Polypeptide-lipid compositions for delivery of nucleic acids, uses thereof and mRNA vaccines

Abstract

The application belongs to the field of polypeptides and vaccines, and particularly relates to a polypeptide with an antigen-presenting cell targeting ligand, a polypeptide-lipid composition for delivering nucleic acid and an mRNA vaccine. The application first provides a polypeptide with an antigen-presenting cell targeting ligand, and a nanoparticle prepared after the polypeptide is loaded with a delivery system composed of a polymer DMG-PEG2000 and mRNA, the nanoparticle has good mRNA delivery capacity, low cytotoxicity, high expression of a target gene, high levels of antigen-specific mucosal immunity and humoral immune response, and provides a reference for developing a novel respiratory mucosal mRNA vaccine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of vaccines, and particularly relates to a polypeptide with antigen-presenting cell targeting ligand, a polypeptide-lipid composition for delivering nucleic acid and an mRNA vaccine. BACKGROUND

[0002] The biopharmaceutical field has also begun a new race to develop a COVID-19 vaccine. Among them, the mRNA vaccines (mRNA-1273 and BNT162b2) of Moderna and Pfizer / BioNTech companies stand out among many candidates due to their strong immune protection, high safety, low cost, simple preparation process, easy scale-up production, short development improvement cycle and strong plasticity.

[0003] The great success of the COVID-19 mRNA vaccine has shown the potential of nucleic acid vaccines for the first time. This milestone technological breakthrough not only benefits from the efficient target protein translation ability of the mRNA platform, but also cannot be separated from the contribution of the mRNA delivery carrier-lipid nanoparticle (LNP). By wrapping the ionizable lipid, cholesterol, PEGylated lipid, and auxiliary lipid 4 components into the target mRNA to form LNP, mRNA can be protected from degradation in complex environments in vitro and in vivo. When the mRNA-LNP is taken up into the endosome by cells, the head group of the ionizable lipid protonates as the pH value in the endosome environment decreases, forms a cationic state and destroys the lysosome membrane, and the "lysosome escape" phenomenon occurs. At the same time, the mRNA is released into the cytoplasm to be translated into the target antigen by ribosomes, and the specific immune response is activated. However, the currently approved COVID-19 vaccines mainly use injection inoculation, which cannot bring specific immune protection to the respiratory mucosa, the area invaded by the virus, and reduce the infectivity of the virus.

[0004] Mucosal-associate lymphoid tissue (MALT) plays an important role in the process of mucosal immune response, after antigen presentation cells display antigen and induce lymphocytes in lymph nodes. B lymphocytes undergo antibody class switching to ultimately produce antigen-specific sIgA, which plays a neutralizing role to reduce the infectivity of pathogens in mucus; T lymphocytes differentiate into antigen-specific effector or memory cell subgroups, clear infected cells and activate acquired immune response. They jointly build a mucosal immune barrier to prevent respiratory pathogens from invading. However, specific serum antibodies are currently used as the evaluation index of SARS-CoV-2 vaccine, but antigen-specific IgG and IgA in serum can only deal with breakthrough infection of viruses invading the circulatory system. Exocrine IgA has not received enough attention because of the limitation of respiratory mucus sampling of subjects. However, studies have found that antigen-specific sIgA was not detected in the nasal swabs and saliva of subjects injected with the mRNA vaccine. However, high levels of antigen-specific sIgA were found in samples of mild and asymptomatic natural infection, and dimerized sIgA had high levels of virus neutralization ability. This shows that effective mucosal immunity not only reduces COVID-19 symptoms, but also reduces the risk of virus transmission through aerosols. Therefore, developing respiratory mucosal vaccines is of great significance to deal with the threat of such highly infectious respiratory pathogens.

[0005] A nucleic acid-polypeptide-poloxamer self-assembled nanoparticle for treating pulmonary cystic fibrosis has been reported, which contains a synthetic polypeptide with three functional motifs: a cationic peptide segment for adsorbing nucleic acids; a hydrophobic peptide segment for anchoring hydrophobic groups; and a targeting peptide segment to improve lung delivery capacity. In addition, poloxamer T704 ( 704), as a derivative of poloxamer with good biocompatibility, its amphiphilicity enables it to bind to the hydrophobic peptide segment of the synthetic polypeptide and increase the solubility of the mixture, thereby improving the stability of the solution.

[0006] Based on this, the application provides a polypeptide with an antigen-presenting cell targeting ligand (sequence: Ac-FAEKFKE AVKDYFAKFWDARLARALARALARHLARALARALRACYTYQGKLC), and an mRNA-lipid-polypeptide ternary complex (mRNA-LPTC) is prepared by mixing the polypeptide with dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) and mRNA. The mRNA-LPTC nanoparticle has good mRNA delivery capacity and low cytotoxicity; can produce high levels of antigen-specific mucosal immune and humoral immune responses; and brings strong respiratory tract antigen-specific cellular immunity, thereby providing a reference for developing a novel respiratory mucosal mRNA vaccine. SUMMARY

[0007] In order to solve the above technical problems, the application aims to provide a polypeptide with an antigen-presenting cell targeting ligand, a polypeptide-lipid composition for delivering nucleic acids and an mRNA vaccine, and specifically includes the following contents:

[0008] In a first aspect, the application provides a polypeptide with an antigen-presenting cell targeting ligand, wherein the polypeptide is a polypeptide represented by the amino acid sequence SEQ ID NO. 1, or a polypeptide derivative of the polypeptide represented by SEQ ID NO. 1 obtained by deletion, insertion or substitution and having the same biological function as the polypeptide represented by SEQ ID NO. 1.

[0009] In a second aspect, the application provides use of the polypeptide of the first aspect in preparation of a nucleic acid vaccine delivery carrier.

[0010] Preferably, the nucleic acid vaccine delivery carrier is a DNA vaccine delivery carrier or an RNA vaccine delivery carrier.

[0011] In a third aspect, the application provides a nucleic acid vaccine delivery carrier, which comprises the polypeptide of the first aspect and a polymer.

[0012] Preferably, the polymer is DMG-PEG2000.

[0013] Preferably, the nucleic acid vaccine delivery carrier is prepared by mixing the polypeptide solution of the first aspect and a DMG-PEG2000 solution.

[0014] In a fourth aspect, the application provides use of the nucleic acid vaccine delivery carrier of the third aspect in preparation of a nucleic acid vaccine.

[0015] In a fifth aspect, the application provides an mRNA vaccine, which comprises the nucleic acid vaccine delivery carrier of the third aspect and mRNA encoding a specific antigen.

[0016] Preferably, the mRNA vaccine is prepared by mixing the nucleic acid vaccine delivery carrier solution of any one of claims 4-6 and the mRNA solution encoding the antigen.

[0017] Preferably, the preparation method of the mRNA vaccine is characterized in that the method is:

[0018] (1) preparing mRNA encoding the antigen;

[0019] (2) placing the DMG-PEG2000 solution and the polypeptide solution of the first aspect above into two syringes respectively, mixing using a microfluidic device to obtain a nucleic acid vaccine delivery carrier;

[0020] (3) placing the nucleic acid vaccine delivery carrier solution and the mRNA solution into two syringes respectively, mixing using a microfluidic device to obtain an mRNA vaccine.

[0021] Preferably, the mRNA vaccine of the novel coronavirus SARS-CoV-2 is prepared by using mRNA encoding the RBD protein of the novel coronavirus SARS-CoV-2 as the antigen.

[0022] The beneficial effects of the present application are: firstly, the present application provides a polypeptide, and the delivery system formed by the polypeptide and the polymer can bind to nucleic acid expressing viral antigens and be delivered in vivo, with high delivery efficiency and good safety, and can realize efficient expression of viral antigen nucleic acid in the lungs, and comprehensively and evenly induce humoral and mucosal immune responses; secondly, the present application provides an mRNA vaccine delivery carrier, which can realize the delivery and expression of the mRNA vaccine and can be used for preparing the mRNA vaccine; finally, the present application prepares the mRNA vaccine of the novel coronavirus SARS-CoV-2 by using mRNA encoding the RBD protein of the novel coronavirus as the antigen, realizes the in vivo delivery of the mRNA vaccine of the novel coronavirus, and can induce high levels of antigen-specific serum IgG and bronchoalveolar lavage fluid sIgA, indicating that it can not only induce humoral acquired immune responses, but also improve the level of respiratory mucosal immunity; lays a foundation for further development of inhaled mRNA vaccines, optimization of biocompatibility and preparation process, etc.; and provides a reference for further research on mRNA delivery mechanisms, nanoparticle structures and assembly principles, respiratory mucosal immune response pathways, etc. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings for illustration:

[0024] Figure 1Nucleic acid gel electrophoresis figure, wherein (A) is a plasmid and a transcription template: lane 1 is DNA Ladder; 2, 4, 6 are RBD, EGFP, F-luc plasmids in turn; 3, 5, 7 are RBD, EGFP, F-luc plasmid double enzyme digestion products; (B) is in vitro transcription and capping product: lane 1 is RNA ladder; 2, 4, 6 are RBD, EGFP, F-luc mRNA after transcription; 3, 5, 7 are RBD, EGFP, F-luc mRNA after 5' end capping;

[0025] Figure 2 Nanoparticle physicochemical property investigation, wherein (A) is the hydrodynamic particle size; (B) is the solution polydispersity coefficient; (C) is the particle Zeta potential; (D) is the mRNA-LPTC morphology under transmission electron microscope, scale = 200 nm;

[0026] Figure 3 F-luc reporter gene in vitro transfection ability and preparation cytotoxicity, wherein (A, B, C) are chemiluminescence intensities of different preparations after transfecting 16HBE, DC2.4, RAW264.7 cells; (D, E, F) are effects of different preparations on 16HBE, DC2.4, RAW264.7 cell viability, ns P>0.05, **P<0.01, ****P<0.0001, n = 5;

[0027] Figure 4 EGFP mRNA-LPTC expression in 16HBE cells; wherein blue is DAPI; green is green fluorescent protein; Merge is two channels combined;

[0028] Figure 5 Preparation in vivo delivery ability, wherein (A) is F-luc reporter gene expression in mouse nose; (B) is F-luc reporter gene expression in mouse lung tissue; (C) is F-luc reporter gene expression in lung tissue at different time points after mRNA-LPTC nose drop, **P<0.01, ****P<0.0001, n = 3;

[0029] Figure 6 Specific serum IgG and bronchoalveolar lavage fluid (BALF) sIgA of mice after nose drop immunization; wherein (A) is a mouse immunization and sampling plan; (B) is RBD-specific serum IgG absorbance at each time point after immunization, n = 8; (C) is RBD-specific serum IgG titer at 28 days after immunization, n = 8; (D) is RBD-specific bronchoalveolar lavage fluid (BALF) sIgA titer at 28 days after immunization, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n = 4;

[0030] Figure 7 ELISPOT cytokine detection results, lung lymphocytes were stimulated by RBD peptide library, (A) IFN-γ+ lymphocyte spots, (B) IL-17+ lymphocyte spots, (C) IL-4+ lymphocyte spots, **P<0.01, ***P<0.001, ns P>0.05, n=3);

[0031] Figure 8 The proportion of effector memory T lymphocytes (Tem) and tissue resident memory T lymphocytes (Trm) in lung tissue, wherein (A) is CD4 + CD44 + CD69 + CD62L - Tem, (B) is CD8 + CD44 + CD69 + CD62L - Tem, (C) is CD4 + CD69 + CD103 + Trm, (D) is CD8 + CD69 + CD103 + Trm, ns P>0.05, *P<0.05, ****P<0.0001, n=3. DETAILED DESCRIPTION

[0032] The application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the application and implement it, but the examples are not limiting to the application.

[0033] In the following examples, mRNA encoding the SARS-CoV-2 RBD protein of the novel coronavirus was used as an antigen to prepare an mRNA vaccine of the novel coronavirus SARS-CoV-2.

[0034] The pUC19 plasmid containing Firefly Luciferase (F-luc), enhanced green fluorescent protein (EGFP), and receptor binding domain (RBD) of SARS-CoV-2 spike protein coding sequences in the following examples were synthesized by Nanjing Jinshui Rui Company; polypeptide KG41 was synthesized by Shanghai Jiersi Biochemical Company; DMG-PEG2000 was purchased from Avanti Polar Lipids Company; 16HBE, DC2.4, and RAW264.7 cell lines were self-prepared by the research group; 6-8-week-old Balb / c female mice were purchased from Beijing Vantoll Life Company, and the research on mice in this paper was approved by the Ethics Committee of the Army Medical University and followed the guidelines of the Army Medical University and the National Research Council on animal experiments and feeding.

[0035] RPMI-1640, DMEM, opti-MEM medium, fetal bovine serum, Pen-Strep double antibody, 0.25% trypsin-EDTA, 1xPBS (purchased from Gibco company); BamH I, Kpn I, NEBufferTM r2.1, DNA loading buffer (purchased from Biolabs company); nuclease-free water, lithium chloride, Lipofectamine 2000 (purchased from invitrogen company); sodium chloride, sodium bicarbonate, sodium carbonate (purchased from Greagent company); proteose peptone, yeast powder (purchased from OXID company); 50xTAE, Tween 20, Firefly Luciferase reporter gene detection kit, immunostaining fixative, washing solution (purchased from Beyotime company); nucleic acid dye, RNA loading buffer, RNA Ladder (purchased from Thermo Scientific company); DNA Ladder, 10xMops (purchased from Solarbio company); low electroendosmosis agarose (purchased from Aladdin company); T7 high yield RNA transcription kit, Cap1 capping kit (purchased from Blue Crane Biopharmaceutical Co., Ltd.); pseudouracil (purchased from Aladdin company); TMB developing solution, 450nm stop solution, goat anti-mouse IgG and IgA (purchased from abcam company); bovine serum albumin (purchased from bioFroxx company); PBS powder (purchased from Biosharp company); isoflurane (purchased from RWD company); D-luciferin potassium salt (purchased from PerkinElmer company); Gel Extraction kit (purchased from Omega company); Spin Mi niprep kit (purchased from QIAGEN company); CCK-8 reagent (purchased from MCE company); ELISPOT kit (purchased from Dakewe company); flow cytometry antibodies and staining buffer (purchased from BD company).

[0036] GraphPad Prism 8.0 was used for statistical analysis in the following examples, and the data were expressed as ±s. One-way ANOVA was used for comparison between groups, and P<0.05 was considered statistically significant.

[0037] Example 1 Preparation of mRNA-LPTC

[0038] In this example, mRNA encoding the SARS-CoV-2 RBD protein of the novel coronavirus was used as an antigen to prepare an mRNA vaccine for the novel coronavirus SARS-CoV-2, as follows:

[0039] 1. Preparation of polypeptide KG41

[0040] The sequence of the designed polypeptide KG41 is: Ac-FAEKFKEAVKDYFAKFWDARLARALARALARHLARALARALRACYTYQGKLC;

[0041] The above polypeptide is prepared by Fmoc solid-phase synthesis: 2-chlorotrityl chloride resin is used as a solid phase, 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) is used as a coupling reagent, and N,N-diisopropylethylamine (DIPEA) is used as a base. Then the synthesized polypeptide is cleaved from the resin by incubation with dichloromethane / trifluoroethanol / acetic acid (70 / 20 / 10, v / v / v) at room temperature for 2 hours. The resin is filtered, and the solvent is removed by evaporation. After deprotection, the final solution is precipitated in ether by adding it to ether on ice. The polypeptide is purified by reverse-phase high-performance liquid chromatography and stored for use.

[0042] 2. Preparation of target mRNA

[0043] 2.1 Plasmid extraction

[0044] Weigh 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride into a triangular flask, add 1 L of pure water, adjust the pH to 7.0 with sodium hydroxide, autoclave for 20 min at 4°C, and store under sterile conditions. Take 10 ml of LB medium and add ampicillin to 50 μg / ml, then add 50 μl of E. coli containing the target plasmid (pUC19 plasmid containing the coding sequences of Firefly Luciferase (F-luc), enhanced green fluorescent protein (EGFP), and receptor binding domain (RBD) of the spike protein of the new coronavirus), and incubate at 37°C, 220 rpm, for 12-16 h. Centrifuge 5 ml of bacterial solution at 8000 rpm for 5 min, and extract the target plasmid according to the instructions of the plasmid extraction kit, and measure the concentration and purity by NanoDrop. The target plasmid is extracted according to the instructions of the plasmid extraction kit, and the concentration and purity are measured by NanoDrop.

[0045] 2.2 Plasmid double digestion and purification

[0046] Mix 3 μg of plasmid with 3 μl of BamH I, 3 μl of Kpn I, and 6 μl of r2.1 buffer, add nuclease-free water to 60 μl, and incubate at 37°C for 1 h in a PCR instrument. According to the instructions of the plasmid extraction kit, extract the target plasmid, and measure the concentration and purity by NanoDrop. The purified linear DNA fragments (transcription templates) were subjected to agarose gel electrophoresis to verify the size of the DNA fragments, and the concentration and purity of the transcription templates were detected by NanoDrop.

[0047] The plasmids and enzyme digestion products were subjected to DNA gel electrophoresis at the same time, and the results are shown in FIG. 2A (A), wherein lanes 2, 4 and 6 are RBD, EGFP and F-Luc plasmids, respectively, and the bands from top to bottom are open circular plasmid, linear plasmid and supercoiled plasmid; the upper bands in lanes 3, 5 and 7 are pUC57 plasmid vector fragments, and the lower bands are RBD, EGFP and F-luc transcription template fragments after double digestion, respectively. According to the DNA Ladder, the three target plasmids and the transcription templates after enzyme digestion are correct. Figure 1

[0048] 2.3 In vitro transcription and 5' end capping

[0049] According to the instructions of the Bluebird in vitro transcription kit, 2 μl of nuclease-free water, 10x transcription buffer, 1 μg of template DNA, 1.5 μl of ATP / GTP / CTP and pseudouracil, and 1 μl of transcription enzyme were sequentially added to a 200 μl PCR tube, with a total reaction volume of 20 μl. The reaction was carried out in a PCR instrument at 37°C for 3 h. After completion, DNase I was added to remove excess template DNA, and lithium chloride was used to purify the mRNA. According to the instructions of the Bluebird capping kit, 50 μg of denatured mRNA was sequentially added to 10x capping reaction buffer 10 μl, GTP 10 μl, SAM donor molecule 2.5 μl, RNAase inhibitor 2.5 μl, methyltransferase 4 μl, and vaccinia virus capping enzyme 4 μl. After mixing, the reaction was carried out at 37°C for 30 min, and lithium chloride was used for purification. Agarose gel electrophoresis was used to verify the size of the mRNA, and the purity and concentration of the final mRNA product were detected.

[0050] The in vitro transcribed mRNA and 5' end capped mRNA were subjected to RNA gel electrophoresis at the same time, and the results are shown in FIG. 2B (B), wherein lanes 2, 4 and 6 are RBD, EGFP and F-luc mRNA after transcription; lanes 3, 5 and 7 are RBD, EGFP and F-luc mRNA after 5' end capping; according to the RNA Ladder, the three target mRNAs and the final products after 5' end capping were successfully prepared and can be used for subsequent experiments. Figure 1

[0051] 3. Preparation of mRNA vaccine delivery vector

[0052] ​​The same volume of 10 mg / ml DMG-PEG2000 solution and 0.667 mg / ml polypeptide KG41 solution were placed in two syringes, mixed using a microfluidic device (NanoFac A, Sichuan Zhuxue Technology Co., Ltd.). The mixed solution was prepared into mRNA-LPTC ternary preparation with the above prepared 0.2 mg / mL mRNA solution in the same way, and stored at 4°C for standby.

[0053] The above prepared 0.667 mg / ml polypeptide KG41 solution and 0.2 mg / mL mRNA solution were prepared into mRNA-KG41 binary preparation in the same way, and stored at 4°C for standby.

[0054] The above prepared 10 mg / ml DMG-PEG2000 solution and 0.2 mg / mL mRNA solution were prepared into mRNA-DMGp binary preparation in the same way, and stored at 4°C for standby.

[0055] 4. Investigation of nanoparticle particle size, PDI, zeta potential and morphology

[0056] 100 μl of the above prepared ternary and binary preparations were diluted 10 times with nuclease-free water, and the nanoparticle hydrodynamic particle size, solution polydispersity coefficient and particle zeta potential were detected using a Malvern particle size analyzer (Malvern Zetasizer) in a glass cuvette. The preparation suspension was dropped on a membrane-supported copper mesh, 3% phosphotungstic acid was added for staining for 2 min, the excess staining solution was absorbed with filter paper, and the transmission electron microscope was used for observation after standing and drying for 4 h.

[0057] The results are shown in Figure 2 (A)-(C), the particle size of DMGp-mRNA binary preparation was 146.51 nm (±1.39), the PDI coefficient was 0.235 (±0.008), and the zeta potential was -12.5 mv (±0.768); the particle size of KG41-mRNA binary preparation was 226.78 nm (±5.23), the PDI coefficient was 0.166 (±0.053), and the zeta potential was +23.7 mv (±1.792); the particle size of mRNA-LPTC ternary preparation was 95.78 nm (±1.54), the PDI coefficient was 0.223 (±0.021), and the zeta potential was +15.825 mv (±0.463), n=4.

[0058] Through transmission electron microscope observation, mRNA-LPTC was a near-spherical nanoparticle, and the surface was smooth Figure 2 (D) shows).

[0059] The above results show that the mRNA-LPTC prepared in the application is a weak cationic spherical nanoparticle with a particle size of ~100 nm, and the preparation suspension has good dispersibility.

[0060] Example 2 Investigation of the in vitro delivery capacity of mRNA-LPTC

[0061] 1. Investigation of the transfection capacity of the preparation on different cells by chemiluminescence

[0062] The 16HBE, DC2.4, and RAW264.7 cells were resuscitated and plated into 10 mm culture dishes and cultured at 37°C under 5% CO2 conditions until the confluence rate was 60-80%, then the cells were counted and plated into sterile 96-well plates at 2×10 4 μl opti-MEM medium, as the experimental group; 400 ng F-luc mRNA, 0.5 μl Lipofectamine 2000, and 100 μl opti-MEM were mixed, as the positive control group; 400 ng F-luc mRNA and 100 μl opti-MEM were mixed, as the negative control group; and 100 μl mRNA-free medium was used as the background control. Each group was added to the wells, and each group was repeated three times. After incubation at 37°C under 5% CO2 conditions for 4 h, the opti-MEM was replaced with RPMI1640 medium containing 1% double antibodies and 5% FBS, and the chemiluminescence was detected after 24 h of further incubation. The supernatant was collected according to the instructions of the Firefly Luciferase Reporter Gene Detection Kit, and the relative light intensity (RLU) was detected using a multifunctional enzyme label instrument.

[0063] 2. Investigation of the transfection capacity of the preparation in 16HBE cells by fluorescence

[0064] 400 ng EGFP mRNA-containing mRNA-LPTC was used to transfect 16HBE cells according to the grouping and steps in 1. After the culture was completed, the medium in the wells was discarded, 200 μl 1×PBS was added, and the wells were left to stand for 3 min, and the liquid in the wells was discarded, and the above steps were repeated three times. An inverted fluorescence microscope was used for observation.

[0065] 3. Investigation of the cytotoxicity of the preparation by CCK-8

[0066] The 16HBE, DC2.4, and RAW264.7 cell suspensions were inoculated into sterile 96-well plates at 1×10 4 cells / well, and the cells were incubated at 37°C under 5% CO2 conditions for 4 h until they adhered. The naked mRNA, ​2000, binary and ternary formulations as control and experimental groups, each group repeated three times. Continue to culture for 24h, add 10μl CCK-8 reagent in each well, incubate in incubator for 4h, use microplate reader to measure absorbance at 450nm wavelength. Calculate the effect of formulation on cell viability according to the following formula.

[0067] Cell viability (%) = ((A 加药 -A 背景 ) / (A 未加药 -Abackground)) x 100%.

[0068] 4. Results

[0069] After transfection of 16HBE, DC2.4 and RAW264.7 cell lines with binary, ternary formulations containing F-luc reporter gene and each control reagent, in 16HBE cells: LPTC group has stronger chemiluminescence signal than other binary formulation groups (P<0.0001) (shown in (A) of Figure 3 Fig. 2); for DC2.4 cells: LPTC group has no difference in chemiluminescence signal intensity compared with KG41 binary formulation group, but has significant difference compared with DMGp binary formulation group (P<0.01) (shown in (B) of Figure 3 Fig. 2); in RAW264.7 cells: LPTC group has the strongest chemiluminescence signal compared with all control groups (including transfection reagent 2000) (P<0.0001) (shown in (C) of Figure 3 Fig. 2).

[0070] Through CCK-8 experiment, it was found that LPTC formulation had no significant effect on the viability of 16HBE, DC2.4 and RAW264.7 cells (P>0.05) (shown in (D)-(F) of Figure 3 Fig. 2). The above results show that mRNA-LPTC ternary formulation has higher in vitro delivery efficiency on bronchial epithelial cells and dendritic cells than KG41-mRNA or DMGp-mRNA binary formulation, and mRNA-LPTC can be specifically taken up by macrophages and expressed, having certain immune enhancement potential. In addition, mRNA-LPTC has no significant toxicity on the three cell lines.

[0071] As shown in Figure 4 Fig. 3, after transfection of 16HBE cells with mRNA-LPTC containing EGFP reporter gene, green fluorescent protein can be effectively expressed. This result again shows that mRNA-LPTC can effectively transfect bronchial epithelial cells in vitro.

[0072] Example 3 Investigation of in vivo delivery capacity of mRNA-LPTC

[0073] 1. Investigation of respiratory tract delivery capacity of different formulations

[0074] Binary and ternary formulations containing 3 μg F-luc mRNA were used as experimental groups, while 1×PBS and naked mRNA served as control groups. Balb / c female mice, aged 6–8 weeks, were anesthetized with isoflurane. The mice's heads were raised to a 180° angle between the respiratory tract and trunk, and 20 μl of the solution was instilled into each nostril at 8-minute intervals, with three mice per group. 24 hours after administration, each mouse was intraperitoneally injected with 120 μl of 1.1% sodium pentobarbital solution. After complete anesthesia, the skin on the chest of the mice was exposed using depilatory cream, and 100 μl of 30 mg / ml D-fluorescein potassium solution was injected intraperitoneally and 20 μl was instilled into each nostril. Five minutes later, the mice were placed in an in vivo imaging system to detect chemiluminescence signals. After completion, the mice were euthanized by cervical dislocation, and the abdominal and thoracic cavities were opened. The liver, spleen, and whole lung were removed, rinsed with 1×PBS, and placed in an in vivo imaging system to detect chemiluminescence signals. The luminous flux of the selected areas was calculated using Living Image software.

[0075] 2. Investigation of the respiratory delivery capacity of mRNA-LPTC at different time points

[0076] Ten Balb / c female mice were randomly selected, and the F-luc mRNA-LPTC preparation was administered intranasally according to the method in section 1. The liver, spleen, and whole lung of the mice were collected at 6h, 12h, 24h, 48h, and 72h after administration for chemiluminescence signal detection.

[0077] 3. Results

[0078] Following intranasal administration of the ternary formulation containing the F-luc reporter gene and the binary control formulation, the LPTC formulation group showed a stronger chemiluminescent signal in the mouse nasal cavity compared to the other control groups (P<0.01). Figure 5 (As shown in Figure (A)). In lung tissue, the LPTC preparation group showed the strongest chemiluminescent signal compared to other control groups (P<0.0001). Figure 5 (As shown in (B)). Furthermore, as... Figure 5 As shown in (C), the chemiluminescence signal intensity in lung tissue reached its peak 24 h after intranasal administration of mRNA-LPTC, and the mRNA could be continuously expressed up to 72 h.

[0079] The above results suggest that mRNA-LPTC nanoparticles, when administered via the respiratory tract, can effectively express the target mRNA in the nasal and lung tissues of mice. Furthermore, they can continuously translate the target protein in lung tissue for 72 hours. However, the KG41-BC binary formulation showed poor in vivo delivery capabilities, and it was not used as an experimental group in subsequent animal immunization experiments.

[0080] Example 4: Investigation of mRNA-LPTC-induced levels of specific antibodies in mice

[0081] 1. Immunization plan for laboratory mice

[0082] Twenty-four female Balb / c mice aged 6–8 weeks were randomly selected and divided into three groups. LPTC ternary formulation containing 3 μg RBD-mRNA, DMGp binary formulation, and 1×PBS solution were administered intranasally as described in section 1.6.1. These groups were designated as the experimental group and the negative control group. Immunization and sampling time points are as follows: Figure 6 As shown in (A).

[0083] 2. Collection of serum and bronchoalveolar lavage fluid samples

[0084] 40 μl of blood was collected from the tail vein of the experimental mice. The blood sample was incubated at 37°C for 1 hour, centrifuged at 8000 rpm for 20 minutes, and the supernatant serum was collected and stored at -80°C for later use. The 28-day-old experimental mice were euthanized by cervical dislocation, their entire bodies were moistened with 75% alcohol, and they were fixed in a supine position. The skin and muscles below the thoracic cavity were opened, and the mediastinum was cut to open the thoracic cavity to the outside. The skin and muscles of the mouse's neck were cut open to expose the tracheal cartilage, and an opening of approximately 2 mm was made. A tracheal tube was introduced using an indwelling needle, and the lung lobes were irrigated with 900 μl of ice-cold 1×PBS and stored at -80°C for later use.

[0085] 3. ELISA

[0086] Add 100 μl of coating buffer containing 2 mg / L RBD protein to each well of a 96-well plate and incubate overnight at 4°C. Discard the liquid in the wells, add 250 μl of 1×PBST to each well to wash the plate, let stand for 2 min, then blot dry. Repeat three times. Add 250 μl of 1×PBST containing 1% BSA to each well, block and incubate overnight at 4°C, then wash the plate. Dilute serum and bronchoalveolar lavage fluid samples as needed using 1×PBST containing 0.5% BSA, adding 100 μl to each well and incubating at 37°C for 1 h. Wash the plate, add 100 μl of diluted goat anti-mouse IgG and IgA to each well of the serum and bronchoalveolar lavage fluid samples, and incubate at 37°C for 40 min. Wash the plate, add 100 μl of TMB chromogenic solution per well in the dark, and after 5 min of chromogenic development, add 100 μl of stop solution to each well. Use an ELISA reader to detect the absorbance at a wavelength of 450 nm.

[0087] 4. Results

[0088] mice Figure 6 Following administration and sampling of the immunization regimen shown in Figure (A), serum samples at time points of 7, 14, 21, 28, 35, 42, and 49 showed significantly higher levels of RBD-specific IgG in the mRNA-LPTC group compared to the PBS group (P<0.05), and serum IgG levels plateaued from day 28 onwards. Figure 6LPTC ternary formulation group 28 days RBD-specific serum IgG titers were significantly higher than DMGp binary formulation group (P < 0.0001) (Fig. 4B). Figure 6 LPTC ternary formulation group 28 days bronchoalveolar lavage fluid RBD-specific sIgA titers were significantly higher than DMGp binary formulation group (P < 0.05) (Fig. 4C). Figure 6 LPTC ternary formulation group 28 days bronchoalveolar lavage fluid RBD-specific sIgA titers were significantly higher than DMGp binary formulation group (P < 0.05) (Fig. 4C).

[0089] The above results suggest that intranasal vaccination with mRNA-LPTC nanoparticles can effectively induce mice to produce high levels of antigen-specific serum IgG and bronchoalveolar lavage fluid sIgA.

[0090] Example 5 Investigation of cellular immune levels of mRNA-LPTC immunized mice

[0091] 1. Isolation of lung tissue lymphocytes from mice

[0092] Under sterile conditions, the lung tissues of the experimental mice in each group were taken out and cut into pieces, and 8 ml of freshly prepared lung tissue digestion solution was added to the lung tissue pieces in each group according to the Lung Lymphocyte Isolation Guide. The mixture was digested at 37°C and 220 rpm for 1 h. After digestion was completed, the lung tissue pieces were poured onto a 200-mesh screen and ground with a rubber grinding rod. The filtrate was centrifuged at 2000 rpm and the supernatant was discarded. 4 ml of RPMI 1640 medium was added to resuspend the cells, and 8 ml of the supernatant was slowly added along the tube wall. Obvious layering was observed, and the mixture was gradient centrifuged at 2500 rpm for 30 min. The upper layer was carefully discarded, and 4 ml of cell suspension in the middle of the two layers was aspirated. The mixture was centrifuged at 2000 rpm and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium and counted, and the concentration was adjusted to 106 cells / ml. The mixture was stored at 4°C for later use. 2. Lung lymphocyte ELISPOT

[0093] According to the ELISPOT kit instructions, 200 μl of RPMI 1640 medium was added to each well of the pre-coated 96-well plate for activation. Lung lymphocyte suspension with a concentration of 1 x 106 cells / well was added to each well. 10 μl of positive stimulant working solution, 10 μl of medium, and 4 μg of RBD peptide library were added to the positive control wells, negative control wells, and experimental wells, respectively. The mixture was incubated at 37°C and 5% CO2 for 36 h. Then, the cells were lysed according to the kit instructions, and enzyme-linked immunosorbent color development was performed using IFN-γ, IL-4, and IL-17 specific antibodies. After drying, ELISPOT spot counting was performed.

[0094] 3. Investigation of memory T lymphocytes in lung tissue by flow cytometry

[0095] 3. Investigation of memory T lymphocytes in lung tissue by flow cytometry ​​

[0096] The lung lymphocytes obtained in Example 4 were centrifuged and resuspended with staining buffer. The cell concentration was adjusted to 10⁶ cells / well and added to 96-well plates. AF700-L / D and FITC-CD3 were added. + Percp-cy5.5-CD4 + PE-cy7-CD8 + APC-CD44 + PE-CD62L + BV421-CD69 + BV510-CD103 + Flow cytometry was used to stain lymphocyte surface antigen differentiation clusters. The staining was performed at 4°C in the dark for 30 min. Excess antibody was washed away with staining buffer, and the cells were then analyzed using a flow cytometer.

[0097] 4. Results

[0098] like Figure 7 As shown in (A), by detecting the number of RBD-specific IFN-γ positive lung lymphocyte spots, the number of spots in the LPTC preparation group of mice was significantly higher than that in the DMGp-BC binary control group and the negative control group (P<0.01), while there was no statistically significant difference between the DMGp-BC binary control group and the negative control group (P>0.05). Figure 7 In the study (B), the number of RBD-specific IL-17 positive lung lymphocyte spots was detected. The number of spots in the LPTC preparation group was significantly higher than that in the DMGp-BC binary control group and the negative control group (P<0.001), while there was no statistically significant difference between the DMGp-BC binary control group and the negative control group (P>0.05). Figure 7 In the study (C), the number of RBD-specific IL-4 positive lung lymphocyte spots was detected. The number of spots in the LPTC preparation group was significantly higher than that in the DMGp-BC binary control group and the negative control group (P<0.01), while there was no statistically significant difference between the DMGp-BC binary control group and the negative control group (P>0.05). These results indicate that after intranasal immunization with mRNA-LPTC nanoparticles, the lung lymphocytes of mice stimulated by the SARS-CoV-2 RBD antigen peptide library significantly increased the secretion of cytokines IFN-γ, IL-17, and IL-4. Simultaneously, IFN-γ... + and IL-17 +A larger number of lymphocyte spots indicates that intranasal immunization with mRNA-LPTC can induce a strong Th1-type acquired immune response in the lower respiratory tract, promote anti-intracellular infection, as well as a strong Th17-type immune response, enhancing the defense against pulmonary pathogens. This helps pulmonary lymphocytes resist the invasion of SARS-CoV-2, greatly enhancing the level of lung tissue-specific cellular immunity, while the DMGp-BC binary control preparation has poor effects.

[0099] In the pulmonary T lymphocytes of immunized mice, as Figure 8 shown in (A), in the LPTC preparation group, CD4 + CD44 + CD69 + CD62L - Tem cell subset had a higher proportion than the DMGp-BC binary control group and the negative control group (P < 0.0001), while there was no statistical difference between the DMGp-BC binary control group and the negative control group (P > 0.05). As Figure 8 shown in (B), in the LPTC preparation group, CD8 + CD44 + CD69 + CD62L - Tem cell subset had a higher proportion than the DMGp-BC binary control group and the negative control group (P < 0.0001), while there was no statistical difference between the DMGp-BC binary control group and the negative control group (P > 0.05). This result indicates that after intranasal immunization with mRNA-LPTC nanoparticles, a large number of CD4+ or CD8+ effector memory T cells can be induced to differentiate in the lungs of mice, thus greatly enhancing the cellular immune level of lung tissue in response to re-exposure. The DMGp-BC binary control preparation has poor effects.

[0100] As Figure 8 shown in (C), in the pulmonary T lymphocytes of immunized mice, in the LPTC preparation group, CD4 + CD69 + CD103 + Trm cell subset had a higher proportion than the DMGp-BC binary control group and the negative control group (P = 0.0001, P < 0.0001). The proportion of CD4 + Trm in the DMGp-BC binary control group had a statistical difference compared with the negative control group (0.01 < P < 0.05). As Figure 8 shown in (D), in the LPTC preparation group, CD8 + CD69 + CD103 +The percentage of Trm cell subsets was significantly higher in the DMGp-BC binary control group and the negative control group (P = 0.0001, P < 0.0001), while there was no statistically significant difference between the two groups in the latter (P > 0.05). These results indicate that intranasal immunization with mRNA-LPTC nanoparticles can induce the production of large amounts of CD4+ in the lungs of mice. + or CD8 + The presence of resident memory T cells significantly enhances the respiratory tract's specific long-term immune protection, potentially enabling a rapid response and control of the disease in the event of secondary exposure. In contrast, the DMGp-BC binary control formulation showed poor efficacy.

[0101] In summary, the mRNA-LPTC nanoparticles described in this application exhibit no significant cytotoxicity, can effectively transfect human bronchial epithelial cells and mouse dendritic cells in vitro, and are readily taken up by mouse macrophages, demonstrating potential immunomodulatory capabilities. In in vivo delivery studies, mRNA-LPTC, administered via nasal drops, significantly increased the expression level of the target mRNA in the nasal and lung tissues of mice, indicating its excellent respiratory mucosal delivery capability and sustained expression within 72 hours. This helps maintain the continuous presentation of the target antigen in the respiratory mucosa, thereby enhancing the adaptive immune response.

[0102] By loading mRNA encoding the SARS-CoV-2 RBD protein into LPTC, an mRNA-LPTC vaccine formulation was obtained. Intranasal immunization of mice induced high levels of antigen-specific serum IgG and bronchoalveolar lavage fluid sIgA, indicating that it can not only induce a systemic adaptive immune response but also enhance respiratory mucosal immunity. Furthermore, high levels of specific sIgA can delay the rate of viral "breakthrough infection," giving the host's immune system more time to initiate an adaptive immune response, thereby reducing the risk of severe COVID-19. It also has the potential to reduce the risk of aerosol transmission, compensating for the deficiency of traditional injectable vaccines in generating specific respiratory immune defense. ELISPOT analysis of lymphocytes in the lung tissue of mice after intranasal immunization revealed that mice immunized with mRNA-LPTC nanoparticles showed significantly increased secretion of IFN-γ, IL-4, and IL-17 cytokines in their lung lymphocytes under RBD peptide library stimulation, with higher levels of IL-4 compared to IL-4. + IFN-γ with more spots + and IL-17 +Lymphocytes, indicating that the lung lymphocytes of the mice after immunization produced IFN-γ-mediated Th1-biased immune response after receiving RBD antigen for re-stimulation, played the role of anti-intracellular infection, and IL-17-mediated Th17 immune response promoted the defense ability of the respiratory tract to the invading pathogens. Through the investigation of T lymphocytes in the lung tissue by flow cytometry, it was found that the mRNA-LPTC nanoparticle nasal immunization could induce significant increase of CD4 + or CD8 + effector memory T lymphocyte subsets (Tem) and CD4 + or CD8 + tissue-resident memory T lymphocyte subsets (Trm) in the lung tissue of the mice, and the specific Tem and Trm memory T lymphocytes in the lung tissue were of great significance to the defense of viral breakthrough infection, the rapid initiation of acquired immune response, the reduction of the risk of severe illness and the prolongation of the vaccine protection period.

[0103] It can be seen that the mRNA-LPTC nanoparticle vaccine nasal immunization described in the present application can not only induce the mice to produce high level of antigen-specific humoral immunity, but also establish strong and durable antigen-specific cellular immunity at the respiratory mucosa, the site of respiratory virus invasion, and further strengthen the defense ability to SARS-CoV-2 and reduce the risk of virus transmission through aerosol.

[0104] In the present study, the particle size, potential, morphology, solution dispersibility, cytotoxicity, in vitro and in vivo delivery capacity, antigen-specific humoral immunity after nasal immunization and the level of cellular immunity in the lung tissue of the mRNA-LPTC nanoparticles were investigated, and the potential of the mRNA-LPTC nanoparticles as a mucosal mRNA vaccine was preliminarily explored. The foundation was laid for the subsequent investigation of antibody neutralization capacity and immune protection rate and the like; the preparation was laid for the further development of inhaled mRNA vaccine, the optimization of its biocompatibility and the preparation process and the like; and the reference was provided for the further research on the mRNA delivery mechanism, the nanoparticle structure and assembly principle, the respiratory mucosal immune response pathway and the like.

[0105] The above-described embodiments are only the preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. The equivalent substitutions or transformations made by the person skilled in the art on the basis of the present application are within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A nucleic acid vaccine delivery vehicle, characterized in that, The nucleic acid vaccine delivery carrier is composed of a polypeptide shown by the amino acid sequence SEQ ID NO. 1 and DMG-PEG2000.

2. The nucleic acid vaccine delivery vector of claim 1, wherein, The nucleic acid vaccine delivery carrier is prepared by mixing a polypeptide solution shown by the amino acid sequence SEQ ID NO. 1 and a DMG-PEG2000 solution.

3. Use of the nucleic acid vaccine delivery carrier according to any one of claims 1-2 in the preparation of an mRNA vaccine.

4. An mRNA vaccine, characterized in that The mRNA vaccine is composed of the nucleic acid vaccine delivery carrier according to any one of claims 1-2 and an mRNA encoding SARS-CoV-2 RBD antigen.

5. The mRNA vaccine as described in claim 4, characterized in that, The mRNA vaccine is prepared by mixing a nucleic acid vaccine delivery carrier solution according to any one of claims 1-2 and an mRNA solution encoding SARS-CoV-2 RBD antigen. 6.The preparation method of the mRNA vaccine according to claim 5, characterized in that, The method is: (1) preparing an mRNA encoding SARS-CoV-2 RBD antigen; (2) placing DMG-PEG2000 solution and polypeptide solution shown by the amino acid sequence SEQ ID NO. 1 into two syringes respectively, mixing using a microfluidic device to obtain a nucleic acid vaccine delivery carrier; (3) placing nucleic acid vaccine delivery carrier solution and mRNA solution into two syringes respectively, mixing using a microfluidic device to obtain an mRNA vaccine.

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