COMPOSITION COMPRISING RECOMBINANT FUSION PROTEIN OF PATHOGENIC ANTIGEN PROTEIN AND FLAGELLIN OF VIBRIO VULNIFICUS FOR PREVENTING, ALLEVIATING, OR TREATING AGING

Abstract

The present invention relates to a composition for preventing, improving, or treating aging, wherein the composition comprises a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen, as an active component. According to the present invention, the recombinant protein of the present invention can improve external and internal aging-related malfunctions and enhance immunity. Also, the composition of the present invention can easily perform immunization through mucosal administration.

Claims

1-11. (canceled)

12. A method for preventing, improving, or treating metabolic disease, the method comprising administering to a subject the composition comprising a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen, as an active ingredient.

13. The method according to claim 12, wherein the flagellin is FlaB, which is flagellin of Vibrio vulnificus.

14. The method according to claim 12, wherein the pathogenic protein antigen is surface protein A (PspA) of Streptococcus pneumonia.

15. The method according to claim 12, wherein the recombinant protein is FlaB-PspA protein having an amino acid sequence of SEQ ID NO: 1.

16. The method according to claim 12, wherein the recombinant protein enhances immunity.

17. The method according to claim 12, wherein the metabolic disease is caused by aging.

18. The method according to claim 12, wherein the metabolic disease is a syndrome in which risk factors, such as obesity, diabetes, hypertriglyceridemia, hypertension, cardiovascular disease, and blood clotting, are shown together.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 shows a schematic view with respect to an experiment schedule and an experiment scheme for immunizing aged mice.

[0062] FIG. 2 shows graphs illustrating measurement results of changes in food intake and body weight in aged mice according to the immunization with FlaB-PspA recombinant protein.

[0063] FIG. 3 shows morphological change results of aged mice through continuous immunization of an antigen.

[0064] FIG. 4 is an IgA reaction graph in aged mice by continuous immunization.

[0065] FIG. 5 illustrates histopathological findings with respect to the cutaneous histological change of aged mice by immunization with FlaB-PspA recombinant protein.

[0066] FIG. 6 shows the changes of hematopoietic stem cells in marrow cells of aged mice by immunization with FlaB-PspA recombinant protein.

[0067] FIG. 7 shows organs illustrating lymphatic system changes of aged mice by immunization with FlaB-PspA recombinant protein.

[0068] FIG. 8 shows the change in bone mineral density of aged mice by immunization with FlaB-PspA recombinant protein.

MODE FOR CARRYING OUT THE INVENTION

[0069] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Materials and Methods

[0070] Aged mice (C57BL/6J, aged at least 23 months) were intranasally immunized with purified protein and recombinant protein continuously at two-week intervals. PspA protein and FlaB-PspA protein were purchased from the laboratory of professor Jun-haeng, Lee of the Clinical Vaccine Development Project Group of Chonnam National University.

[0071] All immunization experiments were conducted in the specific pathogen free (SPF) facility. Young mice (8-10 week old) were used for a control group. A non-immunized aged mouse group, an aged mouse group intranasally immunized with PBS (16 l/mouse) 8 times or more at 2-week intervals, an aged group intranasally immunized with only PspA (2.5 g/16 l/mouse) protein 8 times or more at 2-week intervals, and an aged group intranasally immunized with FlaB-PspA (6.5 g/16 l/mouse) recombinant protein 8 times or more at 2-week intervals were used for immunization experimental groups.

[0072] The morphological and behavioral changes of the aged mice were observed during the continuous immunization.

[0073] Following the immunization, the body weight and feed intake of each mouse were measured every week. In order to accurately measure the changes according to the immunization, the mice were separately managed one by one. In addition, 50 g of feed was provided for each mouse, and then the remainder of the feed was accurately measured at one-week intervals. Also, the body weight of each mouse was accurately measured using an animal scale every week.

[0074] In order to compare morphological changes of aged mice according to the immunization, the appearances of the mice, that is, the hair condition, hair loss, and decoloration was observed and the anus or eyes were also continuously observed, thereby collecting changed patterns.

[0075] In order to compare the behavior changes of the aged mice according to the immunization, the behavioral changes of the mice were observed at a certain point during the immunization. In order to verify the behavioral ability of the mice, the aged mice of each group were placed in a confined space, and the motions of the mice were observed for a period of time. The motions were compared based on general standard items of mouse behavioral ability, that is, the motion, the number of times of standing on hind legs, the number of times of supporting using forelegs, and the number of times of touching the nose.

[0076] During the continuous immunization, in order to verify the change of the immune response depending on the number of times of immunization, blood and feces were collected from some mice, and then the change in antibody production was measured using enzyme-linked immunosorbent assay (ELISA).

[0077] After immunization for an appropriate period of time, several samples were collected from the overall mice, and then the entire changes of the aged mice according to the immunization were measured through various experiments.

[0078] In order to verify the change of the immune response according to the continuous immunization, serum and several mucous samples (feces, saliva, vaginal washing, etc.) were collected from the mice, and in order to compare the mucosal immune response, the change in secretory immunoglobulin A (secretory IgA, slgA) production was measured by ELISA.

[0079] In order to compare the morphological changes of the aged mice according to the immunization, various tissues including the skin tissue were collected, and fixed with formalin prior to making a paraffin-block, and pathological findings were compared through Hematoxylin and Eosin (H&E) staining. In addition, with reference to the effect of immunization and the feed intake through the mucosal immune response, pathological findings of the tissues of small and large intestines, which largely account for the mucosal immunity, were compared and observed through H&E staining.

[0080] In order to compare the antigen-mediated cellular immune response with respect to lymphocytes separated from immune-related tissues, the lymphocytes were separated from the cervical lymph nodes and spleen and then the antigen-mediated cellular response was compared by ELISA. In addition, in order to compare and observe changed patterns of various bone marrow cells with respect to the cells separated from the bone marrow, the bone marrow cells were separated from hind legs of the aged mice, and the comparison was conducted using flow cytometry (FACS, Beckman Coulter). Particularly, the frequency of hematopoietic stem cells essentially associated with T cell differentiation was compared and observed through immuno-staining using CD34, which is a hematopoietic stem cell indicator.

[0081] In order to compare the change in bone mineral density (BMD) of the aged mice according to the immunization with respect to spines separated from the mice, the spines were extracted from the mice, and then the bone mineral density (BMD) according to the continuous immunization and kyphosis according to aging of the aged mice were compared and observed using micro-computer tomography (microCT: Skyscan 1172, Micro Photonics Inc., US).

[0082] Next, for the ongoing study, the morphological and behavioral changes of the aged mice according to the continuous immunization were compared and compared in connection with metabolism. For this, after the blood was collected from the aged mice, the serum or plasma was separated therefrom, and then the changes in hormone-related and metabolism-related genes were compared. In addition, in order to compare and observe the change in mucosal immunity according to the immunization, the intestine-associated microenvironment was compared using normal microbiota. In addition, the blood and feces were collected from the aged mice continuously immunized with antigens, and then the gene expression pattern was compared and observed through advanced analysis methods, such as microarray.

Results

Example 1: Changes in Feed Intake and Body Weight in Aged Mice Immunized with Flagellin-PspA Recombinant Protein

[0083] While the aged mice were intranasally immunized eight times at two-week intervals, the body weight and the feed intake of the mice of each group were measured. The non-immunized aged mice were used as a control group.

[0084] The aged mice were intranasally immunized eight times with phosphate buffered saline (PBS), prepared 2.5 g of PspA, and 6.5 g of FlaB-PspA recombinant protein at two-week intervals. The body weight and the feed intake of the mice of each group were measured every week. The measurement results are shown in FIGS. 2a and 2b.

[0085] As a result of body weight measurement, it was verified that the non-immunized mouse group, the group immunized with PBS, the group immunized with PspA alone, and the group immunized with FlaB-PspA recombinant protein showed no change in body weight due to the immunization (FIG. 2a).

[0086] As a result of comparing the feed intake of the aged mice according to the immunization, the non-immunized mouse group showed no great change in the feed intake over time, and the group immunized with PBS also showed no great change in the feed intake, regardless of eight times of immunization at two-week intervals. On the other hand, the group immunized with PspA alone was verified to show a gradual increase in the feed intake during the continuous immunization. Particularly, it was shown that the group immunized with FlaB-PspA recombinant protein showed an increase in the feed intake through the continuous immunization, and here, the rate in increase of the feed intake was significantly higher than that of the group immunized with PspA alone (FIG. 2b).

[0087] As can be seen from the results of example 1, it can be confirmed that, when the aged mice are continuously immunized with antigens, the body change is not greatly changed, but the feed intake is greatly increased. Particularly, it can be seen that the immunization with a recombinant protein including a vaccine adjuvant fused with a pathogenic antigen significantly increases the feed intake.

Example 2: Observation of Morphological Changes of Mice Through Continuous Immunization with Antigen

[0088] While the aged mice were intranasally immunized eight times at two-week intervals, the morphological changes of the mice according to the immunization were monitored every week. The results are shown in FIG. 3. The non-immunized aged mice were used as a control group.

[0089] The aged mice prior to the immunization had no abnormal findings by appearances. There were no abnormal findings in view of hair condition, hair luster, hair decoloration, hair loss, the anus (colitis or hernia), or the eyes (cataracts).

[0090] However, as a result of observing the aged mice following the immunization, it was verified that the non-immunized aged mouse group had a worse hair condition than the group immunized eight times at two-week intervals, and the appearances became generally worse, such as a severe progression of hair decoloration or hair loss. Besides, normal findings, such as hernia, were severely shown in the anus, and abnormal findings, such as suspected cataracts, were severely shown also in both eyes (O-control). The group immunized with PBS also had a bad hair condition, and the appearances thereof became severely worse, such as severe progressions of hair decoloration and hair loss. In addition, findings of slight colitis or hernia were shown in the anus, and findings of suspected cataracts were severely shown in one eye (O-PBS).

[0091] On the other hand, as a result of eight times of immunization with PspA, slight decoloration was shown but the hair loss was not severe in the appearance of the aged mice, and abnormal findings were not observed in the anus or eyes (O-PspA). Particularly, it was verified that, the group immunized with FlaB-PspA recombinant protein had a good hair condition so that the hair condition of the aged mice of the group was very similar to that of the aged mice prior to the immunization, and abnormal findings were observed in neither the anus nor eyes (O-FlaB-PspA).

[0092] As can be seen from the results of example 2, it can be verified that the appearances of the aged mice are better through the continuous immunization. It is general that the aging causes a severe progression of hair decoloration or hair loss and abnormal findings, such as cataracts occurring in eyes. Rodents showed abnormal findings, such as hernia, in the anus due to the reduction in the muscle amount. However, the continuous immunization with antigens could be verified to prevent the occurrence of such abnormal findings. Particularly, it can be seen that, as for the group immunized with a recombinant protein including a vaccine adjuvant fused with a pathogenic antigen, the appearances of the aged mice are maintained over time or have a better condition.

Example 3: Change in IgA Reaction of Aged Mice Through Continuous Immunization with Antigen

[0093] During the continuous immunization, the feces were collected from the mice of each group after each immunization to verify the IgA reaction of the aged mice according to the immunization by ELISA. The results are shown in FIG. 4. The non-immunized aged mice were used as a control group.

[0094] The non-immunized aged mice had no great difference in the IgA response during the immunization (0-control). It was verified that the group immunized with PBS alone showed no great difference in the IgA response, and then showed a slight increase in the IgA response after the sixth immunization, but there is no great increase in the IgA response (O-PBS). The group treated with PspA alone showed the IgA response after the fourth immunization, but showed no great difference after that (O-PspA). Whereas, as for the group immunized with FlaB-PspA recombinant protein, the IgA response was significantly increased depending on the number of times of immunization (O-FlaB-PspA).

[0095] As can be seen from the results of example 3, it was verified that, as a result of verifying the IgA reaction with respect to the mucosal immune reaction of the aged mice through the continuous immunization, the IgA reaction was significantly increased depending on the immunization in the group immunized with FlaB-PspA recombinant protein while no great change in the IgA reaction or a slight IgA reaction was shown in the other groups. Judging from the results, the mucosal immunity of the aged mice is activated by the recombinant protein.

Example 4: Skin Histological Change of Aged Mice Through Immunization with Recombinant Protein

[0096] The results of example 2 indicated that the hair condition of the aged mice became very favorable in the group continuously immunized with antigens, particularly, the recombinant protein. In order to prove these results in more detail, the results were confirmed through H&E staining. The H&E staining results are shown in FIG. 5. Young mice were used as a control group.

[0097] As a result of verifying H&E staining on the skin tissue of the back of the aged mice, it is general that the aging causes the progression of hair loss and the thinning of the dermis layer. It can be confirmed that, as for the young mice, the dermis layer is thick and a lot of hair follicles, that is, where hairs grow, exist in the dermis layer (Y-control). Whereas, it can be confirmed that, as for the aged mice, the dermis layer become thin and the number of hair follicles is significantly reduced (0-control). It could be verified that, when the aged mice were continuously immunized with an antigen (O-PspA) and a recombinant protein (0-FlaB-PspA), the dermis layer became thickened and the number of hair follicles was increased. Particularly, it could be verified that the number of hair follicles was significantly increased in the group immunized with FlaB-PspA recombinant protein.

[0098] As can be seen from the results of example 4, the histological assay confirmed that the continuous immunization with antigens improved the morphological findings of the aged mice (results of example 2). It could be confirmed through the histological assay that the amelioration of the progression of hair decoloration and hair loss of the aged mice, shown in the results of example 2, is due to the fact that the hair follicles are maintained in the aged mice due to the continuous immunization. In view of the results, it can be seen that the continuous immunization with the recombinant protein slows or prevents the progression of hair decoloration and hair loss occurring due to aging.

Example 5: Change in Bone Marrow Cells in Bone Marrow Through Immunization with Recombinant Protein

[0099] The change in bone marrow cells in the bone marrow of the aged mice through the continuous immunization was verified, and the results are shown in FIG. 6. Non-immunized aged mice were used as a control group.

[0100] As a result of observing the change in bone marrow cells with respect to cells separated from the bone marrow of the aged mice, there is no difference between groups in view of cellularity.

[0101] As a result of confirming the proportions of bone marrow cells (megakaryocyte, myeloid, and erythroid lineages), it was verified that the bone marrow cells were generally well maintained and differentiated in the aged mice.

[0102] In addition, as a result of verifying the frequency of hematopoietic stem cells through immuno-staining using CD34, which is a hematopoietic stem cell indicator, it could be verified that CD34 was increased in the aged mice continuously immunized with FlaB-PspA recombinant protein rather than in the other groups.

[0103] As can be seen from the results of example 5, it could be verified that the continuous immunization with the recombinant protein is associated with not only the changes in the simple appearances but also the immune-related effects in the old mice.

Example 6: Changes of Lymphatic System Organs Through Immunization with Recombinant Protein

[0104] After the continuous immunization for an appropriate period of time, organs were extracted from the mice of each group, and the morphology and weight of each organ were measured. The measurement results are shown in FIG. 7. Young mice were used as a control group.

[0105] The tissues were extracted from the aged mice, and the morphological change and weight of each of the organs were measured. As a result, the difference caused by the continuous immunization was observed in the lymphatic system-related organs rather than in the other organs. Particularly, it was verified that the morphology and weight of the organs in the group immunized with FlaB-PspA recombinant protein were similar to those of the young mice.

[0106] Thymic involution with age is a general phenomenon, but it was verified that the thymus weight was increased in the aged mouse groups continuously immunized with antigens, and particularly, the thymus weight was significantly increased in the group immunized with FlaB-PspA recombinant protein. Besides, even though the mesenteric lymph nodes (MLNs) or the spleen, which are frequently used for systemic inflammation responses, mostly undergo a very hypertrophic morphology with age due to the continuous infection, it was verified that the thymus weight was larger in the aged mice continuously immunized with FlaB-PspA recombinant protein rather than in the other groups; and the spleen weight was smaller in the aged mice continuously immunized with FlaB-PspA recombinant protein rather than in the other groups, and the spleen morphology of the aged mice continuously immunized with FlaB-PspA recombinant protein was also similar to that of the young mice.

[0107] As can be seen from the results of example 6, it could be verified that the aged mice continuously immunized with antigens exhibited improved morphological features of the immune-related organs. Particularly, the aged mice immunized with FlaB-PspA recombinant protein were observed to have very similar organ morphological findings to the young mice.

Example 7: Change in Bone Mineral Density of Aged Mice Through Immunization with Recombinant Protein

[0108] Spines were extracted from the aged mice, and the change in bone mineral density through the immunization with the recombinant protein of the aged mice was measured using micro-computer tomography (microCT). The measurement results are shown in FIG. 8. Young mice were used as a control group.

[0109] After eight times of continuous immunization, the spines were extracted from the aged mice, and the change in bone mineral density through immunization was measured using microCT. As a result, it could be verified that the bone mineral density was significantly improved in the group immunized with the recombinant protein. In connection with example 1, it could be thought that the facts that the body weight was not changed depending on the number of times of immunization and the feed intake was increased in only the group immunized with the recombinant protein are associated with the motion quantity of the aged mice, and it could be explained that, in this regard, the bone mineral density was more significantly increased in the group continuously immunized with the recombinant protein rather than in the other aged mouse groups.

[0110] Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.