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Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols. [ABSTRACT FROM AUTHOR]

Background: The introduction of component-resolved diagnosis was a great advance in diagnosis of allergy. In particular, molecular allergy techniques allowed investigation of the association between given molecular profiles and clinical expression of allergy. We evaluated the possible correlation between the level of specific IgE (sIgE) to single components of Phleum pratense and clinical issues such as the severity of allergic rhinitis (AR) and the presence or absence of asthma. Methods: The study included 140 patients with rhinitis and/or asthma caused by sensitization to grass pollen. sIgE to Phl p 1, Phl p 5, Phl p 7, and Phl p 12 from Phleum pratense were measured, and the correlation between the stage of AR according to Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines and the presence of asthma was studied by multivariate logistic regression in terms of sIgE and ARIA stage, while univariate logistic regression was used for IgE and a dichotomic classification of asthma as present or absent. Results: Ten patients had intermittent AR, 48 had mild persistent AR, and 82 had severe persistent AR. Asthma was present in 86 patients and absent in 54. A significant correlation was found between severe persistent AR and presence of asthma (p < 0.01). The only significant correlation between clinical data and sIgE values was that of low values of sIgE to Phl p 5 and absence of asthma (p < 0.01). Conclusions: This preliminary finding suggests that low values of sIgE to Phl p 5 are correlated with the absence of asthma in patients with grass-pollen induced allergy. The data, provided they are confirmed by further studies, could be useful when selecting patients who are candidates for allergen immunotherapy, since a higher risk of asthma could be used as a selection criterion for using this approach. [ABSTRACT FROM AUTHOR]

Background: Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions. Methods: All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays. Results: Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen. Conclusions: The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Grasses belong to major sources of inhaled allergens. The knowledge of particular molecules responsible for hypersensitivity is of crucial importance for better understanding of individual differences among single allergic subjects and allergic populations living in various world-areas. Methods: Specific-IgE-antibodies against Phl p 1, Phl p 5, Phl p 7, Phl p 12 were detected in a group of 130 Phleum-allergic-subjects (82 children, 48 adults). Results: Phl p 1 antibodies were detected in most pediatric and adult patients, however, the children were associated with higher RAST classes more often. Anti-Phl p 5-antibodies were found more frequently in adults. An increase was observed in the number of pediatric patients reacting to Phl p 7 and Phl p 12. There were no differences in concentrations of specific-IgE against Phl p 5, Phl p 7 and Phl p 12 depending on age. Almost 10% of allergic children produced antibodies directed exclusively against minor allergens or did not produce specific-IgE-antibodies against tested molecules. Part of the patients reacted to profilin and calcium-binding protein originating from only one source (Phl p 12/Bet v 2 and Phl p 7/Bet v 4). Conclusions: Antibodies against Phl p 1 and Phl p 5 can be used as a marker of allergy to grasses in adult patients. Children reacted exclusively to minor allergens more frequently than adults. Prolonged allergen exposure is evidently necessary to induce sensitization to Phl p 5. A high level of homology between profilins and calcium-binding proteins enables only one allergen to be used for diagnostic purposes but a possibility of a reaction to species-bound epitopes should be taken into account. [ABSTRACT FROM AUTHOR]

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 IgG1 heavy chain was excised and replaced by the sequence coding for the human e constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG1 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-FcϵRI 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. [ABSTRACT FROM AUTHOR]

Abstract: In this study we have elucidated the effects of allergen-specific antibodies on human DCs and T-cells. Monocyte-derived DCs from allergic patients were exposed to Phl p 5 alone or in complex with Phl p 5-specific human IgG1, IgG4 or IgE and further co-cultured with autologous memory CD4+ T-cells. We demonstrate that DCs treated with Phl p 5/IgE-complexes secrete higher levels of IL-1α, IL-6, VEGF and MCP-3 compared to Phl p 5 alone. Furthermore, we show that the ability of DCs to present allergen to memory CD4+ T-cells and induce a Th2 cytokine profile is significantly augmented when the uptake is mediated by specific IgE antibodies, whereas IgG1 and IgG4 have no such effect. The differences in cytokine profiles depending on the antibody subtype could partly explain the ability of allergic individuals to amplify allergen-specific immune response and could thus be involved in the etiology of allergic responses. [Copyright &y& Elsevier]

Background: Replicase-based DNA vaccines stimulate TH1-biased immune responses at ultralow doses and induce self-removal of transfected cells through apoptosis. Both aspects are important requirements for efficient and safe DNA-based immunotherapy of type I allergies. Objective: A Sindbis virus replicon-based DNA vaccine encoding the major timothy grass pollen allergen Phl p 5 was evaluated for its antiallergic potential compared with a conventional DNA vaccine in a BALB/c mouse model of allergy. Methods: Mice were intradermally prevaccinated with plasmid DNA, followed by sensitization and intranasal allergen provocation with recombinant Phl p 5. In vitro proliferation and cytokine secretion was measured in splenocyte cultures. Distribution of IgG1, IgG2a, and IgE antibody subclasses was determined by means of ELISA. IgE-mediated degranulation was measured with the basophil release assay. Bronchoalveolar lavage fluid was analyzed for eosinophils, IL-4, IL-5, IL-13, and IFN-γ. Mucus production, inflammatory infiltrates, and epithelial damage were determined in lung sections. Results: Both vaccines induced TH1-biased immune responses, resulting in suppression of functional IgE, reduction of eosinophilia in bronchoalveolar lavage fluid, and alleviation of lung pathology. However, immunization with the replicon DNA vaccine elicited these effects at a 100-fold lower dose compared with the conventional DNA vaccine. Conclusions: The increased immunogenicity of replicon-based DNA vaccines allows for application of extremely low doses, thereby eliminating the concerns associated with conventional DNA vaccines, which have to be administered at milligram amounts to induce immune reactions in human subjects. Clinical implications: Their high safety profile makes replicon-based DNA vaccines promising candidates for treatment of type I allergies in the clinic. [Copyright &y& Elsevier]

Background: At present, recombinant allergens are considered for the diagnosis and treatment of atopic allergies. To evaluate the theoretical impact of isoallergen variation on the selection of isoallergens for specific allergy vaccination, we characterized the T–cell response of allergic patients to the Phleum pratense major allergen, Phl p 5, and five of its recombinant isoallergens. Methods: Phl p 5–specific T cell lines (TCLs) were isolated from the peripheral blood of 3 allergic rhinitis patients, and their reactivity patterns were studied in detail. Results: The TCLs were highly crossreactive with related grasses. The crossreactivity with Poa pratensis was more extensive than with Lolium perenne, directly reflecting the sequence identity between Phl p 5, Poa p 5, and Lol p 5. The TCLs produced IFN–γ and IL–4 simultaneously, resembling a Th0–like cytokine production profile. Interestingly, when T cell clones were tested with natural Phl p 5 and five rPhl p 5 isoallergens, a differential recognition pattern was found. One of the TCLs exclusively reacted with Phl p 5b, another reacted with all isoforms tested, and the third reacted strongly with native purified Phl p 5, but only weakly with all five recombinant isoallergens. Conclusion: These results indicate that Phl p 5–specific T cells are highly heterogeneous, and that they differentially recognize isoallergens. This indicates that when recombinant Phl p 5 is considered for allergy vaccination, a mixture of isoallergens representing the different isoallergen groups may clinically prove to be more effective than single isoallergens. [ABSTRACT FROM AUTHOR]