-like sequence, one or more nucleotides selected from the group consisting of uracil (U), cytosine (C) and guanine (G) other than adenine are inserted between multiple adenines or at the end of the poly (A) tail. An mRNA construct as described in claim 1, wherein the mRNA construct comprises one or more backbone-modified, sugar-modified or base-modified nucleic acids. A vaccine composition comprising the mRNA construct of any one of claims 1 to 22. A vaccine composition as described in claim 23, wherein the mRNA construct is complexed with one or more lipids to form lipid nanoparticles or liposomes. A vaccine composition as described in claim 24, wherein the lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols and non-cationic lipids. A vaccine composition as described in claim 23, wherein the vaccine composition further comprises one or more adjuvants or active agents.