A comparative evaluation of the biological action of allergens and allergoids prepared from plant pollen by the double radial immunodiffusion method

Antisera to allergens and allergoids prepared from timothy, orchard grass, birch and wormwood pollen have been obtained and used in the double radial immunodiffusion test. The preparations of the allergoid row have been found capable of inducing immune response in laboratory animals (rabbits). Both forms of pollen preparations, allergens and allergoids, have been shown to possess common antigenic determinants reacting with antibodies present in antisera to allergens and allergoids. The absence of identity in the ratio of manifestations of gel precipitation reactions for allergoid with respect to the initial forms of allergens of individual pollen preparation has been noted.     

T cell reactivity with allergoids: influence of the type of APC

The use of allergoids for allergen-specific immunotherapy has been established for many years. The characteristic features of these chemically modified allergens are their strongly reduced IgE binding activity compared with the native form and the retained immunogenicity. T cell reactivity of chemically modified allergens is documented in animals, but in humans indirect evidence of reactivity has been concluded from the induction of allergen-specific IgG during immunotherapy. Direct evidence of T cell reactivity was obtained recently using isolated human T cells. To obtain further insight into the mechanism of action of allergoids, we compared the Ag-presenting capacity of different APC types, including DC and macrophages, generated from CD14+ precursor cells from the blood of grass pollen allergic subjects, autologous PBMC, and B cells. These APC were used in experiments together with Phl p 5-specific T cell clones under stimulation with grass pollen allergen extract, rPhl p 5b, and the respective allergoids. Using DC and macrophages, allergoids exhibited a pronounced and reproducible T cell-stimulating capacity. Responses were superior to those with PBMC, and isolated B cells failed to present allergoids. Considerable IL-12 production was observed only when using the DC for Ag presentation of both allergens and allergoids. The amount of IL-10 in supernatants was dependent on the phenotype of the respective T cell clone. High IL-10 production was associated with suppressed IL-12 production from the DC in most cases. In conclusion, the reactivity of Th cells with allergoids is dependent on the type of the APC.     

Characterization of Phleum pratense pollen extracts by 2-D DIGE and allergen immunoreactivity

The allergen content of standardized pollen material is crucial for an effective diagnosis and treatment. However, variations in IgE reactivities of allergic patients to different preparations of Phleum pratense pollen have been reported. In order to define and directly compare the allergen composition of pollen preparations provided by different suppliers, a comprehensive proteome analysis of three different timothy grass pollen extracts was performed. More than 140 proteins were annotated comprising the pollen proteome/allergome in a global 2-D map. With regard to the individual pollen preparations, several major differences in the overall protein composition were detected that also affected known Phleum allergens and their isoforms. Importantly, these differences were also reflected at the level of antibody reactivities in 1-D and 2-D immunoblots. As a consequence, it is suggested that the observed differences should be taken into consideration aiming for a standardized diagnosis and therapy of grass pollen allergies as recommended by international medical agencies.     

A comparative immunochemical analysis of allergoids and allergens]

In comparison with allergens having protein fragments with a molecular weight not exceeding 110 kD, allergoids have been found to consist of larger fragments with a molecular weight of 10-150 kD. Allergoids have less charged components than initial allergens and less antigenic components. Allergoids retain their capacity for stimulating the production of antibodies, specific to all antigenic components.     

Immunochemical and genetic studies of Amb.t. V (Ra5G), an Ra5 homologue from giant ragweed pollen

Giant ragweed pollen allergen Amb.t. V (Ra5G), a homologue of short ragweed pollen Amb.a. V (Ra5S), was isolated in ultrapure form from a 16-min extract of ragweed pollen by a combination of molecular sieving through an Amicon hollow fiber cartridge (H1P5), cation-exchange chromatography, and gel filtration. The size was found to be 4400 daltons (D) by amino acid analysis and 6000 D by SDS-PAGE, and the pI was 8.3 as determined by isoelectric focusing. There was no cross-reactivity detected between the two Amb. V antigens by immunodiffusion and IEP with the use of hyperimmune antisera raised against crude or highly purified antigens. Cross-reactivity between the two Amb. V antigens was further investigated by inhibition double antibody radioimmunoassay by using the sera of nine selected ragweed-allergic patients who had recently been immunized with either mixed short-giant ragweed pollen extract or with short ragweed extract alone and who had IgG antibodies (Ab) to Amb.t. V and generally to Amb.a. V. Unlabeled Amb.t. V did not inhibit the binding of 125I-Amb.a. V to the IgG Ab in any of the sera tested. Conversely, unlabeled Amb.a. V produced some inhibition of the binding of 125I-Amb.t. V to the patients' IgG Ab, primarily in those patients who had received immunotherapy with short ragweed alone. This weak cross-reactivity was probably a result of the primary structural homology between the two protein allergens. The sera from two groups of ragweed-allergic individuals were investigated for the presence of IgG and IgE Ab to Amb.t. V. The presence of IgG Ab was found to be associated both with previous (or current) immunotherapy with giant ragweed extract and with HLA-Dw2. The HLA association is of interest in view of the previously established association between Dw2 and response toward the homologue Amb.a. V. The result suggests the existence of a similar genetic control at the primary level of antigenic recognition of the two Amb. V antigens.     

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.     

Structural studies of novel glycoconjugates from polymerized allergens (allergoids) and mannans as allergy vaccines

Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy. Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells. Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde. The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (non-oxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and ¹H-¹³C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition.     

Atmospheric pollen and fungal spores in Hamilton in 1972 estimated by the Hirst automatic volumetric spore trap

A knowledge of the atmospheric pollen and fungal spores is necessary for the diagnosis and management of extrinsic rhinitis and asthma. The Hirst automatic volumetric spore trap has been used for the first time in Canada to identify the quantitative and seasonal incidence of these particles. The trap is easy to operate and has several advantages over the previously used gravity samplers. Tree, grass and ragweed pollens occurred in short, well-defined seasons. Fungal spores greatly outnumbered pollen by 120 to one, and occurred in long, ill-defined seasons. They included large numbers of small basidiospores and ascospores which have previously not been detected in Canada. The latter have not been considered as potential allergens; their clinical importance requires investigation.     

Expression and analysis of recombinant Amb a V and Amb t V allergens. Comparison with native proteins by immunological assays and NMR spectroscopy.

The Amb V allergens are small, highly disulfide-bonded ragweed pollen allergens that serve as useful models for understanding the molecular basis of the human immune response. We have produced recombinant Amb a V and Amb t V (from short and giant ragweed pollens, respectively) in Escherichia coli and have compared their structural and functional characteristics to those of the native proteins. Recombinant Amb t V was indistinguishable from native Amb t V as determined by NMR spectroscopy and antibody-binding studies. Whereas inhibition analysis showed that recombinant Amb a V possessed only approximately 50% of the antibody-binding activity of native Amb a V, the two proteins were similarly effective in stimulating Amb a V-specific T-cells. Our results demonstrate that even highly homologous proteins exhibit different abilities to fold into their native three-dimensional conformations and establish the potential and limits of expressing the recombinant Amb V allergens intracellularly in E. coli.     

Multiple Amb a I allergens demonstrate specific reactivity with IgE and T cells from ragweed-allergic patients.

The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels.     

Localization and release of allergens from tapetum and pollen grains ofBetula pendula

Summary Although intact pollen grains are assumed to be the primary carrier of pollen allergens, specific immunoreactive components have been found in other aerosol fractions, e.g., starch grains and remains of tapetal cells Cryo-scanning-electron-microscopy results demonstrate the presence of a clear network of strands connecting the tapetum with the microspores. The distribution of protein in tapetal orbicules, pollen wall, and pollen cytoplasm was tested by histochemical stains for light microscopy and transmission electron microscopy. The protein is mainly localized at the apertures and starch grains in the cytoplasm of pollen and in the core and on the surface of tapetal orbicules. Monoclonal antibodies Bv-10, BIP3, and BIP4 have been used to locate the cellular sites of pollen and tapetal allergens inBetula pendula (syn.B. verrucosa). The application of rapid-freeze fixation prevented relocation of allergens from their native sites. The allergens are predominantly found in the starch grains and to lesser extent in the exine. We also tested interactions between mature birch pollen and human fluids: saliva, nostrils fluid, and eyes solution. The aim was to mimic more closely the in vivo situation during allergenic response. In all cases we observed several pollen grains that were burst and had released their cytoplasmic contents. In the nose the allergens are released from the pollen within minutes. In rhinitis, nasal pH is increased from the normal pH 6.0 to 8.0. When we used nasal fluid at pH 8.0, the number of ruptured pollen grains increased. The mechanism that might induce formation of small allergen-bearing particles from living plant cells is discussed.     

The contribution of long-distance transport to the presence of <Emphasis Type="Italic">Ambrosia</Emphasis> pollen in central northern Italy

Ragweed is an allergenic weed of public health concern in several European countries. In Italy ragweed occurs prevalently in north-north-eastern regions, where sensitization is increasing. Because of the small diameter of pollen grains, ragweed pollen is often involved in episodes of long-range transport, as already shown in central Italy. The objective of this study was to evaluate the extent of such transport by comparing pollen and meteorological data for two northern Italian cities (Parma and Mantova) with data from Pistoia and Florence in central Italy. In 2002 and 2004 peaks in ragweed pollen levels were detected in these four cities on the same day, and concentrations of the grains were above clinical thresholds. Weather-map analysis and computation of back-trajectories showed that air masses from eastern Europe might carry ragweed pollen to a wide area of central and northern Italy. These findings suggest that episodes of long-range transport of ragweed pollen could be clinically relevant, resulting in sensitization of a large number of people. The results might provide a basis for monitoring and forecasting periods of long-distance transport with the objective of reducing their effects on allergic patients.     

Ragweed (Ambrosia) sensitisation rates correlate withthe amount of inhaled airborne pollen. A 14-year studyin Vienna, Austria

Ragweed pollen have been monitored since 1976 inVienna. Since 1984, the outdoor patients of theallergy department of the ear-nose-and-throatUniversity Clinic underwent both Skin Prick Test andRAST/CAP test with a standard series of commoninhalant allergens, ragweed (Ambrosia elatiorL.) included. Both the ragweed counts and the number of positiveRAST results showed a significant increase by time.Furthermore, a clear correlation between the number ofairborne pollen and the percentage of positiveRAST/CAP results is evident.The immune-response in the Viennese population ofatopic subjects is dependent on the amount of inhaledpollen.     

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29–31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more α-arabinofuranosyl residues with some β-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked β-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.     

The isolation and composition of helical protein microfibrils from Hevea brasiliensis latex

1. The microfibrils contained within the lutoid particles of Hevea brasiliensis latex obtained from young tissue have been isolated by methods based on low-speed centrifugation, isoelectric precipitation and gel filtration. 2. The isolated microfibrils behave as a single protein having an isoelectric point of about 4 as determined by paper electrophoresis. 3. The only components so far detected in the microfibrils are protein and possibly carbohydrate; nucleic acid appears to be absent. 4. The amino acid composition of the microfibril protein shows no unusual features. 5. In latex from the more mature laticiferous tissues of H. brasiliensis, the lutoid particles appear to be devoid of microfibrils or their protein decomposition products.     

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.     

Pectate Lyase Pollen Allergens: Sensitization Profiles and Cross-Reactivity Pattern

BackgroundPollen 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.MethodsThe 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.ResultsIn 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.ConclusionWe 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.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.