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.

Different allergenic activity of grass pollen allergens revealed by skin testing.

BACKGROUND: Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens. MATERIAL AND METHODS: Eighty-two grass pollen allergic patients were skin prick tested with serial dilutions of approximately equimolar concentrations of the purified allergens in a double-blind study. RESULTS: Phl p 4 and Phl p 13 were identified as major grass pollen allergens according to IgE binding frequency (Phl p 4: 85%; Phl p 13: 56%), but exhibited a five to nine-fold lower allergenic skin reactivity compared to Phl p 1, Phl p 2 or Phl p 5. CONCLUSION: Our results indicate that Phl p 4 and Phl p 13 are not essential components for a therapeutic grass pollen vaccine and underpin the importance of evaluating the in vivo allergenic activity of individual allergens for the formulation of therapeutic vaccines based on purified allergens.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Characterization of a hypoallergenic recombinant Bet v 1 variant as a candidate for allergen-specific immunotherapy.

BACKGROUND: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. METHODS: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. RESULTS: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. CONCLUSIONS: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.

Generation of a low immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a.

BACKGROUND: Immunotherapy of grass pollen allergy is currently based on the administration of pollen extracts containing natural allergens. Specifically designed recombinant allergens with reduced IgE reactivity could be used in safer and more efficacious future therapy concepts. OBJECTIVES: This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. METHODS: Three deletion mutants were produced in Escherichia coli and subsequently purified. The overall IgE-binding capacity of the mutants was compared with the recombinant wild-type allergen by membrane blot and IgE-inhibition assays. The capacity for effector cell activation was determined in basophil activation assays. T cell proliferation assays with allergen-specific T cell lines were performed to confirm the retention of T cell reactivity. Structural properties were characterized by circular dichroism analysis and homogeneity by native isoelectric focusing. The deletion sites were mapped on homology models comprising the N- and C-terminal halves of Phl p 5a, respectively. RESULTS: The double-deletion mutant rPhl p 5a Delta(94-113, 175-198) showed strongly diminished IgE binding in membrane blot and IgE-inhibition assays. Both deletions affect predominantly alpha-helical regions located in the N- and C-terminal halves of Phl p 5a, respectively. Whereas deletion of Delta175-198 alone was sufficient to cause a large reduction of the IgE reactivity in a subgroup of allergic sera, only the combination of both deletions was highly effective for all the sera tested. rPhl p 5a Delta(94-113, 175-198) consistently showed at least an 11.5-fold reduced capacity to activate basophils compared with the recombinant wild-type molecule, and the T cell proliferation assays demonstrated retention of T cell reactivity. CONCLUSION: The mutant rPhl p 5a Delta(94-113, 175-198) fulfils the basic requirements for a hypoallergenic molecule suitable for a future immunotherapy of grass pollen allergy; it offers substantially reduced IgE binding and maintained T cell reactivity.

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).

Clinical effects of specific immunotherapy: a two-year double-blind, placebo-controlled study with a one year follow-up.

BACKGROUND: A new depot allergoid of house dust mite (Dermatophagoides pteronyssinus - D.pt) has been created in line with the principles and methodology established in the successful development of pollen allergoids. A two-year double-blind placebo-controlled clinical trial, with one further follow-up year of active treatment, has been conducted to assess clinical efficacy and tolerance. METHODS: 40 patients (20 verum and 20 placebo) with IgE-mediated mite allergy and a history of moderate to severe perennial symptoms of rhinoconjunctivitis with or without asthma participated in a 2-year randomized, double-blind, placebo-controlled trial. Actively treated patients were included in a follow-up year. Active treatment was performed with an aluminium hydroxide adsorbed house dust mite allergoid. Parameters for baseline data and clinical efficacy: nasal challenge, quantitative skin prick testing, Visual Analog Scale (VAS), patients' diaries, physician's assessment of patients? health condition, symptoms and use of anti-allergic medication as well as adverse reactions and changes in specific IgG4 and IgE antibodies. RESULTS: The trial detected superiority (p < 0.05) of mite depot allergoid versus placebo with regard to VAS and symptom intensity sum score in patients who needed anti-allergic medication in the baseline period. Significant differences (p < 0.05) between verum and placebo groups were also seen for patients' reactivities to nasal challenges and prick tests with allergen. The blinded assessment by the physician documented a significant difference (p < 0.05) between the groups in favour of active treatment. After reaching the maximum dose as well as after 12 and 24 months, specific IgG4 antibody concentrations were significantly elevated in the verum group (p < 0.05) by comparison with placebo. Local reactions were less frequent in the verum group and no systemic adverse reactions occurred. A third year of active treatment resulted in further improvement and documented the advantage of booster therapy to stabilize the clinical success. CONCLUSION: Specific immunotherapy with a mite depot allergoid induced significant clinical improvements versus placebo. Safety was assessed as excellent, and no systemic adverse reactions occurred.

Vaccination with genetically engineered allergens prevents progression of allergic disease.

IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated contact with the disease-eliciting allergens induces rises of allergen-specific IgE Abs and progression of the disease to more severe manifestations. Our study uses a type of vaccine that is based on genetically modified allergen derivatives to treat allergic patients. We developed hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, by genetic engineering and vaccinated birch pollen-allergic patients (n = 124) in a double-blind, placebo-controlled study. Active treatment induced protective IgG Abs that inhibited allergen-induced release of inflammatory mediators. We also observed a reduction of cutaneous sensitivity as well as an improvement of symptoms in actively treated patients. Most important, rises of allergen-specific IgE induced by seasonal birch pollen exposure were significantly reduced in vaccinated patients. Vaccination with genetically engineered allergen derivatives is a therapy for allergy that not only ameliorates allergic reactions but also reduces the IgE production underlying the disease.

Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1.

BACKGROUND: We have recently engineered recombinant derivatives of the major birch pollen allergen Bet v 1 (rBet v 1 fragments and trimer) with strongly reduced allergenic activity. OBJECTIVE: The aim of this study was the in vivo characterization of potential allergy vaccines based on Al(OH)3-adsorbed genetically modified rBet v 1 derivatives in mice. METHODS: BALB/c mice were immunized either with courses of nine injections of increasing doses of Al(OH)3-adsorbed rBet v 1 wild-type, rBet v 1 fragments, rBet v 1 trimer or Al(OH)3 alone in weekly intervals or with three high-dose injections applied in intervals of 3 weeks. Humoral immune responses to rBet v 1 wild-type and homologous plant allergens were measured by ELISA and Western blotting, and the ability of mouse antibodies to inhibit the binding of allergic patients IgE to Bet v 1 was studied by ELISA competition experiments. RESULTS: In both schemes, hypoallergenic rBet v 1 derivatives induced low IgE but high IgG1 responses against rBet v 1 wild-type. The IgG1 antibodies induced by genetically modified rBet v 1 derivatives cross-reacted with natural Bet v 1 and its homologues from alder (Aln g 1) as well as hazel (Cor a 1) and strongly inhibited the binding of birch pollen allergic patients' IgE to Bet v 1 wild-type. CONCLUSION: Genetically modified hypoallergenic rBet v 1 derivatives induce blocking antibodies in vivo. Their safety and efficacy for the treatment of birch pollen and associated plant allergies can now be evaluated in clinical immunotherapy studies.

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.

Rapid method for arrayed investigation of IgE-reactivity profiles using natural and recombinant allergens.

BACKGROUND: The availability of increasing numbers of purified natural and recombinant allergens offer the possibility for component-resolved characterization of IgE binding. To make use of this potential, fast and simple methods with high capacity have to be developed. METHODS: A laboratory multiscreen device was used in an innovative two-dimensional approach. In the first step, natural and recombinant allergens were immobilized onto the membrane using the sample chambers as application mask and, after blocking and rotating the membrane through 90 degrees, the same device was used to apply and incubate sera of allergic patients. Enzyme-linked immunoassay (ELISA) quantification of specific IgE was performed for purposes of comparison. RESULTS: Proteins were most efficiently bound onto nitrocellulose in 20 mM sodium hydroxide (NaOH). Up to 45 proteins or extracts could be investigated with a maximum of 45 sera in a single application, resulting in a resolution of 2,025 spots on one membrane with a size comparable to a standard Western blot. A high correlation for IgE-binding between natural and recombinant allergens was observed. Development of the membrane resulted in very evenly distributed square patterns. The results corresponded with the conventional ELISA measurements of specific IgE. CONCLUSIONS: The innovative usage of a standard incubation device for both application of proteins as well as screening of sera provides a simple high throughput method for the characterization of IgE binding to allergens. The results are important for component resolved diagnosis of allergy by means of fast monitoring of IgE- and IgG-reactivity spectra. Recombinant allergens may be used as targets for these purposes.

Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability.

BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.

Quantitation of the major fungal allergens, Alt a 1 and Asp f 1, in commercial allergenic products.

BACKGROUND: Alternaria is one of the most important fungi associated with allergic disease, whereas Aspergillus fumigatus is involved in a broad spectrum of pulmonary diseases. Currently, fungal extracts used for diagnosis in the United States are unstandardized, and their allergenic content cannot be compared directly. OBJECTIVE: The goal of this study was to compare the variability of major allergen levels among US allergenic products derived from fungi: specifically, Alt a 1 levels in Alternaria alternata extracts, and Asp f 1 levels in A fumigatus extracts. METHODS: A novel 2-site monoclonal antibody ELISA was used for measuring Alt a 1 using recombinant Alt a 1 as a standard. Asp f 1 was also measured by ELISA. Allergenic products produced by 8 US manufacturers over a 2-year period were compared, as were multiple lots produced by a single company. RESULTS: Alt a 1 levels in Alternaria extracts from 8 companies produced in 1998 and 1999 ranged from less than 0.01 to 6.09 microg/mL (mean 1.4 +/- 1.6 microg/mL, n = 15). In general, Alt a 1 levels were consistent within and between companies (1.4 +/- 1.1 microg/mL, n = 27), with 21 of 32 (66%) of all extracts tested containing 0.7 to 2 microg/mL Alt a 1. Aspergillus extracts showed much greater variability in Asp f 1 levels, with extracts from 8 companies containing from less than 0.1 to 64 microg/mL Asp f 1 (mean 16.3 +/- 23.9 microg/mL, n = 15). Overall variability was greater for Aspergillus products within and between manufacturers (22 +/- 22 microg/mL Asp f 1, n = 20). CONCLUSIONS: ELISA-based assays for specific allergens showed greater consistency among allergenic products derived from Alternaria than from Aspergillus. These assays should facilitate improved quality control and standardization of fungal allergen extracts and lead to the development of more consistent products for clinical use.

The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50-60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13.

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.

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.

Purification and immunobiochemical characterization of folding variants of the recombinant major wasp allergen Ves v 5 (antigen 5).

BACKGROUND: Antigen 5 is one of three major allergens in wasp venoms, but unlike phospholipase A(1) and hyaluronidase, both of which are enzymes, its biological function is unknown. The cDNA coding for this allergen has been isolated and used for recombinant expression. Thorough analysis of the expression product is essential in order to evaluate the usefulness for in vivo or in vitro application. OBJECTIVE: In this study, folding variants of the recombinant major allergen Ves v 5 from Vespula vulgaris were immunologically and biochemically investigated in order to determine their possible applicability for diagnostic or therapeutic purposes. METHOD: The cDNA encoding Ves v 5 was cloned into the expression vector pSE420 which generates recombinant products lacking a tag sequence. After expression, inclusion bodies were purified, subsequently denatured and dialyzed against different solutions. The structural properties of soluble proteins were analyzed by size exclusion chromatography, non-reducing SDS-PAGE, native PAGE, N-terminal sequencing, proteolytic digestion and ion exchange chromatography. Immunological investigations were performed by using different monoclonal antibodies (mAbs) specific for Ves v 5 and IgE from patients allergic to wasp venom allergens. RESULTS: After dialysis, soluble monomeric recombinant Ves v 5 was more than 95% pure in each case. Using different dialysis solutions, clearly distinguishable folding variants were obtained. In one case, the recombinant allergen was comparable with the natural counterpart in respect of migration in non-reducing SDS-PAGE, native PAGE and IgE reactivity. This variant reacted with two different Ves v 5-specific mAbs and produced a stable fragment after proteolytic digestion. Elution from a cation exchange chromatography column was achieved with 320 mM NaCl. In two other cases, folding variants exhibited a different migration behavior in SDS-PAGE and native PAGE compared with the natural allergen. Also, the mAb 1E11 recognized none of these variants since it presumably detected a conformational epitope. Moreover, the IgE reactivity was clearly reduced and proteolytic digestion effected almost complete degradation. These variants eluted from the cation exchange column with 400 mM NaCl. CONCLUSION: Defined folding strategies resulted in both soluble misfolded variants with reduced IgE reactivity, potentially suitable for immunotherapy, and natural-like folded variants for diagnosis.

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense).

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

Sensitization to different mite species in German farmers: clinical aspects.

Various mite species referred to collectively as house dust and storage mites are recognized worldwide as a cause of allergic airway disease. Our study aimed to investigate the frequency of sensitization and potential importance of mite species in farmers using a broad mite spectrum. A total of 86 German farmers with rhinitis and/or asthma were studied by skin prick testing and/or enzyme allergosorbent test (EAST) with the following mites: Blomia tjibodas, Blomia tropicalis, Blomia kulagini, Glycyphagus domesticus, Thyreophagus entomophagus, Euroglyphus maynei, Chortoglyphus arcuatus, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, Acarus farris and Cheyletus eruditus. Sensitization to at least one mite species was detected in 51 patients (59%) by skin prick testing, and in 31 patients (36%) by EAST. The most frequent sensitizations determined by skin tests were found for the three Blomia species, E. maynei and G. domesticus. Twelve patients (14%) gave a positive EAST with the predator mite C. eruditus. A total of 22 patients gave positive EAST results with the Dermatophagoides species. We were able to document sensitization to C. arcuatus, E. maynei and T. entomophagus for the first time in Germany. A considerable proportion of the German farmers tested were sensitized to storage mites. The allergological potential of various mite species has been recognized, some for the first time. It was concluded that B. tjibodas, G. domesticus, C. arcuatus and C. eruditus in particular should be included in an allergy diagnosis. Further investigations into the clinical relevance of the sensitizations and possible cross-reactivity between the mite species are necessary.

Reactivity of T cells with grass pollen allergen extract and allergoid.

BACKGROUND: Successful allergen-specific immunotherapy is achieved with progressively increasing doses of allergen or allergoid. In order to gain further insight into the mechanism of action of allergoids several in vitro investigations were conducted. METHODS: Peripheral blood mononuclear cells (PBMC) from grass pollen allergic and nonallergic subjects were stimulated with either grass pollen extract or allergoid and the proliferation and cytokine production (IL-5, IFN-gamma) were measured. Similar investigations were performed with Phl p 5-specific T cell lines (TCL) and clones (TCC). Dendritic cells and PBMC were compared in terms of their relative efficacies as antigen-presenting cells. RESULTS: Both allergen and allergoid induced proliferation and Th2 and Th1 cytokine synthesis by PBMC of allergic subjects, whereas PBMC of nonallergic subjects did not produce IL-5. The maximum level of IL-5 was obtained with a lower concentration than was necessary for maximal IFN-gamma production. Higher stimulation doses of allergen and allergoid shifted the cytokine profiles towards a Th1 phenotype. TCL and TCC clearly showed reactivity with both allergen and allergoid when using autologous PBMC for antigen presentation, but compared with the native allergen the reactivity of the allergoid was reduced with most of the TCC. Using dendritic cells for antigen presentation a pronounced increase of stimulation of the TCC especially for the allergoids becomes obvious. CONCLUSION: In common with grass pollen allergen the corresponding allergoids possess a strong allergen-specific T cell-stimulating capacity. However, the degree of T cell stimulation by the allergoid seems to be dependent on the type of the antigen-presenting cell. Both, allergen and allergoid, can modulate T cell responses in a dose-dependent manner.