Novel nano emulsion adjuvant based on MDP as well as preparation method and application of novel nano emulsion adjuvant

Abstract

The invention provides a novel nano-emulsion adjuvant based on MDP and a preparation method and application thereof, the adjuvant is an oil-in-water nano-emulsion, an oil phase contains squalene, Span85 and muramyl dipeptide, and a water phase contains Tween80 and a citric acid buffer solution; wherein the oil phase is prepared from the following components in percentage by mass volume: 3.0 percent to 5.0 percent of squalene, 0.2 percent to 3.0 percent of Span85 and 0.0001 percent to 0.1 percent of muramyl dipeptide. Furthermore, an immunologic stimulant MDP is added into an oil phase of the MF59 nano-emulsion adjuvant, so that organic integration of a nano-emulsion adjuvant delivery function and a training immune induction mechanism is realized, and dual activation of humoral immunity and cellular immunity of a traditional adjuvant MF59 is endowed.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine adjuvants, and particularly relates to a novel nanoemulsion adjuvant based on MDP, and a preparation method and application thereof. Background Art

[0002] The core mechanism of action of traditional vaccine adjuvants (such as aluminum salts and MF59) relies on local inflammatory reactions to enhance adaptive immune responses, but their ability to activate the long-term memory effect of the innate immune system (i.e., "trained immunity") is significantly limited. The discovery of trained immunity breaks through the traditional immunological framework, revealing that innate immune cells (such as monocytes and NK cells) can establish "non-specific memory" against secondary infection by pathogens through epigenetic reprogramming and metabolic remodeling. This mechanism provides an innovative direction for vaccine design: by inducing trained immunity with adjuvants, it is expected to achieve broad-spectrum and long-lasting protection against highly mutated viruses (such as respiratory syncytial virus RSV).

[0003] MF59 is a squalene-based oil-in-water nanoemulsion (particle size of about 160 nm) that has been used in influenza vaccines. Its technical features are as follows:

[0004] Advantages of lymphatic targeted delivery: Nanoemulsions form a stable oil-water interface through the regulation of surfactants, efficiently migrate to the draining lymph nodes through the lymphatic vessels, and promote the internalization of adsorbed antigens by antigen-presenting cells (APCs); Multi-dimensional immune synergy: The emulsion interface can simultaneously load antigens and pattern recognition receptor (PRR) ligands (such as TLR agonists), enhancing the activation efficiency of dendritic cells (DCs) through spatial co-localization effects; Clinical safety verification: 30 years of application data show that its local reactions are controllable (the incidence of redness and swelling is <15%), and there are no reports of systemic toxicity.

[0005] However, the traditional mode of action of MF59 has the following bottlenecks:

[0006] Immune response bias: mainly drives Th2 polarization, with limited affinity and cross-protection effects on B cells; defective trained immune induction: although monocytes can be recruited to the injection site, it lacks epigenetic regulatory modules for innate immune cells and cannot establish long-term immune memory; insufficient antigen-adjuvant synergy: the passive adsorption mode easily leads to antigen dissociation, reducing lymph node targeting efficiency.

[0007] Therefore, how to break through the limitation of traditional adjuvants that only focus on enhancing adaptive immunity is a problem that needs to be solved. Summary of the Invention

[0008] In response to the problems existing in the prior art, the purpose of the present invention is to provide a new nanoemulsion adjuvant based on MDP and its preparation method and application through the integrated design of delivery system and trained immunity. This new adjuvant can simultaneously stimulate the production of large amounts of antibodies and induce T cell activation.

[0009] The study found that the traditional adjuvant MF59 mainly enhances Th2 response and has limited ability to induce trained immunity. In order to improve the shortcomings of MF59 in regulating cell activation, improving immune bias, and extending immune memory, the inventors proposed to organically integrate the delivery function of the nanoemulsion with the trained immune induction mechanism. In previous studies, a series of immunostimulants ATP and MDP were added to the oil phase of the MF59 nanoemulsion adjuvant, and β-glucan, CpG, ATP, and MDP were added to the aqueous phase of MF59 in different combinations. The results of in vitro stimulation of BMDCs showed that the nanoemulsion adjuvant combined with MDP had a better trained immune mechanism. MM59 stimulated the activation of DC cells in trained immunity, enhanced the secretion of IFN-γ cytokines by T cells, and promoted CD4 + and CD8 + Activation of T cells. In traditional immune programs, MM59 also plays an important role in central immune memory T cells and improving immune bias. In view of this, the inventors provide the following solutions of the present invention.

[0010] In order to achieve the above object, the first aspect of the present invention provides a novel nanoemulsion adjuvant based on MDP, wherein the adjuvant is an oil-in-water nanoemulsion, wherein the oil phase comprises squalene, Span85 and muramyl dipeptide (MDP), and the aqueous phase comprises Tween80 and citric acid buffer (pH 6.5-7.0);

[0011] The oil phase comprises, by weight volume percentage, 3.0% to 5.0% squalene, 0.2% to 3.0% Span85, and 0.0001% to 0.1% muramyl dipeptide. Preferably, the weight volume ratio (W / V) of squalene, Span85, and muramyl dipeptide is 4.3%, 0.5%, and 0.0006% respectively.

[0012] Furthermore, the mass volume ratio of the oil phase to the water phase is 3% to 6%: 94% to 97%; preferably, the mass volume ratio of the oil phase to the water phase is 4.8006%: 95.1994%.

[0013] The second aspect of the present invention provides a novel composite nanoemulsion adjuvant that enhances antibody production and trains immunity, which is formed by further adding CpG to the novel nanoemulsion adjuvant described in the first aspect.

[0014] Furthermore, the added amount of CpG is 10 μg per injection.

[0015] The third aspect of the present invention provides a method for preparing the novel nanoemulsion adjuvant based on MDP according to the first aspect, comprising the following steps:

[0016] 1) Preparation of oil phase: Squalene and Span 85 were placed in a beaker and uniformly mixed at 800-1500 rpm to obtain a primary mixed oil phase; MDP was dissolved in DMSO and the dissolved MDP was poured into the primary mixed oil phase and stirred to obtain a mixed oil phase;

[0017] 2) Preparation of aqueous phase: Place Tween 80 and citric acid buffer in a conical flask and mix uniformly at 800-1500 rpm to obtain a mixed aqueous phase;

[0018] 3) mixing the mixed oil phase and the mixed water phase, and uniformly mixing the mixed solution at 800-1500 rpm to obtain an initial emulsion; the initial emulsion is subjected to high-pressure homogenization and membrane filtration to obtain the novel nanoemulsion adjuvant.

[0019] Furthermore, in step 3), the conditions for high-pressure homogenization treatment are 500-1000 bar and 3-5 minutes.

[0020] The fourth aspect of the present invention provides use of the novel nanoemulsion adjuvant described in the first aspect, or the novel composite nanoemulsion adjuvant described in the second aspect, or the novel nanoemulsion adjuvant prepared by the method described in the third aspect in preparing vaccines.

[0021] The fifth aspect of the present invention provides a vaccine, which comprises the novel nanoemulsion adjuvant described in the first aspect, or the novel composite nanoemulsion adjuvant described in the second aspect, or the novel nanoemulsion adjuvant prepared by the method described in the third aspect.

[0022] Furthermore, the administration of the vaccine includes intramuscular injection.

[0023] The beneficial effects of the present invention compared to the prior art are:

[0024] 1. This invention developed a method for preparing a nanoemulsion adjuvant based on engineering technology, successfully preparing MF59, whose efficacy and physicochemical properties are comparable to those of domestic and international products. This engineering preparation method successfully developed a novel MDP-based trained nanoemulsion adjuvant, MM59. This novel nanoemulsion adjuvant, incorporating MDP, retains the antibody-stimulating effect of MF59.

[0025] 2. The novel nanoemulsion adjuvant MM59, prepared by this invention, not only possesses the inherent properties of MF59 but also possesses a training immune mechanism not found in MF59. Following the initial MM59 stimulation, the mouse's DCs and T cells were activated and rejuvenated by a second, homologous MM59+CpG+DS-Cav1 stimulation. MM59 also possesses a mechanism for improving immune tropism, not found in MF59, by enhancing IFN-γ secretion and reducing IL-4 secretion.

[0026] 3. Compared to MF59, the novel nanoemulsion adjuvant MM59 described herein not only retained its ability to produce antibodies but also enhanced the activation of mouse memory cells. In CD8 and CD4 central memory T cells, the MM59 group significantly outperformed MF59. Based on MM59's ability to train immunity and activate memory cells, the MM59 group exhibited slightly higher long-term antibody production than MF59. Antibody production was also slightly faster after the first immunization.

[0027] 4. The novel nanoemulsion adjuvant MM59, successfully developed by this invention, protected Balb / c mice from RSV infection. Daily monitoring of weight change and body temperature after challenge showed that the adjuvant significantly reduced both weight and temperature. HE analysis revealed a significant reduction in left lung lesions in the MM59+CpG+DS-Cav1 group. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below with reference to the accompanying drawings and examples:

[0029] Figure 1 The test results of the nanoemulsion adjuvant MF59 prepared under different conditions described in Example 1 are shown; wherein, A: particle size distribution of MF59 under different treatment methods under dynamic light scattering (DLS), B: particle size distribution characterized by dynamic light scattering (DLS) of MF59 under different high-pressure homogenization conditions, C: transmission electron microscopy characterization image of MF59 under different high-pressure homogenization conditions;

[0030] Figure 2 The test results of the nanoemulsion adjuvant MF59 from different sources described in Example 1 are shown; wherein, A: comparison of dynamic light scattering (DLS) and transmission electron microscopy (TEM) of high pressure 700 bar 4 min with domestic and foreign standards, B: changes in dynamic light scattering (DLS) and transmission electron microscopy (TEM) of samples stored at 4°C for different times at high pressure 700 bar 4 min;

[0031] Figure 3Schematic diagram showing the comparison of the adjuvant effects of MF59 prepared in Example 1 and domestic and foreign standards; A: The adjuvant combined with respiratory syncytial virus pre F antigen was injected intramuscularly to evaluate the level of pre F-specific IgG antibodies elicited in mice; B: The weight change curve of mice after immunization; C: The level of pre F-specific IgG antibodies elicited by MF59 adjuvants from different sources; D: MF59, AddaVax TM The changes in the levels of pre F-specific IgG antibodies induced by combining with CpG;

[0032] Figure 4 The preparation and particle size investigation of nanoemulsion adjuvants MF59, MM59 and MA59 are shown; wherein, A: transmission electron microscopy characterization images of different adjuvants; B: particle size distribution of different nanoemulsion particles characterized by dynamic light scattering (DLS);

[0033] Figure 5 The in vitro BMDCs immunostimulation evaluation of the composite nanoemulsion adjuvant of Example 5 is shown; wherein, A: an immune program of various adjuvant combinations to stimulate BMDC cell maturation and cell viability in vitro; B, C and D: flow cytometry evaluation of CD80, MHC-Ⅱ upregulation and CCK8 cell activity after BMDC was stimulated with adjuvant in vitro for 24 hours;

[0034] Figure 6 The results of the evaluation and training immune mechanism study of the composite nanoemulsion adjuvant in Example 6 are shown; wherein, A: a schematic diagram of the immune program of nanoemulsion adjuvant in inducing trained immunity in mice; B: flow cytometry analysis of CD4 + T cells (CD3 + 、CD4 + ) proliferation (n=5); C and D: Flow cytometric analysis of the expression of maturation markers (CD80, CD86) of DC cells in the spleen of mice (n=5); E: Expression of MHC-Ⅱ molecules of DC presenting antigens (n=5); F: Flow cytometric analysis of CD8 + T cells (CD3 + 、CD8 + ) proliferation (n=5); G: ELISpot analysis of the number of spots of IFN-γ secretion after mouse spleen cells were stimulated with RSV-Pre F antigen (n=4); H: ELISpot analysis of the number of spots of IL-4 secretion after mouse spleen cells were stimulated with RSV-Pre F antigen (n=4); I: ELISpot analysis of the ratio of spots of IFN-γ and IL-4 secretion after mouse spleen cells were stimulated with RSV-Pre F antigen;

[0035] Figure 7The results of the detection and evaluation of humoral immunity and cellular immunity of MM59 in Example 7 are shown; wherein, A: the immunization procedure for evaluating the cellular immunity and humoral immunity induced by intramuscular injection of mice with adjuvant combined with respiratory syncytial virus pre-F antigen; B: the absorbance value of the specific binding antibody IgG against the pre-F antigen at a dilution ratio of 1:5000 of mouse serum three days after the second immunization (n=4); C: the absorbance value of IgG1 (n=4); D: the absorbance value of IgG2a (n=4); E: the absorbance ratio of IgG2a and IgG1 (n=4); F: flow cytometric analysis of CD4 central memory T cells (CD4+CD444) in mouse spleen + 、CD62L + ) proliferation (n=5); G: CD4 effector memory T cells (CD4+CD44 + 、CD62L - ) proliferation (n=5); H: CD8 central memory T cells (CD8 + CD44 + CD62L + ) proliferation (n=5); I: CD8 effector memory T cells (CD8 + 、CD44 + 、CD62L - ) proliferation (n=5); J: Th1 cells (CD3 + 、CD4 + IFN-γ + ) proliferation (n=5); K: Th2 cells (CD3 + 、CD4 + IL-4 + ) proliferation (n=5); L: ratio of Th1 cells to Th2 cells in mouse spleen (n=5);

[0036] Figure 8 Figure 8 shows that MM59 described in Example 8 elicits faster and longer-lasting antibodies; A: Schematic diagram of the immune response to evaluate changes in antibody titers after intramuscular injection of mice with adjuvant combined with respiratory syncytial virus pre-F antigen; B: ELISA detection of antibody binding levels specific to pre-F in mice 7 days after the first immunization; C and D: ELISA detection of the titer trends of IgG (n=5) and IgG2a / IgG1 specific antibodies against pre-F protein at each time period after immunization (n=4);

[0037] Figure 9The evaluation of different adjuvant combinations described in Example 9 on inflammatory factors and lung protection in mice after challenge is shown; wherein, A: immune schematic diagram of challenge evaluation after intramuscular injection of adjuvant combined with respiratory syncytial virus pre-F antigen; B: weight change curve of mice after intranasal challenge (n=5); C: body temperature change curve of mice after intranasal challenge (n=5); D: cell content in alveolar lavage fluid of mice after challenge (n=5); E: IL-6 content in alveolar lavage fluid of mice after challenge (n=5); F: flow cytometry detection of antigen-specific CTL cells (CD3 + 、CD8 + IFN-γ + ) proliferation (n=5); G: HE sections were used to detect pathological changes in mouse lungs under 2X and 10X conditions. DETAILED DESCRIPTION

[0038] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0039] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0040] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not intended to limit the present invention.

[0041] The raw materials involved in the embodiment of the present invention include:

[0042] Squalene was purchased from Baikaisheng (Shanghai) Biotechnology Co., Ltd., Span85 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., Tween80 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., MDP was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., β-glucan was purchased from Invivogen, CpG was purchased from Huapu Shijiazhuang Pharmaceutical Co., Ltd., and ATP was purchased from MCE Biotechnology Co., Ltd.

[0043] Example 1: Preparation of nanoemulsion adjuvant MF59

[0044] This comparative example provides a traditional nanoemulsion adjuvant MF59, the preparation method of which is as follows:

[0045] 1) Preparation of oil phase: 12.9 g of squalene and 1.5 g of Span 85 were placed in a beaker and uniformly mixed at 1000 rpm for 30 min to obtain an oil phase.

[0046] 2) Preparation of aqueous phase: 1.5 g of Tween 80 and 300 L of citric acid buffer (pH 6.5-7.0) were placed in a conical flask and uniformly mixed at 1000 rpm for 30 min to obtain a mixed aqueous phase.

[0047] 3) The oil phase and the mixed aqueous phase were mixed in a weight-to-volume ratio (W / V) of 4.8006%:95.1994%, and the mixed solution was uniformly mixed at 1000 rpm for 30 minutes to obtain an initial emulsion.

[0048] The initial emulsion was subjected to different ultrasonic disruption treatments or different high-pressure homogenization treatments and then filtered through a 0.22 μm filter membrane to obtain sterile traditional nanoemulsion adjuvant MF59 under different conditions.

[0049] The specific ultrasonic disruption conditions include: 195W, ultrasonication for 30s, intermittent for 5s, ultrasonication for 20min or 30min;

[0050] The high-pressure homogenization conditions specifically include: 700 bar homogenization for 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 1000 bar homogenization for 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min.

[0051] The test results are as follows:

[0052] The nanoemulsion adjuvant MF59 prepared under different conditions was tested using a Malvern particle size analyzer:

[0053] 10 μl of the nanoemulsion adjuvant MF59 prepared under various conditions was diluted to 1 ml with PBS, and the cuvette was placed in a Malvern particle size analyzer for dynamic light scattering (DLS) measurement to determine the particle size distribution and polydispersity index (PDI) of different nanoemulsion preparations.

[0054] Transmission electron microscopy (TEM) detection of nanoemulsion adjuvant MF59 prepared under different conditions:

[0055] The nanoemulsion adjuvant MF59 prepared under various conditions was added to a carbon-coated copper grid and then allowed to stand at room temperature for 5 minutes. The excess liquid on the copper grid was absorbed with absorbent paper, and 2% phosphotungstic acid solution (pH 6.5) was prepared for staining and dried for 30 minutes. The prepared electron microscopy samples were placed on a transmission electron microscope (model HITACHI H-7650) for observation at a voltage of 80 kV.

[0056] Different engineering methods formed nanoemulsion particles of different morphologies. Ultrasonication and high-pressure homogenization treatments significantly reduced the size of the formed nanoparticles. The particle sizes of the particles prepared by different engineering methods ranged from 100 to 300 nm ( Figure 1 Among them, the particle size controlled by high-pressure homogenization technology is relatively good, which is around 160nm. By changing the high-pressure homogenization pressure and time, the nanoparticles formed under the high-pressure homogenization pressure of 700bar4min are closest to 160nm, and the PDI index is 0.2175 ( Figure 1 Middle B).

[0057] like Figure 1 As shown in Figure C, the nanoemulsion adjuvant MF59 prepared under different conditions is characterized by foamy aggregates.

[0058] At the same time, the nanoemulsion adjuvant MF59 prepared by the method of this embodiment was compared with the MF59 reference substance from other sources. It was found that the nanoemulsion adjuvant MF59 prepared by high pressure homogenization G700bar 4min in this embodiment was closest to the foreign reference substance AddaVax under electron microscopy. TM ( Figure 2 In addition, it was found that as the nanoemulsion adjuvant MF59 was stored at 4°C for a longer time, the particle size showed a slight increase ( Figure 2 Middle B).

[0059] Example 2: Detection of the effect of traditional nanoemulsion adjuvant MF59

[0060] To explore the adjuvant effect of the traditional nanoemulsion adjuvant MF59 prepared in Example 1, this example uses RSV-pre F protein as an antigen to prepare a respiratory syncytial virus vaccine. The specific method includes:

[0061] The RSV-pre F protein was diluted to 400 μg / ml, 25 μl of the diluted protein solution was mixed with 25 μl of the nanoemulsion adjuvant MF59 described in Example 1 to prepare the pre F protein subunit vaccine. The vaccine was administered according to the immunization flow chart ( Figure 3 Middle A) The antibody was injected intramuscularly into BALB / c mice, and blood was collected after immunization. Antibody levels were measured by ELISA.

[0062] In order to determine the difference in adjuvant effect between the nanoemulsion particles prepared by the nano-engineering method explored in Example 1 and domestic and foreign standard products, this example conducted an immunological study on antibody titers in mice. In the Balb / c mouse model, MF59 (prepared in Example 1), AddaVax TM25 μl of MF59-like adjuvant (MF59-like adjuvant, catalog number: vac-adx-10), BAF03 (MF59-derived adjuvant: Ruike Bio AS03), and L-MF59 (MF59-like adjuvant from Liu Ye's laboratory, Institute of Medical Biology, Chinese Academy of Medical Sciences) were mixed with 25 μl of 400 μg / ml Ag at a volume ratio of 1:1 and then immunized mice ( Figure 3 Middle A). 14 days after the second immunization, there was no significant difference in the body weight of the mice ( Figure 3 Middle B), indicating that the adjuvant has no obvious toxic side effects.

[0063] Among them, MF59 group was compared with domestic and foreign reference products (AddaVax TM , BAF03, L-MF59) had no significant difference ( Figure 3 C), and MF59 and AddaVax TM The combination with CpG significantly enhanced the production of antibodies ( Figure 3 This indicates that the MF59 nanoemulsion particles prepared in Example 1 have comparable adjuvant effects to domestic and foreign reference substances.

[0064] Example 3: Preparation of a novel training nanoemulsion adjuvant MM59

[0065] This embodiment provides a novel nanoemulsion adjuvant based on MDP, the preparation method of which comprises the following steps:

[0066] 1) Preparation of the oil phase: 12.9 g of squalene and 1.5 g of Span 85 were placed in a beaker and mixed uniformly at 1000 rpm for 30 min to obtain a primary mixed oil phase; 3 mg of MDP was dissolved in 100 μl of DMSO, and the dissolved MDP was poured into the primary mixed oil phase and stirred to obtain a mixed oil phase.

[0067] 2) Preparation of aqueous phase: 1.5 g of Tween 80 and 300 L of citric acid buffer (pH 6.5-7.0) were placed in a conical flask and uniformly mixed at 1000 rpm for 30 min to obtain a mixed aqueous phase.

[0068] 3) The mixed oil phase and the mixed aqueous phase were mixed at a mass volume ratio (W / V) of 4.8006%:95.1994%, and the mixed solution was uniformly mixed at 1000 rpm for 30 minutes to obtain an initial emulsion; the initial emulsion was subjected to high-pressure homogenization at 700 bar for 4 minutes and filtered through a 0.22 μm filter membrane to obtain a sterile new nanoemulsion adjuvant MM59.

[0069] Example 4: Preparation of the novel training nanoemulsion adjuvant MA59

[0070] This embodiment provides a novel nanoemulsion adjuvant based on MDP, the preparation method of which comprises the following steps:

[0071] 1) Preparation of the oil phase: 12.9 g of squalene and 1.5 g of Span 85 were placed in a beaker and mixed uniformly at 1000 rpm for 30 min to obtain a primary mixed oil phase; 1.5 mg of ATP was dissolved in 100 μl of DMSO, and the dissolved ATP was poured into the primary mixed oil phase and stirred to obtain a mixed oil phase.

[0072] 2) Preparation of aqueous phase: 1.5 g of Tween 80 and 300 L of citric acid buffer (pH 6.5-7.0) were placed in a conical flask and uniformly mixed at 1000 rpm for 30 min to obtain a mixed aqueous phase.

[0073] 3) The mixed oil phase and the mixed aqueous phase were mixed at a mass volume ratio (W / V) of 4.8003%:95.1997%, and the mixed solution was uniformly mixed at 1000 rpm for 30 minutes to obtain an initial emulsion; the initial emulsion was subjected to high-pressure homogenization at 700 bar for 4 minutes and filtered through a 0.22 μm filter membrane to obtain a sterile novel nanoemulsion adjuvant MA59.

[0074] The test results are as follows:

[0075] Malvern particle size analyzer to detect different nanoemulsion adjuvants:

[0076] 10 μl of the prepared MM59 and MA59 nanoemulsion adjuvants were dissolved in 1 ml of PBS buffer and mixed. 500 μl of the diluted nanoemulsion adjuvants were measured for particle size distribution and PDI using a Malvern particle size analyzer to evaluate the physicochemical properties of the different composite nanoemulsion adjuvants.

[0077] Transmission electron microscopy (TEM) detection of different nanoemulsion adjuvants:

[0078] The MM59 and MA59 nanoemulsion particles prepared in this example were added to a carbon-coated copper mesh and allowed to stand at room temperature for 5 minutes. Excess liquid on the mesh was removed with absorbent paper. The mesh was then stained with a 2% phosphotungstic acid solution (pH 6.5) and dried for 30 minutes. The prepared electron microscopy sample was then observed using a transmission electron microscope (HITACHI H-7650) at 80 kV.

[0079] ATP is an effective adjuvant component, but due to its short half-life and the wide variety of cell types it affects, the targeting mechanism of ATP is relatively weak. MDP is a stimulator for training immune intracellular effects, and nano-delivery of MDP makes it easier to achieve immune training targeting antigen-presenting cells. The samples were tested for particle size and particle distribution under a transmission electron microscope (TEM) and a Malvern particle size analyzer. The particle size of MF59 prepared in Example 1 was about 160 nm, the particle size of MA59 with ATP added to the oil phase was about 184 nm, and the particle size of MM59 with MDP added to the oil phase was about 187 nm ( Figure 4 (A, B).

[0080] Example 5: Evaluation of trained immune nanoemulsion adjuvants on DC activation and cytotoxicity in vitro

[0081] This example evaluates the in vitro activation and cytotoxicity of DCs by the nanoemulsion adjuvants MF59, MM59, and MA59 prepared in Examples 1, 3, and 4. The specific method is as follows:

[0082] Step A) Removal of mouse femurs and tibias: Anesthetize 6-8 week old C57BL / 6J mice, debond them, and sterilize them by immersion in 75% ethanol. Use scissors to make an incision approximately 1 cm in diameter in the mouse abdomen. Remove the femurs and tibias, shave as much muscle as possible around the bones, and soak them in 75% ethanol for 5 minutes.

[0083] Step B) Obtaining bone marrow cells: The isolated mouse femur and tibia were transferred to sterile PBS and washed 2-3 times. The femur and tibia were then cut at both ends with scissors. The bone marrow cells in the femur and tibia were repeatedly rinsed with 1 mL of sterile RPMI-1640 medium. The cells were filtered through a 70 mm cell filter and the collected cell suspension was placed in a 15 mL centrifuge tube. The tube was centrifuged at 500 g for 5 min, and 5 mL of red blood cell lysis buffer was added to lyse the red blood cells. The tube was then added with 5 mL of sterile PBS to terminate the lysis. The tube was centrifuged at 800 g for 5 min to collect the cells.

[0084] Step C) Induction of BMDC differentiation: Resuspend the cells in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% double-antibody and add them to culture dishes at 10 mL / dish. Recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF) was added to the culture medium to a final concentration of 10 ng / mL. The culture dishes were placed in a 37°C, 5% CO2 incubator for static culture. After 3 days of culture, half of the medium was changed, 5 mL of medium was slowly aspirated, and RPMI-1640 complete medium containing 10 ng / mL GM-CSF was added to continue culture (the medium was changed every two days thereafter). After 8 days of culture, BMDCs were collected for subsequent experiments.

[0085] Step D) Stimulation: BMDCs collected above were seeded in 96-well plates, 1×10 7 2.5 μl of each of the nanoemulsion adjuvants MF59, MM59, and MA59 prepared in Examples 1, 3, and 4 were added respectively. Except for the PBS group, no antigen was added. All other groups were added with 1 μg of the antigen DS-Cav1. In addition, LPS, chitosan, CpG, β-glucan, ATP, and MDP were added in the order of 0.15 μg of LPS, 100 μg of chitosan, 25 ng of CpG, 80 μg of β-glucan, 0.65 μg of ATP, and 100 ng of MDP in each horizontal column. 2.5 μl of each of MF59, MM59, and MA59 were added to each vertical column. PBS was used as a negative control. The cells were incubated at 37° C. in a 5% CO 2 incubator for 24 h.

[0086] Step E) Detection of BMDC surface maturation markers by flow cytometry: The cells collected from each well were aliquoted into 15 ml EP tubes, and the corresponding flow cytometry antibodies were added. Blank wells and single-stained wells were set up, and the tubes were incubated in the dark at 4°C for 30 min. The tubes were centrifuged at 500 g for 5 min, and 200 μL of Cell Staining Buffer was added to each well to resuspend the cells. The cells were centrifuged at 500 g for 5 min (repeat twice), and finally, 100 μL of Cell Staining Buffer was used to resuspend the cells and analyzed by flow cytometry.

[0087] In order to compensate for the cellular immune mechanism that MF59 does not possess, in this example, the immunostimulants ATP, CpG, chitosan, LPS and the trained immune stimulants MDP and β-glucan were combined with the viral antigen DS-Cav1 for in vivo and in vitro immune stimulation.

[0088] Bone marrow cells were collected from the tibia and femur of mice and stimulated with GM-CSF in vitro to differentiate into DC cells. The differentiated DC cells were collected and stimulated with different combinations of adjuvants ( Figure 5 Middle A), flow cytometry analysis results showed that MF59, MM59, and MA59 combined with CpG and β-glucan significantly improved DC maturation, MHC-Ⅱ presentation molecule expression, and cell activity compared to other control groups. Among them, MM59 and MA59 were superior to MF59 in DC cell maturation, antigen presentation, and cell activity ( Figure 5 MA59 was slightly weaker than MM59 in stimulating mice to produce binding antibodies against the pre F antigen of respiratory syncytial virus.

[0089] After screening the above-mentioned training immune components, it was determined that further adding MDP to the oil phase of the nanoemulsion adjuvant MF59 to obtain a new nanoemulsion adjuvant MM59 can enhance antibody production.

[0090] Example 6: Evaluation of the trained immune mechanism of the composite nanoemulsion adjuvant MM59

[0091] To explore the evaluation of the effect of vaccines containing different nanoemulsion adjuvants on training innate immunity in vivo, the following experiments were conducted in this example:

[0092] Step A) First, 8-week-old female BALB / c mice were inoculated intramuscularly with PBS, MF59, or MM59.

[0093] Step B) 4 weeks after vaccination, MM59+CpG+DS-Cav1 was administered intramuscularly. Spleens were collected three days after vaccination for flow cytometry analysis.

[0094] Step C) Isolation of splenocytes to detect antigen-specific responses of lymphocytes expressing IFN-γ and IL-4 using ELISpot assay.

[0095] Immunity training is an immunological mechanism that comprehensively activates the body's immune cells such as DC cells, macrophages, T cells and other innate immune cells and acquired immune cells.

[0096] When evaluating the immune mechanism of training in mice, the first injection of this example was 25 μl / mouse PBS, MF59, and MM59 to stimulate the innate and acquired immune cells of mice. Four weeks later, each mouse was injected with 50 μl of the complete vaccine: MM59 (25 μl) + CpG (10 μg) + DS-Cav1 (10 μg) ( Figure 6 Middle A). Flow cytometry analysis of mouse spleens collected three days after immunization showed that MM59 significantly stimulated CD4 + T cells (CD3+ 、CD4 + )( Figure 6 Middle B) and CD8 + T cells (CD3 + 、CD8 + )( Figure 6 In terms of activating DC maturation, MM59 significantly upregulated CD80 compared with the PBS and MF59 groups. + 、CD86 + Upregulation of markers such as Figure 6 C and D). The expression of MHC-Ⅱ molecular markers for antigen presentation by mature DC cells was also significantly upregulated ( Figure 6 MM59 also activated the proliferation and differentiation of T cells. In the ELISpot experiment of RSV-pre F antigen-specific immune response, MM59 significantly increased the release of antigen-specific IFN-γ ( Figure 6 G), but did not increase the secretion of IL-4 ( Figure 6 Therefore, MM59 can differentiate T cells into Th1 cells and activate antigen-specific killer cells CTL cells (CD3 + CD8 + IFN-γ + ) trend( Figure 6 MF59 significantly decreased the activation of DCs and T cells, which is consistent with other related literature reports that MF59 does not have the function of activating DCs and T cells.

[0097] Example 7: MM59 has a dual immune mechanism of activating humoral immunity and cellular immunity

[0098] To explore the immune mechanism of the novel composite nanoemulsion adjuvant MM59, and with the nanoemulsion adjuvant MF59 as a control, the following experiments were conducted in this example:

[0099] Step A) Animal immunization

[0100] The pre-F protein subunit vaccine was prepared by diluting the pre-F protein to 200 μg / ml or 400 μg / ml. 25 μl of the diluted protein solution was mixed with 25 μl of the nanoemulsion adjuvant. The vaccine was injected intramuscularly into BALB / c mice according to the immunization flow chart. Blood was collected after immunization. Antibody levels were measured by ELISA.

[0101] Step B) ELISA analysis of anti-perF protein IgG binding antibody titer

[0102] Dilute 50 ng of pre-F subunit protein with ELISA coating buffer and place it into an ELISA plate, 50 μL per well. Coat overnight at 4°C and wash three times with PBST buffer. Add 200 μL of prepared 2% BSA to each well and incubate at 37°C for 1-2 hours. Wash three times with PBST buffer. Add 50 μL of diluted serum to each well and incubate at 37°C for 1 hour. Wash three times with PBST buffer. Add 50 μL of prepared HRP-Anti Mouse IgG secondary antibody to each well and incubate at 37°C for 1 hour. Wash five times with PBST buffer. Add TMB colorimetric solution and read the OD value at the appropriate time. A well is considered positive when the OD value is greater than the blank control OD value multiplied by 2.1.

[0103] Step C) ELISA analysis of IgG2a and IgG1 binding antibodies against perF protein

[0104] Dilute 50 ng of pre-F subunit protein with ELISA coating buffer and place it into an ELISA plate, 50 μL per well. Coat overnight at 4°C and wash three times with PBST buffer. Add 200 μL of prepared 2% BSA to each well, incubate at 37°C for 1-2 hours, and wash three times with PBST buffer. Add 50 μL of diluted serum to each well, incubate at 37°C for 1 hour, and wash three times with PBST buffer. Add 50 μL of prepared HRP-Anti Mouse IgG2a and IgG1 secondary antibodies to each well, incubate at 37°C for 1 hour, and wash five times with PBST buffer. Add TMB, a colorimetric solution, and read the OD value at the appropriate time.

[0105] Step D) In ​​vitro isolation of mouse spleen lymphocytes

[0106] After the mouse is sacrificed, the spleen is quickly removed from the body and collected into a centrifuge tube. In a biosafety cabinet, the spleen is placed on a 70μm filter and ground. Lymphocyte separation solution is added multiple times to grind until the final volume is 5mL. Then, 500μL of RPMI-1640 medium is slowly added along the wall of the tube to ensure the interface between the two. After centrifugation at 800g for 30 minutes, the tube is slowly removed and the white middle layer (lymphocyte layer) is aspirated into another centrifuge tube. If the middle layer is red, indicating the presence of red blood cells, the red blood cells are lysed with lysate, and then centrifuged at 500g for 5 minutes. The precipitated lymphocytes are collected, the supernatant is discarded, and the cells are resuspended in an appropriate amount of 1640 complete medium. Cell count is then performed.

[0107] Step E) Isolation of mouse lymph node lymphocytes in vitro

[0108] After the mouse was sacrificed, the lymph nodes were removed and placed in a 1.5 mL centrifuge tube on ice. The tube was minced with scissors and 500 μL of collagenase A solution (1 mg / mL) prepared with RPMI-1640 medium was added. The centrifuge tube was placed on a shaker at 37°C and 120 rpm to digest the lymph node tissue for 1 hour. Subsequent operations were basically the same as those for isolating mouse spleen lymphocytes in vitro.

[0109] Step F) Detection of cellular immunity levels by flow cytometry

[0110] After isolating the lymphocytes and counting the cells, the specific operation of the mouse spleen lymphocyte flow cytometry method is as follows:

[0111] (1) Laying the board: 1×10 7 Lymphocytes were plated in a 96-well U-shaped plate. Stimulation with pre-F antigen protein (final concentration 50 μg / mL) was performed. (If measuring cytotoxic T lymphocytes, after incubation for 2 hours, 1 μL of 100× Brefeldin A was added to each well and incubated for 5 hours.) After 7 hours of culture, the cells were washed twice with 200 μL of cell staining buffer, the supernatant was removed, and the prepared anti-mouse CD3, CD4, or CD8 monoclonal antibodies were added. The cells were incubated on ice in the dark for 30 minutes.

[0112] (2) After the antibody incubation is complete, wash the cells twice with 200 μL cell staining buffer, then fix the cells with Fixation buffer. Incubate in the dark for 20 minutes, then wash the cells twice with 200 μL cell staining buffer. After washing, permeabilize the cells with Permeabilization Wash buffer, centrifuge at 500 g for 5 minutes, remove the supernatant, and permeabilize once more with Permeabilization Wash buffer.

[0113] (3) Add the intracellular factor IFN-γ or IL-4 to be stained into the cells, incubate in the dark for 20 min, then wash twice with Permeabilization Wash buffer, and finally resuspend the cells with cell staining buffer, and analyze the lymphocytes by flow cytometry.

[0114] MF59 is a Th2-biased vaccine adjuvant that primarily triggers antigen recognition and presentation by causing local inflammation and recruiting inflammatory cells, particularly monocytes. However, MF59 lacks the ability and properties to trigger a range of cellular immune responses.

[0115] Each mouse was injected intramuscularly with MF59 (25 μl) + DS-Cav1 (5 μg), MF59 (25 μl) + CpG (10 μg) + DS-Cav1 (5 μg), and MM59 (25 μl) + CpG (10 μg) + DS-Cav1 (5 μg) in the thigh. A second injection was performed four weeks later. Three days after the second injection, blood and spleen were collected to examine humoral immunity and cellular immunity ( Figure 7 Middle A). When CpG adjuvant is added to the vaccine, the secretion of IgG antibodies will be significantly enhanced ( Figure 7 B), and improved the production type of antibodies to IgG2a ( Figure 7 Among them, MM59 still maintains efficient antibody production, and the antibody titer is not weaker than MF59, and has a stronger humoral immunity mechanism ( Figure 7 Middle B). MDP, as a training immune stimulator, endows MF59 with the ability to activate memory T cells, which is consistent with the subsequent advantages in secondary immunity and antibody persistence. + 、CD62L + ) and CD8 (CD8 + 、CD44 + 、CD62L + ) The proliferation of central memory T cells Tcm provides mice with sufficient advantages for the next recognition and elimination of pathogens ( Figure 7 Effector memory T cells TEM are mainly distributed in non-lymphoid tissues and peripheral blood. Therefore, the spleen, a lymphoid organ, has fewer effector memory T cells. It is possible that the fewer effector memory T cells there are, the more concentrated they are in peripheral tissues to detect and identify pathogens ( Figure 7 G, I). MM59 significantly enhanced Th1 (CD3 + 、CD4 + IFN-γ + ) immune cell proliferation, reducing Th2 (CD3 + 、CD4 + IL-4 + ) immune cell proliferation, playing an important role in improving immune bias. Therefore, MM59 is a novel nanoemulsion adjuvant that combines humoral and cellular immunity.

[0116] Example 8: MM59 induces antibodies faster and longer-lasting than MF59

[0117] To compare the ability of nanoemulsion adjuvants MM59 and MF59 to elicit antibodies, the following experiments were conducted in this example;

[0118] Step A) Animal immunization

[0119] The pre-F protein subunit vaccine was prepared by diluting the pre-F protein to 200 μg / ml or 400 μg / ml. 25 μl of the diluted protein solution was mixed with 25 μl of the nanoemulsion adjuvant. The vaccine was injected intramuscularly into BALB / c mice according to the immunization flow chart. Blood was collected after immunization. Antibody levels were measured by ELISA.

[0120] Step B) ELISA analysis of anti-perF protein IgG binding antibody titer

[0121] Dilute 50 ng of pre-F subunit protein with ELISA coating buffer and place it into an ELISA plate, 50 μL per well. Coat overnight at 4°C and wash three times with PBST buffer. Add 200 μL of prepared 2% BSA to each well and incubate at 37°C for 1-2 hours. Wash three times with PBST buffer. Add 50 μL of diluted serum to each well and incubate at 37°C for 1 hour. Wash three times with PBST buffer. Add 50 μL of prepared HRP-Anti Mouse IgG secondary antibody to each well and incubate at 37°C for 1 hour. Wash five times with PBST buffer. Add TMB colorimetric solution and read the OD value at the appropriate time. A well is considered positive when the OD value is greater than the blank control OD value multiplied by 2.1.

[0122] Step C) ELISA analysis of IgG2a and IgG1 binding antibodies against perF protein

[0123] Dilute 50 ng of pre-F subunit protein with ELISA coating buffer and place it into an ELISA plate, 50 μL per well. Coat overnight at 4°C and wash three times with PBST buffer. Add 200 μL of prepared 2% BSA to each well, incubate at 37°C for 1-2 hours, and wash three times with PBST buffer. Add 50 μL of diluted serum to each well, incubate at 37°C for 1 hour, and wash three times with PBST buffer. Add 50 μL of prepared HRP-Anti Mouse IgG2a and IgG1 secondary antibodies to each well, incubate at 37°C for 1 hour, and wash five times with PBST buffer. Add TMB, a colorimetric solution, and read the OD value at the appropriate time.

[0124] Each mouse was injected intramuscularly with DS-Cav1 (10 μg), MF59 (25 μl) + DS-Cav1 (10 μg), MM59 (25 μl) + DS-Cav1 (10 μg), MF59 (25 μl) + CpG (10 μg) + DS-Cav1 (10 μg), and MM59 (25 μl) + CpG (10 μg) + DS-Cav1 (10 μg) in the thigh. The second injection was performed four weeks later. Blood samples were collected at regular intervals to detect the IgG response of mice to RSV pre-F protein ( Figure 8 Seven days after the first vaccination, blood tests revealed that the antibody titer of the MM59+CpG+DS-Cav1 group was 48,000, which was higher than the 33,600 of the MM59+CpG+DS-Cav1 group, indicating that MM59 has the effect of accelerating antibody production ( Figure 8 The antibody titers of each vaccine group were tested at regular intervals. The antibody titers of the vaccine group containing CpG were significantly higher than those without CpG. As time passed after the second immunization, the antibody titer of the adjuvant group containing MDP began to decline more slowly. Among them, 40 days after the second immunization, the antibody titer of the MM59+CpG+DS-Cav1 group was higher than that of the MF59+CpG+DS-Cav1 group, and the antibody titer of the MM59+DS-Cav1 group was higher than that of the MF59+DS-Cav1 group ( Figure 8 In terms of antibody types specific to RSV pre-F protein, the adjuvant containing the MDP group also increased the proportion of IgG2a antibodies in all IgG antibodies ( Figure 8 Middle D).

[0125] Example 9: MM59+CpG+DS-Cav1 protects mice from respiratory syncytial virus attack

[0126] Step A) Mouse challenge protection experiment

[0127] Mice that were 56 days after two injections were immunized and then inoculated with 5×10 5 The mice were infected with PFU of A2 strain virus. The weight and body temperature of the mice were monitored every day after infection. The mice were sacrificed 4 days after infection to evaluate the pathological changes in their lungs.

[0128] Step B) Collect bronchoalveolar lavage fluid and left lung of mice

[0129] A 1ml PBS solution was placed in a cannula and the lungs were irrigated. Alveolar lavage fluid was obtained after three repeated aspirations and insufflations. The volume of the lavage fluid was recorded. The supernatant containing the lavage fluid and cells was obtained by centrifugation at 500g for 5 minutes. The cells were suspended in the same volume of PBS. A portion was counted using a red blood cell counter, and 10μl of the remaining portion was spun onto a slide and then stained with Giemsa.

[0130] Step C) After the left lung was isolated, it was fixed with 4% paraformaldehyde and sliced ​​with HE.

[0131] To verify whether the humoral and cellular immunity induced by MM59 can protect mice from RSV attack, we conducted a virus challenge experiment 63 days after the second immunization. We administered 50 μl of 1×10 7 PFU / ml of A2 strain, and monitor the weight, body temperature and other changes of mice every day after infection ( Figure 9The weight of mice in the PBS group dropped sharply after the challenge, and 4 mice died on the second day, while no mice in the other vaccine groups died. The weight of mice in the other vaccine groups first dropped slightly and then recovered ( Figure 9 The body temperature of mice in the PBS group first dropped sharply, and four mice died after the sharp drop, while the condition of another mouse improved slightly. However, the body temperature of mice in the vaccine group did not change significantly ( Figure 9 Middle C).

[0132] In the alveolar lavage fluid of mice, the cell infiltration in the lung space of the PBS group increased significantly, while the vaccine group maintained a normal number ( Figure 9 Flow cytometry analysis of the infiltration and proliferation of antigen-specific T cells in the spleen of mice after challenge showed that the CTL cell infiltration in the adjuvant groups containing MDP was higher than that in the group without MDP, and the CTL cell infiltration in the adjuvant group containing MM59+CpG+DS-Cav1 was the highest ( Figure 9 In Giemsa staining of the alveolar lavage fluid of mice, the infiltration of neutrophils, monocytes, and macrophages in the vaccine group was significantly reduced. HE results showed that the MM59+CpG+DS-Cav1 group had the weakest lung inflammation, congestion, and alveolar septum thickening ( Figure 9 Middle G).

[0133] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and is not limiting. Although the present invention is described in detail with reference to the preferred arrangement scheme, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A novel nanoemulsion adjuvant based on MDP, characterized in that: The adjuvant is an oil-in-water nanoemulsion, the oil phase contains squalene, Span85 and muramyl dipeptide, and the aqueous phase contains Tween80 and citric acid buffer; Calculated by mass volume percentage, the oil phase includes 3.0% to 5.0% of squalene, 0.2% to 3.0% of Span85, and 0.0001% to 0.1% of muramyl dipeptide.

2. The novel nanoemulsion adjuvant based on MDP according to claim 1, characterized in that The mass volume ratio of the oil phase to the water phase is 3% to 6%: 94% to 97%.

3. A novel composite nanoemulsion adjuvant that enhances antibody production and trains immunity, characterized in that: The novel nanoemulsion adjuvant according to claim 1 or 2 is further composed of CpG.

4. The novel composite nanoemulsion adjuvant for enhancing antibody production and training immunity according to claim 3, characterized in that: The amount of CpG added was 10 μg per injection.

5. A method for preparing a novel nanoemulsion adjuvant based on MDP as claimed in claim 1 or 2, characterized in that: The preparation method comprises the following steps: 1) Preparation of oil phase: Squalene and Span 85 were placed in a beaker and uniformly mixed at 800-1500 rpm to obtain a primary mixed oil phase; MDP was dissolved in DMSO and the dissolved MDP was poured into the primary mixed oil phase and stirred to obtain a mixed oil phase; 2) Preparation of aqueous phase: Place Tween 80 and citric acid buffer in a conical flask and mix uniformly at 800-1500 rpm to obtain a mixed aqueous phase; 3) mixing the mixed oil phase and the mixed water phase, and uniformly mixing the mixed solution at 800-1500 rpm to obtain an initial emulsion; the initial emulsion is subjected to high-pressure homogenization and membrane filtration to obtain the novel nanoemulsion adjuvant.

6. The preparation method according to claim 5, characterized in that In step 3), the high-pressure homogenization treatment is carried out at a pressure of 500 to 1000 bar for 3 to 5 minutes.

7. Use of the novel nanoemulsion adjuvant according to claim 1 or 2, or the novel composite nanoemulsion adjuvant according to claim 3 or 4, or the novel nanoemulsion adjuvant prepared by the method according to claim 5 or 6 in the preparation of vaccines.

8. A vaccine, characterized in that The invention comprises the novel nanoemulsion adjuvant according to claim 1 or 2, or the novel composite nanoemulsion adjuvant according to claim 3 or 4, or the novel nanoemulsion adjuvant prepared by the method according to claim 5 or 6.

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