Lupine allergens: Clinical relevance, molecular characterization, cross-reactivity, and detection strategies.

Lupine is commonly utilized as a technological food and ingredient in a great variety of processed products (snacks, bakery, meat, and dairy products) principally owing to its nutritional value and technological properties. However, its ingestion, even at trace amounts (in the range of mg protein per kg of food), can lead to severe adverse reactions in allergic individuals. Lupine belongs to the Leguminosae family, having the conglutins (α-, ß-, δ-, and γ-) as allergens, among other proteins. Cross-sensitization of lupine-sensitized individuals with other legume species, mainly peanut, can occur, but the associated clinical reactivity is still unclear. The protection of the sensitized individuals should depend on an avoidance diet, which should rely on the compliance of food labeling and, as such, on their verification by analytical methods. Food processing, such as heat treatments, has an important influence on the structural properties of lupine proteins, altering their detectability and allergenicity. In this review, different aspects related with lupine allergy are described, namely, the overall prevalence, clinical relevance, diagnosis, and treatment. The characterization of lupine allergens and their potential cross-reactivity with other legumes are critically discussed. The effects of food matrix, processing, and digestibility on lupine proteins, as well as the available analytical tools for detecting lupine at trace levels in foods, are also herein emphasized.

Allergen Immunization Induces Major Changes in Microbiota Composition and Short-Chain Fatty Acid Production in Different Gut Segments in a Mouse Model of Lupine Food Allergy.

BACKGROUND: The incidence of food allergies in western countries has increased in recent decades. OBJECTIVES: To study the association between gut bacterial microbiota composition, short-chain fatty acids (SCFAs) and food allergy in a mouse model. METHODS: After oral immunizations with the human food allergen lupine with the adjuvant cholera toxin (CT) (or buffer in controls), sensitization and anaphylactic responses were determined. Gastrointestinal content was collected from the distal ileum, cecum, colon, and fecal pellets, and the bacterial diversity and composition was determined by deep sequencing of the 16S rRNA gene. SCFAs in gastrointestinal content supernatants were determined by gas chromatography. RESULTS: The microbiota signatures were profoundly affected by allergen immunization. Ten operational taxonomic units (OTUs) were significantly different between immunized and control animals for at least one of the intestinal segments; eight of these OTUs belonged to the Clostridia class. Although consistent across all four gut segments, the colon showed the highest number of OTUs significantly associated with allergic immunization. SCFA levels in the cecum were also altered by immunization. CONCLUSIONS: Allergen immunization with CT in the present food allergy model induced profound changes in the microbiome composition and SCFA production. The result suggests that the colon may be the most sensitive gut segment for investigating changes in the gut microbiome.

Proteomic analysis of Lupinus angustifolius (var. Zeus and Bojar) and Lupinus luteus (var. Lord and Parys) seed proteins and their hydrolysates.

BACKGROUND: Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. RESULTS: The comparisons of 2-DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as ß-conglutin. CONCLUSIONS: The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry.

Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice.

BACKGROUND: In genetically modified (GM) crops there is a risk that the inserted genes may introduce new allergens and/or adjuvants into the food and feed chain. The MON810 maize, expressing the insecticidal Cry1Ab toxin, is grown in many countries worldwide. In animal models, intranasal and intraperitoneal immunisations with the purified Cry1Ab proteins have induced immune responses, and feeding trials with Cry1Ab-containing feed have revealed some altered immune responses. Previous investigations have primarily measured antibody responses to the protein, while investigations of clinical food allergy symptoms, or allergy promotion (adjuvant effect) associated with the Cry1Ab protein are largely missing. We aimed to investigate immunogenic, allergenic and adjuvant properties of purified Cry1Ab toxin (trypCry1Ab, i.e., trypsin activated Cry1Ab) in a mouse model of food allergy. METHOD: Female C3H/HeJ mice were immunized by intragastric gavage of 10 µg purified, trypsin activated Cry1Ab toxin (trypCry1Ab) alone or together with the food allergen lupin. Cholera toxin was added as a positive control for adjuvant effect to break oral tolerance. Clinical symptoms (anaphylaxis) as well as humoral and cellular responses were assessed. RESULTS: In contrast to results from previous airway investigations, we observed no indication of immunogenic properties of trypCry1Ab protein after repeated intragastric exposures to one dose, with or without CT as adjuvant. Moreover, the results indicated that trypCry1Ab given by the intragastric route was not able to promote allergic responses or anaphylactic reactions against the co-administered allergen lupin at the given dose. CONCLUSION: The study suggests no immunogenic, allergenic or adjuvant capacity of the given dose of trypCry1Ab protein after intragastric exposure of prime aged mice.

Lupin and Other Potentially Cross-Reactive Allergens in Peanut Allergy.

PURPOSE OF REVIEW: The presence of IgE cross-reactivity between peanut allergens and allergens from other legumes and tree nuts has been demonstrated, but the identification of the involved individual allergens is still limited. The aim of this review is to describe new allergenic findings, of potential relevance for cross-reactivity among peanut and lupin. RECENT FINDINGS: Seventeen allergens of peanut have been included in the official allergen nomenclature database to date. Lupin sensitization has been observed in 15-20% of individuals with known peanut allergy, The majority of lupin seed proteins are comprised of α-conglutins (legumin-like) and ß-conglutins (vicilin-like), and to a lesser extent γ-conglutins (vicilin-like) and δ-conglutins (2S albumins). Several molecules may fuel peanut-lupin cross-reactivity. Awareness among physicians and general public could avoid unexpected allergic reactions. However, these do not appear frequent and no data suggest a precautionary labelling of lupin in foods.

IgE sensitization to lupine in bakers - cross-reactivity or co-sensitization to wheat flour?

BACKGROUND: Food allergy to lupine has frequently been reported in patients allergic to peanut or soy, and cross-reactivity between these legumes is known. Moreover, respiratory allergy to lupine has been described after inhalation, mostly at workplaces. Our aim was to study the frequency of lupine sensitization in European bakers with suspected bakers' allergy. Furthermore, associations between sensitizations to lupine and other plant allergens were investigated. METHODS: One hundred and sixteen bakers with work-related allergic symptoms but without known food allergies were examined. Specific IgE (sIgE) antibodies to wheat flour, rye flour, lupine, peanut, soy and the recombinant single birch protein rBet v 1 were quantified. Selected sera were tested for cross-reactivity using ImmunoCAP inhibition and ISAC microarrays. RESULTS: Whereas 67% of bakers were sensitized to wheat and/or rye flour, 35% showed sIgE to peanut and 33% to lupine. All lupine-positive bakers also had sIgE to either wheat flour (89%) and/or peanut (92%), and lupine sIgE correlated significantly with sIgE to peanut, soy, wheat and rye flour. Used as an inhibitor, wheat flour inhibited IgE binding to lupine in 4 out of 8 sera, indicating cross-reactivity. In microarrays, these sera showed IgE binding to lipid transfer proteins, profilins and/or cross-reactive carbohydrate determinants. Further inhibition experiments suggest that these single allergens are involved in cross-reactivity. CONCLUSION: One third of 116 symptomatic bakers showed sIgE to lupine. At least some of these sensitizations were based on cross-reactivity between lupine and wheat flour. However, the considerable sensitization rate could also be a sign that the use of lupine flour in bakeries may be of occupational relevance.

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding.

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F(8) recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F(8) population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of "false positives" (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.

Cross-allergic reactions to legumes in lupin and fenugreek-sensitized mice.

Several legumes may induce allergy, and there is extensive serological cross-reactivity among legumes. This cross-reactivity has traditionally been regarded to have limited clinical relevance. However, the introduction of novel legumes to Western countries may have changed this pattern, and in some studies cross-allergy to lupin has been reported in more than 60% of peanut-allergic patients. We wanted to explore cross-reactions among legumes using two newly established mouse models of food allergy. Mice were immunized perorally with fenugreek or lupin with cholera toxin as adjuvant. The mice were challenged with high doses of fenugreek, lupin, peanut or soy, and signs of anaphylactic reactions were observed. Cross-allergic mechanisms were investigated using serum mouse mast cell protease-1 (MMCP-1), antibody responses, immunoblotting and ex vivo production of cytokines by spleen cells. Signs of cross-allergy were observed for all the tested legumes in both models. The cross-allergic symptoms were milder and affected fewer mice than the primary allergic responses. The cross-allergy was reflected to a certain extent in the antibody and T-cell responses, but not in serum MMCP-1 levels. Cross-allergy to peanut, soy, fenugreek and lupin was observed in lupin-sensitized and fenugreek-sensitized mice. Differences in serological responses between primary allergy and cross-allergy might be due to mediation through different immune mechanisms or reflect different epitope affinity to IgE. These differences need to be further investigated.

Sensitization to lupine flour: is it clinically relevant?

BACKGROUND: Lupinus angustifolius (blue lupine) is used for human and animal consumption. Currently, the lupine content in bread varies from 0% to 10% and from 0.5% to 3% in pastry. Although lupine flour is present in many products, anaphylaxis on lupine flour is rarely seen. OBJECTIVE: The aim of our study was to determine the clinical relevance of sensitization to lupine flour. METHODS: From October 2004 until October 2005, we performed skin prick tests (SPT) with lupine flour, peanut and soy extracts in consecutive patients attending our allergy clinic with a suspected food allergy. In patients sensitized to lupine flour, double-blind placebo-controlled food challenges (DBPCFC) were performed and specific IgE was measured. RESULTS: We tested 372 patients. SPTs with peanut, soy and lupine flour were positive in 135, 58 and 22 patients, respectively. Nine patients with sensitization to lupine flour underwent DBPCFC, which was negative in eight cases. In contrast, one patient experienced significant symptoms. Four of these nine patients suspected lupine by history. Two other patients with a positive history to lupine declined from challenges. In these patients, a 3-day dietary record showed that they could consume lupine without symptoms. Specific IgE in the serum was positive for L. angustifolius, peanut and soy in all nine patients. CONCLUSION: These results demonstrate that clinical lupine allergy is very uncommon, even in the presence of sensitization to lupine flour. The estimated prevalence of lupine allergy, among patients with a suspected food allergy, referred to a tertiary allergy centre in the Netherlands is 0.27-0.81%. In most, although not all cases, sensitization is not clinically relevant and is most likely caused by cross-sensitization to peanut. In selected cases, eliciting doses are low, making significant reactions possible.

Recurrent anaphylaxis due to lupin flour: primary sensitization through inhalation.

Allergic reactions to lupin have increased in parallel with the growing use of lupin flour by food manufacturers. We studied a patient with recurrent anaphylaxis to manufactured foods and a history of rhinitis-asthma related to lupin inhalation and legume tolerance. Skin prick tests with airborne and food allergens, specific immunoglobulin (Ig) E determinations, and an inhalation exposure test to ground lupin were carried out. Lupin allergens and cross-reactivity with other legumes were also studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting/immunoblotting inhibition. The skin tests and specific IgE were positive for lupin and vetchling and negative for other legumes. The presence of lupin flour in the implicated foods was confirmed. Immunoblotting showed multiple IgE-binding bands (10-40 kDa) for lupin and vetchling but not for peanut, pea, or soy extracts. Immunoblotting inhibition demonstrated intense lupin-vetchling cross-reactivity. We present a case of recurrent anaphylaxis due to lupin flour as a hidden food allergen with primary sensitization due to exposure to ground lupin via inhalation. We found cross-reactivity between lupin and vetchling but not other legumes.

[Sweet lupin--a new food allergen]. / Makea lupiini--uusi ruoka-allergeeni.

Lupin, a legume with good nutritional value, is used in food production today, most often in bakery products. In Finland, lupin is a labelled ingredient in very few products. Clinically relevant lupin allergy, even anaphylaxis, often occurs in patients without atopic background or other food allergies, whereas lupin sensitization without clinical relevancy most commonly seems to represent cross reactivity to other legumes. Lupin allergy should be suspected and studied in patients with adverse reactions to food, and patients with allergy to other legumes should be advised about possible lupin allergy, as well.

Assessment of the tolerance to lupine-enriched pasta in peanut-allergic children.

BACKGROUND: Reports of allergy to lupine derivatives (as de novo sensitization or cross-reactivity in subjects allergic to peanut) are increasing as their use in food products increases. OBJECTIVES: The aim of this study was to assess: (1) lupine tolerance in a group of children allergic to peanut, using lupine enriched-pasta instead of raw flour as has been done in previous clinical studies; (2) whether technological treatments of lupine modify its cross-reactivity or co-sensitization with peanut; (3) the role of lupine seed proteins in sensitization, and (4) to identify the eliciting doses (EDs) by using double-blind, placebo-controlled food challenges (DBPCFC). METHODS: Twelve patients with a history of clinical allergic reactions to peanut were evaluated by skin prick tests (SPTs), the ImmunoCAP test, immunoblotting, and DBPCFC. The 12 selected subjects were included in a trial where lupine-enriched pasta and placebo pasta were administered in a DBPCFC protocol. RESULTS: Positive clinical reactions were observed in two children, the EDs being 0.2 and 6.4 g of pasta, corresponding to 50 mg and 1.6 g of lupine proteins, respectively. Beta-conglutin was the protein most involved in SPT positivity. CONCLUSION: Lupine-enriched pasta can be tolerated by most subjects suffering from peanut allergy, but a sizeable minority (2/12 of them in this case) can develop potentially dangerous clinical reactions. Information about possible reactions to lupine derivatives by those allergic to peanuts must be included in the labelling of lupine-enriched products to protect consumers at risk.

A mouse model of lupin allergy.

BACKGROUND: Lupin has been introduced as a new food ingredient in an increasing number of European countries, resulting in reports of allergic reactions mostly due to cross-reactions in peanut-allergic individuals. Some cases of primary lupin allergy have also been reported. OBJECTIVE: The aim of our study was to develop a food allergy model of lupin in mice with anaphylaxis as the endpoint and further, to develop an approach to estimate the allergen dose inducing maximal sensitization using a statistical design requiring a limited number of animals. METHODS: Mice were immunized by intragastric gavage using cholera toxin as an adjuvant. A two-compartment response surface design with IgE as the main variable was used to estimate the maximal sensitizing dose of lupin in the model. This estimated dose was further used to evaluate the model. The mice were challenged with a high dose of lupin and signs of an anaphylactic reaction were observed. Antibody reactions (IgE and IgG2a), serum mast cell protease [mouse mast cell protease-1 (MMCP-1)] and ex vivo production of cytokines (IL-4, IL-5 and IFN-gamma) by spleen cells were measured. An immunoblot with regard to IgE binding was also performed. RESULTS: The dose that elicited the maximal sensitization measured as IgE was 5.7 mg lupin protein per immunization. Mice that received this dose developed anaphylactic reactions upon challenge, IgE against several proteins in the lupin extract, and high levels of MMCP-1, and showed a general shift towards a T-helper type 2 response. Post-challenge serum MMCP-1 levels corresponded to the seriousness of the anaphylactic reactions. CONCLUSION: We have established a mouse model with clinical symptoms of lupin allergy, with an optimized dose of lupin protein. A statistical design that can be used to determine an optimal immunization dose with the use of a minimum of laboratory animals is described.

Clinical relevance of sensitization to lupine in peanut-sensitized adults.

BACKGROUND: The use of lupine in food has been increasing during the last decade and allergic reactions to lupine have been reported, especially in peanut-allergic patients. The frequency and the degree of cross-reactivity to other legumes are not known. The aim of the study was to investigate the frequency of sensitization to lupine, and in addition to pea and soy, and its clinical relevance, in peanut-sensitized patients. Furthermore, to determine the eliciting dose (ED) for lupine using double-blind placebo-controlled food challenges (DBPCFC). METHODS: Thirty-nine unselected peanut-sensitized patients were evaluated by skin prick tests (SPT) and ImmunoCAP to lupine, pea, and soy. Clinical reactivity was measured by DBPCFC for lupine, and by history for pea and soy. RESULTS: Eighty-two percent of the study population was sensitized to lupine, 55% to pea, and 87% to soy. Clinically relevant sensitization to lupine, pea, or soy occurred in 35%, 29%, and 33% respectively of the study population. None of the patients was aware of the use of lupine in food. The lowest ED for lupine, inducing mild subjective symptoms, was 0.5 mg, and the no observed adverse effect level (NOAEL) was 0.1 mg. No predictive factors for lupine allergy were found. CONCLUSION: In peanut-sensitized patients, clinically relevant sensitization to either lupine or to pea or soy occurs frequently. The ED for lupine is low (0.5 mg), which is only fivefold higher than for peanut. Patients are not aware of lupine allergy and the presence of lupine in food, indicating that education is important to build awareness.

Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins.

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.

Immunoglobulin E cross-reactivity between lupine conglutins and peanut allergens in serum of lupine-allergic individuals.

BACKGROUND: Lupine is used increasingly in food products. The development of lupine allergy in peanut-allergic patients is believed to occur as a result of cross-reactivity between lupine and peanut proteins. OBJECTIVE: To investigate the degree of immunoglobulin (Ig) E cross-reactivity between allergens in lupine and peanut. METHODS: We investigated IgE cross-reactivity between lupine alpha-, beta-, gamma-, and delta-conglutins and the major peanut allergens Ara h 1, Ara h 2 and Ara h 3 using enzyme-linked immunosorbent assay with sera from patients with coexisting peanut and lupine allergy. RESULTS: Peanut proteins inhibited IgE binding towards alpha- conglutins, delta-conglutins, and, to a lesser degree, beta-conglutins, while no IgE cross-reaction with delta-conglutin was observed. Ara h 2 most potently inhibited IgE binding to lupine and delta-conglutins, while Ara h 1 most potently cross-reacted with beta-conglutin. Ara h 3 was apparently not involved in these mechanisms. CONCLUSIONS: The present study reveals IgE cross-reactivity between the 2S albumins Ara h 2 and delta-conglutin, and the 7S vicilin-like Ara h 1 and beta-conglutin, which are possibly based on homologies between phylogenetically related proteins. Ara h 2 was the most potent inhibitor of IgE binding to lupine conglutins.