Resistance of parvalbumin to gastrointestinal digestion is required for profound and long‐lasting prophylactic oral tolerance

Abstract Background Early introduction of food allergens into children's diet is considered as a strategy for the prevention of food allergy. The major fish allergen parvalbumin exhibits high stability against gastrointestinal digestion. We investigated whether resistance of carp parvalbumin to digestion affects oral tolerance induction. Methods Natural Cyp c 1, nCyp c 1, and a gastrointestinal digestion‐sensitive recombinant Cyp c 1 mutant, mCyp c 1, were analyzed for their ability to induce oral tolerance in a murine model. Both antigens were compared by gel filtration, circular dichroism measurement, in vitro digestion, and splenocyte proliferation assays using synthetic Cyp c 1‐derived peptides. BALB/c mice were fed once with high doses of nCyp c 1 or mCyp c 1, before sensitization to nCyp c 1. Immunological tolerance was studied by measuring Cyp c 1‐specific antibodies and cellular responses by ELISA, basophil activation, splenocyte proliferations, and intragastric allergen challenge. Results Wild‐type and mCyp c 1 showed the same physicochemical properties and shared the same major T‐cell epitope. However, mCyp c 1 was more sensitive to enzymatic digestion in vitro than nCyp c 1. A single high‐dose oral administration of nCyp c 1 but not of mCyp c 1 induced long‐term oral tolerance, characterized by lack of parvalbumin‐specific antibody and cellular responses. Moreover, mCyp c 1‐fed mice, but not nCyp c 1‐fed mice developed allergic symptoms upon challenge with nCyp c 1. Conclusion Sensitivity to digestion in the gastrointestinal tract influences the capacity of an allergen to induce prophylactic oral tolerance.


| INTRODUC TI ON
Food allergy represents one of the important clinical manifestations of IgE-associated allergy. It often starts in early childhood and can induce severe and life-threatening anaphylaxis. Potent allergen sources are peanuts, tree nuts, cow's milk, egg, soy, wheat, shellfish, and fish. 1,2 Diagnosis of the disease-causing food allergens is extremely important because it guides allergen-specific forms of treatment, such as avoidance, diet, introduction of hypoallergenic formulas, and allergen-specific immunotherapy often performed by the oral route (ie, oral allergen-specific immunotherapy, OIT). [3][4][5] In addition, several clinical studies indicate that early introduction of allergen-containing food into the diet of sensitized but not yet allergic children may prevent the development of food allergy. 6,7 The development of early allergen-specific forms for the prevention of allergy such as oral tolerance induction and/or early allergen-specific immunotherapy (AIT) has become an important topic because it may prevent allergic sensitization, the transition from silent sensitization to symptomatic allergy and the progression from mild to severe forms of allergy especially early in childhood. [8][9][10][11] Fish represents one of the most important food allergen sources which can induce severe anaphylactic reactions. 12 The calcium-binding protein parvalbumin has been identified as the major and cross-reactive allergen in different fish species and is available as recombinant allergen to identify individuals with specific IgE sensitization. 13 We have developed a recombinant mutant of carp parvalbumin, mCyp c 1, which differs from the wild-type allergen only in four amino acids but shows strongly reduced allergenic activity. 14,15 mCyp c 1 has been used for subcutaneous AIT (SCIT) and induced allergen-specific blocking antibodies (Clinicaltrials.gov identifier: NCT02017626 and NCT02382718). [16][17][18] Using a mouse model for fish allergy, we have recently shown that the passive administration of mCyp c 1specific IgG antibodies reduced symptoms of fish allergy 19 similar as has been shown in a clinical trial for Fel d 1-specific IgG antibodies in cat allergic patients. 20 Passive immunization with Bet v 1, Phl p 1-, and Phl p 5-specific IgG antibodies prevented the development of pollen allergy but the duration of the effect has not been investigated. 21 parvalbumin-specific antibody and cellular responses. Moreover, mCyp c 1-fed mice, but not nCyp c 1-fed mice developed allergic symptoms upon challenge with nCyp c 1.
Conclusion: Sensitivity to digestion in the gastrointestinal tract influences the capacity of an allergen to induce prophylactic oral tolerance.

K E Y W O R D S
allergen, allergy, food allergy, oral tolerance induction, parvalbumin

G R A P H I C A L A B S T R A C T
The calcium-binding protein parvalbumin, a major and cross-reactive allergen for fish allergic patients, induces robust and long-lasting immunological and clinical oral tolerance in a murine model of fish allergy. A recombinant parvalbumin mutant, that resembled the wild-type parvalbumin regarding biochemical and immunological properties, but was more sensitive to in vitro digestion, failed to induce oral tolerance. Sensitivity to digestion in the gastrointestinal tract influences the capacity of an allergen to induce prophylactic oral tolerance.
In this study, we used wild-type Cyp c 1 and mCyp c 1 to investigate if early oral administration of the antigens can induce robust and long-lasting immunological and clinical tolerance in the murine model of fish allergy. In particular, we were interested to study if sensitivity to digestion of the tolerogens may affect the outcome of tolerance induction.

| Natural and recombinant antigens, synthetic peptides
Carp extract was prepared from homogenized carp muscle tissue by extraction in phosphate-buffered saline (pH 7.4) at 4°C. 22 For enrichment of natural Cyp c 1 (nCyp c 1), the raw extract was boiled for 30 minutes 22 and precipitated proteins were removed by filtration.
The presence of the Cyp c 1.01 isoform in nCyp c 1 was confirmed by electrospray ionization-liquid chromatography (mass spectrometry LC-ESI-MS/MS). 23 The recombinant mutant Cyp c 1 (mCyp c 1), based on the Cyp c 1.01 sequence, was expressed in E coli BL21 and purified by ion exchange and hydrophobic interaction chromatography as previously described. 14 The amino acid sequence of recombinant mutant

Cyp c 1 differs from wild-type Cyp c 1 by 4-point mutations (D → A)
in the calcium-binding sites of the protein ( Figure 1A). 14 Recombinant wild-type Cyp c 1.01 (rCyp c 1) was obtained from Biomay AG. rCyp c 1 was expressed in E coli and purified by conventional biochemical methods. 13 Endotoxin levels for nCyp c 1 (<1.35 EU/µg), mCyp c 1 (>100 EU/µg), and rCyp c 1 (0.155 EU/µg) were measured on an Endosafe-PTS detection system (Charles River Laboratories Int.). An E coli-expressed, recombinant hypoallergenic hybrid molecule derived from the major timothy grass pollen allergens Phl p 2 and Phl p 6 (hP62) served as control antigen. 24 Synthetic peptides spanning the Cyp c 1.01 sequence (Table S1) were produced by solid phase peptide chemistry, purified to homogeneity, and characterized by mass spectrometry as described. 19

| In vitro digestion assays
Enzyme cleavage sites in nCyp c 1 and mCyp c 1 amino acid sequence were analyzed using ExPASy-PeptideCutter program. Positions for cleavage sites for pepsin (pH > 2, n = 28, black), trypsin (n = 12, blue), chymotrypsin high specificity (n = 11, gray) are indicated in Gastric and pancreatic digestion was mimicked in vitro as previously described. [27][28][29] Briefly, 600 µg of nCyp c 1 and mCyp c 1 were incubated with gastric (pepsin) (Enzynorm; Casella-Med) or pancreatic (trypsin, chymotrypsin, carboxypeptidase A and B) enzyme solution (Solvay Pharma) at 37°C/300 rpm to mimic peristalsis in the digestive tract. Samples were taken at different time points, aliquots of 5 µg protein were loaded on SDS-gels for analysis of degradation and gels were stained with Coomassie Brilliant Blue.

| Statistical analysis
Data were analyzed using GraphPad Prism software 5.0 (GraphPad Software). Significant differences between 2 groups were calculated using a Mann-Whitney U test. Significant differences between more than 2 groups were calculated using a Kruskal-Wallis test and

| nCyp c 1 shows higher resistance to pancreatic digestion as compared to mCyp c 1
The resistance of nCyp c 1 and mCyp c 1 to digestion was stud-  Figure 1A). Interestingly, mice having received mCyp c 1 i.g. before sensitization were not protected from the development of Cyp c 1-specific antibodies (group 3; Figure 3A,B). Feeding of nCyp c 1 or mCyp c 1 alone did not induce an allergen-specific antibody response because mice which were fed but not sensitized did not mount Cyp c 1-specific IgE or IgG 1 responses (groups 4 and 5; Figure 3A,B).
Next, we studied the effect of prophylactic feeding of nCyp c 1 and mCyp c 1 on IgE-mediated immediate allergic reactions in basophil activation assays on day 80. RBL-2H3 cells loaded with IgE from group 1 which had received only PBS i.g. and challenged with rCyp c 1 showed a mean ß-hexosaminidase release of 33%, while there was almost no specific mediator release in group 2 which had been tolerized with nCyp c 1 (mean release: 10%) ( Figure 3E). Loading of sera from mice having received mCyp c 1 before sensitization on RBL cells resulted in a mean mediator release of 45% (group 3; Figure 3E) showing that the i.g. application of mCyp c 1 did not suppress allergic sensitization. The analysis of basophil release induced with the control antigen showed that there was no significant difference between mice having received only PBS i.g. or nCyp c 1 demonstrating that the suppression of effector cell activation was indeed allergen-specific ( Figure 3F).

| Oral tolerance induction with nCyp c 1 but not with mCyp c 1 protects against anaphylactic symptoms upon allergen challenge
In order to investigate the effects of sensitization and/or tolerance   Figure S2).

| Oral tolerance induction with nCyp c 1 but not with mCyp c 1 induces long-lasting prevention of allergen-specific T-cell and antibody responses
The Cyp c 1-specific T-cell response was investigated on day 204. We found that no relevant nCyp c 1-and rCyp c 1-specific Tcell proliferation was observed in mice of group 2 which had been F I G U R E 3 Suppression of Cyp c 1-specific antibody responses and basophil activation by oral tolerance induction. Comparison of A, rCyp c 1specific IgE and B, IgG 1 and C, control antigen-specific IgE and D, IgG 1 levels (y-axes: OD levels, means ± SD) before sensitization (left, gray triangles) and after sensitization (right, black circles) on day 36 in the mouse groups (x-axes: 1-5). E,F, ß-hexosaminidase release (y-axis: percentages of total release, means ± SD) from RBL cells loaded with sera from sensitized mice (day 80) and challenged with E, rCyp c 1 and F, control antigen for the mouse groups (x-axis: 1-5). Significant differences between the sensitized groups are indicated. (*) P-value < 0.05, (**) Pvalue < 0.01, (***) P-value < 0.001  (Figure 6A,B). The nCyp c 1-specific T-cell proliferation in mice of group 1 was significantly higher than that in mice of group 2 ( Figure 6A). Only partial suppression of splenocyte proliferation in mice of group 3 was observed ( Figure 6A,B).

| D ISCUSS I ON
Our study is the first to demonstrate that the intrinsic sensitivity of an allergen to gastrointestinal digestion affects the ability of the antigen to induce robust and long-lasting immunological and "clinical" oral tolerance. Using two forms of the major fish allergen, the F I G U R E 5 Time courses of rCyp c 1specific IgE and IgG 1 antibody responses in mouse groups 1-5. A, IgE and B, IgG 1 antibody levels (OD, y-axes: mean ± SD) of the individual mouse groups 1-5 were measured in sera obtained at different time points (days, x-axes). Interventions are indicated: P, prophylactic feeding; S, sensitization; C, challenge Group digestion-resistant wild-type Cyp c 1 and a digestion-sensitive mutant, mCyp c 1, for oral tolerance induction in a murine model of fish allergy, we found that only the wild-type allergen but not the mutant form induced robust and long-lasting immunological tolerance.
In our murine study, a single high-dose regimen was used which, of course, may need to be adapted to the human situation.
Tolerance was demonstrated by lack of allergen-specific antibody and cellular responses as well as of "clinical" tolerance as shown by lack of anaphylactic symptoms. The specificity of oral tolerance was shown by the fact that tolerized mice lacking Cyp c 1-specific adaptive immune responses mounted specific IgE and IgG responses against an unrelated control antigen which was used for sensitization at the same time as Cyp c 1. Therefore, bystander suppression which might be mediated by cytokines like IL-10 and TGF-ß secreted from regulatory T cells does not seem to play a major role.
Several arguments support the assumption that the different sensi- sites. We therefore think that the exchange of 4 amino acids destabilized the protein because they affect the protein's ability to bind calcium which is important for the overall stability of the protein and is known to affect the surface exposure of certain amino acids in the calcium-bound and calcium-free apoform of calcium-binding proteins. 32,33 Another observation which supports our assumption that the sensitivity to digestion has affected the ability of the proteins to induce oral tolerance is the earlier finding that protection of allergens against digestion by enteric coating allowed to reduce the dose required for tolerance induction. 34,35 Our finding that sensitivity to digestion affects oral tolerance induction is important for at least two reasons. First, it may explain why not all clinical studies which have investigated oral tolerance induction had identical outcomes. 11 In fact, different food allergen sources contain allergens with different stability and the subjects enrolled in these studies are sensitized against different allergens which may have different stability. Second and importantly, our study identifies sensitivity to digestion as another important factor for successful oral tolerance induction besides dose and timing of administration. Therefore, the development of strategies for protecting allergens or allergen-derived molecules from proteolytic cleavage may facilitate the induction of oral tolerance. 11