USE OF SUPERANTIGENS FOR IMPROVING MUCOSAL ALLERGEN SPECIFIC IMMUNOTHERAPY IN NON-HUMAN MAMMALS

Abstract

Use of a superantigen in mucosal allergen specific immune therapy (ASIT) in a non-human mammal to enhance the effect thereof. In order to enhance the effect of the mucosal ASIT, the superantigen is mucosally administered before, or with, the allergen to the non-human mammal.

Claims

1. A method of enhancing the effect of allergen specific immune therapy (ASIT) in a non-human mammal suffering from hypersensitivity towards an allergen, the method comprising the steps of: administering the allergen to the non-human mammal; and administering a superantigen before, or with, the allergen to the non-human mammal in need thereof.

2. The method according to claim 1, wherein the non-human mammal is a dog, a cat, or a horse.

3. (canceled)

4. The method according to claim 1, wherein the superantigen and the allergen are co-administered.

5. The method according to claim 1, wherein at least one of the superantigen and the allergen is orally administered.

6. The method according to claim 5, wherein at least one of the superantigen and the allergen is sublingually administered.

7. The method according to claim 1, wherein the superantigen is administered less than 18 hours before the administration of the allergen.

8. The method according to claim 1, wherein the steps of administering the superantigen and the allergen are repeated, the subsequent administration being performed is at least 4 hours after the preceding administration but less than 2 weeks after the preceding administration.

9. The method according to claim 1, wherein the allergen is formulated for oral administration.

10. The method according to claim 1, wherein the non-human mammal is at least 6 months old.

11. The method according to claim 1, wherein said superantigen is selected from the group consisting of: SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SER, SEQ, SEU, SEV, TSST-1, and a mixture thereof.

12. The method according to claim 11, wherein said superantigen is selected from the group consisting of: SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SEU, and a mixture thereof.

13. The method according to claim 1, wherein the allergen specific immune therapy (ASIT) targets allergies selected from the group consisting of canine atopic dermatitis (CAD) and food allergy.

14. The method according to claim 1, wherein the allergen is selected from the group consisting of environmental allergens and food allergens.

15. A composition comprising a superantigen, an allergen and at least one pharmaceutical acceptable carrier or excipient.

16. The composition according to claim 15, wherein the composition is formulated for oral administration.

17. The composition according to claim 15, wherein the superantigen is selected from the group consisting of: SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SER, SEQ, SEU, SEV, TSST-1, and a mixture thereof.

18. The composition according to claim 17, wherein the superantigen is selected from the group consisting of: SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SEU, and a mixture thereof.

19. The composition according to claim 15, wherein the allergen is selected from the group consisting of environmental allergens and food allergens.

20. (canceled)

21. (canceled)

22. (canceled)

23. The method according to claim 14, wherein the allergen is selected from the group consisting of tree-pollen, grass-pollen, wood-pollen, house dust mites, mold spores, fleas, beef protein, chicken protein, pork protein, corn protein, wheat protein, soybean protein, and egg protein.

24. The method according to claim 19, wherein the allergen is selected from the group consisting of tree-pollen, grass-pollen, wood-pollen, house dust mites, mold spores, fleas, beef protein, chicken protein, pork protein, corn protein, wheat protein, soybean protein, and egg protein.

25. The method according to claim 4, wherein the superantigen and the allergen are formulated into a single composition with at least one pharmaceutically acceptable carrier or excipient.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0085] These and other aspects, features and advantages of which the invention is capable of will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which

[0086] FIG. 1 depicts over view of the experimental protocol used for evaluating the ability of S. aureus enterotoxin A (SEA) to enhance the tolerogenic processing of ovalbumin (OVA).

[0087] FIG. 2 Cells in bronchoalveolar lavage fluid (BALf) of recipient mice. Bronchoalveolar lavage was performed after sensitization and challenge of recipient mice (see FIG. 1). Infiltrating cells were counted and stained by May-Grünwald Giemsa to distinguish the eosinophils. (A) Concentration of infiltrating cells/ml in BAL fluid. (B) Fraction of eosinophils among infiltrating cells. (C) Concentration of eosinophils/ml.

[0088] FIG. 3 Cytokines in supernatants from in vitro stimulated lung cells. Cells prepared from lung tissue, collected from recipient mice after sensitization and challenge (see FIG. 1), were re-stimulated with ovalbumin in vitro for 48 h. Levels of IL-5 and IL-13 in the culture medium were measured by ELISA. (A) IL-5 (pg/ml) (B) IL-13 (pg/ml).

[0089] FIG. 4A Number of CD8α-positive cells in biopsies from SEA-exposed and control mice (CD8 α.sup.+ cells/mm.sup.2)

[0090] FIG. 4B Relative MHC class II-staining of biopsies from SEA-exposed and control mice (% of the epithelium stained positive).

EXAMPLE NO. 1

[0091] Methods

[0092] Experimental Protocol

[0093] An overview of the experimental protocol is depicted in FIG. 1. In short, donor mice were exposed to S. aureus enterotoxin A (SEA) in the drinking water (daily dose: 4 μg/mouse) for 5 days; controls were given standard drinking water. After a resting phase of 3 days, when all mice received standard drinking water, the mice were starved overnight and fed 50 μg ovalbumin (OVA) in PBS by gavage, or sham treated with PBS only. One hour after feeding, donor mice were sacrificed and bled by cardiac puncture. At this time point, sections of small intestines of the donor mice were prepared, stained and examined (n=8). Serum was transferred into naïve recipient mice (1 ml serum/recipient) by i.p injection (n=16). Recipient mice were tested for reactivity in a model of allergic airway inflammation. The recipient mice were immunized with alum-adsorbed OVA i.p. (10 μg) twice and challenged with repeated intranasal instillation of OVA for 5 consecutive days. The day after the last challenge, the mice were sacrificed and subjected to bronchoalveolar lavage. Lung tissue and blood was also collected for analysis.

[0094] Animals

[0095] BALB/c mice (B & K, Stockholm, Sweden) were housed under specific pathogen-free conditions in the animal facilities of the Medical Faculty of the University of Gothenburg. The experiments were performed with the permission of the Ethics Committee, University of Gothenburg.

[0096] S. aureus Enterotoxin-Exposure and Adoptive Serum Transfer

[0097] For a schematic overview of the protocol, see FIG. 1. Donor mice (6-8 weeks old males) were given drinking water with or without (control mice) 0.8 μg/ml S. aureus enterotoxin A (SEA; Sigma Chemical Co., St. Louis, Mo.) for five days. A mouse drinks about 5 ml of water daily, corresponding to 4 μg SEA. Three days later, they were starved overnight and then fed 0.3 ml phosphate-buffered saline (PBS) with or without 50 mg ovalbumin (OVA; grade V, Sigma). One hour later, the mice were anaesthetized (Isoflurane, Baxter Medical, Kista, Sweden) and bled by cardiac puncture. Blood from each group of mice (SEA-PBS, SEA-OVA, control-PBS or control-OVA, respectively) was pooled, allowed to clot and centrifuged twice at 3,000×g for 10 min. 1 ml serum was injected intraperitoneally (i.p.) into naïve BALB/c recipient mice, matched for sex and age.

[0098] The Ovalbumin-Asthma Model

[0099] Recipient mice were tested for tolerance in a model of ovalbumin-induced allergic airway inflammation the OVA-asthma model. Feeding of OVA is known to reduce airway inflammation in this model, i.e. oral tolerance is induced to the model allergen(37-39). Recipients were sensitized by two i.p. injections of 10 μg ovalbumin (grade V, Sigma), dissolved in 50 μl PBS and mixed with 100 μl of aluminium hydroxide gel (Sigma). Sensitization was performed 7 and 17 days after transfer of serum from fed mice (see FIG. 1). Allergic airway inflammation was elicited by repeated intranasal challenge with ovalbumin. Thus, 100 μg ovalbumin in 25 μl PBS was administered daily on 5 consecutive days to briefly anesthetized mice (Isoflurane, Baxter Medical, Kista, Sweden). The first challenge dose was given day 24 after serum transfer, i.e. 6 days after the second sensitizing i.p. dose of ovalbumin.

[0100] Twenty-four hours after the last challenge dose, recipient mice were anesthetized with xylazine (130 mg/kg, Rompun; Bayer, Leverkusen, Germany) and ketamine (670 mg/kg, Ketalar; Pfizer AB, Täby, Sweden). Blood was obtained by cardiac puncture for determination of total and ovalbumin-specific IgE. Lung lavage was performed to enumerate infiltrating eosinophils. Lung tissue was collected for in vitro restimulation of lung resident immune cells with ovalbumin and determination of cytokine production in response to this antigen stimulation (see below).

[0101] Bronchoalveolar Lavage.

[0102] Lung lavage was performed to enumerate infiltrating eosinophilic polymophonuclear granulocytes (“eosinophils”). PBS (0.4 ml) was instilled twice through a tracheal cannula, followed by gentle aspiration. Cells were counted in a Haemocytometer (Bürker chamber). Aliquots of BAL fluid containing 10.sup.5 cells were cytocentrifuged (Shandon Southern, Runcorn, UK). After staining with May-Giemsa, the proportion of eosinophils was determined among 300 cells examined in high-powered microscopic fields. Counting of bronchoalveolar lavage cells and the proportion of eosinophils were performed by an investigator blinded regarding to the treatment given to the mice.

[0103] Ovalbumin-Induced Cytokine Production.

[0104] After lavage, one lung was excised and cut into pieces. Single-cell suspensions were obtained after digestion with collagenase (1 mg/ml; Sigma) and DNase (0.1 mg/ml; Sigma) for 20 min at 37° C. in Iscove's medium, followed by squeezing through a nylon filter. The cells were washed in medium and red blood cells were lysed with NH.sub.4Cl (5 min, 37° C.). After washing, 5×10.sup.5 cells/well were seeded in 96-well U-bottomed plates (Nunc, Roskilde, Denmark) in Iscove's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 50 μg/ml gentamycin, and 50 μm 2-mercaptoethanol (all from Sigma). The cultures were stimulated with 500 μg/ml ovalbumin, or medium alone (blank). Supernatants were collected after 48 h and stored at −20° C. until analyzed. The levels of IL-10, IL-5, IL-13 and IFN-γ in supernatants were determined by sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems detection kit), performed as follows: Costar plates were coated overnight at room temperature with capture antibody, washed ×3 with PBS and blocked for 1 h with PBS containing 1% BSA. Cytokine standards or sample (diluted 1:2, 1:10 and 1:50) were added and incubated for 2 h at room temperature. After washing ×3 with PBS with 0.05% Tween, detection antibody, diluted in PBS with 1% BSA, was added and incubated for 1 h. The plates were washed and incubated with streptavidin-horseradish peroxidase for 30 min and tetramethylbenzidine (TMB) liquid substrate (Sigma) for 20 minutes in the dark. The reaction was stopped with 1 M H.sub.2SO.sub.4 and the absorption at 450 nm was determined spectrophotometrically (Emax, Molecular Devices, Sunnyvale, Calif.). The detection limits were 70 pg/ml (IL-5) and 200 pg/ml (IL-13).

[0105] Determination of Ovalbumin-Specific IgE in Serum.

[0106] Ovalbumin-specific IgE antibodies were assayed by passive cutaneous anaphylaxis (PCA). Sprague-Dawley rats were anaesthetized (isoflurane inhalation followed by 8 mg/kg xylazine and 40 mg/kg ketamine i.p.). Mouse sera were diluted in twofold steps and 50 μl was injected intradermally into shaved dorsal skin of the rat. After 72 h, the rats were given 5 mg of ovalbumin in 1 ml PBS with 1% Evans' blue (Sigma) as an intravenous injection. They were sacrificed 1 h later. In a positive reaction, anti-ovalbumin antibodies of the IgE isotype are absorbed by Fc-epsilon receptors on tissue-bound mast cells. When ovalbumin is injected, mast cell-bound specific IgE reacts with the antigen, which activates release of histamine and leakage of dye-protein complexes into the tissue, leading to the appearance of a blue spot in the skin. The IgE anti-ovalbumin titer was defined as the reciprocal of the highest dilution giving a blue spot with a diameter of >2 mm.

[0107] Quantification of Total Serum IgE.

[0108] Total IgE concentrations in serum of recipient animals were determined by ELISA. Costar plates were coated with mouse anti-IgE (1 μg/ml; BD Biosciences Pharmingen), washed ×3 with PBS and blocked for 1 h with 1% bovine serum albumin (BSA). 50 μl of samples diluted 1:3-1:81 were added to the coated wells and the plates were incubated for 2 h at room temperature. After washing off non-bound serum constituents, biotinylated anti-mouse IgE (2 μg/ml; BD Biosciences Pharmingen) was added, followed by 1 h of incubation. The plates were washed and incubated with streptavidin-horseradish peroxidase and substrate was added, as described above (see: Ovalbumin induced cytokine production). The limit of detection was 5 ng/ml.

[0109] Examination of Intraepithelial Lymphocytes and MHC II Expression in Donor's Small Intestines

[0110] Mid-jejunal biopsies were excised from donor mice at the time for transfer, three days after the last SEA-treatment. Pieces of small intestines were placed in specimen moulds (Tissue-Tek Cryomould Biopsy; Miles Inc., Elkhart, Ind.) with Tissue-Tek O.C.T. compound (Sakura Finetek Europe BV, Zoeterwoude, the Netherlands), frozen instantly in isopentane cooled by liquid nitrogen, and stored at −70° C. Cryostat sections (6 μm thick) were prepared and fixed in cold acetone 50% for 30 s and 100% for 5 min. Endogenous peroxidase activity was blocked by incubation for 10 min in 1 U/l glucose oxidase (Type V-S; Sigma), 10 mM glucose and 1 mM NaN.sub.3. Sections were incubated overnight at 4° C. with biotinylated monoclonals against I-A.sup.d MHC class II or CD8a (both Pharmingen, San Diego, Calif.), in PBS with 0.1% saponine, followed by avidin-conjugated peroxidase (Vectastain ABC; Vector laboratories, Burlingame, Calif.) for 30 min and amino-ethyl-carbazole. The tissue was counter-stained with Mayer's haematoxylin and examined in a Leica Q500MC microscope using Leica Qwin Software by a group-blinded investigator (Leica, Cambridge, UK). MHC class II staining of epithelium was expressed as relative stained area (%) and intraepithelial lymphocytes as CD8α.sup.+ cells/mm.sup.2 villus area. For both markers, 3 sections were analyzed from 8 each of mice per group.

[0111] Statistical Analysis

[0112] Kruskal-Wallis test was used to confirm significant differences between groups, followed by the Mann-Whitney U-test using Prism (GraphPad Software, San Diego, Calif.).

[0113] Results

[0114] Feeding of a dietary protein results in appearance of a tolerogenic form of the fed antigen in serum. The presence of such tolerogenic antigen can be demonstrated by transfer of serum to naïve recipients which will become actively tolerant to the antigen in question. To investigate the effect of S. aureus enterotoxin on tolerogenic processing, donor mice were exposed to SEA in the drinking water for 5 days, rested for 3 days and fed a tolerizing dose of ovalbumin. Serum collected shortly after feeding was transferred to naïve recipients, which were sensitized and challenged with ovalbumin in a model of Th2-mediated allergic airway inflammation. Tolerance was evaluated as reduction in infiltration of inflammatory cells into the lungs and reduction of ovalbumin-induced cytokine production by the cells extracted from the lung parenchyme. The experimental set-up is shown in FIG. 1 and described below.

[0115] Mice (6-8 weeks old) were given Staphylococcal enterotoxin A (SEA) in the drinking water (0.8 mg/ml) for 5 days. SEA was removed and the mice were left to rest for three days. Thereafter, mice (both SEA exposed and untreated SHAM controls) were fed by gavage either with ovalbumin (OVA; 50 mg) or with PBS (controls). The mice were sacrificed at 1 hour after feeding and blood was collected. Serum was prepared and injected intraperitoneally (i.p) (1 ml) into naïve recipient mice. At seven days after injection with serum all mice were introduced into an airway allergy model.

[0116] Reduced Eosinophil Infiltration in BAL after Transfer of Serum from SEA-Pretreated Donors

[0117] FIG. 2A shows the number of cells in the bronchoalveolar lavage (BAL) fluid in recipient mice sensitized and challenged with ovalbumin. To reveal the effect of SEA pretreatment on tolerogenic processing, we compared ovalbumin-specific tolerance in SEA-pretreated (SEA-OVA and SEA-PBS) and sham-treated (Ctrl-OVA and Ctrl-PBS) mice. Mice that had received serum from ovalbumin-fed donors had significantly fewer cells in the lavage fluid than mice that received serum from sham-fed donors. This was true whether the donors had been exposed to SEA 3 days prior to ovalbumin feeding (black symbols) or not (open symbols). This is in line with recent data from our group, showing that serum-transfer from ovalbumin-fed donors renders naive recipient mice tolerant to subsequent challenge with ovalbumin (44).

[0118] After sensitization and challenge with ovalbumin, the majority of cells in BAL fluid were eosinophils in all groups (FIG. 2B). In mice which had received serum from donor mice exposed to SEA prior to ovalbumin-feeding, the fraction of eosinophils was significantly reduced compared to SEA-pretreated non-fed mice. In contrast, the proportion of eosinophils was not significantly reduced in BAL fluid from mice that had received serum from ovalbumin-fed donors with no prior SEA-exposure. As a result, the fraction of eosinophils was significantly lower in BAL fluid of recipients of SEA-pretreated ovalbumin-fed mice, as compared to sham-treated ovalbumin-fed mice. The total number of infiltrating eosinophils, based on numbers of infiltrating cells and the fraction of eosinophils was correspondingly reduced (FIG. 2C). Recipients of serum from ovalbumin-fed mice had lower numbers of infiltrating eosinophils than recipients of serum from sham-fed mice, but the tolerance was more pronounced if the donors had been treated with SEA before feeding. Thus, the number of infiltrating eosinophils was significantly lower in recipients of serum from SEA-pretreated, ovalbumin-fed donors, than in recipients from sham-pretreated ovalbumin-fed donors (FIG. 2C). Of note, SEA treatment in itself did not significantly reduce cell infiltration or eosinophil proportion (Ctrl-PBS vs. SEA-PBS). Thus, the effect was antigen-specific and could not be due to a general effect of SEA pretreatment on e.g. inflammatory effector cells.

[0119] Decreased Production of IL-5 and IL-13 by Lung Cells after Transfer of Serum from SEA-Pretreated and OVA Fed Donors

[0120] Single cell suspensions, prepared from lung tissue of recipient mice, were re-stimulated in vitro with ovalbumin and the cytokine production in response to this recall antigen was measured. With no prior SEA-treatment of the donors, production of IL-5 (FIG. 3A) and IL-13 (FIG. 3B) did not differ significantly between lung cells of recipients of serum from ovalbumin-fed and sham-fed donors. When donors were treated with SEA prior to ovalbumin-feeding, recipients of their serum showed significantly reduced lung cell IL-5 and IL-13 production compared to recipients of serum from sham-fed donors and ovalbumin-fed donors with no prior SEA-treatment.

[0121] As noted above, the effect was antigen dependent and not due to a general effect of SEA, since SEA pretreatment of the serum donors in itself did not reduce Th2 cytokine production (SEA-PBS vs. Ctrl-PBS) in the recipients. The levels of IL-10 did not differ between groups, and there were no detectable levels of IFN-γ in the cell culture supernatants. The serum IgE-levels did not differ between the groups (data not shown).

[0122] Increased Density of CD8α.sup.+ Intestinal Epithelial Lymphocytes in Small Intestinal Villi of SEA-Exposed Donor Mice

[0123] Small intestinal biopsies were obtained from SEA-treated and control donors at the time of serum transfer three days after the last SEA-exposure. Donor mice exposed to SEA had significantly increased density of CD8α.sup.+ intra-epithelial lymphocytes in the small intestine (FIG. 4A). The intestinal epithelial cells clearly tended to express more MHC class II in SEA treated group, p=0.10 (FIG. 4B).

EXAMPLE NO. 2

[0124] Further, it was investigated whether sublingual immunotherapy (SLIT) treatment is effective in a mouse model of airway sensitization and whether administration of superantigen, staphylococcal enterotoxin A (SEA), together with the model antigen ovalbumin (OVA) has any an additional effect.

[0125] In short, female BALB/c mice, 7-8 weeks old, i.e. post the neonatal stage, were given SLIT treatment by sublingual administration of 100 μg OVA solution alone or together with SEA in various concentrations (0.38, 0.75, 1.5, and 3 μg, respectively). This treatment was given 10 times during two weeks. SLIT treated mice were then sensitized by intraperitoneal injections of alum-adsorbed OVA and subsequently challenged intranasally and analyzed for antibody levels, eosinophilia and cellular response.

[0126] The cellular response was evaluated as IFN-γ secretion from in vitro stimulated spleen cells, 2×10.sup.5 splenocytes were incubated at 37° C. together with OVA (0.5 mg/mL) and after three days of culture, supernatant was collected and analyzed for IFN-g by ELISA.

[0127] Preliminary data show that IFN-γ secretion from in vitro stimulated spleen cells were lower in mice given SEA together with OVA, in a dose-dependent matter, compared to mice given OVA alone. These results confirm that administration of a superantigen in conjunction to existing SLIT treatments has a beneficial effect, improving the efficiency of the SLIT treatment.

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