A hypoallergenic hybrid molecule with increased immunogenicity consisting of derivatives of the major grass pollen allergens, Phl p 2 and Phl p 6

Allergen-specific immunotherapy is currently based on the administration of allergen extracts containing natural allergens. However, its broad application is limited by the poor quality of these extracts. Based on recombinant allergens, well-defined allergy vaccines for allergen-specific immunotherapy can be produced. Furthermore, they can be modified to reduce their allergenic activity and to avoid IgE-mediated side effects. Here, we demonstrate that the immunogenicity of two grass pollen-derived hypoallergenic allergen derivatives could be increased by engineering them as a single hybrid molecule. We used a hypoallergenic Phl p 2 mosaic, generated by fragmentation of the Phl p 2 sequence and reassembly of the resulting peptides in an altered order, and a truncated Phl p 6 allergen, to produce a hybrid protein. The hybrid retained the reduction of IgE reactivity and allergenic activity of its components as shown by ELISA and basophil activation assays. Immunization with the hybrid molecule demonstrated the increased immunogenicity of this molecule, leading to higher levels of allergen-specific IgG antibodies compared to the single components. These antibodies could inhibit patients' IgE binding to the wild-type allergens. Thus, the described strategy allows the development of safer and more efficacious vaccines for the treatment of grass pollen allergy.     

An immunoglobulin-like fold in a major plant allergen: the solution structure of Phl p 2 from timothy grass pollen.

BACKGROUND: Grass pollen allergens are the most important and widespread elicitors of pollen allergy. One of the major plant allergens which millions of people worldwide are sensitized to is Phl p 2, a small protein from timothy grass pollen. Phl p 2 is representative of the large family of cross-reacting plant allergens classified as group 2/3. Recombinant Phl p 2 has been demonstrated by immunological cross-reactivity studies to be immunologically equivalent to the natural protein. RESULTS: We have solved the solution structure of recombinant Phl p 2 by means of nuclear magnetic resonance techniques. The three-dimensional structure of Phl p 2 consists of an all-beta fold with nine antiparallel beta strands that form a beta sandwich. The topology is that of an immunoglobulin-like fold with the addition of a C-terminal strand, as found in the C2 domain superfamily. Lack of functional and sequence similarity with these two families, however, suggests an independent evolution of Phl p 2 and other homologous plant allergens. CONCLUSIONS: Because of the high homology with other plant allergens of groups 1 and 2/3, the structure of Phl p 2 can be used to rationalize some of the immunological properties of the whole family. On the basis of the structure, we suggest possible sites of interaction with IgE antibodies. Knowledge of the Phl p 2 structure may assist the rational structure-based design of synthetic vaccines against grass pollen allergy.     

New biomolecular tools for aerobiological monitoring: Identification of major allergenic Poaceae species through fast real‐time PCR

Grasses (Poaceae) are very common plants, which are widespread in all environments and urban areas. Despite their economical importance, they can represent a problem to humans due to their abundant production of allergenic pollen. Detailed information about the pollen season for these species is needed in order to plan adequate therapies and to warn allergic people about the risks they take in certain areas at certain moments. Moreover, precise identification of the causative species and their allergens is necessary when the patient is treated with allergen‐specific immunotherapy. The intrafamily morphological similarity of grass pollen grains makes it impossible to distinguish which particular species is present in the atmosphere at a given moment. This study aimed at developing new biomolecular tools to analyze aerobiological samples and identifying major allergenic Poaceae taxa at subfamily or species level, exploiting fast real‐time PCR. Protocols were tested for DNA extraction from pollen sampled with volumetric and gravimetric methods. A fragment of the matK plastidial gene was amplified and sequenced in Poaceae species known to have high allergological impact. Species‐ and subfamily‐specific primer–probe systems were designed and tested in fast real‐time PCRs to evaluate the presence of these taxa in aerobiological pollen samples. Species‐specific systems were obtained for four of five studied species. A primer–probe set was also proposed for the detection of Pooideae (a grass subfamily that includes also major cereal grains) in aerobiological samples, as this subfamily includes species carrying both grass allergens from groups 1 and 5. These, among the 11 groups in which grass pollen allergens are classified, are considered responsible for the most frequent and severe symptoms.     

A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen-human IgE antibody interaction

The aim of this study was to investigate the molecular basis of human IgE-allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE-allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents.     

Sensitivity to mugwort pollen allergens (Artemisia vulgaris)

The author has found that 42% of patients with pollinosis had positive skin reactions with mugwort (Artemisia vulgaris) pollen allergens. The majority of tested patients (139 out of 187) were also allergic to grass pollens. However, hypersensitivity to mugwort pollen allergens was isolated and did not accompany grass pollen allergy. The symptoms of pollinosis appeared in this group later than in patients sensitive to grass pollen allergens only (over 21 years of age in 71%). Bronchial asthma was diagnosed in 40% of these patients and allergic skin reactions in 25%. Sensitivity to mugwort pollen allergens was accompanied by the sensitivity to pollen allergens of Graminae family of plants in 80% of cases. The author suggests that sensitivity to mugwort pollen allergens is the second most frequent cause of the pollinosis and is diagnosed too rarely. Failures of desensitization in patients sensitive to pollen allergens of Graminae family of plants may often result from coexisting sensitivity to mugwort pollen allergens as this sensitivity produces not only season but perennial clinical symptoms in nearly 50% of patients. The author discusses also botanical relations and cross-reactions in allergy to mugwort and ragweed pollen allergens.     

Multivariate statistical and other approaches for the separation of cereal from wild Poaceae pollen using a large Holocene dataset

The separation of the pollen of wild Poaceae species from that of domesticated cereal crops is of considerable importance to palynologists studying Holocene vegetational and agricultural change. Studies of the characteristics of modern pollen populations indicate that it may be possible to distinguish cereal pollen from that of many (but not all) undomesticated Poaceae species, though there are few detailed investigations into the applicability of such studies to palaeoecological samples. This paper assesses the reliability of available keys for identifying sub-fossil grass pollen using a large Holocene dataset obtained from a series of well-dated profiles from lowland Yorkshire, England. Pollen within the dataset is classified using the keys of Andersen (Danmarks Geol Undersøgelse, Arbog, 1978, 69–92, 1979) and Küster (1988), and the resulting identifications are compared. The possibilities of combining the two approaches and employing the multivariate statistical techniques of principal component and discriminant analysis to achieve greater confidence of identification are then investigated. Finally, the findings of the above analyses are used to discuss the interpretation of incidences of large Poaceae pollen (i.e. >37 m grain diameter as measured in silicone oil) within the palynological record, particularly during prehistory. The outcomes of this study will be of relevance to other investigations in which careful identification of large grass pollen is desirable, but where preservation or other factors prohibit accurate or confident identification of pollen surface pattern.     

Airspora of Durban: a sub-tropical,coastal South African city

The atmospheric pollen of Durban has been monitored since 1989 using a Burkard sampler. Data are presented here for the three-year period ending in 1991. The results do not reflect the indigenous sub-tropical vegetation of the area, but rather exotic elements that are either naturalised (Morus) or cultivated (Cannabis and Poaceae). The only indigenous pollen that was recorded in any number was that of the lawn grass Cynodon dactylodon and the tree Celtis africana. However, these two plant types are found country-wide, and are not restricted to sub-tropical regions. All major pollen types recorded have been implicated as allergens.     

Comparative genomics of grasses tolerant to aluminum

The family Poaceae includes over 10,000 species, among which are the most economically important cereals: maize, sorghum, rice, wheat, rye, barley, and oat. These cereals are very important components of human and animal food. Although divergence of the members of this family occurred about 40 million years ago, comparative genome analyses demonstrated that gene orders among species of this family remain largely conserved, which can be very useful for understanding their roles and evolution. Even with an intricate evolutionary history in which chromosome fragments, losses and duplications have to be considered at the ploidy level, grasses present a genetic model system for comparative genomics. The availability of mapped molecular markers, rice genome sequences and BAC and EST libraries from several grass species, such as rice, wheat, sorghum, and maize, facilitates biology and phylogeny studies of this group. The value of using information from different species in modern plant genetics is unquestionable, especially in the study of traits such as tolerance to aluminum in soils, which affects plant growth and development. Comparative genomic approaches to aluminum tolerance can identify genomic regions and genes responsible for aluminum tolerance in grasses.     

Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.     

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.     

Differences in atmospheric emissions of Poaceae pollen and Lol p 1 allergen

The allergens of different grass species share similar physicochemical and immunological features that account for the high incidence of allergenic cross-reactivity. We aimed to gain more information on the correlation between Poaceae airborne pollen and allergen concentration and hence make a reliable assessment of true pollen exposure in different bioclimatic areas. The release of Lol p 1 allergen from grass pollen differs between years and areas depending on variables like meteorological factors, biological sources, and cross-reactions with homologous allergens. This study monitored airborne pollen concentrations of grasses and Lol p 1 aeroallergen in León and Ourense, two cities with different climatic conditions located in northwestern Spain. Lol p 1 content in aerosol samples was quantified using specific ELISA antibody plates. Some our results show that Lol p 1 concentration increases when the atmospheric relative humidity is below 70%. This could explain the appearance of protein peaks at times when little or no grass pollen is present, especially after a short and heavy storm.     

Airborne pollen of Brisbane,Australia: A five-year record, 1994 - 1999

The seasonal incidence of pollen in the atmosphere of Brisbane has been established from a near-continuous, volumetric trapping program over the five-year period, July 1994-June 1999. Grass pollen accounts for 71.6% of the average annual pollen load with highest densities (up to 150 grains/m 3 ) recorded in summer and autumn. Significant contributions were also made by taxa of the Cupressaceae (8.7%) and Urticaceae (1.8%) during spring and of the Pinaceae (4.5%) during winter. Pollen seasons of the Casuarinaceae (6.5%) and Myrtaceae (3.2%) are more extended, the former peaking in late winter and the latter in late spring. The onset and duration of the Poaceae and Urticaceae seasons varied from year to year, being later when precipitation levels were low in the late spring-early summer months. Total pollen numbers and grass pollen densities are substantially less than those recorded from southern Australia. Nevertheless, respiratory disease in Brisbane affects up to 10% of the population, and airborne pollen of Poaceae, Urticaceae, Cupressaceae, Pinaceae, and Myrtaceae have been implicated in the release of allergens.     

Use of isoelectric focusing for assessing the composition of pollen allergens

The preparations of allergens and allergoids obtained from ragweed, timothy and wormwood pollen, as well as the preparations of allergens from birch and orchard grass pollen differing in the method of their production, have been studied with the use of analytical isoelectric focusing in a thin gel layer. The composition of the preparations of allergoids differs from that of the allergenic preparations from the pollen of the same plant species by the decreased content of protein components detected in this investigation. The main proteins contained in the preparations of allergoids are distributed in the zone of pH 3.5-4.5. Differences in the composition of different batches of the same allergens, manifested by variations in some protein bands or by their absence, have been noted. Protein components with the isoelectric point in the alkaline zone have been detected only in the preparations of ragweed pollen allergens.     

Pollen Loads and Allergic Rhinitis in Darwin, Australia: A Potential Health Outcome of the Grass-Fire Cycle

Although the prevalence of asthma and allergic rhinitis has been increasing in tropical regions, little is known about the allergenicity of pollens from tropical plant families or the importance of ongoing environmental changes. We investigated associations between daily average pollen counts of several tropical plant families and sales of medications for the treatment of allergic rhinitis in Darwin, Australia—a tropical setting in which grass abundance has increased due to increased fire frequencies and the introduction of African pasture grasses. Daily pollen counts with detailed identification of plant species were undertaken in conjunction with a weekly survey of flowering plant species from April 2004 to November 2005. Five pharmacies provided daily sales data of selected medications commonly used to treat allergic rhinitis. We used generalized linear modeling to examine outcomes. All analyses accounted for the potential confounding effects of time trends, holidays, respiratory viral illnesses, meteorological conditions, and air pollution. The peak total pollen count was 94 grains/m³. Despite the low levels of Poaceae (grass) pollen (maximum daily count, 24 grains/m³), there was a clear association with daily sales of anti-allergic medications greatest at a lag of 1 day. Sales increased by 5% with an interquartile range rise (3 grain/m³) in Poaceae pollen (5.07%, 95%CI 1.04%, 9.25%). No associations were observed with pollen from other plant families. Although further testing is required, we suggest that an overlooked aspect of the “grass-fire cycle” that is degrading many tropical landscapes, could be an increase in the prevalence of allergic rhinitis.     

Biological-based and remote sensing techniques to link vegetative and reproductive development and assess pollen emission in Mediterranean grasses

Connecting the signals of the vegetative and reproductive cycles of plants using large-scale phenological techniques is not always an easy task, and this complexity increases considerably when analysing the plant life cycle in grasses, due to the ubiquity and diversity of this taxonomic family. This work integrates remote sensing techniques (NDVI from satellite remote sensing data and greenness from near-surface imagery) and biological-based techniques (airborne pollen monitoring and field observations and sampling) to analyse phenological patterns and productivity in grass-dominated vegetation types. We aim to answer two main applied and unanswered questions; i) how are the specific phases of vegetative and reproductive cycles in grasses linked at the species and plant community level? and ii) which grass-dominant habitats are the major contributors of grass pollen emission to the atmosphere at the plant community level? The multi-scale integration and validation of large-scale methods such as satellite remote sensing data and aerobiological monitoring using high-resolution or field phenological techniques is recommended. The results clearly support the hypothesis that the highest rates of grass pollen emission are successively produced when the major grass-dominated vegetation types go through the final phases of vegetative development during their biological senescence or equivalent phases. At the plant community level, natural and semi-natural grass-dominated vegetation types, rather than grass cropland habitats, constitute the major sources of pollen emission. The major contributors to the grass pollen emission at the species level are also identified. Finally, a positive relationship between year-to-year primary productivity (measured as annual sum or maximum NDVI) and pollen production (measured as airborne pollen intensity) was observed at the community level. This is a very timely study, as the availability of remote sensing data is increasing interest in generating enhanced forecasting model of allergenic airborne pollen.     

Localisation of allergens in ryegrass pollen and in airborne micronic particles

Summary In Melbourne, Australia, grass pollen allergens, especially from ryegrass, are a major cause of allergic hayfever and asthma. This review outlines recent developments in our understanding of how grass pollen allergens find their way into the atmosphere and how they are transported in particulate form. Much of this work has relied on antibody technology in immunological and immunocytochemical investigations. The localisation of allergens in situ has proved difficult due to their water-soluble character. Recently, allergens have been localised in developing ryegrass pollen by dryfixation, rapid-freeze and freeze-substitution techniques. This involved anthers being substituted in a mixture of aldehydes, organic solvents, and 2,2-dimethoxypropane. Incubation in dimethylsulfoxide prior to embedding in LR Gold resin provided good infiltration with freeze-substituted material. Immunogold-labelled sections show that the major allergens, Lol p 1 and Lol p 5, are synthesised in the pollen cytoplasm from the early bicellular stage, soon after the first starch granules are formed. From the early tricellular stage, Lol p 5 moves into the starch granules where it remains until maturity. Lol p 1 is localised in the cytoplasm of mature pollen grains. The incidence of airborne grass pollen, as measured in pollen traps, correlates with hayfever symptoms. Forecasting models which rely on rainfall and temperature data have been produced for the grass pollen (daily and seasonal) counts in Melbourne. Research over the past six years has shed light on the causes of grass-pollen-induced asthma. Micronic particles in the atmosphere may be starch granules originating from pollen grains osmotically ruptured by rainwater. Ultrastructural and immunological characterisation of micronic particles collected from outdoor air filters confirm the presence of airborne starch granules. These are loaded with grass pollen allergens, occur in the atmosphere especially after rainfall, and correlate significantly with instances of allergic asthma. Diesel particles might also play a role in the transmission of grass pollen allergens and thus become an extra asthma trigger. A variation in the mode of release of micronic particles occurs in other species, such as birch, where such particles are derived from burst birch pollen tubes. These particles are positive for Bet v 1 and are starch granules which are released into the atmosphere after light rain as a result of pollen germination on, e.g., leaves. After subsequent rupture of pollen tubes their contents are released when conditions become drier.Abbreviations DECP diesel exhaust carbon particles - DMP 2,2-dimethoxypropane - GPC grass pollen count - IgE immunoglobulin E - IgG immunoglobulin G - OGPS onset of the grass pollen season     

Poaceae pollen grain size as a tool to distinguish past grasslands in South America: a new methodological approach

Despite the dominance of grasslands during the last glacial period, especially in South America, the highly uniform morphology of Poaceae pollen grains has so far allowed only very few palynological studies based on Poaceae pollen. In our study we compare two methods of distinguishing between South American grassland ecosystems based on quantitative morphology of Poaceae pollen grains. We investigated data sets from Páramo in southern Ecuador, Campos de Altitude and Campos in south-eastern and southern Brazil as well as data sets from the Pampa in Argentina by measuring the pollen grain length, grain width, pore diameter and annulus width. Firstly we investigated the potential influence of chemical treatment of pollen grains on pollen grain size as well as the measurement setting for defining the boundary conditions for using Poaceae pollen grains in a palaeoecological investigation. Finally the measured pollen grain parameters were analyzed by comparison of average grain length using statistical tests. This approach reveals highly significant differences in average grain size between all grassland ecosystems. Assuming that a certain grain size range can be assigned to a certain Poaceae taxon, conclusions about differences and similarities in taxa composition can be derived. We used two methods of multivariate data analysis. One uses the pollen grain parameters directly for a Principle Component Analysis (PCA). The other is an already established method in grassland ecology which defines parameter based pollen grain types to investigate similarities between grassland ecosystems. Both approaches confirm the results of the grain length analysis. In this work we demonstrate that the method we developed has the potential to provide acquisition of so far inaccessible information on spatial and temporal patterns and dynamics of South American grasslands.     

Phenological analysis of grasses (Poaceae) as a support for the dissection of their pollen season in Perugia (Central Italy)

Grasses (Poaceae) pollen is a major cause for allergic diseases worldwide. Pollen monitoring in the atmosphere is of primary importance for symptoms interpretation and therapy planning. Microscopic pollen identification and counts do not allow the detection at species or genus level because of the stenopalynous nature of the family. Nevertheless, the assessment of the flowering phenology of different species would be important, because not all grass allergens are cross-reacting and allergic patients could be differentially sensitized. In this work, a phenological survey was carried out in five stations located on the urban territory of Perugia (Central Italy), from April to September 2015, recording the alternation between flowering phenophases of 19 grass species and estimating their contribution to the airborne pollen load of the area through the calculation of a Phenological Index. Moreover, pollen grains of the different species were collected and observed, confirming the impossibility to make a discrimination during microscope pollen counts. The prevailing grasses in terms of contribution to the pollen detection in the studied area resulted to be Dactylis glomerata and Lolium perenne during spring and early summer, and Cynodon dactylon during late summer. Data should be validated repeating the survey in successive years and possibly using biomolecular tools, but the obtained information could be relevant for diagnosis and treatment of grass pollen allergies.