A novel coronavirus vaccine vector and application thereof

Abstract

The application provides a new crown vaccine carrier and application thereof. Composed of SARS-CoV-2 RBD as an antigen and STING agonist diABZI coupled with yeast beta-glucan particles. Compared with the diABZI-RBD vaccine alone, the GP-diABZI-RBD vaccine shows sustained vaccine release and elicits a more persistent immune response in mice. Intraperitoneal injection of GP-diABZI-RBD in mice can cause significant cellular and humoral immune responses. Using a replicable virus-like particle system based on pseudovirus particles, we determined that this vaccine can effectively induce effective and persistent neutralizing antibodies against the prototype and VOC, respectively. In particular, the prototype RBD antigen provided by the GP-diABZI-RBD vaccine shows a stronger and longer humoral immune response and antiviral protection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, in particular relates to a new crown vaccine carrier and its application. BACKGROUND

[0002] Omicron contains 15 amino acid mutations in the receptor binding domain (RBD), resulting in the inability of most therapeutic monoclonal antibodies and clinically available vaccines to be used. Therefore, there is an urgent need to develop a universal and durable vaccine against a wide range of SARS-CoV-2 variants.

[0003] SARS-Cov-2 is a positive-strand RNA virus with an envelope, surrounded by a trimer of spike (S) protein, forming a characteristic bulbous halo on the outer surface. The S protein contains two subunits: S1 and S2. The S1 subunit mediates recognition and entry into human cells through the binding of RBD and human angiotensin-converting enzyme 2 (hACE2). Subsequently, the S protein undergoes a conformational change, participating in the fusion of the viral envelope and the cell membrane. So far, most clinical COVID-19 vaccines have been designed against the S protein or RBD. Serological studies have shown that most of the neutralizing activity in COVID-19 convalescent serum is produced by RBD. Since some RBD-specific human neutralizing monoclonal antibodies exhibit broad protection against SARS-CoV-2 variants, RBD has proven to be an interesting target for developing broad-spectrum vaccines.

[0004] Adjuvants are considered effective tools for inducing antigen-specific cellular immunity. Aluminum vaccine adjuvants are widely used due to their safety. Aluminum has been shown to preferentially induce Th2 responses for antibody production. However, activation of virus-specific T cell responses is needed to enhance broad protection against omicron and other VOCs. Therefore, there is an urgent need for vaccines that can elicit effective T cell responses against SARS-CoV-2 and OVCs. In the past few decades, small molecule agonists of several pattern recognition receptors (PRRs) have been developed as effective adjuvants to activate cellular immune responses and optimize the efficacy of vaccines. Among these molecules, stimulator of interferon genes (STING) agonists have been reported as attractive adjuvants to induce type I interferon (IFN-I) responses and inflammatory cytokines. In addition to producing protective antibodies and stimulating the host's immune response to SARS-CoV-2, recently, STING agonists have been shown to inhibit SARS-CoV-2 replication in lung epithelial cells. Despite these advantages, there are still many challenges in directly utilizing STING agonists in vivo. The widespread spread of COVID-19 has created an urgent need for STING agonists to develop a safe and effective delivery platform that targets immune induction sites with extended half-life and improved stability to induce long-lasting immune memory in vivo.

[0005] Delivery of antigens to dendritic cells (DCs) provides an advantageous strategy for priming antigen-specific cellular immunity. DCs, as the most potent antigen presenting cells (APCs), link innate and adaptive immune responses by targeting antigens for uptake, processing, and priming of T cell responses. As a natural extract of Saccharomyces cerevisiae, yeast beta-glucan particles (GPs) form 2-4 pm sized particles that can be taken up by phagocytic cells. GPs are specifically recognized by the PRR Dectin-1, which is mainly expressed on the surface of monocytes / macrophages, as a pathogen-associated molecular pattern (PAMP). After internalization and cross-presentation by DCs, GPs have been shown to promote mature DC stimulation and drive T cell clonal expansion. Furthermore, as the safety of GPs has been approved by the FDA, GPs can be designed as an ideal delivery platform for STING agonists. SUMMARY

[0006] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provide a new crown vaccine carrier.

[0007] Another object of the present application is to provide the application of the above-mentioned new crown vaccine carrier.

[0008] The object of the present application is achieved by the following technical solutions:

[0009] A new crown vaccine carrier, comprising an antigen protein, a polysaccharide, and a stimulator of interferon genes (STING) agonist.

[0010] The antigen protein, the polysaccharide, and the stimulator of interferon genes agonist are coupled together.

[0011] The antigen protein and the stimulator of interferon genes agonist are coupled together.

[0012] The antigen protein includes but is not limited to: novel coronavirus RBD (SARS-COV-2 Spike Protein RBD), novel coronavirus delta variant RBD (SARS-COV-2 Spike Protein RBD-delta), novel coronavirus gamma variant RBD (SARS-COV-2 Spike Protein RBD-gamma), and novel coronavirus omicron variant RBD (SARS-COV-2 Spike Protein RBD-omicron).

[0013] The polysaccharide is preferably yeast beta-glucan (GP).

[0014] The interferon gene stimulator agonist is preferably diABZI.

[0015] The preparation method of the new crown vaccine carrier preferably comprises the following steps:

[0016] (1) preparing polysaccharide microspheres;

[0017] (2) soaking the polysaccharide microspheres obtained in step (1) in an aqueous solution containing an interferon gene stimulator agonist and an antigen protein, centrifuging to remove the supernatant, resuspending the obtained precipitate, then centrifuging again, resuspending the obtained precipitate in a crosslinking agent for crosslinking, and after the crosslinking is completed, washing to obtain a new crown vaccine carrier.

[0018] The polysaccharide microspheres in step (1) are preferably yeast β-glucan particle (GP) microspheres, and the specific preparation steps are as follows:

[0019] The washed yeast is suspended in an alkaline solution and heated and stirred, then centrifuged, the precipitate is resuspended in water, then the pH is adjusted using an acid solution, incubated, centrifuged, washed with water, isopropanol, and acetone, and dried to obtain yeast β-glucan particle microspheres.

[0020] The heating and stirring in step (1) is heating to 85-95°C and stirring for 0.5-2 hours.

[0021] The mass ratio of the polysaccharide microspheres and the antigen protein is preferably 20:1-30:1.

[0022] The mass ratio of the interferon gene stimulator agonist and the antigen protein is preferably 1:1-3:1.

[0023] The soaking time in step (2) is 1-3 hours.

[0024] The centrifugation condition in step (2) is 2000-4000 rpm for 10-30 minutes.

[0025] The resuspension condition in step (2) is resuspension at room temperature for 0.5-2 hours using a 0.1-0.3% mass concentration chitosan solution.

[0026] The re-centrifugation condition in step (2) is 2000-4000 rpm for 10-30 minutes.

[0027] The crosslinking condition in step (2) is crosslinking for 0.5-2 hours using a 0.05-0.2% mass concentration genipin crosslinking solution.

[0028] The washing in step (2) is washing 1-3 times using PBS.

[0029] Use of the new coronavirus vaccine carrier in the preparation of a new coronavirus vaccine.

[0030] The present application has the following advantages and effects relative to the prior art:

[0031] The present application provides a new coronavirus vaccine carrier and its application. The carrier is composed of SARS-CoV-2 RBD as an antigen and STING agonist diABZI coupled with yeast β-glucan particles (GP-diABZI-RBD). Compared with the diABZI-RBD vaccine alone, the GP-diABZI-RBD vaccine shows sustained vaccine release and elicits a more persistent immune response in mice. Intraperitoneal injection of GP-diABZI-RBD in mice can cause significant cellular and humoral immune responses. In particular, the prototype RBD antigen provided by the GP-diABZI-RBD vaccine shows stronger and longer humoral immune responses and antiviral protection. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a confocal laser scanning microscope result map.

[0033] Figure 2 is a Western blot experiment electrophoresis result map.

[0034] Figure 3 is a Western blot experiment electrophoresis result map showing that the GP-diABZI-RBD vaccine activates the STING signaling pathway of BMDCs cells.

[0035] Figure 4 is a confocal laser scanning microscope result map showing that the GP-diABZI-RBD vaccine can be quickly recognized and taken up by BMDCs cells.

[0036] Figure 5 is an RT-PCR result analysis chart; the GP-diABZI-RBD vaccine activates the STING pathway and shows a more persistent immune response; RT-PCR analysis shows the expression of the indicated pro-inflammatory cytokine and interferon-stimulated related gene transcripts in mouse BMDCs cells.

[0037] Figure 6 is a result chart showing that GP-diABZI-RBD vaccine inoculation promotes T cell immune response in Balb / c mice; A-B: flow cytometry shows that mice under GP-diABZI-RBD or diABZI-RBD / GP-RBD treatment + CD4 + T(A) and IFN-γ + CD8 +Ratio of T(B); C: ELISA shows IFN-g, IL-2, IL-6 and IL-10 production in mice under GP-diABZI-RBD or diABZI-RBD / GP-RBD treatment.

[0038] Figure 7 Figure 1 is the results of GP-diABZI-RBD vaccination induced effective humoral immune response in Balb / c mice; A: flow chart demonstrates the vaccine strategy; B: ELISA analysis shows IgGl and IgG2a production in mice immunized with GP-diABZI-RBD or diABZI-RBD; C: ELISA analysis shows RBD-specific IgG production in mice immunized with GP-diABZI-RBD or diABZI-RBD. DETAILED DESCRIPTION

[0039] The application will be further described in conjunction with the following examples and drawings, but the implementation of the application is not limited thereto.

[0040] Experimental materials:

[0041] DMEM (Invitrogen, Carlsbad, CA, USA). RPMI 1640, fetal bovine serum, Penicillin, Streptomycin, β-mercaptoethanol, Gibco (Grand Island, NY, USA), recombinant mouse GM-CSF, recombinant mouse IL-4 (HyClone, South Logan, UT, USA), DMSO (St. Louis, MO, USA).

[0042] IFNy ELISA kit (Cat# 430804), IL6 ELISA kit (Cat# 431304), IL10 ELISA kit (Cat# 431417), IL2 ELISA kit (Cat# 431004) were all from BioLegend (San Diego, CA, USA). PE anti-mouse CDllc antibody (Cat# 117307), PerCP anti-mouse CD3 antibody (Cat# 100325), PE anti-mouse CD4 antibody (Cat# 100407), APC anti-mouse CD8 antibody (Cat# 100711), PE-Cy7 anti-mouse IFN-γ antibody (Cat# 505825) were purchased from BioLegend (San Diego, CA, USA). Mounting medium containing DAPI-Aqueous, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgGl (Cat# ab97240), IgG2a (Cat# ab97245) antibodies were purchased from Abeam (Cambridge, England). The following antibodies were used for Western blotting: anti-TBK1 (Cat# 38066), anti-IRF3 (Cat# 4302), anti-NF-κΒ (Cat# 8242), anti-STING (Cat# 13647), anti-phospho-STING (Ser366) (Cat# 50907), anti-phospho-TBK1 (Ser172) (Cat# 5483), anti-phospho-IRF3 (Ser396) (Cat# 37829), anti-phospho-NF-κΒ p65 (Ser536) (Cat# 3033) were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-SARS-COV-2 Spike RBD antibody (Cat# GT5449) was ordered from GeneTex. diABZI STING agonist (Cat# S8796) and Genipin (Cat# S2412) were purchased from Selleck Chemicals (Houston, Texas, USA). All other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0043] Specific pathogen-free (SPF) 6-8 weeks old BALB / c mice were obtained from Guangdong Medical Laboratory Animal Center, Guangzhou, Guangdong, China. All animal experiments were approved by the Institutional Animal Care and Use Committee of Jinan University. Animals were housed in a specific pathogen-free environment and mouse experiments were performed in accordance with the guidelines of the ethical committee.

[0044] Example 1

[0045] Preparation of yeast β-glucan particles (GP) microspheres

[0046] 100 g of Saccharomyces cerevisiae (Angel high activity dry yeast, Hubei, China) was washed twice with 1.0 L of deionized water to remove additives. The washed Saccharomyces cerevisiae was suspended in 1.0 L of NaOH (1.0 M) and stirred at 90 °C for 1.0 h. Then centrifuged at 2000 g for 10 minutes. Then the precipitate was suspended in 1.0 L of deionized water, the pH value was adjusted to 4.5 with HC1, and incubated at 55 °C for 1.5 hours. Then the precipitate was centrifuged at 3000 rpm for 10 minutes. The precipitate was washed twice with 1.0 L of deionized water, washed 4 times with 250 ml of isopropanol, and washed twice with 250 ml of acetone. The precipitate was dried at room temperature into a powder to obtain GP microspheres.

[0047] The preparation steps of GP microspheres can refer to the literature (Aouadi M, Tesz G J, Nicoloro S M, et al. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. [J]. Nature, 2009, 458(7242): 1180.).

[0048] Example 2

[0049] Preparation of GP-diABZI-RBD particles and diABZI-RBD particles, GP-RBD particles

[0050] GP microspheres (5 mg) prepared in Example 1 were soaked in 100 μl of SARS-CoV-2 wild type RBD protein (purchased from Huawmei Bioengineering Co., Ltd., SARS-CoV-2 coronavirus spike glycoprotein RBD) solution (2 mg / ml) containing 200 μg of diABZI (Selleck Chemicals, Houston, USA) for 2 hours. Then the supernatant was discarded by centrifugation (3000 rpm, 10 minutes) to remove the excess solution outside the GP. The residue was resuspended with 0.2% (w / v) chitosan solution at room temperature for 1 h, while shaking and centrifugation (3000 rpm, 10 minutes) again. Subsequently, the particles were resuspended in 5 ml of 0.1% (w / v) genipin crosslinking solution for 1 hour. Finally, the obtained GP-diABZI-RBD particles were washed twice with PBS and stored at -80 °C.

[0051] The preparation of diABZI-RBD particles is the same as the above steps, with the only difference being that GP microspheres are not added.

[0052] The preparation of GP-RBD particles is the same as the above steps, with the only difference being that diABZI is not added.

[0053] Example 3

[0054] Preparation of GP-diABZI-RBD-FITC particles

[0055] FITC fluorescent label was used to label RBD. 5 mg of RBD protein solution was added to 10 ml of 5 mM EDTA solution and 1.2 ml of carbonate buffer (0.1 M, pH 9.2) to disperse and mix evenly. Then 400 μl of 2.5 mg / ml FITC in DMSO solution was added to the solution, and incubated overnight at 4°C in the dark. Finally, 2 ml of Tris-HCl (1 M, pH 8.3) was added and reacted for 15 min. The unbound fluorescein was removed by dialysis, and the FITC fluorescently labeled RBD fluorescent protein powder was prepared by freeze-drying at low temperature and stored at -80°C in the dark.

[0056] GP-diABZI-RBD-FITC particles with fluorescent labels were then prepared by the same steps as Example 2.

[0057] Example 4

[0058] Confocal imaging analysis of GP-diABZI-RBD particles

[0059] The size, dispersity and morphology of the particles prepared in Examples 2 and 3 were analyzed using a TCS SP8 confocal laser scanning microscope (Leica, Germany). For confocal imaging analysis, we labeled RBD with FITC and encapsulated it with diABZI into GP particles to prepare GP-diABZI-RBD-FITC particles, and the results are shown in Figure 1

[0060] Example 5

[0061] Generation of mouse bone marrow-derived DCs (BMDCs)

[0062] ​Bone marrow cells were flushed out from femur and tibia of 6-8 weeks old BALB / c mice with PBS. Cells were treated with erythrocyte lysis buffer for 3 minutes and then washed with RPMI-1640. Then, they were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 50 mM β-mercaptoethanol, 100 U / ml penicillin, 100 U / ml streptomycin, recombinant mouse IL-4 (10 ng / ml) and recombinant mouse. 1*10 6 cells / ml concentration in a 10 cm tissue culture plate and added mouse GM-CSF (20 ng / ml) to a final volume of 10 ml of complete RPMI 1640 medium. On day 3 and day 5, half of the medium was replaced with an equal volume of complete RPMI 1640 medium and cultured to day 7 to obtain mature BMDCs.

[0063] Example 6

[0064] BMDCs uptake of GP-diABZI-RBD in vitro

[0065] GP-diABZI-RBD-FITC carrier (2.5 μg) prepared using Example 3 was incubated with 2 ml of DCs (BMDCs) (1*10 6 cells) prepared in Example 5 in a 37°C cell incubator for 2 h (BMDCs were cultured in complete medium containing 10% serum in RPMI-1640).

[0066] After incubation, BMDCs cells were centrifuged at 300g, the supernatant was discarded, and the cells were washed twice with 2 ml of PBS. Then the cells were resuspended with 100 ul containing 2 μl of anti-mouse CD11c-PE antibody, and stained at room temperature for 1 h in the dark. After staining, PBS was washed twice, and the cells were resuspended with PBS containing 5 μg / ml of DAPI dye, and stained for 5 min. Then the cells were washed again with PBS twice, and resuspended with 200 ul of PBS.

[0067] The stained cells were taken 10 μl and placed on a glass slide, and gently covered with a cover glass, and observed for the uptake of GP-diABZI-RBD carrier by BMDCs cells under a TCS SP8 confocal laser scanning microscope (Leica, Germany), and the results are shown in Figure 4 .

[0068] Example 7

[0069] RT-PCR determination of GP-diABZI-RBD vaccine activated STING pathway immune response

[0070] GP-diABZI-RBD vector (2.5 μg) prepared using Example 2 was incubated with 2 ml of DCs (BMDCs) (1*10 6 cells) prepared using Example 5 in a 37 °C cell incubator (BMDCs were cultured in complete medium RPMI-1640 with 10% serum); and a control group was set up, using pure diABZI (2.5 μg) to incubate with BMDCs cells, and samples were taken at 0 h, 6 h, 12 h, 24 h for RT-PCR detection.

[0071] cDNA synthesis and real-time PCR from total RNA extracted from experimental cells

[0072] Experimental cells: BMDCs cells sampled after incubation

[0073] RNA extraction:

[0074] 1) Add 1 ml Trizol to 1*10 7 cells, blow several times with a pipette, and shake well

[0075] 2) Place the mixed sample at 15-30 °C for 5 min to completely separate the nucleic acid protein complex.

[0076] 3) Add 0.2 ml chloroform, cover the tube cap, shake on a vortex shaker for 15 s, and place at room temperature for 3 min. If vortex mixing is not possible, manually invert and mix for 2 min instead.

[0077] 4) Centrifuge at 12000 rpm for 10-15 min at 4 °C, and the sample will separate into three layers: red organic phase, middle layer, and upper colorless aqueous phase, and the RNA is mainly in the aqueous phase. Transfer the aqueous phase (about 600 μl, about 60% of the Trizol reagent used) to a new tube.

[0078] 5) Add an equal volume (about 600 μl) of isopropanol to the obtained aqueous phase solution, invert and mix, and place at -20 °C for 20-30 min.

[0079] 6) Centrifuge at 12000 rpm for 10 min at 4 °C, and discard the supernatant.

[0080] 7) Add 1 ml 75% ethanol (DEPC water treated water preparation) to wash the precipitate. After adding, tap the tube to make the RNA precipitate float, and add 1 ml ethanol.

[0081] 8) Centrifuge at 12000 rpm for 5 min at 4 °C, discard the supernatant; carefully aspirate the supernatant with a pipette, and note that the precipitate should not be aspirated.

[0082] 9) Let the precipitate stand at room temperature to dry, add 50 μl DEPC water, and use a gun head to beat several times to fully dissolve the RNA. Incubate at 50°C for 1 hour. Dissolve and store at -70°C.

[0083] cDNA was obtained by reverse transcription of 1 μg of RNA using a cDNA synthesis kit (Takara). Then, 20 μL of cDNA was subjected to quantitative PCR (RT-qPCR) analysis using SYBR Green Supermix reagent (Bio-Rad). The gene expression level was standardized to GAPDH. Relative mRNA expression was calculated as 2 -ΔΔC(t) The specificity of RT-qPCR amplification was evaluated by melt curve analysis. The primer sequences used in this study are as follows:

[0084] Ifnb1 F, 5'-atgaactccaccagcagaca-3';

[0085] Ifnb1 R, 5'-cctgaagatctctgctcgga-3'.

[0086] Ifnλ1 F, 5'-cactgagccacattcactcc-3';

[0087] Ifnλ1 R, 5'-caggttggaggtgacagagt-3'.

[0088] Ifit1 F, 5'-gcatcaccttcctctggcta-3';

[0089] Ifit1 R, 5'-tggtgaatttctgcctgcac-3'.

[0090] Il-6 F, 5'-gccagagtccttcagagaga-3';

[0091] Il-6 R, 5'-atggtcttggtccttagcca-3'.

[0092] Mx2 F, 5'-accagagttcagggaagagc-3';

[0093] Mx2 R, 5'-cagacctaccccagcaatga-3'.

[0094] Cxcl10 F, 5'-ccaagtgctgccgtcatttt-3';

[0095] Cxcl10 R, 5'-aatgatctcaacacgtgggc-3'.

[0096] Gapdh F, 5'-aacgaccccttcattgacct-3';

[0097] Gapdh R, 5'-atgttagtggggtctcgctc-3'.

[0098] The synthesized cDNA was detected by fluorescence quantitative PCR using SYBR Green Supermix reagent (Bio-Rad) to detect the relative expression changes of IFNβ, IFNλ, IFIT1, MX2, IL-6 and CXCR10 relative to the housekeeping gene GAPDH, and the specific operation was as follows:

[0099] Q-PCR system:

[0100]

[0101] (1) The target gene PCR solution was prepared according to the above Q-PCR system (3 duplicate wells per sample), and was added into 8 PCR tubes according to the proportion;

[0102] (2) Shake and mix in the shaker, and centrifuge;

[0103] (3) PCR detection was performed using Bio-Rad real-time fluorescence quantitative PCR instrument, and the specific procedure was as follows:

[0104]

[0105] (4) The Ct values of each group of samples were counted, and the relative expression of each group of target genes was calculated with GAPDH gene control.

[0106] The experimental results are shown in Figure 5 .

[0107] Example 8

[0108] Western blotting experiment

[0109] For Figure 2 , we added 500ug of GP-diABZI-RBD particles into 100ul RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150mM NaCl, with protease inhibitor cocktail (Roche)) and broke it in an ultrasonic disruptor for 3min, followed by lysis on ice for 30min.

[0110] The lysate was centrifuged at 12000g, 4°C for 15 minutes. The supernatant was mixed with 5X SDS-PAGE loading buffer and denatured at 100°C for 10 minutes, then loaded into 10% SDS-PAGE gel for electrophoresis to analyze protein samples (10 ug of purified RBD protein as a positive control sample). Then the gel was transferred to a PVDF membrane using a Bio-Rad protein transfer system, and the PVDF membrane was blocked with PBS-0.05% Tween (PBST) and 5% BSA at room temperature for 1 hour. The primary antibody anti-SARS-COV-2 RBD antibody (rabbit anti) was added at a volume ratio of 1:1000 and incubated at 4°C overnight. After incubation with the primary antibody, the PVDF membrane was washed with PBST 4 times. The secondary antibody anti-rabbit HRP (CST) was added at a volume ratio of 1:5000 and incubated at room temperature for 1 h. The PVDF membrane was developed using ECL (Pierce) on a Bio-Rad ChemiDoc imaging system, and the developed results were analyzed using ImageJ (v1.51p) software, and the results are shown in Figure 2

[0111] For Figure 3 As a result, we added 10ul DMSO, 10uM diABZI or 50ug GP-diABZI-RBD particles to the culture plate containing 1*10 7 BMDCs cells, respectively, and collected the cells after co-culturing for 3h. Then PBS was washed once, and the cells were obtained by centrifugation at 500g. The cells were resuspended with 80ul RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150mM NaCl, with protease inhibitor cocktail (Roche)), and then lysed on ice for 30 minutes.

[0112] ​The lysate was centrifuged at 12000g, 4°C for 15 minutes. The supernatant was mixed with 5X SDS-PAGE loading buffer and denatured at 100°C for 10 minutes, and then added to 10% SDS-PAGE gel for electrophoresis of protein samples. Then the gel was transferred to a PVDF membrane using a Bio-Rad protein transfer system, and the PVDF membrane was blocked with PBS-0.05% Tween (PBST) and 5% BSA at room temperature for 1 hour. The primary antibody (anti-phospho-STING, anti-STING, anti-TBK1, anti-IRF3, anti-NF-κB, anti-phospho-TBK1, anti-phospho-IRF3, anti-phospho-NF-κB p65) was added at a volume ratio of 1:1000, and incubated at 4°C overnight. After incubation with the primary antibody, the PVDF membrane was washed with PBST 4 times. The secondary antibody anti-rabbit or mouse HRP (CST) was added at a volume ratio of 1:5000, and incubated at room temperature for 1 hour. The PVDF membrane was developed using ECL (Pierce) on a Bio-Rad ChemiDoc imaging system, and the developed results were analyzed using ImageJ (v1.51p) software, and the results are shown in Figure 3

[0113] Example 9

[0114] T cell immune response experiment

[0115] (1) Take healthy 6-8 week old BALB / c mice, and use the GP-diABZI-RBD, diABZI-RBD and GP-RBD prepared in Example 2 to perform a T cell immune response experiment, with each carrier as an experimental group (the total RBD content of each group is 2.5 ug per mouse), and the total volume is adjusted to 100 ul using PBS; use 100 ul / mouse of PBS as a control group, with three mice in each group;

[0116] (2) The day of injection is recorded as the first day, and the mice are injected intraperitoneally once on the left side of the lower abdomen according to the grouping.

[0117] (3) After 2 weeks of immunization, the spleen of the mouse was removed and ground into a single cell suspension, resuspended with 10 ml of PBS, filtered through a 70 um filter screen, and the tissue debris was removed;

[0118] (4) Centrifuge at 1400 rpm / min for 5 min, add 2 ml of red blood cell lysis solution (Biyun Tian) and lyse for 3 min, then add 8 ml of RMPI-1640 medium, centrifuge at 1400 rpm / min for 5 min, discard the supernatant, and count the cells.

[0119] ​(5) 1*10 7 Cells were resuspended with 1 ml of complete RPMI 1640 medium containing 5 pg / ml RBD and added to the culture dish for further incubation for 24 h.

[0120] (6) After 24 h of incubation, cells were collected by centrifugation at 1400 rpm / min, and the supernatant was transferred to a new ep tube and stored at -80 °C;

[0121] (7) Cells were fixed and permeabilized with Cytofix / Cytoperm kit (BD Biosciences) and stained with anti-CD3, anti-CD4, anti-CD8, anti-IFNy flow cytometry antibodies.

[0122] (8) Stained cells were analyzed using a BD FACS Verse flow cytometer for data acquisition, and then analyzed using FlowJo software (version 10, Tree Star).

[0123] (9) IL-6, IL-10, IFN-γ and IL-2 ELISA kits were used to measure the secretion of IL-6, IL-10, IFN-γ and IL-2 cytokines in the supernatant of spleen cells. Experimental data are shown in Figure 6 IFN-γ, IL-2, IL-6 and IL-10 ELISA experiments were performed strictly according to the kit instructions.

[0124] Experimental results are shown in Figure 6

[0125] Example 10

[0126] Humoral immune response experiment

[0127] To compare the immunogenicity of different SARS VOCs, we further developed six GP-diABZI vaccine vectors to deliver delta (GP-diABZI-RBD-delta, diABZI-RBD-delta), gamma (GP-diABZI-RBD-gamma, diABZI-RBD-gamma) and omicron (GP-diABZI-RBD-omicron, diABZI-RBD-omicron) VOCs, respectively. The preparation steps of the mutant strain vector are referred to Example 2, wherein the RBD is replaced with the RBD of the corresponding mutant strain.

[0128] ​(1) Take healthy BALB / c mice, and use the original strain carrier GP-diABZI-RBD, diABZI-RBD prepared in Example 2 and the mutant strain carrier prepared above to carry out a humoral immune response experiment. Each carrier is set up as an experimental group, and a PBS control group is set up. There are three mice in each group.

[0129] (2) All mice are intraperitoneally inoculated with vaccine carriers containing 2.5ug RBD on day 0 and day 14. Figure 7 A). The serum is collected on day 10, day 28 and day 42 after the first immunization, respectively, and the mice are euthanized after the serum is collected on day 42;

[0130] (3) The levels of SARS-CoV-2 or mutant RBD-specific IgG, IgG1 and IgG2 in the serum of mice are measured by ELISA method, and the specific steps are as follows: ① 200ul of 1.5ug / ml original or mutant RBD protein (Huami Bioengineering Co., Ltd.) is added to a 96-well ELISA plate, and incubated overnight at 4℃. ② The solution in the plate is aspirated, 200ul of PBS is added and washed twice, and then 5% BSA-containing PBS is added for blocking for 1h. ③ Gradient-diluted mouse serum samples 200ul are added to the plate, and incubated at room temperature for 2h. ④ Discard the serum diluent, and wash with PBST solution for 4 times. ⑤ Add 1:10000 diluted HRP-conjugated goat anti-mouse secondary antibody solution, incubate at room temperature for 1h, and wash with PBST for 4 times again. ⑥ Add 50ul of 3,3',5,5'-tetramethylbenzidine (TMB) substrate, and after 15min of reaction, add 50ul of 2M H2SO4 to terminate the reaction. ⑦ Finally, the ELISA plate is read by spectrophotometer at 450nm absorbance, and the data is analyzed by non-linear regression analysis using GraphPad Prism 8.0 software to calculate the endpoint titer. The experimental data is shown in Figure 7 .

[0131] Results

[0132] 1. Confocal immunofluorescence proves that GP-diABZI-RBD particles can be quickly taken up by antigen-presenting cells. RBD is labeled with FITC, and it is found that diABZI-RBD can be quickly taken up by antigen-presenting cells after being wrapped and coupled in GP particles Figure 1 .

[0133] 2. Western blotting experiment proves that RBD protein is successfully wrapped into GPs carrier Figure 2 .

[0134] 3、GP-diABZI-RBD particles can activate the STING pathway in vitro, inducing STING pathway-related protein phosphorylation and STING multimerization in human epithelial cells Figure 3

[0135] 4、GP-diABZI-RBD particles can be rapidly taken up by mouse BMDC cells in vitro Figure 4

[0136] 5、GP-diABZI-RBD vaccine can effectively induce interferon-related gene expression and show a more durable immune response. RT-PCR results show that the expression of proinflammatory cytokine and interferon-stimulated related gene transcripts in mouse BMDCs cells is significantly up-regulated, especially at 12h and later, proving that the GP-diABZI-RBD vaccine activates the STING pathway and shows a more durable immune response Figure 5

[0137] 6、GP-diABZI-RBD vaccination promotes T cell immune response in Balb / c mice

[0138] To investigate whether GP-diABZI-RBD vaccination can drive a strong T cell immune response, we first examined the type 1 T helper cell (Th1) immune response in the spleens of vaccinated mice.

[0139] The experimental results show that the percentage of IFN-γ + CD4 + T cells in GP-diABZI-RBD immunized mice is up-regulated by 5 times Figure 6 A) compared with the PBS control group, and the secretion of IFN-γ + and IL-2 is induced by 15 times by GP-diABZI-RBD immunization. diABZI-RBD / GP-RBD alone only induces moderate Th1 cell activation, as shown by the percentage of IFN-γ + CD4 + T cells and IFN-γ + , IL-2 secretion Figure 6 C).

[0140] We next analyzed the immune response of CD8 T cells in the spleens of vaccinated mice. We observed that the GP-diABZI-RBD vaccine can up-regulate the IFN-γ + CD8 + T population by 10 times Figure 6 B). While the diABZI-RBD vaccine only induces a moderate CD8 T cell response, as shown by the percentage of IFN-γ + CD8​​​+ T cells and the percentage of IL-6, IL-10 secretion Figure 6 C). GP-RBD vaccine alone upregulated CD8 T cells that moderately expressed IFN-γ, however, its efficacy on IL-6, IL-10 production was similar to GP-diABZI-RBD vaccine. In summary, these results indicated that GP-diABZI-RBD vaccination can promote CD4 and CD8 T cell immune responses in Balb / c mice. + T cells and the percentage of IL-6, IL-10 secretion

[0141] 7. GP-diABZI-RBD vaccination induced potent humoral immune responses in Balb / c mice

[0142] Since GP-diABZI-RBD and diABZI-RBD exhibited different activation abilities on T cell immune responses, we utilized these two strategies to test the immunogenicity of Balb / c mice Figure 7 A). Vaccination of GP-diABZI-RBD or diABZI-RBD alone increased IgG1 and IgG2a levels in mice serum by more than 10000-fold Figure 7 B-C). Particularly, GP-diABZI-RBD vaccine vector vaccination induced 10-fold higher IgG1 and IgG2a levels than diABZI-RBD. Antibody detection was performed on sera collected at different time periods in mice immunized with GP-diABZI-RBD or diABZI-RBD, and it was found that GP-diABZI-RBD vector-packaged nanovaccines could produce higher RBD-specific IgG antibodies than vaccines without carrier coating Figure 7 D-F). At the early stage of immunization (day 10), the GP-diABZI-RBD vaccine group had low levels of specific RBD IgG antibody production. However, after two immunizations (days 28 and 42), the specific RBD IgG antibody levels were significantly increased, while vaccines without GP carrier coating could not induce strong specific antibody production. In addition, our results also found that GP carrier vaccine coated with different RBD variants could induce high-titer specific RBD IgG antibodies in mice compared to vaccines without carrier coating.

[0143] It can be seen that compared with diABZI-RBD vaccine alone, GP-diABZI-RBD vaccine showed sustained vaccine release and induced a more persistent immune response in mice. Intraperitoneal injection of GP-diABZI-RBD in mice can cause significant cellular and humoral immune responses.

[0144] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement modes, and all shall be included in the protection scope of the present application. <110> Jinan University <120> A new crown vaccine carrier and application thereof <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ifnb1 F <400> 1 atgaactcca ccagcagaca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ifnb1 R <400> 2 cctgaagatc tctgctcgga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ifnλ1 F <400> 3 cactgagcca cattcactcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ifnλ1 R <400> 4 caggttggag gtgacagagt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ifit1 F <400> 5 gcatcacctt cctctggcta 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ifit1 R <400> 6 tggtgaattt ctgcctgcac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Il-6 F <400> 7 gccagagtcc ttcagagaga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Il-6 R <400> 8 atggtcttgg tccttagcca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mx2 F <400> 9 accagagttc agggaagagc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mx2 R <400> 10 cagacctacc ccagcaatga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cxcl10 F <400> 11 ccaagtgctg ccgtcatttt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cxcl10 R <400> 12 aatgatctca acacgtgggc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh F <400> 13 aacgacccct tcattgacct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh R <400> 14 atgttagtgg ggtctcgctc 20

Claims

1. A COVID-19 vaccine vector, characterized in that... It is prepared through the following steps: (1) Wash the brewing yeast with water, suspend the washed yeast in an alkaline solution and heat and stir, then centrifuge, resuspend the precipitate with water, then adjust the pH with an acid solution, incubate, centrifuge again, wash with water, wash with isopropanol and acetone, and then dry to obtain yeast β-glucan microspheres. (2) The yeast β-glucan microspheres obtained in step (1) are soaked in an aqueous solution containing interferon gene stimulating factor agonist and antigen protein, centrifuged to remove the supernatant, the resulting precipitate is resuspended, centrifuged again, and the resulting precipitate is resuspended in a cross-linking agent for cross-linking. After cross-linking is completed, the precipitate is washed to obtain the COVID-19 vaccine vector. The heating and stirring mentioned in step (1) involves heating to 85-95°C and stirring for 0.5-2 hours; The antigen protein mentioned in step (2) includes at least one of the following: the original strain of novel coronavirus RBD, the delta variant of novel coronavirus RBD, the gamma variant of novel coronavirus RBD, and the omicron variant of novel coronavirus RBD; The interferon gene stimulating factor agonist mentioned in step (2) is diABZI; The mass ratio of yeast β-glucan microspheres to antigen protein in step (2) is 20:1 to 30:1; The mass ratio of the interferon gene stimulating factor agonist to the antigen protein in step (2) is 1:1 to 3:1; The soaking time mentioned in step (2) is 1 to 3 hours; The centrifugation conditions described in step (2) are 2000–4000 rpm for 10–30 minutes; The resuspension conditions described in step (2) are to use a chitosan solution with a mass concentration of 0.1-0.3% to resuspend at room temperature for 0.5-2 hours; The conditions for the second centrifugation described in step (2) are 2000-4000 rpm for 10-30 minutes; The crosslinking conditions described in step (2) are: suspension crosslinking in a genipin crosslinking solution with a mass concentration of 0.05-0.2% for 0.5-2 hours; The washing described in step (2) involves washing with PBS 1 to 3 times.

2. The application of the COVID-19 vaccine vector according to claim 1 in the preparation of a COVID-19 vaccine.

Images