PEDV (porcine epidemic diarrhea virus) subunit vaccine as well as preparation method and application thereof

Abstract

The invention discloses a PEDV (porcine epidemic diarrhea virus) subunit vaccine, a preparation method and application, and belongs to the technical field of preparation of PEDV vaccines.The PEDV subunit vaccine is prepared by mixing and emulsifying PEDVS recombinant protein and MONTANIDEISA201VG adjuvant, the vaccine can effectively prevent PEDV viruses and reduce the morbidity, and the challenge protection rate is 100%.

Description

Technical Field

[0001] This invention relates to the field of PEDV vaccine preparation technology, and more specifically to a PEDV subunit vaccine, its preparation method, and its application. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is the main pathogen of porcine epidemic diarrhea (PED), primarily causing intestinal-mediated infectious diseases in pigs, characterized by vomiting, diarrhea, and dehydration. PEDV belongs to the order Nidovirales, family Coronaviridae, and genus Coronavirus. It is a single-stranded, positive-sense infectious RNA virus with only one serotype. It was first discovered in 1971 in UK finishing pig farms, significantly impacting pre-weaning piglets aged 4-5 weeks. Subsequently, it spread to many countries in Europe and Asia. An outbreak began in North America in 2013, and PEDV became a globally prevalent virus, severely damaging the global livestock industry.

[0003] In recent years, the continuous mutation of circulating PEDV strains has rendered existing vaccines insufficient to provide adequate protection. Furthermore, the increasing number of porcine infectious diseases that can co-infect PEDV has further complicated the epidemic, making prevention and treatment more difficult. Therefore, efficient and accurate diagnosis of PEDV is crucial for the effective implementation of disease control measures. These measures help reduce the risk of further virus transmission and buy valuable time for subsequent treatment.

[0004] Therefore, how to provide a subunit vaccine with good immunogenicity that can prevent multiple PEDV variants is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a PEDV subunit vaccine, a preparation method thereof, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A PEDV subunit vaccine comprising: PEDVS recombinant protein and

[0008] MONTANIDEISA201VG adjuvant.

[0009] As an inventive concept with the same technical solution as above, this invention also claims protection for a method for preparing a PEDV subunit vaccine, the process of which is as follows: using MONTANIDEISA201VG as an adjuvant, using purified PEDVS recombinant protein as an aqueous phase, emulsifying according to the method in the instruction manual to prepare a PEDV subunit vaccine.

[0010] As having the same inventive concept as the above-described technical solution, this invention also seeks protection for the use of the described PEDV subunit vaccine in the prevention of PEDV disease.

[0011] As can be seen from the above technical solution, compared with the prior art, this invention selects the PEDVS57-280 protein fragment for protein expression and establishes an ELISA method, which can be used for the clinical detection of PEDV serum antibodies. Simultaneously, the PEDV subunit vaccine prepared using this protein as the antigen protein exhibits good immunogenicity in immunized mice and piglets. Specific antibody level detection, T lymphocyte subtype analysis, lymphocyte proliferation assay, and cytokine detection results all show that this vaccine can stimulate the body to produce a high level of immune response and high levels of specific S protein antibodies. The results of the swine immunoprotective experiment show that this vaccine can provide good immunoprotection against PEDV variant strains, with a challenge protection rate of 100%. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0013] Figure 1 The attached figure shows the results of serum-specific antibody detection in mice immunized with PEDV subunit vaccine.

[0014] Figure 2 The attached figure shows CD4+ levels in peripheral blood of mice immunized with PEDV subunit vaccine 28 days later. 3+ CD 4+ Lymphocyte subset detection results; A: 28-day control; B: 28-day adjuvant; C: 28-day S protein;

[0015] Figure 3 The attached figure shows CD4+ levels in the peripheral blood of mice immunized with PEDV subunit vaccine. 3+ CD 4+ Image showing the results of lymphocyte subset detection;

[0016] Figure 4 The attached figure shows CD4+ levels in peripheral blood of mice immunized with PEDV subunit vaccine 28 days later. 3+ CD 8+ Lymphocyte subset detection results; A: 28-day control, B: 28-day adjuvant, C: 28-day S protein;

[0017] Figure 5 The attached figure shows CD4+ levels in the peripheral blood of mice immunized with PEDV subunit vaccine. 3+CD 8+ Image showing the results of lymphocyte subset detection;

[0018] Figure 6 The attached image shows a graph of serum-specific antibodies detected in piglets immunized with PEDV subunit vaccine.

[0019] Figure 7 The attached figure shows CD4+ levels in the peripheral blood of piglets immunized with PEDV subunit vaccine on day 36. 3+ CD 4+ Lymphocyte subset detection results; A: 36-day control; B: 36-day adjuvant; C: 36-day S protein; D: 36-day commercial vaccine;

[0020] Figure 8 The attached figure shows CD4+ levels in the peripheral blood of piglets immunized with PEDV subunit vaccine. 3+ CD 4+ Lymphocyte subset detection results;

[0021] Figure 9 The attached figure shows CD4+ levels in the peripheral blood of piglets immunized with PEDV subunit vaccine on day 36. 3+ CD 8+ Lymphocyte subset detection results; A: 36-day control; B: 36-day adjuvant; C: 36-day S protein; D: 36-day commercial vaccine;

[0022] Figure 10 The attached figure shows CD4+ levels in the peripheral blood of piglets immunized with PEDV subunit vaccine. 3+ CD 8+ Lymphocyte subset detection results;

[0023] Figure 11 The attached figure shows a peripheral blood T lymphocyte proliferation assay in piglets immunized with PEDV subunit vaccine.

[0024] Figure 12 The attached figure shows a peripheral blood B lymphocyte proliferation assay in piglets immunized with PEDV subunit vaccine.

[0025] Figure 13 The attached figure shows the IL-2 level in the serum of piglets immunized with PEDV subunit vaccine;

[0026] Figure 14 The attached figure shows the IL-4 level in the serum of piglets immunized with PEDV subunit vaccine;

[0027] Figure 15 The attached figure shows the IL-10 level in the serum of piglets immunized with PEDV subunit vaccine;

[0028] Figure 16 The attached figure shows the TNF-α level in the serum of piglets immunized with PEDV subunit vaccine;

[0029] Figure 17 The attached figure shows the IFN-γ level in the serum of piglets immunized with PEDV subunit vaccine;

[0030] Figure 18 The attached image shows the changes in body temperature of piglets after viral challenge;

[0031] Figure 19 The attached figure shows the weight changes of piglets after viral challenge. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1: Preparation of PEDV subunit vaccine

[0033] Using MONTANIDEISA201VG as adjuvant and purified PEDVS recombinant protein as the aqueous phase, the prepared subunit vaccine was emulsified according to the manufacturer's instructions, resulting in a milky white, milky liquid appearance. The finished subunit vaccine was an oil-in-water formulation; centrifugation at 3000 rpm for 15 min showed no stratification, indicating formulation stability. A small amount of vaccine dropped onto a clean, cold water surface diffused in a cloud-like manner, meeting the required standards. Sterility testing, conducted according to the current Chinese Veterinary Pharmacopoeia, showed no bacterial growth.

[0034] Example 2: Evaluation of the immunogenicity of PEDV subunit vaccine in mice

[0035] Thirty 6-week-old SPF-grade female BALB / c mice were randomly divided into three groups of 10 mice each. Group A received an intraperitoneal injection of 25 μg / mouse of the S protein subunit vaccine, Group B received an intraperitoneal injection of 0.1 mL / mouse of adjuvant, and Group C received an intraperitoneal injection of 0.1 mL / mouse of DMEM. A booster immunization was administered 21 days after the initial immunization using the same method. The mice were observed daily, and their feed, water, and bedding were changed regularly. Blood samples were collected from the mice at 0, 14, 21, and 28 days for specific antibody levels and T lymphocyte subtype analysis.

[0036] 1) Specific antibody levels in immunized mice

[0037] PEDV antibody levels in mouse serum collected at 0d, 14d, 21d, and 28d were detected using a self-developed indirect ELISA method. The results showed that, compared with the control group and the adjuvant group, the serum antibody level in the S protein group was significantly higher; compared with 21d, the serum antibody level in the S protein group was higher at 28d. Figure 1 The results showed that the PEDV subunit vaccine could stimulate the body to produce specific antibodies.

[0038] 2) Subtype analysis of T lymphocytes in immunized mice

[0039] CD4+ levels in the peripheral blood of immunized mice were analyzed by flow cytometry at 14, 21, and 28 days. 3+ CD 4+ and CD 3+ CD 8+ Lymphocyte subsets were analyzed, and the results showed that CD4+ levels in the peripheral blood of mice immunized with the S protein group were significantly lower from day 14 to day 28. 3+ CD 4+ Lymphocyte levels were significantly increased; at 21 days post-immunization, the S protein level was significantly higher in the group than in the control and adjuvant groups; at 28 days post-immunization, the S protein level was significantly higher in the group than in the control and adjuvant groups, but the differences were not statistically significant. Figure 2 , Figure 3 CD 3+ CD 8+ Lymphocyte counts showed that, compared to 14 days and 21 days, the S proteome CD45% was significantly lower at 28 days. 3+ CD 8+ Lymphocyte levels were significantly increased and significantly higher than in the control and adjuvant groups. Figure 4 , Figure 5 ).

[0040] Example 3: Evaluation of the immunization effect of PEDVS protein subunit vaccine on piglets

[0041] Twenty healthy 10-day-old piglets were randomly divided into four groups of five each. Group A received an intramuscular injection of 60 μg / pig of the S protein subunit vaccine via the neck; Group B received an intramuscular injection of 2 mL / pig of a commercial vaccine via the neck; Group C received an intramuscular injection of 1 mL / pig of adjuvant via the neck; and Group D received an intramuscular injection of 1 mL / pig of DMEM via the neck. A booster immunization was administered 21 days after the initial immunization using the same method. The piglets were observed daily, fed regularly with feed and water, and blood samples were collected at 0, 14, 28, and 36 days for specific antibody level testing, T lymphocyte subtype analysis, lymphocyte proliferation assay, and cytokine detection. An immune challenge protection test was conducted 14 days after the second immunization. The challenge strain was the PEDV variant strain FJLY01-2021 (virus titer of 105.23 TCID50 / mL, isolated, identified and preserved by the Fujian Provincial Key Laboratory of Livestock Infectious Disease Prevention and Biotechnology). The challenge method was oral administration, and the challenge dose was 3 mL / head. Clinical symptoms and mortality were observed and recorded daily after challenge, and the observation period was 7 days.

[0042] 1) Specific antibody levels in immunized piglets

[0043] Blood samples were collected from piglets at 0, 14, 28, and 36 days post-immunization to separate serum. Serum-specific antibody levels in piglet serum were detected using a self-developed indirect ELISA. Results showed that serum antibody levels in the S protein group were significantly higher than those in the control and adjuvant groups. At 36 days post-immunization, the S protein group antibody level was slightly higher than that in the commercial vaccine group. Figure 6 ).

[0044] 2) Subtype analysis of T lymphocytes in immunized piglets

[0045] CD42 analysis was performed on blood samples collected from immunized piglets at 0, 14, 28, and 36 days using flow cytometry. 3+ CD 4+ and CD 3+ CD 8+ Lymphocyte subset detection. Results showed that 14-28 days post-immunization, S proteome CD40 levels were significantly elevated. 3+ CD 4+ Lymphocyte levels were not significantly different between the control group, adjuvant group, and commercial vaccine group; CD40 levels in the S protein group at 36 days were not significantly different. 3+ CD 4+ Lymphocyte levels were significantly higher than in the control group, adjuvant group, and commercial vaccine group. Figure 7 , Figure 8 CD 3+ CD 8+ Lymphocyte subset analysis results showed that, compared with 28 days, the S protein group of immunized piglets at 36 days had significantly higher CD4 counts. 3+ CD 8+ Lymphocyte levels were significantly elevated and significantly higher than those in the control group, adjuvant group, and commercial vaccine group. Figure 9 , Figure 10 The results above show that the PE DV subunit vaccine can effectively stimulate T lymphocyte activation.

[0046] 3) Lymphocyte proliferation experiment in immunized piglets

[0047] Lymphocyte proliferation experiments were performed on blood samples collected from immunized piglets at 0, 14, 28, and 36 days. The results showed that the T lymphocyte stimulation index of piglets immunized with the S protein group was significantly higher than that of the control group, adjuvant group, and commercial vaccine group from 28 to 36 days. Figure 11 The B lymphocyte proliferation assay showed that, between 28 and 36 days, the B lymphocyte stimulation index in piglets immunized with the S protein group was significantly higher than that in the adjuvant group, but there was no significant difference compared to the commercial vaccine group. Figure 12 This indicates that the PEDV subunit vaccine can enhance the body's cellular immune response.

[0048] 4) Detection results of relevant cytokines in the serum of immunized piglets

[0049] The levels of the cytokine IL-2 in blood collected from immunized piglets at different time points were detected using a porcine interleukin-2 (IL-2) quantitative detection kit. The results showed that the IL-2 level in the S protein group consistently increased after immunization, and was significantly higher in the S protein group than in the control and adjuvant groups, but showed no significant difference compared to the commercial vaccine group. Figure 13 ).

[0050] The levels of the cytokine IL-4 in collected blood samples were measured at different time points using a porcine interleukin-4 (IL-4) quantitative detection kit. The results showed that after immunization, the IL-4 level in the S protein group was significantly higher than that in the control group and the adjuvant group, while there was no significant difference compared to the commercial vaccine group. Figure 14 ).

[0051] The levels of the cytokine IL-10 in immunized piglets were detected using a porcine interleukin-10 (IL-10) quantitative detection kit. Results showed that, 28-36 days post-immunization, the IL-10 level in piglets immunized with the S protein group was higher than that in the control group, adjuvant group, and commercial vaccine group. Figure 15 ).

[0052] The levels of the cytokine TNF-α in blood samples collected from piglets at 0, 14, 28, and 36 days post-immunization were detected using a porcine interferon-alpha (TNF-α) quantitative detection kit. Results showed that TNF-α levels in the S protein group gradually decreased after immunization; TNF-α levels in piglets immunized with the S protein group were lower than those in the control and adjuvant groups; compared to 14-28 days, the TNF-α level in the S protein group at 36 days was lower than that in the commercial vaccine group. Figure 16 ).

[0053] The levels of the cytokine IFN-γ were detected using a porcine interferon-gamma (IFN-γ) quantitative detection kit. Results showed that 28-36 days post-immunization, the IFN-γ level in the S protein group was higher than that in the control group, adjuvant group, and commercial vaccine group. Figure 17 ).

[0054] The above results indicate that the PEDV subunit vaccine can induce the secretion of TH1 and TH2 cytokines while inhibiting the secretion of tumor necrosis factor, thereby enhancing the body's cellular and humoral immune response.

[0055] (3) Virus challenge protection test in immunized piglets

[0056] On the second day after challenge, the piglets in all experimental groups were in good spirits, with a slight decrease in appetite. One piglet in each of the control and adjuvant groups developed loose stools. On the third day after challenge, three piglets in the control group and two piglets in the adjuvant group developed loose stools. The piglets in the commercial vaccine group and the S protein immunization group were in relatively normal spirits and did not develop loose stools or watery diarrhea. By the end of the experiment, the body temperature of all piglets in all experimental groups was normal. Figure 18 , Figure 19 After the experiment, pathological necropsy was performed on piglets in each group. Intestinal tympany was observed in the small intestine of piglets in the control group and adjuvant group, while the intestines of piglets in the commercial vaccine immunization group and S protein immunization group were normal. The results indicate that the vaccine provides good immunoprotection against the PEDV variant strain, with a 100% protection rate against challenge.

[0057] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0058] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A PEDV subunit vaccine, characterized in that, include: PEDVS recombinant protein and MONTANIDEISA201VG adjuvant.

2. The method for preparing a PEDV subunit vaccine according to claim 1, characterized in that, The process was as follows: using MONTANIDEISA201VG as an adjuvant and purified PEDVS recombinant protein as the aqueous phase, the PEDV subunit vaccine was prepared by emulsification according to the instructions.

3. The use of the PEDV subunit vaccine according to claim 1 in the prevention of PEDV disease.

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