A h1n1 broad-spectrum mRNA vaccine targeting HA and NA proteins in series and a preparation method thereof

Abstract

The application provides an H1N1 broad-spectrum mRNA vaccine taking HA and NA proteins in series as targets and a preparation method thereof, relates to the technical field of vaccine preparation, and the amino acid sequence of the mRNA vaccine is shown as SEQ ID NO. 1, and the nucleic acid sequence is shown as SEQ ID NO. 2, that is, the hemagglutinin HA and the neuraminidase NA conservative amino acid sequence are connected by GGGSGGGSGGGSGGGS. The application overcomes the defects of the prior art, improves the broad spectrum and protection effect of the vaccine, and makes the vaccine better cope with the immune escape of the latest emerging mutant strain.

Description

Technical Field

[0001] This invention relates to the field of vaccine preparation technology, specifically to an H1N1 broad-spectrum mRNA vaccine targeting the tandem of HA and NA proteins and its preparation method. Background Technology

[0002] Influenza virus belongs to the Orthomyxoviridae family and is a segmented, negative-sense RNA virus. Its genome consists of eight segments that encode viral RNA polymerase complexes (PB1, PB2, PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M1), ion channel protein (M2), and a series of non-structural proteins (NS1, NS2, NS3). HA and NA are two important glycoproteins on the viral surface and are also the most common vaccine immunogens. HA is responsible for binding the virus to receptors on the host cell surface, mediating viral entry into the host cell; NA is responsible for hydrolyzing sialic acid on the host cell surface, promoting viral release from the host cell.

[0003] Influenza viruses have been a major focus of attention since the 1918 Spanish flu pandemic. At least 18 different subtypes of influenza A virus (IAV) circulate in animal hosts, occasionally entering the human population and causing pandemics. Currently, H1N1 and H3N2 IAVs, as well as one or two antigenically distinct influenza B virus (IBV) lineages, circulate seasonally in the human population. Although studies using various epidemiological surveillance programs and models have increased our understanding of influenza pandemics, it remains impossible to accurately predict which influenza subtype will cause the next pandemic. Therefore, research on universal influenza vaccines has been a key focus in the vaccine community. However, due to the significant differences between different types of influenza viruses, coupled with the high frequency of mutations through antigenic drift and antigenic shift, it is difficult to achieve broad-spectrum coverage with a single monovalent vaccine and a single immunogen. Therefore, the design of broad-spectrum mRNA vaccines is crucial. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a broad-spectrum H1N1 mRNA vaccine targeting the tandem of HA and NA proteins, and its preparation method, thereby improving the vaccine's broad-spectrum nature and protective efficacy, enabling it to better cope with immune evasion from the latest emerging variant strains.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A broad-spectrum H1N1 mRNA vaccine targeting the tandem HA and NA proteins, wherein the amino acid sequence of the mRNA vaccine is shown in SEQ ID NO.1 and the nucleic acid sequence is shown in SEQ ID NO.2.

[0006] The method for preparing the mRNA vaccine includes the following steps: S1. By linking the conserved amino acid sequences of hemagglutinin HA and neuraminidase NA with a flexible peptide, two major influenza antigens can be linked, enhancing the immunogenicity and protective effect of the vaccine. On the other hand, by adding four sets of repeating GGGS amino acids between the two proteins during the synthesis of the sequence, a flexible linker peptide is formed, which provides greater spatial freedom and helps the protein form the correct spatial conformation. In addition, the advantage of flexible peptide linking two antigens compared to cleavable 2A peptides is that it avoids the incomplete or even incorrect cleavage that may occur when 2A peptides are cleaved in vivo. S2. The obtained amino acid sequence is codon optimized according to human codon preference to obtain a DNA sequence, such as SEQ ID NO.2. This sequence is constructed into plasmid PUC57 containing T7 promoter, 5'UTR, 3'UTR and 100 PolyA by enzyme digestion and ligation, which is PUC57-T7-5UTR-HA-NA-3UTR-PolyA, and the plasmid construction is completed. S3. The plasmid successfully constructed above is amplified and transcribed to obtain an mRNA vaccine; S4. The mRNA obtained above is prepared into lipid nanoparticles (LNPs) using a microfluidic method.

[0007] Preferably, the flexible peptide is four repeating GGGS, that is, the amino acid sequence of the flexible peptide is: GGGSGGGSGGGSGGGS.

[0008] Preferably, the nucleotide sequence of the 5'UTR in step S2 is shown in SEQ ID NO.3, and the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.4.

[0009] Preferably, in step S3, plasmid amplification is performed using TSINGKE TSC-C01 Trelief® 5α ChemicallyCompetent Cell strain, and after plasmid extraction, the DNA template is linearized by BsaI restriction enzyme digestion. The linearized template is precipitated with 70% volume isopropanol at -80°C and washed with 70% ethanol.

[0010] Preferably, in step S3, the linearized template is transcribed in vitro using the commercially available kit Novizan. Before transcription, the transcription raw material UTP is completely replaced with N1-methylpseuuridine triphosphate N1-Me-pUTP.

[0011] Preferably, the specific method for preparing lipid nanoparticles (LNPs) in step S4 includes the following steps: S4-1. Dissolve the mRNA in a 50 mM citrate buffer solution with a pH of 4 to obtain an mRNA-citric acid solution with a final concentration of 108 ng / μL. S4-2. Prepare an anhydrous ethanol solution with 50% SM102, 1.5% DMG-PEG2000, 10% DSPC, and 38.5% cholesterol to obtain a lipid mixed solution. S4-3. The lipid mixture and mRNA-citric acid solution were filtered separately through a 0.22 μm microporous membrane and mixed using a microfluidic instrument to obtain lipid nanoparticles (LNP).

[0012] Preferably, in step S4-3, the flow rate ratio of the microfluidic instrument for mixing is 15 mL / min for mRNA-citric acid solution and 5 mL / min for lipid mixture.

[0013] This invention provides a broad-spectrum H1N1 mRNA vaccine targeting the tandem HA and NA proteins and its preparation method, which has the following advantages compared with the prior art: The influenza mRNA vaccine designed based on conserved sequences and dual targets of HA and NA has certain immunogenicity and can induce a good T cell immune response, exhibiting good protective efficacy. It addresses the problem of insufficient protection of existing influenza vaccines due to the rapid mutation of influenza viruses, improves the broad spectrum and protective effect of the vaccine, and enables it to better cope with the immune escape of the latest variant strains in the future. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the C-1 mRNA vaccine in an embodiment of the present invention; Figure 2 The following is a diagram showing the experimental results of the vaccine being qualified in an embodiment of the present invention, wherein (a) is an in vitro transcribed RNA electrophoresis (lane 1); (b) is an encapsulation verification, lane 1: mRNA-LNP, lane 2: mRNA-LNP after being treated with Tritonx-100 to fully release mRNA; (c) is a schematic diagram of the particle size distribution of the prepared mRNA-LNP. Figure 3 This is a schematic diagram comparing the expression of C-1 vaccine in 293T cells with that of RNA transfected using the Mirus kit in an embodiment of the present invention. Figure 4 This is a schematic diagram illustrating the effect of vaccine transfection into 293T cells on the inhibitory activity of neuraminidase in an embodiment of the present invention.

[0015] Figure 5 This is a schematic diagram of the immunization cycle of the C-1 vaccine in an embodiment of the present invention; Figure 6This is a schematic diagram of the level of C-1 vaccine immune binding antibodies in an embodiment of the present invention; Figure 7 These are representative images and counting results of the number of secreted IFN-γ spots in the ELISPOT experiment in this embodiment of the invention; Figure 8 These are representative images and counting results of the number of IL-2 spots secreted in the ELISPOT experiment in this embodiment of the invention; Figure 9 These are representative images and counting results of the number of IL-4 spots secreted in the ELISPOT experiment in this embodiment of the invention; Figure 10 The level of neutralizing antibodies produced against the vaccine design reference strain (A / Michigan / 45 / 2015 (H1N1)) after immunization with the C-1 vaccine in this embodiment of the invention; Figure 11 The level of neutralizing antibodies produced after C-1 immunization in this embodiment of the invention against a strain (Influenza AH1N1 (A / Victoria / 25 / 2570 / 2019)) not included in the reference vaccine design; Figure 12 This is a graph showing the changes in body temperature and weight within 7 days after infection with the A / Michigan / 45 / 2015 (H1N1) strain in an embodiment of the present invention. Figure 13 The change in viral load in mouse nasopharyngeal swabs within seven days after challenge with the A / Michigan / 45 / 2015 (H1N1) strain in this embodiment of the invention; Figure 14 This is a schematic diagram showing the viral load levels in the main tissues of the lungs, nasal turbinates, and trachea after sampling on the seventh day following challenge with the A / Michigan / 45 / 2015 (H1N1) strain in this embodiment of the invention. Figure 15 HE staining (left) and pathological score (right) of lung tissue after challenge with the / Michigan / 45 / 2015 (H1N1) strain in this embodiment of the invention; Figure 16 This is a graph showing the changes in body temperature and weight of mice within seven days after challenge with the Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain in an embodiment of the present invention.

[0016] Figure 17 The viral load in nasopharyngeal swabs of mice within seven days after challenge with the Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain in this embodiment of the invention. Figure 18The viral load levels in the main tissues of the lungs, nasal turbinates, and trachea were measured on the seventh day after challenge with the Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain in this embodiment of the invention. Figure 19 HE staining (left) and pathological score (right) of lung tissue after challenge with Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain in this embodiment of the invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In the following examples, the detection was performed with 12 μg as the high-dose group, 5 μg as the low-dose group, and blank solvent (Tirs HCl (pH=6.8)) as the control group. Example 1

[0019] Preparation of a broad-spectrum mRNA vaccine (C-1) for H1N1 influenza virus targeting the tandem of influenza hemagglutinin and neuraminidase proteins: Using the H1N1 strains (cell culture and recombinant protein vaccines) recommended by the WHO for influenza in the Northern Hemisphere from 2015 to 2025, the conserved amino acid sequences of hemagglutinin (HA) and neuraminidase (NA) were obtained, as follows: HA amino acid sequence: MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKINQTYINDKGKEVLVLWGIHHPPTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI; NA amino acid sequence: MNPNQKIITIGSICMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNKSVITYENNTWVNQTYVNISNTNFAARQSVASVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVI REPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGTNWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVN GSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFK YGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVDSDIVGWSWPDGAELPFTIDK; The amino acid sequence is shown in SEQ ID NO.1, obtained by linking HA and NA with the flexible peptide GGGSGGGSGGGSGGGS. The DNA sequence is obtained by codon optimization according to human codon preference (as shown in SEQ ID NO.2). The vector and the target fragment are digested with SpeⅠ and XhoⅠ double enzymes, and the plasmid PUC57 (PUC57-T7-5UTR-HA-NA-3UTR-PolyA) containing the T7 promoter, 5'UTR (nucleotide sequence shown in SEQ ID NO.3), 3'UTR (nucleotide sequence shown in SEQ ID NO.4), and 100 PolyA molecules is constructed with T4 DNAase. The plasmid is then sequenced for verification.

[0020] The constructed transformation was introduced into competent Escherichia coli (TSINGKE TSC-C01 Trelief® 5α Chemically Competent Cell) using the heat shock method. Single colonies were selected for culture. After the bacterial culture was correctly sequenced, the culture was expanded for plasmid amplification. After plasmid extraction, the DNA template was linearized by BsaI restriction enzyme digestion. The linearized template was precipitated with 70% volume isopropanol at -80℃ and washed with 70% ethanol. In vitro transcription of the linearized template was performed using a commercial kit (Novizan). Before transcription, the transcriptional precursor UTP was completely replaced with N1-methylpseudouridine triphosphate (N1-Me-pUTP). After transcription, the mRNA vaccine was obtained as follows: Figure 1 As shown; The mRNA was dissolved in a 50 mM citrate buffer solution at pH 4 to obtain a final concentration of 108 ng / μL. Next, a lipid mixture was prepared: SM102, DMG-PEG2000, DSPC, and cholesterol were mixed and dissolved in anhydrous ethanol solution at a ratio of 50%, 1.5%, 10%, and 38.5%, respectively, to obtain a lipid mixture. The lipid mixture and the mRNA-citrate solution were then filtered separately through a 0.22 μm microporous membrane. The mixture was then microfluidically mixed according to a 1:8 ratio of phosphorus content in the mRNA to nitrogen content in the SM102, at a flow rate of 15 mL / min:5 mL / min for the mRNA-citrate solution to the lipid mixture, yielding mRNA-LNP. Detection:

[0021] 1. Immediately dilute the obtained mRNA-LNP with 15 ml of the above citrate buffer, and ultrafilter using a 100 kDa ultrafiltration tube at a centrifugation force of 3000 g. After ultrafiltration to 1 / 4 volume, add 50 mM Tris-HCl buffer (pH=7.5) to 15 mL. Repeat twice, and then test whether the vaccine is qualified. Specific results are as follows Figure 2 As shown, where Figure 2 (a) Lane 1 is an electrophoresis diagram of in vitro transcribed RNA. Figure 2 (b) is an electrophoresis diagram for encapsulation verification. Lane 1 is mRNA-LNP, and lane 2 is mRNA-LNP after being treated with 1% tritonx-100 to fully release mRNA. Figure 2 (c) shows the particle size distribution of the prepared mRNA-LNP; Depend on Figure 2 It can be seen that the transcribed RNA did not show obvious degradation or extraneous bands, the LNP was successfully encapsulated and the particle size was about 100nm; the mRNA vaccine quality is qualified.

[0022] The in vitro expression of mRNA-LNP (i.e., C-1 vaccine) in 293T cells was verified: 293T cells were cultured in DMEM complete medium (with 10% FBS and 1% penicillin-streptomycin) at 37°C and 5% CO2. 293T cells in logarithmic growth phase were passaged into 12-well plates at a ratio of 1:2 and cultured overnight. When the cell confluence reached 80%, the complete medium was discarded, and 1 ml of Opti-MEM low-serum medium was added to each well. The positive control wells were transfected with mRNA using the Mirus MIR2225 kit (according to the instructions). The vaccine group was treated with an appropriate amount of mRNA-LNP. Six hours after transfection, the low-serum medium was replaced with complete medium, and the cells were cultured for another 36 hours. Cell proteins were extracted on ice using RIPA lysis buffer and Western blot experiments were performed to detect the in vitro expression level of the target protein.

[0023] Specific results are as follows Figure 3 As shown, Figure 3 After transfection of cells with the C-1 vaccine, the expression of RNA in 293T cells was compared with that of RNA transfected with the Mirus kit. It can be seen that the C-1 vaccine was well expressed in 293T cells after transfection, while the untransfected negative control (-) did not show the target band.

[0024] 3. The influence of tandem strategies on the independent functionality of tandem antigens in experiments The vaccine was transfected into 293T cells. Cell proteins were collected 24 hours later for neuraminidase inhibition assays. Simultaneously, 100 TCID50 A / Michigan / 45 / 2015 (H1N1) strain virus was used as a positive control, and the untransfected group served as a negative control, as detailed below: 1) Seed 293T cells into 6-well plates. After they adhered to the plates, replace the complete culture medium with an equal volume of low serum culture medium. Then, transfect each well with 3 μg of encapsulated mRNA vaccine and incubate at 37°C and 5% CO2 for 24 h.

[0025] 2) After washing the 6-well plate with PBS, place it on ice, add 200 μL of RIPA lysis buffer (add protease inhibitor at a ratio of 1:100), and let it stand on ice for 30 min.

[0026] 3) Collect the protein and add it to an ELISA plate coated with good fetal protein (MedChemExpress, #HY-P2352). At the same time, add 100 TCID50 A / Michigan / 45 / 2015 (H1N1) live virus as a positive control and an equal volume of blank culture medium as a negative control. Incubate overnight.

[0027] (4) The next day, thoroughly wash the wells with PBST. Dilute HRP-labeled peanut lectin (HRPO, 1 mg / mL, Sigma #L0881) to a concentration of 1:2000 with sample buffer and add it to each well. Incubate at room temperature in the dark for 2 hours. Wash the wells again with PBST, and develop the color according to the standard ELISA procedure, i.e., add TMB substrate and incubate in the dark for 15 minutes, then terminate the reaction with stop solution (SolarBio, C1058). Measure the absorbance at wavelengths of 450 nm and 630 nm.

[0028] The results showed that after the vaccine was transfected into 293T cells, it could express the antigen normally, and the tandem component NA could have neuraminidase inhibitory activity similar to that of the virus (e.g., Figure 19 (As shown).

[0029] BALB / c mice were immunized with C-1 vaccine, and the level of binding antibodies in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA): ELISA antigen coating buffer was used to coat the antigen protein at a concentration of 0.8 μg / well (A / Wisconsin / 67 / 2022). (H1N1) strain HA and NA proteins), incubated overnight at 4°C; washed with PBST (PBS solution containing 0.05% Tween 20), then blocked with 2% BSA solution (PBS prepared) for 1 h; washed three times with PBST, serially diluted serum (1:400) and incubated for 1 h; washed three times, diluted secondary antibody at 1:30000 and incubated for 1 h; added TMB substrate (3,3′,5,5′-tetramethylbenzidine) and incubated in the dark for 15 min, then added stop solution, and detected absorbance values ​​at 450 nm and 630 nm. The absorbance value at 450 nm minus the absorbance value at 630 nm > 0.1 was considered positive. The reciprocal of the maximum dilution factor of the positive well represents the binding antibody level.

[0030] Specifically, after immunization of BALB / c mice with the C-1 vaccine, the serum antibody titers against HA and NA protein antigens significantly increased; the C-1 immunization cycle was as follows: Figure 5 As shown in the C-1 vaccine immunization cycle, a booster immunization is administered 28 days after the initial immunization. Starting 14 days later, whole blood is collected every 7 days, and serum is separated to detect the binding antibody levels. Figure 5 shows the C-1 vaccine binding antibody levels. It is evident that binding antibodies against HA and NA proteins are significantly produced from day 14 after C-1 vaccine immunization. Thereafter, antibody levels gradually increase, and after the 28-day booster immunization, antibody levels are maintained at 10. 5 above.

[0031] in addition Figure 6 The level of C-1 vaccine-binding antibodies was shown.

[0032] Following immunization of BALB / c mice with C-1 vaccine, the ELISPOT experiment was conducted. According to the mouse immunization protocol, spleen tissue was harvested 49 days after the initial immunization to isolate splenic lymphocytes for the ELISPOT experiment. 1) Add 1 ml of whole blood and tissue diluent to a petri dish. Under aseptic conditions, remove the spleen from the mouse, remove the spleen capsule, and cut the spleen into small pieces using ophthalmic scissors in the petri dish. Ensure that the spleen and the obtained cells are always in a liquid environment.

[0033] 2) Place the cell sieve on a 50 ml centrifuge tube, place the spleen on the sieve, and grind the spleen with the injection plunger until no red particles are visible.

[0034] 3) Rinse the cell sieve with 1 ml of whole blood and tissue diluent.

[0035] 4) Take a centrifuge tube and add a separation solution equal in volume to the spleen single-cell suspension. The separation solution should be at least 3 ml and the total volume should not exceed two-thirds of the centrifuge tube.

[0036] 5) Aspirate a single-cell suspension from the spleen and slowly add it to the surface of the separation liquid. Due to the difference in density, a distinct stratification interface is formed between the two.

[0037] 6) Centrifuge at 500 g for 20 min at room temperature using a horizontal rotor.

[0038] 7) After centrifugation, the liquid in the centrifuge tube is divided into four layers from top to bottom: the plasma layer, the milky white lymphocyte layer, the separation liquid layer, and the red blood cell layer at the bottom.

[0039] 8) Carefully aspirate the second ring-shaped milky white lymphocyte layer into another clean 15 ml centrifuge tube, add 10 ml of cell washing buffer, and mix by inverting the tube. Centrifuge at 250 g for 5 min using a horizontal rotor.

[0040] 9) Discard the supernatant, add 5 ml of PBS, and resuspend the cell pellet by pipetting. Centrifuge at 250 g for 5 min using a horizontal rotor.

[0041] 10) Calculate the number of wells to be seeded for each cell line, and resuspend the cells in ELISPOTS medium at a rate of 50 μl per well plus an additional 10 μl.

[0042] 11) Take 10 μl of resuspended cells, add 90 μl of PBS and mix well. Then take 10 μl of cell suspension for cell counting (note that the value obtained here is the value after dilution by 10 times).

[0043] 12) Wash the plates four times with 200 μl of sterile PBS, allowing them to stand for 2 min each time. After washing, add 200 μl of ELISPOTS-specific medium to each well and incubate at room temperature for 30 min to equilibrate the plates.

[0044] 13) Remove the culture medium from the plate, and dilute the plate with ELISPOTS-specific medium according to the cell count, taking the number of cells that most people can reach. Generally, the cell number for platemaking is 3.0 × 10⁶ cells / cm². 5 -6.0×10 5 Cells / well. Add 50 μl of cell suspension to each well.

[0045] 14) Add the stimulant to the reaction wells, 1 μg or 2 μg / well, 50 μl per well. Simultaneously set up a PHA positive stimulant control and a blank control without added stimulant.

[0046] 15) Place the plate in a 37 ℃, 5% CO2 incubator and incubate for 24 h-48 h. Do not move it during the incubation period.

[0047] 16) Remove the culture medium from the plate, add 200 μl of pre-cooled deionized water at 4 ℃, soak for 2 min, and then remove the plate.

[0048] 17) Add 200 μl / well of PBS to the wash plate, soak for 2 min, and wash 5 times.

[0049] 18) Dilute the detection antibody (IFN-γ-biotin) to 1 μg / ml with 0.5% FBS in PBS, add 100 μl to each well, and incubate at room temperature for 2 h or at 37 °C for 1 h.

[0050] 19) Washing plate. 20) Dilute Streptavidin-ALP at a ratio of 1:1000 with PBS containing 0.5% FBS, add 100 μl to each well, and incubate at room temperature for 1 h.

[0051] 21) Wash the flat plate, the steps are the same as (9).

[0052] 22) Filter the colorimetric solution using a 0.45 μm filter, add 100 μl to each well until obvious spots appear. The colorimetric time is generally 5-10 min.

[0053] 23) Rinse the plate with tap water until the color stops developing and no longer darkens. Let the plate air dry in a well-ventilated area. Store the plate in a dark place.

[0054] For details, please see [link / details]. Figures 7-9 ,Depend on Figure 7 The image on the left shows representative results of ELISPOT assays to detect the number of cells secreting the cytokine IFN-γ, using HA and NA proteins of strain A / Wisconsin / 67 / 2022 (H1N1 strain) as individual or co-stimuli. Figure 7The right side shows the results of IFN-γ spot count; Figure 8 The image on the left shows representative results of ELISPOT assays to detect the number of cells secreting the cytokine IL-2, using HA and NA proteins of strain A / Wisconsin / 67 / 2022 (H1N1) as individual or co-stimuli. Figure 8 The right side shows the results of IFN-γ spot count; Figure 9 The image on the left shows representative results of ELISPOT assays to detect the number of cells secreting the cytokine IL-4, using HA and NA proteins of strain A / Wisconsin / 67 / 2022 (H1N1) as individual or co-stimuli. Figure 9 IL-4 spot count results on the right side; It is known that immunizing mice with the C-1 vaccine can induce a strong T-cell immune response.

[0055] Antibody production in BALB / c mice after immunization with C-1 vaccine against the vaccine design reference strain and strains not included in the reference range during design: 1) Add 4 times the volume of RDE (receptor-destroying enzyme) to the serum and treat at 37°C for 16 hours.

[0056] 2) Inactivate at 56℃ for 30 minutes.

[0057] 3) Add 25 μL of PBS to the first column of the U-shaped disposable reaction plate, and add 50 μL of serum and RDE mixture to the first column, and then dilute stepwise in 25 μL increments.

[0058] 5) Add 4 units of hemagglutination virus solution and incubate at room temperature for 20 minutes.

[0059] 6) Add 50 μL of 1% guinea pig red blood cells, let stand at room temperature for 1 hour, and then observe the agglutination.

[0060] Forty-two days after the initial immunization, whole blood was collected from mice, serum was separated, and the level of neutralizing antibodies was detected by hemagglutination inhibition assay. According to the relevant guidelines of the National Influenza Center Standard Operating Procedures, a titer of ≥40 against the reference antigen in the test serum was considered to have a protective effect. Specific results are as follows Figure 10 , Figure 11 As shown; where by Figure 10 It can be known that after immunization with the C-1 vaccine, the level of neutralizing antibodies produced against one of the vaccine's design reference strains (A / Michigan / 45 / 2015 (H1N1)) was determined; Figure 11 It can be seen that after C-1 immunization, the level of neutralizing antibodies produced against the strain (Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019)) not included in the vaccine design; That is, C-1 immunization can produce neutralizing antibodies sufficient to provide protection against both strains.

[0061] Protection against virus challenge in BALB / c mice after immunization After completing the immunization process, mice underwent challenge experiments. To verify the broad-spectrum protection of the vaccine against H1N1, challenge protection experiments were conducted with two strains: the vaccine design reference strain A / Michigan / 45 / 2015 (H1N1) and the strain Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019), which was not included in the vaccine design reference. The challenge dose was 256 HAU, and the viral fluid volume was 50 μL, which was administered via nasal drops.

[0062] The challenge protection experiment examined changes in body temperature and weight, viral load in nasopharyngeal swabs, viral load levels in the lungs, nasal turbinates, and trachea on day 7, and pathological damage in lung tissue during the challenge period in mice. Specific results are as follows: Figure 12-19 As shown, the C-1 vaccine has a good protective effect against both strains.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A broad-spectrum H1N1 mRNA vaccine targeting the tandem HA and NA proteins, characterized in that, The amino acid sequence of the mRNA vaccine is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.

2.

2. A method for preparing an mRNA vaccine as described in claim 1, characterized in that, The preparation method includes the following steps: S1, a conserved amino acid sequence of hemagglutinin HA and neuraminidase NA is linked by a flexible peptide. S2. The obtained amino acid sequence is codon optimized according to human codon preference to obtain a DNA sequence, such as SEQ ID NO.

2. This sequence is constructed into plasmid PUC57 containing T7 promoter, 5'UTR, 3'UTR and 100 PolyA by enzyme digestion and ligation, which is PUC57-T7-5UTR-HA-NA-3UTR-PolyA, and the plasmid construction is completed. S3. The plasmid successfully constructed above is amplified and transcribed to obtain an mRNA vaccine; S4. The mRNA obtained above is prepared into lipid nanoparticles (LNPs) using a microfluidic method.

3. The preparation method according to claim 2, characterized in that: The flexible peptide is four repeating GGGS, that is, the amino acid sequence of the flexible peptide is: GGGSGGGSGGGSGGGS.

4. The preparation method according to claim 2, characterized in that: The nucleotide sequence of the 5'UTR in step S2 is shown in SEQ ID NO.3, and the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.

4.

5. The preparation method according to claim 2, characterized in that: In step S3, TSINGKE TSC-C01Trelief® 5α Chemically Competent Cell Escherichia coli was used for transformation and plasmid amplification. After plasmid extraction, the DNA template was linearized by BsaI restriction enzyme digestion. The linearized template was precipitated with 70% volume isopropanol at -80°C and washed with 70% ethanol.

6. The preparation method according to claim 2, characterized in that: In step S3, the linearized template is transcribed in vitro using the commercially available kit Novizan. Before transcription, the transcription raw material UTP is completely replaced with N1-methylpseuuridine triphosphate N1-Me-pUTP.

7. The preparation method according to claim 2, characterized in that, The specific method for preparing lipid nanoparticles (LNPs) in step S4 includes the following steps: S4-1. Dissolve the mRNA in a 50mM citrate buffer solution with pH=4. The phosphorus content and nitrogen content in the mRNA are in a ratio of 1:8 to obtain a final concentration of 108 ng / μL for the mRNA-citrate buffer solution. S4-2. Prepare an anhydrous ethanol solution with 50% SM102, 1.5% DMG-PEG2000, 10% DSPC, and 38.5% cholesterol to obtain a lipid mixed solution. S4-3. The lipid mixture and mRNA-citric acid solution were filtered separately through a 0.22 μm microporous membrane and mixed using a microfluidic instrument to obtain lipid nanoparticles (LNP).

8. The preparation method according to claim 7, characterized in that: In step S4-3, the flow rate ratio for mixing using the microfluidic instrument is 15 mL / min for mRNA-citric acid solution and 5 mL / min for lipid mixture.

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