Grantee Research Project Results

Final Report: Clinically Relevant IgE-Cross-Reactivity of Nut Allergens

Objective:

Summary/Accomplishments (Outputs/Outcomes):

Methodology: We used sera from the blood of peanut and or walnut allergic individuals that were selected by clinical history of anaphylaxis to peanuts, walnuts or both. Western blotting was used to show the different allergenic proteins from walnut and peanut that reacted with IgE in sera from the patients. The patients also were characterized initially for their ability to bind 6 experimental epitopes on SPOTs strips; all sera recognized a previously established, immunodominant IgE epitope from Ara h 2 that had many peptides similar to it (according to the PD scale in SDAP) in other peanut and walnut allergens. The property distance (PD) scale in the Structural Database of Allergenic Proteins (SDAP) recognizes sequences in other allergens that are similar to experimentally defined IgE epitopes.

 

Large SPOTs membranes were designed to include ~300 peptides, including known linear IgE epitopes, and sequences that were similar to them (low PD value) from nut allergens. Mimetic peptides, designed based on 3D-structures of the allergens, also were included, that used the surface presentation of the linear peptides to determine surface exposed residues that were likely to be involved in antibody binding. Smaller membranes then were used to retest the positive peptides, to insure the results were reproducible. A few peptides were made in solution to test the results in competitive ELISA experiments. In later stages of the experiment, antibodies were prepared to a consensus peptide based on repeated sequences in the N-terminal pro-region of the allergen Jug r 2. These antibodies were used to probe nut extracts for proteins that also were recognized by IgE antibodies in the sera of patients with allergy to almonds and other nuts.

 

Overall Results: The immunoassays were designed to test for IgE-binding to protein fragments (or peptides) from walnut and peanut allergens. Sequences from a major walnut allergen Jug r 2, which were predicted to be similar to an immunodominant epitope of the peanut allergen Ara h 2, bound IgE in sera from five patients who reacted to either walnut, peanut or both. These findings demonstrate that our computer software is capable of accurately predicting the amino acid sequences that are similar between different nuts and which of these amino acids is most likely to be recognized by IgE antibodies from the blood of allergic individuals. Competitive ELISA was used to show that peanut and predicted walnut amino acid sequences compete with a purified peanut allergen, Ara h 2 for binding to IgE in serum from a patient that reacts to both peanut and walnut, thus indicating they are true cross reactive antigens.

 

The antibodies to the consensus peptide detected proteins in Western blotting of extracts from various nuts and seeds that were predicted computationally to contain sequences similar (PD value < 8) to the consensus peptide. The same proteins were recognized by IgE in sera of patients allergic to almond. The consensus peptide antibodies also detected bands in walnut extracts that could be residual N-terminal proregion of walnut. The following abstracts of our publications illustrate these results.

 

1) Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts (paper published in Allergy, 2011)

Background: Cross reactivity between peanuts and tree nuts implies that similar IgE epitopes are present in their proteins.

Objective: To determine whether walnut sequences similar to known peanut IgE binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins (SDAP), react with IgE from sera of patients with allergy to walnut and/or peanut.

Methods: Patient sera were characterized by Western blotting for IgE-binding to nut protein extracts, and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive ELISA was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient.

Results: Sequences from the vicilin walnut allergen Jug r 2, which had low PD values to epitopes of the peanut allergen Ara h 2, a 2s-albumin, bound IgE in sera from five patients who reacted to either walnut, peanut or both. A walnut epitope recognized by six patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. A predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2.

Conclusions: Sequences with low PD value (<8.5) to known IgE epitopes could contribute to cross-reactivity between allergens. This further validates the PD scoring method for predicting cross-reactive epitopes in allergens.

 

2) Structural and roasting-induced alterations affect IgE binding to Ara h 1 (paper is being revised for Molecular Nutrition)

Scope: Ara h 1 from roasted peanut bind higher levels of serum IgE than raw peanuts and this is likely due to the Maillard reaction. While Ara h 1 linear IgE epitopes have been mapped, the presence and importance of structural epitopes is not clear.

Methods and results: Immunoblot, ELISA, circular dichroism (CD) and molecular modeling were used to compare and monitor structural and subsequent IgE binding differences in Ara h 1 purified from raw (NA1) and roasted peanuts (RA1) and denatured Ara h 1(DA1). NA1 and RA1 had the same secondary structure, the latter bound significantly higher IgE. A concurrent drop in IgE binding was seen with a loss in the secondary structure. This same IgE binding pattern [roasted > raw > denatured] was seen for 10 patient sera. While the majority of linear epitopes are located on surface and structured regions of Ara h 1, our study shows that structural epitopes of Ara h 1 bind more strongly to IgE than linear epitopes.

Conclusions: Enhanced IgE binding to roasted Ara h 1 could be due to alterations such as chemical modifications to individual amino acids or increased epitope exposure. IgE binding is significantly reduced with loss of structure.

 

3) Novel nut allergen IgE epitopes include a discrete group that correlates with cross-reactivity between peanut and walnut (paper in preparation)

Background. About 30% of peanut allergic patients report a co-allergy to walnut. The Property distance (PD) scale in the Structural Database of Allergenic Proteins (SDAP) recognizes sequences in other allergens that are similar to experimentally defined IgE epitopes .

Objective: Determine IgE epitopes distinctive for peanut or walnut allergy, and those that correlate with clinically significant cross reactivity.

Methods: Previously identified IgE epitopes of the peanut allergens Ara h 1 and Ara h 2, and walnut sequences that were similar (low PD) to these were identified with the PD tool in SDAP. Mimotopes were designed using 3D-strucures. Selected sequences were synthesized on SPOTs membranes and tested for reactivity with IgE in sera from six patients with clinically significant allergy (anaphylactic response) to peanut, walnut or both.

Results: All six sera bound to seven peptides, of which four were from the allergens Ara h 1, Ara h 3, and Jug r 2, and three were mimotopes of known epitopes. Patients with severe allergy only to peanut recognized three additional peptides from Ara h 1 and Ara h 4. Those cross reactive to peanuts and walnuts recognized the first group of peptides, four additional peptides from Ara h 1, Ara h 2, Ara h 4, and four from Jug r 2, where three of these had a low PD to a known Ara h 1 epitope.

Conclusions: These selected IgE epitopes could aid in the design of allergen vaccines or targeted elimination to reduce the allergenicity of nut proteins.

 

 

Figure 1. A Spots membrane was developed with controls and several patient sera
to identify peptides that bound IgE from patients with cross-reactivity between 
peanuts and walnuts. This membrane retested peptides that had been positive
with at lest one of these sera on a first, larger membrane. The membrane was stripped 
and regenerated before each new patient tested (in the order shown here). Sera
from 6 peanut and/or walnut allergic patients were used to identify three different 
types of peptides, and specifically a group of peptides that were only recognized
by the cross-reactive patients. Further confirmation of these results, using peptide
microarrays and many more patient sera, would be the goal of a future study. 

 

The complete details of all technical aspects of the project--both negative and positive--the recipient's findings, conclusions, and results, including the associated quality assurance results. The PI and co-investigators were responsible for quality control of distinct project elements:
-Dr. Catherine H. Schein, PI, was responsible for the overall quality assurance (QA) and quality control (QC) aspects of the research and for smooth integration of the computational and experimental aspects of the program.  She is an Associate Professor with primary appointment in the Department of Biochemistry and Molecular Biology, with a secondary appointment in Microbiology and Immunology. She is an expert in the sequence and structure of allergenic proteins, was one of the founders of the Structural Database of Allergenic Proteins (SDAP), and is experienced in using the tools constructed for that site. She and her group determined sequences likely to be novel epitopes using the well defined Property Distance (PD) tool that is incorporated in the SDAP, and compared the surface properties of the sequences and structures of epitopes of the nut proteins. She designed the membranes, ordered from a commercial source that had with differing versions of the IgE epitopes to be tested. She also supervised the synthesis of individual peptides for antibody binding experiments and determined their concentration and methods to solubilize them for the studies.

- Serum samples were primarily from the serum bank at UC-Davis, under the direction of Dr. Susanne Teuber, MD, professor of Medicine and expert in tree nut allergies.  Dr. Teuber related the clinical phenotype of the patient sera to immunoblotting, and later for correlated this with the results of the individual epitopes detected. The basophil activation assays were also done in Dr. Teuber’s group.

- The immunoblotting and competitive ELISA experiments were carried out in the group of Dr. Soheila Maleki, an expert in peanut allergy and in processing effects on peanut allergenicity.  She and her technical assistant used procedures already in place to insure that data were well maintained (by photographing the blots and storing them as a PowerPoint file) and freely available to the other group members.

Conclusions:

1. One of the most dangerous and least understood factors in food allergy is how high the degree of sequence or structural similarity must be in order to have a high likelihood of cross-reactivity between food sources. While some have suggested that allergens in foods should have a high percent identity in their amino acid sequences, we have demonstrated that small segments of identity, if these are in an IgE binding area, could lead to cross reactivity.

   In this research we chose to compare walnut and peanut allergens, as cross reactivity often is observed between these "nuts", even though they come from quite different botanical sources and their allergenic proteins have a low degree of identity. We used computational methods to identify areas of walnut allergens that were similar in their physicochemical properties and surface exposure to IgE epitopes of peanut proteins. We then made these peptides and showed that certain of the predicted epitopes in walnut allergens bound to IgE in sera of cross-reactive patients as well as previously identified epitopes of peanut. We also found that the cross-reactive sequences tended to be repeats of similar sequences, and that a sequence that was a consensus of these similar repeats detected proteins in many different nut extracts. These last findings suggest that certain determinants of cross reactivity must be ancient sequences that have been maintained in many different tree nuts and seeds perhaps due to their specific properties.

   The methods we used can now be applied directly to many different patient sera, using a new experimental method, peptide microarrays. We obtained a great deal of information from these preliminary trials, and found that the SPOTs method gave reproducible results that could be enlarged upon using another method, competitive ELISA. However, we were limited in the number of patient sera we could characterize due to the cost of the SPOTs membranes, and the need to wash and regenerate them between tests of each patient sera. In future studies, assuming we can find additional funding, we plan to use peptide microarrays, where we can test hundreds of different peptides, using sera from hundreds of patients. It is this type of assay that will become the standard for testing the spectrum of allergens a patient reacts to specifically in the next few years.

2. The primary benefits of this research are both in limiting exposure to allergens in the environment, and in guidelines for handling patients with severe allergy. Distinguishing allergens from related proteins that present little danger to the environment is a major goal of our research groups. There are many proteins similar to known allergens in all common foods and in other environmental aerosols, such as tree pollens, dust and molds. Simply removing these homologous proteins from all these different sources is not possible, but these results may guide the production of hypoallergenic proteins. However, we can counsel patients to avoid certain foods that they are most likely to react to, based on the presence in their sera of IgEs that recognize common sequences. Similarly, many companies now are using proteins in foods to enhance their properties, or even preparing protein preparations and vaccines for injection into humans. Before these products are approved, it is vital to be able to determine their allergenic potential. We have established methods to compare novel proteins to known allergens, so as to evaluate the risks to the public and to those directly involved in producing or processing them. Here we have further identified specific markers in the sequences of these proteins that might classify them as particularly virulent allergens.

   Our promising results, which show that there are certain common areas of allergenic proteins that could mediate cross-reactions, will be most valuable if we are able to verify them in future studies with tests of many more patient sera. We have shown that we can distinguish IgE epitopes that are common to many nuts. 

   We used protein models for some of the parts of the allergens, and for comparison of peanut and walnut allergen structures in our papers and posters. All protein models for this project either used the ones that are available through the SDAP website ( SDAP 2.0 - Structural Database of Allergenic Proteins) or were done using the MPACK Suite as described in the references.

   Model quality was routinely checked with Procheck. The % Identity of the template to the model was given in papers.

References:

Supplemental Keywords:

Relevant Websites:

SDAP 2.0 - Structural Database of Allergenic Proteins Exit

Progress and Final Reports: