Method for inducing oral tolerance via administration of beta-lactoglobulin derived peptides in combination with probiotic

Abstract

The invention pertains to the use of a probiotic and a beta-lactoglobulin-derived peptide in the manufacture of a product for use in inducing oral tolerance, and/or treatment, prevention or reducing the risk of allergy in a subject, in particular cow's milk protein allergy.

Claims

1. A method for inducing oral tolerance, and/or treating, or reducing the risk of allergy in a subject, comprising administering to the subject a composition comprising: (i) 10.sup.2-10.sup.13 cfu, per g dry weight of the composition, of a probiotic Bifidobacteria breve, and (ii) 2 to 10 distinct beta-lactoglobulin-derived peptides comprising an amino acid sequence consisting of 14 to 25 consecutive amino acids from amino acid nos. 13 to 48 of the beta-lactoglobulin protein represented by SEQ ID No. 1.

2. The method according to claim 1, wherein the beta-lactoglobulin-derived peptide consists of an amino acid sequence selected from the group consisting of SEQ ID Nos. 2-5, optionally coupled to 1-6 further amino acids at its C- and/or N-terminus.

3. The method according to claim 1, wherein the composition further comprises a prebiotic.

4. The method according to claim 3, wherein the prebiotic is selected from the group consisting of fructo-oligosaccharide, non-digestible dextrin, galacto-oligosaccharide, xylo-oligosaccharide, arabino-oligosaccharide, arabino-galacto-oligosaccharide, gluco-oligosaccharide, glucomanno-oligosaccharide, galactomanno-oligosaccharide, mannan-oligosaccharide, chito-oligosaccharide, uronic acid oligosaccharide, sialyl-oligosaccharide and fuco-oligosaccharide.

5. The method according to claim 3, wherein the prebiotic comprises a galacto-oligosaccharide and/or a fructo-oligosaccharide.

6. The method according to claim 4, wherein the prebiotic comprises a mixture of a short-chain oligosaccharide having an average degree of polymerisation of 2-8 and a long-chain oligosaccharide having an average degree of polymerisation of 10-60.

7. The method according to claim 1, comprising a further probiotic strain selected from the group consisting of Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus lactis and Streptococcus thermophiles.

8. The method according to claim 1, wherein the comprises Bifidobacterium breve and Bifidobacterium longum.

9. The method according to claim 1, wherein the composition comprises 10-50 μg beta-lactoglobulin-derived peptides per gram total protein.

10. The method according to claim 1, wherein the allergy is cow's milk protein allergy.

11. The method according to claim 1, wherein the Bifidobacterium breve is the sole probiotic.

12. The method according to claim 3, wherein the composition comprises 0.5-2 wt % of the prebiotic.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the skin response (ear swelling) results of example 1. The tested groups are: 1=negative control; 2=positive control; 3=peptide-diet group; 4=synbiotics-diet group; 5=peptide+synbiotics-diet group.

(2) FIG. 2 shows the anaphylactic shock scores of example 1. The tested groups are: 1=negative control; 2=positive control; 3=peptide-diet group; 4=synbiotics-diet group; 5=peptide+synbiotics-diet group.

(3) FIG. 3 shows the T cell subset analysis results of example 3. Lymphocytes isolated from the small intestine lamina propria (SI-LP) were analysed by flow cytometry for T.sub.h1 and T.sub.h2 phenotypes. The tested groups are: 1=negative control; 2=positive control; 3=peptide-diet group; 4=synbiotics-diet group; 5=peptide+synbiotics-diet group. Test groups fed a synbiotics diet are shown with a solid grey bar. For each tested group the percentage of CD4.sup.+ cells with an activated T.sub.h1 phenotype (Graph A) or with an activated T.sub.h2 phenotype (Graph B) are shown, as well as the T.sub.h1/T.sub.h2 ratios within an individual CD4.sup.+ population (Graph C). Data are presented as the mean±SEM of n=4 in the negative control group and n=6-8 in all other groups. * p<0.05, ** p<0.01 as analysed with ANOVA followed by Bonferroni post hoc test for selected groups.

(4) FIG. 4 shows the ex vivo splenocyte cytokine production results of example 3. The tested groups are: 1=negative control; 2=positive control; 3=peptide-diet group; 4=synbiotics-diet group; 5=peptide+synbiotics-diet group. Test groups fed a synbiotics diet are shown with a solid grey bar. The tested cytokines are: IL-13 (Graph A), IL-10 (Graph B), IL-5 (Graph C), IL-17A (Graph D) and IFN-γ (Graph E). Data are presented as the mean±SEM of n=4 in the PBS/CT group and n=6-8 in all other groups. * p<0.05, ** p<0.01 as analysed with Kruskal-Wallis non-parametric test, followed by Dunn's post hoc test for selected groups.

(5) FIG. 5 shows the skin response (ear swelling) results of example 4. The tested groups are: 1=negative control; 2=positive control; 3=peptide-diet group (solid grey bar); 4=probiotics-diet group (diagnonally striped bar); 5=peptide+probiotics-diet group (diagnonally striped bar and a grey background). Data are presented as the mean±SEM of n=6-8 in all other groups. **** p<0.0001, ** p<0.01 as analysed with ANOVA followed by Dunnette's post hoc test for selected groups.

EXAMPLES

Example 1

(6) Material and Methods

(7) Peptides:

(8) 18-amino acid (AA)-long peptides from beta-lactoglobulin were synthetically produced by JPT Peptide Technologies (Berlin, Germany). The synthetic peptides contained a 12-AA-long overlap and their sequence was spanning the B variant of beta-lactoglobulin. The four peptides were previously screened in an assay with human T cell lines by Meulenbroek et al. (Pediatr. Allergy Immunol. 2013:7:656-664) and were selected based on their T cell reactivity for further testing in animal models. Prior to the animal experiment peptides were dissolved in PBS and a mixture of all four peptides was prepared in which each peptide was at a concentration of 0.08 mg/ml (“PepMix”). Peptide sequences are given in Table 1.

(9) TABLE-US-00002 TABLE 1 Peptide sequences Peptide Sequence 1 (18 AA) SEQ ID No. 2 QKVAGTWYSLANIAASDIS 2 (18 AA) SEQ ID No. 3 WYSLANIAASDISLLDAQS 3 (18 AA) SEQ ID No. 4 AASDISLLDAQSAPLRVY 4 (18 AA) SEQ ID No. 5 LLDAQSAPLRVYVEELKP
Diets:

(10) Semipurified cow's milk protein-free standard mouse chow was composed based on AIN-93G recipe (control diet) and supplemented with synbiotics (synbiotic diet) (Research Diet Services, Wijk bij Duurstede, The Netherlands). The synbiotic supplementation consisted of 1 wt % of non-digestible short-chain (sc-) and long-chain (lc-) fructo-oligosaccharides (FOS) (Raftilose P95 (Orafti) and Raftiline HP, respectively) in a ratio scFOS/lcFOS=9:1 and 2 wt % 2×10.sup.9 CFU/g Bifidobacterium breve M-16V (Morinaga Milk Industry Co., Ltd, Tokyo, Japan). The synbiotic components were mixed through the diet and the mixture was pressed into pellets. Diets were stored at −4° C. prior to use.

(11) Animals:

(12) Three-week-old pathogen-free female C3H/HeOuJ mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and were maintained on a cow's milk protein-free standard mouse chow (AIN-93G soy, Research Diet Services). Mice were housed in the animal facility at Utrecht University. Animal care and use was in accordance with the guidelines of the Dutch Committee of Animal Experiments.

(13) Study Protocol:

(14) In order to investigate the tolerogenic properties of the synthetic peptides in combination with the synbiotic diet, a murine model for cow's milk allergy was used as described by Van Esch et al. (Pediatr Allergy Immunol 2011; 22:820-826). Mice were orally exposed (using a blunt needle) to 0.5 ml of the PepMix or phosphate buffered saline (PBS, Lonza, Walkerville, Md., USA) prior to sensitization (daily; from day −7 to day −2). In the same week (from day −9 to day 0) mice were fed the control diet or the synbiotic diet ad libitum. Subsequently, on day 0, 7, 14, 21 and 28, mice were sensitized orally with 20 mg whey protein (DMV International, Veghel, The Netherlands) homogenized in 0.5 ml PBS and mixed with 10 μg cholera toxin (CT; List Biological Laboratories, Inc. California, USA) as an adjuvant. The non-sensitized mice received 10 μg cholera toxin in 0.5 ml PBS only. Table 2 summarizes the five groups tested.

(15) Five days after the last sensitization, mice underwent an intradermal whey challenge (injection in the ear pinnae with 10 μg whey protein in 20 μl PBS) and the acute allergic skin response was recorded. The ear thickness was measured in duplicate before and 1 h after the intradermal challenge using a digital micrometer (Mitutoyo, Veenendaal, The Netherlands). The allergen-specific ear swelling is the difference between the average ear thickness at 1 h and the average basal ear thickness (Δ=ear thickness at 1 h−basal ear thickness) and is expressed in micrometer. The ear swelling due to the local injection is reflected in the “Δ ear swelling” of the non-sensitized mice (Group 1). Next to the ear swelling, clinical symptoms, such as anaphylactic shock, were monitored and scored according to a table as previously described Van Esch et al. (Pediatr Allergy Immunol 2011; 22:820-826).

(16) TABLE-US-00003 TABLE 2 Interventions in the different groups Group Pre-treatment Sensitization Challenge 1 negative control PBS + control diet PBS + CT whey 2 positive control PBS + control diet whey + CT whey 3 peptide PepMix + control diet whey + CT whey 4 synbiotics PBS + synbiotic diet whey + CT whey 5 peptide + PepMix + synbiotic whey + CT whey synbiotics diet
Statistical Analysis:

(17) All statistical analyses were conducted using GraphPad Prism 6.0c software. Data was analysed with one-way ANOVA and post hoc Bonferroni's multiple comparison test. The anaphylactic shock scores were analysed using Kruskal-Wallis test because of the non-parametric nature of the data. All data is presented as mean±SEM of 5-8 animals per group. P<0.05 was considered of statistical significance.

(18) Results

(19) Skin response results are summarized in FIG. 1 and the results on anaphylactic shock scores in FIG. 2. Sensitized, but untreated animals (Group 2) developed a significantly higher allergic response compared to the non-sensitised ones (Group 1). The allergic response was significantly reduced after pre-exposing the animals to a mixture of tolerogenic peptides combined with a diet containing synbiotics (Group 5). The treatments with the peptide mixture (Group 3) and with synbiotic diet (Group 4) were insufficient to reduce the allergic response when applied alone and only the combined exposure showed to be effective (Group 5). Furthermore, sensitized animals developed significant anaphylactic shock symptoms 20-40 min after the intradermal injection with the allergen. However, only the combined peptides-synbiotics (Group 5) exposure prevented from developing significantly higher anaphylactic shock symptoms when compared to the non-sensitized controls (Group 1). Anaphylactic shock scores were significantly increased in Groups 2, 3 and 4, compared to Group 1.

(20) The above experiment was repeated with the exception that the 1% wt % sc-FOS/1c-FOS was substituted with 1 wt % of sc-galacto-oligosaccharides (GOS) and lc-FOS (Vivinal® GOS and Raftiline HP, respectively) in a ratio scGOS/lcFOS=9:1 for the synbiotic that was fed to the animals. The results from this experiment suggest that pre-exposure of animals with a combination of this synbiotic and the tolerogenic peptide mixture also reduces the allergic response to intradermal whey challenged, as compared to a control.

Example 2

(21) An infant formula for infants at risk of cow's milk protein allergy or for infants allergic to cow's milk protein: Energy density: 0.6-0.77 kcal/ml. Protein is present in the form of free amino acids and the beta-lactoglobulin-derived peptides of the present invention. Per g protein about 100 μg peptide mix as tested in example 1 is present. Further characteristics are given in Table 3.

(22) TABLE-US-00004 TABLE 3 Nutritional information per 100 ml* Energy (kJ) 293 Energy (kcal) 70 protein (g) 1.9 (11 en %) carbohydrate (g) 7.9 (45 en %) lipids (g) 3.4 (44 en %) LA (g) 0.6 ALA (mg) 60 AA (mg) 12 DHA (mg) 7 Pepmix of example 1 (μg) 190 B. breve 2 × 10.sup.7 cfu *or per 14.7 g powder to be reconstituted in water to a total of 100 ml

(23) The composition further comprises minerals, vitamins as prescribed for infant formulae, and has an osmolarity of 324 mOsm/L.

Example 3

(24) Materials and Methods

(25) Peptides, Diets, Animals and Treatment Protocol: Same as in Example 1.

(26) Cell Isolation from Tissues:

(27) Lymphocytes were isolated from spleen, mesenteric lymph nodes (MLN) and small intestine lamina propria. Spleens and MLN were crushed through 70 μm cell strainers. Splenocyte suspension was incubated for 5 min. on ice with lysis buffer to remove red blood cells. Both splenocytes and MLN cells were taken up in RPMI 1640 supplemented with 10% FCS and penicillin (100 U/mL)/streptomycin (100 μg/mL). For the isolation of lamina propria cells, the whole small intestine was removed, cleared of Peyer's patches (PP), washed in cold PBS, opened longitudinally, and minced in 0.5 cm fragments. Samples were then washed in Hank's Balanced Salt Solution (HBSS; Invitrogen, Life Technologies, Carlsbad, Calif., USA) supplemented with 15 μM HEPES (Gibco, Life Technologies, Carlsbad, Calif., USA), pH=7.2 followed by 4×15 min incubations with HBSS supplemented with 15 μM HEPES, 5 μM Naz-EDTA, 10% FCS and penicillin (100 U/mL)/streptomycin (100 μg/mL), pH=7.2. The fragments were then washed in RPMI 1640 supplemented with 5% FCS and penicillin (100 U/mL)/streptomycin (100 μg/mL) and incubated 2×45 min with an enzyme solution containing RPMI 1640, 5% FCS, penicillin (100 U/mL)/streptomycin (100 μg/mL) and 0.25 mg/mL Collgenase type VIII (Sigma-Aldrich). In order to collect the small intestine lamina propria cells, fragments were vortexed for 10 s after each incubation and poured over a 70 μm cell strainer. Cell were washed once and used for flow cytometry.

(28) Flow Cytometry Analysis of T Cell Subsets:

(29) Phenotypic characterisation of T cell subsets was performed by means of flow cytometry. Cells were resuspended in PBS/1% BSA and were incubated for 15 min with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Pharmingen, San Jose, Calif., USA). For determining the T.sub.h1/T.sub.h2 subsets, cells were extracellularly stained with CD4-PerCp-Cy5.5, CD69-APC, CXCR3-PE (eBiosciences, San Diego, Calif., USA) and T1ST2-FITC (MD Biosciences, St. Paul, Minn., USA). After staining extracellular markers, cells were stained with a fixable viability dye AlexaFluor780 (eBioscience). Results were collected with BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, N.J., USA) and were analysed with FlowLogic software (Inivai Technologies, Mentone, VIC, Australia).

(30) Ex Vivo Re-Stimulation Assay and Cytokine Levels:

(31) After sacrifice, spleens were removed and a single cell suspension was obtained. Then splenocytes (6×10.sup.5 cells) were cultured either with medium or with 500 μg/mL whey protein at 37° C., 5% CO.sub.2. After 5 days of incubation, supernatants were collected and stored at −20° C. until further analysis. Cytokine quantification of IL-5, IL-13, IL-10, IL-17A and IFN-γ was performed by means of a Cytometric Bead Array (CBA) Flex Set assay (BD Biosciences) following manufacturer's instructions. Beads were analysed with BD FACSCanto II flow cytometer and results were obtained in FCAP v. 3.0 software (Becton Dickinson).

(32) Statistical Analysis:

(33) All statistical analyses used GraphPad Prism 6.0c software for Macintosh (GraphPad Software, San Diego, Calif., USA). All data was analysed for normality and equality of variance. One-way ANOVA, followed by a Bonferroni's multiple comparison post hoc test for selected groups (7 pre-selected comparisons) was used when possible. When data was not normally distributed, as in the case of cytokines levels, it was first LOG-transformed and tested again. If LOG transformation did not improve normality, then the non-parametric Kruskal-Wallis test was used, followed by a Dunn's post hoc for selected groups and 7 pre-selected comparisons. All data is presented as mean±SEM of 4-8 animals per group. P<0.05 was considered of statistical significance.

(34) Results

(35) In order to investigate the local effects in the intestine, lamina propria lymphocytes from the small intestine (SI-LP) were isolated and analysed by flow cytometry for the different T cell subsets. In line with the paradigm that allergy influences the balance between T.sub.h1 and T.sub.h2 lymphocytes skewing it toward the T.sub.h2 environment (Cox, H E, J Pediatr Gastroenterol Nutr 2008; 47 Suppl 2, S45-48), it was observed that allergic mice showed significantly lower numbers of activated T.sub.h1 cells, while activated T.sub.h2 cells appeared to increase in number (FIG. 3). As a result, the ratio of activated T.sub.h1/T.sub.h2 cells was shifted in favour of T.sub.h2 in the allergic control mice (Group 2), while prior feeding with peptides and synbiotics (Group 5) prevented this shift. Feeding mice only the synbiotics-supplemented diet (Group 4) tended to preserve the T.sub.h1/T.sub.h2 balance but less pronounced compared to the combination with peptides, suggesting that the synbiotics ensure a favourable milieu during the presentation of the peptides by antigen presenting cells.

(36) To investigate whether the preventive treatments affect the functionality of cells in the systemic compartment, spleens from the treated groups were collected 18 h after the oral challenge and splenic lymphocytes were stimulated ex vivo with allergen for 5 days in order to determine their capacity to produce cytokines. The results presented in FIG. 4 show that the functionality of splenocytes was affected by pre-exposure to the combination of peptides and synbiotics-enriched diet (Group 5). Functionality was assessed by assaying allergy-related T.sub.h2 cytokines (IL-13, IL-5, IL-10) as well as T.sub.h1-(IFN-γ) and Th17-assosiated ones (IL-17A). Notably, cells from allergic controls (Group 2) markedly produced all these cytokines; not only the T.sub.h2-associated cytokines. However, cells from animals treated with a combination of peptide mixture and synbiotics (Group 5) tended to induce less IL-17A, IL-10 and IL-13, as compared to the peptide mixture alone (Group 3).

(37) These results indicate that combined exposure to peptide mixture and synbiotics reduces allergen-induced cytokine production and prevents unfavourable shift in the T.sub.h1/T.sub.h2 balance in the intestinal lamina propria.

Example 4

(38) Materials and Methods

(39) Peptides:

(40) Same as example 1 except that prior to the animal experiment peptides were dissolved in PBS and a mixture of all four peptides was prepared in which each peptide was at a concentration of 0.8 mg/mL (“PepMix”)

(41) Animals:

(42) Same as in example 1.

(43) Statistical Analysis:

(44) Statistical analyses were conducted using GraphPad Prism 6.0c software for Macintosh (GraphPad Software, San Diego, Calif., USA). Data was analysed with one-way ANOVA and post hoc Dunnett's multiple comparison test. Data is presented as mean±SEM of 6-8 animals per group. P<0.05 was considered of statistical significance.

(45) Diets:

(46) Semipurified cow's milk protein-free standard mouse chow was composed based on AIN-93G recipe (control diet) and supplemented with 2 wt % 2×10.sup.9 CFU/g Bifidobacterium breve M-16V (Morinaga Milk Industry Co., Ltd, Tokyo, Japan) (probiotic diet). The probiotic component was mixed through the diet and the mixture was pressed into pellets. Diets were stored at −4° C. prior to use.

(47) Study Protocol:

(48) Mice were orally exposed (using a blunt needle) to 0.5 mL of the PepMix or phosphate buffered saline (PBS, Lonza, Walkerville, Md., USA) prior to sensitization (daily; from day −7 to day −2). In the same week (from day −9 to day 0) mice were fed the control diet or the probiotic diet ad libitum. Subsequently, on day 0, 7, 14, 21 and 28, mice were sensitized orally with 20 mg whey protein (DMV International, Veghel, The Netherlands) homogenized in 0.5 mL PBS and mixed with 10 μg cholera toxin (CT; List Biological Laboratories, Inc. California, USA) as an adjuvant. The non-sensitized mice received 10 μg cholera toxin in 0.5 mL PBS only. Table 3 summarizes the five groups tested.

(49) Five days after the last sensitization, mice underwent an intradermal whey challenge (injection in the ear pinnae with 10 μg whey protein in 20 μl PBS) and the acute allergic skin response was recorded, as described in example 1.

(50) TABLE-US-00005 TABLE 3 Interventions in the different groups Group Pre-treatment Sensitization Challenge 1 negative control PBS + control diet PBS + CT whey 2 positive control PBS + control diet whey + CT whey 3 peptide PepMix + control diet whey + CT whey 4 probiotics PBS + probiotic diet whey + CT whey 5 peptide + PepMix + probiotic whey + CT whey probiotics diet
Results

(51) Skin response results are summarized in FIG. 5. Sensitized, but untreated animals (Group 2) developed a significantly higher allergic response compared to the non-sensitised ones (Group 1). The treatment with probiotic alone (Group 4) did not significantly reduce the allergic response. However, the allergic response was significantly reduced after pre-exposing the animals to a mixture of tolerogenic peptides alone (Group 3) and this reduction was more pronounced when the tolerogenic peptides were combined with a diet containing probiotics (Group 5).