Particle carrier sustained-release vaccine and application thereof

Abstract

The invention discloses a particle carrier sustained-release vaccine and application thereof. The particle carrier sustained-release vaccine is a self-healing microsphere combined with an aluminum adjuvant; antigens are loaded inside and outside the self-healing microspheres. The preparation method of the particle carrier sustained-release vaccine comprises the following steps: preparing blank porous microspheres by a multiple emulsion emulsification solvent volatilization method, a microfluidic technology, a spray drying method, a salt dissolution method and the like, and loading an aluminum adjuvant adsorbed with an antigen into the blank porous polymer microspheres in a physical diffusion and electrostatic adsorption manner, so as to prepare the particle carrier sustained-release vaccine. Finally, the microspheres are self-healed through the characteristics of the polymer, so that the antigen is divided into an inner part and an outer part. After the particle carrier sustained-release vaccine enters a body, relatively continuous antigen sustained release is respectively carried out in different time periods; the prepared vaccine can effectively collect and activate antigen-presenting cells, release antigens and be taken in by the antigen-presenting cells, so that immune response is caused, generation of specific antibodies is induced, and immune memory is formed.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a particle carrier sustained-release vaccine and application thereof. Background Art

[0002] Vaccines, especially preventive vaccines, have been widely used as the main response method to infectious diseases. Antigen delivery vectors are the key components of new vaccines, which continuously improve the efficacy of vaccines and promote the replacement of vaccines, as the existing adjuvants are not effective and difficult to promote the long-term protective effect of vaccines.

[0003] Anthrax is an infectious disease of animal origin caused by Bacillus anthracis. Bacillus anthracis mainly invades the skin and causes cutaneous anthrax, which forms eschar ulcers with surrounding abscesses and toxemia. It can also cause inhalation anthrax or gastrointestinal anthrax, both of which can be complicated by sepsis. Bacillus anthracis may be used as a biological weapon by terrorists, and has therefore received widespread attention from the international community in recent years. In 1970, the United States approved the anthrax vaccine adsorbed (AVA). In 1979, the United Kingdom approved the use of anthrax vaccine precipitated (AVP) and has been using it for many years. The above two vaccines are adsorbed or precipitated by aluminum adjuvants and are prepared from the cell-free culture supernatant of Bacillus anthracis lacking the pXO2 plasmid. Their main active ingredients are the protective antigen PA. The initial immunization program for the AVA vaccine was 6 injections of subcutaneous administration. In 2008, based on the results of the non-inferiority evaluation of the peak immune response, it was changed to 5 injections of intramuscular administration. In 2012, its primary immunization program was approved as 3 injections at 0, 1, and 6 months. The AVA immunization program is complex, and it is difficult to ensure the consistency of effectiveness between different batches. It may not be protective against strains that lack anthrax toxins. Although the attenuated live anthrax vaccine has good protection, it has a certain residual toxicity, and the scratch method on the skin makes it difficult to guarantee the inoculation amount. Therefore, the current anthrax vaccines have limitations, and researchers are constantly applying new technologies and platforms to develop safe and effective improved anthrax vaccines.

[0004] Poly lactide-co-glycolide (PLGA) is a random polymerization of lactic acid and glycolic acid. Due to its good biocompatibility and biodegradability, it has been approved by many countries for in vivo delivery of active ingredients. Microparticles and nanoparticles made of PLGA are also used in the field of vaccines, playing a role in vaccines through controlled release of antigens and immune adjuvant functions. Based on this, the present invention uses PLGA microparticles as delivery carriers, utilizes its slow-release and weak immune adjuvant capabilities, combines aluminum adjuvants to fix recombinant anthrax antigens, and cooperates with small molecule adjuvants to develop a new anthrax vaccine with high efficiency and long-term effects, so as to solve the problems of poor immune effect and cumbersome immune procedures of existing anthrax vaccines. Summary of the invention

[0005] The purpose of the present invention is to provide a particle carrier sustained-release vaccine that can achieve slow and sustained release of antigens, by loading antigen proteins into microspheres and injecting them into the body intramuscularly to induce an immune response in the body. The vaccine provided by the present invention has mild preparation conditions, no obvious toxicity and good immune effect.

[0006] The particle carrier sustained-release vaccine provided by the present invention is a self-healing microsphere combined with an aluminum adjuvant for adsorbing antigens; the antigens are loaded both inside and outside the self-healing microsphere.

[0007] Furthermore, the particle carrier sustained-release vaccine is prepared by first preparing blank porous microspheres, then loading the aluminum adjuvant adsorbed with the antigen into the blank porous microspheres by physical diffusion and electrostatic adsorption, and then allowing the microspheres to self-heal based on the characteristics of the polymer itself, thereby separating the antigen into two parts inside and outside the microsphere.

[0008] Specifically, the particle carrier sustained-release vaccine is prepared according to a method comprising the following steps: using aluminum adjuvant to adsorb antigens; after adsorption, incubating and co-loading with blank porous microspheres; and then heating the porous microspheres to allow them to self-heal, thereby obtaining the particle carrier sustained-release vaccine.

[0009] In the above method, the mass ratio of the aluminum adjuvant to the antigen can be 2-8:1, preferably 2-4:1.

[0010] According to an embodiment of the present invention, the mass ratio of the aluminum adjuvant to the antigen is 2:1, 4:1, 6:1 or 8:1.

[0011] In the above method, the conditions for the aluminum adjuvant to adsorb the antigen are: incubation at 2-8°C for 1-5h.

[0012] According to an embodiment of the present invention, the conditions for the aluminum adjuvant to adsorb antigens are: incubation on a horizontal shaker at 4°C for 1 hour, followed by standing at 4°C for 4 hours.

[0013] In the above method, the temperature for incubation with blank porous microspheres can be 2-8°C, and the time can be 6-18 hours.

[0014] According to an embodiment of the present invention, the incubation temperature is 4° C. and the time is 12 h.

[0015] In the above method, the self-healing temperature is 38-45°C and the time is 12-18 hours.

[0016] According to an embodiment of the present invention, the self-healing temperature is 42° C. and the time is 18 hours.

[0017] In the above method, the blank porous microspheres have a porosity of 50-85%, a pore size of 0.05-5 μm, and a particle size of 1-160 μm, preferably 10-70 μm.

[0018] The blank porous microspheres used in the present invention can be prepared by methods such as double emulsion emulsification solvent evaporation method, microfluidics technology, membrane emulsification method, spray drying method and salt dissolution method, preferably by double emulsion emulsification solvent evaporation method;

[0019] The polymer material for preparing the blank porous microspheres is poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyacrylate (PAA), polystyrene (PS), polyvinyl ester (PEV) and other materials, preferably poly(lactide-co-glycolide) (PLGA).

[0020] Furthermore, the intrinsic viscosity of the lactide-glycolide copolymer is 0.1-2.0 dL / g, and the weight average molecular weight is 7-100 kDa.

[0021] The blank porous microspheres were prepared by double emulsion solvent evaporation method as an example, and the specific preparation steps are as follows:

[0022] The PLGA is formulated into an oil phase, and the oil phase is mixed with an inner water phase to obtain colostrum; the colostrum is then transferred to an outer water phase to obtain a double emulsion; and finally the double emulsion is solidified and then evaporated to remove the organic solvent.

[0023] Furthermore, the oil phase can be prepared using the following organic solvents: organic solutions that can dissolve lactide-glycolide copolymers, such as ethyl acetate, acetone, dichloromethane, and chloroform, preferably dichloromethane;

[0024] Further, the internal aqueous phase is an aqueous solution of a porogen;

[0025] Further, the porogen may be sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium chloride, etc., preferably sodium bicarbonate;

[0026] Furthermore, the mass concentration of the porogen in the inner water phase is 0.2-2.0%;

[0027] Further, the external aqueous phase is an aqueous solution of polyvinyl alcohol (PVA), Tween, sodium dodecyl sulfate or Pluronic, and its mass volume concentration may be 0.5-3.0%, specifically 1%;

[0028] Furthermore, the volume ratio of the inner water phase to the oil phase may be 1:2-10, specifically 1:4;

[0029] Furthermore, the volume ratio of the oil phase to the external water phase may be 1:10-20, such as 1:12.

[0030] In the present invention, the antigens include but are not limited to recombinant anthrax protective antigens, new coronavirus RBD antigens, respiratory syncytial virus antigens, influenza virus antigens, etc.

[0031] In the present invention, the aluminum adjuvant includes but is not limited to aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum and potassium alum.

[0032] In the present invention, when using aluminum adjuvant to adsorb antigen, small molecule adjuvant can also be added; specifically, the small molecule adjuvant can be dispersed in the antigen and the aluminum adjuvant, and the small molecule adjuvant includes but is not limited to CpG, Toll-like receptor agonist, squalene and the like.

[0033] The sustained-release vaccine provided by the present invention is a preventive or therapeutic vaccine.

[0034] The sustained-release vaccine provided by the present invention is a subunit vaccine.

[0035] Based on the self-healing technology, the release of the vaccine antigen provided by the present invention is mainly divided into two times: the first is the release of the antigen outside the microspheres to achieve primary immunization; as the microspheres degrade, the antigen is released from the inside of the microspheres to form a second slow and continuous release, achieving subsequent antigen stimulation to the body for a longer period of time.

[0036] The present invention uses porous microspheres as carriers, combines aluminum adjuvants and antigens into microspheres by physical diffusion and electrostatic adsorption, closes the microsphere pores by self-healing, and separates aluminum adjuvants and antigen proteins inside and outside the microspheres. After entering the body, the microspheres of the present invention release antigens more continuously at different time periods; the prepared vaccine carrier can effectively recruit and activate antigen-presenting cells, release antigens, be taken up by antigen-presenting cells, cause immune responses, induce the production of specific antibodies, and form immune memory. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The morphology, particle size distribution and pore size distribution of the porous microspheres in Example 1 of the present invention.

[0038] Figure 2The morphology of the porous microspheres after self-healing under different conditions in Example 2 of the present invention.

[0039] Figure 3 This is a diagram of the distribution of antigens in the delivery vector in Example 3 of the present invention.

[0040] Figure 4 The in vitro release curve and scanning electron microscopy image of the delivery vector in Example 4 of the present invention are shown.

[0041] Figure 5 The in vivo release curve of the delivery vector in Example 5 of the present invention and the in vivo imaging of small animals are shown.

[0042] Figure 6 This is a diagram of in vitro BMDC activation in different groups in Example 6 of the present invention.

[0043] Figure 7 This is a diagram of antigen presenting cell activation in different groups in Example 7 of the present invention.

[0044] Figure 8 Graph showing the proportion of germinal center B cells in spleen tissues of different groups in Example 8 of the present invention.

[0045] Fig. 9 This is the in vitro antigen-stimulated proliferation of spleen cells from immunized mice in Example 9 of the present invention.

[0046] Fig.10 is the rPA-specific CD8 + T cell activation status.

[0047] Fig.11 This is the secretion of rPA-specific binding antibodies in vivo after mice were immunized with the vaccine in Example 11 of the present invention.

[0048] Fig.12 This is the secretion of rPA-specific binding antibodies in mice after double injection vaccination in Example 12 of the present invention.

[0049] Fig.13 This is the secretion of neutralizing antibodies in the mice after immunization with the vaccine in Example 13 of the present invention.

[0050] Fig.14 This is an investigation of the cytotoxicity of the preparation immunized against in Example 14 of the present invention.

[0051] Fig.15 The figure shows the changes in body weight and body temperature of mice immunized with the preparation in Example 14 of the present invention.

[0052] Fig.16 The toxic reactions of the main organs of mice immunized with the preparation in Example 14 of the present invention are shown.

[0053] Fig.17 This is an investigation of the blood biochemical indices of the main organs of the mice immunized with the preparation in Example 14 of the present invention. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0056] The rPA antigen used in the following examples is described in the following literature (Polysaccharide PCP-I isolated from Poria cocos enhances the immunogenicity and protection of ananthrax protective antigen-based vaccine. Hum Vaccin Immunother. 2020 Jul 2; 16 (7): 1699-1707. doi: 10.1080 / 21645515.2019.1675457

[0057] Protection against anthrax and plague by a combined vaccine in mice and rabbits.Vaccine.2009Dec 9;27(52):7436-41.doi:10.1016 / j.vaccine.2009.07.015)

[0058] Example 1: Preparation of porous microspheres

[0059] The method used in this example is the double emulsion solvent volatilization method, that is, the oil phase (PLGA solution) and the inner water phase (sodium bicarbonate aqueous solution) are mixed and homogenized to prepare colostrum, the colostrum and the outer water phase are homogenized to prepare double emulsion, the double emulsion is dispersed in the solidification water for solidification, and the organic solvent is volatilized. The inner water phase reacts with the acidic environment to allow the gas to escape and form pores. The specific steps are as follows:

[0060] Preparation method of PLGA porous microspheres: weigh 400 mg PLGA, add 2 ml dichloromethane solution to prepare the oil phase, use 0.5% NaHCO3 solution with a mass volume ratio as the inner water phase, add 0.5 ml of the inner water phase to the oil phase, use a homogenizer, homogenize at 12,000 rpm, and prepare colostrum for 2 minutes; transfer the prepared colostrum to 30 ml of PVA (viscosity 18.8 mPa·s) with a mass volume ratio of 1% external water phase, homogenize at 6,200 rpm, and prepare a double emulsion for 90 seconds. After the preparation is completed, the double emulsion is transferred to 30 ml of curing water, cured for 30 minutes, and then transferred to 500 ml of curing water, evaporate the solvent for 4-5 hours, and the curing stirring speed is 300 rpm. Then, the microspheres are washed with distilled water until the PVA solution is clean.

[0061] The PLGA used in this example is PLGA (7525DLG 2CA) with an intrinsic viscosity of 0.16-0.24 dL / g and a weight average molecular weight of about 15 kDa.

[0062] The scanning electron microscope image of the porous microspheres prepared in this example is as follows Figure 1 As shown, Figure 1 A is the morphology of porous microspheres; Figure 1 B is the particle size distribution of porous microspheres, which is 10-50 μm; Figure 1 C is the pore size distribution, which is 1-5 μm. The porosity of the porous microspheres prepared in this embodiment is about 80%.

[0063] Example 2: Drug loading and self-healing of porous microspheres

[0064] Dissolve 1 mg of OVA antigen in distilled water, and add 2 mg, 4 mg, 6 mg, and 8 mg of aluminum hydroxide solution respectively, with a total volume of 2 ml; add the aluminum hydroxide solution dropwise and continue to shake slightly;

[0065] The mixture of aluminum hydroxide and antigen protein was placed in a 4°C refrigerator, incubated on a horizontal shaker for 1 hour, and then allowed to stand at 4°C for 4 hours;

[0066] To 230 mg of the PLGA porous microspheres prepared in Example 1, 2 ml of the static aluminum hydroxide and antigen mixture was added, and the mixture was incubated on a horizontal shaker at 4° C. for 12 h. The mixture was then transferred to a water bath heating device and heated at 42° C. for 18 h. The supernatant was collected, the residual antigen concentration was detected, and the encapsulation efficiency was calculated.

[0067] In this embodiment, the antigen is first adsorbed on aluminum hydroxide by electrostatic adsorption. The aluminum hydroxide particles adsorbing the antigen are then loaded into the microspheres by physical diffusion and electrostatic adsorption, and separated into two parts of the antigen, the inner and outer parts, by self-healing.

[0068] In this embodiment, the amount of antigens loaded inside and outside the microspheres can be adjusted by adjusting the concentration of the antigen protein in the aluminum hydroxide and antigen mixture, and the concentration of the antigen solution is directly proportional to the amount of antigens loaded on the microspheres.

[0069] In this embodiment, the selected self-healing heating temperature is related to the solution medium in which the antigen is located (the antigen protein in this experiment is solid, and the solution medium is an aqueous solution of protein aluminum hydroxide; but some proteins are in solution and exist in PBS or other buffered salt solution media). Different solution media require different self-healing temperatures and times, but in general, they are close to the glass transition temperature of PLGA7525 2CA.

[0070] In this embodiment, the experimental results are as follows: Figure 2 As shown in AB, Figure 2 The self-healing conditions of A were 42°C, 12h, and 30rpm (the mass ratio of aluminum hydroxide to antigen protein was 2:1), and tiny holes still existed on the surface of the microspheres under an electron microscope; Figure 2 The self-healing conditions of B were 42°C, 18h, and 30rpm (the mass ratio of aluminum hydroxide to antigen protein was 2:1). The surface of the microspheres was smooth and round under the electron microscope, and the self-healing was good. This condition was subsequently used as the self-healing condition. Figure 2 CF shows electron micrographs of the self-healing microspheres with different masses of aluminum hydroxide added. The mass ratio of aluminum hydroxide to antigen protein was 2:1, 4:1, 6:1, and 8:1, and self-healing could occur, indicating that the presence of aluminum hydroxide did not interfere with the self-healing of the microspheres.

[0071] Example 3: Characterization of antigen distribution of vaccine delivery vector

[0072] Weigh 400 mg of PLGA, add 2 ml of dichloromethane solution to prepare the oil phase, use 0.5% NaHCO3 solution with a mass volume ratio as the internal aqueous phase, add 0.5 ml of the internal aqueous phase to the oil phase, use a homogenizer, and homogenize at 12,000 rpm for 2 min to prepare colostrum; transfer the prepared colostrum to 30 ml of PVA (viscosity 18.8 mPa·s) with a mass volume ratio of 1% external aqueous phase, homogenize at 6,200 rpm for 90 s to prepare a double emulsion; transfer the double emulsion to 30 ml of curing water, cure for 30 min, and then transfer to 500 ml of curing water, evaporate the solvent for 4-5 h, and the curing stirring speed is 300 rpm, and then wash the microspheres with distilled water until the PVA solution is clean.

[0073] 17.24 mg of fluorescent gallium was added to 1 ml of 4 mg / ml aluminum hydroxide solution, shaken at 24°C at 125 rpm overnight; centrifuged overnight, the supernatant was discarded, and 1 ml of water was added for resuspending. Take 0.5 ml of the mixed solution (aluminum hydroxide 4 mg / ml, fluorescent gallium 50 μM), add 1 mg Cy5-OVA; mix on a 4°C horizontal shaker for 1 hour, let stand at 4°C for 4 hours, transfer to porous microspheres (230 mg) with the supernatant discarded, incubate on a 4°C horizontal shaker for 12 hours, self-heal at 42°C for 18 hours, and store at 4°C after self-healing is completed. Protect from light throughout the experiment, wrap the centrifuge tube with tin foil, and photograph the prepared samples with a laser confocal microscope.

[0074] In this embodiment, the experimental results are as follows: Figure 3 As shown, the color of aluminum hydroxide is red, and the antigen inside the microsphere is Cy5-OVA, which is green. From the results of laser scanning confocal microscopy, part of the antigen is combined with aluminum hydroxide inside the microsphere, showing yellow, and part of the antigen is combined with aluminum hydroxide and attached to the surface of the microsphere, showing yellow, indicating that the antigen is combined with aluminum hydroxide and is divided inside and outside the microsphere.

[0075] Example 4: In vitro release study

[0076] Using 0.01M PBS (pH 7.4) as the release medium, 240 mg of the prepared OVA-Al-MPs (2 ml of a standing mixed solution containing 2 mg of aluminum hydroxide and 15 mg of OVA protein was added to 230 mg of the PLGA porous microspheres prepared in Example 1, incubated on a horizontal shaker at 4°C for 12 h, and then heated at 42°C for 18 h until self-healing) was centrifuged and the supernatant was discarded. 5 ml of the release medium was added, the temperature was set to 37°C, the rotation speed was 120 rpm, and the release medium was replaced when sampling; while replacing the medium, samples were prepared and the morphology of the microspheres was observed under an electron microscope.

[0077] In this embodiment, the experimental results are as follows: Figure 4 As shown in A, in the in vitro release, there was a burst release on the first day, a second concentrated release peak appeared on the 7th to 28th day, and the second release peak was on the 14th day. The final cumulative release amount in 63 days was 93.10±3.30%. Figure 4 B Electron microscopy image, the microspheres collapsed and broke into pieces on the 14th day, which coincided with the arrival of the second peak of release, indicating that the antigens were released from the microspheres.

[0078] Example 5: In vivo release study

[0079] BALB / c male mice aged 6-8 weeks and weighing 18-22 g were randomly divided into 3 groups, with 3 mice in each group. Cy5-OVA (Xi'an Ruixi Biotechnology Co., Ltd., catalog number: R-OF-003), Cy5-OVA-Al, and Cy5-OVA-Al-MPs were injected into the thigh muscle of each group (the amount of antigen injected in each group was 10 μg, and the aluminum adjuvant was 20 μg). In vivo imaging of small animals was performed on days 1, 4, 7, 14, 21, 28, and 35 after immunization.

[0080] The preparation method of Cy5-OVA-Al used above is as follows: 1 mg Cy5-OVA and 2 mg aluminum hydroxide are incubated at 4° C. on a horizontal shaker for 1 h, and then allowed to stand at 4° C. for 4 h.

[0081] The preparation method of the Cy5-OVA-Al-MPs used above is as follows: add 2 ml of a standing mixed solution containing 2 mg aluminum hydroxide and 1 mg Cy5-OVA protein to 230 mg of the PLGA porous microspheres prepared in Example 1, incubate on a horizontal shaker at 4°C for 12 h, and heat at 42°C for 18 h for self-healing.

[0082] In this embodiment, the experimental results are as follows: Figure 5 As shown in AB, the antigen fluorescence of the Cy5-OVA group basically disappeared on the 7th day; the antigen fluorescence of the Cy5-OVA-Al group basically disappeared on the 14th day; the antigen fluorescence intensity of the Cy5-OVA-Al-MPs group basically disappeared on the 21st day, but the fluorescence intensity was still statistically different from that of the Cy5-OVA group and the Cy5-OVA-Al group. This result is consistent with the in vitro release results.

[0083] Example 6: In vitro activation of mouse bone marrow-derived dendritic cells (BMDC)

[0084] Male C57BL / 6 mice aged 6-8 weeks were selected and killed. After disinfection, the femur and tibia were removed from the clean bench and washed with PBS 2-3 times. The two ends of the bone were cut with scissors, and RPMI 1640 complete medium was blown into the cell culture dish using a 1ml sterile syringe until the bone shaft turned white. The cells were collected by filtering with a 70μm cell sieve, centrifuged at 1,000rpm for 3min to remove the supernatant, and 2ml of red blood cell lysis buffer was added. After lysis for 2-3min, 10ml of RPMI 1640 complete medium was added to stop; centrifugation, supernatant was discarded, RPMI 1640 complete medium was used to wash twice, and RPMI 1640 complete medium containing GM-CSF was resuspended, and the cells were counted and the cell concentration was adjusted to 1×10 6 / ml, inoculated in a 6-well plate, 4ml per well, for culture in a carbon dioxide incubator; half of the medium was replaced every other day, and all suspended cells were collected on the 6th day; the concentration of BMDC was adjusted to 2×106 / ml, 0.5ml was inoculated in a 12-well plate, and OVA, OVA-Al, OVA-Al-MPs, and OVA were added to each well at 2μg; after culturing in the incubator for 24h, the cells were collected and washed three times with PBS; BV421 anti-mouse CD11c antibody, APC anti-mouse CD40 antibody, PE anti-mouse CD80 antibody, and FITC anti-mouse CD86 antibody were used to stain the cells, and the staining was carried out on ice in the dark for 30min. After the staining, the cells were washed twice with pre-cooled PBS, and finally 300μl 1% paraformaldehyde was added to resuspend the cells, and the activation effect of BMDC was detected by flow cytometry.

[0085] The preparation method of the OVA-Al used above is as follows: 1 mg of OVA and 2 mg of aluminum hydroxide are incubated on a horizontal shaker at 4° C. for 1 hour and allowed to stand for 4 hours.

[0086] The preparation method of the OVA-Al-MPs used above is as follows: 2 ml of a static mixture containing 2 mg aluminum hydroxide and 1 mg OVA protein was added to 230 mg of the PLGA porous microspheres prepared in Example 1, incubated on a 4°C horizontal shaker for 12 h, and then heated at 42°C for 18 h for self-healing.

[0087] In this embodiment, the experimental results are as follows: Figure 6 As shown, the activation molecules CD40, CD80, and CD86 on the surface of BMDC cells were higher in the microsphere group than in the aluminum adjuvant group and higher than in the simple antigen group. This indicates that in vitro, BMDC cells are more likely to be activated after contacting the microsphere carrier than in the other two groups, and BMDC activation is an important step in the beginning of immunity. Therefore, the presence of the microsphere carrier is conducive to immune activation, thereby facilitating the generation of a stronger immune response.

[0088] Example 7: Activation of Draining Lymph Node Antigen Presenting Cells in Vivo

[0089] Male BALB / c mice aged 6-8 weeks and 18-22g were selected and randomly divided into 4 groups, 3 mice in each group, and each group was injected with saline, OVA, OVA-Al (same as Example 6), and OVA-Al-MPs (same as Example 6) in the thigh muscle; the immunization scheme is shown in Table 1, but only one immunization was performed on day 0. After 3 days of immunization, the lymph nodes of each group of mice were taken in PBS, ground through a 70μm cell sieve, and prepared as a single cell suspension. After washing with PBS 2-3 times, APC anti-mouse CD11c antibody, PE anti-mouse CD80 antibody, PerCP anti-mouse CD86 antibody, and BV421 anti-mouse F4 / 80 antibody were added, and incubated on ice in the dark for 30 minutes. After the incubation, PBS was added for washing 2-3 times, and then 1% paraformaldehyde was added to resuspend the cells, and the difference in the number of dendritic cells and macrophages and the maturation and activation were detected by flow cytometry.

[0090] In this embodiment, the experimental results are as follows: Figure 7 As shown in the figure, the cell markers of DC cells and macrophages were detected respectively, and the results were consistent. The expression of CD80 and CD86 of DC cells and macrophages was the highest in the OVA-Al-MPs group, followed by the OVA-Al group. This shows that the activation effect of antigen presenting cells in the OVA-Al-MPs group is better than that in the OVA-Al group and the OVA group, indicating that the presence of microspheres is conducive to the enhancement of immune response.

[0091] Table 1 Different vaccine formulations and immunization schedules in Examples 7-8

[0092]

[0093] Example 8: Germinal Center B Cell Detection

[0094] Male BALB / c mice aged 6-8 weeks and weighing 18-22g were selected and randomly divided into 5 groups, with 3 mice in each group. Each group was injected with saline, OVA, OVA-Al, OVA20-Al-MPs-1Dose, and OVA10-Al-MPs-2Doses into the thigh muscle. Two weeks later, except for the OVA20-Al-MPs-1Dose group, two immunizations were performed. The immunization scheme is shown in Table 1. Three weeks after immunization, the lymph nodes of mice in each group were taken in PBS, ground through a 70μm cell sieve, and prepared into a single cell suspension. After washing with PBS for 2-3 times, PE-Cy7 anti-mouse CD19 antibody, PerCP anti-mouse CD45R antibody, and PE anti-mouse GL7 antibody were added, and incubated on ice in the dark for 30 minutes. After the incubation, PBS was added to wash 2-3 times, and then 1% paraformaldehyde was added to resuspend the cells, and the flow cytometer was used for detection.

[0095] In this embodiment, the experimental results are as follows: Figure 8 As shown, the proportion of germinal center B cells in the OVA20-Al-MPs-1Dose group was the largest, followed by the OVA10-Al-MPs-2Doses group, and both were better than the OVA-Al group and the OVA group; this indicates that the presence of microspheres is conducive to the production of germinal center B cells, and the germinal center B cells will further differentiate into plasma cells that secrete specific antibodies and then produce antibodies, and a few differentiate into memory B cells. Therefore, the microsphere preparation vaccine is beneficial to promoting the production of specific antibodies and the subsequent formation of immune memory cells.

[0096] Example 9: Splenocyte proliferation

[0097] Male BALB / c mice aged 6-8 weeks and weighing 18-22g were selected and randomly divided into 5 groups, and inoculated with saline, rPA-Al, rPA-Al-CpG, rPA10-Al-MPs, and rPA10-Al-MPs-CpG20, respectively. Each mouse in different groups was intramuscularly injected with rPA10μg and aluminum hydroxide 50μg according to the dose; each mouse in the group containing CpG (vaccine adjuvant) was intramuscularly injected with CpG 20μg. In the third week, the non-microsphere group was immunized for the second time in the outer thigh muscle according to the above dose. The immunization scheme is shown in Table 2. The spleen was removed on the 35th day after inoculation and prepared into a single cell suspension. Under sterile conditions, the spleen was removed, placed on a 70 μm cell sieve, ground with a tissue grinder, and prepared into a single cell suspension using RPMI 1640 medium. After washing once with RPMI 1640, 3 ml of red blood cell lysis buffer was added and lysed for 3 min. After lysis, 12 ml of PBS was added to terminate the lysis. After washing twice with RPMI 1640, the cells were resuspended in RPMI 1640 complete medium, and the cells were counted and stained with trypan blue. 10 μl was added to detect cell activity and the cell concentration was adjusted to 5×10 6 cells, 100 μL per well, i.e. 5×10 5 Each well was cultured with 1 μg of rPA antigen for in vitro stimulation. Negative control (only cells were added without stimulants) and blank control (only RPMI 1640 complete medium was added). The cells were cultured in a 5% incubator at 37°C. 1 / 10 volume of CCK-8 solution was added to each well. The cells were cultured in a 5% incubator for 1-4 hours. The value at 450 nm was measured by an ELISA reader. The formula for spleen cell proliferation is cell proliferation rate = (OD 疫苗 -OD 空白 ) / (OD 阴性 -OD 空白 )×100%.

[0098] The preparation method of the rPA-Al used above is as follows: rPA and aluminum hydroxide are mixed in a mass ratio of 1:5 (eg, 10 μg rPA, 50 μg aluminum hydroxide), and incubated at 4° C. on a horizontal shaker for 1 hour.

[0099] The preparation method of the rPA-Al-CpG used above is as follows: rPA, aluminum hydroxide and CpG are mixed in a mass ratio of 1:5:2 (eg, rPA 10 μg, aluminum hydroxide 50 μg, CpG 20 μg), and incubated at 4°C on a horizontal shaker for 1 hour.

[0100] The preparation method of the rPA-10-Al-MPs used above is as follows: its preparation method is similar to that of OVA-Al-MPs in Example 6, except that: first, OVA is replaced with rPA; second, after the preparation of rPA-Al-MPs (rPA: aluminum hydroxide mass ratio is 1:2), aluminum hydroxide is supplemented to achieve the same aluminum hydroxide adjuvant content as the rPA-Al control group (rPA: aluminum hydroxide mass ratio is 1:5), so that the final rPA: aluminum hydroxide mass ratio is 1:5, and incubated at 4°C horizontal shaker for 1 hour.

[0101] The preparation method of the rPA-10-Al-MPs-CpG20 used above is as follows: after rPA-10-Al-MPs (rPA: aluminum hydroxide mass ratio is 1:2) completes self-healing, additional aluminum hydroxide and CpG are added, and incubated on a horizontal shaker at 4°C for 1 hour, so that the mass ratio of rPA, aluminum hydroxide and CpG is 1:5:2 (for example, 10μg of rPA is first added with 20μg of aluminum hydroxide, added to porous microspheres and incubated at 4°C for 12h, heated at 42°C for 18h for self-healing, and then 30μg of aluminum hydroxide and 20μg of CpG are added, and incubated on a horizontal shaker at 4°C for 1h).

[0102] In this embodiment, the experimental results are as follows: Fig. 9 As shown, the results of spleen lymphocyte proliferation in the fifth week showed that the rPA-Al group and the rPA10-Al-MPs group were similar, the rPA10-Al-MPs-CpG20 group was better than the rPA-Al-CpG group, and the rPA10-Al-MPs-CpG20 group was the highest among the experimental groups, indicating that when the spleen lymphocytes in this group encountered the rPA antigen again, the proliferation reaction was better than that of other groups, indicating that the microsphere preparation vaccine can enable the body to induce an immune response more quickly when it encounters the same antigen again.

[0103] Table 2 Different vaccine formulations and immunization regimens in Examples 9-14

[0104]

[0105] Example 10: rPA-specific T cell activation

[0106] Male BALB / c mice aged 6-8 weeks and weighing 18-22 g were selected and randomly divided into 5 groups, and inoculated with saline, rPA-Al, rPA-Al-CpG, rPA10-Al-MPs, and rPA10-Al-MPs-CpG20, respectively. Each mouse in the different groups was intramuscularly injected with rPA10μg and aluminum hydroxide 50μg according to the dose; each mouse in the group containing CpG was intramuscularly injected with 20μg. In the third week, the non-microsphere group was immunized for the second time in the outer thigh muscle according to the above dose. The immunization scheme is shown in Table 2. The spleen was removed on the 35th day after inoculation and prepared into a single cell suspension. Under sterile conditions, the spleen was removed, placed on a 70 μm cell sieve, ground with a tissue grinder, and prepared into a single cell suspension using RPMI 1640 medium. After washing once with RPMI 1640, 3 ml of red blood cell lysis solution was added and lysed for 3 min. After lysis, 12 ml of PBS was added to terminate the lysis. After washing twice with RPMI 1640, the cells were resuspended in RPMI1640 complete medium, and the cells were counted and stained with trypan blue. 10 μl was added to detect cell activity. The number of cells seeded per well was 1×10 6 cells, add 1.0 μg of rPA antigen to each well, culture in a 5% incubator at 37°C for 68 hours, then take out for further lymphocyte activation experiments. After collecting spleen cells by blowing, centrifuge and discard the supernatant, wash 3 times with PBS, resuspend in PBS, and stain each sample with BV421 anti-mouse CD3 antibody, PerCP anti-mouse CD8 antibody, and PE anti-mouse CD69 antibody. After adding the sample, gently shake and mix, and keep it in an ice box away from light for 30 minutes. After staining, wash 3 times with PBS, fix the cells with 1% paraformaldehyde, and detect CD69 by flow cytometry. + The proportion of cells.

[0107] In this embodiment, the experimental results are as follows: Fig.10 As shown, the results of spleen lymphocyte activation on the 35th day showed that the rPA-Al group and the rPA10-Al-MPs group were similar, and the rPA10-Al-MPs-CpG20 group was similar to the rPA-Al-CpG group, but the activation levels of the latter two groups were higher than those of the former two groups, indicating that the single-injection microsphere group achieved the same immune effect as the double-injection group. However, the microsphere group reduced the number of injections and the total antigen and adjuvant inoculation doses.

[0108] Example 11: Specific binding antibody detection

[0109] Male BALB / c mice of 18-22 g at 6-8 weeks of age were selected and randomly divided into 9 groups, 6 mice in each group, and inoculated with saline, rPA-Al, and rPA-Al-CpG, respectively. These three groups were double-injection groups, and a second immunization was performed in the third week; rPA10-Al-MPs, rPA10-Al-MPs-CpG20, rPA10-Al-MPs-CpG40, rPA20-Al-MPs, rPA20-Al-MPs-CpG20, and rPA20-Al-MPs-CpG40 were single-injection groups, and only immunized once. The immunization doses are shown in Table 2. After immunization, blood was collected from the eye sockets of the mice, and the mouse serum was tested, weekly for the first 3 months, and monthly for the next 3 months, for a total of 6 months. ELISA: Use coating solution to prepare rPA solution with a concentration of 2μg / ml, add 100μl to each well, and incubate overnight at 4℃; Blocking: Wash three times with PBST washing solution (0.01mol / L, pH 7.4 PBS with 0.05% Tween 20), then add 150μl blocking solution (2% BSA, PBST) to each well, and block at 37℃ for 1h; Add serum: Add 200μl PBST washing solution to each well of the 96-well plate and wash the plate three times, dilute the serum sample with diluent (0.1% BSA, PBST), 1 / 100 in the first well, and then dilute it by 2 times, 100μl in each well. Control group, 1:1000 diluted mouse blank negative serum, incubated at 37°C for 30min; add enzyme-labeled secondary antibody: add 200μl PBST washing solution to each well of the 96-well plate for washing 5 times, add HRP-labeled secondary antibody diluted by diluent, 100μl per well, incubated at 37°C for 30min, IgG (1:10000) diluent; wash the plate 5 times with PBST, add 200μl of color developer TMB to each well, incubate at room temperature in the dark for 5-10min; stop: add stop solution 2M H2SO4, 50μl per well; serum titer determination: use a microplate reader to measure the absorbance value (OD value) at 450nm, where the OD value is higher than twice the OD value of the negative serum and is positive, and the maximum dilution multiple is the titer value of the antigen-specific antibody in the sample serum.

[0110] In this embodiment, the experimental results are as follows: Fig.11As shown, the binding antibody titers of rPA20-Al-MPs-CpG20, rPA20-Al-MPs-CpG40 and rPA-Al-CpG groups were the highest, followed by rPA10-Al-MPs-CpG20 and rPA10-Al-MPs-CpG40, rPA-Al and rPA20-Al-MPs groups, and rPA10-Al-MPs group was the lowest. The data showed that the presence of Al-MPs can reduce the number of immunization injections and achieve the same binding antibody titer as double-injection immunization. The rPA20-Al-MPs-CpG20 group reduced the amount of CpG and achieved the same binding antibody titer as the double-injection rPA-Al-CpG group. In addition, in the early 2-5W binding antibody titers, the group containing Al-MPs was generally higher than the double-injection group, indicating that the Al-MPs group can produce higher binding antibody titers earlier, which is conducive to achieving early protection after vaccination.

[0111] Example 12: Detection of specific binding antibodies at different injection times

[0112] 18-22g male BALB / c mice aged 6-8 weeks were selected and randomly divided into 2 groups, 6 mice in each group, and inoculated with rPA10-Al-MPs-2Doses and rPA10-Al-MPs-CpG20-2Doses, respectively. The second administration was performed in the third week. The immunization dose is shown in Table 2. After immunization, blood was collected from the eye sockets of the mice, and the mouse serum was tested weekly for the first 3 months and monthly for the next 3 months, for a total of 6 months. The binding antibody titer was tested by ELISA, and the antibody titer was the same as that in Example 11.

[0113] In this example, the experimental group of this example was compared with the double-needle rPA-Al group and rPA-Al-CpG group in Example 11. The experimental results are as follows: Fig.12 As shown, while the binding antibody titers of the rPA10-Al-MPs-CpG20-2Doses group and the rPA-Al-CpG group were similar, the rPA10-Al-MPs-CpG20-2Doses group produced binding antibodies earlier and maintained high binding antibody titers for a longer time. The rPA10-Al-MPs-2Doses group was significantly higher than the rPA-Al group, and the antibody titer was close to the rPA-Al-CpG group after 7 weeks. This shows that when the same number of injections is maintained, the presence of Al-MPs is more conducive to the enhancement of binding antibody titers and immune effects.

[0114] Example 13: Detection of rPA-specific neutralizing antibodies

[0115] The mouse sera in Example 11 and Example 12 were collected and frozen at -80°C. Neutralizing antibody detection was performed on the mouse sera at 5W, 9W, and 12W after immunization. J774A.1 cells were cultured to the logarithmic growth phase and inoculated in a 96-well plate with 12,000 cells per well in a carbon dioxide cell culture incubator overnight. 150ng / ml of rPA and 150ng / ml of rLF were mixed and mixed with serum of different dilution ratios (serum was diluted 2-fold using DMEM complete medium), incubated at 37°C for 1h, the J774A.1 cell supernatant was discarded, and the mixture of serum and toxin was added to the J774A.1 cells and cultured for 24h. Cell viability was detected using CCK-8.

[0116] In this embodiment, the experimental results are as follows: Fig.13 As shown, the neutralizing antibody titer of the rPA10-Al-MPs-CpG20-2Doses group was the highest, followed by the rPA20-Al-MPs-CpG40 group and the rPA20-Al-MPs-CpG20 group. The neutralizing antibody titer of the group without CpG was lower, which indicates that the production of neutralizing antibodies depends on CpG adjuvant to a certain extent, and the presence of CpG adjuvant is more conducive to the enhancement of the immune effect of the microspheres of this preparation. The neutralizing antibody titer of the rPA20-Al-MPs-CpG20 group was higher than that of the rPA10-Al-MPs-CpG20 group, which indicates that the increase in the content of rPA-Al-MPs is also conducive to the production of neutralizing antibodies and the enhancement of immune effects.

[0117] Example 14: Vaccine vector safety evaluation

[0118] Cytotoxicity: DC2.4 cells were seeded in 96-well plates at 5 × 10 4 cells, incubated overnight in a carbon dioxide cell culture incubator, and prepared 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / ml Al-MPs suspensions (the preparation method of Al-MPs is as follows: add 2 ml of an aqueous solution containing 2 mg of aluminum hydroxide after standing to 230 mg of the PLGA porous microspheres prepared in Example 1, incubate for 12 h on a horizontal shaker at 4°C, and freeze-dry after self-healing for 18 h at 42°C). The above suspensions were added separately, the negative control was a simple culture medium, and the positive control was a 50% DMSO solution. After 6 h, 12 h and 24 h of co-culture, the culture medium was discarded, the cells were washed 3 times with PBS, and then fresh culture medium DMEM and CCK-8 solution were added, and the absorbance at 450 nm was detected. The calculation formula of cell survival rate was: cell survival rate (%) = OD (experimental group) / OD (control group) × 100%.

[0119] The weight and body temperature of mice were monitored after immunization of the mice of Example 11 and Example 12. The monitoring period was one month after immunization, and the monitoring was performed once every 2 days.

[0120] Toxicity monitoring of major organs: 6-8 week old male BALB / c mice weighing 18-22 g were immunized with intramuscular injections of normal saline, rPA-Al, rPA-Al-CpG, rPA10-Al-MPs, and rPA10-Al-MPs-CpG20. Six months after immunization, the main organs of the mice, including heart, liver, spleen, lung, and kidney, were obtained for H&E staining analysis.

[0121] Blood biochemical test: 6-8 week old male BALB / c mice weighing 18-22 g were immunized with intramuscular injection of normal saline, rPA-Al, rPA-Al-CpG, rPA10-Al-MPs, and rPA10-Al-MPs-CpG20. The serum of mice 6 months after immunization was collected for blood biochemical test. The main test indicators included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and blood urea (UREA).

[0122] In this embodiment, the experimental results are as follows: Fig.14 As shown in the figure, DC2.4 cells were used as a model to investigate the different concentrations of the preparation and the cells were incubated for 6, 12 and 24 hours. The results showed that the cells were in good condition and the preparation was highly safe. The body weight and temperature of the immunized mice were monitored. The experimental results are shown in Fig.15 As shown in the figure, after vaccination with this preparation, the body weight and body temperature of mice did not change abnormally. The results of the main organ toxicity experiment are shown in the figure. Fig.16 As shown in the figure, after mice were immunized with this preparation, no abnormal pathological structures were found in the heart, liver, spleen, lung, and kidney of the mice. Fig.17 As shown, after mice were immunized with the preparation, the ALT, AST, ALP, and UREA of the mice were all normal. In summary, the vaccine delivery vector has good safety.

[0123] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A particle carrier sustained-release vaccine, which is a self-healing microsphere combined with an aluminum adjuvant that adsorbs antigens; the antigens are loaded both inside and outside the self-healing microspheres.

2. The particle carrier sustained-release vaccine according to claim 1, characterized in that: The particle carrier sustained-release vaccine is prepared according to a method comprising the following steps: using aluminum adjuvant to adsorb antigens; after adsorption, incubating and co-loading with blank porous microspheres; and then heating to allow the porous microspheres to self-heal, thereby obtaining the particle carrier sustained-release vaccine.

3. The particle carrier sustained-release vaccine according to claim 2, characterized in that: The mass ratio of the aluminum adjuvant to the antigen is 2-8:1, preferably 2-4:1; and / or, the condition for the aluminum adjuvant to adsorb the antigen is incubation at 2-8°C for 1-5h; and / or, the incubation temperature is 2-8°C and the time is 10-14h; and / or, the self-healing temperature is 38-45°C and the time is 12-18h.

4. The particle carrier sustained-release vaccine according to claim 2 or 3, characterized in that: The blank porous microspheres have a porosity of 50-85%, a pore size of 0.05-5 μm, and a particle size of 1-160 μm, preferably 10-70 μm.

5. The particle carrier sustained-release vaccine according to any one of claims 1 to 4, characterized in that: The antigens include but are not limited to recombinant anthrax protective antigens, new coronavirus RBD antigens, respiratory syncytial virus antigens, influenza virus antigens, etc.

6. The particle carrier sustained-release vaccine according to any one of claims 1 to 5, characterized in that: The aluminum adjuvant includes, but is not limited to, aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum and potassium alum.

7. The particle carrier sustained-release vaccine according to any one of claims 1 to 6, characterized in that: When using aluminum adjuvant to adsorb antigen, small molecule adjuvant can also be added; specifically, the small molecule adjuvant is dispersed in the antigen and the aluminum adjuvant, and the small molecule adjuvant includes but is not limited to CpG, Toll-like receptor agonist, squalene and the like.

8. The particle carrier sustained-release vaccine according to any one of claims 1 to 7, characterized in that: The particle carrier sustained-release vaccine is a preventive or therapeutic vaccine.

9. The particle carrier sustained-release vaccine according to any one of claims 1 to 7, characterized in that: The particle carrier sustained-release vaccine is a subunit vaccine.

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