Method for and use of blocking immunosuppressive functions of pathogens

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.

Claims

1. A method for increasing immunity of a living body against H. pylori, comprising: preparing a composition that blocks an immunosuppressive function of H. pylori; and applying the composition to a living body colonized by H. pylori, wherein the immunosuppressive function is provided by heat shock protein 60, or a fragment thereof, secreted or produced by H. pylori, and wherein the composition comprises a function inhibitor comprising an antibody binding to the sequence of SEQ. NO. 1.

2. The method according to claim 1, wherein the composition is applied to an internal of the living body.

3-8. (canceled)

9. The method according to claim 1, wherein the function inhibitor comprises a humanized product of an antibody that identifies the sequence of SEQ. NO. 1.

10. (canceled)

11. (canceled)

12. The method according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier or diluents.

13. The method according to claim 1, wherein the composition further comprises an adjuvant that increases immunestimulating effects.

14. (canceled)

15. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows experimental results on effects of HpHSP60 on PBMC proliferation.

[0020] FIG. 2 shows experimental results on effects of HpHSP60 on T-cell proliferation.

[0021] FIGS. 3A-3C respectively show experimental results on effects of HpHSP60 on PBMC cell cycles.

[0022] FIG. 4 shows experimental results on Treg cells in vitro induction by HpHSP60.

[0023] FIG. 5 shows experimental results on effects of HpHSP60 on Treg cell proliferation.

[0024] FIG. 6 shows experimental results on effects of HpHSP60-induced Treg cells on T-cell proliferation.

[0025] FIG. 7 shows experimental results on inhibition of H. pylori in vivo growth due blockage of HpHSP60 immunosuppressive functions.

[0026] FIG. 8 shows results of another experiment on inhibition of H. pylori in vivo growth due blockage of HpHSP60 immunosuppressive functions.

[0027] FIG. 9 shows experimental results on inhibition of Treg cells due blockage of HpHSP60 immunosuppressive functions.

[0028] FIG. 10 shows experimental results on active sequence in HpHSP60 that induces Treg cells growth.

[0029] FIG. 11 shows quantization of experimental results shown in FIG. 10.

[0030] FIG. 12 shows experimental results on a study of the immunological mechanism of anti-HpHSP60 antibodies.

[0031] FIG. 13 shows experimental results on effects of anti-HpHSP60 antibodies on Treg cells expressions in mice gastric mucosa.

[0032] FIG. 14 shows experimental results on effects of anti-HpHSP60 antibodies on IL-10 expressions in mice gastric mucosa.

[0033] FIG. 15 shows experimental results on recognition of HpHSP60 fragments by LHP-1 (9E4) antibody.

[0034] FIG. 16 shows results of further experiments on recognition of HpHSP60 fragments by LHP-1 (9E4) antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Although it is not intended to limit the present by any theory, according to this invention, certain pathogens are capable of suppressing the immunity of their hosts, by secreting or producing immunosuppressive substances, so to proliferate or to cause disease to the host. Examples of such pathogens include H. pylori and other similar bacteria such as Arcobacter suis, Tannerella forsythia, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Helicobacter felis. The inventors found that heat shock protein is one of such immunosuppressive substances. According to embodiments of this invention, H. pylori heat shock protein 60 (HpHSP60) is capable of reacting with monocytes to stimulate the production of immunosuppressive hormones, such as IL-10 and TGF-β, and induce the proliferation of Treg cells. As a result, the immunity of the host is suppressed, making the host unable to resist the chronic infections of H. pylori.

[0036] The present invention has developed a novel method to increase the immunity of hosts. A function inhibitor that blocks the functions of the immunosuppressive substances is used to shut down the functions of the immunosuppressive substances. Proliferation of Treg cells is thus effectively inhibited and immunosuppressive response of the host is eliminated.

[0037] A function inhibitor of this invention includes a substance that is capable of identifying the immunosuppressive substance or a fragment thereof and blocking functions of the immunosuppressive substance. When the immunosuppressive substance is a heat shock protein, the function inhibitor is an antibody capable of identifying the amino acid sequence, or a fragment thereof, of the heat shock protein.

[0038] The function inhibitor may be prepared in the form of a monoclonal antibody, to facilitate mass production. The function inhibitor may be prepared as a pharmaceutical composition, along with pharmaceutically acceptable carriers or diluent. The composition may further include an adjuvant that increases immunestimulating effects. One example of the adjuvant is the immunestimulating agent disclosed in the above-mentioned U.S. Pat. No. 6,403,099. The pharmaceutical composition may be used as a drug that stimulates or improve immune response to the pathogens. It may also be used as a drug that treats or prevents a disease or disorder relating to the existence or abnormal expressions of the pathogens.

[0039] In the followings, certain examples will be described by referring to the drawings, in order to illustrate the method for and use of blocking immunosuppressive functions of pathogens of this invention. It is however appreciated that the scope of this invention is not limited to any of the embodiments described. For example, although in the detailed description the pathogenesis mechanism of H. pylori and function inhibitor for HpHSP60 are used as examples in the description of the invention, the heat shock protein of other bacteria, such as Helicobacter felis (known cause of chronic enteritis) and Arcobacter suis (known cause of periodontal disease), also include an identical fragment of HpHSP60, such as HSP60 101-200. Therefore, the method and use of this invention are also useful in these and other pathogenesis bacteria, and other pathogens that have similar pathogenesis mechanism.

Embodiment 1: Cell Culture and Isolation of PBMC and T-Cells

[0040] Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and resuspended in RPMI-1640 with 10% inactivated fetal calf serum and 1% penicillin-streptomycin. For monocyte depletion, PBMCs were cultured in 10-cm dishes at a density of 10.sup.6/ml overnight for monocyte attachment. The suspended cells were then collected by centrifugation at 1500 rpm for 15 min. Total T-cells were isolated from PBMCs by negative selection using a magnetic sorting device (Miltenyi Biotec, MA, USA). Briefly, PBMCs were incubated with a cocktail of biotin-conjugated antibodies, followed by microbead-conjugated anti-biotin Abs for magnetic depletion. T-cells were eluted according to the manufacturer's protocols.

Embodiment 2: The Effect of HpHSP60 on PBMC Proliferation

[0041] Proliferation of anti-CD3 mAb-stimulated PBMCs treated with HpHSP60, rGFP or boiled HpHSP60 at different doses was monitored by a cell proliferation assay. To measure cell proliferation, 0.2 ml of cells at 1×10.sup.6 cells/ml were seeded in each well of an anti-CD3 mAb-precoated 96-well microplate. Cell proliferation was determined by an MTT assay after 96 hours. Results are shown in FIG. 1: experimental results on effects of HpHSP60 on PBMC proliferation. Data shown therein are reported as the proliferation index.

[0042] The cell proliferation index was calculated as follows: Proliferation index (100%)=(OD.sub.595 of the anti-CD3+HpHSP60-treated cells)/(OD.sub.595 of the anti-CD3-treated cells)*100%. The results that differ significantly from the untreated group are indicated by *(p<0.05) (n=15).

[0043] In FIG. 1, (.diamond-solid.) shows proliferation of T-cells is inhibited, after HpHSP60 is added into the PBMC. (.square-solid.) shows rGFP, being a control protein in this experimental system, does not influence T-cell proliferation. The results of this control unit show that not any protein is capable of inhibiting T-cell proliferation. (.box-tangle-solidup.) represents boiled HpHSP60, which includes the sequence of HpHSP60, while its protein structure has been destructed. The results show that boiled HpHSP60 does not influence T-cell proliferation.

Embodiment 3: Influence of HpHSP60 on T-Cell Proliferation in PBMC

[0044] After treatment with anti-CD3 mAb, PBMCs were treated with or without HpHSP60 (200 ng). T-cells or non-T-cells in PBMCs were identified by CD3 surface marker staining. Cell number was then calculated following a flow cytometer analysis.

[0045] For CD3 surface marker staining, cells were harvested and stained with 1 μg mouse anti-human CD3 IgG mAbs (OKT3), followed by 0.5 μg rabbit anti-mouse IgG-FITC secondary Abs (Biolegend, CA, USA). For FoxP3 intracellular staining, the cells were harvested and stained with mouse anti-human CD4-FITC mAbs (Biolegend, CA, USA) prior to fixing and permeabilization, followed by intracellular staining with mouse anti-human FoxP3-PE mAbs (BD Biosciences, MA, USA) according to the manufacturer's protocol. For the cell cycle assay, cells were harvested after 72 hours and 10.sup.6 cells were fixed with 70% ice-cold ethanol. DNA was stained with DNA staining buffer (5% Triton-X 100, 0.1 mg/ml RNase A, and 4 μg/ml propidium iodide) for 30 min at room temperature. Changes in the DNA content were then detected. Fluorescence was analyzed using a FACS flow cytometer (Becton Dickinson, Heidelberg, Germany) and CELLQuest Pro software (Becton Dickinson, Heidelberg, Germany). Results are shown in FIG. 2: experimental results on effects of HpHSP60 on T-cell proliferation.

[0046] In FIG. 2, the proliferation index is calculated as: proliferation index (fold)=(number of T- or non-T-cells in the anti-CD3/HpHSP60-treated group)/(number of T- or non-T-cells in the untreated control). A significant difference is indicated by *(p<0.05) (n=4). The results show that HpHSP60 is capable of inhibiting the proliferation of T-cells. In this figure, (□) represents T-cells in PBMC. (.square-solid.) represents non-T-cells in PBMC. Clearly, what HpHSP60 inhibits is the proliferation of T-cells.

Embodiment 4: Effects of HpHSP60 on the Cell Cycle

[0047] The effects of HpHSP60 on the cell cycle of PBMC were determined. From the PBMC products of Embodiment 3, PBMCs alone, CD3-activated PBMCs and PBMCs treated with anti-CD3 and HpHSP60 are obtained respectively. The percentages of cells in the sub-G1, G1, S, and G2/M phases are observed and presented in histogram plots, as shown in FIG. 3A-3C. FIGS. 3A-3C respectively show experimental results on effects of HpHSP60 on PBMC cell cycles. The figures are representative of three replicates.

[0048] FIGS. 3A-3C show that HpHSP60 inhibits the proliferation of T-cells, rather than causing them death. FIG. 3A shows T-cells without CD3 activation (Cell alone) remain in their dormant phases (G0/G). FIG. 3B shows that, after activation by CD3, the T-cells' growth was activated and the typical cell cycle graphics are formed. FIG. 3C shows no substantial difference with FIG. 3B. PBMCs treated with anti-CD3 and HpHSP60 (Anti-CD3+HpHSP60) exhibit the same ratio at the sub G0/G1 phases (representing death of cells) as that of the Anti-CD3 group. The experimental results show that the role of HpHSP60 is to inhibit the growth of T cells, rather than resulting in death.

Embodiment 5: Results of Treg Cells In Vitro Induction by HpHSP60

[0049] The proportions of CD4.sup.+FoxP3.sup.+ cells in HpHSP60-treated PBMCs were measured over time. A significant difference compared to the anti-CD3 control is indicated by *(p<0.05) (n=5). The results are shown in FIG. 4: experimental results on Treg cells in vitro induction by HpHSP60.

[0050] Since CD4 and FoxP3 are markers for Treg cells, it is possible to identify HpHSP60's effects in T-cell growth from FIG. 4. In this figure, “() cell alone” expresses the original growth curve of T-cells. “(.square-solid.) anti-CD3” expresses the growth curve of T-cells activated by CD3. “(.box-tangle-solidup.) Anti-CD3+HpHSP60” shows a significant proliferation of T-cells. The experimental results show that HpHSP60 is capable of enhancing Treg cell proliferation.

Embodiment 6: HpHSP60 Enhances Tree Cell Proliferation

[0051] Following Embodiment 5, cells were harvested after 72 hours for total RNA isolation. Real-time PCR was used to measure the expression of FoxP3 mRNA. A significant difference compared to the anti-CD3 control is indicated by *(p<0.05) (n=4). The results are shown in FIG. 5: experimental results on effects of HpHSP60 on Treg cell proliferation.

[0052] Since FoxP3 is marker for Treg cells. When Treg cells are activated, expression of FoxP3 also increases. The mRNA assay results of FIG. 5 show that, following the addition of HpHSP60, FoxP3 expression significantly increases. This experiment further supports the fact that adding HpHSP60 enhances Treg cell proliferation.

Embodiment 7: The Activity of HpHSP60-Induced Treg Cells on T-Cell Proliferation

[0053] Functional assay is used to measure the activity of HpHSP60-induced Treg cells on cell proliferation. The results are shown in FIG. 6. FIG. 6 shows experimental results on effects of HpHSP60-induced Treg cells on T-cell proliferation. Numbers on the histogram plots indicate the percentage of proliferative cells. The histogram plot is representative of three replicates.

[0054] The experimental results show that, when number of Treg cells increases, activities of T-cells are correspondingly inhibited. This proves that when HpHSP60 is added in the PBMC, activities of T-cells are inhibited, due to increase of Treg cells.

Embodiment 8: Preparation of Anti-HpHSP60 Serum and HpHSP60 Monoclonal Antibodies

[0055] C3H/HeN mice were purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, and maintained in pathogen-free isolators. All food, water, caging, and bedding were sterilized before use. Male 5-week-old mice were i.v. injected with HpHSP60 to generate immunization reactions. After repeated boost of HpHSP60, blood of the mice is collected. Serum is isolated to obtain anti-HpHSP60 antibody containing serum, referred to as “anti-HpHSP60 serum.” The products of this step are the polyclonal antibody.

[0056] The spleen cells of the mice were fused with mouse myeloma cells to form a hybridoma. The products are further screened by enzyme immunoassay (ELISA) to isolate specific antibodies.

[0057] The resulting cell lines were diluted and re-distributed into a cell culture plate with 96 wells. Calculate to ensure that each well contains only one cell. After the cells grow to form colonies, the colonies are again screened by ELISA to obtain specific antibodies. Monoclonal antibodies are thus obtained.

Embodiment 9: Assessment of the Eradication of H. pylori by Blockage of HSP60 In Vivo

[0058] C3H/HeN mice were purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, and maintained in pathogen-free isolators. All food, water, caging, and bedding were sterilized before use. Male 5-week-old mice were i.v. injected with 0.1 ml anti-HSP60 serum obtained from Embodiment 8 before H. pylori inoculation. At 24 hours after the anti-serum treatment, the mice were infected 0.5 mL live H. pylori (ATCC 15415 strain, approximately 10.sup.9 colony-forming units) in BHI broth by oral gavage twice within a 3-day period. After infection with H. pylori was established, the mice were then i.v. injected with 0.1 ml anti-HSP60 serum every 3 days.

[0059] At the 8th weeks after H. pylori inoculation, all of the mice were sacrificed aseptically and the intact stomachs were opened along the lesser curvature. Each stomach was dissected into two equal longitudinal specimens, containing the gastric body and antrum. The eradication of H. pylori was analyzed by H. pylori re-culture and immunohistochemistry staining for the expression of FoxP3.

[0060] The results are presented as the mean±SEM. The statistical significance was evaluated using the one-tailed Student's t-test; p<0.05 was considered significant. The results are shown in FIGS. 7, 8 and 9. Among them. FIGS. 7 and 8 respectively show results of several experiments on inhibition of H. pylori in vivo growth due blockage of HpHSP60 immunosuppressive functions. FIG. 9 shows experimental results on inhibition of Treg cells due blockage of HpHSP60 immunosuppressive functions.

[0061] FIGS. 7 and 8 show that the anti-HpHSP60 serum significantly reduces the re-culture of H. pylori colonies from a gastric tissue lysate at the 8.sup.th week after H. pylori inoculation. To determine the mechanism of colony decrease by antibodies, the expression of Treg cells in H. pylori-infected gastric tissues was evaluated. FIG. 9 reveals that the anti-HpHSP60 serum treatments significantly reduce the expression of Treg cells in the gastric mucosa. These results indicate that chronic H. pylori infection is correlated with HpHSP60 and that the blockage of HpHSP60 can decrease H. pylori colonization and the generation of Treg cells.

Embodiment 10: Positions of Treg Cell Inducible Sequence in HpHSP60

[0062] In order to allocate the position of active sequence in HpHSP60 that induces Treg cells, anti-HpHSP60 mAbs that recognize the full sequence or fragments of HpHSP60 are prepared. The method of Embodiment 9 is used. After 24-h treatment with the anti-HpHSP60 serum, the mice were infected by H. pylori, whereby infection of H. pylori Is established. The mice were then i.v. injected with 0.1 ml PBS, serum, anti-HSP60 serum, LHP-1 (9E4) mAb and LHP-2 (5A8) mAb, respectively, every 3 days. Mice were sacrificed after 8 weeks. The gastric wall was ground and the obtained gastric homogenates were incubated in H. pylori incubated isolation medium (EYE agar), to confirm H. pylori parasites in stomach. The results are shown in FIG. 10: experimental results on active sequence in HpHSP60 that induces Treg cells growth.

[0063] As shown in FIG. 10, the red spots on the plate are colonies of H. pylori. This experiment reveals that, while anti-HpHSP60 serum inhibits growth of H. pylori, LHP-1 (9E4) antibody is capable of completely eliminating H. pylori.

[0064] Number of H. pylori colonies (CFU) was determined by counting the red colonies on the EYE plate. A significant different is indicated by *(p<0.05). The results are shown in FIG. 11. FIG. 11 shows quantization of experimental results shown in FIG. 10. As shown in FIG. 11, H. pylori was completely eliminated after the LHP-1 (9E4) antibody was added.

Embody 11: Immunological Mechanisms of Anti-HpHSP60 Antibody

[0065] In order to understand immunological mechanisms of anti-HpHSP60 antibody, gastric urease activity of the mice according to Embodiment 10 at the 2.sup.nd, 3.sup.rd and 8.sup.th week after H. pylori inoculation was measured. The urease activity is normalized to the gastric urease activity of the control mice (without H. pylori infection). The results are shown in FIG. 12: experimental results on a study of the immunological mechanism of anti-HpHSP60 antibodies. This figure shows that the LHP-1 (9E4) antibody inhibits growth of H. pylori, or even eliminates H. pylori, by inhibiting the activity of HpHSP60.

[0066] The LHP-1 (9E4) antibody was generated by using the amino acid sequence of positions 101 to 200 of HpHSP60 as antigen. Hybridomas including this antibody, LHP-1 (9E4), were deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Va., 20110, USA, ATCC Designation: PTA-122900 (ATCC accession number: ______). The date of deposit is Mar. 2, 2016.

Embodiment 12: Evaluation of Expression of Treg Cells in Gastric Mucosa

[0067] In order to understand anti-HpHSP60 antibody's effects in the expression of Treg cells in gastric mucosa, mouse stomachs obtained in Embodiment 10 were fixed with neutral buffered 10% formalin and embedded in paraffin. Five-micrometer sections were stained with H&E stain, followed by immunohistochemistry staining of FoxP3. The results are shown in FIG. 13. FIG. 13 shows experimental results on effects of anti-HpHSP60 antibodies on Treg cells expressions in mice gastric mucosa. In this figure, all pictures are representative of the mice sacrificed at the 8.sup.th week (200 μm original magnification ×100). The results show that no Treg expressions are observed in the mice gastric mucosa that was treated with LHP-1 (9E4) antibodies.

Embodiment 13: Effects of HpHSP60 in Expression of IL-10 in Mice Gastric Mucosa

[0068] Mouse stomachs obtained in Embodiment 10 were fixed with neutral buffered 10% formalin and embedded in paraffin. Five-micrometer sections were stained with H&E stain, followed by immunohistochemistry staining of IL-10. The results are shown in FIG. 14. FIG. 14 shows experimental results on effects of anti-HpHSP60 antibodies on IL-10 expressions in mice gastric mucosa. In this figure, all pictures are representative of the mice sacrificed at the 8.sup.th week (200 μm original magnification ×100; 100 μm original magnification ×200). The results show that no IL-10 expressions are observed in the mice gastric mucosa that was treated with LHP-1 (9E4) antibodies.

Embodiment 14: Fragments of HpHSP60 Identifiable by LHP-1 (9E4) Antibody

[0069] The LHP-1 (9E4) antibody is used to identify different lengths of fragments of HpHSP60, to determine fragments of HpHSP60 that can be recognized by the LHP-1 (9E4) antibody. The results are shown in FIG. 15. FIG. 15 shows experimental results on recognition of HpHSP60 fragments by LHP-1 (9E4) antibody. In this figure, dark spots represent positive identifications. Fragments being identified include the followings, while IgK is used as positive controls, since most mice mAbs are kappa type:

[0070] Whole—the full length of HpHSP60. i.e. positions 1-547.

[0071] 1-200—fragment including positions 1-200 of HpHSP60.

[0072] 101-200—fragment including positions 101-200 of HpHSP60.

[0073] 1-250—fragment including positions 1-250 of HpHSP60.

[0074] 200-300—fragment including positions 200-300 of HpHSP60.

[0075] 300-547—fragment including positions 300-547 of HpHSP60.

[0076] The results show that the HpHSP60 fragment identifiable by the LHP-1 (9E4) antibody is the sequence of positions 101-200, which amino acid sequence (Sequence No. 1) is:

TABLE-US-00001 EGLRNITAGANPIEVKRGMDKAAEAIINELKKASKKVGGKEEITQVAFIS ANSDHNIGKLIADAMEKVGKDGVITVEEAKGIEDELDVVEGMQFDRGYLS

Embodiment 15: Further Limitation of HpHSP60 Fragment Identifiable by LHP-1 (9E4) Antibody

[0077] Following the method of Embodiment 14, use the LHP-1 (9E4) antibody to identify different fragments with shorter lengths. The results are shown in FIG. 16. FIG. 16 shows results of further experiments on recognition of HpHSP60 fragments by LHP-1 (9E4) antibody. In this figure, dark spots represent positive identifications. Δ[1] represents positions 134-200 of HpHSP60, with positive results, while Δ[5] represents positions 101-168 of HpHSP60, with negative results. It can therefore be concluded that the HpHSP60 fragment identifiable by the LHP-1 (9E4) antibody includes amino acid positions 169-200 of HpHSP60, which are:

TABLE-US-00002 (Sequence No. 2) KDGVITVEEAKGIEDELDVVEGMQFDRGYLS