Application of nano-selenium as inactivated virus vaccine adjuvant

Abstract

This invention discloses the application of nano-selenium as an adjuvant in inactivated virus vaccines. The application of nano-selenium as an adjuvant in this invention enhances the immunogenicity of inactivated viruses to prevent various viral infectious diseases in aquaculture. This invention utilizes biomineralization technology to grow nano-selenium in situ on the surface of inactivated virus antigens. The adjuvant effect of nano-selenium enables efficient presentation of inactivated virus antigens, improving the immunogenicity of the inactivated virus vaccine and inducing a strong and sustained cellular immune response. The nano-selenium adjuvant obtained by this invention uses inexpensive and readily available raw materials, and the synthesis and purification process is simple and controllable. By optimizing the production process and appropriately scaling up the synthesis, the commercial application of nano-selenium vaccine adjuvants can be realized.

Description

Technical Field

[0001] This invention belongs to the field of nano-selenium vaccines, and particularly relates to the application of inactivated virus vaccine adjuvants for the prevention of viral infections in livestock and poultry in the livestock industry. Background Technology

[0002] In the current livestock and poultry farming industry, viral infectious diseases have directly or indirectly caused huge economic losses to individuals and enterprises. Examples include classical swine fever virus (CSFV) and porcine epidemic diarrhea virus (PEDV) in pig farms, Newcastle disease virus (NDV) in chicken farms, bovine viral diarrhea virus (BVDV) in cattle farms, and sheep border disease virus (BDV) in sheep farms.

[0003] Currently, the main prevention and control measure for these viral infectious diseases is vaccination. Compared with traditionally used attenuated vaccines, inactivated virus vaccines, although having lower immunogenicity, are safer, and their immunogenicity can be enhanced by introducing vaccine adjuvants. As the most commonly used adjuvant, aluminum adjuvants can only enhance the immunogenicity of inactivated vaccines and induce potent antibody production, but they do not affect the triggering of CD8 cytotoxicity. + T-lymphocyte-mediated cellular immunity does not enhance the immune response, which is crucial for combating viral infections. Therefore, there is an urgent need to develop safe, cost-effective, and efficient inactivated virus vaccines to enhance cellular immunity and prevent various viral infectious diseases in aquaculture. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the primary objective of this invention is to provide a method for preparing an inactivated virus vaccine using nano-selenium as an adjuvant.

[0005] Another object of the present invention is to provide an inactivated virus vaccine using nano-selenium as an adjuvant.

[0006] Another object of the present invention is to provide the application of nano-selenium as an adjuvant for inactivated virus vaccines to prevent various viral infectious diseases in the aquaculture industry.

[0007] This invention provides a method for preparing an inactivated virus vaccine using nano-selenium as an adjuvant, comprising the following steps:

[0008] (1) Prepare stock solutions: Dissolve sodium selenite and vitamin C separately in autoclaved water to prepare sodium selenite stock solution and vitamin C stock solution respectively.

[0009] (2) To prepare a mixed solution, add the inactivated virus antigen to the sodium selenite stock solution and stir, then add autoclaved water and continue stirring to form the mixed solution.

[0010] (3) Add vitamin C stock solution dropwise to the mixed solution and stir at room temperature to fully biomineralize it to prepare a solution containing inactivated virus vaccine. Then, perform dialysis and filtration to obtain an inactivated virus vaccine with nano-selenium as an adjuvant.

[0011] Furthermore, the concentrations of the sodium selenite stock solution and the vitamin C stock solution mentioned in step (1) are both 50-400 mM.

[0012] Furthermore, the sodium selenite stock solution and vitamin C stock solution described in step (1) should be stored in a refrigerator at 2-6 degrees Celsius for use after preparation.

[0013] Furthermore, in the mixed solution described in step (2), the final concentration of sodium selenite reaction is 1-50 mM, preferably 1-30 mM; in the solution containing the inactivated virus vaccine described in step (3), the final concentration of vitamin C is 1-150 mM, preferably 1-100 mM.

[0014] Further, step (2) includes: adding 1-2 mg of inactivated viral antigen and 50-400 mM sodium selenite stock solution to 0.5-2 ml of autoclaved water, stirring at room temperature for 10-60 minutes, and then adding 0.5-2 ml of the stock solution of the nano selenium stabilizer and stirring for 1-3 hours to form the mixed solution.

[0015] Furthermore, the molar ratio of sodium selenite to vitamin C is 1:2-20, preferably 1:2-10; the mass ratio of selenium in sodium selenite to inactivated viral antigen is 1:1-30, preferably 1:1-20.

[0016] Furthermore, the dialysis process described in step (3) involves using a sterile dialysis bag and dialysis in autoclaved water at a pressure of 3000-8000 kDa for 12-36 hours.

[0017] The present invention also provides an inactivated virus vaccine, which is prepared by the above-described preparation method and is an inactivated virus vaccine using nano-selenium as an adjuvant.

[0018] This invention also provides an inactivated virus vaccine prepared by the above method using nano-selenium as an adjuvant, which has the application in the biomedical field. Specifically, it uses nano-selenium as an antigen carrier to enhance the immunogenicity of the inactivated virus antigen, and then enhances potent cellular immunity by regulating redox and selenoproteins, thereby achieving the purpose of preventing various viral infectious diseases in poultry and livestock in the breeding industry.

[0019] This invention offers the following advantages and effects compared to existing technologies: It utilizes a biomineralization technique to naturally grow nano-selenium on the surface of inactivated viral antigens for efficient presentation, enhancing the immunogenicity of inactivated viral vaccines and inducing a strong and sustained cellular immune response. The nano-selenium adjuvant obtained by this invention uses inexpensive and readily available raw materials, and its synthesis and purification processes are simple and controllable. The synthesis scale can be appropriately expanded by optimizing the production process. Furthermore, nano-selenium is a food-grade substance with good biosafety, offering better prospects for clinical translational applications. Attached Figure Description

[0020] Figure 1 This is a TEM morphology characterization image of the Se@PEDV nanovaccine in the embodiments of this application.

[0021] Figure 2 This is a hydration particle size diagram of the nanoparticles of the individual PEDV and Se@PEDV nanovaccines in the embodiments of this application.

[0022] Figure 3 This is a Zeta potential diagram of the nanoparticles of the individual PEDV and Se@PEDV nanovaccines in the embodiments of this application.

[0023] Figure 4 This illustrates the uptake of PEDV and Se@PEDV nanovaccines alone in mouse dendritic cells DC2.4 in the embodiments of this application.

[0024] Figure 5 This illustrates the localization of PEDV and Se@PEDV nanovaccines alone in mouse dendritic cells DC2.4 in the embodiments of this application.

[0025] Figure 6 This is a flow cytometry analysis of the maturation of mouse bone marrow-derived macrophages (BMDCs) induced by the Se@PEDV nanovaccine in the embodiments of this application.

[0026] Figure 7 The cellular immune markers in the spleen cells and serum of mice 7 days after the third immunization with the Se@PEDV nanovaccine in this application embodiment are as follows: (a) phagocytosis by macrophages, (b) NK, (c) NKT, and (d) CD8. + Changes in T lymphocyte expression, serum (e) changes in TNF-α levels, and (f) the ratio of antibody titers IgG2a and IgG1.

[0027] Figure 8 The expression level of the inflammatory factor IFN-γ in porcine serum 7 days after the third dose of the Se@PEDV nanovaccine in this embodiment of the application.

[0028] Figure 9This study presents a semi-quantitative fluorescence analysis of immune cells in the spleen of pigs 7 days after the third dose of the Se@PEDV nanovaccine administered in this application embodiment. Figure 9 af represent NK cells, macrophages, γδT cells, and CD4, respectively. + T cells, CD8 + Expression of T cells and activated B cells (plasma cells).

[0029] Figure 10 This is a semi-quantitative fluorescence analysis of immune cells in the mesenteric lymph nodes of pigs 7 days after the third dose of the Se@PEDV nanovaccine in this application embodiment. Figure 10 af represent NK cells, macrophages, γδT cells, and CD4, respectively. + T cells, CD8 + Expression of T cells and activated B cells (plasma cells).

[0030] Figure 11 High-resolution digital image of the particle size of the Se@PEDV nanovaccine. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0032] Example 1: The application of the porcine epidemic diarrhea virus vaccine with nano-selenium as an adjuvant provided by the present invention includes the following steps:

[0033] (1) Preparation of sodium selenite stock solution and vitamin C stock solution: Weigh 138 mg of sodium selenite (Na2SeO3) and 140 mg of vitamin C (Vc) and dissolve them in 2 mL of autoclaved water to prepare sodium selenite stock solution and vitamin C stock solution with a concentration of 400 mM respectively, and store them in a refrigerator at 5℃ for later use.

[0034] (2) Preparation of mixed solution: Take 1 mg of porcine epidemic diarrhea virus inactivated antigen PEDV and add it to 1.835 mL of autoclaved water and mix well. Then add 33 μL of sodium selenite stock solution. Stir the reaction flask magnetically at room temperature for 30 minutes.

[0035] (3) Preparation of Se@PEDV nanovaccine: Then, 132 μL of vitamin C stock solution was added dropwise to the above mixture and stirred at room temperature for 2 hours to allow it to be fully biomineralized, thus obtaining a solution containing Se@PEDV nanovaccine.

[0036] (4) Dialysis: Place the reacted solution in a sterile dialysis bag with a molecular weight of 6000 kDa and dialyze in autoclaved water for 12 hours. The autoclaved water needs to be changed 3-5 times during the dialysis process to fully remove unreacted sodium selenite and vitamin C.

[0037] (5) Storage: Finally, collect the Se@PEDV nano vaccine in centrifuge tubes, seal them, and store them in a refrigerator at 5°C for later use.

[0038] (6) Detection: The hydrated particle size was measured and photographed using a Malvern laser particle size analyzer. After the Se@PEDV nanovaccine was digested with aqua regia according to the national standard method (GB5009.93-2017), the selenium content was determined by inductively coupled plasma mass spectrometry (ICP-MS), and the protein content of PEDV was determined by BCA protein concentration assay kit (Beyotime Biotechnology, P0010S).

[0039] (7) Experimental results: Figure 11 The results showed that the hydrated particle size of the synthesized Se@PEDV nanovaccine was approximately 255 nm. The prepared Se@PEDV nanovaccine was a clear, transparent red liquid. These results indicate that PEDV interacts with nano-selenium, and this example successfully prepared an inactivated porcine epidemic diarrhea virus vaccine using nano-selenium as an adjuvant.

[0040] Example 2: The application of the porcine epidemic diarrhea virus vaccine with nano-selenium as an adjuvant provided by the present invention includes the following steps:

[0041] (1) Preparation of sodium selenite stock solution and vitamin C stock solution: Weigh 56.2 mg of sodium selenite (Na2SeO3) and 57.24 mg of vitamin C (Vc) and dissolve them in 2 mL of autoclaved water to prepare sodium selenite stock solution and vitamin C stock solution with a concentration of 160 mM respectively, and store them in a refrigerator at 4℃ for later use.

[0042] (2) Preparation of mixed solution: Take 1 mg of porcine epidemic diarrhea virus inactivated antigen PEDV and add it to 1.835 mL of autoclaved water and mix well. Then add 33 μL of sodium selenite stock solution. Stir the reaction flask magnetically at room temperature for 30 minutes.

[0043] (3) Preparation of Se@PEDV nanovaccine: Then, 132 μL of vitamin C stock solution was added dropwise to the above mixture and stirred at room temperature for 2 hours to allow it to be fully biomineralized, thus obtaining a solution containing Se@PEDV nanovaccine.

[0044] (4) Dialysis: Place the reacted solution in a sterile dialysis bag with a molecular weight of 5000 kDa and dialyze in autoclaved water for 12 hours. The autoclaved water needs to be changed 3-5 times during the dialysis process to fully remove unreacted sodium selenite and vitamin C.

[0045] (5) Storage: Finally, collect the Se@PEDV nano vaccine in centrifuge tubes, seal them, and store them in a refrigerator at 4°C for later use.

[0046] (6) Detection: The hydrated particle size was measured and photographed using a Malvern laser particle size analyzer. After the Se@PEDV nanovaccine was digested with aqua regia according to the national standard method (GB5009.93-2017), the selenium content was determined by inductively coupled plasma mass spectrometry (ICP-MS), and the protein content of PEDV was determined by BCA protein concentration assay kit (Beyotime Biotechnology, P0010S).

[0047] (7) Comparison of experimental results

[0048] (i) Grouping of experimental animals

[0049] Twenty female C57BL / 6 mice were divided into four groups: saline group, PEDV antigen group, Se@PEDV vaccine group, and aluminum adjuvant (aluminum adjuvant + PEDV) group, with five mice in each group.

[0050] (ii) Administration

[0051] After the drug was autoclaved and dispersed in water, 150 microliters of the drug were administered to each mouse via intradermal injection.

[0052] (iii) Experiment Content

[0053] Each mouse was administered the vaccine at a dose of 75 μg PEDV and 30 μg nano-selenium. Dosage was repeated every two weeks for a total of three administrations. Seven days after the third vaccine injection, serum was collected from the mice via ocular sampling. After the blood samples were allowed to stand at room temperature for 1.5 hours, they were centrifuged at 3000 rpm and 4°C for 10 minutes. The collected serum was transferred to 500 μL small EP tubes and stored at -80°C.

[0054] PEDV was plated at a rate of 10 μg / mL, 100 μL / well in 96-well plates and incubated overnight at 4°C. The next day, the 96-well plates were removed, the supernatant was discarded, and the plates were blotted dry. The plates were then washed with PBST (PBS containing 0.5% Tween 20) for 5 minutes at a time, for a total of 4 washes. The plates were then blocked with 1% BSA solution into each well and incubated with shaking at room temperature for 2 hours. After washing the plates three times with PBST, 100 μL of serially diluted serum samples (e.g., 100-, 200-, 400-, 800-, 1600-, 3200-, 6400-, 12800-, 25600-, 51200-) were added to each well and incubated at room temperature for 2 hours. Then, wash the plate three times with PBST, add 100 μL of 10,000-fold diluted HRP-conjugated goat anti-mouse IgG1 or IgG2a antibody (Abcam, Cambridge, USA) to each well, and incubate at room temperature with shaking for 1 hour. After washing the plate three times with PBS-T, add 100 μL of freshly mixed TMB substrate solution (BD, San Diego, USA) to each well and incubate in the dark for 10 min. Finally, terminate the reaction with 1% NH2SO4 solution, record the absorbance at 450 nm, and detect the PEDV-specific antibody titer using a microplate reader.

[0055] (iv) Experimental Results

[0056] Figure 1-3 The results showed that the hydrated particle size of PEDV alone was 116.94 nm. After biomineralization, the hydrated particle size of the Se@PEDV nanovaccine increased from 116.9 nm to 266.4 nm, and the surface charge decreased from -12.1 mV to -31.2 mV after PEDV modification. The hydrated particle size of the prepared Se@PEDV nanovaccine was consistent with the TEM electron microscopy results. These results indicate that PEDV interacts with nano-selenium, and this example successfully prepared an inactivated porcine epidemic diarrhea virus vaccine using nano-selenium as an adjuvant.

[0057] To further verify the activity of the Se@PEDV nanovaccine, its immunogenicity was monitored in C57BL / 6 mice via intradermal injection. Figure 7 The results showed that, compared with the PEDV antigen group alone, the Se@PEDV nanovaccine enhanced the phagocytic activity of macrophages and upregulated the levels of immune cells NK, NKT, and CD8. + The expression of T lymphocytes and the level of TNF-α in serum. Meanwhile, such as... Figure 7 As shown in f, the ratio of IgG2a to IgG1 compared to the commercial aluminum adjuvant group confirms that the Se@PEDV nanovaccine can trigger a unique Th1-biased cellular immunity.

[0058] Compared to other nanomaterials used as vaccine adjuvants, such as aluminum adjuvants, nano-selenium provides adjuvant activity by enhancing antigen delivery to the immune system and by enhancing innate and adaptive immune responses. Simultaneously, due to its affinity for selenate (Se... 4+ ) or selenite (Se 2+ Compared to nano-sized selenium (Se), 0 Selenium nanoparticles (SeNPs) exhibit low toxicity, making them a strong candidate to replace other forms of selenium in nutritional supplements or pharmaceutical formulations. More importantly, SeNPs can be converted into selenoproteins in the body, some of which are involved in cell activation and differentiation and are important for innate and adaptive immune responses.

[0059] Studies have shown that selenium supplementation plays a vital role in livestock health and immune system function. Selenium supplementation can effectively enhance humoral immunity after influenza vaccination by inhibiting ferroptosis of follicular helper T (TFH) lymphocytes. Previous research by the inventors revealed that functional selenium nanoparticles (SeNPs) can effectively enhance the cytotoxicity of cytokine-induced killer cells against tumor cells by regulating selenoproteins in CIK cells and tumor cells, and promote the elimination of tumor cells by adaptive immune cells such as γδT cells. This evidence suggests that selenium nanoparticles with redox regulatory and immune-enhancing properties are ideal adjuvants for developing vaccines against inactivated viruses.

[0060] Currently, there are no literature reports on the application of nano-selenium in the field of inactivated virus vaccines. Taking porcine epidemic diarrhea virus (PEDV) as an example, the inventors previously discovered that using nano-selenium as an adjuvant for inactivated PEDV virus can significantly enhance the immunogenicity of inactivated PEDV virus alone. Simultaneously, selenium can be metabolized into selenoproteins in the body, maintaining redox levels and inducing a strong and sustained cellular immune response. The inventors' research found that compared to the PEDV antigen group alone, the Se@PEDV vaccine group can promote higher maturation of mouse bone marrow-derived macrophages (BMDCs) and induce higher IgG1 and IgG2a antibody titers in mice. Furthermore, in the spleen and mesenteric lymph nodes of pigs, the Se@PEDV vaccine group can upregulate various immune cells (such as NK cells, macrophages, γδT cells, CD4+). + T cells, CD8 + Expression of T cells and activated B cells.

[0061] Example 3: Nano-selenium as an adjuvant for porcine epidemic diarrhea virus inactivated vaccine promotes DC2.4 cell uptake and cellular localization.

[0062] PEDV was first labeled with N-hydroxysuccinimide-terminated fluorescein isothiocyanate FITC-NHS. Simultaneously, FITC-labeled inactivated PEDV (PEDV-FITC) was used to synthesize a fluorescently labeled Se@PEDV nanovaccine (Se@PEDV-FITC). DC2.4 cells were then treated with PBS, PEDV-FITC, and the Se@PEDV-FITC nanovaccine for 6 hours, with the latter two groups having a working PEDV concentration of 10 μg / mL. Subsequently, DC2.4 cells from different treatments were collected, and flow cytometry and FlowJo software were used to analyze and determine cellular uptake. Figure 4 As shown, DC2.4 cells uptake the Se@PEDV nanovaccine significantly higher than the PEDV antigen group alone, demonstrating that the nano-selenium adjuvant can enhance the uptake of PEDV antigen.

[0063] For cell localization, DC2.4 cells from different treatment groups were washed three times with PBS buffer, then incubated with the lysosomal probe LysoTracker RedDND-99 and the nuclear dye Hoechst 33342. Finally, the cells were observed and photographed using a confocal laser scanning microscope. Figure 5 As shown in the fluorescence images, DC2.4 cells exhibited significantly higher uptake of the Se@PEDV nanovaccine compared to the PEDV antigen group alone, and the vaccine was localized in lysosomes, similar to... Figure 4 The results were consistent.

[0064] Example 4: Study on the induction of BMDC cell maturation using nano-selenium as an adjuvant for porcine epidemic diarrhea virus inactivated vaccine.

[0065] First, bone marrow-derived dendritic cells (BMDCs) were obtained by culturing monodisperse cells from the femoral and tibial bone marrow of female C57BL / 6 mice (6-8 weeks old) in RPMI1640 medium containing 10% fetal bovine serum, granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng / mL) and β-mercaptoethanol (5 ng / mL) for 6-8 days.

[0066] BMDC cells were then treated for 12 hours with PEDV (10 μg / mL), Se@PEDV nanovaccine (10 μg / mL PEDV), or lipopolysaccharide (LPS, 1 μg / mL, as a positive control). Afterwards, cells were stained with fluorescently labeled antibodies (including CD11c-FITC, CD86-APC-Cy7, and CD80-PE) to assess BMDC maturity, and analysis was performed using flow cytometry. Figure 6As shown, compared with the untreated group (i.e., control), the inactivated PEDV antigen group alone did not show significant activation of BMDC maturation (CD80 and CD86 are typical markers of maturation), indicating its low immunogenicity. However, the Se@PEDV nanovaccine can efficiently induce BMDC maturation.

[0067] Example 5: Regulation of the immunogenicity of porcine epidemic diarrhea virus inactivated vaccine with nano-selenium as adjuvant provided by the present invention on pigs.

[0068] (1) Experimental Grouping

[0069] To evaluate the immunogenicity of the developed nanovaccine in pigs, nine sows (Landrace, 30 kg) were divided into three groups: a saline group (control group), a Se@PEDV vaccine group, and a commercial aluminum adjuvant (aluminum adjuvant + PEDV) group.

[0070] (2) Administration method

[0071] The vaccine was administered intramuscularly, once every two weeks, for a total of three injections. In the control group of Se@PEDV vaccine and the commercial aluminum adjuvant (aluminum adjuvant + PEDV) group, the PEDV dose was 1 mg / animal and the selenium content dose was 0.4 mg / animal.

[0072] (3) Experimental Content

[0073] Seven days after the final immunization, immunized pigs were sacrificed, and spleens, mesenteric lymph nodes, and serum were collected for subsequent immunological analysis. Specifically, immunofluorescence staining was used to determine the effects of the Se@PEDV nanovaccine on NK cells, macrophages, γδT cells, and CD4+ in the pig spleen and mesenteric lymph nodes. + T cells, CD8 + The effects of T cells and activated B cells (plasma cells) were investigated. Simultaneously, IFN-γ levels in the serum of immunized pigs were measured using ELISA.

[0074] like Figure 8 As shown, compared with commercial aluminum adjuvant group vaccination, Se@PEDV nanovaccine can effectively increase the level of IFN-γ in the serum of immunized pigs. Figure 9-10 The results showed that the Se@PEDV nanovaccine could indeed induce more innate immune-related immune cells (including NK cells and macrophages) and cell-mediated immune-related cells (including γδT cells and CD4 cells) in the spleen and mesenteric lymph nodes. + and CD8 + Proliferation of T cells. As for humoral immunity, the number of plasma cells in the spleen or mesenteric lymph nodes of pigs immunized with the Se@PEDV nanovaccine was comparable to that of commercially available aluminum adjuvant vaccines.

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an inactivated virus vaccine with nano-selenium as an adjuvant, characterized in that, Includes the following steps: (1) Prepare stock solutions: Dissolve sodium selenite and vitamin C separately in autoclaved water to prepare sodium selenite stock solution and vitamin C stock solution respectively; (2) To prepare a mixed solution, add the inactivated virus antigen to the sodium selenite stock solution and stir, then add autoclaved water and continue stirring to form the mixed solution; (3) Add vitamin C stock solution dropwise to the mixed solution and stir at room temperature to fully biomineralize it to prepare a solution containing inactivated virus vaccine. Then, perform dialysis and filtration to obtain an inactivated virus vaccine with nano selenium as an adjuvant. The inactivated virus antigen is a porcine epidemic diarrhea virus inactivated antigen.

2. The method of claim 1, wherein: The sodium selenite and vitamin C are in a molar ratio of 1:2-10, and the mass ratio of selenium in the sodium selenite to the inactivated viral antigen is 1:1-20.

3. The method of claim 1, wherein: The concentrations of sodium selenite stock solution and vitamin C stock solution mentioned in step (1) are both 50-400 mM.

4. The method of claim 1, wherein: In step (2), the final concentration of sodium selenite in the mixed solution is 1-50 mM; in step (3), the final concentration of vitamin C in the solution containing the inactivated virus vaccine is 1-150 mM.

5. The method of claim 1, wherein: The dialysis process described in step (3) involves using a sterile dialysis bag and dialysis in autoclaved water at a pressure of 3000-8000 kDa for 12-36 hours.

6. An inactivated virus vaccine using nano-selenium as an adjuvant, characterized in that, The inactivated virus vaccine prepared according to any one of claims 2-5 is used to prevent porcine epidemic diarrhea virus.

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