Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System.

Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from Brachypodium distachyon as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in B. distachyon. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific cis-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species.

Molecular evolution of hypoallergenic hybrid proteins for vaccination against grass pollen allergy.

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.

High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes.

Levan, a type of fructan, is an oligomer or polymer with mainly a ß(2,6)-linked fructose chain attached to sucrose. We introduced two timothy genes, PpFT1 and PpFT2, coding for two homologous sucrose:fructan 6-fructosyltransferases into sugar beet. Sugar beet produces a high concentration of sucrose, a starting substrate in fructan synthesis, in the root. Among transgenic T1 lines, we obtained sugar beet transformants that accumulated large amounts of ß(2,6)-linked levans (about 20 to 75mgg(-1) FW) in the roots. The transformed sugar beet plants possessing PpFT1 or PpFT2 produced linear levans with different degrees of polymerization (DP). Namely, the PpFT1 transformants accumulated mainly high DP levans including those with DP>40, while the PpFT2 transformants accumulated levans with DP between 3 and 40. Chromatograms showed that PpFT2 produces pure ß(2,6)-linked linear levans compared with fructans synthesized by PpFT1. These levans belong to the high DP class of plant fructans, but have much shorter DP than that of levans generally produced by microorganisms.

Hypoallergenic mutants of the Timothy grass pollen allergen Phl p 5 generated by proline mutations.

BACKGROUND: Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions. METHODS: All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays. RESULTS: Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen. CONCLUSIONS: The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens.

A mimotope gene encoding the major IgE epitope of allergen Phl p 5 for epitope-specific immunization.

A gene vaccine based on a mammalian expression vector containing the sequence of a peptide mimotope of Phl p 5 was constructed. To test whether mimotope gene vaccines can induce allergen-specific antibody responses via molecular mimicry, BALB/c mice were immunized using the mimotope construct with or without a tetanus toxin T-helper epitope. Moreover, intradermal injection was compared to epidermal application via gene gun immunization. Immunization with both mimotope gene constructs elicited allergen-specific antibody responses. As expected, gene gun bombardment induced a Th2-biased immune response, typically associated with IgG1 and IgE antibody production. In contrast, intradermal injection of the vaccine triggered IgG2a antibody expression without any detectable IgE levels, thus biasing the immune response towards Th1. In an RBL assay, mimotope-specific IgG antibodies were able to prevent cross-linking of allergen-specific IgE by Phl p 5. A construct coding for the complete Phl p 5 induced T-cell activation, IFN-gamma and IL-4 production. In contrast, the mimotope-DNA construct being devoid of allergen-specific T-cell epitopes had no capacity to activate allergen-specific T cells. Taken together, our data show that it is feasible to induce blocking IgG antibodies with a mimotope-DNA construct when applied intradermally. Thus the mimotope-DNA strategy has two advantages: (1) the avoidance of IgE induction and (2) the avoidance of triggering allergen-specific T-lymphocytes. We therefore suggest that mimotope gene vaccines are potential candidates for epitope-specific immunotherapy of type I allergy.

Tolerization of a type I allergic immune response through transplantation of genetically modified hematopoietic stem cells.

Allergy represents a hypersensitivity disease that affects >25% of the population in industrialized countries. The underlying type I allergic immune reaction occurs in predisposed atopic individuals in response to otherwise harmless Ags (i.e., allergens) and is characterized by the production of allergen-specific IgE, an allergen-specific T cell response, and the release of biologically active mediators such as histamine from mast cells and basophils. Regimens permanently tolerizing an allergic immune response still need to be developed. We therefore retrovirally transduced murine hematopoietic stem cells to express the major grass pollen allergen Phl p 5 on their cell membrane. Transplantation of these genetically modified hematopoietic stem cells led to durable multilineage molecular chimerism and permanent immunological tolerance toward the introduced allergen at the B cell, T cell, and effector cell levels. Notably, Phl p 5-specific serum IgE and IgG remained undetectable, and T cell nonresponsiveness persisted throughout follow-up (40 wk). Besides, mediator release was specifically absent in in vitro and in vivo assays. B cell, T cell, and effector cell responses to an unrelated control allergen (Bet v 1) were unperturbed, demonstrating specificity of this tolerance protocol. We thus describe a novel cell-based strategy for the prevention of allergy.

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity.

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.

A pollen-specific polygalacturonase from lily is related to major grass pollen allergens.

A pollen-specific gene from lily (Lilium longiflorum Thunb. cv. Snow Queen), designated LLP-PG, was characterized. Southern blots of lily genomic DNA indicated that LLP-PG is a member of a small gene family. A thorough sequence analysis revealed that the LLP-PG gene is interrupted by two introns and encodes a protein of 413 amino acids, with a calculated molecular mass of 44 kDa, and a pI of 8.1. Evaluation of the hydropathy profile showed that the protein has a hydrophobic segment at the N-terminus, indicating the presence of a putative signal peptide. A sequence similarity search showed a significant homology of the encoded protein to pollen polygalacturonases (PGs) from various plant species and to an important group (group 13) of grass pollen allergens. The LLP-PG transcript is pollen-specific and it accumulates only at the latest stage during pollen development, in the mature pollen. In contrast to other "late genes" LLP-PG transcript can neither be induced by abscisic acid (ABA) nor by dehydration. Immunoblot analyses of pollen protein extracts from lily, timothy grass and tobacco with IgG antibodies directed against LLP-PG and against the timothy grass pollen allergen, Phl p 13, indicated that lily LLP-PG shares surface-exposed epitopes with pollen PGs from monocotyledonous and dicotyledonous plants. Enzyme-linked immunosorbent assay (ELISA) analyses and inhibition ELISA assays with patients' IgE demonstrated a very low IgE reactivity of lily rLLP-PG and a lack of cross-reactivity between rLLP-PG and the timothy grass pollen allergen, rPhl p 13. These data demonstrated that despite the significant sequence homology and the conserved surface-exposed epitopes LLP-PG represents a low-allergenic member of pollen PGs.

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.