WO2024131726A1 - Broad-spectrum influenza mrna vaccine - Google Patents

Broad-spectrum influenza mrna vaccine Download PDF

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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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polynucleotide
sequence
rna
composition
lipid
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PCT/CN2023/139522
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French (fr)
Chinese (zh)
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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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  • 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

The present invention relates to the field of biological pharmaceuticals and virology, and in particular, to an mRNA vaccine for preventing or treating influenza virus infection. Provided is a polynucleotide, such as mRNA, encoding influenza virus NP and M2e, which is optimized by means of a human cell preferred codon sequence. Further provided are a composition and a vaccine comprising the polynucleotide, and a method for treating or preventing influenza virus infection using the polynucleotide, the composition, or the vaccine.

Description

一种广谱流感mRNA疫苗A broad-spectrum influenza mRNA vaccine 技术领域Technical Field
本发明涉及生物医药和病毒学领域，具体涉及预防或治疗流感病毒感染的mRNA疫苗。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.
背景技术Background technique
流感对全球公共卫生造成严重威胁，对人类健康和世界经济造成严重危害。接种疫苗是预防流感最有效的措施之一。现用流感疫苗包括裂解疫苗、全病毒灭活疫苗、减毒活疫苗、亚单位疫苗等，主要通过诱导针对流感病毒囊膜上的血凝素(hemagglutinin，HA)与神经氨酸酶(neuraminidase，NA)特异性中和抗体发挥保护作用。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.
流感病毒的HA型多易变，HA的抗原漂移(antigenic drift)和抗原转变(antigenic shift)常导致出现流感季节性流行和大流行。在疫苗株和流行毒株不匹配的情况下，缺乏保护效力。目前季节性流感疫苗主要是针对HA、NA抗原，几乎每年需要根据预测毒株进行新疫苗的制备，为了减少疫苗重新制备的频率，需要研制广谱流感疫苗，例如靶向流感病毒保守的抗原，如基质蛋白2胞外域(Matrix 2 extracellular domain，M2e)、基质蛋白1(Matrix 1，M1)和核蛋白(nucleoprotein，NP)，的疫苗。The HA type of influenza virus is variable, and the antigenic drift and antigenic shift of HA often lead to seasonal and pandemic influenza. In the case of mismatch between vaccine strains and epidemic strains, there is a lack of protective efficacy. At present, seasonal influenza vaccines mainly target HA and NA antigens, and new vaccines need to be prepared almost every year based on predicted strains. In order to reduce the frequency of vaccine re-preparation, it is necessary to develop 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公开了一种编码甲型流感病毒NP蛋白和M2e多肽的融合基因。可以在大肠杆菌中高效表达甲型流感病毒NP和M2e融合蛋白NM2e，使用纯化的NM2e融合蛋白制备蛋白亚单位疫苗。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.
然而，蛋白亚单位疫苗的制备涉及蛋白的表达、纯化，周期长，在出现突发疫情时难以及时制备，因而需要建立制备更加高效、快速的疫苗，例如可以在对象中实现高表达的mRNA疫苗。However, the preparation of protein subunit vaccines involves protein expression and purification, which takes a long time and is difficult to prepare in a timely manner when an outbreak occurs. Therefore, it is necessary to establish a more efficient and rapid vaccine preparation method, such as an mRNA vaccine that can achieve high expression in subjects.
发明内容Summary of the invention
一方面，本发明提供一种多核苷酸，其包含编码SEQ ID NO:1的融合蛋白的核苷酸序列，其中所述核苷酸序列与选自5、6、7、8、15、16、17和18的核苷酸序列具有至少80％的相同性。On the one hand, 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.
在一些实施方案中，所述多核苷酸为RNA。在一些实施方案中，所述RNA是mRNA。在一些实施方案中，所述mRNA还包含5’UTR、3’UTR和polyA。在一些实施方案中，所述5’UTR包含SEQ ID NO:2的核苷酸序列。在一些实施方案中，所述3’UTR包含SEQ ID NO:3的核苷酸序列。在一些实施方案中，所述polyA包含75个腺苷酸残基。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 polyA. In some embodiments, the 5'UTR comprises a nucleotide sequence of SEQ ID NO:2. In some embodiments, the 3'UTR comprises a nucleotide sequence of SEQ ID NO:3. In some embodiments, the polyA comprises 75 adenylate residues.
在一些实施方案中，所述多核苷酸包含与SEQ ID NO:10-13之一具有至少80％的相同性的核苷酸序列。In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80% identical to one of SEQ ID NO:10-13.
第二方面，本发明提供一种组合物，其包含本发明的多核苷酸。在一些实施方案中，所述组合物包含包封所述多核苷酸的脂质。在一些实施方案中，所述组合物包含脂 质多聚复合物。在一些实施方案中，包封所述多核苷酸的脂质包含阳离子脂质、非阳离子脂质和聚乙二醇修饰的脂质；任选地，所述组合物还包含阳离子聚合物，其中所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在脂质中形成脂质多聚复合物。In a second aspect, the present invention provides a composition comprising a polynucleotide of the present invention. In some embodiments, the composition comprises a lipid encapsulating the polynucleotide. In some embodiments, the composition comprises a lipid In some embodiments, 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.
第三方面，本发明提供一种疫苗制剂，其包含本发明的多核苷酸或组合物。在一些实施方案中，所述疫苗制剂中包封所述多核苷酸的脂质包含10-70摩尔％的M5、10-70摩尔％的1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、10-70摩尔％的胆固醇和0.05-20摩尔％的1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)2000；优选地，所述脂质为摩尔比为40:15:43.5:1.5的M5、DOPE、胆固醇和(DMG-PEG)2000，
In a third aspect, the present invention provides a vaccine formulation comprising a polynucleotide or composition of the present invention. In some embodiments, 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,
在一些实施方案中，所述疫苗制剂为液态制剂或冻干制剂。在一些实施方案中，所述疫苗制剂通过肌肉注射施用。在一些实施方案中,所述疫苗制剂粘膜内施用,例如鼻腔喷雾。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 intramucosally, such as a nasal spray.
第四方面，本发明提供一种预防或治疗有需要的对象中流感病毒感染的方法，所述方法包括给有需要的对象施用本发明的多核苷酸、组合物或疫苗制剂。In a fourth aspect, 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.
本发明还提供本发明的多核苷酸、组合物或疫苗制剂在制备用于预防和/或治疗有需要的对象中流感病毒感染的药物中的用途。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.
本发明还提供本发明的多核苷酸、组合物或疫苗制剂在制备，其用于预防和/或治疗有需要的对象中流感病毒感染。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.
在一些实施方案中，所述对象是人或非人动物。In some embodiments, the subject is a human or a non-human animal.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示构建mRNA的脂质多聚复合物(LPP)的流程图。FIG1 shows a flow chart for constructing lipid polyplexes (LPPs) of mRNA.
图2显示通过western blot分析用LPP制剂转染的293T细胞中NP蛋白(图2A)、M2e蛋白(图2B)表达的结果，以及用不同剂量(1和10μg)的LPP免疫的小鼠体内针对NM2e的抗体的滴度。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 μg) of LPP.
图3显示LPP制剂在小鼠中诱导的抗NP蛋白和抗M2e IgG滴度。Figure 3 shows the anti-NP protein and anti-M2e IgG titers induced by LPP preparation in mice.
图4和5显示LPP制剂在小鼠中诱导的针对NP蛋白和M2e的细胞免疫应答。Figures 4 and 5 show the cellular immune responses against NP protein and M2e induced by LPP preparations in mice.
图6显示LPP制剂免疫后对小鼠用流感病毒毒株X31(图6A)、PR8(图6B)和AH(图6C)进行攻毒实验的结果。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 ).
发明详述DETAILED DESCRIPTION OF THE INVENTION
一、一般定义1. General Definition
本文引用的所有专利、专利申请、科学出版物、制造商的说明书和指南等，无论上文或下文，均整体援引加入本文。本文中的任何内容均不应理解为承认本公开无权先于 这样的公开。All patents, patent applications, scientific publications, manufacturer's instructions and guides, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate Such disclosure.
除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白和核酸化学、分子生物学、细胞和组织培养、微生物学相关术语均为相应领域内广泛使用的术语(参见，例如，Molecular Cloning:A Laboratory Manual,2nd Edition,J.Sambrook et al.eds.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。Unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology related terms used herein are widely used terms in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). At the same time, in order to better understand the present invention, the definitions and explanations of the related terms are provided below.
如本文所用，表述“包括”、“包含”、“含有”和“具有”是开放式的，表示包括所列举的元素、步骤或组分但不排除其他未列举的元素、步骤或组分。表述“由……组成”不包括未指定的任何元素、步骤或组分。表述“基本上由……组成”是指范围限于指定的元素、步骤或组分，加上不显著影响要求保护的主题的基本和新颖性质的任选存在的元素、步骤或组分。应当理解，表述“基本上由……组成”和“由……组成”涵盖在表述“包含”的含义之内。As used herein, 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."
如本文所用，除非上下文另外指明，单数形式的表述“一个”、“一种”或“这个”包括复数指代。术语“一个或多个”或者“至少一个”涵盖1、2、3、4、5、6、7、8、9个或更多个。As used herein, the singular forms "a", "an" or "the" include plural references unless the context indicates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.
本文中值的范围的列举仅为了用作单独提到落在所述范围内的每个不同值的速记方法。除非本文另有说明，否则每个单独的值如其在本文中单独列举地加入本说明书。除非明确指出相反，在本文示出的数值或范围均由“约”修饰，表示所列举或声称的数值或范围±20％、±10％、±5％或±3％。Recitation of ranges of values herein is intended merely to serve as a shorthand method of referring individually to each different 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 expressly indicated to the contrary, values or ranges set forth herein are modified by "about", meaning ±20%, ±10%, ±5%, or ±3% of the recited or claimed value or range.
除非另有说明，否则本文描述的所有方法可以以任何合适的顺序进行。Unless otherwise stated, all methods described herein can be performed in any suitable order.
如本文所用，术语“野生型”表示该序列是天然存在的并且未经人为修饰的，包括天然存在的突变体。As used herein, the term "wild type" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.
如本文所用，关于序列的术语“％相同性”是指在待比较的序列之间的最佳比对中相同的核苷酸或氨基酸的百分比。两个序列之间的差异可以分布在待比较序列的局部区域(区段)或整个长度上。通常在对区段或“比较窗口”最佳比对之后，确定两个序列之间的相同性。最佳比对可以手动进行，或者借助于本领域已知算法，包括但不限于Smith and Waterman,1981,Ads App.Math.2,482和Neddleman and Wunsch,1970,J.Mol.Biol.48,443描述的局部同源性算法，Pearson and Lipman,1988,Proc.Natl Acad.Sci.USA 88,2444描述的相似性搜索方法，或使用计算机程序，例如Wisconsin Genetics Software Package,Genetics Computer Group,575 Science Drive,Madison,Wis.中的GAP、BESTFIT、FASTA、BLAST P、BLAST N和TFASTA进行。例如，可以利用美国国家生物技术信息中心(NCBI)网站公共可用的BLASTN或BLASTP算法确定两个序列的百分比相同性。As used herein, the term "% identity" with respect to a sequence 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. Sci. USA 88, 2444, or using computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, 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).
通过确定待比较的序列对应的相同位置的数目，用这个数目除以比较的位置数目(例如，参考序列中的位置数目)，并将这个结果乘以100，获得％相同性。在一些实施方案中，至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％或约100％的区域给出相同性程度。在一些实施方案中，对参考序列的整个长度给出相同性程度。可以用本领域已知的工具 进行确定序列相同性的比对，优选利用最佳序列比对，例如，利用Align，利用标准设置，优选EMBOSS::needle、Matrix:Blosum62、Gap Open 10.0、Gap Extend 0.5。% 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, 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%. In some embodiments, 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.
在本文中，“核苷酸”包括脱氧核糖核苷酸和核糖核苷酸及其衍生物。如本文所用，“核糖核苷酸”是核糖核酸(RNA)的构成物质，由一分子碱基，一分子五碳糖，一分子磷酸组成，其是指在β-D-呋喃核糖(β-D-ribofuranosyl)基团的2’位置具有羟基的核苷酸。而“脱氧核糖核苷酸”是脱氧核糖核酸(DNA)的构成物质，也由一分子碱基，一分子五碳糖，一分子磷酸构成，其是指在β-D-呋喃核糖(β-D-ribofuranosyl)基团的2’位置的羟基被氢取代的核苷酸，是染色体的主要化学成分。“核苷酸”通常由代表其中碱基的单字母来指代：“A(a)”指含有腺嘌呤的脱氧腺苷酸或腺苷酸，“C(c)”指含有胞嘧啶的脱氧胞苷酸或胞苷酸，“G(g)”指含有鸟嘌呤的脱氧鸟苷酸或鸟苷酸，“U(u)”指含有尿嘧啶的尿苷酸，“T(t)”指含有胸腺嘧啶的脱氧胸苷酸。In this article, "nucleotide" includes deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "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 β-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 β-D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes. "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.
如本文所用，术语“多核苷酸”和“核酸”可以互换使用，用于指脱氧核糖核苷酸的聚合物(脱氧核糖核酸，DNA)或核糖核苷酸的聚合物(核糖核酸，RNA)。“多核苷酸序列”、“核酸序列”和“核苷酸序列”可以互换使用，用来表示多核苷酸中核苷酸的排序。本领域人员应当理解，DNA编码链(有义链)与其编码的RNA可以看作具有相同的核苷酸序列，DNA编码链序列中的脱氧胸苷酸对应其编码的RNA序列中的尿苷酸。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 order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that a DNA coding strand (sense strand) and the 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.
如本文所用，“编码序列”是指多核苷酸中可以作为模板用于在生物过程中合成具有确定的核苷酸序列(例如tRNA和mRNA)或确定的氨基酸序列的核苷酸序列。编码序列可以是DNA序列或RNA序列。如果对应于DNA序列(包括与mRNA序列相同的编码链和与之互补链的模板链)的mRNA在生物过程中翻译成多肽，可以认为所述DNA序列或mRNA序列编码所述多肽。As used herein, "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.
如本文所用，“密码子”指多核苷酸中三个连续的核苷酸序列(又称三联体密码)，其编码特定的氨基酸。同义密码子(编码相同氨基酸的密码子)在不同物种中使用的频率不同，称为“密码子偏好性”。通常认为，对于给定物种，使用其偏好的密码子的编码序列可以在该物种表达***中具有较高的翻译效率和准确率。因此，可以对多核苷酸进行“密码子优化”，即改变多核苷酸中的密码子以反映宿主细胞偏好的密码子，而优选不改变其编码的氨基酸序列。本领域技术人员会理解，由于密码子的简并性，本发明的多核苷酸可以包含这样的编码序列，其与本文所述编码序列不同(例如与本文所述编码序列具有约70％、75％、80％、85％、90％、95％、96％、97％、98％、99％相同性)但编码相同的氨基酸序列。在特定实施方案中，本发明的RNA包含针对宿主(例如受试者，特别是哺乳动物)细胞优化的密码子，使得本发明的多肽在受试者，如哺乳动物、禽类或人中最佳表达。As used herein, "codon" 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. It will be understood by those skilled in the art that due to the degeneracy of codons, 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. In a specific embodiment, 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.
如本文所用，术语“表达”包括核苷酸序列的转录和/或翻译。因此，表达可以涉及转录物和/或多肽的产生。术语“转录”涉及将DNA序列中的遗传密码转录为RNA(转录物)的过程。术语“体外转录”指在不含细胞的***中(例如在适当的细胞提取物中)体外合成RNA，特别是mRNA(参见，例如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.)。可以用 于产生转录物的载体又称为“转录载体”，其中包含转录所需的调控序列。术语“转录”涵盖“体外转录”。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" refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "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. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). It may be expressed as A vector for producing transcripts is also called a "transcription vector", which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription".
如本文所用，“编码”是指多核苷酸中特定核苷酸序列的固有特性，比如基因，cDNA或者mRNA都能作为模板去合成其他生物过程中的多聚物和大分子，只要已经有明确的核苷酸序列或者有明确的氨基酸序列。因此一个基因编码蛋白是指基因的mRNA通过转录和翻译在细胞中或其它生物***中产生蛋白。As used herein, "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.
如本文所用，术语“多肽”指包含通过肽键共价连接的两个以上氨基酸的聚合物。“蛋白”可以包含一条或多条多肽，其中多肽之间通过共价或非共价方式相互作用。除非另有说明，“多肽”和“蛋白”可以互换使用。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 covalently or non-covalently. Unless otherwise indicated, "polypeptide" and "protein" may be used interchangeably.
如本文所用，术语“宿主细胞”指用于接受、保持、复制、表达多核苷酸或载体的细胞。在一些实施方案中，宿主细胞可以是在其中表达本发明的多肽的细胞。As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, express a polynucleotide or vector. In some embodiments, the host cell may be a cell in which a polypeptide of the present invention is expressed.
如本文所用，“抗原”是指进入机体后可以引起机体获得性免疫应答的分子，此免疫应答可能涉及抗体的产生，或特定的免疫原活性细胞，或者两者同时发生。本领域技术人员应当理解任何大分子，几乎包括所有蛋白质或肽段可以作为抗原。更进一步，抗原可以来自重组或者基因组DNA或RNA。本领域技术人员应当理解任何在本文的DNA或RNA，它们的核苷酸序列或者部分核苷酸序列可以编码能引起机体获得性免疫的蛋白。更进一步，本领域技术人员应当理解一个抗原不需要单独地只编码一个基因的全长核苷酸序列。显而易见地，本发明包括但不限于使用超过一个基因的部分核苷酸序列，并且这些核苷酸序列形成不同的混合物去诱导应答的发生。更进一步，本领域技术人员应当理解抗原不需要完全被一个基因所编码。显而易见地，抗原可以被合成生成，也可以来源于生物样本。生物样本包括但不限于组织样本，肿瘤样本，细胞或生物流体。As used herein, "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. Obviously, 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. Furthermore, those skilled in the art will understand that antigens do not need to be completely encoded by a gene. Obviously, 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.
如本文所用，“抗体”是指机体由于抗原的刺激而产生的具有保护作用的蛋白质。它是一种免疫球蛋白，由B淋巴细胞产生。抗体的单体是一个Y形的分子，有4条多肽链组成。其中包括两条相同的重链，以及两条相同的轻链，之间由双硫键连接在一起。每条重链50kDa，每条轻链25kDa，轻重链间存在二硫键链接。其独特之处在于对结合伙伴的高亲和力和特异性。As used herein, "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.
如本文所用，“疫苗”是指包含活性成分(例如本发明的多核苷酸)的组合物，它能通过接种引起接种对象免疫应答。在具体实施方案中，它诱导的免疫应答能提供免疫保护，并足以预防和/或减轻与病原体或疾病感染相关的至少一种症状。根据本发明，本文所述的多核苷酸或组合物可以作为疫苗，用于在有需要的受试者中提供针对流感病毒的预防性和/或治疗性免疫。As used herein, "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. In a specific embodiment, 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. According to the present invention, 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.
如本文所用，术语“中和抗体”是指能够中和，即防止、抑制、降低或干扰病原体在宿主(例如宿主生物体或宿主细胞)中引发和/或保持感染的能力的抗体或其片段。根据本发明，用本发明的疫苗进行接种的受试者中可以产生针对流感病毒的中和抗体，例如在受试者的免疫血清中。可以使用本领域已知的方法测量免疫血清中的中和抗体滴度水平。As used herein, the term "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). According to the present invention, 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.
如本文所用，“免疫应答”是指涉及到效应功能激活和/或诱导的过程，效应功能发生在不限于以下举例中，如T细胞、B细胞、自然杀伤性细胞和/或抗原呈递细胞。因此，免疫应答可以被本领域技术人员理解为包括但不限于任何可检测的T辅助性细胞抗 原特异性激活和/或诱导，细胞毒性T细胞活性或应答，抗体的产生，抗原呈递细胞活性或浸润，巨噬细胞活性或浸润，中性粒细胞活性或浸润，或者类似反应。As used herein, "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. Thus, 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.
如本文所用，“Th1”是指初始CD4+T细胞在干扰素-γ(IFN-γ)的诱导下可分化为Th1细胞，分泌IFN-γ，参与细胞介导的免疫应答和单核细胞或巨噬细胞介导的炎症反应；在IL-4的诱导下可分化为Th2细胞，分泌IL-4、IL-5等细胞因子，参与体液免疫应答，能刺激B细胞促进抗体产生，并促进肥大细胞和嗜酸细胞的增殖和功能。As used in this article, "Th1" means that the initial CD4+ T cells can differentiate into Th1 cells under the induction of interferon-γ (IFN-γ), secrete IFN-γ, 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.
如本文所用，“流感病毒”是正粘病毒科成员，是一种有包膜的负链RNA病毒。流感病毒基因组RNA与核蛋白(nucleoprotein，NP)结合形成核糖核蛋白(ribonucleoprotein，RNP)复合物。流感病毒还包含基质蛋白、血凝素和神经酰胺酶。血凝素(hemagglutinin，HA)和神经氨酸酶(neuraminidase，NA)，是流感病毒包膜中的糖蛋白，负责病毒和宿主的表面接触。病毒进入宿主需要HA的调控，它与细胞受体结合并促进病毒膜与内体膜的融合。流感病毒可以根据HA和NA的不同而分为多种亚型。As used herein, "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.
“NP蛋白”是具有498个氨基酸的基本蛋白。在它的N端有RNA结合域和两个NP-NP自相互作用区域。它们对病毒核糖核蛋白的维持至关重要，可以与多种宿主蛋白发生相互作用，在流感病毒复制周期中起着十分重要的作用。NP蛋白具有在不同流感病毒中都高度保守的区域。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”指基质蛋白2的胞外域。M2蛋白是流感病毒的基质蛋白，全长是97个氨基酸，包括N端24个氨基酸的胞外域，19个氨基酸跨膜结构域，以及C端54个氨基酸的胞内域。M2蛋白的胞外域在流感病毒中高度保守。"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.
如本文所用，术语“NM2e”是指NP蛋白和M2e的融合多肽，包含全长NP蛋白(SEQ ID NO:1的位置1-498)、其变体或片段和M2蛋白的残基2-24(SEQ ID NO:1的位置499-521)。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), its variants or fragments and residues 2-24 of M2 protein (positions 499-521 of SEQ ID NO: 1).
如本文所用，“NP55–69”是指由NP蛋白的残基55-69组成的肽，其为H-2d限制性Th表位，其氨基酸序列为RLIQNSLTIERMVLS。As used herein, "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.
如本文所用，“NP147–155”是指由NP蛋白的残基147-155组成的肽，其为H-2d限制性CTL表位，其氨基酸序列为TYQRTRALV。As used herein, "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.
如本文所用，“M2e肽池”是指M2e蛋白的混合肽，其中包含分别对应于M2蛋白的残基1-15(MSLLTEVETPIRNEW)，残基5-19(TEVETPIRNEWGCRC)和残基9-23(TPIRNEWGCRCNDSS)的三条肽。As used herein, "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.
如本文所用，术语“脂质”是指包含疏水部分并且任选地还包含亲水部分的有机化合物。脂质通常难溶于水但可溶于许多有机溶剂。通常，包含疏水部分和亲水部分的两亲性脂质可以在水环境中组织为脂质双层结构，例如以囊泡形式存在。脂质可以包括但不限于：脂肪酸、甘油酯、磷脂、鞘脂、糖脂和类固醇和胆固醇酯等。As used herein, the term "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.
如本文所用，术语“阳离子聚合物”涉及在指定pH下能够带有净正电荷从而与核酸静电结合的任何离子聚合物。阳离子聚合物的实例包括但不限于：聚-L-赖氨酸、鱼精蛋白和聚乙烯亚胺(PEI)。聚乙烯亚胺可以是线性或支化的聚乙烯亚胺。As used herein, the term "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). The polyethyleneimine can be linear or branched polyethyleneimine.
术语“鱼精蛋白”是指富含精氨酸的低分子量碱性蛋白，其存在于各种动物(特别是鱼)的***中并代替组蛋白与DNA结合。在一优选实施方案中，阳离子聚合物为鱼 精蛋白(例如硫酸鱼精蛋白)。The term "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. In a preferred embodiment, the cationic polymer is fish Protamine (eg protamine sulfate).
二、多肽2. Peptide
本发明涉及NM2e多肽。在一些实施方案中，所述NM2e多肽包含与SEQ ID NO:1具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的氨基酸序列。在一些实施方案中，所述NM2e包含NP蛋白的变体和/或片段，以及M2e，其中所述NP蛋白的变体和/或片段包含NP蛋白的保守区。在一些实施方案中，所述保守区不包含突变(包括氨基酸的取代、缺失和***)。在一些实施方案中，所述保守区包含保守性取代。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 higher identity to SEQ ID NO: 1. In some embodiments, 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. In some embodiments, the conserved region does not comprise mutations (including substitutions, deletions and insertions of amino acids). In some embodiments, the conserved region comprises conservative substitutions.
在一些实施方案中，所述NM2e的NP区段中包含至少一个氨基酸修饰，例如***、取代和/或缺失。在一些实施方案中，所述NM2e的NP区段中包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸的取代、***和/或缺失。In some embodiments, 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.
三、多核苷酸3. Polynucleotides
本发明还涉及编码所述NM2e多肽的多核苷酸。多核苷酸可以是单链的或双链的。多核苷酸包括但不限于DNA、cDNA、RNA(例如mRNA)、重组产生和化学合成的多核苷酸。多核苷酸可以包含在载体中。本发明的多核苷酸可以包括天然存在的、合成的和修饰的核苷酸。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.
在一些实施方案中，本发明的多核苷酸用于在细胞中表达本文所述的多肽以提供多肽抗原。在一些实施方案中，所述多肽抗原可以在合适的受试者中诱导针对流感病毒的免疫应答，例如细胞免疫应答和抗体应答。In some embodiments, the polynucleotides of the present invention are used to express the polypeptides described herein in cells to provide polypeptide antigens. In some embodiments, 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.
所述多核苷酸可以包含一个或多个区段(核苷酸片段)(例如1、2、3、4、5、6、7、8个区段)。多核苷酸可以包含编码感兴趣多肽(例如本文所述多肽和多肽抗原)的区段。在特定实施方案中，多核苷酸可以包含感兴趣多肽的编码序列以及调控序列(包括但不限于转录和翻译调控序列)。在一实施方案中，调控序列包含以下的一个或多个：启动子序列、5’非翻译区(5’UTR)序列、3’非翻译区(3’UTR)序列和poly(A)序列。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). In a specific embodiment, 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). In one embodiment, 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.
在一些实施方案中，本发明的多核苷酸包含如本文所述多肽抗原的编码序列。在一实施方案中，本发明的多核苷酸包含与本文所述编码序列互补的核苷酸序列。在一些实施方案中，本发明的多核苷酸包含如本文所述多肽的编码序列。在一实施方案中，编码序列在其5’端包含起始密码子，并且在其3’端包含终止密码子。在一实施方案中，编码序列包含本文所述的开放阅读框(ORF)。In some embodiments, 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.
在一些实施方案中，所述多核苷酸包含与SEQ ID NO:4具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的核苷酸序列。在一些实施方案中，所述多核苷酸编码的NM2e包含与SEQ ID NO:1具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的氨基酸序列。在一些实施方案中，所述NM2e包含NP蛋白的变体和/或片段，以及M2e，其中所述NP蛋白的变体和/或片段包含NP蛋白的保守区。在一些实施方案中，所述保守区不包含突变(包括氨基酸的取代、缺失 和***)。在一些实施方案中，所述保守区包含保守性取代。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 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. In some embodiments, 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. In some embodiments, the conserved region does not comprise a mutation (including amino acid substitutions, deletions, In some embodiments, the conserved region comprises a conservative substitution.
在一些实施方案中，所述NM2e的NP区段中包含至少一个氨基酸修饰，例如***、取代和/或缺失。在一些实施方案中，所述NM2e的NP区段中包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸的取代、***和/或缺失。在一些实施方案中，所述NP区段包含NP蛋白的保守区，所述保守区不包含所述氨基酸修饰。In some embodiments, 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.
在一些实施方案中，本发明的多核苷酸是RNA。如本文所用，“RNA”的定义涵盖单链、双链、线性和环状RNA。本发明的RNA可以是通过化学合成的、重组产生的和体外转录的RNA。在一实施方案中，本发明的RNA用于在宿主细胞中表达本发明的多肽。In some embodiments, the polynucleotide of the present invention is RNA. As used herein, the definition of "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. In one embodiment, the RNA of the present invention is used to express the polypeptide of the present invention in a host cell.
在一些实施方案中，本发明的RNA是单链RNA。在一实施方案中，本发明的RNA是体外转录的RNA(IVT-RNA)。IVT-RNA可以通过RNA聚合酶利用DNA模板进行体外转录获得(例如如本文所述)。In some embodiments, the RNA of the present invention is a single-stranded RNA. In one embodiment, 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).
在一些实施方案中，本发明的RNA是信使RNA(mRNA)。一般而言，mRNA可以包含5’-UTR序列、多肽的编码序列、3’-UTR序列和任选存在的poly(A)序列。mRNA可以例如通过体外转录或化学合成产生。在一实施方案中，本发明的mRNA通过RNA聚合酶(例如T7RNA聚合酶)利用DNA模板进行体外转录获得。在一实施方案中，本发明的mRNA包含(1)5’-UTR、(2)编码序列、(3)3’-UTR和(4)任选存在的poly(A)序列。所述5’-UTR、编码序列、3’-UTR和poly(A)序列如本文所述。在一实施方案中，本发明的mRNA是核苷修饰的mRNA。在一实施方案中，本发明的mRNA包含任选存在的5’帽。In some embodiments, the RNA of the present invention is a messenger RNA (mRNA). In general, 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. In one embodiment, 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). In one embodiment, 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. 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.
在一些实施方案中，本发明的RNA包含如本文所述多肽抗原的编码序列。在一些实施方案中，本发明的RNA包含如本文所述多肽的编码序列。In some embodiments, 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.
在一些实施方案中，本发明的RNA还包含有助于提高RNA的稳定性和/或翻译效率的结构元件，包括但不限于5’帽、5’-UTR、3’-UTR和poly(A)序列。In some embodiments, 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.
如本文所用，术语“非翻译区(UTR)”一般指RNA中(如mRNA)中不翻译为氨基酸序列的区域(非编码区)，或者DNA中的相应区域。通常，位于开放阅读框(起始密码子)的5’端(上游)的UTR可以称为5’非翻译区 5’-UTR；位于开放阅读框(终止密码子)的3’端(下游)的UTR可以称为3’-UTR。在5’帽存在的情况下，5’-UTR位于5’帽的下游，例如，与5’帽直接相邻。在特定实施方案中，可以在5’-UTR中，例如在临近起始密码子的位置，包含优化的“Kozak序列”以提高翻译效率。在poly(A)序列存在的情况下，3’-UTR位于poly(A)序列的上游，例如与poly(A)序列直接相邻。As used herein, 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. Generally, 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. In the presence of a 5’ cap, the 5’-UTR is located downstream of the 5’ cap, for example, directly adjacent to the 5’ cap. In a specific embodiment, an optimized “Kozak sequence” may be included in the 5’-UTR, for example, near the start codon, to improve translation efficiency. In the presence of a poly(A) sequence, the 3’-UTR is located upstream of the poly(A) sequence, for example, directly adjacent to the poly(A) sequence.
在一些实施方案中，本发明的RNA包含5’-UTR。在一优选实施方案中，5’-UTR包含SEQ ID NO:2的核苷酸序列。在一优选实施方案中，3’-UTR包含SEQ ID NO:3的核苷酸序列。在一些实施方案中，本发明的RNA包含5’-UTR和3’-UTR。在一具体实施方案中，5’-UTR包含SEQ ID NO:2的核苷酸序列，3’-UTR包含SEQ ID NO:3的核苷酸序列。In some embodiments, the RNA of the present 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 present 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.
在一些实施方案中，本发明的RNA包含poly(A)序列。“poly(A)序列”或“poly(A)尾” 是指包含连续或不连续腺苷酸的核苷酸序列。poly(A)序列通常位于RNA的3’端，例如3’-UTR的3’端(下游)。在一些实施方案中，poly(A)序列在其3’端不包含腺苷酸以外的核苷酸。Poly(A)序列可以在制备IVT-RNA期间，由DNA依赖性RNA聚合酶根据DNA模板的编码序列转录产生，或者通过不依赖于DNA的RNA聚合酶(poly(A)聚合酶)连接至IVT-RNA的游离3’端，例如3’-UTR的3’端。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 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. In some embodiments, 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).
在一实施方案中，poly(A)序列包含连续的腺苷酸。在一实施方案中，poly(A)序列可以包含至少20、30、40、50、60、70、75、80、85、95或100以及多达120、150、180、200、300个腺苷酸。在一实施方案中，poly(A)序列中的连续腺苷酸序列被包含U、C或G核苷酸的序列中断。优选地，poly(A)序列包含75个腺苷酸。In one embodiment, the poly(A) sequence comprises consecutive adenylic acids. In one embodiment, 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. In one embodiment, the consecutive adenylic acid sequence in the poly(A) sequence is interrupted by a sequence comprising U, C or G nucleotides. Preferably, the poly(A) sequence comprises 75 adenylic acids.
poly(A)序列可以包含至少20、30、40、50、60、70、75、80、85、95或100以及多达120、150、180、200、300个核苷酸。在一实施方案中，poly(A)序列包含至少50个核苷酸。在一实施方案中，poly(A)序列包含至少80个核苷酸。在一实施方案中，poly(A)序列包含至少100个核苷酸。在一些实施方案中，poly(A)序列包含约70、80、90、100、120或150个核苷酸。在具体实施方案中，poly(A)序列包含75个核苷酸。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.
如本文所用，术语“5’帽”一般涉及通过5’至5’三磷酸键连接至mRNA的5’端的N7-甲基鸟苷结构(又称为“m7G帽”、“m7Gppp-”)。5’帽可以在体外转录中共转录加至RNA中(例如使用抗反向帽类似物“ARCA”)，或者可以利用加帽酶在转录后连接至RNA。As used herein, 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.
在一些实施方案中，本发明的RNA包含SEQ ID NO:10、11、12或13的核苷酸序列。在一些实施方案中，本发明的RNA包含(a)包含与SEQ ID NO:10、11、12或13的核苷酸序列具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的核苷酸序列；并且(b)编码与SEQ ID NO:1的氨基酸序列具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的氨基酸序列。在一些实施方案中，本发明的RNA所编码的多肽包含NP蛋白的保守区。在一些实施方案中，所述保守区不包含突变(包括氨基酸的取代、缺失和***)。在一些实施方案中，所述保守区包含保守性取代。In some embodiments, the RNA of the present invention comprises the nucleotide sequence of SEQ ID NO:10, 11, 12 or 13. In some embodiments, 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. In some embodiments, the polypeptide encoded by the RNA of the present invention comprises a conserved region of the NP protein. In some embodiments, the conserved region does not comprise a mutation (including substitutions, deletions and insertions of amino acids). In some embodiments, the conserved region comprises a conservative substitution.
在一些实施方案中，本发明的多核苷酸是DNA。这样的DNA可以是例如用于在体外转录本发明的RNA的DNA模板或者用于在宿主细胞中表达多肽抗原的DNA疫苗。DNA可以是双链、单链、线性和环状DNA。In some embodiments, the polynucleotide of the present invention is DNA. Such 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.
DNA模板可以在合适的转录载体中提供。一般而言，DNA模板可以是双链复合物，其包含与本文所述编码序列相同的核苷酸序列(编码链)和与本文所述编码序列互补的核苷酸序列(模板链)。如本领域技术人员已知的，DNA模板可以包含启动子、5’-UTR、编码序列、3’-UTR和任选存在的poly(A)序列。启动子可以是本领域技术人员已知的合适RNA聚合酶(特别是DNA依赖性RNA聚合酶)可用的启动子，包括但不限于SP6、T3和T7RNA聚合酶的启动子。在一些实施方案中，DNA模板中的5’-UTR、编码序列、3’-UTR和poly(A)序列为本文所述RNA中包含的相应序列或者与之互补。作为DNA疫苗的多核苷酸可以在质粒载体(例如环状质粒载体)中提供。The DNA template can be provided in a suitable transcription vector. In general, 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). As known to those skilled in the art, 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. In some embodiments, 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. As a polynucleotide of a DNA vaccine, it can be provided in a plasmid vector (e.g., a circular plasmid vector).
在一些实施方案中，本发明的DNA包含如本文所述多肽抗原的编码序列。在一些 实施方案中，本发明的DNA包含如本文所述多肽的编码序列。在一些实施方案中，本发明的DNA从5’端至3’端包含如本文所述的(1)T7启动子、(2)5’-UTR、(3)编码序列、(4)3’-UTR和(5)任选存在的poly(A)序列。In some embodiments, 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.
四、组合物和疫苗制剂IV. Compositions and Vaccine Formulations
本发明还提供一种组合物，其包含本发明的多核苷酸(特别是RNA)。在一实施方案中，本发明的组合物用于在受试者中提供针对流感病毒的预防性和/或治疗性免疫。在一些实施方案中，本发明的组合物包含本发明的多核苷酸。在一些实施方案中，本发明的组合物包含本发明的DNA。在一些实施方案中，本发明的组合物包含本发明的RNA。在一实施方案中，所述RNA为体外转录的RNA。在一实施方案中，所述RNA为mRNA。The present invention also provides a composition comprising a polynucleotide of the present invention (particularly RNA). In one embodiment, the composition of the present invention is used to provide preventive and/or therapeutic immunity against influenza virus in a subject. In some embodiments, the composition of the present invention comprises a polynucleotide of the present invention. In some embodiments, the composition of the present invention comprises a DNA of the present invention. In some embodiments, the composition of the present invention comprises an RNA of the present invention. In one embodiment, the RNA is an RNA transcribed in vitro. In one embodiment, the RNA is an mRNA.
在一些实施方案中，本发明的组合物包含如本文所述的多核苷酸(特别是RNA，例如mRNA)以及包封所述多核苷酸的脂质。In some embodiments, the compositions of the invention comprise a polynucleotide (particularly RNA, such as mRNA) as described herein and a lipid encapsulating the polynucleotide.
特别优选的核酸组合物可以是例如本文所述的脂质纳米颗粒(LNP)和脂质多聚复合物(LPP)。制备这类组合物的方法可以参见例如Kaczmarek,J.C.et al.,2017,Genome Medicine 9,60或者如本文所述。在一些实施方案中，本发明的组合物包含脂质纳米颗粒(LNP)或脂质多聚复合物(LPP)。在一些实施方案中，本发明的组合物为包含本发明的RNA的脂质纳米颗粒(LNP)或脂质多聚复合物(LPP)。Particularly preferred nucleic acid 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. In some embodiments, the composition of the present invention comprises lipid nanoparticles (LNP) or lipid polymer complexes (LPP). In some embodiments, the composition of the present invention is a lipid nanoparticle (LNP) or lipid polymer complex (LPP) comprising the RNA of the present invention.
在一些实施方案中，包封多核苷酸的脂质包含阳离子脂质和非阳离子脂质。在一优选实施方案中，所述阳离子脂质为可电离阳离子脂质。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.
在一实施方案中，阳离子脂质包含DOTMA、DOTAP、DDAB、DOSPA、DODAC、DODAP、DC-Chol、DMRIE、DMOBA、DLinDMA、DLenDMA、CLinDMA、DMORIE、DLDMA、DMDMA、DOGS、N4-胆固醇基-精胺、DLin-KC2-DMA、DLin-MC3-DMA或其组合。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-spermamine, DLin-KC2-DMA, DLin-MC3-DMA, or a combination thereof.
在一实施方案中，阳离子脂质包含M5，其具有如下结构：
In one embodiment, the cationic lipid comprises M5, which has the structure:
在一实施方案中，阳离子脂质包含DOTMA。在一实施方案中，阳离子脂质包含DOTAP。在一实施方案中，阳离子脂质包含DOTMA和DOTAP。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.
在一实施方案中，非阳离子脂质包含如本文所述的磷脂。在一实施方案中，非阳离子脂质包含如本文所述的类固醇。在一实施方案中，非阳离子脂质包含如本文所述的磷脂和类固醇。在一实施方案中，所述磷脂包含DSPC、DPPC、DMPC、DOPC、POPC、DOPE、DOPG、DPPG、POPE、DPPE、DMPE和DSPE或其组合。在一实施方案中，所述类固醇为胆固醇。在一实施方案中，非阳离子脂质包含DOPE。在一实施方案中，非阳离子脂质包含胆固醇。在一实施方案中，非阳离子脂质包含DOPE和胆固醇。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.
在一实施方案中，阳离子脂质包含M5，非阳离子脂质包含DOPE和胆固醇。In one embodiment, the cationic lipid comprises M5 and the non-cationic lipid comprises DOPE and cholesterol.
在一些实施方案中，包封多核苷酸的脂质进一步包含聚乙二醇修饰的脂质。在一实施方案中，聚乙二醇修饰的脂质包含DMG-PEG(例如DMG-PEG 2000)、DOGPEG和 DSPE-PEG或其组合。在一实施方案中，聚乙二醇修饰的脂质包含DSPE-PEG。在一实施方案中，聚乙二醇修饰的脂质包含DMG-PEG(例如DMG-PEG 2000)。In some embodiments, the lipids encapsulating the polynucleotide further comprise polyethylene glycol-modified lipids. In one embodiment, the polyethylene glycol-modified lipids comprise 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 (eg, DMG-PEG 2000).
在一些实施方案中，本发明的组合物进一步包含阳离子聚合物，所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在所述脂质中。In some embodiments, 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.
在一实施方案中，阳离子聚合物包含聚-L-赖氨酸、鱼精蛋白、聚乙烯亚胺(PEI)或其组合。在一实施方案中，阳离子聚合物为鱼精蛋白。在一实施方案中，阳离子聚合物为聚乙烯亚胺。In one embodiment, 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.
在一实施方案中，组合物中脂质的量以摩尔百分比(摩尔％)来计算，所述摩尔百分比基于组合物中脂质的总摩尔来确定。In one embodiment, 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.
在一实施方案中，组合物中阳离子脂质的量为约10-约70摩尔％。在一些实施方案中，组合物中阳离子脂质的量为约20-约60摩尔％、约30-约50摩尔％、约35-约45摩尔％、约38-约45摩尔％、约40-约45摩尔％、约40-约50摩尔％或约45-约50摩尔％。In one embodiment, 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%.
在一实施方案中，组合物中磷脂的量为约10-约70摩尔％。在一实施方案中，组合物中磷脂的量为约20-约60摩尔％、约30-约50摩尔％、约10-约30摩尔％、约10-约20摩尔％或约10-约15摩尔％。In one embodiment, 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%.
在一实施方案中，组合物中胆固醇的量为约10-约70摩尔％。在一实施方案中，组合物中胆固醇的量为约20-约60摩尔％、约30-约50摩尔％、约35-约40摩尔％、约35-约45摩尔％、约40-约45摩尔％或约45-约50摩尔％。In one embodiment, 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%.
在一实施方案中，组合物中聚乙二醇修饰的脂质的量为约0.05-约20摩尔％。在一实施方案中，组合物中聚乙二醇修饰的脂质的量为约0.5-约15摩尔％、约1-约10摩尔％、约5-约15摩尔％、约1-约5摩尔％、约1.5-约3摩尔％或约2-5摩尔％。In one embodiment, 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%.
在一些实施方案中，本发明的RNA(特别是mRNA)配制为脂质纳米颗粒(LNP)。如本文所用，“脂质纳米颗粒”或“LNP”涉及由脂质形成的颗粒，核酸(例如mRNA)被包封在脂质中。In some embodiments, RNA (particularly mRNA) of the present invention is formulated as lipid nanoparticle (LNP). As used herein, "lipid nanoparticle" or "LNP" refers to a particle formed by lipid, in which nucleic acid (e.g., mRNA) is encapsulated.
在一实施方案中，LNP包含本发明的RNA以及包封RNA的脂质，其中所述包封RNA的脂质包含阳离子脂质、磷脂、胆固醇和聚乙二醇修饰的脂质。在一实施方案中，所述阳离子脂质为M5。在一实施方案中，所述磷脂为DSPC。在一实施方案中，所述聚乙二醇修饰的脂质为DMG-PEG 2000。在一实施方案中，所述阳离子脂质为M5，所述磷脂为DSPC，所述聚乙二醇修饰的脂质为DMG-PEG 2000。In one embodiment, 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. 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.
在一实施方案中，所述包封RNA的脂质包含50摩尔％的M5、10摩尔％的DSPC、38.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG 2000。In one embodiment, the lipids encapsulating the RNA comprise 50 mol% M5, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG 2000.
在一些实施方案中，本发明的RNA(特别是mRNA)配制为脂质多聚复合物(lipopolyplex，LPP)。如本文所用，“脂质多聚复合物”或“LPP”是指包含由脂质外壳包封的核酸内核的核壳结构，所述核酸内核包含与聚合物缔合的核酸(例如mRNA)。In some embodiments, the RNA (particularly mRNA) of the present invention is formulated as a lipid polyplex (LPP). As used herein, "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.
在一实施方案中，LPP包含本发明的RNA，其与阳离子聚合物缔合为复合物；以及包封所述复合物的脂质，其中所述包封复合物的脂质包含阳离子脂质、非阳离子脂质和聚乙二醇修饰的脂质。在一实施方案中，所述非阳离子脂质包含磷脂和类固醇。在一实施方案中，所述非阳离子脂质包含选自1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、二 硬脂酰基磷脂酰胆碱(DSPC)或其组合的磷脂以及胆固醇。在一实施方案中，所述阳离子聚合物包含鱼精蛋白。在一实施方案中，所述聚乙二醇修饰的脂质包含DMG-PEG 2000。In one embodiment, 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. 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), di In one embodiment, the cationic polymer comprises protamine. In one embodiment, the polyethylene glycol-modified lipid comprises DMG-PEG 2000.
在一实施方案中，所述阳离子脂质包含M5，其具有如下结构：
In one embodiment, the cationic lipid comprises M5, which has the following structure:
所述非阳离子脂质包含选自1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、二硬脂酰基磷脂酰胆碱(DSPC)或其组合的磷脂以及胆固醇；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;
所述聚乙二醇修饰的脂质包含1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG 2000)；The polyethylene glycol-modified lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000);
所述阳离子聚合物包含鱼精蛋白。The cationic polymer comprises protamine.
在一实施方案中，所述阳离子聚合物为鱼精蛋白，所述阳离子脂质为M5，所述磷脂为DOPE，所述聚乙二醇修饰的脂质为DMG-PEG 2000。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.
在一实施方案中，所述包封复合物的脂质包含40摩尔％的M5、15摩尔％的DOPE、43.5摩尔％的胆固醇和1.5摩尔％的DMG-PEG 2000。In one embodiment, the lipids of the encapsulation complex comprise 40 mol% M5, 15 mol% DOPE, 43.5 mol% cholesterol and 1.5 mol% DMG-PEG 2000.
在一些实施方案中，本发明的疫苗制剂包含本文所述的多核苷酸。In some embodiments, vaccine formulations of the invention comprise a polynucleotide described herein.
在一些实施方案中，本发明的疫苗制剂包含本文所述的组合物，其中所述脂质包含10-70摩尔％的M5、10-70摩尔％的DOPE、10-70摩尔％的胆固醇和0.05-20摩尔％的DMG-PEG 2000，
In some embodiments, 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,
其中所述多核苷酸编码本文所述的多肽。wherein the polynucleotide encodes a polypeptide as described herein.
在一些实施方案中，所述多核苷酸包含与SEQ ID NO:4具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的核苷酸序列。在一些实施方案中，所述多核苷酸编码的NM2e包含与SEQ ID NO:1具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的氨基酸序列。在一些实施方案中，所述NM2e包含NP蛋白的变体和/或片段，以及M2e，其中所述NP蛋白的变体和/或片段包含NP蛋白的保守区。在一些实施方案中，所述保守区不包含突变(包括氨基酸的取代、缺失和***)。在一些实施方案中，所述保守区包含保守性取代。In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the conserved region does not comprise a mutation (including substitutions, deletions and insertions of amino acids). In some embodiments, the conserved region comprises a conservative substitution.
在一些实施方案中，所述NM2e的NP区段中包含至少一个氨基酸修饰，例如***、取代和/或缺失。在一些实施方案中，所述NM2e的NP区段中包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸的取代、***和/或缺失。在一些实施方案中，所述NP区段包含NP蛋白的保守区，所述保守区不包含所述氨基酸修饰。In some embodiments, 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.
在一些实施方案中，本发明的多核苷酸是RNA。如本文所用，“RNA”的定义涵盖单链、双链、线性和环状RNA。本发明的RNA可以是通过化学合成的、重组产生的和 体外转录的RNA。在一实施方案中，本发明的RNA用于在宿主细胞中表达本发明的多肽。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 RNA. The RNA of the invention can be chemically synthesized, recombinantly produced, and In vitro transcribed RNA. In one embodiment, the RNA of the invention is used to express a polypeptide of the invention in a host cell.
在一些实施方案中，本发明的RNA是单链RNA。在一实施方案中，本发明的RNA是体外转录的RNA(IVT-RNA)。IVT-RNA可以通过RNA聚合酶利用DNA模板进行体外转录获得(例如如本文所述)。In some embodiments, the RNA of the present invention is a single-stranded RNA. In one embodiment, 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).
在一些实施方案中，本发明的RNA是信使RNA(mRNA)。一般而言，mRNA可以包含5’-UTR序列、多肽的编码序列、3’-UTR序列和任选存在的poly(A)序列。mRNA可以例如通过体外转录或化学合成产生。在一实施方案中，本发明的mRNA通过RNA聚合酶(例如T7RNA聚合酶)利用DNA模板进行体外转录获得。在一实施方案中，本发明的mRNA包含(1)5’-UTR、(2)编码序列、(3)3’-UTR和(4)任选存在的poly(A)序列。所述5’-UTR、编码序列、3’-UTR和poly(A)序列如本文所述。在一实施方案中，本发明的mRNA是核苷修饰的mRNA。在一实施方案中，本发明的mRNA包含任选存在的5’帽。In some embodiments, the RNA of the present invention is a messenger RNA (mRNA). In general, 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. In one embodiment, 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). In one embodiment, 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. 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.
在一些实施方案中，本发明的RNA包含如本文所述多肽抗原的编码序列。在一些实施方案中，本发明的RNA包含如本文所述多肽的编码序列。In some embodiments, 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.
在一些实施方案中，本发明的RNA还包含有助于提高RNA的稳定性和/或翻译效率的结构元件，包括但不限于5’帽、5’-UTR、3’-UTR和poly(A)序列。In some embodiments, 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.
如本文所用，术语“非翻译区(UTR)”一般指RNA中(如mRNA)中不翻译为氨基酸序列的区域(非编码区)，或者DNA中的相应区域。通常，位于开放阅读框(起始密码子)的5’端(上游)的UTR可以称为5’非翻译区 5’-UTR；位于开放阅读框(终止密码子)的3’端(下游)的UTR可以称为3’-UTR。在5’帽存在的情况下，5’-UTR位于5’帽的下游，例如，与5’帽直接相邻。在特定实施方案中，可以在5’-UTR中，例如在临近起始密码子的位置，包含优化的“Kozak序列”以提高翻译效率。在poly(A)序列存在的情况下，3’-UTR位于poly(A)序列的上游，例如与poly(A)序列直接相邻。As used herein, 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. Generally, 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. In the presence of a 5’ cap, the 5’-UTR is located downstream of the 5’ cap, for example, directly adjacent to the 5’ cap. In a specific embodiment, an optimized “Kozak sequence” may be included in the 5’-UTR, for example, near the start codon, to improve translation efficiency. In the presence of a poly(A) sequence, the 3’-UTR is located upstream of the poly(A) sequence, for example, directly adjacent to the poly(A) sequence.
在一些实施方案中，本发明的RNA包含5’-UTR。在一优选实施方案中，5’-UTR包含SEQ ID NO:2的核苷酸序列。在一优选实施方案中，3’-UTR包含SEQ ID NO:3的核苷酸序列。在一些实施方案中，本发明的RNA包含5’-UTR和3’-UTR。在一具体实施方案中，5’-UTR包含SEQ ID NO:2的核苷酸序列，3’-UTR包含SEQ ID NO:3的核苷酸序列。In some embodiments, the RNA of the present 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 present 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.
在一些实施方案中，本发明的RNA包含poly(A)序列。In some embodiments, the RNA of the invention comprises a poly(A) sequence.
在一实施方案中，poly(A)序列包含连续的腺苷酸。在一实施方案中，poly(A)序列可以包含至少20、30、40、50、60、70、75、80、85、95或100以及多达120、150、180、200、300个腺苷酸。在一实施方案中，poly(A)序列中的连续腺苷酸序列被包含U、C或G核苷酸的序列中断。优选地，poly(A)序列包含75个腺苷酸。In one embodiment, the poly(A) sequence comprises consecutive adenylic acids. In one embodiment, 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. In one embodiment, the consecutive adenylic acid sequence in the poly(A) sequence is interrupted by a sequence comprising U, C or G nucleotides. Preferably, the poly(A) sequence comprises 75 adenylic acids.
poly(A)序列可以包含至少20、30、40、50、60、70、75、80、85、95或100以及多达120、150、180、200、300个核苷酸。在一实施方案中，poly(A)序列包含至少50个核苷酸。在一实施方案中，poly(A)序列包含至少80个核苷酸。在一实施方案中，poly(A)序列包含至少100个核苷酸。在一些实施方案中，poly(A)序列包含约70、80、90、100、 120或150个核苷酸。在具体实施方案中，poly(A)序列包含75个核苷酸。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.
如本文所用，术语“5’帽”一般涉及通过5’至5’三磷酸键连接至mRNA的5’端的N7-甲基鸟苷结构(又称为“m7G帽”、“m7Gppp-”)。5’帽可以在体外转录中共转录加至RNA中(例如使用抗反向帽类似物“ARCA”)，或者可以利用加帽酶在转录后连接至RNA。As used herein, 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.
在一些实施方案中，本发明的RNA包含SEQ ID NO:10、11、12或13的核苷酸序列。在一些实施方案中，本发明的RNA包含(a)包含与SEQ ID NO:10、11、12或13的核苷酸序列具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的核苷酸序列；并且(b)编码与SEQ ID NO:1的氨基酸序列具有至少60％、70％、80％、90％、95％、96％、97％、98％、99％或更高的相同性的氨基酸序列。在一些实施方案中，本发明的RNA所编码的多肽包含NP蛋白的保守区。在一些实施方案中，所述保守区不包含突变(包括氨基酸的取代、缺失和***)。在一些实施方案中，所述保守区包含保守性取代。In some embodiments, the RNA of the present invention comprises the nucleotide sequence of SEQ ID NO:10, 11, 12 or 13. In some embodiments, 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. In some embodiments, the polypeptide encoded by the RNA of the present invention comprises a conserved region of the NP protein. In some embodiments, the conserved region does not comprise a mutation (including substitutions, deletions and insertions of amino acids). In some embodiments, the conserved region comprises a conservative substitution.
五、脂质5. Lipids
1、阳离子脂质1. Cationic lipids
阳离子脂质是在指定pH下带有净正电荷的脂质。带有净正电荷的脂质可以通过静电相互作用与核酸缔合。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.
阳离子脂质的实例包括但不限于 1,2-二-O-十八烯基-3-三甲基铵丙烷(1,2-di-O-octadecenyl-3-trimethylammonium-propane，DOTMA)、1,2-二油酰基-3-三甲基铵-丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、双十烷基二甲基溴化铵(Didecyldimethylammonium bromide，DDAB)、2,3-二油酰基氧基-N-[2(精胺羧酰胺)乙基]-N,N-二甲基-l-丙胺鎓三氟乙酸盐(2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate，DOSPA)、双十八烷基二甲基氯化铵(dioctadecyldimethyl ammonium chloride，DODAC)、1,2-二油酰基-3-二甲基铵-丙烷(1,2-dioleoyl-3-dimethylammonium-propane，DODAP)、3-(N—(N′,N′-二甲基氨基乙烷)-氨甲酰基)胆固醇(3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol，DC-Chol)、2,3-二(十四烷基氧基)丙基-(2-羟基乙基)-二甲基氨鎓(2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium，DMRIE)、N,N-二甲基-3,4-二油基氧基苄胺(N,N-dimethyl-3,4-dioleyloxybenzylamine，DMOBA)、1,2-二亚油基氧基-N,N-二甲基氨基丙烷(1,2-dilinoleyloxy-N,N-dimethylaminopropane，DLinDMA)、1,2-二亚油烯基氧基-N,N-二甲基氨基丙烷(1,2-dilinolenyloxy-N,N-dimethylaminopropane，DLenDMA)、3-二甲基氨基-2-(胆甾-5-烯-3-β-氧基丁烷-4-氧基)-1-(顺式,顺式-9,12-十八碳二烯基氧基)丙烷(3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane，CLinDMA)、N-(2-氨基乙基)-N,N-二甲基-2,3-双(十四烷基氧基)丙烷-1-胺鎓溴化物(N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide，DMORIE)、N,N-二甲基-2,3-双(十二烷基氧基)丙烷-1-胺(N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine，DLDMA)、N,N-二甲基-2,3-双(十四烷 基氧基)丙烷-1-胺(N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine，DMDMA)、双十八烷基酰氨基甘氨酰基精胺(dioctadecylamidoglycyl spermine，DOGS)、N4-胆固醇基-精胺(N4-cholesteryl-spermine)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧戊环(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane，DLin-KC2-DMA)、三十七烷基-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate，DLin-MC3-DMA)、十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((癸氧基)己基)氨基)辛酸酯)(heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate)、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)。Examples of 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)-dimethylazanium (DMRIE), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane ... -dimethylaminopropane, DLinDMA), 1,2-dilinolenyloxy-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-octadecadienoxy)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)propan-1-aminium bromide ( bromide, DMORIE), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), dioctadecylamidoglycyl spermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate utanoate, DLin-MC3-DMA), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).
在一些实施方案中，阳离子脂质优选为可电离阳离子脂质。可电离阳离子脂质在例如酸性pH下带有净正电荷，而在较高pH(例如生理pH)下是中性的。可电离阳离子脂质的实例包括但不限于：双十八烷基酰氨基甘氨酰基精胺(dioctadecylamidoglycyl spermine，DOGS)、N4-胆固醇基-精胺(N4-cholesteryl-spermine)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧戊环(2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane，DLin-KC2-DMA)、三十七烷基-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate，DLin-MC3-DMA)、十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((癸氧基)己基)氨基)辛酸酯)(heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate)、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)。In some embodiments, 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). Examples of 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)octanoate), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).
在一实施方案中，阳离子脂质包含M5，其具有以下结构：
In one embodiment, the cationic lipid comprises M5, which has the following structure:
2、非阳离子脂质2. Non-cationic lipids
在本文中，“非阳离子脂质”是指在指定pH下不带有净正电荷的脂质，例如阴离子脂质和中性脂质。术语“中性脂质”指在生理pH下以不带电、中性或两性离子形式存在的脂质。中性脂质可以包括但不限于磷脂和类固醇。In this article, "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. The term "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.
磷脂的实例包括但不限于：1,2-二油酰-sn-甘油-3-磷酸乙醇胺(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine，DOPE)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(1-palmitoyl-2-oleoylphosphatidylethanolamine，POPE)、二硬脂酰基磷脂酰胆碱(distearoylphosphatidylcholine，DSPC)、二硬脂酰基-磷脂酰乙醇胺(distearoyl-phosphatidylethanolamine，DSPE)、二油酰基磷脂酰胆碱(dioleoylphosphatidylcholine，DOPC)、二肉豆蔻酰基磷脂酰胆碱(dimyristoylphosphatidylcholine，DMPC)、二棕榈酰磷脂酰胆碱(dipalmitoylphosphatidylcholine，DPPC)、二花生四烯酰基磷脂酰胆碱(diarachidoylphosphatidylcholine，DAPC)、二二十二酰基磷脂酰胆碱 (dibehenoylphosphatidylcholine，DBPC)、二二十三酰基磷脂酰胆碱(ditricosanoylphosphatidylcholine，DTPC)、二二十四酰基磷脂酰胆碱(dilignoceroylphatidylcholine，DLPC)、棕榈酰油酰基-磷脂酰胆碱(palmitoyloleoyl-phosphatidylcholine，POPC)、二棕榈酰-磷脂酰乙醇胺(dipalmitoyl-phosphatidylethanolamine，DPPE)、二肉豆蔻酰基-磷脂酰乙醇胺(dimyristoyl-phosphatidylethanolamine，DMPE)和二月桂酰基-磷脂酰乙醇胺(dilauroyl-phosphatidylethanolamine，DLPE)。Examples of 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 ... sphatidylethanolamine, DSPE), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine The following are some of the most common phosphatidylcholine compounds: dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) and dilauroyl-phosphatidylethanolamine (DLPE).
类固醇的实例包括但限于例如胆固醇、胆甾烷醇、胆甾烷酮、胆甾烯酮、胆固醇基-2'-羟基乙基醚、胆固醇基-4'-羟基丁基醚、生育酚及其衍生物。Examples of steroids include, but are not limited to, for example, cholesterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and derivatives thereof.
3、聚乙二醇修饰的脂质3. Polyethylene glycol-modified lipids
如本文所用，术语“聚乙二醇修饰的脂质”是指包含聚乙二醇部分和脂质部分的分子。聚乙二醇修饰的脂质的实例包括但不限于：1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol，DMG‐PEG)、1,2-二油酰基-rac-甘油,甲氧基-聚乙二醇(1,2-Dioleoyl-rac-glycerol,methoxypolyethylene Glycol，DOGPEG))和1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-聚(乙二醇)(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol)，DSPE‐PEG)。As used herein, the term "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.
在一实施方案中，聚乙二醇修饰的脂质为DMG-PEG，例如DMG-PEG 2000。在一实施方案中，DMG-PEG 2000具有以下结构：
In one embodiment, the polyethylene glycol-modified lipid is DMG-PEG, such as DMG-PEG 2000. In one embodiment, DMG-PEG 2000 has the following structure:
其中n的平均值为44。The average value of n is 44.
六、预防和治疗流感病毒感染VI. Prevention and treatment of influenza virus infection
本发明提供本发明的多核苷酸(特别是RNA)、组合物或疫苗制剂，用于预防和/或治疗有需要的受试者流感病毒感染。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.
本发明提供本发明的多核苷酸(特别是RNA)、组合物或疫苗制剂在制备用于预防和/或治疗有需要的受试者流感病毒感染的药物中的用途。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.
本发明提供一种用于在有需要的受试者中预防和/或治疗流感病毒感染的方法，所述方法包括给受试者施用治疗有效量的本发明的多核苷酸(特别是RNA)、组合物或疫苗制剂。在一实施方案中，所述方法包括施用治疗有效量的包含本发明的mRNA的组合物，特别是包含如本文所述的LPP的组合物。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. In one embodiment, 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.
术语“预防或治疗有效量”是指足以预防或抑制疾病或症状的发生和/或减缓、减轻、延迟疾病或症状的发展或严重程度的量。预防或治疗有效量受到包括但不限于以下因素的影响：疾病或症状的发展速度和严重程度，受试者的年龄、性别、体重和生理状况，治疗的持续时间以及具体施用途径。预防或治疗有效量可以在一个或多个剂量中施用。预防或治疗有效量可以通过持续或间断施用实现。 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 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.
在一些实施方案中，预防或治疗有效量在一次或多次施用中提供。在一些实施方案中，预防或治疗有效量在两次施用中提供。在一些实施方案中，预防或治疗有效量在三次施用中提供。In some embodiments, 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.
在一些实施方案中，本发明的组合物或疫苗制剂可以通过本领域技术人员已知的任何方法施用受试者，例如肠胃外、经口、经粘膜、经皮、肌肉内、静脉内、皮内、皮下或腹膜内。优选地，本发明的组合物或疫苗制剂通过肌肉注射施用。In some embodiments, the 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. Preferably, the composition or vaccine formulation of the present invention is administered by intramuscular injection.
如本文所用，术语“受试者”描述了可以对其提供使用本发明多核苷酸或组合物的治疗的生物体，例如人、非人哺乳动物(如猪)或禽类(如鸡)。As used herein, 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.
七、有益效果VII. Beneficial Effects
本发明的多核苷酸、组合物、疫苗制剂和方法实现了在细胞中表达比现有技术更高水平的NM2e多肽，并在动物中诱导了显著的细胞和抗体应答，并提供了针对不同毒株(同型毒株和异型毒株)的改进的保护，可在体内诱发针对保守抗原的广谱交叉免疫保护。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.
实施例Example
通过参考以下实施例进一步描述本发明。应当理解，这些实施例仅作为示例，而不对本发明构成限制。以下材料和仪器均是可商购的或根据本领域公知的方法制备。以下实验均按照制造商的说明书或根据本领域公知的方法和步骤进行。The present invention is further described by reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the present invention. The following materials and instruments are commercially available or prepared according to methods known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.
实施例1、mRNA的制备Example 1. Preparation of mRNA
1.1.DNA模板的设计和合成1.1. Design and synthesis of DNA template
为了在实现最佳表达，发明人对编码SEQ ID NO:1的NM2e融合蛋白的核苷酸序列进行密码子优化，得到如表1所示的序列。其中NM2e-Ori代表野生型序列，NM2e-1、NM2e-2、NM2e-3、NM2e-4代表优化的序列。In order to achieve optimal expression, the inventors codon-optimized the nucleotide sequence encoding the NM2e fusion protein of SEQ ID NO: 1, and obtained the sequence shown in Table 1. NM2e-Ori represents the wild-type sequence, and NM2e-1, NM2e-2, NM2e-3, and NM2e-4 represent optimized sequences.
还设计T7启动子序列(TAATACGACTCACTATA)、5’-UTR序列(SEQ ID NO:19)、3’-UTR序列(SEQ ID NO:20)和poly(A)序列(75个腺苷核苷酸)。在5’UTR序列中包含Kozak序列“GCCACC”。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.
然后按照T7启动子序列、5’-UTR序列、DNA ORF序列、3’-UTR序列和poly(A)序列的顺序连接。以pUC57为载体进行全基因合成(苏州金唯智生物科技有限公司)，获得质粒DNA模板。Then, the 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.
利用一对加尾PCR引物(上游引物：5’TTGGACCCTCGTACAGAAGCTAATACG 3’；和下游poly(T)长引物：5’TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTTCCTACTCAGGCTTTATTCAAAGACCA 3’)和基于高保真DNA聚合酶的PCR扩增试剂盒(宝日医生物技术(北京)有限公司)进行PCR扩增获得DNA模板。A pair of tailing PCR primers (upstream primer: 5’TTGGACCCTCGTACAGAAGCTAATACG 3’; and downstream poly (T) long primer: 5’TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTTCCTACTCAGGCTTTATTCAAAGACCA 3’) and a high-fidelity DNA polymerase-based PCR amplification kit (Baori Medical Biotechnology (Beijing) Co., Ltd.) were used to obtain the DNA template by PCR amplification.
1.2.从DNA模板体外转录mRNA1.2. In vitro transcription of mRNA from DNA template
以PCR产物纯化试剂盒(Takara)纯化实施例1.1制备的PCR产物。以纯化后的PCR 产物为模板，通过T7RNA聚合酶进行共转录加帽反应，进行体外转录RNA，从而产生Cap1 mRNA。体外转录中使用1-甲基-假尿苷-三磷酸代替三磷酸尿苷(UTP)，因此，体外转录的Cap1 mRNA中1-甲基-假尿嘧啶的修饰比例为100％。转录结束后，使用DNaseI(赛默飞世尔科技有限公司)消化DNA模板，以降低残余DNA模板带来的风险。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%. After the transcription was completed, the DNA template was digested with DNaseI (Thermo Fisher Scientific) to reduce the risk of residual DNA template.
使用DynabeadsMyone(赛默飞世尔科技有限公司)对mRNA进行纯化。将纯化的mRNA溶解于1mM柠檬酸钠缓冲液(pH6.5)中，无菌过滤，并在-80℃下冷冻保存直至使用。所获得的mRNA序列如表1所示。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.
表1.核酸的序列
Table 1. Nucleic acid sequences
实施例2、LPP制剂的制备Example 2: Preparation of LPP preparation
2.1.实验材料2.1 Experimental Materials
阳离子脂质M5为斯微生物合成；辅助磷脂(DOPE)采购自CordenPharma；胆固醇采购于Sigma-Aldrich；mPEG2000-DMG(即DMG-PEG 2000)采购于Avanti Polar Lipids,Inc.；PBS采购于Invitrogen；硫酸鱼精蛋白采购自北京斯利安药业有限公司。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.
2.2.mRNA的脂质多聚复合物(LPP)的制备2.2. Preparation of mRNA lipid polyplex (LPP)
如图1所示，制备LPP。具体而言，所示制备包括以下步骤。As shown in Figure 1, LPP is prepared. Specifically, the preparation includes the following steps.
mRNA水溶液的配制：用10mM柠檬酸-柠檬酸钠缓冲液(pH 4.0)将如实施例1.2制备的每种mRNA稀释为0.2mg/mL的mRNA溶液。Preparation of 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).
脂质溶液的配制：将阳离子脂质(M5)：DOPE：胆固醇：DMG-PEG 2000以40:15:43.5:1.5的摩尔比溶解于无水乙醇，配制成10mg/mL的脂质溶液。Preparation of 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.
硫酸鱼精蛋白溶液的配制：将硫酸鱼精蛋白溶解于无核酸酶水中配制成工作浓度为0.25mg/mL的硫酸鱼精蛋白溶液。Preparation of 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.
核纳米颗粒(core nanoparticle)溶液的制备：使用微流控技术(迈安纳(上海)科技股份有限公司，型号：Inano D)，在以下条件将硫酸鱼精蛋白溶液与mRNA溶液混合获得由鱼精蛋白和mRNA形成的核纳米颗粒溶液：Volume＝4.0mL；Flow rate ratio＝5(mRNA):1(鱼精蛋白溶液)，Total flow rate＝12mL/min，前废(start waste)＝0.35mL，后废(end waste)＝0.1mL，室温。Preparation of core nanoparticle solution: Using microfluidic technology (Myana (Shanghai) Technology Co., Ltd., model: Inano D), the protamine sulfate solution and the mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution formed by protamine and mRNA: Volume = 4.0 mL; Flow rate ratio = 5 (mRNA): 1 (protamine solution), Total flow rate = 12 mL/min, start waste = 0.35 mL, end waste = 0.1 mL, room temperature.
LPP的制备：在以下条件下将核纳米颗粒溶液与脂质溶液进行二次混合：Volume＝4.0mL，Flow rate ratio＝1(脂质溶液):3(核纳米颗粒溶液)，Total flow rate＝12mL/min，前废＝0.35mL，后废＝0.1mL，室温，使用PBS溶液稀释，获得LPP溶液。Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed for a second time under the following conditions: Volume = 4.0 mL, Flow rate ratio = 1 (lipid solution): 3 (core nanoparticle solution), Total flow rate = 12 mL/min, front waste = 0.35 mL, rear waste = 0.1 mL, room temperature, diluted with PBS solution to obtain LPP solution.
离心超滤：将LPP溶液通过超滤离心去除乙醇(转速3000rpm，离心时间60min， 温度4℃)，获得LPP-NM2e制剂LPP-NPM2e-Ori、LPP-NPM2e-1、LPP-NPM2e-2、LPP-NPM2e-3和LPP-NPM2e-4。Centrifugal ultrafiltration: The LPP solution was centrifuged to remove ethanol (speed 3000 rpm, centrifugation time 60 min, Temperature 4°C) to obtain LPP-NM2e preparations LPP-NPM2e-Ori, LPP-NPM2e-1, LPP-NPM2e-2, LPP-NPM2e-3 and LPP-NPM2e-4.
实施例3、mRNA在细胞中的表达Example 3. Expression of mRNA in cells
将未经优化的LPP-NM2e-Ori和经优化的LPP-NM2e-1、LPP-NM2e-2、LPP-NM2e-3和LPP-NM2e-4转染293T细胞，24小时后进行收集细胞进行Western blot分析。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.
如图2A和2B所示，优化后LPP-NM2e的表达明显提高。As shown in Figures 2A and 2B, the expression of LPP-NM2e was significantly increased after optimization.
实施例4、检测LPP制剂的免疫原性Example 4: Detection of immunogenicity of LPP preparations
4.1.将5周龄BALB/c小鼠(n＝8，雌性，上海灵畅生物科技有限公司)，通过肌肉注射分别施用未经优化的LPP-NM2e-Ori和经优化的LPP-NM2e-1、LPP-NM2e-2、LPP-NM2e-3和LPP-NM2e-4，免疫剂量分别为1μg(低剂量组)、10μg(高剂量组)，各两剂，间隔三周。对初免后2周和加强后2周的小鼠进行眼眶后取血以收集血清并进行NPM2e结合抗体检测。4.1. 5-week-old BALB/c mice (n=8, female, Shanghai Lingchang Biotechnology Co., Ltd.) were administered non-optimized LPP-NM2e-Ori and optimized LPP-NM2e-1, LPP-NM2e-2, LPP-NM2e-3 and LPP-NM2e-4 by intramuscular injection, with immunization doses of 1 μg (low-dose group) and 10 μg (high-dose group), respectively, two doses each, three weeks apart. Mice were bled retro-orbitally 2 weeks after the primary immunization and 2 weeks after the boost to collect serum and perform NPM2e binding antibody detection.
如图2C所示，无论是1μg组还是10μg组，经优化的LPP-NPM2e-1、LPP-NM2e-2、LPP-NM2e-3和LPP-NM2e-4免疫的小鼠中的抗体滴度显著高于未经优化的LPP-NM2e-Ori免疫的小鼠。其中LPP-NM2e-3诱导的体液免疫比其它经优化的mRNA诱导的体液免疫强度更高，低剂量组初免后2周的抗体滴度达到70,400，加强后2周的抗体滴度达到525,000；高剂量组初免后2周的抗体滴度达到128,000，加强后2周的抗体滴度达到1,600,000(图2C)。因此，我们选择LPP-NPM2e-3(后续称为LPP-NPM2e)进一步深入表征其体液免疫和细胞免疫，并进行攻毒实验。As shown in Figure 2C, whether it is the 1μg group or the 10μg group, 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. Among them, the humoral immunity induced by LPP-NM2e-3 is stronger than the humoral immunity induced by other optimized mRNAs. The antibody titer of the low-dose group reached 70,400 2 weeks after the initial immunization, and the antibody titer reached 525,000 2 weeks after the boost; the antibody titer of the high-dose group reached 128,000 2 weeks after the initial immunization, and the antibody titer reached 1,600,000 2 weeks after the boost (Figure 2C). Therefore, we chose LPP-NPM2e-3 (hereinafter referred to as LPP-NPM2e) to further characterize its humoral immunity and cellular immunity, and conduct a challenge experiment.
4.2.表征LPP-NM2e诱导的免疫应答4.2. Characterization of LPP-NM2e-induced immune responses
将5周龄BALB/c小鼠(雌性，上海灵畅生物科技有限公司)随机分配为3组，通过肌肉注射分别施用(初免-加强)不包含mRNA的LPP(100μl，Mock组，n＝10)、1μg LPP-NM2e(低剂量组，n＝15)和10μg LPP-NM2e(高剂量组，n＝15)，各两剂，间隔三周。每剂施用后两周从眼眶取血进行抗体检测，并取小鼠脾脏制备脾单个核细胞进行细胞免疫应答检测。Five-week-old BALB/c mice (female, Shanghai Lingchang Biotechnology Co., Ltd.) were randomly divided into three groups and administered (primary immunization-boosting) LPP without mRNA (100μl, Mock group, n=10), 1μg LPP-NM2e (low-dose group, n=15) and 10μg LPP-NM2e (high-dose group, n=15) by intramuscular injection, two doses each, three weeks apart. Two weeks after each dose, blood was collected from the eye sockets for antibody detection, and spleen mononuclear cells were prepared from the mouse spleen for cellular immune response detection.
抗体检测的结果如图3所示，施用第一剂LPP-NM2e制剂后，检测到NP蛋白特异性抗体(IgG)滴度分别为6.28×103(低剂量组)和2.88×104(高剂量组)，显示出显著的剂量依赖性(P<0.0001)。施用第二剂LPP-NM2e制剂后，NP蛋白特异性抗体滴度分别升高至5.60×104(低剂量组)和1.34×105(高剂量组)，显著高于施用第一剂诱导的抗体滴度(P<0.05)，且显示出显著的剂量依赖性(P<0.0001)(图3A)。The results of antibody detection are shown in Figure 3. After the first dose of LPP-NM2e preparation was administered, the titers of NP protein-specific antibodies (IgG) were 6.28×10 3 (low-dose group) and 2.88×10 4 (high-dose group), respectively, showing significant dose-dependence (P<0.0001). After the second dose of LPP-NM2e preparation was administered, the titers of NP protein-specific antibodies increased to 5.60×10 4 (low-dose group) and 1.34×10 5 (high-dose group), respectively, which were significantly higher than the antibody titers induced by the first dose (P<0.05), and showed significant dose-dependence (P<0.0001) (Figure 3A).
施用第一剂LPP-NM2e制剂后，检测到M2e特异性抗体(IgG)滴度分别为1.2×102(低剂量组)和1.86×102(高剂量组)。施用第二剂LPP-NM2e制剂后，M2e特异性抗体滴度分别升高至9.70×102(低剂量组)与3.98×103(高剂量组)，高剂量组的滴度显著高于施用第一剂LPP-NM2e制剂后检测到的M2e特异性抗体滴度(P<0.05)(图3B)。After the first dose of LPP-NM2e preparation, the M2e-specific antibody (IgG) titers were 1.2×10 2 (low-dose group) and 1.86×10 2 (high-dose group). After the second dose of LPP-NM2e preparation, 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).
此外，对LPP-NM2e疫苗免疫(n＝4)后抗体IgG2a、IgG1亚型进行检测，结果如 表2和3所示，低剂量和高剂量组的NP蛋白、M2e特异性IgG2a:IgG1比值基本上大于1(只有一只小鼠小于1)，提示诱导了偏向Th1型免疫应答。In addition, the IgG2a and IgG1 subtypes of antibodies were detected after immunization with LPP-NM2e vaccine (n=4). As shown in Tables 2 and 3, 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.
表2 NP抗原特异性IgG2a/IgG1比值
Table 2 NP antigen-specific IgG2a/IgG1 ratio
表3 M2e抗原特异性IgG2a/IgG1比值
Table 3 M2e antigen-specific IgG2a/IgG1 ratio
用NP55-69、NP147-155和M2e肽池刺激所制备的脾单个核细胞。采用IFN-γELISPOT对LPP-NM2e免疫的小鼠所诱导的细胞免疫应答进行检测，结果如图3所示。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-γ ELISPOT, and the results are shown in FIG3 .
第一次施用后，来自低剂量组和高剂量组的脾单个核细胞中NP55-69、NP147-155和M2e特异性斑点形成(IFN-γ分泌)细胞数量平均密度分别为24SFC(spot-forming cells)/106SMNC(spleen mononuclear cells)、448SFC/106SMNC、10SFC/106SMNC和10SFC/106SMNC、383SFC/106SMNC和25SFC/106SMNC，均与对照组无显著差异。After the first administration, the average densities of NP 55-69 , NP 147-155 and M2e-specific spot-forming (IFN-γ-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.
第二次施用后，细胞免疫应答增强。低剂量组中NP55-69、NP147-155和M2e特异性SFC平均密度分别为196SFC/106SMNC、1943SFC/106SMNC、98SFC/106SMNC，显著高于第一次施用后的水平(P<0.01，P<0.05和P<0.05)；高剂量组中NP55-69、NP147-155和M2e特异性SFC平均密度分别为278SFC/106SMNCs、2950SFC/106SMNCs、128SFC/106SMNCs，NP55-69、NP147-155特异性细胞免疫应答显著高于第一次施用后的水平(P<0.0001)，M2e特异性免疫应答无统计显著性差异，但显示剂量依赖性趋势。After the second administration, the cellular immune response was enhanced. In the low-dose group, 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); in the high-dose group, 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.
此外，对LPP-NM2e疫苗免疫后5个月的小鼠(n＝4)脾脏细胞进行流式分析，采用NM2e肽池进行刺激，结果如图4和5所示，抗原特异性T细胞反应强烈，主要表现为分泌细胞因子IFN-γ和TNF-α的CD8和CD4的增加，并且T细胞反应仍然呈现出剂量依赖的趋势。In addition, flow cytometry analysis was performed on spleen cells of mice (n=4) 5 months after immunization with LPP-NM2e vaccine and stimulated with NM2e peptide pool. The results are shown in Figures 4 and 5. The antigen-specific T cell response was strong, mainly manifested by an increase in CD8 and CD4 that secrete cytokines IFN-γ and TNF-α, and the T cell response still showed a dose-dependent trend.
结果显示，本发明的mRNA的LPP制剂诱导了显著的针对流感病毒NP蛋白和M2e的细胞和抗体应答，抗体应答显示显著的剂量依赖性，而针对NP和M2e蛋白的细胞应答也显示出剂量依赖的趋势。The results showed that the LPP preparation of the mRNA of the present invention induced significant cellular and antibody responses against influenza virus NP protein and M2e, the antibody response showed significant dose-dependency, and the cellular response against NP and M2e proteins also showed a dose-dependent trend.
实施例5、LPP制剂在小鼠中诱导的免疫保护Example 5: Immune protection induced by LPP preparation in mice
本实施例采用来自中国疾病预防控制中心，病毒病预防控制所的流感病毒毒株X31(H3N2)、PR8(H1N1)和AH(H7N9)进行攻毒实验，以检测LPP制剂在小鼠中诱导的免疫保护。In this example, influenza virus strains X31 (H3N2), PR8 (H1N1) and AH (H7N9) from the Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention were used for challenge experiments to detect the immune protection induced by LPP preparations in mice.
如实施例4所述对小鼠进行免疫，在第一剂施用5周后，给小鼠滴鼻施用流感病毒(5xLD50剂量的毒株X31(n＝10)、3xLD50剂量的毒株PR8(n＝10)和3xLD50剂量的毒株AH(n＝10(高剂量组),n＝11(低剂量组))进行攻毒实验，以检测LPP制剂在小鼠中诱导的 免疫保护。在接种流感病毒后，每天观察并记录小鼠体重变化及存活情况，持续两周。Mice were immunized as described in Example 4. Five weeks after the first dose, influenza virus (5xLD50 dose of strain X31 (n=10), 3xLD50 dose of strain PR8 (n=10), and 3xLD50 dose of strain AH (n=10 (high dose group), n=11 (low dose group)) was administered intranasally to the mice for a challenge experiment to detect the effects of LPP preparations on the immune response in mice. Immune protection. After influenza virus inoculation, the weight changes and survival of mice were observed and recorded every day for two weeks.
体重变化如图6A、B和C左幅所示，低剂量和高剂量组的小鼠均在攻毒后5天体重下降至最低，之后体重逐渐升高恢复至Day0相当的水平。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.
小鼠存活情况如图6A、B和C右幅所示，攻毒后5天(X31)、6天(PR8)、5天(AH)，Mock组小鼠全部死亡。对于施用了LPP制剂的小鼠，接种毒株X31的小鼠存活率分别为60％(低剂量组)和100％(高剂量组)(图6A)；接种毒株PR8的小鼠存活率分别为90％(低剂量组)和100％(高剂量组)(图6B)；接种毒株AH的小鼠存活率分别为82％(低剂量组)和90％(高剂量组)(图6C)。The survival of mice is shown in the right panels of Figures 6A, B and C. All mice in the Mock group died 5 days (X31), 6 days (PR8) and 5 days (AH) after the challenge. For mice administered with LPP preparations, the survival rates of 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).
结果表明，本发明的LPP制剂不仅可有效保护小鼠抵御同型流感病毒X31(H3N2)的攻击，同时还可以有效保护抵御异型流感病毒毒株PR8(H1N1)，以及高致病性禽流感病毒毒株AH(H7N9)的攻击。The results show that the LPP preparation of the present invention can effectively protect mice not only against the attack of the same type of influenza virus X31 (H3N2), but also against the attack of the heterotypic influenza virus strain PR8 (H1N1) and the highly pathogenic avian influenza virus strain AH (H7N9).
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明精神和范围内，都可以做各种的改动与修饰，因此，本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
实施例6、材料和方法Example 6. Materials and methods
1、细胞、蛋白、抗体1. Cells, proteins, antibodies
HEK293细胞培养在补充有10％FBS(Gibco)、100U/mL盘尼西林和100mg/mL链霉素(Gibco)的Dulbecco’s modified Eagle’s medium(DMEM)中，培养条件为37℃和5％CO2。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 .
用于ELISA测定的重组NP、M2e和NPM2e蛋白均购自义翘神州，在大肠杆菌或杆状病毒－昆虫细胞中表达。The recombinant NP, M2e, and NPM2e proteins used in ELISA assays were purchased from Sino Biological and expressed in Escherichia coli or baculovirus-insect cells.
用于蛋白免疫印迹的抗体包括：抗甲型流感M2蛋白抗体购自Abcam；抗流感NP蛋白抗体购自义翘神州，抗小鼠辣根过氧化物酶(HRP)-缀合的抗体和抗兔辣根过氧化物酶(HRP)-缀合的抗体，购自Abbkine。The antibodies used for western blotting included: 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.
2、结合IgG抗体检测2. Combined with IgG antibody detection
将NP、M2e或NPM2e蛋白分别用0.05M碳酸钠缓冲液稀释至1μg/ml，加入96孔ELISA板(Greiner)中(100μl/孔)于4℃包被过夜。用PBS-T(磷酸缓冲盐水+0.05％Tween-20)洗涤板，之后用2％BSA(配制在PBS-T中)在37℃封闭60分钟。将2倍连续稀释的小鼠血清样品添加至包被的板中并在37℃孵育60分钟。然后将板用HRP缀合的二抗在37℃孵育60分钟。将板洗涤3次后，添加TMB底物(碧云天生物技术)。反应终止后，使用酶标仪(BioTek)读取450波长处的光密度(OD)。样品的吸光度值高于2.1倍阴性对照样品的最高稀释度的倒数值判定为最终滴度。NP, M2e or NPM2e protein were diluted to 1 μg/ml with 0.05 M sodium carbonate buffer and added to a 96-well ELISA plate ( Greiner) (100 μl/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. After the plates were washed 3 times, 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.
3、细胞转染3. Cell transfection
转染前一天将1.0×106细胞/孔HEK293细胞接种于6孔细胞培养皿中，次日细胞融 合至70-90％时，用新鲜培养基换液，将2.5μg的NPM2e mRNA-LPP与细胞共同孵育。24小时后收集细胞，用1×SDS-PAGE上样缓冲液(Beyotime)裂解LPP转染的细胞，进行SDS-PAGE和Western blot检测。One day before transfection, 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 μg 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.
4、酶联免疫斑点(ELISpot)测定4. ELISpot assay
按照制造商的说明，使用IFN-γELISpotPLUS试剂盒(Mabtech)进行小鼠IFN-γELISpot测定。简而言之，将板在RPMI 1640培养基(补充有10％FBS)中封闭并孵育30分钟。取下的小鼠脾脏，对脾脏进行研磨和过滤，并经红细胞裂解液处理后，对获得的细胞(即脾单个核细胞)计数，以3×105细胞/孔铺板，并用8μg/ml NP55-69肽段、8μg/ml NP14-155肽段和8μg/ml M2e肽池和10μg/ml NM2e肽池(均购自上海吉尔生化有限公司)，phytohemagglutinin(PHA)+ionomycin(阳性对照)或者仅RPMI 1640培养基(阴性对照)体外刺激，在37℃，5％CO2孵育20小时。之后用生物素化的IFN-γ-检测抗体和链霉亲和素-碱性磷酸酶(ALP)检测，加入BCIP/NBT-plus(5-溴-4-氯-3-吲哚-磷酸盐/硝基蓝四氮唑-plus)底物显色并用ELISpot读板仪(ImmunoSpot S6 Core Analyzer(CTL))计数。Mouse IFN-γ ELISpot assay was performed using the IFN-γ 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 μg/ml NP55-69 peptide, 8 μg/ml NP14-155 peptide, 8 μg/ml M2e peptide pool, and 10 μg/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. Afterwards, biotinylated IFN-γ-detection antibody and streptavidin-alkaline phosphatase (ALP) 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)).
肽池序列：Peptide pool sequence:
NP55–69氨基酸序列为RLIQNSLTIERMVLS。The amino acid sequence of NP 55–69 is RLIQNSLTIERMVLS.
NP147–155氨基酸序列为TYQRTRALV。The amino acid sequence of NP 147–155 is TYQRTRALV.
M2e肽池是M2e蛋白的混合肽，其中包含分别对应于M2蛋白的残基1-15(MSLLTEVETPIRNEW)，残基5-19(TEVETPIRNEWGCRC)和残基9-23(TPIRNEWGCRCNDSS)的三条肽。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.
NM2e肽池是由对应于全长NM2e蛋白，长度为15个氨基酸，互相间重叠11个氨基酸的肽段所构成的肽库。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.
5、动物研究5. Animal research
通过双侧肌肉内注射分别于第0天(D0)和21天(D21)用100μL的mRNA-LPP制剂免疫5周的雌性BALB/c小鼠。所有血液样本通过眼眶后采血采集，约200μL血液/次，在4℃1,500g条件下离心10分钟以进行血清分离。Female BALB/c mice aged 5 weeks were immunized with 100 μL 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 μL of blood/time was centrifuged at 1,500 g for 10 minutes at 4°C for serum separation.
6、统计分析6. Statistical analysis
动物研究的统计分析使用GraphPad Prism 8.0软件进行。数据表示为平均值±SEM。使用双尾T检验比较两个实验组。对于多于两个实验组的比较，使用One-way ANOVA。认为小于0.05的P值具有显著性。*，P<0.05；**，P<0.01；***，P<0.001；****，P<0.0001。 Statistical analysis of animal studies was performed using GraphPad Prism 8.0 software. Data are expressed as mean ± SEM. Two experimental groups were compared using a two-tailed T test. For comparisons of more than two experimental groups, one-way ANOVA was used. P values less than 0.05 were considered significant. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P < 0.0001.
序列sequence
SEQ ID NO:1 NM2e融合多肽的氨基酸序列
SEQ ID NO:1 Amino acid sequence of NM2e fusion polypeptide
SEQ ID NO:2 5’UTR
SEQ ID NO:2 5'UTR
SEQ ID NO:3 3’UTR
SEQ ID NO:3 3'UTR
SEQ ID NO:4未优化的编码DNA序列NM2e-Ori
SEQ ID NO:4 Unoptimized coding DNA sequence NM2e-Ori
SEQ ID NO:5优化的编码DNA序列NM2e-1
SEQ ID NO:5 Optimized coding DNA sequence NM2e-1
SEQ ID NO:6优化的编码DNA序列NM2e-2

SEQ ID NO:6 Optimized coding DNA sequence NM2e-2

SEQ ID NO:7优化的编码DNA序列NM2e-3
SEQ ID NO:7 Optimized coding DNA sequence NM2e-3
SEQ ID NO:8优化的编码DNA序列NM2e-4

SEQ ID NO:8 Optimized coding DNA sequence NM2e-4

SEQ ID NO:9mRNA序列NM2e-Ori
SEQ ID NO:9 mRNA sequence NM2e-Ori
SEQ ID NO:10 mRNA序列NM2e-1
SEQ ID NO:10 mRNA sequence NM2e-1
SEQ ID NO:11 mRNA序列NM2e-2

SEQ ID NO:11 mRNA sequence NM2e-2

SEQ ID NO:12 mRNA序列NM2e-3
SEQ ID NO:12 mRNA sequence NM2e-3
SEQ ID NO:13 mRNA序列NM2e-4
SEQ ID NO:13 mRNA sequence NM2e-4
SEQ ID NO:14未优化的编码RNA序列NM2e-Ori

SEQ ID NO: 14 Unoptimized coding RNA sequence NM2e-Ori

SEQ ID NO:15优化的编码RNA序列NM2e-1
SEQ ID NO: 15 Optimized coding RNA sequence NM2e-1
SEQ ID NO:16优化的编码RNA序列NM2e-2

SEQ ID NO: 16 Optimized coding RNA sequence NM2e-2

SEQ ID NO:17优化的编码RNA序列NM2e-3
SEQ ID NO: 17 Optimized coding RNA sequence NM2e-3
SEQ ID NO:18优化的编码RNA序列NM2e-4
SEQ ID NO: 18 Optimized coding RNA sequence NM2e-4
SEQ ID NO:19 5’UTR DNA序列
SEQ ID NO:19 5'UTR DNA sequence
SEQ ID NO:20 3’UTR DNA序列
SEQ ID NO:20 3'UTR DNA sequence

Claims (23)

  1. 一种多核苷酸，其包含编码SEQ ID NO:1的融合蛋白的核苷酸序列，其中所述核苷酸序列与选自SEQ ID NO:5、6、7、8、15、16、17和18的核苷酸序列具有至少80％的相同性。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 SEQ ID NO:5, 6, 7, 8, 15, 16, 17 and 18.
  2. 权利要求1的多核苷酸，其为RNA。The polynucleotide of claim 1, which is RNA.
  3. 权利要求2的多核苷酸，其中所述RNA是mRNA。The polynucleotide of claim 2, wherein the RNA is mRNA.
  4. 权利要求3的多核苷酸，其中所述mRNA还包含5’UTR、3’UTR和polyA。The polynucleotide of claim 3, wherein the mRNA further comprises a 5'UTR, a 3'UTR and polyA.
  5. 权利要求4的多核苷酸，其中所述5’UTR包含SEQ ID NO:2的核苷酸序列。The polynucleotide of claim 4, wherein the 5’UTR comprises the nucleotide sequence of SEQ ID NO:2.
  6. 权利要求4的多核苷酸，其中所述3’UTR包含SEQ ID NO:3的核苷酸序列。The polynucleotide of claim 4, wherein the 3’UTR comprises the nucleotide sequence of SEQ ID NO:3.
  7. 权利要求4的多核苷酸，其中所述polyA包含75个腺苷酸残基。The polynucleotide of claim 4, wherein the polyA comprises 75 adenylate residues.
  8. 权利要求1-7任一项的多核苷酸，其包含与SEQ ID NO:10-13之一具有至少80％的相同性的核苷酸序列。A polynucleotide according to any one of claims 1-7, comprising a nucleotide sequence having at least 80% identity with one of SEQ ID NO:10-13.
  9. 一种组合物，其包含权利要求1-8中任一项的多核苷酸。A composition comprising the polynucleotide of any one of claims 1-8.
  10. 权利要求9的组合物，其包含包封所述多核苷酸的脂质。The composition of claim 9, comprising a lipid encapsulating the polynucleotide.
  11. 权利要求9或10的组合物，其包含脂质多聚复合物。The composition of claim 9 or 10, comprising a lipid polymer complex.
  12. 权利要求10或11的组合物，其中包封所述多核苷酸的脂质包含阳离子脂质、非阳离子脂质和聚乙二醇修饰的脂质；任选地，所述组合物还包含阳离子聚合物，其中所述阳离子聚合物与所述多核苷酸缔合为复合物，共同包封在脂质中形成脂质多聚复合物。The composition of claim 10 or 11, wherein the lipids encapsulating the polynucleotide comprise cationic lipids, non-cationic lipids and polyethylene glycol-modified lipids; optionally, the composition further comprises a cationic polymer, wherein the cationic polymer is associated with the polynucleotide as a complex and is co-encapsulated in the lipid to form a lipid polymer complex.
  13. 权利要求12的组合物，其中所述阳离子聚合物选自聚-L-赖氨酸、鱼精蛋白和聚乙烯亚胺；优选地，所述阳离子聚合物为鱼精蛋白。The composition of claim 12, wherein the cationic polymer is selected from the group consisting of poly-L-lysine, protamine and polyethyleneimine; preferably, the cationic polymer is protamine.
  14. 一种疫苗制剂，其包含权利要求1-8中任一项的多核苷酸或权利要求9-13中任一项的组合物。 A vaccine preparation comprising the polynucleotide of any one of claims 1 to 8 or the composition of any one of claims 9 to 13.
  15. 权利要求14的疫苗制剂，其中包封所述多核苷酸的脂质包含10-70摩尔％的M5、10-70摩尔％的1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、10-70摩尔％的胆固醇和0.05-20摩尔％的1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(DMG-PEG)2000；优选地，所述脂质为摩尔比为40:15:43.5:1.5的M5、DOPE、胆固醇和(DMG-PEG)2000，
    The vaccine formulation of claim 14, wherein the lipid encapsulating the polynucleotide 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,
  16. 权利要求14或15的疫苗制剂，其中所述疫苗制剂为液态制剂或冻干制剂。The vaccine preparation of claim 14 or 15, wherein the vaccine preparation is a liquid preparation or a freeze-dried preparation.
  17. 权利要求14-16中任一项的疫苗制剂，其中所述疫苗制剂通过肌肉注射施用或粘膜内施用。The vaccine formulation according to any one of claims 14 to 16, wherein the vaccine formulation is administered by intramuscular injection or intramucosal administration.
  18. 权利要求1-8中任一项的多核苷酸、权利要求9-13中任一项的组合物、或权利要求14-17中任一项的疫苗制剂在制备用于预防和/或治疗有需要的对象中流感病毒感染的药物中的用途。Use of the polynucleotide of any one of claims 1 to 8, the composition of any one of claims 9 to 13, or the vaccine formulation of any one of claims 14 to 17 in the preparation of a medicament for preventing and/or treating influenza virus infection in a subject in need thereof.
  19. 权利要求18的用途，其中所述对象是人或非人动物。The use of claim 18, wherein the subject is a human or a non-human animal.
  20. 预防或治疗有需要的对象中流感病毒感染的方法，所述方法包括:A method for preventing or treating influenza virus infection in a subject in need thereof, the method comprising:
    给有需要的对象施用权利要求1-8中任一项的多核苷酸，权利要求9-13中任一项的组合物，或权利要求14-17中任一项的疫苗制剂。Administering 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 to a subject in need thereof.
  21. 权利要求20的方法，其中所述有需要的对象是人或非人动物。The method of claim 20, wherein the subject in need thereof is a human or a non-human animal.
  22. 权利要求1-8中任一项的多核苷酸、权利要求9-13中任一项的组合物、或权利要求14-17中任一项的疫苗制剂，其用于预防或治疗有需要的对象流感病毒感染的用途。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 preparation according to any one of claims 14 to 17, for use in preventing or treating influenza virus infection in a subject in need thereof.
  23. 权利要求22的用于所述用途的多核苷酸、组合物或疫苗制剂，其中所述对象是人或非人动物。 The polynucleotide, composition or vaccine formulation for use according to claim 22, wherein the subject is a human or a non-human animal.
PCT/CN2023/139522 2022-12-19 2023-12-18 Broad-spectrum influenza mrna vaccine WO2024131726A1 (en)

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CN202211634439.0 2022-12-19

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