Aqueous composite adjuvants and their use in the preparation of vaccines

Abstract

The present application relates to the field of biological vaccine, and especially relates to an aqueous compound adjuvant and application thereof in preparation of vaccine. The present application independently develops an aqueous adjuvant which is efficient, safe, easy to absorb and non-stress, solves the stress and abscess problem caused by application of current oil emulsion vaccine in fattening group and breeding group, develops an aqueous compound adjuvant which is simple to produce, good in stability, low in cost and good in application effect, reduces the dependence on imported adjuvant and the production cost of vaccine, and adopts the aqueous compound adjuvant which is compounded by Carbopol 934P adjuvant, PIKA compound and yeast beta-glucan, and is simple to prepare, good in stability, low in cost and convenient to expand production. The components and dosage of the aqueous compound adjuvant are safe, and no stress, abscess and abnormal side reaction is caused in the application process. The antibody triggered after immunization of the inactivated vaccine prepared by the aqueous compound adjuvant and porcine pseudorabies virus is better than that of a PRV commercial inactivated vaccine, and good protection can be generated, which proves that the adjuvant has good immune enhancement effect.

Description

Technical Field

[0001] This invention relates to the field of biological vaccines, and more particularly to aqueous compound adjuvants and their application in vaccine preparation. Background Technology

[0002] Immunologic adjuvants are an important component in the preparation of inactivated vaccines. They can significantly enhance the induction of specific immune responses in the body by substances with weak antigenicity, such as polysaccharides or peptides. By stimulating the body with the minimum amount of antigen and the minimum number of vaccinations, sufficient immune responses and high-titer antibodies can be generated and maintained for a long time, resulting in a lasting effect. They are also safe, non-toxic, and biodegradable.

[0003] Currently, the most commonly used aqueous adjuvants in inactivated vaccines include: aluminum hydroxide gel, Montanide IMS1313VG, Montanide GEL01, Montanide GEL02, Astragalus polysaccharide, levamisole, and acrylic polymer Carbopol.

[0004] However, this adjuvant component requires a large dosage, has high preparation costs, and can cause side effects such as stress and abscesses. Therefore, there is an urgent need to independently develop efficient, safe, easily absorbed, and stress-free aqueous adjuvants to solve the stress and abscess problems caused by the current oil emulsion vaccines when applied to fattening and breeding populations. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an aqueous compound adjuvant and its application in inactivated vaccines, wherein the compound adjuvant is highly efficient, safe, easily absorbed, and stress-free.

[0006] The present invention provides a water-soluble complex adjuvant, which is composed of Carbopol 934P, PIKA complex and yeast β-glucan; wherein the mass ratio of Carbopol 934P:PIKA complex:yeast β-glucan is (1000~2000):(0~200):(0~100).

[0007] The water-soluble compound adjuvant of this invention comprises a Carbopol 934P:PIKA complex:yeast β-glucan mass ratio of 2000:100:(25-50). In some embodiments, the mass ratio of Carbopol 934P:PIKA complex:yeast β-glucan in the water-soluble compound adjuvant of this invention is 2000:100:25. In other embodiments, the mass ratio of Carbopol 934P:PIKA complex:yeast β-glucan is 2000:100:50.

[0008] In some embodiments, the water-soluble complex adjuvant of the present invention contains 20 mg / ml of Carbopol 934P and 250 μg / ml of the PIKA complex; in other embodiments, the Carbopol 934P contains 20 mg / ml and the PIKA complex contains 500 μg / ml; in some specific embodiments, the Carbopol 934P contains 20 mg / ml and the PIKA complex contains 1000 μg / ml; in other specific embodiments, the Carbopol 934P contains 20 mg / ml and the β-glucan contains 150 μg / ml; in one embodiment, the Carbopol 934P contains 20 mg / ml and the β-glucan contains 250 μg / ml; in another embodiment, the Carbopol 934P contains 20 mg / ml and the β-glucan contains 500 μg / ml.

[0009] In the water-soluble compound adjuvant of this invention, Carbopol 934P is the basic water-based adjuvant, relative to MONTANIDE. TM GEL02, MONTANIDE TM Adjuvants such as GEL01, aluminum hydroxide gel, Carbopol 971p or Carbopol 974p, and Carbopol 934P used in combination with immunostimulants such as PIKA complex, polyIC, R848, VSP, DDA or levamisole can more effectively reduce adverse reactions and side effects during vaccine use, and significantly enhance the degree of immune effect.

[0010] Experiments show that, compared to combined use with polysaccharides such as Astragalus polysaccharide, Ganoderma lucidum polysaccharide, DEAE glucan, or Lentinan, the combination of Carbopol 934P, the PIKA complex, and yeast β-glucan can significantly enhance the immune effect and reduce adverse reactions and side effects during vaccine use. This indicates that yeast β-glucan can exert a superior synergistic effect with Carbopol 934P and the PIKA complex.

[0011] This invention utilizes Carbopol 934P, the PIKA complex, and yeast β-glucan as a water-soluble adjuvant to reduce adverse reactions and side effects during vaccine use, and also exhibits good immune-enhancing effects. Compared to the control group using Carbopol 934P, the PIKA complex, and yeast β-glucan alone, or the control group using Carbopol 934P with either the PIKA complex or yeast β-glucan, the composition provided by this invention significantly reduces adverse reactions and side effects during vaccine use and enhances immune efficacy. This indicates that at the same dosage, the composition of this invention achieves superior effects, and the components of the composition achieve a good synergistic effect through reasonable combination.

[0012] The aqueous composite adjuvant described in this invention exhibits excellent reproducibility and stability, superior to commercially available adjuvants. The three components—Carbopol 934P, the PIKA complex, and yeast β-glucan—and their concentrations provided by this invention play a crucial role in improving stability and reproducibility. Through optimization of the water-soluble composite adjuvant formulation, the optimized adjuvant features rationally selected components at appropriate concentrations. These components functionally support each other, exhibiting interactive relationships and synergistic effects, thus achieving excellent reproducibility and stability.

[0013] The present invention provides a method for preparing the water-soluble complex adjuvant, the method comprising mixing Carbopol934P, PIKA complex and yeast β-glucan.

[0014] The preparation method described in this invention specifically includes:

[0015] Carbopol 934P powder was dissolved in water, and the pH was adjusted to 6.8–7.2 to prepare Carbopol 934P mother liquor.

[0016] PIKA complex powder was dissolved in PBS solution to prepare PIKA complex stock solution;

[0017] Yeast β-glucan stock solution was prepared by dissolving dextran powder in PBS solution.

[0018] The water-soluble composite adjuvant was prepared by mixing Carbopol 934P stock solution, PIKA complex stock solution and yeast β-glucan stock solution; wherein the volume ratio of Carbopol 934P stock solution, PIKA complex stock solution and yeast β-glucan stock solution was 1:0.5:0.5.

[0019] The method for preparing the water-soluble compound adjuvant of this invention includes first preparing Carbopol 934P stock solution, PIKA complex stock solution, and yeast β-glucan stock solution separately, and then mixing the three stock solutions to maintain a 1:0.5:0.5 ratio. This three-step preparation process is simple, the resulting vaccine is highly stable, maintains the excellent properties of the water-soluble compound adjuvant, and achieves good immune enhancement effects when used in conjunction with it.

[0020] This invention provides the application of the aforementioned water-soluble compound adjuvant and the water-soluble compound adjuvant prepared by the aforementioned method in the preparation of vaccines.

[0021] This invention also provides a vaccine comprising the aforementioned water-soluble compound adjuvant and antigen. The vaccine of this invention is primarily intended for use on animals, specifically livestock, including pigs, cattle, sheep, horses, donkeys, and mules. Pigs are particularly emphasized.

[0022] In the vaccine of this invention, the antigen comprises inactivated porcine pseudorabies virus 10^ 8.0 TCID 50 / mL.

[0023] In the vaccine described in this invention, the dosage is as follows: the concentration of the water-soluble compound adjuvant is 50 wt%; the concentration of the antigen is 10 wt%. 8.0 TCID 50 / mL.

[0024] The method for preparing the vaccine according to the present invention includes mixing a water-soluble compound adjuvant and an antigen.

[0025] The present invention also provides a treatment method using the aforementioned water-soluble compound adjuvant, the method comprising mixing the antigen with a PIKA complex and a β-glucan solution, maintaining a final concentration of 1 mg / dose, and then mixing the mixture with Carbopol 934P and stirring.

[0026] In the treatment method described in this invention, the concentration of Carbopol 934P is 20 mg / ml, and the pH value is 6.8–7.2.

[0027] This invention independently developed a water-soluble adjuvant composed of Carbopol 934P, PIKA complex, and yeast β-glucan. This highly efficient, safe, easily absorbed, and stress-free water-based adjuvant solves the stress and abscess problems that occur after application of current oil-emulsion vaccines in fattening and breeding populations. The developed water-based adjuvant is simple to produce, has good stability, low cost, and good application effects, reducing vaccine production costs and facilitating large-scale production. No stress, abscesses, or abnormal side effects were observed during application. The inactivated vaccine prepared with porcine pseudorabies virus (PRV) exhibits superior antibody stimulation after immunization compared to commercial PRV inactivated vaccines, providing better protection and enhancing immunity. The antibody effects of vaccines B1 and B2 of this invention are comparable to those of commercial pseudorabies vaccines, both producing a high antibody duration of at least 60 days. Vaccine B2 shows better protection against challenge than B1 and commercial pseudorabies vaccines, with better results in antigen detection rate and fever rate after challenge. Therefore, the PRV composite water adjuvant inactivated vaccine developed in this invention to address the shortcomings of current vaccines is feasible, and the composite water adjuvant has good prospects for commercial development and clinical application. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort:

[0029] Figure 1 This shows the results of PRV neutralizing antibody testing;

[0030] Figure 2 Showing the positive rate of neutralizing antibodies;

[0031] Figure 3 Analysis of gB detection results;

[0032] Figure 4 This indicates the positive detection rate of gE antigen;

[0033] Figure 5 The percentage of patients who developed fever after infection was shown. Detailed Implementation

[0034] This invention provides an aqueous compound adjuvant and its application in a porcine pseudorabies inactivated vaccine. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0035] 1. Adjuvant preparation:

[0036] 1) From MONTANIDE TM GEL02, MONTANIDE TM GEL01, aluminum hydroxide gel, Carbopol, etc., with Carbopol as the preferred aqueous adjuvant; and Carbopol934P is preferred from among many Carbopol types such as Carbopol 934P, Carbopol940, Carbopol 971p, and Carbopol974p, with the dosage added at 20% of the total vaccine volume.

[0037] 2) Immunostimulants: Among the more studied immunostimulants are PIKA, polyI:C, R848, VSP, DDA, levamisole, etc. This study prefers the PIKA complex, and the final concentration of PIKA used is 200-2000 μg / dose.

[0038] 3) Polysaccharides: Currently, the most studied polysaccharides include yeast β-glucan, astragalus polysaccharide, Ganoderma lucidum polysaccharide, DEAE glucan, and lentinan. This invention prefers yeast β-glucan, and the final concentration for application is 100-1000 μg / dose.

[0039] 2. Preparation of porcine pseudorabies antigen:

[0040] Extract toxic substances with a titer ≥10 from the reactor. 8.0 TCID 50 PRV stock solution was centrifuged at 6000 rpm for 10 min and filtered through a 0.8 μm filter to remove cell debris. The supernatant was then concentrated by 500 kD hollow fiber ultrafiltration and sent for virus titer determination. The concentrated virus solution was inactivated with 0.25% formaldehyde at 37°C for 72 h. After complete inactivation, inactivation and sterility tests were performed.

[0041] 3. Seedling preparation:

[0042] 1) First, add a certain amount of potency >10 8.0 TCID 50 / mL of PRV inactivated antigen was diluted with sterile PBS buffer to maintain the vaccine titer at 10. 8.0 TCID 50 / Toufen.

[0043] 2) Then, add PIKA and β-glucan stock solution, stir well, and keep the final concentration at 1 mg / dose.

[0044] 3) Finally, add 20% by volume of Carbopol 934P adjuvant with a pH value of 6.8 to 7.2, and stir at 37°C for 30 minutes under sterile conditions.

[0045] 4) The prepared vaccine is repackaged, stored at 4°C, and subjected to animal safety testing and efficacy evaluation tests.

[0046] 5) Conduct safety inspections and efficacy verifications in accordance with the requirements of the Veterinary Pharmacopoeia.

[0047] The experimental materials for this invention are sourced from:

[0048] The strain: The pseudorabies strain is the classic Bartha strain.

[0049] Cells: BHK-21 cells, VeroT cells (cells with good viability stored in the laboratory).

[0050] Attack venom: Wild-type venom isolated and purified from pseudorabies-positive diseased pig tissues from a pig farm in Zhongxiang City.

[0051] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0052] The PIKA complex, also written as PIKA in this invention, is a complex of polyinosine monophosphate (polyI), polycytosine monophosphate (polyC), kanamycin (K), and calcium chloride (Ca). It was purchased from Hebei Yidekangsheng Traditional Chinese Medicine Technology Co., Ltd.

[0053] Example 1: Optimal application and content optimization of immune enhancers

[0054] 1. Preparation of compound adjuvants

[0055] Preparation of Carbopol 934P stock solution: Weigh 1g of Carbopol 934P powder (purchased from Lubrizol, France) and soak it in 100ml of ultrapure water overnight to ensure complete dissolution. Then adjust the pH to 6.8-7.2 with 1M sodium hydroxide solution; autoclave the prepared 10mg / ml Carbopol 934P solution at 121℃ for 30min, and it will be colorless and transparent after cooling.

[0056] Preparation of PIKA complex stock solution: Weigh an appropriate amount of PIKA powder (purchased from Hebei Yidekangsheng Traditional Chinese Medicine Technology Co., Ltd.) and dissolve it in sterile PBS solution. Then, use a disposable pinhole filter with a pore size of 0.22 μm to filter and sterilize the high-concentration stock solution, and then dispense it into containers and store it at -20℃ for later use.

[0057] Preparation of yeast β-glucan stock solution: Weigh a small amount of glucan powder (purchased from Yuanye Biotechnology Co., Ltd.) and dissolve it in sterile PBS solution to prepare a high-concentration stock solution. Then, filter it with a disposable pinhole filter with a pore size of 0.22 μm for sterilization and store it at -20℃ for later use.

[0058] 2. Vaccine preparation

[0059] Preparation of porcine pseudorabies antigen

[0060] 1) Inoculation: First, BHK-21 cells with a growth density of 70%–80% were digested into single cells using trypsin, and then inoculated at a rate of 2 × 10⁻⁶ cells / year. 5 Cells were transferred at a density of cells / mL to 75 cm⁻¹. 2 In cell flasks, culture to a density of 60%–70%. Inoculate with a virus titer of 10^6 at a dose of 0.05 MOI. 8.0 TCID 50 / ml of live PRV virus, according to the formula PFU = number of cells × MOI = 0.7 × TCID 50 Calculate the required virus solution. After adsorption at 37°C for 1 hour in a carbon dioxide incubator, discard the virus culture solution and replace it with a 3% fetal bovine serum culture medium as a maintenance solution.

[0061] 2) Virus collection: Observe cytopathic effect (CPE). When cells show signs of degeneration, shrinkage, fusion, floating, or netting, it indicates successful infection. When the CPE ratio reaches 70% to 80%, place the cell flask in a -80°C freezer and freeze-thaw it three times to allow the cells to fully lyse and release the virus.

[0062] 3) Virus purification: Collect the viral fluid and centrifuge at 6000 rpm for 10 min at 4°C, collecting the supernatant. Then, remove some debris and impurities using a 0.8 μm pore size filter membrane. Finally, concentrate the harvested viral fluid using a 500 KD hollow fiber column for ultrafiltration.

[0063] 4) TCID 50 Detecting the price of poison

[0064] Passaging: One day in advance, VeroT cells were digested and diluted to a density of 1.5 × 10⁶ cells with 8% DMEM serum medium. 4 Cells / mL. In a biosafety cabinet, cells were transferred from the sample tray to a 96-well cell plate using an 8-well pipette and incubated overnight at 37°C in a 5% CO2 mammalian incubator until they adhered to the plate.

[0065] Virus reception: The next day, after removing the purified virus sample from the -80℃ freezer, thaw it on ice, shake well, and then begin the serial dilution of the sample, starting from 10... -1 Up to 10 -8 Eight dilution gradients were used. After vortexing, the 96-well plate was removed from the incubator and inoculated with the virus in a biosafety cabinet. 100 μL of virus dilution was added to each well, with each column representing the same dilution. The last two columns of normal cells served as normal control wells, with 100 μL of medium supplemented with 3% fetal bovine serum added to each well to ensure normal cell growth. The 96-well plate was then placed in a CO2 cell culture incubator at 37°C for 1 hour (Note: the inoculated plate should be placed on the bottom shelf). The medium was then replaced with 3% serum in the biosafety cabinet. Cytopathic effect (CPE) was observed daily from inoculation, and the number of CPE wells in each column was recorded, continuing until day 5. Finally, the data were compiled and the viral titer was calculated using the Karber formula.

[0066] 5) Inactivation and verification

[0067] The ultrafiltration concentrate was inactivated with formaldehyde at a final concentration of 0.25% at 37°C for 48 hours. The inactivated antigen solution was then stored at 4°C. One mL of the inactivated virus solution was used for blind passage through three generations according to the veterinary pharmacopoeia method to verify inactivation.

[0068] Vaccine preparation: As shown in Table 1, appropriate amounts of vaccines with a potency >10 were added to different groups of vaccines. 8.0 The PRV antigen ultrafiltration dilution was diluted to a concentration of ≥10 / mL to ensure a potency of ≥10 per dose (2mL) of vaccine. 8.0 TCID 50 2) Add PIKA and β-glucan complex, and stir evenly at 300 rpm / min in a sterile environment. 3) Finally, add 20% volume of Carbopol 934P adjuvant concentrate and stir at low speed for 30 min. 4) Fill the prepared PRV aqueous adjuvant vaccine into vaccine bottles and store temporarily at 4℃.

[0069] Table 1: Preparation of vaccines with different components

[0070]

[0071]

[0072] Note: The reason for this grouping design is that some studies have directly used 1000μg of PIKA adjuvant, and the inventors also found that doses of 200μg to 500μg had very good antibody effects in mice and pigs during the experiment, but no comparison was made; while there is less research data on β-glucan as an adjuvant, and there is no recommended optimal dose; and the effects of different adjuvants are not necessarily concentration gradient dependent, and the concentration effect of some adjuvants shows a trend of first increasing and then decreasing. Therefore, it is necessary to explore the application dosage of PIKA and β-glucan separately to determine the optimal application amount and safety of the two.

[0073] After determining the optimal concentration of PIKA to be 1000 μg / ml in Example 1, the two were combined in Example 2 to optimize the concentration of dextran and screen for water adjuvant combinations that could produce better results.

[0074] 3. Immunization effects of different vaccine doses

[0075] 1) Security check: Five healthy mice weighing 18g-20g were selected, injected intraperitoneally with 0.5mL of commercially available vaccine, and fed and managed in a separate room. The survival and abnormal behavior of the mice were observed for 14 days.

[0076] 2) Immunization schedule: Mice were intraperitoneally immunized twice with the finished vaccines A1-A7, 0.2 ml / vaccination, with a 14-day interval between the two immunizations. Blood samples were collected from the orbital venous plexus at 14, 28, and 42 days post-immunization to detect changes in neutralizing antibodies. On day 28 post-immunization, 5 mice from each group were challenged intraperitoneally with PRV wild-type virus, each injected with 0.5 ml (10... 6.0 TCID 50 The optimal dose of immune enhancer in each component vaccine was screened by the increase of PRV neutralizing antibodies and the survival rate after challenge ( / ml).

[0077] 3) Inspection results: All mice survived within 14 days, ate normally, and showed no abnormal behavior.

[0078] 4) Efficacy Assessment: The changes in neutralizing antibodies in mice after immunization are shown in Table 2 below. Neutralizing antibodies against vaccines A3, A5, and A6 increased rapidly, reaching their peak at 28 days. The viability of vaccines A1, A2, A3, A5, and A6 after challenge was >80%. Therefore, it can be concluded that PIKA immunization is concentration-gradient dependent, with the optimal response concentration being 1000 μg / ml. The effective protective concentration range for β-glucan is 250 μg / ml to 500 μg / ml. However, the safe dose of PIKA adjuvant in mice should not exceed 200 μg, and the recommended safe dose in pigs is 1000 μg to 2000 μg. Therefore, the optimal immunization dose of PIKA is 1000 μg / ml.

[0079] Table 2: Mean antibody levels and challenge survival rates in mice after immunization with each component of the vaccine

[0080] Vaccine A1 10 0 102 147 180 80％ Vaccine A2 10 0 147 256 205 80％ Vaccine A3 10 0 154 512 333 100％ Vaccine A4 10 0 64 141 179 50％ Vaccine A5 10 0 109 256 256 80％ Vaccine A6 10 0 128 256 256 80％ Vaccine A7 10 0 32 90 67 20％ Blank control 10 0 0 0 0 0％

[0081] Example 2: Immunization experiment of optimized drug combination on piglets

[0082] 1. Experimental grouping and vaccine preparation

[0083] Table 3: Preparation of combined adjuvant vaccines

[0084] Vaccine B1 pseudorabies 20％ 1000μg / ml 250μg / ml Vaccine B2 pseudorabies 20％ 1000μg / ml 500μg / ml commercial seedlings pseudorabies —— —— —— Challenge to control —— —— —— —— Blank control —— —— —— ——

[0085] Note: The antigens in vaccines B1, B2 and the commercial vaccine are completely identical. The adjuvant in the commercial vaccine is an imported biphasic adjuvant, while the adjuvants in vaccines B1 and B2 are provided by this invention.

[0086] 2. Vaccine safety inspection: Select 5 healthy Class A piglets of uniform weight at 30 days old from the same batch of pigs. After quarantine, they are free from PRV and other antigen infections. After marking the ear tags, inject 4mL of finished vaccine into the triangular area behind the ear. Observe and record the survival, stress and abnormal behavior within 14 days after vaccination.

[0087] 3. Vaccine Evaluation: Ten healthy 60-day-old weaned piglets without antigen infection were immunized with the prepared PRV aqueous adjuvant inactivated vaccines B1 and B2 and the commercially available pseudorabies inactivated vaccine, respectively. A positive challenge control group and a negative challenge control group were also established. Immunization was performed in two steps, with a 14-day interval between immunizations. 5ml of venous blood was collected before immunization, and 14 and 28 days after immunization. After centrifugation, the supernatant serum was collected to detect neutralizing antibodies and gB antibodies, evaluating the antibody efficacy of the vaccine. Challenge: 28 days after the second immunization, 2ml of porcine pseudorabies wild-type virus was injected intramuscularly into each of the three vaccine groups and the positive challenge group. The challenge dose was 10^ 6.0 TCID 50Body temperature was measured before and within 1 to 14 days after the challenge to determine the proportion of pigs with fever. Blood samples were collected at 0, 7, 14, 21 and 28 days after the challenge to detect antigen positivity and viral load. After the challenge, antigen-positive pigs from each group were selected for necropsy to compare the lesions in the lymph nodes, trigeminal nerve, lungs, brain and other parts of the body.

[0088] 4. Security check results

[0089] All piglets survived within 14 days, ate normally, and showed no stress or abnormal behavior.

[0090] 5. Immune effect

[0091] 1) Neutralizing antibody detection: ① Dilute serum: Serially dilute the serum to be tested at ratios of 1:21, 1:22, 1:23, etc.; ② Mix antibody and virus: Mix quantitative virus solution with serum diluted at different ratios and incubate at 37°C for 1 hour; ③ Inoculate cells: Inoculate the incubated mixture onto a monolayer of cells at a certain density and incubate at 37°C for 1 hour; ④ Observe lesions: Add 3% serum culture medium to the cells to maintain cell growth and continue culturing in a 37°C incubator, observing the lesions daily; ⑤ Calculate neutralizing antibody value: The highest serum dilution that can protect 50% of cells is the neutralizing antibody value.

[0092] PRV neutralizing antibody test results showed that... Figure 2 and Figure 3 As shown.

[0093] After the second immunization, vaccines B1 and B2 can rapidly produce high levels of antibodies, and the average value and positive rate of neutralizing antibodies are greater than those of commercial vaccines. 60 days after the immunization, the antibody levels of vaccines B1 and commercial vaccines began to decline, while vaccine B2 still maintained a high level, proving that the duration of immunity in the vaccine group met the standards of commercial vaccines, and vaccine B2 was better.

[0094] 2) gB antibody detection: Follow the instructions for the BioChek porcine pseudorabies gB antibody ELISA kit as follows:

[0095] ① Remove the reaction plate coated with PRV gB from the sealed bag and record the sample position on the recording paper;

[0096] ② Add 100 μl of negative control to wells A1 and B1;

[0097] ③ Add 100 μl of positive control to wells C1 and D1;

[0098] ④ Add 100 μl of the diluted sample to the corresponding well, cover the reaction plate with the cap, and incubate at room temperature (22℃~27℃) for 30 minutes;

[0099] ⑤ Pour out the contents of the wells and wash 4 times with washing solution (350 μl per well). For the last wash, invert the reaction plate and gently tap it on absorbent paper;

[0100] ⑥ Add 100 μl of enzyme-labeled secondary antibody to each well, cover the reaction plate with the cap, and incubate at room temperature (22℃~27℃) for 30 minutes;

[0101] ⑦ Repeat step 5;

[0102] ⑧ Add 100 μl of substrate reagent to each well, cover the reaction plate with the lid, and incubate at room temperature (22℃~27℃) for 15 minutes;

[0103] ⑨ Add 100 μl of stop solution to each well to stop the reaction and read the results within 30 minutes;

[0104] ⑩ Zero the microplate reader with air and measure the absorbance of each well at a wavelength of 405 nm.

[0105] Analysis of gB test results as follows: Figure 3 As shown:

[0106] As shown in the figure, the gB antibody positivity rate of the second immunization group, including vaccine B1, vaccine B2, and commercial vaccine, all reached 100%.

[0107] 3) gE antigen detection

[0108] Blood samples were collected from the neck before and at 1, 3, 5, 7, 11, 14, 21, and 28 days after viral challenge. The positivity for gE antigen in the blood after viral challenge was quantitatively detected using qPCR. The results are as follows: Figure 4 As shown:

[0109] No gE antigen was detected in the pig herd from before challenge to 6 days after challenge. The antigen began to be detected on day 7, with the highest positivity rate (71.4%) observed in the challenge-positive control group. The antigen positivity rate of vaccine B1 was higher than that of vaccine B2 and the commercial vaccine group.

[0110] The negative control group of pigs remained antigen-negative throughout, and both the commercial vaccine group and the positive challenge group showed an intermittent viral shedding trend.

[0111] 4) Fever after virus challenge

[0112] Results: Temperature changes in pigs were measured within 14 days post-challenge. The positive challenge group had the highest proportion of fever, which steadily increased from day 3 to day 9. Pigs in the vaccine B1 group developed fever from day 3 to day 7 post-challenge, with the highest proportion on day 5, and their temperature returned to normal by day 11. Pigs in the vaccine B2 group showed a lower proportion of fever from day 5 to day 9 post-challenge, with their temperature returning to normal by day 11. The commercial vaccine group developed fever on day 3 post-challenge, with the highest proportion on day 9, followed by a slow decline. The negative control group showed no fever throughout. Figure 5As shown.

[0113] in conclusion:

[0114] 1) Vaccines B1 and B2 have comparable antibody effects to commercial pseudorabies vaccines, and both can produce a high antibody duration of at least 60 days.

[0115] 2) Vaccine B2 has a better protective effect against viral challenge than B1 and commercial pseudorabies vaccines, and is more effective in terms of antigen detection rate and fever rate after viral challenge.

[0116] Therefore, the PRV composite water adjuvant inactivated vaccine developed in this invention to address the shortcomings of current vaccines is feasible, and the composite water adjuvant has good development and application prospects.

[0117] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A water-soluble compound adjuvant, characterized in that, It is composed of Carbopol 934P, PIKA complex and yeast β-glucan; wherein the mass ratio of Carbopol 934P:PIKA complex: yeast β-glucan is 2000:100:(25~50); wherein the PIKA complex is a complex of polyxanthine nucleotide, polycytosine nucleotide, kanamycin and calcium chloride.

2. The water-soluble compound adjuvant according to claim 1, characterized in that, in, The mass ratio of Carbopol 934P:PIKA complex:yeast β-glucan is 2000:100:25; Alternatively, Carbopol 934P:PIKA complex: yeast β-glucan in a mass ratio of 2000:100:

50.

3. The method for preparing the water-soluble composite adjuvant according to claim 1 or 2, characterized in that, The method involves mixing Carbopol 934P, the PIKA complex, and yeast β-glucan.

4. The preparation method according to claim 3, characterized in that, The method specifically includes: Carbopol 934P powder was dissolved in water, and the pH was adjusted to 6.8-7.2 to prepare Carbopol 934P mother liquor; PIKA complex powder was dissolved in PBS solution to prepare PIKA complex stock solution; Yeast β-glucan stock solution was prepared by dissolving dextran powder in PBS solution. The water-soluble composite adjuvant was prepared by mixing Carbopol 934P stock solution, PIKA complex stock solution and yeast β-glucan stock solution; wherein the volume ratio of Carbopol 934P stock solution, PIKA complex stock solution and yeast β-glucan stock solution was 1:0.5:0.

5.

5. The application of the water-soluble compound adjuvant according to claim 1 or 2, or the water-soluble compound adjuvant prepared by the preparation method according to claim 3 or 4, in the preparation of vaccines.

6. A vaccine, characterized in that, The vaccine comprises the water-soluble compound adjuvant and antigen as described in claim 1.

7. The vaccine according to claim 6, characterized in that, The antigen includes inactivated porcine pseudorabies virus 10^ 8.0 TCID 50 / mL.

8. The vaccine according to claim 6, characterized in that, In the vaccine, the concentration of the water-soluble adjuvant is 50 wt%; the concentration of the antigen is 10^ 8.0 TCID 50 / mL.

9. A method for preparing the vaccine according to any one of claims 6 to 8, characterized in that, This involves mixing water-soluble adjuvants and antigens.

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