MXPA01007225A - A hypoallergenic composition containing tolerogenic peptides inducing oral tolerance - Google Patents

Abstract

A hypoallergenic composition for the induction of protein tolerance in at risk individuals of protein allergy, consisting of (i) a"non allergenic"protein extensively hydrolysed basis and/or of (ii) a free amino acid basis, said composition comprising as the active ingredient at least one tolerogenic peptide of the allergenic protein.

Description

A HYPOALERGIC COMPOSITION CONTAINING TOLEROGENIC PEPTIDES THAT INDUCE ORAL TOLERANCE FIELD OF THE INVENTION The present invention relates to a hypoallergenic composition containing protein-specific or protein-specific peptides, said composition being able to induce oral tolerance to natural proteins. It is also related to the use of peptides t or 1 e r or gé n i c o s of the milk protein for the preparation of a composition that induces the immunological tolerance of milk proteins.

BACKGROUND OF THE INVENTION During the common nutrition process, dietary proteins are presented to the immune system through the intestine, followed by an immune insensitivity to the ingested nutrients. This vital phenomenon, called oral tolerance, is efficient for most people. If oral tolerance fails, a food allergy is present, and it is necessary to strictly avoid the incriminated food.

Avoiding marked food represents a difficult task for adult patients with food allergies. For example, strict elimination of cow's milk from the diet of allergic babies could be even more difficult, especially if breastfeeding is not possible or is not desired. Allergies to cow's milk and formulas containing cow's milk adapted to the needs of babies are due to the fact that the proteins in cow's milk differ from the proteins in the mother's milk and can constitute allergens. Among the whey proteins, ß-lactoglobulin is the main component and is a strong allergen. In addition to breastfeeding, which is the primary recommendation for prevention, hypoallergenic formulas are systematically prescribed to "at-risk" newborns, that is, asymptomatic babies with atopic parents. In contrast to the adapted formulas, cow's milk proteins have been hydrolyzed in hypoallergenic formulas, to reduce potential allergenicity. It has been shown that this approach is efficient to prevent sensitization by natural proteins present in the adapted formulas. Accordingly, in US Pat. No. 4,293,571 a protein hydrolyzate was prepared by pancreatic hydrolysis, coagulation of non-hydrolysed proteins by thermal treatment and ultrafiltration to remove the coagulated residual proteins and the macropipes that could be allergens. Also, US No. 5,039,532 provides an improved process for the preparation of an animal milk protein hydrolyzate substantially reduced in allergens, in which a whey product was subjected to enzymatic hydrolysis. Two different types of hypoallergenic formulas are proposed for babies at high risk: partially and extensively hydrolyzed formulas, differentiated by the hydrolysis measurement of natural proteins. However, as is clear from the aforementioned technique, the primary focus to date in the treatment of cow's milk allergies has been to find preparations that do not induce an allergic response, i.e., provide non-allergic formulations .

However, while these formulations have allowed a person allergic to cow's milk to avoid an allergic response, they do not solve the problem of allowing a person to drink unaltered dairy products. More recently, it has been investigated that partially hydrolyzed milk formulations not only have a reduced allergenicity, but can also induce an unological tolerance to milk proteins (EP 0827 679). The widely hydrolyzed formulas are specially designed to treat patients allergic to cow's milk proteins, but their capacity for induced long-term oral tolerance is questioned. Accordingly, EP 0 629 350 discloses the use of non-allergenic whey protein hydrolysates that are said to induce protein tolerance of cow's milk. Although this patent application indicates that whey protein hydrolysates substantially free of allergenic proteins could be used to induce protein tolerance of cow's milk in children at risk of suffering an allergy to cow's milk, the present inventors discovered when analyzing other non-allergenic whey protein hydrolysates that non-allergenicity does not necessarily translate into the ability to induce tolerance to the proteins of cow's milk. In fact, it was discovered that some of the formulations that exhibit the highest degree of non-allergenicity are not suitable for inducing a tolerance to the proteins of cow's milk. Atough, Lo, C.W and Kleiman, R.E. (American Journal of Clinical Nutrition 1996, 63 (4), 646S-650S) suggests that the baby formula contains tolerogenic peptides that can be used to treat allergic diseases or that suppresses the development of autoimmune diseases, it is clear that the technique lacked the information to provide formulas that were most suitable for the induction of tolerance.

DESCRIPTION OF THE INVENTION The present invention provides a hypoallergenic composition for the induction of protein tolerance in persons at risk of allergy to proteins containing (i) a "non-allergenic" base of extensively hydrolyzed proteins and / or (ii) a base of free amino acids, said composition comprising as active ingredient at least one tolerogenic peptide of the allergenic protein. In a preferred embodiment, said tolerogenic peptides are present in the form of (i) tolerogenic peptide fractions isolated from hydrolysis of proteinaceous material containing the allergenic protein and / or (ii) synthetically prepared tolerogenic peptides. The present composition contains a source of nitrogen that could provide 7 to 25% of the total energy, a source of carbohydrates that could provide at least 28 to 66% of the total energy, a source of lipids that could provide at least 25 to 60% of the total energy and at least one tolerogenic peptide of the different proteins. A main advantage of the present composition is to induce an oral tolerance in people "at risk", with the aim of avoiding an eventual sensitization by the use of natural tolerogens. In addition, tolerogenic peptides derived from protein hydrolysis offer both hypoallergenic and tolerogenic properties and induce oral tolerance at humoral and cellular levels. The present composition is particularly intended for people at risk of allergy to milk protein. Another aspect of the present invention is the use of tolerogenic peptides of milk proteins for the preparation of a hypoallergenic composition designed for mammals susceptible to an allergy to cow's milk. In a preferred embodiment, the tolerogenic peptides are of milk origin and particularly have their origin in β-lactoglobulin (β-LG), α-lactalbumin, bovine serum albumin or casein. For the preparation of said composition, tolerogenic peptides in the form of a peptide moiety containing the following peptides can be used: H2N-IDALNENK-COOH, H2N- -LVLD-T-DYK, -K-COOH or H2N-TPEVDDEALEKFDK-COOH of β-lactoglobulin. In another aspect, the invention provides a method for the preparation of tolerogenic peptides useful in the induction of a tolerance to proteins in persons at risk of protein allergy, in which: (i) a proteinaceous material containing the allergenic protein at a degree of hydrolysis of about 10 to 50%; (ii) it is then treated by inactivating the residual enzymatic activity; (iii) the protein hydrolyzate solution is clarified and subjected to a precipitation treatment or passed through a chromatography column filled with the appropriate resin to extract the tolerogenic peptide fractions.

DETAILED DESCRIPTION OF THE INVENTION In the present invention, the term tolerance is to be understood as a state of specific immunological insensitivity. Both humoral (antibodies) and cell-mediated (lymphocytes ...) pathways of the immune response can be suppressed by the induction of tolerance. A failure of oral tolerance is considered to be the underlying cause of food allergy. The term "allergen" is to be understood as a protein or acropeptide capable of initiating allergic reactions in humans, particularly in infants or infants at risk. Babies are considered to be "at risk" of a food allergy when one of the parents or a sibling is atopic. The term tolerogenic peptides is to be understood as protein fragments, which correspond to parts of the natural protein, of a size of 200 to 6000 Da (3 to 50 amino acids), and preferably, between 500 and 3000 Da and which are capable of induce a specific oral tolerance to natural proteins. The term "non-allergenic" is to be understood as a source of nitrogen containing a well-balanced amino acid composition. "Non-allergenicity" is defined in the case of milk proteins, as a residual allergenicity of individual whey proteins not exceeding 1 ppm and as a residual allergenicity of total caseins not exceeding 10 ppm. The hypoallergenic composition can contain as a source of nitrogens, peptides or free amino acids and particularly of milk proteins such as whey proteins, α-lactalbumin, β-lactoglobulin, bovine serum albumin, casein acid, caseinates, or α, β , k-casein, for example. The nitrogen source can provide at least 7 to 25% of the total energy. As a carbohydrate source, lactose, sucrose, starch or maltodextrin can be used. Carbohydrates can provide at least 28 to 66% of the total energy. Preferably, vegetable or butter oil agents are used as a source of lipids that can provide at least 25 to 60% of the total energy. You can add vitamins, or 1 igoelementos and minerals in a sufficient amount to meet daily demands. The composition according to the present invention comprises as an active ingredient at least one tolerogenic peptide of the allergenic protein, said tolerogenic peptide having been selected for its ability to induce oral tolerance. The tolerogenic peptides can be obtained by the enzymatic hydrolysis of the proteinaceous material containing the allergenic proteins that are responsible for the allergies in people at risk, followed by the isolation of tolerogenic peptide fractions.

These peptide fractions enriched in said tolerogenic peptides can be obtained by separation of the protein hydrolyzate. Likewise, the tolerogenic peptides may also be present in the composition in the form of synthetically prepared tolerogenic peptides. The composition contains a quantity sufficient to induce oral tolerance, which is preferably the one that allows a induction of complete oral tolerance, that is, one that avoids any reaction after performing the DBPCFC (double blind placebo controlled food challenge) with the milk of the cow. Accordingly, the tolerogenic peptides can be present in an amount of about 0.01 to 10% (nitrogen source of the protein), for example and preferably, about 0.1 to 0.2% of total peptides. In the particular case of tolerance to milk proteins, the composition may contain tolerogenic peptides of milk origin such as, for example, β-lactoglobulin or caseins. Accordingly, the tolerogenic peptides may be in the form of a peptide moiety comprising at least one of the following peptides: H2N-I -DALNENK-COOH, H2N-VLVLDT -DYK, -K-COOH or H2N-TPEVDDEALEKFDK-COOH of β-lactoglobulin. In a preferred embodiment, a method for the preparation of tolerogenic peptides comprises the following steps: (i) a proteinaceous material containing the allergenic protein is hydrolysed to a degree of hydrolysis of about 10 to 50%; (ii) it is then treated by inactivating the residual enzymatic acti; (iii) the protein hydrolyzate solution is clarified and subjected to a precipitation treatment or passed through a chromatography column filled with the appropriate resin and the tolerogenic peptide fractions are recovered. This preferred method is well suited for the treatment of prepared hydrolysates of various protein concentrations (Ntot% = N * 6.38) to modify the proportion of tolerogenic acti and residual antigenicity of the proteinaceous material. If it is arbitrarily defined that the antigenicity of a natural protein is 106 (as 106 μg / g of protein), and the tolerogenic response is 1, then, for the natural protein, this ratio is 10-6. Therefore, the proportion that qualifies the tolerogenic acti of a certain fraction or tolerogenic peptide should be at least 2 X 1Q ~ 2. The proteinaceous material to be treated can be any composition containing protein material and in particular a solution or dispersion of milk proteins: whey proteins, acid whey proteins, sweet whey proteins, whey protein concentrates, whey protein isolate, demineralized whey powder or caseinate, for example. In general, the protein content can vary within the range of about 70 to 95% by weight, but the raw material is preferably as rich in protein as possible. The proteins present in the proteinaceous material can be modified with proteolytic enzymes in protein hydrolyzate having a degree of hydrolysis (a-amino-N / Nto t) preferably around 10 to 50%. Proteolytic enzymes may be, for example, of animal or vegetable origin (pepsin, chymotrypsin, trypsin, intestinal mucosa extract, pancreatic extracts, chymosin, papain, bromelain, ficin), of bacterial or fungal origin (serine and metalloproteases of Bacillus subtilis , Bacillus licheniformis, Aspergillus orysae, Aspegillus wentii and acidogenic proteases of ß.spergillus orizae, Aspergillus wentíi, Mucor miehei, Mucor pusillus, Endothia parasitica) or a combination thereof. During hydrolysis, the concentration of the proteinaceous material in a solution or in a suspension is preferably about 5 to 20% by weight and could be theurized before introducing the proteases. The ratio between enzyme / protein can be from 0.1 to 10% w / w and, preferably, from about 0.25 to 4%. The hydrolysis can be carried out at a temperature of about 35 ° C to 65 ° C, for 30 minutes to 10 hours, preferably 30 minutes to 4 hours, at pH values in the range of 2.5 to 11, preferably 4.5 , 7.0, 8.0 and 8.5. If desired, the pH value of the solution can be adjusted and adjusted with citric acid, HCl or food grade NaOH, NH40H, KOH, Ca (0H) 2, for example, in a pure 2N concentration or as a mixture . Then, the protein hydrolyzate can be subjected to a heat treatment of about 0.1 to 10 minutes at a temperature of about 70 to 110 ° C to inactivate the residual enzymes (ie, proteases). The protein hydrolyzate solution obtained in this way can be clarified by centrifugation and / or ultrafiltration to remove insoluble and intact proteins respectively, and the clear solution is recovered. It is possible to use on an industrial scale different types of membranes (spiral, tubular, flat, fibrous) made with different materials (minerals, polysulfone, ...) and having different cut-off limits between 1,000 and 100,000 Daltons. Depending on the type of enzyme, the hydrolysis conditions and the type of membranes, the modification of tolerogenic fractions could be sufficient at this stage. The recovered clear hydrolyzate solution may be concentrated, if desired, by evaporation to a dry solid content of 10 to 50% for further treatment or may be spray dried if enrichment in tolerogenic peptides is sufficient. The protein hydrolyzate solution obtained in this way can be subjected to a precipitation treatment by solvent, acid, or salts, for example, followed by centrifugation. In the precipitation treatment, the concentration of the hydrolyzate solution increases the production and reduces the amounts of solvent. For example, ethanol can be added to obtain a final concentration within 15 to 60% volume / volume at a temperature of about 4 ° C to 25 ° C. After one hour of incubation, a centrifugation (30 minutes at 4500 g) may allow the separation of the soluble and insoluble peptides. Depending on the process, acid (phosphoric or hydrochloric, for example) or focal calcium precipitation may be used. Then, the solvents can be removed by evaporation and the salts by electrodialysis, for example. Preferably, the clear solution and the insoluble fraction are recovered. The protein hydrolyzate solution obtained in this way can be passed through a column filled with adsorption, ion exchange or hydrophobic resin at a flow rate of 0.1 to 4 column volumes per hour at a temperature between about 4 ° C and 60 ° C. Prior to the chromatography treatment, the protein hydrolyzate can be concentrated to provide a solution having a dry solid content of 8 to 35% by weight. During chromatography, a peptide fraction is absorbed into the resin by passing the hydrolyzate solution through a column filled with the suitable support at a rate of 0.1 to 4 column volumes per hour. It is possible to use different types of chromatography on an industrial scale such as: ion exchange, hydrophobic interactions, reverse phases, adsorption (hydroxyapatite, activated carbon, polystyrene-based hydrophobic resins or covalent chromatography, for example.) In chromatography treatment, the amount of hydrolyzate solution per liter of column filled with resin can be 5 liters with respect to dry solids of 10%, preferably a solution of hydrolyzate having 20 to 1000 g of dry solid per liter of resin is passed through the column filled with resin The treatment can be carried out by chromatography at a pH of about 2 to 10, preferably 6 to 8, for the clarified hydrolyzate solution.The treatment can be carried out by chromatography at a temperature of about 4 °. C at 60 ° C.

For example, treatment by chromatography to select the tolerogenic fractions of β-lactoglobulin may consist of using: a strong cationic resin equilibrated with 0.1 N HCl at a flow rate of 1 volume / hour. The non-retained fraction was eluted with 3 volumes of water; the second fraction (fraction containing tolerogenic peptides) was eluted with 0 to 0.5 N NaOH and the third fraction with 0.1 N HCl, a reverse phase resin (C18) equilibrated with pure water. The non-retained fraction was eluted with water, then stepwise (20% and 40% ethanol); the second and third fractions were recovered. - a strong anionic resin equilibrated with 0.1 N NaOH. The non-retained fraction was eluted in 3 volumes of water. The second fraction was eluted with 0.5 N HCl, the third with 0.1 N NaOH. The most preferred method is to treat a neutral solution with resin; in that case, a pH adjustment is not required after the hydrolysis step and the salt content of the product will be lower.

To finish the treatment by chromatography, the column can be eluted with pure water, then water containing salts, buffer, acids, bases, or organic solvents at a temperature of 4 to 60 ° C. The elution is carried out step by step or by means of a concentration gradient. The solutions that have passed through the column are recovered. If necessary, the salts, solvents, acids, bases of the recovered solution are removed, and the recovered solutions can be concentrated in a dry solid content of 35 to 65% and spray dried. These peptides are then specific fragments corresponding to a part of the natural protein sequence or to a part of the specific tryptic peptides of the hydrolyzed protein. These tolerogenic peptides can be used for the preparation of a composition that induces oral tolerance to natural proteins; said composition is intended for mammals susceptible to allergy to proteins and particularly humans and pets. The following examples are provided by way of illustration only and in no way should be construed as limiting the subject of the present application.

Figures Figure 1 represents a specific oral tolerance induction in mice fed once with TTH ß-LG, F2 or F (7 + 9). A: IgE anti-ß-LG humoral response (black bars) and ucosal (gray bars). B: DTH response ß-LG-specific. (•) are individual pad increases and (-) are means of 10 mice per group. C: Specific responses (ß-LG) and proliferati vas (PHA) nonspecific splenic cells. Splenic lymphocytes were isolated from mice fed with (F), TTH ß-LG («), F2 (A), or F (7 + 9) (•) and subsequently stimulated with decreasing concentrations of antigens. The incorporation of 3H thymidine was measured after 120 hours of culture. The results of the incorporation of (3H) Tdr were expressed in cpm, as a means of triplicate cultures, then the mean subtracted blank was plotted respectively against a concentration of ß-LG or PHA. Figure 2 depicts primary sequences assigned to triptych peptides identified in β-Lactoglobulin hydrolyzate. The vertical bar represents a disulfide bond.

EXAMPLES Example 1: Selection and characterization of the tolerogenic peptides of β-Lactoglobulin In the first example below, experiments were carried out to select the tolerogenic peptides of β-LG. The ß-LG was digested by trypsin treated with TPCK under selected conditions to cause its gentle digestion. The size and molecular weight distribution of the resulting peptides of our ß-LG hydrolysis model ranged from two to twenty-three amino acids, and from 247 to 2719 Da respectively. The tolerogenic properties of the resulting peptides have been tested in an experimental oral tolerance mouse model.

Methods a) Animals Female Balb / c mice were obtained from IFFA-Crédo (L 'Abresle, France). All of them were raised on a milk-free diet. The mice had 3 weeks of life at the beginning of the experiments. b) Preparation of tolerogenic fractions In order to be digested, ß-LG is dissolved (220 grams.) In bi-distilled water at a final concentration of 5% (w / w). ß-LG was digested by trypsin treated with TPCK, using an Enzyme / Substrate (E / S) ratio. of 1/100 (w / w) at 40 ° C, pH 7.6 under constant stirring After a one hour hydrolysis, the same amount of enzyme was added to provide a final I / O ratio of 2/100 (weight After a hydrolysis of 4 hours, the reaction was stopped by inactivating the trypsin at 85 ° C for 5 minutes, then the total tryptic hydrolyzate of β-lactoglobulin (TTH β-LG) was lyophilized. The products of ß-LG digested by preparative chromatography on a cationic resin, 15 different fractions were obtained, individually nano fi ltered to concentrate and eliminate salts, diafiltered, dialyzed, lyophilized and stored dry at room temperature until experiments were performed. í n vi vo.

In addition, each fraction was characterized by its peptide content using reverse phase High Performance Liquid Chromatography. Considering TTH ß-LG as a reference, the enrichment and depletion of peptides in each fraction per area detected at 214 nm was appreciated using injections of iso-nitrogen. c) Procedure for induction of oral tolerance and immunization Oral priming was administered to the mice at 22 days of age, by gastric feeding of natural ß-LG (5 mg / g of body weight), various amounts of ß-LG TTH, or various amounts of the different peptide fractions of ß-LG. The control mice were fed with saline water. Five days later, all mice were immunized with ß-LG and OVA (Ovalbumin, quality V, Sigma), as an unrelated antigen to test the specificity of the immune response, 21 days after the systemic challenge, a Hypersensitivity evaluation was performed. of Delayed Type (DTH) by measures of duplicated thickness of the left posterior plantar pad before, and after, immunization with ß-LG. d) Evaluation of the immune response 24 hours after the DTH immunization, the individual increases in thickness of the plantar pad of those injected the previous day were measured. The differences between these two measurements were expressed in thickness? (millimeters) and were used in group comparisons. Then, from all the mice, blood samples were taken, the spleens were taken and combined according to the treatment group. Splenocyte specific proliferation assays were performed for each group. The intestinal content was collected individually. Serum and intestinal samples were rapidly frozen at -80 ° C, until the tests were performed. The specific IgE against ß-LG and against OVA was determined in both the serum and intestinal samples. e) Specific IgE antibody assays Dilutions of serum and intestinal fluid in duplicate were tested for anti-ß-LG and anti-OVA IgE antibodies by ELISA-, Combined samples of twenty non-immunized female mice were used as Negative controls on each of the cymbals. The titers were determined by calculating the dilution of the sample that provided the absorbance of the negative control. The titles were expressed as the logio of the reciprocal of the dilution. f) Cell cultures Soluble cell solutions were homogenized and purified. Cells were cocultivated in the presence of ß-LG or in vitro fitohaemaglut A. A (3H) Tdr (Amersham, Zurich) was added in the final 6 hours of culture and the dishes were harvested and analyzed by scintillation counting. The stimulation indices were calculated as the proportion of the test subtracted blank and the count values were expressed as the average incorporation of (3H) Tdr average cpm by triplicate cultures. g) Determination of the binding capacity of IgG of ß-LG An inhibition by ELISA was used to determine the epitopes of ß-LG in the hydrolyzate of ß-LG and in the fractions of ß-LG. The ß-lactoglobulin titers were calculated from a standard ß-LG curve of 5-part dilution developed in each dish. DTH responses, serum and intestinal IgE responses were compared using single factor ANOVA tests.

Results a) Characterization of the obtained fractions of ß-LG TTH In order to isolate the tolerogenic peptides of the ß-LG TTH, large quantities of tryptic fractions of ß-LG have been produced by preparative chromatography. From each of them, 15 fractions (F1-F15) were collected and combined respectively. Previously, the assigned sequences of the tryptic peptides of ß-LG were determined (Figure 2). • No specific peptide enrichment could be detected in fractions Fl, F5 and F15. • 3 different peptides T6, T17 and T18 present in fraction F2 were enriched; but they did not concentrate on any of the other fractions. • Similarly, T7 was specifically enriched in fraction F6. Apart from these peptides, concentrated in only a certain fraction, some others were enriched in several fractions. This was the case of T21, for example, clearly detected at high speeds in fractions F7, F9 and FIO (Table 1).

Table 1: Mass fractions of ß-LG and significant enrichment of peptides within the fractions. b) Two different fractions of the tryptic hydrolyzate of ß-LG presented tolerogenic properties: The fractions were combined according to their similarities of enriched peptides and in proportion to their respective masses, and then they were tested i n vi vo. - It was considered that Fl, F2, F5 and F15 were administered individually, because they did not share the peptide enrichment with the other fractions.

- F3 and F4 were combined based on the highest concentration of T23; - F8 and F9 were mixed to test the tolerogenic activity of T12; - F7 and F9 were combined according to the enrichment of T20 and T21; - F6, Fll, F12, F13 and F14 were combined, each one enriched in T9, UNCLE and TIL. The qualitative responses of the tolerogenic properties generated from the feeding were evaluated with the different groups of fractions in two different concentrations (0.5 or 0.125 mg / g of body weight). The induction or absence of induction of tolerogenic responses to ß-LG was determined by the statistical significance of the immune response. Two of the eight fractions tested appeared to be active in reducing anti-ß-LG IgE responses and splenocyte-specific proliferation: - F2, when given orally at 0.125 mg / g body weight, - F (7 + 9), in both doses tested. c) Humoral, mucosal and cellular tolerance induced by the oral administration of tryptic fractions of ß-LG F2 and F (7 + 9) According to our quantitative criteria, serum and intestinal anti-ß-LG IgE responses, DTH and proliferation Specific splenocytes, oral tolerance has been induced in mice either with F2 or with F (7 + 9) (Figure 1). For the two groups fed with fractions, serum anti-ß-LG IgE titers, 3.58 +/- 0.18 and 3.21 +/- 0.29 respectively for F2 and F (7+ 9), were not as low as when induced by TTH ß-LG feed (2.68 +/- 0.05) (Figure 1A). However, the specific levels of IgE were considerably lower than the titers of the control mice (4.16 +/- 0.05) (p <0.05). Likewise, the specific intestinal IgE was reduced in mice fed with TTH ß-LG (1.66 +/- 0.32) or fed with the two tryptic fractions of ß-LG (1.1 +/- 0.34 and 1.9 +/- 0.21 respectively for F2 and F (7 + 9)), evaluating downward regulation induced orally compared to the control group (2.32 +/- 0.64). Figure IB shows that the specific DTH was clearly reduced in the mice fed with TTH ß-LG) 0.074 mm) or each of the two tolerogenic fractions (0.063 mm and 0.062 mm respectively for F2 and F (7 + 9)) in Comparison with local hypersensitivity measured in control mice fed only saline (0.164 mm). To confirm the successful induction of oral tolerance in cellular immunity, assays of specific and non-specific proliferation of splenocytes were carried out. Figure 1C and D show that feeding the mice with either of the two ß-LG fractions chosen as well as with ß-LG TTH caused a severe decrease in the proliferative response to ß-LG compared to that observed in splenocytes isolated from control mice fed with saline. In fact, the stimulation indices were 0.225, 0.174 and 0.341, respectively for mice fed with TTH ß-LG, F2 and F (7 + 9). As expected, the proliferative to non-specific response due to PHA stimulation was not affected by the different feeding regimen. d) Antigenicity of ß-LG hydrolyzate and ß-LG fractions The measurement of the residual ß-LG epitopes performed for the different fractions by ELISA inhibition showed that the amounts of ß-LG epitopes present per g of proteins were extremely different from one fraction to another, fluctuating from 15 μg / g of protein for Fl up to 9240 μg / g of protein for F13. These results also indicate that the antigenic and tolerogenic sites could be clearly located, indicating that allergenicity of tolerogenicity could be disengaged. In schematic form, three different types of fractions can be described: - a first type, represented by fraction F2, including basic peptides with a high tolerogenic potential associated with a very low antigenicity, - at the other extreme, a fraction F13 type that it contains acidic peptides, highly antigenic and lacking tolerogenic activity and, finally, in a medium position, the peptides present in the tolerance inrs of fractions F7 and F9, but which still have a remarkable antigenicity.

The fractions F2 and the mixture F (7 + 9) are both tolerogenic. The antigenicity of the tolerogenic fractions F2, F7 and F9 was lower than that of the total hydrolyzate. Surprisingly, it was found that the antigenicity of the tolerogenic F2 was 53 times lower than the antigenicity of the ß-LG TTH. In these two tolerogenic fractions, the sizes of the potentially tolerogenic peptides were distributed between 8 amino acids for T6, and 23 amino acids for T21, and respective molecular weights between 915 and 2719 Da. To be tolerogenic, the peptides seem to require an accurate balance between dose, size, sequence and structure. However, until now, the correlation between peptide structure and tolerogenic properties has not been described in the literature.

EXAMPLE 2: Obtaining Tolerogenic Peptides from Whey Protein In order to obtain tolerogenic peptides, 1 kg of whey protein isolate was dissolved in 7 liters of water at 50 ° C. The pH was adjusted to 7.5 with 2N KOH. The volume was adjusted to 14 liters with water at 50 ° C to obtain a 7% solution with respect to the powder. 10 g of Trips ina + Quimo trypsin enzyme were added and the solution was hydrolyzed for 120 minutes. The pH was maintained with an alkaline solution of 2N KOH and 2N NaOH. After hydrolysis, the solution was heated at 80 ° C for 10 minutes. The solution was cooled to 40 ° C and ultrafiltered with 40,000 Da cut membranes. The clear permeate was treated with cationic resin in the following manner in a final resin volume of about 180 ml. The resin was washed and equilibrated with 0.1N NaOH at a flow rate of 300 ml / h. 100 ml of 40,000 Da of clear permeate were passed through the column. The non-retained fraction was eluted in 540 ml of pure water. The second and third fraction were eluted in 900 ml of 0.5 N HCl and 540 ml of 0.1 N NaOH respectively. Tolerogenic peptides are present in fraction F2, for example, in an amount sufficient to prevent any reaction after performing DBPCFC with cow's milk.

EXAMPLE 3: Obtaining Tolerogenic Caseinate Peptides 1 kg of sodium caseinate was dissolved in 7 liters of water at 50 ° C. The pH was adjusted to 7.5 with 2N KOH. The volume was adjusted to 10 liters with water to 50 ° C to obtain a 10% solution with respect to the powder. 40 g of Alcalase enzyme was added and the solution was hydrolyzed for 120 minutes. The pH was maintained with an alkaline solution of 2N KOH and 2N of NaOH. After hydrolysis, the solution was heated at 80 ° C for 10 minutes. The solution was cooled to 40 ° C and ultrafiltered with polyethylene phona membranes of 10,000 Da cut. Reverse phase chromatography: 300 ml of clear permeate was passed through the equilibrated column at a flow rate of 3 ml / minutes. The non-retained fraction was eluted with 300 ml of water, then the second fraction was eluted with 200 ml of 0-20% ethanol gradient. Finally, the third fraction was eluted with 200 ml of 20-40% ethanol gradient. The column was regenerated by 400 ml of ethanol gradient 40-80% v / v and equilibrated with 500 ml of pure water.

EXAMPLE 4 The procedure was as described in Example 3, except that the amount of the enzyme and the membrane cut were changed by Alcalase 20 g and 40,000 Da respectively. Subsequent chromatography on cationic resin was performed as follows: 150 ml of resin was washed 3 times with 500 ml of pure water, then poured into a column whose final resin volume was 180 ml. The resin was washed and equilibrated with 0.1N NaOH at a flow rate of 300 ml / h. 100 ml of 40,000 Da of clear permeate was passed through the column at a flow rate of 300 ml / h. The non-retained fraction was eluted with 540 ml of pure water. The second and third fractions were eluted with 900 ml of 0.5 N HCl and 540 ml of 0. N NaOH respectively.

EXAMPLE 5 The procedure was as described in example 3, except that the reversed phase treatment was changed by chromatography on a strong anionic resin as follows: 150 ml of resin was washed 3 times with 500 ml of pure water, then poured into a column where the resin was washed and equilibrated with 0. IN HCl at a flow rate of 180 ml / h. 100 ml of ultrafiltrate was passed through the column at a flow rate of 180 ml / h. The non-retained fraction was eluted with 540 ml of water. The second and third fractions were eluted with 3 column volumes of 0.5 N NaOH and 5 column volumes of 0. I N of HCl respectively.

EXAMPLE 6: Tolerogenic Formula for Babies The tolerogenic peptides T6 and T17 contained in the tolerogenic F2 fraction were prepared synthetically. T13 contained in a non-tolerogenic fraction was also synthesized as a negative control. The induction of oral tolerance of these peptides was tested in mice as described in example le) except that the dose is 20 meg / g of body weight.

The oral administration of T6 or T17 induces a cellular tolerance (specific inhibition of lymphocyte proliferation). T17 also induces a humoral tolerance by a significant IgE anti-b-LG specific decrease. Oral administration of T13 (negative control) does not induce any tolerance. These two T6 peptides (H2N-I-D-A-L-N-E-N-K-COOH) and T17 (H2N-V-L-V-L-D-T-D-Y-K, -K-COOH) have a high tolerogenic potential associated with a very low antigenicity. They can be used in food or pharmaceutical compositions to induce a tolerance of proteins in individuals at risk of protein allergy.

Example 7: Tolerogenic formula for babies The composition for 100 g of powder contains 12.5% of peptides (the tolerogenic peptides synthesized as prepared in example 2, represent approximately 0.1 to 0.2% of the total peptides), 26% of fats, 56% carbohydrates (including 39% lactose, 11% maltodextrin, 6% starch), traces of vitamins and trace elements to meet daily demands, 2.5% minerals and 3% humidity. 13 g of this powder are mixed in 100 ml of water. The composition obtained is a formula for babies particularly designed for babies at risk of suffering from cow's milk allergy.

EXAMPLE 8: Tolerogenic Formula for Babies To obtain a tolerogenic infant formula, the following mixture containing per 100 ml of the formula is prepared, 1.6% of peptides (the tolerogenic peptides as prepared in example 2, represent approximately 0.1). to 0.2% of total peptides), 3.4% fat, 7.4% carbohydrates (including 5.2% lactose, 1.4% maltodextrin, 0.8% starch), traces of vitamins and trace elements to comply with the daily demands, 0.3% minerals and 79.9% water.

Example 9: To obtain a tolerogenic preparation of a small volume, the following mixture containing 30 ml of preparation was prepared, from about 0.003% to about 0.015% of tolerogenic peptides (tolerogenic peptides as prepared in example 2, represent 100 % of total peptides), 7.4% of carbohydrates (including 1.4% maltodextrin and 6% sucrose), 92.5% of water and aroma. To induce oral tolerance, this preparation is administered 1 to 5 times daily, to newborns < < at risk > > of cow's milk allergy either hypoallergenic formula feed or lactation feed.

Claims (5)

1. Hypoallergenic composition for the induction of protein tolerance in persons at risk of protein allergy, contains (i) a base of "non-allergenic" protein material extensively hydrolyzed and / or (ii) a base of free amino acids, comprising said composition as an active ingredient for at least one tolerogenic peptide of the allergenic protein wherein the tolerogenic peptides are present in the form of (i) tolerogenic peptide moieties isolated from layrolysis of proteinaceous material containing the allergic protein and / or (ii) peptides tolerogens prepared synthetically, in such quantity that the ratio of tolerogenic activity by residual antigenicity is at least 2 x 10 ~ 2.

2. Composition according to claim 1, which contains an amount of tolerogenic peptide from about 0.01% to 10%, and preferably, from about 0.1% to 0.2% of the total nitrogen source of the proteins in the composition.

3. composition according to claims 1 or 2, intended for mammals susceptible to suffering cow's milk allergy, containing a peptide fraction comprising H2N-I-D-A-L-N-E-N-K-COOH, H2N-v-L-V-L-D-T-D-Y-K, -K-COOH or H2N-T-P-E-V-D-D-E-A-L-E-K-F-D-K-COOH of β-lactoglobulin.

4. Composition according to any of claims 1 to 3, which contains a source of nitrogen that provides 7 to 25% of the total energy, a source of carbohydrates that provides 28 to 66% of the total energy, a source of lipids that provides 25 to 60% of the total energy, minerals and vitamins to meet the daily demands and at least one of said tolerogenic peptides that are added in an efficient amount in a manner that induces oral tolerance.

5. Β-lactoglobulin tolerogenic peptide which has the ability to induce oral tolerance to milk proteins, selected from the group comprising H2N-IDALNENK-COOH, H2N-VLVLDTDYK, -K-COOH or H2N-T -PEVDDEALEK- FDK-COOH .