Use of a type 2 streptococcus suis membrane vesicle in the preparation of a vaccine and / or an immunopotentiator

Abstract

This invention belongs to the field of vaccine preparation technology, and discloses the application of Streptococcus suis type 2 membrane vesicles in the preparation of vaccines and / or immune enhancers. This invention is the first to use Streptococcus suis type 2 membrane vesicles as a vaccine for animal immunization. Animal experiments have demonstrated that the Streptococcus suis type 2 membrane vesicles of this invention can induce high levels of antibodies and have a significant effect in preventing Streptococcus suis type 2 infection. It can be developed and applied as a novel vaccine to prevent Streptococcus suis type 2 disease and avoid bacterial resistance, demonstrating good practical application value. Simultaneously, this invention also discloses a vaccine for preventing Streptococcus suis type 2 and an immune enhancer.

Description

Technical Field

[0001] This invention relates to the field of vaccine preparation technology, and in particular to the application of a type 2 Streptococcus suis membrane vesicle in the preparation of vaccines and / or immune enhancers. Background Technology

[0002] Streptococcus suis, a Gram-positive bacterium, is a significant zoonotic bacterial pathogen that seriously threatens the pig industry. Serotype 2, in particular, is the most widespread and pathogenic, causing enormous economic losses to the global pig industry and seriously endangering human health. Vaccination is an effective strategy for controlling Streptococcus suis. While traditional vaccines (such as inactivated vaccines) have some effect, they have inherent drawbacks such as poor immunization efficacy. At the same time, traditional antibiotic therapy faces serious drug resistance problems, necessitating the development of safer and more effective new vaccine strategies.

[0003] Outer membrane vesicles (MVs) are nanoscale, spherical vesicles secreted by bacteria during their growth. They represent a unique and novel secretory system in bacteria, carrying a variety of bacterial antigens and pathogen-associated molecular patterns, such as lipopolysaccharides, outer membrane proteins, lipoproteins, nucleic acids, and virulence factors. This endows them with natural immunogenicity and self-adjuvant properties, enabling them to induce a strong and effective innate immune response in the host. The nanoparticle nature of vesicles facilitates uptake by antigen-presenting cells, simultaneously inducing humoral, cellular, and mucosal immunity, thus achieving highly efficient vaccine delivery and immune activation.

[0004] Research on Gram-negative bacterial membrane vesicles is extensive and in-depth. In recent years, significant progress has been made in immunological studies of Gram-negative bacterial vesicles. The delivery of Acinetobacter baumannii outer membrane proteins via bacterial biomimetic vesicles significantly enhances humoral immunity; Salmonella typhimurium outer membrane vesicles activate the host immune system and reverse the immunosuppressive tumor microenvironment; Klebsiella pneumoniae outer membrane vesicles recruit tumor-associated macrophages, triggering an immune response. MVs have been successfully applied to vaccine development against serogroup B meningococci, and the European Medicines Agency and the US Food and Drug Administration have approved vaccines containing serogroup B meningococcal MVs for marketing, providing protection against serogroup B meningococci.

[0005] Research on Gram-positive bacterial membrane vesicles has lagged behind. Due to their thick cell walls, they were long considered incapable of vesicle production until the last decade or so. Although some studies have reported on the immunogenicity of Gram-positive bacterial membrane vesicles, current research mainly focuses on a few pathogens such as Staphylococcus aureus and Streptococcus pneumoniae. In-depth exploration of the immunogenicity of membrane vesicles from other bacterial species, especially zoonotic pathogens, such as Streptococcus suis type 2 vesicles, remains limited.

[0006] Currently, the control of Streptococcus suis mainly relies on vaccination and antibiotics. Most existing Streptococcus suis vaccines are inactivated vaccines, which are cumbersome to prepare, costly, and have poor immunizing efficacy. Furthermore, Streptococcus suis exhibits a very serious drug resistance situation, displaying multidrug resistance. To address the increasingly serious clinical challenges posed by Streptococcus suis infection, especially by drug-resistant strains, research on membrane vesicle immunoprotection is urgently needed. Summary of the Invention

[0007] The purpose of this invention is to provide an application of Streptococcus suis type 2 membrane vesicles in the preparation of vaccines and / or immune enhancers. These Streptococcus suis type 2 membrane vesicles can induce the production of high levels of antibodies and have a significant effect in preventing Streptococcus suis type 2 infection. They have a high challenge protection rate and can be developed and applied as a novel vaccine to prevent Streptococcus suis type 2 and avoid bacterial resistance.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] Application of a type 2 Streptococcus suis membrane vesicle in the preparation of vaccines and / or immune enhancers.

[0010] Preferably, the vaccine is a vaccine for preventing Streptococcus suis type 2.

[0011] Preferably, the immune enhancer is an immune enhancer against Streptococcus suis type 2.

[0012] Preferably, the type 2 streptococcal membrane vesicles have a particle size of 10-100 nm and a protein band size of 62-75 kDa.

[0013] In addition, the present invention discloses a vaccine for the prevention of Streptococcus suis type 2, the vaccine comprising Streptococcus suis type 2 membrane vesicles.

[0014] Preferably, the dose of type 2 Streptococcus suis membrane vesicles in the vaccine is 150 μg.

[0015] Finally, the present invention discloses an immune enhancer against Streptococcus suis type 2, wherein the immune enhancer comprises Streptococcus suis type 2 membrane vesicles.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention provides the application of Streptococcus suis type 2 membrane vesicles in the preparation of vaccines and / or immune enhancers. For the first time, Streptococcus suis type 2 membrane vesicles are used as vaccines to immunize animals. Animal experiments have demonstrated that these Streptococcus suis type 2 membrane vesicles can induce high levels of antibodies and have a significant effect in preventing Streptococcus suis type 2 infection. They have a high challenge protection rate and can be developed and applied as a novel vaccine to prevent Streptococcus suis type 2 and avoid bacterial resistance. Attached Figure Description

[0018] Figure 1 The particle size distribution of type 2 streptococcal membrane vesicles extracted in this embodiment of the invention;

[0019] Figure 2 This is an SDS-PAGE electrophoresis image of proteins from type 2 Streptococcus suis membrane vesicles extracted in an embodiment of the present invention.

[0020] Figure 3 This is a transmission electron microscope image of type 2 streptococcal membrane vesicles extracted in an embodiment of the present invention;

[0021] Figure 4 This is a standard curve of protein concentration versus absorbance.

[0022] Figure 5 This is a graph showing the serum IgG, IgG1, IgG2a, and IgG2b antibody expression levels in mice after immunization with type 2 Streptococcus suis membrane vesicles extracted in this embodiment of the invention.

[0023] Figure 6 This is a graph showing the serum cytokine IL-4 and TNF-α expression levels in mice after immunization with Streptococcus suis membrane vesicles extracted in this embodiment of the invention. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. It should be noted that, where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Unless otherwise specified, all parts used in the embodiments of the present invention are parts by weight, and % are weight percentages.

[0025] The type 2 Streptococcus suis involved in the examples has been described in reference ( Keda Shi # Yan Li #, MinshengXu, et al., Membrane vesicles derived from streptococcus suis serotype 2induce cell pyroptosis in endothelial cells via the NLRP3 / Caspase-1 / GSDMD pathway, Journal of Integrative Agriculture, 2024, 23(4):1338-1353. ② Shi Keda, Zang Ying'an, Li Yan et al., Construction of mouse model of streptococcal meningitis type 2 and its transcriptomic analysis, Bulletin of Microbiology, 2023, 50(12):5413-5426.)

[0026] Example 1: Extraction of Streptococcus suis membrane vesicles

[0027] Includes the following steps:

[0028] (1) Resuscitation of Streptococcus suis type 2;

[0029] (2) Bacterial passage

[0030] Take the type 2 Streptococcus suis revived in step (1), pick a single colony, and shake it overnight;

[0031] (3) Liquid culture of Streptococcus suis type 2

[0032] Inoculate with a 1:100 ratio of Streptococcus suis type 2 bacterial suspension, and culture in a constant temperature shaking incubator at 37℃ and 225 r / min for 6-8 h to reach the logarithmic growth phase, thus obtaining a Streptococcus suis type 2 bacterial suspension.

[0033] (4) Extraction of Streptococcus suis membrane vesicles

[0034] Take the bacterial culture in the exponential growth phase from step (3), centrifuge at 4°C, 10000×g for 10 min in a low-temperature centrifuge, and remove the precipitate. Filter the supernatant using a 0.45 μm filter, and concentrate the filtrate in a 100 KD ultrafiltration tube, centrifuging at 4°C, 4000×g for 30 min. Take the concentrated supernatant and centrifuge at 4°C, 150000×g for 3 h in a preparative ultracentrifuge. Remove the supernatant, and resuspend the precipitate in sterile 20 mM HEPES to obtain crude membrane vesicles.

[0035] (5) Purification of type 2 streptococcal membrane vesicles

[0036] Different concentrations of OptiPrep separation layers (40%, 30%, 20%, 10%) were prepared using 20 mM HEPES and OptiPrep. These were added sequentially and smoothly from the bottom of the centrifuge tube, allowed to stand for 30 min, and then resuspended membrane vesicles were added to the top layer. The tubes were then centrifuged at 4°C, 268,000 × g for 6 h in a separation-type ultracentrifuge. Each separation layer was collected, centrifuged in a 100 kDa ultrafiltration tube to remove OptiPrep, and resuspended in 20 mM HEPES buffer to obtain purified type 2 Streptococcus suis membrane vesicles.

[0037] Example 2: Characterization and analysis of Streptococcus suis membrane vesicle extract

[0038] (1) Particle size distribution of Streptococcus suis membrane vesicle extracts

[0039] The particle size distribution of the extracted Streptococcus suis membrane vesicle extract was tested, and the results are as follows: Figure 1 As shown, the particle abundance distribution of type 2 streptococcal membrane vesicles extracted after ultracentrifugation is between 10 and 100 nm, with an average diameter of 82.81 nm, which is consistent with the size range of membrane vesicles.

[0040] (2) SDS-PAGE was performed on the extracted type 2 streptococcal membrane vesicles to observe the state of their proteins and the extraction of membrane vesicle proteins. The results are as follows: Figure 2 As shown, the 20% density gradient layer has a higher content of membrane vesicles, while the 30% and 40% density gradient layers have slightly lower content of membrane vesicles, but the purity is relatively higher.

[0041] (3) To further identify the extracted membrane vesicles, the extracted type 2 Streptococcus suis membrane vesicles were observed using transmission electron microscopy. The results are as follows: Figure 3 As shown, the type 2 streptococcal membrane vesicles exhibit a complete shape and a regular spherical structure, with particle sizes concentrated within 100 nm.

[0042] (4) Quantification of membrane vesicle proteins: The concentration of membrane vesicle proteins was determined by the BCA method. Follow the instructions in the manual and establish a protein concentration-absorbance standard curve using protein standard slides (e.g., ...). Figure 4 (See the protein concentration-absorbance standard curve shown). R 2 =0.992, showing a good linear relationship; 20 μL of diluted membrane vesicle sample was added to the sample well, followed by 200 μL of BCA working solution, and incubated at 37℃ for 30 min. The absorbance was measured at 562 nm. The calculated concentration of membrane vesicle protein was 8.7 μg / μL.

[0043] Example 3: Immunoprotective test of Streptococcus suis membrane vesicle extract.

[0044] (1) Immunity

[0045] Six- to eight-week-old SPF-grade female BALB / c mice were selected, with 15 mice in each group. The pure membrane vesicle group and the Freund's adjuvant (FA) + membrane vesicle group received a subcutaneous injection of 150 μg / mouse in the neck, while the control group received sterile PBS. A booster immunization was administered two weeks after the initial immunization. On days 14 and 28 post-immunization, three mice from each group were randomly selected and serum was collected via enucleation (6 mice in total). Antibody levels of IgG, IgG1, IgG2a, and IgG2b, as well as cytokines TNF-α and IL-4, were measured.

[0046] The results of antibody IgG, IgG1, IgG2a, and IgG2b detection are as follows: Figure 5 As shown, the results of detecting IgG, IgG1, IgG2a, and IgG2b antibody levels in mouse serum indicated that 14 days after immunization, the IgG antibody levels in the pure membrane vesicle group and the Freund's adjuvant + membrane vesicle group showed an increasing trend, but the difference was not significant compared with the control group. 28 days after immunization, the IgG antibody levels in both the pure membrane vesicle group and the Freund's adjuvant + membrane vesicle group showed a significant increase, and the IgG antibody level induced by the pure membrane vesicle group was higher than that of the Freund's adjuvant + membrane vesicle group. Fourteen days post-immunization, the levels of IgG1 antibodies in both the pure membrane vesicle group and the Freund's adjuvant + membrane vesicle group were significantly higher than those in the control group. At 28 days post-immunization, the IgG1 antibody level in the pure membrane vesicle group remained significantly elevated, while the IgG1 antibody level in the Freund's adjuvant + membrane vesicle group decreased, but remained higher than that in the control group. Serum IgG2a antibody levels decreased in both groups at both 14 and 28 days post-immunization. At 14 days post-immunization, serum IgG2b antibody levels were significantly elevated in both the pure membrane vesicle group and the Freund's adjuvant + membrane vesicle group. At 28 days post-immunization, the IgG2b antibody level in the pure membrane vesicle group remained significantly elevated, while the IgG2b antibody level in the Freund's adjuvant + membrane vesicle group decreased, but remained higher than that in the control group. These serum IgG and its subtypes IgG1, IgG2a, and IgG2b antibody levels indicate that Streptococcus suis type 2 membrane vesicles can independently induce a Th2 immune response.

[0047] TNF-α and IL-4 detection results are as follows Figure 6As shown, the results of cytokine TNF-α and IL-4 detection indicated that at 14 days post-immunization, both the pure membrane vesicle group and the Freund's adjuvant + membrane vesicle group induced a significant increase in serum IL-4 expression levels in mice. At 28 days post-immunization, the serum IL-4 expression level in the pure membrane vesicle group continued to increase significantly, while the serum IL-4 level in the Freund's adjuvant + membrane vesicle group showed a decreasing trend. IL-4 is mainly produced by Th2 cells and is a key cytokine in Th2-type immune responses. TNF-α is a key inflammatory factor in Th1-type immune responses. In this case, membrane vesicle immunization of mice did not induce an increase in TNF-α, indicating that immunization of mice with Streptococcus suis type 2 membrane vesicles can induce a Th2-biased immune response, successfully inducing a humoral immune response dominated by antibody production, and that membrane vesicle immunization alone can exert a good immune effect.

[0048] (2) Attacking the poison

[0049] Fourteen days after the second immunization, nine mice in each of the immunization groups and the control group were intraperitoneally injected with 500 μL of Streptococcus suis type 2 bacterial suspension (containing 7 × 10⁻⁶ bacteria). 8 Mice were challenged with CFU (carbohydrate, volatile organic compound), and observed for 7 days after challenge, with their symptoms and mortality recorded.

[0050] The results are shown in Table 1;

[0051] Table 1 Results of the challenge protection test

[0052] MVs 9 1 89% MVs-FA 9 3 67% PBS 9 9 0

[0053] As shown in Table 1, the MVs prepared from Streptococcus suis type 2 showed a protection rate of 89% after immunizing mice, demonstrating a protective effect on mice and suggesting its potential for development and application as a novel vaccine.

[0054] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. The use of a type 2 Streptococcus suis membrane vesicle in the preparation of vaccines and / or immune enhancers.

2. The application according to claim 1, characterized in that, The vaccine mentioned is a vaccine to prevent Streptococcus suis type 2.

3. The application according to claim 1, characterized in that, The immune enhancer mentioned is an immune enhancer targeting Streptococcus suis type 2.

4. The application according to claim 1, characterized in that, The type 2 streptococcal membrane vesicles have a particle size of 10-100 nm and a protein band size of 62-75 kDa.

5. A vaccine for the prevention of Streptococcus suis type 2, characterized in that, The vaccine contains type 2 streptococcal membrane vesicles.

6. The vaccine according to claim 4, characterized in that, The dose of type 2 Streptococcus suis membrane vesicles in the vaccine is 150 μg.

7. An immune enhancer against Streptococcus suis type 2, characterized in that, The immune enhancer contains type 2 streptococcal membrane vesicles.

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