Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

BACKGROUND: House dust mite (HDM) allergens are major elicitors of allergic reactions worldwide.
OBJECTIVE: Identification, characterization, and evaluation of diagnostic utility of a new important HDM allergen was performed.
METHODS: A cDNA coding for a new Dermatophagoides pteronyssinus (Dp) allergen, Der p 37, was isolated from a Dp expression library with allergic patients\' IgE antibodies. Recombinant Der p 37 (rDer p 37) expressed in Escherichia coli was purified, then characterized by mass spectrometry, circular dichroism, and IgE reactivity by ImmunoCAP ISAC technology with sera from 111 clinically defined HDM-allergic patients. The allergenic activity of rDer p 37 was studied by basophil activation and CD4+ T-cell responses by carboxyfluorescein diacetate succinimidyl ester dilution assays. Specific antibodies raised against rDer p 37 were used for the ultrastructural localization of Der p 37 in mites by immunogold transmission electron microscopy.
RESULTS: Der p 37, a 26 kDa allergen with homology to chitin-binding proteins, is immunologically distinct from Der p 15, 18, and 23. It is located in the peritrophic membrane of fecal pellets. Der p 37 reacted with IgE antibodies from a third of HDM-allergic patients and induced specific basophil- and CD4+ T-cell activation. Der p 37 IgE-positive patients had significantly higher IgE levels to major HDM allergens, reacted with more HDM allergens, and had a higher risk (odds ratio = 3.1) of asthma compared to Der p 37-negative patients.
CONCLUSIONS: Der p 37, a new Dp allergen recognized by a third of HDM-allergic patients, may serve as a surrogate marker for severe HDM sensitization and asthma.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

Arginine kinase (AK) was first identified as an allergen in the Indian-meal moth and subsequently shown to occur as allergen in various invertebrates and shellfish. The cDNA coding for AK from the house dust mite (HDM) species Dermatophagoides pteronyssinus, Der p 20, has been isolated, but no recombinant Der p 20 (rDer p 20) allergen has been produced and characterized so far. We report the expression of Der p 20 as recombinant protein in Escherichia coli. rDer p 20 was purified and shown to be a monomeric, folded protein by size exclusion chromatography and circular dichroism spectroscopy, respectively. Using AK-specific antibodies, Der p 20 was found to occur mainly in HDM bodies, but not in fecal particles. Thirty percent of clinically well-characterized HDM allergic patients (n = 98) whose immunoglobulin E (IgE) reactivity profiles had been determined with an extensive panel of purified HDM allergens (Der f 1, 2; Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 and 37) showed IgE reactivity to Der p 20. IgE reactivity to Der p 20 was more frequently associated with lung symptoms. AKs were detected in several invertebrates with specific antibodies and Der p 20 showed IgE cross-reactivity with AK from shrimp (Litopenaeus vannamei). Thus, Der p 20 is a cross-reactive HDM allergen and may serve as a diagnostic marker for HDM-induced lung symptoms such as asthma.

Storage mites, such as Tyrophagus putrescentiae, are an important source of allergens that cause allergic diseases in humans. It has previously been indicated that T. putrescentiae has a high sensitization rate as an allergen in some Asian and European countries. Identifying and cloning the allergens in this species may enable improved diagnostic and therapeutic approaches. The aim of the present study was to clone and sequence the T. putrescentiae group 13 allergen (Tyr p 13) isolated from storage mites in China, to use bioinformatics tools to model its biophysical characteristics and to induce protein expression to test its IgE-binding activity. The full-length cDNA comprised 486 bp and was predicted to include a signal peptide of 22 amino acids. Its secondary structure was shown to comprise an α-helix (10.79%), extended strand (33.81%) and random coils (55.40%). Using homology modeling, the present study constructed a reasonable tertiary structure of Tyr p 13. Linear Bcell epitopes at amino acids 47-53, 70-76, 81-86, 101-105 and 112120 were predicted. Three discontinuous B-cell epitopes were also predicted: i) 47, 48, 49, 50, 51, 52, 53, 70, 71, 72 and 73; ii) 91, 92, 93, 94, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 and 138; and iii) 74, 76, 79, 81, 82, 83, 84, 86, 101, 102, 103, 104 and 105. SDS-PAGE identified a specific band at the predicted molecular weight of the recombinant Tyr p 13 (rTyr p 13), demonstrating its successful expression. The rTyr p 13 bound to IgE in the serum of 13.2% (5/38) of patients allergic to T. putrescentiae, according to ELISA. The successful cloning of Tyr p 13 and basic bioinformatics analysis of the protein provided a foundation for the further study of this allergen with regards to the diagnosis and treatment of patients allergic to storage mites. These results provided a theoretical basis for the design of rTyr p 13 with modified B-cell epitopes.

Severe allergy to fruits mediated by a 7 kDa allergen belonging to the gibberellin-regulated protein (GRP) family is known to be associated with Cupressaceae pollinosis.
To identify and characterize Cupressaceae pollen allergens involved in GRP-related fruit allergy.
Pru p 7-related proteins from pollen of Cupressus sempervirens, Juniperus ashei and Cryptomeria japonica were identified using a rabbit anti-Pru p 7 antiserum, purified chromatographically and sequenced by mass spectrometry and bioinformatic comparisons. The C sempervirens protein was produced as a recombinant allergen in Pichia pastoris. IgE antibody binding to pollen GRP proteins was analysed in a peach allergic (n = 54) and a cypress pollen allergic (n = 88) patient population from southern France using ImmunoCAP.
In each of the three Cupressaceae species studied, a 7 kDa pollen protein related to Pru p 7 was identified and found to comprise an amino acid sequence of 63 residues in length, 92%-98% identical to each other and 67%-68% identical to Pru p 7. The C sempervirens, J ashei and C japonica GRP allergens have been officially recognized by the WHO/IUIS Allergen Nomenclature Sub-Committee and named Cup s 7, Jun a 7 and Cry j 7, respectively. Recombinant Cup s 7 showed IgE antibody binding capacity comparable to that of the purified natural allergen. Among 51 peach allergic subjects sensitized to Pru p 7, substantially higher levels of IgE to Cup s 7 than to Pru p 7 were found. Further, the pollen protein was able to completely outcompete IgE binding to Pru p 7, while the reverse competition effect was modest, consistent with primary sensitization by the pollen allergen.
Pru p 7-related pollen allergens from three Cupressaceae species have been characterized and may become useful for the identification of pollinosis patients at risk of developing severe fruit allergy.

Poultry meat can induce severe allergic reactions. So far, the molecules causing poultry meat allergy are largely unknown.
Our aim was to identify and characterize poultry meat allergens.
Profiles of patients\' IgE reactivity to chicken muscle were analyzed in immunoblots, and proteins recognized by the majority of patients were subjected to peptide mass fingerprinting. A 23-kDa IgE-reactive protein was identified as myosin light chain 1, designated Gallus domesticus 7 (Gal d 7). Recombinant Gal d 7 was produced in Escherichia coli. The protein\'s IgE reactivity was analyzed in ELISA experiments, and cross-reactivity with allergens of other poultry species was assessed in inhibition immunoblots. Fold and thermal stability were evaluated by circular dichroism analysis, and enzymatic stability was investigated using in vitro gastrointestinal digestion assays.
Recombinant Gal d 7 represents a properly folded, predominantly α-helical protein and displays IgE-binding activity comparable to that of its natural counterpart. IgE reactivity analysis in 28 patients allergic to chicken meat revealed that Gal d 7 is a major allergen for patients primarily sensitized to chicken meat. Furthermore, Gal d 7-cross-reactive allergens were also detected in other poultry species, suggesting that recombinant Gal d 7 can be used as a diagnostic marker allergen for poultry meat allergy. The high thermal stability, refolding capacity, and resistance to gastrointestinal enzymes might explain why Gal d 7 can act as a potent sensitizing agent.
Gal d 7 represents a novel major chicken meat allergen. Recombinant Gal d 7 could be used for diagnosis of genuine poultry meat sensitization.

Four recombinant (r) allergens (rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101) were successfully produced and used for sensitization studies. The allergens belong to the profilin family which is one of the most numerous allergen families. These four proteins represent allergens originating from pollen of weeds (rAmb a 8.0101 and rArt v 4.0101), tree (rBet v 2.0101) and grass (rPhl p 12.0101). The recombinant allergens were characterized using various biochemical and biophysical methods and tested for their ability to bind patient-derived antibodies. One hundred patients aged 2 to 50 years sensitized to pollen and plant-derived food allergens (IgE > 0.35 kU/L) were included. Sensitization to individual allergen sources and components of birch and timothy pollens was evaluated using multiparameter immunoblots. The presence of IgE to pollen-derived recombinant profilins rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101 in serum was evaluated using ELISA method. The presence of IgE against pollen profilins was detected in 20 out of 100 studied patients. High correlation was seen between IgE ELISA results with individual pollen profilins. In summary, it was shown that the recombinant versions of the four allergenic profilins can be used for sensitization studies and for component-resolved allergy diagnostics.

OBJECTIVE: The prevalence of fish allergy has increased in recent years. The parvalbumin Gad c 1 is a major cod allergen that is used as a follow-up marker in patients with fish allergy. Objectives: To determine the clinical and laboratory characteristics of a population of patients with fish allergy. To analyze the role of the specific IgE (sIgE) of recombinant Gad c 1 (rGad c 1) and skin prick tests (SPTs) in confirming the acquisition of tolerance to fish.
METHODS: We performed a retrospective study of patients with fish allergy from July 1, 2005 to December 31, 2016. The population was characterized according to demographic data, species of fish associated with allergic reactions, and symptoms. The SPT wheal diameter and sIgE for fish and rGad c 1 were evaluated before acquisition of tolerance (T0) and afterwards (T1).
RESULTS: The study population comprised 81 patients (68% male). Most reactions were triggered by hake (51%), mackerel (30%), and cod (26%). The most frequent manifestations were urticaria/angioedema (72%), gastrointestinal symptoms (35%), and eczema (33%); 42% of patients experienced anaphylaxis. At T0, the average sIgE values were as follows: cod, 32.2 kUA/L; sardine, 18.4 kUA/L; hake, 17.5 kUA/L; salmon, 13.9 kUA/L; tuna, 4.5 kUA/L; and rGad c 1, 22.9 kUA/L. In patients who acquired tolerance to at least 1 fish species (n=60; 74%), the mean value of rGad c 1 at T1 (5.1 kUA/L) was significantly lower than at T0 (16.8 kUA/L) (P=.001). Significant values were also recorded for the average diameter of the SPT wheal and the evaluations at T0 and T1 for hake (9.42 mm/3.79 mm) and salmon (7.8 mm/2.8 mm) (P=.002 and P=.026, respectively).
CONCLUSIONS: The decrease in sIgE to rGad c 1 and the mean wheal diameter of SPT for hake and salmon can be used as markers of prognosis in the acquisition of tolerance by fish-allergic patients.