COMPOSITIONS FOR ENHANCING IMMUNE FUNCTION OF T CELLS AND PREPARATION METHODS THEREFOR

Abstract

Provided is a composition comprising Bacteroides fragilis. The composition is used for enhancing the immune function of the T cells in a body, or for preventing and/or treating tumors.

Claims

1. A composition for enhancing immune function of T cells in a body, comprising Bacteroides fragilis.

2. The composition according to claim 1, wherein the Bacteroides fragilis is one or more selected from living Bacteroides fragilis; at least one of inactivated, genetically recombined, altered or modified, attenuated, chemically treated, physically treated or inactivated Bacteroides fragilis; Bacteroides fragilis lysate; and Bacteroides fragilis culture supernatant.

3. The composition according to claim 1, wherein the Bacteroides fragilis is inactivated by any one or more of methods of dry heating, moist heating, filtration, organic solvent, chemical reagent, ultraviolet ray, infrared ray, fermentation, freeze-drying, genetic recombination, genetic modification and genetic alternation.

4. The composition according to claim 1, wherein the composition is any one of pharmaceutical compositions, foods, health products, and food additives.

5. A preparation method of the composition according to claim 1, comprising steps of: (1) collecting living Bacteroides fragilis cultures; (2) washing the Bacteroides fragilis with 15% of glycerol/saline solution, wherein the 15% of glycerol/saline solution refers to the saline solution containing 15 vol. % glycerol; (3) resuspending the Bacteroides fragilis in the 15% of glycerol/saline solution; (4) heating the Bacteroides fragilis for 30 minutes by using a 70 C. water bath to obtain inactivated Bacteroides fragilis.

6. A composition for w preventing at least one of preventing and treating a tumor, comprising Bacteroides fragilis.

7. The composition according to claim 6, wherein the Bacteroides fragilis is one or more selected from living Bacteroides fragilis; inactivated, genetically recombined, altered or modified, attenuated, chemically treated, physically treated or inactivated Bacteroides fragilis; Bacteroides fragilis lysate; and/or Bacteroides fragilis culture supernatant.

8. The composition according to claim 6, wherein the Bacteroides fragilisis inactivated by any one or more of methods of dry heating, moist heating, filtration, organic solvent, chemical reagent, ultraviolet ray, infrared ray, fermentation, freeze-drying, genetic recombination, genetic modification and genetic alternation.

9. The composition according to claim 6, wherein the composition is any one of pharmaceutical compositions, foods, health products and food additives.

10. The composition according to claim 6, wherein the tumor is one or more of breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, prostatic cancer, carcinoma of urinary bladder, melanoma, lung cancer, stomach cancer, liver cancer, cancer of biliary duct, glioma, squamous cell carcinoma of the head or neck, cervical cancer, renal cell carcinoma, thyroid cancer, acute myeloid leukemia, myeloma, esophagus cancer, lymphoma, and skin cancer.

11. A preparation method of the composition according to claim 6, comprising steps of: (1) collecting living Bacteroides fragilis cultures; (2) washing the Bacteroides fragilis with 15% of glycerol/saline solution, wherein the 15% of glycerol/saline solution refers to the saline solution containing 15 vol. % glycerol; (3) resuspending the Bacteroides fragilis in the 15% of glycerol/saline solution; (4) heating the Bacteroides fragilis for 30 minutes by using a 70 C. water bath to obtain living Bacteroides fragilis.

12. A method of enhancing the immune function of the T cells in a body, comprising administering the composition comprising Bacteroides fragilis according to claim 1 to a subject in need thereof.

13. A method of preventing or treating a tumor comprising administering the composition comprising Bacteroides fragilis according to claim 6 to a subject in need thereof.

Description

BRIEF DESCRIPTION

[0035] FIG. 1 is a schematic flow chart of an experiment for detecting the therapeutic and/or preventive effect of Bacteroides fragilis and inactivated Bacteroides fragilis on melanoma in mice.

[0036] FIG. 2 shows an in-situ detectionof inhibiting effect of Bacteroides fragilis and inactivated Bacteroides fragilis on the tumor growth in mice, wherein the dotted-line circle indicates the location and volume of the tumor. The bigger the diameter of the circle is, the faster the tumor grows and the larger the tumor volume is. It can be seen from the figure that the murine tumor treated with inactivated Bacteroides fragilis is significantly smaller than that of the other two groups.

[0037] FIG. 3 is the tumor detection photograph showing the inhibiting effect of Bacteroides fragilis and inactivated Bacteroides fragilis on the tumor growth in mice. It can be seen from the figure that the murine tumor volume is significantly decreased after a treatment of inactivated Bacteroides fragilis, compared with the saline control group or non-inactivated control group.

[0038] FIG. 4 is a comparative statistical analysis chart of tumor volume in mice after Bacteroides fragilis and inactivated Bacteroides fragilis are used for prevention and/or treatment. Compared with the saline control group or non-inactivated control group, the inactivated Bacteroides fragilis has a 40-90%, or more, better effect of inhibiting the tumor growth volume. * represents student t-test p<0.05, * represents student t-test p<0.01. Either p<0.05 or p<0.01 indicates a statistic significant difference. There are 12 mice in each group.

[0039] FIG. 5 shows a flow cytometry graph of one mouse for each group, exhibiting the expression of inhibitory molecule Tim-3 on CD4+T cells after Bacteroides fragilis and inactivated Bacteroides fragilis are used for preventing and/or treating melanoma. The number in the right-top quadrant represents the percentage of Tim-3+CD4+T cells (both Tim-3 and CD4 are expressed on the T cells) in total CD4+T cells in the spleen. As seen from the flow cytometry quadrant graph, the inactivated Bacteroides fragilis decreases the expression of Tim-3 by 38-65% or more, compared with the saline control group or inactivated control group.

[0040] FIG. 6 is a statistical analysis chart showing the expression of inhibitory molecule Tim-3 on the CD4+T cells after Bacteroides fragilis and inactivated Bacteroides fragilis are used for preventing and/or treating melanoma. As seen from the statistical chart, the inactivated Bacteroides fragilis significantly decrease the expression of Tim-3 on the CD4+T cells, compared with the saline control group or non-inactivated control group. In the statistical chart, * represents student t-test p<0.01. p<0.01 indicates a statistic significant difference. 12 mice in each group.

[0041] FIG. 7 shows a flow cytometry graph exhibiting the expression of inhibitory molecule Tim-3 on the CD8+T cells after Bacteroides fragilis and inactivated Bacteroides fragilis are used for preventing and/or treating melanoma. The number in the right-top quadrant represents the percentage of Tim-3+CD8+T cells (both Tim-3 and CD8 are expressed on the T cells) in total CD8+T cells in the spleen. As seen from the flow cytometry quadrant graph, the inactivated Bacteroides fragilis decreases the expression of Tim-3 by 35-60% or more, compared with the saline control group or non-inactivated control group.

[0042] FIG. 8 is a statistical analysis chart showing the expression of inhibitory molecule Tim-3 on the CD8+T cells after Bacteroides fragilis and inactivated Bacteroides fragilis are used for preventing and/or treating melanoma. As seen from the statistical chart, the inactivated Bacteroides fragilis significantly decrease the expression of Tim-3 on the CD8+T cells, compared with the saline control group or non-inactivated control group. In the statistical chart, * represents student t-test p<0.05. p<0.05 indicates a statistic significant difference. 12 mice in each group.

DETAILED DESCRIPTION

[0043] The present disclosure will be further described below with reference to the embodiments without any limitation. It should be pointed out that the inactivated Bacteroides fragilis for enhancing the immune function of T cells in the body, and treating and/or preventing a tumor in the present disclosure, or a pharmaceutical composition, a food, a health product and a food additive containing the Bacteroides fragilis of the present disclosure can be applied to the indications described above and exhibits the functions described above, after they are administered to the subject. The tests have been done in all dosage forms within the scope of the present disclosure. In the following, only a small part is described in the examples just for illustration, however, it should not be construed as a limitation of the disclosure.

[0044] The Bacteroides fragilis is inactivated, attenuated, low-contagious or non-contagious, and can decrease the expression of exhausted cells of T-cells on CD4+T cells and/or CD8+T cells. Preferably, the Bacteroides fragilis is inactivated by any one or more of methods of dry heating, moist heating, filtration, organic solvent, chemical reagents, ultraviolet ray, infrared ray, fermentation, freeze-drying, genetic recombination, genetic modification and genetic alternation. The tumor/cancer comprises but is not limited to solid tumor/cancer. In some embodiments, the tumor/cancer may be breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, prostatic cancer, carcinoma of urinary bladder, melanoma, lung cancer, stomach cancer, liver cancer, cancer of biliary duct, glioma, squamous cell carcinoma of the head or neck, cervical cancer, renal cell carcinoma, thyroid cancer, acute myeloid leukemia, myeloma, esophagus cancer, lymphoma, skin cancer, or any combination thereof.

EXAMPLE 1

Bacteroides fragilis Culture

Culture Method

[0045] Step 1: A lyophilized preserved Bacteroides fragilis strain (purchased from ATCC official website) was taken, then 200 L Tryptic Soy Broth (TSB) was added to reconstitute it. Bacterial solution was streaked on the blood agar plate. Then the plate was placed in a biochemical incubator after an air exhaust processing by an anaerobic jar gassing system, and cultured in anaerobic environment at 37 C. for 48 h;

[0046] Step 2: Monoclonal colony was selected and inoculated in 10 mL TSB, followed by being cultured in anaerobic environment at 37 C. for 12 h;

[0047] Step 3: 1% (v/v) of strain was inoculated in 500 mL TSB in a flask and cultured in anaerobic environment at 37 C. for 48 hours.

[0048] Step 4: The bacterial solution was collected and centrifuged at 6000 rpm for 10 min. The bacterial sludge was washed twice with saline, and was finally reconstituted with saline for later use. The viable bacteria were counted.

EXAMPLE 2

Experiment of the Therapeutic Effect of Bacteroides fragilis on Tumors (Melanoma) in Mice

[0049] FIG. 1 is a schematic flow chart of an experiment for detecting the therapeutic and/or preventive effect of Bacteroides fragilis and inactivated Bacteroides fragilis on tumors in mice (melanoma).

[0050] 1. Culture Method

[0051] The culture method of Bacteroides fragilis is the same as that in Example 1.

[0052] 2. Sample Preparation

[0053] 1) Preparation of living Bacteroides fragilis ZY-312

[0054] Step 1: A lyophilized preserved strain (purchased from ATCC official website) was taken, and then 200 L lyophilized preserved bacterial culture medium was added to reconstitute it. Then 20 L bacterial solution was pipetted, and was streaked on the blood agar plate. The plate was placed in a biochemical incubator after an air exhaust processing by an anaerobic jar gassing system, and then was cultured in anaerobic environment at 37 C. for 48 h;

[0055] Step 2: Monoclonal colony was selected and inoculated in 10 mL TSB, and followed by being cultured in anaerobic environment at 37 C. for 12 h;

[0056] Step 3: 1% (v/v) of strain was inoculated in 500 mL TSB in a flask, and cultured in anaerobic environment at 37 C. for 48 hours;

[0057] Step 4: The bacterial solution was taken and centrifuged with a centrifuge at 6000 rpm for 10 minutes. The bacterial sludge was washed twice with saline, and was finally reconstituted with saline for later use. The viable bacteria were counted.

[0058] 2) Inactivated Bacteroides fragilis

[0059] The bacterial solution was heated in a water bath at 70 C. for 30 minutes to obtain inactivated bacterial solution.

[0060] Experimental animals: Thirty-six C57BL/6 mice, 3 to 4 weeks old, in good mental state, were purchased from the Experimental Animal Center of Sun Yat-sen University. The mice were randomly divided into 3 groups, 12 mice for each, and the 3 groups were the saline control group, the Bacteroides fragilis gavage group (living bacteria group or non-inactivated group, for short), and the inactivated Bacteroides fragilis gavage group (inactivated group for short), respectively.

[0061] Experimental Process and Results:

[0062] As shown in FIG. 1, 110.sup.9 CFU of Bacteroides fragilis, 110.sup.9 CFU of inactivated Bacteroides fragilis, and saline control were respectively administered intragastrically to the three groups of mice for 2 weeks, and the body weights of the mice were measured daily. The murine tumor (melanoma) cells B16 were digested with TE after they were grown to logarithmic phase, and the medium was used for neutralizing. The cells were collected through centrifugation, and then washed twice with DPBS to remove the residual serum. The cells were resuspended with DPBS and counted. 110.sup.6B16 cells were inoculated subcutaneously into the right armpit of each mouse, and the intragastrical administration were continued. After 2 weeks, the tumor-bearing mice were killed to obtain the subcutaneous tumors, and tumor volume was measured.

[0063] Considering CD4+T cell and CD8+T cell are two important subgroups of T cells for anti-tumor in a body, anti-CD3, anti-CD4, anti-CD8 and anti-Tim-3 monoclonal fluorescent antibodies were used to label and separate the lymphocyte cells of spleen in mice, and a flow cytometry was used to analyze the percentage of expression of Tim-3 on CD4+T cells and CD8+T cells in the spleen of the mice.

[0064] The results show that both Bacteroides fragilis and its inactivated bacteria can significantly inhibit formation and growth of murine tumor (FIG. 2 and FIG. 3, the tumor is indicated by the arrow in FIG. 2). The experimental results shown in FIG. 2, FIG. 3 and FIG. 4 illustrate that the murine tumor (melanoma) in the saline control group become larger gradually over time, and the tumor in the inactivated Bacteroides fragilis group is significantly smaller than that in the saline control group and non-inactivated control group (there is statistically significant difference). These results (FIG. 2, FIG. 3 and FIG. 4) show that Bacteroides fragilis being activated can significantly enhance the anti-tumor effect of the body and inhibit the tumor growth, which is more effective for preventing and treating tumors (e.g. melanoma).

[0065] Tim-3 is an exhausted molecule to inhibit the anti-tumor function of T cell, and higher expression of Tim-3 indicates that the anti-tumor immune function of the T cells in the body is inhibited, low-active and/or exhausted. It is a key point to control and decrease the expression of Tim-3 on the T cells, in order to improve or enhance the anti-tumor immune function of the T cells in a body. The lower the expression of Tim-3 is, the better the anti-tumor immune function of the T cell has. The results shown in FIG. 5, FIG. 6, FIG. 7 and FIG. 8 indicate that the inactivated Bacteroides fragilis, compared with the saline or non-inactivated Bacteroides fragilis, can inhibit the expression of Tim-3 on CD4+T cell and/or CD8+T cell more effectively and significantly (the effect of inhibiting expression of Tim-3 is statistically significant), and thus enhance the immune function of the T cells and anti-tumor immunity of the body. This result further explains why the inactivated Bacteroides fragilis has better anti-tumor effect, compared with saline or living Bacteroides fragilis.

[0066] The above content is a further detailed description of the technical solution in combination with the preferred embodiments of the present disclosure. It cannot be considered that the specific implementation of the present disclosure is limited to the above descriptions. For those skilled in the art, any simple deductions or replacements without departing from the inventive concept of the invention should all be regarded as belonging to the protection scope of the invention.