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.

Immunoglobulin E antibody reactivity to bacterial antigens in atopic dermatitis patients.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 20% children and 9% adults world-wide. AD patients are often sensitized against a broad variety of allergens and more than 90% of them suffer from skin superinfections with Staphylococcus aureus. OBJECTIVE: In this study, we searched for the presence of specific IgE antibodies against S. aureus and Escherichia coli antigens in AD patients. METHODS: Sera from AD patients (n=79), patients suffering only from allergic rhinoconjunctivitis (n=41) or allergic asthma (n=37) were tested for IgE reactivity to nitrocellulose-blotted S. aureus, E. coli and gut bacterial antigens. IgE-reactive bacterial antigens were affinity purified and identified by mass spectrometry. RESULTS: More than 30% of AD patients but not patients suffering only from allergic rhinoconjunctivitis and asthma or non-allergic persons exhibited IgE binding to several protein antigens among them DNA-binding and ribosomal proteins and flagellin. Patients with severe skin manifestations showed more frequently IgE reactivity to S. aureus compared with AD patients with mild symptoms. Positive immediate and late skin test reactions could be induced in sensitized AD patients with S. aureus extract. CONCLUSION AND CLINICAL RELEVANCE: Specific IgE reactivities against a variety of bacterial antigens were observed in a subgroup comprising a third of AD patients and may contribute to allergic inflammation.

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.

Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen.

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

A comparative analysis of the cross-reactivity in the polcalcin family including Syr v 3, a new member from lilac pollen.

BACKGROUND: Polcalcins are pollen-specific allergens with two EF-hand calcium-binding sites that exhibit strong cross-reactivity. Our objective was to isolate and express the cDNA coding of the EF-hand calcium-binding allergen from lilac pollen and to study cross-reactivity with other polcalcins from related and nonrelated pollen sources with different specific antibodies and sera from two different populations. METHODS: Specific cDNA was amplified by PCR, cloned and expressed in Escherichia coli. Purification was achieved by gel permeation and ion exchange chromatographies. ELISA titration and inhibition assays were performed using the recombinant forms of Syr v 3, Ole e 3, Che a 3 and Phl p 7 with sera from two Spanish regions with different sensitization profiles, as well as Phl p 7- and Ole e 3-specific polyclonal rabbit antisera, and an Ole e 3-specific monoclonal antibody. RESULTS: Syr v 3 displays two EF-hand consensus sites and 8863 Da of theoretical molecular mass. The allergen consists of 80 residues with identities ranging from 66 to 87% with polcalcins included in this study. Syr v 3, Ole e 3, Che a 3 and Phl p 7 showed a similar IgG- and IgE-binding capacity although differences at quantitative level were observed depending on the population of patients' sera. CONCLUSION: Syr v 3 is a polcalcin with structural and antigenic similarities to the members of this family. Diagnosis of polcalcin-sensitized patients could be performed whatever polcalcin used, whereas for immunotherapy, primary sensitization to a particular allergenic source should be considered.

Analysis of the sensitization profile towards allergens in central Africa.

BACKGROUND: Almost no information is available regarding the prevalence of IgE-mediated allergies and the disease-eliciting allergens in tropical Africa. OBJECTIVE: To study IgE-mediated allergies and the allergen profile in allergic patients from Zimbabwe. METHODS: The frequency of sensitization to common environmental allergen sources was determined by skin prick testing in 650 allergic patients from Zimbabwe. Fifty representative sera were analysed for IgE reactivity to 20 respiratory and 20 food allergen extracts by multiallergen extract testing. The IgE reactivity profiles to recombinant pollen and mite allergens were compared between grass pollen- and mite-sensitized patients from Zimbabwe and central Europe. Sera from grass pollen-allergic patients were also analysed for IgE reactivity to nitrocellulose-blotted natural timothy grass and Bermuda grass pollen allergens. RESULTS: IgE-mediated allergies were found to be common in Zimbabwe. Similar to the situation in central Europe, mites and grass pollens represented the most prevalent allergen sources. However, the IgE reactivity profiles determined with single recombinant pollen and mite allergens revealed interesting differences between the European and African patients, which most likely reflect the local allergen exposure. CONCLUSIONS: The striking differences regarding sensitization to grass pollen and mite allergens between African and European patients revealed by recombinant allergen-based testing emphasize the need for component-resolved allergy testing to optimize allergy prevention and therapy in different populations.