A PLGA nanoemulsion, its preparation, and its application in a paratuberculosis vaccine.

Abstract

This invention provides a novel method for preparing PLGA nanoemulsions co-loaded with multiple immunostimulants and antigens. By using PLGA to encapsulate all-trans retinoic acid and coating the surface of the nanoparticles with dopamine hydrochloride, recombinant antigen proteins and CPG oligonucleotides are successfully loaded onto the surface of the nanoparticles, providing a new method for the prevention and treatment of intestinal pathogens and paratuberculosis.

Description

Technical Field

[0001] This application belongs to the field of vaccines in the field of biotechnology. Specifically, this application provides a PLGA nanoemulsion co-loaded with CPG oligonucleotide-all-trans retinoic acid-antigen protein, its preparation, and its application in a paratuberculosis vaccine. Background Technology

[0002] Paratuberculosis is a chronic digestive tract disease in common livestock and ruminants caused by *Mycobacterium avium subsp. paratuberculosis* (MAP), which seriously affects the development of animal husbandry. There is an urgent need to develop new methods for the prevention and control of MAP. Traditional vaccination methods such as subcutaneous, intramuscular, and intradermal injections have been repeatedly proven to fail to activate mucosal immunity due to tissue limitations. To improve the convenience of clinical use, this study aims to develop a novel nanovaccine with a simple immunization route that can activate intestinal mucosal immunity to prevent and treat paratuberculosis infection in livestock.

[0003] CpG ODN is a synthetically produced oligodeoxynucleotide containing unmethylated cytosine and guanine nucleotides as its core. It can mimic bacterial DNA to stimulate immune cells in various mammals, including humans. It is also a Toll-like receptor 9 agonist, directly activating antigen-presenting cells such as B cells, macrophages, and dendritic cells, and indirectly activating NK cells and T cells. It stimulates these immune cells to secrete cytokines such as TNF-α, IFN-γ, and IL-12, inducing a Th1-type immune response and generating strong humoral and cellular immunity. In recent years, the important role and mechanism of all-trans retinoic acid (atRA) in mucosal immune responses have been revealed: it can induce lymphocyte homing to the intestine through multiple pathways, including regulating α4β7 integrin, the cytokine CCR9, thymic stromal lymphopoietin (TSLP), and lactoferrin, thereby increasing the level of secretory sIgA in the intestinal mucosa. However, atRA has poor water solubility and photostability, severely limiting its application as an adjuvant.

[0004] PLGA is a biodegradable high-molecular-weight organic compound formed by the random polymerization of lactic acid and glycolic acid monomers. It is non-toxic, has good biocompatibility, and exhibits encapsulation and film-forming properties. Due to its excellent safety in humans and animals, PLGA has been approved by the Food and Drug Administration (FDA) as a delivery carrier for vaccines and drugs. Summary of the Invention

[0005] This application successfully loaded antigens and CPG oligonucleotides onto the surface of PLGA nanoparticles by encapsulating lipid-soluble atRA and coating the nanoparticle surface with dopamine hydrochloride, thereby introducing active groups. This co-loading of atRA, CPG oligonucleotides, and antigen proteins allows for the simultaneous induction of humoral, cellular, and mucosal immunity via conventional injection immunization, demonstrating the feasibility and advantages of this co-loaded nanosphere as an adjuvant for paratuberculosis vaccines. This application also provides a novel method for preparing PLGA nanoemulsions co-loaded with multiple immunostimulants and antigens. By encapsulating all-trans retinoic acid in PLGA and coating the nanoparticle surface with dopamine hydrochloride, recombinant antigen proteins and CPG oligonucleotides are loaded onto the nanoparticle surface, providing a new method for the prevention and treatment of intestinal pathogen paratuberculosis.

[0006] On the one hand, this application provides a PLGA nanoemulsion loaded with CPG oligonucleotide-all-trans retinoic acid-antigen protein.

[0007] Furthermore, the PLGA nanoemulsion is prepared by loading lipid-soluble all-trans retinoic acid, water-soluble CPG oligonucleotides, and antigen proteins onto PLGA.

[0008] Furthermore, the antigen protein is a Mycobacterium paratuberculosis antigen.

[0009] Furthermore, the antigen protein is a fusion protein HBHA-Ag85B-Bfra.

[0010] On the other hand, this application provides a method for preparing the PLGA nanoemulsion, comprising:

[0011] 1) Weigh out an appropriate amount of PLGA and all-trans retinoic acid, dissolve them in the oil phase, and shake to mix until completely dissolved;

[0012] 2) Add the solution prepared in 1) dropwise into the PVA solution and emulsify by ultrasonication under ice bath conditions;

[0013] 3) Pour the solution obtained in 2) into the PVA solution and stir for 4 hours to evaporate the oil phase;

[0014] 4) After centrifuging and washing the PLGA nanoemulsion loaded with atRA prepared in 3), it was resuspended in Tris buffer containing dopamine hydrochloride and mixed and reacted at room temperature.

[0015] 5) After centrifuging and washing the prepared nanoemulsion coated with dopamine hydrochloride, it was resuspended in PBS solution containing CPG oligonucleotides and antigen protein and mixed at room temperature;

[0016] 6) The prepared nanoparticle solution is centrifuged and washed in a high-speed refrigerated centrifuge. After washing, the supernatant is discarded, and the nanoparticles are dried in a vacuum freeze dryer at -80°C to obtain microsphere powder.

[0017] Furthermore, the mass ratio of PLGA to trans-retinoic acid is 0.5%.

[0018] Furthermore, PLGA is coated with dopamine hydrochloride to enable it to adsorb antigens and CPG.

[0019] Furthermore, the nanoemulsion coated with dopamine hydrochloride was resuspended in a PBS solution containing 100 μg / mL CPG oligonucleotides and 40 μg / mL antigen protein.

[0020] Furthermore, the oil phase is ethyl acetate or dichloromethane.

[0021] Furthermore, in the Tris buffer containing dopamine hydrochloride, the concentration of dopamine hydrochloride is 2 mg / mL, the concentration of Tris is 10 M, and the pH is 8.5.

[0022] On the other hand, this application provides the application of the above-mentioned PLGA nanoemulsion or the PLGA nanoemulsion prepared according to the above method in the preparation of paratuberculosis vaccine.

[0023] Furthermore, the vaccine can reduce the bacterial load in recipients after infection with Mycobacterium paratuberculosis.

[0024] Furthermore, the vaccine can enhance the secretion of TNF-α, IL-10, IFN-γ, antibody IgG, and intestinal mucosal IgA in vaccinated individuals after immunization.

[0025] On the other hand, this application provides a paratuberculosis vaccine comprising the above-described PLGA nanoemulsion or a PLGA nanoemulsion prepared according to the above method.

[0026] The antigenic protein used in this application is not limited to HBHA-Ag85B-Bfra; other MAP antigenic proteins may also be used.

[0027] The CPG oligonucleotide described in this application is a type B CpG ODN, which is a fully thiolated linear CpG ODN that has strong immunostimulatory activity against B cells. It can be obtained commercially or in-house by those skilled in the art.

[0028] The nanoparticles / vaccines in this application are preferably used orally, but this does not preclude administration by injection, nasal spray, or other methods after selecting a suitable carrier and formulation.

[0029] The excipients used in this application can be selected by those skilled in the art based on conventional knowledge in the vaccine field, including but not limited to adjuvants, solvents, cosolvents, buffers, antioxidants, and preservatives. Attached Figure Description

[0030] Figure 1 The purified recombinant fusion protein HBHA-Ag85B-Bfra was validated by SDS-PAGE and Western blot.

[0031] Figure 2 Characterization of novel co-loaded nanoparticles;

[0032] Figure 3 For the evaluation of the immunogenicity of nanoparticles;

[0033] Figure 4 This represents the bacterial load in mouse livers. Detailed Implementation

[0034] Example 1: Expression of recombinant fusion protein

[0035] E. coli transformed with the HBHA-AG85B-Bfra-pET-30a(+) prokaryotic expression plasmid were revived and preserved. The bacteria were cultured in LB medium containing kanamycin (final concentration 50 μg / mL) to the logarithmic growth phase (OD600nm 0.6-0.8). IPTG (final concentration 1 mM) was added, and expression was induced for 4 h at 30°C and 160 rpm on a shaker. The cells were collected by centrifugation at 4°C and 8000 rpm for 3 min, and washed twice with pre-cooled PBS. The cells were resuspended in PBS, sonicated, lysed, and centrifuged at 10000 rpm for 10 min to obtain the lysis supernatant. The lysis supernatant was purified by Ni column affinity chromatography to obtain the recombinant fusion protein HBHA-AG85B-Bfra.

[0036] The recombinant fusion protein nucleic acid sequence is as follows:

[0037]

[0038] The purified recombinant protein was subjected to SDS-PAGE gel electrophoresis and Western blot analysis using MAP-positive serum from mice. The results showed a single band on the NC membrane, consistent with the expected size. Figure 1 ).

[0039] Example 2: Preparation of co-loaded nanoparticles

[0040] 1) Weigh 0.1g PLGA and 500μg all-trans retinoic acid, dissolve them in 5ml dichloromethane, and shake to mix until completely dissolved;

[0041] 2) Add the solution prepared in the previous step to 10 mL of 1% PVA, and sonicate under ice bath conditions. The sonication conditions are 300 W, the total time is 6 min, the working time is 2 s and the stop time is 3 s.

[0042] 3) Pour the solution obtained in step 2 above into 10 mL of 1% PVA solution, stir at 300-400 rpm at room temperature for 4 h to evaporate the oil phase;

[0043] 4) After centrifuging and washing the PLGA nanoemulsion loaded with atRA obtained in step 3 above, resuspend it in Tris buffer containing 2 mg / mL dopamine hydrochloride and react at room temperature for 3 h.

[0044] 5) After centrifuging and washing the prepared nanoemulsion coated with dopamine hydrochloride, it was resuspended in PBS solution containing 100 μg / mL CPG oligonucleotides and 40 μg / mL antigen protein and mixed at room temperature for 3 h.

[0045] 6) The prepared nanoparticle solution is centrifuged and washed in a high-speed refrigerated centrifuge. After washing three times, the supernatant is discarded. The solution is stored at 4°C and used the next day, or dried in a vacuum freeze dryer at -80°C to obtain microsphere powder.

[0046] Example 3 Characterization of co-loaded nanoparticles

[0047] Take an appropriate amount of microsphere powder, disperse it with a small amount of deionized water, and evenly spread it on the corresponding metal plate of the instrument. Dry it at room temperature, sputter-coated with gold, and observe the morphology of the microspheres under a scanning electron microscope. Take a small amount of microsphere powder, redissolve it in deionized water to make it evenly dispersed, and put it into the dynamic optical particle analyzer according to the instructions. Analyze the data using Malvern Instrument software.

[0048] like Figure 2 As shown, the prepared nanoparticles have an average particle size of approximately 700 nm and a potential of -29.8 mV. Scanning electron microscopy results show that the co-loaded nanoparticles are relatively uniform in size and have a smooth spherical shape.

[0049] Example 4: Evaluation of the immunogenicity and protective effect of co-loaded nanoparticles

[0050] C57BL / 6 mice were randomly divided into four groups of 12 mice each: PBS (control), antigen protein + atRA (Ag-atRA), CPG + antigen protein + atRA (Ag-atRA-CPG), aluminum adjuvant + Ag + atRA (Ag-atRA-ALum), and co-loaded nanoparticle group (Ag-PLPCa). Immunization was administered intramuscularly, with each group receiving three immunizations of 10 mg, spaced two weeks apart. After the final immunization, three mice from each group were randomly selected for relevant immune marker testing. Challenge was performed two weeks after the final immunization by intraperitoneal injection of 100 μl of PBS into each group. 8 CFU / mouse MAP (2015WD-1 strain); 8 weeks after challenge, 5 mice from each group were randomly selected for necropsy and sampling for subsequent testing.

[0051] Figure 3 The results showed that Bfra-PLGA immunization significantly promoted the secretion of intestinal mucosal IgA and the proliferation of splenic T cells. Figure 3 ).

[0052] Eight weeks after MAP challenge, the bacterial load in the liver of mice was measured, and the results were as follows: Figure 4 The results showed that intramuscular injection of co-loaded nanoparticles significantly reduced bacterial load in mouse livers.

Claims

1. A type of PLGA nanoparticle that reduces bacterial load after infection with Mycobacterium paratuberculosis, characterized in that, The PLGA nanoparticles were prepared by the following method: 1) Weigh 0.1g PLGA and 500μg all-trans retinoic acid, dissolve them in 5ml dichloromethane, and shake to mix until completely dissolved; 2) Add the solution obtained in step 1) dropwise to 10 mL of 1% PVA, and sonicate under ice bath conditions. The sonication conditions are 300 W, the total time is 6 min, the working time is 2 s and the stop time is 3 s. 3) Pour the solution obtained in step 2) into 10 mL of 1% PVA solution, stir at 300-400 rpm at room temperature for 4 h, evaporate the oil phase, and obtain PLGA nanoemulsion loaded with all-trans retinoic acid. 4) After centrifuging and washing the PLGA nanoemulsion loaded with all-trans retinoic acid obtained in step 3), it was resuspended in Tris buffer containing 2 mg / mL dopamine hydrochloride and mixed and reacted at room temperature for 3 h to obtain a nanoemulsion with dopamine hydrochloride coating on the surface. 5) After centrifuging and washing the nanoemulsion with dopamine hydrochloride surface coating obtained in step 4), it is resuspended in PBS solution containing 100 μg / mL CPG oligonucleotide and 40 μg / mL antigen protein, and mixed at room temperature for 3 h to obtain nanoparticle solution; the antigen protein is HBHA-Ag85B-Bfra, and its amino acid sequence is SEQ ID NO.1; 6) The nanoparticle solution obtained in step 5) is centrifuged and washed in a high-speed refrigerated centrifuge. After washing 3 times, the supernatant is discarded, and the nanoparticles are dried in a vacuum freeze dryer at -80°C to obtain microsphere powder.

2. A method for preparing PLGA nanoparticles that reduce bacterial load after infection with Mycobacterium paratuberculosis, characterized in that, The preparation method includes: 1) Weigh 0.1g PLGA and 500μg all-trans retinoic acid, dissolve them in 5ml dichloromethane, and shake to mix until completely dissolved; 2) Add the solution obtained in step 1) dropwise to 10 mL of 1% PVA, and sonicate under ice bath conditions. The sonication conditions are 300 W, the total time is 6 min, the working time is 2 s and the stop time is 3 s. 3) Pour the solution obtained in step 2) into 10 mL of 1% PVA solution, stir at 300-400 rpm at room temperature for 4 h, evaporate the oil phase, and obtain PLGA nanoemulsion loaded with all-trans retinoic acid. 4) After centrifuging and washing the PLGA nanoemulsion loaded with all-trans retinoic acid obtained in step 3), it was resuspended in Tris buffer containing 2 mg / mL dopamine hydrochloride and mixed and reacted at room temperature for 3 h to obtain a nanoemulsion with dopamine hydrochloride coating on the surface. 5) After centrifuging and washing the nanoemulsion with dopamine hydrochloride surface coating obtained in step 4), it is resuspended in PBS solution containing 100 μg / mL CPG oligonucleotide and 40 μg / mL antigen protein, and mixed at room temperature for 3 h to obtain nanoparticle solution; the antigen protein is HBHA-Ag85B-Bfra, and its amino acid sequence is SEQ ID NO.1; 6) The nanoparticle solution obtained in step 5) is centrifuged and washed in a high-speed refrigerated centrifuge. After washing 3 times, the supernatant is discarded, and the nanoparticles are dried in a vacuum freeze dryer at -80°C to obtain microsphere powder.

3. The application of the PLGA nanoparticles according to claim 1 in the preparation of paratuberculosis vaccine.

4. A paratuberculosis vaccine, characterized in that, The vaccine comprises PLGA nanoparticles according to claim 1.

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