IgG4 but no IgG1 antibody production after intralymphatic immunotherapy with recombinant MAT-Feld1 in human.

Allergy immunotherapy (AIT) mediates protection against allergen exposure in part due to allergen-specific antibodies. While immunization typically stimulated IgG1 and IgG2, AIT is often associated with production of IgG4. Here, twenty cat dander-sensitized patients were randomized to receive three injections of intralymphatic immunotherapy (ILIT) with MAT-Feld1 adsorbed to aluminum hydroxide or just aluminum hydroxide (placebo) in a double-blind setting (ClinicalTrials.gov NCT00718679). Whereas the clinical data, showing benefit of Mat-Feld1 ILIT was published in 2012 (Senti et al., J Allergy Clin Immunol, vol 129(5):1290-1296), the current study investigated the cat allergen-specific antibody responses. Blood was drawn prior to ILIT, as well as 1, 3, and 12 months after first ILIT. The sera were analyzed to characterize all IgG subclasses and IgE antibody responses. ILIT with MAT-Feld1 elicited high levels of total IgG that were maintained for at least 12 months. Interestingly, a strong increase in IgG4 and some increase in IgG2 were observed throughout the study, while production of cat-specific IgG1 and IgG3 was not stimulated by MAT-Feld1 ILIT. The IgE levels remained constant.

The cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1.

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat-sensitized Swedish patients and elucidated its allergenic activity and cross-reactivity with the dog lipocalin Can f 1. Sixty-five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross-reactive antisera. Fel d 7 is a common allergen in a Swedish cat-sensitized population that cross-reacts with Can f 1, and may contribute to symptoms in cat- but also in dog-allergic patients.

Altered immunoregulatory profile during anti-tumour necrosis factor treatment of patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) can be treated effectively by anti-tumour necrosis factor (TNF) therapy. We set out to investigate the unclear immunoregulatory mechanisms of the treatment. Thirty-four patients with IBD treated with anti-TNF were included. Lymphocytes from peripheral blood and intestinal biopsies were analysed by flow cytometry. Regulation of antigen-stimulated proliferation was analysed by blocking of interleukin (IL)-10, transforming growth factor (TGF)-ß or depletion of CD25(+) cells in peripheral blood mononuclear cell cultures. No changes in CD4(+)CD25(+), CD25(+)TNF-RII(+) or CD4(+)CD25(+) forkhead box protein 3 (FoxP3(+)) T cells could be observed in peripheral blood after, in comparison to before, 6 weeks of treatment. The suppressive ability of CD4(+)CD25(+) cells did not change. There was an initial decrease of CD4(+)CD25(+) cells in intestinal mucosa after 2 weeks of treatment, followed by an increase of these cells from weeks 2 to 6 of treatment (P < 0·05). This was accompanied by an increased percentage of CD69(+) cells among these cells after 6 weeks of treatment compared to before treatment (P < 0·01). There was also an increase of mucosal T helper type1 cells from weeks 2 to 6 (P < 0·05). In addition, CD25(+)TNF-RII(+) cells in the mucosa were decreased after 6 weeks of treatment compared to before treatment (P < 0·05). Before treatment, peripheral blood mononuclear cell baseline proliferation was increased when IL-10 was blocked (P < 0·01), but not after. In CD25(+) cell-depleted cultures proliferation increased after treatment (P < 0·05). Our data indicate that anti-TNF treatment leads to an induction of effector T cells. Anti-TNF therapy has no significant impact on regulatory T cells in IBD, although the composition of regulatory T cell subsets may change during treatment.

Treatment with a Fel d 1 hypoallergen reduces allergic responses in a mouse model for cat allergy.

BACKGROUND: A hypoallergen of the major cat allergen Fel d 1, recombinant (r) Fel d 1 (DTE III), was previously shown to have retained T-cell reactivity and strongly reduced IgE-binding capacity compared to unmodified rFel d 1. Here, we evaluated the therapeutic capacity of rFel d 1 (DTE III) in a mouse model for cat allergy. METHODS: Mice were subcutaneously (s.c.) sensitized with rFel d 1 and subsequently treated (s.c.) with 50 or 200 µg rFel d 1 (DTE III), or 50 µg rFel d 1, prior to intranasal challenge with cat dander extract. Airway hyperreactivity (AHR), cells and cytokines in bronchoalveolar lavage fluid, splenocyte in vitro response, and serum immunoglobulins were analyzed. Seven cat-allergic patients and ten healthy controls were tested for skin prick test (SPT) reactivity to rFel d 1 (DTE III) and rFel d 1. RESULTS: Mice treated with 50 and 200 µg rFel d 1 (DTE III), and 50 µg rFel d 1, produced increased serum levels of rFel d 1-specific IgG1 and IgG2a compared to sham-treated mice. IgG from all treatment groups could block binding of patients' IgE to rFel d 1. The 200 µg rFel d 1 (DTE III) treatment tended to reduce AHR. All mice tolerated treatment with rFel d 1 (DTE III), in contrast to only four of ten treated with rFel d 1. Compared to rFel d 1, the hypoallergen showed a tendency of reduced SPT reactivity. CONCLUSION: The rFel d 1 (DTE III) hypoallergen might be a promising candidate for application in immunotherapy of cat allergy with improved safety and efficacy.

Allergen provocation increases TH2-cytokines and FOXP3 expression in the asthmatic lung.

BACKGROUND: Allergic asthma is caused by allergen-specific IgE and T-helper cell (Th) type 2 responses towards airborne allergens. The objective of this study was to investigate local and systemic regulatory mechanisms in the early asthmatic response to bronchial allergen provocation. METHODS: Birch pollen-allergic patients with mild asthma (n = 13) and healthy nonallergic controls (n = 14) were subjected to bronchoalveolar lavage (BAL) and blood sampling. On patients BAL was performed twice: without preceding provocation ('before samples') and 24 h after bronchial provocation with birch pollen allergen. Lymphocytes in BAL and peripheral blood mononuclear cells (PBMCs) were phenotyped by multi-colour flow cytometry and cytokines measured by cytometric bead array. Proliferation and secreted cytokines were analysed in allergen-stimulated PBMCs, CD25(+) depleted PBMCs and PBMCs with IL-10 neutralizing antibodies. RESULTS: The numbers of CD69(+) and FOXP3(+) lymphocytes were higher in BAL after compared with before allergen provocation in asthmatic patients. Moreover, allergen provocation increased expression of FOXP3 in CD4(+)CD25(bright) cells. The cytokine profile in BAL fluid from asthmatics revealed higher levels of IL-5, compared with the controls, and an increase in IL-5, IL-6, IL-9 and IL-10 after allergen provocation. Pollen allergen stimulated PBMC cultures from asthmatic patients produced elevated levels of IL-5 and IL-13 compared with the controls, which were not affected by depletion of CD25(+) cells or IL-10 neutralization. CONCLUSION: Despite an increase in CD4(+)CD25(bright) cells expressing high levels of FOXP3 in response to bronchial allergen provocation, asthmatic patients exhibit enhanced levels of Th2 cytokines in the lung, which may indicate an inability among infiltrating cells to suppress Th2 responses.

The non-proteolytic house dust mite allergen Der p 2 induce NF-kappaB and MAPK dependent activation of bronchial epithelial cells.

BACKGROUND: House dust mites (HDM) are well-known as a source of indoor aeroallergens and for causing allergic airway diseases. Some proteolytic HDM allergens are known to activate respiratory epithelial cells to produce pro-inflammatory mediators, while there is limited knowledge regarding such activity among non-proteolytic HDM allergens. OBJECTIVE: To investigate whether Der p 2, a major non-proteolytic allergen of Dermatophagoides pteronyssinus, activates respiratory epithelial cells to produce mediators involved in asthma pathogenesis and to elucidate the mechanism of such activation. METHODS: The human bronchial epithelial cell line BEAS-2B, normal human bronchial epithelial (NHBE) cells and the alveolar epithelial cell line A549 were exposed to recombinant Der p 2. Following exposure, we analysed a panel of soluble mediators and cell adhesion receptors involved in asthma pathogenesis by promoting recruitment, survival and binding of inflammatory cells. The involvement of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPKs) was studied using specific inhibitors. RESULTS: Der p 2 activated bronchial BEAS-2B and NHBE cells, but not alveolar A549 cells. In BEAS-2B cells Der p 2 induced dose-dependent up-regulation in both mRNA level and protein secretion of granulocyte-macrophage colony-stimulating factor, IL-6, IL-8, monocyte-chemotactic protein-1 and macrophage inflammatory protein-3alpha. Secretion as well as surface expression of intercellular adhesion molecule (ICAM)-1 was also up-regulated, which was associated with increased adhesion of monocytes to the epithelial cells. The release of cytokines and chemokines was regulated by NF-kappaB and MAPK activation in different ways, while expression of ICAM-1 was solely dependent on NF-kappaB activation. CONCLUSION: These results show that Der p 2 activates respiratory epithelial cells, indicating that this non-proteolytic allergen, in addition to its immunogenic properties, can aggravate respiratory airway disease by adjuvant-like activation of the lung epithelium.

Prolonged antigen-exposure with carbohydrate particle based vaccination prevents allergic immune responses in sensitized mice.

BACKGROUND: Defined particles carrying tightly bound allergens at high density have been suggested as alternatives in allergy vaccination. Carbohydrate based particles (CBP), sized 2 microm, provide a platform for covalent coupling of allergens. OBJECTIVE: To investigate the mechanisms of antigen presentation by CBP, as well as cellular and humoral responses after vaccination with the major cat allergen Fel d 1, covalently coupled to CBP. METHODS: Mice (n = 10/group) were subcutaneously vaccinated with CBP-rFel d 1, CBP or phosphate buffer saline (PBS) before sensitization with rFel d 1 and challenged with cat dander extract. Fluorescent and (75)Se-radiolabeled tracking of allergens and particles were performed with flow cytometry and whole-body autoradiography. Humoral, cellular and regulatory immune responses were analyzed by ELISA and flow cytometry. Cytokines were measured in bronchoalveolar lavage fluid and splenocyte cultures. RESULTS: CBP-rFel d 1 prevented induction of airway inflammation and induced allergen-specific T-cell anergy. CBP-rFel d 1 also induced rapid IgM and IgG1-responses compared with soluble rFel d 1. Particles were phagocytosed by antigen-presenting cells and transported to draining lymph nodes and spleen. Moreover, antigen coupled to CBP remained longer at the injection site compared with alum. CONCLUSIONS: Covalent coupling of rFel d 1 to CBP induces rapid antibody production, prevents induction of allergic immune responses and systemic allergen spreading. Thus, CBP comprise several attractive adjuvant features for use in allergy vaccination. CLINICAL IMPLICATIONS: Prolonged allergen exposure through covalent coupling to particles suitable for phagocytosis, provides an adjuvant for safer and efficient allergy vaccination.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation.

BACKGROUND: Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. METHODS: The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. RESULTS: Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. CONCLUSIONS: Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy.

BACKGROUND: Allergen-specific immunotherapy (ASIT) is the only treatment of allergic disease that gives long-lasting relief of symptoms. However, concerns for safety and efficiency have highlighted the need for improvement of the therapy. We have previously suggested carbohydrate-based particles (CBPs) as a novel adjuvant and allergen carrier for ASIT. Our aim of this study was to evaluate the therapeutic potential of CBPs in ASIT, employing a mouse model for cat allergy. METHODS: BALB/c mice were subcutaneously immunized with the recombinant (r) cat allergen Fel d 1 followed by intranasal challenge with cat dander extract (CDE). The sensitized mice were therapeutically treated with rFel d 1 covalently coupled to CBPs (CBP-rFel d 1). Airway hyper-reactivity (AHR), infiltration of leucocytes in bronchoalveolar lavage (BAL) fluid, allergen-specific serum immunoglobulin levels and in vitro splenocyte responses were evaluated. RESULTS: Mice treated with CBP-rFel d 1 showed reduced features of allergic inflammation. They responded with (i) significantly decreased AHR and infiltration of eosinophils in BAL fluid after CDE challenge, (ii) the serum level of rFel d 1-specific IgE was reduced and the level of IgG(2)a was more pronounced after CBP-rFel d 1 treatment, and (iii) there was also a tendency of decreased allergen-specific cellular response. CONCLUSIONS: Carbohydrate-based particles are effective tools as adjuvant and allergen carriers for use in ASIT and constitutes a promising strategy to improve allergy treatment.

Immune regulation by CD4+CD25+ T cells and interleukin-10 in birch pollen-allergic patients and non-allergic controls.

BACKGROUND: CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. OBJECTIVE: To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. METHODS: Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. RESULTS: CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. CONCLUSION: We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.

Rational design of hypoallergens applied to the major cat allergen Fel d 1.

BACKGROUND: Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. OBJECTIVE: The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. METHODS: The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. RESULTS: Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. CONCLUSION: By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.

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.

T cell responses to recombinant isoforms, synthetic peptides and a mutant variant of Lep d 2, a major allergen from the dust mite Lepidoglyphus destructor.

BACKGROUND: The dust mite Lepidoglyphus destructor is an important cause of allergic reactions to dust, especially in farming environments. Two isoforms, recombinant (r)Lep d 2.01 and rLep d 2.02, of the major allergen Lep d 2, have previously been expressed as recombinant proteins. These isoforms differ 10.4% at the amino acid level. Furthermore, a mutant form of Lep d 2.01 (rLep d 2.6Cys) with a highly reduced IgE reactivity, has also been produced. OBJECTIVE: To investigate the T cell responses to the recombinant isoforms of Lep d 2, the Lep d 2.6Cys mutant and peptides of Lep d 2, in allergic and non-allergic individuals. METHODS: Peripheral blood mononuclear cells from 18 allergic and 16 non-allergic individuals were stimulated with the different antigens and the proliferative responses were measured. The cytokine production (interleukin (IL)-4, IL-5 and interferon (IFN)-gamma) were measured by ELISA. RESULTS: Higher T cell proliferation was measured to isoform 01 than to 02 in 28/34 subjects. The responses to rLep d 2.6Cys were lower than to isoform 01 in most subjects, but higher than to Lep d 2.02. Two immuno-dominant peptides, corresponding to amino acid residue 11-25 and 61-75 were identified. The atopic subjects produced significantly lower IFN-gamma in response to Lep d 2.01 as compared to the non-atopics. CONCLUSIONS: There was a significant difference in T cell response between the two isoforms of rLep d 2. The hypoallergenic mutant rLep d 2.6Cys was able to evoke a T cell response with a magnitude which is between the two isoforms. Amino acid residue 11-25 and 61-75 are the most frequently recognized parts of Lep d 2 and are likely to contain the immuno-dominant T cell epitopes.

A hypoallergenic derivative of the major allergen of the dust mite Lepidoglyphus destructor, Lep d 2.6Cys, induces less IgE reactivity and cellular response in the skin than recombinant Lep d 2.

BACKGROUND: The major allergen of the dust mite Lepidoglyphus destructor, Lep d 2, has been produced as a recombinant allergen (rLep d 2) with IgE reactivity both in vivo and in vitro. A modified form of rLep d 2 (rLep d 2.6Cys) obtained by site-directed mutagenesis has been shown to have a reduced IgE reactivity in vitro. In this study we have compared the ability of rLep d 2 and rLep d 2.6Cys to elicit positive skin prick tests and cellular responses among L. destructor-sensitized subjects. METHODS: Seventeen subjects were skin prick-tested with rLep d 2, rLep d 2.6Cys, histamine and negative controls and 17-20 h later skin biopsy specimens were taken from the skin prick-tested sites. The biopsy specimens were stained immunohistochemically for EG2+, CD3+, CD1a+, mast cell tryptase+, and IgE+ cells. Dermal cell infiltrates were judged in hematoxylin and eosin staining. Total IgE and allergen-specific IgE were determined by CAP-RAST. RESULTS: Compared to rLep d 2, rLep d 2.6Cys induced significantly smaller and fewer skin prick test reactions (p < 0.001) and dermal cell infiltrates (p < 0.05). Further, rLep d 2.6Cys induced fewer EG2+ cells (p < 0.001) but more tryptase+ cells (p < 0.05) than rLep d 2. A positive RAST to rLep d 2 was obtained for 88.2% of the subjects, while only 35.2% displayed a positive RAST to rLep d 2.6Cys. CONCLUSION: This study demonstrates that rLep d 2.6Cys is less able to evoke IgE-mediated reactions and cellular responses, as measured both in skin and in serum, than rLep d 2. In the future this hypoallergenic derivative may be a promising candidate molecule for immunotherapy of L. destructor-allergic patients.

The molecular basis of antigenic cross-reactivity between the group 2 mite allergens.

BACKGROUND: Mite group 2 allergens Der p 2, Der f 2, and Eur m 2 are 14-kDa proteins of unknown function that share 83% to 85% amino acid sequence identity. Isoforms of the allergens within each genus have been identified which differ by 3 or 4 amino acids, but little is known of the influence of group 2 polymorphisms on human IgE antibody binding. OBJECTIVE: The purpose of this study was to investigate the importance of interspecies and isoform substitutions on murine mAb and IgE antibody binding and on the molecular structure of the group 2 allergens. METHODS: Site-directed mutagenesis was used to incorporate the isoform amino acid substitutions onto the Der p 2.0101 sequence. Recombinant allergens were expressed and purified from Escherichia coli and used to evaluate antibody binding by enzyme-linked immunosorbent assay (ELISA). Molecular modeling of the tertiary structure was used to analyze structural differences between the various group 2 allergens. RESULTS: The substitution of asparagine for aspartic acid at position 114 restored mAb binding of rDer p 2.0101; the other Der p 2 isoforms and the 3 rDer f 2 isoforms also reacted in the 2-site ELISA. The correlation of IgE binding to the Der p 2 isoforms was excellent and tended to be higher in the isoforms with the asparagine 114 substitution (r (2) = 0.87 vs r (2) = 0.95). rEur m 2.0101 bound to all mAb except 7A1; when compared with rDer p 2 for IgE binding, rEur m 2.0101 gave a correlation coefficient of r (2) = 0.68. Molecular modeling revealed that Eur m 2 and the storage mite homologs Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2. Eur m 2 has a conserved surface, whereas Lep d 2 and Tyr p 2 present most of the amino acid substitutions on this surface. Lep d 2 and Tyr p 2 did not react with mAb or with sera from patients with IgE to Dermatophagoides species. CONCLUSION: The isoform substitutions of rDer p 2 can be distinguished by mAb. The allergenic cross-reactivity between Der p 2, Der f 2, and Eur m 2 is a direct result of the conserved antigenic surface, whereas the lack of cross-reactivity with Lep d 2 and Tyr p 2 is a result of the multiple substitutions across this surface.

Cross-reactivity studies of a new group 2 allergen from the dust mite Glycyphagus domesticus, Gly d 2, and group 2 allergens from Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae with recombinant allergens.

BACKGROUND: Dust mites are important inducers of allergic disease. Group 2 allergens are recognized as major allergens in several mite species, including Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae. No allergens have thus far been characterized on the molecular level from the dust mite Glycyphagus domesticus. OBJECTIVE: We sought to examine the cross-reactivity among group 2 allergens of G domesticus, L destructor, T putrescentiae, and D pteronyssinus. METHODS: A group 2 allergen from G domesticus, Gly d 2, was cloned and expressed as a recombinant protein. Cross-reactivity between Gly d 2 and 3 other group 2 allergens, Lep d 2, Tyr p 2, and Der p 2, was studied by using individual sera and a serum pool RAST-positive to G domesticus, L destructor, T putrescentiae, and D pteronyssinus. Recombinant allergens were used as inhibitors of IgE binding in immunoblotting experiments. Molecular modeling on the basis of the Der p 2 structure was carried out for Gly d 2, Lep d 2, and Tyr p 2. RESULTS: Two cDNAs encoding isoforms of Gly d 2 were isolated, but only the Gly d 2.02 isoform was used in this study. Sixteen of 17 subjects had IgE to Gly d 2. The protein sequence of Gly d 2 revealed 79% identity to Lep d 2 and 46% and 41% identity to Tyr p 2 and Der p 2, respectively. Extensive cross-reactivity was demonstrated among Gly d 2, Lep d 2, and Tyr p 2, but little cross-reactivity was found between these allergens and Der p 2. According to the tertiary structure of Der p 2 and 3-dimensional models of Gly d 2, Lep d 2, and Tyr p 2, differences reside mainly in surface-exposed residues. CONCLUSION: Gly d 2 showed high sequence homology to Lep d 2. Cross-reactivity was observed between Gly d 2, Lep d 2, and Tyr p 2, but only limited cross-reactivity was demonstrated between these 3 allergens and Der p 2.

Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology.

The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contributes to dust mite allergy in urban areas. One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in vitro and shown to detect 60% or more of L. destructor-sensitized subjects. Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L. destructor allergy. The aim of this study was to clone and express new allergens from L. destructor and determine their recognition frequency among sensitized individuals. A phage display cDNA expression library was constructed and screened with sera from L. destructor-sensitized individuals. The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C-terminal His6-tagged proteins. Immunoblotting of the recombinant proteins was performed using sera from 45 subjects allergic to L. destructor. Three new allergens from L. destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 (13%) sera from L. destructor-sensitized subjects, respectively.

Skin prick test and serological analysis with recombinant group 2 allergens of the dust mites L. destructor and T. putrescentiae.

BACKGROUND: The dust mites Lepidoglyphus destructor and Tyrophagus putrescentiae are important sources of allergen in farming environments. The major allergens of the dust mites L. destructor and T. putrescentiae have been cloned and expressed as recombinant proteins. OBJECTIVE: To evaluate the use of recombinant group 2 allergens of L. destructor (rLep d 2) and T. putrescentiae (rTyr p 2) in skin prick test (SPT), and serological analysis in sensitized and non-sensitized farmers chronically exposed to dust mites. METHODS: Skin prick test with rLep d 2, rTyr p 2 and the corresponding commercial extracts was performed in 44 farmers sensitized to L. destructor and/or T. putrescentiae, and 38 control farmers. IgE and IgG subclass antibodies to the recombinant allergens were analysed by RAST and ELISA, respectively. RESULTS: Out of the 44 subjects positive in SPT to L. destructor and/or T. putrescentiae extract, 26 (59%) displayed a positive SPT to one or the other of the recombinant allergens, whereas 21 (48%) were positive to both. Significant correlations were registered between the sizes of the weals induced by rLep d 2 and rTyr p 2 and the corresponding RAST values (P < 0.001). A majority of subjects positive in SPT to the recombinant allergens had detectable IgG4 antibodies, and the levels were significantly higher in the dust mite sensitized group than in the controls (P < 0.05). No such differences were found in the IgG1 values (P > 0.05). The results obtained with rLep d 2 and rTyr p 2 correlated relatively well with each other with respect to SPT, RAST and IgG4, suggesting that the allergens have similar or shared IgE epitopes. All the control subjects had a negative SPT and RAST to rLep d 2 and rTyr p 2. CONCLUSION: Recombinant group 2 allergens from the dust mite L. destructor and T. putrescentiae represent useful tools for diagnosis of dust mite allergy.

Evaluation of specific IgE to the recombinant group 2 mite allergens Lep d 2 and Tyr p 2 in the Pharmacia CAP system.

BACKGROUND: Several recombinant allergens have been shown to be potentially useful for diagnosis of IgE-mediated allergy, but only a few recombinant allergens are at present commercially available in serological assays for detection of specific IgE. The aim of this study was to evaluate the IgE binding to the recombinant major dust mite allergens rLep d 2 and rTyr p 2 and compare it with the IgE binding to the commercial mite extracts Lepidoglyphus destructor and Tyrophagus putrescentiae in the Pharmacia RAST CAP System. METHODS: The recombinant allergens rLep d 2 and rTyr p 2 were immobilised on ImmunoCAPs, and sera from 461 Swedish farmers who are frequently exposed to mites were analysed for specific IgE antibodies. Immunoblotting was performed to evaluate discrepancies between the results obtained with the recombinant and the commercial CAP assays. RESULTS: The IgE values of each recombinant assay significantly correlated with the IgE values of the corresponding commercial CAP assay. The sensitivity of the rLep d 2 assay was 73.3% and that of the rTyr p 2 assay, 60.5% of that provided by the commercial L. destructor and T. putrescentiae assays. Two subjects out of 416, who tested negative in the commercial L. destructor assay, were positive to rLep d 2. The corresponding figures for rTyr p 2 and the T. putrescentiae extract were 5/418. The possibility that these subjects were sensitised to L. destructor and T. putrescentiae could not be excluded. CONCLUSIONS: The data suggest that it may be possible to use rLep d 2 and rTyr p 2 on ImmunoCAPs to detect and quantify IgE antibodies to these, the major allergens of L. destructor and T. putrescentiae. It appears likely that the addition of just a few more recombinant L. destructor and T. putrescentiae allergens in the CAP assay will be sufficient for in vitro diagnosis of IgE mediated allergy to L. destructor and T. putrescentiae.