Use of bacillus calmette-guÉrin (BCG) vaccine-based omvs trained immunity inducer in preparation of sepsis immunotherapeutic drug

Abstract

A use of a Bacillus Calmette-Guérin (BCG) vaccine-based OMVs trained immunity inducer in the preparation of a sepsis immunotherapeutic drug. OMVs secreted by BCG bacteria are separated and collected to obtain a BCG vaccine-based OMVs trained immunity inducer (denoted as B-OMVs). In the provided B-OMVs trained immunity inducer, significant advantages of BCG and OMVs are combined, and disadvantages of BCG and OMVs are avoided. The developed B-OMVs trained immunity inducer has characteristics of being non-replicative, non-infectious, and low in toxicity, has minimal impact on bodies, and exhibits high safety. In addition, the provided B-OMVs trained immunity inducer exhibits a mild and effective immunomodulatory capability, and can induce trained immunity, enhance resistance of bodies against microorganisms, reduce antibiotic usage, and more effectively improve sepsis prognosis.

Description

Application of a BCG-based OMV training immune inducer in the preparation of sepsis immunotherapy drugs Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of an OMVs training immune inducer based on BCG in the preparation of immunotherapeutic drugs for sepsis. Background Technology

[0002] Sepsis is a clinical syndrome characterized by multiple organ dysfunction caused by a dysregulated host response to infection. Severe immune dysregulation and the disruption of homeostasis due to the imbalance between pro-inflammatory and anti-inflammatory mechanisms are major causes of disease progression and even death in sepsis patients. Sepsis poses a serious threat to human life and health, but currently, effective treatments are lacking. Besides anti-infective therapy targeting the pathogenic microorganisms, other treatments are mostly non-specific symptomatic and supportive. Furthermore, the overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, which are on the rise clinically, and even superbugs completely resistant to existing antibiotics have appeared, posing a significant threat to global public health security. Therefore, developing a safe and effective new drug for the treatment of sepsis is crucial.

[0003] Training immunity (TI) is an immunomodulatory strategy in which innate immune cells, represented by monocytes-macrophages, NK cells, and ILC cells, interact with pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs). This leads to epigenetic and metabolomics reprogramming of innate immune cells, resulting in a faster or stronger immune response upon re-exposure to unspecified stimuli, thereby eliminating the stimulus and restoring homeostasis. This memory function of innate immunity is called training immunity. TI possesses a strong ability to resist pathogenic microorganisms such as viruses, bacteria, and fungi, and can rapidly restore homeostasis. Therefore, TI has broad application prospects in the prevention and treatment of sepsis. Studies have shown that inducing TI in mice can enhance their pro-inflammatory response and bacterial clearance function after sepsis modeling, thereby rapidly eliminating pathogenic microorganisms and improving sepsis prognosis.

[0004] Live attenuated bovine tuberculosis bacillus (BCG) can provide good protection against infectious diseases and tumors by inducing tuberculosis induction (TI). However, because BCG is a live attenuated vaccine, it is immunogenic and can cause BCG-induced tuberculosis, which in severe cases can even lead to 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 structure and composition similar to the bacterial outer membrane, contain abundant PAMPs, and can interact with immune cells to modulate the immune system. However, OMVs derived from Gram-negative bacteria are rich in endotoxins, which can elicit a strong regulatory response from the immune system and, in some cases, even assist the bacterial infection process, triggering sepsis. Therefore, before applying Gram-negative bacteria-derived OMVs to clinical treatment, they must be attenuated to ensure their safety. Although various attenuation techniques, such as mineralization and genetic engineering, are currently employed, these methods still struggle to completely remove the endotoxin components from OMVs. Technical issues

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an application of BCG-based OMVs training immune inducer in the preparation of sepsis immunotherapy drugs. The present invention aims to combine the advantages of both BCG and OMVs, reduce their toxicity, and more safely and effectively induce training immunity, thereby improving the prognosis of sepsis. Technical solutions

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0009] S1. Add BCG bacteria to liquid culture medium for culture, collect bacterial solution, then centrifuge the bacterial solution, take the supernatant, filter to remove bacteria and bacterial debris, and obtain filtrate.

[0010] S2. Ultrafiltration is performed on the filtrate to remove small molecule proteins. Then the filtrate is concentrated. The concentrated liquid is then centrifuged to remove bacterial debris. The supernatant is centrifuged again to obtain a sterile supernatant.

[0011] S3. Centrifuge the sterile supernatant at high speed to remove the supernatant. Resuspend the resulting precipitate in PBS buffer, then centrifuge and wash to obtain the BCG-based OMVs training 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℃ and the rotation speed is 200-300rpm.

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

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

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

[0017] As a preferred technical solution of the present invention, in step S3, the speed of ultracentrifugation is 100,000g-200,000g, and the time is 1-3h.

[0018] Secondly, the present invention also provides the application of the above-mentioned BCG-based OMVs training 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 an injection, tablet, capsule, granule, pill, oral liquid or emulsion.

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

[0021] Specifically, the carrier includes one or more of the following: disintegrant, diluent, lubricant, binder, humectant, flavoring agent, suspending agent, surfactant, and preservative. Beneficial effects

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

[0023] 1. This invention obtains a BCG-based OMV training and immune inducer (denoted as B-OMVs) by isolating and collecting OMVs secreted by BCG bacteria. The B-OMVs training and immune inducer provided by this invention combines the significant advantages of BCG and OMVs while avoiding their disadvantages. The developed B-OMVs training and immune inducer has the characteristics of non-replication, non-infectiousness, and low toxicity, with minimal impact on the body and high safety.

[0024] 2. The B-OMVs training immune inducer provided by this invention exhibits a milder yet more effective immunomodulatory ability, which can induce training immunity, enhance the body's resistance to microorganisms, reduce the use of antibiotics, and more effectively improve the prognosis of sepsis. Attached Figure Description

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

[0026] Figure 2 shows the comparison of safety effects among different groups of mice. In the figure, A is a comparison of survival rates among different groups of mice; B is a comparison of organ damage-related markers among different groups of mice; and C is a comparison of serum inflammatory factors among different groups of mice. In the figure, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0027] Figure 3 shows a comparison of the prognostic improvement of sepsis in different groups of mice. A is a comparison of the survival rate of mice in different groups; B is a comparison of the serum inflammatory factor levels of mice in different groups; C is a comparison of organ damage-related markers of mice in different groups; D is a comparison of bacterial load in organs of mice in different groups. In the figure, *P< 0.05, **P< 0.01, ***P< 0.001. Embodiments of the present invention

[0028] The present invention will be 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, all chemical reagents involved in this invention were purchased through commercial channels.

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

[0031] Example 1

[0032] A method for preparing an OMV training immune inducer based on BCG includes the following steps:

[0033] S1. Add the BCG culture to M7H9 liquid medium and incubate at 37°C and 250 rpm on a shaking incubator. When the bacterial culture OD... 600 Once the pH reaches 0.8, collect the bacterial culture, centrifuge at 3450g for 15 minutes, collect the supernatant, and filter the supernatant using a 0.45um filter to completely remove bacteria and large fragments, obtaining the filtrate.

[0034] S2. Use a 100kd ultrafiltration tube to ultrafilter the filtrate to remove small molecule proteins. Then concentrate the liquid volume by 20 times. Centrifuge the concentrated liquid at 4000g for 15min to remove large fragments and collect the supernatant. Centrifuge the supernatant again at 15000g for 30min and collect the supernatant to obtain sterile supernatant.

[0035] S3. Place the sterile supernatant in an ultracentrifuge and centrifuge at 150,000g for 2 hours. Remove the supernatant and resuspend the resulting precipitate in 500 μL of PBS buffer. Centrifuge at 20,000g for 15 minutes to obtain the BCG-based OMVs training immune inducer (denoted as B-OMVs).

[0036] Example 2

[0037] A method for preparing an OMV training immune inducer based on BCG includes the following steps:

[0038] S1. Add the BCG culture to M7H9 liquid medium and incubate at 37°C and 250 rpm on a shaking incubator. When the bacterial culture OD... 600 Once the pH reaches 0.9, collect the bacterial culture, centrifuge at 3450g for 15 minutes, collect the supernatant, and filter the supernatant using a 0.45um filter to completely remove bacteria and large fragments, obtaining the filtrate.

[0039] S2. Use a 100kd ultrafiltration tube to ultrafilter the filtrate to remove small molecule proteins. Then concentrate the liquid volume by 20 times. Centrifuge the concentrated liquid at 4000g for 15min to remove large fragments and collect the supernatant. Centrifuge the supernatant again at 15000g for 30min and collect the supernatant to obtain sterile supernatant.

[0040] S3. Place the sterile supernatant in an ultracentrifuge and centrifuge at 150,000g for 2 hours. Remove the supernatant and resuspend the resulting precipitate in 500 μL of PBS buffer. Centrifuge at 20,000g for 15 minutes to obtain the BCG-based OMVs training immune inducer.

[0041] Example 3

[0042] A method for preparing an OMV training immune inducer based on BCG includes the following steps:

[0043] S1. Add the BCG culture to M7H9 liquid medium and incubate at 37°C and 250 rpm on a shaking incubator. When the bacterial culture OD... 600Once the value reaches 1.0, collect the bacterial culture, centrifuge at 3450g for 15min, collect the supernatant, filter the supernatant using a 0.45um filter to completely remove bacteria and large fragments, and obtain the filtrate.

[0044] S2. Use a 100kd ultrafiltration tube to ultrafilter the filtrate to remove small molecule proteins. Then concentrate the liquid volume by 20 times. Centrifuge the concentrated liquid at 4000g for 15min to remove large fragments and collect the supernatant. Centrifuge the supernatant again at 15000g for 30min and collect the supernatant to obtain sterile supernatant.

[0045] S3. Place the sterile supernatant in an ultracentrifuge and centrifuge at 150,000g for 2 hours. Remove the supernatant and resuspend the resulting precipitate in 500 μL of PBS buffer. Centrifuge at 20,000g for 15 minutes to obtain the BCG-based OMVs training immune inducer.

[0046] Comparative Example 1

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

[0048] S1. Add the Escherichia coli strain to M7H9 liquid culture medium and incubate at 37°C and 250 rpm on a shaking incubator. When the bacterial culture OD... 600 Once the pH reaches 0.8, collect the bacterial culture, centrifuge at 3450g for 15 minutes, collect the supernatant, and filter the supernatant using a 0.45um filter to completely remove bacteria and large fragments, obtaining the filtrate.

[0049] S2. Use a 100kd ultrafiltration tube to ultrafilter the filtrate to remove small molecule proteins. Then concentrate the liquid volume by 20 times. Centrifuge the concentrated liquid at 4000g for 15min to remove large fragments and collect the supernatant. Centrifuge the supernatant again at 15000g for 30min and collect the supernatant to obtain sterile supernatant.

[0050] S3. Place the sterile supernatant in an ultracentrifuge and centrifuge at 150,000g for 2 hours. Remove the supernatant and resuspend the resulting precipitate in 500 μL of PBS buffer. Centrifuge at 20,000g for 15 minutes to obtain the E. coli-based OMVs biological agent (denoted as E-OMVs).

[0051] Compared with Comparative Example 1 and Example 1, Escherichia coli was used instead of BCG bacteria.

[0052] Figure 1 shows an electron microscope image of the B-OMVs prepared in Example 1 of the present invention. As can be seen from the figure, the B-OMVs prepared in Example 1 of the present invention are nanostructures with a particle size of less than 200 nm.

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

[0054] Experimental Example 1: Safety Assessment of B-OMVs

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

[0056] B-OMVs were administered at doses of 5 mg / kg, 12.5 mg / kg, 25 mg / kg, and 50 mg / kg; E-OMVs at doses of 5 mg / kg, 7.5 mg / kg, and 10 mg / kg; and BCG (Bacillus Calmette-Guérin) was administered at a dose of 1 x 10⁻⁶. 6 CFU / mouse. Mice were divided into groups of 10 mice each and injected intraperitoneally at the above dosage. The mortality of mice was observed every 24 hours after the injection. The treatment was terminated after 7 days, and the mortality rate of each group was recorded.

[0057] B-OMVs were administered at doses of 5 mg / kg, 12.5 mg / kg, 25 mg / kg, and 50 mg / kg; E-OMVs at 5 mg / kg; and BCG (Bacillus Calmette-Guérin) at 1 x 10^6 doses. 6 CFU was administered to mice in groups of 5, and the mice were intraperitoneally injected with the doses described above. Serum was collected 24 hours after injection, and IL-6 and TNF-α were detected by ELISA. AST and CR were detected by a detection instrument.

[0058] The results are shown in Figure 2. As can be seen from Figure 2A, the safety of B-OMVs is far better than that of E-OMVs. High dose (50 mg / kg) of B-OMVs did not cause death in mice. As can be seen from Figure 2B 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 low dose (5 mg / kg), causing severe inflammatory response 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 this invention has the characteristics of high safety and has good clinical application prospects.

[0059] Experiment 2: Evaluation of the effect of B-OMVs-induced training on improving the prognosis of sepsis in mice.

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

[0061] The specific experimental steps are as follows:

[0062] Grouping and treatment of C57BL / 6 mice:

[0063] Fifty-four 6-8 week old C57BL / 6 mice were randomly divided into three groups: PBS-CLP, BCG-CLP, and B-OMVs-CLP, with 18 mice in each group. The PBS-CLP group received an intraperitoneal injection of 0.5 ml PBS as a control, while the BCG-CLP group received 1*10 ml of PBS. 6 The CFU / kg dose was administered intraperitoneally, while the B-OMVs-CLP group received an intraperitoneal injection of 5 mg / kg. After administration, the patients rested for 3 days.

[0064] Establishment of a mouse sepsis model and drug administration:

[0065] To induce a mouse sepsis model, CLP was performed on mice in the above-mentioned groups. The modeling method was as follows: Mice were anesthetized, the hair on the left lower abdomen was shaved, disinfected with povidone-iodine, a small incision (about 1 cm) was made, the cecum was gently pulled out with forceps, 50% of the cecum was ligated with surgical sutures, a needle was punctured, and an appropriate amount of intestinal contents was squeezed out from the puncture site. The cecum was then returned to the abdominal cavity and sutured. Mice were subcutaneously injected with 1 mL of pre-warmed 37°C sterile saline for fluid resuscitation.

[0066] Twenty-four hours after modeling, the survival status of C57BL / 6 mice in each group was recorded every 24 hours, and the survival curves of each group of mice were statistically analyzed using the Log-Rank test. The experimental results are shown in Figure 3A.

[0067] Whole blood was collected from mice 24 hours after CLP modeling and serum was separated. The levels of IL-6 and TNF-α in the serum were detected by ELISA. The results are shown in Figure 3B. The levels of AST and CR were detected by the instrument. The results are shown in Figure 3C.

[0068] Liver, spleen, lung and other organs of mice were collected 24 hours after CLP modeling. Each organ was ground under sterile conditions and resuspended with an equal volume of PBS. The ground organ suspension was then spread on LB plates. Colony growth was observed 24 hours after spreading. The experimental results are shown in Figure 3D.

[0069] As shown in Figure 3, 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 reduced, organ damage in mice was significantly alleviated, and 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 training immunity, thereby killing pathogens early and rapidly, 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 way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. The use of OMVs trained immunity inducer based on Bacillus Calmette-Guerin (BCG) in the preparation of immunotherapeutic drugs for sepsis, characterized in that, The preparation method of the BCG-based OMV training immune inducer is as follows: S1. Add BCG bacteria to liquid culture medium for culture, collect bacterial solution, then centrifuge the bacterial solution, take the supernatant, filter to remove bacteria and bacterial debris, and obtain filtrate. S2. Ultrafiltration is performed on the filtrate to remove small molecule proteins. Then the filtrate is concentrated. The concentrated liquid is then centrifuged to remove bacterial debris. The supernatant is centrifuged again to obtain a sterile supernatant. S3. Centrifuge the sterile supernatant at high speed to remove the supernatant. Resuspend the resulting precipitate in PBS buffer, then centrifuge and wash to obtain the BCG-based OMVs training immune inducer.

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

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

4. Use according to claim 1, characterized in that, In step S3, the ultracentrifugation speed is 100,000g-200,000g, and the time is 1-3h.

5. The use according to claim 1, characterized in that, The dosage forms of the drug are injections, tablets, capsules, granules, pills, oral liquids, or emulsions.

6. Use according to claim 1, characterized in that, Drugs also include pharmaceutically or pharmacologically acceptable carriers.

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

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