A d-type influenza virus mRNA vaccine and a preparation method thereof

Abstract

This invention discloses an mRNA vaccine for influenza D virus and its preparation method, belonging to the field of biomedical technology. Using the receptor-binding domain of the hemagglutinin-esterase-fusion protein of the Yamagata 2019 lineage of influenza D virus as the antigen, this invention designs a codon-optimized mRNA sequence based on its encoding gene and prepares it into a lipid nanoparticle mRNA vaccine. Experimental results show that the mRNA vaccine prepared by this invention can efficiently express the target protein in 293T cells; after intramuscular immunization of BALB / c mice, it can induce the production of specific IgG antibodies and neutralizing antibodies, while simultaneously activating CD4+ and CD8+ T cell immune responses; challenge protection experiments show that the vaccine-immunized mice can effectively resist lethal challenges from influenza D virus, significantly reducing viral load in the heart, lungs, and brain tissues. The mRNA vaccine of this invention is simple to prepare, has good immunogenicity, and strong protective effect, providing a new technical solution for the prevention and control of influenza D virus and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an mRNA vaccine against influenza D virus, its preparation method, and its application in preventing influenza D virus infection. Background Technology

[0002] Influenza D virus (IDV) is a new genus, officially classified in 2016, within the family Orthomyxoviridae, and represents the genus *Deltainfluenzavirus*. IDV was first isolated in 2011 from pigs suffering from respiratory disease in Oklahoma, USA, and has since been detected in numerous countries and regions worldwide. Epidemiological surveys indicate that IDV has a wide host range, having been detected in various animals including cattle, pigs, horses, alpacas, small ruminants, wild boars, and dogs. Serological evidence also suggests that its host range continues to expand. Domestic cattle are identified as the primary natural host of IDV; in serological surveys in Italy, the seropositivity rate against the D / 660 lineage in cattle reached as high as 87%; in Poland, the IDV seropositivity rate in cattle also reached 45.2%. IDV is one of the pathogenic factors of bovine respiratory disease syndrome (BRS), and co-infection with other pathogens can exacerbate respiratory diseases, causing economic losses to the cattle industry.

[0003] IDV poses a risk of cross-species transmission and infection in humans. Studies have detected IDV-specific antibodies in the serum of farm workers who have been in contact with cattle. A 2026 study showed that IDV isolates from cattle and pigs replicated in human respiratory cells and lung tissue with capabilities comparable to influenza A virus. Based on the distribution of IDV in livestock farming and its potential zoonotic risks, the World Health Organization and public health agencies in some countries have listed it as a priority emerging respiratory pathogen for surveillance.

[0004] The continued spread and evolution of IDV (Influenza Virus) poses challenges to vaccine development. The hemagglutinin-esterase-fusion protein of IDV is the main protective antigen on the viral surface. Based on the HEF coding sequence, IDV can be divided into five genetic lineages, with antigenic variations existing between these lineages. Vaccines designed for a single lineage are unlikely to provide cross-protection against heterologous lineages. Furthermore, antigenic drift of influenza viruses and potential gene reassortment during cross-species transmission increase the likelihood of the virus evading immune recognition.

[0005] To date, there are no commercially available IDV vaccines approved globally for human or animal use. Studies have reported on β-propiolactone-inactivated IDV vaccine candidates. These inactivated vaccines can induce antibody responses and provide protection against homologous strain challenge in guinea pig models, but their protective spectrum is narrow and they struggle to address antigenic variations between lineages. Inactivated vaccines primarily induce humoral immune responses, with limited activation of cellular immunity, and their preparation involves live virus culture and inactivation, posing biosafety risks and long production cycles. Another IDV single-cycle replication vaccine candidate based on matrix protein deletion still involves live virus manipulation, carrying the risk of virulence reversion and requiring complex manufacturing processes. Other platforms, such as adenovirus vector vaccines and subunit vaccines, also face limitations such as difficulties in antigen design and selection, poor immunogenicity due to insufficient antigenic epitope exposure, and vector-pre-existing immunity weakening vaccine efficacy.

[0006] mRNA vaccines have demonstrated certain advantages in the research and application of vaccines against the novel coronavirus. mRNA vaccines have a short development cycle; mRNA can be designed and synthesized immediately after obtaining the pathogen's gene sequence. They do not involve live virus manipulation, eliminating the safety risks of virulence reversion or incomplete inactivation. They can simultaneously activate humoral and cellular immunity in vivo. They are easily updated and can be multivalently combined. Production does not rely on cell culture or chicken embryos, making the process relatively simple and facilitating large-scale production. Therefore, developing mRNA vaccines against influenza D virus can help overcome the limitations of existing vaccine platforms in terms of broad spectrum, safety, production efficiency, and immune protection. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical bottlenecks in the development of existing influenza D virus vaccines and to provide an influenza D virus mRNA vaccine with good immunization effect, broad protection spectrum, high safety and simple preparation, as well as its preparation method and application.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] 1. A type D influenza virus mRNA vaccine, characterized in that the vaccine comprises mRNA expressing a type D influenza virus hemagglutinin-esterase-fusion protein and an mRNA vaccine delivery system, wherein the amino acid sequence of the hemagglutinin-esterase-fusion protein is shown in SEQ ID NO.1.

[0010] 2. The influenza D virus mRNA vaccine as described in item 1, characterized in that the mRNA expressing the hemagglutinin-esterase-fusion protein comprises the RNA sequence shown in SEQ ID NO.2.

[0011] 3. The influenza D virus mRNA vaccine as described in item 1 or 2, characterized in that the RNA sequence of the mRNA further includes a 5'-untranslated region and a 3'-untranslated region sequence, and has a cap modification at the 5' end and a polyadenylated tail at the 3' end.

[0012] 4. The influenza D virus mRNA vaccine as described in item 3, characterized in that the RNA sequence of the 5'-untranslated region is as shown in SEQ ID NO.3, the RNA sequence of the 3'-untranslated region is as shown in SEQ ID NO.4, the RNA sequence of the signal peptide (VSV) is as shown in SEQ ID NO.5, and the RNA sequence of the polyadenylate tail is as shown in SEQ ID NO.6.

[0013] 5. The influenza D virus mRNA vaccine as described in any one of items 1-4, characterized in that the mRNA vaccine delivery system is a lipid nanoparticle.

[0014] 6. The influenza D virus mRNA vaccine as described in item 5, characterized in that the lipid nanoparticles comprise ionizable lipids, non-cationic lipids, cholesterol or polyethylene glycol modified lipids or combinations thereof.

[0015] 7. The type D influenza virus mRNA vaccine as described in item 6, characterized in that the molar ratio of each component in the lipid nanoparticles is ionizable lipid: non-cationic lipid: cholesterol: polyethylene glycol modified lipid = 50:10:38:1.

[0016] 8. The influenza D virus mRNA vaccine as described in item 7, characterized in that the ionizable lipid is SM-102, the non-cationic lipid is DSPC, and the polyethylene glycol modified lipid is DMG-PEG2000.

[0017] 9. The influenza D virus mRNA vaccine as described in any one of items 1-8, characterized in that the vaccine is a multivalent vaccine comprising mRNA molecules encoding two or more different HEF genetic lineage antigen proteins.

[0018] 10. A method for preparing an mRNA vaccine according to any one of items 1-9, characterized in that the method comprises the following steps: constructing an in vitro transcription plasmid containing a nucleotide sequence encoding the hemagglutinin-esterase-fusion protein; linearizing the plasmid to obtain a linearized template; using the linearized template to perform in vitro transcription to synthesize mRNA; purifying the mRNA; and mixing the purified mRNA with a lipid component to encapsulate it to form lipid nanoparticles.

[0019] 11. The method of claim 10, wherein the in vitro transcription is a co-transcriptional capping reaction using a Cap1 structural analog.

[0020] 12. The method as described in item 10, characterized in that the mixing adopts a microfluidic mixing method, and the volumetric flow rate ratio of the aqueous phase to the organic phase is 2:1 to 5:1.

[0021] 13. The use of the influenza D virus mRNA vaccine as described in any one of items 1-9 in the preparation of a drug for preventing influenza D virus infection.

[0022] 14. The use of the influenza D virus mRNA vaccine as described in any one of items 1-9 in the preparation of a medicament for treating diseases caused by influenza D virus infection.

[0023] This application proposes to prepare an mRNA vaccine using mRNA expressing the HEF protein of influenza D virus and an mRNA delivery system, retaining the major neutralizing epitopes of the HEF protein to achieve broad-spectrum protection against multiple genetic lineages. The research in this application demonstrates that the mRNA expressing the HEF protein can successfully express the HEF protein, as verified by in vitro cell transfection experiments. Immunization of mice with the prepared HEF mRNA vaccine produces neutralizing antibodies against influenza D virus and induces a cellular immune response. In vivo challenge experiments show that, compared with the control group, this mRNA vaccine can protect animals from both homologous and heterologous influenza D virus attacks, reduce respiratory viral load, and alleviate lung pathological damage. Therefore, the mRNA vaccine provided in this application has advantages such as good immunogenicity, broad-spectrum protection, and good safety, making it suitable for animal disease prevention and stockpiling vaccines. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. Obviously, the following drawings only show some embodiments of the present invention; those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0025] Figure 1 This is a diagram illustrating the mRNA sequence design pattern in an embodiment of the present invention.

[0026] Figure 2 This is the mRNA length identification result in the embodiments of the present invention.

[0027] Figure 3 The results for LNP particle size / PDI / encapsulation efficiency in the embodiments of the present invention are shown.

[0028] Figure 4 This is the Western blot verification result from the in vitro transcription mRNA protein expression verification experiment in this embodiment of the invention.

[0029] Figure 5This is the fluorescence microscopy verification result from the in vitro transcription mRNA protein expression verification experiment in this embodiment of the invention.

[0030] Figure 6 The results show the levels of specific antibodies in animals after immunization in this embodiment of the invention.

[0031] Figure 7 The results show the neutralizing antibody titers in this embodiment of the invention.

[0032] Figure 8 The results of cell immunity detection are shown in the embodiments of the present invention.

[0033] Figure 9 The results of the virus challenge protection test are shown in the embodiments of the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0035] Example 1: mRNA Design and Synthesis

[0036] 1.1 Antigen sequence selection and codon optimization

[0037] The receptor-binding domain (RBD) of the hemagglutinin-esterase-fusion protein (HEF) of the Yamagata 2019 lineage strain (XLQ29343.1) of influenza D virus was selected as the target antigen. The amino acid sequence of this RBD region is shown in SEQ ID NO.1.

[0038] To adapt to mammalian expression systems (target hosts being cattle or humans), the nucleotide sequence encoding the RBD was codon-optimized. The optimization principles included: using codons preferred by the target host (cattle) and avoiding rare codons; adjusting the GC content to 40%-60%; removing internal repetitive sequences, stem-loop structures, and transcription termination signals; and adding a Kozak sequence (GCCACC) upstream of the start codon.

[0039] 1.2 Plasmid Construction

[0040] The codon-optimized RBD coding sequence was tandemly linked with the following elements: 5'-Cap, 5'-UTR, VSV signal peptide, RBD coding sequence, 3'-UTR, and PolyA tail. The full-length sequence, after artificial synthesis, was ligated into the pET28a vector via double digestion with BamHI and XhoI.

[0041] The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar containing 50 μg / mL kanamycin, incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium with shaking. The recombinant plasmid was extracted using a plasmid extraction kit. Double enzyme digestion and Sanger sequencing confirmed the correct insertion sequence. The sequencing primers were a universal promoter primer and an insertion fragment-specific primer. The correctly sequenced recombinant plasmid was named pET28a-IDV-RBD.

[0042] 1.3 Plasmid linearization

[0043] The pET28a(+) empty vector was double-digested with restriction endonucleases BamHI and XhoI in a water bath at 37°C for 30 min to obtain a linearized vector. The digestion system is shown in Table 1.

[0044] Table 1. Double Enzyme Digestion Reaction System

[0045] 10× FlyCut® Buffer 5 μL FlyCut® BamHI 2 μL FlyCut® XhoI 2 μL empty carrier 10 μL ddH2O 33 μL Total Volume 50 μL

[0046] 1.4 In vitro transcription (IVT) synthesis of mRNA

[0047] mRNA was synthesized using a co-transcriptional capping method, employing RNA polymerase and a Cap1 analog (CleanCap Reagent AG). In a nuclease-free microcentrifuge tube, the following components were added sequentially: linearized template (final concentration 10 μg / 100 μL), 10× transcription buffer, ATP, GTP, CTP, UTP (final concentration 4 mM each), Cap1 analog (final concentration 4 mM), RNA polymerase (final concentration 5 U / μL), pyrophosphatase (final concentration 0.01 U / μL), and RNase inhibitor (final concentration 0.4 U / μL). The volume was then brought to the required level with nuclease-free water. After gentle refluxing and brief centrifugation, the reaction tube was incubated at 37°C in a metal bath for 3 hours. After the reaction, 1 μL of the product was subjected to 1% denaturing agarose gel electrophoresis to preliminarily assess mRNA synthesis.

[0048] 1.5 Template DNA Removal

[0049] Add 2 μL of DNase I to the IVT reaction product, mix well, and incubate at 37°C for 15 minutes to degrade the linearized DNA template.

[0050] 1.6 mRNA purification

[0051] Preliminary purification was performed using lithium chloride (LiCl) precipitation: 1 / 2 volume (approximately 50 μL) of 7.5 M LiCl solution (Sigma, L7026) was added to the reaction system, mixed, and precipitated at -20℃ for 30 minutes; centrifuged at 4℃ and 16000×g for 20 minutes, and the supernatant was discarded; the precipitate was washed once with pre-cooled 70% ethanol, centrifuged at 4℃ and 16000×g for 5 minutes, and the supernatant was discarded; the precipitate was air-dried at room temperature and resuspended in 50 μL of nuclease-free water. To further remove residual short RNA fragments and unincorporated nucleotides, purification was performed using an oligo dT affinity chromatography column (Thermo Fisher, K158001): The LiCl-precipitated mRNA sample was mixed with an equal volume of 2× binding buffer and loaded onto an equilibrated oligo dT column; the column was washed three times with washing buffer; the mRNA was eluted with elution buffer (nuclease-free water), and the eluent was collected.

[0052] 1.7 Sequencing Validation

[0053] The obtained cDNA samples were sent to Jilin Kumei Biotechnology Co., Ltd., where bidirectional sequencing was performed using promoter sequence primers and specific internal primers. The sequencing results were compared with the expected sequence (SEQ ID NO.2) to confirm that the mRNA sequence was correct and without mutations or deletions.

[0054] Example 2: Validation of mRNA expression in vitro

[0055] 2.1 Cell Culture and Transfection

[0056] 293T cells (ATCC, CRL-3216) were seeded into 6-well cell culture plates, with 2 mL of DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator. Transfection was performed when the cells reached 70%-80% confluence.

[0057] One hour before transfection, replace the medium with serum-free Opti-MEM (Gibco, 31985-062), 1.5 mL per well. Take the IDV-RBD mRNA prepared in Example 1 and transfect according to the following steps:

[0058] (1) Prepare transfection complex A solution: Take 2 μg mRNA, add 125 μL Opti-MEM medium, and mix gently.

[0059] (2) Prepare transfection complex B solution: Take 4 μL of Lipofectamine 2000 (Thermo Fisher, 11668019), add 125 μL of Opti-MEM medium, mix gently, and let stand at room temperature for 5 minutes.

[0060] (3) Mix solution A and solution B, gently blow to mix, and incubate at room temperature for 15 minutes to form mRNA-liposome complex.

[0061] (4) Add the complex dropwise to the cells in the 6-well plate and gently shake to mix.

[0062] (5) Set up a negative control group: transfected with an equal volume of nuclease-free water containing Lipofectamine 2000 complex; set up a blank control group: added only an equal volume of Opti-MEM medium.

[0063] (6) Place the culture plate in a 37℃, 5% CO2 incubator for 24-48 hours.

[0064] 2.2 Western blot detection of target protein expression

[0065] 2.2.1 Cell lysis and protein extraction

[0066] Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed twice with pre-chilled PBS buffer. 150 μL of LRIPA lysis buffer (containing a 1× protease inhibitor mixture) was added to each well, and the cells were incubated on ice for 30 minutes, gently shaking every 10 minutes. The lysates were collected using a cell scraper into 1.5 mL centrifuge tubes, centrifuged at 13,000 rpm for 15 minutes at 4°C, and the supernatant was used as the total protein extract for protein concentration determination.

[0067] 2.2.2 SDS-PAGE and Transfer

[0068] Sample preparation: Take 40 μL of the sample to be tested and mix it with 5× protein loading buffer at a volume ratio of 4:1. Boil in a water bath for 10 minutes to fully denature the protein. Then centrifuge at 8000×g for 5 minutes and collect the supernatant for later use.

[0069] Gel preparation: A 10% SDS-PAGE separating gel was prepared using the Yaxin protein gel preparation kit and allowed to stand for 30 minutes for polymerization.

[0070] Sample loading and electrophoresis: Accurately load 10 μL of the treated sample into each well. Initially set the electrophoresis voltage to 80V constant voltage. After the bromophenol blue indicator migrates to the separating gel interface, adjust the voltage to 120V constant voltage and continue electrophoresis until the indicator reaches the bottom of the colloid.

[0071] Transfer: After preparing the gel and performing electrophoresis according to the standard SDS-PAGE procedure, accurately cut out the gel region containing the target protein and protein marker; immerse the gel block, filter paper, and PVDF membrane activated with methanol for 30 seconds in the transfer buffer and equilibrate for 10 minutes; stack the transfer layers in the order of "filter paper-PVDF membrane-gel-filter paper", thoroughly remove air bubbles, and transfer at a constant voltage of 20V for 30 minutes.

[0072] 2.2.3 Antibody incubation and color development

[0073] Blocking and antibody incubation: After the transfer is completed, the PVDF membrane is immersed in 5% skim milk powder (dissolved in PBST) and blocked at room temperature for 1 hour; the membrane is then transferred to a diluted primary antibody solution (1:2000 diluted in 5% skim milk powder) and incubated at 4°C with shaking for 12-16 hours; the membrane is then washed with PBST at room temperature with shaking (5 minutes × 3 times) to remove unbound antibodies.

[0074] Secondary antibody reaction and development: Immerse the membrane in HRP-labeled secondary antibody solution (1:5000 diluted in 5% skim milk powder) and incubate at room temperature with shaking for 40 minutes; after washing three times with PBST, mix ECL chemiluminescence solution A / B at a 1:1 ratio and evenly cover the membrane surface; expose the membrane in a chemiluminescence imaging device in a dark room for 30 seconds to 5 minutes to capture specific signals.

[0075] 2.3 Immunofluorescence detection of target protein expression

[0076] 293T cells were seeded into 12-well plates pre-placed with sterile coverslips, 2 × 10⁶ cells per well. 5 Cells were cultured overnight. Transfection was performed as described in 2.1, with 1 μg mRNA transfected into each well. Immunofluorescence staining was performed 48 hours after transfection.

[0077] 2.3.1 Cell fixation and permeabilization

[0078] Discard the culture medium and wash the cells twice with PBS. Add 500 μL of 4% paraformaldehyde (PFA) solution to each well and fix for 20 minutes at room temperature. Discard the fixative and wash three times with PBS for 5 minutes each time. Add 500 μL of 0.2% Triton X-100 (diluted with PBS) to each well and permeabilize for 10 minutes at room temperature. Wash three times with PBS for 5 minutes each time.

[0079] 2.3.2 Blocking and Antibody Incubation

[0080] Add 500 μL of 5% BSA blocking buffer (diluted with PBS) to each well and block at room temperature for 1 hour. Discard the blocking buffer and add 250 μL of primary antibody (anti-HEF protein polyclonal antibody, 1:200 diluted in blocking buffer) to each well, and incubate overnight at 4°C. Wash three times with PBS for 5 minutes each time. Add fluorescent secondary antibody (Alexa Fluor 488-labeled goat anti-rabbit IgG antibody, Thermo Fisher, A-11008, 1:500 diluted) to each well and incubate at room temperature in the dark for 1 hour. Wash three times with PBS for 5 minutes each time. Add 100 μL of DAPI staining solution (1 μg / mL, diluted with PBS) to each well and incubate at room temperature in the dark for 5 minutes. Wash three times with PBS for 5 minutes each time.

[0081] 2.3.3 Mounting and Observation

[0082] Remove the coverslip with tweezers and mount it on a slide containing an anti-fluorescence quenching mount. Observe and photograph using a fluorescence microscope (e.g., Olympus IX73). Set the excitation wavelengths: DAPI 358 nm, Alexa Fluor 488 495 nm. Acquire images of the DAPI (nucleus, blue) and Alexa Fluor 488 (target protein, green) channels separately and observe them together. Positive cells should show a green fluorescent signal (mainly located in the cytoplasm), while the negative control and blank control groups should show no specific green fluorescence.

[0083] Example 3: LNP Encapsulation and Vaccine Preparation

[0084] 3.1 Lipid composition and molar ratio

[0085] The lipid nanoparticles used in this embodiment are composed of four lipid components:

[0086] Ionizable lipids: SM-102; Non-cationic lipids: DSPC; Cholesterol; Polyethylene glycol-modified lipids: DMG-PEG2000.

[0087] The molar ratio of the four lipids is: SM-102 : DSPC : Cholesterol : DMG-PEG2000 = 50 : 10 : 38 : 1.

[0088] 3.2 Preparation of Aqueous and Organic Phases

[0089] Aqueous phase preparation: The IDV-RBD mRNA purified in Example 1 was dissolved in 10 mM citrate buffer (pH 4.0) to adjust the final mRNA concentration to 0.2 mg / mL. Before use, it was filtered through a 0.22 μm filter membrane for sterilization and stored on ice.

[0090] Organic phase preparation: Weigh SM-102, DSPC, cholesterol, and DMG-PEG2000 according to the above molar ratio, and dissolve them in anhydrous ethanol to make the total lipid concentration 10 mmol / L. Place the mixture on a magnetic stirrer and stir until completely dissolved. Filter through a 0.22 μm filter membrane for sterilization and store at room temperature for later use.

[0091] 3.3 Microfluidic Hybrid Encapsulation

[0092] mRNA-LNP encapsulation was performed using microfluidic equipment.

[0093] Equipment preparation: Clean the microfluidic chip and tubing system with anhydrous ethanol and 10 mM citrate buffer (pH 4.0), respectively.

[0094] Parameter settings: The volumetric flow rate ratio of the aqueous phase to the organic phase is set to 3:1, and the total flow rate is 12 mL / min. The aqueous phase flow rate is 9 mL / min, and the organic phase flow rate is 3 mL / min.

[0095] Mixed collection: Connect the aqueous phase syringe (containing mRNA citrate solution) and the organic phase syringe (containing lipid ethanol solution) to the two injection channels of the microfluidic device, respectively. Start the device, discard the first 0.2 mL of the mixture as waste, and then collect the mixture in a sterile centrifuge tube.

[0096] During the mixing process, lipids rapidly precipitate at the interface between ethanol and aqueous phase, spontaneously assembling into lipid nanoparticles that encapsulate mRNA.

[0097] 3.4 Ultrafiltration Concentration and Buffer Replacement

[0098] The collected LNP mixture was diluted 10-fold with nuclease-free PBS buffer (pH 7.4) to reduce the ethanol concentration and adjust the pH. The diluted solution was transferred to a 100 kDa molecular weight cutoff ultrafiltration centrifuge tube (Millipore, UFC910024) and centrifuged at 3000 g for 30 minutes at 4°C to concentrate to the original volume. Ten-fold volumes of PBS buffer (containing 5% sucrose) were added to the concentrate, and the concentration was repeated twice to fully displace residual ethanol and citrate buffer. Finally, the mRNA-LNP was concentrated to the target mRNA concentration (0.1–0.5 mg / mL), filtered through a 0.22 μm filter for sterilization, aliquoted, and stored at 2–8°C or -80°C for later use.

[0099] 3.5 Particle size and polydispersity index (PDI) detection

[0100] The average particle size and PDI of LNPs were determined using a dynamic light scattering particle size analyzer (Malvern Zetasizer Nano ZS90).

[0101] Sample preparation: Take 20 μL of vaccine stock solution and dilute it to 200 μL with PBS buffer (pH 7.4), and mix gently.

[0102] Detection conditions: Temperature set at 25℃, equilibration time at 60 seconds, detection angle at 173° (backscattering mode), refractive index at 1.47, and absorption coefficient at 0.001. Each sample was tested three times.

[0103] Results Record: Record the average particle size (Z-average) and polydispersity index (PDI). Expected particle size range: 80-120 nm, PDI < 0.2.

[0104] 3.6 Encapsulation efficiency test

[0105] The encapsulation efficiency of mRNA was determined using the RiboGreen fluorescent dye method.

[0106] Standard curve plotting: Prepare a series of mRNA standards (0, 50, 100, 200, 500, 1000 ng / mL) using TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Add 100 μL of each concentration of standard to a black 96-well plate, add 100 μL of RiboGreen working solution (diluted 1:200 with TE buffer), and incubate at room temperature in the dark for 5 minutes. Measure the fluorescence intensity using a fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Plot a standard curve with mRNA concentration on the x-axis and fluorescence intensity on the y-axis.

[0107] Total mRNA concentration (C_total) determination: Take 10 μL of vaccine stock solution, add 190 μL of TE buffer, vortex to mix, and incubate at room temperature for 10 minutes to destroy LNP particles. Take 100 μL of diluted lysis buffer, add 100 μL of RiboGreen working solution, incubate in the dark for 5 minutes, and then detect the fluorescence intensity. Calculate the total mRNA concentration according to the standard curve.

[0108] Cell-free mRNA concentration (C_free) determination: Take another 100 μL of vaccine stock solution, centrifuge at 15000 rpm for 30 minutes at 4℃, and collect the supernatant. Take 100 μL of supernatant, add 100 μL of RiboGreen working solution, incubate in the dark for 5 minutes, and then detect the fluorescence intensity. Calculate the cell-free mRNA concentration according to the standard curve.

[0109] Encapsulation rate calculation: Encapsulation rate (%) = (C_total - C_free) / C_total × 100%.

[0110] 3.7 Zeta potential detection

[0111] The surface zeta potential of LNPs was detected using a Malvern Zetasizer Nano ZS90.

[0112] Sample preparation: Take 50 μL of vaccine stock solution and dilute it to 500 μL with 0.1 mM KCl solution (adjust the conductivity to a suitable range), and mix gently.

[0113] Detection conditions: The sample was injected into the zeta potential sample cell, the temperature was set to 25℃, and the equilibration time was 60 seconds. Each sample was tested three times.

[0114] Results Recording: Record the average zeta potential value. The expected zeta potential range is -5 mV to +10 mV.

[0115] Example 4: Evaluation of immunogenicity in animals

[0116] 4.1 Animal Model and Grouping

[0117] Six- to eight-week-old SPF-grade female BALB / c mice, weighing 18-22 g, were selected. After one week of acclimatization, they were randomly divided into four groups of six mice each.

[0118] Group 1: Intramuscular injection of IDV-RBD mRNA-LNP vaccine, 10 μg per animal.

[0119] Group 2: Intramuscular injection of IDV-RBD mRNA-LNP vaccine, 5 μg per animal.

[0120] Group 3 immunization: Intramuscular injection of IDV-RBD mRNA-LNP vaccine, 2.5 μg per animal.

[0121] Blank control group: Intramuscular injection of an equal volume of PBS

[0122] 4.2 Immunization Program

[0123] Two immunizations were administered, 14 days apart.

[0124] Primary immunization (day 0): The vaccine or corresponding control solution was injected into the lateral thigh muscle of the left hind leg of the mouse.

[0125] Boost immunization (day 14): Inject the same dose of vaccine or control solution into the lateral thigh muscle of the right hind leg of the mouse.

[0126] 4.3 Serum Collection

[0127] Blood was collected from the orbital venous plexus, with approximately 200 μL of whole blood collected from each mouse per collection. The blood was placed in a 1.5 mL sterile centrifuge tube and incubated at room temperature for 1 hour. The blood was then centrifuged at 3000 rpm for 10 minutes at 4°C. The serum was then separated and stored at -80°C for later use.

[0128] 4.4 Antibody detection (ELISA)

[0129] 4.4.1 Detection of serum-specific IgG antibodies

[0130] The level of specific IgG antibody against IDV HEF-RBD protein in mouse serum was detected by indirect ELISA.

[0131] Coating antigen: Dilute the recombinant IDV HEF-RBD protein to a final concentration of 2 μg / mL with coating buffer, add 100 μL to each well of a 96-well microplate, and coat overnight at 4°C.

[0132] Washing and blocking: Discard the coating solution and wash three times with PBST for 3 minutes each time. Add 200 μL of blocking buffer (PBS containing 5% skim milk powder) to each well and block at 37°C for 2 hours. Discard the blocking buffer and wash three times with PBST.

[0133] Sample addition: Dilute the serum sample to be tested serially from 1:100 to 2- or 3-fold using blocking buffer, adding 100 μL to each well, and incubate at 37°C for 1.5 hours. Set up a negative control (negative control serum) and a blank control (no serum added). Wash 5 times with PBST.

[0134] Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (Proteintech, SA00001-1, diluted 1:5000 with blocking buffer) to each well and incubate at 37°C for 1 hour. Wash 5 times with PBST.

[0135] Development and termination: Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 15 minutes. Terminate the reaction by adding 50 μL of 2 M H2SO4 to each well.

[0136] Reading: OD values ​​were read at 450 nm using a microplate reader. Antibody titer was defined as the highest dilution with an OD450 value greater than the mean of the negative control group plus three standard deviations.

[0137] 4.5 Cellular Immunoassay

[0138] Fourteen days after booster immunization (day 28), three mice in each group were sacrificed, and their spleens were aseptically harvested to prepare spleen lymphocyte suspensions for flow cytometry detection of T cell subsets.

[0139] 4.5.1 Splenic lymphocyte isolation

[0140] Mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol, and their spleens were aseptically removed and placed in a sterile culture dish containing 2 mL of RPMI-1640 medium. The spleen was gently ground with a syringe plunger and filtered through a 70 μm cell sieve. The cell suspension was collected into a 15 mL centrifuge tube. The tube was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. 3 mL of erythrocyte lysis buffer (ACK lysis buffer) was added, and the cells were incubated at room temperature for 2-3 minutes to lyse the erythrocytes. 10 mL of RPMI-1640 medium was added to terminate the lysis, and the tube was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 10 mL of RPMI-1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin), counted, and the cell density was adjusted to 2 × 10⁶ cells / mL. 6 per mL.

[0141] 4.5.2 Flow cytometry detection

[0142] Cell surface staining: Take 1×10 6 One spleen lymphocyte was placed in a flow cytometry tube, 1 mL of PBS was added, and the cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 100 μL of PBS (containing 1% BSA), and 0.5 μg each of fluorescently labeled anti-mouse CD3e-FITC (BD Biosciences, 553062), CD4-PE (BD Biosciences, 553730), and CD8a-APC (BD Biosciences, 553035) antibodies were added. The cells were incubated at room temperature in the dark for 30 minutes. The cells were washed once with 2 mL of PBS, centrifuged, the supernatant was discarded, and the cells were resuspended in 300 μL of PBS.

[0143] Analytical assay: Flow cytometry (e.g., BD FACSCanto II) was used. First, the lymphocyte population was gated (FSC-SSC scatter plot), then the ratio of CD4+ to CD8+ T cells in CD3+ T cells was analyzed. At least 10,000 lymphocyte events were collected from each sample.

[0144] Data analysis: FlowJo software was used for analysis. The percentages of CD4+ T cells and CD8+ T cells relative to CD3+ T cells in the spleen of mice in each group were calculated. Differences between the vaccine group and the control group were statistically analyzed using t-tests.

[0145] Example 5: Evaluation of the protective effect against viral infection

[0146] 5.1 Animal Model and Grouping

[0147] Six- to eight-week-old SPF-grade female BALB / c mice, weighing 18-22 g, were selected. After one week of acclimatization, they were randomly divided into two groups of 10 mice each.

[0148] Vaccine challenge group: IDV-RBD mRNA-LNP vaccine was administered intramuscularly at a dose of 5 μg / animal on day 0 and day 14;

[0149] Blank control group: The same volume of PBS was injected intramuscularly on day 0 and day 14.

[0150] 5.2 Dosage and route of infection

[0151] 5.2.1 Virus challenge: A representative strain of the Yamagata 2019 lineage of influenza D virus was used. After amplification in Vero cells, the viral titer was determined and the virus was aliquoted and stored at -80°C.

[0152] 5.2.2 Challenge dose: The median lethal dose (LD50) of this strain for BALB / c mice was determined in advance through preliminary experiments. In this example, 10 times the LD50 was used as the challenge dose, that is, the challenge dose per mouse was 10 × LD50 PFU.

[0153] 5.2.3 Infection route: Infection was performed via nasal instillation. Mice were slowly instilled with 50 μL of viral challenge solution into their nasal cavity using a micropipette, maintaining an upright position to allow the viral solution to be naturally inhaled. An equal volume of PBS was instilled into the blank control group using the same method.

[0154] 5.3 Observation and Sampling

[0155] 5.3.1 Clinical observation: After challenge, mice were observed daily for 14 consecutive days for their mental state, appetite, weight changes, respiratory status, and mortality. The morbidity and mortality rates of each group were recorded.

[0156] 5.3.2 Tissue Sampling: On day 5 post-infection (peak viral load), 5 mice from each group were sacrificed and dissected under biosafety conditions. The following tissues were aseptically collected: heart, trachea, lungs, nasal turbinates, intestines (small intestine segment), and brain.

[0157] 5.4 Tissue viral load detection (qRT-PCR method)

[0158] The viral load of IDV in each tissue was detected by real-time quantitative PCR (qRT-PCR).

[0159] Tissue homogenate preparation: Remove frozen tissue from -80°C and thaw on ice. Weigh approximately 50-100 mg of tissue and place it in a 2 mL sterile centrifuge tube, adding a steel bead. Add 1 mL of sterile PBS (pH 7.4) to each tube and prepare a tissue homogenate using a tissue homogenizer (60 Hz, 3 minutes). Centrifuge the homogenate at 4°C and 5000 rpm for 10 minutes, collect the supernatant, aliquot it, and use it for RNA extraction.

[0160] Total RNA extraction: Take 200 μL of tissue homogenate supernatant and follow the instructions of the RNA extraction kit.

[0161] Virus detection: One-step RT-qPCR was performed using IDV HEF gene-specific primers and probes. Viral load per gram of tissue was calculated using a standard curve. Results are as follows: Figure 9 As shown.

[0162] sequence list

[0163] SEQ ID NO.1:

[0164] MFLLLATITAITACQAERELICIVQRVNESFSLHSGFGGNVYSMKTEPMTGFTNVTKGASVINQKDWIGFGDSRTDLNNDQFPASSDVPLAVAKKFRSLSGASLMLSAFGPPGKVDYLYQ GCGKEKVFYEGVNWSPEAGIDCFGSNWTQTKKDFYSKIYEAARSSTCMTLVNSLDTKISSTTATAGTASSCSSSWMKSPLWYAESSVNPRTKPQVCGTEQSATFTLPTSFGIYKCNKHVVQ LCYFVYENKTAFNTFGCGDYYQNYYDNGGNLIGGIDNRVAAYRGIANVGVKIECPSKILNPGTYSIRSTPRFLLVPKRSYCFDTDGGYPIQVVQSEWSASRRSDNATEEASLQTEGCIFIK KTTPYVGEADDNHGDIEMRQLLSGLGNNDTVCVSQSGYTKGETPFVRDYLSPPKYGRCQLKTDSGRIPTLPSGLIIPQAGTDSLMRTLTPATRIFGIDDLIFGLLFVGFVAGGVAGGYFWG

[0165] SEQ ID NO.2:

[0166]

[0167] SEQ ID NO.3:

[0168] ACACTTGCTTCTGACACAACCGTGTTCACTAGCAACTACACAAACAGACACC

[0169] SEQ ID NO.4:

[0170] GCTCCCTTTCCTGCTTTCCAGGAAAGGTTTTTTCATCCTCAGAGCCCAAAGATTGAATATGGAAAAATTATGAAGTGTTTTGAGCATCTGGCCTCTGCCTAATAAAGACATTTATTTTCATTGCTCACTGACTGGCTCCCTTTCCTGCTTTCCAGGAAAGGTTTTTTCATCCTCAGAGCCCAAAGATTGAATATGGAAAAATTATGAAGTGTTTTGAGCATCTGGCCTCTGCCTAATAAAGACATTTATTTTCATTGC

[0171] SEQ ID NO.5:

[0172] ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGC

[0173] SEQ ID NO.6:

[0174] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

Claims

1. An mRNA vaccine against influenza D virus, characterized in that, Including mRNA and mRNA vaccine delivery systems that express the receptor-binding domain of influenza D virus surface glycoprotein.

2. The mRNA vaccine for influenza D virus according to claim 1, characterized in that, The amino acid sequence of the receptor-binding domain of the surface glycoprotein of the Yamagata 2019 lineage of influenza D virus is shown in SEQ ID NO.

1.

3. An mRNA vaccine for influenza D virus according to claim 1 or 2, characterized in that, The mRNA expressing the surface glycoprotein of the influenza D virus contains the RNA sequence shown in SEQ ID NO.

2.

4. An mRNA vaccine against influenza D virus according to any one of claims 1-3, characterized in that, The mRNA also includes one or more of the following elements: The RNA sequence of the 5'-untranslated region is shown in SEQ ID NO.3; The RNA sequence of the 3'-untranslated region is shown in SEQ ID NO.4; The RNA sequence of the signal peptide is shown in SEQ ID NO.5; The RNA sequence of the polyadenylated tail is shown in SEQ ID NO.

6.

5. An mRNA vaccine for influenza D virus according to any one of claims 1-4, characterized in that, The mRNA vaccine delivery system is composed of lipid nanoparticles.

6. The mRNA vaccine for influenza D virus according to claim 5, characterized in that, The lipid nanoparticles comprise ionizable lipids, non-cationic lipids, cholesterol, and polyethylene glycol-modified lipids; preferably, the ionizable lipid is SM-102, the non-cationic lipid is DSPC, and the polyethylene glycol-modified lipid is DMG-PEG2000; preferably, the ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 is 50:10:38:

1.

7. An mRNA vaccine against influenza D virus according to any one of claims 1-6, characterized in that, The vaccine contains an mRNA molecule that expresses an antigen protein derived from the Yamagata 2019 lineage IDV.

8. A method for preparing an mRNA vaccine of influenza D virus as described in any one of claims 1-7, characterized in that, Includes the following steps: Construct in vitro transcription plasmids containing antigen-coding sequences; Linearize the plasmid and use it as a transcription template; mRNA synthesis via in vitro transcription: The IVT reaction using linearized plasmid as template yielded stable results, with approximately 1.86 ± 0.12 mg of mRNA obtained per 100 μg template, meeting the expected standard; Purify mRNA; mRNA and lipid components were mixed using a microfluidic method and encapsulated to form lipid nanoparticles.

9. The use of the mRNA vaccine of influenza D virus according to any one of claims 1-7 in the preparation of the following medicine: a vaccine for preventing influenza D virus infection.

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