Influenza multivalent mRNA vaccine and use thereof

Abstract

The present application relates to an influenza multivalent mRNA vaccine and uses thereof. In particular, the present application relates to an mRNA molecule having an optimized UTR sequence and / or encoding tandem influenza virus antigens, a composition comprising the above mRNA molecule, and the use of the composition in the preparation of a vaccine for the manufacture of a prophylactic or therapeutic vaccine against influenza.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid vaccines, and more specifically, relates to an mRNA influenza vaccine and its application. Background Technology

[0002] Influenza (flu) is an acute respiratory infectious disease caused by the influenza virus. The influenza virus is prone to mutation, highly contagious, and the general population is susceptible, resulting in a high incidence rate. Therefore, influenza prevention has always been a top priority for countries worldwide. Currently, there are still no effective treatments for influenza in clinical practice; influenza vaccination is the only effective means of prevention.

[0003] Influenza viruses belong to the Orthomyxoviridae family and are single-stranded, negative-sense, segmented RNA viruses. Based on differences in nucleoproteins and matrix proteins, they are classified into four types: A, B, C, and D. Influenza viruses are spherical, while newly isolated strains are mostly filamentous, with diameters ranging from 80 to 120 nm, and filamentous influenza viruses reaching lengths of up to 4000 nanometers. The structure of the influenza virus, from the outside in, can be divided into three parts: the envelope, the matrix protein, and the core. The viral glycoproteins hemagglutinin (HA) and neuraminidase (NA) on the surface are the most important antigenic sites. Anti-hemagglutinin antibodies can neutralize viral infectivity, while anti-neuraminidase antibodies can reduce the severity of disease. Because HA is the main influenza virus antigen recognized by neutralizing antibodies, this glycoprotein has always been a focus of current influenza vaccines.

[0004] Traditional inactivated vaccines and recombinant protein vaccines have long production cycles and complex processes, making them unsuitable for large-scale vaccination needs during sudden outbreaks of epidemics. mRNA, on the other hand, offers a rapid-response vaccine development platform for addressing explosive outbreaks. In recent years, Moderna and BioNTech have initiated clinical trials for their mRNA (messenger ribonucleic acid) vaccines, providing preliminary data on the safety and efficacy of mRNA vaccines. mRNA vaccines are synthesized in vitro using linearized plasmid DNA as a template through enzymatic transcription. This synthesis strategy avoids the problems associated with live cell culture production, safety considerations, and complex manufacturing processes. The mRNA vaccine platform is safe, effective, has a short production cycle, and a simple process, making it particularly suitable for vaccine development. Summary of the Invention

[0005] In a first aspect, the present invention provides an mRNA molecule, wherein the 5'UTR of the mRNA molecule is encoded by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with any of the sequences shown in SEQ ID NO: 1-11, and / or the 3'UTR of the mRNA molecule is encoded by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with any of the sequences shown in SEQ ID NO: 12-24.

[0006] In some embodiments, the 5'UTR of the mRNA molecule is encoded by a sequence shown in any one of SEQ ID NO: 7, 8, and 11 or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in any one of SEQ ID NO: 7, 8, and 11, and / or the 3'UTR of the mRNA molecule is encoded by a sequence shown in any one of SEQ ID NO: 15, 16, 17, 19, 22, and 23 or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in any one of SEQ ID NO: 15, 16, 17, 19, 22, and 23.

[0007] In some embodiments, the mRNA molecule encodes an influenza virus antigen, preferably an influenza virus HA antigen or an influenza virus NA antigen, more preferably selected from influenza A virus HA antigen, influenza A virus NA antigen, influenza B virus HA antigen, influenza B virus NA antigen and their immunogenic fragments or variants, and most preferably H1N1 HA antigen.

[0008] In a second aspect, the present invention provides an mRNA molecule, wherein the mRNA molecule encodes an influenza virus antigen or a fusion protein of two or more influenza virus antigens, preferably the influenza virus antigen being an influenza virus HA antigen or an influenza virus NA antigen, more preferably selected from influenza A virus HA antigen, influenza A virus NA antigen, influenza B virus HA antigen, influenza B virus NA antigen and their immunogenic fragments or variants.

[0009] In some embodiments, the influenza virus antigen is an influenza virus HA antigen or NA antigen having a sequence shown in SEQ ID NO: 26, 28, 30, 32, 34 or 36 or a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in SEQ ID NO: 26, 28, 30, 32, 34 or 36.

[0010] In some embodiments, the mRNA molecule encodes a fusion protein of a first antigen and a second antigen, wherein the first antigen and the second antigen are linked by a peptide bond or a linker.

[0011] In some embodiments, the first antigen is an H1N1 HA antigen and the second antigen is an H3N2 HA antigen; or the first antigen is a B / Victoria HA antigen and the second antigen is a B / Yamagata HA antigen; or the first antigen is a B / Victoria HA antigen and the second antigen is a B / Victoria NA antigen; or the first antigen is a B / Victoria NA antigen and the second antigen is a B / Yamagata NA antigen; or the first antigen is a B / Victoria NA antigen and the second antigen is a B / Yamagata NA antigen.

[0012] In some embodiments, the connector is a P2A connector or a GS connector, such as (G4S)n, where n is an integer from 1 to 30, preferably an integer from 5 to 25, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0013] In some embodiments, the mRNA molecule encodes a sequence represented by any one of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence represented by any one of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58.

[0014] In some embodiments, the coding region of the mRNA molecule comprises a sequence represented by any one of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence represented by any one of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57.

[0015] In some embodiments, the mRNA molecule has a 5'UTR and / or a 3'UTR as defined in the first aspect.

[0016] Thirdly, the present invention provides a composition comprising a first or second aspect of an mRNA molecule encapsulated in an LNP, wherein the LNP comprises an ionizable cationic lipid, phospholipid, steroid, and / or PEG lipid.

[0017] In some embodiments, the LNP comprises about 10 mol% to about 100 mol% of ionizable cationic lipids, about 0 mol% to about 40 mol% of phospholipids, about 0 mol% to about 70 mol% of steroids and / or about 0.01 mol% to about 10 mol% of PEG lipids.

[0018] In some embodiments, the LNP comprises A1-EP10-O18A, DSPC, cholesterol, and DMG-PEG2000.

[0019] In some embodiments, the mass ratio of total lipids to mRNA in the LNP is 1-100:1, preferably 10-80:1, and more preferably 10-60:1.

[0020] Fourthly, the present invention provides the use of the mRNA molecule of the first or second aspect or the composition of the third aspect in the preparation of a vaccine for the treatment or prevention of influenza, such as influenza A and / or influenza B.

[0021] In addition, the present invention provides a method for treating or preventing influenza, such as influenza A and / or influenza B, the method comprising administering to a subject in need the mRNA molecule of the first aspect or the second aspect or the composition described in the third aspect.

[0022] Beneficial effects

[0023] On the one hand, the mRNA molecule of the present invention has an optimized UTR sequence, thereby improving expression. On the other hand, the mRNA molecule of the present invention encodes a tandem influenza virus antigen, thereby improving the immune response and simplifying the subsequent CMC process in vaccines. Attached Figure Description

[0024] Figure 1 Optimization of 5' UTR and 3' UTR sequences for ELISA detection based on HIN1 HA.

[0025] Figure 2 Western blot was used to detect the expression levels of HA protein in four different influenza mRNAs.

[0026] Figure 3a and Figure 3b The expression levels of HA protein in four different influenza mRNAs were detected using FACS. Figure 3a The antibody used was Influenza A Virus Hemagglutinin / HA Antibody, Rabbit Mab (86001-RM01). Figure 3b The antibody used was Influenza B Hemagglutinin / HA Antibody, Rabbit Mab (11053-R004). The horizontal axis represents fluorescence intensity, and the vertical axis represents the number of cells at the corresponding fluorescence intensity. The stronger the fluorescence intensity, the higher the expression level of the corresponding HA protein.

[0027] Figure 4 The titer of 5-week serum IgG antibodies in mice was detected by ELISA.

[0028] Figure 5 Western blot was used to detect the expression level of tandem influenza HA protein.

[0029] Figures 6a-6c The expression level of influenza tandem HA mRNA protein was detected by FACS, among which... Figure 6a The antibody used was Influenza A H1N1 Hemagglutinin / HA Antibody, Mouse Mab (11055-MM04T). Figure 6b The antibody used was Influenza A H3N2 Hemagglutinin / HA Antibody, Mouse mAb (11056-MM03). Figure 6cThe antibody used was Influenza B Hemagglutinin / HA Antibody, Rabbit ab(11053-R004). The horizontal axis represents fluorescence intensity, and the vertical axis represents the number of cells at the corresponding fluorescence intensity. The stronger the fluorescence intensity, the higher the expression level of the corresponding HA protein.

[0030] Figure 7 To detect the serum IgG antibody titer in mice using ELISA;

[0031] Figure 8 For the detection of tandem influenza HA mRNA 5W serum neutralizing antibodies; and

[0032] Figure 9 The titer of IgG antibodies in mouse serum was detected by ELISA. Detailed Implementation

[0033] definition

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the definitions herein shall prevail. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0035] As used herein, the terms “comprising,” “including,” or “having” mean that any of the listed elements must be included, and other elements may optionally be included. The term “consisting of” means that all unlisted elements are excluded. The terms “comprising,” “including,” or “having” cover “consisting of” or “substantially consisting of”.

[0036] As used herein, the term "identity" refers to the relationship between two or more polypeptide (e.g., antigen) or two or more nucleotide sequences as determined by sequence comparison. Sequence identity is determined by comparing the sequences after a given sequence has been optimally aligned with a reference sequence to produce the highest degree of sequence similarity, such as by matching strings of these sequences. Sequence identity can be readily calculated using known methods, such as BLAST. Those skilled in the art can use computer programs well-known in the art (e.g., DNASTAR™ software) to determine which amino acid residues can be replaced, inserted, or deleted without eliminating biological or immune activity. For example, functional variants of the invention may include conserved substitutions. Conserved substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties, or similar side chain volumes. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Exemplary conserved substitutions are well known in the art (see, for example, WO 97 / 09433, page 10, published March 13, 1997; Lehninger, Biochemistry, second edition; Worth Publishers, Inc. NY:NY (1975), pp. 71–77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8). It is well known that conserved substitutions between amino acids of similar properties generally do not affect the activity of the peptide sequence.

[0037] mRNA molecules

[0038] In this document, the term "mRNA" refers to messenger ribonucleic acid, which can be naturally occurring or non-naturally occurring. Non-naturally occurring mRNA may include modified and / or non-naturally occurring components, such as one or more modified nucleotides or linkers. Translation of mRNA can produce polypeptides. Traditionally, the basic components of an mRNA molecule include at least a 5'-cap, a 5'-untranslated region (5'-UTR), a coding region, a 3'-untranslated region (3'-UTR), and a polyA sequence. The "5'-UTR" refers to the region in mRNA located directly upstream (i.e., 5') of the start codon, which does not encode a polypeptide. The "3'-UTR" refers to the region in mRNA located directly downstream (i.e., 3') of the stop codon, which does not encode a polypeptide. A "coding region (CDS)" or "open reading frame (ORF)" is a continuous DNA sequence that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide. The polyA sequence is a polyadenylated sequence located downstream of the 3'-UTR.

[0039] In this paper, the DNA sequence encoding mRNA molecules refers to the nucleotide sequence of the non-template strand (sense strand / coding strand) of double-stranded DNA, whose nucleotide sequence is identical to the sequence of the transcribed mRNA (in RNA, U replaces T in DNA). Correspondingly, the DNA strand in double-stranded DNA that transcribes mRNA molecules is called the template strand.

[0040] Influenza virus

[0041] Seasonal influenza is a serious respiratory viral illness caused by influenza viruses distributed globally. There are four types of influenza viruses: A, B, C, and D. The most life-threatening type is influenza A, which can cause death and various respiratory illnesses. Influenza B viruses are also the main culprits behind human seasonal influenza pandemics. Influenza C viruses do not cause epidemics and usually cause milder flu symptoms. Influenza D viruses have been found to infect animals such as pigs, cattle, and sheep, but not humans.

[0042] Influenza A and B viruses have eight genes that encode ten proteins, including hemagglutinin (HA) and neuraminidase (NA). For influenza A viruses, they can be further subdivided into different subtypes based on the differences in these two surface proteins. To date, 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) have been identified. In this invention, influenza A virus subtypes include A (H1N1) and A (H3N2). Influenza B viruses do not have different subtypes, but can be divided into different lineages, such as the B / Yamagata or B / Victoria lineage, also known as B / Yam or B / Vic. In some embodiments of this invention, B1 refers to B / Vic, and B2 refers to B / Yam.

[0043] In some embodiments, the mRNA molecule of the present invention may encode the HA antigen and / or NA antigen of influenza A virus or influenza B virus. In some embodiments, the mRNA molecule of the present invention may encode the HA antigen and / or NA antigen selected from the group consisting of: H1N1 HA, H1N1 NA, H3N2 HA, H3N2 NA, B / Yamagata HA, B / Yamagata NA, B / Victoria HA, B / Victoria NA, or combinations thereof. In some embodiments, the antigen of the present invention has a sequence shown in SEQ ID NO: 26, 28, 30, 32, 34, or 36, or a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in SEQ ID NO: 26, 28, 30, 32, 34, or 36.

[0044] The "influenza virus antigen" of this invention encompasses naturally isolated influenza virus antigens or immunogenic fragments or variants thereof. An immunogenic fragment or variant of an influenza virus antigen refers to a fragment or variant that, compared to the natural antigen, has substitutions, deletions, and / or insertions at certain positions in its amino acid sequence but retains its immunogenicity. Immunogenic fragments or variants of influenza virus antigens can be prepared using conventional mutagenesis techniques and subjected to appropriate analysis to determine whether they possess the desired properties. Analyses for determining expression levels and immunogenicity are well known in the art, as described in the Embodiments section of this invention.

[0045] connector

[0046] In this invention, the connector can be any connector commonly used in the art, including flexible connectors, rigid connectors, detachable connectors, and non-detachable connectors.

[0047] In some embodiments, the linker of the present invention is a 2A peptide, such as P2A. 2A peptides are peptide fragments of 18-22 amino acid residues in length that can induce self-cleavage of recombinant proteins containing 2A peptides within cells. Currently, there are four commonly used 2A peptides: T2A, P2A, E2A, and F2A, where F2A originates from foot-and-mouth disease virus, E2A from Equine rhinitis A virus, P2A from Porcine teschovirus, and T2A from Thosea asigna virus.

[0048] In some embodiments, the connector of the present invention is a GS connector, such as (G4S)n, where n is an integer from 1 to 30, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably an integer from 5 to 25.

[0049] lipid nanoparticles

[0050] In this invention, "lipid nanoparticles" or "LNPs" refers to particles containing one or more lipids and having a nanoscale size (e.g., 1-1,000 nm). In some embodiments, the lipid nanoparticles of this invention comprise ionizable cationic lipids, phospholipids, steroids, and / or PEG lipids.

[0051] In this invention, the term "ionizable cationic lipid" refers to lipids that are protonated and become positively charged under specific conditions, such as in an acidic environment. Ionizable cationic lipids can remain neutral under other conditions, such as in the circulatory system, thereby improving the biocompatibility of the lipid nanoparticles. Ionizable cationic lipids can be included in the lipid nanoparticles in varying amounts. For example, based on total lipids, the lipid nanoparticles may include about 10 mol% to about 100 mol%, for example, about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol%, about 40 mol% to about 50 mol%, for example, about 46 mol% of ionizable cationic lipids.

[0052] The ionizable cationic lipid of the present invention can be any cationic lipid suitable for use in lipid nanoparticles. In some embodiments, the ionizable cationic lipid of the present invention is the cationic lipid disclosed in PCT / CN2023 / 137998. In some embodiments, the ionizable cationic lipid of the present invention is, for example, A1-EP10-O18A disclosed in PCT / CN2023 / 137998:

[0053] .

[0054] In this invention, examples of phospholipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (Cl6 LysoPC), 1,2-dilinolenoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidonicyl-sn-glycerol-3-phosphocholine, 1,2-bis(eicosahenoyl-sn-glycerol-3-phosphocholine), 1,2-diphytoyl-sn-glycerol-3-phosphoethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(eicosahenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and any mixture thereof. In some embodiments, the molar fraction of phospholipids, based on total lipids, is from about 0 mol% to about 40 mol%, for example, about 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, or 40 mol%. In some embodiments, the phospholipids of the present invention are DSPC. In some embodiments, the phospholipids of the present invention are the phospholipids disclosed in Chinese Patent Application 20241097020.1.

[0055] In this invention, examples of steroids include, but are not limited to, cholesterol and its derivatives, ergosterol, lanosterol, stigmasterol, sitosterol, and any mixture thereof. In some embodiments, the molar fraction of steroids, based on total lipids, is from about 0 mol% to about 70 mol%, for example, about 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, or 70 mol%. In some embodiments, the molar fraction of steroids, based on total lipids, is from about 40 mol% to about 50 mol%, for example, about 42.4 mol%.

[0056] In this invention, the term "PEG lipid" refers to any PEG-modified lipid, examples of which include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, and PEG-modified diacylglycerol. In some embodiments, the PEG lipid is PEG-modified distearylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol-methoxy(polyethylene glycol) MW 2000 (DMG-PEG2000). In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight of the PEG-modified material is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. The PEG lipids may be included in the lipid nanoparticles in different amounts. For example, based on total lipids, the lipid nanoparticles may include about 0 mol% to about 10 mol%, such as about 0.01 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 1 mol% to about 2 mol%, such as about 1.6 mol% of PEG lipids.

[0057] In some embodiments, the mass ratio of total lipids to nucleic acids in the composition of the present invention is 1-100:1, preferably 10-80:1, more preferably 10-60:1, for example 10:1, 20:1, 30:1, 40:1, 50:1, 60:1. In some embodiments, the nucleic acid of the present invention is mRNA.

[0058] Example 1: Optimization of the 5' UTR and 3' UTR sequences of influenza H1N1 HA mRNA

[0059] Based on literature and patents 1-3 Natural UTR sequences with biological activity potential or novel UTR sequences designed and generated were selected to replace the original UTR sequence in the mRNA backbone (H1N1 HA (A / Wisconsin / 588 / 2019(H1N1) pdm09)). Specific information is shown in Tables 1 and 2. The mRNA obtained after in vitro transcription was transfected into 293T cells. 293T cells in logarithmic growth phase were used, and the cell suspension concentration was adjusted to 4 × 10⁻⁶. 5 Cells / mL were seeded at 2 mL per well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow cell adhesion. For transfection, 4 μg mRNA and 8 μL of Lipofectamine 2000 (Thermo Fisher Scientific) were diluted separately in 500 μL of serum-free culture medium (Opti-MEM), mixed thoroughly, and incubated at room temperature for 5 min. Then, the mRNA solution was mixed with the Lipofectamine 2000 solution and incubated at room temperature for 15-18 min before being added to the cell culture plate. 24 h after transfection, cells were lysed using Pierce RIPA Buffer (Thermo, 89901), and the resulting protein lysate was used for ELISA detection. Specific procedures were followed according to the ELISA kit instructions (SinoBiological. KIT11055). Results are as follows: Figure 1 As shown in the figure, the results indicate that partial UTR optimization can effectively enhance H1N1 HA protein expression.

[0060] Table 1. UTR Sequence Information

[0061]

[0062]

[0063]

[0064] Table 2. Optimal Combinations of UTR

[0065]

[0066]

[0067] Example 2: In vitro antigen expression detection of influenza HA mRNA (Western Blot)

[0068] H1N1 HA mRNA, H3N2 HA mRNA, B / Victoria HA mRNA, and B / Yamagata HA mRNA, prepared by in vitro transcription, were transfected into COS7 cells. COS7 cells in logarithmic growth phase were harvested, and the cell suspension concentration was adjusted to 4 × 10⁻⁶. 5 Cells were seeded at 2 mL / well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow cell adhesion. For transfection, 4 μg mRNA and 8 μL of Lipofectamine 2000 (Thermo Fisher Scientific) were diluted separately in 500 μL of serum-free culture medium (Opti-MEM), mixed, and incubated at room temperature for 5 min. The mRNA solution was then mixed with the Lipofectamine 2000 solution and incubated at room temperature for 15-18 min before being added to the cell culture plates. 24 h after transfection, COS7 cells were lysed using Pierce RIPA Buffer (Thermo, 89901), and the resulting protein lysate was used for Western blotting. Specifically: total protein concentration was determined using the BRADFORD method; the total protein concentration of each group was adjusted with 1X PBS; and the gel was run in a Tianneng VE-180 vertical electrophoresis tank. After electrophoresis, the gel was transferred to a Tianneng VE-186 transfer electrophoresis tank. After transfer, the transfer membrane was blocked with Blocking Buffer for 2 h. Diluted primary antibody was added, and the membrane was incubated overnight at 4ºC. The membrane was washed three times with 1X PBST. Diluted secondary antibody was added, and the membrane was incubated at room temperature for 1 h. The membrane was washed three times with 1X PBST. Chemiluminescence detection was performed on the ECL substrate using a Tanon 4200 fully automated chemiluminescence imaging system, followed by grayscale analysis and quantification using Gel-Pro analyzer software. Results are as follows: Figure 2 As shown in the figure, the results indicate that all four influenza HA mRNAs can be well expressed in vitro.

[0069] Example 3: In vitro antigen expression detection of influenza HA mRNA (FACS)

[0070] H1N1 HA mRNA, H3N2 HA mRNA, B / Victoria (B / Vic) HA mRNA, and B / Yamagata (B / Yam) HA mRNA, prepared by in vitro transcription, were transfected into COS7 cells. COS7 cells in logarithmic growth phase were harvested, and the cell suspension concentration was adjusted to 4 × 10⁻⁶. 5 Cells / mL were seeded at 2 mL per well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow cell adhesion. For transfection, 4 μg mRNA and 8 μL of Lipofectamine 2000 (ThermoFisher Scientific) were diluted separately in 500 μL of serum-free culture medium (Opti-MEM), mixed, and incubated at room temperature for 5 min. Then, the mRNA solution was mixed with the Lipofectamine 2000 solution and incubated at room temperature for 15-18 min before being added to the cell culture plate. After 24 h, FACS was used to detect protein expression after transfection. Specifically: transfected cells were removed, the supernatant was discarded, and the cells were washed once with PBS. Trypsin digestion was performed for approximately 1 min; after cell detachment was observed, digestion was stopped with DMEM complete medium, and the cells were collected. The cells were centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were washed twice with 1 mL of FACS buffer (PBS containing 2% FBS). The antibody was diluted 1:200 with FACS Buffer and added to the corresponding cells. The mixture was vortexed and incubated at 4°C for 1 hour. After washing the cells three times with FACS Buffer, the corresponding secondary antibody was diluted 1:2000 and added to the cells. The mixture was vortexed and incubated at 4°C for 1 hour. After washing with FACS Buffer, the cells were resuspended and analyzed. The results were analyzed using FlowJo V10 software. Results are as follows: Figure 3a and Figure 3b As shown: Strong in vitro expression of HA mRNAs from all four influenza strains was detected. The expression levels of H1N1 HA and H3N2 HA were comparable, while the expression level of B / Yam HA was higher than that of B / Vic HA (70% vs 30%).

[0071] Example 4: In vivo immunoassay of influenza HA mRNA

[0072] In this embodiment, four mRNAs—H1N1 HA, H3N2 HA, B / Vic HA, and B / Yam HA—were selected, and SM102 was used to prepare mRNA vaccines for immunization experiments in female Balb / C mice (6 to 8 weeks old). Specific groupings are shown in Table 3. In group G5, the test substances were the four HA antigen mRNAs prepared into different LNPs and mixed in a 1:1:1:1 ratio before administration. The negative control was physiological saline injection. Animals were immunized on days 0 and 21 of the experiment, and mouse serum was collected via the orbital venous plexus on day 35. The IgG antibody titer in the serum was detected by ELISA. The specific detection method was as follows: the corresponding H1N1 HA protein, H3N2 HA protein, B / Vic HA protein, and B / Yam HA protein were diluted to 1 μg / mL with PBS, and 100 μL / well was added to a 96-well ELISA plate and incubated overnight at 4°C. Wash the plate three times with 1× wash buffer (300 μL / well of 20× Washing Buffer diluted to 1× with pure water), and blot dry. Add 300 μL of blocking buffer (2% BSA) to each well and incubate for 2 hours in a constant temperature shaker. Wash the plate three times with 1× wash buffer and blot dry. Add 100 μL of serially diluted serum sample to each well and incubate for 2 hours in a constant temperature shaker. Wash the plate three times with 1× wash buffer and blot dry. Rabbit anti-mouse IgG H&L (HRP) was diluted 1:1000 with PBST (1× PBS with 0.05% TWEEN20) as the enzyme-labeled antibody, and 100 μL / well was added to each well and incubated for 1 hour in a constant temperature shaker. After washing with wash buffer, add 100 μL of TMB equilibrated to room temperature to each well and develop the color at room temperature in the dark. After a certain time, 100 μL of ELISA stop solution was added to each well to stop the color development. Within 20 minutes, the absorbance values ​​at 450 nm and 630 nm were read on a microplate reader. The results were analyzed using GrapgPad Prism 8 software.

[0073] Table 3 Experimental Design of Example 4

[0074]

[0075] The results are as follows Figure 4 As shown, immunization of G1 and G2 mice induced high titers of H1N1 HA IgG and H3N2 HA IgG antibodies. Immunization of G3 and G4 mice induced high titers of B / Vic HA IgG and B / Yam HA IgG antibodies.

[0076] Example 5: Detection of in vitro antigen expression of influenza A and B virus tandem HA mRNA (Western Blot)

[0077] Use (G4S) respectively 10 (G4S) 15 (G4S) 20 Four linkers, including P2A, were used to tandemly bind the HA proteins of two influenza A viruses, H1N1 and H3N2, and two influenza B viruses, B / Vic and B / Yam. The tandemly bound mRNAs were transfected into COS7 cells, and the expression of the tandemly bound proteins was detected by Western blotting, following the same procedure as above. Results are shown below. Figure 5 As shown, the expression of the corresponding HA protein was detected after transfection of COS7 cells with the tandem HA mRNA.

[0078] Example 6: Detection of in vitro antigen expression of influenza A and B virus tandem HA mRNA (FACS)

[0079] Use (G4S) respectively 10 (G4S) 15 (G4S) 20 Four linkers, including P2A, were used to tandemly synthesize the HA proteins of two influenza A viruses, H1N1 and H3N2, and two influenza B viruses, B / Vic and B / Yam. The tandemly synthesized mRNAs were transfected into COS7 cells, and the expression of the tandemly synthesized proteins was detected by FACS, following the same procedure as above. Results are shown below. Figures 6a-6c As shown, the expression of the corresponding HA protein was detected after transfection of COS7 cells with the tandem HA mRNA.

[0080] Example 7 Preparation of LNP

[0081] LNP preparation: Ionizable cationic lipids, phospholipids, steroids, and PEG lipids were dissolved in ethanol according to their respective formulation ratios. mRNA was dissolved in 10 mM sodium citrate solution (pH=4.0). The crude LNP product was prepared by mixing the ethanol phase (four lipids) and the aqueous phase (mRNA) at a volume ratio of 1:3. The crude LNP product was diluted 3 times with PBS and purified using a 100 kD ultrafiltration tube. The ethanol was replaced with PBS. The product was then filtered through a 0.22 μm sterile filter to obtain LNPs encapsulated with mRNA.

[0082] STAR0225 LNP contains four lipids: A1-EP10-O18A, DSPC, cholesterol, and DMG-PEG2000. The molar percentages of the four lipids are 46%, 8%, 42.4%, and 1.6%, respectively; the total lipid to mRNA mass ratio is 29.3:1. The A1-EP10-O18A lipid is a proprietary lipid designed by our company, and its structural formula is as follows:

[0083] .

[0084] Example 8: In vivo immunoassay of tandem influenza HA mRNA

[0085] In this embodiment, H1N1 HA-(G4S) is selected. 10 -H3N2 HA, H1N1 HA-P2A-H3N2 HA and B / VicHA-(G4S) 10 Three HA tandem mRNAs, including -B / Yam HA, were used, and STAR0225 was selected to prepare an mRNA vaccine. Immunization experiments were conducted on female Balb / C mice (6 to 8 weeks old), with specific groupings shown in Table 4. The negative control was physiological saline injection. Animals were immunized on days 0 and 21 of the experiment. Serum was collected from mice via the orbital venous plexus at 2 weeks (14 days), 4 weeks (28 days), and 5 weeks (35 days) after the first immunization, and the IgG antibody titer in the serum was detected by ELISA, following the same procedure as above.

[0086] The results are as follows Figure 7 As shown, in the influenza A tandem sequence, group G2 H1N1 HA-(G4S) 10 The H1N1 HA immunization group induced a high titer of H1N1 HA IgG antibodies. The H1N1 HA-P2A-H3N2 HA titer induced in the G3 group was comparable to that induced in the G1 group by H1N1 HA+H3N2 HA. The G2 group H1N1 HA-(G4S) immunization group... 10 The titers of H3N2 HA IgG antibodies induced by H1N1 HA and H3N2 HA immunization in group G1 were comparable, while those induced by H1N1 HA-P2A-H3N2 HA immunization in group G3 were the second highest, indicating that H1N1 HA-(G4S) 10 Immunization of mice with the -H3N2 HA tandem sequence can induce a strong immune response. In the influenza B tandem sequence, the G5 group B / Vic HA-(G4S) 10 -B / Yam HA induced B / Vic HA IgG antibody titers comparable to those of the G4 group B / Vic HA+B / Yam HA, while simultaneously inducing stronger B / Yam HA IgG antibody titers than those of the G4 group B / Vic HA+B / Yam HA. This indicates that B / Vic HA-(G4S) 10 Immunizing mice with the -B / Yam HA tandem sequence can induce a strong immune response.

[0087] Table 4 Experimental Design of Example 8

[0088]

[0089] Example 9: Detection of tandem influenza HA mRNA neutralizing antibodies

[0090] Serum from 5-week mice in groups G1, G2, G4, and G6 of Example 8 was selected, and the titers of neutralizing antibodies produced were detected. The specific procedures were as follows: First, H1N1 and H3N2 viruses were amplified using chicken embryos, and then analyzed using hemagglutination assays and TCID assays. 50 Virus titer was determined; MDCK cells were resuscitated and passaged 2-3 times for neutralization assay; RDE enzyme was co-incubated with the serum to be tested at 37 ℃ overnight, followed by inactivation at 56 ℃ for 30 min; the virus was removed from the -80 ℃ freezer, thawed on moist ice, and diluted to the working titer using serum-free medium; a 96-well plate confluent with a monolayer of MDCK cells was removed, the supernatant was discarded, and the cells were washed twice with serum-free medium. The diluted 96-well plate mixture was added one-to-one to the 96-well plate containing MDCK cells and incubated at 37 ℃, 5% CO2 for 2 h. The supernatant was discarded, and cell maintenance medium (2% FBS) was added at 200 μL / well; the plate was incubated at 37 ℃, 5% CO2 for another 48 h. Cell control (cells, no sample treatment or virus infection) and virus control (cells infected with virus, no sample treatment) wells were set up. The OD value of each well was detected using ELISA, and the inhibition rate was calculated. The highest dilution of serum with an inhibition rate of 50% was the neutralizing titer of that serum. Data were processed using Office Excel 2013 and GraphPad Prism 6.0, and are expressed as x ± M (standard error). One-way ANOVA was used for analysis, and Tukey's test was used to test the significance of differences between groups. When comparing two groups, a two-tailed t-test was used. A significant difference was considered to be between the two groups when p < 0.05.

[0091] The results are as follows Figure 8 As shown, group G1 contains H1N1 HA + H3N2 HA and group G2 contains H1N1 HA - (G4S). 10 Immunization with H3N2 HA mice induced neutralizing antibodies against both H1N1 and H3N2 strains. Immunization with B / Vic HA+B / Yam HA mice in group G4 induced neutralizing antibodies against both B / Vic and B / Yam strains.

[0092] Example 10: Experiment on combined immunization of mice with influenza HA mRNA and NA mRNA

[0093] In this embodiment, HA tandem mRNA sequences and NA tandem mRNA sequences of influenza A and B were selected, and STAR0225 was used to prepare an mRNA vaccine for immunization experiments in female Balb / C mice (6 to 8 weeks old). Specific groupings are shown in Tables 5-1 and 5-2. The negative control was physiological saline injection. Animals were immunized on days 0 and 21 of the experiment. Serum was collected from mice via the orbital venous plexus at 2 weeks (14 days) and 4 weeks (28 days) after the first immunization, respectively. The IgG antibody titer in the serum was detected by ELISA, following the same procedure as above.

[0094] The results are as follows Figure 9 As shown, each immunization group can induce a high titer of the corresponding IgG antibody, indicating that the above-mentioned tandem combination of HA mRNA and NA mRNA sequences can induce a strong immune response.

[0095] Table 5-1 Experimental Design of Example 10

[0096]

[0097]

[0098] For ease of statistical analysis, abbreviations will be used in the charts, as shown in the table below:

[0099] Table 5-2 Test Substance Codes

[0100]

[0101] Note: A and B refer to influenza A and B viruses, respectively.

[0102] Table 6. Sequence List

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] refer to:

[0133] 1. CN116144651A - A set of improved 5'UTR and 3'UTR based on coronavirus genomes and their applications

[0134] 2.Kozak M. Influences of mRNA secondary structure on initiation byeukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850-4. doi:10.1073 / pnas.83.9.2850. PMID: 3458245; PMCID: PMC323404.

[0135] 3.Babendure JR, Babendure JL, Ding JH, Tsien RY. Control of mammaliantranslation by mRNA structure near caps. RNA. 2006 May;12(5):851-61. doi:10.1261 / rna.2309906. Epub 2006 Mar 15. PMID: 16540693; PMCID: PMC1440912.

Claims

1. An mRNA molecule, wherein the 5'UTR of the mRNA molecule is encoded by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with any of the sequences shown in SEQ ID NO: 1-11, and / or the 3'UTR of the mRNA molecule is encoded by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with any of the sequences shown in SEQ ID NO: 12-24.

2. The mRNA molecule according to claim 1, wherein the mRNA molecule encodes an influenza virus antigen, preferably an influenza virus HA antigen or an influenza virus NA antigen, more preferably selected from influenza A virus HA antigen, influenza A virus NA antigen, influenza B virus HA antigen, influenza B virus NA antigen and their immunogenic fragments or variants, most preferably H1N1 HA antigen.

3. An mRNA molecule, wherein the mRNA molecule encodes an influenza virus antigen or a fusion protein of two or more influenza virus antigens, preferably the influenza virus antigen being an influenza virus HA antigen or an influenza virus NA antigen, more preferably selected from influenza A virus HA antigen, influenza A virus NA antigen, influenza B virus HA antigen, influenza B virus NA antigen and immunogenic fragments or variants thereof.

4. The mRNA molecule of claim 3, wherein the mRNA molecule encodes a fusion protein of a first antigen and a second antigen, and wherein the first antigen and the second antigen are linked by a peptide bond or a linker.

5. The mRNA molecule of claim 4, wherein the first antigen is an H1N1 HA antigen and the second antigen is an H3N2 HA antigen; or the first antigen is a B / Victoria HA antigen and the second antigen is a B / Yamagata HA antigen; or the first antigen is a B / Victoria HA antigen and the second antigen is a B / Victoria NA antigen; or the first antigen is a B / Victoria NA antigen and the second antigen is a B / Yamagata NA antigen; or the first antigen is a B / Victoria NA antigen and the second antigen is a B / Yamagata NA antigen.

6. The mRNA molecule according to claim 4 or 5, wherein the adapter is a P2A adapter or a GS adapter, such as (G4S)n, wherein n is an integer from 1 to 30, preferably an integer from 5 to 25, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

7. The mRNA molecule according to any one of claims 3 to 6, wherein the mRNA molecule encodes a sequence represented by any one of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or a polypeptide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence represented by any one of SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58.

8. The mRNA molecule according to any one of claims 3 to 7, wherein the coding region of the mRNA molecule comprises a sequence represented by any one of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence represented by any one of SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57.

9. The mRNA molecule according to any one of claims 3 to 8, wherein the mRNA molecule has a 5'UTR and / or a 3'UTR as defined in claim 1.

10. A composition comprising an mRNA molecule according to any one of claims 1-9 encapsulated in an LNP, wherein the LNP comprises an ionizable cationic lipid, a phospholipid, a steroid, and / or a PEG lipid.

11. The composition of claim 10, wherein the LNP comprises about 10 mol% to about 100 mol% of an ionizable cationic lipid, about 0 mol% to about 40 mol% of a phospholipid, about 0 mol% to about 70 mol% of a steroid and / or about 0.01 mol% to about 10 mol% of a PEG lipid.

12. The composition according to claim 10 or 11, wherein the LNP comprises A1-EP10-O18A, DSPC, cholesterol, and DMG-PEG2000.

13. The composition according to any one of claims 10 to 12, wherein the mass ratio of total lipids to mRNA in the LNP is 1-100:1, preferably 10-80:1, more preferably 10-60:

1.

14. Use of the mRNA molecule according to any one of claims 1 to 9 or the composition according to any one of claims 10 to 13 in the preparation of a vaccine for the treatment or prevention of influenza, such as influenza A and / or influenza B.

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