Henrik Ipsen

Extensive IgE cross-reactivity towards the Pooideae grasses substantiated for a large number of grass-pollen-sensitized subjects.

Background: Allergy to taxonomically related species is a common phenomenon caused by the same immunological receptor cross-reacting to homologous allergens from different species. Knowledge of patterns of cross-reactivity is crucial for the selection of optimal products for diagnosis and for specific immunotherapy. The objective of this study was to investigate patterns of serum IgE cross-reactivity towards pollens from various grass species. Methods: With grass group 1 allergens as the representative group, amino acid sequence alignment, structural modelling and comparison of 3D surface characteristics were performed to exemplify the molecular basis of IgE cross-reactivity. IgE binding to extracts from ten different grass species was determined (total number of data pairs >19,000), and IgE inhibition experiments using Phleum pratense were performed. Results: Analysis of surface topography for group 1 grass allergens demonstrated ample space for IgE binding epitopes in surface areas conserved among Pooideae grasses. Significant correlation was observed between the serum IgE response to P. pratense extract and extracts from the other Pooideae grasses analyzed. P. pratense extract was demonstrated to inhibit the binding of IgE to the allergens in all of the extracts included in the investigation, indicating patient IgE to be primarily directed towards common epitopes. Conclusion: Extensive IgE cross-reactivity was observed towards the allergens of the Pooideae grasses, meaning that the immune system does not appear to distinguish based on the IgE level between the different species of this subfamily. The data suggest equal effect upon use of any of the Pooideae species for diagnostic as well as therapeutic purposes.

The Significance of Isoallergenic Variations in Present and Future Specific Immunotherapy

The isoallergenic variation of the tree pollen major allergens has been studied by 2D gel electrophoresis, and by analysis of several recombinant clones. The studies have included both antibody-based and T cell stimulation assays. Bet v 1, the major allergen of birch, forms at least 24 spots when conventional extracts are analyzed by 2D gel electrophoresis. Comparison of Bet v 1-encoding DNA sequences reveals a considerable number of amino acid substitutions. This sequence variation can theoretically account for the number of spots observed in 2D gels. Whereas pools of serum from allergic individuals and monospecific antibodies raised in rabbits bind to most if not all spots in 2D gels, analyses of individual serum and/or murine monoclonal antibodies show individual patterns of reactivity with various subsets of spots. These observations point to a model in which amino acid substitutions induce local perturbations of the allergen surface, causing differences in epitope structure. Furthermore, analysis of pollen from individual trees shows that each tree produces individual subsets of Bet v 1 spots. When analyzed in stimulation assays, T cell clones also display differences in reactivity to different isoallergens. In conclusion, we have shown that Bet v 1 is heterogeneous, and that individual trees produce various subsets of isoallergens which display differences in reactivity both towards antibodies and T cells. A careful selection of isoform may therefore be of major importance if recombinant allergens or synthetic peptides are to be used for conventional immunotherapy.

Specific IgE response toward allergenic molecules during perennial hyposensitization: a three-year prospective double-blind study.

Allergenic extracts are multicomponent systems containing a number of potentially allergenic molecules. In a 3 yr prospective double-blind study, sera from 40 patients hyposensitized with either a two-component timothy extract or a 20-component timothy extract were evaluated by means of crossed radioimmunoelectrophoresis. The patients exhibited specific IgE response toward individual allergenic molecules, and IgE with new specificities did not develop after hyposensitization. A high degree of clinical protection could generally be obtained on treatment with the 20-component timothy extract, but this was true only with the two-component timothy extract containing the allergenic molecules that matched the individual IgE response.

Antigenic Similarity among Group 1 Allergens from Grasses and Quantitation ELISA Using Monoclonal Antibodies to Phl p 1

Background: Group 1 allergens elicit a specific IgE response in about 90% of grass pollen-allergic patients. The aim of this work was to study the antigenic similarity among group 1 allergens from different grasses and to develop a monoclonal antibody (MAb)-based quantitation ELISA. Methods: Twenty specific MAbs were produced from BALB/c mice immunized with natural Phl p 1. These MAbs were tested for specificity with thirteen different grass pollen extracts from the Poaceae family and in cross-inhibition experiments for the binding of Phl p 1. Purified group 1 allergens from Poeae grasses (Dactylis glomerata, Lolium perenne, Festuca pratensis and Poa pratensis) were tested for parallelism in quantitation ELISA. Results: Eighteen to nineteen anti-Phl p 1 MAbs recognized the homologous allergen in pollen extracts from grasses of the Poeae tribe. In contrast, only four MAbs recognized group 1 from Cynodon dactylon and Phragmites communis. Four groups of MAbs with different epitope specificity were identified. A grass group 1 quantitation ELISA was developed using a mix of three MAbs on the solid phase and a polyclonal rabbit antibody as the second antibody. The group 1 content could be measured in different batches of Phleum pratense as well as in pollen extracts from Poeae grasses, since they showed parallel dose-response curves. Conclusions: MAbs produced in this work enabled us to show the high antigenic similarity between group 1 allergens from temperate grasses. The results prove the usefulness of the ELISA method developed for standardization of grass allergen products.

Highly heterogeneous Phl p 5-specific T cells from patients with allergic rhinitis differentially recognize recombinant Phl p 5 isoallergens

BACKGROUND The prevalence of atopic allergy to Poaceae grasses poses a serious health problem. OBJECTIVE To evaluate the T cell-activating capacity of recombinant grass pollen allergens suggested for immunotherapy, we characterized the T-cell response of allergic patients to the Phleum pratense (Phl p) major allergen, Phl p 5. METHODS Thirty-eight Phl p 5-specific CD4(+) T-cell clones (TCCs) were isolated from the peripheral blood of 3 patients with allergic rhinitis and were characterized with respect to cross-reactivity, HLA restriction, cytokine profiles, and isoallergen specificity when tested with natural Phl p 5 and 5 rPhl p 5 isoallergens (4 Phl p 5a and 1 Phl p 5b). RESULTS The TCCs were highly cross-reactive with group 5 allergens of related grasses, and the different cross-reactivity patterns found indicate that several T-cell epitopes are present in Phl p 5. Most TCCs displayed a TH0-like cytokine profile, whereas a few TCCs belonged to the TH2 or TH1 subset. The TCCs were restricted by HLA-DR (24/38 TCCs), HLA-DQ (11/38 TCCs), or HLA-DP (3/38 TCCs). Interestingly, 4 of the 34 TCCs tested reacted exclusively with the 4 rPhl p 5a isoforms; 8 of 34 TCCs were rPhl p 5b specific, and 3 of 34 TCCs reacted with all isoforms; 19 of 34 TCCs did not react with any of the rPhl p 5 isoallergens. Moreover, the overall isoform recognition pattern differed considerably among patients. CONCLUSION These results demonstrate that Phl p 5-specific T cells are highly heterogeneous and that individual TCCs, and individual patients, differentially recognize isoallergens. The differential isoallergen recognition for Phl p 5-specific T cells suggests, if confirmed in larger patient groups, that a combination of 2 or more rPhl p 5 isoallergens may be needed to replace the natural grass allergen for immunotherapy.

Epitope grafting: re-creating a conformational bet V 1 antibody epitope on the surface of the homologous apple allergen MAL D 1

Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen from Betula verrucosa (Bet v 1). Still, to what extent two molecular surfaces need to be similar for clinically relevant antibody cross-reactivity to occur is unknown. Here, we describe the grafting of a defined conformational antibody epitope from Bet v 1 onto the surface of the homologous apple allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction to Bet v 1 and to the mutated Mal d 1 variant, respectively, were assessed by Biacore experiments demonstrating indistinguishable binding kinetics. This demonstrates that a conformational epitope defined by a high affinity antibody-allergen interaction can successfully be grafted onto a homologous scaffold molecule without loss of epitope functionality. Furthermore, we show that increasing surface similarity to Bet v 1 of Mal d 1 variants by substitution of 6–8 residues increased the ability to trigger basophil histamine release with blood from birch-allergic patients not responding to natural Mal d 1. Conversely, reducing surface similarity to Bet v 1 of a Mal d 1 variant by substitution of three residues abolished histamine release in one patient reacting to Mal d 1.

Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

Immunochemical characterization of antigens of Parietaria judaica pollen: identification of allergens by means of crossed radio immunoelectrophoresis

A crude extract of Parietaria judaica pollen was obtained by means of extraction, centrifugation and dialysis, and studied by means of quantitative immunoelectrophoresis. Crossed immunoelectrophoresis, using a high-titer purified rabbit antibody fraction, showed that the pollen extract contained at least 26 antigens of which 18 moved towards the anode, 6 moved towards the cathode and 1 moved both towards the anode and the cathode. The allergens in the extract were identified by means of crossed radio immunoelectrophoresis. Nine of the 26 antigens were able to bind specific human IgE to their corresponding immunoprecipitates, and 4 of these antigens can be classified as major allergens. The apparent molecular weights of 17 antigens were determined by a combination of size exclusion chromatography and immunochemical analysis. Ten antigens had Kav values corresponding to molecular weights in the range of 10-40 kilodaltons, 1 antigen possessed an apparent molecular weight of less than 10 kilodaltons, and six antigens had molecular weights above 40 kilodaltons. Most of the antigens, as analysed by column isoelectric focusing, had pI values between 4 and 7. Crossed line immunoelectrophoresis using an extract of Parietaria officinalis shows that the antigens of this extract exhibit a high degree of identity with those of P. judaica.

Bioavailability of House Dust Mite Allergens in Sublingual Allergy Tablets Is Highly Dependent on the Formulation

Background: In sublingual immunotherapy (SLIT), the immune system is addressed by solubilized allergen that interacts with immunocompetent cells of the oral mucosa, the efficiency of which is governed by 2 main factors of SLIT allergen bioavailability: the allergen concentration and the mucosal contact time. Recently, 3 house dust mite (HDM) SLIT tablets were developed that differ with regard to allergen content, nominal strength (maintenance doses: 6 SQ-HDM/10,000 Japanese Allergen Units [JAU], 12 SQ-HDM/ 20,000 JAU, and 300 IR/57,000 JAU), and formulation (freeze-dried/compressed). Here, the importance of the SLIT tablet formulation for HDM major allergen bioavailability is examined. Methods: The HDM major allergen content, tablet disintegration times, and allergen release kinetics were determined. Dissolution kinetics (allergen concentration vs. time) of Der f 1, Der p 1, and Der 2 were measured. Area under the curve (AUC) was used as a surrogate parameter for allergen bioavailability. Results: The release of HDM major allergens from the freeze-dried tablets was complete after 30 s, while only partial release was achieved with the compressed tablets, even after prolonged dissolution. At 1 min, i.e., the recommended sublingual holding time for the freeze-dried tablets, the allergen bioavailability (AUC) of the compressed 300 IR/57,000 JAU tablet was 4.7-fold (Der f 1), 10.8-fold (Der p 1), and 23.6-fold (Der 2) lower than that of the freeze-dried 12 SQ-HDM/20,000 JAU tablet and similar to (Der f 1) and 5.3-fold (Der p 1) and 12.5-fold (Der 2) lower than that of the freeze-dried 6 SQ-HDM/10,000 JAU tablet. Conclusions: SLIT tablet allergen bioavailability depends highly on the tablet formulation. Only the fast-dissolving freeze-dried tablets provide maximal delivery of soluble allergens and achieve allergen concentrations that reflect the nominal tablet strengths within the recommended sublingual holding time.

Structural changes of protein antigens due to adsorption onto and release from aluminium hydroxide using FTIR–ATR

Structural integrity of antigens upon adsorption and release is not only important for investigating vaccine immunogenicity, but also for the epitope specificity of the resulting immune response and hence therapeutic efficacy. Moreover, the structural information is also important for understanding the mechanism of how adjuvants can enhance the immune response. However, little is known about an antigens structure when it is adsorbed on and subsequently released from aluminium adjuvants. In this study, the structures of two protein antigens, bovine serum albumin and β-lactoglobulin, were investigated using Fourier transform infrared–attenuated total reflection (FTIR–ATR) spectroscopy. The secondary structures of both model antigens change when adsorbed to aluminium hydroxide. The structural perturbation depends on the amount of adsorbed protein. Maximal adsorption gives a more native-like structure. This may indicate that protein is adsorbed in different manners depending on the concentration. The adsorbed antigens are released using phosphate buffer pH 7.4 (PB). The recovery is approximate 80% after 40 min in the presence of PB. The recovery curves of both proteins also indicate two different adsorption modes. FTIR–ATR and circular dichroism (CD) spectroscopy yield similar results suggesting that the adsorbed antigens refold to their native-like state after release.