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WO2024131726A1 - Vaccin antigrippal à arnm et à large spectre - Google Patents

Vaccin antigrippal à arnm et à large spectre Download PDF

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Publication number
WO2024131726A1
WO2024131726A1 PCT/CN2023/139522 CN2023139522W WO2024131726A1 WO 2024131726 A1 WO2024131726 A1 WO 2024131726A1 CN 2023139522 W CN2023139522 W CN 2023139522W WO 2024131726 A1 WO2024131726 A1 WO 2024131726A1
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Prior art keywords
polynucleotide
sequence
rna
composition
lipid
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PCT/CN2023/139522
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English (en)
Chinese (zh)
Inventor
方熠
易应磊
尹曼曼
黄雷
沈明云
沈海法
李航文
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斯微(上海)生物科技股份有限公司
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Publication of WO2024131726A1 publication Critical patent/WO2024131726A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • 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
    • A—HUMAN NECESSITIES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the fields of biomedicine and virology, and in particular to an mRNA vaccine for preventing or treating influenza virus infection.
  • Influenza poses a serious threat to global public health and causes serious harm to human health and the world economy.
  • Vaccination is one of the most effective measures to prevent influenza.
  • Current influenza vaccines include split vaccines, whole virus inactivated vaccines, live attenuated vaccines, subunit vaccines, etc., which mainly play a protective role by inducing specific neutralizing antibodies against hemagglutinin (HA) and neuraminidase (NA) on the influenza virus envelope.
  • HA hemagglutinin
  • NA neuraminidase
  • the HA type of influenza virus is variable, and the antigenic drift and antigenic shift of HA often lead to seasonal and pandemic influenza.
  • seasonal influenza vaccines mainly target HA and NA antigens, and new vaccines need to be prepared almost every year based on predicted strains.
  • a broad-spectrum influenza vaccine such as vaccines targeting conserved antigens of influenza viruses, such as matrix protein 2 extracellular domain (Matrix 2 extracellular domain, M2e), matrix protein 1 (Matrix 1, M1) and nucleoprotein (nucleoprotein, NP).
  • CN101899461B discloses a fusion gene encoding influenza A virus NP protein and M2e polypeptide.
  • the influenza A virus NP and M2e fusion protein NM2e can be efficiently expressed in Escherichia coli, and the purified NM2e fusion protein is used to prepare a protein subunit vaccine.
  • the present 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 with a nucleotide sequence selected from 5, 6, 7, 8, 15, 16, 17 and 18.
  • the polynucleotide is RNA.
  • the RNA is mRNA.
  • the mRNA further comprises a 5'UTR, a 3'UTR and polyA.
  • the 5'UTR comprises a nucleotide sequence of SEQ ID NO:2.
  • the 3'UTR comprises a nucleotide sequence of SEQ ID NO:3.
  • the polyA comprises 75 adenylate residues.
  • the polynucleotide comprises a nucleotide sequence that is at least 80% identical to one of SEQ ID NO:10-13.
  • the present invention provides a composition comprising a polynucleotide of the present invention.
  • the composition comprises a lipid encapsulating the polynucleotide.
  • the composition comprises a lipid
  • the lipids encapsulating the polynucleotides include cationic lipids, non-cationic lipids and polyethylene glycol-modified lipids; optionally, the composition further includes a cationic polymer, wherein the cationic polymer is associated with the polynucleotides as a complex, and are co-encapsulated in the lipids to form a lipid polymer complex.
  • the present invention provides a vaccine formulation comprising a polynucleotide or composition of the present invention.
  • the lipid encapsulating the polynucleotide in the vaccine formulation comprises 10-70 mol% of M5, 10-70 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 10-70 mol% of cholesterol and 0.05-20 mol% of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) 2000; preferably, the lipid is M5, DOPE, cholesterol and (DMG-PEG) 2000 in a molar ratio of 40:15:43.5:1.5,
  • 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 intramucosally, such as a nasal spray.
  • the present invention provides a method for preventing or treating influenza virus infection in a subject in need thereof, the method comprising administering the polynucleotide, composition or vaccine formulation of the present invention to a subject in need thereof.
  • the present invention also provides use of the polynucleotide, composition or vaccine preparation of the present invention in the preparation of a medicament for preventing and/or treating influenza virus infection in a subject in need thereof.
  • the present invention also provides the polynucleotide, composition or vaccine formulation of the present invention in preparation for use in preventing and/or treating influenza virus infection in a subject in need thereof.
  • the subject is a human or a non-human animal.
  • FIG1 shows a flow chart for constructing lipid polyplexes (LPPs) of mRNA.
  • Figure 2 shows the results of western blot analysis of the expression of NP protein (Figure 2A) and M2e protein (Figure 2B) in 293T cells transfected with LPP preparations, as well as the titer of antibodies against NM2e in mice immunized with different doses (1 and 10 ⁇ g) of LPP.
  • Figure 3 shows the anti-NP protein and anti-M2e IgG titers induced by LPP preparation in mice.
  • Figures 4 and 5 show the cellular immune responses against NP protein and M2e induced by LPP preparations in mice.
  • FIG6 shows the results of the challenge experiment in which mice were immunized with LPP preparations and then challenged with influenza virus strains X31 ( FIG6A ), PR8 ( FIG6B ), and AH ( FIG6C ).
  • the expressions “comprises,” “comprising,” “containing,” and “having” are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components.
  • the expression “consisting of” excludes any element, step, or component not specified.
  • the expression “consisting essentially of” means that the scope is limited to the specified elements, steps, or components, plus 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.”
  • wild type means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.
  • % identity refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared.
  • the differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared.
  • the identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window".
  • Optimal alignment can be performed manually or with the aid of algorithms known in the art, including but not limited to the local homology algorithm 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.
  • the percent identity of two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the website of the National Center for Biotechnology Information (NCBI).
  • % 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.
  • a degree of identity is given for a region of 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%.
  • a degree of identity is given for the entire length of a reference sequence. The degree of identity can be given using tools known in the art.
  • An alignment to determine sequence identity is performed, preferably using optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • nucleotide includes deoxyribonucleotides and ribonucleotides and their derivatives.
  • ribonucleotide is a constituent substance of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide with a hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group.
  • Deoxyribonucleotide is a constituent substance of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes.
  • DNA deoxyribonucleic acid
  • Nucleotide is usually referred to by a single letter representing the base: "A (a)” refers to deoxyadenosine or adenylic acid containing adenine, “C (c)” refers to deoxycytidine or cytidine containing cytosine, “G (g)” refers to deoxyguanosine or guanylate containing guanine, “U (u)” refers to uridine containing uracil, and “T (t)” refers to deoxythymidylate containing thymine.
  • 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 and “nucleotide sequence” are used interchangeably to refer to the order of nucleotides in a polynucleotide.
  • DNA coding strand sense strand
  • RNA it encodes can be considered to have the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.
  • coding sequence refers to a nucleotide sequence in a polynucleotide that can be used as a template for synthesizing a nucleotide sequence having a determined nucleotide sequence (e.g., tRNA and mRNA) or a determined amino acid sequence in a biological process.
  • the coding sequence can be a DNA sequence or an RNA sequence. If the mRNA corresponding to the DNA sequence (including a coding strand identical to the mRNA sequence and a template strand complementary thereto) is translated into a polypeptide in a biological process, it can be considered that the DNA sequence or mRNA sequence encodes the polypeptide.
  • cognid refers to three consecutive nucleotide sequences (also known as triplet codes) in a polynucleotide, which encode a specific amino acid. Synonymous codons (codons encoding the same amino acid) are used at different frequencies in different species, which is called “codon preference”. It is generally believed that for a given species, the coding sequence using its preferred codon can have higher translation efficiency and accuracy in the species expression system. Therefore, polynucleotides can be "codon optimized", that is, the codons in the polynucleotides are changed to reflect the codons preferred by the host cell, and preferably the amino acid sequence encoded by it is not changed.
  • the polynucleotides of the present invention may include such coding sequences, which are different from the coding sequences described herein (e.g., having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homogeneity with the coding sequences described herein) but encode the same amino acid sequence.
  • the RNA of the invention comprises codons optimized for host (eg, subject, particularly mammalian) cells, such that the polypeptide of the invention is optimally expressed in the subject, such as a mammal, bird or human.
  • the term "expression” includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides.
  • transcription refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript).
  • in vitro transcription refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation.
  • transcripts A vector for producing transcripts is also called a "transcription vector", which contains regulatory sequences required for transcription.
  • transcription encompasses "in vitro transcription”.
  • encoding refers to the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA can be used as a template to synthesize polymers and macromolecules in other biological processes, as long as there is a clear nucleotide sequence or a clear amino acid sequence. Therefore, a gene encodes a protein when the gene's mRNA produces a protein in a cell or other biological system through transcription and translation.
  • 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 covalently or non-covalently. Unless otherwise indicated, “polypeptide” and “protein” may be used interchangeably.
  • the term "host cell” refers to a cell used to receive, maintain, replicate, express a polynucleotide or vector.
  • the host cell may be a cell in which a polypeptide of the present invention is expressed.
  • antigen refers to a molecule that can cause an acquired immune response in the body after entering the body. This immune response may involve the production of antibodies, or specific immunogenic active cells, or both. Those skilled in the art will understand that any macromolecule, including almost all proteins or peptides, can be used as an antigen. Furthermore, antigens can come from recombinant or genomic DNA or RNA. Those skilled in the art will understand that any DNA or RNA herein, their nucleotide sequence or partial nucleotide sequence can encode a protein that can cause acquired immunity in the body. Furthermore, those skilled in the art will understand that an antigen does not need to encode the full-length nucleotide sequence of a gene alone.
  • the present invention includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences form different mixtures to induce the occurrence of a response.
  • antigens do not need to be completely encoded by a gene.
  • antigens can be generated synthetically or derived from biological samples.
  • Biological samples include but are not limited to tissue samples, tumor samples, cells or biological fluids.
  • antibody refers to a protein with a protective effect produced by the body due to the stimulation of an antigen. It is an immunoglobulin produced by B lymphocytes.
  • the monomer of an antibody is a Y-shaped molecule consisting of four polypeptide chains. It includes two identical heavy chains and two identical light chains, which are connected by disulfide bonds. Each heavy chain is 50kDa, each light chain is 25kDa, and there is a disulfide bond between the light and heavy chains. Its uniqueness lies in its high affinity and specificity for binding partners.
  • vaccine refers to a composition comprising an active ingredient (e.g., a polynucleotide of the present invention) that can induce an immune response in a vaccinated subject by vaccination.
  • an active ingredient e.g., a polynucleotide of the present invention
  • the immune response it induces can provide immune protection and is sufficient to prevent and/or alleviate at least one symptom associated with a pathogen or disease infection.
  • the polynucleotides or compositions described herein can be used as vaccines to provide preventive and/or therapeutic immunity against influenza viruses in subjects in need.
  • neutralizing antibody refers to an antibody or fragment thereof that can neutralize, i.e., prevent, inhibit, reduce or interfere with the ability of a pathogen to cause and/or maintain infection in a host (e.g., a host organism or a host cell).
  • a host e.g., a host organism or a host cell.
  • neutralizing antibodies against influenza virus can be produced in a subject vaccinated with the vaccine of the present invention, for example, in the immune serum of the subject.
  • the level of neutralizing antibody titers in immune serum can be measured using methods known in the art.
  • immune response refers to a process involving the activation and/or induction of effector functions, which occur in, for example, T cells, B cells, natural killer cells and/or antigen presenting cells.
  • an immune response may be understood by those skilled in the art to include, but is not limited to, any detectable T helper cell antigen.
  • the invention also includes 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.
  • Th1 means that the initial CD4+ T cells can differentiate into Th1 cells under the induction of interferon- ⁇ (IFN- ⁇ ), secrete IFN- ⁇ , and participate in cell-mediated immune responses and monocyte- or macrophage-mediated inflammatory responses; they can differentiate into Th2 cells under the induction of IL-4, secrete cytokines such as IL-4 and IL-5, participate in humoral immune responses, stimulate B cells to promote antibody production, and promote the proliferation and function of mast cells and eosinophils.
  • IFN- ⁇ interferon- ⁇
  • IL-4 secrete cytokines
  • IL-4 and IL-5 participate in humoral immune responses, stimulate B cells to promote antibody production, and promote the proliferation and function of mast cells and eosinophils.
  • influenza virus is a member of the Orthomyxoviridae family and is an enveloped negative-strand RNA virus.
  • the influenza virus genomic RNA combines with nucleoprotein (NP) to form a ribonucleoprotein (RNP) complex.
  • the influenza virus also contains matrix protein, hemagglutinin and neuraminidase.
  • Hemagglutinin (HA) and neuraminidase (NA) are glycoproteins in the influenza virus envelope, responsible for the surface contact between the virus and the host.
  • the virus enters the host requiring the regulation of HA, which binds to cell receptors and promotes the fusion of the viral membrane with the endosomal membrane.
  • Influenza viruses can be divided into multiple subtypes based on the differences in HA and NA.
  • the "NP protein” is a basic protein with 498 amino acids. At its N-terminus, there is an RNA binding domain and two NP-NP self-interaction regions. They are essential 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 in different influenza viruses.
  • M2e refers to the extracellular domain of matrix protein 2.
  • M2 protein is a matrix protein of influenza virus, with a total length of 97 amino acids, 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 M2 protein is highly conserved in influenza virus.
  • 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), its variants or fragments and residues 2-24 of M2 protein (positions 499-521 of SEQ ID NO: 1).
  • NP 55-69 refers to a peptide consisting of residues 55-69 of the NP protein, which is an H-2d restricted Th epitope, and whose amino acid sequence is RLIQNSLTIERMVLS.
  • NP 147-155 refers to a peptide consisting of residues 147-155 of the NP protein, which is an H-2d restricted CTL epitope, and whose amino acid sequence is TYQRTRALV.
  • M2e peptide pool refers to a mixed peptide of M2e protein, which includes three peptides corresponding to residues 1-15 (MSLLTEVETPIRNEW), residues 5-19 (TEVETPIRNEWGCRC) and residues 9-23 (TPIRNEWGCRCNDSS) of M2 protein, respectively.
  • lipid refers to an organic compound comprising a hydrophobic portion and optionally also a hydrophilic portion. Lipids are generally insoluble in water but soluble in many organic solvents. Typically, amphipathic lipids comprising a hydrophobic portion and a hydrophilic portion can be organized into a lipid bilayer structure in an aqueous environment, for example, in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, steroids, and cholesterol esters, etc.
  • cationic polymer refers to any ionic polymer that can carry a net positive charge at a specified pH, thereby electrostatically binding to nucleic acids.
  • examples of cationic polymers include, but are not limited to, poly-L-lysine, protamine, and polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the polyethyleneimine can be linear or branched polyethyleneimine.
  • protamine refers to a low molecular weight basic protein rich in arginine, which is present in sperm cells of various animals (especially fish) and binds to DNA instead of histones.
  • the cationic polymer is fish Protamine (eg protamine sulfate).
  • the present invention relates to NM2e polypeptides.
  • the NM2e polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 1.
  • the NM2e comprises variants and/or fragments of NP protein, and M2e, wherein the variants and/or fragments of NP protein comprise a conserved region of NP protein.
  • the conserved region does not comprise mutations (including substitutions, deletions and insertions of amino acids).
  • the conserved region comprises conservative substitutions.
  • the NP segment of the NM2e comprises at least one amino acid modification, such as insertion, substitution and/or deletion. In some embodiments, the NP segment of the NM2e comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions and/or deletions.
  • the present invention also relates to polynucleotides encoding the NM2e polypeptide.
  • the polynucleotides 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 polynucleotides may be contained in a vector.
  • the polynucleotides of the present invention may include naturally occurring, synthetic and modified nucleotides.
  • the polynucleotides of the present invention are used to express the polypeptides described herein in cells to provide polypeptide antigens.
  • the polypeptide antigens can 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).
  • the polynucleotide may comprise a segment encoding a polypeptide of interest (e.g., a polypeptide and polypeptide antigen described herein).
  • the polynucleotide may comprise a coding sequence for a polypeptide of interest and a regulatory sequence (including but not limited to transcription and translation regulatory sequences).
  • the regulatory sequence comprises one or more of the following: a promoter sequence, a 5' untranslated region (5'UTR) sequence, a 3' untranslated region (3'UTR) sequence, and a poly (A) sequence.
  • the polynucleotides of the present invention comprise the coding sequence of a polypeptide antigen as described herein. In one embodiment, the polynucleotides of the present invention comprise a nucleotide sequence complementary to the coding sequence as described herein. In some embodiments, the polynucleotides of the present invention comprise the coding sequence of a polypeptide as described herein. In one embodiment, the coding sequence comprises a start codon at its 5' end and a stop codon at its 3' end. In one embodiment, the coding sequence comprises an open reading frame (ORF) as described herein.
  • ORF open reading frame
  • 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.
  • the NM2e encoded by the 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.
  • the NM2e comprises variants and/or fragments of the NP protein, and M2e, wherein the variants and/or fragments of the NP protein comprise a conserved region of the NP protein.
  • the conserved region does not comprise a mutation (including amino acid substitutions, deletions, In some embodiments, the conserved region comprises a conservative substitution.
  • the NP segment of the NM2e comprises at least one amino acid modification, such as insertion, substitution and/or deletion. In some embodiments, the NP segment of the NM2e comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions and/or deletions. In some embodiments, the NP segment comprises a conserved region of the NP protein, and the conserved region does not comprise the amino acid modification.
  • the polynucleotide of the present invention is RNA.
  • RNA encompasses single-stranded, double-stranded, linear and circular RNA.
  • the RNA of the present invention can be RNA produced by chemical synthesis, recombinant production and in vitro transcription.
  • the RNA of the present invention is used to express the polypeptide of the present invention in a host cell.
  • the RNA of the present invention is a single-stranded RNA.
  • the RNA of the present invention is an in vitro transcribed RNA (IVT-RNA).
  • IVT-RNA can be obtained by in vitro transcription using a DNA template by RNA polymerase (e.g., as described herein).
  • the RNA of the present invention is a messenger RNA (mRNA).
  • the mRNA may comprise a 5'-UTR sequence, a coding sequence for a polypeptide, a 3'-UTR sequence, and an optional poly (A) sequence.
  • the mRNA may be produced, for example, by in vitro transcription or chemical synthesis.
  • the mRNA of the present invention is obtained by in vitro transcription using a DNA template by an RNA polymerase (e.g., T7 RNA polymerase).
  • the mRNA of the present invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3'-UTR, and (4) an optional poly (A) sequence.
  • the 5'-UTR, coding sequence, 3'-UTR, and poly (A) sequence are as described herein.
  • the mRNA of the present invention is a nucleoside-modified mRNA.
  • the mRNA of the present invention comprises an optional 5' cap.
  • the RNA of the present invention comprises a coding sequence of a polypeptide antigen as described herein. In some embodiments, the RNA of the present invention comprises a coding sequence of a polypeptide as described herein.
  • the RNA of the present invention further comprises structural elements that help to improve the stability and/or translation efficiency of the RNA, including but not limited to a 5' cap, a 5'-UTR, a 3'-UTR, and a poly (A) sequence.
  • the term “untranslated region (UTR)” generally refers to a region (non-coding region) in RNA (such as mRNA) that is not translated into an amino acid sequence, or a corresponding region in DNA.
  • RNA such as mRNA
  • the UTR located at the 5’ end (upstream) of the open reading frame (start codon) can be referred to as the 5’ untranslated region 5’-UTR;
  • the UTR located at the 3’ end (downstream) of the open reading frame (stop codon) can be referred to as the 3’-UTR.
  • the 5’-UTR is located downstream of the 5’ cap, for example, directly adjacent to the 5’ cap.
  • an optimized “Kozak sequence” may be included in the 5’-UTR, for example, near the start codon, to improve translation efficiency.
  • the 3’-UTR is located upstream of the poly(A) sequence, for example, directly adjacent to the poly(A) sequence.
  • the RNA of the present invention comprises a 5'-UTR.
  • the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 2.
  • the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 3.
  • the RNA of the present invention comprises a 5'-UTR and a 3'-UTR.
  • the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 2
  • the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 3.
  • the RNA of the invention comprises a poly(A) sequence.
  • Poly(A) sequence or “poly(A) tail” refers to a nucleotide sequence containing continuous or discontinuous adenylic acid.
  • the poly(A) sequence is usually located at the 3' end of the RNA, such as the 3' end (downstream) of the 3'-UTR.
  • the poly(A) sequence does not contain nucleotides other than adenylic acid at its 3' end.
  • the poly(A) sequence can be transcribed by a DNA-dependent RNA polymerase according to the coding sequence of the DNA template during the preparation of the IVT-RNA, or can be linked to the free 3' end of the IVT-RNA, such as the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).
  • the poly(A) sequence comprises consecutive adenylic acids.
  • 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 adenylic acids.
  • the consecutive adenylic acid sequence in the poly(A) sequence is interrupted by a sequence comprising U, C or G nucleotides.
  • the poly(A) sequence comprises 75 adenylic acids.
  • 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.
  • the term “5' cap” generally refers to an N7-methylguanosine structure (also known as “m7G cap”, “m7Gppp-”) attached to the 5' end of an mRNA via a 5' to 5' triphosphate bond.
  • the 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or can be attached to the RNA after transcription using a capping enzyme.
  • the RNA of the present invention comprises the nucleotide sequence of SEQ ID NO:10, 11, 12 or 13.
  • the RNA of the present 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) encoding an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:1.
  • the polypeptide encoded by the RNA of the present invention comprises a conserved region of the NP protein.
  • the conserved region does not comprise a mutation (including substitutions, deletions and insertions of amino acids).
  • the conserved region comprises a conservative substitution.
  • the polynucleotide of the present invention is DNA.
  • DNA can be, for example, a DNA template for in vitro transcription of the RNA of the present invention or a DNA vaccine for expressing a polypeptide antigen in a host cell.
  • DNA can be double-stranded, single-stranded, linear, and circular DNA.
  • the DNA template can be provided in a suitable transcription vector.
  • the DNA template can be a double-stranded complex comprising a nucleotide sequence identical to the coding sequence described herein (coding strand) and a nucleotide sequence complementary to the coding sequence described herein (template strand).
  • the DNA template can include a promoter, 5'-UTR, a coding sequence, 3'-UTR, and an optional poly (A) sequence.
  • the promoter can be a promoter available for a suitable RNA polymerase (particularly DNA-dependent RNA polymerase) known to those skilled in the art, including but not limited to promoters of SP6, T3, and T7 RNA polymerases.
  • the 5'-UTR, coding sequence, 3'-UTR, and poly (A) sequence in the DNA template are corresponding sequences included in RNA described herein or are complementary thereto.
  • a polynucleotide of a DNA vaccine it can be provided in a plasmid vector (e.g., a circular plasmid vector).
  • the DNA of the present invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the DNA of the present invention comprises a coding sequence for a polypeptide as described herein. In some embodiments, the DNA of the present 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 optional poly(A) sequence as described herein.
  • the present invention also provides a composition comprising a polynucleotide of the present invention (particularly RNA).
  • the composition of the present invention is used to provide preventive and/or therapeutic immunity against influenza virus in a subject.
  • the composition of the present invention comprises a polynucleotide of the present invention.
  • the composition of the present invention comprises a DNA of the present invention.
  • the composition of the present invention comprises an RNA of the present invention.
  • the RNA is an RNA transcribed in vitro.
  • the RNA is an mRNA.
  • compositions of the invention comprise a polynucleotide (particularly RNA, such as mRNA) as described herein and a lipid encapsulating the polynucleotide.
  • compositions may be, for example, lipid nanoparticles (LNP) and lipid polymer complexes (LPP) as described herein. Methods for preparing such compositions may be found, for example, in Kaczmarek, J.C. et al., 2017, Genome Medicine 9, 60 or as described herein.
  • the composition of the present invention comprises lipid nanoparticles (LNP) or lipid polymer complexes (LPP).
  • the composition of the present invention is a lipid nanoparticle (LNP) or lipid polymer complex (LPP) comprising the RNA of the present invention.
  • the lipid encapsulating the polynucleotide comprises a cationic lipid and a non-cationic lipid.
  • the cationic lipid is an ionizable cationic lipid.
  • the cationic lipid comprises DOTMA, DOTAP, DDAB, DOSPA, DODAC, DODAP, DC-Chol, DMRIE, DMOBA, DLinDMA, DLenDMA, CLinDMA, DMORIE, DLDMA, DMDMA, DOGS, N4-cholesteryl-spermamine, DLin-KC2-DMA, DLin-MC3-DMA, or a combination thereof.
  • the cationic lipid comprises M5, which has the structure:
  • the cationic lipid comprises DOTMA. In one embodiment, the cationic lipid comprises DOTAP. In one embodiment, the cationic lipid comprises DOTMA and DOTAP.
  • 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.
  • the cationic lipid comprises M5 and the non-cationic lipid comprises DOPE and cholesterol.
  • the lipids encapsulating the polynucleotide further comprise polyethylene glycol-modified lipids.
  • the polyethylene glycol-modified lipids comprise DMG-PEG (e.g., DMG-PEG 2000), DOGPEG and DSPE-PEG or a combination thereof.
  • the polyethylene glycol-modified lipid comprises DSPE-PEG.
  • the polyethylene glycol-modified lipid comprises DMG-PEG (eg, DMG-PEG 2000).
  • the composition of the present invention further comprises a cationic polymer, which is associated with the polynucleotide as a complex and is co-encapsulated in the lipid.
  • the cationic polymer comprises poly-L-lysine, protamine, polyethyleneimine (PEI), or a combination thereof. In one embodiment, the cationic polymer is protamine. In one embodiment, the cationic polymer is polyethyleneimine.
  • the amount of lipid in the composition is calculated as a mole percent (mol %), which is determined based on the total moles of lipid in the composition.
  • the amount of cationic lipid in the composition is about 10-about 70 mol%. In some embodiments, the amount of cationic lipid in the composition is about 20-about 60 mol%, about 30-about 50 mol%, about 35-about 45 mol%, about 38-about 45 mol%, about 40-about 45 mol%, about 40-about 50 mol%, or about 45-about 50 mol%.
  • the amount of phospholipids in the composition is about 10 to about 70 mol%. In one embodiment, the amount of phospholipids in the composition is about 20 to about 60 mol%, about 30 to about 50 mol%, about 10 to about 30 mol%, about 10 to about 20 mol%, or about 10 to about 15 mol%.
  • the amount of cholesterol in the composition is about 10 to about 70 mol%. In one embodiment, the amount of cholesterol in the composition is about 20 to about 60 mol%, about 30 to about 50 mol%, about 35 to about 40 mol%, about 35 to about 45 mol%, about 40 to about 45 mol%, or about 45 to about 50 mol%.
  • the amount of the polyethylene glycol-modified lipid in the composition is about 0.05-about 20 mol%. In one embodiment, the amount of the polyethylene glycol-modified lipid in the composition is about 0.5-about 15 mol%, about 1-about 10 mol%, about 5-about 15 mol%, about 1-about 5 mol%, about 1.5-about 3 mol%, or about 2-5 mol%.
  • RNA (particularly mRNA) of the present invention is formulated as lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • nucleic acid e.g., mRNA
  • the LNP comprises the RNA of the present invention and a lipid encapsulating the RNA, wherein the lipid encapsulating the RNA comprises a cationic lipid, a phospholipid, cholesterol, and a polyethylene glycol-modified lipid.
  • the cationic lipid is M5.
  • the phospholipid is DSPC.
  • the polyethylene glycol-modified lipid is DMG-PEG 2000.
  • the cationic lipid is M5, the phospholipid is DSPC, and the polyethylene glycol-modified lipid is DMG-PEG 2000.
  • the lipids encapsulating the RNA comprise 50 mol% M5, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG 2000.
  • the RNA (particularly mRNA) of the present invention is formulated as a lipid polyplex (LPP).
  • lipid polyplex or “LPP” refers to a core-shell structure comprising a nucleic acid core encapsulated by a lipid shell, wherein the nucleic acid core comprises a nucleic acid (e.g., mRNA) associated with a polymer.
  • the LPP comprises the RNA of the present invention, which is 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.
  • the non-cationic lipid comprises a phospholipid and a steroid.
  • the non-cationic lipid comprises a phospholipid selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), di
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • the cationic polymer comprises protamine.
  • the polyethylene glycol-modified lipid comprises DMG-PEG 2000.
  • 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), distearoylphosphatidylcholine (DSPC) or a combination thereof, and cholesterol;
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DSPC distearoylphosphatidylcholine
  • the polyethylene glycol-modified lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000);
  • the cationic polymer comprises protamine.
  • the cationic polymer is protamine
  • the cationic lipid is M5
  • the phospholipid is DOPE
  • the polyethylene glycol-modified lipid is DMG-PEG 2000.
  • the lipids of the encapsulation complex comprise 40 mol% M5, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG 2000.
  • vaccine formulations of the invention comprise a polynucleotide described herein.
  • the vaccine formulation of the invention comprises a composition as described herein, wherein the lipid comprises 10-70 mol% M5, 10-70 mol% DOPE, 10-70 mol% cholesterol, and 0.05-20 mol% DMG-PEG 2000,
  • polynucleotide encodes a polypeptide as described herein.
  • the polynucleotide comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:4.
  • the NM2e encoded by the polynucleotide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:1.
  • the NM2e comprises a variant and/or fragment of the NP protein, and M2e, wherein the variant and/or fragment of the NP protein comprises a conserved region of the NP protein.
  • the conserved region does not comprise a mutation (including substitutions, deletions and insertions of amino acids).
  • the conserved region comprises a conservative substitution.
  • the NP segment of the NM2e comprises at least one amino acid modification, such as insertion, substitution and/or deletion. In some embodiments, the NP segment of the NM2e comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions and/or deletions. In some embodiments, the NP segment comprises a conserved region of the NP protein, and the conserved region does not comprise the amino acid modification.
  • the polynucleotides of the invention are RNA.
  • RNA encompasses single-stranded, double-stranded, linear, and circular RNA.
  • the RNA of the invention can be chemically synthesized, recombinantly produced, and In vitro transcribed RNA.
  • the RNA of the invention is used to express a polypeptide of the invention in a host cell.
  • the RNA of the present invention is a single-stranded RNA.
  • the RNA of the present invention is an in vitro transcribed RNA (IVT-RNA).
  • IVT-RNA can be obtained by in vitro transcription using a DNA template by RNA polymerase (e.g., as described herein).
  • the RNA of the present invention is a messenger RNA (mRNA).
  • the mRNA may comprise a 5'-UTR sequence, a coding sequence for a polypeptide, a 3'-UTR sequence, and an optional poly (A) sequence.
  • the mRNA may be produced, for example, by in vitro transcription or chemical synthesis.
  • the mRNA of the present invention is obtained by in vitro transcription using a DNA template by an RNA polymerase (e.g., T7 RNA polymerase).
  • the mRNA of the present invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3'-UTR, and (4) an optional poly (A) sequence.
  • the 5'-UTR, coding sequence, 3'-UTR, and poly (A) sequence are as described herein.
  • the mRNA of the present invention is a nucleoside-modified mRNA.
  • the mRNA of the present invention comprises an optional 5' cap.
  • the RNA of the present invention comprises a coding sequence of a polypeptide antigen as described herein. In some embodiments, the RNA of the present invention comprises a coding sequence of a polypeptide as described herein.
  • the RNA of the present invention further comprises structural elements that help to improve the stability and/or translation efficiency of the RNA, including but not limited to a 5' cap, a 5'-UTR, a 3'-UTR, and a poly (A) sequence.
  • the term “untranslated region (UTR)” generally refers to a region (non-coding region) in RNA (such as mRNA) that is not translated into an amino acid sequence, or a corresponding region in DNA.
  • RNA such as mRNA
  • the UTR located at the 5’ end (upstream) of the open reading frame (start codon) can be referred to as the 5’ untranslated region 5’-UTR;
  • the UTR located at the 3’ end (downstream) of the open reading frame (stop codon) can be referred to as the 3’-UTR.
  • the 5’-UTR is located downstream of the 5’ cap, for example, directly adjacent to the 5’ cap.
  • an optimized “Kozak sequence” may be included in the 5’-UTR, for example, near the start codon, to improve translation efficiency.
  • the 3’-UTR is located upstream of the poly(A) sequence, for example, directly adjacent to the poly(A) sequence.
  • the RNA of the present invention comprises a 5'-UTR.
  • the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 2.
  • the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 3.
  • the RNA of the present invention comprises a 5'-UTR and a 3'-UTR.
  • the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 2
  • the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 3.
  • the RNA of the invention comprises a poly(A) sequence.
  • the poly(A) sequence comprises consecutive adenylic acids.
  • 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 adenylic acids.
  • the consecutive adenylic acid sequence in the poly(A) sequence is interrupted by a sequence comprising U, C or G nucleotides.
  • the poly(A) sequence comprises 75 adenylic acids.
  • 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.
  • the term “5' cap” generally refers to an N7-methylguanosine structure (also known as “m7G cap”, “m7Gppp-”) attached to the 5' end of an mRNA via a 5' to 5' triphosphate bond.
  • the 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or can be attached to the RNA after transcription using a capping enzyme.
  • the RNA of the present invention comprises the nucleotide sequence of SEQ ID NO:10, 11, 12 or 13.
  • the RNA of the present 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) encoding an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:1.
  • the polypeptide encoded by the RNA of the present invention comprises a conserved region of the NP protein.
  • the conserved region does not comprise a mutation (including substitutions, deletions and insertions of amino acids).
  • the conserved region comprises a conservative substitution.
  • Cationic lipids are lipids with a net positive charge at a given pH. Lipids with a net positive charge can associate with nucleic acids through electrostatic interactions.
  • cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Didecyldimethylammonium bromide (DDAB), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), dioctadecyldimethyl ammonium chloride (DDAB), chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl
  • the cationic lipid is preferably an ionizable cationic lipid.
  • Ionizable cationic lipids carry a net positive charge at, for example, acidic pH, and are neutral at higher pH (e.g., physiological pH).
  • ionizable cationic lipids include, but are not limited to, dioctadecylamidoglycyl spermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptahedral-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butyrate (heptatriaconta-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butyrate aen-19-yl-4-(dimethylamino)butanoate, DLin-MC3-DMA), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octan
  • the cationic lipid comprises M5, which has the following structure:
  • non-cationic lipid refers to lipids that do not have a net positive charge at a specified pH, such as anionic lipids and neutral lipids.
  • neutral lipid refers to lipids that exist in an uncharged, neutral or zwitterionic form at physiological pH. Neutral lipids can include, but are not limited to, phospholipids and steroids.
  • phospholipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidylethanolamine (DSPC), distearoyl-phosphatidylcholine (DSPC), distearoyl-phosphatidylethanolamine (DSPE ...
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine
  • DSPC distearoylphosphatidylcholine
  • DSPC distearoyl-phosphatidylcholine
  • DSPE distearoyl-phosphatidylethanolamine
  • sphatidylethanolamine DSPE
  • dioleoylphosphatidylcholine DOPC
  • dimyristoylphosphatidylcholine DMPC
  • dipalmitoylphosphatidylcholine DPPC
  • diarachidoylphosphatidylcholine DAPC
  • dibehenylphosphatidylcholine DBPC
  • ditricosanoylphosphatidylcholine DTPC
  • dilignoceroylphatidylcholine DLPC
  • palmitoyloleoyl-phosphatidylcholine POPC
  • dipalmitoyl-phosphatidylethanolamine DPPE
  • dimyristoyl-phosphatidylethanolamine DMPE
  • DLPE dimyristoyl-phosphatidylethanolamine
  • steroids examples include, but are not limited to, for example, cholesterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and derivatives thereof.
  • polyethylene glycol-modified lipid refers to a molecule comprising a polyethylene glycol portion and a lipid portion.
  • examples of polyethylene glycol-modified lipids include, but are not limited to, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-dioleoyl-rac-glycerol, methoxypolyethylene Glycol (DOGPEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol), DSPE-PEG.
  • the polyethylene glycol-modified lipid is DMG-PEG, such as DMG-PEG 2000.
  • DMG-PEG 2000 has the following structure:
  • n 44.
  • the present invention provides the polynucleotide (especially RNA), composition or vaccine preparation of the present invention for preventing and/or treating influenza virus infection in a subject in need thereof.
  • polynucleotide especially RNA
  • composition or vaccine preparation of the present invention for preventing and/or treating influenza virus infection in a subject in need thereof.
  • the present invention provides use of the polynucleotide (particularly RNA), composition or vaccine preparation of the present invention in the preparation of a medicament for preventing and/or treating 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 present invention.
  • the method comprises administering a therapeutically effective amount of a composition comprising the mRNA of the present invention, particularly a composition comprising LPP as described herein.
  • 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 down, alleviate, or delay the development or severity of a disease or symptom.
  • the prophylactically or therapeutically effective amount is affected by factors including, but not limited to, the speed and severity of the development of the disease or symptom, the age, sex, weight, and physiological condition of the subject, the duration of treatment, and the specific route of administration.
  • the prophylactically or therapeutically effective amount may be administered in one or more doses.
  • the prophylactically or therapeutically effective amount may be achieved by continuous or intermittent administration.
  • the prophylactic or therapeutically effective amount is provided in one or more administrations. In some embodiments, the prophylactic or therapeutically effective amount is provided in two administrations. In some embodiments, the prophylactic or therapeutically effective amount is provided in three administrations.
  • composition or vaccine formulation of the present invention can be administered to a subject by any method known to those skilled in the art, such as parenteral, oral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous or intraperitoneal.
  • the composition or vaccine formulation of the present invention is administered by intramuscular injection.
  • the term "subject” describes an organism, such as a human, a non-human mammal (such as a pig), or a bird (such as a chicken), to which therapy using a polynucleotide or composition of the invention can be provided.
  • the polynucleotides, compositions, vaccine preparations and methods of the present invention achieve higher levels of NM2e polypeptide expression in cells than the prior art, induce significant cellular and antibody responses in animals, provide improved protection against different strains (homotypic strains and heterotypic strains), and can induce broad-spectrum cross-immune protection against conserved antigens in vivo.
  • NM2e-Ori represents the wild-type sequence
  • NM2e-1, NM2e-2, NM2e-3, and NM2e-4 represent optimized sequences.
  • T7 promoter sequence (TAATACGACTCACTATA), 5'-UTR sequence (SEQ ID NO: 19), 3'-UTR sequence (SEQ ID NO: 20) and poly (A) sequence (75 adenosine nucleotides) were also designed.
  • the Kozak sequence "GCCACC" was included in the 5'UTR sequence.
  • T7 promoter sequence 5’-UTR sequence, DNA ORF sequence, 3’-UTR sequence and poly(A) sequence were connected in order.
  • pUC57 was used as a vector for full gene synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd.) to obtain a plasmid DNA template.
  • a pair of tailing PCR primers (upstream primer: 5’TTGGACCCTCGTACAGAAGCTAATACG 3’; and downstream poly (T) long primer: 5’TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • the PCR product prepared in Example 1.1 was purified using a PCR product purification kit (Takara). The product was used as a template, and a co-transcriptional capping reaction was performed by T7 RNA polymerase to perform in vitro transcription of RNA to produce Cap1 mRNA.
  • 1-Methyl-pseudouridine-triphosphate was used instead of uridine triphosphate (UTP) in the in vitro transcription, so the modification ratio of 1-methyl-pseudouridine in the in vitro transcribed Cap1 mRNA was 100%.
  • the DNA template was digested with DNaseI (Thermo Fisher Scientific) to reduce the risk of residual DNA template.
  • the mRNA was purified using Dynabeads Myone (Thermo Fisher Scientific Inc.). The purified mRNA was dissolved in 1 mM sodium citrate buffer (pH 6.5), sterile filtered, and frozen at -80°C until use. The obtained mRNA sequence is shown in Table 1.
  • Cationic lipid M5 was synthesized by Simicrobial; auxiliary phospholipid (DOPE) was purchased from CordenPharma; cholesterol was purchased from Sigma-Aldrich; mPEG2000-DMG (i.e. DMG-PEG 2000) was purchased from Avanti Polar Lipids, Inc.; PBS was purchased from Invitrogen; and protamine sulfate was purchased from Beijing Silian Pharmaceutical Co., Ltd.
  • DOPE auxiliary phospholipid
  • DMG-PEG 2000 i.e. DMG-PEG 2000
  • PBS was purchased from Invitrogen
  • protamine sulfate was purchased from Beijing Silian Pharmaceutical Co., Ltd.
  • LPP is prepared. Specifically, the preparation includes the following steps.
  • mRNA aqueous solution Dilute each mRNA prepared as in Example 1.2 to a 0.2 mg/mL mRNA solution using 10 mM citric acid-sodium citrate buffer (pH 4.0).
  • lipid solution Dissolve cationic lipid (M5): DOPE: cholesterol: DMG-PEG 2000 in anhydrous ethanol at a molar ratio of 40:15:43.5:1.5 to prepare a 10 mg/mL lipid solution.
  • M5 Dissolve cationic lipid
  • DOPE cholesterol: DMG-PEG 2000 in anhydrous ethanol at a molar ratio of 40:15:43.5:1.5 to prepare a 10 mg/mL lipid solution.
  • protamine sulfate solution Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.25 mg/mL.
  • LPP LPP
  • LPP-NM2e preparations LPP-NPM2e-Ori, LPP-NPM2e-1, LPP-NPM2e-2, LPP-NPM2e-3 and LPP-NPM2e-4.
  • 293T cells were transfected with non-optimized LPP-NM2e-Ori and optimized LPP-NM2e-1, LPP-NM2e-2, LPP-NM2e-3 and LPP-NM2e-4, and the cells were collected 24 hours later for Western blot analysis.
  • the antibody titers in mice immunized with optimized LPP-NPM2e-1, LPP-NM2e-2, LPP-NM2e-3 and LPP-NM2e-4 are significantly higher than those in mice immunized with unoptimized LPP-NM2e-Ori.
  • the humoral immunity induced by LPP-NM2e-3 is stronger than the humoral immunity induced by other optimized mRNAs.
  • LPP-NPM2e-3 (hereinafter referred to as LPP-NPM2e) to further characterize its humoral immunity and cellular immunity, and conduct a challenge experiment.
  • the M2e-specific antibody (IgG) titers were 1.2 ⁇ 10 2 (low-dose group) and 1.86 ⁇ 10 2 (high-dose group).
  • the M2e-specific antibody titers increased to 9.70 ⁇ 10 2 (low-dose group) and 3.98 ⁇ 10 3 (high-dose group), respectively.
  • the titers in the high-dose group were significantly higher than the M2e-specific antibody titers detected after the first dose of LPP-NM2e preparation (P ⁇ 0.05) ( Figure 3B).
  • the NP protein and M2e-specific IgG2a:IgG1 ratios in the low-dose and high-dose groups were substantially greater than 1 (only one mouse had a ratio less than 1), suggesting that a Th1-biased immune response was induced.
  • the prepared spleen mononuclear cells were stimulated with NP 55-69 , NP 147-155 and M2e peptide pool.
  • the cellular immune response induced by mice immunized with LPP-NM2e was detected by IFN- ⁇ ELISPOT, and the results are shown in FIG3 .
  • the average densities of NP 55-69 , NP 147-155 and M2e-specific spot-forming (IFN- ⁇ -secreting) cells in spleen mononuclear cells from the low-dose group and high-dose groups were 24SFC (spot-forming cells)/10 6 SMNC (spleen mononuclear cells), 448SFC/10 6 SMNC, 10SFC/10 6 SMNC and 10SFC/10 6 SMNC, 383SFC/10 6 SMNC and 25SFC/10 6 SMNC, respectively, which were not significantly different from those in the control group.
  • the cellular immune response was enhanced.
  • the average densities of NP 55-69 , NP 147-155 and M2e-specific SFC were 196SFC/10 6 SMNCs, 1943SFC/10 6 SMNCs and 98SFC/10 6 SMNCs, respectively, which were significantly higher than the levels after the first administration (P ⁇ 0.01, P ⁇ 0.05 and P ⁇ 0.05);
  • the average densities of NP 55-69 , NP 147-155 and M2e-specific SFC were 278SFC/10 6 SMNCs, 2950SFC/10 6 SMNCs and 128SFC/10 6 SMNCs, respectively.
  • the specific cellular immune responses of NP 55-69 and NP 147-155 were significantly higher than the levels after the first administration (P ⁇ 0.0001). There was no statistically significant difference in the M2e-specific immune response, but it showed a dose-dependent trend.
  • influenza virus strains X31 H3N2
  • PR8 H1N1
  • AH H7N9
  • the changes in body weight are shown in the left panels of Figures 6A, B, and C.
  • the body weight of mice in both the low-dose and high-dose groups dropped to the lowest level 5 days after the challenge, and then gradually increased and recovered to the level equivalent to Day 0.
  • mice inoculated with strain X31 were 60% (low-dose group) and 100% (high-dose group) ( Figure 6A); the survival rates of mice inoculated with strain PR8 were 90% (low-dose group) and 100% (high-dose group) ( Figure 6B); the survival rates of mice inoculated with strain AH were 82% (low-dose group) and 90% (high-dose group) ( Figure 6C).
  • HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) at 37°C and 5% CO 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • NP, M2e, and NPM2e proteins used in ELISA assays were purchased from Sino Biological and expressed in Escherichia coli or baculovirus-insect cells.
  • anti-influenza A M2 protein antibody purchased from Abcam
  • anti-influenza NP protein antibody purchased from Sino Biological
  • anti-mouse horseradish peroxidase (HRP)-conjugated antibody and anti-rabbit horseradish peroxidase (HRP)-conjugated antibody purchased from Abbkine.
  • NP, M2e or NPM2e protein were diluted to 1 ⁇ g/ml with 0.05 M sodium carbonate buffer and added to a 96-well ELISA plate ( Greiner) (100 ⁇ l/well) at 4°C overnight.
  • the plates were washed with PBS-T (phosphate buffered saline + 0.05% Tween-20) and then blocked with 2% BSA (prepared in PBS-T) at 37°C for 60 minutes. Two-fold serial dilutions of mouse serum samples were added to the coated plates and incubated at 37°C for 60 minutes. The plates were then incubated with HRP-conjugated secondary antibodies at 37°C for 60 minutes.
  • TMB substrate (Biyuntian Biotechnology) was added. After the reaction was terminated, the optical density (OD) at a wavelength of 450 was read using a microplate reader (BioTek). The absorbance value of the sample was higher than the reciprocal value of the highest dilution of 2.1 times the negative control sample as the final titer.
  • 1.0 ⁇ 10 6 cells/well HEK293 cells were seeded in a 6-well cell culture dish. When the confluency reached 70-90%, the medium was replaced with fresh medium, and 2.5 ⁇ g of NPM2e mRNA-LPP was co-incubated with the cells. After 24 hours, the cells were collected, and the LPP-transfected cells were lysed with 1 ⁇ SDS-PAGE loading buffer (Beyotime) for SDS-PAGE and Western blot detection.
  • 1 ⁇ SDS-PAGE loading buffer Beyotime
  • Mouse IFN- ⁇ ELISpot assay was performed using the IFN- ⁇ ELISpot PLUS kit (Mabtech) according to the manufacturer's instructions. Briefly, the plate was blocked and incubated in RPMI 1640 medium (supplemented with 10% FBS) for 30 minutes.
  • the spleen of the removed mouse was ground and filtered, and after treatment with red blood cell lysis buffer, the obtained cells (i.e., spleen mononuclear cells) were counted and plated at 3 ⁇ 10 5 cells/well and stimulated in vitro with 8 ⁇ g/ml NP55-69 peptide, 8 ⁇ g/ml NP14-155 peptide, 8 ⁇ g/ml M2e peptide pool, and 10 ⁇ g/ml NM2e peptide pool (all purchased from Shanghai Jier Biochemical Co., Ltd.), phytohemagglutinin (PHA) + ionomycin (positive control) or only RPMI 1640 medium (negative control), and incubated at 37°C, 5% CO 2 for 20 hours.
  • PHA phytohemagglutinin
  • RPMI 1640 medium negative control
  • biotinylated IFN- ⁇ -detection antibody and streptavidin-alkaline phosphatase were used for detection, and BCIP/NBT-plus (5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium-plus) substrate was added for color development and counted using an ELISpot plate reader (ImmunoSpot S6 Core Analyzer (CTL)).
  • CTL ELISpot plate reader
  • amino acid sequence of NP 55–69 is RLIQNSLTIERMVLS.
  • amino acid sequence of NP 147–155 is TYQRTRALV.
  • the M2e peptide pool is a mixed peptide of the M2e protein, which contains three peptides corresponding to residues 1-15 (MSLLTEVETPIRNEW), residues 5-19 (TEVETPIRNEWGCRC) and residues 9-23 (TPIRNEWGCRCNDSS) of the M2 protein, respectively.
  • the NM2e peptide pool is a peptide library consisting of peptide segments corresponding to the full-length NM2e protein, with a length of 15 amino acids and overlapping with each other by 11 amino acids.
  • mice Female BALB/c mice aged 5 weeks were immunized with 100 ⁇ L of mRNA-LPP preparations on days 0 (D0) and 21 (D21) by bilateral intramuscular injection. All blood samples were collected by retro-orbital bleeding, and about 200 ⁇ L of blood/time was centrifuged at 1,500 g for 10 minutes at 4°C for serum separation.

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Abstract

La présente invention concerne le domaine des produits pharmaceutiques biologiques et de la virologie, et en particulier, un vaccin à ARNm pour la prévention ou le traitement d'une infection par le virus de la grippe. L'invention concerne un polynucléotide, tel que l'ARNm, codant le virus de la grippe NP et M2e, qui est optimisé au moyen d'une séquence de codons préférée de cellule humaine. L'invention concerne en outre une composition et un vaccin comprenant le polynucléotide, et un procédé de traitement ou de prévention d'une infection par le virus de la grippe à l'aide du polynucléotide, de la composition ou du vaccin.
PCT/CN2023/139522 2022-12-19 2023-12-18 Vaccin antigrippal à arnm et à large spectre WO2024131726A1 (fr)

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CN109310751A (zh) * 2015-10-22 2019-02-05 摩登纳特斯有限公司 广谱流感病毒疫苗
CN111526886A (zh) * 2017-10-27 2020-08-11 国家血清研究所 多基因流感疫苗
WO2021202734A2 (fr) * 2020-03-31 2021-10-07 The Trustees Of The University Of Pennsylvania Vaccin universel contre la grippe faisant appel à un arnm modifié par un nucléoside
CN116751306A (zh) * 2023-03-17 2023-09-15 上海生物制品研究所有限责任公司 一种编码通用流感的mRNA疫苗及其用途

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WO2021202734A2 (fr) * 2020-03-31 2021-10-07 The Trustees Of The University Of Pennsylvania Vaccin universel contre la grippe faisant appel à un arnm modifié par un nucléoside
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