Use of extracellular beta-glucan from aureobasidium pullulans for the preparation of a training immunity inducing agent

Abstract

The present application relates to the application of extracellular beta-glucan from Aureobasidium pullulans in preparing training immune inducer. By establishing mouse bone marrow derived macrophage (BMDMs) and zebrafish larvae training immune activation evaluation model, the immune activity of extracellular beta-glucan (eBG) from Aureobasidium pullulans is evaluated. It is found that compared with cell wall derived beta-glucan (cBG), the extracellular beta-glucan (eBG) from Aureobasidium pullulans has more significant training immune activation effect, which proves that the extracellular beta-glucan (eBG) from Aureobasidium pullulans is an excellent training immune inducer, and has good application prospect in the field of animal feed addition and disease immune prevention and control.

Description

Technical Field

[0001] This invention relates to the field of immunomodulatory technology, and in particular to the application of an extracellular β-glucan derived from *Bacillus melanogenis* in the preparation of training immune inducers. Background Technology

[0002] Training immunity is a non-specific immune memory phenomenon. Its basic principle is that the epigenetic modifications produced by immune cells after initial stimulation are retained even after resting. Therefore, when cells are stimulated again by the same or different pathogens, a faster and stronger immune response occurs to clear the pathogen. Based on this principle, activation of training immunity enables the host to possess a longer-term ability to resist pathogenic microorganisms such as bacteria, fungi, viruses, or parasites. Currently, there are anti-infection or disease immune control strategies based on training immunity, such as using training immunity inducers as immunotherapeutic drugs, vaccine adjuvants, feed additives, and immune enhancers. As immunotherapeutic drugs, activation of training immunity can enhance anti-tumor effects, and inhibitors based on the principle of training immunity can also alleviate systemic inflammation. As vaccine adjuvants, the non-specific immune protection generated by training immunity activation can assist the antigen-specific immune protection effect of vaccines. As feed additives or immune enhancers, training immunity inducers can provide long-term protective effects; for the livestock industry, dietary supplementation is not only convenient but also an economical and practical means of immune protection.

[0003] β-glucan is a classic immune-training inducer, commonly found in bacterial or fungal cell walls, oat or wheat germ, and algal cell walls. Based on the main chain linkage, β-glucan is mainly divided into β-1,3-glucan and β-1,4-glucan, and may also have branches primarily linked by β-1,6 glycosidic bonds. However, cell wall-derived β-glucan suffers from low purity and poor batch-to-batch stability due to differences in species and purification methods, and its specific branching structure and mechanism of action remain unclear. Previous studies have found that the genetically engineered melanin-producing *Brachystomiae* strain BZW-Δags2-1 / 2 can stably produce extracellular β-glucan (eBG), significantly reducing the cost of β-glucan extraction and purification. eBG belongs to the β-(1,3 / 1,6)-glucan family with a purity as high as 84.78%; however, there are currently no reports evaluating the activating immune-training effect of eBG. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides an application of extracellular β-glucan derived from *Bacillus melanogenis* in the preparation of training immune inducers.

[0005] To achieve the above objectives, the present invention provides an evaluation of the effectiveness of extracellular β-glucan derived from *Bacillus melanogenis* as a training immune inducer.

[0006] Preferably, the training immune inducer is used as an immunotherapeutic drug, vaccine adjuvant, feed additive, or immune enhancer.

[0007] The animal cell training and immune activation evaluation model of this invention is a hematopoietic development process model. It selects β-glucan treatment during macrophage differentiation to promote the maturation of more macrophages. In this cell model, this invention demonstrates, through the expression level of the pro-inflammatory factor IL-1β, that the training immune effect induced by eBG is superior to that of cell wall β-glucan (cBG) of the same origin and conventional yeast cell wall-derived β-glucan (BG).

[0008] The animal in vivo training immune activation evaluation model of the present invention is based on the model organism zebrafish. In order to match the normal hematopoietic development process of zebrafish, the initial training immune stimulation is carried out at the more lasting stage of targeted hematopoiesis. Furthermore, the adaptive immune system of zebrafish is not yet fully developed throughout the model period.

[0009] As a preferred technical solution of the present invention, the training method on the living model uses yolk sac injection to simulate feeding.

[0010] As a preferred technical solution of the present invention, the infection method used in the live model is cochlear injection of Edwardsiella tumefaciens, with an injection concentration of 50 CFU per fish.

[0011] This invention demonstrates, in this in vivo model, that eBG enhances the host's training immune activation function and produces a tangible anti-infection effect through three aspects: pro-inflammatory factor expression level, anti-infection function evaluation, and macrophage recruitment quantity.

[0012] This invention provides a method for preparing cell wall-derived β-glucan and extracellular β-glucan solutions, and the specific implementation method is shown in S2 of Example 1.

[0013] As a preferred technical solution of the present invention, the concentration of cBG and eBG used is 10 μg / mL per well in the cell model and 10 ng per fish in the zebrafish juvenile model.

[0014] Conventional training immune inducers use cell wall-derived β-glucan. This invention demonstrates that extracellular β-glucan has a more significant training immune induction effect. The examples provided by this invention are significant for the possibility of using extracellular β-glucan to replace cell wall β-glucan in clinical applications: on the one hand, extracellular β-glucan can be used as a vaccine adjuvant to enhance the innate immune function of mammals or aquatic animals, thereby assisting in the long-term protective effect of adaptive immunity; on the other hand, extracellular β-glucan can be used as a feed additive to provide broad-spectrum and long-term anti-infective effects in reducing diseases occurring during the breeding of mammals or aquatic animals.

[0015] The beneficial effects of this invention are as follows: By establishing a training immune activation evaluation model using mouse bone marrow-derived macrophages (BMDMs) and zebrafish larvae, the immune activity of extracellular β-glucan (eBG) derived from *Brachystomum melanogaster* was evaluated. It was found that eBG has a more significant training immune activation effect compared to cell wall-derived β-glucan (BG or cBG), demonstrating that eBG is an excellent training immune inducer with good application prospects in the fields of feed additives and disease immune control in farmed animals. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of Example 1.

[0017] Figure 2 A comparison of IL-1β expression levels in mouse BMDMs induced by eBG, BG, and cBG in response to LPS.

[0018] Figure 3 A to Figure 3 Figure C shows a comparison of the expression levels of pro-inflammatory factors in mouse BMDMs induced by eBG and cBG in response to Pam3CSK4 and Poly(I:C), respectively.

[0019] Figure 4 A to Figure 4 Figure D shows a comparison of the expression levels of pro-inflammatory factors in zebrafish juveniles in response to bacterial infection after treatment with eBG and cBG. Figure 4 A is a schematic diagram of Example 3. Figure 4 B to Figure 4 D is the result of comparing cBG and eBG.

[0020] Figure 5 A comparison of the survival rates of zebrafish juveniles after eBG and cBG treatment under bacterial infection.

[0021] Figure 6Comparative graph showing the bacterial clearance ability of zebrafish juveniles after eBG and cBG treatment.

[0022] Figure 7 A and Figure 7 B represents the macrophage recruitment at the cochlear infection site in juvenile zebrafish after treatment with eBG and cBG. Figure 7 A shows the fluorescence results of the cochlea in zebrafish, where macrophages are labeled with green fluorescence. The scale bar is 200 μm. Figure 7 B is a quantity statistics chart. Detailed Implementation

[0023] To more clearly describe the technical content of the present invention, the following description is provided in conjunction with specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0025] This invention provides an application of extracellular β-glucan derived from *Brachystomum melanogaster* as a training immune inducer. Based on a mouse bone marrow-derived macrophage training immune model and a zebrafish juvenile training immune activation evaluation model, this invention demonstrates, from the perspectives of pro-inflammatory cytokine expression levels, survival rate, bacterial clearance capacity, and immune cell recruitment capacity, that extracellular β-glucan (eBG) from *Brachystomum melanogaster* significantly outperforms cell wall β-glucan (cBG) and conventional yeast cell wall β-glucan (BG) in inducing training immunity. This invention is the first to propose the role of extracellular β-glucan from *Brachystomum melanogaster* as a training immune inducer in enhancing the host's anti-infective immune response, which is of great significance for guiding the development and application of training-based immune enhancers.

[0026] This invention provides an animal cell training immune activation evaluation model, the model establishment method of which is described in Example 1. This invention also provides an animal in vivo training immune activation evaluation model, the model establishment method of which is described in Example 2.

[0027] The fish-killing Edwardsiella tarda used in this invention (EIB202, CCTCC NO: M208068) is preserved by the Ahua Bioengineering Institute of East China University of Science and Technology.

[0028] The eBG and cBG used in this invention are both derived from the melanin-producing Brugia short-stem mold engineered strain BZW-Δags2-1 / 2 (see patent CN117660204A). The conventional yeast-derived BG was purchased commercially.

[0029] Example 1

[0030] eBG enhances mouse BMDM cell training and immune activation.

[0031] Establishing a mouse BMDM training and immune activation evaluation model includes the following steps:

[0032] S1. L929 cells were cultured in DMEM medium containing penicillin (100U / mL), streptomycin (100mg / mL) and 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 cell culture incubator. After 6-7 days, the cell culture supernatant was collected, filtered through a 0.45μm filter membrane, and stored at 4°C for later use.

[0033] S2. Dissolve 5 mg of β-glucan in 1 mL of sterile PBS, sonicate at 100 W, sonicate for 2 seconds with a 4-second interval, for a total sonication time of 1 hour, and store at 4°C for later use.

[0034] S3. Take the hind limbs of 4-6 week old C57 BL / 6 mice, carefully remove the muscles and fascia, leaving the femur and tibia intact, cut off the joints, and use a 1 mL syringe to draw BMDM medium (DMEM + 10% FBS + 1% penicillin-dextrose antibody + 20% L929 supernatant). Preheat to 37°C, insert the needle into the bone marrow cavity and flush the bone marrow into a 50 mL centrifuge tube. Repeat 3 times until no obvious red color is visible in the leg bone. Repeatedly pipette the cells to disperse them, centrifuge at 1000 rpm for 10 min at 4°C, discard the supernatant, add red blood cell lysis buffer (mixed with the medium in equal proportion), let stand for 3 min, centrifuge under the same conditions, discard the supernatant, reselect the medium, and sieve through a 40 μm cell filter at 3 × 10⁻⁶ cells / mL. 5 Cells are seeded at a density of cells / well into 24-well plates and cultured at 37°C in a 5% CO2 cell culture incubator. The adherent cells are called BMDMs.

[0035] S4. On day 3 of culture, the culture medium was replaced with opti-MEM containing 10 μg / mL extracellular β-glucan from *Brachystomia melanin-producing* as the eBG group, and opti-MEM containing 10 μg / mL cell wall β-glucan from *Brachystomia melanin-producing* as the cBG group. The cells were cultured for another 24 hours. The cells were treated with opti-MEM without any additives as the control group. After 24 hours, the cells were washed with PBS and cultured in fresh BMDM medium for another 5 days. Then, the culture medium was replaced with opti-MEM containing 10 μg / mL LPS, 10 ng / mL Pam3CSK4, or 10 ng / mL Poly(I:C) to simulate bacterial infection. After 24 hours, samples were taken for ELISA or qPCR detection.

[0036] S5. Collect the cell culture supernatant and perform ELISA to detect the expression level of IL-1β according to the kit instructions.

[0037] S6. RNA was extracted from cells, reverse transcribed, and then detected by qPCR. The forward primer for the internal reference gene β-actin was 5'-CATTGCTGACAGGATGCAGAAGG-3', and the reverse primer was 5'-TGCTGGAAGGTGGACAGTGAGG-3'. The forward primer for the pro-inflammatory factor TNFA was 5'-GGTGCCTATGTCTCAGCCTCTT-3', and the reverse primer was 5'-GCCATAGAACTGATGAGAGGGAG-3'. The forward primer for IL6 was 5'-TACCACTTCACAAGTCGGAGGC-3', and the reverse primer was 5'-CTGCAAGTGCATCATCGTTGTTC-3'. The forward primer for IL1b was 5'-TGGACCTTCCAGGATGAGGACA-3', and the reverse primer was 5'-GTTCATCTCGGAGCCTGTAGTG-3'.

[0038] from Figure 2 The results showed that eBG induced greater IL-1β secretion after LPS treatment, and there was no significant difference in effect between BG and cBG, indicating that BG and cBG had similar effects on inducing immune activation during training. Therefore, subsequent experiments only compared cBG and eBG. Figure 3 A to Figure 3 The results in C show that the expression levels of pro-inflammatory factors in the eBG group were significantly increased after secondary stimulation. Compared with the cBG group, eBG treatment not only significantly enhanced the inflammatory response after Pam3CSK4 secondary stimulation, but also significantly enhanced the immune response after Poly(I:C) secondary stimulation.

[0039] Example 2

[0040] eBG enhances the immune activation effect in zebrafish larvae during training.

[0041] Establish a training and immune activation evaluation model for zebrafish juveniles, including the following steps:

[0042] S1. Place wild-type AB strain zebrafish into the hatching tank one night in advance, separating the male and female fish. After the light exposure begins, remove the partition to allow the male and female zebrafish to mate freely. Collect the zebrafish embryos and remove impurities. Disinfect with 0.003% sodium hypochlorite for 5 minutes. Rinse the culture water three times for 5 minutes each time. Incubate in a 28℃ constant temperature incubator. Remove dead eggs and change the water daily.

[0043] S2. When the zebrafish embryos develop to the point of 2 days after fertilization, 2 mL of 5 mg / mL cBG or eBG is injected into the zebrafish yolk sac using a microinjector. Sterile PBS is used as a control group. After injection, the zebrafish fry are placed in clean system water for static culture.

[0044] S3. After 4 days of rest, i.e., 6 days post-fertilization, the juvenile zebrafish were anesthetized by aspirating a 200 μg / mL tricaine solution. 50 CFU of *E. piscicida* was injected into the cochlea using a microinjector. RNA was extracted from zebrafish samples at 3, 8, and 24 hours post-infection, with 8-10 juveniles per tube. Reverse transcription was performed followed by qPCR detection. The forward primer for the internal reference gene β-actin was 5'-GAGCGTAAATACTCCGTCTGG-3', and the reverse primer was 5'-GACTCATCGTACTCCTGCTTG-3'. The forward primer for the pro-inflammatory factor TNFA was 5'-TCAACAAGATGGAAGTGTGCTG-3', and the reverse primer was 5'-GTCCTGGTCATCTCTCCAGTC-3'. The forward primer for il6 is 5'-GGCATTTGAAGGGGTCAGGA-3', and the reverse primer is 5'-GCGTTAGACATCTTTCCGTGC-3'. The forward primer for il1b is 5'-ATCAAACCCCAATCCACAGAGT-3', and the reverse primer is 5'-GGCACTGAAGACACCACGTT-3'.

[0045] Figure 4 A to Figure 4 The results from D showed that, compared to cBG, eBG significantly enhanced the expression of pro-inflammatory factors in zebrafish larvae, indicating that eBG had a better training effect on immune activation.

[0046] Example 3

[0047] The evaluation of eBG-induced training to activate immune system and enhance host anti-infection function includes the following steps:

[0048] S1. As in Example 2, 2 mL of 5 mg / mL cBG or eBG was injected into the yolk sac of zebrafish juveniles 2 days after fertilization, with PBS as a control. After resting for 4 days, 50 CFU of Edwardsiella tumefaciens was injected into the cochlea of ​​zebrafish juveniles.

[0049] S2. After infection, observe the survival status of the juvenile fish every 12 hours and count them. Remove dead fish in time and change the water every day for 3 consecutive days.

[0050] S3. At 3, 8, and 24 hours post-infection, normally swimming zebrafish juveniles were collected for bacterial colonization. Three zebrafish juveniles were taken from each tube, the culture water was discarded, and 100 μl of sterile PBS was added to each tube. The mixture was then homogenized with a homogenizer until no obvious tissue fragments were observed. The homogenate was then serially diluted 10⁻⁶ times. 1 10 2 103 10 4 Then, take 10 μl of the stock solution or diluted solution and drop it onto a cholestyrene agar plate. Incubate the plate upside down in a 28°C incubator for 24 hours, and then count the colonies on the plate.

[0051] S4. Calculate the bacterial colonization count results using the following formula:

[0052]

[0053] Figure 5 The results showed that eBG could induce a higher survival rate in zebrafish juveniles under bacterial infection. Figure 6 The results showed that this higher survival rate was associated with stronger bacterial clearance, indicating that eBG-induced training immune activation can enhance the host's anti-infection function.

[0054] Example 4

[0055] eBG-induced training to enhance immune activation and improve macrophages' anti-infection capabilities includes the following steps:

[0056] S1. Place Tg(mpeg1.1:EGFP) zebrafish into the hatching tank one night in advance, separating the male and female fish. After the light exposure begins, remove the partition to allow the male and female zebrafish to mate freely. Collect the zebrafish embryos and remove impurities. Disinfect with 0.003% sodium hypochlorite for 5 minutes. Rinse the culture water three times for 5 minutes each time. Incubate in a constant temperature incubator at 28℃. Remove dead eggs and change the water daily.

[0057] S2. When the zebrafish embryos developed to the point of 2 days after fertilization, 2 mL of 5 mg / mL cBG or eBG was injected into the yolk sac of the zebrafish using a microinjector. Sterile PBS was used as a control group. The injected zebrafish fry were placed in clean system water for static culture.

[0058] S3. After resting for 4 days, i.e., when the zebrafish have developed to the point of 6 days after fertilization, the juvenile fish are anesthetized by aspirating a 200 μg / mL tricaine solution. 50 CFU of Edwardsiella tarda is injected into the cochlea of ​​the zebrafish using a microinjector. Zebrafish samples are collected at 3, 8 and 24 hours after injection for fluorescence imaging.

[0059] S4. For fluorescence imaging, fixation was performed using 0.5% agarose. The infected cochlea was photographed under a 20x microscope using the Leica Thunder imaging system, and the green fluorescent cells were counted using Image J.

[0060] Figure 7 A and Figure 7The results from B showed that, compared to cBG, eBG treatment enabled zebrafish juveniles to recruit more macrophages to the infected site for bactericidal action.

[0061] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. Application of extracellular β-glucan derived from the engineered strain BZW Δ ags2-1 / 2 of melanin-producing brevicorphyromonas in the preparation of adjuvants for drugs or vaccines against Edwardsiella tectoris infection.

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