Homologous tropomyosins from vertebrate and invertebrate: Recombinant calibrator proteins in functional biological assays for tropomyosin allergenicity assessment of novel animal foods

Abstract Background Novel foods may provide new protein sources for a growing world population but entail risks of unexpected food‐allergic reactions. No guidance on allergenicity assessment of novel foods exists, while for genetically modified (GM) crops it includes comparison of sequence identity with known allergens, digestibility tests and IgE serum screening. Objective As a proof of concept, to evaluate non‐/allergenic tropomyosins (TMs) regarding their potential as new calibrator proteins in functional biological in vitro assays for the semi‐quantitative allergy risk assessment of novel TM‐containing animal foods with mealworm TM as an example. Methods Purified TMs (shrimp, Penaeus monodon; chicken Gallus gallus; E coli overexpression) were compared by protein sequencing, circular dichroism analysis and in vitro digestion. IgE binding was quantified using shrimp‐allergic patients' sera (ELISA). Biological activities were investigated (skin testing; titrated basophil activation tests, BAT), compared to titrated biological mediator release using humanized rat basophil leukaemia (RBL) cells. Results Shrimp and chicken TMs showed high sequence homology, both alpha‐helical structures and thermal stability. Shrimp TM was stable during in vitro gastric digestion, chicken TM degraded quickly. Both TMs bound specific IgE from shrimp‐allergic patients (significantly higher for shrimp TM), whereas skin reactivity was mostly positive with only shrimp TM. BAT and RBL cell assays were positive with shrimp and chicken TM, although at up to 100‐ to 1000‐times lower allergen concentrations for shrimp than chicken TM. In RBL cell assays using both TM as calibrators, an activation of effector cells by mealworm TM similar to that by shrimp TM confirmed the already reported high allergenic potency of mealworm TM as a novel protein source. Conclusions & clinical relevance According to current GM crops' allergenicity assessment, non‐allergenic chicken TM could falsely be considered an allergen on a weight‐of‐evidence approach. However, calibrating allergenic potency in functional BAT and RBL cell assays with clinically validated TMs allowed for semi‐quantitative discrimination of novel food protein's allergenicity. With TM calibration as a proof of concept, similar systems of homologous protein might be developed to scale on an axis of allergenicity.


| INTRODUC TI ON
The United Nations forecast a growth of the world population by 50% in the period from 2000 to 2050 which is expected to lead to a pressing need for more food production but also the introduction of novel types of foods in order to address a shortage of protein sources. 1,2 Novel foods, defined differently in different countries but in the European Union as any kind of food not yet been used in human diet to a significant degree before 1997, are entering continuously the market. 3 Prominent examples of novel foods from animal origin are edible insects or exotic meats (eg crocodile or kangaroo meat) and from plant origin, algae-related food (eg spirulina) or novel seeds (eg chia seeds). [4][5][6][7] In the absence of studies on epidemiology and natural history of allergic reactions to novel foods, these foods bear possible risk for food-allergic reactions in allergic patients due to clinical cross-reactions as well as in previously tolerant individuals due to de novo sensitization. 8,9 However, there is no generally accepted strategy for the allergenicity assessment of novel foods prior to market launch. 10 Food allergy emerged over the past thirty years as an important public health problem, in a second wave of the "allergy epidemic" succeeding the first respiratory epidemic. 11 About 20 million people in Europe suffer from food allergies, defined as chronic immune-mediated adverse reactions to otherwise harmless food proteins. 12,13 IgE-mediated reactions are the most common and best-understood food allergy type, manifesting as a multi-organ disease and varying from mild clinical symptoms to severe anaphylaxis. 14,15 Today, effective management of food-allergic patients relies on strict avoidance diet of the culprit food source of origin including other cross-reactive foods. 16 Successful food allergen avoidance is a challenging task involving not only the patient but also the patient's personal/professional environment as well as other stakeholders, public authorities and food industry. 17 Achieving food allergen avoidance becomes even more complex when novel foods suddenly become available on the consumer market. 8 Dietary proteins are the main drivers of food-allergic reactions, being responsible for both allergic sensitization (eg via ingestion, inhalation and cutaneous uptake) as well as clinical manifestations in previously sensitized patients. 14,18 Potent food allergens are often highly stable proteins and beyond, impaired gastric digestion and increased intestinal permeability have been associated with clinical food allergy, suggesting particular bioavailability of food antigens in allergic conditions. 19,20 Homologous panallergens from closely related sources are often cross-reactive, such as crustacean tropomyosins and fish parvalbumins. [21][22][23] However, homologous proteins might also differ by their allergenic potency, especially when they originate from distantly related sources. 24 Tropomyosins (TMs) are known panallergens in arthropods and major food allergens in, for example various species of shellfish, molluscs and the fish parasite Anisakis as well as respiratory minor allergens from environmental origin (eg mites and cockroaches). 23 Fifteen arthropod TMs have been registered as food allergens according to the criteria set by the WHO/IUIS allergen nomenclature sub-committee (www.aller gen.org, retrieved 01 March 2019). They are highly homologous alpha-helical proteins sharing characteristic rod-shaped structures of two parallel helices. Allergenic TMs are strong food allergens and highly cross-reactive within the invertebrate group. IgE reactivity to TM is associated with severe symptoms in shrimp-allergic patients. 25 Recently, TM from yellow mealworm, the larvae of the beetle Tenebrio molitor, was identified as a main allergen from this novel food source. 4,26 Although TMs are highly conserved proteins throughout the animal kingdom, only homologues from invertebrate source are verified allergens while vertebrate TMs, such as mammalian and avian TMs including chicken TM, are generally considered as non-allergenic. 27 Structural relationship of proteins or a certain degree of amino acid (aa) identity increases the probability of allergic cross-reactivity. However, mere aa identity and structural homology, essentially when low thresholds of criteria are applied (eg 35% sequence identity to a known allergen using a sliding window of 80 aa), are weak predictors in allergen risk assessment of novel foods, and additional tests are needed to assess their allergenic potential. 10 For genetically modified (GM) food crops, guidance based on a decision tree for the allergen risk assessment exists and includes, for example analysis whether the source of the protein is known to be allergenic or not, comparison of sequence identity with known allergens, digestibility tests and IgE serum screening (eg with sera of TH, JC, KV, KHS, AK). A supportive grant for laboratory consumables was received by Laboratoires Réunis, Luxembourg (JK, AK, MO). patients allergic to the source of the protein). 28,29 By contrast, there is no generally accepted strategy of how to perform allergenicity assessment of novel foods. 10 Recently, an European food allergy expert consortium (COST action FA1402, ImPARAS, 2014-2018) identified important gaps in the safety assessment of novel foods with major weaknesses in guidance on allergenicity and immunogenicity assessment of food sources. [8][9][10] A multidisciplinary strategy has been identified for future systematic and comprehensive food risk assessment, with emphasis on using well-characterized pairs of homologous proteins as opponents on an allergenicity scale, in order to validate applied methodologies and to calibrate the assays for those cases where the putative novel food allergen is known to have allergenic/non-allergenic homologues. 10 In the present study, we followed the available guidance on the allergenicity assessment of GM crops, extended it for in vitro biological potency assays and used TMs from an invertebrate origin, the well-known food allergen from black tiger shrimp (Penaeus monodon) muscle, and TM from a vertebrate origin, a non-allergenic molecule expressed in chicken (Gallus domesticus) muscle, as a pair of homologous proteins. First, we characterized the two TMs at the biomolecular level and second used them in differential in vitro assays to assess their relative allergenic potency in shrimp-allergic individuals. The overall goal was to evaluate whether the pair of homologous TMs with divergent allergenic potential is suitable to calibrate in vitro biological potency assays in order to assess semi-quantitatively and confirm the allergenic potency of a known novel animal food protein, namely tropomyosin of yellow mealworm. Results are evaluated and discussed in the light of existing allergenicity assessment guidance.

| Shrimp-allergic patients and sera
Twenty shrimp-allergic patients (12 male, 8 female; mean age 18.8 years) were recruited at the National Unit of Immunology and Allergology, Central Hospital of Luxembourg (CHL). Criteria for selection were a convincing clinical history of shrimp allergy and positive prick-to-prick tests (PTP) with shrimp as well as specific IgE higher than 0.1 kU A /L for shrimp extract and tropomyosin Pen a 1 (ImmunoCAP, Phadia-Thermofisher). All patients developed allergic symptoms upon exposure to shrimp (ingestion and inhalation/ skin contact) on several occasions. Shrimp-allergic patients tolerated chicken meat (regular consumption, ≥1×/month). PTP were performed with cooked shrimp muscle and in single cases (randomly selected: patient no. 1,2,6,9), with cooked chicken muscle.
Histamine was used as positive control. A positive PTP response was scored when the weal diameter was 3 mm larger than the negative control (glycerine-containing saline). Patients without severe history of shrimp allergy and positive Pen a 1-titres (1-50 kU A /L) were asked for further skin testings. Single patients (n = 6) agreed to be tested for skin reactivity with increasing concentrations of native, purified TM (0.1, 1, 10, 50 μg allergen/mL) diluted in sterile saline containing 0.03% human serum albumin (ALK, Denmark). 24 Skin testing was stopped at the protein concentration where a positive reaction was scored. All patients were also examined as to house dust mite (HDM) allergy (medical history, specific IgE for HDM extract and Der p 10). Two individuals with allergies other than to shrimp were used as negative controls in IgE testing (IgE ELISA, effector cell reactivity tests) (

| Secondary protein structure determination by circular dichroism
Tropomyosins were measured in 20 mmol/L KH 2 PO 4 pH 7.2 to establish their circular dichroism (CD) spectra using the Chirascan CD spectrometer (Applied Photophysics). Thermal sensitivity was assessed by ramping temperatures (20°C to 95°C to 20°C). Far-ultraviolet CD spectra were recorded at 180-260 nm (1 nm bandwidth, 0.5 seconds interval, 5 repeats). The read-out was converted with respective protein details into degrees*cm 2 *dmol -1 . GLOBAL 3 and DICHROWEB software were used to analyse and interpret CD spectra, measured as a function of temperature.

| In vitro digests
Simulated digestion was performed as reported previously, 35 with further modifications as published for successive gastric and intestinal digests according to the international consensus paper published by Minekus et al, 36 in terms of adjusted pH and applied incubation times. Briefly, protein extracts were incubated with pepsin (Sigma-Aldrich) at a final ratio of 1 U enzyme/µg protein extract in simulated gastric fluid, pH 3 for 2 hours. The last sample from the gastric digest was mixed 1:1 with pancreatin (based on trypsin activity at 100 U/ mL) and 10 mmol/L bile salts (both Sigma-Aldrich) for two hours in simulated intestinal fluid, pH 7. Samples were drawn at T0, T1, T2, T5, T10, T20, T30, T60, T90 and T120 min during each digestion phase, following by analysis on SDS-PAGE and immunoblot.

| Immunoblot and ELISA analyses using commercial antibodies and patient sera
Tropomyosins samples were separated by SDS-PAGE/Coomassie/ silver stain, in order to revise protein purity and protein size. 37 Protein identity was confirmed by immunodetection in immunoblot and enzyme-linked immunosorbent assay (ELISA). Polyclonal rabbit IgG-antibodies were used to detect shrimp (PA-SHM; Indoor Biotech) or chicken TM (ab11190; Abcam), each antibody diluted 1:10 000 in immunoblot and 1:5000 in ELISA analysis, followed by secondary antibody incubation (1:10 000; anti-rabbit IgG-antibody labelled with alkaline phosphatase; Sigma-Aldrich).
IgE ELISA was used to quantify IgE titres in patient sera. 34 In short, patient sera were fivefold, 10-and 20-fold diluted in blocking TA B L E 1 Demographic and clinical data from shrimp-allergic patients buffer containing 3% bovine serum albumin (BSA). Five sera of nonatopic individuals were used as negative controls, resulting in a 10fold lower mean background than the cut-off value (0.1 kU A /L).

| Basophil activation test
Basophil activation was assessed using the Flow-CAST kit ® (BÜHLMANN Laboratories AG, Swiss). 24 We included seven representative patients, patients no. 1-7, Table 1, with various clinical profiles (Pen a 1-specific IgE titres low to high; clinical symptoms mild to severe). Briefly, basophils from fresh blood were stimulated with serial protein concentrations (0.1-10 000 ng/mL) and following, the percentage of activated (CD63 + CCR3 + ) basophils upon stimulation measured by flow cytometry (cut-off for positivity ≥15% of CD63 + basophils compared to total basophils). Data acquisition/analysis was performed using BD FACSDiva software (BD Biosciences) and Kaluza (Beckman Coulter) software, respectively.

| Mediator release assay
Mediator release assays were performed using the rat basophilic leukaemia (RBL)-2H3 cell line transfected with the α-chain of the human high-affinity IgE receptor. 38 Briefly, patient sera (patients no. 1-20, Table 1; 50 μL/well; dilution 1/11) were incubated with the cells (clone RBL-703/21). Serial dilution of all proteins (0.001-10 000 ng/ mL) was used to induce antigen-specific release quantified by measuring the enzymatic activity of β-hexosaminidase. Releases were expressed as the percentage of release from cells sensitized with serum in relation to the total release obtained by lysis of all cells (cutoff for positivity ≥10% of positive release compared to total release).
Adjustment for spontaneous release (serum added without allergen) was carried out.

| Statistical analyses
IgE results were plotted using GraphPad Prism 5 (GraphPad software). Differences in IgE titres were assessed using the nonparametric Kruskal-Wallis test and the Mann-Whitney U test.

| Shrimp-allergic patients tolerate chicken meat
All patients experienced multiple allergic episodes upon shrimp ingestion with clinical symptoms detailed in Table 1 (Table S2).

| In-/vertebrate TM: similar thermal but divergent in vitro digestion stability
Tropomyosins from shrimp and chicken were detected in extracts prepared from muscle using specific anti-TM antibodies ( Figure   S1). Both homologues proved to be heat-stable, being still detecta- F I G U R E 1 Shrimp and chicken tropomyosins, both native and recombinant, were purified to homogeneity. Native shrimp tropomyosin was found to be a homodimer (single band) while chicken tropomyosin from breast muscle was mostly a α/αhomodimer and chicken tropomyosin from leg muscle a α/βheterodimer (double band). r, recombinant protein sequence coverage to P04268.2) (Tables S3-S5). Accordingly, TM purified from chicken leg was concluded to be a TM α/β-heterodimer and TM purified from chicken breast to be mostly an α/α-ho-

| Low IgE titres to chicken proteins and purified chicken TM
Levels of specific IgE to shrimp extract, chicken extract, mealworm extract, natural and recombinant shrimp and chicken TM were compared using IgE ELISA analysis (Table S6). Specific IgE to shrimp extract was found in all study participants by IgE ELISA (0.2-100 kUA/L median 6.3 kUA/L) (Table S6) Very weak anti-TM-sIgE signals were detected for patient no. 16 Table S7.

| Low skin prick reactivity to chicken TM in shrimp-allergic patients
The negative control (saline) did not induce any skin reaction.

| Chicken TM has low capacity to activate basophils in vitro
Tropomyosins from chicken (native, recombinant) showed a drastically lower potency to activate effector cells compared with shrimp homologues ( Figure 4A In comparison with shrimp proteins, chicken TM also revealed low potency to induce β-hexosaminidase release from humanized rat basophils that were passively sensitized with IgE from patient sera. Following stimulation with 100 ng/mL of protein, the median percentage of histamine release was 10%-13% with shrimp TM (range of histamine release: Pen m 1, 0.7%-77.9%; rPen m 1, 0.2%-73.0%) compared to <0.8% with chicken homologues ( Figure 4B). A comparable median mediator release of 10% was obtained after increasing the chicken TM concentration to 10 000 ng/mL in comparison with 100 ng/mL for shrimp TM. Differences in statistical significance, high capacity to induce mediator release using shrimp but not chicken TM, were found for the protein concentrations tested (eg Pen m 1 (r) vs chicken α/α P = .0087 Pen m 1 (r) vs chicken α/β P = .0260; n = 20). No mediator release was induced when using two sera from tolerant controls (Table S1) for rat basophil sensitization (data not shown).

| D ISCUSS I ON
In this study, we evaluated two homologous proteins, a pair of tropomyosins (TM) consisting of the highly allergenic shrimp Pen m 1 and the non-allergenic counterpart from chicken muscle. We investigated their potential use as calibrator molecules for the assessment of protein allergenicity in in vivo skin prick testing, in vitro IgE-binding capacity, ex vivo basophil activation and in vitro mediator release from passively sensitized basophils, respectively. Identification of appropriate calibrators and predictive assays would allow for the future assessment of allergenicity of novel food proteins. Indeed, invertebrate TM is a known food allergen while only few studies relate vertebrate TM to clinical food allergy (eg in fish). [39][40][41] First, we showed that the purified proteins from shrimp and chicken origin exhibit similar biomolecular properties. Allergen Pen m 1 was confirmed as a dimer made of two alpha-helical chains.
TM from chicken breast was found mostly as αα-homodimer and Thus, the thermal unfolding of the αβ-heterodimeric proteins was found to be similar to that of the αα-homodimers. 43 Common food allergens are often more stable during thermal treatment than others. 19,20 However, both allergenic shrimp and non-allergenic chicken TM showed no differences in thermal de-/refolding characteristics.
Simulated digestion experiments revealed clear stability differences in shrimp and chicken TM. Assay conditions were chosen mild (pH 3; 1 U pepsin/µg extract protein) in order to follow the progressive protein digestion. Although in our study resistance to pepsinolysis separated the allergen from the non-allergen, it is important to say that a straightforward correlation between protease stability and allergenicity is not always given, depending on the protein analysed and the assay conditions applied. 35  In terms of exposure levels to shrimp and chicken TM, we estimated the protein content of TMs in total extracts by densitometric band quantification from SDS-PAGE. 29 Similar levels, ca. 10%-12% TM of total extracted protein, were found for both shrimp and chicken TM (data not shown), indicating that exposure levels might be similar as well.
F I G U R E 4 A, Basophil activation is induced efficiently at low protein concentrations (≤100 ng/mL) by shrimp tropomyosin but not chicken homologues (n = 7 shrimp-allergic patients). B, Shrimp tropomyosin has a high capacity to induce at low concentrations (≤10 ng/ mL) histamine release from RBL cells previously sensitized with sera from 20 shrimp-allergic patients, as compared to chicken proteins ng/mL protein concentration to release mediators from basophils. Hence, the RBL cell assay using rPen m 1 and r-chicken αα-TM as calibrators confirmed mealworm TM, as one example of a newly introduced food protein with high allergenic potency that is comparable to that of shrimp tropomyosin. Indeed, yellow mealworm is a novel food that causes allergic reactions as a primary and as a cross-reacting food allergen. 4,26,33 As previously published, for the majority of shrimp-allergic individuals, mealworm TM is a major allergen, and clinical reactivity to mealworm was in the same range as to shrimp, both with regard to severity of allergic reactions and eliciting doses. 26 In this context, the outcome of reported clinical reactivity to mealworm 26  Essentially, only the comparison of the relative potency, by matching effector cell activation versus titrated protein concentrations, revealed the discrimination of the allergen from the non-allergen.
The titrated effector cell reactivity test based on homologues could be proposed to be included in current risk assessment procedures.
For assay calibration, threshold definition for low and high allergenicity would be required. However, it needs to be considered that the performance of the mediator release is always related to the selected patients' sera. Accordingly, appropriate 'high performing' sera would need to be identified, selected and further characterised with regard to repeatability of assay performance. Subsequently, appropriate and relevant cut-off levels for positivity and range of calibrator amount can be defined, though this represents another level of assay validation.
To identify percentages of mediator release reflecting clinical reactivity or oral tolerance, future studies will be required including food challenges that allow to approach threshold reactivity in terms of ingested food protein doses-however, a true prediction of clinical reactivity, based on a cellular test, is expected to be unlikely. Further recommendations for using mediator release assays pertain to the novel food source that shall be tested also as a total extract, as this allows to target allergens beyond the calibrator molecules.
Finally, it is important to point out that the proposed calibration system is TM-specific. With this TM model as a proof of concept, similar systems of homologous proteins might be elaborated based on other allergens/non-allergens, such high and low allergenic nsLTP in plant foods. Taking together the data from our study using the cohort of shrimp-allergic patients including the semi-quantitative cellular test, mealworm (based on its TM) would have been ranked with an allergenic potential that is comparable high to that of shrimp while chicken meat (also, based on its TM) would have been assessed as a food with low allergenic potential for human consumption. The approach of protein pairs represents a perspective for the risk assessment of novel foods where homologous proteins to known strong or anaphylactic food allergens are present, and thus, the risk is related to sensitized/allergic individuals. De novo sensitization, possibly to proteins different from TM, would require further consideration in the allergenicity assessment guidance, a topic that goes beyond the present study.
In summary, this study shows that clinically validated tropomyo-

ACK N OWLED G EM ENTS
We thank T Scheuermann, D Revets, K Benkirane and T Graf for their great technical support.

CO N FLI C T O F I NTE R E S T
All other authors have declared that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.