Determination of Crucial Immunogenic Epitopes in Major Peanut Allergy Protein, Ara h2, via Novel Nanoallergen Platform

Abstract

Current methods for detection and diagnosis of allergies do not provide epitope specific immunogenic information and hence lack critical information that could aid in the prediction of clinical responses. To address this issue, we developed a nanoparticle based platform, called nanoallergens that enable multivalent display of potential allergy epitopes for determining the immunogenicity of each IgE binding epitope. By synthesizing nanoallergens that present various epitopes from the major peanut allergen, Ara h2, we directly determined the immunogenicity of each epitope, alone and in combination with other epitopes, using patient sera. This information provided insights on which epitopes are most critical for physiological responses to Ara h2 and revealed the importance of both high and low affinity epitopes for allergic responses. We anticipate the nanoallergen platform to be used to provide information regarding allergic reactions and therefore potentially aid in more accurate diagnosis and design of personalized treatment options.

Figure 1

Nanoallergen design, synthesis and characterization. (A) Epitope-lipid conjugate design for presenting Ara h 2 epitopes on nanoallergens is shown where epitope is peptide mimetic of IgE binding epitope of Ara h 2. The crystal structure of Ara h 2 with major IgE binding epitopes are color coded. Note that epitope 5 and 8 are not shown due to their presence on the N and C terminus of the protein respectively. (B) Schematic of nanoallergen synthesis. Epitope-lipids were synthesized and purified as stated above and then mixed with a PEG-lipid conjugate and DSPC at a ratio of 2:5:93 and extruded to form 100 nm particles. (C) Particle sizing of the nanoallergens was determined by DLS. Nanoallergen effective diameter was consistently 128 nm for the nanoallergens. A representative data is shown.

Figure 2

Cartoon representation of nanoallergen triggering degranulation compared to natural allergens. (A) Cartoon representation of the crosslinking events induced by the natural peanut allergen protein, Ara h 2 (with different colors labeling potential epitopes on the protein and their corresponding IgEs), leading to mast cell degranulation. (B) Nanoallergens are designed to present immunogenic epitopes to trigger crosslinking and degranulation in a similar fashion as natural allergens. This cartoon was adapted from Deak et al..

Figure 3

Nanoallergens are designed to present Ara h 2 epitopes and trigger degranulation. (A) IgEs specific to Ara h 2 protein and Ara h 2 epitope 2 were identified in sera taken from a peanut sensitive patient using an ELISA assay. Binding to their respective antigens was determined by comparing maximum fluorescence values, which demonstrated that approximately 10% of Ara h 2 IgEs were specific for epitope 2. (B) Epitope 2 was loaded into nanoallergens at a 2% concentration and used to stimulate degranulation in RBL-SX38 cells that were primed with 10% serum from a peanut allergy patient. (C) Epitope 2 loaded nanoallergens were incubated with RBL-SX38 cells and a western blot analysis was performed on cell lysates to examine SHIP phosphorylation. (D) Control experiments performed in the presence of 50 μM free epitope peptide 2 or when blank liposomes were used demonstrated almost complete inhibition of degranulation.

Figure 4

Nanoallergens demonstrate immunogenicity of Ara h2 IgE binding epitopes 1–8 with patient sera. Nanoallergens were loaded with 2% of each Ara h 2 IgE binding epitope and used to trigger degranulation with RBL-SX38 cells primed with 10% serum 1. Colored stars indicate concentration at which degranulation occurred at a significant level above blank liposome control (p < 0.01).

Figure 5

Heterogeneous nanoallergens presenting a combination of Ara h 2 epitopes reveal which epitopes are the most crucial for degranulation. (A) Epitope 2 and 3 were mixed at varying ratios in nanoallergen formulations to examine degranulation responses. (B) Nanoallergens presenting epitope 2 was combined with other immunoreactive epitopes (3,5,6,7) while maintaining a constant total epitope loading of 2% and used to trigger degranulation. (C) A nanoallergen with a mixture of epitope 2,3 and 5 was used to trigger degranulation with epitope 2 or 3 or 5 omitted from the formulation or in the presence of free epitope 2 peptide (50 μM). Stars indicate p < 0.05.

Figure 6

SHIP phosphorylation varies for different nanoallergen formulations. Western blots were performed using RBL-SX38 cells sensitized with patient sera 1. Ara h 2 or nanoallergen formulations as indicated (loaded with 2% total epitope-lipid at 1:1 ratios or 1:1:1 ratios) were added at 100 pM. Graph indicates both EC₅₀ value (on right axis, colored blue) and maximum degranulation observed with nanoallergen formulation (on left axis, colored red). Gels were cropped for clarity, see Fig. S3 for full length blots. Stars indicate p < 0.05, note that for epitope 3,5 nanoallergen EC₅₀ > 1000 pM.

Figure 7

Nanoallergens reveal crucial epitopes in a clinical population. Four patient sera were tested for degranulation response with nanoallergens presenting Ara h2 epitopes. (A) EC₅₀ values of the degranulation assays are shown. Note that epitope 4 and 8 did not demonstrate any degranulation in any of the patient sera. A’s indicate a EC₅₀ values between 5000–2500 pM, B’s indicate a EC₅₀ values > 5000pM. (B) D_max values for degranulation assays.