AU765211B2 - Peanut allergens and methods - Google Patents

Description

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AUSTRALIA

Patents Act 1990 University of Arkansas h

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ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: r Peanut allergens and methods The following statement is a full description of this invention including the best method of performing it known to us:- \p Australia Documents received on. V Bat 8 2 No aD Batch No.

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EPITOPE SPECIFICITY OF THE MAJOR PEANUT ALLERGEN, Ara h II Peanuts are considered one of the most allergenic foods.' Peanut allergy is a significant health problem because of the potential severity of the allergic reaction, the chronicity of the allergic sensitivity, and the ubiquity of peanut products.

Individuals sensitive to peanuts may experience symptoms ranging from mild urticaria to severe, systemic anaphylaxis.' In food-induced, fatal anaphylaxis, peanuts are the food most commonly implicated in causing the reaction.

23 Sensitivity to peanuts often appears early in life, and unlike most other food allergies, tends to persist indefinitely.

4 To elucidate the exact mechanism of IgE-mediated reactions, the identification and purification of the precise allergens are necessary. Significant information has accumulated in allergen. characterization from a wide variety of sources, including pollens, dust mite, animal danders, and insects.

5 In comparison, allergen characterization for even the most common food allergens is much less defined. Despite the significant prevalence of peanut hypersensitivity reactions and several deaths annually, the identification of the clinically relevant antigens and an understanding of the immunobiology of peanut hypersensitivity is just beginning.

.20 Monoclonal antibodies are being increasingly used to define and characterize the allergenic epitopes of many allergens. Multiple allergens including the dust mite allergen, Derf I, 6 and the grass pollen allergen, Lol p I, 7 have been studied by using monoclonal antibodies. Murine monoclonal antibodies to these oo*• allergens have been shown to be quite effective in defining their allergenic 25 epitopes.

In this report we have investigated the epitope specificity of Ara h II, 8 a major peanut allergen, by using monoclonal antibodies as probes for mapping the possible antigenic determinants. We have produced and characterized a panel of monoclonal antibodies specific to Ara h II. The Ara h II monoclonal antibodies allowed us to define at least two antigenic sites on Ara h II. Inhibition assays were used to determine the IgE-binding sites on Ara h II.

METHODS

Patients with positive peanut challenge responses Approval for this study was obtained from the Human Use Advisory Committee at the University of Arkansas for Medical Sciences. Twelve patients with atopic determatitis and a positive immediate prick skin test response to peanut had either a positive response to double-blind placebo-controlled food challenge (DBPCFC) or a convincing history of peanut anaphylaxis (the allergic reaction was potentially life-threatening, that is with laryngeal edema, severe wheezing, and/or hypotension). Details of the challenge procedure and interpretation have been previously discussed.

9 Five milliliters of venous blood was drawn from each patient and allowed to clot, and the serum was collected. An equal volume of serum from each donor was mixed to prepare a peanut-specific IgE antibody pool.

Crude peanut extract 15 Three commercial lots of Southeastern Runners peanuts (Arachis hypogaea), medium grade, from the 1979 crop (North Carolina State University) were used in this study. The peanuts were stored in the freezer at -18 0 C until they were roasted.

The three lots were combined in equal proportions and blended before defatting.

The defatting process (defatted with hexane after roasting for 13 to 16 minutes at 20 163°C to 177 0 C) was done in the laboratory of Dr. Clyde Young (North Carolina State University). The powdered crude peanut was extracted in 1 mol/L NaC1, mmol/L sodium phosphate (pH 7.0)1 and 8 mol/L urea for 4 hours at 4 0 C. The extract was clarified by centrifugation at 20,000 g for 60 minutes at 4°C. The total protein determination was done by the bicinchoninic acid method (Pierce 25 Laboratories, Rockville, ill.).

Monoclonal antibodies Mouse hybridoma cell lines were prepared by standard selection after polyethylene glycol-mediated cell fusion was carried out as previously described.

10 1 4 mouse/myeloma cells were fused with immune splenocytes from female BALB/c mice hyperimmunized with Ara h II. Hybridoma cell supernatants were screened by ELISA and Western blotting, and cell lines were cloned by limiting dilution. The antibodies secreted by the monoclonal hybridoma cell lines were isotyped according the directions provided (Screen Type; Boehringer Mannheim, Indianapolis, Ind.). Ascites fluid produced in BALB/c mice was purified with Protein G Superose, as outlined by the manufacturer (Pharmacia, Uppsala, Sweden). Purified monoclonal antibodies were used in ELISA and ELISA inhibition assays.

ELISA for IgE A biotin-avidin ELISA was developed to quantify IgE anti-peanut protein antibodies with modifications from an assay previously described." The upper 2 rows of a 96-well microtiter plate (Gibco, Santa Clara, Calif.) were coated with 100 il each of equal amounts (1 4g/ml) of anti-human IgE monoclonal antibodies, 7.12 and 4.15 (kindly provided by Dr. Andrew Saxon). The remainder of the plate was coated with the peanut protein at a concentration of 1 Ig /ml in coating buffer (0.1 mol/L sodium carbonate-bicarbonate buffer, pH The plate was incubated at 37 0 C for 1 hour and then washed five times with rinse buffer (phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20, Sigma Chemical Co., St. Louis, Mo.) immediately and between subsequent incubations. A secondary IgE reference 15 standard was added to the upper 2 rows to generate a curve for IgE, ranging from 0.05 to 25 ng/ml.

The serum pool and patient serum samples were diluted (1:20 vol/vol) and dispensed into individual wells in the lower portion of the plate. After incubation for 1 hour at 37 0 C and washing, biotinylated, affinity-purified goat anti-human IgE (KPL, Gaithersburg, Md.) (1:1000 vol/vol bovine serum albumin) was added to all wells. Plates were incubated for 1 hour at 37°C and washed, and 100 pl horseradish peroxidase-avidin conjugate (Vector Laboratories, Burlingame, Calif) was added for 5 minutes. After washing, the plates were developed by the addition of a citrate buffer containing 0-phenylenediamine (Sigma Chemical The reaction was stopped by the addition of 100 pi 2N hydrochloric acid to each well, and absorbance was read at 490 nm (Bio-Rad Microplate reader model 450; Bio-Rad Laboratories Diagnostic Group, Hercules, Calif.). The standard curve was plotted on a log-logit scale by means of simple linear regression analysis, and values for the pooled serum and individual samples were read from the curve." 8 ELISA inhibition An inhibition ELISA was developed to examine the site specificity of the monoclonal antibodies generated to Ara h II. One hundred microliters of Ara h II protein (1 mg/ml) was added to each well of a 96-well microtiter plate (Gibco) in coating buffer (carbonate buffer, pH 9.6) for 1 hour at 370C. Next, 100 ld of differing concentrations (up to 1000-fold excess) of each of the monoclonal antibodies was added to each well for 1 hour at 37°C. After washing, a standard concentration of the biotinylated monoclonal antibody preparation was added for 1 hour at 370C.

The assay was developed by the addition of the avidin substrate as in the ELISA above.

A similar ELISA inhibition was performed with the peanut-positive serum IgE pool instead of the biotinylated monoclonal antibody to determine the ability of each monoclonal antibody to block specific IgE binding.

RESULTS

15 Hybridomas specific for Ara h H Cell fusions between spleen cells obtained from female BALB/c mice immunized with Ara h II and the mouse myeloma cells resulted in a series of Shybridomas specific for Ara h II. Seven monoclonal antibody-producing lines were chosen for further study. In preliminary studies all seven hybridoma-secreting cell 20 lines had antibodies that bound Ara h II, as determined by ELISA and immunoblot analysis.

1 2 1 3 On the basis of different binding studies, four of the hybridomas were used for further analysis. As determined by isotype immunoglobulin-specific ELISA, all four hybridoma-secreting cell lines typed as IgG.

ELISA with monoclonal antibody as solid phase Four monoclonal antibody preparations (4996D6, 4996C3, 5048B3, and 4996D5) were used as capture antibodies in an ELISA with Ara h II as the antigen.

Serum from individual patients, who had positive challenge responses to peanut, was used to determine the amount of IgE binding to each peanut fraction captured by the Ara h II-specific monoclonal antibody (Table A reference peanut-positive seruni pool was used as the control serum for 100% binding. Seven patients who had positive DBPCFC responses to peanut were chose. All seven patients had significant amounts of anti-peanut-specific IgE to the peanut antigen presented by each of the four monoclonal antibodies compared with the control sera (patient 8 without peanut sensitivity who had elevated serum IgE values, patient 9 without peanut sensitivity who had normal serum IgE values). Titration curves were performed to show that limited amounts of antigen binding were not responsible for similar antibody binding. There were no significant differences in the levels of anti-peanut-specific IgE antibody to the peanut antigens presented by each monoclonal antibody. Most patients had their highest value for IgE binding to the peanut antigen presented by either 4996D6 or 4996C3, whereas no patient had his or her highest percent of IgE binding to the peanut antigen presented by monoclonal antibody 4996D5.

Food antigen specificity of monoclonal antibodies to Ara h II To determine whether the Ara h II monoclonal antibodies would bind to only peanut antigen, an ELISA was developed with the pooled peanut-specific IgE from patients who had positive DBPCFC responses to peanut. All four monoclonal antibodies that were fully characterized bound only peanut antigen (Table In the ELISA no binding to soy, lima beans, or ovalbumin occurred. When the normal 15 serum pool was used in the ELISA, no peanut-specific IgE to either Ara h II or crude o*o peanut could be detected.

*'In the United States, three varieties of peanuts are commonly consumed: Virginia, Spanish, and Runner. In an ELISA, we attempted to determine whether there were differences in monoclonal antibody binding to the three varieties of 20 peanuts. There was only a minor variation with the ability of the peanut-specific oooo IgE to bind to the captured peanut antigen (data not shown) Site specificity of four monoclonal antibodies ,An inhibition ELISA was used to determine the site specificity of the four monoclonal antibodies to Ara h II (Table As determined by ELISA inhibition analysis, there are at least two different epitomes on Ara h II, which could be recognized by the various monoclonal antibodies (epitope 1-4996C3, epitope 2- 4996D6, 5048B3, 4996D5). Seven different monoclonal antibodies generated to Ara h I, a 63.5 kd peanut allergen,' were used to inhibit the binding of the four Ara h II monoclonal antibodies to the Ara h II protein. None of the Ara h I monoclonal antibodies inhibited any binding of the Ara h II monoclonal antibodies.

Site specificity of peanut-specific human IgE Results of inhibition assays with monoclonal antibodies to inhibit IgE binding from the IgE pool (from patients with peanut hypersensitivity) to Ara h II are shown in Table 4. Monoclonal antibodies 4996C3 and 4996D5 inhibited the peanutspecific IgE up to approximately 25%. Monoclonal antibodies 4996D6 and 5048B3 did not inhibit peanut-specific IgE binding. These two inhibition sites correspond with the two different IgG epitopes recognized by the monoclonal antibodies in the inhibition experiments.

DISCUSSION

The route of allergen administration, dosage, frequency of exposure, and genetic factors all determine the type and severity of an individual's allergic response.

1 4 To date, no distinct features, which would distinguish allergens as unique antigens, have been identified.

1 4 In contrast, only three foods in the United States (milk, eggs, and peanuts) account for approximately 80% of positive responses to food challenges in children.

Although clinical sensitivity to most foods is typically lost as a patient ages, clinical sensitivity to peanut is rarely lost. For this reason, it is important to examine the peanut allergens to determine whether they have distinct features that 15 would cause the persistence of clinical reactions.

Two major peanut allergens, Ara h I and Ara h II, have recently been identified and characterized.

8 9 Ara h I has two major bands as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis with a mean molecular weight of 63.5 kd and an isoelectric point of 4.55. Ara h II has a mean molecular 20 weight of 17 kd and an isoelectric point of 5.2. Individual sequencing of Ara h I and Ara h II indicates that they are probably isoallergens." Other peanut allergens have been identified including peanut 116 and concanavalin A-reactive glycoprotein.

1 In this study four monoclonal antibodies to Ara h II were extensively characterized. All four monoclonal antibodies produced to Ara h II, when used as 25 capture antibodies in an ELISA, presented antigens that bound IgE from patients "with positive challenge responses to peanut. No significant differences were detected in the binding of IgE from any one patient to the allergen presented by the individual monoclonal antibodies. In separate ELISA experiments, the four monoclonal antibodies generated to Ara h II did not bind to other legume allergens and did not bind to one variety of peanuts preferentially.

To determine the epitope site specificity of these monoclonal antibodies, inhibition ELISAs were done. At least two different and distinct IgG epitopes could be identified in experiments with the allergen, Ara h II. In related experiments done with pooled serum from patients with positive DBPCFC responses to peanut, two similar IgE epitopes were identified. The results of this study are comparable to those with monoclonal antibodies to Der f I' in which five nonoverlapping antigenic sites and three IgE-binding epitopes were identified. In our previous studies with Ara h I monoclonal antibodies," four different antigenic sites were recognized, and three of these sites were IgE-binding epitopes.

In related experiments with other allergens, a variety of solid-phase inhibition assays have been used to block the polyclonal IgE response to the allergen being studied.

6 The interpretation of the level of inhibition that should be regarded as significant has varied from 15% to 80%.

6 The Ara h II monoclonal antibodies inhibited the polyclonal IgE response by up to The characterization of these Ara h II monoclonal antibodies will allow future studies to better define the exact amino acid sequence that is responsible for IgE binding. Additionally, these monoclonal antibodies should make purification of the Ara h II allergen much simpler and more efficient. Immunoaffinity 15 purification of allergens, such as that completed with the cockroach allergens 6 and *with the Ara h I peanut allergen," 9 has produced a technique to purify allergens from a heterogeneous crude source material.

Future studies on the antigenic and allergenic structure of allergens will likely use monoclonal antibody techniques, in addition to recombinant DNA 20 technology. Monoclonal antibodies will be used to map these epitopes and to identify cDNA clones specific for the allergens. Together, recombinant DNA technology and monoclonal antibody production will be used to examine the role of specific T-cell epitopes in the induction and regulation of the allergenic response.

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REFERENCES

25 1. Yunginger Jones RT. A review of peanut chemistry: implications for the standardization of peanut extracts. In: Schaeffer M, Sisk C, Brede HI, eds.

Proceedings of the Fourth International Paul Ehrlich Seminar on the Regulatory Control and Standardization of Allergenic Extracts, October 16-17, 1985; Bethesda, Md. Stuttgart: Gustav Fischer Verlag, 1987;251-64.

2. Yunginger JW, Sweeney KG, Sturner WQ, et al. Fatal food-induced anaphylaxis. JAMA 1988;260:1450-2.

3. Sampson HA, Mendelson L, Rosen JP. Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N Engl J Med 1992;327:380-4.

4. Hoffman DR, Haddad ZH. Diagnosis of IgE-mediated reaction to food antigens by radioimmunoassay. J ALIERGY CIIN IMMUNOL 1974;54:165-73.

Chapman MD. Purification of allergens. Curr Opin Immunol 1989;1:647-53.

6. Chapman MD. Monoclonal antibodies as structural probes for mite, cat, and cockroach allergens. J Immunol 1987; 139:1479-84.

7. Mourad W, Mecheri S, Peltre G, David B, Hebert J. Study of the epitope structure of purified Dac g I and Lol p I, the major allergens of Dactylis glomerate and Lolium perenne pollens, using monoclonal antibodies. J Immunol 1988;141:3486-91.

8. Burks AW, Williams LW, Connaughton C, Cockrell G, O'Brien TJ, Helm RM.

Identification and characterization of a second major peanut allergen, Ara h II, with use of the sera of patients with atopic dermatitis and positive peanut challenges. ALLERGY CLIN IMMUNOL 1992;90:962-9.

9. Burks AW, Williams LW, Helm RM, Connaughton CA, Cockrell G, O'Brien TJ.

15 Identification of a major peanut allergen, Ara h I, in patients with atopic dermatitis and positive peanut challenges. J ALLERGY CLIN IMMUNOL S 1991;88:172-9.

10. Rouse DA, Morris SL, Karpas AB, Probst PG, Chaparas SD. Production, characterization, and species specificity of monoclonal antibodies to 20 mycobacterium avium complex protein antigens. Infect Immun 1990;58:1445- 99.

11. Burks AW, Sampson HA, Buckley RH. Anaphylactic reactions following gammaglobulin administration in patients with hypogammaglobulinemia; detection of IgE antibodies to IgA. N Engl J Med 1986;314:560-4.

25 12. Sutton R, Wrigley CW, Baldo BA. Detection of IgE and IgG binding proteins after electrophoresis transfer from polyacrylamide gels. J Immunol Methods 1982;52:183-6.

13. Towbin H, Staehelin T, Gordan J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350-4.

14. Marsh DG. Allergens and the genetics of allergy. In: Sela M. ed. The antigens. New York: Academic Press. 1975;3:271-359.

Sampson HA, McCaskill CC. Food hypersensitivity in atopic dermatitis: evaluation of 113 patients. J Pediatr 1985; 107:669-75.

16. Sachs MI, Jones RT, Yunginger JW. Isolation and partial characterization of a major peanut allergen. J ALLERGY CLIN IMMUNOL 1981;67:27-34.

17. Barnett D, Howden, MEH, Bonham B, Burley RW. Aspects of legume allergy research. proc Sydney Allergy Group 1985; 4:104-18.

18. Chapman MD, Heyman PW, Platts-Mills TAE. Epitope mapping of two major inhalant allergens, Derp I and DerfI, from mites of the genus Derinatophagoides. J Immunol 1987;139:1479-84.

19. Burks AW, Cockrell G, Connaughton C, Helm RM. Epitope specificity and immunoaffinity purification of the major peanut allergen, Ara h I. J ALLERGY CLIN IMMUNOL 1994;93:743-50.

O'Hehir RE, Young DB, Kay AB, Lamb JR. Cloned human T lymphocytes reactive with Dermatophagoides farina (house dust mite): a comparison of Tand B-cell antigen recognition. Immunology 1987;62:635-40.

ISOLATION, IDENTIFICATION, AND CHARACTERIZATION OF CLONES ENCODING ANTIGENS RESPONSIBLE FOR PEANUT HYPERSENSITIVITY Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Peanut hypersensitivity reactions often tend to be quite severe, sometimes resulting in episodes of fatal anaphylaxis Despite the significant prevalence of peanut hypersensitivity reactions and several fatalities annually, the identification of the clinically relevant antigens and an understanding of the immunobiology of peanut hypersensitivity are just beginning The identification and purification of allergens is essential for the immunological studies necessary to understand their role in stimulating IgE antibody formation. Because of the prevalence and severity of peanut hypersensitivity reactions in both children and adults, coupled with the recent identification of two major peanut allergens that are involved in this process 15 we set out to clone and characterize the Ara h I peanut allergen. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut ScDNA expression library were used to identify clones that encode peanut allergens.

one of the major peanut allergens, Ara h I, was selected from these clones using Ara h I-specific oligonucleotides and polymerase chain reaction technology. Using the 20 oligonucleotide GA(TC)AA(AG)GA(TC)AA(TC)GTNAT(TCA)GA(TC)CA derived from amino acid sequence analysis of the Ara h I (63.5kD) peanut allergen as one primer and a 27-nucleotide-long oligo-dT stretch as the second primer, a portion of the mRNA that encodes this protein was amplified from peanut cDNA. To determine if this clone (5Ala) represented the entire Ara h I, a 2 P-labeled insert 25 from this clone was used as a hybridization probe of a Northern blot containing peanut poly A RNA. This insert hybridized to a single-size mRNA of approximately 2.3 kb. The insert contained 1,360 bases not including the poly A tail. The sequence beginning at position 985 and extending through to position 1032 encodes an amino acid sequence identical to that determined from Ara h I peptide I. DNA sequence analysis of the cloned insert revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants There were 64% homology over more than 1,000 bases when the clone 5Ala sequence was compared with the broad bean and pea vicilins.

IgE immunoblot analysis was performed using serum IgE from patients with peanut hypersensitivity and Ara h I protein expressed from clone 5Ala in Escherichia coli XL1-Blue cells to address the question of how frequently recombinant Ara h I was recognized by these individuals. Figure 1 shows three representative immunoblot strips that have been incubated with different patient sera. Two of the patients showed strong IgE binding to the recombinant Ara h I protein while one patient had no detectable IgE binding to this protein. Of the 11 patient sera tested in this manner, 8 had IgE which recognized recombinant Ara h I (Table We have demonstrated that the cloned Ara h I gene is capable of producing a protein product in prokaryotic cells that is recognized by serum IgE from a large number of individuals with documented peanut hypersensitivity. These results are significant in that they indicate that some of the allergenic epitopes responsible for this reaction are linear amino acid sequences that do not include a carbohydrate component. These findings may provide the basis for improving diagnosis and therapy of persons with food hypersensitivity. With the production of the 15 recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.

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REFERENCES

1. Yunginger JW, Squillace DL, Jones RT, Helm RM: Fatal anaphylactic reactions induced by peanuts. Allergy Proc 1989;10:249-253.

2. Sampson HA, Mendelson L, Rosen JP: Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N Engl J. Med 1992;327:380- 384.

3. Burks AW, Williams LW, Helm RM, Connaughton C, Cockrell G, O'Brien TJ: Identification of a major peanut Allergen, Ara h I, in patients with atopic dermatitis and positive peanut challenges. J Allergy Clin Immunol 1991;88:172-179.

4. Burks AW, Williams LW, Connaughton C, Cockrell G, O'Brien T, Helm RM: Identification and characterization of a second major peanut allergen, Ara h II, utilizing the sera of patients with atopic dermatitis and positive peanut challenge. J Allergy Clin Immunol 1992;90:962-969.

15 5. Chee PP, Slightom JL: Molecular biology of legume vicilintype seed 00 *storage protein genes. Subcell Bioch 1991;17:31-52.

6. Dure L: An unstable domain in the vicilin genes of higher plants. N Biol 1990;2:487-493.

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a a TABLE 1 Peanut-specific IgE to antigen presented by four monoclonal antibodies Patient Capture antibody No 4996D6 4996C3 5048B3 4996D5 1 95% 80% 80% 91% 2 94% 66% 72% 3 96% 114% 87% 96% 4 98% 116% 76% 96% 97% 74% 130% 107% 6 94% 63% 76% 86% 7 109% 123% 104% 116% 8 0% 0% 0% 0% 9 0% 0% 0% 0% Ara h II monoclonal antibodies used as capture antibodies in ELISA with Ara h II as the antigen. Values are expressed as a percent of binding compared with challenge-positive peanut pool. Patients 1 to 7 had positive DBPCFC responses to peanut; patient 8 is the patient without peanut sensitivity with elevated serum IgE; patient 9 is the patient without peanut sensitivity with normal serum IgE.

TABLE 2 IgE-specific binding to legume's captured by Ara h II monoclonal antibodies Ca ture antibod 4996D6 4996C3 5048B3 4996D5 Pooled serum* Ara h II (17 kd) 0.451 0.565 0.235 0.381 Crude peanut 0.137 0.409 0.161 0.170 Soy 0.053 0.055 0.055 0.015 Lima beans 0.033 0.026 0.029 0.025 Ovalbumin 0.028 0.029 0.029 0.035 Normal serum Arah II (17 kd) 0.024 0.031 0.038 0.033 Crude peanut 0.017 0.027 0.028 0.024 Values are expressed as optical density units.

*Pooled serum is from patients with positive responses to peanut challenge.

14 TABLE 3 ELISA inhibition for four monoclonal antibodies to Ara h II Inhibitory antibody 4996C3 4996D6 5048B3 Biotinylated mAb 4996C3 4996D6 5048B3 4996D5 99% 0% 30% 1% 8% 53% 83% 44% 6% 31% 100% 56% 4996D5 3% 18% 100% 64% Altl 1% 9% 3% 8% Site specificity of four Ara h II monoclonal antibodies as determined by ELISA inhibition analysis. Values are expressed as percent inhibition.

mAb, Monoclonal antibody.

TABLE 4 Individual anti-peanut-specific IgE binding to Ara h II Serum dilution 1:320 1:100 1:80 1:40 1:20 4996D6 0% 0% 0% 0% 3% 4996C3 14% 10% 10% 12% 10% 24% 5048B3 0% 5% 5% 5% 7% 11% 4996D5 0% 10% 10% 22% 23% Site specificity of four Ara hII monoclonal antibodies inhibiting anti-peanut-specific IgE (serum pool from patients with peanut hypersensitivity) binding to Ara h II. Values are expressed as percent of anti-peanut-specific IgE binding to Ara h II without inhibiting 15 monoclonal antibody.

TABLE 5 Recognition of Ara h I protein by patient serum IgE from patients with peanut hypersensitivity Patient Recombinant Ara h I Native Ara h I AC BE TL AS KS KF CS SM TM TH JH Recombinant or native Ara h I protein was electrophoresed on denaturing polyacrylamide gels, blotted to nitrocellulose, and then probed with serum IgE from patients with peanut hypersensitivity. Patients were scored for the presence or absence of serum IgE to recombinant or native Ara h I.

Mapping of the B-cell Epitopes on Ara h I, a Legume Vicilin Protein and a Major Allergen in Peanut Hypersensitivity

SUMMARY

Peanut allergy is a significant health problem because of the potential severity of the allergic reaction and the difficulty in the accurate diagnosis of this disease. Serum IgE from patients with documented peanut hypersensitivity reactions and overlapping peptides were used to identify the major IgE binding epitopes on the major peanut allergen, Ara h I. At least twenty-three different linear IgE binding epitopes, located throughout the length of the Ara h I protein, were identified. Two of the peptides appeared to be immunodominant IgE binding epitopes in that they were recognized by serum from >90% of the patients tested. No other peptide was recognized by greater 15 than 50% of the peanut sensitive population tested. Mutational analysis of the immunodominant epitopes revealed that single amino acid changes within these peptides had dramatic effects on IgE binding characteristics. With the identification of the IgE binding epitopes on the Ara h I protein and the determination of the amino acids within these epitopes important to immunoglobulin binding it will now be possible to address the Spathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.

INTRODUCTION

.Approximately 8% of children and 1-2% of adults have some type of food allergy Peanuts, fish, tree nuts, and shellfish account for the majority of food hypersensitivity reactions in adults, while peanuts, milk, and eggs cause over 80% of food hypersensitivity reactions in children Unlike the food hypersensitivity reactions to milk and eggs, peanut hypersensitivity reactions usually persist into adulthood and last for a lifetime In addition, hypersensitivity reactions to peanuts tend to be more severe than those to other food allergens. Allergic reactions to peanuts can produce symptoms ranging from urticaria to anaphylaxis in patients with peanut hypersensitivity.

Several reports have detailed fatal and near-fatal anaphylactic reactions occurring in adolescents and adults following the ingestion of peanuts or peanut products. Diagnosis of individuals with peanut hypersensitivity is often complicated by the presence of cross-reacting antibodies to other legumes Currently, the only effective treatment for patients with peanut hypersensitivity is avoidance of any food products which contain the allergen.

This is becoming more difficult due to the inclusion of peanuts and peanut products as protein extenders in many different foods.

Food hypersensitivity reactions occur shortly after contact of a specific allergen with its corresponding IgE antibodies which are bound to mast cells.

Allergen-specific IgE when cross-linked by the respective allergen activates the mast cells to release histamine, heparin, and other mediators responsible for the clinical symptoms observed. Thus the IgE binding epitopes of the allergens play an important role in the disease process. Their characterization will provide a better understanding of the human immune response involved in food hypersensitivity reactions. If improved diagnostic and therapeutic capabilities are to be developed it is important to determine the primary 15 structure and frequency of recognition of any IgE binding epitopes contained within the allergen.

Various studies have shown that the most allergenic portion of the peanut is the protein fraction of the cotyledon A major allergen found in the cotyledon is the peanut protein, Ara h I This protein is recognized by 20 >90% of peanut sensitive patients, thus establishing it as an important allergen The majority of serum IgE recognition of the Ara h I allergen appears to be due to epitopes within this protein that are linear amino acid sequences that do not contain significant amounts of carbohydrate The Ara h I allergen belongs to the vicilin family of seed storage proteins 25 Previous results have demonstrated similarity between the level of IgE binding **to recombinant Ara h I protein and the native form of this allergen when individual patient serum was tested These results indicated that the recombinant protein could be considered for use in both diagnostic and immunotherapeutic approaches to peanut hypersensitivity.

Because of the prevalence and severity of peanut hypersensitivity reactions in both children and adults, coupled with the difficult nature of diagnosing this food allergy, we set out to map and characterize the major IgE epitopes of the Ara h I allergen. In this communication we report the primary structure of the Ara h I IgE-binding epitopes recognized by peanut hypersensitive individuals. Two epitopes that bound peanut specific serum IgE from >90% of patients tested were identified. The amino acids important to peanut-specific IgE recognition of these epitopes were then determined for the purpose of using them in future diagnostic and immunotherapeutic approaches to this disease.

MATERIALS AND METHODS Patients. Serum from fifteen patients with documented peanut hypersensitivity reactions (mean age, 25 yr) was used to identify the Ara h I IgE binding epitopes. Each of these individuals had a positive immediate prick skin test to peanut and either a positive double blind, placebo controlled, food challenge (DBPCFC) or a convincing history of peanut anaphylaxis (laryngeal edema, severe wheezing, and/or hypotension). one individual with elevated serum IgE levels (who did not have peanut specific IgE or peanut hypersensitivity) was used as a control in these studies. In some instances a 15 serum pool was made by mixing equal aliquot's of serum IgE from each of the 15 patients with peanut hypersensitivity. This pool was then used in immunoblot analysis experiments to determine the IgE binding characteristics 'of the population. At least five mls of venous blood were drawn from each patient and allowed to clot, and the serum collected. All studies were S* 20 approved by the Human Use Advisory Committee at the University of Arkansas for Medical Sciences.

Computer analysis ofAra h I sequence. Sequence analysis of the Ara h I gene and peptide sequences was done on the University of Arkansas for Medical Science's Vax computer using the Wisconsin DNA analysis software 25 package. The predicted antigenic regions on the Ara h I protein are based on S* -algorithms developed by Jameson and Wolf (10) that relates antigenicity to hydrophilicity, secondary structure, flexibility, and surface probability.

Peptide synthesis. Individual peptides were synthesized on a cellulose membrane containing free hydroxyl groups using Fmoc-amino acids according to the manufacturer's instructions (Genosys Biotechnologies, The Woodlands, TX). Synthesis of each peptide was started by esterification of an Fmoc-amino acid to the cellulose membrane. After washing, all residual amino functions on the sheet were blocked by acetylation to render it unreactive during the subsequent steps. Each additional Fmoc-amino acid is esterified to the previous one by this same process. After addition of the last amino acid in the peptide, the amino acid side chains were de-protected using a mixture of dichloromethane/trifluoroacetic acid/triisobutylsilane followed by treatment with dichloromethane and washing with methanol. Membranes containing synthesized peptides were either probed immediately with serum IgE or stored at -20 0 C until needed.

IgE binding assay. Cellulose membranes containing synthesized peptides were incubated with the serum pool or individual serum from patients with peanut hypersensitivity diluted in a solution containing TBS and 1% bovine serum albumin for at least 12 h at 4 0 C or 2 h at room temperature. Detection of the primary antibody was with 2 5 -labeled anti-IgE antibody (Sanofi Pasteur Diagnostics, Chaska, MN).

RESULTS

There are Multiple IgE Binding Regions Throughout the Ara h I 15 Protein. The Ara h I protein sequence was analyzed using a computer program to model secondary structure and predict antigenicity based on the S* parameters of hydrophilicity, secondary structure, flexibility, and surface probability. Eleven antigenic regions, each containing multiple antigenic sites, were predicted by this analysis along the entire length of the molecule (Fig. 1).

Seventy-seven overlapping peptides representing the entire length of the Ara h I protein were synthesized and probed with pooled serum to determine IgE binding to the predicted antigenic regions, or any other regions of the protein. Each peptide was 15 amino acids long and offset from the previous 25 peptide by eight amino acids. These peptides were then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity or with serum IgE from a control patient with no food allergy. Figure 2A shows 12 IgE binding regions along the entire length of the Ara h I protein recognized by this population of peanut hypersensitive patients. Serum IgE from the control patient did not recognize any of the synthesized peptides (data not shown). In general, there was good agreement between the predicted antigenic regions (Fig. 2B, boxed areas P1-P11) and those that were determined (Fig. 2B, shaded areas D1-D12) by actual IgE binding. However, there were two predicted antigenic regions (AA221-230; AA263-278) that were not recognized by serum IgE from peanut hypersensitive individuals. In addition, there were numerous IgE binding regions found in the Ara h I protein between amino acids 450-600 (Fig. 2A) In order to determine the amino acid sequence of the IgE binding sites, small overlapping peptides spanning each of the larger IgE binding regions identified in Figure 2 were synthesized. By synthesizing smaller peptides amino acids long) that were offset from each other by only two amino acids it was possible to identify individual IgE binding epitopes within the larger IgE binding regions of the Ara h I molecule. Figure 3 shows a representative immunoblot and the respective amino acid sequence of the binding region D2- D3 (AA82-133). Four epitopes (Fig. 3, numbers 4-7) were identified in this region. Similar blots were performed for the remaining IgE binding regions to identify the core amino acid sequences for each IgE epitope. Table 6 S. 15 summarizes the 23 IgE epitopes (peptides 1-23) and their respective positions in the Ara h I molecule. The most common amino acids found were acidic and basic residues comprising 40% of all amino acids found in the epitopes. In addition, no obvious amino acid sequence motif was shared by the epitopes.

20 Identification of Common Ara h I Epitopes Recognized by Serum IgE from Patients With Peanut Hypersensitivity.

Each set of twenty-three peptides was probed with serum IgE from individuals to determine which of the twenty-three epitopes were recognized by serum IgE from patients with peanut hypersensitivity. Serum from five 25 individuals randomly selected from the 15 patient serum pool and an additional five sera from peanut hypersensitive patients not represented in the serum pool were used to identify the common epitopes. Figure 4 shows the IgE binding results of the 10 immunoblot strips containing these peptides incubated with individual patient sera. All of the patient sera tested (10/10) recognized multiple epitopes. The average number of epitopes recognized was 6/patient sera, ranging from one serum recognizing only 2 epitopes to another patient's serum recognizing 12 epitopes. The results are summarized in Table 7. Interestingly, epitope 17 was recognized by all patient sera tested (10/10) and epitope 4 was recognized by 90% (9/10) of patient sera tested. No other epitope was recognized by more than 50% of the patient sera tested.

IgE binding Characteristics of Mutated Ara h I Epitopes.

The amino acids essential to IgE binding in epitopes 4 and 17 were determined by synthesizing duplicate peptides with single amino acid changes at each position. The amino acids were changed to either an alanine or glycine residue because these amino acids have small, R groups. These peptides were then probed with pooled serum IgE from 15 patients with peanut hypersensitivity to determine if the change affected peanut specific IgE binding. The results are shown in Figure 5. Clearly, a single amino acid substitution has dramatic effects on the IgE binding characteristics of that peptide. Replacement of any amino acid in the 91-96 region of epitope 4 resulted in almost complete loss of IgE binding to this epitope. In epitope 17, replacement of the tyrosine residue at position 500 or replacement of the 15 glutamic acid residue at position 506 also resulted in dramatic decreases in IgE "binding.

Significant sequence homology between epitopes 4 and 17 and seed storage proteins from other plants could explain the presence of cross-reacting antibodies to other legumes which complicates diagnosis. To assess the S" 20 prevalence of the amino acid sequences of epitope 4 and 17 in other seed storage proteins, the complete Ara h I amino acid sequence was first used to select all plant proteins that shared sequence homology with the peanut ~vicilin. There were 93 entries selected on this basis, representing amino acid sequences deposited in the protein data base from a variety of seed storage proteins. The amino acid sequence for epitope 17 was present in many of these proteins with sequence identity ranging from 20-60%. Interestingly, even in those proteins with only 20% identity the tyrosine at position 500 and the glutamic acid residue at position 506 were almost always conserved (Table The amino acid sequence for epitope 4 was present in fewer of these proteins with sequence identity ranging from 20-30%. In every case, at least one of the amino acids at positions 91-96 were different from the peanut vicilin (Table 8).

DISCUSSION

The development of an IgE response to an allergen involves a series of interactions between T cells and B cells. B cells bearing appropriate antigenspecific receptors interact with proliferating allergen specific T-cells which leads to isotype switching and the generation of antigen-specific IgE. The antigen-specific IgE then binds to surface receptors of mast cells, basophils, macrophages, and other APCs enabling the immune system to respond to the next encounter with the specific antigen (B-cell epitope). Because antigen specific IgE plays such a critical role in the etiology of allergic disease, determination of allergen-specific, IgE binding epitopes is an important first step toward a better understanding of this complex disease process.

The vicilins are seed storage proteins found in most higher plants (11).

A comparison of the vicilin amino acid sequences from different plant sources reveals that considerable sequence homology exists between the carboxyl twothirds of all these molecules. The major difference between the vicilins is 15 found in the amino terminal end of these proteins where little sequence homology is detected In sequence comparison studies with other legumes, the peanut vicilin, Ara h I conforms to this general rule with the highest similarity being found in the carboxyl two-thirds of this molecule.

In the present study we have determined that there were multiple 20 antigenic sites predicted for the Ara h I allergen.- In general, as has been found with other allergens (12,13), there was good agreement between those residues "predicted by computer analysis and B-cell epitopes determined by experimental analysis of overlapping peptides. This strofi-g correlation between predicted and determined epitopes is probably due to the ability of the computer model to predict which regions of the molecule are exposed on the surface of the allergen, making them accessible to immunoglobulin interactions. There are at least 23 different IgE recognition sites on the major peanut allergen Ara h I. These sites are distributed throughout the protein.

The identification of multiple epitopes on a single -allergen is not novel.

Allergens from cow milk codfish hazel, soy (17) and shrimp (18) have all been shown to contain multiple IgE binding epitopes. The observation that most of these proteins have multiple IgE binding sites probably reflects the polyclonal nature of the immune response to them and may be a necessary step in establishing a protein as an allergen.

The elucidation of the major IgE binding epitopes on Ara h I may also enable us to better understand the immunopathogenic mechanisms involved in peanut hypersensitivity. Recent evidence suggests that there is a preferential variable heavy chain usage in IgE synthesis and a direct switching from IgM production to IgE synthesis This would suggest that epitopes responsible for antigen-specific IgE antibody production may differ from those promoting antigen-specific IgG antibodies. Immuno-therapeutic approaches utilizing peptides representing IgG epitopes may be able to shift the balance of antigen-specific antibody production from IgE to IgG. We are currently identifying which of the IgE binding epitopes also bind IgG to determine if this would be a feasible strategy for patients with peanut hypersensitivity.

Two of the Ara h I peptides appear to be immunodominant IgE binding epitopes in that they are recognized by >90% of patient sera tested.

Interestingly, epitope 17 which is located in the carboxyl end of the protein 15 (AA 498-507), is in a region that shares significant sequence homology with vicilins from other legumes. The amino acids important to IgE binding also I appear to be conserved in this region and may explain the possible crossreacting antibodies to other legumes that can be found in sera of patients with a positive DBPCFC to peanuts. Epitope 4, located in the amino terminal 20 portion (AA 89-98) of the protein, appears to be unique to this peanut vicilin and does not share any significant sequence homology with vicilins from other legumes. In addition, the amino acids important to IgE binding in this region are not conserved. These findings may enable us to develop more sensitive and specific diagnostic tools and lead to the design of novel therapeutic agents 25 to modify the allergic response to peanuts.

The only therapeutic option presently available for the prevention of a food hypersensitivity reaction is food avoidance. Unfortunately, for a ubiquitous food such as peanut, the possibility of an inadvertent ingestion is great, one therapeutic option used extensively for patients with allergic reactions to various aeroallergens and insect sting venoms is allergen desensitization immunotherapy. Allergen immunotherapy consists of injections of increasing amounts of allergens to which a patient has Type I immediate hypersensitivity (20,21). Allergens for immunotherapy are usually extracted from natural sources and represent mixtures of several different 23 proteins, to many of which the patient is not allergic. These non-allergenic components could induce an IgE-response in hyposensitized patients (22) thus complicating their use as a therapeutic tool. One of the major improvements in allergen immunotherapy has been the use of standardized allergenic extracts which has been made possible by the use of recombinant allergens (23,24).

While the absolute mechanism of immunotherapy is unknown, an increase in IgG or IgG4 antibody activity, a decrease in allergen-specific IgE levels, and a decrease in basophil activity have all been implicated (25-28) in mediating this response. Because allergen immunotherapy has been proven efficacious for treatment of some allergies, treatment with peanut immunotherapy is now being studied as a possible option Our work showing the IgE binding epitopes of a major peanut allergen may allow for the use of immunodominant epitopes in this approach. One possible advantage of using peptides over using the whole allergen is the reduced danger of anaphylaxis. The degranulation of mast cells requires the cross-linking of IgE antibodies bound to the high affinity FceR I receptors Peptides containing single IgE epitopes would be unable to bind to more than one IgE antibody and therefore unable to cross-link the bound IgE. We are currently exploring this possibility in vitro and in vivo models.

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Teigland, M. Bray, P.A. Benson, J.A. York, Biedrzycki, D.L. Squillace, et al. 1988. Fatal foodinduced anaphylaxis. JAMA, 260:1450-1452.

15 6. Bernhisel-Broadbent, S. Taylor, and H.A. Sampson. 1989. Cross- Sallergenicity in the legume botanical family in children with food hypersensitivity. II. Laboratory correlates J. Allergy Clin. Immunol; 84:701-709.

7. Taylor, W.W. Busse, M.I. Sachs, J.L. Parker, and J.W. Yunginger.

1981. Peanut oil is not allergenic to peanut sensitive individuals. J.

Allergy Clin. Immunol. 68:372-375.

8. Burks L.W. Williams, R.M. Helm, C. Connaughton, G. Cockrell, T.

S O'Brien. 1991. Identification of a major peanut allergen Ara h I, in patients with atopic dermatitis and positive peanut challenge. J. Allergy Clin.

25 Immunol. 88:172-179.

9. Burks G. Cockrell, J.S. Stanley, R.M. Helm, G.A. Bannon. 1995.

Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity. J. Clin. Invest. 96:1715-1721.

Jameson,B.A, and H. Wolf. 1988. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput. Appl. Biosci. 4:181-186.

11. Gibbs, K.B. Strongin, and A. McPherson. 1989. Evolution of legume seed storage proteins a domain common to legumins and vicilins is duplicated in vicilins. Mol. Biol. Evol. 6:614-623.

12. Van Kampen, W.M. Becker, Z. Chen, H.P. Rihs, G. Mazur, M. Raulf, V.

Liebers, S. Isringhausen-Bley, and X. Baur. 1994. Analysis of B-cell epitopes in the N-terminal region of Chi t I component III using monoclonal antibodies. Molecular Immunol., 31:1133-1140.

13. Breiteneder, F. Ferreira, A. Reikerstorfer, M. Duchene, R. Valenta, K.

Hoffman-Sommergruber, C. Ebner, M.Breitenbach, D. Kraft, 0. Scheiner.

1992. Complementary DNA cloning and expression in Escherichia coli of Aln g I, the major allergen in pollen of alder (Alnus glutinosa). J. Allergy Clin. Immunol., 90:909-917.

14. Ball M.J. Shelton, B.J. Walsh, D.J. Hill, C.S. Hosking, and M.E.

Howden. 1994. A major continuous allergenic epitope of bovine batalactoglobulin recognized by human IgE binding. Clinical and Experimental Allergy. 24:758-764.

15. Aas, K. and S. Elsayed. 1975. Physico-chemical properties and specific 15 activity of a purified allergen (codfish). Developments in Biological Standardization. 29:90-98.

16. Elsayed, E. Holen, and T. Dybendal. 1989. Synthetic allergenic epitopes from the amino-terminal regions of the major allergens of hazel and birch pollen. Int'l. Archives of Allergy Applied Immunology, 20 89:410-415.

17. Herian, S.L. Taylor, and R.K. Bush. 1990. Identification of soybean allergens by immunoblotting with sera from soy-allergic adults. Int. Arch.

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18. Shanti, B.M. Martin, S. Nagpal, D.D. Metcalf, and P.V. Rao. 1993.

25 Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes. J. of Immunology. 151:5354- 5363.

19. Van der Stoep, W. Korver, and T. Logtenberg. 1994. In vivo and in vitro IgE isotype switching in human B lymphocytes: evidence for a predominantly direct IgM to IgE class switch program. European J. of Immunol., 24:1307-1311.

Reisman, R.E. 1994. Fifteen years of hymenoptera venom immunotherapy: changing concepts and lessons. Allergy Proceedings, 15:61-63.

21. Fitzsimons, and L.C. Grammer. 1990. Immunatherapy-definition and mechanism. Allergy Proc., 11:156.

22. Birkner, H. Rumpold, E. Jarolim, H. Ebner, M. Breitenbach, O.Scheiner, and D. Kraft. Evaluation of immunotherapy-induced changes in specific IgE, IgG, and IgG-subclasses in birch pollen-allergic patients by means of immuno-blotting. Correlation with clinical response. Allergy, 45:418-426.

23. Scheiner, 0. 1992. Recombinant allergens: biological, immunological and practical aspects. Int Arch Allergy Immunol., 98:93-96.

24. Gordon, 1995. Future immunotherapy: what lies ahead? Otolaryngol Head Neck Surg., 113:603-605.

Sparholt, O.T. Olsen, and C. Schou. 1992. The allergen specific Bcell response during immunotherapy. Clinical and Experimental Allergy, 22:648-653.

26. Gieni, R. X. Yang, and K.T. Hayglass. 1993. Allergenspecific 15 modulation of cytokine synthesis patterns and IgE responses in vivo with chemically modified allergen. The Journal of Immunol., 150:302-310.

27. Secrist, C.J. Chelen, Y. Wen, J.D. Marshall, and D.T. Umetsu. 1993.

Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals. J. Exp. Med., 178:2123-2130.

20 28. Garcia, N.R. Lynch, M.C. Di Prisco, and R.I. Lopez. 1995.

Nonspecific changes in immunotherapy with house dust extract. J Invest.

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29. Oppenheimer, H.S. Nelson, S.A. Bock, F. Christensen, and D.Y.,Leung.

1992. Treatment of peanut allergy with rush immunotherapy. J. Allergy 25 Clin. Immunol., 90:151-152.

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Ngo, J.A. Panakos, E. Chourmouzis, F.T. Liu, and C.Y. Lau. 1996.

Transgenic mice expressing the human high-affinity immunoglobulin (Ig) E receptor alpha chain respond to human IgE in mast cell degranulation and in allergic reactions. J. of Exp. Med., 183:49-56.

FIGURE LEGENDS FIGURE 1. There Are Multiple Predicted Antigenic Sites on the Ara h I Allergen.

The amino acid sequence of the Ara h I protein was analyzed for potential antigenic sites by the Jameson and Wolf (1988) algorithm. These predictions are based on a model that relates antigenicity to hydrophilicity, secondary structure, flexibility, and surface probability. There were 11 (1-11) predicted regions that contained multiple antigenic sites (octagons) along the entire length of the molecule. Amino acid residues (small numbers) are represented as alpha-helical (sinusoidal curve), Beta-sheet (saw tooth curve), and coil (flat sinusoidal curve) conformations. Beta turns are denoted by chain reversals.

FIGURE 2. Multiple IGE Binding Regions Identified on the Ara h I Allergen.

Fig. 2A; Upper Panel: Epitope mapping was performed on the Ara h I 15 allergen by synthesizing the entire protein in 15 amino acid long overlapping peptides that were offset from each other by 8 amino acids. These peptides were then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity. The position of the peptides within the Ara h I protein are indicated on the left hand side of this panel.

20 Fig 2.B; Lower Panel: The amino acid sequence of the Ara h I protein is shown in the lower panel. The numbered boxes correspond to the predicted antigenic regions (P1-P11). The hatched boxes (D1-D12) correspond to the IgE binding regions shown in Figure 2A.

FIGURE 3. Core IgE Binding Epitopes identified on the Ara h I Allergen.

25 Panel A: Detailed epitope mapping was performed on IgE binding regions identified in Fig. 2 by synthesizing 10 amino acid long peptides offset from each other by two amino acids. These peptides were then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity.

The data shown represents regions D2 and a portion of D3 encompassing amino acid residues 82-133. Numbers correspond to peptides as shown in Table 6. Panel B: The amino acid sequence (residues 82-133) of Ara h I that was tested in Panel A is shown. Shaded areas of boxes correspond to IgE binding peptides in Panel A.

FIGURE 4. Commonly Recognized Ara h I Epitopes.

Core IgE binding epitopes were synthesized (10 amino acids long) and then probed individually with serum IgE from 10 patients with documented peanut hypersensitivity. The top panel represents where each of the Ara h I peptides (1-23) were placed on the membrane. Panels A-J show the peptides that bound serum IgE from patients with peanut hypersensitivity. The control panel was probed with sera from a patient with elevated IgE but who does not have Peanut hypersensitivity.

FIGURE 5. Amino Acids Involved in IgE Binding.

Epitope 4 and 17 were synthesized with a glycine or alanine (A) substituted for one of the amino acids in each of these peptides and then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity. The letters across the top of each panel indicate the one letter amino acid code for the residue normally at that position and the amino 15 acid that was substituted for it. The numbers indicate the position of each residue in the Ara h I protein.

V. ftftftf Table 6. Ara h I IgE binding epitopes.

PEPTIDE

AA SEQUENCE AKSSPYOK T

QEPDDLKOK

LEYDPRLVYD

GERTRGROPG

PGDYDDDRRQ

PRREEGGRWG

REREEDWRQP

EDWRRPSHO

OPRKIRPEGR

TPGOED FFP

SYLQEFSRNT

FNAEFNEIRR

EOEERGORRW

DITNPINLRE

NNFGKLFEVK

GTGNLEL VA V

RRYTARLKEG

ELHLLGFGIN

Ara h I POSITION 25-34 48-5 7 65-74 89-98 97-105 107-116 123-13 2 134-143 143-152 294-303 3 11-320 325-3 34 344-353 393-402 409-418 461-470 498-507 525-534 00*6 00 0 S a*.0 19 HRIFLAGDKD 539-548 IDO0IEK0AKD 551-560 21 KDLAFPGSGE 559-568 22 KESHFVSARP. 578-587 23 PEKESPEKED 597-606 The underlined portions of each peptide are the smallest IgE binding sequences as determined by the analysis as described in Fig. 3.

t,

S.

S TABLE 7 IgE binding of core Ara h I epitopes by serum from peanut hypersensitive individuals.

Epitopes/ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Patient A X X X X X X 6 B X X X X X X 6 C X X X X X X X X X X X X 12 D X X X X X X 6 E X X X X X X X X X X X 11 F X X X X 4 G X X X X X X 6 H X X X 3 I X X X X X X X X 8 X XX X X. X X 7 Pts/ 4 5 3 9 4 1 0 3 4 2 1 3 3 1 3 1 10 2 1 4 1 3 1 Epitope Patients are indicated by letters on the left hand side of the table. Arah I peptides are indicated by number (1-23) across the top of the table. The number of epitopes recognized by each patient (epitopes/patient) is shown on the right hand side of the table. The number of patients that recognized each epitope (pts/epitope) is shown across the bottom of the table. An X indicates that a peptide bound IgE.

TABLE 8 Amino acid comparison of Ara h I epitopes 4 and 17 with similar regions in other see storage proteins.

EPITOPE 4 EPITOPE 17 Ara h I GERRTRGRQPG Ara h I RRYTARLKEG Soy FPRPQPMQEE SOy RKYRAELSEQ Cacao -EQCEQRCER lack bean RRYAATLSEG Pea EEHEEEK.QKY Pea QRYEARLADG Ma~ize WEDDNHI-HH Fava bean QNYKAKLSPG The peptides representing Ara h I epitopes 4 and 17 were compared with similar regions from other seed storage proteins. The amino acids residues important to IgE binding are indicated as bold underlined letters. Those amino acids that are identical to the Ama h I sequence are underlined.

9*

C.

C

C

*Ce.

C.

*CC.

C THE MAJOR PEANUT ALLERGEN ARA H II IS A SEED STORAGE PROTEIN WITH MULTIPLE IGE-BINDING EPITOPES

INTRODUCTION

Immediate hypersensitivity reactions to foods occur in about 4% of children and 1% of adults and are mediated by the production of IgE antibodies to glycoproteins of very high abundance present in the food.

Peanuts are a major cause of serious allergic reactions in both children and adults. The hypersensitivity to peanuts often starts in childhood and continues throughout life. This is in contrast to other childhood food allergies such as to milk and eggs which generally resolve spontaneously with age. In addition, peanut allergy is more likely to cause fatal anaphylaxis than any other food allergy. Currently, avoidance is the only effective means of dealing with food allergy, but the use of peanuts and peanut by-products as supplements in many different foods makes accidental consumption almost inevitable. Thus, the prevalence and chronic nature of peanut allergy, the potential severity of the allergic reaction, and the widespread use of peanuts in consumer foods necessitates improved methods for managing peanut hyper-sensitivity.

Food hypersensitivity reactions occur shortly after contact of a specific allergen with its corresponding IgE antibodies which are botLnd to mast cells.

Cross-linking of the allergen-specific IgE by the respective allergen activates the mast cells to release histamine, heparin, and other mediators responsible for the clinical symptoms observed. Thus, the IgE binding epitopes of the allergens play an important role in the disease process. Their characterization will provide a better understanding of the human immune response involved in food hypersensitivity reactions. If improved diagnostic and therapeutic capabilities are to be developed it is important to determine the primary structure of the major allergens, the IgE binding sites of these allergens, and the frequency of recognition of any IgE binding epitopes that are identified.

Various studies have shown that the most allergenic portion of the peanut is the protein fraction of the cotyledon. Two highly abundant glycoproteins found in the cotyledon are the peanut allergens, Ara h I and Ara h I. These proteins are recognized by serum IgE from >90% of peanut sensitive patients, thus establishing them as important allergens. The majority of serum IgE recognition of the Ara h I and Ara h II allergens appear to be due to epitopes within these proteins that are linear amino acid sequences that do not contain significant amounts of carbohydrate. The gene encoding the Ara h I allergen has been cloned, sequenced, and identified as a seed storage protein belonging to the vicilin family of legume storage proteins.

The major peanut allergen, Ara h II, has now been cloned and the nucleotide sequence determined. The derived amino acid sequence has been used to construct synthetic peptides and perform a detailed examination of the linear IgE binding epitopes of this protein.

EXPERIMENTAL PROCEDURES Patients. Serum from 15 patients with documented peanut hypersensitivity (mean age, 25 yr) was used to identify peanut allergens.

"Each of these individuals had a positive immediate skin prick test to peanut and either a positive double-blind, placebo controlled, food challenge or a convincing history of peanut anaphylaxis (laryngeal edema, severe wheezing, and/or hypotension). Details of the challenge procedure and interpretation 20 have been discussed previously. One individual with elevated serum IgE levels (who did not have peanut specific IgE or peanut hypersensitivity) was used as a control in these studies. At least five mis of venous blood were drawn from each patient and allowed to clot, and the serum was collected.

All studies were approved by the Human Use Advisory Committee at the University of Arkansas for Medical Sciences. Isolation and amino acid sequence analysis of peanut allergen Ara h II.

Ara h II was purified to near homogeneity from whole peanut extracts according to the methods of Burks et al. Purified Ara h II was electrophoresed on 12.5% acrylamide mini-gels (Bio-Rad. Hercules, CA) in Tris glycine buffer. The gels were stained with 0.1% Coomassie blue in acetic acid, 50% methanol, and 40%. water for 3 h with continuous shaking.

Gel slices containing Ara h II were sent to the W.M. Keck Foundation (Biotechnology Resource Laboratory, Yale University, New Haven CT) for amino acid sequencing. Amino acid sequencing of intact Ara h II and tryptic peptides of this protein was performed on an Applied Biosystems sequencer with an on-line HPLC column that was eluted with increasing concentrations of acetonitrile.

Peanut RNA isolation and northern (RNA) gels. Three commercial lots from the 1979 crop of medium grade peanut species, Arachis hypogaea (Florunner) were obtained from North Carolina State University for this study. Total RNA was isolated from one gram of this material according to procedures described by Larsen. Poly A+ RNA was isolated using a purification kit supplied by collaborative Research (Bedford MA) according Sto manufacturer's instructions. Poly A+ RNA was subjected to electrophoresis in 1.2% formaldehyde agarose gels, transferred to .9 nitrocellulose, and hybridized with 3 P-labeled probes according to the methods of Bannon et al.

IComputer analysis of Ara h II sequence. Sequence analysis of the Ara h II gene was done on the University of Arkansas for Medical Science's Vax .computer using the.Wisconsin DNA analysis software package. The predicted Ara h II epitopes are based on a algorithms developed by Jameson and Wolf (1988) that relates antigenicity to hydophilicity, secondary structure, flexibility, and surface probability.

cDNA expression library construction and screening. Peanut poly A+ RNA was used to synthesize double-stranded cDNA according to the methods of Watson and Jackson and Huynh et al. The cDNA was treated with EcoRI methylase and then ligated with EcoRI and Xhol linkers. The DNA was then ligated with EcoRI-XhoI cut, phosphatase treated Lambda ZAP XR phage arms (Stratagene, Lajolla, CA) and in vitro packaged. The library was recombinants carrying insert sizes >400 bp. The library was screened using an IgE antibody pool consisting of an equal volume of serum from each patient with peanut hypersensitivity. Detection of primary antibody was with 2 I-labeled anti-IgE antibody performed according to the manufacturer's instructions (Sanofi, Chaska, MN).

PCR amplification of the Ara h II mRNA sequence. Using the oligonucleotide CA(AG)CA(AG)TGGGA(AG)TT(AG)CA(AG)GG(N)GA(TC)AG derived from amino acid sequence analysis of the Ara h II peanut allergen as one primer and a 23 nucleotide long primer which hybridizes to the Bluescript vector, the cDNA that encodes Ara h II was amplified from the IgE positive clones. Reactions were carried out in a buffer containing 3 mM MgC1, 500 mM KC1, 100 mM Tris-HC1, pH 9.0. Each cycle of the polymerase chain reaction consisted of 1 min at 94°C, followed by 2 min at 42 0 C, and three minutes at 72 0 C. Thirty cycles were performed with both primers 15 present in all cycles. From this reaction, a clone carrying an approximately 700 bp insert was identified.

DNA sequencing and analysis. DNA Sequencing was done according to the methods of Sanger et al. using either 32 P-hnd labeled oligonucleotide primers or on a automated ABI model 377 DNA sequencer using fluorescent tagged nucleotides. Most areas of the clone were sequenced at least twice and in some cases in both directions to ensure an accurate nucleotide sequence for the Ara h II gene.

Peptide synthesis. Individual peptides were synthesized on a derivatised cellulose membrane using Fmoc amino acid active esters according to the manufacturer's instructions (Genosys Biotechnologies, Woodlands, TX). Fmoc-amino acid derivatives were dissolved in l-methyl-2pirrolidone and loaded on marked spots on the membrane. Coupling reactions were followed by acetylation with a solution of 4% acetic anhydride in N,N-Dimethylformamide (DMF). After acetylation, Fmoc groups were removed by incubation of the membrane in 20% piperdine in DMF. The membrane was then stained with bromophenol blue to identify the location of the free amino groups. Cycles of coupling, blocking, and deprotection were repeated until the peptides of the desired length were synthesized. After addition of the last amino acid in the peptide, the amino acid side chains were deprotected using a solution containing a 1/1/0.5 mixture of dichloromethane/ trifluoroacetic acid/triisobutlysilane.

Membranes were either probed immediately or stored at -20 0 C until needed.

IgE binding assay. Cellulose membranes containing synthesized peptides were washed with Tris-buffered saline (TBS) and then incubated with blocking solution overnight at room temperature. After blocking, the membranes were incubated with serum from patients with peanut hypersensitivity diluted in a solution containing TBS and 1% bovine serum albumin for at least 12 h at 4°C or 2 h at room temperature. Detection of the primary antibody was with 2 I-labeled anti-IgE antibody (Sanofi, Chaska, MN).

RESULTS

Isolation and partial amino acid sequence determination of the Ara h IIprotein. The amino terminus of the purified Ara h II protein, or peptides resulting from trypsin digestion of this protein, were used for amino acid sequence determination. It was possible to determine the first 17 residues from peptide I and the first 13 residues from peptide II of the major peptide in each fraction. The amino acid sequence representing the amino terminus of the Ara h II protein (peptide I) and a tryptic peptide fragment (peptide II) is noted in Table 9. These results confirm and extend previous amino acid sequence analysis of the Ara h II protein.

Identification and characterization of clones that encode peanut allergen Ara h II. RNA isolated from the peanut species, Arachis hypogaea (Florunner) was used to construct an expression library for screening with serum IgE from patients with peanut hypersensitivity. Numerous IgE binding clones were isolated from this library after screening 106 clones with serum IgE from a pool of patients with reactivity to most peanut allergens by Western blot analysis. Since the number of plaques reacting with serum IgE was too large to study all in detail we randomly selected a small portion of the positive clones for further analysis.

To identify which of the clones encoded the Ara h II allergen, a hybridization probe was constructed using an oligonucleotide developed from Ara h II amino acid sequence and PCR technology. The oligonucleotide sequence CA(AG)CA(AG)TGGGA (AG)TT(AG)CA(AG)GG(N)GA(TC)AG was derived from the amino terminus of the Ara h II peanut allergen (peptide I).

15 Utilizing this oligonucleotide, an -700-bp cDNA clone was identified. DNA sequence revealed that the selected clone carried a 741-base insert which included a 21-base poly A tail and a 240 base 3' non-coding region. This insert contained a large open reading frame starting with an ACG codon and ending with a TAA stop codon at nucleotide position 480 (Fig The 20 calculated size of the protein encoded by this open reading frame was -17.5 kD, which is in good agreement with the molecular weight ofAra h II that has been determined experimentally. The amino acid sequence that was determined from the amino terminus.and a tryptic peptide from purified Ara ~h II (Table 9) were found in this clone. The additional, coding region on the amino terminal end of this clone probably represents a signal peptide which would be cleaved from the mature Ara h II allergen.

To determine what size mRNA this clone identified, 3 P-labeled insert was used as a hybridization probe of a Northern blot containing peanut poly(A)+ RNA. This insert hybridized to an -0.7-kb mRNA. Since the size of the cloned insert and the size of the mRNA were in good agreement, coupled with the good agreement in both the calculated and determined size of the Ara h II protein and the identity of the determined amino acid sequence with that which was determined from the clone, we concluded that an Ara h II specific clone had been isolated.

Peanut allergen Ara h II is a conglutin-like seed storage protein. A search of the GenBank database revealed significant amino acid sequence homology between the Ara h II protein and a class of seed storage proteins called conglutins. There was -32% identity with the Ara h II protein and a delta conglutin from the lupin seed. These results indicate that the Ara h II allergen belongs to a conglutin-like family of seed storage proteins.

Multiple IgE binding epitopes on the Ara h II protein. The Ara h II protein sequence was analyzed for potential antigenic epitopes by algorithms designed to determine which portion(s) of this protein could be responsible for antibody binding. There were four possible antigenic regions predicted by this analysis along the entire length of the molecule (Fig. 7).

Nineteen overlapping peptides representing the derived amino acid sequence of the Ara h II protein were synthesized to determine if the predicted antigenic regions, or any other regions, were recognized by serum IgE. Each peptide was 15 amino acids long and was offset from the previous peptide by eight amino acids. In this manner, the entire length of the Ara h II protein could be studied in large overlapping fragments. These peptides 'were then probed with a pool of serum from 12 patients with documented peanut hypersensitivity or serum from a control patient with no peanut 20 hypersensitivity. Serum IgE from the control patient did not recognize any of-the synthesized peptides (data not shown). In contrast, Figure 8 shows that there are five IgE binding regions along the entire length of the Ara h II protein that are recognized by this population of patients with peanut "hypersensitivity. These IgE binding regions were amino acid residues 17-38, 41-62, 57-78, 113-134, and 129-154..

In order to determine the exact amino acid sequence of the IgE binding epitopes, small peptides (10 amino acids long offset by two amino acids) representing the larger IgE binding regions were synthesized. In this manner it was possible to identify individual IgE binding epitopes within the larger IgE binding regions of the Ara h II molecule (Fig. The ten IgE binding epitopes that were identified in this manner are shown in Table 10. The size of the epitopes ranged from 6-10 amino acids in length. Three epitopes (aa17-26, aa23-32, aa29-38), which partially overlapped with each other, were found in the region of amino acid residues 17-38. Two epitopes (aa41-50, aa51-60) were found in region 41-62. Two epitopes (aa59-68, aa67-76) were also found in region 57-78. Finally, three epitopes (aa117-126, aa129-138, aa145-154) were found in the overlapping regions represented by amino acid residues 113-134 and 129-154. Sixty-three percent of the amino acids represented in the epitopes were either polar or apolar unchanged residues.

There was no obvious amino acid sequence motif that was shared by all the epitopes, with the exception that epitopes 6 and 7, which contained the sequence DPYSPS.

In an effort to determine which, if any, of the ten epitopes were recognized by the majority of patients with peanut hypersensitivity each set of ten peptides was probed individually with serum IgE from 10 different patients. Five patients were randomly selected from the pool of 12 patients used to identify the common epitopes and five patients were selected from outside this pool. An immunoblot strip containing these peptides was incubated with an individual's patient serum. The remaining patients were tested in the same manner and the intensity of IgE binding to each spot was determined as a function of that 10 patient's total IgE binding to these ten Sepitopes (Fig. 10). All of the patient sera tested (10/10) recognized multiple epitopes (Table 11). The average number of epitopes recognized was about 4- 20 5/patient ranging from two sera recognizing only 3 epitopes and one patients' sera recognizing as many as 7 epitopes. Interestingly, epitopes 3, 6, and 7 were recognized by all patients tested (10/10). No other epitope was recognized by more than 50% of the patients tested.

DISCUSSION

Peanuts are one of the most common food allergens in both children and adults. In addition, peanut hypersensitivity is less likely to resolve spontaneously and more likely to result in fatal anaphylaxis. Because of the significance of the allergic reaction and the widening use of peanuts as protein extenders in processed food, the risk to the peanut-sensitive individual is increasing.

Various studies over the last several years have examined the nature and location of the multiple allergens in peanuts. Taylor et al. demonstrated that the allergenic portion of peanuts was in the protein portion of the cotyledon. Our laboratory recently identified two major allergens from peanut extracts, designated Ara h I and Ara h II. Greater than 90% of our patients who were challenge positive to peanut had specific IgE to these proteins. The Ara h I allergen has been identified as a seed storage protein with significant homology to the vicilins, a family of proteins commonly found in many higher plants. The Ara h II nucleotide sequence identified in this report has significant sequence homology with another class of seed storage proteins called conglutins. It is interesting to note that two of the major peanut allergens thus far identified are seed storage proteins that have significant sequence homology with proteins in other plants. This may explain the cross-reacting antibodies to other legumes that are found in the sera of patients that manifest clinical symptoms to only one member of the legume family.

In the present study we have determined that there were multiple antigenic sites predicted for the Ara h II allergen. As has been found for 15 another peanut allergen Ara h I, and other allergens in general, there was good agreement between those residues predicted by computer analysis and S" B-cell epitopes determined by experimental analysis of overlapping peptides.

This strong correlation between predicted and determined epitopes is probably due to the ability of the computer model to predict which regions of 20 the molecule are accessible to immunoglobulin interactions. In fact, 3-D *structural models of the Ara h I protein indicate that most of the peptides identified by computer modeling and experimental analysis as IgE binding epitopes are located on the surface of the molecule (unpublished observation).

There are at least 10 IgE recognition sites distributed throughout the major peanut allergen Ara h II. The identification of multiple epitopes on a single allergen is not novel, there being reports of multiple IgE binding epitopes on allergens from many foods that cause immediate hypersensitivity reactions. The observation that most of these proteins have multiple IgE binding sites probably reflects the polyclonal nature of the immune response to them and may be a necessary step in establishing a protein as an allergen.

Recent evidence suggests that there is a preferential variable heavy chain usage in IgE synthesis and a direct switching from IgM production to IgE synthesis. This would suggest that epitopes responsible for antigenspecific IgE antibody production may differ from those promoting antigenspecific IgG antibodies and that there may be some structural similarity between peptides that elicit IgE antibody production. However, there was no obvious sequence motif that was shared by the 23 different IgE binding epitopes of the peanut allergen Ara h I. In the present study, two epitopes share a hexameric peptide (DPYSPS). It is significant to note that these peptides are recognized by serum IgE from all the peanut-hypersensitive patients tested in this study. In addition, serum IgE that recognize these peptides represent the majority ofAra h II specific IgE found in these patients. Whether there is any further structural similarity between the IgE binding epitopes of Ara h II remains to be determined.

The elucidation of the major IgE binding epitopes on Ara h II may enable us to design better therapeutic options for the prevention of anaphylaxis as a result of peanut hypersensitivity. The only therapeutic 15 option presently available for the prevention of a food hypersensitivity reaction is food avoidance. Unfortunately, for a ubiquitous food such as peanut, the possibility of an inadvertent ingestion is great. One therapeutic option used extensively for patients with allergic reactions to various aeroallergens and insect sting venoms is allergen desensitization I 20 immunotherapy. Allergen immunotherapy consists of injections of increasing amounts of allergens to which a patient has Type I immediate hypersensitivity. While the absolute mechanism of immunotherapy is unknown, an increase in IgG or IgG 4 antibody activity, a decrease in allergenspecific IgE levels, and a decrease in basophil activity have all been implicated in mediating this response. Because allergen immunotherapy has been proven efficacious for treatment of some allergies, treatment with peanut immunotherapy is now being studied as a possible option. Our work showing the IgE binding epitopes of a major peanut allergen may allow for the use of immunodominant epitopes in this approach.

Another potential immunotherapeutic approach that has recently attracted much attention is the use of DNA vaccines. In this approach a promoter region is placed 5' to the cDNA encoding the allergen and then introduced to the whole animal via intramuscular injection or intradermal application. Early work with a dust mite allergen, Derp 1, indicates that this approach can both prevent the development of an immunogenic response to a specific protein and dampen the response to a protein to which the animal has already been sensitized. We are currently exploring this possibility with the Ara h II allergen.

FIGURE LEGENDS FIGURE 6. Nucleotide Sequence of an Ara h II cDNA Clone. The nucleotide sequence is shown on the first line. The second line is the derived amino acid sequence. Bold amino acid residues are those areas which correspond to the determined amino acid sequence of peptide I and II ofAra h II (Table The numbers on the right of the figure indicate the nucleotide sequence.

An Ara h II Clone Hybridizes to a 700 bp Peanut mRNA. Peanut poly A+ RNA was isolated from Arachis hypogaea (Florunner) species and 10 pg were electrophoresed on formaldehyde denaturing agarose gels. Insert from an Ara h II clone was purified, labeled with alpha- 2 P-dCTP, and used as a 15 hybridization probe for Northern blot analysis of this gel. Sizes of known RNA species are expressed in kilobases along the right side of the figure.

FIGURE 7. Multiple Predicted Antigenic Sites are Present in the Ara h II Allergen. The amino acid sequence of the Ara h II protein was analyzed for potential antigenic epitopes by the Jameson and Wolf (1988) algorithm.

20 These predictions are based on a model that relates antigenicity to hydrophilicity, secondary structure, flexibility, and surface probability.

There were 4 predicted regions that contained multiple antigenic sites (octagons) along the entire length of the molecule. Amino acid residues (small numbers) are represented as alpha-helical (sinusoidal curve), Betasheet (saw tooth curve), and coil (flat sinusoidal curve) conformations. Beta turns are denoted by chain reversals.

FIGURE 8. Multiple IgE Binding Sites Identified in the Ara h II Allergen.

Epitope analysis was performed on the Ara h II allergen by synthesizing amino acid long peptides, offset from each other by 8 amino acids for the entire protein molecule. These peptides, represented as spots 1-19, were then probed with a serum pool consisting of 15 patients with documented peanut hypersensitivity.

43 FIGURE 9. Core IgE Binding Epitopes Identified on the Ara h II Allergen.

Epitope analysis was performed on the IgE binding sites identified in Fig. 8 by synthesizing 10 amino acid long peptides offset by two amino acids.

These peptides were then probed with the 18 patient serum pool. Figure 9 is the peptide analysis ofAra h II amino acid residues 49-70. Figure 9 identifies the amino acid sequence of this region.

FIGURE 10. Of the 10 patients five were selected at random from the 18 patient serum pool and five were patients with peanut hypersensitivity that were not included in the pool. Patient K represents a non-peanut sensitive (negative) control.

o *6 44 Characterization of a Major Peanut Allergen: Mutational Analysis of the Ara h I IgE binding epitopes Immediate hypersensitivity reactions to foods occur in about 6-8% of young children and 1% of adults. These reactions are mediated by the production of IgE antibodies to glycoproteins found in the food. Peanuts are a major cause of serious allergic reactions in both adults and children. Ara h I, a major peanut allergen, has been extensively characterized and shown to contain 23 linear IgE binding epitopes. We set out to determine the amino acids critical to their binding and to determine the location of these epitopes on the 3-D structure of the Ara h I molecule. To accomplish this, mutational analysis of each epitope was performed by synthesizing 10 amino acid long peptides with single amino acids changed at each position to alanine, followed by determination of the IgE binding capacity of each mutated epitope relative to wild-type. It was determined that changes in those amino 15 acids located at positions, 4, 5, and 6 of the epitope have a greater influence than residues located on either end. In addition, the substitution of most apolar, charged residues resulted in the loss of IgE. More importantly, 21/23 epitopes could be mutated to non-IgE binding by a single amino acid substitution. The 3-D model of the Ara h I protein indicates that the majority 20 of the IgE binding epitopes are located on the surface of the molecule.

"Currently, we are determining what effect the amino acid substitutions that lead to loss of IgE binding will have on the tertiary structure of the protein.

e TABLE 9 AMINO ACID SEQUENCE OF Ara h HI PEPTIDES Peptide Amino Acid Seqiuence I X-Q-Q-W-E-L-Q-G-D-R-R-R-Q-S-Q-L-E-R 11 A-N-L-R-P-C-E-O-H-L-M-O-K The amino acid sequence of the amino terminus and a tryptic peptide (II) derived from Ara h IH protein was determined. The sequence is shown as the one letter amino acid code.

TABLE 10 Ara h H ISE BINDING EPITOPES PEPTIDE AA SEQUENCE Ara h HI POSITION 1 HASARQQWEL 17-26 2 QWELQGDRRC 23-32 3 DRRCQSQLER 29-38 4 LRPCEQHLMQ 41-50 5 2 KIQRDEDSY-Ej 51-60 6 YERDPYSPSQ 59-68 7 SQDPYSPSPY 6 7-76 8 DRLQGRQQEQ 117-126 9 KRELRNLPQQ 129-138 QRCDLDVESG 145-154 46 TABLE 11 IgE BINDING TO Ara h II EPITOPES Epitopes/ 1 2 3 4 5 6 7 8 9 10 Pt A X X X X 4 B X X X X X C X X X X X D X X X X 4 E X X X X 4 F X X X X X X X 7 G X X X 3 H X X X X X X 6 I X X X 3 I X X X X X Pts/ 4 1 10 4 1 10 10 4 1 1 Epitope 5 Patients are indicated by letters on the left hand side of the table. Ara h II peptides are indicated by number (1-10) across the top of the table. The number of epitopes recognized by each patient (epitopes/patient) is shown on the right hand side of the table. The number of patients that recognized each epitope is shown across the bottom of the table. An X indicates that a peptide bound IgE.

a 4 a Cloning, epitope mapping, and mutational analysis of Ara h II, a major peanut allergen A major peanut allergen, Ara h II, is recognized by serum IgE from of patients with peanut hypersensitivity. Biochemical characterization of this allergen indicates that it is a glycoprotein of -17.5 kDa. Using N-terminal amino acid sequence data from purified Ara h II, oligonucleotide primers were synthesized and used to identify a clone (700 bp) from a peanut cDNA library.

This clone was capable of encoding a 17.5 kDa protein with homology to the conglutin family of seed storage proteins. The major linear IgE binding epitopes of this allergen were mapped using overlapping peptides synthesized on an activated cellulose membrane and pooled serum IgE from 15 peanut sensitive patients. Ten IgE binding epitopes were identified, distributed throughout the length of the Ara h II protein. The size of the epitopes ranged 15 from 6-10 amino acids in length. Sixty-three percent of the amino acids represented in the epitopes were either polar or apolar unchanged residues. In an effort to determine which, if any, of the ten epitopes were recognized by the majority of patients with peanut hypersensitivity, each set of ten peptides was probed individually with serum IgE from 10 different patients. All of the 20 patient sera tested recognized multiple epitopes. Three epitopes (aa29-38, aa59-68, and 67-76) were recognized by all patients tested. Mutational analysis of these immunodominant epitopes indicate that single amino acid changes result in loss of IgE binding. Both epitopes contained in region aa59- 76 contained the amino acid sequence DPYSPS that appears to be necessary 25 for IgE binding. These results may allow for the design of improved diagnostic and therapeutic approaches to peanut hypersensitivity.

and therapeutic approaches to peanut hypersensitivity.

Ara h 3, a peanut allergen identified by using peanut sensitive patient sera adsorbed with soy proteins Peanuts and soybeans are members of the legume family and share several common antigenic fractions. Patients allergic to one of these foods have serum IgE antibodies which immunologically cross-react with other legumes. However, ingestion of other legumes generally does not induce an allergic reaction, suggesting that cross-reacting antibodies to soy were removed from the sera of patients clinically allergic to peanuts. Adsorbed sera were then used to identify specific IgE binding to peanut immunoblots. Several peanut proteins ranging in size from 5 kDa to 49 kDa, were identified. A -14 kDa protein identified in this manner was purified and prepared for amino acid sequence analysis. Amino terminal sequencing determined the first 23 amino acids of this protein. A search of the Genbank protein database with this peptide revealed that it had 61% identity with a soybean gene for glycinin subunit G3. A degenerate oligonucleotide primer was then designed from this data to use in conjunction with vector primers to amplify the clones that encode this protein from a peanut cDNA library. DNA sequencing of these clones also revealed -70% homology with the soybean gene for glycinin subunit G3. These data indicate that while there is significant homology 20 between the peanut and soybean glycinins 177 there must be peanut-specific epitopes responsible for the binding of the soy-adsorbed serum IgE.

Subsequent characterization of this allergen will include determination of the IgE binding epitopes and testing of the clinical relevance of this protein in :peanut hypersensitivity. If this strategy is successful it will not only identify proteins that bind IgE but also those allergens and -epitopes important in the disease process.

MAPPING OF THE B-CELL EPITOPES ON Ara h I AND Ara h II LEGUME STORAGE PROTEINS AND MAJOR ALLERGENS INVOLVED IN PEANUT HYPERSENSITIVITY Approximately 8% of children and 1-2% of adults suffer from some form of food allergy. Reactions to peanuts are more likely than other food allergies to give rise to fatal or near fatal anaphylaxis in sensitized patients.

Ara h I (MW=63.5 kD) and Ara h II (MW=17 kD) are peanut proteins recognized by serum IgE from 90% of peanut sensitive patients, thus establishing them as clinically important allergens. Overlapping peptides representing the entire Ara h I and Ara h II molecules were constructed and IgE immunoblot analysis performed to determine which portions of these allergens were responsible for IgE binding. Utilizing a pool (n=15) of patients with peanut hypersensitivity, 23 IgE binding epitopes were identified on Ara h 609. 6I and 6 epitopes were identified on Ara h II. Even though there were multiple 15 epitopes identified on each allergen, two epitopes on Ara h I and one epitope on Ara h II were recognized by 90% of individual patient sera tested The amino acids important for IgE binding in these immunodominant epitopes were determined by mutational analysis. The identification of the major Ara h I and Ara h II IgE binding epitopes may lead to improved diagnosis of peanut 20 hypersensitivity and eventually to an improved therapeutic regimen for this disease. SUPPORTED IN PART BY THE NATIONAL INSTITUTE OF HEALTH, CLARISSA SOSIN ALLERGY RESEARCH FOUNDATION, AND ARKANSAS SCIENCE AND TECHNOLOGY AUTHORITY.

INTRODUCTION

25 Approximately 1-2% of the USA population suffers from some for of food allergy. Peanuts, fish, tree nuts, and shell fish account for the majority of food hypersensitivity reactions in adults; while peanuts, milk, and eggs cause over of food hypersensitivity reactions in children. Unlike the food hypersensitivity reactions to milk and eggs, peanut hypersensitivity reactions usually persist into adulthood and last for a lifetime. In addition, hypersensitivity reactions to peanuts tend to be more severe than those to other food allergens, sometimes resulting in death. Several reports have detailed the fatal and near-fatal anaphylactic reactions occurring in adolescents and adults. Currently, avoidance is the only effective means of dealing with food allergy, but the use of peanuts and peanut by-products as supplements in many different foods makes accidental consumption almost inevitable.

Two major allergens involved in peanut hypersensitivity are the peanut proteins, Ara h I and Ara h II. These proteins are recognized by 90% of peanut positive patients, thus establishing them as clinically important allergens.

Both proteins are seed storage proteins. Ara h I shares significant sequence homology with vicilin proteins from other plants while Ara h II is a conglutin like protein.

Food hypersensitivity reactions occur shortly after contact of a specific allergen with its corresponding IgE antibodies which are bound to mast cells.

IgE, when complexed with antigen, will activate mast cells to release histamine, heparin, and other substances which are responsible for the clinical symptoms observed. Thus the IgE binding epitopes of the allergens play an 15 important role in the disease process and their elucidation will lead to a better understanding of the human immune response involved in food hypersensitivity reactions and to improved diagnostic, and therapeutic capabilities.

Figures 1 and 7. Multiple Predicted Antigenic Sites 20 in the Ara h I and Ara h II Allergens The amino acid sequences of the Ara h I and Ara h II proteins were S. analyzed for potential antigenic epitopes.: These predictions are based on a model that relates antigenicity to hydrophilicity, secondary structure, flexibility, and surface probability. There were 11 (1-11) predicted regions "25 that contained multiple antigenic sites (octagons) along the entire length of the Ara h I protein and 4 predicted regions on the Ara h II protein. Amino acid residues (small numbers) are represented as alpha-helical (sinusoidal curve), Beta sheet (saw tooth curve), and coil (flat sinusoidal curve) conformations. Beta turns are denoted by chain reversals.

Figures 2 and 8. Multiple IgE Binding Regions Identified in the Ara h I and Ara h II Allergens Upper Panels: Epitope mapping was performed on the Ara h I and Ara h II allergens by synthesizing each of these proteins in 15 amino acid long overlapping peptides that were offset from each other by 8 amino acids. These peptides were then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity. The position of the peptides within the Ara h I and Ara h II proteins are indicated on the left hand side of each panel.

Lower Panels: The amino acid sequences of the Ara h I and Ara h II proteins are shown in the lower panels. The numbered boxes correspond to the predicted antigenic regions (P1-P11; P1-P4). The hatched boxes (D1-D12; D1- 4) correspond to the IgE binding regions shown in the upper panels.

Figures 3 and 9. Core IgE Binding Epitopes Identified in the Ara h I and Ara h II Allergens Detailed epitope mapping was performed on IgE binding regions 15 identified in Fig. 2 and 8 by synthesizing 10 amino acid long peptides offset from each other by two amino acids. These peptides were then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity.

The data shown represents regions D2 and a portion of D3 from Ara h I and region D2 from Ara h II. Numbers correspond to peptides as shown in Table 20 12. The amino acid sequences of Ara h I and Ara h II that were tested in the upper panels are shown. Shaded areas of boxes correspond to IgE binding peptide.

Figures 4 and Commonly Recognized Ara h I Epitopes 25 (e Core IgE binding epitopes were synthesized (10 amino acids long) and then probed individually with serum IgE from 10 patients with documented peanut hypersensitivity. The top panels represent where each of the Ara h I peptides (1-23) and Ara h II peptides were placed on the membrane.

Panels A-J show the peptides that bound serum IgE from each patient. The control panels were probed with sera from a patient with elevated IgE but who does not have peanut hypersensitivity.

Figures 5 and 11.

Amino Acids Involved in IgE Binding 52 Epitopes 4 and 17 from Ara h I and epitope 3 from Ara h II were synthesized with a glycine or alanine residue substituted for one of the amino acids in each of these peptides and then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity. The letters across the top of each panel indicate the one letter amino acid code for the residue normally at that position and the amino acid that was substituted for it. The numbers indicate the position of each residue in the Ara h I and Ara h II proteins.

.e The major peanut allergens Ara h I and Ara h II have been cloned, sequenced, and identified as seed storage proteins.

B-cell epitopes of Ara h I and 6 B-cell epitopes of Ara h II were mapped using synthetic peptides probed with serum IgE from a population of peanut hypersensitive patients.

Epitope #4 (AA89-98) and #17 (AA498-507) of Ara h I and epitope #3 (AA59-66) of Ara h II were recognized by 90% of peanut hypersensitive patients tested.

Amino acids important to IgE binding of the immunodominant epitopes of Ara h I and Ara h II were determined.

Figure 12 highlights peptides I, II, and III of the Ara h I protein.

Table 16 is a partial Ara h I Beta sequence (clone Table 17 is an Ara h I Alpha sequence (clone p17).

15 Table 18 is the Ara h II sequence (clone Ara h II p38).

Table 19 is the Ara h I Beta sequence (clone p41b).

Table 20 is the Ara h II p38 translation of Ara h II p38.

In accordance with the present invention, it is contemplated that the discovery or identification of particular peptides or epitopes which bind IgE 20 and cause an IgE response by a person having an allergy or sensitivity to that particular protein may be used to produce a DNA vaccine for immunization therapy to hopefully reduce the IgE response and thereby eliminate or reduce the negative effects of the allergy or sensitivity. For example, a protein, peptide, or epitope can be produced and injected into a patient as a DNA 25 vaccine which hopefully will have an immuno modulation effect of the IgE response and stimulate a different response, such as IgG, IgM, IgA, etc. and thereby down regulate IgE senthesis to the specific allergen.

Also in accordance with the present invention, similar peptides, epitopes and IgE binding proteins from other legumes, herbs, oil, seeds, and the like, for example soybeans or wheat, can be isolated and identified, mutated so that they do not bind IgE, and used in a mutated DNA vaccine for immunization therapy.

53A In accordance with the present invention, it is contemplated that the discovery or identification of particular peptides or epitopes which bind IgE and cause an IgE response by a person having an allergy or sensitivity to that particular protein may be used to produce a DNA vaccine for immunization therapy to hopefully reduce the IgE response and thereby eliminate or reduce the negative effects of the allergy or sensitivity. For example, a protein, peptide, or epitope can be produced and injected into a patient as a DNA vaccine which hopefully will have an immuno modulation effect of the IgE response and stimulate a different response, such as IgG, IgM, IgA, etc. and thereby down regulate IgE senthesis to the specific allergen.

Also in accordance with the present invention, similar peptides, epitopes and IgE binding proteins from other legumes, herbs, oil seeds, and the like, for example soybeans or wheat, can be isolated and identified, mutated so that they do not bind IgE, and used in a mutated DNA vaccine for immunization therapy.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a •o0• context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

*oo -11i.

Table 12 Ara h I IgE Binding Epitopes PEPTIDE AA SEQUENCE* Ara h I POSITION I AKSSPYQKKT 25-34 2 OEPDDLKOA 48-5 7 3 LEYDPRLVYD 65-74 4 GERTRGROPG 89-98 PGDYDDDRRQ 97-105 6 PRREEGGRWG 107-116 7 REREEDWROP 123-132 8 EDWRRPSHOO 134-143 9 OPRKIRPEGR 143-152 TPGOFEDFFP 294-303 11 SJLOEFSRNT 311-32.0 12 FNAEFNEIRR 325-334 13 EOEERGORRW 344-353 14 DITNPINLRE 393-402 NNFGKLFETK 409-418 16 GTGNLELVAV 461-470 17 RRYTARLKEG 498-507 18 ELHLLGFGIN 525-534 19 HRIFLAGDKD 539-548 IDOIEKOAKD 551-560 21 KDFLAFPGSGE 559-568 22 KESHFVSARP 5 78-587 23 PEKESPEKED 597-6Q6 Ar-a h I IgE Binding Epitopes PEPTIDE AASEQUENCE* Ara h II POSITION 1 LLAAHASARQ 14-23 2 QGDRRCOSQL 27-36 3 YERDPYSPSQ 60-69 4 AGSS0140ERC 8 1-90 CNELNEFENN 91-100 6 ORCDLDVESG 105-159 *The underlined portions of each peptide 6fe the smallest IgE binding sequences as determined by the analysis as described in FIG. 9 a a a 9 99 .9 9 9 9 9 9 9 9* C 9 *9 TABLE 13 Ara h I Epitopes IgE binding of core Ar h I epitopes by serum from peanut hypersensitive individuals.

Epitopes/ Patients 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Patient A X X X X X X 6 B X X X X X X 6 C X X X X X X X X X X X X 12 D X X X X X X 6 E X X X X X X X X X X X 11 F X X X X 4 G X X X X X X 6 H X X X 3 I X X X X X X X X 8 J X X X X X X X 7 Patients/ Epitope 4 5 4 9 4 1 0 3 4 2 1 3 3 1 3 10 2 1 4 1 3 1 Ara h H Epitopes IgE binding of core Ara h II epitopes by serum from peanut hypersensitive individuals Patients 1 2 3 4 5 Epitopes/ Patient 1 2 2 2 3 1

J

Patients/ Epitopes Patients are indicated by letters on the left hand side of the table. Am h I II peptides are indicated by number across the top of the table. The number of epitopes recognized by each patient (epitopes/patient) is shown on the right hand side of the table. The number of patients that recognized each epitope (patients/epitope) is shown across the bottom of the table. An X indicates that a peptide bound IgE.

56 Table 14 IDENTIFICATION OF NATIVE AMINO ACID SEQUENCES IN THE DEDUCED AMINO ACID SEQUENCE OF CLONE ARA H II P38 THE FOLLOWING AMINO ACID SEQUENCE WAS TRANSLATED FROM THE ARA H II P39 GENE (NUCLEOTIDE SEQUENCE) ISOLATED FROM OUR cDNA

LIBRARY.

TRANSIATION of GENE: arah1Iv38 1 LTTLVALALF pe 51 QRIJEDSYERD 101 CMCEALQQIM N-terminal sequence LLAAHASARQ QWELQGDRRC ptide 37 PYSPSQDPYS PSPYDRRGAG

QSQLERANLR

peptide

SSQHQERGCN

RNLPQQCGLR

PCEQHLMQII

ELNEFENNQR

APQRCDLDVE

peptide QQEQQFKREL ENQSDRLQGR 151 SGGRDRY Table The following information was obtained by physicochemical measures and used to confirm the deduced amino acid sequence from clone Ara h II p38.

17.5 kD N-TERMINAL SEQUENCE: gene sequence 19 48 1 2 3 4 5 6 7 8 9 GLY GLN GLN TRP GLU LEU GLN GLY ASP ARG Q Q W E L Q G D R 14 15 16 17 18 19 20 21 22 23 SER GLN LEU GLU ARG ALA ASN LEU X PRO S Q L E R A N L R P 26 27 28 29 GLN X LEU MET X Q H K M PEPTIDE 20: Identified in gene sequence 121---128: 1 2 3 4 5 6 7 8 GLN GLN GLU GLN GLN PHE LYS ARG Q Q E Q Q F K R PEPTIDE 37: identified in gene sequence 60-76: 1 2 3 4 5 6 7 8 9 10 11 12 13 ASP PRO TYR SER PRO SER GLN ASP PRO TYR SER PRO SER P Y S P S Q "D P- Y S P S 14 15 16 17 PRO TYR ASP ARG P Y D R PEPTIDE 45: identified in gene sequence 37-49: 1 2 3 4 5 6 7 8 9 10 11 12 13 ALA ASN LEU ARG PRO CYS GLU GLN HIS LEU MET GLN LYS A N L R P C E Q H L M Q K 58 TABLE 16

LOCUS

DEFINITION

ACCESSION

KEYWORDS

SOURCE

ORGAN ISM

REFERENCE

AUTHORS

TITLE

JOURNAL

STANDARD

COMMENT

FEATURES

sourcf 25 kRQARAHI 1340 bp ss-rnRNA P krachis hypogea (clone 5Ala) Ara h I mRNA, :omplete cds.

,34402 Arachis hypogea (strain Florunner) seed cDNA to mRNA.

Arachis hypogea Eukaryota; Plantae; Exbryobionta; Magnoliophyta; Magnoliopsida; Rosidae; Fabales; Fabaceae 1 (bases 1 to 1340).

Burks,A.W., Cockrell,G., Stanley,J.S., Helm,R.M. and Bannon, G.A Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitinity Unpublished (1994) full autoftc-tic NCBI gi: 508640 Location/Qualifiers 1. .1340 /or.ganism="Arachis hypogea" /strain="Florunner" /dev stage="seed" /sequenced-mol="cDNA to mRNA" 231. .1238 /gene="Ara h I" /note="NCBI gi: 508641" /codon start=l /trans lation="MPVNTPGQFEDFFPASSRDQSSYLQGFSRNTLEAAFNAEFNEIRRVLLEENA

GGEQEERGQRRWSTRSSENNEGVIVKVSKEHVEELTKHAKSVSKKGSEEEGDITNPINLREGEPDL

SNNFGKLFEVKPDKKNPQLQDLDMMLTCVEI KEGALMLPHFNSKAMVIVVVNKGTGNLELVAVRKE

QQQRGRREEEEDEDEEEEGSNREVRRYTARLKEGDVFIMPAAHPVAINASSELHLLGFGINAENNH

RI FLAGDKDNVIDQIEKQAKDLAFPGSGEQVEKLIKNQKESHFVSAQSQSQSPSSPEKESPEKEDQ EEENQGGKGPLLSILKAFN" f BASE COUNT

ORIGIN

422 a 296 c 340 g 282 t Arqarahi Length: 1340 05:04 Type: N Check: 8329 1 GTATTGTGCA GATCGAGGCC AAACCTAACA CTCTTGTTCT TCCCAAGCAC 51 GCTGATGCTG ATAACATCCT TGTTATCCAG CAAGGGCAAG CCACCGTGAC 101 CGTAGCAAAT GGCAATAACA GAAAGAGCTT TAATCTTGAC GAGGGCCATG 151 CACTCAGAAT CCCATCCGGT TTCATTTCCT ACATCTTGAA CCGCCATGAC 201 AACCAGAACC TCAGAGTAGC TAAAATCTCC ATGCCCGTTA ACACACCCGG 251 CCAGTTTGAG GATTTCTTCC CGGCGAGCAG CCGAGACCAA TCATCCTACT 301 TGCAGGGCTT CAGCAGGAAT ACGTTGGAGG CCGCCTTCAA TGCGGAATTC 351 AATGAGATAC GGAGGGTGCT GTTAGAAGAG AATGCAGGAG GTGAGCAAGA 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 30 1101 1151 1201 1251 1301

GGAGAGAGGG

GAGTGATAGT

GCTAAATCCG

CCCAATCAAC

AGTTATTTGA

CACATGATGC

ACACTTCAAC

GAAACCTTGA

CGCGAAGAAG

GGTGCGTAGG

CAGCAGCTCA

GGCTTCGGTA

TAAGGACAAT

TCCCTGGGTC

TCTCACTTTG

GAAAGAGTCT

AGGGTCCACT

CTTGTTATGT

AACTTATCAA

CAGAGGCGAT

CAAAGTGTCA

TCTCAAAGAA

TTGAGACAAG

GGTGAAGCCA

TCACCTGTGT

TCAAAGGCCA

GGAGTACTCG

AAGGAGCACG

AGGCTCCGAA

GC GAG CCC GA

GACAAGAAGA

AGAGATCAAA

TGGTTATCGT

GAGTAGTGAG

TTGAAGAACT

GAAGAGGGAG

TCTTTCTAAC

ACCCCCAGCT

GAAGGAGCTT

CGTCGTCAAC

AGCAACAACA

GAGGAGGGAAL

AGGCGATGTG

CCTCCGAACT

AGAATCTTCC

GCAPLGCGAAG

AACAATGAAG

TACTAAGCAC

ATATCACCAA

AACTTTGGGA

TCAGGACCTG

TGATGCTCCC

AAAGGAACTG

GAGGGGACGG

GTAACAGAGA

TTCATCATGC

CCATCTGCTT

TTGCAGGTGA

GATTTAGCAT

ACTCGTGGCT GTAAGAAAAG

AGGAGGACGA

TACACAGCGA

TCCAGTAGCC

TCAACGCTGA

GTGATAGACC

GGGTGAACAA

TGAGTGCTCA

CCTGAGAAAG

CCTTTCAATT

ATCGATAATA

TAAATAAAAA

AGACGAAGAA

GGTTGAAGGA

ATCAACGCTT

AAACAACCAC

AGATAGAGAA

GTTGAGAAGC

AT CTCAAT CT

AGGATCAAGA

TTGAAGGCTT

AGATCACGCT

CGTTTGTGCG

TCATCAAAAA CCAGAAGGAA CAATCTCCGT CGTCTCCTGA GGAGGAAAAC CAAGGAGGGA TTAACTGAGA ATGGAGGCAA TTTGTACTCT ACTATCCAAA

TTGTTTCTCC

a TABLE 17

LOCUS

DEFINITION

ACCESSION

NID

KEYWORDS

SOURCE

ORGANISM

REFERENCE

AUTHORS

TITLE

JOURNAL

COMMENT

FEATURES

25 30 ARQARAH 1949 bp MRNA PLN Arachis hypogea (clone P17) Ara h I MPNA, complete cds.

L38 853 g62 0024 peanut hypersensitivity Arachis hypogea (strain Florunner) Seed cDNA to rnRNA Arachis hypogaea Eukaryota; Plantae; Embryobionta; Magnoliophyta; Magnoliopsida; Rosidae; Fabales; Fabaceae 1 (bases 1 to 1949) Burks,A.W., Cockrell,G., Stanley,J.S., Helm,R.M. and Bannon, G.A.

Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity Unpublished (1994) NCBI gi: 620024 Location/Quali fiers source 1. .1949 /organism=-"Arachis hypogea" /strain="Florunner" /dev-stage=" Seed" /sequenced-mol='cDNA to mRNA" 51 UTR 1. .2 CDS 3. .1847 /gene="Ara h I"' /note="NCBI gi. 620025" /codon start=l /db-xref="PID:g620025"

V

V

V V

V

/translation="MRGRVSPLMLLLGILVLASVSATQAKSPYRKTENPCAQRCLQSC

QQEPDDLKQKACESRCTKLEYDPRCVYDTGATNQRHPPGERTRGRQPGDYDDDRRQPR

35 REEGGRWGPAEPREREREEDWRQPREDWRRPSHQQPRKIRPEGREGEQEWGTPGSEVR EETSRNNPFYFPSRRFSTRYGNQNGRIRVLQRFDQRSKQFQNLQNHRIVQI EARPNTL VLPKHADADNILVIQQGQATVTVANGNNRKS FNLDEGHALRI PSGFI SYILNRHDNQN LRVAKI SMPVNTPGQFEDFFPAS SRDQS SYLQGFS RNTLEAAFNAEFNEI RRVLLEEN AGGEQEERCQRRRSTRS SDNEGVIVKVSKEHVQELTKHAKSVS.KKGSEEEDITNPINL

RDGEPDLSNNFGRLFEVKPDKKNPQLQDLDMMLTCVEIKEGAL~MLPHFNISKAMVIVVV

NKGTGNLELVAVRKEQQQRGRREQEWEEEEEDEEEEGSNREVRRYTARLKEGDVFIMP

AAHPVAINASSELHLLGFGINAeNNHRI FLAGDKDNVIDQIEKQAKDLAFPGSGFQVE KLI KNQRESHFVSARPQSQS P55PEKEDQEEENQGGKGPLLS ILKAFN" 3'UTR 1848., .1949 45 polya.site 1 94 9 BASE COUNT

ORIGIN

599 a 455 c 517 g 378 t 1996 16:44 Type: N Check: 6409 Arqarah Length: 1949 1 CAATGAGAGG GAGGGTTTCT CCACTGATGC TGTTGCTTGG GATCCTTGTC 51 CTGGCTTCAG TTTCTGCAAC GaCAGGCCAAG TCACCTTACC GGAAAACAGA 101 GAACCCCTGC GCCCAGAGGT GCCTCCAGAG TTGTCAACAG GAACCGGACG 151 ACTTGAAGCA AAAGGCATGC GAGTCTCGCT GCACCAAGCT CGAGTATGAT 201 CCTCGTTGTG TCTATGACAC TGGCGCCACC AACCAACGTC ACCCTCCAGG 251 GGAGCGGACA CGTGGCCGCC AACCCGGAGA CTACGATGAT GACCGCCGTC Veeov

C*

CCC

600.

301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651

AACCCCGAAG

CGTGAAAGAG

AAGTCATCAG

AAGAGTGGGG

AAC CCTTT CT

AAACGGTAGG

TTCAGAATCT

ACTCTTGTTC

GCAAGGACAA

TT PAT CTTGA

TACATCTTGA

CAT GCCCGTT

GCCGAGACCA

GCCGCCTTCA

GAATGCAGGA

GGAGTAGTGA

CAAGAACTTA

GGAAGATATC

CTAACAACTT

CAGCTTCAGG

AGCTTTGATG

TCAACAAAGG

CAACAGAGGG

AGAAGAG GAG

AGGAAGGCGA

GCTTCCTCCG

CCACAGAATC

AGAAGCAAGC

AGAGGAAGGA

AAGAAGACTG

CAGCCACGGA

AACACCAGGT

ACT TCC CGT C

ATCCGCGTCC

CCAGAATCAC

TTCCCAAGCA

GCCACCGTGA

CGAGGGCCAT

AT CGACAT GA

AACACGCCCG

ATCATCCTAC

ATGCGGAATT

GGAGAGCAAG

TAATGAAGGA

CTAAGCACGC

ACCAACCCAA

TGGGAGGTTA

ACCTGGACAT

CTCCCACACT

AACTGGAAAC

GACGGCGGGA

GGAAGTAACA

TGTGTTCATC

AACT CCAT CT

TTCCTTGCAG

GAAGGATTTA

GGCCGATGGG

GAGACAACCA

AAATAAGGCC

AGCGAGGTGA

AAGGCGGTTT

TGCAGAGGTT

CGTATTGTGC

CGCTGATGCT

CCGTAGCAAA

GCACTCAGAA

CAACCAGAAC

GCCAGTTTGA

TTGCAGGGAT

CAATGAGATA

AGGAGAGAGG

GTGATAGTCA

TAAATCCGTC

TCAACTTGAG

TTTGAGGTGA

GATGCTCACC

TCAACTCAAA

CTTGAACTCG

ACAAGAGTGG

GAGAGGTGCG

ATGCCAGCAG

GCTTGGCTTC

GTGATAAGGA

G CAT TCCCT G

GACCAGCTGA

AGAGAAGATT

CGAAGGAAGA

GGGAAGAAAC

AG CAC CC GC T

TGACCAAAGG

AGATCGAGGC

GATAACATCC

TGGCAATAAC

T CC CAT CCGG

CTCAGAGTAG

GGATTTCTTC

TCAGCAGGAA

CGGAGGGTGC

GCAGAGGCGA

AAGTGTCAAA

TCAAAGAAAG

AGATGGCGAG

AG CCAGACAA

TGTGTAGAGA

GGCCATGGTC

TAGCTGTAAG

GAAGAAGAGG

TAGGTACACA

CT CAT CCAGT

GGTATCAACG

CAATGTGATA

GTTCGGGTGA

ACCGAGGGAG

GGAGGCGACC

GAAGGAGAAC

ATCACGGAAC

ACGGGAACCA

TCAAAGCAGT

CAGACCTAAC

TTGTTATCCA

AGAAAGAGCT

TTTCATTTCC

CTAAAAT CT C

CCGGCGAGCA

TACTTTGGAG

TGTTAGAAGA

CGGAGTACTC

GGAGCACGTT

GCTCCGAAGA

CCC GAT CTTT

GAAGAACCCC

TCAAAGAAGG

ATCGTCGTCG

AAAAGAGCAA

AGGAAGATGA

GCGAGGTTGA

AGC CAT CAAC

CTGAAAACAA

GACCAGATAG

ACAAGTTGAG

1701 AAGCTCATCA AAAACCAGAG GGAGTCTCAC TTTGTGAGTG CTCGTCCTCA 1751 ATCTCAATCT CCGTCGTCTC CTGAAAAAGA GGATCAAGAG GAGGAAAACC 1801 AAGGAGGGAA GGGTCCACTC CTTTCAATTT TGAAGGCTTT TAACTGAGAA 1851 TGGAGGAAAC TTGTTATGTA TCCATAATAA GATCACGCTT TTGTAATCTA 1901 CTATCCAAAA ACTTATCAAT AAATAAAAAC GTTTGTGCGT TGTTTCTCC *0OS 90 0* S 00 S@ S

S.

S

0@ @5 0 0

S

05*5

S

.55.55 9 0

OSS(

S

5.

0O S 0* 0 0*9S Table 18 LOCUS ARQALLII 717 bp DNA PLN DEFINITION Arachis hypogaea (clone Ara h II p 38 allergen II gene, polyA signal.

ACCESSION L77197 NID g1236995 KEYWORDS allergen; conglutin; seed storage protein.

SOURCE Arachis hypogea (strain Florunner) (clone: Axa h II p38) DNA.

ORGANISM Arachis hypogaea Eukaryotae; mitochondrial eukaryotes; Viridiplantae; Charophyta/Embryophyta group; Embryophyta; Magnaliophyta; Magnoliopsida; Rutanae; Sapindales; Fabaceae; Papilionoideae; Arachis.

REFERENCE 1 (bases 1 to 717) AUTHORS Stanley,J.S.

TITLE The major peanut allergen Ara h II is a seed storage protein with multiple IgE-binding epitopes JOURNAL Unpublished (1996) FEATURES Location/Oualifier3 source 1. .717 /organism--"Arachis hypogaea" /strain="Florunner' /clone="Ara h II p 38 polyA_signal 562. .567 BASE COUNT 217 a 152 c 184 g 164 t

ORIGIN

Arqallii Length: 1 GCTCACCATA 51 CTGCGAGGCA 101 CTCGAGAGGG 151 CCAACGTGAC 40 201 ATCCGTACAG 251 CACCAAGAGA 301 GTGCATGTGC 351 TGCAGGGGAG 401 CCTCAACAGT 451 AAGTGGCGGC 501 AAAGAAAAGA 551 GTTTTGGTAA 601 ACTAAGGCAA 651 TTGTCTATGT 701 AAAGATCATG 717

CTAGTAGCCC

GCAGTGGGAA

CGAACCTGAG

GAGGATTCAT

CCCTAGTCCA

GGTGTTGCAA

GACGCATTGC

GCAACAGGAG

GCGGCCTTAG

AGAGACAGAT

AAATAGCTTA

TAATAAAGAT

CCTTAGGTTA

TTTGTTGCTG

TTTTGTT

1996 14:32 Type: N Check: 3606 TCGCCCTTTT CCTCCTCGCT GCCCACGCAT

CTCCAAGGAG

GCCCTGCGAG

ATGAACGGGA

TATGATCGGA

TGAGCTGAAC

AACAGATCAT

CAACAGTTCA

GGCACCACAG

ACTAAACACC

TATATAAGCT

CATCACTATA

TATGAGCACC

CAGAGTTGTA

ACAGAAGATG

CAACATCTCA

CCCGTACAGC

GAGGCGCTGG

GAGTTTGAGA

GGAGAACCAG

AGAGGGAGCT

CGTTGCGACT

TATCTCAAAA

ATTATCTATG

TGAATGTGTT

TTTAGAGTGC

ACCATCTTGA

CCAGAGCCAG

TGCAGAAGAT

CCTAGTCAGG

ATCCTCTCAG

ACAACCAAAG

AGCGATAGGT

CAGGAACTTG

TGGACGTCGA.

AAAGAAAAGA

GTTATGTTTA

GATCGTGTTA

TTTTATGGCG

AATAATATAA

64 Table 19

LOCUS

DEFINITION

ACCESSION

IIID

KEYWORDS

SOURCE

ORGAN ISM

REFERENCE

AUTHOP,S

TITLE

JOURNAL

COMMENT

FEATURES

source 25 ARQARA-I 2032 bp mRNA PLN Arachis hypogea (Arch I beta) Ara h I mRNA, complete cds.

L34402 g602435 allergen Arachis hypogea (strain Florunner) seed cDNA to mRNA.

Arachis hypogaea Eukaryota; Plantae; Exbryobionta; Magnoliophyta; Magnoliopsida; Rosidae; Fabales; Fabaceae.

1 (bases 1 to 2032) Burks,A.W., Cockrell,G., Stanley,J.S., Helm,R.M. and Bannon, G.A.

Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity Unpublished (1994) NCBI gi: 602435 Location/Qualifiers 1. .2032 /organism: "Arachis hypogea" /strain=" Florunner" /dev stage="seed" /sequenced mol="cDNA to mRNA." cl one=" p4Th" .49 50. .1930 CDS /gene="Ara h I" /note="NCBI gi: 602436" /codon start=1 1db -xref="PID:g60243611"' /translation="MRGRVSPLMLLLGILVIASVSATIHAKSSPYQKKTENPCAQRCLQ 35 SCQQEPDDLKQKACESRCTKLEYDPRCVYDPRGHTGTTNQRSPPGERTRGRQPGDYDD DRRQPRREEGGRWGPAGPREREREEDWRQPREDWRRPSHQQPRKI RPEGREGEQEWGT PGSHVREETSRNNPFYFPSRRFSTRYGNQNGRIRVLQRFDQRSRQFQNLQNHRIVQI E AKPNTLVLPKHADADNILVIQQGQATVTVANGNNRKSFNLDEGHALRI PSGFI SYILNI RHDNQNLRVAKI SMPVNTPGQFEDFFPASSRDQSSYLQGFSRNTLEAAFNAEFNEIRR 40 VLLEENAGGEQEERGQRRWSTRSSENNEGVIVKVSKEHVEELTKHAKSVSKKGSEEEG DITNPINLREGEPDLSNNFGKLFEVKPDKKNPQLQDLDMMLTCVEI KEGAIIMLPHFNS

KAM'VIVVVNKGTGNLELVAVRKEQQQRGRREEEEDEDEEEEGSNREVRRYTARLKEGD

VFIMPAAH-PVAINASSELHLLGFGINAENNHRI FLAGDKDNVI DQIEKQAKDLAFPGS GEQVEKLIKNQKSEHFVSARPQSQS.QSPSSPEKES PEKEDQEEENQGGKGPLLSILKA

FNVI

3 'UTR polyA,_signal polyA_site 1931. .2032 2 00 5-2 010 2032 BASE COUNT 628 a 473 c 530 g 401 t

ORIGIN

Arqarahi Length: 2032 1996 16:36 Type: N Check: 8370 1 AATAATCATA TATATTCATC AATCATCTAT ATAAGTAGTA GCAGGAGCAA 51 TGAGAGGGAG GGTTTCTCCA CTGATGCTGT TGCTAGGGAT CCTTGTCCTG 101 GCTTCAGTTT CTGCAACGCA TGCCAAGTCA TCACCTTACC AGAAGAAAAC 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001

AGAGAACCCC

ATGACTTGAA

GATCCTCGTT

ACGTTCCCCT

ATGATGACCG

G CT GGAC CGA

AGATTGGAGG

GAAGAGAAGG

GAAACATCTC

CCGCTACGGG

AAAGGTCAAG

GAGGCCAAAC

CATCCTTGTT

ATAACAGAAA

TCCGGTTTCA

AGTACCTAAA

TCTTCCCGGC

AGGAATACGT

TGCGCCCAGA

GCAAAAGGCA

GTGTCTATGA

CCAGGGGAGC

CCGTCAACCC

GGGAGCGTGA

CGACCAAGTC

AGAACAAGAG

GGAACAACC C

AACCAAAACG

GCAGTTTCAG

CTAACACTCT

AT CCAGCAAG

GAGCTTTAAT

TTTCCTACAT

AT C TCCAT GC

GAGCAGCCGA

TGGAGGCCGC

GGTGCCTCCA

TGCGAGTCTC

TCCTCGAGGA

GGACACGTGG

CGAAGAGAGG

AAGAGAAGAA

ATCAGCAGCC

TGGGGAACAC

TTTCTACTTC

GTAGGATCCG

AATCTCCAGA

TGTTCttCCC

GGCAAGCCAC

CTTGACGAGG

CTTGAACCGC

CCGTTAACAC

GACCAATCAT

CTTCAATGCG

GAGTTGTCAA

GCTGCACCAA

CACACTGGCA

CCGCCAACCC

AAGGAGGCCG

GACTGGAGAC

ACCGAAAATA

CAGGTAGCCA

CCGTCAAGGC

GGTCCTGCAG

ATCACCGTAT

AAGCACGCTPG

CGTGACCGTA

GCCATGCACT

CAT GACAAC C

ACCCGGCCAG

CCTACTTGCA

GAATTCAATG

CAGGAACCGG

GCTCGACTAT

CCACCAACCA

GGAGACTACG

ATGGGGACCA

AACCAAGAGA

AGGCCCGAAG

TGTGAGGGAA

GGTTTAGCAC

AGGTTTGACC

TGTGCAGATC

ATGCTGATAA

GCAAATGGCA

CAGAAT CCCA

AGAACCTCAG

TTTGAGGATT

GGGCTTCAGC

AGATACGGAG

1051 GGTGCTGTTA GAAGAGAATG CAGGAGGTGA GCAAGAGGAG AGAGGGCAGA a 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001

GGCGATGGAG

GTGTCAAAGG

AAAGAAAGGC

GAGAAGGCGA

AAGCCAGACA

CTGTGTAGAG

AGGCCATGGT

GTGGCTGTAA

GGACCAACAC

CAGC GAG GTT

GTAGCCATCA

CGCTGAAAAC

TAGACCAGAT

GAACAAGTTG

TGCTCGTCCT

CTCCTGACAA

CTCCTTTCAA

GTATCGATAA

AATAAATAAA

TACTCGGAGT

ACCACGTTGA

TCCGAAGAAG

GCCCGATCTT

AGAAGAACC C

ATCAAAGAAG

TATCGTCGTC

CAAAAGAGCA

CAAGAAGAGG

GAAGGAAGGC

ACGCTTCCTC

AACCACAGAA

AGAGAAGCAA

AGAAGCT CAT

CAATCTCAAT

AGAGGATCAA

TTTTGAAGGC

TAAGATCACG

AACGTTTGTG

66

AGTGAGAACA

AGAACTTACT

AGGGAGATAT

TCTAACAACT

CCAGCTTCAG

GAGCTTTGAT

GTCAACAAAG

ACAACAGAGG

AGGGAAGTAA

GATGTGTTCA

C GAACT CCAT

TCTTCCTTGC

GCGAAGGATT

CAAAAACCAG

CT CAAT CT CC

GAGGACGAAA

TTTTAACTGA

CTTTTGTACT

CGTTGTTTCT

ATGAAGGAGT

AAGCACGCTA

CACCAACCCA

TTGGGAAGTT

GACCTGGACA

CCTCCCACAC

GAACTGGAAA

GGACGGCGGG

CAGAGAGGTG

TCATGCCACC

CTGCTTGGCT

ACGTGATAAG

TAGCATT CCC

AAGGAATCTC

GTCGTCTCCT

ACCAAGGAGG

GAATGGAGGC

CTACTAT CCA cc

GATAGTCAAA

AATCCGTCTC

AT CAACTT GA

ATTTGAGGTG

TGATGCTCAC

TTCAACTCAA

CCTTGAACTC

AAGAAGAGGA

CGTAGGTACA

AGCT CAT CCA

TCGGTATCAA

GACAATGTGA

TGGGTCGGGT

ACTTTGTGAG

GAGAAAGAGT

GAAGGGTCCA

AACTTGTTAT

AAAACTTATC

Table 67 GENIE> type arahIlp38.pep TRANSLATE of: arahIlp38.final check: 9822 from: 4 to: 480 generated symbols 1 to: 159.

Arah2p38.Pep Length: 157 1996 15:24 Type: P- Check:2859 1 LTILVALALF LLAAHASARQ QWELQGDRRC QSQLERANLR PCEQHLMQKI 51 QRDEDSYERD PYSPSQDPYS PSPYDRRGAG SSQHQERCCN ELNEFENNQR 101 CMCEALQQIM ENQSDRLQGR QQEQQFKREL RNLPQQCGLR APQRCDLDVE 151 SGGRDRY EDITORIAL NOTE APPLICATION NUMBER 43769/01 The following Sequence Listing pages 68 to 78 are part of the description. The claims pages follow on pages 79 to 79.

a SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: UNIVERSITY OF ARKANSAS (ii) TITLE OF INVENTION: PEANUT ALLERGENS AND METHODS (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Patrea L. Pabst STREET: 2800 One Atlantic Center 1201 West Peachtree Street CITY: Atlanta STATE: GA COUNTRY: USA ZIP: 30309-3450 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PCT/US96/15,222 FILING DATE: 23 SEPTEMBER 1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Pabst, Patrea L.

REGISTRATION NUMBER: 31,284 REFERENCE/DOCKET NUMBER: HS 103 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (404)-873-8794 TELEFAX: (404)-873-8795 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 474 nucleotides TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA DESCRIPTION: identified as Ara h II cDNA clone (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No FRAGMENT TYPE: Not applicable (vi) ORIGINAL SOURCE: ORGANISM: Arachis hypogaea STRAIN: Florunner INDIVIDUAL ISOLATE: DEVELOPMENT STAGE: seed HAPLOTYPE: Not applicable TISSUE TYPE: seed cDNA (vii) IMMEDIATE SOURCE: LIBRARY: florunner seed cDNA expression library in Uni-ZAP XR vector (ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: By agreement with protein information and established consensus sequence OTHER INFORMATION: Seed storage protein and allergen (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 69 cTCAccATAC TAGTAGCc'r CGCCCTTTTC CTCCTCQCTG CCCACGCATC T~cGAGGCAG CAGTGGGANAC TCCAAGGAX3A CAGAAGATGC CAGAGCCAGC TCGAGAGGGC GAACCTOAG 120 CCCTGCGAGC AACATC1'CAT GCAGAAGATC CAACGTGACG AGGA1"ICATA TGAhCGGGAC 180 CCGTACAGCC CTAOTCAGGA TCCGTAcA~c CCTAG3TCCAT ATQATCGGAG AGGC3CTG. 240 TCCTCTCAGC ACCAAGAGAG GTGTTGCAAT GAGCTGAACG AGTTTGAGAA CAACCAAAGG 300.

TGCATGTGCG AGOCATTGCA ACAGATCATG GAGAACCAGA G 0GA TAGGTT GCAGGGGN3G :360 CAACAGGAGC AACAGTTCAA GAGGGGCTC AGGAACTI'GC CTCAACAGTG CGGCCTTAGG 420 GCACCACAGC GTTGCGACTT GGACGTCOAA AGTGGCGGCA GAGACAGATA CTAA 474 INFORMATION FOR SEO ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 157 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: amino acid sequence DESCRIPTION: identified as Ara h II cDNA clone derived amino acid sequence (vi) ORIGINAL SOURCE: oRGmism: Arachis hypogaea STRAIN: Florunner IXDIVIDUAL ISOLATE- DEVELOPMENT STAGE: seed

FEATURE:

NA.MR/KEY:

LOCATION-

IDENTIFICATION METHOD: By ag'reement With protein information and established consensus sequence OTHER INFORMATION: Seed storage protein and allergen *UKi) FEATURE:

NAME/KEY:

LOCATIQN: amino acids 15-24 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h. 2 IgE bindingepitope, peptide 1

FEATURE:

blAMS/KEY: LOCATION: amino acids 21-30' IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h 2 IgE binding epitope, peptide 2 (ix) FEATURE:

NAE/KEY:

LOCATION: amino acids 27-36 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h 2 1gE binding epitope, peptide 3 (ix) FEATURE:

NAME/KEY:

LsoCATION: amino acids 39-48 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h 2 IgE binding epitope, peptide 4 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 49-58

U

IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h 2 ISE binding epitope, peptide S (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 60-69 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h It ISE binding epitope, peptide 3 (ix) FETURE:

NAME/KEY;

LOCATION: amino acids S7-66 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h 2 ISE binding epitope, peptide 6 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 65-74 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h 2 1gE binding epitope. peptide 7 (ix) FEATURE.

NAME/KEY:

LOCATION: amino acids 81-90 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h it 19E binding epitope, peptide 4 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 91-100 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara. h II IgE binding epitope. peptide S (ix) FEAUE:

NAME/KEY:

LOCATION: amino acids 105-159 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h II IgE binding epitope, peptide 6 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 415-124

IDENTIFICATION*METHOD:'

OTHER INFORMATION: identified as Ara h 2 IgE binding epitope, peptide 8 (ix) FEATURE:-

NAME/KEY:

LOCATION: amirfo acids 127-136 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara hi 2 IgE binding epitope, peptide 9 (ix) FEATURE:

NAME/KEY:-

LOCATION: amino acids 143-152 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h 2 1gE binding epitope, peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Leu Thr Ile Leu Val Ala Leu Ala Leu Phe Leu tLeu Ala Ala His Ala 1 10 Ser Ala Arg Gin Gin Trp Giu Leu Gin ciiy Asp Arg Arg Cyr. Gin Ser 25 s.

C

a a a a a Gin Leu Glu Arg Ala Asn Leu Arg Pro Cys Giu Gin His Leu Met Gin 40 Lys Ile Gin Arg Asp Giu Asp Ser Tyr GlU Arg Asp Pro Tyr Set Pro 55 Ser Gin Asp Pro Tyr Ser Pro Ser Pro Tyr Asp Arg Arg Gly Ala Gly 70 75 Ser Ser Gin His Gin Glu Arg Cys Cys Asn Glu Leu Asn Glu Phe Giu as 90 Asn Asn Gin Arg Cys Met Cys Giu Ala Leu Gin Gin le Met Giu Asn 100 105 110 Gin Ser Asp Arg Leu Gin Gly Arg Gin Gin Glu Gin Gin Phe Lys Arg 115 120 125 Giu Leu Arg Asn Leu Pro Gin Gin Cys Gly Leu. Arg Ala Pro Gin Arg 130 135 140 Cys Asp Leu Asp.Val Glu Ser Gly Gly Arg Asp Arg Tyr 145 i5o iss INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARPACTERISTICS: LENGTH: 1930 TYPE% nucleic acid STRANDEDNESS: double TOPOLOGY: iinear (ii) MOLECULE TYPE: cDNA DESCRIPTION: identified as Ara h I cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AATAATCALTA TATATTCATc AATCATCTAT ATAAGTAGTA. GCAGGAGCA ATG AGA Met Arg 000 AGG OTT TCT CCA CTG ATG CTG TTG CTA 000 ATC CTT GTC CTG GCT Giy Arg Val Ser Pro Leu. Met Leu Leu Leu Gly Ile Leu Val Leu Ala S 10 TCA G=r TC'r GCA ACG CAT GCC Am3 TCA TCA CCT TAC.'CAG AAG AAA -ACA Set Val Set Ala Thr His Ala Lys Ser Ser Pro Tyr Gin Lys Lys-Thr 20 25 GAG AAC CCC TGC GCC CAG AGO T GC CTC CAG AGT TOGT CAA CAG GAA CCG Giu Asn Pro Cys Ala Gin Arg Cys Leu. Gin Set Cys, Gin Gin Giu Pro 35 40 45 s0 GAT GAC TTG AAG CAA AAG GCA TOC GAG TCT CGC TGC ACC AAG CTC GAG Asp Asp Leu Lys Gin Ly's Ala Cys Giu Ser Arg Cys Thr Lys Leu Giu 60 TAT OAT CCT COT TGT GTC TAT OAT CCT CGA 00k CAC ACT GCC ACC ACC Tyr Asp Pro Arg Cys Val Tyr Asp Pro Arg Gly His Thr Gly Thr Thr 75 AAC CAA CGT TOO CCT OCA 000 GAG COG ACA COT GOC CGC CAA CCC OGA Asn Gin Arg Ser Pro Pro Gly OiU Arg Thr Arg Gly Arg Gin Pro Gly 90 103 151 199 247 295 343 GAC TAC GAT OAT Asp Tryr Asp Asp 100 TOGG OGA CCA OCT Trp Gly Pro Ala 115 CAM CCA AGA GAM Gin Pro Arg Glu ATA AGG CCC GAA Ile Arg Pro (flu 1So AGC CAT GTG AGG Ser His Val Arg 165 TCA AGG CGG TTT Ser Arg Arg Phe 180 GTC CTo CAG AGO Val Lieu Gin Arg 195 AAT CAC COT ATT Asn His Arg Ile CCC AAG CAC GCT Pro Lys His Ala 230 0CC ACC GTG ACC Ala Thr Val Thr 245 GAC GAG GGC CAT Asp Giu Gly His 000*: *6 0** 04 66.0 COT CAA CCC CGA AGA GAG GAA GGA Arg Gin Pro Arg Arg Glu Giu Gly 105 110 AGO GAG COT GAA AGA GMA GMA GAG Arg Giu Arg Glu Arg Giu Giu Asp 125 AGO CGA CCA AOT CAT CAG CAG CCA Arg Arg Pro Ser His Gin Gin Pro 14 0 GMA GGA GMA CMA GAG TGG GGA ACA Giu Gly Glu Gin Giu Trp Gly Thx 155 160 ACA TCT COO MAC AAC COT TTC TAC Thr Ser Arg Asn Asn Pro Phe Tyr 170 175 CGC TAC 000 AAC CMA AC GOT AGG Arg Tyr Giy Asn Gin Asn Gly Arg 185 190 CMA AGO TCA AGO CAG TTT CG A T Gin Arg Ser Arg Gin Phe Gin Asn 205 ATC GAG 0CC MAA GOT MAC ACT OTT le Glu Ala Lys Pro Asn Thr Leu 220 GAT MAC ATC OTT OTT ATC CAG CAA Asp Asn Ile Leu Vai le Gin Gin 235 240 MAT GOC MAT AC AGA MOG AGC TTT Asn Gly Asn Asn Arg Lys Ser Phe 250 255 AGA ATC CCA TCC GOT TTC ATT TCC Arg le Pro-Ser Gly Phe le-Ser 265 270 CAG MAC CTC AGA OTA GOT AMA ATC Gin Asn Leu-Arg-Val-Ala-Lys Ile 285 CAG TTT GAG GAT TTC TTC CCG GCG Gin Phe Giu-Asp Phe Phe Pro.Ala 300 i-rO GAG 0CC TTC AGO AGO MAT AG Lieu Gin Gly Phe Ser Arg Asn Thr 315 1 320 TTC MAT GAG ATA COG AGO GTG CTG Phe Asn Glit Ile Arg Arg Val Leu 330 335 GGC CGA Giy Arg TO;G AGA Trp Arg 130 COO MA Arg Lys 145 CGA GGT Pro Giy TTC CCO Phe Pro ATC CGO le Arg CTC GAG Lieu Gin 210 GTT CTT Val Leu 225 000 CMA Gly Gin MAT CTT Asn Lieu TAC ATC Tyr Ile TCG ATG -Ser -Met 290 AGO AGC Set Ser 305 TTG GAG Lieu Oilu TTA GMA Lieu Glii 391 439 487 535 583 631 679 727 775 823 871 967 1015 1063 1111 CCA CTC Ala Lieu

TT(;

Leu 275

CCC

Pro

CGA

Arg 0CC Ala 260

AAC

Asn

GTT

Val

GAG

Asp

GCC

Ala

GAC

Asp

CCC

Pro 295

TCG

Ser GcG Ala

MAC

Asn 280

GOC

Giy

TAG

Tyr

GMA

Giu GAG MAT GCA GGA Glu Asn Ala Oly 340 GOT GAG CMA GAG GAG AGA 000 CAG AGO Gly Giu Gin Glu Glu Arg Gly Gin Arg .345 350 CGA TGG AGT Arg Trp Ser ACT CGG AG? AGT GAG AAC Thr Arg Ser Ser Glu Asn 355 360 GAG CAC GTT GAA GAA CTT Giu His Val Giu Giu Leu 37S GGC TCC GAAL GMA GAG GGA Gly Ser Giu Giu GiU Gly 390 CCC GAG CCC GAT CT? TCT Gly Glu Pro Asp Leu Ser 405 OCA CAC MAG AAG MAC CCC Pro Asp Lys Lys Asn Pro 420 TGT GTA GAG ATC AAA GMA Cys Val Giu Ile Lys Glu 435 440 MAG GCC ATG GTT ATC GTC Lys Ala Met Val Ile Val 455 CTC GTG GCT GTA AGA A Leu Val Ala Val Arg Lys 470 GAG GAG GAC GMk GAC GMA Glu Giu Asp Glu Asp Glu 485 AGG, TAC ACA GCG AGG TTG Arg Tyr Thr Ala Arg Leu S00 GCT CAT CCA GTA GCC ATC Ala His Pro Val Ala Ile 515 520 TTC GGT ATC MAC GCT GMA Phe Gly Ile Asn Ala Glu 535 MAG GAC MAT GTG ATA GAC Lys Asp Asn Val Ile Asp 550 TTC CC? GGG TCG GGT GMA Phe Pro Gly Ser Gly Giu 565 GMA TCT CAC TI'? GTG AG? Giu Ser His Phe Val Ser 580 MAT GMA Asn Glu ACT MAG Thr Lys GAT ATC Asp Ile MAC MAC Asn Asn 410.

GAG CT? Gin Leu 425 GGA GCT Gly Ala GTC GTC Val Val GAG CMA Giu Gin GMA GAG Giu Giu 490 MAG GMA Lys Giu 505 MAC C Asn Ala MAC AMC Asn An GAG ATA Gin Ile CMA OTT Gin Val 570 GCT CGT Ala Arg 585 GGA GTG ATA GTC MAA GTG TCA MAG Gly Val Ile Val Lys Val Ser Lys 365 370 CAC GCT MAA TCC GTC TCA MAG MA His Ala Lys Ser Val Ser Lys Lys 380 385 ACC MAC CCA ATC MAC TTG AGA GMA Thr Asn Pro Ile Asn Leu Arg Giu 395 400 TI'? GGG MAG TTA TTT GAG GTG MAG Phe Gly Lys Leu Phe Giu Vai Lys 415 GAG GAC CTG GAC ATG ATG CTC ACC Gin Asp Leu Asp Met Met Leu Thz 430 TTG ATG CTC CCA CAC TTC MAC TCA Leu Met Leu Pro His Phe Asn Ser 445 450 MC MAA OGA AC? GGA MAC CTT GMA Asn Lys Gly Thr Gly Ann Leu Glu 460 465 CMA GAG AGG GGA CGG CGG GMA GMA Gin Gin Arg Gly Arg Arg Giu Oiu 475 480 GAG GGA AG? MAC AGA GAG GTG. CGT Giu Gly Ser Asn Arg Giu Vai Arg 495 GGC GAT GTG TTC ATC ATG CCA GCA Gly Asp Vai Phe lie Met Pro.Ala 510 TCC TCC GMA CTC CAT CTG CTT GCC Ser Ser Giu Leu His Leu Leu Gly 525 530 CAC AGA ATC TTC CTT GCA GGT-GAT His Arg Ile Phe- Leu Ala Giy Asp- 540 545 GAG MAG CMA GCG MAG GAT TTA GCA Giu Lys Gin Ala-Lys Asp Leu:Ala 555 560 GAG MAG CTC ATC MAA MAC GAG MAG Giu Lys Leu Ile Lys Asn Gin Lys 575 CC? CAA TCT CAA TCT CMA TCT CCG Pro Gin Ser Gin Ser Gin Ser Pro 590 1159 1207 1255 1303 1351 1399 1447 1495 1543 1591 1639 1687 1735 1783 1831 1879 TCG TCT CCT Ser Ser Pro 595 GAG MAA GAG TCT Giu Lys Glu Ser 600 CC? GAG MA Pro Glu Lys GAT CAA GAG GAG GMA Asp Gln Giu Glu Glu AAC CAA GGA Azn GIn Gly

TGA

GGG AAG GGT CCA CTC CTT TCA ATT TTG AAG GCT Gly Lys Gly Pro Leu Leu Ser Ile Leu Lys Ala 615 620 TTT AA~C Phe Asn 625 1927 1930 INFORMATION FOR, SEQ ID NO: 4: Wi SEQUENCE CHARACTERISTICS: LENGTH: 626 TYPE: amino acid TOPOLOGY: unknown (ii) M4OLECULjE TYPE: glycoprotein (vi) ORIGINAL SOURCE: ORGANISM: Arachis hypogaea STRAIN: Florunner IMDIVIDUAL ISOLATE: Ara h I (ix) FAUE

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: By agreement with protein information and established consensus sequxence OTHER INFORMATION: Seed storage protein and allergen (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 25-34 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h I 1g] epitope, peptide I (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 48-57 IDENTIFICATION METHOD: OTE INFORMATION: identified as Ara h I Ig~ epitope, peptide 2 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 65-74 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara ha I Ig epitope, peptide 3 (ix) FEATURE:

NAME/KEY:

LOCATION; amino acids 69-98 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara h I Ig epitope, peptide 4 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 97-105 IDEN4TIFICATION METHOD: OTHER INFORMATION: identified as Ara h I Ig epitope, peptide (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 107-116.

IDENTIFICATION METHOD: OTHER INFORMATION. identified as Ara h I 112 epitope, peptide 6 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 123-132 -IDENTIFICATION METHOD: binding Ebinding Ebinding E binding E binding rE binding 0 OTHER INFORMATION: identified as Ara epitope, peptide 7

FEATURE:

NAME/KEY:

LOCATION: amino acids 134-143 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara epitope, peptide 8 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 143-152 IDENTIFICATION METHOD: OTHER INFORMATION.- identified as Ara epitope, peptide. 9 FEATUREi

NAMEKEY:

LOCATION: amino acids 294-303 IDENTIFICATION METHOD: OTHER IN~FORMATION: identified as Ara epitope, peptide (ix) FEATURE:

NAM/KEY:

LOCATION: amino acids 311-320 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara epitope, peptide 11 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 325-334 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara epitope, peptide 12 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 344-353 IDENTIFICATION METHOD: OTHER INFOMATION: identified as Ara epitope, peptide 13 (ix) FEATURE:

NAM/KEY:

LOCATION: amino acids 393-402 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ara epitope, peptide 14 (ix) FEATURE:

NAM/KEY:

LOCATION: amino acids 409-418 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ar4 epitope, peptide

IS

(ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 461-470 IDENTIFICATION METHOD: OTHER INFORMATION: identified as Ar~ epitope, peptide 16 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 498-507 IDENTIFICATION METHOD.

OTHER INFORMATION: identified as Ar epitope, peptide 17 (ix) FEATURE.- NAME/ KEY: h I IgE binding h I IgE binding h I IgE binding h I IgE binding h I IgE binding h I IgE binding h I IgE binding h I IgE binding hi I 1gB binding i h I IgE binding a hi I IgE binding LOCATION: amino acids 52S-534 IDENTIFICATION METHOD: OTHER INFORMATION: identified epitope, peptide 18 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 539-548 IDENTIFICATION METHOD: OTHER INFORMATION: identified epitope, peptide 19 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 551-560 IDENTIFICATION METHOD: OTHER INFORMATION: identified epitope, peptide (ix) FEATURE-

NAME/KEY:

LOCATION. amino acids 59-568 IDENTIFICATION METHOD: OTHER INFORMATION: identified epitope, peptide 21 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 578-587 IDENTIFICATION METHOD: OTHER INFORMATION: identified epitope, peptide 22 (ix) FEATURE:

NAME/KEY:

LOCATION: amino acids 597-606 IDENTIFICATION M=TOD: OTHER INFORMATION: identified as Ara h I IgE binding as Ara h I IgE binding as Ara h I IgR binding as Ara h I IgE binding as Arah I IgE binding as Ara h I IgE binding (xi) met Leu Lys Giu Leu 65 Thr Pro Gly epitope, peptide 23 SEQUENCE DESCRIPTION: SEQ ID NO: 4: Arg Gly Arg Val Ser Pro Lieu Met Lieu Lei S Ala Ser Val Ser Ala Thr His Ala Lys Se, 25 Thr Glu Asn Pro Cys Ala Gin Arg Cys Le' 3S 40 Pro Asp Asp Lieu Lys Gin Lys Ala- Cys Gi so 55 Glu Tyr Asp Pro Arg Cys Val Tyr Asp Pr 70 75 Thr Asn Gin Arg Ser Pro Pro Giy Glu Ar 90 Gly Asp Tyr Asp Asp Asp Arg Arg Gin Pr 3.00 105 Arg Trp Gly Pro Ala Gly Pro Arg Glu Ar 115 120 ar

U-

0 g Leu Set Gin Ser Arg Thr Arg Glu Gly Pro Ser Arg Gly Arg Arg Arg 125 Ile Tyr Cys Cys His Gly Glu 110 Glu Lieu Val Gin Lys.

Gin Gin Thr Lys- Thr Gly Arg Gin Glu Gly Glu Asp Trp Arg Gin Pro Arg 130 Glu Asp Trp Arg Arg Pro Ser His Gin Gin Pro 135 140 Arg Lys Ilie Arg Pro Glu Gly Arg Glu Gly Glu Gin Glu Trp Gly Thr 77 145 150 155 160 Pro Gly Ser His Val Arg Glu Gin Thr Ser Arg Asn Asn Pro Phe Tryr 165 170 175 Phe Pro Ser Arg Arg Phe Ser Thr Arg Tyr Gly Asn Gin Asn Gly Arg 180 185 190 Ile Arg Val Leu Gin Arg Phe Asp Gin Arg Ser Arg Gin Phe.Gin Asn 195 200 205 Leu Gin Asn His Arg Ile Val Gin Ile Giu Ala Lys Pro Asn Thr Leu 210 215 220 Val Leu Pro Lys His Ala Asp Ala Asp Asn Ile Leu Vai Ile Gin Gin 225 230 235 240 Gly Gin Ala Thr Val Thr Val Ala Asn Gly Asn Asn Arg Lys Ser Phe 245 250 255 Asn Len Asp Giu Gly His Ala Leu Arg Ile Pro Ser Gly Phe Ile Ser 260 265 270 Tyr Ile Leu Asn Arg His Asp Asn Gin Asn Leu Arg Val Ala Lys Ile 275 280 285 Ser Met Pro Val Asn Thr Pro Gly Gin Phe Giu Asp Phe Phe Pro Ala *290 295 3400 .Ser Ser Arg Asp Gin Ser Ser Tyr Leu Gin Giy Phe Ser Arg Asn Thr 305 310 315 320 *Leu Gin Ala Ala Phe Asn Ala Giu Phe Asn Giu Ile Arg Arg Val Leu *325 330 335 Len Giu Gin Asn Ala Gly Gly Giu Gin Gin Giu Arg Gly Gin Arg Arg 340 345 350 Trp Ser Thr Arg Ser Ser Giu Asn Asn Giu Gly Val Ile Val Lys Val *355 360 365 Ser Lys Gin His Vai Gin Giu Leu Thr Lys His Ala Lys Ser Val Ser 370 375 380 Lys Lys Gly Ser Gin Glu Gin Gly Asp Ile Thr Asn Pro Ile Asn Len o385 390 395 400 Arg Gin Giy Gin Pro Asp Leu Ser Asn Asn Phe Gly Lys Len Phe Gin 405 410 415 *Val Lys Pro Asp Lys Lys Asn Pro Gin Len Gin Asp Len Asp Met Met 420 425 430 Leu Thr Cys Val Giu Ile Lys Gin Gly Ala Len Met Len Pro His Phe 435 440 445 Asn Ser Lys Ala Met Val Ile Val Vai.Val Asn Lys Giy Thr Gly Asn .450 455 460 Leu Gin Len Val Ala Val Arg Lys Giu Gin Gin Gin Arg Gly Arg Arg 465 470 475 480 Giu Giu Giu Gin Asp Gin Asp Glu Giu Gin Giu Gly Ser Asn Arg Gin 485 490 495 Val Pro Leu Gly 545 Leu Gin Ser Glu Phe 625 Arg Ala Gly 530 Asp Ala Lys Pro Giu 610 Asn Arg Ala 515 Phe Lys Phe Glu Ser 595 Asa Tyr S00 His Gly Asp Pro Ser 580 Ser Gin Thr Ala Pro Val Ile Asn Asn Val 550 Gly Ser 565 His Phe Pro Giu Gly Gly Arg Ala Ala 535 Ile Gly Val Lys Lys 615 Val Phe 510 Glu Leu 525 Ile Phe Gin Ala Leu Ile Ser Gin 590 G1u Asp 605 le Leu Ile His Leti

LYS

Lys 575 Ser Gin

LYS

Claims (6)

1. A peptide comprising an IgE binding epitope of Ara hi.

2. A peptide comprising an immunodominant IgE binding epitope of Ara hi.

3. A peptide comprising an IgE binding epitope of Ara hi that has been modified to reduce binding of IgE.

4. A peptide comprising an IgE binding epitope of Ara hII. A peptide comprising an immunodominant IgE binding epitope of Ara hII.

6. A peptide comprising an IgE binding epitope of Ara hII that has been modified to reduce binding of IgE.

7. A peptide substantially a hereinbefore described with reference to the examples, excluding comparative examples. Dated this 14th day of July 2003 University of Arkansas Patent Attorneys for the Applicant: F B RICE CO *lo•