CN118256522A - Broad-spectrum influenza mRNA vaccine - Google Patents
Broad-spectrum influenza mRNA vaccine Download PDFInfo
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- CN118256522A CN118256522A CN202211634439.0A CN202211634439A CN118256522A CN 118256522 A CN118256522 A CN 118256522A CN 202211634439 A CN202211634439 A CN 202211634439A CN 118256522 A CN118256522 A CN 118256522A
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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Abstract
The invention relates to the fields of biological medicine and virology, in particular to an mRNA vaccine for preventing or treating influenza virus infection. The present invention provides polynucleotides encoding influenza virus NPs and M2e, e.g., mRNAs, optimized for human cell-preferred codon sequences. The invention also provides compositions and vaccines comprising the polynucleotides, and methods of using the polynucleotides, compositions or vaccines to treat or prevent influenza virus infection.
Description
Technical Field
The invention relates to the fields of biological medicine and virology, in particular to an mRNA vaccine for preventing or treating influenza virus infection.
Background
Influenza poses a serious threat to public health worldwide and a serious hazard to human health and world economy. Vaccination is one of the most effective measures for preventing influenza. The existing influenza vaccines comprise split vaccines, whole virus inactivated vaccines, attenuated live vaccines, subunit vaccines and the like, and mainly exert a protective effect by inducing specific neutralizing antibodies against hemagglutinin (hemagglutinin, HA) and Neuraminidase (NA) on influenza virus envelope.
HA type is highly variable in influenza virus, and antigenic drift (ANTIGENIC DRIFT) and antigenic shift (ANTIGENIC SHIFT) of HA often lead to seasonal influenza epidemics and pandemics. In the event of a mismatch between vaccine strains and epidemic strains, the protective efficacy is lacking. Current seasonal influenza vaccines are mainly directed against HA, NA antigens, and almost every year new vaccine preparations are required based on predicted strains, in order to reduce the frequency of vaccine re-preparation, broad-spectrum influenza vaccines, such as vaccines targeting influenza virus conserved antigens, such as Matrix protein 2 ectodomain (Matrix 2extracellular domain,M2e), matrix protein 1 (Matrix 1, M1) and nucleoprotein (nucleoprotein, NP), are required.
CN101899461B discloses a fusion gene encoding an influenza a NP protein and an M2e polypeptide. The protein subunit vaccine can be prepared by efficiently expressing influenza A virus NP and M2e fusion protein NM2e in escherichia coli and using the purified NM2e fusion protein.
However, the preparation of protein subunit vaccines involves the expression and purification of proteins, and has a long period, and is difficult to prepare in time when an epidemic situation occurs, so that more efficient and rapid vaccines, such as mRNA vaccines capable of realizing high expression in a subject, need to be established.
Disclosure of Invention
In one aspect, the invention provides a polynucleotide comprising a nucleotide sequence encoding a fusion protein of SEQ ID NO. 1, wherein the nucleotide sequence has at least 80% identity to a nucleotide sequence selected from the group consisting of 5, 6, 7, 8, 15, 16, 17 and 18.
In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the mRNA further comprises a 5'utr, a 3' utr, and a polyA. In some embodiments, the 5' UTR comprises the nucleotide sequence of SEQ ID NO. 2. In some embodiments, the 3' UTR comprises the nucleotide sequence of SEQ ID NO. 3. In some embodiments, the polyA comprises 75 adenylate residues.
In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% identity to one of SEQ ID NOs 10-13.
In a second aspect, the invention provides a composition comprising a polynucleotide of the invention. In some embodiments, the composition comprises a lipid encapsulating the polynucleotide. In some embodiments, the composition comprises a lipopolysaccharide complex. In some embodiments, the lipid encapsulating the polynucleotide comprises a cationic lipid, a non-cationic lipid, and a polyethylene glycol modified lipid; optionally, the composition further comprises a cationic polymer, wherein the cationic polymer associates with the polynucleotide as a complex, and is co-encapsulated in a lipid to form a lipopolysaccharide complex.
In a third aspect, the invention provides a vaccine formulation comprising a polynucleotide or composition of the invention. In some embodiments, the lipid encapsulating the polynucleotide in the vaccine formulation comprises 10-70 mole% M5, 10-70 mole% 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 10-70 mole% cholesterol, and 0.05-20 mole% 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG) 2000; preferably, the lipids are M5, DOPE, cholesterol and (DMG-PEG) 2000 in a molar ratio of 40:15:43.5:1.5,
In some embodiments, the vaccine formulation is a liquid formulation or a lyophilized formulation. In some embodiments, the vaccine formulation is administered by intramuscular injection. In some embodiments, the vaccine formulation is administered intramuscularly, such as by nasal spray.
In a fourth aspect, the present invention provides a method of preventing or treating an influenza virus infection in a subject in need thereof, the method comprising administering to a subject in need thereof a polynucleotide, composition or vaccine formulation of the present invention.
The invention also provides the use of a polynucleotide, composition or vaccine formulation of the invention in the manufacture of a medicament for the prevention and/or treatment of influenza virus infection in a subject in need thereof.
The invention also provides for the preparation of a polynucleotide, composition or vaccine formulation of the invention for use in the prevention and/or treatment of influenza virus infection in a subject in need thereof.
In some embodiments, the subject is a human or non-human animal.
Drawings
FIG. 1 shows a flow chart for constructing a lipid multimeric complex (LPP) of mRNA.
FIG. 2 shows the results of expression of NP protein (FIG. 2A), M2e protein (FIG. 2B) in 293T cells transfected with LPP preparations by western blot analysis, and the titers of antibodies against NM2e in mice immunized with different doses (1 and 10 μg) of LPP (FIG. 2C).
Figure 3 shows anti-NP protein and anti-M2 e IgG titers induced by LPP formulations in mice.
Figures 4 and 5 show the cellular immune response induced by LPP formulations in mice against NP protein and M2 e.
Fig. 6 shows the results of challenge experiments with influenza virus strains X31 (fig. 6A), PR8 (fig. 6B) and AH (fig. 6C) in mice after immunization with LPP formulations.
Detailed Description
1. General definition
All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, and the like, cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure.
Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology related terms as used herein are terms that are widely used in the corresponding field (see, e.g., ,Molecular Cloning:ALaboratory Manual,2nd Edition,J.Sambrook et al.eds.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989)., while, for a better understanding of the present invention, definitions and explanations of related terms are provided below.
As used herein, the terms "comprises," "comprising," "includes," "including," "having" and "containing" are open-ended, meaning the inclusion of the stated elements, steps or components, but not the exclusion of other non-recited elements, steps or components. The expression "consisting of … …" does not include any elements, steps or components not specified. The expression "consisting essentially of … …" means that the scope is limited to the specified elements, steps, or components, plus any optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of … …" and "consisting of … …" are encompassed within the meaning of the expression "comprising".
As used herein, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. The term "one or more" or "at least one" encompasses 1,2, 3,4, 5, 6, 7, 8, 9 or more.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless specifically indicated to the contrary, the numerical values or ranges set forth herein are modified by "about" to mean the enumerated or claimed values or ranges are + -20%, + -10%, + -5%, or + -3%.
All methods described herein can be performed in any suitable order unless otherwise indicated.
As used herein, the term "wild-type" means that the sequence is naturally occurring and not artificially modified, including naturally occurring mutants.
As used herein, the term "% identity" with respect to sequences refers to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences to be compared. The difference between the two sequences may be distributed over a local area (section) or the entire length of the sequences to be compared. The identity between two sequences is typically determined after optimal alignment of the segments or "comparison windows". The optimal alignment may be performed manually or by means of algorithms known in the art, including but not limited to the local homology algorithms described by SMITH AND WATERMAN,1981,ADS APP.MATH.2,482 and NEDDLEMAN AND Wunsch,1970, j.mol. Biol.48,443, the similarity search method described by Pearson AND LIPMAN,1988,Proc.Natl Acad.Sci.USA 88,2444, or using a computer program, such as GAP, BESTFIT, FASTA, BLAST P, BLAST N and tfast a in Wisconsin Genetics Software Package, genetics Computer Group,575Science Drive,Madison,Wis. For example, the percent identity of two sequences may be determined using the BLASTN or BLASTP algorithm commonly available at the National Center for Biotechnology Information (NCBI) website.
The% identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the regions give a degree of identity. In some embodiments, the degree of identity is given to the entire length of the reference sequence. Alignment for determining sequence identity can be performed using tools known in the art, preferably using optimal sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS:: needle, matrix: blosum62, gap Open 10.0, gap extension 0.5.
Herein, "nucleotide" includes deoxyribonucleotides and ribonucleotides and derivatives thereof. As used herein, a "ribonucleotide" is a constituent material of ribonucleic acid (RNA) and consists of one molecule of base, one molecule of pentose, and one molecule of phosphate, which refers to a nucleotide having a hydroxyl group at the 2' -position of the β -D-ribofuranose (β -D-ribofuranosyl) group. The "deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), and also comprises one molecule of base, one molecule of pentose and one molecule of phosphoric acid, and refers to a nucleotide in which the hydroxyl group at the 2' -position of the beta-D-ribofuranose (beta-D-ribofuranosyl) group is replaced by hydrogen, and is a main chemical component of a chromosome. "nucleotide" is generally referred to by the single letter representing the base therein: "A (a)" means adenine-containing deoxyadenylate or adenylate, "C (C)" means cytosine-containing deoxycytidylate or cytidylate, "G (G)" means guanine-containing deoxyguanylate or guanylate, "U (U)" means uracil-containing uridylate, "T (T)" means thymine-containing deoxythymidylate.
As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the ordering of nucleotides in a polynucleotide. It will be appreciated by those skilled in the art that the coding strand (sense strand) of DNA can be considered to have the same nucleotide sequence as the RNA it encodes, with deoxythymidylate in the sequence of the coding strand of DNA corresponding to uridylate in the sequence of the RNA it encodes.
As used herein, "coding sequence" refers to a nucleotide sequence in a polynucleotide that can be used as a template for synthesis of a polypeptide having a defined nucleotide sequence (e.g., tRNA and mRNA) or a defined amino acid sequence in a biological process. The coding sequence may be a DNA sequence or an RNA sequence. If an mRNA corresponding to a DNA sequence (including the same coding strand as the mRNA sequence and the template strand complementary thereto) is translated into a polypeptide in a biological process, the DNA sequence or mRNA sequence may be considered to encode the polypeptide.
As used herein, "codon" refers to three consecutive nucleotide sequences (also known as triplet codes) in a polynucleotide that encode a particular amino acid. Synonymous codons (codons encoding the same amino acid) are used differently in different species, termed "codon bias". It is generally believed that for a given species, coding sequences using codons that are favored by it can have higher translational efficiency and accuracy in the expression system of that species. Thus, a polynucleotide may be "codon optimized," i.e., codons in the polynucleotide are altered to reflect codons favored by the host cell, preferably without altering the amino acid sequence it encodes. One of skill in the art will appreciate that due to the degeneracy of the codons, a polynucleotide of the invention may comprise a coding sequence which differs from (e.g., has about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to) a coding sequence described herein but encodes the same amino acid sequence. In certain embodiments, the RNA of the invention comprises codons optimized for the host (e.g., subject, particularly mammalian) cell, such that the polypeptide of the invention is optimally expressed in the subject, such as a mammal, bird, or human.
As used herein, the term "expression" includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides. The term "transcription" relates to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the synthesis of RNA, in particular mRNA, in vitro in a cell-free system (e.g. in a suitable cell extract) (see, e.g. Pardi N.,Muramatsu H.,Weissman D.,KarikóK.(2013).In:Rabinovich P.(eds)Synthetic Messenger RNA and Cell Metabolism Modulation.Methods in Molecular Biology(Methods and Protocols),vol 969.Humana Press,Totowa,NJ.). vectors which may be used for the production of transcripts are also referred to as "transcription vectors", which contain the regulatory sequences required for transcription.
As used herein, "encoding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, that can be used as a template to synthesize polymers and macromolecules in other biological processes, provided that there is a defined nucleotide sequence or a defined amino acid sequence. Thus, a gene encodes a protein, meaning that mRNA of the gene is transcribed and translated to produce the protein in a cell or other biological system.
As used herein, the term "polypeptide" refers to a polymer comprising two or more amino acids covalently linked by peptide bonds. A "protein" may comprise one or more polypeptides, wherein the polypeptides interact with each other by covalent or non-covalent means. Unless otherwise indicated, "polypeptide" and "protein" may be used interchangeably.
As used herein, the term "host cell" refers to a cell that is used to receive, hold, replicate, express a polynucleotide or vector. In some embodiments, the host cell may be a cell in which a polypeptide of the invention is expressed.
As used herein, "antigen" refers to a molecule that upon entry into the body can elicit an immune response that is acquired by the body and that can be directed to the production of antibodies, or to specific immunogenically active cells, or both. It will be appreciated by those skilled in the art that any macromolecule, including almost all proteins or peptide fragments, may act as an antigen. Still further, the antigen may be from recombinant or genomic DNA or RNA. It will be appreciated by those skilled in the art that any of the DNA or RNA herein, the nucleotide sequence or portions thereof, may encode a protein capable of eliciting an acquired immunity in the body. Still further, it will be understood by those skilled in the art that an antigen need not solely encode the full length nucleotide sequence of only one gene. It will be apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences form different mixtures to induce the onset of a response. Still further, it will be appreciated by those skilled in the art that the antigen need not be encoded entirely by one gene. Obviously, the antigen may be synthetically produced or may be derived from a biological sample. Biological samples include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.
As used herein, "antibody" refers to a protein that has a protective effect by the body as a result of stimulation by an antigen. It is an immunoglobulin produced by B lymphocytes. The monomer of the antibody is a Y-shaped molecule and consists of 4 polypeptide chains. The chain comprises two identical heavy chains and two identical light chains, which are connected together by disulfide bonds. Each heavy chain is 50kDa, each light chain is 25kDa, and disulfide bonds exist between the light and heavy chains. It is unique in high affinity and specificity for binding partners.
As used herein, "vaccine" refers to a composition comprising an active ingredient (e.g., a polynucleotide of the invention) that is capable of eliciting an immune response in an vaccinated subject upon vaccination. In particular embodiments, the immune response it induces provides immune protection and is sufficient to prevent and/or ameliorate at least one symptom associated with a pathogen or disease infection. According to the invention, the polynucleotides or compositions described herein may be used as vaccines for providing prophylactic and/or therapeutic immunity against influenza virus in a subject in need thereof.
As used herein, the term "neutralizing antibody" refers to an antibody or fragment thereof that is capable of neutralizing, i.e., preventing, inhibiting, reducing, or interfering with the ability of a pathogen to initiate and/or maintain an infection in a host (e.g., host organism or host cell). According to the invention, neutralizing antibodies against influenza virus can be raised in a subject vaccinated with a vaccine of the invention, e.g., in the immune serum of the subject. Neutralizing antibody titer levels in immune serum can be measured using methods known in the art.
As used herein, "immune response" refers to a process involving activation and/or induction of effector functions that occur in, but not limited to, examples such as T cells, B cells, natural killer cells, and/or antigen presenting cells. Thus, an immune response may be understood by those of skill in the art to include, but is not limited to, any detectable T helper cell antigen specific activation and/or induction, cytotoxic T cell activity or response, antibody production, antigen presenting cell activity or infiltration, macrophage activity or infiltration, neutrophil activity or infiltration, or the like.
As used herein, "Th1" refers to the fact that naive CD 4+ T cells can differentiate into Th1 cells under the induction of interferon-gamma (IFN-gamma), secrete IFN-gamma, participate in cell-mediated immune responses and monocyte-or macrophage-mediated inflammatory responses; can differentiate into Th2 cells under the induction of IL-4, secrete cytokines such as IL-4, IL-5 and the like, participate in humoral immune response, stimulate B cells to promote antibody production, and promote proliferation and functions of mast cells and eosinophils.
As used herein, an "influenza virus" is a member of the orthomyxoviridae family, and is an enveloped negative-strand RNA virus. Influenza genomic RNA binds to nucleoprotein (nucleoprotein, NP) to form Ribonucleoprotein (RNP) complexes. Influenza virus also comprises matrix proteins, hemagglutinin and ceramidase. Hemagglutinin (hemagglutinin, HA) and Neuraminidase (NA), are glycoproteins in the influenza envelope responsible for surface contact of the virus and host. The viral entry into the host requires regulation by HA, which binds to cellular receptors and promotes fusion of the viral membrane with the endosomal membrane. Influenza viruses can be divided into subtypes based on HA and NA.
"NP protein" is a basic protein having 498 amino acids. At its N-terminus there is an RNA binding domain and two NP-NP self-interacting regions. They are critical for the maintenance of viral ribonucleoproteins, can interact with a variety of host proteins, and play a very important role in the influenza virus replication cycle. The NP protein has regions that are highly conserved among different influenza viruses.
"M2e" refers to the extracellular domain of matrix protein 2. The M2 protein is the matrix protein of influenza virus, and is 97 amino acids in full length, including an extracellular domain of 24 amino acids at the N-terminus, a transmembrane domain of 19 amino acids, and an intracellular domain of 54 amino acids at the C-terminus. The extracellular domain of the M2 protein is highly conserved among influenza viruses.
As used herein, the term "NM2e" refers to a fusion polypeptide of NP protein and M2e, comprising the full length NP protein (positions 1-498 of SEQ ID NO: 1), variants or fragments thereof, and residues 2-24 of the M2 protein (positions 499-521 of SEQ ID NO: 1).
As used herein, "NP 55–69" refers to a peptide consisting of residues 55-69 of NP protein, which is an H-2d restricted Th epitope, with an amino acid sequence of RLIQNSLTIERMVLS.
As used herein, "NP 147–155" refers to a peptide consisting of residues 147-155 of NP protein, which is an H-2d restricted CTL epitope, the amino acid sequence of which is TYQRTRALV.
As used herein, "pool of M2e peptides" refers to a mixed peptide of M2e proteins comprising three peptides corresponding to residues 1-15 (MSLLTEVETPIRNEW), residues 5-19 (TEVETPIRNEWGCRC) and residues 9-23 (TPIRNEWGCRCNDSS), respectively, of M2 protein.
As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic moiety and optionally also a hydrophilic moiety. Lipids are generally poorly soluble in water but soluble in many organic solvents. Generally, amphiphilic lipids comprising a hydrophobic portion and a hydrophilic portion may be organized in an aqueous environment as a lipid bilayer structure, for example in the form of vesicles. Lipids may include, but are not limited to: fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, and steroids and cholesterol esters, and the like.
As used herein, the term "cationic polymer" refers to any ionic polymer capable of carrying a net positive charge at a specified pH to electrostatically bind nucleic acids. Examples of cationic polymers include, but are not limited to: poly-L-lysine, protamine and Polyethylenimine (PEI). The polyethyleneimine may be a linear or branched polyethyleneimine.
The term "protamine" refers to arginine-rich low molecular weight basic proteins that are present in sperm cells of various animals (particularly fish) and bind to DNA in place of histones. In a preferred embodiment, the cationic polymer is protamine (e.g., protamine sulfate).
2. Polypeptides
The present invention relates to NM2e polypeptides. In some embodiments, the NM2e polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO. 1. In some embodiments, the NM2e comprises a variant and/or fragment of an NP protein, and M2e, wherein the variant and/or fragment of an NP protein comprises a conserved region of an NP protein. In some embodiments, the conserved regions do not comprise mutations (including amino acid substitutions, deletions, and insertions). In some embodiments, the conserved regions comprise conservative substitutions.
In some embodiments, the NP segment of NM2e comprises at least one amino acid modification, e.g., an insertion, substitution, and/or deletion. In some embodiments, the NP segment of NM2e comprises substitutions, insertions, and/or deletions of 1,2,3, 4, 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids.
3. Polynucleotide
The invention also relates to polynucleotides encoding the NM2e polypeptides. The polynucleotide may be single-stranded or double-stranded. Polynucleotides include, but are not limited to DNA, cDNA, RNA (e.g., mRNA), recombinantly produced, and chemically synthesized polynucleotides. The polynucleotide may be contained in a vector. Polynucleotides of the invention may include naturally occurring, synthetic, and modified nucleotides.
In some embodiments, the polynucleotides of the invention are used to express a polypeptide described herein in a cell to provide a polypeptide antigen. In some embodiments, the polypeptide antigen may induce an immune response, such as a cellular immune response and an antibody response, against influenza virus in a suitable subject.
The polynucleotide may comprise one or more segments (nucleotide fragments) (e.g., 1, 2,3,4, 5, 6, 7, 8 segments). Polynucleotides may comprise segments encoding polypeptides of interest (e.g., polypeptides and polypeptide antigens described herein). In particular embodiments, the polynucleotide may comprise coding sequences for the polypeptide of interest as well as regulatory sequences (including but not limited to transcriptional and translational regulatory sequences). In one embodiment, the regulatory sequence comprises one or more of the following: promoter sequence, 5 'untranslated region (5' UTR) sequence, 3 'untranslated region (3' UTR) sequence, and poly (A) sequence.
In some embodiments, the polynucleotides of the invention comprise a coding sequence for a polypeptide antigen as described herein. In one embodiment, the polynucleotides of the invention comprise a nucleotide sequence complementary to the coding sequences described herein. In some embodiments, a polynucleotide of the invention comprises a coding sequence for a polypeptide as described herein. In one embodiment, the coding sequence comprises an initiation codon at its 5 'end and a termination codon at its 3' end. In one embodiment, the coding sequence comprises an Open Reading Frame (ORF) as described herein.
In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID No. 4. In some embodiments, the NM2e encoded by said polynucleotide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO. 1. In some embodiments, the NM2e comprises a variant and/or fragment of an NP protein, and M2e, wherein the variant and/or fragment of an NP protein comprises a conserved region of an NP protein. In some embodiments, the conserved regions do not comprise mutations (including amino acid substitutions, deletions, and insertions). In some embodiments, the conserved regions comprise conservative substitutions.
In some embodiments, the NP segment of NM2e comprises at least one amino acid modification, e.g., an insertion, substitution, and/or deletion. In some embodiments, the NP segment of NM2e comprises substitutions, insertions, and/or deletions of 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the NP segment comprises a conserved region of NP protein that does not comprise the amino acid modification.
In some embodiments, the polynucleotides of the invention are RNA. As used herein, the definition of "RNA" encompasses single-stranded, double-stranded, linear, and circular RNAs. The RNA of the invention may be RNA produced by chemical synthesis, recombination and in vitro transcription. In one embodiment, the RNA of the invention is used to express a polypeptide of the invention in a host cell.
In some embodiments, the RNA of the invention is single stranded RNA. In one embodiment, the RNA of the invention is in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription with a DNA template by RNA polymerase (e.g., as described herein).
In some embodiments, the RNA of the invention is messenger RNA (mRNA). In general, mRNA can comprise a 5'-UTR sequence, a coding sequence for a polypeptide, a 3' -UTR sequence, and optionally a poly (a) sequence. mRNA can be produced, for example, by in vitro transcription or chemical synthesis. In one embodiment, the mRNA of the invention is obtained by in vitro transcription by RNA polymerase (e.g., T7 RNA polymerase) using a DNA template. In one embodiment, the mRNA of the invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3' -UTR, and (4) optionally, a poly (A) sequence. The 5'-UTR, coding sequence, 3' -UTR and poly (A) sequences are as described herein. In one embodiment, the mRNA of the present invention is a nucleoside modified mRNA. In one embodiment, the mRNA of the present invention comprises an optional 5' cap.
In some embodiments, the RNA of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the RNA of the invention comprises a coding sequence for a polypeptide as described herein.
In some embodiments, the RNAs of the invention further comprise structural elements that help to improve stability and/or translation efficiency of the RNAs, including, but not limited to, 5' caps, 5' -UTRs, 3' -UTRs, and poly (a) sequences.
As used herein, the term "untranslated region (UTR)" generally refers to a region in RNA (e.g., mRNA) that is not translated into an amino acid sequence (non-coding region), or a corresponding region in DNA. In general, the UTR located 5' to (upstream of) the open reading frame (start codon) may be referred to as the 5' -UTR of the 5' untranslated region; UTRs located 3 'to (downstream of) the open reading frame (stop codon) may be referred to as 3' -UTRs. In the presence of a 5 'cap, the 5' -UTR is located downstream of the 5 'cap, e.g., immediately adjacent to the 5' cap. In particular embodiments, an optimized "Kozak sequence" may be included in the 5' -UTR, e.g., adjacent to the start codon, to increase translation efficiency. In the presence of a poly (A) sequence, the 3' -UTR is located upstream of the poly (A) sequence, e.g., immediately adjacent to the poly (A) sequence.
In some embodiments, the RNA of the invention comprises a 5' -UTR. In a preferred embodiment, the 5' -UTR comprises the nucleotide sequence of SEQ ID NO. 2. In a preferred embodiment, the 3' -UTR comprises the nucleotide sequence of SEQ ID NO. 3. In some embodiments, the RNA of the invention comprises a 5'-UTR and a 3' -UTR. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO. 2 and the 3' -UTR comprises the nucleotide sequence of SEQ ID NO. 3.
In some embodiments, the RNA of the invention comprises a poly (a) sequence. "poly (A) sequence" or "poly (A) tail" refers to a nucleotide sequence comprising continuous or discontinuous adenylates. The poly (A) sequence is typically located at the 3' end of the RNA, e.g., 3' end (downstream) of the 3' -UTR. In some embodiments, the poly (a) sequence does not comprise nucleotides other than adenylate at its 3' end. Poly (A) sequences can be transcribed from the coding sequence of a DNA template by a DNA-dependent RNA polymerase during the preparation of IVT-RNA or can be linked to the free 3' end of IVT-RNA, e.g., the 3' end of the 3' -UTR, by a DNA-independent RNA polymerase (Poly (A) polymerase).
In one embodiment, the poly (A) sequence comprises contiguous adenylates. In one embodiment, the poly (a) sequence can comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 and up to 120, 150, 180, 200, 300 adenylates. In one embodiment, the contiguous adenylate sequence in the poly (a) sequence is interrupted by a sequence comprising U, C or G nucleotides. Preferably, the poly (a) sequence comprises 75 adenylates.
The poly (a) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 and up to 120, 150, 180, 200, 300 nucleotides. In one embodiment, the poly (a) sequence comprises at least 50 nucleotides. In one embodiment, the poly (a) sequence comprises at least 80 nucleotides. In one embodiment, the poly (a) sequence comprises at least 100 nucleotides. In some embodiments, the poly (a) sequence comprises about 70, 80, 90, 100, 120, or 150 nucleotides. In a specific embodiment, the poly (a) sequence comprises 75 nucleotides.
As used herein, the term "5 'cap" generally refers to an N7-methylguanosine structure (also known as "m7G cap", "m7 Gppp-") attached to the 5' end of an mRNA by a 5 'to 5' triphosphate bond. The 5' cap may be co-transcribed into the RNA in vitro transcription (e.g., using an anti-reverse cap analogue "ARCA") or may be post-transcriptionally linked to the RNA using a capping enzyme.
In some embodiments, the RNA of the invention comprises the nucleotide sequence of SEQ ID NO 10, 11, 12 or 13. In some embodiments, the RNA of the invention comprises (a) a nucleotide sequence comprising at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleotide sequence of SEQ ID NO. 10, 11, 12 or 13; and (b) encodes an amino acid sequence that has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the polypeptide encoded by the RNA of the invention comprises a conserved region of NP protein. In some embodiments, the conserved regions do not comprise mutations (including amino acid substitutions, deletions, and insertions). In some embodiments, the conserved regions comprise conservative substitutions.
In some embodiments, the polynucleotides of the invention are DNA. Such DNA may be, for example, a DNA template for in vitro transcription of the RNA of the invention or a DNA vaccine for expression of a polypeptide antigen in a host cell. The DNA may be double-stranded, single-stranded, linear and circular.
The DNA template may be provided in a suitable transcription vector. In general, a DNA template may be a double-stranded complex comprising a nucleotide sequence (coding strand) identical to a coding sequence described herein and a nucleotide sequence (template strand) complementary to a coding sequence described herein. As known to those skilled in the art, a DNA template may comprise a promoter, a 5'-UTR, a coding sequence, a 3' -UTR, and optionally a poly (a) sequence. Promoters may be available to suitable RNA polymerases (particularly DNA-dependent RNA polymerases) known to those skilled in the art, including but not limited to promoters of SP6, T3 and T7 RNA polymerases. In some embodiments, the 5'-UTR, coding sequence, 3' -UTR, and poly (a) sequences in the DNA templates are or are complementary to the corresponding sequences contained in the RNAs described herein. Polynucleotides as DNA vaccines may be provided in plasmid vectors (e.g., circular plasmid vectors).
In some embodiments, the DNA of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the DNA of the invention comprises a coding sequence for a polypeptide as described herein. In some embodiments, the DNA of the invention comprises, from the 5 'end to the 3' end, (1) a T7 promoter, (2) a 5'-UTR, (3) a coding sequence, (4) a 3' -UTR, and (5) an optionally present poly (a) sequence as described herein.
4. Composition and vaccine formulation
The invention also provides a composition comprising a polynucleotide (particularly RNA) of the invention. In one embodiment, the compositions of the invention are used to provide prophylactic and/or therapeutic immunity against influenza virus in a subject. In some embodiments, the compositions of the invention comprise a polynucleotide of the invention. In some embodiments, the compositions of the invention comprise a DNA of the invention. In some embodiments, the compositions of the invention comprise an RNA of the invention. In one embodiment, the RNA is in vitro transcribed RNA. In one embodiment, the RNA is mRNA.
In some embodiments, the compositions of the invention comprise a polynucleotide (particularly RNA, e.g., mRNA) as described herein and a lipid encapsulating the polynucleotide.
Particularly preferred nucleic acid compositions can be, for example, lipid Nanoparticles (LNPs) and lipid multimeric complexes (LPPs) as described herein. Methods of preparing such compositions can be found, for example, in Kaczmarek, j.c.et al, 2017,Genome Medicine 9,60 or as described herein. In some embodiments, the compositions of the invention comprise Lipid Nanoparticles (LNPs) or lipid multimeric complexes (LPPs). In some embodiments, the compositions of the invention are Lipid Nanoparticles (LNPs) or lipid multimeric complexes (LPPs) comprising the RNAs of the invention.
In some embodiments, the lipid encapsulating the polynucleotide comprises a cationic lipid and a non-cationic lipid. In a preferred embodiment, the cationic lipid is an ionizable cationic lipid.
In one embodiment, the cationic lipid comprises DOTMA、DOTAP、DDAB、DOSPA、DODAC、DODAP、DC-Chol、DMRIE、DMOBA、DLinDMA、DLenDMA、CLinDMA、DMORIE、DLDMA、DMDMA、DOGS、N4- cholesteryl-spermine, DLin-KC2-DMA, DLin-MC3-DMA, or a combination thereof.
In one embodiment, the cationic lipid comprises M5, which has the following structure:
In one embodiment, the cationic lipid comprises DOTMA. In one embodiment, the cationic lipid comprises DOTAP. In one embodiment, the cationic lipid comprises DOTMA and DOTAP.
In one embodiment, the non-cationic lipid comprises a phospholipid as described herein. In one embodiment, the non-cationic lipid comprises a steroid as described herein. In one embodiment, the non-cationic lipid comprises a phospholipid and a steroid as described herein. In one embodiment, the phospholipid comprises DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE and DSPE or a combination thereof. In one embodiment, the steroid is cholesterol. In one embodiment, the non-cationic lipid comprises DOPE. In one embodiment, the non-cationic lipid comprises cholesterol. In one embodiment, the non-cationic lipid comprises DOPE and cholesterol.
In one embodiment, the cationic lipid comprises M5 and the non-cationic lipid comprises DOPE and cholesterol.
In some embodiments, the polynucleotide-encapsulating lipid further comprises a polyethylene glycol-modified lipid. In one embodiment, the polyethylene glycol modified lipid comprises DMG-PEG (e.g., DMG-PEG 2000), DOGPEG, and DSPE-PEG, or a combination thereof. In one embodiment, the polyethylene glycol modified lipid comprises DSPE-PEG. In one embodiment, the polyethylene glycol modified lipid comprises DMG-PEG (e.g., DMG-PEG 2000).
In some embodiments, the compositions of the invention further comprise a cationic polymer associated with the polynucleotide as a complex, co-encapsulated in the lipid.
In one embodiment, the cationic polymer comprises poly-L-lysine, protamine, polyethylenimine (PEI), or a combination thereof. In one embodiment, the cationic polymer is protamine. In one embodiment, the cationic polymer is a polyethyleneimine.
In one embodiment, the amount of lipid in the composition is calculated as mole percent (mole%) based on the total moles of lipid in the composition.
In one embodiment, the amount of cationic lipid in the composition is from about 10 to about 70 mole%. In some embodiments, the amount of cationic lipid in the composition is from about 20 to about 60 mole%, from about 30 to about 50 mole%, from about 35 to about 45 mole%, from about 38 to about 45 mole%, from about 40 to about 50 mole%, or from about 45 to about 50 mole%.
In one embodiment, the amount of phospholipid in the composition is from about 10 to about 70 mole%. In one embodiment, the amount of phospholipid in the composition is from about 20 to about 60 mole%, from about 30 to about 50 mole%, from about 10 to about 30 mole%, from about 10 to about 20 mole%, or from about 10 to about 15 mole%.
In one embodiment, the amount of cholesterol in the composition is from about 10 to about 70 mole%. In one embodiment, the amount of cholesterol in the composition is from about 20 to about 60 mole%, from about 30 to about 50 mole%, from about 35 to about 40 mole%, from about 35 to about 45 mole%, from about 40 to about 45 mole%, or from about 45 to about 50 mole%.
In one embodiment, the amount of polyethylene glycol modified lipid in the composition is from about 0.05 to about 20 mole%. In one embodiment, the amount of polyethylene glycol modified lipid in the composition is from about 0.5 to about 15 mole%, from about 1 to about 10 mole%, from about 5 to about 15 mole%, from about 1 to about 5 mole%, from about 1.5 to about 3 mole%, or from about 2 to 5 mole%.
In some embodiments, the RNA (particularly mRNA) of the invention is formulated as Lipid Nanoparticles (LNP). As used herein, "lipid nanoparticle" or "LNP" refers to particles formed from lipids in which nucleic acids (e.g., mRNA) are encapsulated.
In one embodiment, the LNP comprises an RNA of the invention and an RNA-encapsulating lipid, wherein the RNA-encapsulating lipid comprises a cationic lipid, a phospholipid, cholesterol, and a polyethylene glycol modified lipid. In one embodiment, the cationic lipid is M5. In one embodiment, the phospholipid is DSPC. In one embodiment, the polyethylene glycol modified lipid is DMG-PEG 2000. In one embodiment, the cationic lipid is M5, the phospholipid is DSPC, and the polyethylene glycol modified lipid is DMG-PEG 2000.
In one embodiment, the RNA-encapsulating lipid comprises 50 mole% M5, 10 mole% DSPC, 38.5 mole% cholesterol, and 1.5 mole% DMG-PEG 2000.
In some embodiments, the RNAs (particularly mrnas) of the invention are formulated as lipid multimeric complexes (lipopolyplex, LPP). As used herein, "lipid multimeric complex" or "LPP" refers to a core-shell structure comprising a nucleic acid core encapsulated by a lipid outer shell, the nucleic acid core comprising a nucleic acid (e.g., mRNA) associated with a polymer.
In one embodiment, the LPP comprises an RNA of the invention, associated with a cationic polymer as a complex; and a lipid encapsulating the complex, wherein the lipid encapsulating the complex comprises a cationic lipid, a non-cationic lipid, and a polyethylene glycol modified lipid. In one embodiment, the non-cationic lipid comprises a phospholipid and a steroid. In one embodiment, the non-cationic lipid comprises a phospholipid selected from 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), or a combination thereof, and cholesterol. In one embodiment, the cationic polymer comprises protamine. In one embodiment, the polyethylene glycol modified lipid comprises DMG-PEG 2000.
In one embodiment, the cationic lipid comprises M5, which has the following structure:
The non-cationic lipid comprises a phospholipid selected from 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), or a combination thereof, and cholesterol;
the polyethylene glycol modified lipid comprises 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000);
the cationic polymer comprises protamine.
In one embodiment, the cationic polymer is protamine, the cationic lipid is M5, the phospholipid is DOPE, and the polyethylene glycol modified lipid is DMG-PEG 2000.
In one embodiment, the lipid of the encapsulation complex comprises 40 mole% M5, 15 mole% DOPE, 43.5 mole% cholesterol, and 1.5 mole% DMG-PEG 2000.
In some embodiments, the vaccine formulations of the invention comprise a polynucleotide described herein.
In some embodiments, the vaccine formulations of the present invention comprise a composition as described herein, wherein the lipid comprises 10-70 mole% M5, 10-70 mole% DOPE, 10-70 mole% cholesterol, and 0.05-20 mole% DMG-PEG 2000,
Wherein the polynucleotide encodes a polypeptide described herein.
In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID No. 4. In some embodiments, the NM2e encoded by said polynucleotide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO. 1. In some embodiments, the NM2e comprises a variant and/or fragment of an NP protein, and M2e, wherein the variant and/or fragment of an NP protein comprises a conserved region of an NP protein. In some embodiments, the conserved regions do not comprise mutations (including amino acid substitutions, deletions, and insertions). In some embodiments, the conserved regions comprise conservative substitutions.
In some embodiments, the NP segment of NM2e comprises at least one amino acid modification, e.g., an insertion, substitution, and/or deletion. In some embodiments, the NP segment of NM2e comprises substitutions, insertions, and/or deletions of 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the NP segment comprises a conserved region of NP protein that does not comprise the amino acid modification.
In some embodiments, the polynucleotides of the invention are RNA. As used herein, the definition of "RNA" encompasses single-stranded, double-stranded, linear, and circular RNAs. The RNA of the invention may be RNA produced by chemical synthesis, recombination and in vitro transcription. In one embodiment, the RNA of the invention is used to express a polypeptide of the invention in a host cell.
In some embodiments, the RNA of the invention is single stranded RNA. In one embodiment, the RNA of the invention is in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription with a DNA template by RNA polymerase (e.g., as described herein).
In some embodiments, the RNA of the invention is messenger RNA (mRNA). In general, mRNA can comprise a 5'-UTR sequence, a coding sequence for a polypeptide, a 3' -UTR sequence, and optionally a poly (a) sequence. mRNA can be produced, for example, by in vitro transcription or chemical synthesis. In one embodiment, the mRNA of the invention is obtained by in vitro transcription by RNA polymerase (e.g., T7 RNA polymerase) using a DNA template. In one embodiment, the mRNA of the invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3' -UTR, and (4) optionally, a poly (A) sequence. The 5'-UTR, coding sequence, 3' -UTR and poly (A) sequences are as described herein. In one embodiment, the mRNA of the present invention is a nucleoside modified mRNA. In one embodiment, the mRNA of the present invention comprises an optional 5' cap.
In some embodiments, the RNA of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the RNA of the invention comprises a coding sequence for a polypeptide as described herein.
In some embodiments, the RNAs of the invention further comprise structural elements that help to improve stability and/or translation efficiency of the RNAs, including, but not limited to, 5' caps, 5' -UTRs, 3' -UTRs, and poly (a) sequences.
As used herein, the term "untranslated region (UTR)" generally refers to a region in RNA (e.g., mRNA) that is not translated into an amino acid sequence (non-coding region), or a corresponding region in DNA. In general, the UTR located 5' to (upstream of) the open reading frame (start codon) may be referred to as the 5' -UTR of the 5' untranslated region; UTRs located 3 'to (downstream of) the open reading frame (stop codon) may be referred to as 3' -UTRs. In the presence of a 5 'cap, the 5' -UTR is located downstream of the 5 'cap, e.g., immediately adjacent to the 5' cap. In particular embodiments, an optimized "Kozak sequence" may be included in the 5' -UTR, e.g., adjacent to the start codon, to increase translation efficiency. In the presence of a poly (A) sequence, the 3' -UTR is located upstream of the poly (A) sequence, e.g., immediately adjacent to the poly (A) sequence.
In some embodiments, the RNA of the invention comprises a 5' -UTR. In a preferred embodiment, the 5' -UTR comprises the nucleotide sequence of SEQ ID NO. 2. In a preferred embodiment, the 3' -UTR comprises the nucleotide sequence of SEQ ID NO. 3. In some embodiments, the RNA of the invention comprises a 5'-UTR and a 3' -UTR. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO. 2 and the 3' -UTR comprises the nucleotide sequence of SEQ ID NO. 3.
In some embodiments, the RNA of the invention comprises a poly (a) sequence.
In one embodiment, the poly (A) sequence comprises contiguous adenylates. In one embodiment, the poly (a) sequence can comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 and up to 120, 150, 180, 200, 300 adenylates. In one embodiment, the contiguous adenylate sequence in the poly (a) sequence is interrupted by a sequence comprising U, C or G nucleotides. Preferably, the poly (a) sequence comprises 75 adenylates.
The poly (a) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 and up to 120, 150, 180, 200, 300 nucleotides. In one embodiment, the poly (a) sequence comprises at least 50 nucleotides. In one embodiment, the poly (a) sequence comprises at least 80 nucleotides. In one embodiment, the poly (a) sequence comprises at least 100 nucleotides. In some embodiments, the poly (a) sequence comprises about 70, 80, 90, 100, 120, or 150 nucleotides. In a specific embodiment, the poly (a) sequence comprises 75 nucleotides.
As used herein, the term "5 'cap" generally refers to an N7-methylguanosine structure (also known as "m7G cap", "m7 Gppp-") attached to the 5' end of an mRNA by a 5 'to 5' triphosphate bond. The 5' cap may be co-transcribed into the RNA in vitro transcription (e.g., using an anti-reverse cap analogue "ARCA") or may be post-transcriptionally linked to the RNA using a capping enzyme.
In some embodiments, the RNA of the invention comprises the nucleotide sequence of SEQ ID NO 10, 11, 12 or 13. In some embodiments, the RNA of the invention comprises (a) a nucleotide sequence comprising at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleotide sequence of SEQ ID NO. 10, 11, 12 or 13; and (b) encodes an amino acid sequence that has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the polypeptide encoded by the RNA of the invention comprises a conserved region of NP protein. In some embodiments, the conserved regions do not comprise mutations (including amino acid substitutions, deletions, and insertions). In some embodiments, the conserved regions comprise conservative substitutions.
5. Lipid
1. Cationic lipids
Cationic lipids are lipids that carry a net positive charge at a specified pH. Lipids with a net positive charge can associate with nucleic acids through electrostatic interactions.
Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (1, 2-di-O-octadecenyl-3-trimethylammonium-propane, DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP), bisdecanyl dimethylammonium bromide (Didecyldimethylammonium bromide, DDAB), 2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl ] -N, N-dimethyl-l-propylamine onium trifluoroacetate (2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate,DOSPA)、 dioctadecyl dimethyl ammonium chloride (dioctadecyldimethyl ammonium chloride, DODAC), 1,2-dioleoyl-3-dimethyl ammonium-propane (1, 2-dioleoyl-3-dimethylammonium-propane, DODAP), 3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol, DC-Chol), 2,3-di (tetradecyloxy) propyl- (2-hydroxyethyl) -dimethylaminoonium (2, 3-di (tetradecoxy) propyl- (2-hydroxyethyl) -dimethylazanium, DMRIE), N-dimethyl-3,4-dioleyloxybenzylamine (N, N-dimethyl-3,4-dioleyloxybenzylamine, DMOBA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (1, 2-dilinoleyloxy-N, N-dimethylaminopropane, DLinDMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (1, 2-dilinolenyloxy-N, N-dimethylaminopropane, DLenDMA), 3-dimethylamino-2- (cholest-5-en-3- β -oxybutan-4-oxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane,CLinDMA)、N-(2- aminoethyl) -N, N-dimethyl-2,3-bis (tetradecyloxy) propane-1-aminium chloride (N- (2-aminoethyl) -N, N-dimethyl-2,3-bis (tetradecyloxy) propan-1-aminium bromide, DMORIE), N-dimethyl-2,3-bis (dodecyloxy) propane-1-amine (N, N-dimethyl-2,3-bis (dodecyloxy) propan-1-amine, DLDMA), N-dimethyl-2,3-bis (tetradecyloxy) propane-1-amine (N, N-dimethyl-2,3-bis (tetradecyloxy) propan-1-amine, DMDMA), dioctadecyl amidoglycyl spermine (dioctadecylamidoglycyl spermine, DOGS), N4-cholesteryl-spermine (N4-cholesteryl-spermine), 2-diiodol-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxane, DLin-KC 2-DMA), triacontanyl-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate (heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethyl-amine) butanoate, DLin-MC 3-DMA), heptadec-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate) (heptadecan-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate), ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate).
In some embodiments, the cationic lipid is preferably an ionizable cationic lipid. The ionizable cationic lipid carries a net positive charge at, for example, an acidic pH, and is neutral at a higher pH (e.g., physiological pH). Examples of ionizable cationic lipids include, but are not limited to: dioctadecyl amidoglycyl spermine (dioctadecylamidoglycyl spermine, DOGS), N4-cholesteryl-spermine (N4-cholesteryl-spermine), 2-dioleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1,3] -diolide, DLin-KC 2-DMA), triacontanyl-6,9,28,31-tetralin-19-yl-4- (dimethylamino) butyrate (heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethyllamino) butanoate, DLin-MC 3-DMA), heptadec-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate) (heptadecan-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (hexyl) amino) octanoate, ((4-hydroxybutyl) azepine-19-yl-4- (dimethylamino) butyrate) bis (1-864-dihexyl) bis (24-864-2-bis (864-2-8654).
In one embodiment, the cationic lipid comprises M5, which has the following structure:
2. Non-cationic lipids
Herein, "non-cationic lipids" refers to lipids that do not carry a net positive charge at a specified pH, such as anionic lipids and neutral lipids. The term "neutral lipid" refers to a lipid that exists in an uncharged, neutral or zwitterionic form at physiological pH. Neutral lipids may include, but are not limited to, phospholipids and steroids.
Examples of phospholipids include, but are not limited to: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (1-palmitoyl-2-oleoylphosphatidylethanolamine, POPE), distearoyl phosphatidylcholine (distearoylphosphatidylcholine, DSPC), distearoyl-phosphatidylethanolamine (distearoyl-phosphotidylinothiolamine, DSPE), dioleoyl phosphatidylcholine (dioleoylphosphatidylcholine, DOPC), dimyristoyl phosphatidylcholine (dimyristoylphosphatidylcholine, DMPC), dipalmitoyl phosphatidylcholine (dipalmitoylphosphatidylcholine, DPPC), ditetraenoyl phosphatidylcholine (diarachidoylphosphatidylcholine, DAPC), didodecyl phosphatidylcholine (dibehenoylphosphatidylcholine, DBPC), ditridecyl phosphatidylcholine (ditricosanoylphosphatidylcholine, DTPC), ditetradecyl phosphatidylcholine (dilignoceroylphatidylcholine, DLPC), palmitoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylcholine, POPC), ditolyphosphatidylethanolamine (dButyl-93-phosphotidyline, DPPE), and ditolyphosphatidylethanolamine (dipalmitoyl-phosphotidyline, DPPE).
Examples of steroids include, but are not limited to, for example, cholesterol, cholestanol, cholestanone, cholestenone, cholestyl-2 '-hydroxyethyl ether, cholestyl-4' -hydroxybutyl ether, tocopherol, and derivatives thereof.
3. Polyethylene glycol modified lipids
As used herein, the term "polyethylene glycol modified lipid" refers to a molecule comprising a polyethylene glycol moiety and a lipid moiety. Examples of polyethylene glycol modified lipids include, but are not limited to: 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol, DMG-PEG), 1,2-dioleoyl-rac-glycerol, methoxy-polyethylene glycol (1, 2-Dioleoyl-rac-glycerol, methoxypolyethylene Glycol, DOGPEG)) and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-poly (ethylene glycol) (1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol), DSPE-PEG).
In one embodiment, the polyethylene glycol modified lipid is DMG-PEG, such as DMG-PEG 2000. In one embodiment, the DMG-PEG 2000 has the following structure:
Wherein n has an average value of 44.
6. Preventing and treating influenza virus infection
The present invention provides a polynucleotide (particularly RNA), composition or vaccine formulation of the invention for use in the prevention and/or treatment of influenza virus infection in a subject in need thereof.
The present invention provides the use of a polynucleotide (particularly RNA), composition or vaccine formulation of the invention in the manufacture of a medicament for the prevention and/or treatment of influenza virus infection in a subject in need thereof.
The present invention provides a method for preventing and/or treating influenza virus infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polynucleotide (particularly RNA), composition or vaccine formulation of the invention. In one embodiment, the method comprises administering a therapeutically effective amount of a composition comprising an mRNA of the present invention, particularly a composition comprising LPP as described herein.
The term "prophylactically or therapeutically effective amount" refers to an amount sufficient to prevent or inhibit the occurrence of a disease or symptom and/or to slow, alleviate, delay the progression or severity of a disease or symptom. A prophylactically or therapeutically effective amount is affected by factors including, but not limited to: the rate and severity of the disease or condition, the age, sex, weight and physiological condition of the subject, the duration of the treatment, and the particular route of administration. A prophylactically or therapeutically effective amount may be administered in one or more doses. A prophylactically or therapeutically effective amount may be achieved by continuous or intermittent administration.
In some embodiments, a prophylactically or therapeutically effective amount is provided in one or more administrations. In some embodiments, a prophylactically or therapeutically effective amount is provided in two administrations. In some embodiments, a prophylactically or therapeutically effective amount is provided in three administrations.
In some embodiments, the compositions or vaccine formulations of the invention may be administered to a subject by any method known to those of skill in the art, such as parenteral, oral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, or intraperitoneal. Preferably, the composition or vaccine formulation of the invention is administered by intramuscular injection.
As used herein, the term "subject" describes an organism, e.g., a human, a non-human mammal (e.g., pig) or an avian (e.g., chicken), to which treatment using a polynucleotide or composition of the present invention may be provided.
7. Advantageous effects
The polynucleotides, compositions, vaccine formulations and methods of the invention achieve expression of higher levels of NM2e polypeptide in cells than prior art, and induce significant cellular and antibody responses in animals, and provide improved protection against different strains (homotypic and heterotypic strains), which can induce broad-spectrum cross-immune protection against conserved antigens in vivo.
Examples
The invention is further described by reference to the following examples. It should be understood that these embodiments are by way of example only and are not limiting of the invention. The following materials and instruments are commercially available or prepared according to methods well known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures well known in the art.
Example 1 preparation of mRNA
Design and Synthesis of DNA templates
In order to achieve optimal expression, the inventors codon-optimized the nucleotide sequence encoding the NM2e fusion protein of SEQ ID NO. 1 to obtain the sequences shown in Table 1. Wherein NM2e-Ori represents wild-type sequence, NM2e-1, NM2e-2, NM2e-3, NM2e-4 represents optimized sequence.
T7 promoter sequences (TAATACGACTCACTATA), 5'-UTR sequences (SEQ ID NO: 19), 3' -UTR sequences (SEQ ID NO: 20) and poly (A) sequences (75 adenosine nucleotides) were also designed. The Kozak sequence "GCCACC" is contained in the 5' utr sequence.
Then ligated in the order of T7 promoter sequence, 5'-UTR sequence, DNAORF sequence, 3' -UTR sequence and poly (A) sequence. The plasmid DNA template was obtained by total gene synthesis (Scoring Jin Weizhi Biotechnology Co., ltd.) using pUC57 as a vector.
The DNA template was obtained by PCR amplification using a pair of tailing PCR primers (upstream primer: 5'TTGGACCCTCGTACAGAAGCTAATACG 3'; and downstream poly (T) long primer :5'TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTACTTCCTACTCAGGCTTTATTCAAAGACCA 3')) and a high-fidelity DNA polymerase-based PCR amplification kit (Bao Ri doctor materials technology (Beijing) Co., ltd.).
1.2. In vitro transcription of mRNA from DNA templates
The PCR product prepared in example 1.1 was purified with a PCR product purification kit (Takara). And (3) taking the purified PCR product as a template, performing co-transcription capping reaction by using T7 RNA polymerase, and performing in vitro transcription of RNA, thereby generating Cap1 mRNA. In vitro transcription 1-methyl-pseudouridine triphosphate was used instead of Uridine Triphosphate (UTP), and therefore the modification ratio of 1-methyl-pseudouracil in vitro transcribed Cap1 mRNA was 100%. After transcription, the DNA template was digested with dnaseli (sameil technologies limited) to reduce the risk of residual DNA template.
MRNA was purified using DynabeadsMyone (Semer Feishul technologies Co., ltd.). Purified mRNA was dissolved in 1mM sodium citrate buffer (pH 6.5), sterile filtered, and cryopreserved at-80℃until use. The mRNA sequences obtained are shown in Table 1.
TABLE 1 sequence of nucleic acids
Example 2 preparation of LPP formulation
2.1. Experimental materials
Cationic lipid M5 is a s microbial synthesis; helper phospholipids (DOPE) were purchased from CordenPharma; cholesterol was purchased from Sigma-Aldrich; mPEG2000-DMG (i.e., DMG-PEG 2000) was purchased at Avanti Polar Lipids, inc; PBS was purchased from Invitrogen; protamine sulfate was purchased from Beijing Lian pharmaceutical Co.
Preparation of lipid multimeric complexes (LPP) of mRNA
LPP was prepared as shown in fig. 1. Specifically, the preparation shown includes the following steps.
Preparation of an aqueous mRNA solution: each mRNA prepared in example 1.2 was diluted to 0.2mg/mL of mRNA solution with 10mM citric acid-sodium citrate buffer (pH 4.0).
Preparation of lipid solution: cationic lipid (M5): DOPE: cholesterol: DMG-PEG 2000 was dissolved in absolute ethanol at a molar ratio of 40:15:43.5:1.5 to prepare a lipid solution of 10 mg/mL.
Preparing a protamine sulfate solution: the protamine sulfate is dissolved in water without the nucleotidase to prepare the protamine sulfate solution with the working concentration of 0.25 mg/mL.
Preparation of core nanoparticle (core nanoparticle) solution: using microfluidic technology (micana (Shanghai) technologies, inc.: inano D), a solution of protamine sulfate was mixed with a solution of mRNA under the following conditions to obtain a solution of nuclear nanoparticles formed from protamine and mRNA: volume=4.0 mL; flow rate ratio=5 (mRNA): 1 (protamine solution), total Flow rate=12 mL/min, front waste (START WASTE) =0.35 mL, rear waste (end waste) =0.1 mL, room temperature.
Preparation of LPP: the core nanoparticle solution was secondarily mixed with the lipid solution under the following conditions: volume= 4.0mL,Flow rate ratio =1 (lipid solution) 3 (core nanoparticle solution), total flow rate=12 mL/min, front waste=0.35 mL, back waste=0.1 mL, room temperature, diluted with PBS solution to obtain LPP solution.
Centrifugal ultrafiltration: the LPP solution was subjected to ultrafiltration centrifugation to remove ethanol (rotation speed 3000rpm, centrifugation time 60min, temperature 4 ℃ C.) to obtain LPP-NM2e preparations LPP-NPM2e-Ori, LPP-NPM2e-1, LPP-NPM2e-2, LPP-NPM2e-3 and LPP-NPM2e-4.
EXAMPLE 3 expression of mRNA in cells
The non-optimized LPP-NM2e-Ori and the optimized LPP-NM2e-1, LPP-NM2e-2, LPP-NM2e-3 and LPP-NM2e-4 were transfected into 293T cells, and the cells were collected 24 hours later for Western blot analysis.
As shown in FIGS. 2A and 2B, the expression of optimized LPP-NM2e was significantly improved.
Example 4 detection of immunogenicity of LPP formulations
4.1. 5-Week-old BALB/c mice (n=8, female, shanghai Ling Biotech Co., ltd.) were given non-optimized LPP-NM2e-Ori and optimized LPP-NM2e-1, LPP-NM2e-2, LPP-NM2e-3 and LPP-NM2e-4, respectively, by intramuscular injection at 1 μg (low dose group), 10 μg (high dose group), two doses, three weeks apart. Mice 2 weeks post priming and 2 weeks post boost were bled retroorbital to collect serum and assayed for NPM2e binding antibodies.
As shown in FIG. 2C, the antibody titers in the optimized LPP-NPM2e-1, LPP-NM2e-2, LPP-NM2e-3 and LPP-NM2e-4 immunized mice were significantly higher than the non-optimized LPP-NM2e-Ori immunized mice, whether in the 1 μg group or the 10 μg group. Wherein the LPP-NM2e-3 induced humoral immunity is higher than that induced by other optimized mRNA, the antibody titer of the low dose group reaches 70,400 weeks after the initiation and the antibody titer reaches 525,000 weeks after the enhancement; the antibody titre reached 128,000 2 weeks after priming and 1,600,000 at 2 weeks after boosting in the high dose group (fig. 2C). Therefore, we selected LPP-NPM2e-3 (hereinafter referred to as LPP-NPM2 e) to further characterize its humoral and cellular immunity and conduct an challenge experiment.
4.2. Characterization of LPP-NM2e induced immune response
BALB/c mice (female, shanghai Ling Biotechnology Co., ltd.) of 5 weeks old were randomly assigned to 3 groups, and LPP (100 μl, mock group, n=10), 1 μ gLPP-NM2e (low dose group, n=15) and 10 μg LPP-NM2e (high dose group, n=15) each containing no mRNA were administered (prime-boost) by intramuscular injection, two doses each, three weeks apart. Two weeks after each dose, blood was taken from the orbit for antibody detection, and spleen mononuclear cells were prepared from the spleen of the mice for cellular immune response detection.
The results of the antibody detection are shown in fig. 3, and the NP protein specific antibodies (IgG) titers of 6.28×10 3 (low dose group) and 2.88×10 4 (high dose group), respectively, were detected after administration of the first dose of LPP-NM2e formulation, showing a significant dose dependence (P < 0.0001). The NP protein-specific antibody titers increased to 5.60×10 4 (low dose group) and 1.34×10 5 (high dose group), respectively, following administration of the second dose of LPP-NM2e formulation, were significantly higher than the antibody titers induced by administration of the first dose (P < 0.05), and showed significant dose dependence (P < 0.0001) (fig. 3A).
After administration of the first dose of LPP-NM2e formulation, M2 e-specific antibodies (IgG) titers of 1.2×10 2 (low dose group) and 1.86×10 2 (high dose group), respectively, were detected. After administration of the second dose of LPP-NM2e formulation, the M2 e-specific antibody titers increased to 9.70×10 2 (low dose group) and 3.98×10 3 (high dose group), respectively, with titers significantly higher than the M2 e-specific antibody titers detected after administration of the first dose of LPP-NM2e formulation (P < 0.05) (fig. 3B).
In addition, the detection of the IgG2a, igG1 subtype of antibodies after immunization (n=4) with LPP-NM2e vaccine, the results are shown in tables 2 and 3, with NP protein, M2e specific IgG2a to IgG1 ratio of substantially greater than 1 (only one mouse is less than 1), suggesting that a biased Th1 type immune response is induced.
TABLE 2NP antigen-specific IgG2a/IgG1 ratio
TABLE 3M2e antigen-specific IgG2a/IgG1 ratio
The prepared spleen mononuclear cells were stimulated with NP 55-69、NP147-155 and M2e peptide pools. The cell immune response induced by LPP-NM2e immunized mice was examined using IFN-. Gamma.ELISPOT, and the results are shown in FIG. 3.
After the first administration, the average densities of numbers of NP 55-69、NP147-155 and M2 e-specific spot forming (IFN- γ secretion) cells in spleen mononuclear cells from the low dose and high dose groups were 24SFC(spot-forming cells)/106SMNC(spleen mononuclear cells)、448SFC/106SMNC、10SFC/106SMNC and 10SFC/10 6SMNC、383SFC/106 SMNC and 25SFC/10 6 SMNC, respectively, each without significant differences from the control group.
After the second administration, the cellular immune response is enhanced. Average densities of NP 55-69、NP147-155 and M2e specific SFC in the low dose group were 196SFC/10 6SMNC、1943SFC/106SMNC、98SFC/106 SMNC, respectively, significantly higher than levels after the first administration (P <0.01, P <0.05 and P < 0.05); the average densities of NP 55-69、NP147-155 and M2e specific SFC in the high dose group were 278SFC/10 6SMNCs、2950SFC/106SMNCs、128SFC/106SMNCs,NP55-69、NP147-155 specific cellular immune responses, respectively, significantly higher than the level after the first administration (P < 0.0001), with no statistically significant differences in the M2e specific immune response, but showed a dose-dependent trend.
In addition, spleen cells of mice 5 months after immunization with LPP-NM2e vaccine (n=4) were subjected to flow analysis, stimulated with NM2e peptide pool, and as shown in fig. 4 and 5, antigen-specific T cell responses were strong, mainly manifested as an increase in CD8 and CD4 secreting cytokines IFN- γ and TNF- α, and the T cell responses still exhibited a dose-dependent trend.
The results show that LPP preparations of mRNA of the present invention induce significant cellular and antibody responses against the influenza NP proteins and M2e, with the antibody responses showing significant dose-dependence, and the cellular responses against the NP and M2e proteins also showing a dose-dependent trend.
EXAMPLE 5 LPP formulation induced immunoprotection in mice
In this example, influenza virus strains X31 (H3N 2), PR8 (H1N 1) and AH (H7N 9) from the chinese disease prevention and control center, the virus disease prevention and control center, were used for challenge experiments to detect the immunoprotection induced by LPP formulations in mice.
Mice were immunized as described in example 4 and, after 5 weeks of the first dose administration, mice were nasally given influenza virus (5 xLD doses of strain X31 (n=10), 3xLD doses of strain PR8 (n=10) and 3xLD doses of strain AH (n=10 (high dose group), n=11 (low dose group)) for a challenge experiment to detect LPP formulation induced immune protection in mice.
Body weight changes as shown in figures 6A, B and C panels, the low and high dose groups each had a minimum body weight 5 days after challenge, after which the body weight gradually increased back to Day 0.
Mice survived, and Mock mice all died 5 days (X31), 6 days (PR 8), 5 days (AH) after challenge, as shown in figures 6A, B and right panel C. For mice administered LPP formulation, the survival rate of mice vaccinated with strain X31 was 60% (low dose group) and 100% (high dose group), respectively (fig. 6A); mice vaccinated with strain PR8 had viability of 90% (low dose group) and 100% (high dose group), respectively (fig. 6B); the mice vaccinated with strain AH had viability of 82% (low dose group) and 90% (high dose group), respectively (fig. 6C).
The result shows that the LPP preparation of the invention can not only effectively protect mice from being attacked by homotypic influenza virus X31 (H3N 2), but also can effectively protect mice from being attacked by heterotypic influenza virus strain PR8 (H1N 1) and highly pathogenic avian influenza virus strain AH (H7N 9).
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Example 6, materials and methods
1. Cells, proteins, antibodies
HEK293 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (Gibco), 100U/mL penicillin and 100mg/mL streptomycin (Gibco) at 37℃and 5% CO 2.
Recombinant NP, M2e, and NPM2e proteins for ELISA assays were purchased from Yinqiao and expressed in E.coli or baculovirus-insect cells.
Antibodies for western blotting include: anti-influenza a M2 protein antibodies were purchased from Abcam; anti-influenza NP protein antibodies were purchased from the state of sense, anti-mouse horseradish peroxidase (HRP) -conjugated antibodies and anti-rabbit horseradish peroxidase (HRP) -conjugated antibodies, from Abbkine.
2. Detection of bound IgG antibodies
NP, M2e, or NPM2e protein was diluted to 1. Mu.g/ml with 0.05M sodium carbonate buffer, respectively, and added to 96-well ELISA platesGreiner) was coated overnight at 4 ℃. The plates were washed with PBS-T (phosphate buffered saline+0.05% Tween-20) followed by blocking with 2% BSA (formulated in PBS-T) for 60 min at 37 ℃. A 2-fold serial dilution of mouse serum samples was added to the coated plates and incubated for 60 minutes at 37 ℃. The plates were then incubated with HRP conjugated secondary antibody for 60 minutes at 37 ℃. After washing the plates 3 times, TMB substrate (bi yun sky biotechnology) was added. After the reaction was terminated, the Optical Density (OD) at 450 wavelengths was read using a microplate reader (BioTek). The reciprocal value of the highest dilution of the sample with absorbance value higher than 2.1 times negative control sample was determined as the final titer.
3. Cell transfection
1.0X10 6 cells/well HEK293 cells were seeded into 6-well cell culture dishes the day before transfection, and 2.5. Mu.g of NPM2e mRNA-LPP was incubated with cells with fresh medium exchange when the cells were confluent to 70-90% the next day. After 24 hours, cells were collected, and LPP transfected cells were lysed with 1 XSDS-PAGE loading buffer (Beyotime) for SDS-PAGE and Western blot detection.
4. ELISA spot (ELISPot) assay
The mouse IFN-. Gamma.ELISpot assay was performed using the IFN-. Gamma.ELISpot PLUS kit (Mabtech) according to the manufacturer's instructions. Briefly, plates were blocked in RPMI 1640 medium (supplemented with 10% fbs) and incubated for 30min. The spleens of the mice were removed, ground and filtered, and after treatment with red blood cell lysates, the cells obtained (i.e., spleen mononuclear cells) were counted, plated at 3×10 5 cells/well, and incubated with 8 μg/ml NP55-69 peptide, 8 μg/ml NP14-155 peptide, and 8 μg/ml M2e peptide pool and 10 μg/ml NM2e peptide pool (both available from shanghai Ji Biochemical limited), phytohemagglutinin (PHA) + ionomycin (positive control) or RPMI 1640 medium alone (negative control) for 20 hours at 37 ℃. After that, with biotinylated IFN-. Gamma. -detection antibody and streptavidin-alkaline phosphatase (ALP), BCIP/NBT-plus (5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium-plus) substrate was added for color development and counted with an ELISPOT reader (ImmunoSpot S6 Core Analyzer (CTL)).
Peptide pool sequence:
The NP 55–69 amino acid sequence was RLIQNSLTIERMVLS.
The NP 147–155 amino acid sequence was TYQRTRALV.
The M2e peptide pool is a mixed peptide of M2e protein, which contains three peptides corresponding to residues 1-15 (MSLLTEVETPIRNEW), residues 5-19 (TEVETPIRNEWGCRC) and residues 9-23 (TPIRNEWGCRCNDSS) of M2 protein, respectively.
The NM2e peptide pool is a peptide pool consisting of peptide fragments corresponding to the full-length NM2e protein, 15 amino acids in length, overlapping each other by 11 amino acids.
5. Animal study
Female BALB/c mice were immunized with 100. Mu.L of mRNA-LPP preparation for 5 weeks on day 0 (D0) and 21 (D21), respectively, by double-sided intramuscular injection. All blood samples were collected by retroorbital blood sampling, about 200 μl blood/time, and centrifuged at 1,500g at 4 ℃ for 10 minutes for serum separation.
6. Statistical analysis
Statistical analysis of animal studies was performed using GRAPHPAD PRISM 8.0.0 software. Data are expressed as mean ± SEM. Two experimental groups were compared using a two-tailed T-test. For comparison of more than two experimental groups, one-way ANOVA was used. P values less than 0.05 are considered significant. * P <0.05; * P <0.01; * P <0.001; * P <0.0001.
Sequence(s)
Amino acid sequence of SEQ ID NO. 1NM2e fusion polypeptide
MASQGTKRSYEQMETDGERQNATEIRASVGKMIDGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYKRVDGRWMRELVLYDKEEIRRIWRQANNGDDATAGLTHMMIWHSNLNDTTYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMIKRGINDRNFWRGENGRKTRSAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFSARSALILRGSVAHKSCLPACVYGPAVSSGYNFEKEGYSLVGIDPFKLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIASNENMDNMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFEKSTVMAAFTGNTEGRTSDMRAEIIRMMEGTKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDNSLLTEVETPIRNEWGCRCNDSSD
SEQ ID NO:2 5’UTR
AGGAAAUUCCAUUUGGCUGCAGCUUCUGGAGGGAGCCGACAGGAGACGUGGGGAGACGGCCACC
SEQ ID NO:3 3’UTR
GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGU
SEQ ID NO. 4 non-optimized coding DNA sequence NM2e-Ori
ATGGCGTCCCAAGGCACCAAACGGTCTTATGAACAGATGGAAACTGATGGGGAACGCCAGAATGCAACTGAGATTAGGGCATCCGTCGGGAAGATGATTGATGGAATTGGGCGATTCTACATCCAAATGTGCACTGAACTTAAACTCAGTGATTATGAAGGGCGGTTGATCCAGAACAGCTTGACAATAGAGAGAATGGTGCTCTCTGCTTTTGATGAGAGAAGGAATAGATATCTGGAAGAACACCCCAGCGCGGGGAAAGATCCTAAGAAAACTGGAGGGCCCATATACAAGAGAGTAGATGGAAGATGGATGAGGGAACTCGTCCTTTATGACAAAGAAGAAATAAGGCGAATCTGGCGCCAAGCCAACAATGGTGATGATGCGACAGCTGGTCTAACTCACATGATGATCTGGCATTCCAATTTGAATGATACAACATACCAGAGGACAAGAGCTCTTGTTCGCACCGGAATGGACCCCAGGATGTGCTCTCTGATGCAGGGTTCGACTCTCCCTAGAAGGTCCGGAGCTGCAGGCGCTGCAGTCAAAGGAATCGGGACAATGGTGATGGAGCTGATCAGAATGATCAAACGGGGGATCAACGATCGAAATTTCTGGAGAGGTGAGAATGGGCGGAAAACAAGGAGTGCTTATGAGAGAATGTGCAACATTCTTAAAGGAAAATTTCAAACAGCTGCACAAAGAGCAATGATGGATCAAGTGAGAGAAAGCCGGAACCCAGGAAATGCTGAGATCGAAGATCTCATATTTTCGGCAAGATCTGCACTAATATTGAGAGGGTCAGTTGCTCACAAATCTTGCCTACCTGCCTGTGTGTATGGACCTGCAGTATCCAGTGGGTACAACTTCGAAAAAGAGGGATATTCCTTAGTGGGAATAGACCCTTTCAAACTACTTCAAAATAGCCAAGTATACAGCCTAATCAGACCGAACGAGAATCCAGCACACAAGAGTCAGCTGGTGTGGATGGCATGCCATTCTGCTGCATTTGAAGATTTAAGATTGTTAAGCTTCATCAGAGGGACCAAAGTATCTCCGCGGGGGAAACTTTCAACTAGAGGAGTACAAATTGCTTCAAATGAGAACATGGATAATATGGAATCAAGTACTCTTGAACTGAGAAGCAGGTACTGGGCCATAAGGACCAGGAGTGGAGGAAACACTAATCAACAGAGGGCCTCTGCAGGCCAAATCAGTGTGCAACCTACGTTTTCTGTACAAAGAAACCTCCCATTTGAAAAATCAACCGTCATGGCAGCATTCACTGGAAATACGGAGGGAAGAACCTCAGACATGAGGGCAGAAATCATAAGGATGATGGAAGGTACAAAACCAGAAGAAGTGTCCTTCCGTGGGCGGGGAGTTTTCGAGCTCTCAGACGAAAAGGCAACGAACCCGATCGTGCCCTCTTTTGACATGAGTAATGAAGGATCTTATTTCTTCGGAGACAATGCAGAAGAGTACGACAATAGCCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGGTGCAGATGCAACGATTCAAGTGAC
SEQ ID NO. 5 optimized coding DNA sequence NM2e-1
ATGGCCAGCCAGGGCACCAAGAGGAGCTACGAGCAGATGGAGACCGACGGCGAGAGACAGAACGCCACCGAGATCAGGGCCAGCGTGGGCAAGATGATCGACGGCATCGGCAGATTCTACATCCAGATGTGCACCGAGCTGAAGCTGAGCGACTACGAGGGCAGGCTGATCCAGAACAGCCTGACCATCGAGAGGATGGTGCTGAGCGCCTTCGACGAGAGGAGAAACAGGTACCTGGAGGAGCACCCCAGCGCCGGCAAGGACCCCAAGAAGACCGGCGGCCCCATCTACAAGAGGGTGGACGGCAGGTGGATGAGGGAGCTGGTGCTGTACGACAAGGAGGAGATCAGGAGGATCTGGAGGCAGGCCAACAACGGCGACGACGCCACCGCCGGCCTGACCCACATGATGATCTGGCACAGCAACCTGAACGACACCACCTACCAGAGGACCAGGGCCCTGGTGAGAACCGGCATGGACCCCAGGATGTGCAGCCTGATGCAGGGCAGCACCCTGCCCAGGAGAAGCGGCGCCGCCGGCGCCGCCGTGAAGGGCATCGGCACCATGGTGATGGAGCTGATCAGGATGATCAAGAGAGGCATCAACGACAGGAACTTCTGGAGGGGCGAGAACGGCAGGAAGACCAGAAGCGCCTACGAGAGAATGTGCAACATCCTGAAGGGCAAGTTCCAGACCGCCGCCCAGAGGGCCATGATGGACCAGGTGAGGGAGAGCAGGAACCCCGGCAACGCCGAGATCGAGGACCTGATCTTCAGCGCCAGGAGCGCCCTGATCCTGAGGGGCAGCGTGGCCCACAAGAGCTGCCTGCCCGCCTGCGTGTACGGCCCCGCCGTGAGCAGCGGCTACAACTTCGAGAAGGAGGGCTACAGCCTGGTGGGCATCGACCCCTTCAAGCTGCTGCAGAACAGCCAGGTGTACAGCCTGATCAGGCCCAACGAGAACCCCGCCCACAAGAGCCAGCTGGTGTGGATGGCCTGCCACAGCGCCGCCTTCGAGGACCTGAGACTGCTGAGCTTCATCAGGGGCACCAAGGTGAGCCCCAGGGGCAAGCTGAGCACCAGAGGCGTGCAGATCGCCAGCAACGAGAACATGGACAACATGGAGAGCAGCACCCTGGAGCTGAGGAGCAGATACTGGGCCATCAGGACCAGGAGCGGCGGCAACACCAACCAGCAGAGGGCCAGCGCCGGCCAGATCAGCGTGCAGCCCACCTTCAGCGTGCAGAGGAACCTGCCCTTCGAGAAGAGTACCGTGATGGCCGCCTTCACCGGCAACACCGAGGGCAGGACCAGCGACATGAGAGCCGAGATCATCAGGATGATGGAGGGCACCAAGCCCGAGGAGGTGAGCTTCAGGGGCAGAGGCGTGTTCGAGCTGAGCGACGAGAAGGCCACCAACCCCATCGTGCCCAGCTTCGACATGAGCAACGAGGGCAGCTACTTCTTCGGCGACAACGCCGAGGAGTACGACAACAGCCTGCTGACCGAGGTGGAGACCCCCATCAGGAACGAGTGGGGCTGCAGGTGCAACGACAGCAGCGAC
SEQ ID NO. 6 optimized coding DNA sequence NM2e-2
ATGGCCAGCCAAGGCACCAAGAGAAGCTACGAGCAGATGGAGACAGACGGCGAACGGCAGAACGCCACAGAGATCAGGGCCAGCGTGGGAAAGATGATCGATGGAATCGGAAGATTCTACATCCAGATGTGCACCGAGCTGAAGCTGTCTGATTACGAGGGCCGGCTGATCCAGAACTCTCTGACAATCGAGAGAATGGTCCTGAGCGCCTTCGACGAGAGACGGAATAGATACCTGGAGGAGCACCCCAGCGCCGGCAAGGACCCCAAGAAGACCGGGGGCCCTATCTACAAGCGGGTGGACGGCAGATGGATGAGAGAGCTGGTGCTGTACGACAAGGAAGAAATCAGACGGATCTGGAGACAGGCCAACAACGGCGACGACGCCACCGCCGGCCTGACTCACATGATGATCTGGCACAGCAACCTGAACGACACCACCTACCAGCGGACCAGGGCCCTGGTGAGAACAGGCATGGATCCTCGTATGTGCAGCCTGATGCAGGGCAGCACCCTGCCCAGACGGAGCGGCGCCGCCGGCGCCGCTGTTAAGGGCATCGGCACAATGGTGATGGAGCTGATTCGGATGATCAAGAGGGGCATCAACGATAGAAACTTCTGGCGGGGCGAGAACGGCAGAAAGACCAGATCCGCCTACGAGCGGATGTGTAACATCCTGAAAGGCAAGTTCCAGACCGCCGCTCAGAGGGCCATGATGGACCAGGTGCGGGAAAGCAGAAACCCCGGCAATGCCGAGATCGAGGATCTGATCTTCAGCGCCAGATCCGCCTTAATTCTGCGGGGATCTGTGGCCCACAAGTCCTGCCTGCCAGCCTGCGTGTACGGCCCCGCCGTGTCTTCTGGATACAACTTCGAAAAGGAAGGCTACAGCCTGGTGGGCATCGACCCTTTTAAGCTGCTGCAGAATAGCCAAGTGTATAGCCTGATTCGGCCTAACGAGAATCCTGCTCATAAGAGCCAGCTGGTGTGGATGGCTTGTCACAGCGCCGCTTTTGAGGACCTGAGACTGCTCAGCTTCATCAGAGGCACCAAAGTGTCCCCTAGAGGCAAACTGTCCACCCGGGGCGTGCAGATCGCCAGTAATGAGAACATGGACAACATGGAATCTTCTACACTTGAACTGAGATCTAGATACTGGGCCATCAGAACCAGAAGCGGCGGAAACACAAACCAGCAGAGAGCTAGCGCCGGACAGATCAGCGTCCAACCTACATTCAGCGTGCAGAGAAATCTGCCTTTCGAAAAAAGCACCGTGATGGCCGCCTTTACAGGCAACACCGAGGGCAGAACCAGCGATATGAGAGCAGAGATCATCCGGATGATGGAAGGCACCAAGCCTGAGGAAGTGTCTTTCAGAGGACGGGGTGTTTTCGAACTGTCTGACGAGAAAGCCACCAACCCAATCGTGCCAAGCTTTGATATGAGCAACGAGGGAAGCTATTTCTTCGGCGACAACGCTGAGGAATACGATAATAGCCTGCTGACCGAGGTGGAAACCCCTATCCGCAACGAGTGGGGCTGCAGATGCAACGACAGCTCCGAC
SEQ ID NO. 7 optimized coding DNA sequence NM2e-3
ATGGCATCCCAGGGAACCAAGCGGTCTTACGAGCAGATGGAGACAGATGGCGAGAGACAGAACGCCACCGAGATCAGGGCCTCTGTGGGCAAGATGATCGACGGCATCGGCAGATTCTACATCCAGATGTGCACCGAGCTGAAGCTGAGCGATTATGAGGGCCGCCTGATCCAGAACTCCCTGACAATCGAGCGGATGGTGCTGTCTGCCTTTGACGAGCGGAGAAATCGGTACCTGGAGGAGCACCCTAGCGCCGGCAAGGACCCCAAGAAGACCGGAGGCCCCATCTACAAGAGGGTGGACGGCAGGTGGATGAGGGAGCTGGTGCTGTATGATAAGGAGGAGATCAGGCGCATCTGGCGCCAGGCCAACAATGGCGACGATGCAACAGCAGGACTGACCCACATGATGATCTGGCACTCCAACCTGAATGACACCACATACCAGAGGACACGCGCCCTGGTGAGAACCGGAATGGACCCCAGGATGTGCTCCCTGATGCAGGGCTCTACCCTGCCACGGAGAAGCGGAGCAGCAGGAGCAGCCGTGAAGGGCATCGGCACAATGGTCATGGAGCTGATCCGGATGATCAAGAGAGGCATCAACGACAGGAATTTCTGGAGGGGAGAGAACGGAAGGAAGACCAGATCCGCCTATGAGAGAATGTGCAATATCCTGAAGGGCAAGTTTCAGACAGCCGCCCAGAGGGCCATGATGGACCAGGTGAGGGAGAGCCGCAACCCAGGCAATGCCGAGATCGAGGATCTGATCTTTTCTGCCCGCAGCGCCCTGATCCTGAGGGGCAGCGTGGCACACAAGTCCTGCCTGCCTGCATGCGTGTACGGACCAGCCGTGTCTAGCGGCTACAACTTCGAGAAGGAGGGCTATTCCCTGGTGGGCATCGATCCCTTTAAGCTGCTGCAGAACAGCCAGGTGTATTCTCTGATCAGGCCAAACGAGAATCCCGCCCACAAGAGCCAGCTGGTGTGGATGGCATGCCACTCCGCCGCCTTCGAGGACCTGAGACTGCTGTCCTTTATCAGGGGCACAAAGGTGAGCCCTCGCGGCAAGCTGTCCACCAGAGGCGTGCAGATCGCCTCTAACGAGAATATGGATAACATGGAGTCCTCTACCCTGGAGCTGCGGTCTAGATACTGGGCCATCAGGACACGCAGCGGCGGCAACACCAATCAGCAGAGGGCATCTGCCGGACAGATCAGCGTGCAGCCAACATTCTCCGTGCAGCGGAACCTGCCCTTTGAGAAGTCTACCGTGATGGCCGCCTTCACAGGCAATACCGAGGGCCGGACAAGCGACATGAGAGCCGAGATCATCAGGATGATGGAGGGCACCAAGCCTGAGGAGGTGAGCTTCCGGGGCAGAGGCGTGTTTGAGCTGTCCGACGAGAAGGCCACAAACCCCATCGTGCCTAGCTTTGATATGTCCAATGAGGGCTCTTACTTCTTTGGCGACAACGCCGAGGAGTATGATAATTCCCTGCTGACAGAGGTGGAGACCCCTATCCGCAACGAGTGGGGCTGCCGGTGTAATGACAGCTCCGAT
SEQ ID NO. 8 optimized coding DNA sequence NM2e-4
ATGGCCTCTCAGGGCACCAAGAGAAGCTACGAGCAGATGGAAACCGACGGCGAGAGACAGAACGCCACCGAGATTAGAGCCAGCGTGGGCAAGATGATCGACGGCATCGGCCGGTTCTACATCCAGATGTGCACCGAGCTGAAGCTGAGCGACTACGAGGGCAGACTGATCCAGAACAGCCTGACCATCGAGCGGATGGTGCTGAGCGCCTTCGACGAGCGGAGAAACAGATACCTGGAAGAACACCCCAGCGCCGGCAAGGACCCCAAGAAAACAGGCGGCCCTATCTACAAGCGCGTGGACGGCAGATGGATGAGAGAACTGGTGCTGTACGACAAAGAGGAAATCCGGCGGATCTGGCGGCAGGCCAACAATGGGGATGATGCCACAGCCGGCCTGACACACATGATGATCTGGCACAGCAACCTGAACGACACCACCTACCAGCGGACAAGAGCCCTCGTCAGAACCGGCATGGACCCTAGAATGTGCAGCCTGATGCAGGGCAGCACCCTGCCTAGAAGATCTGGTGCTGCTGGCGCTGCCGTGAAAGGCATCGGCACAATGGTCATGGAACTGATCCGGATGATCAAGCGGGGAATCAACGACCGGAACTTTTGGAGAGGCGAGAACGGCAGAAAGACCCGCAGCGCCTACGAGAGGATGTGCAATATCCTGAAGGGCAAGTTCCAGACCGCCGCTCAGAGGGCCATGATGGATCAAGTGCGCGAGAGCAGAAACCCCGGCAATGCCGAGATCGAGGACCTGATCTTTAGCGCCAGAAGCGCCCTGATCCTGAGAGGATCTGTGGCCCACAAGAGCTGTCTGCCTGCCTGTGTTTATGGCCCTGCCGTGTCCAGCGGCTACAACTTCGAGAAAGAGGGCTACAGCCTCGTCGGCATCGACCCCTTTAAGCTGCTGCAGAACTCCCAGGTGTACAGCCTGATCAGACCCAACGAGAACCCCGCTCACAAGAGCCAGCTTGTGTGGATGGCCTGTCACAGCGCCGCCTTCGAAGATCTGAGACTGCTGAGCTTCATCCGGGGCACAAAGGTGTCCCCAAGAGGCAAGCTGAGCACCAGAGGCGTGCAGATCGCCAGCAACGAGAATATGGACAACATGGAAAGCAGCACACTGGAACTGCGGAGCCGGTACTGGGCCATCAGAACAAGAAGCGGCGGCAACACCAACCAGCAGAGAGCTTCTGCCGGACAGATCAGCGTGCAGCCTACCTTTAGCGTGCAGAGAAACCTGCCTTTCGAGAAGTCCACCGTGATGGCCGCCTTCACCGGCAATACCGAAGGCAGAACCAGCGACATGCGGGCCGAGATCATCAGAATGATGGAAGGCACCAAGCCTGAGGAAGTGTCCTTCAGAGGCAGGGGCGTGTTCGAGCTGTCCGACGAGAAAGCCACCAATCCTATCGTGCCCAGCTTCGACATGAGCAATGAGGGCAGCTACTTCTTCGGCGACAACGCCGAGGAATACGACAACAGCCTGCTGACCGAGGTGGAAACCCCTATCAGAAACGAGTGGGGCTGCAGATGCAACGACAGCAGCGAT
SEQ ID NO. 9mRNA sequence NM2e-Ori
AGGAAAUUCCAUUUGGCUGCAGCUUCUGGAGGGAGCCGACAGGAGACGUGGGGAGACGGCCACC
AUGGCGUCCCAAGGCACCAAACGGUCUUAUGAACAGAUGGAAACUGAUGGGGAACGCCAGAAUGCAACUGAGAUUAGGGCAUCCGUCGGGAAGAUGAUUGAUGGAAUUGGGCGAUUCUACAUCCAAAUGUGCACUGAACUUAAACUCAGUGAUUAUGAAGGGCGGUUGAUCCAGAACAGCUUGACAAUAGAGAGAAUGGUGCUCUCUGCUUUUGAUGAGAGAAGGAAUAGAUAUCUGGAAGAACACCCCAGCGCGGGGAAAGAUCCUAAGAAAACUGGAGGGCCCAUAUACAAGAGAGUAGAUGGAAGAUGGAUGAGGGAACUCGUCCUUUAUGACAAAGAAGAAAUAAGGCGAAUCUGGCGCCAAGCCAACAAUGGUGAUGAUGCGACAGCUGGUCUAACUCACAUGAUGAUCUGGCAUUCCAAUUUGAAUGAUACAACAUACCAGAGGACAAGAGCUCUUGUUCGCACCGGAAUGGACCCCAGGAUGUGCUCUCUGAUGCAGGGUUCGACUCUCCCUAGAAGGUCCGGAGCUGCAGGCGCUGCAGUCAAAGGAAUCGGGACAAUGGUGAUGGAGCUGAUCAGAAUGAUCAAACGGGGGAUCAACGAUCGAAAUUUCUGGAGAGGUGAGAAUGGGCGGAAAACAAGGAGUGCUUAUGAGAGAAUGUGCAACAUUCUUAAAGGAAAAUUUCAAACAGCUGCACAAAGAGCAAUGAUGGAUCAAGUGAGAGAAAGCCGGAACCCAGGAAAUGCUGAGAUCGAAGAUCUCAUAUUUUCGGCAAGAUCUGCACUAAUAUUGAGAGGGUCAGUUGCUCACAAAUCUUGCCUACCUGCCUGUGUGUAUGGACCUGCAGUAUCCAGUGGGUACAACUUCGAAAAAGAGGGAUAUUCCUUAGUGGGAAUAGACCCUUUCAAACUACUUCAAAAUAGCCAAGUAUACAGCCUAAUCAGACCGAACGAGAAUCCAGCACACAAGAGUCAGCUGGUGUGGAUGGCAUGCCAUUCUGCUGCAUUUGAAGAUUUAAGAUUGUUAAGCUUCAUCAGAGGGACCAAAGUAUCUCCGCGGGGGAAACUUUCAACUAGAGGAGUACAAAUUGCUUCAAAUGAGAACAUGGAUAAUAUGGAAUCAAGUACUCUUGAACUGAGAAGCAGGUACUGGGCCAUAAGGACCAGGAGUGGAGGAAACACUAAUCAACAGAGGGCCUCUGCAGGCCAAAUCAGUGUGCAACCUACGUUUUCUGUACAAAGAAACCUCCCAUUUGAAAAAUCAACCGUCAUGGCAGCAUUCACUGGAAAUACGGAGGGAAGAACCUCAGACAUGAGGGCAGAAAUCAUAAGGAUGAUGGAAGGUACAAAACCAGAAGAAGUGUCCUUCCGUGGGCGGGGAGUUUUCGAGCUCUCAGACGAAAAGGCAACGAACCCGAUCGUGCCCUCUUUUGACAUGAGUAAUGAAGGAUCUUAUUUCUUCGGAGACAAUGCAGAAGAGUACGACAAUAGCCUUCUAACCGAGGUCGAAACGCCUAUCAGAAACGAAUGGGGGUGCAGAUGCAACGAUUCAAGUGACUAA
GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGU
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO. 10mRNA sequence NM2e-1
AGGAAAUUCCAUUUGGCUGCAGCUUCUGGAGGGAGCCGACAGGAGACGUGGGGAGACGGCCACC
AUGGCCAGCCAGGGCACCAAGAGGAGCUACGAGCAGAUGGAGACCGACGGCGAGAGACAGAACGCCACCGAGAUCAGGGCCAGCGUGGGCAAGAUGAUCGACGGCAUCGGCAGAUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGAGCGACUACGAGGGCAGGCUGAUCCAGAACAGCCUGACCAUCGAGAGGAUGGUGCUGAGCGCCUUCGACGAGAGGAGAAACAGGUACCUGGAGGAGCACCCCAGCGCCGGCAAGGACCCCAAGAAGACCGGCGGCCCCAUCUACAAGAGGGUGGACGGCAGGUGGAUGAGGGAGCUGGUGCUGUACGACAAGGAGGAGAUCAGGAGGAUCUGGAGGCAGGCCAACAACGGCGACGACGCCACCGCCGGCCUGACCCACAUGAUGAUCUGGCACAGCAACCUGAACGACACCACCUACCAGAGGACCAGGGCCCUGGUGAGAACCGGCAUGGACCCCAGGAUGUGCAGCCUGAUGCAGGGCAGCACCCUGCCCAGGAGAAGCGGCGCCGCCGGCGCCGCCGUGAAGGGCAUCGGCACCAUGGUGAUGGAGCUGAUCAGGAUGAUCAAGAGAGGCAUCAACGACAGGAACUUCUGGAGGGGCGAGAACGGCAGGAAGACCAGAAGCGCCUACGAGAGAAUGUGCAACAUCCUGAAGGGCAAGUUCCAGACCGCCGCCCAGAGGGCCAUGAUGGACCAGGUGAGGGAGAGCAGGAACCCCGGCAACGCCGAGAUCGAGGACCUGAUCUUCAGCGCCAGGAGCGCCCUGAUCCUGAGGGGCAGCGUGGCCCACAAGAGCUGCCUGCCCGCCUGCGUGUACGGCCCCGCCGUGAGCAGCGGCUACAACUUCGAGAAGGAGGGCUACAGCCUGGUGGGCAUCGACCCCUUCAAGCUGCUGCAGAACAGCCAGGUGUACAGCCUGAUCAGGCCCAACGAGAACCCCGCCCACAAGAGCCAGCUGGUGUGGAUGGCCUGCCACAGCGCCGCCUUCGAGGACCUGAGACUGCUGAGCUUCAUCAGGGGCACCAAGGUGAGCCCCAGGGGCAAGCUGAGCACCAGAGGCGUGCAGAUCGCCAGCAACGAGAACAUGGACAACAUGGAGAGCAGCACCCUGGAGCUGAGGAGCAGAUACUGGGCCAUCAGGACCAGGAGCGGCGGCAACACCAACCAGCAGAGGGCCAGCGCCGGCCAGAUCAGCGUGCAGCCCACCUUCAGCGUGCAGAGGAACCUGCCCUUCGAGAAGAGUACCGUGAUGGCCGCCUUCACCGGCAACACCGAGGGCAGGACCAGCGACAUGAGAGCCGAGAUCAUCAGGAUGAUGGAGGGCACCAAGCCCGAGGAGGUGAGCUUCAGGGGCAGAGGCGUGUUCGAGCUGAGCGACGAGAAGGCCACCAACCCCAUCGUGCCCAGCUUCGACAUGAGCAACGAGGGCAGCUACUUCUUCGGCGACAACGCCGAGGAGUACGACAACAGCCUGCUGACCGAGGUGGAGACCCCCAUCAGGAACGAGUGGGGCUGCAGGUGCAA
CGACAGCAGCGACUAA
GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGU
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO. 11mRNA sequence NM2e-2
AGGAAAUUCCAUUUGGCUGCAGCUUCUGGAGGGAGCCGACAGGAGACGUGGGGAGACGGCCACC
AUGGCCAGCCAAGGCACCAAGAGAAGCUACGAGCAGAUGGAGACAGACGGCGAACGGCAGAACGCCACAGAGAUCAGGGCCAGCGUGGGAAAGAUGAUCGAUGGAAUCGGAAGAUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGUCUGAUUACGAGGGCCGGCUGAUCCAGAACUCUCUGACAAUCGAGAGAAUGGUCCUGAGCGCCUUCGACGAGAGACGGAAUAGAUACCUGGAGGAGCACCCCAGCGCCGGCAAGGACCCCAAGAAGACCGGGGGCCCUAUCUACAAGCGGGUGGACGGCAGAUGGAUGAGAGAGCUGGUGCUGUACGACAAGGAAGAAAUCAGACGGAUCUGGAGACAGGCCAACAACGGCGACGACGCCACCGCCGGCCUGACUCACAUGAUGAUCUGGCACAGCAACCUGAACGACACCACCUACCAGCGGACCAGGGCCCUGGUGAGAACAGGCAUGGAUCCUCGUAUGUGCAGCCUGAUGCAGGGCAGCACCCUGCCCAGACGGAGCGGCGCCGCCGGCGCCGCUGUUAAGGGCAUCGGCACAAUGGUGAUGGAGCUGAUUCGGAUGAUCAAGAGGGGCAUCAACGAUAGAAACUUCUGGCGGGGCGAGAACGGCAGAAAGACCAGAUCCGCCUACGAGCGGAUGUGUAACAUCCUGAAAGGCAAGUUCCAGACCGCCGCUCAGAGGGCCAUGAUGGACCAGGUGCGGGAAAGCAGAAACCCCGGCAAUGCCGAGAUCGAGGAUCUGAUCUUCAGCGCCAGAUCCGCCUUAAUUCUGCGGGGAUCUGUGGCCCACAAGUCCUGCCUGCCAGCCUGCGUGUACGGCCCCGCCGUGUCUUCUGGAUACAACUUCGAAAAGGAAGGCUACAGCCUGGUGGGCAUCGACCCUUUUAAGCUGCUGCAGAAUAGCCAAGUGUAUAGCCUGAUUCGGCCUAACGAGAAUCCUGCUCAUAAGAGCCAGCUGGUGUGGAUGGCUUGUCACAGCGCCGCUUUUGAGGACCUGAGACUGCUCAGCUUCAUCAGAGGCACCAAAGUGUCCCCUAGAGGCAAACUGUCCACCCGGGGCGUGCAGAUCGCCAGUAAUGAGAACAUGGACAACAUGGAAUCUUCUACACUUGAACUGAGAUCUAGAUACUGGGCCAUCAGAACCAGAAGCGGCGGAAACACAAACCAGCAGAGAGCUAGCGCCGGACAGAUCAGCGUCCAACCUACAUUCAGCGUGCAGAGAAAUCUGCCUUUCGAAAAAAGCACCGUGAUGGCCGCCUUUACAGGCAACACCGAGGGCAGAACCAGCGAUAUGAGAGCAGAGAUCAUCCGGAUGAUGGAAGGCACCAAGCCUGAGGAAGUGUCUUUCAGAGGACGGGGUGUUUUCGAACUGUCUGACGAGAAAGCCACCAACCCAAUCGUGCCAAGCUUUGAUAUGAGCAACGAGGGAAGCUAUUUCUUCGGCGACAACGCUGAGGAAUACGAUAAUAGCCUGCUGACCGAGGUGGAAACCCCUAUCCGCAACGAGUGGGGCUGCAGAUGCAACGACAGCUCCGACUAA
GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGU
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO. 12mRNA sequence NM2e-3
AGGAAAUUCCAUUUGGCUGCAGCUUCUGGAGGGAGCCGACAGGAGACGUGGGGAGACGGCCACC
AUGGCAUCCCAGGGAACCAAGCGGUCUUACGAGCAGAUGGAGACAGAUGGCGAGAGACAGAACGCCACCGAGAUCAGGGCCUCUGUGGGCAAGAUGAUCGACGGCAUCGGCAGAUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGAGCGAUUAUGAGGGCCGCCUGAUCCAGAACUCCCUGACAAUCGAGCGGAUGGUGCUGUCUGCCUUUGACGAGCGGAGAAAUCGGUACCUGGAGGAGCACCCUAGCGCCGGCAAGGACCCCAAGAAGACCGGAGGCCCCAUCUACAAGAGGGUGGACGGCAGGUGGAUGAGGGAGCUGGUGCUGUAUGAUAAGGAGGAGAUCAGGCGCAUCUGGCGCCAGGCCAACAAUGGCGACGAUGCAACAGCAGGACUGACCCACAUGAUGAUCUGGCACUCCAACCUGAAUGACACCACAUACCAGAGGACACGCGCCCUGGUGAGAACCGGAAUGGACCCCAGGAUGUGCUCCCUGAUGCAGGGCUCUACCCUGCCACGGAGAAGCGGAGCAGCAGGAGCAGCCGUGAAGGGCAUCGGCACAAUGGUCAUGGAGCUGAUCCGGAUGAUCAAGAGAGGCAUCAACGACAGGAAUUUCUGGAGGGGAGAGAACGGAAGGAAGACCAGAUCCGCCUAUGAGAGAAUGUGCAAUAUCCUGAAGGGCAAGUUUCAGACAGCCGCCCAGAGGGCCAUGAUGGACCAGGUGAGGGAGAGCCGCAACCCAGGCAAUGCCGAGAUCGAGGAUCUGAUCUUUUCUGCCCGCAGCGCCCUGAUCCUGAGGGGCAGCGUGGCACACAAGUCCUGCCUGCCUGCAUGCGUGUACGGACCAGCCGUGUCUAGCGGCUACAACUUCGAGAAGGAGGGCUAUUCCCUGGUGGGCAUCGAUCCCUUUAAGCUGCUGCAGAACAGCCAGGUGUAUUCUCUGAUCAGGCCAAACGAGAAUCCCGCCCACAAGAGCCAGCUGGUGUGGAUGGCAUGCCACUCCGCCGCCUUCGAGGACCUGAGACUGCUGUCCUUUAUCAGGGGCACAAAGGUGAGCCCUCGCGGCAAGCUGUCCACCAGAGGCGUGCAGAUCGCCUCUAACGAGAAUAUGGAUAACAUGGAGUCCUCUACCCUGGAGCUGCGGUCUAGAUACUGGGCCAUCAGGACACGCAGCGGCGGCAACACCAAUCAGCAGAGGGCAUCUGCCGGACAGAUCAGCGUGCAGCCAACAUUCUCCGUGCAGCGGAACCUGCCCUUUGAGAAGUCUACCGUGAUGGCCGCCUUCACAGGCAAUACCGAGGGCCGGACAAGCGACAUGAGAGCCGAGAUCAUCAGGAUGAUGGAGGGCACCAAGCCUGAGGAGGUGAGCUUCCGGGGCAGAGGCGUGUUUGAGCUGUCCGACGAGAAGGCCACAAACCCCAUCGUGCCUAGCUUUGAUAUGUCCAAUGAGGGCUCUUACUUCUUUGGCGACAACGCCGAGGAGUAUGAUAAUUCCCUGCUGACAGAGGUGGAGACCCCUAUCCGCAACGAGUGGGGCUGCCGGUGUAA
UGACAGCUCCGAUUAA
GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGU
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO. 13mRNA sequence NM2e-4
AGGAAAUUCCAUUUGGCUGCAGCUUCUGGAGGGAGCCGACAGGAGACGUGGGGAGACGGCCACC
AUGGCCUCUCAGGGCACCAAGAGAAGCUACGAGCAGAUGGAAACCGACGGCGAGAGACAGAACGCCACCGAGAUUAGAGCCAGCGUGGGCAAGAUGAUCGACGGCAUCGGCCGGUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGAGCGACUACGAGGGCAGACUGAUCCAGAACAGCCUGACCAUCGAGCGGAUGGUGCUGAGCGCCUUCGACGAGCGGAGAAACAGAUACCUGGAAGAACACCCCAGCGCCGGCAAGGACCCCAAGAAAACAGGCGGCCCUAUCUACAAGCGCGUGGACGGCAGAUGGAUGAGAGAACUGGUGCUGUACGACAAAGAGGAAAUCCGGCGGAUCUGGCGGCAGGCCAACAAUGGGGAUGAUGCCACAGCCGGCCUGACACACAUGAUGAUCUGGCACAGCAACCUGAACGACACCACCUACCAGCGGACAAGAGCCCUCGUCAGAACCGGCAUGGACCCUAGAAUGUGCAGCCUGAUGCAGGGCAGCACCCUGCCUAGAAGAUCUGGUGCUGCUGGCGCUGCCGUGAAAGGCAUCGGCACAAUGGUCAUGGAACUGAUCCGGAUGAUCAAGCGGGGAAUCAACGACCGGAACUUUUGGAGAGGCGAGAACGGCAGAAAGACCCGCAGCGCCUACGAGAGGAUGUGCAAUAUCCUGAAGGGCAAGUUCCAGACCGCCGCUCAGAGGGCCAUGAUGGAUCAAGUGCGCGAGAGCAGAAACCCCGGCAAUGCCGAGAUCGAGGACCUGAUCUUUAGCGCCAGAAGCGCCCUGAUCCUGAGAGGAUCUGUGGCCCACAAGAGCUGUCUGCCUGCCUGUGUUUAUGGCCCUGCCGUGUCCAGCGGCUACAACUUCGAGAAAGAGGGCUACAGCCUCGUCGGCAUCGACCCCUUUAAGCUGCUGCAGAACUCCCAGGUGUACAGCCUGAUCAGACCCAACGAGAACCCCGCUCACAAGAGCCAGCUUGUGUGGAUGGCCUGUCACAGCGCCGCCUUCGAAGAUCUGAGACUGCUGAGCUUCAUCCGGGGCACAAAGGUGUCCCCAAGAGGCAAGCUGAGCACCAGAGGCGUGCAGAUCGCCAGCAACGAGAAUAUGGACAACAUGGAAAGCAGCACACUGGAACUGCGGAGCCGGUACUGGGCCAUCAGAACAAGAAGCGGCGGCAACACCAACCAGCAGAGAGCUUCUGCCGGACAGAUCAGCGUGCAGCCUACCUUUAGCGUGCAGAGAAACCUGCCUUUCGAGAAGUCCACCGUGAUGGCCGCCUUCACCGGCAAUACCGAAGGCAGAACCAGCGACAUGCGGGCCGAGAUCAUCAGAAUGAUGGAAGGCACCAAGCCUGAGGAAGUGUCCUUCAGAGGCAGGGGCGUGUUCGAGCUGUCCGACGAGAAAGCCACCAAUCCUAUCGUGCCCAGCUUCGACAUGAGCAAUGAGGGCAGCUACUUCUUCGGCGACAACGCCGAGGAAUACGACAACAGCCUGCUGACCGAGGUGGAAACCCCUAUCAGAAACGAGUGGGGCUGCAGAUGCAACGACAGCAGCGAUUAA
GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGU
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO. 14 non-optimized coding RNA sequence NM2e-Ori
AUGGCGUCCCAAGGCACCAAACGGUCUUAUGAACAGAUGGAAACUGAUGGGGAACGCCAGAAUGCAACUGAGAUUAGGGCAUCCGUCGGGAAGAUGAUUGAUGGAAUUGGGCGAUUCUACAUCCAAAUGUGCACUGAACUUAAACUCAGUGAUUAUGAAGGGCGGUUGAUCCAGAACAGCUUGACAAUAGAGAGAAUGGUGCUCUCUGCUUUUGAUGAGAGAAGGAAUAGAUAUCUGGAAGAACACCCCAGCGCGGGGAAAGAUCCUAAGAAAACUGGAGGGCCCAUAUACAAGAGAGUAGAUGGAAGAUGGAUGAGGGAACUCGUCCUUUAUGACAAAGAAGAAAUAAGGCGAAUCUGGCGCCAAGCCAACAAUGGUGAUGAUGCGACAGCUGGUCUAACUCACAUGAUGAUCUGGCAUUCCAAUUUGAAUGAUACAACAUACCAGAGGACAAGAGCUCUUGUUCGCACCGGAAUGGACCCCAGGAUGUGCUCUCUGAUGCAGGGUUCGACUCUCCCUAGAAGGUCCGGAGCUGCAGGCGCUGCAGUCAAAGGAAUCGGGACAAUGGUGAUGGAGCUGAUCAGAAUGAUCAAACGGGGGAUCAACGAUCGAAAUUUCUGGAGAGGUGAGAAUGGGCGGAAAACAAGGAGUGCUUAUGAGAGAAUGUGCAACAUUCUUAAAGGAAAAUUUCAAACAGCUGCACAAAGAGCAAUGAUGGAUCAAGUGAGAGAAAGCCGGAACCCAGGAAAUGCUGAGAUCGAAGAUCUCAUAUUUUCGGCAAGAUCUGCACUAAUAUUGAGAGGGUCAGUUGCUCACAAAUCUUGCCUACCUGCCUGUGUGUAUGGACCUGCAGUAUCCAGUGGGUACAACUUCGAAAAAGAGGGAUAUUCCUUAGUGGGAAUAGACCCUUUCAAACUACUUCAAAAUAGCCAAGUAUACAGCCUAAUCAGACCGAACGAGAAUCCAGCACACAAGAGUCAGCUGGUGUGGAUGGCAUGCCAUUCUGCUGCAUUUGAAGAUUUAAGAUUGUUAAGCUUCAUCAGAGGGACCAAAGUAUCUCCGCGGGGGAAACUUUCAACUAGAGGAGUACAAAUUGCUUCAAAUGAGAACAUGGAUAAUAUGGAAUCAAGUACUCUUGAACUGAGAAGCAGGUACUGGGCCAUAAGGACCAGGAGUGGAGGAAACACUAAUCAACAGAGGGCCUCUGCAGGCCAAAUCAGUGUGCAACCUACGUUUUCUGUACAAAGAAACCUCCCAUUUGAAAAAUCAACCGUCAUGGCAGCAUUCACUGGAAAUACGGAGGGAAGAACCUCAGACAUGAGGGCAGAAAUCAUAAGGAUGAUGGAAGGUACAAAACCAGAAGAAGUGUCCUUCCGUGGGCGGGGAGUUUUCGAGCUCUCAGACGAAAAGGCAACGAACCCGAUCGUGCCCUCUUUUGACAUGAGUAAUGAAGGAUCUUAUUUCUUCGGAGACAAUGCAGAAGAGUACGACAAUAGCCUUCUAACCGAGGUCGAAACGCCUAUCAGAAACGAAUGGGGGUGCAGAUGCAACGAUUCAAGUGAC
SEQ ID NO. 15 optimized coding RNA sequence NM2e-1
AUGGCCAGCCAGGGCACCAAGAGGAGCUACGAGCAGAUGGAGACCGACGGCGAGAGACAGAACGCCACCGAGAUCAGGGCCAGCGUGGGCAAGAUGAUCGACGGCAUCGGCAGAUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGAGCGACUACGAGGGCAGGCUGAUCCAGAACAGCCUGACCAUCGAGAGGAUGGUGCUGAGCGCCUUCGACGAGAGGAGAAACAGGUACCUGGAGGAGCACCCCAGCGCCGGCAAGGACCCCAAGAAGACCGGCGGCCCCAUCUACAAGAGGGUGGACGGCAGGUGGAUGAGGGAGCUGGUGCUGUACGACAAGGAGGAGAUCAGGAGGAUCUGGAGGCAGGCCAACAACGGCGACGACGCCACCGCCGGCCUGACCCACAUGAUGAUCUGGCACAGCAACCUGAACGACACCACCUACCAGAGGACCAGGGCCCUGGUGAGAACCGGCAUGGACCCCAGGAUGUGCAGCCUGAUGCAGGGCAGCACCCUGCCCAGGAGAAGCGGCGCCGCCGGCGCCGCCGUGAAGGGCAUCGGCACCAUGGUGAUGGAGCUGAUCAGGAUGAUCAAGAGAGGCAUCAACGACAGGAACUUCUGGAGGGGCGAGAACGGCAGGAAGACCAGAAGCGCCUACGAGAGAAUGUGCAACAUCCUGAAGGGCAAGUUCCAGACCGCCGCCCAGAGGGCCAUGAUGGACCAGGUGAGGGAGAGCAGGAACCCCGGCAACGCCGAGAUCGAGGACCUGAUCUUCAGCGCCAGGAGCGCCCUGAUCCUGAGGGGCAGCGUGGCCCACAAGAGCUGCCUGCCCGCCUGCGUGUACGGCCCCGCCGUGAGCAGCGGCUACAACUUCGAGAAGGAGGGCUACAGCCUGGUGGGCAUCGACCCCUUCAAGCUGCUGCAGAACAGCCAGGUGUACAGCCUGAUCAGGCCCAACGAGAACCCCGCCCACAAGAGCCAGCUGGUGUGGAUGGCCUGCCACAGCGCCGCCUUCGAGGACCUGAGACUGCUGAGCUUCAUCAGGGGCACCAAGGUGAGCCCCAGGGGCAAGCUGAGCACCAGAGGCGUGCAGAUCGCCAGCAACGAGAACAUGGACAACAUGGAGAGCAGCACCCUGGAGCUGAGGAGCAGAUACUGGGCCAUCAGGACCAGGAGCGGCGGCAACACCAACCAGCAGAGGGCCAGCGCCGGCCAGAUCAGCGUGCAGCCCACCUUCAGCGUGCAGAGGAACCUGCCCUUCGAGAAGAGUACCGUGAUGGCCGCCUUCACCGGCAACACCGAGGGCAGGACCAGCGACAUGAGAGCCGAGAUCAUCAGGAUGAUGGAGGGCACCAAGCCCGAGGAGGUGAGCUUCAGGGGCAGAGGCGUGUUCGAGCUGAGCGACGAGAAGGCCACCAACCCCAUCGUGCCCAGCUUCGACAUGAGCAACGAGGGCAGCUACUUCUUCGGCGACAACGCCGAGGAGUACGACAACAGCCUGCUGACCGAGGUGGAGACCCCCAUCAGGAACGAGUGGGGCUGCAGGUGCAACGACAGCAGCGAC
SEQ ID NO. 16 optimized coding RNA sequence NM2e-2
AUGGCCAGCCAAGGCACCAAGAGAAGCUACGAGCAGAUGGAGACAGACGGCGAACGGCAGAACGCCACAGAGAUCAGGGCCAGCGUGGGAAAGAUGAUCGAUGGAAUCGGAAGAUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGUCUGAUUACGAGGGCCGGCUGAUCCAGAACUCUCUGACAAUCGAGAGAAUGGUCCUGAGCGCCUUCGACGAGAGACGGAAUAGAUACCUGGAGGAGCACCCCAGCGCCGGCAAGGACCCCAAGAAGACCGGGGGCCCUAUCUACAAGCGGGUGGACGGCAGAUGGAUGAGAGAGCUGGUGCUGUACGACAAGGAAGAAAUCAGACGGAUCUGGAGACAGGCCAACAACGGCGACGACGCCACCGCCGGCCUGACUCACAUGAUGAUCUGGCACAGCAACCUGAACGACACCACCUACCAGCGGACCAGGGCCCUGGUGAGAACAGGCAUGGAUCCUCGUAUGUGCAGCCUGAUGCAGGGCAGCACCCUGCCCAGACGGAGCGGCGCCGCCGGCGCCGCUGUUAAGGGCAUCGGCACAAUGGUGAUGGAGCUGAUUCGGAUGAUCAAGAGGGGCAUCAACGAUAGAAACUUCUGGCGGGGCGAGAACGGCAGAAAGACCAGAUCCGCCUACGAGCGGAUGUGUAACAUCCUGAAAGGCAAGUUCCAGACCGCCGCUCAGAGGGCCAUGAUGGACCAGGUGCGGGAAAGCAGAAACCCCGGCAAUGCCGAGAUCGAGGAUCUGAUCUUCAGCGCCAGAUCCGCCUUAAUUCUGCGGGGAUCUGUGGCCCACAAGUCCUGCCUGCCAGCCUGCGUGUACGGCCCCGCCGUGUCUUCUGGAUACAACUUCGAAAAGGAAGGCUACAGCCUGGUGGGCAUCGACCCUUUUAAGCUGCUGCAGAAUAGCCAAGUGUAUAGCCUGAUUCGGCCUAACGAGAAUCCUGCUCAUAAGAGCCAGCUGGUGUGGAUGGCUUGUCACAGCGCCGCUUUUGAGGACCUGAGACUGCUCAGCUUCAUCAGAGGCACCAAAGUGUCCCCUAGAGGCAAACUGUCCACCCGGGGCGUGCAGAUCGCCAGUAAUGAGAACAUGGACAACAUGGAAUCUUCUACACUUGAACUGAGAUCUAGAUACUGGGCCAUCAGAACCAGAAGCGGCGGAAACACAAACCAGCAGAGAGCUAGCGCCGGACAGAUCAGCGUCCAACCUACAUUCAGCGUGCAGAGAAAUCUGCCUUUCGAAAAAAGCACCGUGAUGGCCGCCUUUACAGGCAACACCGAGGGCAGAACCAGCGAUAUGAGAGCAGAGAUCAUCCGGAUGAUGGAAGGCACCAAGCCUGAGGAAGUGUCUUUCAGAGGACGGGGUGUUUUCGAACUGUCUGACGAGAAAGCCACCAACCCAAUCGUGCCAAGCUUUGAUAUGAGCAACGAGGGAAGCUAUUUCUUCGGCGACAACGCUGAGGAAUACGAUAAUAGCCUGCUGACCGAGGUGGAAACCCCUAUCCGCAACGAGUGGGGCUGCAGAUGCAACGACAGCUCCGAC
SEQ ID NO. 17 optimized coding RNA sequence NM2e-3
AUGGCAUCCCAGGGAACCAAGCGGUCUUACGAGCAGAUGGAGACAGAUGGCGAGAGACAGAACGCCACCGAGAUCAGGGCCUCUGUGGGCAAGAUGAUCGACGGCAUCGGCAGAUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGAGCGAUUAUGAGGGCCGCCUGAUCCAGAACUCCCUGACAAUCGAGCGGAUGGUGCUGUCUGCCUUUGACGAGCGGAGAAAUCGGUACCUGGAGGAGCACCCUAGCGCCGGCAAGGACCCCAAGAAGACCGGAGGCCCCAUCUACAAGAGGGUGGACGGCAGGUGGAUGAGGGAGCUGGUGCUGUAUGAUAAGGAGGAGAUCAGGCGCAUCUGGCGCCAGGCCAACAAUGGCGACGAUGCAACAGCAGGACUGACCCACAUGAUGAUCUGGCACUCCAACCUGAAUGACACCACAUACCAGAGGACACGCGCCCUGGUGAGAACCGGAAUGGACCCCAGGAUGUGCUCCCUGAUGCAGGGCUCUACCCUGCCACGGAGAAGCGGAGCAGCAGGAGCAGCCGUGAAGGGCAUCGGCACAAUGGUCAUGGAGCUGAUCCGGAUGAUCAAGAGAGGCAUCAACGACAGGAAUUUCUGGAGGGGAGAGAACGGAAGGAAGACCAGAUCCGCCUAUGAGAGAAUGUGCAAUAUCCUGAAGGGCAAGUUUCAGACAGCCGCCCAGAGGGCCAUGAUGGACCAGGUGAGGGAGAGCCGCAACCCAGGCAAUGCCGAGAUCGAGGAUCUGAUCUUUUCUGCCCGCAGCGCCCUGAUCCUGAGGGGCAGCGUGGCACACAAGUCCUGCCUGCCUGCAUGCGUGUACGGACCAGCCGUGUCUAGCGGCUACAACUUCGAGAAGGAGGGCUAUUCCCUGGUGGGCAUCGAUCCCUUUAAGCUGCUGCAGAACAGCCAGGUGUAUUCUCUGAUCAGGCCAAACGAGAAUCCCGCCCACAAGAGCCAGCUGGUGUGGAUGGCAUGCCACUCCGCCGCCUUCGAGGACCUGAGACUGCUGUCCUUUAUCAGGGGCACAAAGGUGAGCCCUCGCGGCAAGCUGUCCACCAGAGGCGUGCAGAUCGCCUCUAACGAGAAUAUGGAUAACAUGGAGUCCUCUACCCUGGAGCUGCGGUCUAGAUACUGGGCCAUCAGGACACGCAGCGGCGGCAACACCAAUCAGCAGAGGGCAUCUGCCGGACAGAUCAGCGUGCAGCCAACAUUCUCCGUGCAGCGGAACCUGCCCUUUGAGAAGUCUACCGUGAUGGCCGCCUUCACAGGCAAUACCGAGGGCCGGACAAGCGACAUGAGAGCCGAGAUCAUCAGGAUGAUGGAGGGCACCAAGCCUGAGGAGGUGAGCUUCCGGGGCAGAGGCGUGUUUGAGCUGUCCGACGAGAAGGCCACAAACCCCAUCGUGCCUAGCUUUGAUAUGUCCAAUGAGGGCUCUUACUUCUUUGGCGACAACGCCGAGGAGUAUGAUAAUUCCCUGCUGACAGAGGUGGAGACCCCUAUCCGCAACGAGUGGGGCUGCCGGUGUAAUGACAGCUCCGAU
SEQ ID NO. 18 optimized coding RNA sequence NM2e-4
AUGGCCUCUCAGGGCACCAAGAGAAGCUACGAGCAGAUGGAAACCGACGGCGAGAGACAGAACGCCACCGAGAUUAGAGCCAGCGUGGGCAAGAUGAUCGACGGCAUCGGCCGGUUCUACAUCCAGAUGUGCACCGAGCUGAAGCUGAGCGACUACGAGGGCAGACUGAUCCAGAACAGCCUGACCAUCGAGCGGAUGGUGCUGAGCGCCUUCGACGAGCGGAGAAACAGAUACCUGGAAGAACACCCCAGCGCCGGCAAGGACCCCAAGAAAACAGGCGGCCCUAUCUACAAGCGCGUGGACGGCAGAUGGAUGAGAGAACUGGUGCUGUACGACAAAGAGGAAAUCCGGCGGAUCUGGCGGCAGGCCAACAAUGGGGAUGAUGCCACAGCCGGCCUGACACACAUGAUGAUCUGGCACAGCAACCUGAACGACACCACCUACCAGCGGACAAGAGCCCUCGUCAGAACCGGCAUGGACCCUAGAAUGUGCAGCCUGAUGCAGGGCAGCACCCUGCCUAGAAGAUCUGGUGCUGCUGGCGCUGCCGUGAAAGGCAUCGGCACAAUGGUCAUGGAACUGAUCCGGAUGAUCAAGCGGGGAAUCAACGACCGGAACUUUUGGAGAGGCGAGAACGGCAGAAAGACCCGCAGCGCCUACGAGAGGAUGUGCAAUAUCCUGAAGGGCAAGUUCCAGACCGCCGCUCAGAGGGCCAUGAUGGAUCAAGUGCGCGAGAGCAGAAACCCCGGCAAUGCCGAGAUCGAGGACCUGAUCUUUAGCGCCAGAAGCGCCCUGAUCCUGAGAGGAUCUGUGGCCCACAAGAGCUGUCUGCCUGCCUGUGUUUAUGGCCCUGCCGUGUCCAGCGGCUACAACUUCGAGAAAGAGGGCUACAGCCUCGUCGGCAUCGACCCCUUUAAGCUGCUGCAGAACUCCCAGGUGUACAGCCUGAUCAGACCCAACGAGAACCCCGCUCACAAGAGCCAGCUUGUGUGGAUGGCCUGUCACAGCGCCGCCUUCGAAGAUCUGAGACUGCUGAGCUUCAUCCGGGGCACAAAGGUGUCCCCAAGAGGCAAGCUGAGCACCAGAGGCGUGCAGAUCGCCAGCAACGAGAAUAUGGACAACAUGGAAAGCAGCACACUGGAACUGCGGAGCCGGUACUGGGCCAUCAGAACAAGAAGCGGCGGCAACACCAACCAGCAGAGAGCUUCUGCCGGACAGAUCAGCGUGCAGCCUACCUUUAGCGUGCAGAGAAACCUGCCUUUCGAGAAGUCCACCGUGAUGGCCGCCUUCACCGGCAAUACCGAAGGCAGAACCAGCGACAUGCGGGCCGAGAUCAUCAGAAUGAUGGAAGGCACCAAGCCUGAGGAAGUGUCCUUCAGAGGCAGGGGCGUGUUCGAGCUGUCCGACGAGAAAGCCACCAAUCCUAUCGUGCCCAGCUUCGACAUGAGCAAUGAGGGCAGCUACUUCUUCGGCGACAACGCCGAGGAAUACGACAACAGCCUGCUGACCGAGGUGGAAACCCCUAUCAGAAACGAGUGGGGCUGCAGAUGCAACGACAGCAGCGAU
SEQ ID NO. 19' UTR DNA sequence
AGGAAATTCCATTTGGCTGCAGCTTCTGGAGGGAGCCGACAGGAGACGTGGGGAGACGGCCACC
SEQ ID NO. 20' UTR DNA sequence
GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGT
Claims (23)
1. A polynucleotide comprising a nucleotide sequence encoding a fusion protein of SEQ ID No. 1, wherein said nucleotide sequence has at least 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID nos. 5, 6, 7, 8, 15, 16, 17 and 18.
2. The polynucleotide of claim 1 which is RNA.
3. The polynucleotide of claim 2, wherein the RNA is mRNA.
4. The polynucleotide of claim 3, wherein the mRNA further comprises a 5'utr, a 3' utr, and a polyA.
5. The polynucleotide of claim 4, wherein the 5' UTR comprises the nucleotide sequence of SEQ ID NO. 2.
6. The polynucleotide of claim 4, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO. 3.
7. The polynucleotide of claim 4, wherein the polyA comprises 75 adenylate residues.
8. The polynucleotide according to any one of claims 1-7 comprising a nucleotide sequence having at least 80% identity to one of SEQ ID NOs 10-13.
9. A composition comprising the polynucleotide of any one of claims 1-8.
10. The composition of claim 9, comprising a lipid encapsulating the polynucleotide.
11. The composition of claim 9 or 10, comprising a lipopolysaccharide complex.
12. The composition of claim 10 or 11, wherein the lipid encapsulating the polynucleotide comprises a cationic lipid, a non-cationic lipid, and a polyethylene glycol modified lipid; optionally, the composition further comprises a cationic polymer, wherein the cationic polymer associates with the polynucleotide as a complex, and is co-encapsulated in a lipid to form a lipopolysaccharide complex.
13. The composition of claim 12, wherein the cationic polymer is selected from the group consisting of poly-L-lysine, protamine, and polyethylenimine; preferably, the cationic polymer is protamine.
14. A vaccine formulation comprising the polynucleotide of any one of claims 1-8 or the composition of any one of claims 9-13.
15. The vaccine formulation of claim 14, wherein the lipid encapsulating the polynucleotide comprises 10-70 mole% M5, 10-70 mole% 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 10-70 mole% cholesterol, and 0.05-20 mole% 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) 2000; preferably, the lipids are M5, DOPE, cholesterol and (DMG-PEG) 2000 in a molar ratio of 40:15:43.5:1.5,
16. The vaccine formulation of claim 14 or 15, wherein the vaccine formulation is a liquid formulation or a lyophilized formulation.
17. The vaccine formulation of any one of claims 14-16, wherein the vaccine formulation is administered by intramuscular injection or intramuscularly.
18. Use of the polynucleotide of any one of claims 1-8, the composition of any one of claims 9-13, or the vaccine formulation of any one of claims 14-17 in the manufacture of a medicament for preventing and/or treating influenza virus infection in a subject in need thereof.
19. The use of claim 18, wherein the subject is a human or non-human animal.
20. A method of preventing or treating an influenza virus infection in a subject in need thereof, the method comprising:
Administering to a subject in need thereof a polynucleotide according to any one of claims 1 to 8, a composition according to any one of claims 9 to 13, or a vaccine formulation according to any one of claims 14 to 17.
21. The method of claim 20, wherein the subject in need thereof is a human or non-human animal.
22. Use of the polynucleotide of any one of claims 1-8, the composition of any one of claims 9-13, or the vaccine formulation of any one of claims 14-17 for preventing or treating an influenza virus infection in a subject in need thereof.
23. The polynucleotide, composition or vaccine formulation for use of claim 22, wherein the subject is a human or non-human animal.
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CN202211634439.0A CN118256522A (en) | 2022-12-19 | 2022-12-19 | Broad-spectrum influenza mRNA vaccine |
PCT/CN2023/139522 WO2024131726A1 (en) | 2022-12-19 | 2023-12-18 | Broad-spectrum influenza mrna vaccine |
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CN202211634439.0A CN118256522A (en) | 2022-12-19 | 2022-12-19 | Broad-spectrum influenza mRNA vaccine |
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Family Cites Families (6)
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CN101899461B (en) * | 2010-05-14 | 2012-10-03 | 中国疾病预防控制中心病毒病预防控制所 | Fusion gene capable of encoding influenza A virus NP protein and M2e polypeptide |
EP3103474A1 (en) * | 2015-06-12 | 2016-12-14 | Institut Pasteur | Live recombinant measles-m2 virus and its use in eliciting immunity against influenza viruses |
WO2017070620A2 (en) * | 2015-10-22 | 2017-04-27 | Modernatx, Inc. | Broad spectrum influenza virus vaccine |
US11389525B2 (en) * | 2017-10-27 | 2022-07-19 | Statens Serum Institut | Polygene influenza vaccine |
MX2022012251A (en) * | 2020-03-31 | 2023-03-13 | Univ Pennsylvania | Universal influenza vaccine using nucleoside-modifield mrna. |
CN116751306A (en) * | 2023-03-17 | 2023-09-15 | 上海生物制品研究所有限责任公司 | mRNA vaccine for encoding universal influenza and application thereof |
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