A paratuberculosis vaccine

Abstract

The present application provides a chitosan-sodium alginate-transmembrane peptide ternary polyelectrolyte complex. The ternary polyelectrolyte complex is formed by electrostatic mixing after coating chitosan and sodium alginate with different charge characteristics on the surface of PLGA nanoparticles; the PLGA nanoparticles encapsulate a complex of antigen protein and transmembrane peptide. The ternary polyelectrolyte complex of the present application can successfully overcome the gastrointestinal barrier, protect the antigen protein from being degraded by the gastrointestinal environment and enhance its ability to penetrate the intestinal barrier, activate an efficient mucosal immune response, and can be used as an oral vaccine delivery carrier. After encapsulating the antigen protein of Mycobacterium avium subsp. paratuberculosis, it can effectively prevent and control Mycobacterium avium subsp. paratuberculosis.

Description

Technical Field

[0001] This application belongs to the vaccine technology field in the pharmaceutical and biological field. Specifically, this application provides a chitosan-sodium alginate-penetratin ternary polyelectrolyte complex, its preparation, and its application in paratuberculosis vaccines. Background Art

[0002] Paratuberculosis is a chronic digestive tract disease of common livestock and ruminants caused by Mycobacterium avium subsp paratuberculosis (MAP), which seriously affects the development of the livestock industry. There is an urgent need to develop new methods for preventing and treating Mycobacterium paratuberculosis. MAP is an intestinal pathogen, and the host-pathogen interaction of the intestinal mucosa is the primary determinant of the final disease process. The mucosal immune system has the largest immune cell pool in the body. Therefore, the main purpose of this study is to use mucosal immunity to improve the immune effect.

[0003] Oral vaccines can trigger systemic and mucosal immune responses, effectively resist most pathogenic bacterial infections, and significantly reduce bacterial colonization of the gastrointestinal mucosa. Compared with other potential routes of entry into the mucosal surface (such as nasal or rectal administration), the oral route is still more attractive considering the convenience of administering a large number of animals. However, subunit vaccines face some challenges when immunized via the oral route. Factors affecting oral drugs or vaccines include: biochemical barriers, namely pH barriers, from strongly acidic gastric juice to weakly alkaline intestinal juice; protease barriers; mucus barriers; intestinal epithelial cell barriers. Summary of the Invention

[0004] Based on the above considerations, the present application designs a pH-sensitive chitosan / sodium alginate / transmembrane peptide polyelectrolyte complex to protect antigens through a nanodelivery system, enabling the antigen components to fully exert their functions in the gastrointestinal tract. Among them, chitosan is a natural cationic polysaccharide composed of randomly arranged glucosamine and N-acetylglucosamine, with advantages such as low toxicity, low biodegradability, and low immunogenicity, and is considered a promising drug sustained-release carrier. In addition, chitosan has a unique property that it can adhere to the mucosal surface and open the tight junctions between epithelial cells; sodium alginate is a water-soluble natural linear anionic polysaccharide that can contract at a lower pH, retaining the encapsulated drug in the stomach while protecting the drug from inactivation by enzymes, and thus has been widely used in pH-responsive polymers. After coating chitosan and sodium alginate on the surface of PLGA nanoparticles respectively, the two nanoparticles with opposite charges are mixed, and through intermolecular hydrogen bonds and electrostatic interactions between amino and carboxyl groups, the two nanoparticles are cross-linked with each other to form a network composite system with nanoparticles as units, and this polyelectrolyte complex is used as a carrier to orally deliver drugs, improving the stability and sustained-release property of the encapsulated drugs. Furthermore, we use the transmembrane peptide R8 with strong cell membrane penetration ability and strong cationic properties to bind to the antigen protein through electrostatic self-assembly, which can further increase the chance of the antigen protein being taken up by intestinal immune cells.

[0005] In the present application, the transmembrane peptide R8 is bound to the antigen protein through electrostatic self-assembly to form a transmembrane peptide R8-antigen protein complex. Then, the above R8-antigen protein complex is embedded using the polymer material PLGA, and chitosan or sodium alginate is respectively coated on the surface of the PLGA nanoparticles loaded with the antigen protein to form two nanoparticles with opposite charges on the surface. After that, these two nanoparticles are mixed to prepare a chitosan-sodium alginate-transmembrane peptide ternary polyelectrolyte complex. The ternary polyelectrolyte complex of the present invention can successfully overcome the gastrointestinal barrier, protect the antigen protein from degradation in the gastrointestinal environment and enhance its ability to penetrate the intestinal barrier, activate an efficient mucosal immune response, and can be used as an oral vaccine delivery carrier. By encapsulating the paratuberculosis antigen protein, it is expected to become a new method for preventing and treating the intestinal pathogen paratuberculosis.

[0006] On the one hand, the present application provides a ternary polyelectrolyte complex, and the ternary polyelectrolyte complex is a chitosan-sodium alginate-transmembrane peptide ternary polyelectrolyte complex.

[0007] Furthermore, the ternary polyelectrolyte complex is a polyelectrolyte complex formed by electrostatic mixing after coating chitosan and sodium alginate with different charge characteristics on the surface of PLGA nanoparticles; the PLGA nanoparticles encapsulate a complex of an antigen protein and a transmembrane peptide.

[0008] Furthermore, the cell-penetrating peptide is cell-penetrating peptide R8.

[0009] Furthermore, the antigen protein is Mycobacterium paratuberculosis antigen.

[0010] Furthermore, the antigen protein is the fusion protein HBHA-Ag85B-Bfra.

[0011] On the other hand, the present application provides a method for preparing the ternary polyelectrolyte complex, including:

[0012] 1) Mix the antigen protein with cell-penetrating peptide R8, and form an antigen-cell-penetrating peptide (Ag-R8) complex through electrostatic self-assembly;

[0013] 2) Prepare chitosan-coated PLGA nanoparticles:

[0014] Pour the Ag-R8 solution prepared in the above step 1) into the oil phase in which PLGA is dissolved, and perform ultrasonic emulsification to obtain primary emulsion; add the primary emulsion to the mixed solution of PVA and chitosan, and perform ultrasonic emulsification to obtain multiple emulsion; add the multiple emulsion to the PVA solution, stir, and volatilize the organic phase; centrifuge and wash;

[0015] 3) Prepare sodium alginate-coated PLGA nanoparticles:

[0016] Pour the Ag-R8 solution prepared in the above step 1) into the oil phase solution in which PLGA is dissolved, and perform ultrasonic emulsification to obtain primary emulsion; add the primary emulsion to the sodium alginate solution, and perform ultrasonic emulsification to obtain multiple emulsion; add the multiple emulsion to the poloxamer F68 solution, stir, and volatilize the organic phase; centrifuge and wash;

[0017] 4) Resuspend the nanoparticles prepared in steps 2) and 3) in PBS solution and mix them to obtain the chitosan-sodium alginate-cell-penetrating peptide ternary polyelectrolyte complex.

[0018] Furthermore, the oil phase is ethyl acetate or dichloromethane.

[0019] Furthermore,

[0020] On the other hand, the method provided by the present application includes:

[0021] 1) Mix the antigen protein with cell-penetrating peptide R8 at a mass ratio of 1:1, dissolve them in PBS solution, and form an antigen-cell-penetrating peptide complex through electrostatic self-assembly;

[0022] 2) Prepare chitosan-coated PLGA nanoparticles:

[0023] 500 μL of the 2 mg / mL Ag-R8 solution prepared in the above step 1 was poured into 5 mL of a dichloromethane solution dissolving 100 mg of PLGA, and ultrasonic emulsification was carried out to obtain primary emulsion; the primary emulsion was added to 10 mL of a mixed solution of 2% PVA and 0.5% chitosan, and ultrasonic emulsification was carried out to obtain double emulsion; the double emulsion was added to 10 mL of a 0.5% PVA solution, stirred at room temperature for 5 hours, and the organic phase was volatilized; centrifuged and washed;

[0024] 3) Preparation of alginate-coated PLGA nanoparticles:

[0025] 500 μL of the 2 mg / mL Ag-R8 solution prepared in the above step 1 was poured into a dichloromethane solution dissolving 100 mg of PLGA, and ultrasonic emulsification was carried out to obtain primary emulsion; the primary emulsion was added to 10 mL of a 0.5% sodium alginate solution, and ultrasonic emulsification was carried out to obtain double emulsion; the double emulsion was added to 10 mL of a 2% poloxamer F68 solution, stirred at room temperature for 5 hours, and the organic phase was volatilized; centrifuged and washed;

[0026] 4) The nanoparticles prepared in step 2) and step 3) were resuspended in PBS solution and mixed to obtain a chitosan-alginate-transmembrane peptide ternary polyelectrolyte complex. The application of the above ternary polyelectrolyte complex or the ternary polyelectrolyte complex prepared according to the above method in the preparation of paratuberculosis vaccine.

[0027] Furthermore, the vaccine can reduce the bacterial load after the vaccinated subject is infected with Mycobacterium paratuberculosis.

[0028] Furthermore, the vaccine can enhance the intestinal mucosal IgA secretion and spleen T cell proliferation after immunization of the vaccinated subject.

[0029] On the other hand, the present application provides a paratuberculosis vaccine, which contains the above ternary polyelectrolyte complex or the ternary polyelectrolyte complex prepared according to the above method.

[0030] The antigen protein in the present application is not limited to HBHA-Ag85B-Bfra, and other antigen proteins of MAP can also be used.

[0031] The transmembrane peptide R8 in the present application is a functional short peptide composed of 8 arginines, that is, octaarginine, which can be obtained commercially or prepared by those skilled in the art themselves.

[0032] The nanoparticles / vaccine in the present application are preferably used orally, but it does not exclude administration by injection, nasal spray, etc. after selecting a suitable carrier and formulation.

[0033] Those skilled in the art can select known or in-research varieties of excipients in the present application according to the general knowledge in the vaccine field, including but not limited to adjuvants, solvents, cosolvents, buffers, antioxidants, preservatives. Brief Description of the Drawings

[0034] Figure 1 To verify the purified recombinant fusion protein HBHA-Ag85B-Bfra by SDS-PAGE and Western blot;

[0035] Figure 2 To characterize the novel co-loaded nanoparticles;

[0036] Figure 3 To study the morphology of ternary polyelectrolyte complexes under different conditions;

[0037] Figure 4 To evaluate the immunogenicity of the nanoparticles;

[0038] Figure 5 To determine the bacterial load in the mouse liver. Detailed Description of the Invention

[0039] Example 1 Expression of the Recombinant Fusion Protein

[0040] Resuscitate the cryopreserved Escherichia coli transfected with the HBHA-AG85B-Bfra-pET-30a(+) prokaryotic expression plasmid, culture the bacteria in LB medium containing kanamycin (final concentration: 50 μg / mL) until the logarithmic growth phase (OD600nm is 0.6 - 0.8), add IPTG (final concentration: 1 mM), incubate at 30 °C on a shaker at 160 rpm for 4 h, centrifuge at 4 °C and 8000 rpm for 3 min to collect the bacterial cells, wash the cells twice with pre-cooled PBS, resuspend the cells in PBS and sonicate to lyse them, centrifuge at 10000 rpm for 10 min to obtain the lysate supernatant. Purify the lysate supernatant by Ni column affinity chromatography to obtain the recombinant fusion protein HBHA-AG85B-Bfra. The nucleic acid sequence of the recombinant fusion protein is as follows:

[0041]

[0042] The purified recombinant protein was subjected to SDS-PAGE gel electrophoresis and Western blot identification using mouse anti-MAP positive serum. The results showed that a single band was visible on the NC membrane, which was consistent with the expected size( Figure 1 ).

[0043] Example 2 Preparation of Nanoparticles

[0044] 1) The antigen protein was mixed with the cell-penetrating peptide R8 at a mass ratio of 1:1 and dissolved in PBS solution. The antigen-cell-penetrating peptide (Ag-R8) complex was formed by electrostatic self-assembly;

[0045] 2) Preparation of chitosan-coated PLGA nanoparticles:

[0046] The Ag-R8 solution (500 μL, 2 mg / mL) prepared in the first step was poured into 5 mL of dichloromethane solution containing dissolved PLGA (100 mg,). It was emulsified by ultrasound (300 W, 6 min, ultrasound for 2 s, pause for 3 s) to obtain the primary emulsion. The primary emulsion was added to a mixed solution of 2% PVA and 0.5% chitosan (10 mL) and emulsified by ultrasound (300 W, 8 min, ultrasound for 2 s, pause for 3 s) to obtain the secondary emulsion. The secondary emulsion was added to 0.5% PVA solution (10 mL), stirred at room temperature for 5 hours, and the organic phase was evaporated; centrifuged and washed.

[0047] 3) Preparation of sodium alginate-coated PLGA nanoparticles:

[0048] The Ag-R8 solution (500 μL, 2 mg / mL) prepared in the first step was poured into dichloromethane solution containing dissolved PLGA (100 mg,). It was emulsified by ultrasound (300 W, 6 min, ultrasound for 2 s, pause for 3 s) to obtain the primary emulsion. The primary emulsion was added to 0.5% sodium alginate solution (10 mL) and emulsified by ultrasound (300 W, 8 min, ultrasound for 2 s, pause for 3 s) to obtain the secondary emulsion. The secondary emulsion was added to 2% poloxamer (F68) solution (10 mL), stirred at room temperature for 5 hours, and the organic phase was evaporated; centrifuged and washed.;

[0049] 4) The nanoparticles prepared in the second and third steps were resuspended in PBS solution and mixed to obtain the chitosan-sodium alginate-cell-penetrating peptide ternary polyelectrolyte complex.

[0050] Example 3 Characterization of Nanoparticles

[0051] Take an appropriate amount of microsphere powder, disperse it with a small amount of deionized water, evenly spread it on the corresponding metal plate of the instrument, dry it at room temperature, sputter gold, and observe the microsphere morphology under a scanning electron microscope. Take a small amount of microsphere powder, redissolve it with deionized water to make it evenly dispersed, and put it into a dynamic light particle analyzer according to the instructions, and analyze the data using Malvern Instrument software.

[0052] As Figure 2 shown, the average particle size of the prepared chitosan-PLGA nanoparticles is about 429 nm, and the potential is +25.6 mv; the average particle size of the prepared chitosan-PLGA nanoparticles is about 432 nm, and the potential is -30.3 mv. As Figure 3 shown, the scanning electron microscope results show that under acidic conditions (pH 1.2), the polyelectrolyte complex is a cross-linked network. Under simulated intestinal fluid (pH 6.8) conditions, the polyelectrolyte complex begins to dissociate. In simulated blood (pH 7.4), the complex has dissociated into individual nanoparticles.

[0053] Evaluation of the immunity and protection of nanoparticles in Example 4

[0054] Randomly divide C57BL / 6 mice into 4 groups, with 12 mice in each group, namely the PBS group (control), the antigen protein + cell-penetrating peptide + chitosan PLGA group (Ag / R8-CSPLGA), the antigen protein + chitosan / sodium alginate / PLGA group (Ag-PEC), the Ag + ternary polyelectrolyte complex group (Ag / R8-PEC), and the antigen protein group (Ag). The immunization method is oral gavage (10 mg / mouse), and each group is immunized three times at two-week intervals. After the last immunization, 3 mice are randomly selected from each group for detection of relevant immune indexes. The challenge method is: two weeks after the last immunization, each mouse in each group is intraperitoneally injected with 100 μl of 10 8 CFU / mouse MAP (2015WD-1 strain); 8 weeks after the challenge, 5 mice are randomly selected from each group for autopsy and sampling for subsequent detection.

[0055] Figure 3 The results show that after immunization with Bfra-PLGA, it can significantly promote the secretion of intestinal mucosal IgA and the proliferation of spleen T cells ( Figure 4 ).

[0056] Detect the bacterial load in the mouse liver 8 weeks after the MAP challenge. The results ( Figure 5 ) show that after gavage with this ternary polyelectrolyte complex, the bacterial load in the mouse liver can be significantly reduced.

Claims

1. A paratuberculosis vaccine, characterized in that, the preparation method of the paratuberculosis vaccine is as follows: 1) Mix the paratuberculosis mycobacterium antigen protein HBHA-Ag85B-Bfra encoded by the nucleotide sequence shown in SEQ ID NO.1 and the transmembrane peptide R8 in a mass ratio of 1:1, dissolve them in PBS solution, and form a complex of the paratuberculosis mycobacterium antigen protein HBHA-Ag85B-Bfra and the transmembrane peptide R8 through electrostatic self-assembly; 2) Prepare chitosan-coated PLGA nanoparticles: Pour 500 μL of the complex solution of the paratuberculosis mycobacterium antigen protein HBHA-Ag85B-Bfra and the transmembrane peptide R8 prepared in step 1) with a concentration of 2 mg / mL into 5 mL of a dichloromethane solution dissolved with 100 mg of PLGA, and perform ultrasonic emulsification to obtain primary emulsion; Add the primary emulsion to 10 mL of a mixed solution of 2% PVA and 0.5% chitosan, and perform ultrasonic emulsification to obtain double emulsion; Add the double emulsion to 10 mL of 0.5% PVA solution, stir at room temperature for 5 hours, volatilize the organic phase; Centrifuge and wash; Obtain chitosan-coated PLGA nanoparticles; 3) Prepare sodium alginate-coated PLGA nanoparticles: Pour 500 μL of the complex solution of the paratuberculosis mycobacterium antigen protein HBHA-Ag85B-Bfra and the transmembrane peptide R8 prepared in step 1) with a concentration of 2 mg / mL into a dichloromethane solution dissolved with 100 mg of PLGA, and perform ultrasonic emulsification to obtain primary emulsion; Add the primary emulsion to 10 mL of 0.5% sodium alginate solution, and perform ultrasonic emulsification to obtain double emulsion; Add the double emulsion to 10 mL of 2% poloxamer F68 solution, stir at room temperature for 5 hours, volatilize the organic phase; Centrifuge and wash; Obtain sodium alginate-coated PLGA nanoparticles; 4) Resuspend the chitosan-coated PLGA nanoparticles prepared in step 2) and the sodium alginate-coated PLGA nanoparticles prepared in step 3) in PBS solution and mix them to obtain the paratuberculosis vaccine; the paratuberculosis vaccine is an oral vaccine.

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