CN119055685A - Application of Parabacteroides faecalis in the preparation and isolation of graft-versus-leukemia effect and graft-versus-host disease drugs - Google Patents
Application of Parabacteroides faecalis in the preparation and isolation of graft-versus-leukemia effect and graft-versus-host disease drugs Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
本发明涉及粪副拟杆菌在制备分离移植物抗白血病效应与移植物抗宿主病药物中的应用。粪副拟杆菌在体外和动物模型中均显示出抑制异基因T细胞增殖及炎症反应的作用，显著改善GVHD小鼠的生存率和肠道微环境。此外，粪副拟杆菌具有潜在的移植物抗白血病效应。本发明采用一株粪副拟杆菌作为粪菌移植的单一菌株用于治疗肠道GVHD，同时增强移植物抗白血病效应，这为其在白血病患者中的应用提供了更广阔的前景。与传统的FMT相比，单一菌株的粪副拟杆菌治疗具有成分明确、可标准化、风险低等优点，展现出良好的应用前景。可标准化、风险低等优点，显示出良好的应用前景。The present invention relates to the use of Parabacteroides faecalis in the preparation and separation of graft-versus-leukemia effect and graft-versus-host disease drugs. Parabacteroides faecalis showed the effect of inhibiting allogeneic T cell proliferation and inflammatory response in vitro and in animal models, and significantly improved the survival rate and intestinal microenvironment of GVHD mice. In addition, Parabacteroides faecalis has a potential graft-versus-leukemia effect. The present invention uses a strain of Parabacteroides faecalis as a single strain for fecal microbiota transplantation to treat intestinal GVHD, while enhancing the graft-versus-leukemia effect, which provides a broader prospect for its application in leukemia patients. Compared with traditional FMT, the treatment of Parabacteroides faecalis with a single strain has the advantages of clear ingredients, standardization, low risk, etc., showing good application prospects. It has the advantages of standardization and low risk, showing good application prospects.
Description
Technical Field
The invention relates to application of bacteroides faecalis, in particular to application of bacteroides faecalis in preparation of a medicament for separating graft-versus-leukemia effect from graft-versus-host disease.
Background
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a key method for treating malignant blood tumors currently, and the hematopoietic stem cells of healthy donors are transplanted to reconstruct the hematopoietic and immune systems of patients so as to achieve the aim of curing the malignant blood tumors. In this process, graft versus leukemia effect (GvL) and Graft Versus Host Disease (GVHD) are two key immune responses. GvL effect refers to the process by which donor immune cells recognize and kill malignant blood tumor cells, and is of great importance in preventing recurrence of malignant blood tumors. GVHD is an immune response caused by donor immune cells attacking normal tissue of the recipient, and is a common and serious complication after allo-HSCT, severely affecting the quality of life and survival rate of patients. GVHD is a complex and severe immune response that often involves multiple organs such as the skin, liver and intestines. GVHD in the intestine is particularly severe and clinically manifested as diarrhea, abdominal pain, intestinal bleeding and the like. Currently, there is no standard two-line regimen for the treatment of hormone refractory intestinal GVHD. Although glucocorticoids are the first line therapeutic agents for GVHD, about 20% of patients are ineffective in their treatment, eventually developing hormone refractory intestinal GVHD with a very poor prognosis and a total survival rate of only 5-30%. However, maintaining GvL effects while inhibiting GVHD is a significant challenge clinically, as excessive immunosuppression may impair GvL effects, increasing the risk of recurrence of hematological malignancies.
In recent years, studies have shown that intestinal flora plays an important role in the development and progression of GVHD and GvL. Pretreatment measures such as radiotherapy and chemotherapy after transplantation seriously damage the balance of intestinal flora, reduce diversity of intestinal flora, and further aggravate GVHD symptoms. The composition and diversity of intestinal flora directly influence the immune function of the host, and healthy intestinal flora can regulate immune response, promote repair and maintenance of intestinal barrier, and thus reduce the severity of GVHD. Meanwhile, the change of intestinal flora can also influence the GvL effect, and specific flora can enhance the recognition and killing ability of donor immune cells to leukemia cells through metabolites or direct immunoregulation. However, existing FMT treatment has some technical drawbacks. First, FMT-transplanted flora is complex in composition, difficult to normalize, and affects the consistency of therapeutic effects. Secondly, FMT may introduce pathogenic bacteria or induce immune rejection, and the former treatment mode has certain potential safety hazard. The accurate treatment of the fecal fungus transplantation with definite components has the advantages of safety, definite curative effect and wide application prospect. Finally, different sources of healthy donors of FMT will affect the therapeutic efficacy, whereas standardized in vitro amplified strains have more consistent efficacy as therapeutic applications.
Disclosure of Invention
The invention aims to provide a novel application of bacteroides faecalis (Parabacteroides merdae, P.merdae).
In particular, the invention provides an application of parabacteroides faecalis P.merdae in preparing a medicament for separating a graft versus leukemia effect from a graft versus host disease.
The inventor discovers that P.merdae shows the effect of inhibiting the proliferation and inflammatory reaction of the allogeneic T cells in vitro and animal models, obviously improves the survival rate and intestinal microenvironment of GVHD mice, shows that the parabacteroides faecalis strain has the functions of high-efficiency repair and inflammation inhibition, particularly has the obvious effect of inhibiting intestinal inflammation caused by GVHD, can effectively improve systemic GVHD symptoms, including acute GVHD and chronic GVHD, can improve the survival rate after transplantation, and can enhance the graft-versus-leukemia effect. In addition, the P.merdae strain can enhance GvL effect, reduce GVHD diseases, realize the effect of separating GVL from GVHD, and have potential graft-versus-leukemia effect, thus providing wider prospect for application in leukemia patients. Compared with the traditional FMT, the P.merdae treatment of the single strain has the advantages of definite components, standardization, low risk and the like, and has good application prospect. Can be standardized, has low risk and the like, and has good application prospect.
Drawings
FIG. 1 shows the results of the identification and comparison of the P.merdae 16s RNA sequences.
FIG. 2 is a comparison of P.merdae bacteria treatment with acute GVHD transplant model.
FIG. 3 is a comparison of P.merdae bacteria treatment chronic GVHD transplant models.
FIG. 4 shows that P.merdae bacteria can improve the symptoms of GVHD in the intestinal tract by promoting the balance of Th1 and Treg, inhibiting the proliferation and activation of T cells.
Figure 5 is a graph showing that p.merdae treatment enhances survival and decreases GVHD scores in mice with a model of a20 hematological tumor transplantation.
Figure 6 is that p.merdae treatment enhances survival, reduces GVHD score and tumor burden in Baf3 hematological tumor engrafting model mice.
Detailed Description
1. Separation, preservation and in vitro culture method of bacteroides faecalis
The strain is separated and purified from the feces of healthy people, belongs to gram-negative bacteria, has short rod-shaped thalli, is often arranged singly or in pairs, strictly anaerobic, has positive reaction in tests of indole, hydrogen sulfide, methyl red, voges-Proskauer, nitrate reduction and the like, and has negative reaction in tests of contact enzyme, oxidase, citrate utilization, gelatin liquefaction, urease and the like. After 48 hours of culture on Mianyang blood agar medium, the colony is round, smooth, neat in edge, about 1-2mm in diameter and milky to pale yellow.
The preparation method of the parabacteroides faecalis sequentially comprises the following steps:
(1) Separation
Fecal samples were taken from healthy human subjects, suspended in sterile saline and mixed at a ratio of 1:10. Shaking, mixing, standing, taking supernatant, serial diluting by 10 times, coating the diluted solution on Mianyang blood agar plate, and anaerobic culturing at 37deg.C for 48-72 hr.
(2) Purification
The colonies which were initially isolated were picked and purified by plate streaking. Single colonies were picked and inoculated onto new BHI agar plates, and the culture and selection were repeated until pure strains were obtained. The inclined plane is stored in a blood plate of Mianyang and is placed in a refrigerator at 4 ℃ for standby. After 16S ribosomal DNA identification high-throughput sequencing, the NCBI database is compared, and the strain is finally determined to be the bacteroides faecalis (Parabacteroides merdae), as shown in figure 1.
(3) Preservation method of bacteroides faecalis
The culture medium for preserving the Brucella faecalis strain comprises 37.0g of brain heart extract, 10.0g of peptone, 2.0g of glucose, 5.0g of sodium chloride, 2.5g of potassium dihydrogen phosphate, 1000ml of distilled water and pH value of pH7.4. The glycerol was preserved at-80℃using the glycerol preservation method (25% glycerol to medium ratio).
(4) In-vitro amplification culture method of bacteroides faecalis
The frozen Paramycolatopsis strains were cultured at 37℃in thioglycolate medium for 24-48 hours to restore their activity. And transferring the activated bacterial liquid into a deoxidized BHI culture medium, shaking uniformly, and inoculating the bacterial liquid on a blood agar plate by streaking by using a disposable sterile inoculating loop. Blood agar plates were placed in an anaerobic incubator and anaerobically cultured at 37 ℃ for 48-72 hours using an oxygen-scavenging bag. Colony morphology, color and size were observed in the plates. The purity of the flora was monitored dynamically using fluorescent real-time quantitative polymerase chain reaction (qPCR) using primer pairs F: AGGGTGCGTAGGTGGTGAT and R: TTCACCGCTACACCACGC. The Thermo Fisher's PowerUp SYBR TM GREEN MASTER Mix kit was selected. In a 20. Mu.L qPCR reaction system, 10. Mu.L of 2X PowerUp SYBR TM GREEN MASTER Mix, 0.5. Mu.M forward primer (F), 0.5. Mu.M reverse primer (R), 2ng of template DNA were prepared and deionized water was added to the total reaction volume. qPCR amplification conditions were as follows:
| Step (a) | Temperature (temperature) | Time of | Number of cycles |
| Pre-denaturation | 95°C | 2 Minutes | 1 |
| Denaturation (denaturation) | 95°C | 15 Seconds | 40 |
| Annealing/extension | 60°C | For 1 minute | 40 |
| Melting curve analysis | 95°C | 15 Seconds | 1 |
| 60°C | For 1 minute | 1 |
The qPCR signal strongest colony on the plate is selected and inoculated in deoxygenated BHI culture medium, and the culture is anaerobic cultured, and the liquid is centrifugally changed every 48-72 hr. Viable bacteria numbers were detected by flow cytometry using SYT09 and propidium iodide staining.
2. Isolation of graft versus leukemia Effect and graft versus host disease by Paramycolatopsis
The experimental method comprises the following steps:
GVHD model construction Balb/C (H-2 d) mice and FVB (H-2 q) mice were used as donors, C57BL/6 (H-2 b) mice were used as recipients, and after receiving whole body irradiation (800-1000 cGy), bone marrow and T cells from the donors were reinfused via the tail vein to induce GVHD. In the chronic GVHD (cGVHD) model, the procedure of transplantation is similar to aGVHD, but chronic GVHD is induced by decreasing the input of naive T cells. During the experiment, mice were randomized into PBS control and P.Merdae treatment groups for intervention, and GVHD severity was scored by symptoms of skin, weight, hair, motility, etc. of the mice.
2. A method for culturing mesenteric lymph node bacteria comprises aseptically isolating lymph node from mouse mesenterium, and preparing suspension in PBS. Lymph nodes were homogenized using a tissue grinder, and the suspension was spread on LB agar plates (purchased from Soy Corp.) and incubated at 37℃for 24-48 hours. After incubation, colony Forming Units (CFU) on each plate were counted and the number of colonies was calculated based on the dilution factor. The results can be used to evaluate intestinal barrier function and flora translocation, and further to analyze the protective effect of the treatment on the intestinal barrier.
HE staining to assess histopathology tissue was fixed in 4% paraformaldehyde, dehydrated and paraffin embedded and sectioned. The sections were dewaxed with xylene, subsequently hydrated with gradient alcohol, stained with hemaloxylin for 5-10 minutes, rinsed and blued in ammonia. Then dyeing with Eosin for 30 seconds to 1 minute, and finally sealing in neutral gum after dehydration and transparentizing steps. The stained sections were observed under a microscope, the hemanoxylin stained nuclei blue, the Eosin stained cytoplasm and stroma pink for evaluation of histopathological changes.
4. Sodium fluorescein staining to observe the superficial eye surface 0.5% sodium fluorescein drop was applied to the lower eyelid to ensure uniform coverage of the cornea and conjunctiva surface with dye. The dye was adhered to the damaged area of the anterior ocular surface, and the damaged area was observed after a short period of eye closure using a slit-lamp microscope and a blue-light filter, and showed green fluorescent spots or streaks, thereby evaluating the degree of damage of cornea and conjunctiva.
5. Tear measurement standard Schirmer test paper (available from minoxidil medical instruments, se.) was placed inside the lower eyelid of the mouse for 5 minutes, and the length of the test paper soaked was representative of the amount of tear secretion.
6. CFSE staining and flow cytometry of donor T cells murine T cells were sorted with magnetic bead antibodies, added with CFSE at a concentration of 5 μm, incubated at room temperature for 10 min, and after incubation with serum-containing medium, staining was stopped, followed by washing the cells twice with PBS. Donor T cells labeled CFSE were reinfused via the tail vein back into C57 mice that were myeloablative by radiation therapy, and after one week the tissue organs were collected and ground into cell suspensions. The cell suspension was resuspended in PBS and then surface antibodies pre-labeled with fluorescent dyes (e.g., antibodies against CD4, CD8 or other markers) were added, incubated at 4℃for 30 minutes and protected from light. After incubation, cells were washed with PBS to remove unbound antibody. Finally, the fluorescence intensity of the cell surface antibody markers is detected by using a flow cytometer, and the expression of specific molecules or cell populations is analyzed.
A20 and Baf3 leukemia mice were subjected to oncological and in vivo imaging experiments first, the C57 mice were cleaned using radiotherapy, and then hematopoietic function was reconstituted by tail vein injection of donor Balb/C bone marrow cells. And simultaneously, injecting a proper amount of A20 or Ba/F3 leukemia cells into a mouse through tail vein. During the experiment, the mice are randomly grouped into PBS control groups and P.Merdae treatment groups for intervention, and the in vivo diffusion and tumor growth conditions of leukemia cells are tracked in real time through an IVIS and other living imaging systems, so that the model and the treatment effect are evaluated.
Experimental results after application of the strain, the survival rates on day 20, day 30, day 40, and day 80 were increased by 21.0%, 44.7%, 52.0%, and 52.0% respectively compared to the control in an aGVHD transplanted mouse model with C57 as donor and Balb/C as murine subject (fig. 2A and B; P < 0.05). To exclude species differences, we performed strain verification in the aGVHD transplanted mouse model with FVB as donor and C57 as murine, and the results showed that survival rates on day 20, day 30, day 40, day 80 were still improved by 25.6%, 42.6%, 48.0%, 51.2% compared to the control (fig. 2C; p < 0.05). Pathological observation of intestinal target organ in aGVHD model shows that the length of intestinal organ treated by P.Merdae strain is obviously longer than that of control group, which suggests that organ inflammation is lighter and no contracture is obvious. Colony colonies were determined in mesenteric lymph nodes and found to significantly decrease in the number of colonic invasive regional lymph nodes following treatment with the P.Merdae strain (FIG. 2D, P.Merdae group: bacterial colony mean 5CFU/plate vs control group: 35CFU/plate; P < 0.05). HE pathology staining of sections of GVHD target organs showed that lymphocyte infiltration was reduced in various target organs after p.merdae bacterial treatment, especially intestinal improvement was most pronounced, and the structures of small intestine and colon were significantly restored (fig. 2E). In addition, in the C57 donor, balb/C, murine cGVHD model, p.merdae bacterial treatment was able to reduce cGVHD symptoms, with 1, 2 and 3 clinical symptoms (P < 0.05) reduced over the control at 30, 40 and 50 days, respectively. In the detection of ocular fundus and lacrimal gland functions, the P.Merdae bacteria were found to reduce the occurrence of ocular fundus neovascularization, and lacrimal gland secretion function was significantly improved (figures 3A-C, mean of lacrimal fluid content about P.Merdae group: 2mm vs control group: 1mm; P < 0.05). The results show that the strain plays an important positive role in improving acute GVHD and chronic GVHD and has a remarkable improvement effect on improving survival rate after transplantation. The result of the treatment of GVHD model by P.Merdae bacteria was analyzed by flow cytometry, and the result shows that the P.Merdae bacteria significantly inhibit proliferation and activation of T cells, regulate balance of Th1 cells and Treg cells, reduce attack of immune cells on intestinal epidermal cells (figure 4), and promote improvement of GVHD symptoms of a recipient.
In the study of the GVL transplantation model of hematological tumor mice of A20, it was found that the P.Merdae strain treated GVHD while having no effect on GVL response (FIG. 5). In the study of the Ba/f3 blood tumor mice GVL model, the survival rate of the mice is improved by 15.7 percent, 20.2 percent and 40.8 percent respectively in 21 days, 35 days and 49 days compared with the control group, the GVHD score is reduced by 1,3 and 4 percent, and the blood tumor signal is reduced by 25 percent. These results indicate that the use of the p.merdae strain can enhance GvL effect while reducing GVHD disease, achieving the effect of separating GvL from GVHD (fig. 6).
Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the method and product of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods described herein without departing from the spirit and scope of the invention.
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| WO2020079021A1 (en) * | 2018-10-15 | 2020-04-23 | Pharmabiome Ag | Consortia of living bacteria useful for treatment of cancer related diseases, particularly graft versus host disease |
| CN115427583A (en) * | 2020-04-17 | 2022-12-02 | 玛阿特制药公司 | FMT Performance prediction test to guide and optimize treatment management in GVHD patients |
| CN118593464A (en) * | 2024-06-05 | 2024-09-06 | 福建医科大学附属第一医院 | Application of palmitoleic acid in the preparation of drugs for regulating intestinal flora and improving acute graft-versus-host disease after hematopoietic stem cell transplantation |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020079021A1 (en) * | 2018-10-15 | 2020-04-23 | Pharmabiome Ag | Consortia of living bacteria useful for treatment of cancer related diseases, particularly graft versus host disease |
| CN115427583A (en) * | 2020-04-17 | 2022-12-02 | 玛阿特制药公司 | FMT Performance prediction test to guide and optimize treatment management in GVHD patients |
| CN118593464A (en) * | 2024-06-05 | 2024-09-06 | 福建医科大学附属第一医院 | Application of palmitoleic acid in the preparation of drugs for regulating intestinal flora and improving acute graft-versus-host disease after hematopoietic stem cell transplantation |
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| GAVRIILAKI E等: "Alteration of Gut Microbiota Composition and Diversity in Acute and/or Chronic Graft-versus-Host Disease Following Hematopoietic Stem Cell Transplantation: A Prospective Cohort Study", INT J MOL SCI., vol. 25, no. 11, 26 May 2024 (2024-05-26), pages 1 - 15 * |
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