Fusion proteins consisting of Bet v 1 and Phl p 5 form IgE-reactive aggregates with reduced allergenic activity.

The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.

Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Possible effect of landscape design on IgE recognition profiles of two generations revealed with micro-arrayed allergens.

The aim of this study was to investigate possible effects of landscape design on the IgE sensitization profile toward inhalant allergens in patients with respiratory allergy from Uzbekistan where green areas have been changed during the last two decades by a State program. Sera from two different generations of Uzbek (n=58) and, for control purposes, from two generations of Austrian (n=58) patients were analyzed for IgE reactivity to 112 different micro-arrayed allergen molecules by ImmunoCAP ISAC technology. Changes in molecular IgE sensitization profiles to pollen allergens in the young vs the middle-aged Uzbek population were associated with replanting, whereas those in the Vienna populations reflected natural changes in plant growth. Our data indicate that anthropologic as well as natural changes in the biome may have effects on IgE sensitization profiles already from one to another generation.

Anti-OX40L alone or in combination with anti-CD40L and CTLA4Ig does not inhibit the humoral and cellular response to a major grass pollen allergen.

BACKGROUND: IgE-mediated allergy is a common disease characterized by a harmful immune response towards otherwise harmless environmental antigens. Induction of specific immunological non-responsiveness towards allergens would be a desirable goal. Blockade of costimulatory pathways is a promising strategy to modulate the immune response in an antigen-specific manner. Recently, OX40 (CD134) was identified as a costimulatory receptor important in Th2-mediated immune responses. Moreover, synergy between OX40 blockade and 'conventional' costimulation blockade (anti-CD40L, CTLA4Ig) was observed in models of alloimmunity. OBJECTIVE: We investigated the potential of interfering with OX40 alone or in combination with CD40/CD28 signals to influence the allergic immune response. METHODS: The OX40 pathway was investigated in an established murine model of IgE-mediated allergy where BALB/c mice are repeatedly immunized with the clinically relevant grass pollen allergen Phl p 5. Groups were treated with combinations of anti-OX40L, CTLA4Ig and anti-CD40L. In selected mice, Tregs were depleted with anti-CD25. RESULTS: Blockade of OX40L alone at the time of first or second immunization did not modulate the allergic response on the humoral or effector cell levels but slightly on T cell responses. Administration of a combination of anti-CD40L/CTLA4Ig delayed the allergic immune response, but antibody production could not be inhibited after repeated immunization even though the allergen-specific T cell response was suppressed in the long run. Notably, additional blockade of OX40L had no detectable supplementary effect. Immunomodulation partly involved regulatory T cells as depletion of CD25(+) cells led to restored T cell proliferation. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, our data provide evidence that the allergic immune response towards Phl p 5 is independent of OX40L, although reduction on T cell responses and slightly on the asthmatic phenotype was detectable. Besides, no relevant synergistic effect of OX40L blockade in addition to CD40L/CD28 blockade could be detected. Thus, the therapeutic potential of OX40L blockade for IgE-mediated allergy appears to be ineffective in this setting.

Epitope specificity determines cross-protection of a SIT-induced IgG4 antibody.

BACKGROUND: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT). METHODS: We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments. RESULTS: Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity. CONCLUSIONS: Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.

Poor association of allergen-specific antibody, T- and B-cell responses revealed with recombinant allergens and a CFSE dilution-based assay.

BACKGROUND: The adaptive immunity underlying allergy comprises two components, the allergen-specific antibody (i.e. IgE, IgG) and the T-cell response. These two components are responsible for different disease manifestations and can be targeted by different therapeutic approaches. Here, we investigated the association of allergen-specific antibody and T- as well as B-cell responses in pollen-allergic patients using recombinant (r) major birch pollen allergen rBet v 1 and major timothy grass pollen allergen rPhl p 5 as defined antigens. METHODS: Allergen-specific IgE and IgG antibody responses were determined by ELISA, and allergen-specific T- and B-cell responses were measured in peripheral blood mononuclear cells using a carboxyfluorescein-diacetate-succinimidylester (CFSE) dilution assay. RESULTS: CFSE staining in combination with T-cell- and B-cell-specific gating allowed discriminating between allergen-specific T-cell and B-cell responses. Interestingly, we identified patients where mainly T cells and others where mainly B cells proliferated in response to allergen stimulation. No association between the level of allergen-specific Ig responses and B- or T-cell proliferation was observed. CONCLUSION: Purified recombinant allergens in conjunction with CFSE staining allow the dissection of allergen-specific B- and T-cell responses. The dissociation of allergen-specific antibody, and B- and T-cell responses may explain the occurrence of selective IgE- and T-cell-mediated manifestations of allergic inflammation and may be important for the development of diagnostic and therapeutic strategies selectively targeting B cells and T cells.

Multiple independent IgE epitopes on the highly allergenic grass pollen allergen Phl p 5.

BACKGROUND: Group 5 allergens are small proteins that consist of two domains. They belong to the most potent respiratory allergens. OBJECTIVE: To determine the binding sites and to study allergic patients' IgE recognition of the group 5 allergen (Phl p 5) from timothy grass pollen using human monoclonal IgE antibodies that have been isolated from grass pollen allergic patients. METHODS: Using recombinant isoallergens, fragments, mutants and synthetic peptides of Phl p 5, as well as peptide-specific antibodies, the interaction of recombinant human monoclonal IgE and Phl p 5 was studied using direct binding and blocking assays. Cross-reactivity of monoclonal IgE with group 5 allergens in several grasses was studied and inhibition experiments with patients' polyclonal IgE were performed. RESULTS: Monoclonal human IgE showed extensive cross-reactivity with group 5 allergens in several grasses. Despite its small size of 29 kDa, four independent epitope clusters on isoallergen Phl p 5.0101, two in each domain, were recognized by human IgE. Isoallergen Phl p 5.0201 carried two of these epitopes. Inhibition studies with allergic patients' polyclonal IgE suggest the presence of additional IgE epitopes on Phl p 5. CONCLUSIONS & CLINICAL RELEVANCE: Our results reveal the presence of a large number of independent IgE epitopes on the Phl p 5 allergen explaining the high allergenic activity of this protein and its ability to induce severe allergic symptoms. High-density IgE recognition may be a general feature of many potent allergens and form a basis for the development of improved diagnostic and therapeutic procedures in allergic disease.

High-resolution crystal structure and IgE recognition of the major grass pollen allergen Phl p 3.

BACKGROUND: Group 2 and 3 grass pollen allergens are major allergens with high allergenic activity and exhibit structural similarity with the C-terminal portion of major group 1 allergens. In this study, we aimed to determine the crystal structure of timothy grass pollen allergen, Phl p 3, and to study its IgE recognition and cross-reactivity with group 2 and group 1 allergens. METHODS: The three-dimensional structure of Phl p 3 was solved by X-ray crystallography and compared with the structures of group 1 and 2 grass pollen allergens. Cross-reactivity was studied using a human monoclonal antibody which inhibits allergic patients' IgE binding and by IgE inhibition experiments with patients' sera. Conformational Phl p 3 IgE epitopes were predicted with the algorithm SPADE, and Phl p 3 variants containing single point mutations in the predicted IgE binding sites were produced to analyze allergic patients' IgE binding. RESULTS: Phl p 3 is a globular ß-sandwich protein showing structural similarity to Phl p 2 and the Phl p 1-C-terminal domain. Phl p 3 showed IgE cross-reactivity with group 2 allergens but not with group 1 allergens. SPADE identified two conformational IgE epitope-containing areas, of which one overlaps with the epitope defined by the monoclonal antibody. The mutation of arginine 68 to alanine completely abolished binding of the blocking antibody. This mutation and a mutation of D13 in the predicted second IgE epitope area also reduced allergic patients' IgE binding. CONCLUSION: Group 3 and group 2 grass pollen allergens are cross-reactive allergens containing conformational IgE epitopes. They lack relevant IgE cross-reactivity with group 1 allergens and therefore need to be included in diagnostic tests and allergen-specific treatments in addition to group 1 allergens.

Cloning, expression in E. coli and immunological characterization of Par j 3.0201, a Parietaria pollen profilin variant.

Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.

Multiple grass mixes as opposed to single grasses for allergen immunotherapy in allergic rhinitis.

Grass pollen allergy affects approximately 40% of allergic patients. Subcutaneous allergen immunotherapy (SCIT) is the only allergen-specific and disease-modifying treatment available. Currently available therapeutic vaccines for the treatment of grass pollen allergy are based on natural grass pollen extracts which are either made from pollen of one cross-reactive grass species or from several related grass species. Clinical studies have shown that SCIT performed with timothy grass pollen extract is effective for the treatment of grass pollen allergy. Moreover, it has been demonstrated that recombinant timothy grass pollen allergens contain the majority of relevant epitopes and can be used for SCIT in clinical trials. However, recent in vitro studies have suggested that mixes consisting of allergen extracts from several related grass species may have advantages for SCIT over single allergen extracts. Here, we review current knowledge regarding the disease-relevant allergens in grass pollen allergy, available clinical studies comparing SCIT with allergen extracts from timothy grass or from mixes of several related grass species of the Pooideae subfamily, in vitro cross-reactivity studies performed with natural allergen extracts and recombinant allergens and SCIT studies performed with recombinant timothy grass pollen allergens. In vitro and clinical studies performed with natural allergen extracts reveal no relevant advantages of using multiple grass mixes as opposed to single grass pollen extracts. Several studies analysing the molecular composition of natural allergen extracts and the molecular profile of patients' immune responses after SCIT with allergen extracts indicate that the major limitation for the production of a high quality grass pollen vaccine resides in intrinsic features of natural allergen extracts which can only be overcome with recombinant allergen-based technologies.

Recombinant allergen-based monitoring of antibody responses during injection grass pollen immunotherapy and after 5 years of discontinuation.

BACKGROUND: Subcutaneous injection immunotherapy (SCIT) is considered as antigen-specific and disease-modifying treatment with long-lasting effect. METHODS: We used a panel of recombinant grass pollen allergens for analyzing allergen-specific IgE, IgG(1) -IgG(4) , IgM, IgA, and light-chain (kappa, lambda) responses in grass pollen-allergic patients who had received one course of injection immunotherapy (SCIT) with an aluminum hydroxide-adsorbed grass pollen extract or only anti-inflammatory treatment. Serum samples were analyzed before and after 5 months of treatment as well as after 5 years. RESULTS: After 5 months of SCIT but not of anti-inflammatory treatment, IgG(1) > IgG(4) > IgG(2) > IgA antibody responses using both kappa and lambda light chains specific for major grass pollen allergens (Phl p 1, Phl p 5, Phl p 6, Phl p 2) increased significantly, whereas specific IgM or IgG(3) levels were unaltered. Allergen-dependent basophil degranulation was only inhibited with SCIT sera containing therapy-induced allergen-specific IgG antibodies. Likewise, decreases in Phl p 1- and Phl p 5-specific IgE levels and significant (P<0.001) reduction in symptom and medication scores were found only in the SCIT group but not in the group of patients receiving anti-inflammatory treatment. After 5 years, allergen-specific IgG antibody levels in the SCIT group had returned to baseline levels and there was no significant difference regarding symptoms between the SCIT and non-SCIT groups. CONCLUSION: The results from our observational study demonstrate that only SCIT but not anti-inflammatory treatment induces allergen-specific IgG and reduces boosts of allergen-specific IgE production but that one SCIT course was not sufficient to achieve long-term immunological and clinical effects.

Recombinant monoclonal human immunoglobulin E to investigate the allergenic activity of major grass pollen allergen Phl p 5.

BACKGROUND: Allergen recognition by IgE antibodies is a key event in allergic inflammation. OBJECTIVE: To construct a plasmid for the expression of human monoclonal IgE antibodies of any desired specificity and to express IgE specific for the major timothy grass pollen allergen Phl p 5. METHODS: In a first step, the DNA sequence coding for the IgG(1) heavy chain was excised and replaced by the sequence coding for the human ɛ constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG(1) heavy chain. Then, this construct together with a second plasmid expressing the corresponding Phl p 5-specific light chain was co-expressed in COS-7 cells. The Phl p 5-specific IgE (rhuMabEP5) was analysed for allergen-specificity and isotype by ELISA. Cross-reactivity of rhuMabEP5 was investigated by immunoblotting using pollen extracts from various grass species. The allergenic activity of Phl p 5 was studied by exposing rat basophil leukaemia (RBL) cells expressing human-FcɛRI to rhuMabEP5 and Phl p 5. RESULTS: We report the construction of vector pLNOH2-P5IgE, for the expression of human IgE and exemplify its usefulness by the production of a complete and functional human monoclonal IgE (rhuMabEP5). rhuMabEP5 is specific for the grass pollen allergen Phl p 5 and cross-reacts with group 5 allergens in natural grass pollen extracts. RBL-release assays with rhuMabEP5 demonstrated that oligomerization does not contribute to the high allergenic activity of Phl p 5. CONCLUSION AND CLINICAL RELEVANCE: Plasmid pLNOH2-P5IgE allowed the production of a fully functional human monoclonal IgE antibody specific for Phl p 5. Recombinant human IgE antibodies of defined specificity represent useful tools to investigate mechanisms underlying IgE-mediated allergies.

Analysis of the antibody responses induced by subcutaneous injection immunotherapy with birch and Fagales pollen extracts adsorbed onto aluminum hydroxide.

BACKGROUND: Allergen-specific subcutaneous immunotherapy (SCIT) is an antigen-specific therapy of IgE-mediated allergies. In the present study, we analyze the epitope specificities of antibody responses induced by SCIT with allergen extracts from pollen of trees belonging to the order Fagales (birch, alder, hazel) adsorbed onto aluminum hydroxide. METHODS: The IgE, IgG1-4 and IgA responses to defined recombinant allergens (birch pollen: Bet v 1; alder pollen: Aln g 1; hazel pollen: Cor a 1; apple: Mal d 1) as well as to Bet v 1-derived recombinant fragments and synthetic peptides were analyzed in sera from patients who had undergone SCIT for different periods of time. RESULTS: Long-term SCIT (>1 year; cumulative dose >1,000,000 SQ units) induced more pronounced IgG1, IgG2 and IgG4 responses to Bet v 1 and Bet v 1-related allergens according to the degree of sequence homology (Bet v 1>Aln g 1>Cor a 1>Mal d 1) than short-term SCIT (<1 year; cumulative dose <1,000,000 SQ units). In contrast to patients treated for <1 year, patients treated for >1 year mounted distinct IgG1, IgG2 and IgG4 responses against sequential Bet v 1 epitopes. No relevant allergen-specific IgA or IgG3 responses were induced by short- or long-term SCIT. Using a competitive ELISA assay, it could be shown that serum IgG from patients undergoing long-term SCIT inhibited IgE reactivity to Bet v 1 better than IgG from patients undergoing short-term SCIT. CONCLUSION: SCIT with allergen extracts adsorbed onto aluminum hydroxide induces IgG responses against new epitopes that block IgE binding and cross-react with structurally related allergens depending, among other factors, on duration of treatment and cumulative injected dose.

Molecular composition and biological activity of commercial birch pollen allergen extracts.

BACKGROUND: Commercial extracts used for diagnosis and treatment of allergy are currently prepared from natural allergen sources. The aim of this study was to analyse birch pollen allergen extracts produced for in vivo diagnosis of birch pollen allergy regarding their contents of individual birch pollen allergens (Bet v 1, Bet v 2 and Bet v 4). METHODS: Protein contents were measured and the allergen composition was analysed by immunoblotting using antibody probes specific for Bet v 1, Bet v 2 and Bet v 4 in birch pollen extracts from five manufacturers of allergen extracts. The contents of the major birch pollen allergen, Bet v 1, were quantified with a specific two-site binding enzyme-linked immunosorbent assay with nanogram sensitivity for Bet v 1. The biological activities of the allergen extracts were evaluated by skin prick testing in birch pollen allergic patients and compared with their sensitization profiles. RESULTS: A more than 10-fold variation regarding total protein contents (23.1-314 microg mL(-1)) and also regarding the amounts of the major birch pollen allergen, Bet v 1 (1.62-19.6 microg mL(-1)) was found. The highly cross-reactive Bet v 4 allergen was absent in three of the five tested extracts. Furthermore, varying skin test results were obtained in birch pollen allergic patients with the allergen extracts. CONCLUSIONS: Commercial birch pollen extracts exhibit a considerable variability regarding allergen contents and hence deliver varying in vivo test results. These problems might be overcome with recombinant allergen-based preparations.

Micro-arrayed wheat seed and grass pollen allergens for component-resolved diagnosis.

BACKGROUND: Wheat is a potent allergen source and can cause baker's asthma, food and pollen allergy. The aim of the study was to develop an allergen micro-array for differential diagnosis of baker's asthma, wheat-induced food allergy and grass pollen allergy. METHODS: We analysed the immunoglobulin-E reactivity profiles of patients suffering from baker's asthma, wheat-induced food allergy and grass pollen allergy to micro-arrayed recombinant wheat flour allergens and grass pollen allergens and compared these results with clinical results and diagnostic tests based on crude wheat flour, wheat pollen and grass pollen allergen extracts. RESULTS: We identified recombinant wheat flour allergens, which are specifically recognized by patients suffering from baker's asthma, but not from patients with food allergy to wheat or pollen allergy. rPhl p 1 and rPhl p 5 were identified as marker allergens specific for grass pollen allergy. They can be used to replace grass pollen extracts for allergy diagnosis and to identify grass pollen allergic patients among patients suffering from baker's asthma and wheat-induced food allergy. Profilin was identified as a cross-reactive allergen recognized by patients suffering from baker's asthma, food and pollen allergy. CONCLUSIONS: Our results indicate that it will be possible to design serological tests based on micro-arrayed recombinant wheat seed and grass pollen allergens for the discrimination of baker's asthma, wheat-induced food allergy and grass pollen allergy.

The allergen profile of beech and oak pollen.

BACKGROUND: Beech and oak pollen are potential allergen sources with a world-wide distribution. OBJECTIVE: We aimed to characterize the allergen profile of beech and oak pollen and to study cross-reactivities with birch and grass pollen allergens. METHODS: Sera from tree pollen-allergic patients with evidence for beech and oak pollen sensitization from Basel, Switzerland, (n=23) and sera from birch pollen-allergic patients from Vienna, Austria, (n=26) were compared in immunoblot experiments for IgE reactivity to birch (Betula pendula syn. verrucosa), beech (Fagus sylvatica) and oak (Quercus alba) pollen allergens. Subsequently, beech and oak pollen allergens were characterized by IgE inhibition experiments with purified recombinant and natural allergens and with allergen-specific antibody probes. Birch-, beech- and oak pollen-specific IgE levels were determined by ELISA. RESULTS: Beech and oak pollen contain allergens that cross-react with the birch pollen allergens Bet v 1, Bet v 2 and Bet v 4 and with the berberine bridge enzyme-like allergen Phl p 4 from timothy grass pollen. Sera from Swiss and Austrian patients exhibited similar IgE reactivity profiles to birch, beech and oak pollen extracts. IgE levels to beech and oak pollen allergens were lower than those to birch pollen allergens. CONCLUSION: IgE reactivity to beech pollen is mainly due to cross-reactivity with birch pollen allergens, and a Phl p 4-like molecule represented another predominant IgE-reactive structure in oak pollen. The characterization of beech and oak pollen allergens and their cross-reactivity is important for the diagnosis and treatment of beech and oak pollen allergy.

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.

Heterogeneity of commercial timothy grass pollen extracts.

BACKGROUND: The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources. OBJECTIVE: To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity. METHODS: Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients. RESULTS: The allergen extracts showed broad variations in protein compositions and amounts (24.1-197.7 microg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32-384 ng/mL; Phl p 2: 1128-6530 ng/mL, Phl p 5: 40-793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients. CONCLUSIONS: Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.

A high-affinity monoclonal anti-IgE antibody for depletion of IgE and IgE-bearing cells.

BACKGROUND: We have identified a monoclonal anti-human immunoglobulin E (IgE) antibody, which recognizes FcepsilonRI-bound IgE and prevents binding of IgE to FcepsilonRI. In this study, we assessed the binding kinetics and affinity of monoclonal antibody 12 (mAb12) for IgE and investigated whether mAb12 can be used for depletion of IgE and isolation of IgE-bearing cells from peripheral blood. METHODS: Binding kinetics and affinity for IgE were studied using Biacore surface plasmon resonance technique experiments. IgE antibodies were depleted from serum using sepharose-coupled mAb12 and IgE-bearing cells were enriched from heparinized blood samples with mAb12. The extent and biological relevance of IgE depletion were studied by quantitative IgE measurements and basophil histamine release experiments. Specific binding of mAb12 to IgE-bearing cells (basophils, mast cells, IgE-secreting plasma cells) was demonstrated by FACS. RESULTS: Monoclonal antibody 12 shows rapid association (k(a) = 5.46e5/Ms) with IgE, almost no dissociation (k(d) = 8.8e-5/s) and an affinity for IgE (K(D) = 1.61e-10 M), which is as high as that of FcepsilonRI. Immobilized mAb12 could be used to deplete IgE antibodies and isolate IgE-bearing cells from peripheral blood in a single-step procedure. CONCLUSIONS: Monoclonal antibody 12 is a high affinity anti-human IgE antibody, which efficiently removes IgE and IgE-bearing cells from peripheral blood and may thus be used for extracorporeal depletion of IgE and IgE-bearing cells.

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.