Application of tamibarotene and Toll-like receptor stimulant in preparation of vaccine adjuvant

Abstract

The invention belongs to the technical field of vaccine adjuvants, and particularly relates to application of tamibarotene and a Toll-like receptor stimulant in preparation of a vaccine adjuvant. The invention relates to a tamibarotene vaccine adjuvant composition, which is prepared by combining tamibarotene and a Toll-like receptor (TLRS) agonist, and the tamibarotene vaccine adjuvant composition is prepared by combining tamibarotene and the TLRs agonist. The composition can significantly promote antigen-specific mucosal immune response, and especially has a prominent effect on gastrointestinal mucosa parts; meanwhile, an organism can be effectively stimulated to generate specific systemic humoral immunity and cellular immune response. In an enterohemorrhagic Escherichia coli challenge model experiment, the vaccine adjuvant composition can be used for remarkably improving the immune protection effect induced by the recombinant intimin antigen, and a new effective strategy is provided for vaccine research and development and immune enhancement.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine adjuvants, and particularly relates to the application of tamibarotene and Toll-like receptor agonists in the preparation of vaccine adjuvants. Background Art

[0002] Infectious diseases have seriously affected human health. Vaccination is an effective and economical medical strategy for preventing infectious diseases. Most pathogens invade the body through mucosal sites. Currently, the marketed intramuscular vaccines perform well in inducing systemic immunity, but they are unable to effectively induce mucosal immunity, which fails to fully exert the immune protection effect of the vaccines. An adjuvant is a substance that can non-specifically change or enhance the specific immune response induced by antigenic substances. Adjuvants can also enhance the activity and scope of action of immune cells and play an immune response enhancing role through various pathways. A suitable adjuvant can regulate the intensity and type of the immune response, enabling the vaccine to produce an immune response rapidly at the initial site of infection while providing systemic protection, thereby enhancing the overall immune effect of the vaccine. Therefore, adding a suitable adjuvant to the vaccine is not only a feasible way to improve the vaccine effect but also an effective measure to enhance the public health's ability to respond to major epidemics.

[0003] Therefore, developing a good vaccine adjuvant is of great significance for the application of vaccines. Summary of the Invention

[0004] The present invention discovers that the combination of tamibarotene and Toll-like receptors (TLRs) agonists can promote the immune response ability of the body, especially the gastrointestinal mucosal response ability of the body. Therefore, it can be used as a vaccine adjuvant or applied in the preparation of vaccines.

[0005] To achieve the above object, the present invention can adopt the following technical solutions:

[0006] On the one hand, the present invention provides a vaccine adjuvant, which comprises tamibarotene and a Toll-like receptor agonist.

[0007] Preferably, in the above vaccine adjuvant, the Toll-like receptor agonist is selected from one or more combinations of agonist R848, agonist Pam2CSK4, agonist Poly(I:C), agonist MPLA, or agonist CpG.

[0008] More preferably, in the above vaccine adjuvant, the Toll-like receptor agonist is selected from agonist Pam2CSK4.

[0009] Preferably, in the above vaccine adjuvant, the mass ratio of tamibarotene to the Toll-like receptor agonist is (20 - 80):1.

[0010] Preferably, the above vaccine adjuvant is loaded using nanoemulsion.

[0011] Preferably, in the above vaccine adjuvant, the nanoemulsion comprises squalene, span 85, tween 80 and sterile water, and the mass ratio thereof is (2 - 4):(1 - 3):(9 - 11):(34 - 36).

[0012] On the other hand, the present invention provides a vaccine comprising the vaccine adjuvant in the present invention.

[0013] Preferably, in the above vaccine, the vaccine further comprises an immunogenic antigen, and the immunogenic antigen comprises a bacterial antigen or a model antigen OVA.

[0014] More preferably, in the above vaccine, the bacterial antigen is an enterohemorrhagic Escherichia coli antigen.

[0015] More preferably, in the above vaccine, the bacterial antigen is the intimin of enterohemorrhagic Escherichia coli membrane protein.

[0016] Preferably, in the above vaccine, the mass ratio of tamibarotene to the immunogenic antigen is (4 - 13):1; the mass ratio of the Toll - like receptor agonist to the immunogenic antigen is (0.15 - 0.5):1.

[0017] On the other hand, the present invention provides an application of tamibarotene and TLRs agonist in the preparation of a vaccine adjuvant.

[0018] Preferably, in the above application, the vaccine adjuvant comprises one or more of the following functions:

[0019] (i) Having the function of promoting the generation of mucosal immune response in the body through intramuscular immunization, and the mucosa includes gastric mucosa, intestinal mucosa or genital mucosa;

[0020] (ii) Having the function of promoting the activation of dendritic cells;

[0021] (iii) Having the function of promoting the systemic humoral immune response of the body;

[0022] (iv) Having the function of promoting the cellular immune response of the body;

[0023] (v) Having the function of promoting the generation of germinal centers, TFH cells and sIgA - secreting cells in the inguinal lymph nodes of the body.

[0024] On the other hand, the present invention provides an application of the vaccine adjuvant in the present invention in the preparation of a vaccine.

[0025] The beneficial effects of the present invention at least include: Tamiapalatin combined with Toll-like receptor agonists (such as agonist Pam2CSK4) can enhance the antigen presentation ability of dendritic cells in the body, promote the generation of germinal centers, TFH cells, IgA-secreting cells in the body, and can promote the generation of mucosal (especially gastrointestinal) immune responses, systemic humoral immune and cellular immune responses in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the screening process of tamiapalatin;

[0027] Figure 2 It is the humoral immune response of the complex adjuvant of tamiapalatin combined with TLR7 / 8 agonist R848;

[0028] Figure 3 It is the cellular immune response of the complex adjuvant of tamiapalatin combined with TLR7 / 8 agonist R848;

[0029] Figure 4 It is a schematic diagram of the screening process of TLRs agonists;

[0030] Figure 5a It is the OVA-specific slgA titer in the vaginal lavage fluid of mice immunized with the complex adjuvant of tamiapalatin combined with different TLRs agonists;

[0031] Figure 5b It is the OVA-specific slgA titer in the small intestine lavage fluid of mice immunized with the complex adjuvant of tamiapalatin combined with different TLRs agonists;

[0032] Figure 5c It is the OVA-specific slgA titer in the gastric lavage fluid of mice immunized with the complex adjuvant of tamiapalatin combined with different TLRs agonists;

[0033] Figure 5d It is the OVA-specific slgA titer in the gastric homogenate of mice immunized with the complex adjuvant of tamiapalatin combined with different TLRs agonists;

[0034] Figure 5e It is the OVA-specific slgA level in the alveolar lavage fluid of mice immunized with the complex adjuvant of tamiapalatin combined with different TLRs agonists;

[0035] Figure 5f It is the OVA-specific slgA level in the nasal lavage fluid of mice immunized with the complex adjuvant of tamiapalatin combined with different TLRs agonists;

[0036] Figure 5g It is the OVA-specific IgG titer in the serum of mice immunized with the complex adjuvant of tamiapalatin combined with different TLRs agonists;

[0037] Figure 6 The cellular immune response of the combined adjuvant of tamibarotene and different TLR agonists;

[0038] Figure 7 The cytotoxicity test of the combined adjuvant of tamibarotene and TLR2 / 6 agonist Pam2CSK4 (TB / P2C-NE);

[0039] Figure 8 The detection of the effects of the combined adjuvant of tamibarotene and TLR2 / 6 agonist Pam2CSK4 (TB / P2C-NE) on the biochemical indexes of liver and kidney toxicity;

[0040] Figure 9 The detection of the damage to the main organs by the combined adjuvant of tamibarotene and TLR2 / 6 agonist Pam2CSK4 (TB / P2C-NE);

[0041] Figure 10 The detection of the particle size, dispersity and potential of TB-NE and TB / P2C-NE adjuvants;

[0042] Figure 11 The transmission electron microscopy detection of TB-NE and TB / P2C-NE adjuvants;

[0043] Figure 12 The detection of the cytokine expression promoted by TB-NE and TB / P2C-NE adjuvants in BMDCs;

[0044] Figure 13 The flow cytometry representative diagram of the co-stimulatory factor expression promoted by TB-NE and TB / P2C-NE adjuvants in BMDCs;

[0045] Figure 14 The bar chart of the co-stimulatory factor expression promoted by TB-NE and TB / P2C-NE adjuvants in BMDCs;

[0046] Figure 15 The bar chart of the flow cytometry detection of the effect of TB-NE and TB / P2C-NE adjuvants on the phagocytosis of BMDCs;

[0047] Figure 16 The confocal microscopy observation of the effect of TB-NE and TB / P2C-NE adjuvants on the phagocytosis of DC2.4 cells;

[0048] Figure 17 The flow cytometry representative diagram of the effect of TB-NE and TB / P2C-NE adjuvants on the activation of dendritic cells in vivo;

[0049] Figure 18Bar graph of the effect of TB-NE and TB / P2C-NE adjuvants on the activation of dendritic cells in vivo detected by flow cytometry;

[0050] Figure 19 Schematic diagram of the time points for evaluating the adjuvant effect of TB / P2C-NE;

[0051] Figure 20 Immunofluorescence of the promotion of antigen-induced germinal centers and IgA-secreting cells in the inguinal lymph nodes of mice by TB-NE and TB / P2C-NE adjuvants;

[0052] Figure 21 Bar graph of the promotion of antigen-induced TFH cell production in the inguinal lymph nodes of mice by TB-NE and TB / P2C-NE adjuvants;

[0053] Figure 22 Situation of the promotion of antigen-specific IgA secretion by splenic lymphocytes induced by TB-NE and TB / P2C-NE adjuvants;

[0054] Figure 23a Situation of the promotion of IFN-γ secretion by splenic lymphocytes by TB-NE and TB / P2C-NE adjuvants;

[0055] Figure 23b Situation of the promotion of IL-4 secretion by splenic lymphocytes by TB-NE and TB / P2C-NE adjuvants;

[0056] Figure 24 Situation of the promotion of mucosal immunity and systemic humoral immune responses induced by antigens in the gastrointestinal tract by TB-NE and TB / P2C-NE adjuvants;

[0057] Figure 25 Schematic diagram of the process of the challenge test of mice immunized with TB-NE and TB / P2C-NE adjuvants;

[0058] Figure 26 Situation of the induction of humoral immunity in mice immunized with TB-NE and TB / P2C-NE adjuvants;

[0059] Figure 27 Quantification of bacteria in the feces of mice immunized with TB-NE and TB / P2C-NE adjuvants;

[0060] Figure 28 Bacterial loads in the cecum, colon and small intestine of mice immunized with TB-NE and TB / P2C-NE adjuvants;

[0061] Figure 29 Situation of intestinal inflammation infiltration and structural changes in mice immunized with TB-NE and TB / P2C-NE adjuvants;

[0062] Figure 30Changes in the level of sIgA in the feces of mice immunized with TB-NE and TB / P2C-NE adjuvants;

[0063] Figure 31 Lethal challenge experiments on mice immunized with TB-NE and TB / P2C-NE adjuvants. Specific embodiments

[0064] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art can make non-essential improvements and adjustments to the implementation scheme based on the above-mentioned invention content, which still fall within the protection scope of the present invention.

[0065] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having an obviously different meaning in the context, the expressions in the singular form include those in the plural form. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility of the existence or addition of one or more other features, numbers, operations, components, parts, elements, materials or their combinations. As used herein, depending on the situation, " / " can be interpreted as "and" or "or".

[0066] An embodiment of the present invention provides a vaccine adjuvant, which includes tamibarotene and Toll-like receptor agonists.

[0067] It should be noted that in the present invention, it is found that the combination of tamibarotene and Toll-like receptor agonists (TLRs agonists) can enhance the immune response ability of the body's mucosa (such as vaginal mucosa or gastrointestinal mucosa), so it can be prepared into a vaccine adjuvant; specifically, the structural formula of tamibarotene is shown as follows:

[0068]

[0069] In addition, tamibarotene can also be used in the vaccine adjuvant in the form of a salt, and the form of the salt can be well-known in the art, and generally hydrochloride is selected. In addition, Toll-like receptor agonists are well-known in the art.

[0070] In some specific examples, the above Toll-like receptor agonists can be selected from one or more combinations of TLR7 / 8 agonist R848 (CAS No.: 144875-48-9), TLR2 / 6 agonist Pam2CSK4 (CAS No.: 868247-72-7), TLR3 agonist Poly(I:C) (CAS No.: 24939-03-5), TLR4 agonist MPLA (monophosphoryl lipid A, CAS No.: 1246298-63-4), or TLR9 agonist CpG.

[0071] It should be noted that the Toll-like receptor agonists in the present invention can be those well-known in the art, such as the agonists listed above. Among them, TLR2 / 6 agonist Pam2CSK4 is preferred. The immune effect of the combination of tamibarotene and TLR2 / 6 agonist Pam2CSK4 (Pam2CSK4 as an adjuvant cannot induce mucosal immunity in the body through intramuscular injection) is better than that of other TLRs agonists. Moreover, the nanoemulsion composite adjuvant formed by tamibarotene and Pam2CSK4 can promote strong mucosal immunity of protein vaccines in the gastrointestinal tract through intramuscular injection, and this effect is significantly stronger than that of the currently recognized drug RA that promotes gastrointestinal mucosal immunity. At the same time, it can also promote systemic immune responses.

[0072] In some specific examples, in the above vaccine adjuvant, the mass ratio of tamibarotene to the Toll-like receptor agonist is (20-80):1.

[0073] It should be noted that when tamibarotene and the Toll-like receptor agonist are used in combination, the mass ratio of tamibarotene to the Toll-like receptor agonist can be (20-80):1, such as 23:1, 40:1, or 70:1, etc.

[0074] In some specific examples, the above vaccine adjuvant is loaded using nanoemulsion.

[0075] It should be noted that the vaccine adjuvant in the present invention can be in various forms well-known in the art. In the present invention, nanoemulsion form is preferred. Nanoemulsion has a high drug loading rate, which improves the stability and bioavailability of the adjuvant. In addition, nanoemulsion itself has the ability to enhance the body's immune response. When combined with the above vaccine adjuvant, it can further enhance the immune response ability of the vaccine adjuvant. In addition, it should be understood that nanoemulsion is used to encapsulate the vaccine adjuvant, or is combined with the vaccine adjuvant in the form of electrostatic adsorption, chemical bonding, or physical mixing. The preparation method of nanoemulsion is well-known in the art.

[0076] In some specific examples, among the above vaccine adjuvants, the nanoemulsion includes squalene, span 85, tween 80 and sterile water, and their mass ratio is (2 - 4):(1 - 3):(9 - 11):(34 - 36).

[0077] The embodiment of the present invention also provides a vaccine, which includes the vaccine adjuvant in the present invention.

[0078] It should be noted that the vaccine adjuvant in the present invention can be prepared into a vaccine together with an immune stimulant. The immune stimulant can be an immunogenic antigen or a viral vector; in addition, the vaccine can also contain auxiliary materials such as preservatives and stabilizers, and both the preservatives and stabilizers are well known in the art.

[0079] In some specific examples, in the above vaccine, the vaccine further includes an immunogenic antigen, and the immunogenic antigen includes a bacterial antigen or a model antigen OVA.

[0080] It should be noted that the bacterial antigen in the present invention is well known in the art, such as enterohemorrhagic Escherichia coli antigen (such as intimin antigen, etc.).

[0081] In some specific examples, in the above vaccine, preferably, in the above vaccine, the mass ratio of tamibarotene to the immunogenic antigen is (6 - 26):1; the mass ratio of Pam2CSK4 to the immunogenic antigen is (0.5 - 0.15):1.

[0082] It should be noted that when the vaccine adjuvant and the immunogenic antigen are prepared into a vaccine in the present invention, the mass ratio of tamibarotene to the immunogenic antigen in the vaccine adjuvant can be (4 - 13):1, such as 5:1, 7:1, 10:1 or 12:1, etc.; the mass ratio of the Toll-like receptor agonist to the immunogenic antigen can be (0.15 - 0.5):1, such as 0.4:1, 0.3:1 or 0.2:1, etc.

[0083] The embodiment of the present invention also provides an application of tamibarotene and a Toll-like receptor agonist in the preparation of a vaccine adjuvant.

[0084] In some specific examples, in the above application, the vaccine adjuvant includes one or more of the following functions:

[0085] (i) It has the function of promoting the body to produce a mucosal immune response through intramuscular injection, and the mucosa includes gastric mucosa, intestinal mucosa or genital mucosa;

[0086] (ii) It has the function of promoting the activation of dendritic cells;

[0087] (iii) It has the function of promoting the systemic humoral immune response of the body;

[0088] (iv) It has the function of promoting the cellular immune response of the body;

[0089] (v) It has the function of promoting the generation of germinal centers, TFH cells, and sIgA-secreting cells in the inguinal lymph nodes of the body.

[0090] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0091] In the following examples, the ELISA detection method for antibodies is carried out according to the following steps: Each well of the flat-bottom 96-well plate is incubated overnight at 4 °C with 100 μL of OVA at a concentration of 2 μg / mL, then the plate is washed 4 times with a plate washer, and PBST containing 1% BSA is used as the blocking solution. 200 μL of the blocking solution is added to each well and incubated at 37 °C for 2 h, and then washed 4 times continuously. After diluting the samples at different concentrations (when detecting antibodies of IgG, IgG1, and IgG 2a in mouse serum, first dilute the mouse serum with PBST containing 1% BSA at a ratio of 1:1000, then add it to the 96-well ELISA plate and perform serial dilutions from top to bottom. At the same time, set two wells in each 96-well plate, add naive mouse serum diluted 1:1000, and use the absorbance values corresponding to these two wells as the cutoff value. Incubate at 37 °C for 1 h, then wash the plate 4 times. After preparing the horseradish peroxidase-labeled secondary antibody (HRP-labeled goat anti-mouse IgG) with PBST at a ratio of 1:10000, add 100 μL of it to each well of the 96-well plate and incubate at 37 °C for 40 min. After washing the plate 4 times, develop the color with 50 μL of TMB per well until it reaches an appropriate degree, then terminate the color development with 50 μL of the termination solution, and finally read the absorbance of each well at 450 nm with an enzyme-linked immunosorbent assay reader. Take 2.1 times the absorbance value of the corresponding well of the mouse sample as the cutoff value. If the absorbance of the sample well after gradient dilution is greater than the cutoff value, it is considered positive. Take the logarithm to the base 2 of the dilution factor corresponding to the smallest value among the absorbance values greater than the cutoff value to obtain the corresponding titer. Take 2.1 times the absorbance of the corresponding well of the mouse sample as the cutoff value. If the absorbance of the sample well after gradient dilution is greater than the cutoff value, it is considered positive. Take the logarithm to the base 2 of the dilution factor corresponding to the smallest value among the absorbance values greater than the cutoff value to obtain the corresponding titer.

[0092] In the following examples, the ELISpot assay was performed using an ELISpot kit (for IFN-γ, the catalog number of the ELISpot kit is: 3321-4AST-10; for IL-4, the catalog number of the ELISpot kit is: 3311-4APW-2; for IgA, the catalog number of the ELISpot kit is: 3865-2H). The detection method was carried out with reference to the instruction manual or the following steps: The ELISpot plate was pre-activated with complete RPMI 1640 medium (including RPMI 1640 medium (C11875500BT, from Gibco), 10% fetal bovine serum (10099-141), and 1% penicillin-streptomycin double antibody (BL505A)) for 30 min. Then, 100 μL of cell suspension was added to the corresponding wells to make the cell number in each well 1×10 6 cells. In the wells of the stimulated group, 100 μL of 1640 complete medium with an OVA concentration of 40 μg / mL was added, so that the working concentration of OVA was 20 μg / mL, while 100 μL of 1640 complete medium was added to the non-stimulated wells; the 96-well plate was transferred to a cell culture incubator and cultured for another 36 h. Then, the medium in the plate was discarded and the plate was washed 5 times with PBS. Then, 100 μL of detection antibody with a concentration of 1 μg / mL (from the ELISpot kit) was added to each well and incubated at room temperature for 2 h. Then, the plate was washed 5 times again and 100 μL of secondary antibody with a concentration of 1 μg / mL (HRP-labeled streptavidin in the ELISpot kit) was added to each well and incubated at room temperature for 1 h. Finally, the plate was washed 5 times again. After patting dry the PBS in the plate, 100 μL of chromogenic solution was added to each well. When it reached the appropriate degree, the plate was rinsed with tap water to terminate the chromogenesis. After it was air-dried, the number of spots in the plate was read.

[0093] In the following examples, the vaginal lavage fluid was obtained as follows: The mouse was fixed with the left hand with its abdomen facing up, and 75 μL of ELISA primary antibody (test sample) diluent was aspirated with a 200 μL pipette and used to repeatedly rinse the genital tract of the mouse. The rinsed liquid was transferred to an EP tube; the genital tract rinsing operation for each mouse was repeated four times, and finally the combined samples were frozen in an -80 °C refrigerator.

[0094] In the following examples, the ocular serum was obtained as follows: The mouse was grasped with the left hand to make its eyeball protrude; the mouse eyeball was clamped with a curved forceps and then quickly removed. The mouse ocular blood was collected with a 1.5 mL EP tube, and then the collected ocular blood was placed in a 4 °C refrigerator for 2 hours; the placed ocular blood was transferred to a low-temperature centrifuge and centrifuged at a speed of 4000 r / min for 10 min. Finally, the upper-layer serum was aspirated with a pipette into a new EP tube and stored in an -80 °C refrigerator.

[0095] In the following example, the method for obtaining small intestine lavage fluid is as follows: Prepare a 15 mL centrifuge tube and label it. Add 3 mL of small intestine lavage fluid buffer (the buffer is prepared from 100 mL of PBS, 1.8612 g of EDTA, 0.0174 g of PMSF, and 10 mg of trypsin inhibitor) to the centrifuge tube. Fix the mouse on a foam board, cut open the abdominal cavity with ophthalmic scissors, take the section of intestine from the duodenum to the colon, and place the opened intestine in a 15 mL centrifuge tube containing the small intestine lavage fluid buffer after cutting it open with ophthalmic scissors. After shaking the sample, transfer the centrifuge tube to a 4°C refrigerator and let it stand for 2 h, then centrifuge at 4000 r / min for 20 min, collect the supernatant and store it in an -80°C refrigerator.

[0096] In the following example, the method for obtaining gastric lavage fluid is as follows: After taking the small intestine from the mouse, cut off the mouse's stomach with ophthalmic scissors and place it in a 1.5 mL EP tube, then add 1 mL of ELISA primary antibody dilution solution. Fix the EP tube on a small shaker and let it turn over in a 4°C environment for 4 h, centrifuge at 4°C and 5000 r / min for 10 min, collect the supernatant and freeze it in an -80°C refrigerator.

[0097] In the following example, the method for obtaining gastric homogenate is as follows: After the gastric lavage fluid is collected, transfer the mouse gastric sample to a homogenization tube, add 3 ceramic beads to each homogenization tube, and homogenize the mouse gastric sample using a homogenizer. After homogenization is completed, take out the sample and homogenize it at 4°C and 2000 r / min for 10 min (the centrifuge speed should not be too high here to prevent the ceramic beads from piercing through the homogenization tube). Collect the supernatant and store it in an -80°C refrigerator.

[0098] In the following example, the method for collecting fecal samples to be tested is as follows: Centrifuge the prepared fecal buffer (the buffer is prepared from 5 mL of PBS, 5 mL of glycerol, 1 mg of trypsin inhibitor, 73.06 mg of EDTA, 1.74 mg of PMSF, and 100 mg of BSA), and take 300 μL of the supernatant into a 600 μL EP tube. Hold the mouse with the left hand so that its abdomen is facing up and wait for the mouse to defecate. Pick up the fresh feces with forceps and put them into the above EP tube. Collect three feces from each mouse. Place the collected feces in a 4°C environment for 2 h to soften them, then shake them on a vortex mixer to make them into a homogenate, and then centrifuge at 4°C and 8000 r / min for 10 min to collect the supernatant for subsequent experiments.

[0099] In the following example, the preparation method of bronchoalveolar lavage fluid is as follows: 14 days after the last immunization, the mice were sacrificed by cervical dislocation. The fur was moistened with 75% alcohol, and the mice were fixed on a foam board with a syringe needle. The skin and tissues of the neck of the mice were cut open with ophthalmic scissors to expose the trachea. An opening was made in the trachea and a special soft needle was inserted into the trachea of the mice. 500 μL of PBST containing 1% BSA was aspirated with a 1 mL syringe and injected into the lungs of the mice through the soft needle. The piston of the syringe was repeatedly pulled to thoroughly wash the lungs with the lavage fluid. Finally, the fluid was aspirated to obtain the bronchoalveolar lavage fluid.

[0100] In the following example, the preparation method of nasal lavage fluid is as follows: After the collection of bronchoalveolar lavage fluid from the mice was completed, the mice were turned around, and then a flexible hose needle was inserted into the trachea towards the nasal cavity and the mice were placed upside down. 500 μL of PBST containing 1% BSA was aspirated with a 1 mL syringe and injected into the nasal cavity of the mice through the flexible hose needle. The lavage fluid flowing out of the nasal cavity of the mice was collected into a 1.5 mL EP tube to obtain the nasal lavage fluid.

[0101] I. Screening of tamibarotene

[0102] In the following example, the preparation method of TB&R848-NE nanoemulsion adjuvant is as follows:

[0103] (1) Span 85 and Tween 80 were preheated in a water bath at 65 °C for 10 min;

[0104] (2) 4 mg of R848 and 10 mg of tamibarotene were weighed separately and placed in a small beaker. Then 0.3 g of squalene was added to dissolve the two drugs. At the same time, 1 g of Tween 80 and 0.2 g of Span 85 were added and stirred well;

[0105] (3) After the drugs were dissolved, ultrapure water was slowly added while stirring with a glass rod until the nanoemulsion was uniform and clear;

[0106] (4) Finally, the volume of the nanoemulsion was fixed to 5 mL, and the TB&R848-NE nanoemulsion adjuvant containing R848 and tamibarotene at concentrations of 800 μg / mL and 2000 μg / mL respectively was prepared.

[0107] The preparation method of RA&R848-NE is the same as the above preparation method of TB&R848-NE nanoemulsion adjuvant, and the RA&R848-NE nanoemulsion adjuvant containing R848 and RA at concentrations of 800 μg / mL and 2000 μg / mL respectively was prepared.

[0108] The preparation method of RA-NE is similar to the above preparation method of RA&R848-NE nanoemulsion adjuvant, and the difference is that R848 is not added.

[0109] In the following example, the vaccine system is prepared by mixing nanoemulsion adjuvant (hereinafter referred to as adjuvant), OVA solution (hereinafter referred to as OVA, with a concentration of 1 mg / mL), and normal saline in a certain proportion.

[0110] (I) Animal Immunization

[0111] Mice (purchased from Vital River Laboratories, Beijing, 6 - 8 weeks old, female Balb / c, 18 - 20 g) were divided into 4 groups (OVA, OVA + RA - NE, OVA + RA&R848 - NE, and OVA + TB&R848 - NE), with 5 mice in each group. As Figure 1 shown, on days 0, 14, and 28, the mice were injected with 100 μL (a total of 200 μL) of the corresponding vaccine system into the left and right thigh muscles respectively (in the 200 μL vaccine system, the doses of RA and tamibarotene were 200 μg / mouse, the dose of R848 was 80 μg / mouse, and the dose of OVA was 15 μg / mouse); and immunological tests were performed on the mice on day 42; among them, the components of the vaccine systems in different groups are shown in Table 1 below, and the preparation method is to mix evenly.

[0112] Table 1 Components of Vaccine Systems in Different Groups

[0113] Group OVA Adjuvant Saline OVA 105 μL 0 1295 μL OVA + RA - NE 105 μL 700 μL 595 μL OVA + RA&R848 - NE 105 μL 700 μL 595 μL OVA + TB&R848 - NE 105 μL 700 μL 595 μL

[0114] (II) Antibody Detection

[0115] On day 42, vaginal lavage fluid, small intestine lavage fluid, gastric lavage fluid, and gastric homogenate were collected from the mice and the antibody levels were detected by ELISA; the detection results are as Figure 2 shown. The results showed that high levels of sIgA titers were detected in the vaginal lavage fluid, intestinal lavage fluid, and gastric homogenate of the mice in the OVA + TB&R848 - NE group, indicating that this composite adjuvant can promote obvious mucosal immune responses of the vaccine in the vaginal mucosa, intestinal mucosa, and gastric mucosa; in addition, it was found that all adjuvants can significantly promote the humoral immune response induced by the vaccine.

[0116] (III) Detection of Cellular Immune Response

[0117] On day 42, the spleens of the mice were taken and ground, then treated with erythrocyte lysate (TIANGEN; RT122 - 02) for 5 min, and then the cells were counted and the cell concentration was adjusted to 1×10 7 cells / mL; then an ELISpot experiment was performed; the experimental results are as Figure 3 shown. The results showed that only the ability of the spleen lymphocytes of the mice in the OVA + RA&R848 - NE group and the OVA + TB&R848 - NE group to secrete IFN - γ was significantly stronger than that of the single antigen group, indicating that both of these two composite adjuvants can promote the T - cell immune response induced by the vaccine.

[0118] II. Screening of TLRs Agonists

[0119] (1) Animal Immunization

[0120] The TLR2 / 6 agonist Pam2CSK4, TLR3 agonist Poly(I:C), TLR4 agonist MPLA, and TLR9 agonist CpG were respectively combined with tamibarotene, and nanoemulsion was still used as the delivery system and named TB / P2C-NE, TB / Poly(I:C)-NE, TB&MPLA-NE, and TB / CpG-NE respectively; the preparation methods of TB / P2C-NE, TB / Poly(I:C)-NE, TB&MPLA-NE, and TB / CpG-NE were the same as those of the above-mentioned TB&R848-NE nanoemulsion adjuvant (TB&R848-NE). In addition, the preparation method of TB-NE was the same as that of RA-NE above; and different vaccine systems were prepared according to the above preparation methods, and the components of the corresponding vaccine systems are shown in Table 2 below.

[0121] Table 2 Components of Vaccine Systems in Different Groups

[0122] Group OVA Adjuvant Saline OVA 105 μL 0 1295 μL OVA + TB - NE 105 μL 700 μL 595 μL OVA + TB&R848 - NE 105 μL 700 μL 595 μL OVA + TB&P2C - NE 105 μL 700 μL 595 μL OVA + TB&MPLA - NE 105 μL 700 μL 595 μL OVA + TB / Poly(I:C) 105 μL 700 μL 595 μL OVA + TB / CpG - NE 105 μL 700 μL 595 μL

[0123] The mice were divided into 7 groups (OVA, OVA+TB-NE, OVA+TB&R848-NE, OVA+TB / P2C-NE, OVA+TB&MPLA-NE, OVA+TB / Poly(I:C)-NE, and OVA+TB / CpG-NE), with 5 mice in each group. As Figure 4 shown, on the 0th, 14th, and 28th days, the mice were injected with 100 μL of the corresponding vaccine system into the left and right thigh muscles respectively, and the mice were subjected to immunological detection on the 42nd day.

[0124] (2) Antibody Detection

[0125] On the 42nd day after the first immunization of the mice, vaginal lavage fluid, small intestine lavage fluid, gastric lavage fluid, gastric homogenate, alveolar lavage fluid, nasal lavage fluid, and serum (ocular serum) of the mice were collected and the antibody levels were detected by ELISA; the detection results are as Figures 5a to 5gAs shown, the vaginal mucosa of all adjuvant groups produced a comparable mucosal response level. In the gastrointestinal tract, the OVA+TB / P2C-NE group of mice showed the highest sIgA antibody level. However, none of the adjuvants could promote mucosal immunity induction by the vaccine in the respiratory tract. In addition, OVA+TB / P2C-NE simultaneously induced a more robust humoral immune response than the OVA+TB&R848-NE adjuvant. The above results indicate that TB / P2C-NE is an adjuvant that can promote both gastrointestinal mucosal immunity and systemic immune responses.

[0126] (III) Detection of cellular immune response

[0127] On the 42nd day, the spleens of mice were taken and ground, and then treated with red blood cell lysate (TIANGEN, RT122-02) for 5 min. Then the cells were counted and the cell concentration was adjusted to 10 7 cells / mL; then an ELISpot assay was performed; the experimental results are as Figure 6 shown. The splenocytes of the OVA+TB / P2C-NE group of mice showed the highest ability to secrete IFN-γ and were significantly stronger than those of the OVA+TB&R848-NE group. The OVA+TB / Poly(I:C)-NE and OVA+TB / CpG-NE groups also had the effect of promoting cellular immunity, but the effect was less than that of OVA+TB / P2C-NE; the above results indicate that TB / P2C-NE can be used as an adjuvant to promote a strong cellular immune response.

[0128] III. Safety detection

[0129] (I) Cytotoxicity experiment

[0130] The cytotoxic effect of the TB / P2C-NE adjuvant on BMDCs cells was determined by the CCK-8 method. Specifically, the tibias and femurs of the mice in the OVA+TB / P2C-NE group were isolated, and then the bone marrow cavities were rinsed with sterile PBS. The cells were collected and treated with red blood cells for 5 min to obtain cells. Then the cells were cultured in 1640 complete medium containing 20 ng / mL PMSF and 10 ng / mL IL-4, and induced in a cell culture incubator (37 °C, 5% CO2) for seven days. The medium was changed on the second and fourth days, and the BMDCs cells were collected on the sixth day for subsequent experiments; the BMDCs cell concentration was adjusted to 10 6 cells / mL to obtain a cell suspension. 100 μL of the cell suspension was added to each well of a 96-well round bottom plate, so that the number of cells in each well was 10 5Prepare several dilutions of TB / P2C-NE to form a concentration gradient from 0.25 - 128 mg / mL in 1640 complete medium. Then, add 100 μL of each dilution to a 96-well plate. Incubate the cells in a cell culture incubator for 24 h. After that, add 10 μL of CCK-8 to each well and continue incubating for 2 h. Finally, measure the absorbance at 450 nm using a microplate reader. In the formula, the absorbance of the control group refers to the absorbance of cells without drug treatment after incubation with CCK-8 under the same conditions as the experimental wells.

[0131] The detection results are as Figure 7 shown. When the concentration of tamibarotene is no higher than 8 μg / mL and the concentration of Pam2CSK4 is no higher than 0.3 μg / mL, TB / P2C-NE has no obvious toxicity to BMDCs cells.

[0132] (II) In vivo safety detection experiment

[0133] Mice were immunized three times by intramuscular injection with OVA, OVA+TB-NE, and OVA+TB / P2C-NE vaccine systems (the vaccine systems and injection methods were in accordance with the "Screening of TLRs agonists" in item "II" above). Fourteen days after the third immunization, the heart, liver, spleen, lungs, and kidneys were collected and fixed in paraformaldehyde for 72 h. At the same time, serum was collected and stored at -80 °C.

[0134] The serum was sent to the clinical laboratory of Southwest Hospital to detect biochemical indices for evaluating the liver and kidney toxicity of the TB / P2C-NE adjuvant. The detection results are as Figure 8 shown. The results showed that there were no significant differences in the levels of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and blood urea nitrogen in the serum of all groups, and all were within the normal range (it should be noted that the normal range values of ALP, ALT, AST, LDH, and BUN in mice are 60 - 209, 17 - 77, 54 - 298, 215 - 1024, and 8 - 33, respectively).

[0135] In addition, pathological sections were prepared by dehydrating, clearing, embedding, sectioning, and staining different organs, and then the sections were scanned using a slide scanner. The results are as Figure 9 shown. The results showed that there were no obvious structural changes in the main organs of mice in each group compared with the PBS group.

[0136] The above results indicate that the TB / P2C-NE adjuvant does not cause obvious damage to the mouse body.

[0137] IV. Pharmaceutical characterization

[0138] In the following examples, the TB-NE and TB / P2C-NE composite adjuvants were prepared according to the method described in the "Screening of TLRs agonists" in "II" above.

[0139] (I) Detection of the particle size, dispersibility and potential of the adjuvant

[0140] Add 990 μL of ultrapure water to a sterile 1.5 mL EP tube, and then add 10 μL of TB-NE and 10 μL of the TB / P2C-NE composite adjuvant respectively. After vortex mixing, a nanoemulsion adjuvant sample diluted at a ratio of 1:100 is obtained. The particle size, dispersion coefficient and potential of the nanoemulsion adjuvant are detected using a Malver particle size analyzer.

[0141] The test results are as Figure 10 shown. The particle sizes of the two nanoemulsions, TB-NE and TB / P2C-NE, are normally distributed and stable below 100 nm. The average potential of TB-NE is -19 mV, while the potential of TB / P2C-NE is -11 mV. The PDI values of the two adjuvants are both around 0.242.

[0142] (II) Transmission electron microscopy for detecting the morphology of the nanoemulsion adjuvant

[0143] Precisely weigh 1.0 g of phosphotungstic acid solid and add it to 100 mL of ultrapure water to prepare a 1% phosphotungstic acid solution; respectively take 10 μL of the TB-NE and TB / P2C-NE composite adjuvants and add them to 490 μL of ultrapure water, and mix evenly to obtain a 1:50 diluted sample; respectively take 20 μL of the diluted sample and drop it on a wax sheet, then gently take out the copper mesh with forceps and slowly put it into the droplet, and let it stand for 10 min; take 20 μL of the 1% phosphotungstic acid solution and drop it on the wax sheet, take out the copper mesh from the sample and blot it dry with filter paper, then place it in the 1% phosphotungstic acid droplet for staining for 30 s, then take it out and blot it dry with filter paper, and place it in a petri dish lined with filter paper for waiting to be observed.

[0144] The results are as Figure 11 shown. After observation, it is found that both nanoemulsions are spherical and evenly distributed, and no aggregation of nanoemulsion particles is seen.

[0145] V. Effects of TB / P2C-NE on BMDCs

[0146] In the following examples, the TB-NE and TB / P2C-NE composite adjuvants and the corresponding vaccine systems were prepared according to the method described in the "Screening of TLRs agonists" in "II" above.

[0147] (I) Detection of the expression levels of co-stimulatory factors

[0148] Bone marrow cavities of femurs and tibias of female BALB / c mice were flushed to obtain cells, which were induced to differentiate for 7 days in RPMI 1640 medium containing IL-4 (10 ng / mL), GM-CSF (20 ng / mL) and 10% FBS to obtain BMDCs; After BMDCs were treated with adjuvant TB-NE and adjuvant TB / P2C-NE for 24 h respectively, the culture medium supernatant was collected and the inflammatory factors were detected using an ELISA kit (all ELISA kits were from Dakewei) according to the instructions. The ELISA results were as Figure 12 shown that the TB / P2C-NE complex adjuvant could promote BMDCs to secrete inflammatory factors IL-1β and IL-6, but the level of the anti-inflammatory factor IL-10 was also significantly up-regulated, which might be the result of feedback regulation.

[0149] In addition, the cells were incubated with FITC-CD11c, PE-CD40, Percp / Cy5.5-CD86 and APC-CD80 at 4 °C for 30 min, then centrifuged to remove the supernatant, and the cells were resuspended with PBS and processed by flow cytometry (from BD, model Fortessa); The experimental results were as Figure 13 and Figure 14 shown that the TB / P2C-NE complex adjuvant could promote BMDCs to express co-stimulatory factors CD40, CD80 and CD86.

[0150] (2) Effects of TB / P2C-NE on the phagocytosis of dendritic cells

[0151] BMDCs were treated with TB-NE and TB / P2C-NE complex adjuvant respectively for 24 h, then Cy5-labeled OVA protein was added to make the working concentration 5 μg / mL and incubation continued for 6 h, then the cells were collected and incubated with FITC-CD11c antibody at 4 °C for 30 min, then centrifuged to remove the supernatant, resuspended with PBS and processed by flow cytometry; DC2.4 cells in the logarithmic growth phase were seeded in confocal dishes, with 3×10 5 cells per dish. After the cells adhered, they were treated with TB-NE and TB / P2C-NE for 24 h, then Cy5-labeled OVA was added and its working concentration was 5 μg / mL, and incubation continued for 3 h. The cell supernatant was discarded and the cells were washed three times with PBS. Paraformaldehyde was added to fix the cells for 20 min. After washing the cells, PBS containing phalloidin was added and incubation continued for 10 min. After washing the cells, DAPI was used to stain the nuclei. Finally, observation and photographing were performed using a confocal microscope.

[0152] The flow cytometry results were as Figure 15 shown that the ability of BMDCs to uptake antigens was significantly enhanced after treatment with the TB / P2C-NE adjuvant; The confocal results were as Figure 16As shown, the ability of DC2.4 cells to phagocytose OVA antigen was significantly improved.

[0153] (III) Effect of TB / P2C-NE on the activation of dendritic cells in vivo

[0154] Six- to eight-week-old BALB / c mice were divided into 4 groups (PBS, OVA, OVA+TB-NE, and OVA+TP / P2C-NE), with 5 mice in each group. Each mouse in each group was injected with 100 μL of PBS, OVA, OVA+TB-NE, and OVA+TP / P2C-NE (the vaccine system was prepared according to the above preparation method) into the left and right thigh muscles respectively; at 24 h after immunization, inguinal lymph nodes were taken and ground into single-cell suspensions, and then the cells were incubated with live / dead dyes, APC / Cy7-CD45, FITC-CD11c, APC-CD80, Percp / Cy5.5-CD86, and PE-CD40 antibodies at 4 °C for 30 min. After washing the cells, they were processed by flow cytometry.

[0155] The results are as Figure 17 and Figure 18 shown. At 24 h after immunizing mice with OVA combined with TB / P2C-NE, the proportions of dendritic cells of CD40 + , CD80 + and CD86 + in the inguinal lymph nodes were significantly increased, indicating that the TB / P2C-NE adjuvant can promote the maturation of dendritic cells in the inguinal lymph nodes.

[0156] VI. Evaluation of the adjuvant effect of TB / P2C-NE

[0157] In the following examples, the TB-NE and TB / P2C-NE composite adjuvants were prepared according to the method described in "Screening of TLRs agonists" in "II" above; the vaccine systems of OVA combined with TB-NE or TB / P2C-NE respectively were the same as those in Table 1 above.

[0158] (I) Immunization protocol and sample collection

[0159] Mice were divided into 4 groups (PBS, OVA, OVA+TB-NE, and OVA+TP / P2C-NE), with 5 mice in each group; the mice were immunized on days 0, 14, and 28 respectively (the immunization method was to inject 100 μL of PBS, OVA, OVA+TB-NE, and OVA+TP / P2C-NE into the left and right thigh muscles of each mouse); samples were collected at 28 days and 42 days after the first immunization for immunological evaluation, and the experimental time points are as Figure 19 shown.

[0160] (II) Detection of germinal centers and follicular helper T cells (TFH)

[0161] Mice were sacrificed 14 days after the second immunization, and inguinal lymph nodes were collected. The lymph nodes were fixed in 4% paraformaldehyde and ground into single-cell suspensions for immunofluorescence detection of germinal centers and flow cytometry detection of TFH cell levels. After being fixed in paraformaldehyde for 72 hours, the lymph nodes were dehydrated, cleared, embedded in paraffin, and sectioned. After blocking, the sections were incubated overnight at 4°C with AF647-GL7 and PE-IgA antibodies, washed, and then incubated with FITC-B220 at room temperature for 2 hours. Confocal microscopy was used to observe the generation of germinal centers and IgA-secreting cells.

[0162] After the lymph nodes were made into single-cell suspensions, they were co-incubated with live / dead dye, PE-CD3, Percp / Cy5.5-CD4, PE / Cy7-CD44, BV421-ICOS, FITC-PD-1, biotinylated CXCR5, and APC-avidin at 4°C for 30 minutes. Then the cells were collected, washed, and processed for flow cytometry.

[0163] The results of the immunofluorescence experiment are as Figure 20 shown. The results showed that the number of germinal centers in the inguinal lymph nodes of mice in the TB / P2C-NE adjuvant group was significantly increased compared with those in the single antigen group and the TB-NE group. In addition, the IgA signal in the inguinal lymph nodes was also significantly upregulated, indicating that the TB / P2C-NE adjuvant can promote the induction of more germinal centers and IgA antibodies by the vaccine in the draining lymph nodes of mice.

[0164] The results of flow cytometry detection of TFH levels are as Figure 21 shown. The results showed that both the TB-NE and TB / P2C-NE adjuvants can promote the induction of TFH cells by the vaccine in the inguinal lymph nodes of mice.

[0165] (3) Detection of cytokine secretion in the spleen

[0166] (1) Detection of IgA secretion

[0167] The 96-well PVDF plates were pre-coated with 2 μg / mL OVA overnight at 4°C, then washed 5 times. 100 μL of cell suspension at a concentration of 1×10 7 cells / mL was added to each well so that there were 1×10 6 cells per well. Then the 96-well plates were placed in a cell culture incubator and cultured for another 36 hours. After the culture, the liquid in the plates was discarded, and the plates were washed 5 times with PBS. Detection antibodies were added and incubated at room temperature for 2 hours. After incubation, the plates were washed 5 times again, secondary antibodies were added and incubated at room temperature for 1 hour. After washing 5 times, the plates were developed. When the color developed to an appropriate degree, the development was terminated by rinsing with tap water. After drying, the plates were read.

[0168] The detection results are as follows Figure 22 shown. Both TB-NE and TB / P2C-NE adjuvants can promote the secretion of antigen-specific IgA by splenic lymphocytes induced by the vaccine, but the effect of TB / P2C-NE is better.

[0169] (2) Detect the secretion of IFN-γ and IL-4

[0170] The 96-well plates pre-coated with IFN-γ and IL-4 were washed five times with sterile PBS, then 200 μL of RPMI 1640 complete medium was added to each well and activated at room temperature for 30 min. Then, the medium in the plates was discarded, and 100 μL of cell suspension with a concentration of 10 7 cells / mL was added to each well to make the number of cells in each well 10 6 cells. In the control group, an additional 100 μL of RPMI 1640 complete medium was added. In the stimulation group, 100 μL of RPMI 1640 complete medium containing 40 μg / mL of OVA was added to make the working concentration of OVA 20 μg / mL. The 96-well plates were transferred to the cell culture incubator and continued to be cultured for 36 h. After the culture was completed, the liquid in the plates was discarded and washed five times with PBS. After adding the detection antibody, it was incubated at room temperature for 2 h. After the incubation was completed, it was washed five times again, and the secondary antibody was added and incubated at room temperature for 1 h. After washing five times, it was subjected to a display treatment. After the color development reached an appropriate degree, it was rinsed with tap water to terminate the color development. After drying, it was subjected to a plate reading treatment.

[0171] The detection results are as follows Figure 23a and Figure 23b shown. Compared with the group with antigen alone (OVA), the ability of splenic lymphocytes of mice in the OVA+TB-NE and OVA+TB / P2C-NE groups to secrete IFN-γ and IL-4 was significantly improved, and the effect of the TB / P2C-NE adjuvant was better than that of TB-NE, indicating that the TB / P2C-NE adjuvant can promote both Th1 and Th2 immune responses.

[0172] (IV) Detection of antibody levels

[0173] Mice were sacrificed 14 days after the third immunization to collect feces, small intestine lavage fluid, gastric lavage fluid, gastric homogenate, and ocular serum, and the levels of antigen-specific antibodies in the relevant samples were detected by ELISA.

[0174] The results are as follows Figure 24 shown. The results showed that the highest level of sIgA antibody titer was detected in the feces and small intestine lavage fluid of mice in the OVA+TB / P2C-NE group, while the sIgA antibody levels in the gastric homogenate and gastric lavage fluid were comparable between the OVA+TB-NE group and the OVA+TB / P2C-NE group. In addition, the antibody levels of IgG and its subtypes in the serum of mice in the OVA+TB / P2C-NE group were also the highest, IgG 2aThe ratio of / IgG1 reflects that the OVA+TB / P2C-NE adjuvant promotes a greater degree of Th1 immune response.

[0175] The above data indicate that the TB / P2C-NE adjuvant can simultaneously promote a strong mucosal immune and systemic humoral immune response induced by the vaccine in the gastrointestinal tract.

[0176] VII. Vaccine efficacy verification experiment

[0177] (I) Animal immunization

[0178] Mice were immunized with intimin of enterohemorrhagic Escherichia coli O157 (EHEC O157:H7) membrane protein (obtained by prokaryotic expression) combined with the TB-NE or TB / P2C-NE composite adjuvant. The TB-NE or TB / P2C-NE composite adjuvant was prepared according to the method described in "Screening of TLRs agonists" in "II" above. The components of the vaccine system are the same as those shown in Table 1 above. The mice were divided into 3 groups (intimin, intimin+TB-NE, and intimin+TB / P2C-NE), with 5 mice in each group. The treatment time points for each mouse were as follows Figure 25 As shown, on days 0, 14, and 28, each mouse was injected with 100 μL (a total of 200 μL) of the corresponding vaccine system into the left and right thigh muscles, and samples were collected on day 41 for detection.

[0179] (II) Sample collection and detection

[0180] On day 41 after the first immunization, mouse vaginal lavage fluid, feces, and tail vein blood were collected. A 96-well ELISA plate was coated overnight at 4°C with 2 μg / mL of intimin. The next day, after washing four times with a plate washer, 200 μL of PBST containing 1% BSA was added to each well and incubated at 37°C for 2 h for blocking. After washing again, the samples were added and incubated at 37°C for 1 h. After washing four times, HRP-labeled secondary antibody was added and incubated at 37°C for 40 min. After washing, 50 μL of TMB was added to each well for color development, and then 50 μL of stop solution was added to terminate the color development. The absorbance at 450 nm was read with an enzyme-linked immunosorbent assay reader. The 2.1 times the absorbance of the corresponding well of the mouse sample was used as the cut-off value.

[0181] The detection results are as Figure 26As shown, the results showed that the intimin+TB-NE and intimin+TB / P2C-NE groups induced comparable levels of mucosal immunity in the vaginal mucosa of mice. High levels of sIgA antibodies were detected in the feces of both the intimin+TB / P2C-NE group and the intimin+TB-NE group, but the intimin+TB / P2C-NE group was significantly higher than the intimin+TB-NE group. The levels of IgG in the feces of the two groups of mice were comparable to those in the single antigen group, indicating that the TB / P2C-NE adjuvant induced strong mucosal immunity in the mouse intestine. In addition, it was found that the intimin+TB / P2C-NE group could also promote the humoral immunity induced by the vaccine, and the effect was better than that of the intimin+TB-NE group.

[0182] (III) Sublethal challenge experiment

[0183] Mice were gavaged with streptomycin at a dose of 5 mg / kg / day for three consecutive days before challenge to disrupt the homeostasis of the intestinal flora, and then the diet was stopped 12 h before challenge. A sublethal model was established by gavaging 1×10 10 CFU of EHEC O157:H7. Mouse feces were collected 1-4 days after challenge, then homogenized with sterile PBS and centrifuged, and the bacterial load of EHEC O157:H7 was colonized on MacConkey medium. On the fifth day after challenge, equal masses of mouse colon, cecum, and small intestine were taken and homogenized, and the load of EHEC O157:H7 in the intestinal tract was measured. The small intestine was fixed in paraformaldehyde for 72 h and then used for HE staining.

[0184] The detection results are as Figure 27 shown. Compared with the non-immunized group of mice, the quantification of bacteria in the feces of the intimin+TB / P2C-NE group of mice was significantly reduced, while there was a tendency for a decrease in the intimin+TB-NE group without statistical difference; the bacterial colonization in the intestine is as Figure 28 shown. The bacterial load in the cecum of the intimin+TB / P2C-NE group of mice was significantly lower than that of other groups, and although there was no statistical difference in the colon and small intestine, there was a tendency for a decrease; the HE staining results are as Figure 29 shown. A large number of inflammatory infiltrations and structural changes were observed in the small intestines of the non-immunized group and the single antigen group of mice, while the small intestines of the intimin+TB-NE and intimin+TB / P2C-NE groups of mice still maintained normal structures; in addition, the levels of sIgA in the feces of mice were detected before and after challenge, and the results are as Figure 30 shown. After challenge, the levels of sIgA in the feces of the intimin+TB-NE and intimin+TB / P2C-NE groups of mice both increased, suggesting that the mice produced specific protective effects under the attack of pathogens.

[0185] (4) Lethal challenge experiment

[0186] Mice were gavaged with streptomycin at a dose of 5 mg / kg / day for three consecutive days before challenge to disrupt the intestinal flora homeostasis. Then, food was withheld 12 h before challenge. A lethal model was established by gavaging each mouse with 1×10 11 CFU of EHEC O157:H7. The mice were divided into 4 groups (intimin, intimin+TB-NE, and intimin+TB / P2C-NE), with 7 mice in each group. The survival of the mice was observed continuously for ten days and the survival rate was calculated. The survival rate is shown as Figure 31 follows. The mice in the intimin+TB / P2C-NE group showed the highest survival rate, reaching 71% (i.e., 5 mice survived), indicating that the TB / P2C-NE adjuvant has a good immune protection promotion effect.

[0187] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A vaccine adjuvant, characterized in that, It includes tamibarotene and Toll-like receptor agonists.

2. The vaccine adjuvant according to claim 1, wherein The Toll-like receptor agonist is selected from one or more combinations of R848, Pam2CSK4, Poly(I:C), MPLA or CpG.

3. The vaccine adjuvant according to claim 2, characterized in that, The Toll-like receptor agonist is Pam2CSK4.

4. The vaccine adjuvant according to any one of claims 1 to 3, characterized in that, The mass ratio of tamibarotene to Toll-like receptor agonist is (20 - 80):

1.

5. The vaccine adjuvant according to any one of claims 1 to 3, characterized in that, The vaccine adjuvant is loaded using nanoemulsion.

6. The vaccine adjuvant according to claim 5, characterized in that, The nanoemulsion includes squalene, span 85, tween 80 and sterile water, and their mass ratio is (2 - 4):(1 - 3):(9 - 11):(34 - 36).

7. A vaccine, characterized in that, It includes the vaccine adjuvant according to any one of claims 1 to 6.

8. The vaccine according to claim 7, characterized in that, The vaccine also includes an immunogenic antigen, and the immunogenic antigen includes a bacterial antigen or a model antigen OVA.

9. The antigen according to claim 8, wherein The bacterial antigen is an enterohemorrhagic Escherichia coli antigen.

10. The vaccine according to claim 9, wherein, The bacterial antigen is the intimin of enterohemorrhagic Escherichia coli membrane protein.

11. The vaccine according to any one of claims 7 to 10, characterized in that, The mass ratio of tamibarotene to immunogenic antigen in the vaccine adjuvant is (4 - 13):1; the mass ratio of Toll-like receptor agonist to immunogenic antigen is (0.15 - 0.5):

1.

12. Use of tamibarotene and Toll-like receptor agonists in the preparation of a vaccine adjuvant.

13. The application according to claim 12, characterized in that, The vaccine adjuvant has one or more of the following functions: (i) It has the function of promoting the body to produce a mucosal immune response through intramuscular injection, and the mucosa includes gastric mucosa, intestinal mucosa or genital mucosa; (ii) It has the function of promoting the activation of dendritic cells (iii) It has the function of promoting the systemic humoral immune response of the body; (iv) It has the function of promoting the cellular immune response of the body; (v) It has the function of promoting the generation of germinal centers, TFH cells and sIgA-secreting cells in the inguinal lymph nodes of the body.

14. Use of the vaccine adjuvant according to any one of claims 1 to 6 in the preparation of a vaccine.

Images