H1N1 broad-spectrum mRNA vaccine with HA and NA proteins connected in series as target based on key amino acid mutation and preparation method of H1N1 broad-spectrum mRNA vaccine

Abstract

The invention provides an H1N1 broad-spectrum mRNA vaccine with HA and NA proteins connected in series as a target based on key amino acid mutation and a preparation method of the H1N1 broad-spectrum mRNA vaccine, and relates to the technical field of vaccine preparation. The amino acid sequence of the mRNA vaccine is as shown in SEQ ID NO.1, and the nucleotide sequence of the mRNA vaccine is as shown in SEQ ID NO.2, namely on the basis of an HA conserved sequence, variation sites with amino acid property crossing near a main antigen site of an HA head and at a receptor binding part are reserved, an amino acid sequence containing a key mutation site HA'is obtained, and GGGSGGSGGSGGGGS is adopted to connect HA 'and NA conserved amino acid sequences, so that the mRNA vaccine is obtained. The defects in the prior art are overcome, the broad-spectrum protection effect on the H1N1 type influenza virus is improved by reserving the key amino acid mutation site in the HA conserved sequence, the vaccine production cost is reduced, the immune cycle is shortened, and an effective coping scheme is provided for the influenza outbreak season.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular to an H1N1 broad-spectrum mRNA vaccine targeting HA and NA proteins with key amino acid mutations in series and a preparation method thereof. Background Art

[0002] Influenza virus (Irfluenza virus) belongs to the Orthomyxoviridae family and is a segmented, negative-strand RNA virus. The eight negative-strand RNA segments of its genome encode the viral RNA polymerase complex (PB1, PB2, PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M1), ion channel protein (M2), and a series of nonstructural proteins (NS1, NS2, and NS3). HA and NA are two important glycoproteins on the viral surface and are the most popular vaccine immunogens. HA is responsible for binding to host cell surface receptors and mediating viral entry; NA hydrolyzes sialic acid on the host cell surface, facilitating viral release from the cell.

[0003] Influenza viruses have been of widespread concern since the Spanish flu outbreak of 1918. At least 18 distinct influenza A virus (IAV) subtypes circulate in animal reservoirs, occasionally crossing into humans and causing pandemics. Currently, H1N1 and H3N2 IAVs, along with one or two antigenically distinct influenza B virus (IBV) lineages, circulate seasonally in humans. Although various epidemic surveillance programs and modeling studies have improved our understanding of influenza pandemics, it remains impossible to accurately predict which influenza subtype will cause the next pandemic. Therefore, the development of a universal influenza vaccine has been a key research priority in the vaccine community. However, due to the significant differences between different influenza virus types and the high frequency of mutation through antigenic drift and antigenic shift, achieving broad coverage with a monovalent vaccine or a single immunogen is challenging. Therefore, the design of a broad-spectrum mRNA vaccine is crucial. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides an H1N1 broad-spectrum mRNA vaccine targeting HA and NA proteins in tandem with key amino acid mutations and a preparation method thereof. By retaining key amino acid mutation sites in the conserved HA sequence, the broad-spectrum protection against H1N1 type influenza viruses is improved, the vaccine production cost and immunization cycle are reduced, and an effective response plan is provided for the influenza outbreak season.

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

[0006] The preparation method of mRNA vaccine includes the following steps: S1. Based on the conserved HA sequence, the variant sites near the main antigenic site of the HA head and the receptor binding site where the amino acid properties span are retained to obtain the HA' amino acid sequence containing the key mutation sites, wherein the HA' amino acid sequence is as shown in SEQ ID NO. 3; S2. Connecting the HA' amino acid sequence and the conserved NA amino acid sequence via a flexible peptide to obtain the amino acid sequence shown in SEQ ID NO. 1; S3. The obtained amino acid sequence was codon-optimized according to human codon preference to obtain a DNA sequence, such as SEQ ID NO. 2. This sequence was then constructed into the plasmid PUC57 containing a T7 promoter, 5'UTR, 3'UTR, and 100 PolyA residues by enzyme digestion and ligation, namely PUC57-T7-5UTR-HA-NA-3UTR-PolyA, completing the plasmid construction; S4. Amplify and transcribe the successfully constructed plasmid to obtain an mRNA vaccine; S5. The mRNA obtained above is prepared into lipid nanoparticles LNP by a microfluidic method.

[0007] Preferably, in step S2, the flexible peptide is four repeated GGGS, that is, the amino acid sequence of the flexible peptide is: GGGSGGGSGGGSGGGS.

[0008] Preferably, the nucleotide sequence of the 5'UTR in step S3 is shown as SEQ ID NO.4, and the nucleotide sequence of the 3'UTR is shown as SEQ ID NO.5.

[0009] Preferably, in step S4, TSINGKE TSC-C01 Trelief® 5α Chemically Competent Cell is used for transformation and amplification, and after plasmid extraction, BsaI is used to enzymatically linearize the DNA template, and the linearized template is precipitated at -80°C using 70% volume of isopropanol and washed with 70% ethanol.

[0010] Preferably, in step S4, the linearized template is transcribed in vitro using a commercial kit Novozymes, and before transcription, the raw material UTP is completely replaced with N1-methylpseudouridine triphosphate N1-Me-pUTP.

[0011] Preferably, the specific method of preparing lipid nanoparticles LNP in step S5 includes the following steps: S5-1. Dissolve the mRNA in 50 mM citric acid buffer at pH 4, using a ratio of 1:8 between the P and N content in the mRNA to obtain a final concentration of 108 ng / μL mRNA-citric acid solution. S5-2, prepare an anhydrous ethanol solution with a 50% SM102 mass concentration, a 1.5% DMG-PEG2000 mass concentration, a 10% DSPC mass concentration, and a 38.5% cholesterol mass concentration to obtain a 16 mM lipid mixed solution; S5-3. The lipid mixed solution and the mRNA-citric acid solution were filtered through a 0.22 μm microporous filter membrane respectively, and mixed using a microfluidic instrument to obtain lipid nanoparticles LNP.

[0012] Preferably, in step S5-3, the flow rate ratio of the mixing using the microfluidic instrument is 15 mL / min:5 mL / min for the mRNA-citric acid solution:lipid mixed solution.

[0013] The present invention provides a broad-spectrum mRNA vaccine for H1N1 based on tandem targeting of HA and NA proteins with key amino acid mutations, which has the following advantages over the prior art: The present invention designs a vaccine that exerts immune protection through dual targets of HA and NA, and obtains HA' based on key amino acid mutations. The influenza mRNA vaccine designed with dual targets of HA' and NA has certain immunogenicity and can induce a good T cell immune response, with good protective efficacy. By retaining the key amino acid mutation sites in the original conserved sequence of HA', the broad-spectrum protection effect against H1N1 type influenza viruses is improved, the vaccine production cost and the immunization cycle are reduced, and an effective response plan is provided for the influenza outbreak season. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the mRNA vaccine in an embodiment of the present invention; Figure 2 Figures 1 and 2 show the experimental results for testing the eligibility of vaccines in the examples of the present invention, including (a) electrophoresis of in vitro transcribed RNA (lane 2); (b) encapsulation verification: lane 1: mRNA-LNP, lane 2: mRNA-LNP after treatment with Triton X-100 to fully release mRNA; (c) particle size distribution of the prepared mRNA-LNP. Figure 3 The expression of K-1 vaccine in 293T cells after cell transfection is compared with RNA transfection with Mirus kit in the examples of the present invention; Figure 4 The K-1 vaccine immunization cycle in the embodiment of the present invention; Figure 5 is the level of K-1 vaccine immune binding antibodies in the examples of the present invention; Figure 6 Representative images and counting results of the number of IFN-γ secreted spots in the ELISPOT experiment in the embodiment of the present invention; Figure 7 Representative images and counting results of IL-2 secretion in ELISPOT experiments in the examples of the present invention; Figure 8 Representative images and counting results of IL-4 secretion in ELISPOT experiments in the examples of the present invention; Figure 9 The neutralizing antibody level produced by the K-1 vaccine against the reference strain for vaccine design (A / Michigan / 45 / 2015 (H1N1)) after immunization in the embodiment of the present invention; Figure 10 This is the neutralizing antibody level produced after K-1 immunization in the examples of the present invention against a strain not included in the reference vaccine design (Influenza AH1N1 (A / Victoria / 25 / 2570 / 2019)); Figure 11 This is a graph showing changes in body temperature and body weight within 7 days after challenge with the A / Michigan / 45 / 2015 (H1N1) strain in the examples of the present invention; Figure 12 The figure shows the changes in viral load in nasopharyngeal swabs of mice within seven days after challenge with the A / Michigan / 45 / 2015 (H1N1) strain in the examples of the present invention; Figure 13 The viral load levels in the lungs, nasal conchae, and trachea were measured seven days after challenge with the A / Michigan / 45 / 2015 (H1N1) strain in the examples of the present invention. Figure 14 HE staining (left) and pathological scoring (right) of mouse lung tissue after challenge with the A / Michigan / 45 / 2015 (H1N1) strain in the examples of the present invention; Figure 15 This is a graph showing 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 the examples of the present invention; Figure 16 This is the viral load level of nasopharyngeal swabs in mice within seven days after challenge with the Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain in the examples of the present invention; Figure 17 The viral load levels in the lungs, nasal conchas, and trachea of ​​the main tissues were measured after the seventh day of challenge with the Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain in the present invention. Figure 18 HE staining (left) and pathological scoring (right) of mouse lung tissue after challenge with the Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019) strain in the examples of the present invention. DETAILED DESCRIPTION

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0016] In the following examples, the test 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:

[0017] Preparation of a broad-spectrum mRNA vaccine (K-1) targeting influenza hemagglutinin HA' and neuraminidase proteins that retain key mutant genes: The conserved amino acid sequences of hemagglutinin (HA) and neuraminidase (NA) were obtained from the recommended H1N1 influenza strains (cell culture and recombinant protein vaccines) for the Northern Hemisphere from 2015 to 2025 published by the WHO: Conserved HA amino acid sequence: MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKINQTYINDKGKEVLVLWGIHHPPTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI; Conserved NA amino acid sequence: MNPNQKIITIGSICMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNKSVITYENNTWVNQTYVNISNTNFAARQSVASVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVI REPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGTNWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVN GSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFK YGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVDSDIVGWSWPDGAELPFTIDK; On the basis of the conserved HA sequence, the variant sites near the main antigenic site of the HA head and the receptor binding site where the amino acid properties span are retained to obtain the HA' sequence containing the key mutation sites: HA' sequence: ; HA' and NA were linked by a flexible peptide (GGGSGGGSGGGSGGGS) to obtain the amino acid sequence shown in SEQ ID NO.1. Codon optimization was performed according to human codon preference to obtain a DNA sequence (SEQ ID NO.2). The vector and target fragment were double-digested with SpeⅠ and XhoⅠ, and the sequence was ligated with T4 DNA enzyme to construct the plasmid PUC57 (PUC57-T7-5UTR-HA-NA -3UTR-PolyA) containing a T7 promoter, a 5'UTR (nucleotide sequence shown in SEQ ID NO.3), a 3'UTR (nucleotide sequence shown in SEQ ID NO.4), and 100 PolyA residues. After the plasmid construction was completed, sequencing verification was performed.

[0018] The constructed construct was transformed into competent E. coli (TSINGKE TSC-C01 Trelief®5α Chemically Competent Cell) using the heat shock method. Monoclonal colonies were selected for culture. After the bacterial solution was sequenced correctly, the culture was expanded for plasmid amplification. After the plasmid was extracted, the DNA template was linearized with BsaI. The linearized template was precipitated with 70% isopropanol at -80°C and washed with 70% ethanol. The linearized template was transcribed in vitro using a commercial kit (Novozymes). Before transcription, the raw material UTP was completely replaced with N1-methyl pseudouridine triphosphate (N1-Me-pUTP). After transcription, the mRNA vaccine was obtained. Figure 1 As shown; mRNA was dissolved in 50 mM citric acid buffer at pH 4 to obtain an mRNA-citric acid solution with a final concentration of 108 ng / μL. A lipid mixture solution was then prepared: SM102, DMG-PEG2000, DSPC, and cholesterol were dissolved in anhydrous ethanol at ratios of 50%, 1.5%, 10%, and 38.5% to obtain a lipid mixture solution. The lipid mixture solution and the mRNA-citric acid solution were filtered through a 0.22 μm microporous membrane and mixed using a microfluidic instrument at a flow rate of 15 mL / min:5 mL / min, based on the phosphorus content of the mRNA and the nitrogen content of SM102, to obtain mRNA-LNP.

[0019] Detection: 1. Immediately dilute the obtained mRNA-LNP with 15 ml of the above-mentioned citric acid buffer and ultrafilter using a 100KD ultrafiltration tube at a centrifugal force of 3000 g. After ultrafiltration to 1 / 4 volume, add 50 mM Tris-HCl buffer (PH = 7.5) to 15 mL. Repeat twice to test whether the vaccine is qualified: Specific results such as Figure 2 As shown, Figure 2 Lane 2 in (a) is the electrophoresis diagram of in vitro transcribed RNA. Figure 2 (b) Electrophoresis diagram for encapsulation verification, lane 1 is mRNA-LNP, lane 2 is mRNA-LNP after treatment with 1% tritonx-100 to fully release mRNA; Figure 2 (c) Particle size distribution of prepared mRNA-LNPs; Depend on Figure 2 It can be seen that the transcribed RNA has no obvious degradation and impurity bands, LNP is successfully encapsulated and the particle size is about 100nm; the quality of the mRNA vaccine is qualified.

[0020] 2. Verify the in vitro expression effect of mRNA-LNP (i.e., K-1 vaccine) in 293T cells: 293T cells were cultured in DMEM complete medium (supplemented with 10% FBS and 1% penicillin-streptomycin) at 37°C and 5% CO2. 293T cells in the logarithmic growth phase were transferred to a 12-well plate 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). An appropriate amount of mRNA-LNP was added to the vaccine group. 6 h after transfection, the low-serum medium was replaced with complete medium. After a further 36 h of culture, cellular 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.

[0021] Specific results such as Figure 3 As shown, after K-1 vaccine was transfected into cells, its expression in 293T cells was compared with RNA transfected with Mirus kit. It can be seen that K-1 vaccine was well expressed in 293T cells after transfection, and the untransfected negative control (-) had no target band.

[0022] BALB / c mice were immunized with K-1 vaccine, and the level of binding antibodies in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA): antigen protein (A / Wisconsin / 67 / 2022) was coated at 0.8 μg / well using ELISA antigen coating solution. (H1N1) strain HA and NA proteins), incubated at 4°C overnight; washed with PBST (PBS solution containing 0.05% Tween 20), blocked with 2% BSA solution (prepared in PBS) for 1 hour; washed three times with PBST, incubated with serially diluted serum (1:400) for 1 hour; washed three times, incubated with secondary antibody diluted 1:30000 for 1 hour; added TMB substrate (3,3',5,5'-tetramethylbenzidine) and incubated in the dark for 15 minutes, then added stop solution, and the absorbance at 450nm and 630nm was detected. Positive was determined when the absorbance at 450nm minus the absorbance at 630nm was greater than 0.1. The reciprocal of the maximum dilution factor of the positive well represented the level of bound antibody.

[0023] The specific results showed that after BALB / c mice were immunized with K-1 vaccine, the titers of antibodies against HA and NA protein antigens in the mouse serum increased significantly; the K-1 immunization cycle was as follows: Figure 4 As shown, booster immunization was performed 28 days after the initial immunization, and whole blood was collected and serum was separated to detect the binding antibody level starting from the 14th day with a 7-day cycle. Figure 5The K-1 vaccine immune combined antibody level shows that after K-1 vaccine immunization, the combination antibodies against HA and NA proteins are obviously produced from day 14, and the antibody level gradually increases thereafter. After the booster immunization on day 28, the antibody level can be maintained at 10 5 above.

[0024] 4. ELISPOT assay after immunization of BALB / c mice with K-1 vaccine: According to the mouse immunization process, spleen tissue was collected 49 days after the initial immunization to separate spleen lymphocytes for ELISPOT assay: 1) Add 1 ml of whole blood and tissue diluent to a dish. Aseptically remove the mouse spleen, remove the spleen capsule, and cut the spleen into small pieces using ophthalmic scissors in the dish. Ensure that the spleen and the harvested cells remain in a liquid environment.

[0025] 2) Place a cell strainer on a 50 ml centrifuge tube, place the spleen on the strainer, and grind the spleen using a syringe plunger until no red particles are visible.

[0026] 3) Rinse the cell sieve with 1 ml of whole blood and tissue diluent.

[0027] 4) Take a centrifuge tube and add an amount of separation buffer equal to the amount of spleen single cell suspension. The separation buffer should be at least 3 ml and the total volume should not exceed two-thirds of the centrifuge tube.

[0028] 5) Aspirate the spleen single cell suspension and slowly add it to the surface of the separation solution. The difference in density between the two will form a clear layered interface.

[0029] 6) Centrifuge at 500 g for 20 min at room temperature in a swing-out rotor.

[0030] 7) After centrifugation, the liquid in the centrifuge tube is divided into four layers from top to bottom: plasma layer, milky white lymphocyte layer, separation fluid layer, and red blood cell layer at the bottom.

[0031] 8) Carefully transfer the second layer of circular milky white lymphocytes to another clean 15 ml centrifuge tube. Add 10 ml of cell wash buffer and mix by inverting. Centrifuge at 250 g in a swing-out rotor for 5 min.

[0032] 9) Discard the supernatant, add 5 ml of PBS, and resuspend the cell pellet by pipetting. Centrifuge at 250 g in a swinging rotor for 5 min.

[0033] 10) Calculate the number of wells to be plated for each cell line and resuspend the cells in ELISPOTS-specific medium at a rate of 50 μl per well plus an additional 10 μl.

[0034] 11) Take out 10 μl of the resuspended cells, add 90 μl of PBS and mix well. Then take 10 μl of the cell suspension to count the cells (note that the value obtained here is the value after 10-fold dilution).

[0035] 12) Wash the plate four times with 200 μl of sterile PBS, allowing it to rest for 2 minutes each time. Add 200 μl / well of ELISPOTS medium to the washed plate and incubate at room temperature for 30 minutes to equilibrate the plate.

[0036] 13) Remove the culture medium from the plate and dilute the plate with ELISPOTS-specific culture medium according to the count value. Take the number of cells that can meet the requirements of most of the cells. Generally, 3.0×10 cells are plated. 5 -6.0×10 5 Add 50 μl of cell suspension to each well.

[0037] 14) Add stimulants to the reaction wells at 1 μg or 2 μg / well, adding 50 μl to each well. Also set up a PHA-positive stimulant control and a blank control without stimulant addition.

[0038] 15) Place the plate in a 37°C, 5% CO2 incubator and incubate for 24-48 hours. Do not move the plate during incubation.

[0039] 16) Discard the culture medium from the plate, add 200 μl of 4°C pre-cooled deionized water, soak for 2 min, and then discard.

[0040] 17) Wash the plate by adding 200 μl / well PBS, soaking for 2 minutes, and washing five times.

[0041] 18) Dilute the detection antibody (IFN-γ-biotin) to 1 μg / ml in PBS with 0.5% FBS, add 100 μl to each well, and incubate at room temperature for 2 h or at 37°C for 1 h.

[0042] 19) Wash the plate. 20) Dilute Streptavidin-ALP at a ratio of 1:1000 using PBS containing 0.5% FBS, add 100 μl to each well, and incubate at room temperature for 1 h.

[0043] 21) Wash the plate, following the same steps as (9).

[0044] 22) Filter the colorimetric solution through a 0.45 μm filter and add 100 μl to each well until distinct spots appear. The color development time generally takes 5-10 minutes.

[0045] 23) Stop the color development by rinsing the plate with tap water until the color no longer darkens. Allow the plate to air dry in a well-ventilated area. Store the plate in a dark place.

[0046] Specific results can be found in Figure 6-Figure 8 ,Depend on Figure 6 On the left are representative photos of the results of an ELISPOT assay using the HA and NA proteins of the A / Wisconsin / 67 / 2022 (H1N1) strain as individual or co-stimulators to detect the number of cells secreting the cytokine IFN-γ; Figure 6 On the right are the results of counting secreted IFN-γ spots; Figure 7 On the left are representative photos of the results of ELISPOT assays using HA and NA proteins of the A / Wisconsin / 67 / 2022 (H1N1) strain as single or co-stimulators to detect the number of cells secreting the cytokine IL-2; Figure 7 On the right are the results of counting secreted IFN-γ spots; Figure 8 On the left are representative photos of the results of ELISPOT assays using HA and NA proteins of the A / Wisconsin / 67 / 2022 (H1N1) strain as single or co-stimulators to detect the number of cells secreting the cytokine IL-4; Figure 8 The right side shows the results of counting secreted IL-4 spots; It can be seen that K-1 vaccine can induce a strong T cell immune response after immunization of mice.

[0047] 5. Antibody production in BALB / c mice after K-1 vaccine immunization against the reference strains and strains not included in the reference range during vaccine design: 1) Add 4 volumes of RDE (receptor destroying enzyme) to the serum and treat at 37°C for 16 hours.

[0048] 2) Inactivate at 56°C for 30 minutes.

[0049] 3) In a U-shaped disposable reaction plate, add 25ul of PBS to the first column, add 50ul of serum and RDE mixture to the first column, and dilute serially in 25ul increments.

[0050] 4) Add 4 hemagglutination units of virus solution and incubate at room temperature for 20 minutes.

[0051] 5) Add 50 μl of 1% guinea pig red blood cells and let the tube stand at room temperature for 1 hour before observing the aggregation.

[0052] 42 days after the first immunization, the whole blood of the mice was collected to separate the serum and the neutralizing antibody level was tested by hemagglutination inhibition test. According to the relevant guidelines of the "National Influenza Center Standard Operating Procedures", the inhibitory titer of the test serum against the reference antigen ≥40 was considered to have a protective effect. Figure 9After immunization with K-1 vaccine, the neutralizing antibody levels against one of the reference strains for vaccine design (A / Michigan / 45 / 2015(H1N1)) and Figure 10 After K-1 immunization, the neutralizing antibody levels produced against the strain that was not included in the reference design of the vaccine (Influenza A H1N1 (A / Victoria / 25 / 2570 / 2019)) can be seen from the two figures that after K-1 immunization, neutralizing antibodies sufficient to exert protective effects can be produced against both strains.

[0053] 6. Challenge and protection experiment of BALB / c mice after immunization After the mice completed the immunization process, a challenge experiment was performed. To verify the vaccine's broad spectrum against H1N1 types, challenge protection experiments were conducted with two strains, including 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 used was 256 HAU, and the virus liquid volume was 50ul for the challenge experiment via nasal drops.

[0054] The challenge protection experiment detected the changes in body temperature and weight of mice during the challenge period, the changes in viral load of nasopharyngeal swabs, the viral load levels of the lungs, nasal concha and trachea on the seventh day, and the pathological damage of lung tissue. The specific results are as follows Figure 11-18 As shown, the results showed that K-1 vaccine had a good protective effect against both strains.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A broad-spectrum mRNA vaccine for H1N1 targeting HA and NA proteins with key amino acid mutations, 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 the mRNA vaccine according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Based on the conserved HA sequence, the variant sites near the main antigenic site of the HA head and the receptor binding site where the amino acid properties span are retained to obtain the HA' amino acid sequence containing the key mutation sites, wherein the HA' amino acid sequence is as shown in SEQ ID NO.3; S2. Connecting the HA' amino acid sequence and the conserved NA amino acid sequence via a flexible peptide to obtain the amino acid sequence shown in SEQ ID NO. 1; S3. The obtained amino acid sequence was codon-optimized according to human codon preference to obtain a DNA sequence, such as SEQ ID NO.

2. This sequence was then constructed into the plasmid PUC57 containing a T7 promoter, 5'UTR, 3'UTR, and 100 PolyA residues by enzyme digestion and ligation, namely PUC57-T7-5UTR-HA-NA-3UTR-PolyA, completing the plasmid construction; S4. Amplify and transcribe the successfully constructed plasmid to obtain an mRNA vaccine; S5. The mRNA obtained above is prepared into lipid nanoparticles LNP by a microfluidic method.

3. The preparation method according to claim 2, wherein: In step S2, the flexible peptide is four repeated GGGS, that is, the amino acid sequence of the flexible peptide is: GGGSGGGSGGGSGGGS.

4. The preparation method according to claim 2, wherein: The nucleotide sequence of the 5'UTR in step S3 is shown as SEQ ID NO.4, and the nucleotide sequence of the 3'UTR is shown as SEQ ID NO.

5.

5. The preparation method according to claim 2, wherein: In step S4, the TSINGKE TSC-C01 Trelief® 5α Chemically Competent Cell strain was used for transformation, the plasmid was amplified, and after the plasmid was extracted, the linearized DNA template was digested with BsaI. The linearized template was precipitated with 70% volume of isopropanol at -80°C and washed with 70% ethanol.

6. The preparation method according to claim 2, wherein: In step S4, the linearized template is transcribed in vitro using a commercial kit, Novozymes. Before transcription, the raw material UTP is completely replaced with N1-methylpseudouridine triphosphate N1-Me-pUTP.

7. The preparation method according to claim 2, characterized in that The specific method for preparing lipid nanoparticles LNP in step S5 includes the following steps: S5-1. Dissolve the mRNA in 50 mM citric acid buffer at pH 4, using a ratio of 1:8 between the phosphorus content and the nitrogen content in the mRNA to obtain a final concentration of 108 ng / μL mRNA-citric acid solution. S5-2, prepare an anhydrous ethanol solution with a SM102 mass concentration of 50%, a DMG-PEG2000 mass concentration of 1.5%, a DSPC mass concentration of 10%, and a cholesterol mass concentration of 38.5% to obtain a lipid mixed solution; S5-3. The lipid mixed solution and the mRNA-citric acid solution were filtered through a 0.22 μm microporous filter membrane respectively, and mixed using a microfluidic instrument to obtain lipid nanoparticles LNP.

8. The preparation method according to claim 7, characterized in that: The flow rate ratio of the mRNA-citric acid solution to the lipid mixed solution in step S5-3 using a microfluidic instrument is 15 mL / min:5 mL / min.

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