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.

Boiling down the cysteine-stabilized LTP fold - loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation.

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are important allergens in fruits, pollen, vegetables, nuts and latex. Due to their compact structure, LTPs are highly resistant to heat treatment. Here, Art v 3 from mugwort pollen and Pru p 3 from peach were used as model allergens to in-depth investigate structural and immunological properties upon thermal treatment at different buffer conditions. METHODS: Recombinant Art v 3 and Pru p 3 were purified from E. coli and incubated at 95 °C up to 120 min using sodium phosphate buffer pH 3.4 or 7.3. Physicochemical properties of allergens were analyzed in circular dichroism spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, size exclusion chromatography, and mass spectrometry. The crystal structure of Art v 3.0201 was determined to 1.9 Šresolution. IgG and IgE binding was investigated in ELISA using murine and LTP allergic patients' sera. RESULTS: Highly pure and homogenous recombinant allergens were obtained from bacterial production. The crystal structure of Art v 3.0201 revealed an antiparallel four helix bundle with a C-terminal extension mediating an asymmetric, transient dimer interface and differently sized cavities. Both allergens showed high thermal stability at acidic conditions. In contrast, extensive heat treatment in neutral buffer induced irreversible structural changes due to lanthionine-based cysteine rearrangement. This fostered loss of the typical α-helical structure, increased molecular size and abrogation of IgG and IgE binding epitopes. Pru p 3 lost its structural integrity at shorter heat stress duration than Art v 3, which did however only partially affect the molecule's IgE binding epitopes. CONCLUSION: During thermal treatment, susceptibility to structural changes of the LTP-fold is highly dependent on the surrounding environment but also on intrinsic features of individual LTPs. This is a crucial fact to consider when processing LTP-containing food or food products as this will directly influence their allergenic potential.

Expression and characterization of recombinant Par j 1 and Par j 2 resembling the allergenic epitopes of Parietaria judaica pollen.

The weed wall pellitory, Parietaria judaica, is one the most important pollen allergen sources in the Mediterranean area causing severe symptoms of hay fever and asthma in allergic patients. We report the expression of the major Parietaria allergens, Par j 1 and Par j 2 which belong to the family of lipid transfer proteins, in insect cells. According to circular dichroism analysis and gel filtration, the purified allergens represented folded and monomeric proteins. Insect cell-expressed, folded Par j 2 exhibited higher IgE binding capacity and more than 100-fold higher allergenic activity than unfolded Escherichia coli-expressed Par j 2 as demonstrated by IgE ELISA and basophil activation testing. IgE ELISA inhibition assays showed that Par j 1 and Par j 2, contain genuine and cross-reactive IgE epitopes. IgG antibodies induced by immunization with Par j 2 inhibited binding of allergic patients IgE to Par j 1 only partially. IgE inhibition experiments demonstrated that insect cell-expressed Par j 1 and Par j 2 together resembled the majority of allergenic epitopes of the Parietaria allergome and therefore both should be used for molecular diagnosis and the design of vaccines for allergen-specific immunotherapy of Parietaria allergy.

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.

Isolation of cypress gibberellin-regulated protein: Analysis of its structural features and IgE binding competition with homologous allergens.

The presence in cypress pollen of an important allergen, belonging to the gibberellin-regulated protein (GRP) family, has been suggested for many years. However, it has never been isolated and sometimes the homologous peach allergen, Pru p 7, has been used as a surrogate to perform immunological investigations. The aim of this study has been the isolation and molecular characterization of the GRP contained in the Cupressus sempervirens pollen. This protein, named Cypmaclein, has been purified from the natural source using conventional biochemical methods consisting in different chromatographic separations. Cypmaclein has been identified by direct protein sequencing of the N-terminal region and of internal fragments of the molecule. In SDS-PAGE, its apparent molecular mass is slightly higher than that of Pru p 7. Nevertheless, the mass spectrometry experiments reveal that the exact molecular mass of Cypmaclein (6821.88 Da) is very close to that of Pru p 7 (6909.90 Da). Two regions of Cypmaclein have been sequenced providing 50% of its primary structure. A high overall sequence identity of Cypmaclein with all the analyzed GRP has been observed, although in the N-terminal region the high identity is limited to the homolog of Cryptomeria japonica. In circular dichroism experiments Cypmaclein produced a spectrum overlapping that of Pru p 7. However, the comparative analysis of Cypmaclein, Pru p 7 and Pun g 7 IgE reactivity revealed a behavior that was not completely overlapping, thus suggesting that the IgE epitopes are only partially shared. In single point highest inhibition achievable assays performed with the FABER test, Cypmaclein efficiently competed with the allergenic peach and pomegranate GRP in the binding of specific IgE of patients sensitized to Pru p 7. In conclusion, the natural cypress pollen GRP has been isolated for the first time, its structural features have been investigated and its cross-reactivity with Pru p 7 and Pun g 7 has been demonstrated. This protein is now available for further investigations aimed at understanding its clinical relevance in the allergy to cypress pollen. In addition, the prevalence of sensitization directly to Cypmaclein, and not limited to the homologs, can be defined.

Pectate lyase pollen allergens: sensitization profiles and cross-reactivity pattern.

BACKGROUND: Pollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions. METHODS: The clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients' sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization. RESULTS: In ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity. CONCLUSION: We could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy diagnosis and therapy.

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.

Differential IgE sensitization to cypress pollen associated to a basic allergen of 14 kDa.

The common cypress (Cupressus sempervirens) (Cups) pollen represents the first cause of respiratory allergies in the Mediterranean basin. The aim of this study was to characterize a novel 14-kDa cypress pollen allergen (BP14) allowing a clear dissociation of IgE sensitization patterns among allergic patients. The biochemical and immunochemical characterization of BP14 included determination of its isoelectric point, molecular mass, extraction kinetics, IgE binding prevalence, the presence of bromelain-type cross-reactive carbohydrate determinant and its IgE reactivity under reducing conditions. The presence of potential cross-reactive homologues in closely related cypress species, i.e. Cupressus arizonica (Cupa) and Cryptomeria japonica (Cryj), as well as in several taxonomically unrelated species was also investigated. According to our results, BP14 is easily and quickly solubilized in phosphate-buffered saline and exhibits several allergenic isoforms covering a broad range of pI (6.5-10.5). This allergen displays heat-stable conformational epitopes and does not include cross-reactive carbohydrate determinants, in contrast to high molecular weight cypress allergens. BP14 is expressed at higher levels in Cups than in Cupa and Cryj. No IgE cross-reactivity was found between the 14-kDa Cups pollen protein and proteins from some other non-Cupressaceae pollen allergenic sources such as orchard, timothy, wheat, maize, birch, ash and pine. Thus, IgE reactivity to BP14 is specific to Cupressaceae and discriminates two groups of patients allergic to cypress pollen. It might correspond to a relevant marker in relation to the sensitization process and/or the symptoms observed in some cypress-pollen-allergic patients. Furthermore, the description of BP14 should improve the diagnosis of cypress pollinosis.

Sensitization prevalence, antibody cross-reactivity and immunogenic peptide profile of Api g 2, the non-specific lipid transfer protein 1 of celery.

BACKGROUND: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. METHODOLOGY: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. RESULTS: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. CONCLUSIONS: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

The role of lipid transfer proteins in allergic diseases.

Nonspecific lipid transfer proteins (LTPs) are important allergens in fruits, vegetables, nuts, pollen, and latex. Despite their wide distribution throughout the plant kingdom, their clinical relevance is largely confined to the Mediterranean area. As they can sensitize via the gastrointestinal tract, LPTs are considered true food allergens, and IgE reactivity to LTPs is often associated with severe systemic symptoms. Although Pru p 3 represents the predominant LTP in terms of patients' IgE recognition, the contribution of pollen LTPs in primary sensitization cannot be ruled out. Due to structural homology, LTPs from different allergen sources are generally IgE cross-reactive. However, sensitization profiles among allergic patients are extremely heterogeneous, and individual cross-reactivity patterns can be restricted to a single LTP or encompass many different LTPs. Molecule-based approaches in allergy research and diagnosis are important for better understanding of LTP allergy and could assist clinicians with providing adequate patient-tailored advice.

Isoform identification and characterization of Art v 3, the lipid-transfer protein of mugwort pollen.

Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.

Influence of the natural ripening stage, cold storage, and ethylene treatment on the protein and IgE-binding profiles of green and gold kiwi fruit extracts.

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili.

BACKGROUND: Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. RESULTS: We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. CONCLUSION: Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.

Kiwellin, a modular protein from green and gold kiwi fruits: evidence of in vivo and in vitro processing and IgE binding.

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.

A hypoallergenic vaccine obtained by tail-to-head restructuring of timothy grass pollen profilin, Phl p 12, for the treatment of cross-sensitization to profilin.

Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive alpha-helical contents of the protein but retained its beta-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients' IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.

Allergy multivaccines created by DNA shuffling of tree pollen allergens.

BACKGROUND: The major allergens of trees belonging to the Fagales order are collectively known as the Bet v 1 family. Members of the Fagales order have distinct geographic distribution, and it is expected that depending on the exposure pattern of the individual, inclusion of other Bet v 1 family members might increase the efficacy of the treatment. OBJECTIVE: We aimed to generate molecules that are suitable for specific immunotherapy not only against birch pollen allergy but also against allergies caused by other cross-reactive tree pollens. METHODS: Fourteen genes of the Bet v 1 family were randomly recombined in vitro by means of DNA shuffling. This library of chimeric proteins was screened for molecules displaying low capacity to induce release of inflammatory mediators but with T-cell immunogenicity higher than that of the parental allergens. RESULTS: Two chimeric proteins were selected from the library of shuffled clones displaying low allergenicity and high immunogenicity, as determined in in vitro assays using human and animal cells and antibodies, as well as in vivo in animal models of allergy. CONCLUSION: Our results show that it is possible to randomly recombine in vitro T- and B-cell epitopes of a family of related allergens and to select chimeric proteins that perfectly match the criteria presently thought to be relevant for improving allergen-specific immunotherapy. CLINICAL IMPLICATIONS: The hypoallergenic chimeras described here recombine epitopes of the major Fagales pollen allergens and thus can efficiently substitute a mixture of extracts used for treating patients with tree pollen-induced spring pollinosis worldwide.

Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7.

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

IgE to cross-reactive carbohydrate determinants: analysis of the distribution and appraisal of the in vivo and in vitro reactivity.

BACKGROUND: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. METHODS: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. RESULTS: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. CONCLUSIONS: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.