Food Contaminants - American Chemical Society

Chapter 24

Molecular Allergology of Egg White Ovomucoid P. Rupa and Y. Mine*

Downloaded by UNIV OF GUELPH LIBRARY on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch024

Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Allergens could be altered by genetic modifications to reduce their ability to initiate an allergic reaction. Ovomucoid is a dominant allergen in hen's egg-white. In the present study, we investigated the efficacy of a genetic variant of ovomucoid (GMFA) to modulate the allergic response in mice and characterized the role of GMFA in desensitizing ovomucoidsensitized mice. The results suggest that GMFA could suppress allergic reactions in mice and desensitization with GMFA in ovomucoid-sensitized mice, significantly decreased allergic symptoms and histamine levels, suppressed production of ovomucoid-specific IgE antibodies, and modulated the T cell response, when compared to a control group treated with intact ovomucoid. This study indicates that GMFA can be used to potentially provide targeted immunotherapy for egg-allergic patients.

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© 2008 American Chemical Society

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


383 Food allergy is an immunologic disease responsible for substantial morbidity and mortality. Food allergy occurs in 7-8% of children and 4% of adults (1,2) and the prevalence is increasing. Severe food allergic reactions may cause anaphylaxis and death. Food allergies cause approximately 30,000 anaphylactic episodes 'and 200 deaths per year in the U.S. According to the Asthma and Allergic Foundation of America (2002), allergies are the sixth leading cause of chronic diseases in the U.S. and the annual cost of dealing with them is estimated at $18 billion. Very often food-induced anaphylaxis following exposure to the ingestion of the so-called "hidden allergen" sources is the most common reason for someone with food allergy to report to a hospital emergency department with an anaphylactic reaction. While allergic reactions can occur with any food or food components, some are more common than others. Common food allergens include milk, egg, peanuts, tree nuts, wheat, fish, shellfish, and soy (3). Food allergens are mostly water-soluble glycoproteins that are 10 to 70 kDa (kilodalton) in size and fairly stable to heat, acid, and proteases. Food allergic disorders encompass IgE-mediated hypersensitivity and cellmediated hypersensitivity. Both immune mechanisms contribute to gastrointestinal (GI), cutaneous, respiratory, and systemic symptoms associated with food allergy. The GI tract is responsible for digestion and absorption of essential nutrients in the body. Delayed maturation of GI mucosal immunity and oral tolerance in newborns is linked to high incidence of food allergy in children under 5 years of age. Despite the evolution of the complex mucosal barrier in adults, about 2 % of ingested food antigens are absorbed and transported throughout the body in an intact form, even through the normal mature gut (4). Regional dietary habits and methods of food preparation play a role in the prevalence of specific food allergies. Since there are many hidden ingredients in food that could contain egg constituents and could trigger allergic reactions, an immediate need for treatment against egg-induced allergic reactions requires attention. The most effective strategy to prevent an allergic episode is strict food avoidance. Food avoidance is complicated by the ubiquitous distribution of peanut, soy, milk, egg, and other allergens in processed foods, poor food labeling practices, and cross-contamination of food products during processing. Hen's egg is one of the most commonly implicated cause of food allergies, representing the second major agent (30%) next to peanut (5). Type I allergic reactions to egg (IgE-antibody mediated) usually begin within minutes to one to two hours after ingestion. Eggs are composed of 56-61 % of egg white and 27-32 % of egg yolk. Egg yolk is considered less allergenic than the egg white (albumin) (6). Two-thirds of children diagnosed with food allergy are reactive to egg white (7). Allergic reactions to egg are more prevalent in children than in adults (8). Egg was reported as the cause of 11.6% of food induced anaphylaxis incidents (9). The symptoms associated with egg allergy include allergic rhinitis, asthma, dermatitis, diarrhea, gastrointestinal symptoms, hives, nausea, oral



384 allergy syndrome, vomiting, wheezing, and, in some cases, anaphylaxis. Currently, there is no cure for egg-induced allergic reactions. Immune modulation offers the only opportunity to modify the underlying disease processes of allergy in a long-term cure, as no pharmacologic therapeutic agent has been shown to do this. Avoidance may not be an explicit solution in case of egg allergy (70), due to the fact that eggs are important ingredients, associated with most of the food preparations. Foods containing eggs are very difficult to avoid, unlike peanuts, which could be avoided. Allergen-immunotherapy offers an alternative approach to controlling food allergy, by modulating the immune system. Immunotherapy may be used to treat food allergies by modifying the immune response to allergenic foods. Novel immunotherapeutic strategies include peptide immunotherapy, traditional Chinese medicine, mutated or homologous protein immunotherapy, DNA immunization, and immunization with immunostimulatory sequences, which all strive to elicit a decreased T helper cell type 2like response or tolerance by the immune system in response to a specific food allergen (77). The major egg white allergens are ovomucoid (Gal dl), ovalbumin (Gal d2), ovotransferrin (Gal d3), and lysozyme (Gal d4) (72). Ovalbumin was considered the major egg white allergen earlier (73) but recently ovomucoid has been shown to be the dominant allergen in hen's egg white (14). Ovomucoid is a highly glycosylated protein containing 20-25% carbohydrate, with a molecular weight of 28 kDa and an isoelectric point of 4.1 (75). It consists of 186 amino acids with three structurally independent tandem homologous domains (Domain I, II, and III) that are interconnected with peptide bonds. The domains could be cleaved by limited proteolysis or by cyanogen bromide or by a combination of both. Each domain is cross-linked within each other by intradomain disulfide bridges and behaves as a native globular protein, but there are no interdomain disulphide bridges (16). The carbohydrate moiety of ovomucoid seems to contribute to the thermal stability of its structure, since deglycosylation resulted in higher sensitivity to trypsin inhibition after heat denaturation (77). Carbohydrate moieties were reported to contribute to allergenicity of ovomucoid earlier (18), but in contrast they seem to have an inhibitory effect on IgE binding activity of ovomucoid, as shown recently (19). The third domain of ovomucoid (Dili) was reported to be more allergenic with greater IgE binding activity than the first and the second domain using sera from egg allergic patients (19). Detailed B cell epitope mapping of the entire ovomucoid molecule has been reported and amino acids that are critical to IgE binding activity have been identified (20-22). Substitution of a single amino acid was shown to drastically reduce the ability to bind IgE, in the case of an apple allergen (23). A similar effect has been observed with the two site mutation of



385 the ovomucoid gene (where glycine was substituted with methionine and phenylalanine was substituted with alanine (GMFA)), which led to complete loss of IgE response against sera from egg allergic patients (24). Use of hypoallergenic formulas is an alternate strategy to avoid food allergies. Traditional allergy shots have not been proven practical in case of food allergic reactions, one of the disadvantages being the use of natural extracts which could trigger allergic reactions and cause severe side effects. Promising therapeutic modalities for the treatment and eventual prevention of food allergy are being developed and are in focus in recent years (25). Recent advances in food allergy research suggest that new research directions focused at the molecular level will advance our understanding of the mechanisms of food allergy and translate these findings into new options for identification and treatment of susceptible individuals. Conformational variants of allergens, displaying reduced allergenicity accompanied by retained IgG and T cell recognition, offer a safe, specific, and flexible approach to immunotherapy of type I allergy (26). The current principal approach to allergen modification is to modify B cell epitopes to prevent IgE binding, while preserving T cell epitopes to retain the capacity of inducing tolerance. In this way, the modified allergen will be directed to T cells by a phagocytosis-mediated antigen uptake mechanism, bypassing IgE cross-linking and IgE-dependent antigen presentation (27). Informative peptides that are predictive for the persistence of food allergic reactions have to be identified initially. Vaccination with genetically engineered allergen was recently shown to prevent progression of allergic diseases (28). Many studies on allergen immunotherapy have shown modulation of T-cell response with inhibition of Th2 cell response (Interleukin (IL) -4) and induction of more Thl-like response with increased allergen-induced inteferon (IFN)-y and interleukin-12 (29). A variety of avenues is being explored on the basis that many clinically important allergens have been identified, purified, cloned, epitope-mapped, produced as biologically active recombinant proteins, and administered safely by mucosal and cutaneous routes (30). Based on sequence information, protein engineering could be used to design homologous, nonallergenic proteins with similar structures, stability, and biologic/functional properties. The research work presented here describes characterization of a genetic variant of ovomucoid third domain (GMFA) in alteration of allergic response for immunotherapeutic treatment of egg-induced allergic reactions in a mouse model system. The present study was carried out to investigate possible mechanisms to suppress egg white allergy by a genetically modified variant of ovomucoid (GMFA) and to study the role of GMFA to alter and desensitize ovomucoid-induced allergic response in Balb/c mice.


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Strategic Approaches for Specific Immunotherapy by Sitedirected Mutagenesis Allergen-specific immunotherapeutic studies and novel immunotherapeutic strategies designed to alter the immune response to food allergens are being examined as a potential treatment modality for food allergy (31). Successful immunotherapy is associated with a modulation of the immune response to allergens at die level of Th cells (32, 33). Recombinant allergens offer a safe alternative and can be modified to reduce the risk of IgE-mediated side effects (34, 35). Modified allergens should aim at the production of molecules with reduced IgE binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes) and for induction of IgG antibodies reacting with the natural allergen (blocking antibodies). Many hypoallergens in food have been studied for targeted protein modification, in order to alter protein function or properties in a predictable manner (36-38). Engineering of hypoallergens usually requires knowledge of B and T cell epitopes and in some cases of the three-dimensional structure of the native allergen (39, 40). The knowledge of the major IgE binding epitopes creates the possibility for a specific vaccination or immunotherapy in allergic patients. Targeted immunotherapy has been the only curative approach for type I allergies (41, 42). Most of the treatment procedures using specific immunotherapy are accompanied with the risk of IgE-mediated side effects and shows variable clinical efficacy. For example, initial trials of immunotherapy for food allergens have demonstrated an unacceptable safety to efficacy ratio (43-45). Molecular modification of food allergens are an alternative option for promising therapeutic modalities in the treatment and prevention of food allergy. The above mentioned results have prompted investigators to seek alternative forms of immunotherapy, as well as other forms of interventions for treatment of food allergy. Modification of the IgE binding sites (epitopes) of allergens and production of hypoallergenic proteins are an alternative approach to attenuate hypersensitivity reactions for an improved immunotherapy of type I allergy. In this study we investigated the effect of the allergic response elicited by a genetic variant of the third domain of ovomucoid (GMFA), in comparison to the native third domain of ovomucoid without the carbohydrate chain (DIII-) in Balb/c mice. It was shown earlier that the engineered mutant (GMFA) had low IgE binding reactivity, upon replacement of the two critical IgE binding amino acids ( G and F ) on Western blot and ELISA, using sera from patients with egg allergy (24). The epitope sequence where the mutation was constructed was also predominantly recognized with Balb/c mice sera, raised against the recombinant wild type of the third domain of ovomucoid (46). We mapped the IgE binding epitopes of ovomucoid in mice and studied the effect of the mutant isoform (GMFA) in comparison to the native form of the allergen (Dili) in mice. We 162

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387 evaluated in detail, the allergen-specific immune response elicited in Balb/c mice by both the antigens, for specific antibody levels in vivo, and cytokine levels in vitro. These findings would draw attention to a potential use of this "hypoallergen" of ovomucoid as an agent in specific-immunotherapy to prevent egg allergy. To further evaluate if this "hypoallergen" was capable of desensitizing intact ovomucoid-sensitized mice, we characterized the potential role of the efficacy of this variant in desensitization of ovomucoid-allergic mice, using intraperitoneal injections. The clinical symptoms in the intact ovomucoidsensitized (Fovm) and GMFA-desensitized animals were compared with control mice. Specific antibody and histamine levels were evaluated and the cellular response to both the sensitized and the desensitized groups were compared in order to analyze the efficacy of GMFA to inhibit allergic reactions.

Mapping of IgE Binding Epitopes of Ovomucoid in Balb/c Mice At least two IgE-binding epitopes are required to activate mast cells and basophils, via cross-linking of IgE bound to Fc -receptors. In order to evaluate the immunological response of the modulated derivative of the ovomucoid gene in a murine model system (Balb/c), the IgE binding epitopes of ovomucoid in mice need to be identified. The T cell epitope mapping of ovomucoid in mice has been recently reported (47). Arrays of synthetic peptides representing the entire ovomucoid gene were synthesised on a derivatised cellulose membrane (SPOTs Kit, Genosys Biotechnologies, the Woodlands, TX) by Fmoc chemistry (using N-9-fluorenylmethoxycarbonyl chloroformate) as previously described (22). The peptides synthesized were 10 amino acids long and had an offset of 5 amino acids. The membrane was visualized under a light imager (EG & G Berthold, Bad Wildbad, Germany). The image processing was done using Win Light software (EG & G Berthold). The obtained results demonstrated that the linear B-cell epitope recognized by the BALB/c mice, corresponds to the following sequence from the second domain - G K V M V L C N R A F N P V C and Y G N K C N F C N A W E S N from the third domain as shown in Figure 1. Two adjacent epitopes were recognized by the sera from the BALB/c mice in the second domain (II) region and two major epitopes were recognized in the third domain (III) region which were also adjacent to each other. Interestingly, the major epitope which is recognized by the Balb/c mice in the third domain, was also the major epitope recognized by the egg allergic human patient's sera. Substitution of the critical amino acid at this epitope in the third domain of ovomucoid at position 32, where glycine was substituted by methionine (G to M) and phenylalanine at position 37 was substituted by alanine (F to A), led to complete loss of activity of IgE binding with patient's sera. The signal (relative intensity) of the spot was very strong e


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with the epitopes in the third domain of ovomucoid and were very faint with .the epitopes in the second domain, which agrees with our previous data (19) that the third domain is more allergenic than the first and the second domains. Incubation with normal murine serum alone revealed no non-specific binding of the murine immunoglobulins or of the secondary antibodies to die peptide sequences.

AEVDCSRFPNATDKEGKDVLVCNKDLRPIC GTDGVTYNNE CLLCAYSIEF GTNISKEHDG ECKETVPMNC SSYANTTSED GKVMVLCNRAFNPVCGTO^ VDKRHDGECRKELAAVSVDCSEYPKPDCTAEDRPLCGSDN KTYGNKCNFCNA WESNGTLTL SHFGKC Domain I and II

Domain III

Figure 1. IgE epitope mapping of ovomucoid in mice sera. The epitopes identified in Balb/c mice are represented in bold and underlined on the primary sequence of the ovomucoid gene.

Animals, Experimental Design and Modulation of Allergic Response Balb/c mice (four per group) (6-8 weeks old) were sensitized with the native third domain ovomucoid antigen without carbohydrate (DÜI-), mutant ovomucoid antigen (GMFA), and PBS; 100 \i% of the antigens were emulsified with 100 ^iL of complete Freund's adjuvant (CFA) and injected subcutaneously. Animals were frequently monitored for stress, pain, and lesions. Four weeks later, a booster was given with the same amount of antigens in Incomplete Freund's adjuvant (IFA). The control animals were immunized with PBS containing adjuvant. Two weeks later, all the animals were euthanized and blood samples and spleen were collected. Sera collected from mice at the end point were used to determine antigen-specific and total antibody levels. We evaluated



389 in detail the allergen-specific immune response elicited in Balb/c mice by both the antigens. The total and specific IgE and IgG antibodies in mice sera were measured according to a previously described procedure (48). For specific IgGl and IgG2a determinations, an indirect ELISA was performed. Sensitization of ovomucoid antigen Dili- in BALB/c mice resulted in high levels of specific IgE antibodies. On the contrary, the GMFA treated mice maintained significantly low serum specific IgE antibodies. Subtyping of the IgG class revealed that the mice immunized with the native third domain of ovomucoid (DIII-) showed detectable levels of specific IgGl, and low levels of specific IgG2a. On the contrary, in the sera sample collected from the mice immunized with GMFA protein, the specific levels of IgGl were considerably lower when compared to the native third domain (DIII-), but a significant increase in the level of specific IgG2a was observed. GMFA was thus shown to inhibit IgGl levels and up-regulate IgG2a production. In order to determine if the IgE suppression effect of the mutant antigen had any influence on the cytokine profile, we determined the levels of Thl and Th2 cytokines, secreted by the culture supernatants from splenocytes of sensitized mice (the native third domain ovomucoid (Dili), the mutant ovomucoid (GMFA), and the control group of mice). We detected elevated levels of IL-4 in mice sensitized with Dili antigen and significantly reduced levels of IL-4 in mice sensitized with GMFA. Considerable increase in the levels of INF-y and IL-12 was observed with GMFA when compared to Dili antigen. This result suggests that the suppression effect of the mutant antigen (GMFA) is due to modulation of cytokines from an existing allergic Th2 to anti-allergic Thl skewed pathway. To test whether IL-12 is involved in the IL-4-inhibitory activity of supernatants in GMFA samples, we examined the effect of neutralization of GMFA on the production of IL-4, INF-y and IL-12, using different concentrations of monoclonal anti-IL-12 antibodies along with the control GMFA antigen to stimulate the culture supernatants. Addition of anti-IL-12 antibody abrogated the suppression of IL-4 in the case of the GMFA treated samples. The suppressed IL-4 levels were restored and INF-y and IL-12 levels were suppressed with monoclonal anti-IL-12 antibodies tested at various concentrations. Our results show that the combination of the hypoallergen concept with the Thl-inducing approach, which is cytokine driven, would pave a way for therapeutic treatment of egg-allergy. Desensitization (specific immunotherapy) helps in making an allergic substance less sensitive and is the only disease-modifying treatment or therapeutic option available today for a cure against allergic diseases, other than avoidance of a particular food. We have characterized and reported desensitization of allergic response to ovomucoid-sensitized mice. The rationale behind the use of a recombinant hypoallergenic variant of ovomucoid third domain described here is to modify the surface topography to reduce IgE binding activity, causing a partial disruption of a-helix, while retaining the folding



390 pattern of the backbone to preserve surface structures capable of generating an IgG response. Loss of epitope for the specific IgE antibody and exclusive induction of Thl cell differentiation and probable disruption of the a-helix in the tertiary structure of the third domain of ovomucoid, collectively make GMFA an ideal antigen for hyposensitization therapy. G32 which is located in P-bend of the third domain could have synergistic effect on antigenicity and F37, a core of the a-helix structure, has an important role in allergenicity; a combined effect of these two mutations causes complete abrogation of ovomucoid-induced allergic response. In order to dissect the underlying mechanism of protective response induced by the modified form of ovomucoid (GMFA) in desensitization, we analyzed the hypersensitivity symptoms of both the sensitized (Full Ovomucoid, FOvm) and the desensitized groups (sensitized with Fovm and desensitized with GMFA), along with the negative control groups (PBS). Acute symptoms (wheezing and breathing problems) scoring level 3 were observed in some mice sensitized with the intact ovomucoid antigen, whereas the desensitized group hardly showed any sign of hypersensitivity reaction except for slow movement in themice attributed to the injection pain (Figure 2A). Control groups treated with PBS did not show any symptom. We conclude that the modified ovomucoid (GMFA) protected our mouse model against an otherwise severe challenge of anaphylactic shock induced by intact ovomucoid in the desensitized group. Because histamine is one of the major mediators for anaphylactic reaction, we analyzed serum histamine levels in samples. Serum histamine secretion was blocked, indicating very low allergen-induced histamine release in desensitized mice, whereas intact ovomucoid sensitized mice showed significantly higher levels of serum histamine levels. The control group showed low or decreased levels of histamine (Figure 2B). IgE plays a vital role in allergic reactions. Ovomucoid-antigen specific IgE antibodies were significantly reduced in desensitized mice (P < 0.001) when compared to the Fovm sensitized group. Specific IgE levels are direct indications for antigen-induced allergic response and it was significant from the data obtained that GMFA desensitization causes suppression of ovomucoid-specificIgE antibodies, when compared to the high levels of specific IgE in the sensitized group. The PBS control sera elicited minimal or only very low antibody response (Figure 3A). IL-4, a T helper Th2 cytokine, regulates the switch from IgM/D to IgGl and IgE in activated B cells (49). Type 1 cytokines, such as interferon gamma (IFN-y) and interleukin 12 (IL12), steer the immune response towards a phenotype characterized by the production of immunoglobulin (Ig) isotype IgG2 in mice. Type 2 cytokines, such as IL-4 promote the production of Ig isotype IgGl. The Thl cytokine IFN-y is important in vitro and in vivo for enhancement of IgG2a secretion. Thl and Th2 cytokines also function to cross-regulate Ig isotypes. For example, IFN-y antagonizes IL-4-induced IgGl responses at the level of IgGl transcription (50), whereas IL-4 has the ability to suppress IFN-y-driven IgG2a responses. The


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Figure 2A. Score of the hypersensitivity signs. Mice were sensitized, desensitized, and challengedfor induction ofsystemic anaphylaxis as described in Methods. Thirty to 40 minutes later, the symptoms of hypersensitivity were scored blinded on a scalefrom0 (no symptoms) to 5 (death). Each * symbol indicates an individual mouse.

isotyping of mice sera samples from the sensitized and the desensitized groups revealed that the IgGl level was markedly increased in the intact ovomucoid sensitized group, which indicated a Th2 augmented reaction; the IgG2a level was enhanced in the desensitized group, reflecting a more Thl favoring response in the desensitized group (Figure 3B). Control mice sensitized with PBS showed low levels of both IgGl and IgG2a. We hypothesized that desensitization with GMFA may promote antigenspecific Thl response and consequently inhibit Th2 cytokine production. In order to test this hypothesis, we analyzed the levels of cytokines in culture supernatant of splenocyte samples from the sensitized and the desensitized group of mice. Spleen was isolated from individual mice, stimulated with intact ovomucoid antigen and incubated for 72 h. Cytokine levels were analyzed with reference to standard cytokines. It was observed that the desensitized group showed significantly low levels of IL-4 and increased levels of IFN-y secretion in the culture supernatant samples, which were significantly different when compared against control sensitized mice, suggesting the occurrence of a discrete



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Figure 2B. Serum histamine levels in mice exposed to Fovm or GMFA antigens. The level of histamine was measured by inhibition ELISA and calculated in comparison to a standard curve. Values are expressed as mean ± SEM. Fovm results were significantly higher (P < 0.001) than results for GMFA and control groups.

and prominent Thl response, whereas significantly higher levels of IL-4 and lower levels of INF-y were seen in the intact ovomucoid sensitized group (Figure 4A). Control group supernatant samples showed low levels of IL-4 and INF-y production. The levels of IL-10 and EL-12 in culture supernatants were also quantified in comparison to standard cytokine curves (Figure 4B), to clarify whether GMFA desensitization induced these cytokines in vitro. It was found that significantly higher levels of IL-10 and IL-12 were secreted in the desensitized group, as opposed to low levels of IL-10 and IL-12 in the sensitized group. The low levels of IL-4 found in the desensitized mice culture supernatant may be the result of an Ig-class switching attributed to the inhibition of specific IgE levels and bias towards a Thl pathway. This



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Figure 3A. Specific IgE levels in mice exposed to Fovm (Sensitized), GMFA (Desensitized), or Control (PBS) antigens. Levels of serum specific IgE were determined at the end point (day 42) in each individual mouse serum by ELISA. The specific levels of antibodies are represented as absorbance (OD) at 405 nm. Values are expressed as mean ± SEM. Significantly higher (P < 0.001) than control group. Significantly higher (P < 0.001) than GMFA group. 0

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mechanism is further confirmed by high levels of INF-y in culture supernatants of the desensitized group and low levels in the sensitized group. IL-12p70 is the most potent signal directing force for T cells towards an interferon y-producing Thl phenotype. Our study confirms this hypothesis and we observed an increase in IL-12 which could possibly be driving the production of INF-y towards a Thl bias in the desensitized group.

Conclusion This research focused on characterization and evaluation of a hypoallergenic variant of ovomucoid third domain in suppression, and desensitization of ovomucoid-induced allergic reactions for immunotherapy. Egg allergies are the most common type of food allergy that occurs in people sensitive to one or more components of eggs. In such people, an allergic reaction occurs after coming into


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Figure 3B. Determination of specific IgGl and IgG2a levels by ELISA in Fovm (Sensitized), GMFA (Desensitized), or Control (PBS) treated groups. Mice were sensitized, desensitized, and challenged and sera were collected and analyzed as described in Methods. Values are expressed as means ± SEM (n = 5 per group). Significantly higher (P < 0.01) than Fovm group. Significantly higher (P < 0.001) than GMFA group. Significantly higher (P < 0.001) than control group. a

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Figure 4A. Cytokine production in splenocytesfromthe sensitized, desensitized, and the control groups. Intact ovomucoid-activated splenocyte supernatants from all the three groups were collected and analyzedfor the levels ofIL-4 and INF-y as described in Methods. Values are expressed as mean ± SEMof 5 mice. Significantly higher (P < 0.05) than Fovm group, Significantly higher (P < 0.01) than control group, Significantly higher (P < 0.001) than GMFA group. Significantly higher (P < 0.001) than control group. (Reproduced with permissionfromClin. Exp. Immunol. 2006, 145(3), 493-501. Copyright 2006 Wiley-Blackwell Publishers.) a

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Figure 4B. Cytokine production in splenocytesfromthe sensitized, desensitized, and control groups. Intact ovomucoid-activated splenocyte supernatants were collectedfromall the three groups and analyzedfor the levels ofIL-10 and IL-12 as described in Methods. Values are expressed as mean ± SEMof 5 mice. Significantly higher (P < 0.01) than Fovm group, Significantly higher (P < 0.001) than control group. (Reproduced with permissionfromClin. Exp. Immunol. 2006, 145(3), 493-501. Copyright 2006 Wiley-Blackwell Publishers.) a

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397 contact with eggs (usually by ingestion or consumption), resulting in allergic symptoms such as itchiness, rash, hives, stomach cramps, nausea, respiratory problems, and in some cases severe anaphylaxis. Most people with egg allergies are allergic to the egg white and ovomucoid is the most dominant allergen in the egg white. Since ovomucoid is responsible for most egg allergies, there is a need for prophylactic and therapeutic strategies for ovomucoid-induced allergic reactions. Hypoallergenic mutants are potential candidates for future use in immunotherapy. They produce modified molecules with reduced IgE-binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes). Hypoallergens are designed by using structural information and knowledge of B and T cell epitopes of the allergen of interest. IgE-epitope mapping of ovomucoid in Balb/c mice was done and the amino acid sequence revealed homologous epitopes in both man and mice. This validated the Balb/c mouse system as a model for testing the response of GMFA and indicated the possibility, that it could be used in man as an immunotherapeutic vaccine. In the present study, the results of screening GMFA and DIII for allergenic reactivity in mice revealed that GMFA suppressed allergic response in mice by lowering specific IgE levels, and changing an existing Th2 pathway to a Th1 skewed cellular pathway. Desensitization of ovomucoid-sensitized mice with GMFA revealed that this hypoallergen is capable of elimination of anaphylaxis symptoms, reducing histamine levels, lowering specific IgE levels, and modulating the allergen-specific Th2-dominated cellular response into a more Th1 phenotype, accompanied by enhanced production of IL-10. The present finding suggests that it is possible to develop a novel prophylactic and therapeutic form of hypoallergen for egg allergy for the prevention and/or reduction of allergic responses to ovomucoid.

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