Complete immune activated holoantigen nano vaccine as well as preparation method and application thereof

Abstract

The invention discloses a full immune activation holoantigen nano vaccine and a preparation method and application thereof, and relates to the technical field of vaccine preparation, the nano vaccine is obtained by using mesoporous nanoparticles as a carrier to load a bacterial plasm antigen and then coating the surface with a bacterial membrane antigen, the particle size of the nano vaccine is 20-200nm, and the particle size of the nano vaccine is 20-200nm. The problems that the existing mesoporous silica loaded single antigen for preventing staphylococcus aureus infection is poor in effect and unstable in structure are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological vaccine preparation, and in particular to a preparation method and application of a fully immune-activated full-antigen nano-vaccine delivery system. Background Art

[0002] Staphylococcus aureus is a pathogenic bacterium with high pathogenicity and wide colonization range, which has posed a major threat to human life and health. The birth of antibiotics once brought unlimited hope for the treatment of bacterial infections, but with the emergence of drug-resistant bacteria and "super bacteria" caused by the abuse of antibiotics, the treatment of Staphylococcus aureus has once again fallen into crisis. Therefore, it is urgent to find new treatment strategies for Staphylococcus aureus infection. In recent years, nanovaccine delivery systems have attracted widespread attention due to their unique immune activation function.

[0003] A large number of studies have shown that mesoporous silica has a high loading rate, large specific surface area, adjustable particle size and pores, an easily functionalized structural framework, and good biocompatibility, and is considered to be a promising nanoadjuvant development platform. Although studies have used mesoporous silica to load single antigens to prevent Staphylococcus aureus infection, these methods have limitations in terms of immune activation efficacy and serotype protection spectrum, and are limited in preventing Staphylococcus aureus infection. Therefore, the development of a full-antigen nanovaccine delivery system that can achieve full immune activation to combat bacterial mutations and broaden the serotype protection spectrum has become a scientific problem that needs to be solved urgently. Summary of the invention

[0004] In response to the above problems, the present invention provides a fully immune-activated full-antigen nanovaccine and a preparation method and application thereof, which solves the problem that the current mesoporous silica loaded with a single antigen to prevent Staphylococcus aureus infection has poor effect and unstable structure.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A fully immune activated full antigen nano vaccine, wherein the nano vaccine is obtained by using mesoporous nanoparticles as carriers to load bacterial antigens and then coating the surface with bacterial membrane antigens, and the particle size of the nano vaccine is 20-200nm, preferably 90-150nm.

[0007] Furthermore, the mesoporous nanoparticles are one of mesoporous silica or mesoporous polydopamine with a pore size of 5-30 nm, preferably 15-25 nm.

[0008] The present invention uses mesoporous silica as a carrier, loads the cytoplasmic antigen of Staphylococcus aureus in the pores, and coats the surface with the bacterial membrane antigen of Staphylococcus aureus, preparing a fully immunologically activated whole antigen nano-vaccine. Compared with traditional nano-vaccines, the present invention has a more abundant antigen delivery system, can use mesoporous silica to co-transport the cytoplasmic antigen and the bacterial membrane antigen to immune tissues and cells, activate a highly efficient immune activation reaction, and produce specific antibodies against Staphylococcus aureus, thereby achieving the prevention and treatment effects on the body.

[0009] The present invention also discloses a preparation method of a fully immunologically activated whole antigen nano-vaccine, and the preparation method is as follows:

[0010] (1) Collect bacteria in the logarithmic growth phase, wash and resuspend them, then perform repeated freezing and thawing and ultrasonic disruption treatments. Centrifuge the obtained suspension, and the obtained precipitate is the bacterial membrane antigen BM, and the suspension is the cytoplasmic antigen BC;

[0011] (2) Add cetyltrimethylammonium p-toluenesulfonate and triethanolamine to water and mix evenly, then slowly dropwise add tetraethyl orthosilicate, and stir at 80 °C and a rate of 1000 - 1500 rpm for 12 h. Centrifuge to collect the precipitate, wash it, place the obtained product in absolute ethanol, slowly dropwise add 3-aminopropyltriethoxysilane, stir again, centrifuge to collect the precipitate, wash it, and freeze-dry it to obtain mesoporous silica MSNs;

[0012] (3) Place the mesoporous silica in water, ultrasonically disperse it evenly, and mix it with the cytoplasmic antigen, and oscillate at a rate of 0 - 4 °C and 500 - 1000 rpm for 12 h. Centrifuge to collect the precipitate, wash it, and then freeze-dry it to obtain MSNs-BC;

[0013] (4) After mixing the obtained MSNs-BC with the bacterial membrane antigen solution, extrude it through a polycarbonate porous membrane using a liposome microextruder, wash it after extrusion, and freeze-dry it to obtain the whole antigen nano-vaccine MSNs-BC@BM.

[0014] Furthermore, the antigen molecular weight of the nano-vaccine is 20 - 150 kDa.

[0015] Furthermore, the bacteria include one of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus.

[0016] Furthermore, the specific operation of the repeated freezing and thawing in (1) is: freeze at -80 °C and then dissolve at 37 °C, and the number of times is 2 - 5 times; in the ultrasonic disruption treatment, the ultrasonic power used is 400 - 600 W, and the time is 30 - 120 min.

[0017] Further, the mass-volume ratio of cetyltrimethylammonium p-toluenesulfonate, triethanolamine and tetraethyl orthosilicate in (2) is (0.15 - 0.3) g : (0.03 - 0.09) g : (1 - 2) ml.

[0018] Further, the mass ratio of the mesoporous nano-silica to the bacterial cytoplasmic antigen is 1 : (1 - 4), and the mass ratio of the MSNs-BC to the bacterial membrane antigen is 1 : (1 - 3).

[0019] For the optimization of the preparation method of the nano-vaccine, the mass-volume ratio of cetyltrimethylammonium p-toluenesulfonate, triethanolamine and tetraethyl orthosilicate is 0.263 g : 0.04 g : 2 ml; the mass ratio of the mesoporous silica to the bacterial cytoplasmic antigen is 1 : 1, and the mass ratio of the MSNs-BC to the bacterial membrane antigen is 1 : 1.

[0020] When the present invention prepares the nano-vaccine related to Staphylococcus aureus, it has a more abundant antigen delivery system. However, during the preparation, it means that it is more difficult to load the double-layer antigen, and the loading efficiency of the external bacterial membrane antigen is low. Therefore, in the process of preparing the nano-vaccine of the present invention, by adjusting the raw material ratio and operation parameters for preparing MSNs-BC, MSNs-BC and the subsequent bacterial membrane antigen can be better loaded together. At the same time, it is also necessary to adjust the mass ratio of MSNs-BC to the bacterial membrane antigen and the loading operation to avoid adverse reactions caused by over-loading of antigens leading to over-immunization.

[0021] The nano-vaccine prepared by the present invention is applied to the treatment of bacterial infections, such as the treatment and immunization of Staphylococcus aureus-related infections.

[0022] Beneficial effects:

[0023] (1) The present invention prepares a fully antigen nano-vaccine that can achieve sufficient immune activation, has a more abundant antigen delivery system to fully activate the in vivo immune efficacy, and can effectively inhibit wound bacterial infection and promote wound healing by preventing over-immunization through a suitable antigen ratio.

[0024] (2) The present invention simply separates the bacterial cytoplasmic antigen and the bacterial membrane antigen, and the subsequent nano-vaccine preparation process is simple and controllable, the obtained nano-vaccine has a stable structure, and there is no aggregation between the vaccines. Description of the drawings

[0025] Figure 1 : Transmission electron micrograph (A); particle size (B); Zeta potential (C); nitrogen adsorption isotherm and pore size distribution of MSNs (D) during the preparation of the nano-vaccine MSNs-BC@BM;

[0026] Figure 2: SDS-PAGE protein gel electrophoresis and LC-MS / MS analysis during the preparation of the nano-vaccine MSNs-BC@BM;

[0027] Figure 3 : (A) Process of extracting immune cells BMDCs; (B) In vitro activation of immune cell marker FITC CD 86 by the nano-vaccine MSNs-BC@BM; (C) APC CD 80; (D) PE MHC-I; (E) FITC MHC-II;

[0028] Figure 4 : IgG antibody titer in the immune serum activated by the nano-vaccine MSNs-BC@BM;

[0029] Figure 5 : Activation of immune cell markers in the spleen in vivo by the nano-vaccine MSNs-BC@BM (A) FITC CD 4; (B) FITC CD 8; (C) APC CD 19;

[0030] Figure 6 : (A) Photos of the infected wounds of mice at different times after different treatments; (B) Photos of the bacterial colonies formed by bacteria in the wound tissues of mice; (C) Relative wound area of the infected wounds of mice at different times; (D) Number of viable bacteria in the wound tissues of mice. Detailed implementation mode

[0031] The present invention will be described in detail below in conjunction with specific embodiments and drawings:

[0032] Example 1: Preparation of nano-vaccine

[0033] (1) After Staphylococcus aureus grew to the logarithmic growth phase, the bacteria were collected by centrifugation at a rate of 6000 rpm for 5 min and washed twice with cold sterile 1×PBS. After resuspending the bacteria in 1×PBS containing a protease inhibitor mixture, they were frozen at -80°C and then thawed at 37°C, and the bacteria were repeatedly frozen and thawed 3 times. Then, the suspension was sonicated for 30 min (on for 5 s, off for 5 s, 540 W) using an ultrasonic homogenizer. The sonicated suspension was centrifuged at 8000 rpm for 35 min, and the obtained precipitate was the bacterial membrane antigen BM, and the suspension was the bacterial cytoplasmic antigen BC. Finally, the BC in the supernatant was filtered through a 0.22 μm filter to remove residual cell debris, and then BC was collected by centrifugation at 12000 rpm for 100 min.

[0034] (2) 0.263 g of cetyltrimethylammonium p-toluenesulfonate and 0.04 g of triethanolamine (TEA) were added to 20 ml of water and mixed evenly. Then, 2 ml of tetraethyl orthosilicate (TEOS) was slowly added dropwise, and the mixture was stirred at 80 °C and a rate of 1000 rpm for 12 h. The resulting product was centrifuged and washed three times with ethanol, and then extracted with 100 ml of ethanol solution at 80 °C for 12 h to remove the template. 1 g of the above product was added to 60 ml of anhydrous ethanol with 1 ml of 3-aminopropyltriethoxysilane (APTES), refluxed for 10 h, and finally freeze-dried for subsequent experiments.

[0035] (3) First, 2 mg / mL of bacterial cytoplasmic antigen BC and 2 mg / mL of mesoporous silica MSNs were oscillated at 4 °C and 500 rpm for 12 h to obtain MSNs-BC.

[0036] (4) The 2 mg MSNs-BC solution prepared above was mixed with 2 mg of bacterial membrane antigen BM, and then extruded through a 200 nm polycarbonate porous membrane using a liposome mini-extruder. After extrusion, the excess BM and BC were separated by dialysis, the precipitate was collected by centrifugation, washed, and then freeze-dried to obtain the whole antigen nano-vaccine (MSNs-BC@BM).

[0037] The nano-vaccine obtained above was observed by electron microscopy, and the results are as Figure 1 、 Figure 2 shown:

[0038] Transmission electron microscopy images showed that BC and BM could be successfully separated by the method of step (1) of the present invention ( Figure 1 -A). On the basis of successfully separating BC and BM, during the preparation process of the MSNs-BC@BM nano-vaccine, the hydrodynamic diameter ( Figure 1 -B) and Zeta potential ( Figure 1 -C) of each step product were detected. The changes in hydrodynamic diameter and Zeta potential supported the successful preparation of the MSNs-BC@BM nano-vaccine, indicating that the surface was successfully coated with bacterial membrane antigen after loading the bacterial cytoplasmic antigen. In addition, the nitrogen adsorption isotherm further showed that MSNs had a mesoporous structure ( Figure 1 -D), with a pore diameter of 22.33 nm and a specific surface area of 1406.81 m 2 / g. The above results showed that MSNs were successfully prepared, providing a carrier for the subsequent successful preparation of the nano-vaccine. Transmission electron microscopy showed that BC was attached in the pores of MSNs ( Figure 1 -A), and a viscous BM layer covered the surface of MSNs-BC ( Figure 1-A) also indicates the successful preparation of MSNs-BC@BM nanovaccine. SDS-PAGE protein gel electrophoresis and LC-MS / MS analysis ( Figure 2 ), indicating that the prepared nanovaccine is rich in loaded antigens.

[0039] Experiment 1: In vitro activation of immunity by nanovaccine

[0040] Mouse myeloid dendritic cells BMDCs were extracted from the bone marrow of 6-week-old male C57 BL / 6 mice. Immature BMDCs were seeded in 12-well plates at a density of 10 6 cells per well. Then, the cells were stimulated with various formulations containing BC+BM and MSNs-BC@BM (both containing 2.5 μg total protein) at 37 °C for 24 h, and mice treated with PBS were used as controls. Subsequently, the cells were stained with fluorescently labeled antibodies FITC CD86, APC CD80, PE MHC-I, and FITC MHC-II for 30 min. Finally, flow cytometry analysis was performed on all samples, and the results were as Figure 3 shown:

[0041] The experimental results showed that: compared with free BC+BM and PBS, the expression levels of the mature characteristic markers CD86, CD80, MHC-I, and MHC-II of BMDCs were significantly increased in the MSNs-BC@BM nanovaccine group. It can be thus shown that BMDCs cells possess the ability of antigen presentation, can effectively activate and regulate the immune response of T cells, and promote wound healing.

[0042] Experiment 2: In vivo activation of immune response by nanovaccine

[0043] Five-week-old male BALB / c mice were randomly divided into 3 groups of 5 mice each. The mice were immunized on days 1, 8, and 15: (1) NaCl, (2) BC+BM, (3) MSNs-BC@BM. The total protein concentration of all samples was 25 μg / mL total protein, and the volume was 100 μL. Mouse jugular venous blood samples were taken on days 0, 7, 14, and 21 to collect serum. The serum was collected, and the IgG antibody titer was quantitatively detected by enzyme-linked immunosorbent assay (ELISA). The mice were sacrificed 7 d after the last immunization. The spleens and sera of each group of mice were collected. The excised spleens were mechanically digested into single-cell suspensions. Flow cytometry was used to detect APC CD 3, FITC CD4, FITC CD 8, and APC CD 19 fluorescently labeled anti-mouse antibodies in 1×10 6 spleen cells of each group. The results were as Figure 4 , Figure 5 shown:

[0044] Experimental results: The IgG antibody titer in serum continuously increased during the immunization cycle, and the MSNs-BC@BM nano-vaccine group had the strongest ability to increase the IgG antibody titer ( Figure 4 ); compared with the NaCl group and the BC+BM group, the percentages of CD4 + T cells, CD8 + T cells, and CD19 + B cells in the spleens of mice immunized with the MSNs-BC@BM nano-vaccine were significantly increased ( Figure 5 ). It can be thus shown that the prepared nano-vaccine performs excellently in inducing immune responses and has potential application value in vaccine development.

[0045] Experiment 3: Protective efficacy of nano-vaccine against wound infection

[0046] Referring to Experiment 2, on the 7th day after the last immunization, a 6-mm infected wound model was established on the backs of male BALB / c mice. The wounds were injected with Staphylococcus aureus cells (5×10 7 CFU, 60 μL). The wounds of each group were measured and photographed every day to monitor the healing process. After 7 days, the infected wound tissues of the mice were homogenized and placed in 1 mL of sterile physiological saline and incubated at 37 °C for 24 h. The plate colony method was used to analyze the bacterial content at the infected site. The results obtained are as Figure 6 shown:

[0047] Experimental results: As shown in Figure 6 A, 6C, compared with the NaCl group and the BC+BM group, the wound areas of the mice treated with the MSNs-BC@BM nano-vaccine were significantly reduced, and were basically completely healed on the 7th day of treatment, and new pink skin could be observed. The plate colony counting method was used to determine the number of bacteria on the wounds of the mice. The results are as Figure 6 shown in B, 6D. After treatment with the MSNs-BC@BM nano-vaccine, the survival rate of bacteria decreased significantly, which was significantly lower than that of other treatment groups. It shows that the MSNs-BC@BM nano-vaccine group can effectively activate the body's immune response, inhibit Staphylococcus aureus infection, and promote wound healing.

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention and not 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 within the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. A fully immune activated full antigen nano vaccine, characterized in that: The nano vaccine is obtained by using mesoporous nano particles as carriers to load bacterial antigens and then coating the surface with bacterial membrane antigens. The particle size of the nano vaccine is 20-200 nm.

2. A fully immune activated full antigen nanovaccine according to claim 1, characterized in that: The mesoporous nanoparticles are mesoporous silica or mesoporous polydopamine with a pore size of 5-30 nm.

3. The method for preparing a fully immune activated full antigen nano vaccine according to claim 2, characterized in that: The preparation method is as follows: (1) Collect bacteria that have grown to the logarithmic growth phase, freeze-thaw repeatedly, and ultrasonically disrupt them, then centrifuge the resulting suspension. The resulting precipitate is the bacterial membrane antigen BM, and the suspension is the bacterial plasma antigen BC; (2) adding hexadecyltrimethylammonium p-toluenesulfonate and triethanolamine to water and mixing them evenly, then slowly adding tetraethyl orthosilicate, stirring at 80°C and 1000-1500 rpm for 12 hours, collecting the precipitate by centrifugation, washing, placing the obtained product in anhydrous ethanol, slowly adding 3-aminopropyltriethoxysilane, stirring again, collecting the precipitate by centrifugation, washing, and freeze-drying, and the obtained product is mesoporous silica MSNs; (3) placing mesoporous silica in water, uniformly dispersing it by ultrasonication, and mixing it with bacterial antigens, shaking it at 0-4°C and 500-1000 rpm for 12 h, collecting the precipitate by centrifugation, washing it, and freeze-drying it to obtain MSNs-BC; (4) The obtained MSNs-BC was mixed with a bacterial membrane antigen solution and extruded through a polycarbonate porous membrane using a liposome microextruder. After extrusion, the mixture was washed and freeze-dried to obtain the full antigen nanovaccine MSNs-BC@BM.

4. The method for preparing a fully immune activated full antigen nano vaccine according to claim 3, characterized in that: The bacteria include one of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus.

5. The method for preparing a fully immune activated full antigen nano vaccine according to claim 3, characterized in that: The specific operation of repeated freezing and thawing of (1) is as follows: freezing at -80°C and then thawing at 37°C for 2-5 times; in the ultrasonic crushing treatment, the ultrasonic power used is 400-600W and the time is 30-120min.

6. The method for preparing a fully immune activated full antigen nano vaccine according to claim 3, characterized in that: The mass volume ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine and tetraethyl orthosilicate in (2) is (0.15-0.3) g: (0.03-0.09) g: (1-2) ml.

7. The method for preparing a fully immune activated full antigen nano vaccine according to claim 6, characterized in that: The mass ratio of the mesoporous nano-silica to the bacterial antigen is 1:(1-4), and the mass ratio of the MSNs-BC to the bacterial membrane antigen is 1:(1-3).

8. The method for preparing a fully immune activated full antigen nano vaccine according to claim 7, characterized in that: The mass volume ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine and tetraethyl orthosilicate is 0.263g:0.04g:2ml; the mass ratio of the mesoporous silica to the bacterial antigen is 1:1, and the mass ratio of the MSNs-BC to the bacterial membrane antigen is 1:

1.

9. The nanovaccine prepared according to any one of claims 3 to 8, characterized in that: The nano vaccine is used to treat bacterial infection.

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