TW201740974A - Method for and use of blocking immunosuppressive functions of pathogens - Google Patents

Abstract

The present invention provides a method for blocking immunosuppressive functions of pathogens, comprising: applying to an environment where the pathogens exist a composition that blocks immunosuppressive functions of the pathogens. The immunosuppressive functions are provided by an immunosuppressive substance secreted or produced by the pathogens. The composition includes a function inhibitor that comprises an antibody identifying the immunosuppressive substance or a fragment thereof. Use of the function inhibitor composition is also disclosed.

Description

Method for closing immunosuppressive function of pathogen and use thereof

The present invention relates to a method for shutting down the immunosuppressive function of a pathogen and its use, and more particularly to a method for shutting down an immunosuppressive substance secreted by a pathogen to enhance the immunity of the host and its use.

Helicobacter Pylori ( H. pylori ) is a Gram-negative bacterium that has been infected in half of the world. Chronic inflammation caused by H. pylori can lead to several outcomes ranging from peptic ulcers to gastric cancer, depending on the extent and extent of gastric inflammation caused. Although the host mainly produces a mucosal immune response that tends to Th1 type, it is unable to achieve a degree sufficient to protect the host from H. pylori infection, resulting in the formation of a chronic infection, and will develop into a gastric cancer lesion in some patients. Previous studies have demonstrated that H. pylori lysates inhibit the proliferation of T cells that induce mitogens. It is shown that there is a factor associated with immunosuppressive activity in the lysate. These factors can attenuate the activity of T cells, and their mechanism of action is independent of the bacterial virulence factors CagA and VacA. Researchers have proposed several mechanisms to explain why H. pylori can directly or indirectly inhibit the immune response of T-cells. Including: Helicobacter pylori inhibits T cell proliferation and receptor expression on T cells by arginase; H. pylori stimulates release of immunosuppressive hormone TGF-β; H. pylori interferes with VacA The expression of the antigen without a change chain; H. pylori negatively regulates the function of DCs to phosphorylate intracellular proteins by CagA; or H. pylori inhibits the phagocytic function of macrophages via VirB7 and VirB11. and many more.

Although the above various mechanisms may be justified, the current industry believes that regulatory T cells (regulatory T-cells) are the main regulatory factors that inhibit T cell activity and balance inflammation and bacterial persistent infection. It was reported in 2003 that CD4 ⁺ CD25 ⁺ T cells are associated with H. pylori-induced immunosuppression and its parasitism. Further studies have shown that host Treg cells are the primary key to protecting H. pylori-infected hosts from excessive gastric inflammation and disease symptoms, but also promote bacterial parasites on the stomach and duodenal mucosa. In addition, the co-stimulatory factor B7-H1 exhibited by gastric epithelial cells in patients also promotes the development of gastric epithelial cells of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Treg cells after H. pylori infection. This means that this pathogen will promote the induction of host Treg cells. Subsequent studies examined the function of these Helicobacter pylori-induced Treg cells. The results showed that this Treg cell can inhibit the action of H. pylori-specific T cells and disable them. In addition, gastritis caused by Helicobacter pylori often coincides with the infiltration of FoxP3 ⁺ Treg cells, and the degree of bacterial parasitism is related to the degree of expression of TGF-β in the mucosa. The above various studies have shown that the host Treg response induced by H. pylori is an important regulatory factor for the host H. pylori immune response and the pathogenic mechanism of H. pylori-associated diseases.

H. pylori heat shock protein 60 (HpHSP60) can induce the secretion of proinflammatory cytokines and TGF-β1 by monocytes. It has been reported that HpHSP60 is expressed in the cell wall of bacteria together with urease, and can be used as an adhesion molecule of gastric Helicobacter pylori to gastric epithelial cells. In addition, studies have found that administration of anti-HpHSP60 antibodies can interfere with the growth of H. pylori. Therefore, HpHSP60 is not only an important factor affecting the viability of H. pylori, but also provides the Helicobacter pylori required for human gastric parasitism. However, many studies have also shown that HpHSP60 is an immunogen that strongly stimulates the production of pro-inflammatory cytokines such as TNF-α, IL-8 and IL-6. These cytokines cause an inflammatory response at the site of infection, and this HpHSP60-induced inflammatory response promotes tumor malignancy, including vascular proliferation and cancer cell migration. HpHSP60 is also an important virulence factor for human Helicobacter pylori infection in human hosts.

Due to the complex relationship between HpHSP60 and Treg cells, the study of the relationship between the two has become an important issue in this industry. However, past studies have focused on the inflammatory response induced by HpHSP60. Little is known about the relationship between HpHSP60 and host immunosuppression.

U.S. Patent No. 6,403,099 is directed to a conjugated compound formed by a heat-stressed protein and a polysaccharide or oligosaccharide. The compound induces the formation of an anti-polysaccharide antibody and can be used as a vaccine for humans and animals. Wherein, the heat-stressing protein comprises H. pylori heat-stressing protein.

It is an object of the present invention to provide a method of shutting down the immunosuppressive function of a pathogenic agent. It is also an object of the present invention to provide a use for shutting down the immunosuppressive function of a pathogenic agent.

It is also an object of the present invention to provide a method of shutting down the action of an immunosuppressive substance secreted by a pathogen, and the use of the method.

It is also an object of the present invention to provide a novel method of improving immunity in vivo and its use.

According to the present invention, several pathogens produce a function of inhibiting host immunity. This function is provided in particular by immunosuppressive substances secreted or produced by the pathogen. By shutting down the action of the immunosuppressive substance, the immunosuppressive function of the pathogen can be turned off. Due to this immunosuppressive function By the time of closure, the host's own immune function is not inhibited. The purpose of reducing or even eliminating the pathogen can be achieved by the uninhibited immune function.

According to one aspect of the present invention, there is provided a novel method for improving immunity in a living body, and the use of the method for preparing a medicament for preventing or treating a disease.

According to the present invention, the method for improving the immunity of a living body comprises directly or indirectly administering an immunosuppressive function capable of blocking the bacterial pathogen to a bacterial pathogen hosted in the living body or in vitro. The steps of the composition. In a particular embodiment of the invention, the composition is administered to a living host to which the pathogen is attached. In such and other instances, the immunosuppressive function of the pathogenic agent is provided by an immunosuppressive substance secreted or produced by the pathogenic agent. Therefore, the composition for shutting down the immunosuppressive function of the pathogenic agent includes a functional inhibitor that shuts down the action of the immunosuppressive substance. Wherein, the functional inhibitor may comprise an antibody that recognizes the immunosuppressive substance or a part thereof. The immunosuppressive substance can be a protein, a portion of which is part of the amino acid sequence of the protein. The antibody may be an antibody naturally produced by an immune response to the immunosuppressive substance in vivo. The antibody may be an antibody to which the aforementioned antibody is humanized.

The composition can include an effective amount of the functional inhibitor and a pharmaceutically acceptable carrier or diluent thereof. Adjuvants that increase the effectiveness of immunostimulation may also be included.

Uses of the invention include the use of such a functional inhibitor to prepare a medicament that stimulates or enhances an immune response against the pathogenic agent. The use of the present invention also encompasses the use of the functional inhibitor to prepare a medicament for treating or preventing a disease or condition associated with the presence or abnormal expression of the pathogenic agent.

According to a particular embodiment of the invention, a method of enhancing the immunity of a living organism comprises administering to the environment in which the pathogen is present a functional inhibitor composition to shut down the action of the heat-stressing protein secreted or produced by the pathogenic agent. In this type of embodiment, the pathogen environment includes the The living host is attached to the pathogen. The pathogen may include H. pylori and other bacteria attached to the surface of the living body.

In such and other instances, the functional inhibitor can include an antibody that recognizes the heat-stressing protein or a portion thereof of the amino acid sequence. The antibody may be an antibody naturally produced by an immunological reaction of the heat-stressed protein or a portion of the amino acid sequence thereof in vivo. The antibody may be an antibody to which the aforementioned antibody is humanized.

The composition can include an effective amount of the functional inhibitor and a pharmaceutically acceptable carrier or diluent thereof. Adjuvants that increase the effectiveness of immunostimulation may also be included.

Uses of the invention include the use of such a functional inhibitor to prepare a medicament that stimulates or enhances an immune response against the pathogenic agent. The use of the present invention also encompasses the use of the functional inhibitor to prepare a medicament for treating or preventing a disease or condition associated with the presence or abnormal expression of the pathogenic agent.

Figure 1 shows the experimental results of the effect of HpHSP60 on the proliferation of PBMC.

Figure 2 shows the results of an experiment in which HpHSP60 affects T cell proliferation in PBMC.

Figure 3 shows the results of an experiment to determine the effect of HpHSP60 on the cell cycle of PBMC.

Figure 4 shows the results of in vitro induction of Treg cells by HpHSP60.

Figure 5 shows the results of an experiment to test that HpHSP60 promotes Treg proliferation.

Figure 6 shows the results of an experiment in which HpHSP60-inducible Treg cells inhibit T cell proliferation.

Figure 7 shows the results of an experiment in which H. pylori is inhibited by HSP60 in a living animal to inhibit cell growth.

Figure 8 also shows the results of an experiment in which H. pylori is inhibited by HSP60 in a living animal to inhibit cell growth.

Figure 9 shows the results of an experiment in which Treg was inhibited by inhibition of HSP60 in a living animal.

Figure 10 shows the results of detection of HpHSP60 with the sequence of activity that induces Treg cells.

Figure 11 shows the results of the data of the experimental results of Figure 10.

Figure 12 shows the results of an experiment exploring the immune mechanism of the anti-HpHSP60 antibody.

Figure 13 shows the results of detection of the expression of Treg cells in the gastric mucosa by an anti-HpHSP60 antibody.

Figure 14 shows the results of detection of the expression of IL-10 in the gastric mucosa by an anti-HpHSP60 antibody.

Figure 15 shows the results of an experiment to detect a fragment of HpHSP60 recognizable by the LHP-1 (9E4) antibody.

Figure 16 shows the results of an experiment to further detect the HpHSP60 fragment recognizable by the LHP-1 (9E4) antibody.

While not wishing to be bound or limited by any theory, in accordance with the present invention, several pathogens may produce or secrete an immunosuppressive substance to inhibit the host's ability to immunosist, as a result of causing pathogenic hyperplasia to cause host disease. Such pathogens include Helicobacter pylori and other similar bacteria, such as Arcobacter suis , Tannerella forsythia, Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , Helicobacter felis and the like. The present inventors have found that a heat-stressing protein is one of such immunosuppressive substances. According to an embodiment of the present invention, H. pylori heat-stressing protein (HpHSP60) can stimulate the production of immunosuppressive hormones IL-10 and TGF-β by acting with monocytes, thereby inducing proliferation of Treg cells, resulting in a host. Immunosuppression, resulting in a chronic infection against H. pylori.

The invention further develops a new method for improving the immunity of the host, and uses the functional inhibitor which can shut down the action of the immunosuppressive substance to close the action of the immunosuppressive substance, effectively inhibit the proliferation of Treg cells, and eliminate the phenomenon of immunosuppression.

The functional inhibitor proposed by the present invention comprises a substance capable of recognizing the immunosuppressive substance or a part thereof and blocking the action of the immunosuppressive substance. When the immunosuppressive substance is a heat-stressing protein, the functional inhibitor includes an antibody which recognizes the amino acid sequence of the heat-impressed protein or a part thereof.

The functional inhibitor can form a form of monoclonal antibody for mass production. The functional inhibitor can be combined with a pharmaceutically acceptable carrier or diluent to form a pharmaceutical composition. The composition may also contain an adjuvant that increases the effectiveness of the immunostimulatory. The adjuvant may include the immunostimulating agent shown in the aforementioned U.S. Patent No. 6,403,099. The pharmaceutical composition is useful as a medicament for stimulating or enhancing an immune response against the pathogenic agent. It is also useful as a medicament for treating or preventing a disease or condition associated with the presence or abnormal expression of the pathogenic agent.

The method of the immunosuppressive function of the off-pathogen of the present invention and its use will be described below by way of examples and with reference to the accompanying drawings. However, it should be understood that the scope of the invention is not limited by the scope of the embodiments. For example, although this example is exemplified by the pathogenic mechanism of H. pylori and a functional inhibitor of H. pylori heat-stressing protein 60 (HpHSP60), for example, Helicobacter felis (which can cause chronic enteritis in humans) and Arcobacter suis (can be used) The heat-stressed protein amino acid sequence leading to human periodontal disease also contains the same fragment as HpHSP60, such as HSP 60 101-200. The methods and uses of the present invention are applicable to such and other pathogenic bacteria, as well as other pathogens having the same pathogenic mechanisms.

Example 1: Cell culture, separation of PBMC from T-cells

Human peripheral blood mononuclear cells (PBMC) supplied by healthy donors were separated by density gradient centrifugation using Ficoll-Paque Plus (trademark, GE Healthcare, Uppsala, Sweden), and resuspended in 10% fetal bovine serum and 1% penicillin-streptomycin in RPMI-1640 medium. For removal of monocytes, PBMCs were incubated overnight in a 10-cm culture dish at a density of 10 ⁶ /mL to allow monocytes to attach. The suspended cells were collected after centrifugation at 1500 rpm for 15 minutes. All T cells obtained from the PBMC were subjected to negative screening separation using a magnetic sorting device (Miltenyi Biotec, Inc., Massachusetts, USA). Briefly, PBMCs were co-cultured with a mixture of non-T cell antibodies (the antibody mixture was grafted onto Biotin), followed by reaction with microbeads containing anti-Biotin antibodies, and non-T cells-Biotin-anti using a magnetic base. - Biotin-magnetic bead complex adsorption, T cells are collected after the bank is washed by the manufacturer's levee.

Example 2: Effect of HpHSP60 on proliferation of PBMC

The cell proliferation was analyzed by cell proliferation assay using PBMC activated with CD3 mAb, different doses of HpHSP60, rGFP and boiled HpHSP60. 0.2 ml of each group of cells was seeded at each well at a concentration of 1 × 10 ⁶ cells/ml in each well of a 96-well microplate pretreated with anti-CD3 monoclonal antibody. Cell proliferation was measured by MTT assay after 96 hours. Figure 1 shows the experimental results. The values shown in the figure are the proliferation index. The cell proliferation index was calculated as follows: Hyperplasia index (100%) = (OD ₅₉₅ value of cells treated with anti-CD3 + HpHSP60) / (OD ₅₉₅ value of cells treated with anti-CD3-) x 100%. Compared with the control group not added with HpHSP60, if there was a significant difference, it was expressed by * (P < 0.05) (n = 15).

Figure 1 shows that (◆) T cell proliferation is inhibited after HpHSP60 is added to human peripheral mononuclear cells (PBMC). (■) rGFP is an experimental system for controlling histones and has no effect on T cell proliferation. It can be seen from the control group that not all proteins can inhibit T cell proliferation. (▲) Boiled HpHSP60 indicates that only the HpHSP60 sequence is boiled to lose protein structure, which denatures the protein. The experimental results show that the HpHSP60 sequence after boiling does not affect T cell proliferation.

Example 3: Effect of HpHSP60 on T cell proliferation in PBMC

PBMCs containing T-cells or T-free cells were treated with anti-CD3 monoclonal antibody, with or without HpHSP60 (200 ng), and the number of cells was calculated after staining with CD3 surface marker. When CD3 surface marker staining was performed, the collected cells were stained with CD3 monoclonal antibody (OKT3). This was followed by staining with IgG FITC fluorescent secondary antibody (Biolegend, Inc., California, USA). For intracellular staining of FoxP3, cells were collected and stained with CD4 FITC fluorescent monoclonal antibody (Biolegend, Inc., California, USA). The treated cells were fixed and the cell membrane was holed. Intracellular staining was then performed using FoxP3-PE monoclonal antibody (BD Biosciences, Inc., Massachusetts, USA) according to the manufacturer's specifications. Cell cycle analysis was then performed: cells were fixed with 70% ethanol after 72 hours. Further, DNA staining buffer (5% Triton-X 100, 0.1 mg/ml of RNase A and 4 μg/ml of PI) was stained for 30 minutes at room temperature, after which the change in DNA content was examined. Fluorescence analysis was performed using a FACS flow cytometer (Becton Dickinson, Heidelberg, Germany) and a CELLQUEST Pro program (Becton Dickinson, Heidelberg, Germany).

The experimental results are shown in Figure 2. The numerical proliferation index (multiples) in Figure 2 = (number of T cells or non-T cells in the anti-CD3/HpHSP60 treated group) / (number of T cells or non-T cells in the untreated control group). If there is a significant difference, it is represented by * (P < 0.05), (N = 4). The experimental results show that HpHSP60 can inhibit the proliferation of T cells in PBMC. In the figure, (□) shows the part of T cells in PBMC; (■) shows the part of non-T cells in PBMC. From the results, it was found that HpHSP60 is inhibited against T cells.

Example 4: Determination of the effect of HpHSP60 on cell cycle

PBMC cells were taken, and the percentages of subG1, G1, S and G2/M phases were observed by PBMC cells themselves, CD3-activated PBMC cells, and Anti-CD3+HpHSP60-treated PBMC. The results are shown in the histogram of Fig. 3. The figure shows the results of three replicate experiments.

Figure 3 shows that HpHSP60 inhibits T cell proliferation rather than causing T cell death. In the figure, Cell alone in Fig. 3A shows that normal T cells are in a dormant phase (G0/G1) without CD3 activation. Anti-CD3 of Figure 3B shows that after CD3 activation, T cells begin to grow and grow, forming a typical cell cycle pattern. The Anti-CD3+HpHSP60 of Figure 3C showed no change compared to Figure 3B, and the ratio in the Sub G0/G1 phase (the DNA fragment representing cell death) did not increase compared to the Anti-CD3 group. The experimental results show that the effect of HpHSP60 is to inhibit the growth of T cells, rather than causing T cell death.

Example 5: In vitro induction of Treg cells by HpHSP60

For PBMC cells treated with HpHSP60, the ratio of CD4 ⁺ FoxP3 ⁺ cells was measured over time. Compared with the control group treated only with anti-CD3, if there is a significant difference, it is represented by * (P < 0.05) (n = 5). The results are shown in Figure 4.

Since CD4 and FoxP3 are markers of Treg cells, the effect of HpHSP60 on the growth of Treg cells can be directly identified from Figure 4. In the figure, (●) cell alone indicates the original growth of Treg cells. (■) anti-CD3 indicates the growth of Treg cells after activation with CD3. (▲) Anti-CD3+HpHSP60 indicates that Treg cells proliferate significantly. The experimental results show that HpHSP60 can promote the proliferation of Treg cells.

Example 6: HpHSP60 promotes Treg cell proliferation

Following Example 5, after 72 hours of cell collection, total RNA was isolated. The amount of FoxP3 mRNA expression was determined using real-time PCR. Compared with the control group treated with the anti-CD3 antibody, if there is a significant difference, it is represented by * (P < 0.05), (n = 4). The results are shown in Figure 5.

Since FoxP3 is a marker for Treg cells, the amount of FoxP3 expression is also increased when Treg cells are activated. The figure shows that the mRNA expression results showed that the expression of FoxP3 was significantly increased after the addition of HpHSP60, further demonstrating that the addition of HpHSP60 promoted Treg proliferation.

Example 7: Effect of HpHSP60-inducible Treg cells on inhibition of T cell proliferation

The effect of HpHSP60-induced Treg cells on T cell proliferation was measured by functional assay. The results obtained are shown in the histogram of FIG. The numbers in the figure indicate the percentage of proliferating cells. This figure represents the result of three repetitions.

The experimental results show that when Treg cells increase, they will inhibit T cell activation. It was demonstrated that the addition of PBMC to HpHSP60 inhibited the activation of T cells due to proliferation of Treg cells.

Example 8: Preparation of anti-HpHSP60 serum (anti-HpHSP60 serum) and HpHSP60 monoclonal antibody

H. pylori heat-stressing protein 60 (HpHSP60) was injected into mice to cause an immunological reaction. After multiple administrations of HpHSP60 (boost), antibodies against HpHSP60 were produced in mice. After the blood of the mouse was collected, the serum was taken to obtain a serum containing an anti-HpHSP60 antibody, which was called anti-HpHSP60 serum. The antibody obtained in this step is a polyclonal antibody.

The mouse spleen cells are fused with bone marrow cancer cells to form a hybridoma. Further screening, specific antibodies were picked using enzyme immunoassay (ELISA).

The obtained cell strain was diluted and re-aliquoted into a 96-well cell culture dish, and it was calculated that only one cell per cell was allowed to grow into a colony, and the specific antibody was again screened by ELISA. A monoclonal antibody was obtained.

Example 9: Effect of H. pylori inhibiting cell growth due to inhibition of HSP60 in vivo

C3H/HeN mice were purchased from the National Laboratory Animal Breeding Research Center in Taipei, Taiwan, and kept free of pathogen isolation. All food, water and cage supplies are disinfected prior to use. Five-week-old male mice were intravenously injected with 0.1 ml of anti-HSP60 serum (anti-HpHSP60 serum) obtained in Example 8 before inoculation of H. pylori. After 24 hours of antiserum administration, the mice were infected with 0.5 mL of live Helicobacter pylori (a strain with an ATCC number of 15,415, about ¹⁰⁹ colony forming units). The BHI liquid medium was fed twice by a period of 3 days via oral tube feeding. After confirming infection with H. pylori, the mice were intravenously injected with 0.1 ml of the anti-HSP60 serum obtained in Example 8, once every 3 days.

After the 8th week of infection with H. pylori, all mice were sacrificed under sterile conditions. Cut the entire stomach along a small bend. Each stomach is divided into two equal longitudinal samples, each containing a stomach and an antrum. The gastric tissue was ground and the Helicobacter pylori in the stomach tissue was cultured, and the performance of FoxP3 was analyzed by immunohistochemical staining to evaluate the state of Helicobacter pylori parasitization in the stomach. The results of the evaluation are expressed as mean ± SEM. Statistical significance was assessed using the one-tailed Student's t-test method. P < 0.05 was considered to be a significant difference. The results are shown in Figures 7, 8, and 9.

Figures 7 and 8 show that anti-HpHSP60 serum significantly reduced the number of colonies of H. pylori obtained from its gastric tissue lysate after 8 weeks of vaccination with H. pylori in mice. To investigate the mechanism by which antibodies reduce the parasitic activity of H. pylori in the stomach, the amount of Treg cells in the stomach tissue infected with H. pylori was evaluated. Figure 9 shows that treatment with anti-HpHSP60 serum significantly reduced the amount of Treg cells expressed in the gastric mucosa. The above results indicate that chronic infection of H. pylori is associated with HpHSP60, and inhibition of HpHSP60 can reduce the parasitic amount of H. pylori and also reduce the production of Treg cells.

Example 10: HpHSP60 has an active sequence for inducing Treg cells

In order to find out the active sequence of HpHSP60-inducible regulatory T cells, monoclonal antibodies against HpHSP60 were prepared, including monoclonal antibodies containing the entire sequence of HpHSP60 and fragments thereof. According to the method of Example 9, after the mice were treated with anti-HpHSP60 serum for 24 hours, the mice were fed with H. pylori and confirmed to be infected with H. pylori, the mice were intravenously injected with 0.1 ml, and PBS was administered once every 3 days. Serum, anti-HSP60 serum, LHP-1 (9E4) monoclonal antibody and LHP-2 (5A8) monoclonal antibody. After 8 weeks, the mice were sacrificed, and the stomach wall of the mice was ground, and the gastric homogenate was further cultured on the Helicobacter pylori isolation medium (EYE agar) to confirm the parasite of H. pylori in the stomach. The results are shown in Figure 10.

The red dot in Fig. 10 is a Helicobacter pylori colony, which shows that anti-HSP60 serum has an effect of inhibiting the growth of H. pylori, but LHP-1 (9E4) antibody can effectively eliminate H. pylori completely.

The number of colored colonies in the Helicobacter pylori isolation medium plate was calculated, and the number of Helicobacter pylori colonies (CFU) was determined. If there is a significant difference, it is represented by * (P < 0.05). The results are shown in Figure 11. Figure 11 shows that H. pylori can be completely eliminated after administration of the LHP-1 (9E4) antibody.

Example 11: Immune mechanism of anti-HpHSP60 antibody

To understand the immune mechanism of the anti-HpHSP60 antibody, for the mouse of Example 10, the urease activity of H. pylori was measured at 2, 3, and 8 weeks after inoculation of H. pylori to determine the Helicobacter pylori secretase. Urease B activity. Urease activity was normalized to the urease activity of control mice (not infected with H. pylori). The results are shown in Figure 12. The figure shows that the LHP-1 (9E4) antibody achieves an effect of inhibiting the growth of H. pylori and even eradicating H. pylori by inhibiting HpHSP60.

The LHP-1 (9E4) antibody is an amino acid sequence located at HpHSP60 101-200 as an antigen. The fusion tumor LHP-1 (9E4) containing the antibody was deposited on January 8, 2015 and deposited at the Food Industry Development Research Institute under the registration number BCRC 960493.

Example 12: Evaluation of anti-HpHSP60 antibody on the expression of Treg cells in gastric mucosa

The results of the evaluation of the anti-HpHSP60 antibody on the expression of Treg cells in the gastric mucosa were confirmed. The mouse stomach of Example 10 was fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with H&E, followed by immunohistochemical staining of FoxP3. The results are shown in Fig. 13 (original 200 μm, magnification 100 times). The graph shows the amount of Treg cells present in the gastric wall sections of the sacrificed mice at week 8. Treg expression was not found in the gastric mucosa of mice treated with LHP-1 (9E4).

Example 13: Evaluation of the performance of IL-10 in gastric mucosa by anti-HpHSP60 antibody

The mouse stomach of Example 10 was fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with H&E followed by immunohistochemical staining of IL-10. The results are shown in Fig. 14 (original 200 μm, magnification 100 times). The figure shows the amount of IL-10 cells expressed in the gastric wall sections of the sacrificed mice at week 8. It was shown that IL-10 expression was not found in the gastric mucosa of mice treated with LHP-1 (9E4).

Example 14: HHPSP60 fragment recognizable by LHP-1 (9E4) antibody

In order to clarify the HpHSP60 fragment recognizable by the 8LHP-1 (9E4) antibody, the different fragments of HpHSP60 were identified by the 8LHP-1 (9E4) antibody. The results are shown in Figure 15. The black dot portion of the figure represents a fragment of HpHSP60 that can be recognized by the LHP-1 antibody. The identified fragments included the following, and IgK was used as the positive control group of this experiment, since most of the mouse monoclonal antibodies were kappa species: Whole table HpHSP60 1-547 full length.

1-200 table HpHSP60 1-200 fragment

101-200 table HpHSP60 101-200 fragment

1-250 table HpHSP60 1-250 fragment

200-300 table HpHSP60 200-300 fragment

300-547 table HpHSP60 300-547 fragment

From the results, it was found that the LHP-1 antibody recognizes that the fragment of HpHSP60 is 101-200. The amino acid sequence of this fragment is (SEQ ID NO: 1): EGLRNITAGANPIEVKRGMDKAAEAIINELKKASKKVGGKEEITQVATISANSDHNIGKLIADAMEKVGKDGVITVEEAKGIEDELDVVEGMQFDRGYLS

Example 15: LHP-1 (9E4) antibody recognizable HpHSP60 fragment further defined

The sequence recognized by the LHP-1 (9E3) antibody was reduced to 31 amino acid sequences by the method of Example 14. The results are shown in Figure 16. In the figure, the black dot portion represents a fragment in which HHPSP60 can be recognized by the LHP-1 (9E3) antibody. The figure shows that Δ[1] is a fragment of HpHSP60 134-200. Black spots were observed and were identifiable. While Δ[5] is a fragment of HpHSP60 101-168, no black spots were observed and were unrecognizable. This experiment can be inferred that the LHP-1 (9E3) antibody recognizes that the sequence of HpHSP60 is a 169-200 fragment. The amino acid sequence of this fragment is (SEQ ID NO: 2): KDGVITVEEAKGIEDELDVVEGMQFDRGYLS

【Biomaterial Storage】

TW Republic of China, Food Industry Development Institute, 2015/01/08, BCRC960493.

<110> Shuo Ying Sheng Medical Co., Ltd.

<120> A method for shutting down an immunosuppressive function of a pathogen and use thereof

<160> 2

<210> 1

<211> 100

<212> Amino acid

<213> Helicobacter pylori

<400> 1

<210> 2

<211> 31

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<213> Helicobacter pylori

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Claims (15)

A method for improving immunity in a living body, comprising: directly or indirectly administering a composition capable of blocking an immunosuppressive function of the bacterial pathogen to a bacterial pathogen hosted in the living body or in vitro A step of. The method of claim 1, wherein the composition is administered to a living host to which the pathogen is attached. The method of claim 1, wherein the bacterial pathogen comprises one of a Helicobacter pylori, an Arcobacter suis , a Tannerella forsythia , a Porphyromonas gingivalis , an Aggregatibacter actinomycetemcomitans , and a Helicobacter felis . The method of claim 1, wherein the immunosuppressive function of the bacterial pathogen is provided by an immunosuppressive substance secreted or produced by the pathogenic agent, and the immunosuppressive function of the pathogen is turned off The composition includes a functional inhibitor that shuts down the action of the immunosuppressive substance. The method of claim 4, wherein the immunosuppressive substance secreted or produced by the bacterial pathogen comprises a heat-stressing protein or a fragment thereof. The method of claim 5, wherein the heat-stressing protein comprises heat-stressing protein 60 or a fragment thereof. The method of claim 6, wherein the heat-stressing protein 60 comprises the sequence of SEQ ID NO: 1. The method of claim 4, wherein the functional inhibitor comprises a compound or antibody that recognizes the immunosuppressive substance or a portion thereof of an amino acid sequence. The method of claim 4, wherein the functional inhibitor comprises an antibody that recognizes the immunosuppressive substance or a portion thereof of an amino acid sequence, a humanized product. The method of claim 5, wherein the functional inhibitor comprises an antibody that recognizes the heat-stressing protein or a portion thereof of an amino acid sequence. The method of claim 5, wherein the functional inhibitor comprises an antibody naturally produced by an immunological reaction of the heat-stressing protein or a portion of the amino acid sequence thereof by the host. The method of claim 4, wherein the composition comprises an effective amount of the functional inhibitor and a pharmaceutically acceptable carrier or diluent thereof. The method of claim 12, wherein the composition further comprises an adjuvant that enhances the effectiveness of the immunostimulatory. A use of a functional inhibitor composition according to claims 8-11 of the patent application to prepare a medicament for stimulating or enhancing an immune response against the pathogenic agent. A use of a functional inhibitor composition according to claims 8-11 of the patent application for the manufacture of a medicament for the treatment or prevention of a disease or condition associated with the presence or abnormal expression of the pathogenic agent.