A mucosal immune enhancer for improving intestinal DC targeting, and its preparation method and application

Abstract

The present invention discloses a mucosal immune enhancer for improving intestinal DC targeting, wherein the mucosal immune enhancer for improving intestinal DC targeting contains 0.01-1 mg / mL of cyclic di-AMP and 0.4-2 mg / mL of CTA-CD154. The synergistic effect of cyclic di-AMP and CTA-CD154 is utilized to improve the ability to target intestinal DC, while significantly enhancing the body's mucosal immune response, and can increase intestinal SIgA by more than 5 times. The present invention also discloses the use of the mucosal immune enhancer for improving intestinal DC targeting in an inactivated vaccine. When used in an inactivated vaccine for porcine epidemic diarrhea, it can increase the serum neutralizing antibodies of the animal body by more than 6 times. The preparation method is simple and has value for large-scale promotion and application.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and in particular relates to a mucosal immunopotentiator for improving targeted intestinal DCs, a preparation method and application thereof. Background Art

[0002] The main characteristics of coronaviruses are high recombination rates, rapid mutation, and the inability of vaccine strains to provide adequate protection against newly circulating strains. Porcine epipedemic diarrhea (PED), belonging to the Coronaviridae family and the genus Coronavirus, primarily infects the intestinal tract of pigs. Pigs of all stages are susceptible, with infection being particularly severe in suckling piglets, where mortality rates can reach 100%. Currently, no effective vaccine can provide adequate immune protection for piglets because existing vaccines fail to elicit an effective mucosal immune response. Circulating antibodies or serum neutralizing antibodies alone are inadequate to protect against viral infection, leading to diarrhea, dehydration, or even death. Research has shown that SIgA plays a crucial role in mucosal immune responses. It can bind to pathogens and prevent them from adhering to cell surfaces, making it the body's "border defense force" against infection. Therefore, clinical production urgently requires an immunopotentiator that can increase the titer of mucosal immune antibodies in existing PED vaccines.

[0003] The intestine is a target organ for PEDV infection. As an immune organ, the intestine is closely linked to antigen-presenting cells (APCs), a key factor in regulating immune responses. Dendritic cells (DCs) are recognized as the most powerful professional APCs in the body, involved in antigen recognition, processing, and presentation. They are widely distributed in various tissues, such as the gastrointestinal epithelium, the lamina propria, and associated lymphoid tissues, and serve as the primary guardians of the intestinal mucosal immune system. As the link between innate and adaptive immunity, DCs can activate Th cells and B cells, promote the differentiation of effector T / B cells, and induce a cascade of specific immune responses. Therefore, designing an immunopotentiator that can enhance the ability of DCs to target the intestine and effectively boost mucosal immune responses would provide new technical support for the prevention and treatment of diseases that primarily induce mucosal immune responses. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a mucosal immune enhancer that improves the targeting of intestinal DCs.

[0005] The technical problem that the present invention also aims to solve is to provide the application of the above-mentioned immunopotentiator in inactivated vaccines.

[0006] The technical problem that the present invention also aims to solve is to provide a method for preparing an inactivated porcine epidemic diarrhea vaccine.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a mucosal immune enhancer for improving targeted intestinal DCs, wherein the mucosal immune enhancer for improving targeted intestinal DCs contains 0.01~1 mg / mL of cyclic diadenylic acid and 0.4~2 mg / mL of CTA-CD154.

[0008] The present invention also provides the use of the above-mentioned mucosal immunity enhancer for improving intestinal DC targeting in inactivated vaccines.

[0009] The present invention also provides an inactivated vaccine comprising the above-mentioned immunopotentiator.

[0010] Furthermore, the inactivated vaccine also includes an oil phase solution.

[0011] Furthermore, the oil phase solution is ISA206 adjuvant.

[0012] Furthermore, the inactivated vaccine also includes an inactivated antigen solution.

[0013] Furthermore, the antigen solution is an inactivated porcine epidemic diarrhea virus solution.

[0014] The present invention also provides a method for preparing an inactivated porcine epidemic diarrhea vaccine, comprising uniformly mixing an immunopotentiator containing cyclic diadenylic acid and CTA-CD154 with an inactivated antigen solution to obtain an aqueous solution; and mixing and emulsifying the aqueous solution with an oily solution to obtain the inactivated vaccine.

[0015] Furthermore, the preparation method of the immunopotentiator is to mix cyclic di-AMP and CTA-CD154, and let it stand overnight to obtain the immunopotentiator.

[0016] Furthermore, the volume ratio of the aqueous phase solution to the oil phase solution is 1:1.

[0017] Furthermore, the antigen solution is an inactivated porcine epidemic diarrhea virus solution.

[0018] Beneficial Effects: Compared with existing technologies, the present invention offers the following significant advantages: The immunopotentiator disclosed herein contains cyclic di-AMP (c-di-AMP) and CTA-CD154. Leveraging their synergistic effect, c-di-AMP effectively enhances the ability of CTA-CD154 to target intestinal DCs, while significantly boosting the body's mucosal immune response, increasing intestinal SIgA by more than fivefold. The inactivated porcine epidemic diarrhea vaccine disclosed herein can increase serum neutralizing antibodies in animals by more than sixfold. Furthermore, the preparation method is simple, making it promising for large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The number of DCs in the mesenteric lymph nodes and intestinal lamina propria after immunization with the inactivated vaccine containing c-di-AMP and CTA-CD154 of the present invention is counted; wherein A is the total DCs in the mesenteric lymph nodes, B is the total DCs in the mesenteric lymph nodes + DC, C is the total DC in the intestinal lamina propria, D is the CD103 + DC;

[0020] Figure 2 The results of intestinal sIgA antibody detection after immunization with the inactivated vaccine containing c-di-AMP and CTA-CD154 of the present invention;

[0021] Figure 3 The results are serum neutralizing antibody detection results after immunization with the inactivated vaccine containing c-di-AMP and CTA-CD154 of the present invention. Implementation Method

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] Experimental Materials

[0024] c-di-AMP was purchased from InvivoGen; enhancer CTA-CD154 (laboratory homemade, published in ZL201710969969.3); inactivated PEDV-NJ strain (seed virus titer 10 7.0 TCID 50 / mL) and VERO cells were provided by the Animal Vaccine Immunity Technology Innovation Team of the Institute of Animal Immunoengineering, Jiangsu Academy of Agricultural Sciences; 6-week-old ICR mice were purchased from the Center for Comparative Medicine of Yangzhou University; ISA206 adjuvant was purchased from SEPPIC, France; FITC anti-mouse CD11c, APC anti-mouse MHCII, and PE anti-mouse CD103 were purchased from BD Biosciences; and the mouse (sIgA) ELISA detection kit was purchased from ELISA-linked (Shanghai) Bioreagent Technology Co., Ltd.

[0025] 2. Vaccine preparation

[0026] The preparation method of inactivated vaccine A is as follows: inactivated porcine epidemic diarrhea virus liquid (the strain is PEDV-NJ strain, the virus titer before inactivation is 10 7.0 TCID 50 / mL) was mixed with adjuvant ISA 206 at a volume ratio of 1:1, and emulsified using an emulsifier to obtain inactivated vaccine A.

[0027] Prepare an inactivated vaccine with the following final concentration of immunopotentiator (mix different concentrations of cyclic di-AMP and CTA-CD154, let stand overnight to obtain the immunopotentiator):

[0028] Inactivated vaccine B: 0.01 mg / mL c-di-AMP, 0.4 mg / mL CTA-CD154;

[0029] Inactivated vaccine C: 0.02 mg / mL c-di-AMP, 1 mg / mL CTA-CD154;

[0030] Inactivated vaccine D: 0.5 mg / mL c-di-AMP, 1.5 mg / mL CTA-CD154;

[0031] Inactivated vaccine E: 5 mg / mL c-di-AMP, 10 mg / mL CTA-CD154;

[0032] Inactivated vaccine F: 0.01 mg / mL c-di-AMP;

[0033] Inactivated vaccine G: 0.4 mg / mL CTA-CD154;

[0034] Inactivated vaccine H: 0.04 mg / mL c-di-AMP;

[0035] Inactivated vaccine I: 2 mg / mL CTA-CD154;

[0036] The preparation method of blank vaccine J is as follows: phosphate buffer PBS (PH=7.4) and ISA 206 adjuvant are mixed in a volume ratio of 1:1, and emulsified using an emulsifier to obtain inactivated vaccine J.

[0037] 3. Animal grouping and immunization

[0038] One hundred healthy ICR mice were randomly divided into 10 groups of 10 mice each. Each group was immunized with inactivated epidemic diarrhea vaccines A, B, C, D, E, F, G, H, and I, and PBS control vaccine J, by subcutaneous injection at a dose of 50 μL per mouse.

[0039] Preparation and detection of lymphocytes from intestinal lamina propria and mesenteric lymph nodes

[0040] 24 hours after immunization, three mice per group were sacrificed by cervical dislocation. After disinfection with alcohol, the small intestine was removed and immediately placed in pre-chilled PBS to remove mesenteric fat and connective tissue. The mesenteric lymph nodes were gently removed, placed through a 70 µm strainer, and gently ground to prepare a cell suspension.

[0041] Isolation of intestinal lamina propria lymphocytes: Rinse the intestine with pre-chilled PBS, dissect the intestine longitudinally, and cut into 1-2 cm pieces. Place the pieces in 30 mL of extraction buffer (RPMI1640 + 5% DTT + 0.5M EDTA + 5% FBS) and incubate at 37°C at 500 rpm for 15 minutes. After incubation, filter through a 70 µm filter. The supernatant is the intestinal epithelial cells. Transfer the pellet to a new beaker and add 25 mL of digestion buffer (RPMI1640 + 20 µg / mL DNase I + 1 mg / mL collagenase II + 5% FBS). Digest at 37°C at 200 rpm for 30 minutes. During digestion, pipette repeatedly to break up any large pieces of tissue. Filter through a 70 µm filter, collect the filtrate, centrifuge at 500g for 10 minutes, remove the supernatant, and resuspend the pellet in RPMI1640 containing 10% FBS. This is the intestinal lamina propria lymphocytes.

[0042] Flow cytometry analysis: Mesenteric lymph node cells and intestinal lamina propria lymphocytes were collected and the cell density was adjusted to 1×10 6 FITC anti-mouse CD11c, APC anti-mouse MHCII, and PE anti-mouse CD103 antibodies were added to 100 μL of culture medium, respectively, and stained at 4°C for 30 min. After centrifugation, the cells were washed twice with PBS, resuspended in 400 μL of PBS, and then detected by flow cytometry.

[0043] The test results of each group are as follows Figure 1 As shown, the number of total and migratory DCs in the mesenteric lymph nodes and intestinal lamina propria of inactivated vaccine groups B, C, and D was significantly higher than that of inactivated vaccine groups A, E, F, G, H, and I, as well as the PBS control group. The results indicate that the combination of c-di-AMP and CTA-CD154 enhances the ability of CTA-CD154 to target intestinal DCs and significantly increases the number of total and migratory DCs in the intestine of mice immunized with the inactivated PEDV-NJ vaccine. Compared with the doses reported in existing articles and patents, c-di-AMP did not demonstrate significant intestinal DC targeting efficacy (Group H). While CTA-CD154 demonstrated intestinal DC targeting efficacy (Group I), this ability was significantly diminished at a reduced dose (Group G). When the two formulations were used together, the lowest-dose group (Group B) demonstrated the greatest intestinal DC targeting efficacy, followed by the medium-dose group (Group C) and the high-dose group (Group D). However, the highest-dose group (Group E) demonstrated no significant difference in intestinal DC targeting efficacy compared to the inactivated vaccine group (Group A).

[0044] Intestinal sIgA antibody testing

[0045] 28 days after immunization, three mice were selected from each group and killed by cervical dislocation. 5-8 cm of small intestine were removed under sterile conditions, and the intestinal debris was removed and minced. 500 μL of PBS was added and thoroughly shaken to mix. The mixture was centrifuged at 4000 g for 15 min at 4°C, and the supernatant was collected. The intestinal mucosal SIgA level was detected using a mouse SIgA ELISA kit.

[0046] The results of intestinal mucosal SIgA level test in each group were as follows: Figure 2 As shown, the levels of SIgA antibodies in the inactivated vaccine groups B, C, and D were significantly higher than those in the inactivated vaccine groups A, E, F, G, H, and I, as well as the PBS control group. The results indicate that the combination of c-di-AMP and CTA-CD154 significantly increased SIgA levels in the intestinal mucosa of mice immunized with the inactivated PEDV-NJ vaccine. The combination showed a clear dose-dependent effect, with the lowest-dose inactivated vaccine group (Group B) producing the highest mucosal antibody levels, followed by the medium-dose (Group C) and high-dose (Group D) groups. However, the highest-dose inactivated vaccine group (Group E) showed no significant difference in SIgA levels compared to the vaccine control group (Group A), indicating that the inactivated vaccine did not effectively increase SIgA antibody levels in the intestinal mucosa. Compared to the dose groups (Groups H and I) reported in existing articles and patents, the lowest-dose inactivated vaccine group (Group B) produced mucosal antibody levels more than five times higher.

[0047] 6. Neutralizing antibody detection

[0048] Blood was collected on day 28 after immunization and serum was separated. The PEDV-NJ virus stock solution was diluted to contain 200 TCID per unit dose. 50 , mixed with an equal amount of serially diluted serum to be tested and incubated at 37°C for 1 hour. Each dilution was inoculated into 6 wells of VERO cells, and the neutralizing titer of the serum was calculated by the Karber method after 72 hours.

[0049] The serum neutralization titer level of each group was Figure 3As shown, the average neutralizing antibody level in inactivated vaccine group A was 6.58±1.94, the average neutralizing antibody level in inactivated vaccine group B was 64±3.87, the average neutralizing antibody level in inactivated vaccine group C was 60±3.76, the average neutralizing antibody level in inactivated vaccine group D was 55.87±2.32, the average neutralizing antibody level in inactivated vaccine group E was 6.57±1.12, the average neutralizing antibody level in inactivated vaccine group F was 6.86±1.05, the average neutralizing antibody level in inactivated vaccine group G was 7.02±1.18, the average neutralizing antibody level in inactivated vaccine group H was 10.35±1.26, the average neutralizing antibody level in inactivated vaccine group I was 10.6±1.21, and the average neutralizing antibody level in PBS control group J was 0. The neutralizing antibody levels in inactivated vaccine groups B, C, and D were significantly higher than those in inactivated vaccine groups A, E, F, G, H, I, and the PBS control group. The results showed that the combination of c-di-AMP and CTA-CD154 can significantly increase the neutralizing antibody level in mice after immunization with PEDV-NJ inactivated vaccine, and the combination showed obvious dose-dependence. The neutralizing antibody level of the group with the lowest dose of inactivated vaccine (Group B) was the highest, followed by the medium dose (Group C) and high dose groups (Group D); but the neutralizing antibody level of the group with the highest dose of inactivated vaccine (Group E) was not significantly different from that of the vaccine control group (Group A), and could not effectively increase the neutralizing antibody level of the inactivated vaccine; compared with the dosage groups (Group H and Group I) reported in existing articles and patents, the neutralizing antibody level of the group with the lowest dose of inactivated vaccine (Group B) in the combination was more than 6 times higher.

Claims

1. An inactivated vaccine, characterized in that: The method comprises an aqueous phase, wherein the aqueous phase comprises 0.01 mg / mL cyclic di-AMP, 0.4 mg / mL CTA-CD154, and an inactivated antigen solution; the antigen solution is an inactivated porcine epidemic diarrhea virus solution.

2. The inactivated vaccine according to claim 1, characterized in that The inactivated vaccine also includes an oil phase solution.

3. The inactivated vaccine according to claim 2, characterized in that The oil phase solution is ISA206 adjuvant.

4. A method for preparing the inactivated porcine epidemic diarrhea vaccine according to claim 1, characterized in that: The cyclic di-AMP, CTA-CD154 and inactivated antigen solution are mixed evenly to obtain an aqueous phase solution; the aqueous phase solution is mixed with the oil phase solution and emulsified to obtain the inactivated vaccine.

5. The method for preparing an inactivated vaccine according to claim 4, wherein The volume ratio of the aqueous phase solution to the oil phase solution is 1:

1.

6. The method for preparing an inactivated vaccine according to claim 4, wherein The antigen solution is inactivated porcine epidemic diarrhea virus solution.

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