Ophiopogonin D' with enhanced immunity effect and application thereof in viral vaccine adjuvant

Abstract

This invention belongs to the field of viral vaccine preparation technology, and discloses an ophiopogon saponin D' with immune-enhancing effects and its application in viral vaccine adjuvants. The coronavirus RBD vaccine includes coronavirus RBD antigen and vaccine adjuvant, with the adjuvant being ophiopogon saponin D'. The specific preparation method of the coronavirus RBD vaccine involves mixing the coronavirus RBD protein and the vaccine adjuvant, and then suspending them at low temperature. When ophiopogon saponin D' of this invention is used in combination with a vaccine, it can enhance cellular immune response and / or humoral immune response, increase serum-specific IgG, IgG1, and IgG2a titers, and increase the secretion levels of specific IL-1β, TNF-α, and IFN-γ in vivo. Simultaneously, it can promote spleen cell proliferation, lymphocyte subtype differentiation, regulate cytokines, and enhance humoral and cellular immunity. Furthermore, it has advantages such as long-lasting immune protection, no strong immune response, and high safety.

Description

Technical Field

[0001] This invention belongs to the field of viral vaccine preparation technology, specifically relating to an ophiopogon saponin D' with immune-enhancing effects and its application in viral vaccine adjuvants. Background Technology

[0002] Vaccines are the most effective way to prevent, control, and even eradicate infectious diseases. Currently, vaccines based on the coronavirus spike protein (RBD) subunit have clearly defined antigenic components, high purity, and good safety profiles. They also reduce the adverse reactions that may occur with artificial attenuation or inactivation methods used in vaccine preparation, such as low-grade fever, nausea, and dizziness, thus attracting wider attention from researchers. However, these vaccines still have certain limitations, such as weak immunogenicity, requiring adjuvants to produce effective protection in the population.

[0003] Naturally derived plant immunostimulant molecules possess numerous unique advantages, such as wide availability, good biocompatibility, and the ability to promote dendritic cell maturation and immune cytokine secretion, significantly enhancing the immune response. These advantages make them a current hot topic in adjuvant research. Ophiopogon japonicus, a traditional Chinese medicine, is known for its yin-nourishing, fluid-generating, lung-moistening, and heart-clearing effects. Ancient medical texts such as the *Shennong Bencao Jing* and *Yixue Zhongzhong Canxi Lu* praise it highly, stating that "long-term consumption of Ophiopogon japonicus leads to a light body, longevity, and no hunger." Saponins are composed of a lipophilic triterpenoid core with one or more oligosaccharide chains attached to its sides. They possess the ability to regulate the cell-mediated immune system and exhibit low-dose adjuvant activity. Ophiopogon japonicus saponins are important bioactive substances in Ophiopogon japonicus extract, exhibiting immunomodulatory, antioxidant, and anti-inflammatory effects.

[0004] Ophiopogonin D'(OPD'), a rare naturally occurring C29 steroidal glycoside isolated from the tubers of Ophiopogon japonicus, is an isomer of Ophiopogonin D(OPD) and is a naturally occurring plant-derived immunostimulatory molecule with vaccine adjuvant potential. However, there are currently no research reports, either domestically or internationally, on the immune response and mechanism of Ophiopogonin D' enhancing coronavirus protein vaccines (RBD). Summary of the Invention

[0005] This invention discovers that the naturally derived plant immunostimulant molecule Ophiopogon japonicus saponin D' can significantly enhance the immune response of coronavirus RBD vaccines and can be used as an adjuvant for coronavirus RBD antigens to prepare coronavirus RBD vaccines.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] One aspect of the present invention provides a coronavirus RBD vaccine, which includes a coronavirus RBD antigen and a vaccine adjuvant, wherein the vaccine adjuvant is Ophiopogon japonicus saponin D'.

[0008] Preferably, the mass fraction ratio of coronavirus RBD antigen to vaccine adjuvant is 1:1, and the adjuvant range is 20-200 μg / dose.

[0009] Preferably, the coronavirus RBD vaccine is in the form of an injection, an oral dose, an inhalation dose, or a skin patch.

[0010] The ophiopogonin D' in this invention is isolated from the tuber of Ophiopogon japonicus and is a rare naturally occurring C29 steroidal glycoside. OPD' has the English name Ophiopogonin D', CAS number 65604-80-0, molecular weight 855.017, and density 1.4±0.1 g / cm³. 3 The molecular formula is C 44 H 70 O 16 OPD' is an isomer of Ophiopogon saponin D (OPD), which is an existing structure, and its structural formula is consistent with the structure disclosed in patent publication number CN110652519A.

[0011] The coronavirus RBD antigen, or receptor-binding domain (RBD) antigen of the coronavirus spike protein (S protein), is an important structural protein fragment of the coronavirus. The RBD is the primary region that induces neutralizing antibodies. Neutralizing antibodies specifically recognize and bind to the RBD, thereby preventing the virus from binding to host cells and inhibiting viral infection.

[0012] Another aspect of the present invention provides a method for preparing a coronavirus RBD vaccine, the specific steps of which are: mixing coronavirus RBD protein and vaccine adjuvant, and then suspending them at low temperature.

[0013] Preferably, the concentration of coronavirus RBD protein is 450–550 μg / mL.

[0014] In another aspect, the present invention provides the application of a coronavirus RBD vaccine in the preparation of a formulation that enhances immune response.

[0015] In another aspect, the present invention provides the use of a coronavirus RBD vaccine in the preparation of a formulation that enhances the titers of serum-specific IgG, IgG1 and IgG2a.

[0016] In another aspect, the present invention provides the application of Ophiopogon japonicus saponin D' in the preparation of viral vaccine adjuvants, wherein the viral vaccine adjuvant is a vaccine adjuvant based on coronavirus antigens.

[0017] In another aspect, this invention provides the application of Ophiopogon japonicus saponin D' in the preparation of a viral vaccine, wherein the viral vaccine is a vaccine based on coronavirus antigens.

[0018] In another aspect, the present invention provides the use of Ophiopogon japonicus saponin D' in the preparation of a formulation for enhancing the immune response of a viral vaccine, wherein the viral vaccine is a coronavirus antigen-based vaccine.

[0019] The beneficial effects of this invention are as follows: When the Ophiopogon japonicus saponin D' of this invention is used in combination with a vaccine, it can enhance the cellular immune response and / or humoral immune response, increase the serum specific IgG, IgG1 and IgG2a titers, and increase the secretion levels of specific IL-1β, TNF-α and IFN-γ in the body. At the same time, it can promote spleen cell proliferation, lymphocyte subtype differentiation, regulate cytokines, and enhance the body's humoral and cellular immunity. It also has the advantages of long-lasting immune protection, not inducing strong immune responses and high safety. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. The drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort:

[0021] Figure 1 Figure showing the effect of OPD' on the viability of BMDCs;

[0022] Figure 2 This is a graph showing the changes in body weight of mice after immunization.

[0023] Figure 3 Comparison of BUN levels in the serum of mice in different groups after immunization;

[0024] Figure 4 Comparison of SCr levels in the serum of mice in different groups after immunization;

[0025] Figure 5 Comparison of AST levels in the serum of mice in different groups after immunization;

[0026] Figure 6 A comparison of ALT levels in the serum of mice in different groups after immunization;

[0027] Figure 7 This is a comparison chart of IgG levels 21 days after secondary immunization;

[0028] Figure 8 A comparison chart of IgG levels 14 days after the last immunization;

[0029] Figure 9A comparison chart of IgG1 levels 14 days after the last immunization;

[0030] Figure 10 A comparison chart of IgG2a levels 14 days after the last immunization;

[0031] Figure 11 A comparison chart of IgG2a / IgG1 ratios 14 days after the last immunization;

[0032] Figure 12 Comparison of IL-1β detection results in the supernatant of spleen lymphocyte stimulation in mice after immunization;

[0033] Figure 13 Comparison of TNF-α detection results in the supernatant of spleen lymphocyte stimulation in mice after immunization;

[0034] Figure 14 Comparison of IFN-γ detection results in the supernatant of spleen lymphocyte stimulation in mice after immunization;

[0035] Figure 15 Comparison of IFN-γ spot-forming cells in each group;

[0036] Figure 16 A comparison of the number of IFN-γ spot-forming cells in spleen cells as detected by ELISpot on day 14 post-immunization;

[0037] Figure 17 CLSM diagram of APC-OVA taken by BMDCs;

[0038] Figure 18 Flow cytometry plot of APC-OVA uptake by BMDCs;

[0039] Figure 19 A comparison of the average fluorescence intensity of OVA and OVA / RBD within BMDCs;

[0040] Figure 20 Clustering heatmap of differentially expressed genes;

[0041] Figure 21 Volcanic diagrams of the RBD / OPD' group and the DEGs of the RBD group;

[0042] Figure 22 This is a graph showing the enrichment of allogeneic GO genes.

[0043] Figure 23 This is a graph showing the KEGG enrichment analysis of differentially expressed genes. Detailed Implementation

[0044] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0045] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of a feature, number, operation, material, or combination thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0046] A coronavirus RBD vaccine comprises a coronavirus RBD antigen and a vaccine adjuvant, wherein the vaccine adjuvant is Ophiopogon japonicus saponin D' (hereinafter referred to as OPD'). In this invention, the mass fraction ratio of the coronavirus RBD antigen to the vaccine adjuvant is 1:1 (w / w, 1:1).

[0047] In practical use, coronavirus RBD vaccines are available in injection, oral, inhalation, or transdermal patch formulations. Currently, commercially available viral vaccines are administered via injection, oral, inhalation, or transdermal routes. Therefore, the viral vaccine in this invention can be administered as an injection, oral, inhalation, or transdermal patch, and the preparation methods for different formulations can follow those known in the art. Compared to other formulations, injection formulations offer advantages such as precise drug delivery, rapid onset of action, avoidance of the first-pass effect, and good stability. Therefore, in this invention, the preferred formulation for the coronavirus RBD vaccine is an injection.

[0048] In this invention, the coronavirus RBD antigen can be SARS-CoV-2 (2019-nCoV) RBD antigen, SARS-CoV RBD antigen, MERS-CoV RBD antigen, HCoV-HKU1 RBD antigen or HCoV-229E RBD antigen.

[0049] The preparation method of the aforementioned coronavirus RBD vaccine is as follows: A method for preparing a coronavirus RBD vaccine includes the following steps: mixing coronavirus RBD protein at a concentration of 500 μg / mL and OPD' at a mass fraction ratio of 1:1, and then suspending them at 4°C using a rotary suspending apparatus for 12 hours. After suspension, the mixture is placed in a refrigerator and refrigerated at 2–8°C for later use.

[0050] The coronavirus RBD vaccine of this invention can be used in the preparation of formulations that enhance immune responses. The immune response capabilities in this invention include both cellular and humoral immune responses.

[0051] It should be noted that the OPD' in the coronavirus RBD vaccine of this invention has an adjuvant effect. OPD' can effectively promote the body to produce RBD-specific IgG, IgG1, and IgG2a, increase the serum-specific IgG, IgG1, and IgG2a titers, promote Th1 and Th2 responses, and stimulate the body to produce cellular and humoral immunity.

[0052] It should be noted that the OPD' in the coronavirus RBD vaccine of this invention has an adjuvant effect. The coronavirus RBD vaccine prepared by combining OPD' with RBD can induce high levels of Th1 cell immune responses, including interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), and interferon-gamma (IFN-γ). The number of cells that promote the secretion of IFN-γ by lymphocytes was significantly increased in the group.

[0053] In some embodiments, OPD' can be used in the preparation of viral vaccine adjuvants, which are vaccine adjuvants based on coronavirus antigens.

[0054] In some embodiments, OPD' can be used in the preparation of viral vaccines, which are vaccines based on coronavirus antigens.

[0055] In some embodiments, OPD' can be used in the preparation of formulations for enhancing the immune response of viral vaccines, which are based on coronavirus antigens.

[0056] OPD can significantly enhance the immune response stimulated by viral antigens (such as coronavirus RBD), so it can be applied in the preparation of viral vaccines. In this invention, the coronavirus antigen is preferably the coronavirus RBD antigen. Furthermore, this viral vaccine can be a preventative vaccine or a therapeutic vaccine.

[0057] In practice, the aforementioned coronavirus RBD antigens are SARS-CoV-2 RBD antigen, SARS-CoV RBD antigen, MERS-CoV RBD antigen, HCoV-HKU1 RBD antigen, or HCoV-229E RBD antigen.

[0058] It should be noted that the viral vaccine in this invention is based on the coronavirus RBD antigen, so the virus is a coronavirus that includes the RBD protein, and it is particularly effective against SARS-CoV-2 virus.

[0059] It should be noted that OPD' in this invention has an adjuvant effect, which helps to promote BMDCs to take up more antigens and significantly improve the antigen uptake rate of BMDCs.

[0060] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0061] experiment

[0062] I. Experimental Materials

[0063] 1. Laboratory animals

[0064] Female Balb / c mice, SPF grade, 6-8 weeks old, weighing 18-20g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Animal Ethics Committee of Army Medical University, approval number: AMUWEC20223007.

[0065] 2. Main reagents and instruments

[0066] RBD antigen was provided by Chongqing Zhifei Biological Products Co., Ltd.; RBD peptide library was provided by Shanghai Botai Biotechnology Co., Ltd.; Ophiopogon japonicus saponin D' was purchased from Chengdu Purifa Technology Development Co., Ltd.; AL(OH)3 adjuvant (aluminum adjuvant, i.e., Alum) was purchased from Invivogen; enzyme-linked immunosorbent assay (ELISA) plate was purchased from Corning; IFN-γ ELISA kit, TNF-α ELISA kit, IL-17A ELISA kit, and ELISpot Plus: Mouse IFN-γ (ALP) kit were purchased from Daco Biotechnology Co., Ltd.; full-wavelength microplate reader was purchased from Bio-Rad; ELISpot plate reader was purchased from AID GmbH, Germany; all instruments used were provided by the National Engineering Research Center for Immunobiological Products and the University Central Laboratory of the Department of Pharmacy and Laboratory Medicine, Army Medical University.

[0067] II. Experimental Methods and Results

[0068] 1. In vitro and in vivo safety evaluation of OPD immunostimulatory molecules

[0069] (1) In vitro safety evaluation experimental process

[0070] Mice euthanized under anesthesia were disinfected by immersion in 75% alcohol. The tibia and femur were removed using sterile instruments. Bone marrow was repeatedly washed out using 1 mL of sterile PBS. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded. The cells were resuspended in erythrocyte lysis buffer and lysed for 5 min. Lysis was then terminated by adding PBS, followed by centrifugation at 1500 rpm for 5 min and discarding the supernatant. The cells were resuspended in RPMI 1640 complete medium containing 20 ng / mL GM-CSF and 10 ng / IL-4 and cultured. Half the medium was replaced on days 2 and 4. On day 6, mature bone marrow-derived dendritic cells (BMDCs) were collected and counted, with a count of 2.5 × 10⁻⁶. 4 Cells were added to 96-well plates and allowed to rest for 6 hours. Then, different concentrations of OPD were added and the cells were cultured for 48 hours. The supernatant was discarded, and 100 μL of 10% CCK-8 reagent was added to each well. The plates were incubated in the dark for 2 hours, and the OD values ​​were read using a microplate reader. 450nm Statistical analysis was then performed, and cell viability was calculated using the following formula:

[0071] Cell viability (%) = [OD 实验 -OD 空白 ] / [OD 对照 -OD 空白 ]×100

[0072] OD 实验 : Indicates the absorbance of the well containing cells, CCK-8 reagent, and OPD' solution.

[0073] OD 空白 : Indicates the absorbance of wells containing culture medium and CCK-8 reagent but without cells.

[0074] OD 对照 : Indicates the absorbance of wells containing cells and CCK-8 reagent but not OPD' solution.

[0075] (2) In vivo safety evaluation experimental process

[0076] Mice were observed for survival and weight monitoring every morning after a single injection of the vaccine. On the third day, mouse serum was collected and blood urea nitrogen (BUN) and creatinine (Scr) were measured using an automated biochemical analyzer to evaluate kidney function, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to evaluate liver function.

[0077] (3) Safety evaluation results

[0078] First, BMDCs were used to detect the cytotoxicity of OPD', and the specific results are as follows: Figure 1As shown. According to Figure 1 It is evident that when the dose of OPD' is less than 5 μg / mL, it is non-toxic to BMDCs, and the survival rate is 100%.

[0079] Next, the in vivo toxicity of OPD was evaluated. Weight monitoring of mice after a single intramuscular injection yielded the following results: Figure 2 As shown. According to Figure 2 It can be seen that the body weight of mice decreased slightly within 72 hours after OPD' immunization, but rebounded after 96 hours.

[0080] The levels of blood urea nitrogen, creatinine, alanine aminotransferase, and aspartate aminotransferase in the serum of immunized mice were detected using a biochemical analyzer to reflect kidney and liver damage and toxicity. Figure 3-6 As shown. According to Figure 3-6 As can be seen, all four biochemical indicators were within the normal safety range, and there were no significant differences between the groups (P>0.05). These results indicate that OPD' has good safety and can be applied to subsequent vaccine development.

[0081] 2. Preparation of Coronavirus RBD Vaccine

[0082] The coronavirus RBD protein at a concentration of 500 μg / mL was mixed with OPD' and then suspended at 4°C for 12 h using a rotary suspending apparatus. After suspension, it was placed in a refrigerator and refrigerated at 5°C for later use.

[0083] 3. Animal immunization

[0084] Twenty experimental mice were randomly divided into four groups of five each, named: PBS group (PBS), RBD group (RBD), RBD / OPD' group, and RBD / Alum group. RBD was administered at 5 μg / mouse, and adjuvant at 100 μg / mouse. Each group was injected with 100 μL of a different type of injection solution (PBS solution or vaccine). Immunization was performed intramuscularly on days 0, 21, and 42. Mice were sacrificed on day 56. The specific solutions or vaccines injected into each group are as follows:

[0085] The specific sources or preparation methods of the above-mentioned injection solutions are as follows:

[0086] The RBD vaccine of the RBD group is a vaccine prepared from the coronavirus RBD protein of the present invention. The specific preparation method is as follows: the coronavirus RBD protein with a concentration of 500 μg / mL is suspended in a rotary suspending apparatus at a temperature of 4°C for 12 h.

[0087] The preparation method of the coronavirus RBD vaccine of the RBD / OPD' group is as follows: the coronavirus RBD protein with a concentration of 500 μg / mL and OPD' are mixed at a mass fraction ratio of 1:1, and then suspended at 4°C using a rotary suspending apparatus for 12 h.

[0088] The preparation method of the coronavirus RBD vaccine for the RBD / Alum group is as follows: coronavirus RBD protein with a concentration of 500 μg / mL and aluminum adjuvant are mixed at a mass ratio of 1:1, and then suspended at 4°C using a rotary suspending apparatus for 12 h.

[0089] 4. ELISA detection of serum RBD-specific antibodies and their subtypes.

[0090] (1) Detection process

[0091] Based on the animal immunization procedure described in point 3 above, blood was collected from the tail vein of mice on day 21 after the second immunization and day 14 after the last immunization. Whole blood was collected from each group of mice. The collected whole blood was then centrifuged at 3000 rpm for 10 min, and the serum was separated and stored at -80℃. RBD antigen was diluted to 10 μg / mL with coating buffer and added to ELISA plates at 100 μL / well. The plates were coated overnight at 4℃ and then washed. Blocking buffer (1% BSAPBST) was added to 250 μL / well, and the plates were blocked at 37℃ for 2 h and then washed. Post-immunization mouse serum was diluted and added to the wells, followed by serial dilutions. The plates were incubated at 37℃ for 1 h and then washed. HRP-labeled goat anti-mouse IgG, IgG1, and IgG were added to each well at 100 μL / well. 2a Add (1:10000) to the well plate, incubate at 37°C for 40 min, then wash the plate; add 100 μL / well TMB to the well plate, incubate in the dark for 10 min, then add 50 μL / well stop solution; read the OD value using a microplate reader. 450nm Statistical analysis was then performed. Positive reference serum OD 450nm ≥0.5; negative reference serum OD 450nm ≤0.1; Cut-off value = negative reference serum OD 450nm ×2.1; with OD 450nm The ≥Cut-off value is used as the threshold for determining a positive result, and the corresponding dilution is the antibody titer of the sample.

[0092] (2) Test results

[0093] To investigate the ability of OPD' to induce antigen-specific immune responses in vivo, serum titers of RBD-specific IgG, IgG1, and IgG2a antibodies were measured on day 21 after the second immunization and day 14 after the last immunization. Specific results are as follows: Figure 7-11 As shown. According to Figure 7-10The results show that OPD' effectively promoted the production of RBD-specific IgG, IgG1, and IgG2a in mice, with significant differences compared to the RBD group (P < 0.05, P < 0.01, P < 0.0001). The levels of RBD-specific IgG and IgG1 induced by OPD' were comparable to those in the Alum group, while the level of RBD-specific IgG2a induced by OPD' was significantly higher than that in the Alum group (P < 0.05), and the IgG2a / IgG1 ratio was also significantly higher in the OPD' group than in the Alum group (P < 0.01). Figure 11 The above results indicate that OPD' has an adjuvant effect, which can promote Th1 and Th2 responses and stimulate the body to produce cellular and humoral immunity.

[0094] 5. ELISA detection of specific IL-1β, TNF-α, and IFN-γ secretion levels in spleen lymphocyte culture supernatant.

[0095] (1) Detection process

[0096] Following the animal immunization procedure described in point 3 above, 14 days post-immunization, the supernatant of mouse spleen lymphocyte culture was collected, and the total amount of cytokines secreted by the spleen cells was detected using ELISA. Specifically, the cell concentration was 1×10⁻⁶ cells / mL. 7 On day 14 post-last immunization, 500 μL / well of mouse spleen lymphocyte suspension was added to a 24-well plate, followed by 500 μL / well of RBD peptide library, bringing the final concentration to 20 μg / mL. The 24-well plates were placed in a cell culture incubator containing 5% CO2 at 37°C. After 48 h of culture, the cell supernatant was collected, centrifuged at 1500 rpm for 5 min, and the cell pellet was separated. The supernatant was then stored at -80°C. The levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-gamma (IFN-γ) were detected using an ELISA kit.

[0097] (2) Test results

[0098] The specific detection results of the above ELISA kit are as follows: Figure 12-14 As shown, according to Figure 12-14 The results showed that the levels of specific IL-1β, TNF-α, and IFN-γ in mice were significantly increased after immunization with RBD / OPD' (P<0.01, P<0.001, P<0.0001), indicating that OPD' can effectively induce the production of cytokines for Th1 cell immunity, further demonstrating that OPD' can induce a strong T cell immune response.

[0099] 6. ELISpot measurement of IFN-γ secretion levels

[0100] (1) Detection process

[0101] To assess the cellular immune response in mice after immunization, a suspension of mouse spleen lymphocytes was isolated 14 days after the final immunization, and the number of IFN-γ-secreting cells was measured using ELISApot. Specifically, following the animal immunization procedure described in point 3 above, mice were anesthetized with isoflurane and then sacrificed 14 days after the final immunization; a suspension of spleen lymphocytes was isolated, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 cells / mL. Add 100 μL of cell suspension to a pre-activated ELISpot-IFN-γ plate, then add 100 μL of 40 μg / mL RBD peptide library and mix. Inject 2 μL of phorbol ester solution into the positive control wells and 100 μL of RPMI 1640 complete medium into the negative control wells. Then, incubate the plates in a cell culture incubator containing 5% CO2 at 37°C for 48 h. Discard the medium, wash five times with 250 μL / well PBS solution, add 100 μL / well of detection antibody (R4-6A2-biotin, 1:1000 dilution), and incubate at room temperature for 2 h. Wash five times with 250 μL / well PBS solution, add 100 μL / well of streptavidin-horseradish peroxidase (1:1000), and incubate at room temperature for 1 h. Wash five wells with 250 μL / well PBS solution. Add 100 μL / well TMB chromogenic solution. When obvious spots appear, rinse the plate with distilled water to stop the chromogenic reaction. After the plate has dried, place it in an ELISpot plate reader to read the spots and acquire images.

[0102] (2) Test results

[0103] The specific results of the above ELISpot measurement are as follows: Figure 15 As shown, according to Figure 15 The results show that, after stimulation with the RBD peptide library, the RBD / OPD' group had a significantly higher number of cells secreting IFN-γ compared to the RBD group in the immunoblotting patterns of each group. Figure 16 The bar chart, generated after counting the number of spots, shows that the number of IFN-γ-secreting splenic lymphocytes in the RBD / OPD' group was significantly higher than that in the RBD group (P < 0.001). These results indicate that OPD', as an adjuvant, can significantly enhance the antigen-specific T-cell immune response in the spleen of mice after immunization.

[0104] 7. Evaluation of in vitro antigen uptake by DCs

[0105] (1) Evaluation process

[0106] DC2.4 with 2×10 5Cells were seeded into confocal culture dishes, and then APC-OVA and APC-OVA / OPD' were added (APC-OVA was purchased from MCE, catalog number: HY-NP053; APC-OVA / OPD' was prepared by physically mixing APC-OVA and OPD'). The dishes were then incubated in a cell culture incubator containing 5% CO2 at 37°C for 24 hours in the dark. After discarding the culture medium, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min; washed twice with PBS and permeabilized with 0.2% Triton-X100 solution for 10 min; washed twice again with PBS and incubated with 100 nmol / L phalloidin for 10 min; washed twice with PBS and incubated with DAPI staining solution for 10 min; then washed three times with PBS and added with an appropriate amount of PBS solution for observation using a confocal microscope.

[0107] BMDCs with 1×10 6 Cells were seeded into 24-well plates, and PBS, APC-OVA, and APC-OVA / OPD' were added respectively. The plates were then incubated in a cell culture incubator containing 5% CO2 at 37°C in the dark for 24 hours. After washing the cells twice with 1 mL PBS, the cells were resuspended in 0.5 mL PBS in 1.5 mL EP tubes, and the uptake of antigens by BMDCs was detected by flow cytometry.

[0108] (2) Test results

[0109] To investigate the mechanism by which OPD' exerts its adjuvant effect, this invention hypothesizes that OPD' facilitates antigen uptake by dendritic cells (DCs). Therefore, flow cytometry and laser confocal microscopy were used to study the effect of OPD' on the antigen uptake capacity of BMDCs. Intracellular tracking was performed using APC-labeled model antigen OVA (APC-OVA), such as... Figure 17 As shown ( Figure 17 In this study, Phalloidin represents the cytoskeleton; DAPI represents the cell nucleus; and APC-OVA represents the antigen. Free APC-OVA has limited effective entry into cells, but co-incubation of OPD, APC-OVA, and BMDCs resulted in more antigen being efficiently taken up by the BMDCs. Flow cytometry was then used to quantitatively compare the differences in antigen phagocytosis capacity of BMDCs, and the results are as follows: Figure 18 As shown in Figure 18, the antigen uptake rate of BMDCs significantly increased after treatment with the OVA / OPD group, and the difference was statistically significant (P < 0.01). The average fluorescence intensity of OVA and OVA / RBD in BMDCs is shown in Figure 18. Figure 19 As shown.

[0110] 8. Transcriptome sequencing

[0111] (1) Sequencing process

[0112] To explore the mechanism by which OPD' enhances the immune effect, this invention utilizes transcriptomics. Specifically, six experimental mice were randomly divided into two groups: the RBD group and the RBD / OPD' group, with three mice in each group. The RBD group received 5 μg / mouse, and the RBD / OPD' group received 100 μg / mouse. Forty-eight hours after intramuscular injection, the mice were sacrificed, and bilateral inguinal lymph nodes were collected. The samples were sent to Guangzhou Epigenetics Technology Co., Ltd. for transcriptome sequencing. The distribution of differentially expressed genes in GeneOntology was studied using the GO database to elucidate the manifestation of gene function differences in the experimental samples. Using the KEGG database, pathway significant enrichment was used to identify the most important biochemical metabolic pathways and signal transduction pathways involved in the differentially expressed genes.

[0113] (2) Sequencing results

[0114] The results of differentially expressed genes (DEGs) screening are as follows: Figure 20 As shown. According to Figure 20 The results showed that, compared with the RBD group, the RBD / OPD' group had 83 differentially expressed genes, of which 67 were upregulated (LFC>1, P<0.05) and 16 were downregulated (LFC<-1, P<0.05). The differentially expressed gene clustering diagram is shown below. Figure 21 As shown, it visually displays the different gene expression profiles between the RBD / OPD' group mice and the RBD group mice. The number of genes corresponding to GO annotations was counted, and then plotted according to biological processes, cellular components, and molecular functions. The results are as follows: Figure 22 As shown. Then, through analysis of the Kyotoencyclopedia of genes and genomes (KEGG) pathway, such as... Figure 23 As shown, the RBD group alone showed an enrichment of PPAR signals compared to the RBD / OPD' group, suggesting that OPD' may have activated the PPAR signaling pathway.

[0115] 9. Statistical Analysis

[0116] Statistical analysis was performed using GraphpadPrism 9.5, and the data are presented as follows: The t-test was used to compare two groups of data, and the one-way ANOVA was used to compare multiple groups of data. P < 0.05 was considered statistically significant.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A coronavirus RBD vaccine, characterized in that, This includes coronavirus RBD antigen and vaccine adjuvant, with the vaccine adjuvant being Ophiopogon japonicus saponin D'.

2. The coronavirus RBD vaccine according to claim 1, characterized in that, The mass fraction ratio of coronavirus RBD antigen to vaccine adjuvant is 1:1, and the adjuvant range is 20-200 μg / dose.

3. The coronavirus RBD vaccine according to claim 1 or 2, characterized in that, The coronavirus RBD vaccine is available in injection, oral, inhalation, or skin patch formulations.

4. The method for preparing the coronavirus RBD vaccine according to any one of claims 1 to 3, characterized in that, The specific steps are as follows: mix the coronavirus RBD protein and the vaccine adjuvant, and then suspend them at low temperature.

5. The method for preparing the coronavirus RBD vaccine according to claim 4, characterized in that, The concentration of coronavirus RBD protein is 450–550 μg / mL.

6. The use of the coronavirus RBD vaccine as described in any one of claims 1 to 3 in the preparation of formulations that enhance immune response.

7. Use of the coronavirus RBD vaccine according to any one of claims 1 to 3 in the preparation of formulations that enhance serum-specific IgG, IgG1 and IgG2a titers.

8. Application of Ophiopogon japonicus saponin D' in the preparation of viral vaccine adjuvants, which are vaccine adjuvants based on coronavirus antigens.

9. Application of Ophiopogon japonicus saponin D' in the preparation of viral vaccines, which are vaccines based on coronavirus antigens.

10. Application of Ophiopogon japonicus saponin D' in the preparation of formulations for enhancing the immune response of viral vaccines, which are based on coronavirus antigens.

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