Highly effective subunit vaccine against porcine deltacoronavirus

Abstract

The present invention provides a highly efficient subunit vaccine against porcine deltacoronavirus, which relates to the field of biological vaccine manufacturing. The highly efficient subunit vaccine against porcine deltacoronavirus is prepared by the following method: (1) Mix an aqueous solution of polylysine with an aqueous solution of flagellin, stir for 5-10 h, and obtain freeze-dried powder of an immune enhancer nanoparticle after freeze-drying; the amino acid sequence of the flagellin is as shown in SEQ ID NO: 1; (2) Add silicic acid to the aqueous solution of the immune enhancer nanoparticle, react for 4-6 h, and obtain a silicified immune enhancer nanoparticle solution; (3) Mix the silicified immune enhancer nanoparticle solution with a PDCoV antigen solution with a HIS tag in a volume ratio of 2-4:1, centrifuge, take the precipitate, and suspend it with a solvent to obtain the vaccine. The vaccine of the present invention can efficiently stimulate the immune response of animals against antigens, significantly improve the antibody level, and significantly prolong the antibody persistence period.

Description

Technical Field

[0001] This invention relates to the field of biological vaccine manufacturing, specifically to a highly effective subunit vaccine against porcine deltacoronavirus. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) can cause vomiting, watery diarrhea, and dehydration in pigs, with a high mortality rate in newborn piglets. As a newly emerging coronavirus infectious disease, there are currently no commercially available vaccines or effective treatments. PDCoV is an enveloped positive-sense RNA virus with a genome structure similar to other coronaviruses. Its spike protein (S) contains two functional subunits, S1 and S2, and contains the main neutralizing antigenic epitopes, serving as the primary target for inducing the production of neutralizing antibodies. The S protein has also been a focus of subunit vaccine research in recent years. Because subunit vaccines only contain the main antigenic targets, they generally suffer from poor immunogenicity, usually requiring the addition of immunostimulants to enhance their specific immune response. However, currently available technologies lack highly effective subunit vaccines for porcine deltacoronavirus. Summary of the Invention

[0003] The purpose of this invention is to provide a highly effective subunit vaccine against porcine delta coronavirus, which can efficiently stimulate an animal's immune response to the antigen, significantly increase antibody levels, and significantly prolong the duration of antibody activity.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A highly effective subunit vaccine against porcine deltacoronavirus was prepared using the following method.

[0006] (1) Mix polylysine aqueous solution with flagellin aqueous solution, stir for 5-10 h, freeze dry to obtain immune enhancer nanoparticle freeze-dried powder; the amino acid sequence of the flagellin is shown in SEQ ID NO: 1;

[0007] (2) Add silicic acid to the aqueous solution of immune enhancer nanoparticles and react for 4-6 hours to obtain a silanized immune enhancer nanoparticle solution.

[0008] (3) The siliconized immune enhancer nanoparticle solution and the HIS-tagged PDCoV antigen solution are mixed at a volume ratio of 2-4:1, centrifuged, the precipitate is collected, and the precipitate is resuspended in a solvent to obtain the vaccine.

[0009] In this invention, the PDCoV antigen is the PDCoV S protein.

[0010] In this invention, the concentration of the polylysine aqueous solution in step (1) is 0.4-0.6 mg / mL, and the concentration of the flagellin aqueous solution is 0.15-0.3 mg / mL; the volume ratio of the polylysine aqueous solution to the flagellin aqueous solution is 1:0.8-1.2.

[0011] In this invention, the concentration of silicic acid in step (2) is 8-10 mg / mL, the concentration of the immune enhancer nanoparticle solution is 0.008-0.012 mg / mL, and the volume ratio of silicic acid to the immune enhancer nanoparticle aqueous solution is 1:0.8-1.2.

[0012] In this invention, the PDCoV S protein in step (3) is labeled with HIS, and the concentration of the PDCoV S protein solution is 450-550 μg / mL.

[0013] In this invention, the solvent in step (3) is PBS buffer or physiological saline.

[0014] In this invention, the pH of the PBS buffer is 6.8-7.5.

[0015] In this invention, the flagellin is obtained by inserting the gene shown in SEQ ID NO: 2 into the pET32a vector, introducing it into Escherichia coli, inducing expression with IPTG, lysing the resulting bacterial cells, and purifying the supernatant using a nickel column.

[0016] In this invention, the PDCoV S protein is obtained by inserting the PDCoV S protein gene into the pET32a vector, introducing it into Escherichia coli, inducing expression with IPTG, lysing the resulting bacterial cells, and then purifying the supernatant using a nickel column.

[0017] Beneficial Effects: The porcine delta-coronavirus highly efficient subunit vaccine prepared by this invention simultaneously possesses the characteristics of immune enhancement, antigen purification, and immune targeting. This invention nanoparticles the immune enhancer and, using coating technology, coats its surface with a layer of silica. Simultaneously, it utilizes the principle of forming hydrogen bonds between silica and histidine (HIS) imidazole groups to purify the HIS-tagged antigen, displaying the antigen of the highly efficient porcine delta-coronavirus subunit vaccine on the enhancer surface. This achieves structural integration of the immune enhancer and antigen, fundamentally solving the key problem of poor immunogenicity in subunit vaccines, while simultaneously achieving highly efficient antigen purification. The porcine delta-coronavirus highly efficient subunit vaccine of this invention can efficiently stimulate the animal's immune response to the antigen, significantly increase antibody levels, and significantly prolong the antibody duration. Attached Figure Description

[0018] Figure 1 These are electron microscope images of an immune enhancer nanoparticle solution.

[0019] Figure 2 These are electron microscope images of siliconized immune enhancer nanoparticles.

[0020] Figure 3 1. SDS-PAGE electrophoresis images of purified silanized immunostimulant nanoparticle solution and PDCoV S protein solution at different volume ratios; M - protein standard marker, 1 - PDCoV S protein solution, 2 - supernatant after binding of silanized immunostimulant nanoparticle solution and PDCoV S protein solution at a volume ratio of 1:1, 3 - precipitation after binding of silanized immunostimulant nanoparticle solution and PDCoV S protein solution at a volume ratio of 1:1, 4 - supernatant after binding of silanized immunostimulant nanoparticle solution and PDCoV S protein solution at a volume ratio of 2:1, 5 - precipitation after binding of silanized immunostimulant nanoparticle solution and PDCoV S protein solution at a volume ratio of 2:1, 6 - supernatant after binding of silanized immunostimulant nanoparticle solution and PDCoV S protein solution at a volume ratio of 3:1, 7 - precipitation after binding of silanized immunostimulant nanoparticle solution and PDCoV S protein solution at a volume ratio of 3:1.

[0021] Figure 4 This is a schematic diagram showing the detection results of specific IgG antibodies in the serum of immunized mice.

[0022] Figure 5 This is a schematic diagram showing the detection results of neutralizing antibodies in the serum of immunized mice.

[0023] Figure 6 CD8 in the draining lymph nodes of immunized mice + A diagram illustrating the percentage of T cells. Detailed Implementation

[0024] The polylysine used in these embodiments of the invention has a weight-average molecular weight of 150,000 and was purchased from Sigma-Aldrich Shanghai Trading Co., Ltd. Tetramethoxysilane was purchased from Beijing Bailingwei Technology Co., Ltd.

[0025] Example 1

[0026] I. Vaccine Preparation

[0027] 1. Preparation of vaccine A

[0028] (1) Preparation of immune enhancer nanoparticles

[0029] A 0.5 mg / mL aqueous solution of polylysine and a 0.25 mg / mL aqueous solution of flagellin were mixed at a volume ratio of 1:1 and stirred continuously at 28 °C and 1000 rpm for 8 h to obtain an immunostimulant nanoparticle solution with an average particle size of 20 nm. Figure 1The nanoparticles exhibit good dispersibility and uniform size. After lyophilizing, the immunostimulant nanoparticles are obtained as lyophilized powder. 1 mg of the lyophilized powder is dissolved in 100 mL of deionized water to obtain a 0.01 mg / mL immunostimulant nanoparticle solution.

[0030] The amino acid sequence of flagellin is shown in SEQ ID NO: 1, and its encoding gene sequence is shown in SEQ ID NO: 2. Flagellin was obtained by inserting the gene shown in SEQ ID NO: 2 between the NdeⅠ and XhoⅠ restriction sites of the pET32a vector, introducing it into E. coli, inducing expression with IPTG, and purifying the supernatant after bacterial lysis using a nickel column.

[0031] (2) Preparation of silanized immune enhancer nanoparticles

[0032] Take 0.15g of methyl silicate (TMOS) and add it to 1mL of deionized water. After hydrolysis, silicic acid is obtained. Then, dilute with deionized water to obtain 9mg / mL of silicic acid.

[0033] 9 mg / mL of silicic acid was added to an equal volume of 0.01 mg / mL immunostimulant nanoparticle solution, and the reaction was allowed to proceed for 5 hours to obtain a silanized immunostimulant nanoparticle solution. The average particle size of these silanized immunostimulant nanoparticles was 70 nm. Electron microscopy observation showed the following results: Figure 2 It was discovered that the surface of the immune enhancer nanoparticles was coated with a layer of silica.

[0034] (3) Siliconized immune enhancer nanoparticles purify antigens

[0035] The silanized immune enhancer nanoparticle solution obtained in step (2) was mixed with 500 μg / mL PDCoV S protein solution at a volume ratio of 3:1. The PDCoV S protein with HIS tag was bound to the silanized immune enhancer nanoparticles. After centrifugation, the precipitate was collected and resuspended in PBS buffer (pH 7.4, concentration 0.01M) of equal volume to the PDCoV S protein solution to obtain vaccine A.

[0036] Depend on Figure 3 It is evident that when the silanized immune enhancer nanoparticle solution is mixed with a 500 μg / mL PDCoV S protein solution at a product ratio of 3:1, the PDCoV S protein is completely bound to the silanized immune enhancer nanoparticles.

[0037] The amino acid sequence of the PDCoV S protein is shown in SEQ ID NO:3, and the coding gene sequence is shown in SEQ ID NO:4. The PDCoV S protein was prepared as follows: the coding gene of the PDCoV S protein was inserted between the BamHI and XhoHI restriction sites in the pET32a vector, introduced into *E. coli*, and induced to express using IPTG. After lysis of the bacterial cells, the supernatant was purified using a nickel column. Because the pET32a vector carries the HIS tag coding sequence (CACCACCACCACCACCAC), the expressed and purified PDCoV S protein carries the HIS tag (HHHHHH).

[0038] In vaccine A, the concentration of PDCoV S protein is 500 μg / mL, and the concentration of immune enhancer nanoparticles is 0.015 mg / mL.

[0039] 2. Preparation of other vaccines

[0040] Vaccine B: 1 mL of an aqueous solution of PDCoV S protein at a concentration of 500 μg / mL (the solvent being 0.01 M, pH 7.4 PBS buffer) was mixed with 10.38 mg / mL of commercially available silica (silica, Beijing Deco Island Gold Technology Co., Ltd., product number JL-SiO2-S30) and 15 μg of the lyophilized nanoparticles of the immunostimulant prepared in Title 1(1) of this embodiment. The mixture was stirred until homogeneous to obtain vaccine B. In vaccine B, the concentration of silicon in the commercially available silica was the same as that in vaccine A.

[0041] Vaccine C: Mix 1 mL of an aqueous solution of PDCoV S protein at a concentration of 500 μg / mL (solvent is PBS buffer at pH 7.4, concentration of 0.01 M) with 10.38 mg / mL of commercial silica, and stir until homogeneous to obtain vaccine C.

[0042] Vaccine D: Mix 1 mL of PDCoV S protein aqueous solution with a concentration of 500 μg / mL (solvent is PBS buffer with pH 7.4 and a concentration of 0.01 M) with 15 μg of the lyophilized nanoparticles of the immune enhancer prepared in Title 1(1) of this embodiment, stir evenly, and obtain vaccine D.

[0043] Vaccine E (antigen control group): 500 μg / mL of PDCoV S protein in aqueous solution (solvent is 0.01M, pH 7.4 PBS buffer).

[0044] Vaccine F (blank control group): 0.01M PBS buffer at pH 7.4.

[0045] Example 2: Immunization Experiment and Efficacy of Highly Effective Subunit Vaccine Against Porcine δ-Coronavirus 1. Animal Grouping and Immunization

[0046] Sixty healthy ICR mice were randomly divided into 6 groups of 10 mice each. Each group of mice was immunized with vaccines A, B, C, D, E, and F via subcutaneous injection at a dose of 100 μL per mouse.

[0047] III. Evaluation of Vaccine Immunization Efficacy

[0048] 2. Specific antibody detection

[0049] Serum samples were collected at 14, 28, 42, 70, 84, 98, 120, 150, 180, and 210 days post-immunization. The IgG antibody levels in the serum were detected using an indirect ELISA method. PDCoV S protein (Example 1) was diluted to 100 ng / mL with coating buffer (0.848 g Na₂CO₃ and 1.428 g NaHCO₃ dissolved in deionized water and brought to a final volume of 500 mL). 100 μL / well was added to each well and incubated overnight at 4°C. The coating buffer was discarded, and the plate was washed three times with PBST (0.01 M, pH 7.4 PBS buffer containing 0.5% Tween-20) for 5 min each time, then patted dry. PBST containing 1% BSA was added to each well. 200 μL, block at 37℃ for 1 h; wash plate 3 times with PBST, 5 min each time, and pat dry; dilute the serum to be tested with PBST containing 1% BSA at a volume ratio of 1:800, 100 μL / well, incubate at 37℃ for 1 h; wash plate 3 times with PBST, 5 min each time, and pat dry; dilute HRP-labeled goat anti-mouse IgG (Beyotime Biotech Reagent, product number A0216) with PBST containing 1% BSA at a volume ratio of 1:5000, 100 μL / well, incubate at 37℃ for 1 h; wash plate 3 times with PBST, 5 min each time, and pat dry; add TMB chromogenic solution, react at 37℃ in the dark for 15 min; add 2M H2SO4 aqueous solution, 100 μL / well, to stop the reaction; place the microplate in a microplate reader and read the OD value at a wavelength of 450 nm.

[0050] The results of the detection of specific IgG antibodies in the serum of immunized mice are as follows: Figure 4As shown, an OD450nm ≥ 0.5 is considered positive. In vaccine group A mice, the level of specific IgG antibodies in serum was significantly higher than in all control groups 14 days post-immunization, continued to rise until 70 days post-immunization, then plateaued until 120 days post-immunization, and remained at a high level until the monitoring period of 210 days. In vaccine group B mice, the level of specific antibodies in serum reached its peak 28 days post-immunization, followed by a downward trend. In vaccine groups C and D mice, the level of specific antibodies in serum reached its peak 42 days post-immunization, followed by a downward trend. In vaccine group E (antigen control group) mice, the level of specific antibodies in serum reached its peak 42 days post-immunization, and remained at a low level throughout the entire duration. The level of specific IgG antibodies in mice immunized with vaccines other than vaccine A turned negative 70 days after immunization. The experimental results show that vaccine A can significantly increase antibody levels in mice immunized with PDCoV S protein, and the high antibody levels are maintained for up to 210 days after immunization, confirming that the siliconized immune enhancer nanoparticles can effectively improve the humoral immunity efficacy and duration of immunity.

[0051] 3. Neutralizing antibody detection

[0052] Serum was collected 28 days post-immunization and inactivated in a 56°C water bath for 30 min. The serum was then serially diluted with DMEM medium in a 2-fold gradient. The diluted serum was then mixed with a solution containing 200 TCID50. 50 PDCoV-NJ21 virus solution was mixed with DMEM medium at a 1:1 volume ratio and incubated at 37°C for 1 hour. The cell culture medium in the 96-well plate confluent with PK cells was discarded, and 100 μL of the serum-virus mixture was added to each well. DMEM medium served as a negative control, and the medium containing the virus solution served as a positive control. After 72 hours, the serum neutralizing titer was calculated using the Reed-Muench method.

[0053] Results of neutralizing antibody detection in immunized mouse serum as follows Figure 5 As shown, the serum neutralizing antibody level in mice treated with vaccine A was 1:588, in vaccine B it was 1:36, in vaccine C it was 1:5.6, in vaccine D it was 1:32, in vaccine E it was 1:4, and in vaccine F (the blank control group) it was 0. The neutralizing antibody level in vaccine A was significantly higher than that in the other vaccine control groups. These results indicate that mice vaccinated with a vaccine containing siliconized immune-enhancing nanoparticles exhibit a good neutralizing effect on PDCoV-infected PK cells.

[0054] 5. CD8 + T-cell detection

[0055] Seven days after immunization, inguinal lymph nodes were isolated from mice in each group, and lymphocyte suspensions were prepared. CD8+ cells were detected by flow cytometry (specific detection methods are disclosed in Hou L, Yu X, Zhang Y, et al. Enhanced Immune Responses in Mice Induced by the c-di-GMP Adjuvanted Inactivated Vaccine for Pseudorabies Virus. Front Immunol. 2022 Mar 31; 13:845680.). + T cell activation status.

[0056] Immunizing mice CD8 + The activation results of T cells are as follows: Figure 6 As shown, CD8 lymph nodes of mice in vaccine group A + The T-cell activation rate was significantly higher than that of other vaccine control groups, and the difference was highly significant. These results indicate that vaccines containing siliconized immune-enhancing nanoparticles can effectively activate the CTL response in mice, helping the body to rapidly exert a cellular immune response, thereby achieving an antiviral effect.

Claims

1. A highly effective subunit vaccine against porcine deltacoronavirus, characterized in that Prepared by the following method (1) Mix the polylysine aqueous solution with the flagellin aqueous solution, stir for 5 - 10 h, and freeze-dry to obtain the freeze-dried powder of immune enhancer nanoparticles; the amino acid sequence of the flagellin is as shown in SEQ ID NO: 1; (2) Add silicic acid to the aqueous solution of immune enhancer nanoparticles, react for 4 - 6 h to obtain a silicated immune enhancer nanoparticle solution; (3) Mix the silicated immune enhancer nanoparticle solution with the PDCoV antigen solution with HIS tag in a volume ratio of 2 - 4:1, centrifuge, take the precipitate, and suspend it with a solvent to obtain the vaccine; The PDCoV antigen is PDCoV S protein; in step (1), the concentration of the polylysine aqueous solution is 0.4 - 0.6 mg / mL, and the concentration of the flagellin aqueous solution is 0.15 - 0.3 mg / mL; the volume ratio of the polylysine aqueous solution to the flagellin aqueous solution is 1:0.8 - 1.2; in step (2), the concentration of silicic acid is 8 - 10 mg / mL, the concentration of the immune enhancer nanoparticle solution is 0.008 - 0.012 mg / mL, and the volume ratio of the silicic acid to the aqueous solution of immune enhancer nanoparticles is 1:0.8 - 1.2; in step (3), the PDCoV S protein has a HIS tag, and the concentration of the PDCoV S protein solution is 450 - 550 μg / mL.

2. The vaccine according to claim 1, wherein In step (3), the solvent is PBS buffer or physiological saline.

3. The vaccine according to claim 2, wherein The pH of the PBS buffer is 6.8 - 7.

5.

4. The vaccine according to claim 3, characterized in that The flagellin is obtained by inserting the gene shown in SEQ ID NO: 2 into the pET32a vector, introducing it into Escherichia coli, inducing expression with IPTG, lysing the obtained bacterial cells, and taking the supernatant for purification with a nickel column.

5. The vaccine according to claim 4, wherein The PDCoV S protein is obtained by inserting the gene of the PDCoV S protein into the pET32a vector, introducing it into Escherichia coli, inducing expression with IPTG, lysing the obtained bacterial cells, and taking the supernatant for purification with a nickel column.

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