Irradiation does not impact the immunogenicity of food flours used for oral immunotherapy

Abstract

Background

Peanut and tree nut flours are considered drug products by the US Food and Drug Administration when used during oral immunotherapy clinical trials and oral food challenges and must meet specific standards for microbial contamination and allergen content. Gamma irradiation of food flours can reduce microbial contamination, but its effects on food allergen content and immunogenicity remain unclear.

Objective

We sought to investigate whether gamma irradiation of peanut and tree nut flours alters allergen content and immunogenicity.

Methods

Commercially available peanut, cashew, walnut, hazelnut, and almond flours underwent gamma irradiation. Allergen content and stability, as well as microbial contamination, in irradiated and nonirradiated flours were measured over a 12-month period. The immunogenicity of flour extracts was assessed by basophil activation tests, T-cell activation-induced marker assays, and T_H cytokine production using whole blood or PBMCs from peanut- or tree nut–sensitized individuals.

Results

Irradiation of peanut and tree nut flours decreased microbial contamination while not affecting allergen content or stability for 12 months. Irradiated and nonirradiated flours were similar in their ability to activate basophils and allergen-reactive CD4⁺ effector and regulatory T cells from sensitized individuals. Irradiated and nonirradiated flours induced comparable T_H1, T_H2, T_H17, and regulatory T- cell cytokine production by PBMCs from sensitized individuals.

Conclusions

Irradiation of peanut and tree nut flours decreases microbial contamination without significantly altering allergen content or immunogenicity. Irradiated food flours are likely effective for use in oral immunotherapy studies and oral food challenges.

IgE-mediated peanut and tree nut allergies affect roughly 1% to 2% of the US population and are among the most common causes of anaphylaxis in children and young adults.^1,2 Peanut and tree nut allergies are usually lifelong and pose a significant clinical and financial burden for patients and their families.^3,4 Oral immunotherapy (OIT) using peanut and tree nut flours is highly efficacious at desensitizing patients with peanut^5,6 and tree nut7, 8, 9 allergies. Peanut and tree nut flours are also routinely used during oral food challenges (OFCs) required for inclusion and demonstration of clinical efficacy in therapeutic trials for food allergy.¹⁰ Food flours are considered oral drug products by the US Food and Drug Administration (FDA) when used for OIT or OFC in clinical trials and must meet specific standards for microbial contamination and allergen content.¹¹ Although Peanut Allergy Powder-dnfp (Palforzia) is an OIT product approved by the FDA, there are currently no FDA-approved tree nut products for oral delivery. The lack of standardized tree nut flours that meet FDA requirements for oral drugs is a barrier to conducting OIT trials and other interventional studies for food allergy.

We have previously described a process for preparing and characterizing peanut and tree nut flours for use in OIT clinical trials that meets FDA standards for allergen stability and bioburden testing for investigational drugs.^11,12 To address high bioburden levels of bacteria, yeast, and mold found in commercially sourced tree nut flours, we subjected the flours to gamma irradiation, a technique commonly used to reduce microbial contamination and prolong the shelf life of foods.¹³ Gamma irradiation significantly reduced microbial contamination in tree nut flours without affecting total protein or allergen content over a 24-month period.¹² Although our findings indicated that irradiation could generate food flours that met FDA standards for oral drug products, it is possible that irradiation may affect food allergen immunogenicity by altering protein structure and modifying antigen epitopes.¹⁴ Irradiation-induced alterations in food protein structure could reduce IgE binding, resulting in decreased mast cell or basophil activation and degranulation.¹⁵ Irradiation could also induce oxidative and structural modifications in food proteins that affect antigen processing and presentation, thereby affecting recognition by allergen-specific T cells.¹⁶ Indeed, gamma irradiation has been reported to diminish the immunogenicity of peanut and tree nut allergens by altering B-and T-cell epitopes.17, 18, 19 Although reducing allergen immunogenicity may be beneficial for decreasing clinical reactivity to foods, it could result in inconsistent or inaccurate findings when using irradiated food flours for OFCs in clinical studies. Furthermore, altering the immunogenicity of peanut or tree nut flour could inadvertently affect the efficacy of OIT, which is thought to rely on the stimulation and modulation of allergen-specific CD4⁺ T_H cells.²⁰ There is currently no FDA-specific guidance for assessing the effects of irradiation on allergens. Therefore, we investigated whether gamma irradiation of peanut and tree nut flours, including cashew, walnut, hazelnut, and almond, altered their immunogenicity using ex vivo human basophil activation tests (BATs) and T-cell stimulation assays.

Methods

Irradiation of peanut and tree nut flours

Light roast (12% fat) peanut flour was purchased from Hoosier Hill Farm (Middleton, Wis). Defatted hazelnut flour was purchased from Holmquist Hazelnut Orchards, LLC (Lynden, Wash). Blanched, extra-fine almond flour was purchased from Honeyville (Spanish Fork, Utah). Raw cashew flour was purchased from Nuts.com. Raw, defatted walnut flour was purchased from Live Oil by Lesna Corporation. All bulk flours were accepted after visual inspection and review of the accompanying certificate of analysis from the vendors to confirm the flour identity. Flours were stored at 2°C to 8°C. Fourteen pounds of each flour sample was sent to Sterigenics (Haw River, NC) for gamma irradiation at 2.0 to 10.0 kGy. Following irradiation, the flours were shipped back to our laboratory and prepared into aqueous extracts within 2 weeks of receipt. Aqueous extracts from the matching nonirradiated flour fractions were also prepared at the same time. For preparation of aqueous extracts, irradiated or nonirradiated flours were mixed with PBS at a defined ratio of weight to volume (1:4 wt/vol for peanut flour and 1:5 wt/vol for cashew, walnut, hazelnut, and almond flours). Walnut was mixed with a PBS solution containing an additional 1 mol NaCl to improve extraction efficiency. Suspensions were stirred for 90 minutes while maintaining a constant pH of approximately 8.5 using 6 molar NaOH. The resulting extracts were centrifuged at 30,000g at 4°C for 45 minutes. Supernatants were filtered using 0.45-μm and then 0.2-μm filters. Finally, protein concentrations were determined by bicinchoninic acid assay. The aqueous extracts were then analyzed for allergen content immediately (0-month time point).

Bioburden, toxin, and total protein content testing

A 160-g sample of each flour was weighed in the Good Manufacturing Practice environment and shipped for overnight delivery to Deibel Laboratories (Niles, Ill), where it was tested at 0, 3, 6, 9, and 12 months following irradiation. All bioburden, toxin, and total protein content evaluations were conducted at Diebel Laboratories using United States Pharmacopeia guidelines for defining the acceptance criteria for the preparation of an orally administered drug as required by our Investigational New Drug application with the FDA (see Table E1 in this article’s Online Repository at www.jaci-global.org).^11,12,21 Specifically, the following parameters were measured by Diebel Laboratories: aerobic plate count, yeast, mold, Escherichia coli, Salmonella, aflatoxin, moisture, water activity, and total protein content.

Measurement of allergen content

Densitometric scanning from SDS-PAGE of key allergens was used to evaluate allergen content between irradiated and nonirradiated samples at 0, 3, 6, 9, and 12 months following irradiation. Allergenic proteins used for analysis included Ara h 1 and 2 from peanut, Cor a 11 and 14 from hazelnut, Pru du 6 from almond, Ana o 1 and 3 from cashew, and Jug r 1 and 2 from walnut flour samples. SDS-PAGE was conducted with 10 μg protein extract per lane. An image of the gel was captured with a camera system, and densitometric scanning was performed on the image using iBright Analysis Software, version 5.4.0 (Thermo Fisher Scientific). The absolute percent variance between irradiated and nonirradiated samples was calculated.

Allergen content from the flours was evaluated by quantifying the allergen components Ara h 1, Ara h 3, Ara h 6, Ana o 3, Cor a 9, Jug r 1, and Pru du 6 using the MARIA Custom 8-plex Multiplex Array (InBio, Charlottesville, Va) according to the manufacturer’s instructions. Ara h 2 was quantified using the InBio Ara h 2 ELISA kit (EL-AH2) according to the manufacturer’s instructions. The allergen extracts were diluted to fall within the linear range of the assays, as recommended by the manufacturer’s instructions.

Blood samples and processing

Whole blood was collected in sodium heparin tubes by venipuncture from peanut- and tree nut–sensitized individuals who participated in the institutional review board–approved Blood Repository for Food Allergic Patients study at the University of North Carolina at Chapel Hill. Sensitized individuals were defined as having allergen-specific IgE greater than or equal to 0.35 kU/L, as determined by ImmunoCAP (Thermo Fisher Scientific), or a skin prick test mean wheal size of 3 mm or higher (after subtraction of the saline control) to peanut, cashew, walnut, hazelnut, or almond. Whole blood was used for either BATs or isolation of PBMCs within 24 hours of collection. For PBMC isolation, whole blood was first treated with RosetteSep Human Granulocyte Depletion Cocktail (STEMCELL Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’s instructions. PBMCs were then isolated by density centrifugation with Ficoll-Paque PLUS (Cytiva, Uppsala, Sweden) and cryopreserved in heat-inactivated human AB serum (BioIVT, Hicksville, NY) containing 10% dimethyl sulfoxide in liquid nitrogen.

Basophil activation tests

Whole blood was used for BATs as previously described.²² Briefly, 50 μL of whole blood was incubated with the same volume of basophil media (RPMI with 2 ng/mL human IL-3; Invitrogen, Carlsbad, Calif), polyclonal anti-IgE (1 μg/mL; Bethyl Labs, Montgomery, Tex), N-formyl-methionyl-leucyl-phenylalanine (1 μM; Tocris Biosciences, Avonmouth, Bristol, United Kingdom), or irradiated or nonirradiated peanut or tree nut flour extracts diluted in basophil media. The peanut and tree nut extracts were prepared from the food flour materials at 0 months and stored at −20°C until use. Stimulations were performed with 10,000, 1,000, 100, 10, or 1 ng/mL of each extract, or the controls, for 30 minutes at 37°C and 5% CO₂, and then stopped with 20 mM EDTA. Cells were stained for 30 minutes with anti-CD123–BV421 (clone 9F5; BD Biosciences, San Jose, Calif), anti-CD203c–phycoerythrin (clone 97A6; Beckman Coulter, Brea, Calif), and anti-CD63–fluorescein isothiocyanate (clone H5C6; BD Biosciences). Data were acquired on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed with FlowJo software (Ashland, Ore). Basophils were identified as CD123⁺CD203c⁺ cells within the lymphocyte gate, and activation was expressed as the percentage of CD63⁺ basophils. Area under the dose-response curve was calculated using GraphPad Prism 10.1.1 (GraphPad Software, San Diego, Calif).

T-cell assays

Cryopreserved PBMCs were thawed, washed, resuspended in AIM (activation-induced marker) V medium (Gibco, Waltham, Mass) containing 5% human AB serum and rested overnight at 37°C and 5% CO₂. The following morning, cells were collected and resuspended in fresh media for use in AIM assays or T_H cytokine studies.

For AIM assays, 1 × 10⁶ PBMCs were cultured with allergen extracts (200 μg/mL) prepared from nonirradiated or irradiated peanut or tree nut flours, media alone, or concanavalin A (10 μg/mL) in 96-well round-bottom plates at 37°C and 5% CO₂. Human anti-CD40 mAb (clone HB14; BioLegend, San Diego, Calif) was added at a concentration of 1 μg/mL to maintain CD154 at the cell surface. After 24 hours, the cells were harvested and stained with Zombie Aqua Fixable Viability dye (diluted at 1:2500 in PBS) for 15 minutes. The cells were then washed, blocked with Human TruStain Fcx (BioLegend), and stained with anti-CD3–Alexa Fluor 488 (clone SK7), anti-CD4–PerCP/Cy5.5 (clone RPA-T4), anti-CD45RA–Spark UV 387 (clone HI100), anti-CD154–phycoerythrin (clone 24-31), anti-CD69–BV421 (clone FN50), and anti-CD137–allophycocyanin (clone 4B4-1) antibodies (all purchased from BioLegend) for 30 minutes at 4°C. The cells were then washed, fixed with 4% paraformaldehyde, and acquired using an LSRFortessa cytometer (BD Biosciences, Franklin Lakes, NJ). Flow-cytometric data were analyzed using FlowJo software. Allergen-reactive T effector (T_eff) cells and regulatory T (T_reg) cells were identified as CD154⁺CD69⁺ cells and CD137⁺CD154⁻ cells, respectively, after gating on viable CD3⁺CD4⁺CD45RA⁻ cells. PBMCs from nonsensitized individuals were included as controls and did not significantly upregulate AIMs following stimulation with nonirradiated or irradiated allergen extracts.

For allergen-induced T_H cytokine studies, 0.5 × 10⁶ PBMCs were cultured with allergen extracts (200 μg/mL) prepared from nonirradiated or irradiated peanut or tree nut flours, media alone, or concanavalin A (10 μg/mL) in 96-well round-bottom plates at 37°C and 5% CO₂ for 72 hours. Cell culture supernatants were then collected, and T_H cytokines (IFN-γ, TNF, IL-2, IL-4, IL-5, IL-9, IL-13, IL-17A, IL-17F, IL-22, and IL-10) were measured using the Meso Scale Discovery U-PLEX T-cell Combo (human) multiplex assay kit (Meso Scale Technologies, Rockville, Md).

Statistical analysis

Comparisons between irradiated and nonirradiated flours were made using paired parametric t tests, with P values less than .05 considered significant. On the basis of pilot-derived estimates of variability for our BATs and AIM assays, sample sizes of 4 and 7 paired donor blood samples, respectively, would provide approximately 80% power to detect a 50% difference between nonirradiated and irradiated groups. Multiplicity corrections were not performed intentionally to allow for the most generous definition of significance in comparisons between the irradiated and nonirradiated flours. Statistical analyses were performed using GraphPad Prism 10.1.1.

Results

Gamma irradiation of peanut and tree nut flours diminishes microbial contamination without affecting allergen content or stability

We first verified that gamma irradiation did not alter protein or major allergen content in peanut and tree nut flours. Commercially available peanut, cashew, walnut, hazelnut, and almond flours were subjected to gamma irradiation or left untreated. Aqueous protein extracts were then prepared from nonirradiated and irradiated food flours and analyzed by SDS-PAGE to quantify major allergen content. Consistent with our previous report,¹² we found that irradiation did not appear to alter major allergen content in peanut or tree nut flours (Fig 1, A). We also measured the levels of major food allergens using multiplex immunoassays and found no differences between nonirradiated and irradiated food flours (Fig 1, B). We next assessed the stability of total protein and major allergens in irradiated flours stored at 2°C to 8°C for 12 months by SDS-PAGE analysis. The percent variance (ie, percent difference using nonirradiated flours as the reference) in total protein and major allergen levels between nonirradiated and irradiated flours remained stable (≤30%) over 12 months as previously defined in our Investigational New Drug application with the FDA^11,12 (Table I).

Fig 1.

Irradiation of food flours does not alter allergen protein content. A, Protein extracts of nonirradiated and irradiated food flours were analyzed by SDS-PAGE in duplicate. L, Molecular weight ladder. B, Major allergen concentrations were measured by immunoassays in protein extracts of nonirradiated and irradiated food flours. Bars represent mean (duplicate samples) ± SD.

Table I.

Percent variance of total protein and major allergens in irradiated flours compared with nonirradiated flours at 0-12 mo following irradiation

Food flour	0 mo	3 mo	6 mo	9 mo	12 mo	Average
Peanut
Total protein	0.13	0.83	6.77	10.15	12.21	6.02
Ara h 1	3.34	7.83	3.98	12.91	4.66	6.54
Ara h 2	6.42	19.13	14.69	11.45	15.52	13.44
Cashew
Total protein	2.96	2.31	28.07	9.08	5.59	9.60
Ano o 1	1.03	3.89	8.87	15.50	8.56	7.57
Ano o 3	3.84	7.32	0.95	1.87	5.34	3.87
Walnut
Total protein	0.00	1.19	1.81	2.42	16.53	4.39
Jug r 1	4.72	10.37	16.11	5.43	7.50	8.83
Jug r 2	0.09	2.80	5.31	6.38	19.61	6.54
Hazelnut
Total protein	2.15	1.83	30.05	0.96	1.12	7.22
Cor a 11	7.13	15.51	3.89	5.99	11.07	8.72
Cor a 14	11.35	12.85	3.30	0.53	12.49	8.10
Almond
Total protein	1.99	0.14	3.57	1.38	6.68	2.75
Pru du 6	0.54	2.52	0.83	3.52	2.92	2.07

We next assessed the effects of irradiation on FDA-required bioburden testing (aerobic bacteria plate count, yeast, mold, E coli, Salmonella, and aflatoxin) for orally delivered drug products. Nonirradiated tree nut flours had detectable aerobic plate counts, mold, and yeast throughout the 12-month period (Table II). In contrast, these microbes were largely undetectable in irradiated food flours over a 12-month period (Table II). In summary, gamma irradiation of peanut and tree nut flours reduced bioburden without affecting the stability of major food allergens over a 12-month period.

Table II.

Bioburden analysis of nonirradiated and irradiated flours at 0-12 mo following irradiation

Food flour	Nonirradiated					Irradiated
	0 mo	3 mo	6 mo	9 mo	12 mo	0 mo	3 mo	6 mo	9 mo	12 mo
Peanut
APC (cfu/g)	<10	<10	<10	<10	<10	<10	<10	<10	<10	<10
Mold (cfu/g)	<10	<10	<10	<10	<10	<10	<10	<10	<10	<10
Yeast (cfu/g)	<10	<10	<10	<10	<10	<10	<10	<10	<10	<10
E coli	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Salmonella	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Aflatoxin (ppb)	<5	<5	<5	<5	<5	<5	<5	<5	<5	5.1
Cashew
APC (cfu/g)	<10	30	60	30	20	40	<10	<10	<10	<10
Mold (cfu/g)	<10	<10	<10	10	<10	10	<10	<10	<10	<10
Yeast (cfu/g)	<10	20	10	<10	<10	10	<10	<10	<10	<10
E coli	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Salmonella	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Aflatoxin (ppb)	<5	<5	<5	<5	<5	<5	<5	<5	<5	<5
Walnut
APC (cfu/g)	140	100	150	110	80	10	<10	<10	<10	<10
Mold (cfu/g)	40	20	30	40	10	<10	<10	<10	<10	<10
Yeast (cfu/g)	<10	<10	20	<10	<10	<10	<10	<10	<10	<10
E coli	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Salmonella	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Aflatoxin (ppb)	7.7	7.8	6.5	11.7	12.6	12.7	7.1	7.8	10.2	5.1
Hazelnut
APC (cfu/g)	20	50	10	210	70	<10	<10	<10	<10	<10
Mold (cfu/g)	10	30	<10	10	10	<10	<10	<10	<10	<10
Yeast (cfu/g)	<10	<10	10	<10	<10	<10	<10	<10	<10	<10
E coli	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Salmonella	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Aflatoxin (ppb)	<5	<5	<5	<5	<5	<5	<5	<5	<5	<5
Almond
APC (cfu/g)	>2500	>2500	>2500	>2500	1310	10	<10	<10	<10	<10
Mold (cfu/g)	<10	10	130	<10	<10	<10	<10	<10	<10	<10
Yeast (cfu/g)	<10	<10	10	<10	<10	<10	<10	<10	<10	<10
E coli	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Salmonella	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
Aflatoxin (ppb)	<5	<5	<5	<5	5.1	7.2	<5	<5	<5	<5

APC, Aerobic plate count.

Gamma irradiation does not significantly alter the ability of peanut and tree nut flours to trigger IgE-dependent basophil activation

Gamma irradiation of allergens may alter their ability to bind IgE and trigger basophil or mast cell degranulation,^19,23 potentially affecting clinical reactivity during OFCs using irradiated food flours. We therefore investigated whether irradiation of peanut and tree nut flours altered their ability to bind to and cross-link membrane-bound IgE using BATs. Whole blood samples from individuals sensitized to peanut (n = 10), cashew (n = 11), walnut (n = 6), hazelnut (n = 5), or almond (n = 4) were treated with the respective allergen extract prepared from nonirradiated or irradiated flours, and basophil activation based on CD63 expression was determined by flow cytometry. Both nonirradiated and irradiated flour extracts triggered basophil activation in a dose-dependent manner (Fig 2, A). There were no statistically significant differences between nonirradiated and irradiated flours in their ability to activate basophils as determined by calculating the area under the dose-response curve (Fig 2, B). We also did not detect a statistically significant difference in the maximal basophil activation, as measured by the percentage of CD63⁺ cells (see Fig E1 in this article’s Online Repository at www.jaci-global.org).²⁴ Thus, we did not detect any negative effects of irradiation on the ability of peanut or tree nut allergens in food flours to bind to IgE and induce basophil activation.

Fig 2.

Irradiation does not alter the ability of food flours to cross-link IgE on basophils. A, Representative results of BATs performed using whole blood from sensitized individuals stimulated with nonirradiated or irradiated flours. B, AUC of the BAT dose-response curve for nonirradiated and irradiated flours. Comparisons were performed using paired t tests. AUC, Area under the curve.

Nonirradiated and irradiated peanut and tree nut flours stimulate allergen-specific T cells similarly

Gamma irradiation of food allergens has been reported to modify T-cell responses in mice.^16,17,25 However, whether irradiation affects human allergen-specific T-cell responses remains unclear. We therefore investigated the ability of irradiated food flours to stimulate allergen-reactive T cells from sensitized individuals using flow-cytometric AIM assays.^26,27 PBMCs from individuals sensitized to peanut (n = 16), cashew (n = 17), walnut (n = 8), hazelnut (n = 10), or almond (n = 9) were stimulated with allergen extracts prepared from nonirradiated or irradiated flours in the presence of anti-CD40 antibody. After 24 hours, the activation of allergen-reactive CD4⁺ T effector cells (T_eff) was determined by the upregulation of CD154 and CD69, as measured by flow cytometry (Fig 3, A). We also assessed the ability of irradiated flours to stimulate allergen-reactive CD4⁺ T_reg cells by measuring upregulation of CD137 expression on CD154⁻ cells using flow cytometry (Fig 3, A). We did not detect any statistically significant differences between nonirradiated and irradiated peanut and tree nut flours in their ability to stimulate allergen-reactive T_eff and T_reg as determined by AIM assays (Fig 3, B). Thus, irradiated peanut and tree nut flours activate human allergen-specific CD4⁺ T cells in a similar manner.

Fig 3.

Nonirradiated and irradiated food flours similarly stimulate allergen-reactive CD4⁺ T_H cells. AIM assays were performed on PBMCs from sensitized subjects stimulated for 24 hours with nonirradiated or irradiated flours. A, Representative AIM assay results using PBMCs from a sensitized subject stimulated with nonirradiated flours (gated on CD4⁺CD45RA⁻ T cells). Allergen-reactive T_eff and T_reg cells were identified as CD154⁺CD69⁺ and CD137⁺CD154⁻ cells, respectively. B, Stimulation of allergen-reactive T_eff (top graphs) and T_reg (bottom graphs) cells by nonirradiated and irradiated flours. The cell percentages shown are after subtraction of media-only controls. Comparisons were performed using paired t tests.

The ability of OIT to modulate allergen-specific CD4⁺ T_H cytokine responses is considered an important mechanism for inducing desensitization to food allergens.²⁰ Therefore, we investigated whether gamma irradiation of peanut or tree nut flours altered allergen-induced production of T_H cytokines. PBMCs from the same peanut- or tree nut–sensitized individuals analyzed in the AIM assays were cultured with allergen extracts prepared from nonirradiated and irradiated flours. After 72 hours, cell culture supernatants were collected and T_H1 (IFN-γ, TNF, and IL-2), T_H2 (IL-4, IL-5, IL-9, and IL-13), T_H17 (IL-17A, IL-17F, and IL-22), and T_reg (IL-10) cytokines were measured by a multiplex immunoassay. Overall, nonirradiated and irradiated peanut and tree nut flours induced similar T_H cytokine production by PBMCs (Fig 4). Irradiated peanut flour induced a slight increase in IL-13 production but did not affect the production of other T_H2 cytokines. Interestingly, irradiated cashew flour induced a moderate increase in IL-17F production, but not other T_H17 cytokines. Irradiated cashew also induced a slight decrease in IL-10 production. In summary, these results indicate that irradiation of food flours had a minimal effect on allergen-induced T_H cytokine production by PBMCs from sensitized individuals.

Fig 4.

Irradiation of food flours has minimal effect on allergen-induced T_H cytokine production. T_H cytokine levels were measured in supernatants from PBMCs stimulated with nonirradiated or irradiated flours for 72 hours. The numbers indicate the mean cytokine concentrations (after subtracting media-only controls). Comparisons were performed using paired t tests, with P values highlighted as indicated in the legend.

Discussion

Peanut and tree nut flours are administered for both therapeutic and diagnostic purposes during OIT trials and associated OFCs. Because OIT trials can extend for several months to years, food flour preparations must maintain FDA specifications for bioburden testing while also demonstrating allergen stability during long-term storage. We previously demonstrated that gamma irradiation of tree nut flours can reduce microbial bioburden to FDA-mandated levels without compromising protein stability.¹² However, the effects of irradiation on the immunogenicity of food flour were not assessed. In the present study, we confirmed that gamma irradiation of peanut and tree nut flours inhibits microbial growth without affecting the stability of major food allergens over a 12-month period. Importantly, we show that gamma irradiation of peanut and tree nut flours does not alter their immunogenicity as determined by their ability to stimulate basophils and T cells from allergen-sensitized individuals. Taken together, these findings indicate that gamma irradiation of food flours is an effective method for reducing microbial bioburden without affecting the stability or immunogenicity of food allergens.

Irradiation of foods is considered a safe and effective method for eliminating potentially harmful microbes and extending shelf life.¹³ Gamma irradiation is regularly used during the commercial production of peanuts and tree nuts, which are frequently contaminated with aflatoxin-producing molds and microbial pathogens, including E coli and Salmonella.²⁸ Indeed, we found that tree nut flours contained high levels of aerobic bacteria, mold, and yeast, which were dramatically decreased for 12 months following gamma irradiation. Although E coli or Salmonella was not detected in the peanut or tree nut flours used in the present study, we have previously detected these pathogens in other tree nut flour preparations and demonstrated that they are also reduced following gamma irradiation.¹² Although gamma irradiation decreased microbial contamination, it had little effect on the total protein or major allergen content of peanut and tree nut flours over a 12-month period. The stability of allergens in irradiated food flours is particularly important for OIT and OFC protocols, because significant changes over time could result in subtherapeutic dosing or inaccurate challenge results.

Although gamma irradiation is effective at reducing microbial contamination in food products, it has also been used to reduce the immunogenicity of food allergens.^14,29 Gamma irradiation can alter secondary and tertiary protein structure, potentially disrupting antigenic epitopes recognized by the adaptive immune system.¹⁴ Exposure of whole peanut extract to gamma irradiation at doses similar to those used in our studies (1-10 kGy) was reported to disrupt the molecular structure of the major peanut allergen Ara h 6, thereby reducing its binding to specific IgG.¹⁸ Gamma irradiation (3-10 kGy) of the major cashew allergen Ano o 3 was also reported to decrease its ability to bind IgE and trigger degranulation of a human basophil cell line.¹⁹ However, another study found that gamma irradiation of peanut powder at 25 kGy did not reduce the binding of specific IgE from peanut-allergic individuals.³⁰ We also found that gamma irradiation (2-10 kGy) did not alter IgE binding to allergens, because irradiated and nonirradiated flours induced similar activation of basophils from allergen-sensitized individuals. The differing results across studies may be due to the physical state of the allergen during irradiation, because gamma irradiation was shown to have a greater effect on aqueous protein extracts than on solid proteins.²³ Gamma irradiation causes the formation of reactive oxygen species from water molecules, which can alter allergen structure by disrupting peptide bonds¹⁴; thus, differences in water content in peanut and tree nut flour preparations may also explain the disparate results between studies.

The modulation of allergen-specific CD4⁺ T-cell responses is likely an essential mechanism of OIT. Suppression of food-specific T_H2 responses is associated with clinical remission during OIT.^26,31, 32, 33 The induction and expansion of food-specific T_reg cells is also thought to occur during OIT, although evidence from human OIT trials has been mixed.^27,31,33, 34, 35 Animal studies suggest that gamma irradiation of proteins may alter antigen-specific T-cell responses, which could have implications for OIT using irradiated food flours. Splenocytes from mice immunized with irradiated ovalbumin showed reductions in ovalbumin-specific T-cell proliferation, secretion of T_H1 and T_H2 cytokines, and expression of T-cell activation markers.¹⁶ In addition, splenocytes from peanut-sensitized mice stimulated with gamma-irradiated peanut protein showed increased IFN-γ production, but decreased IL-4 production.¹⁷ In contrast, using AIM assays, we did not detect statistically significant differences in the ability of irradiated and nonirradiated peanut and tree nut flours to stimulate human allergen-reactive CD4⁺ T_eff and T_reg cells. Furthermore, irradiated and nonirradiated peanut and tree nut flours induced similar levels of T_H cytokine production in PBMCs from sensitized individuals. The few observed differences in T_H cytokine production were generally of low magnitude. One exception was the moderately increased levels of IL-17F in PBMC cultures stimulated with irradiated cashew flour. IL-17F binds to the same receptor complex as IL-17A, albeit with less signaling potency, and primarily contributes to fungal immunity at barrier surfaces.³⁶ The clinical significance of increased IL-17F levels is unclear, given that other T_H17 cytokines, such as IL-17A and IL-22, were not increased. Further studies are needed to determine the mechanisms by which irradiated cashew flour induces IL-17F and to assess its clinical significance.

Our study has several limitations. In our BATs and T-cell stimulation assays, we had a small sample size for some tree nuts, which may have made certain comparisons underpowered to detect differences. We also did not perform a complete characterization of peanut and tree nut flours for contaminating aeroallergens, such as mites, cockroaches, or pollens, nor did we assess all donors for sensitization to aeroallergens. It is possible that aeroallergen contamination of the peanut and tree nut flours could affect the BAT and T-cell stimulation assays in donors sensitized to aeroallergens. Future studies using inhibition microarray assays would be helpful for assessing aeroallergen contamination while also measuring major food allergen content in peanut and tree nut flours. It is possible that irradiation may have altered the integrity of allergen epitopes or the protein structure in ways that could not be detected by SDS-PAGE densitometry or allergen-specific immunoassays. Moreover, irradiation could have altered other facets of immunogenicity that were not measured in our cell-based assays, such as modification of IgG- or IgA-binding epitopes and antigen processing and presentation by dendritic cells. Finally, although our in vitro studies suggest that irradiated peanut and tree nut flours should be suitable for use in OIT and OFCs, this must ultimately be confirmed in vivo through clinical studies.

We found that irradiated peanut and tree nut flours retained their capacity to activate basophils and stimulate allergen-specific CD4⁺ T-cell responses, indicating they should be comparable with nonirradiated flours when used for OIT or OFCs. Indeed, OIT using irradiated wheat flour induced desensitization in individuals with wheat allergy,³⁷ providing further evidence that irradiated food products should be effective for OIT. Overall, our findings suggest that gamma irradiation of peanut and tree nut flours is an effective strategy to reduce microbial contamination without compromising their efficacy in clinical studies.

Key messages.

• •Gamma irradiation of peanut and tree nut flours diminishes microbial contamination to levels required by the FDA without affecting allergen content or stability.
• •Irradiation of peanut and tree nut flours does not alter their immunogenicity as demonstrated in ex vivo human BATs and T-cell stimulation assays.
• •Irradiated peanut and tree nut flours are likely effective for use in OIT clinical trials and OFCs.

Disclosure statement

This work was supported by the National Institutes of Health (NIH) (grant no. 5-UM1-AI130936-07). The University of North Carolina Flow Cytometry Core Facility is supported in part by a Cancer Center Core Support Grant to the University of North Carolina Lineberger Comprehensive Cancer Center (grant no. P30 CA016086). The study was also supported by the National Institute of Environmental Health Sciences (T32 Toxicology Training Grant no. 2T32ES007126-41 to J.H.). P.C.F.’s coauthorship of this publication does not constitute endorsement by the National Institute of Allergy and Infectious Diseases, the NIH, or any other agency of the US government.

Disclosure of potential conflict of interest: T. P. Moran receives research funding from the NIH and the American Heart Association. M. D. Kulis reports consultancy with Alpina Biotechnology, Inc.; and receives research funding from the NIH and the Department of Defense. Y. V. Virkud reports consultancy with the Food Allergy Research and Education advocacy organization; and receives research funding from the NIH. E. H. Kim reports consultancy with ALK-Abelló, Allergy Therapeutics, Cellergy Pharma, DBV Technologies, Genentech, GrowHappy, Hanimmune Therapeutics, IgGenix, Phylaxis BioScience, and Ukko; and receives funding from the NIH and the National Institute of Allergy and Infectious Diseases. The rest of the authors declare that they have no relevant conflicts of interest.