Pattern of sensitization to Juniperus oxycedrus 4EF-hand polcalcin, Jun o 4, compared with the 2EF-hand grass homolog Phl p 7 in a general Italian population of subjects suffering from pollinosis.

Cupressaceae pollen causes allergic reactions worldwide with long-lasting symptomatic periods. Currently, no cypress polcalcin is available for diagnostic purposes. With the aim to investigate the pattern of sensitization to a cypress polcalcin, a synthetic gene of Jun o 4, the Juniperus oxycedrus 4EF-hand polcalcin, was cloned and expressed in Escherichia coli. Its features were investigated in comparison with the grass 2EF-hand Phl p 7. Rhinitis was the symptom most frequently reported in a cohort of Italian patients sensitized to rJun o 4 and/or rPhl p 7. The detection of many pollen allergic patients sensitized to the cypress polcalcin, but negative to Phl p 7, indicates that Phl p 7 cannot be further considered a marker of sensitization towards all the polcalcins. A 4EF-hand cypress polcalcin claims the inclusion in allergy diagnostic tests. In addition, the sensitivity of polcalcins to gastrointestinal digestion is reported and discussed for the first time.

Molecular profile of specific IgE to allergenic components in allergic adults using allergen nano-bead array.

BACKGROUND: An increasing interest in the field of molecular diagnosis of allergy has been developed in recent years and it goes to be as the routine in vitro protocol in allergy diagnosis. friendly allergen nano-bead array (FABER) is a new multiplex assay for the evaluation of specific IgE against 244 allergens including whole extracts and allergenic molecules. The research intended to assess the pattern of IgE sensitization to allergenic components of allergens in allergic adults using FABER 244. METHODS: Sixty patients with allergic diseases entered this cross-sectional study. Specific IgE to 122 whole allergens extracts and 122 allergenic components were assessed using an allergen nano-bead array (FABER) for all patients. This test includes inhalant and food allergens. RESULTS: Thirty-seven patients were male (61.7%). The mean (SD) age of patients was 30.73(±6.87) years. As the allergen nano-bead array results showed, Lolium perenne (63.3%), Phleum pratense (60%) and Platanus acerifolia (51.7%) were considered as the most common IgE sensitizations to the aeroallergen extracts. Moreover, Lol p 1, Phl p 1.0102 and Cup a 1 were found as the most frequent allergenic components in our allergic patients. Among protein families, CCD-bearing proteins, expansin, cysteine protease and profilin families illustrated the highest allergic sensitization. CONCLUSIONS: The results of the present study demonstrated that despite the higher prevalence of sensitization to Salsola kali (47.2%) using extract-based assays in the previous phase of this research, allergenic components of grasses (Lol p 1, Phl p 1.0102), Cup a 1 as well as Sal k1 as the major components of Cupressuss arizonica and Salsola kali showed the higher sensitization, respectively, in adults' allergic patients using FABER test.

Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

Potential allergenicity of Medicago sativa investigated by a combined IgE-binding inhibition, proteomics and in silico approach.

BACKGROUND: Alfalfa (Medicago sativa L) is one of the most planted crops worldwide primarily used to feed animals. The use of alfalfa in human diet as sprouts, infusions and nutritional supplements is rapidly gaining popularity. Despite this, allergenicity assessment of this novel plant food is largely lacking. RESULTS: Here, leaf protein extract of alfalfa was studied using a combined proteomics, Immunoglobulin E (IgE)-binding inhibition assay and in silico approach to find potential allergens. We have identified and annotated 129 proteins using in-gel digestion proteomics and Blast2Go suit. A search against COMPARE database, using the identified proteins as query sequences, revealed high similarity with several allergenic proteins. The Single Point Highest Inhibition Achievable assay (SPHIAa) performed on the multiplex FABER® allergy testing system confirmed the in silico results and showed some additional potential allergens. This approach allowed the detection of proteins in alfalfa leaves cross-reacting with plant allergens from three different allergen families such as lipid transfer, thaumatin-like and Bet v 1-like protein families. In addition, the absence of structural determinants cross-reacting with seed storage allergenic proteins and with animal allergens was recorded. CONCLUSION: This study reports for the first time potential allergenic proteins in alfalfa. The results suggest that this plant food can be safely introduced, as a protein-rich supplement, in the diet of patients allergic to animal food allergens. Allergic patients towards certain plant food allergens need to be careful about consuming alfalfa because they might have allergic symptoms. © 2020 Society of Chemical Industry.

Similar Allergenicity to Different Artemisia Species Is a Consequence of Highly Cross-Reactive Art v 1-Like Molecules.

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin-proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy.

BACKGROUND: Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood. OBJECTIVE: We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT. METHODS: We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT. RESULTS: Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. CONCLUSION: In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions.

BACKGROUND: Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. OBJECTIVE: We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense). METHODS: The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. RESULTS: Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. CONCLUSION: Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.

Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. METHODS: Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. RESULTS: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. CONCLUSION: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

Structure, stability, and IgE binding of the peach allergen Peamaclein (Pru p 7).

Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D-structure up to 90°C, but it becomes unfolded at temperatures of 100-120°C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE-binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein.

Molecular and immunological characterization of Tri a 36, a low molecular weight glutenin, as a novel major wheat food allergen.

Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ∼80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.

Plant and Arthropod IgE-Binding Papain-like Cysteine Proteases: Multiple Contributions to Allergenicity.

Papain-like cysteine proteases are widespread and can be detected in all domains of life. They share structural and enzymatic properties with the group's namesake member, papain. They show a broad range of protein substrates and are involved in several biological processes. These proteases are widely exploited for food, pharmaceutical, chemical and cosmetic biotechnological applications. However, some of them are known to cause allergic reactions. In this context, the objective of this review is to report an overview of some general properties of papain-like cysteine proteases and to highlight their contributions to allergy reactions observed in humans. For instance, the literature shows that their proteolytic activity can cause an increase in tissue permeability, which favours the crossing of allergens through the skin, intestinal and respiratory barriers. The observation that allergy to PLCPs is mostly detected for inhaled proteins is in line with the reports describing mite homologs, such as Der p 1 and Der f 1, as major allergens showing a frequent correlation between sensitisation and clinical allergic reactions. In contrast, the plant food homologs are often digested in the gastrointestinal tract. Therefore, they only rarely can cause allergic reactions in humans. Accordingly, they are reported mainly as a cause of occupational diseases.

Wheat IgE profiling and wheat IgE levels in bakers with allergic occupational phenotypes.

OBJECTIVES: To characterise occupational wheat allergic phenotypes (rhino-conjunctivitis, asthma and dermatitis) and immunoglobulin (IgE) sensitisation to particular wheat allergens in bakers. METHODS: We conducted clinical and immunological evaluations of 81 consecutive bakers reporting occupational symptoms using commercial tests (skin prick test (SPT), specific IgE, ISAC microarray) and six additional dot-blotted wheat allergens (Tri a 39, Tri a Trx, Tri a GST, Tri a 32, Tri a 12, Tri a DH). RESULTS: Wheat SPT resulted positive in 29 bakers and was associated with work-related asthma (p<0.01). Wheat IgE was detected in 51 workers and was associated with work-related asthma (p<0.01) and rhino-conjunctivitis (p<0.05). ISAC Tri a 30 was positive in three workers and was associated with work-related dermatitis (p<0.05). Wheat dot-blotted allergens were positive in 22 bakers. Tri a 32 and Tri a GST were positive in 13 and three bakers, respectively, and both were associated with work-related dermatitis (p<0.05). This association increased (p<0.01) when Tri a 32, Tri a GST and Tri a 30 were analysed together (p<0.01). Wheat IgE levels were associated with work-related dermatitis (p<0.01). CONCLUSIONS: Wheat IgE levels and wheat microarrayed allergens may be associated with some occupational allergic phenotypes. The extension of the panel of wheat allergens may be promising for discriminating the clinical manifestations of baker's allergy.

Comparative Analysis of the Immune Response and the Clinical Allergic Reaction to Papain-like Cysteine Proteases from Fig, Kiwifruit, Papaya, Pineapple and Mites in an Italian Population.

Several plant papain-like cysteine proteases are exploited by the food, cosmetic, pharmaceutical and textile industries. However, some of these enzymes can cause allergic reactions. In this context, we investigated the frequency of sensitization and allergic reactions to some fruit and/or latex cysteine proteases, which are used as additives by the food industry to improve and modify the quality of their products. The FABER test was used to analyse the patients' sensitization towards five plants and, for comparison, two homologous mite cysteine proteases. In an Italian population of 341 allergic patients, 133 (39%) had IgE specific for at least one of the seven cysteine proteases under investigation. Most of the patients were IgE positive for Der p 1 and/or Der f 1 (96.38%) reported a clinical history suggestive of respiratory allergy to mites, whereas none of the subjects sensitized to the homologs from papaya, pineapple and fig reported allergy symptoms following ingestion of these foods. Only one patient referred symptoms from ingesting kiwifruit. Therefore, the obtained results showed that sensitization to the fruit enzymes was only rarely concomitant with allergic reactions. These observations, together with the literature reports, suggest that the allergy to plant papain-like cysteine proteases might mainly be an occupational disease.

Specific IgE toward allergenic molecules is a new prognostic marker in patients with Sézary syndrome.

BACKGROUND: Sézary syndrome (SS) is the aggressive leukemic form of cutaneous T cell lymphoma characterized by erythroderma, the presence of a malignant circulating T memory cells with skin homing potential, and the ability to produce a variety of Th2 soluble factors, such as IL-4 and IL-5. We measured total and specific IgE in SS patients as a further parameter of a Th2-skewed immune system, and studied their clinical impact. METHODS: Specific IgE production in a cohort of 55 SS patients was evaluated by the molecule-based ISAC microarray system. We then evaluated survival times and the hazard ratios in this cohort by Kaplan-Meier and Cox methods. RESULTS: Twenty-four (43.6%) SS patients had specific IgE to both environmental and food allergens. For survival analysis, patients found positive to at least one allergen were defined as IgE+. By comparing IgE+ versus IgE- we found a significant difference in the median survival times, 2.9 versus 8.9 years (p < 0.001). Conversely, no survival difference could be observed when total IgE levels were considered. IgE+ patients had higher levels of CD60+CD49-CD4+ T cells, which also represent another worse prognostic index recently identified. CONCLUSION: SS patients had specific IgE to both environmental and food allergens. IgE+ SS patients had a significant lower survival rate. High levels of CD60-CD49+CD4+ T cells associated with an IgE- phenotype allow the identification of a restricted group of long survivor SS patients. Therefore, specific measurement of IgE seems to be useful in discriminating survival.

Varying allergen composition and content affects the in vivo allergenic activity of commercial Dermatophagoides pteronyssinus extracts.

BACKGROUND: Diagnosis and immunotherapy of house-dust mite (HDM) allergy is still based on natural allergen extracts. The aim of this study was to analyze commercially available Dermatophagoides pteronyssinus extracts from different manufacturers regarding allergen composition and content and whether variations may affect their allergenic activity. METHODS: Antibodies specific for several D. pteronyssinus allergens (Der p 1, 2, 5, 7, 10 and 21) were used to analyze extracts from 10 different manufacturers by immunoblotting. Sandwich ELISAs were used to quantify Der p 1 and Der p 2 in the extracts. Mite-allergic patients (n = 45) were skin-tested with the extracts and tested for immunoglobulin E (IgE) reactivity to a panel of 10 mite allergens (Der p 1, 2, 4, 5, 7, 8, 10, 14, 20 and 21) by dot blot. RESULTS: Only Der p 1 and Der p 2 were detected in all extracts but their concentrations and ratios showed high variability (Der p 1: 6.0-40.8 µg ml(-1); Der p 2: 1.7-45.0 µg ml(-1)). At least 1 out of 4 allergens (i.e. Der p 5, 7, 10 and 21) was not detected in 8 of the studied extracts. Mite-allergic subjects showed different IgE reactivity profiles to the individual mite allergens, the extracts showed different allergenic activity in skin-prick tests and false-negative results. CONCLUSIONS: Commercially available D. pteronyssinus extracts lack important allergens, show great variability regarding allergen composition and content and some gave false-negative diagnostic test results in certain patients.

In planta expression of a mature Der p 1 allergen isolated from an Italian strain of Dermatophagoides pteronyssinus.

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.

Carrier-bound, nonallergenic Ole e 1 peptides for vaccination against olive pollen allergy.

BACKGROUND: Trees of the family Oleaceae (olive and ash) are important allergen sources in Mediterranean countries, Northern and Central Europe, and North America. The major olive pollen allergen Ole e 1 represents the majority of allergenic epitopes in olive pollen and cross-reacts with Fra e 1, the major ash pollen allergen. OBJECTIVE: We sought to develop a safe vaccine for the treatment of Oleaceae pollen allergy. METHODS: We synthesized 5 peptides ranging from 32 to 36 amino acids, which covered the whole sequence of Ole e 1. The IgE and T-cell reactivity of the peptides was compared with that of Ole e 1 by means of dot blot experiments, as well as ELISA, and in proliferation assays. Rabbits were immunized with non-IgE-reactive, keyhole limpet hemocyanin-coupled peptides or Ole e 1. The reactivity of the IgG antibodies with Ole e 1 and their ability to inhibit IgE binding to nOle e 1 was evaluated by means of ELISA. RESULTS: Only the C-terminal Ole e 1 peptide showed IgE binding, whereas the other peptides were nonallergenic. Immunization of rabbits with Ole e 1-derived peptides bound to the carrier molecule keyhole limpet hemocyanin induced in rabbits the production of Ole e 1-specific IgG antibodies, which cross-reacted with Fra e 1, and inhibited olive and ash pollen-sensitized patients' IgE binding to Ole e 1. CONCLUSION: Two non-IgE-binding peptides with low T-cell reactivity from the N-terminus of Ole e 1 were identified that might represent safe vaccine candidates for immunotherapy of Oleaceae pollen allergy.