Application of an OMVs-trained immune inducer based on BCG in the preparation of immunotherapy drugs for sepsis

Abstract

The present invention discloses an application of an OMVs-trained immune inducer based on BCG in the preparation of a sepsis immunotherapy drug. The present invention obtains an OMVs-trained immune inducer based on BCG (denoted as B‑OMVs) by separating and collecting OMVs secreted by BCG bacteria. The B‑OMVs-trained immune inducer provided by the present invention combines the significant advantages of BCG and OMVs, while avoiding the disadvantages of both. The developed B‑OMVs-trained immune inducer has the characteristics of non-replication, non-infectivity, low toxicity, etc., has little effect on the body, and is highly safe. In addition, the B‑OMVs-trained immune inducer provided by the present invention exhibits a milder and more effective immunoregulatory ability, can induce trained immunity, enhance the body's resistance to microorganisms, reduce the use of antibiotics, and more effectively improve the prognosis of sepsis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of an OMVs-trained immune inducer based on Bacillus Calmette-Guérin (BCG) in the preparation of a sepsis immunotherapy drug. Background Art

[0002] Sepsis is a clinical syndrome of multiple organ dysfunction caused by a dysregulated host response to infection. The occurrence and pathogenesis of sepsis are accompanied by severe immune disorders. The disruption of body homeostasis caused by the imbalance of pro-inflammatory and anti-inflammatory mechanisms is the main cause of disease progression and even death in patients with sepsis. Sepsis has seriously threatened human life and health, but there is currently a lack of effective treatment methods. In addition to anti-infection treatment for pathogenic microorganisms, other treatments are mostly non-specific symptomatic supportive treatments. In addition, the overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, which is on the rise clinically, and even super bacteria that are completely resistant to existing antibiotics have emerged, posing a huge threat to global public health security. Therefore, it is crucial to develop a safe and effective new drug for the treatment of sepsis.

[0003] Trained immunity (TI) is an immunomodulatory strategy. Innate immune cells represented by monocytes-macrophages, NK cells, and ILC cells interact with pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), thereby causing epigenetic and metabolomics reprogramming of innate immune cells. When they are stimulated again, they produce a faster or stronger immune response, thereby eliminating the stimulus and restoring the body to homeostasis. This memory effect of innate immunity is called trained immunity. TI has a strong ability to resist pathogenic microorganisms such as viruses, bacteria, and fungi, and can quickly restore the body to homeostasis. Therefore, TI has broad application prospects in the prevention and treatment of sepsis. Studies have shown that by inducing TI in mice, their proinflammatory response and bacterial clearance function after sepsis modeling can be enhanced, thereby quickly eliminating pathogenic microorganisms and improving the prognosis of sepsis.

[0004] Attenuated bovine tuberculosis bacillus Calmette–Guérin (BCG) can have a good protective effect against infectious diseases and tumors by inducing TI. However, since BCG is a live attenuated vaccine, it is immunogenic and can cause BCG disease, and in severe cases, even systemic disseminated tuberculosis. These factors further limit the clinical application of BCG-induced TI.

[0005] Bacterial outer membrane vesicles (OMVs) are nanoparticles naturally secreted by bacteria through outward budding. They have a similar structure and composition to the bacterial outer membrane, are rich in PAMPs, and can interact with immune cells for immune regulation. However, OMVs derived from Gram-negative bacteria are rich in endotoxins, which can produce a strong regulatory response to the immune system and even in some cases, can assist the bacterial infection process and trigger the occurrence of sepsis. In view of this, before Gram-negative bacteria-derived OMVs are used in clinical treatment, they must be attenuated to ensure their safety. Although a variety of attenuation technologies are currently used, such as mineralization and genetic engineering regulation, these methods still find it difficult to completely remove the endotoxin components in OMVs. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an application of an OMVs trained immune inducer based on BCG for the preparation of sepsis immunotherapy drugs. The present invention aims to integrate the advantages of BCG and OMVs, reduce their toxicity, and induce trained immunity more safely and effectively, thereby improving the prognosis of sepsis.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] In a first aspect, the present invention provides an OMVs training immune inducer based on BCG, and the preparation method thereof is as follows:

[0009] S1. Adding BCG strains to a liquid culture medium for cultivation, collecting the bacterial solution, then centrifuging the bacterial solution, taking the supernatant, filtering to remove bacteria and bacterial fragments, and obtaining a filtrate;

[0010] S2, ultrafiltration of the filtrate to remove small molecule proteins, followed by concentration, centrifugation of the concentrated liquid to remove bacterial debris, and centrifugation of the supernatant again to obtain a sterile supernatant;

[0011] S3. Ultracentrifuge the sterile supernatant, remove the supernatant, resuspend the obtained precipitate with PBS buffer, and then wash it by centrifugation to obtain the BCG-based OMVs trained immune inducer.

[0012] As a preferred technical solution of the present invention, in step S1, the liquid culture medium is selected from M7H9 liquid culture medium.

[0013] As a preferred technical solution of the present invention, in step S1, the culture temperature is 35-37°C and the rotation speed is 200-300 rpm.

[0014] As a preferred technical solution of the present invention, in step S1, the bacterial solution OD 600The value is 0.8-1.

[0015] As a preferred technical solution of the present invention, in step S1, the centrifugal speed is 3000-4000g, and the centrifugal time is 10-20min.

[0016] As a preferred technical solution of the present invention, in step S2, the concentration multiple is 10-30 times.

[0017] As a preferred technical solution of the present invention, in step S3, the speed of ultracentrifugation is 100000g-200000g, and the time is 1-3h.

[0018] In a second aspect, the present invention also provides the use of the above-mentioned BCG-based OMVs trained immune inducer in the preparation of sepsis immunotherapy drugs.

[0019] As a preferred technical solution of the present invention, the dosage form of the drug is injection, tablet, capsule, granule, pill, oral liquid or emulsion.

[0020] As a preferred technical solution of the present invention, the drug also includes a pharmaceutically or pharmacologically acceptable carrier.

[0021] Specifically, the carrier includes one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant and a preservative.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention obtains an OMVs training immune inducer (denoted as B-OMVs) based on BCG by separating and collecting OMVs secreted by BCG bacteria. The B-OMVs training immune inducer provided by the present invention combines the significant advantages of BCG and OMVs, while avoiding the disadvantages of both. The developed B-OMVs training immune inducer has the characteristics of non-replication, non-infectivity, low toxicity, etc., has little effect on the body, and is highly safe;

[0024] 2. The B-OMVs trained immune inducer provided by the present invention exhibits a milder and more effective immunoregulatory ability, can induce trained immunity, enhance the body's resistance to microorganisms, reduce the use of antibiotics, and more effectively improve the prognosis of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an electron micrograph of the B-OMVs trained immune inducer prepared in Example 1 of the present invention.

[0026] Figure 2The following are comparisons of the safety effects of different groups of mice, where A is a comparison of the survival rates of different groups of mice; B is a comparison of the organ damage-related markers of different groups of mice; C is a comparison of the serum inflammatory factors of different groups of mice; ** P < 0.01,*** P < 0.001, **** P < 0.0001.

[0027] Figure 3 The following are comparisons of the improvement effects of sepsis prognosis in different groups of mice, where A is a comparison of the survival rates of mice in different groups; B is a comparison of the levels of serum inflammatory factors in mice in different groups; C is a comparison of organ damage-related markers in mice in different groups; D is a comparison of the bacterial loads in the organs of mice in different groups; * P < 0.05, ** P < 0.01,*** P <0.001. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0029] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0030] The BCG strain used in the present invention was donated by Suzhou Center for Disease Control and Prevention, and the Escherichia coli model is ATCC8099.

[0031] Example 1

[0032] A method for preparing an OMVs training immune inducer based on BCG, comprising the following steps:

[0033] S1. Add BCG strains to M7H9 liquid culture medium and culture at 37°C and 250 rpm shaking in a shaker. When the bacterial solution OD 600 When the value reaches 0.8, the bacterial solution is collected and centrifuged at 3450g for 15 min. The supernatant is collected and filtered using a 0.45um filter to completely remove bacteria and large debris to obtain a filtrate.

[0034] S2. The filtrate was ultrafiltered using a 100 kd ultrafiltration tube to remove small molecular weight proteins, and then the liquid volume was concentrated 20 times, and the ultrafiltered and concentrated liquid was centrifuged at 4000 g for 15 min to remove large debris, and the supernatant was collected, and the supernatant was centrifuged again at 15000 g for 30 min to obtain a sterile supernatant;

[0035] S3. Place the sterile supernatant in an ultracentrifuge and perform ultracentrifugation at 150,000 g for 2 h. Remove the supernatant and resuspend the precipitate in 500 μL PBS buffer and centrifuge at 20,000 g for 15 min to obtain BCG-based OMVs trained immune inducer (denoted as B-OMVs).

[0036] Example 2

[0037] A method for preparing an OMVs training immune inducer based on BCG, comprising the following steps:

[0038] S1. Add BCG strains to M7H9 liquid culture medium and culture at 37°C and 250 rpm shaking in a shaker. When the bacterial solution OD 600 When the value reaches 0.9, the bacterial solution is collected and centrifuged at 3450g for 15 min. The supernatant is collected and filtered using a 0.45um filter to completely remove bacteria and large debris to obtain a filtrate.

[0039] S2. The filtrate was ultrafiltered using a 100 kd ultrafiltration tube to remove small molecular weight proteins, and then the liquid volume was concentrated 20 times, and the ultrafiltered and concentrated liquid was centrifuged at 4000 g for 15 min to remove large debris, and the supernatant was collected, and the supernatant was centrifuged again at 15000 g for 30 min to obtain a sterile supernatant;

[0040] S3. Place the sterile supernatant in an ultracentrifuge and perform ultracentrifugation at 150,000 g for 2 h. Remove the supernatant and resuspend the precipitate in 500 μL PBS buffer and centrifuge at 20,000 g for 15 min to obtain BCG-based OMVs trained immune inducer.

[0041] Example 3

[0042] A method for preparing an OMVs training immune inducer based on BCG, comprising the following steps:

[0043] S1. Add BCG strains to M7H9 liquid culture medium and culture at 37°C and 250 rpm shaking in a shaker. When the bacterial solution OD 600 When the value reaches 1.0, the bacterial solution is collected and centrifuged at 3450g for 15 min. The supernatant is collected and filtered using a 0.45um filter to completely remove bacteria and large debris to obtain a filtrate.

[0044] S2. The filtrate was ultrafiltered using a 100 kd ultrafiltration tube to remove small molecular weight proteins, and then the liquid volume was concentrated 20 times, and the ultrafiltered and concentrated liquid was centrifuged at 4000 g for 15 min to remove large debris, and the supernatant was collected, and the supernatant was centrifuged again at 15000 g for 30 min to obtain a sterile supernatant;

[0045] S3. Place the sterile supernatant in an ultracentrifuge and perform ultracentrifugation at 150,000 g for 2 h. Remove the supernatant and resuspend the precipitate in 500 μL PBS buffer and centrifuge at 20,000 g for 15 min to obtain BCG-based OMVs trained immune inducer.

[0046] Comparative Example 1

[0047] A method for preparing an OMVs biological preparation based on Escherichia coli comprises the following steps:

[0048] S1. Add E. coli to M7H9 liquid culture medium and culture at 37°C and 250 rpm shaking. 600 When the value reaches 0.8, the bacterial solution is collected and centrifuged at 3450g for 15 min. The supernatant is collected and filtered using a 0.45um filter to completely remove bacteria and large debris to obtain a filtrate.

[0049] S2. The filtrate was ultrafiltered using a 100 kd ultrafiltration tube to remove small molecular weight proteins, and then the liquid volume was concentrated 20 times, and the ultrafiltered and concentrated liquid was centrifuged at 4000 g for 15 min to remove large debris, and the supernatant was collected, and the supernatant was centrifuged again at 15000 g for 30 min to obtain a sterile supernatant;

[0050] S3. Place the sterile supernatant in an ultracentrifuge and perform ultracentrifugation at 150,000 g for 2 h. Remove the supernatant and resuspend the resulting precipitate in 500 μL PBS buffer and centrifuge at 20,000 g for 15 min to obtain the Escherichia coli-based OMVs biological preparation (denoted as E-OMVs).

[0051] Compared with Example 1, Comparative Example 1 used Escherichia coli to replace BCG bacteria.

[0052] The electron microscope image of the B-OMVs prepared in Example 1 of the present invention is as follows: Figure 1 As shown in the figure, it can be seen that the B-OMVs prepared in Example 1 of the present invention are nanostructured and have a particle size of less than 200 nm.

[0053] The B-OMVs prepared in Example 1 of the present invention were tested as follows:

[0054] Experimental Example 1 Evaluation of the safety of B-OMVs

[0055] The present invention uses C57 BL / 6 mice to intraperitoneally inject different doses of B-OMVs and E-OMVs (the biological preparation prepared in Comparative Example 1), and evaluates the safety of B-OMVs by comparing the survival rate of mice, organ damage-related markers, and serum inflammatory factors. The specific experimental steps are as follows:

[0056] B-OMVs were administered at 5 mg / kg, 12.5 mg / kg, 25 mg / kg, and 50 mg / kg; E-OMVs were administered at 5 mg / kg, 7.5 mg / kg, and 10 mg / kg; and BCG (bacillus Calmette-Guérin) was administered at a dose of 1*10 6 cfu / mouse. The mice were grouped according to the above dose, with 10 mice in each group, and injected intraperitoneally according to the above dose. The death of mice was observed every 24 hours after the injection. The injection was terminated after 7 days, and the mortality rate of each group was counted.

[0057] B-OMVs were administered at 5 mg / kg, 12.5 mg / kg, 25 mg / kg, 50 mg / kg, E-OMVs at 5 mg / kg, and BCG (bacillus Calmette-Guérin) at 1*10 6 The mice were grouped with 5 mice in each group and injected intraperitoneally at the above doses. Serum was collected 24 hours after injection, and IL-6 and TNF-α were detected by ELISA, and AST and CR were detected by a detection instrument.

[0058] The results are as follows Figure 2 As shown, from Figure 2 As can be seen from Figure A, the safety of B-OMVs is much better than that of E-OMVs. A high dose (50 mg / kg) of B-OMVs did not cause death in mice. Figure 2 As can be seen from Figures B and C, the inflammatory factors and organ damage-related markers of mice in the B-OMVs group and the BCG group were basically normal, while the E-OMVs group showed obvious toxicity at a low dose (5 mg / kg), causing severe inflammatory reactions in mice, leading to the death of some mice, and elevated inflammatory factors and organ damage-related markers. It can be seen that the B-OMVs provided by the present invention are highly safe and have good clinical application prospects.

[0059] Experimental Example 2 Evaluation of the effect of B-OMVs-induced trained immunity on improving the prognosis of sepsis in mice

[0060] C57 BL / 6 mice were intraperitoneally injected with B-OMVs or BCG to induce mouse training immunity, and PBS injection was used as a control. Then, cecal ligation and puncture (CLP) sepsis model was established. The survival rate, inflammatory factor levels, organ damage-related markers and bacterial load of mice were compared to verify the protective effect of pre-injection of B-OMVs on septic mice.

[0061] The specific experimental steps are as follows:

[0062] C57BL / 6 mice grouping and treatment:

[0063] Fifty-four C57BL / 6 mice aged 6-8 weeks were randomly divided into three groups: PBS-CLP, BCG-CLP, and B-OMVs-CLP, with 18 mice in each group. The PBS-CLP group was intraperitoneally injected with 0.5 ml PBS as a control, and the BCG-CLP group was injected with 1*10 6 The B-OMVs-CLP group was injected intraperitoneally at a dose of 5 mg / kg, and the patients rested for 3 days after administration.

[0064] Construction of mouse sepsis model and drug administration:

[0065] In order to induce a mouse sepsis model, mice in the above groups were subjected to CLP modeling. The modeling method was as follows: anesthetize the mice, shave the hair on the left lower abdomen, disinfect with iodine, make a small incision (about 1 cm), gently pull out the cecum with forceps, ligate 50% of the cecum with surgical sutures, puncture with a needle, squeeze out an appropriate amount of intestinal contents from the puncture site, put the cecum back into the abdominal cavity, and suture it. Mice were subcutaneously injected with pre-warmed 37°C sterile saline (1 mL) for fluid resuscitation.

[0066] After 24 hours of modeling, the survival status of C57BL / 6 mice in each group was recorded every 24 hours, and the Log-Rank test was used to perform statistical analysis on the survival curves of mice in each group. Figure 3 As shown in A;

[0067] 24 hours after CLP modeling, whole blood was collected from mice and serum was separated. ELISA was used to detect the levels of IL-6 and TNF-α in serum. The test results are shown in Figure 3 As shown in B; AST and CR levels were detected using a testing instrument, and the test results were as follows Figure 3 As shown in C.

[0068] The liver, spleen, lung and other organs of mice were collected 24 hours after CLP modeling. Each organ was ground under sterile conditions and resuspended in an equal amount of PBS. The ground organ suspension was plated on an LB plate and the colony growth was observed 24 hours after the plate was plated. The test results are shown in the figure. Figure 3 As shown in D.

[0069] from Figure 3 It can be seen that compared with the BCG group, the survival rate of mice in the B-OMVs group was significantly increased, the level of inflammatory factors was significantly decreased, the organ damage of mice was significantly alleviated, and the bacterial load was significantly reduced. It can be seen that the B-OMVs provided by the present invention can enhance the early pro-inflammatory and phagocytic functions of mice by inducing trained immunity, thereby killing pathogens early and quickly, preventing the spread of infection, and thus improving the prognosis of septic mice.

[0070] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. An application of an OMVs-trained immune inducer based on BCG in the preparation of a sepsis immunotherapy drug, characterized in that: The preparation method of the BCG-based OMVs training immune inducer is as follows: S1. Adding BCG strains to a liquid culture medium for cultivation, collecting the bacterial solution, then centrifuging the bacterial solution, taking the supernatant, filtering to remove bacteria and bacterial fragments, and obtaining a filtrate; S2, ultrafiltration of the filtrate to remove small molecule proteins, followed by concentration, centrifugation of the concentrated liquid to remove bacterial debris, and centrifugation of the supernatant again to obtain a sterile supernatant; S3. Ultracentrifuge the sterile supernatant, remove the supernatant, resuspend the obtained precipitate with PBS buffer, and then wash it by centrifugation to obtain the BCG-based OMVs trained immune inducer.

2. The use according to claim 1, characterized in that: In step S1, the OD of the bacterial solution 600 The value is 0.8-1.

3. The use according to claim 1, characterized in that: In step S2, the concentration factor is 10-30 times.

4. The use according to claim 1, characterized in that: In step S3, the speed of ultracentrifugation is 100000g-200000g, and the time is 1-3h.

5. The use according to claim 1, characterized in that: The dosage form of the drug is injection, tablet, capsule, granule, pill, oral solution or emulsion.

6. The use according to claim 1, characterized in that: The medicament also includes a pharmaceutically or pharmacologically acceptable carrier.

7. The use according to claim 6, characterized in that: The carrier includes one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant and a preservative.

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