Siniperca chuatsi and frog iridovirus oral composite vaccine and preparation method thereof

Abstract

The invention belongs to the technical field of antivirus, and particularly discloses a siniperca chuatsi frog iridovirus oral composite vaccine and a preparation method thereof.The composite vaccine comprises an antigen core, a mucous membrane penetrating layer, a pH sensitive protective shell layer and a targeted modification layer, and the antigen core is formed by compounding a siniperca chuatsi frog iridovirus inactivated vaccine and an immunologic adjuvant; the mucous membrane penetrating layer is a pegylated chitosan-sodium alginate compound; the pH sensitive protective shell layer is a poly (beta-amino ester)-trehalose copolymer, and the targeted modification layer is wheat lectin. According to the siniperca chuatsi and frog iridovirus oral compound vaccine and the preparation method thereof, the technical problems that oral vaccines are easily degraded by digestive enzymes in aquatic animal bodies and the mucous membrane absorption efficiency is low are solved through the multi-layer structural design, the immune protection effect of the vaccines is remarkably improved, and the preparation method is simple and controllable and suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of antiviral vaccine technology, and in particular to an oral combined vaccine against mandarin frog iridovirus and its preparation method. Background Technology

[0002] Mandarin frog iridovirus (MRV) is a virus that seriously harms freshwater farmed fish such as mandarin fish and snakehead fish. It spreads rapidly and has a high mortality rate, causing huge economic losses to the aquaculture industry. At present, the prevention and control of MRV mainly relies on disinfection of the aquaculture environment and the use of antibiotics. However, the overuse of antibiotics can easily lead to problems such as drug resistance and environmental pollution. Therefore, the development of safe and effective vaccines has become the key to the prevention and control of this virus.

[0003] Oral vaccines have become a research hotspot in aquatic vaccines due to their advantages such as ease of use, no need for injection, and minimal damage to the fish. However, the digestive system of aquatic animals has strong acidity and digestive enzyme activity. After oral vaccines enter the body, they are easily degraded by gastric acid and proteases, leading to antigen inactivation. At the same time, the barrier function of the intestinal mucosa makes it difficult for vaccine antigens to be absorbed into the body, resulting in low delivery efficiency and seriously affecting the immunoprotective effect of oral vaccines.

[0004] In existing technologies, to improve the stability and delivery efficiency of oral vaccines, a single carrier is often used to encapsulate the antigen. However, a single carrier cannot simultaneously achieve functions such as gastric acid protection, mucosal penetration, and targeted delivery. Furthermore, existing vaccines have limited immune-enhancing effects, making it difficult to elicit a comprehensive immune response. Therefore, developing an oral composite vaccine that combines gastric acid protection, mucosal penetration, and targeted delivery functions, along with a highly effective adjuvant, is of great significance for improving the delivery efficiency and immunization efficacy of MRV oral vaccines. Summary of the Invention

[0005] The purpose of this invention is to provide an oral combined vaccine against mandarin frog iridovirus and its preparation method. Through a multi-layered structural design and the combination of a highly efficient adjuvant, it achieves efficient antigen protection, mucosal penetration, and targeted delivery, significantly improving the immunoprotective effect of the oral vaccine. This addresses the technical problems of existing MRV oral vaccines, such as easy degradation, low delivery efficiency, and poor immunization efficacy.

[0006] To achieve the above objectives, the present invention provides an oral composite vaccine against mandarin frog iridovirus, comprising an antigen core, a mucosal penetration layer, a pH-sensitive protective shell, and a targeting modification layer. The antigen core is composed of a mandarin frog iridovirus inactivated vaccine and an immune adjuvant. The mucosal penetration layer is a polyethylene glycol-chitosan-sodium alginate complex. The pH-sensitive protective shell is a poly(β-amino ester)-trehalose copolymer. The targeting modification layer is wheat lectin.

[0007] Preferably, the mass ratio of the mandarin frog iridovirus inactivated vaccine to the immunizing adjuvant is 1:0.2-0.5.

[0008] Preferably, the immune adjuvant comprises yeast β-glucan and lactoferrin, wherein the mass ratio of yeast β-glucan to lactoferrin is 3-6:1.

[0009] Preferably, the mass ratio of polyethylene glycol to chitosan in the mucosal penetrating layer is 1:3-5, and the mass ratio of chitosan to sodium alginate is 2-3:1.

[0010] This invention also provides a method for preparing an oral combined vaccine against mandarin frog iridovirus, comprising the following steps: Step 1, Preparation of antigen core: The inactivated vaccine of mandarin frog iridovirus and the adjuvant are added to phosphate buffer and stirred at 200-300 rpm for 1-2 hours at 4°C. Then, the mixture is homogenized 3-5 times by a high-pressure homogenizer to form an antigen core suspension with a particle size of 100-200 nm. Step 2, coating of the mucosal penetration layer: The antigen core suspension from Step 1 is slowly added dropwise to the polyethylene glycol-chitosan-sodium alginate complex solution, wherein the mass ratio of the antigen core to the polyethylene glycol-chitosan-sodium alginate complex is 1:1-2. The mixture is stirred at 150-200 rpm at 25°C for 2-3 hours to form a complex that coats the mucosal penetration layer. Step 3, pH-sensitive protective shell encapsulation: Adjust the pH of the complex suspension from Step 2 to 5.5-6.0, slowly add a poly(β-amino ester)-trehalose copolymer solution, with a mass ratio of complex to poly(β-amino ester)-trehalose copolymer of 1:1.5-2, stir at 100-150 rpm at 37°C for 1-2 hours, and dialyze to remove unreacted copolymer to obtain double-layer encapsulated particles; Step 4, grafting of the targeted modification layer: The bilayer-encapsulated particles from Step 3 were dispersed in 2-(N-morpholino)ethanesulfonic acid buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1:0.8-1.2. After activation for 30-60 min, wheat lectin solution was added, and the mixture was stirred and reacted at 4°C in the dark for 4-6 h. After centrifugation and washing three times, the oral combined vaccine was obtained by freeze-drying.

[0011] Preferably, in step 2, the preparation method of polyethylene glycol-modified chitosan is as follows: chitosan is dissolved in 1% acetic acid solution, activated polyethylene glycol is added, the reaction is carried out at 60-70℃ for 8-12 hours, purified by dialysis, and then freeze-dried to obtain the product.

[0012] Preferably, in step 3, the preparation method of the poly(β-amino ester)-trehalose copolymer is as follows: 1,4-butanediol diacrylate and diethanolamine are reacted at 50°C for 6-8 hours in a molar ratio of 1:1.2 to obtain poly(β-amino ester), then trehalose is added and reacted at 70-80°C for 4-6 hours, and the copolymer is obtained after purification.

[0013] Preferably, in step 4, the concentration of wheat lectin is 1-2 mg / mL, and the concentration of 2-(N-morpholino)ethanesulfonic acid buffer is 20-50 mmol / L.

[0014] The advantages and beneficial effects of the above-mentioned oral combined vaccine of mandarin frog iridovirus and its preparation method are as follows: 1. The pH-sensitive protective shell of this invention can effectively protect antigens in the acidic environment of the stomach and improve antigen retention rate.

[0015] 2. The mucosal penetration layer and the targeted modification layer of this invention work synergistically to improve the intestinal mucosal adhesion rate and the antigen absorption efficiency, thus solving the technical problem of low mucosal absorption efficiency of oral vaccines.

[0016] 3. The composite adjuvant and multilayer delivery system of this invention work synergistically to improve the relative protection rate of mandarin fish after immunization. All vaccine carrier materials exhibit good biocompatibility and no significant toxicity, and oral administration has no significant impact on the growth performance and liver and kidney function of mandarin fish. The entire preparation process requires no complex equipment, uses mild reaction conditions, is suitable for large-scale production, and has broad application prospects.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a bar chart showing the results of the immune protection effect in Example 3; Figure 2 The bar chart shows the immune protection rates of Example 2 and the comparative example. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] The following examples are not intended to limit the invention, but are only for illustration. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] Example 1 The mandarin frog iridovirus oral composite vaccine comprises an antigen core, a mucosal penetration layer, a pH-sensitive protective shell, and a targeting modification layer. The antigen core is composed of a mandarin frog iridovirus inactivated vaccine and an adjuvant; the mucosal penetration layer is a polyethylene glycol-chitosan-sodium alginate complex; the pH-sensitive protective shell is a poly(β-amino ester)-trehalose copolymer; and the targeting modification layer is wheat lectin (WGA).

[0023] The mass ratio of inactivated mandarin frog iridovirus vaccine to immunizing adjuvant is 1:0.2-0.5.

[0024] The immune adjuvants include yeast β-glucan and lactoferrin, with a mass ratio of yeast β-glucan to lactoferrin of 3-6:1.

[0025] The mass ratio of polyethylene glycol to chitosan in the mucosal penetrating layer is 1:3-5, and the mass ratio of chitosan to sodium alginate is 2-3:1.

[0026] The preparation method of the oral combined vaccine against mandarin frog iridovirus includes the following steps: Step 1, Preparation of antigen core: Inactivated Mandarin frog iridovirus (MRV) vaccine and adjuvant are added to phosphate buffer (PBS) and stirred at 200-300 rpm for 1-2 hours at 4°C. Then, the mixture is homogenized 3-5 times using a high-pressure homogenizer (pressure 80-100MPa) to form an antigen core suspension with a particle size of 100-200nm.

[0027] Step 2, Encapsulation of the mucosal penetration layer: The antigen core suspension from Step 1 is slowly added dropwise to a polyethylene glycol-modified chitosan-sodium alginate complex solution, wherein the mass ratio of the antigen core to the polyethylene glycol-modified chitosan-sodium alginate complex is 1:1-2. The mixture is stirred at 150-200 rpm for 2-3 hours at 25°C to form a complex that encapsulates the mucosal penetration layer. The polyethylene glycol-modified chitosan is prepared by dissolving chitosan in a 1% acetic acid solution, adding activated polyethylene glycol, reacting at 60-70°C for 8-12 hours, dialysis purification, and freeze-drying.

[0028] Step 3, pH-sensitive protective shell encapsulation: Adjust the pH of the complex suspension from Step 2 to 5.5-6.0, and slowly add a poly(β-amino ester)-trehalose copolymer solution. The mass ratio of the complex to the poly(β-amino ester)-trehalose copolymer is 1:1.5-2. Stir at 100-150 rpm at 37℃ for 1-2 hours. Dialyze (molecular weight cutoff 10 kDa) to remove unreacted copolymer, obtaining double-layer encapsulated particles. The preparation method of poly(β-amino ester)-trehalose copolymer is as follows: react 1,4-butanediol diacrylate and diethanolamine at a molar ratio of 1:1.2 at 50℃ for 6-8 hours to obtain poly(β-amino ester), then add trehalose and react at 70-80℃ for 4-6 hours. After purification, the copolymer is obtained.

[0029] Step 4, Grafting of the Targeted Modification Layer: The bilayer-encapsulated particles from Step 3 were dispersed in 2-(N-morpholino)ethanesulfonic acid (MES) buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added. The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1:0.8-1.2. After activation for 30-60 min, wheat lectin (WGA) solution was added, and the reaction was carried out at 4°C in the dark with stirring for 4-6 h. The mixture was centrifuged and washed three times (8000-10000 rpm, 10-15 min), and then freeze-dried to obtain the oral combined vaccine. The concentration of wheat lectin was 1-2 mg / mL, and the concentration of 2-(N-morpholino)ethanesulfonic acid (MES) buffer was 20-50 mmol / L.

[0030] The oral combined vaccine adopts a four-layer structure design, with each layer working synergistically, as detailed below: (1) Antigen core: The MRV inactivated vaccine is used as the core antigen, which retains the complete antigenic epitopes of the virus and can stimulate the body's comprehensive humoral and cellular immune responses; combined with yeast β-glucan and lactoferrin complex adjuvant, the immunogenicity of the vaccine is enhanced synergistically. The inactivated vaccine and the complex adjuvant are mixed and homogenized under high pressure to form nanoscale complex particles, ensuring uniform dispersion and tight binding of antigen and adjuvant.

[0031] (2) Mucosal penetration layer: A polyethylene glycol-modified chitosan-sodium alginate complex. Polyethylene glycol can reduce the immunogenicity and clearance rate of the carrier, while the composite structure of chitosan and sodium alginate (forming a stable gel network through electrostatic interaction) can enhance the adhesion of the vaccine to the intestinal mucosa and improve the mucosal penetration ability of the antigen. The antigen core suspension is mixed with polyethylene glycol-modified chitosan-sodium alginate solution and reacted to form a mucosal penetration layer with strong adhesion and low immunogenicity through electrostatic interaction.

[0032] (3) pH-sensitive protective shell: In the acidic environment of the stomach (pH<3.0), the poly(β-amino ester)-trehalose copolymer protonates to form a dense protective shell, preventing the antigen from being degraded by gastric acid and digestive enzymes; in the neutral environment of the intestine (pH>6.0), the copolymer deprotonates and undergoes a hydrophobic-hydrophilic transition, rapidly degrading and releasing the antigen. By adjusting the pH, the surface of the composite particles becomes positively charged, interacting with the negatively charged poly(β-amino ester)-trehalose copolymer to form a pH-sensitive protective shell.

[0033] (4) Targeted Modification Layer: Wheat lectin (WGA) can specifically bind to N-acetylglucosamine residues on the surface of fish intestinal epithelial cells, achieving targeted delivery of antigens and improving the absorption efficiency of antigens in the intestinal mucosa and the intensity of local immune response. The carboxyl groups on the surface of the composite particles are activated by the EDC / NHS activation method, and an amidation reaction occurs with the amino groups of WGA, achieving stable grafting of the target molecule.

[0034] Inactivated vaccine against mandarin frog iridovirus: The virus was cultured in CPB cells with a viral titer ≥10. 9 TCID 50 / ml, centrifuge at low speed to remove cell debris, then inactivate with formaldehyde (final concentration 0.2-0.4%), and inactivate by low-speed shaking at 37°C for 16-20 hours to ensure complete inactivation of the virus and preservation of the integrity of the antigen epitopes. Virus solution that passes the inactivation test can be stored at 2-8°C for short-term use, and for long-term (more than 2 weeks) storage, it is recommended to freeze at -20°C or below.

[0035] Immunoadjuvants: Yeast β-glucan can activate macrophages and dendritic cells, enhancing antigen presentation ability; lactoferrin has antibacterial, anti-inflammatory and immunomodulatory functions, and can promote lymphocyte proliferation and differentiation. When the two are mixed at a mass ratio of 3-6:1, their synergistic effect significantly enhances the immunogenicity of the vaccine, which is superior to the effect of a single adjuvant.

[0036] Optimization of carrier material ratio: The mass ratio of polyethylene glycol to chitosan is 1:3-5, which ensures the immunogenicity shielding effect of polyethylene glycol without affecting the mucosal adhesion of chitosan; the molar ratio of poly(β-amino ester) to trehalose is 1:1.5-2.5, and trehalose can enhance the biocompatibility and pH-sensitive degradation performance of the copolymer.

[0037] Targeted modification amount: The modification amount of WGA is 50-100 μg grafted onto the surface of the compound vaccine carrier per milligram. This modification amount can ensure the specific binding of the vaccine to intestinal epithelial cells, while avoiding the increase of carrier toxicity and immunogenicity due to excessive modification amount.

[0038] Example 2 The preparation method of the oral combined vaccine against mandarin frog iridovirus includes the following steps: Step 1, preparation of immune adjuvant: Yeast β-glucan (molecular weight 80kDa) and lactoferrin (molecular weight 78kDa, purity 96%) were mixed at a mass ratio of 4:1, and PBS buffer (pH 7.4) was added. After stirring evenly, the adjuvant concentration was 10mg / mL.

[0039] Step 2, Preparation of antigen core: Take 20 mL of mandarin frog iridovirus inactivated vaccine, add 2.4 mg of adjuvant, stir and mix at 250 rpm for 1.5 h at 4 °C, and then homogenize 4 times by a high pressure homogenizer (pressure 90 MPa) to form an antigen core suspension with a particle size of 150 ± 20 nm.

[0040] Step 3, Preparation of polyethylene glycol-modified chitosan: Dissolve 2g of chitosan in 100mL of 1% acetic acid solution, add 0.5g of activated PEG (molecular weight 3000Da), and react at 65℃ for 10h. Then dialyze in deionized water for 3 days using a dialysis bag with a molecular weight cutoff of 5kDa, changing the dialysate twice a day. After freeze-drying, polyethylene glycol-modified chitosan is obtained.

[0041] Step 4, coating the mucosal penetration layer: Dissolve 1g of polyethylene glycol-modified chitosan and 0.4g of sodium alginate in 100mL of PBS buffer (pH 7.4), and slowly add the above antigen core suspension dropwise. The mass ratio of antigen core to polyethylene glycol-modified chitosan-sodium alginate is 1:1.5. Stir at 180rpm at 25℃ for 2.5h to form composite particles that coat the mucosal penetration layer.

[0042] Step 5, Preparation of poly(β-amino ester)-trehalose copolymer: 10 mmol of 1,4-butanediol diacrylate and 12 mmol of diethanolamine were added to a reaction flask and stirred at 50 °C for 7 h to obtain poly(β-amino ester); then 20 mmol of trehalose was added, and the temperature was raised to 75 °C and the reaction was continued for 5 h. After the reaction was completed, the poly(β-amino ester)-trehalose copolymer was obtained by column chromatography purification.

[0043] Step 6, pH-sensitive protective shell coating: Adjust the pH of the composite particle suspension to 5.8 with 1 mol / L HCl, and slowly add a poly(β-amino ester)-trehalose copolymer solution (concentration 10 mg / mL). The mass ratio of composite particles to copolymer is 1:1.8. Stir at 120 rpm at 37℃ for 1.5 h. Then dialyze for 2 days using a dialysis bag with a molecular weight cutoff of 10 kDa to remove unreacted copolymer and obtain double-layer coated particles.

[0044] Step 7, Grafting of the Target Modification Layer: The bilayer-coated particles were dispersed in 50 mmol / L LMES buffer (pH 5.2) at a concentration of 10 mg / mL. EDC and NHS were added at a molar ratio of 1:1. After activation for 45 min, WGA solution (concentration 1.5 mg / mL) was added at a rate of 80 μg of WGA per milligram of composite particle graft. The mixture was stirred and reacted at 4 °C in the dark for 5 h. Then, it was centrifuged at 9000 rpm for 12 min, washed 3 times, and freeze-dried to obtain the oral composite vaccine.

[0045] Example 3 Performance testing of oral combined vaccines.

[0046] Particle size and Zeta potential: The particle size and Zeta potential of the vaccine were detected using a Malvern laser particle size analyzer. The results showed that the average particle size of the vaccine was 380±30nm, the Zeta potential was -18±2mV, and the particle size distribution was uniform (PDI=0.21±0.03), which meets the delivery requirements of oral vaccines.

[0047] pH sensitivity: The vaccine was placed in simulated gastric fluid (pH 2.0, containing 0.3% pepsin) and simulated intestinal fluid (pH 7.4, containing 0.1% trypsin) and incubated at 37°C for 2 hours. Antigen retention and particle size changes were then measured. Results showed that in simulated gastric fluid, the vaccine particle size did not change significantly (390±25 nm), and the antigen retention rate was 92.5±3.1%. In simulated intestinal fluid, the vaccine particle size rapidly increased to 850±50 nm, and the antigen release rate reached 89.7±2.8%, indicating that the protective shell has good pH-sensitive degradation performance.

[0048] Mucosal adhesion: An in vitro intestinal mucosal tissue adhesion experiment was conducted. The vaccine was incubated with the intestinal mucosal tissue of mandarin fish at 37°C for 1 hour. After washing, the amount of vaccine residue on the surface of the mucosal tissue was detected by ELISA. The results showed that the adhesion rate of the composite vaccine of the present invention was 68.5±4.2%, which was significantly higher than that of the unmodified WGA vaccine (adhesion rate 32.1±3.5%).

[0049] Immunoprotective effect: 150 healthy mandarin fish (weight 20±2g) were randomly divided into 3 groups of 50 fish each: Experimental group: The compound vaccine of Example 2 was administered orally. 1g of compound vaccine powder was dissolved in water and mixed with 200g of feed and fed continuously for 4 days. After an interval of 14 days, it was administered orally for another 3 days. The amount of feed given each time was calculated as 2% of the body weight of the experimental fish.

[0050] Control group: The fish were given an oral compound vaccine of unmodified WGA (with the same structure in other aspects). 1g of compound vaccine powder was dissolved in water and mixed with 200g of feed and fed for 4 consecutive days. After an interval of 14 days, the fish were given the vaccine orally for another 3 consecutive days. The amount of feed given each time was calculated as 2% of the body weight of the experimental fish.

[0051] Blank control group: Orally administered feed containing an equal volume of PBS buffer mixed with water.

[0052] On day 21 after the immunization period, use mandarin frog iridovirus (MRV) virus solution (10... 6 TCID 50 Intraperitoneal injection of 0.1 ml / tail ( / mL) was administered to the infected animal. The animal was observed for 14 days, and the mortality rate was recorded and the relative protection rate (RPS) was calculated using the following formula: ; like Figure 1 As shown, the mortality rate in the experimental group was 13.3%, and the RPS was 85.2%; the mortality rate in the control group was 36.7%, and the RPS was 59.2%; the mortality rate in the blank control group was 90%. This indicates that the combined vaccine of the present invention has a significantly better immune protection effect than existing vaccines.

[0053] Biosafety: The growth performance of the experimental group of mandarin fish was monitored for 30 consecutive days. The results showed that there was no significant difference in feeding and growth compared with the blank control group. The liver function indicators such as alanine aminotransferase and aspartate aminotransferase in serum, as well as the kidney function indicators such as blood urea nitrogen and creatinine, were all within the normal range, indicating that the vaccine had no obvious toxicity and good biosafety.

[0054] Comparative Example 1 Vaccine preparation lacking a pH-sensitive protective shell.

[0055] The vaccine was prepared according to the method in Example 2, except that steps 5 and 6 (encapsulation of the pH-sensitive protective shell) were omitted, and targeted modification was performed directly after encapsulation of the mucosal penetrating layer. After incubation in simulated gastric fluid for 2 hours, the antigen retention rate of this vaccine was only 21.7%, and the relative immune protection rate was only 43.3%, indicating that the pH-sensitive protective shell is crucial for preventing the antigen from being degraded by gastric acid and digestive enzymes, and is a key structure for ensuring the immunization effect of the vaccine.

[0056] Comparative Example 2 Vaccine preparation lacking a targeted modification layer.

[0057] The vaccine was prepared according to the method of Example 2, except that step 7 (grafting of the targeted modification layer) was omitted. The intestinal mucosal adhesion rate of this vaccine was 32.1%, and the relative immune protection rate was 59.2%, which was significantly lower than that of the composite vaccine of the present invention. This indicates that the targeted modification layer can significantly improve the adhesion and absorption efficiency of the vaccine on the intestinal mucosa, thereby enhancing the immune protection effect.

[0058] Comparative Example 3 Vaccine preparation using a single adjuvant.

[0059] The vaccine was prepared according to the method of Example 2, except that only yeast β-glucan (without lactoferrin) was used as the adjuvant. After immunization, the relative protection rate of this vaccine was 58.3%, significantly lower than that of the vaccine in Example 2 (relative protection rate 85.2%). Figure 2 As shown in the figure, the combined adjuvant of yeast β-glucan and lactoferrin enhances the immunogenicity of the vaccine through synergistic effect, which is superior to the immunogenicity enhancement effect of a single adjuvant.

[0060] Comparative Example 4 Preparation of vaccines without polyethylene glycol modification.

[0061] The vaccine was prepared according to the method in Example 2, except that the mucosal penetration layer used an unmodified chitosan-sodium alginate complex (without polyethylene glycol modification). This vaccine exhibited high immunogenicity, causing mild intestinal inflammation (mucosal congestion) in mandarin fish after oral administration, and showed rapid clearance from the body, with a relative protection rate of 53.3%. This indicates that polyethylene glycol modification can reduce the immunogenicity of the carrier, prolong the circulation time of the vaccine in the body, and enhance the immunization effect.

[0062] Therefore, this invention employs the aforementioned oral composite vaccine against mandarin frog iridovirus and its preparation method. The core of the vaccine antigen is a composite particle of inactivated mandarin frog iridovirus vaccine and an adjuvant; the mucosal penetration layer uses a polyethylene glycol-modified chitosan-sodium alginate complex, which enhances the vaccine's adhesion and penetration ability in the intestinal mucosa; the pH-sensitive protective shell is a poly(β-amino ester)-trehalose copolymer, which can remain stable in the acidic environment of the stomach and degrade to release antigens in the neutral environment of the intestine; the targeting modification layer is a fish intestinal epithelial cell-specific lectin, which enables targeted delivery of antigens. This invention solves the technical problems of easy degradation by digestive enzymes and low mucosal absorption efficiency of oral vaccines in aquatic animals through multi-layer structural design, significantly improving the immunoprotective effect of the vaccine. The preparation method is simple and controllable, suitable for large-scale production.

[0063] Finally, it should be noted that 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An oral combined vaccine against mandarin frog iridovirus, characterized in that: It includes an antigen core, a mucosal penetration layer, a pH-sensitive protective shell, and a targeting modification layer. The antigen core is composed of an inactivated mandarin frog iridovirus vaccine and an immune adjuvant. The mucosal penetration layer is a polyethylene glycol-chitosan-sodium alginate complex. The pH-sensitive protective shell is a poly(β-amino ester)-trehalose copolymer. The targeting modification layer is wheat lectin.

2. The oral combined vaccine against mandarin frog iridovirus according to claim 1, characterized in that: The mass ratio of the inactivated mandarin frog iridovirus vaccine to the immunizing adjuvant is 1:0.2-0.

5.

3. The oral combined vaccine against mandarin frog iridovirus according to claim 1, characterized in that: The immune adjuvant comprises yeast β-glucan and lactoferrin, with a mass ratio of yeast β-glucan to lactoferrin of 3-6:

1.

4. The oral combined vaccine against mandarin frog iridovirus according to claim 1, characterized in that: The mass ratio of polyethylene glycol to chitosan in the mucosal penetrating layer is 1:3-5, and the mass ratio of chitosan to sodium alginate is 2-3:

1.

5. The method for preparing the oral combined vaccine for mandarin frog iridovirus according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1, Preparation of antigen core: The inactivated vaccine of mandarin frog iridovirus and the adjuvant are added to phosphate buffer and stirred at 200-300 rpm for 1-2 hours at 4°C. Then, the mixture is homogenized 3-5 times by a high-pressure homogenizer to form an antigen core suspension with a particle size of 100-200 nm. Step 2, coating of the mucosal penetration layer: The antigen core suspension from Step 1 is slowly added dropwise to the polyethylene glycol-chitosan-sodium alginate complex solution, wherein the mass ratio of the antigen core to the polyethylene glycol-chitosan-sodium alginate complex is 1:1-2. The mixture is stirred at 150-200 rpm at 25°C for 2-3 hours to form a complex that coats the mucosal penetration layer. Step 3, pH-sensitive protective shell encapsulation: Adjust the pH of the complex suspension from Step 2 to 5.5-6.0, slowly add a poly(β-amino ester)-trehalose copolymer solution, with a mass ratio of complex to poly(β-amino ester)-trehalose copolymer of 1:1.5-2, stir at 100-150 rpm at 37°C for 1-2 hours, and dialyze to remove unreacted copolymer to obtain double-layer encapsulated particles; Step 4, grafting of the targeted modification layer: The bilayer-encapsulated particles from Step 3 were dispersed in 2-(N-morpholino)ethanesulfonic acid buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1:0.8-1.

2. After activation for 30-60 min, wheat lectin solution was added, and the mixture was stirred and reacted at 4°C in the dark for 4-6 h. After centrifugation and washing three times, the oral compound vaccine was obtained after freeze-drying.

6. The method for preparing the mandarin frog iridovirus oral combined vaccine according to claim 5, characterized in that, In step 2, the preparation method of polyethylene glycol-modified chitosan is as follows: chitosan is dissolved in 1% acetic acid solution, activated polyethylene glycol is added, the reaction is carried out at 60-70℃ for 8-12 hours, purified by dialysis, and then freeze-dried to obtain the product.

7. The method for preparing the mandarin frog iridovirus oral combined vaccine according to claim 5, characterized in that, In step 3, the preparation method of poly(β-amino ester)-trehalose copolymer is as follows: 1,4-butanediol diacrylate and diethanolamine are reacted at 50°C for 6-8 hours in a molar ratio of 1:1.2 to obtain poly(β-amino ester), then trehalose is added and reacted at 70-80°C for 4-6 hours. After purification, the copolymer is obtained.

8. The method for preparing the mandarin frog iridovirus oral combined vaccine according to claim 5, characterized in that: In step 4, the concentration of wheat lectin is 1-2 mg / mL, and the concentration of 2-(N-morpholino)ethanesulfonic acid buffer is 20-50 mmol / L.

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