IgE and IgG antibodies of patients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa*

IgE and IgG antibody response to birch pollen antigens were studied by means of immunoblotting experiments testing 58 sera from patients with Type I allergy to birch pollen. 56/58 patients showed IgE antibodies reactive with Bet v I, a 17 kilodalton (kD) pollen protein. 2D-electrophoresis/immunoblot revealed a heterogeneity of that protein. Ten spots (pH 4.9-5.9) could be detected, presumably representing differentially glycosylated isoallergens. In 33/58 patients, there was no evidence of IgE antibodies directed against allergens other than Bet v I. However, in 25/58 of patients' sera, 11 minor allergens (13, 15, 18, 27, 29, 32, 39, 44, 57, and 68 kD) with individual incidences from 1.7% to 17.2% were identified. All proteins were also recognized by the patients' IgG antibodies: in the case of Bet v I recognition was weak, whereas the IgG response to the minor allergens was pronounced. Sera from healthy individuals showed similar IgG antibody responses, but no IgG to the 15, 27, and 29 kD proteins. Our results suggest that IgG directed against minor allergens may function as trapping antibodies in healthy individuals. Too low or lacking amounts of anti-Bet v I IgG may facilitate an allergic reaction.     

Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen

Background Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer froin allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis. leading to death.
Objective The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy.
Methods A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradennal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting.
Results Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA. Westernblot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v I.
Conclusion Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.     

In vitro and in vivo allergenicity of recombinant Bet v 1 compared to the reactivity of natural birch pollen extract

BACKGROUND : Diagnostic procedures using natural extracts show only limited quantitative correlation between in vivo and in vitro results. Highly pure recombinant allergens might show more predictive findings. OBJECTIVE : The aim of this study was to compare natural birch pollen extract (BPE) and recombinant Betula verrucosa (rBet v 1) for their diagnostic value comparing skin prick tests (SPTS) and nasal provocation tests (NPTS) with specific IgE in the serum. METHODS : Thirty-four patients allergic to birch pollen and five healthy controls were investigated. SPT and NPT were performed with BPE and rBet v 1 at different concentrations. Specific serum IgE was measured by the Pharmacia CAP system. RESULTS : Commercial BPE and rBet v 1 (10 micro g/mL) were able to elicit similar allergenic reactions in vivo and IgE binding in vitro. SPT reflects immediate-type allergy as determined by NPT to a higher degree than specific IgE, for both reagents. To cause allergic reactions in NPT, higher amounts of rBet v 1 were needed than for skin tests and the sensitivity was lower than with BPE. CONCLUSION : rBet v 1 alone is sufficient for a reliable diagnosis of birch pollen allergy in most patients and induces comparable skin test reactivity as BPE, but less allergic reactions in nasal provocations.     

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives

Background Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe. Objective To investigate the clinical effects of injection immunotherapy with genetically modified derivatives of major birch pollen allergen Bet v 1 on pollen‐induced allergic symptoms. Methods A three‐arm double‐blind placebo‐controlled immunotherapy study was conducted with one pre‐seasonal course of treatment using two derivatives of Bet v 1, namely a recombinant Bet v 1 trimer and an equimolar mixture of two recombinant Bet v 1 fragments together representing the whole protein sequence. Analysis of local and systemic adverse events was performed for 124 patients who had received at least one dose of medication. Clinical efficacy was monitored by symptom medication scores and interval scoring in the per protocol‐treated population (n=84). In addition, skin and nasal provocation responses and allergen‐specific antibodies were assessed. Results There were trends towards improvement in the subjects' well‐being and clinical symptoms (nasal scores), although comparisons with a placebo group did not show statistical significance in the main end‐point, the combined symptom medication score. Reductions in skin and nasal sensitivity were observed for some subjects with a trend for the Bet v 1 trimer to be more effective than the fragments. Treatment induced strong IgG1 and IgG4 allergen‐specific antibody responses. Local injection‐site reactions were most frequent in the trimer group affecting 59.5% of patients as opposed to 37.8% and 30.6% in the fragment and placebo groups, respectively. Systemic reactions were elicited more frequently by fragments. A large proportion of adverse side‐effects appeared hours following injections, and might be attributable to concurrent exposure to related pollens. Conclusion Single courses of injection immunotherapy with Bet v 1 allergen derivatives showed trends towards improved well‐being and reduced reactivity to specific allergen provocation, but did not yield significant improvement in the combined symptom medication score in this study.     

Allergenicity, immunogenicity and dose-relationship of three intact allergen vaccines and four allergoid vaccines for subcutaneous grass pollen immunotherapy

Different vaccines containing intact allergens or chemically modified allergoids as active ingredients are commercially available for specific immunotherapy. Allergoids are claimed to have decreased allergenicity without loss of immunogenicity and this is stated to allow administration of high allergoid doses. We compared the allergenicity and immunogenicity of four commercially available chemically modified grass pollen allergoid products with three commercially available intact grass pollen allergen vaccines. The allergenicity was investigated with immunoglobulin (Ig)E-inhibition and basophil activation assays. Human T cell proliferation and specific IgG-titres following mouse immunizations were used to address immunogenicity. Furthermore, intact allergen vaccines with different contents of active ingredients were selected to study the influence of the allergen dose. In general, a lower allergenicity for allergen vaccines was clearly linked to a reduced immunogenicity. Compared with the vaccine with the highest amount of intact allergen, the allergoids caused reduced basophil activation as well as diminished immunogenicity demonstrated by reduced T cell activation and/or reduced induction of murine grass-specific IgG antibodies. Interestingly, intact allergen vaccines with lower content of active ingredient exhibited similarly reduced allergenicity, while immunogenicity was still higher or equal to that of allergoids. The low allergenicity observed for some allergoids was inherently linked to a significantly lower immunogenic response questioning the rationale behind the chemical modification into allergoids. In addition, the linkage between allergenicity, immunogenicity and dose found for intact allergen vaccines and the immunogen as well as allergenic immune responses observed for allergoids suggest that the modified allergen vaccines do not contain high doses of immunologically active ingredients.     

Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation: a study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin E detection system measuring immunoglobulin E capable of binding to FcɛRI

BACKGROUND: Results from several studies indicate that the magnitude of immediate symptoms of type I allergy caused by allergen-induced cross-linking of high-affinity Fc epsilon receptors on effector cells (mast cells and basophils) is not always associated with allergen-specific IgE levels. OBJECTIVE: To investigate the association of results from intradermal skin testing, basophil histamine release and allergen-specific IgE, IgG1-4, IgA and IgM antibody levels in a clinical study performed in birch pollen-allergic patients (n = 18). METHODS: rBet v 1-specific IgEs were measured by quantitative CAP measurements and by using purified Fc epsilon RI-derived alpha-chain to quantify IgE capable of binding to effector cells. Bet v 1-specific IgG subclasses, IgA and IgM levels were measured by ELISA, and basophil histamine release was determined in whole blood samples. Intradermal skin testing was performed with the end-point titration method. RESULTS: Our study demonstrates on the molecular level that the concentrations of allergen-specific IgE antibodies capable of binding to Fc epsilon RI and biological sensitivities are not necessarily associated. A moderate association was found between cutaneous and basophil sensitivity. CONCLUSION: Our results highlight the quantitative discrepancies and limitations of the present diagnostic tools in allergy, even when using a single allergenic molecule. The quantity of allergen-specific serum IgE is only one component of far more complex cellular systems (i.e. basophil-based tests, skin tests) used as indirect diagnostic tests for IgE-mediated allergic sensitivity.     

Basophil histamine release in patients with birch pollen hypersensitivity with and without allergic symptoms to fruits

Histamine release (HR) studies were performed in 40 birch pollen-allergic patients (positive case history, positive SPT, positive birch pollen-specific serum IgE: RAST > or = 3) with (n = 20, A) and without (n = 20, B) fruit hypersensitivity, and 10 nonatopic volunteers (C). Several fruit allergens were used and characterized by protein determination and immunoblot techniques. Dose-dependent HR (apple peel = apple pulp > peach = cherry) was demonstrated in both allergic groups, but to a higher extent in patients with fruit allergy (P < 0.01). Increased basophil sensitivity to birch pollen was found in the group with fruit allergy (P < 0.001). Strong correlations between the mediator response induced by several fruits indicate common allergens within the extracts. We conclude that fruit-related symptoms require not only high specific serum IgE, but a strong cellular sensitization to birch pollen allergens together with an increased cellular reactivity to fruit allergens.     

Effects of air pollution and other environmental factors on birch pollen allergens

To determine the effects of anthropogenic pollution on water-soluble proteins and specifically allergens in birch ( Betula pendula and B. pubescens ) pollen, we analyzed extracts of pollen from the pollution gradient around a factory complex (emitting sulfur oxides and heavy metals) by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE) and IgE immunoblotting. In addition, tree density-associated shading of the tree habitat, and quantity and quality of proteins and allergens in pollen of the two birch species were studied. The two studied birch species gave identical allergen profiles even though their protein profiles differed. Distance from the factory did not affect the amount of birch pollen major allergen. Bet v 1 (17 kDa), or of two other strong allergens (23 and 36 kDa). Trees growing in shaded places had significantly stronger responses to Bet v 1 and to the 23-kDa allergen than trees growing in open or half-open environments. Thus, we propose that combined heavy metal and sulfur dioxide pollution does not have an important effect on birch pollen allergens. Instead, other factors, e.g., shading and soil properties of the tree habitat, as well as the genetic background of the tree, may have a stronger influence on the quantity and relative composition of allergens.     

Efficacy and safety of preseasonal-specific immunotherapy with an aluminium-adsorbed six-grass pollen allergoid

BACKGROUND: The clinical efficacy and safety of a six-grass pollen allergoid has been studied. The advent of more exacting clinical guidelines and a better appreciation of the possible mechanisms of treatment prompted this reappraisal. METHODS: A 2-year double-blind multicentre placebo-controlled phase 3 clinical trial was undertaken in 154 patients suffering symptoms of rhinoconjunctivitis with or without asthma (GINA I or II). Therapy comprised two consecutive preseasonal short-courses of subcutaneous injections using a grass pollen allergoid adsorbed to aluminium hydroxide. RESULTS: A combined symptom and medication score (SMS) was used as the primary end-point for clinical efficacy. SMS from the first year showed a significant difference of 26.6% between the two study groups (P=0.026) and this was improved after the second year when there was a 48.4% difference in SMS between active and placebo treatment in favour of the allergoid (P = 0.018). Highly significant increases in grass pollen allergen-specific IgG1 and IgG4 antibody concentrations were measured in association with active treatment. Allergen tolerance was increased as judged by a conjunctival provocation test and significant improvements in quality of life were documented using a standardized questionnaire. The allergoid was well tolerated. CONCLUSIONS: The grass pollen allergoid was shown to be safe and clinically efficacious in the management of hay fever with or without asthma (GINA I or II).     

Apple allergy: the IgE-binding potency of apple strains is related to the occurrence of the 18-kDa allergen

Low-temperature, acetone powder extracts were prepared from mature fruit of 16 apple strains. SDS-PAGE and immunoblot analysis revealed great variation in the relative amounts of the 18-kDa apple allergen in these extracts. EAST (RAST) scores, measured with individual and pool sera from patients allergic to birch pollen and apples, ranged from 0.2 to 4.0 and were related to the relative amount of the 18-kDa protein. These findings were confirmed by ELISA-inhibition assays, dose-related histamine release, semiquantitative evaluation of immunoblots by absorption/reflection densitometry, and skin prick tests with extracts of Golden Delicious, Boskoop, and Jamba apples (corresponding to a high, low, and very low 18-kDa allergen content). Additional open oral challenge tests were performed with two apple-allergic patients and 15 and 16 apple strains. With all methods, the deduced allergenic potency decreased in the following order: Golden Delicious>Boskoop>Jamba. Therefore, we concluded that the IgE-binding potency of apple strains depends on the occurrence of the 18-kDa allergen.     

Determination of the allergenic activity of birch pollen and apple prick test solutions by measurement of β‐hexosaminidase release from RBL‐2H3 cells. Comparison with classical methods in allergen standardization

BACKGROUND: A murine in vitro model of the allergic type I reaction was set up to determine the biologic activity of extracts without involvement of human beings. It is based on beta-hexosaminidase release from passively sensitized RBL cells after allergen challenge. The intended application of this RBL cell assay in the field of quality control of allergenic extracts requires its comparison with established methods. METHODS: The activity of five standardized birch-pollen prick test solutions was determined in parallel by RBL assay, direct IgE binding, IgE-binding inhibition, major allergen content, histamine-release assay, and skin testing. RESULTS: The RBL cell-release assay corresponded well to other methods if a reagin raised against natural birch-pollen extract was used for passive sensitization. However, in the case of a reagin against recombinant Bet v 1, only a decreased activity was observed, presumably because a reduced number of epitopes were recognized by the monospecific reagin. In contrast to standardized birch-pollen extracts, nonstandardized apple extracts showed poor activity in all assays. CONCLUSIONS: This murine model might be a useful tool in the quality control of allergenic extracts. It combines properties of assays based on standardized antisera and of assays that consider IgE cross-linking properties.     

Effects of birch pollen and traffic particulate matter on Th2 cytokines, immunoglobulin E levels and bronchial hyper-responsiveness in mice

BACKGROUND: Health effects due to air pollution arising from motor vehicles are a major public and political concern world-wide. Epidemiological studies have shown that the manifestations of asthma are increased by air pollution in already affected individuals. OBJECTIVE: To investigate the potential role of air-polluted tunnel dust (traffic particulate matter, TPM) or pure carbon core particles in the initiation and persistence of experimental allergic inflammation. METHODS: BP2 mice were immunized with birch pollen alone (group B) or pollen together with TPM (group A), or with birch pollen and Al(OH)3 (group C), or with birch pollen and carbon core particles (group D). Before methacholine challenge they were challenged intranasally and thereafter bronchial hyper-reactivity (BHR) was evaluated in a whole-body plethysmograph. Levels of Th2 cytokines, fibronectin and lactate dehydrogenase (LDH) were determined, and differential counts were performed in the bronchoalveolar lavage (BAL) fluid. Sera were collected for determination of antibody titres and cytokine levels. RESULTS: Specific IgE titres, BHR, the number of recruited eosinophils and levels of fibronectin and LDH in BAL were increased in mice immunized and challenged with a mixture of birch pollen and TPM. However, mice immunized with birch pollen alone and challenged intranasally with pollen or a mixture of pollen and TPM demonstrated the highest levels of IL-4 and IL-5. CONCLUSION: This study highlights the importance of the exposure to a combination of particulate matters and pollen allergens, in the induction of allergic disease in the airways, and we have demonstrated that polluted tunnel dust has an effect on both the inflammatory and immunological components of experimental allergy. Immunization and challenge with carbon core particles together with birch pollen increased neither the BHR nor the specific IgE production significantly. Our results therefore strongly suggest that it is most likely to be the organic phase bound to the carbon core of the diesel exhaust particles that might have an important adjuvant effect in the induction of experimental allergy.     

Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2

BACKGROUND: Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen. To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen. OBJECTIVES: Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin. METHODS: Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5'RACE method for the identification of the unknown 5'-end of the cDNA. Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a. The recombinant profilin was purified by affinity chromatography on poly L-proline coupled to sepharose. Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. RESULTS: The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71-82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was similar to that of natural protein. Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting. Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen. Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay. CONCLUSIONS: Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.