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The proportion of patients with type I allergy in the world population has been increasing and with it the number of people suffering from allergic symptoms. Recently we showed that prophylactic cell therapy employing allergen-expressing bone marrow (BM) cells or splenic B cells induced allergen-specific tolerance in naïve mice. Here we investigated if cell therapy can modulate an established secondary allergen-specific immune response in pre-immunized mice. We sensitized mice against the grass pollen allergen Phl p 5 and an unrelated control allergen, Bet v 1, from birch pollen before the transfer of Phl p 5-expressing BM cells. Mice were conditioned with several combinations of low-dose irradiation, costimulation blockade, rapamycin and T cell-depleting anti-thymocyte globulin (ATG). Levels of allergen-specific IgE and IgG1 in serum after cell transfer were measured via ELISA and alterations in cellular responses were measured via an in vitro proliferation assay and transplantation of Phl p 5+ skin grafts. None of the tested treatment protocols impacted Phl p 5-specific antibody levels. Transient low-level chimerism of Phl p 5+ leukocytes as well as a markedly prolonged skin graft survival were observed in mice conditioned with high numbers of Phl p 5+ BMC or no sensitization events between the day of cell therapy and skin grafting. The data presented herein demonstrate that a pre-existing secondary allergen-specific immune response poses a substantial hurdle opposing tolerization through cell therapy and underscore the importance of prophylactic approaches for the prevention of IgE-mediated allergy. [ABSTRACT FROM AUTHOR]

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]

The proportion of patients with type I allergy in the world population has been increasing and with it the number of people suffering from allergic symptoms. Recently we showed that prophylactic cell therapy employing allergen-expressing bone marrow (BM) cells or splenic B cells induced allergen-specific tolerance in naïve mice. Here we investigated if cell therapy can modulate an established secondary allergen-specific immune response in pre-immunized mice. We sensitized mice against the grass pollen allergen Phl p 5 and an unrelated control allergen, Bet v 1, from birch pollen before the transfer of Phl p 5-expressing BM cells. Mice were conditioned with several combinations of low-dose irradiation, costimulation blockade, rapamycin and T cell-depleting anti-thymocyte globulin (ATG). Levels of allergen-specific IgE and IgG1 in serum after cell transfer were measured via ELISA and alterations in cellular responses were measured via an in vitro proliferation assay and transplantation of Phl p 5+ skin grafts. None of the tested treatment protocols impacted Phl p 5-specific antibody levels. Transient low-level chimerism of Phl p 5+ leukocytes as well as a markedly prolonged skin graft survival were observed in mice conditioned with high numbers of Phl p 5+ BMC or no sensitization events between the day of cell therapy and skin grafting. The data presented herein demonstrate that a pre-existing secondary allergen-specific immune response poses a substantial hurdle opposing tolerization through cell therapy and underscore the importance of prophylactic approaches for the prevention of IgE-mediated allergy.

Protein modifications such as oligomerization and tyrosine nitration alter the immune response to allergens and may contribute to the increasing prevalence of allergic diseases. In this mini-review, we summarize and discuss relevant findings for the major birch and grass pollen allergens Bet v 1 and Phl p 5 modified with tetranitromethane (laboratory studies), peroxynitrite (physiological processes), and ozone and nitrogen dioxide (environmental conditions). We focus on tyrosine nitration and the formation of protein dimers and higher oligomers via dityrosine cross-linking and the immunological effects studied.

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.

The Biological Standardization Project BSP090 has been successfully concluded in 2021. As a result, two standard methods for quantification of the major allergens Bet v 1 and Phl p 5 will be implemented in the European Pharmacopoeia (Ph. Eur.). The General Chapter describing the protocol of the respective Bet v 1‐specific ELISA has already been adopted by the Ph. Eur. Commission and will become an official part of the Ph. Eur. in the beginning of 2023. As this will be the first allergen‐specific standard method in the EU, this paper intends to summarize the preceding process and outline the measures necessary to comply with the new regulatory requirement. [ABSTRACT FROM AUTHOR]

The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO–) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO– and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO–-induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment.

Background: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5‐specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. Methods: A Phl p 5‐specific ELISA system was assessed with respect to accuracy, precision, inter‐assay (within laboratory) and inter‐laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. Results: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter‐assay variation (max. GCV 24%) and especially inter‐laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. Conclusions: Based on the collaborative study results, the assessed Phl p 5‐specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare airborne levels of Phl p 1 and Phl p 5, with Poaceae pollen concentrations inside and outside of the pollen season, and to evaluate their association with symptoms in grass allergic patients and the influence of climate and pollution. The Hirst and the Burkard Cyclone samplers were used for pollen and allergen quantification, respectively. The sampling period ran from 23 March 2009 to 27 July 2010. Twenty-three patients with seasonal allergic asthma and rhinitis used an electronic symptom card. The aerosol was extracted and quantified for Phl p 1 and Phl p 5 content. Descriptive statistics, non-parametric paired contrast of Wilcoxon, Spearman's correlations, and a categorical principal component analysis (CatPCA) were carried out. Significant variations in pollen, aeroallergen levels, pollen allergen potency, and symptoms score were observed in this study. Phl p 5 pollen allergen potency was higher at the beginning of the 2010 grass pollen season. Presence of Phl p 1 outside the pollen season with positive O3 correlation was clinically relevant. 45.5% of the variance was explained by two dimensions in the CatPCA analysis, showing the symptom relationships dissociated in two dimensions. In the first one, the more important relationship was with grass pollen grains concentration and Phl p 5 and to a lesser extent with Phl p 1 and levels of NO2 and O3, and in the second dimension, symptoms were associated with humidity and SO2. Clinically relevant out-season Phl p 1 was found with a positive O3 correlation. The effect of climate and pollution may have contributed to the higher seasonal allergic rhinitis symptom score recorded in 2009. [ABSTRACT FROM AUTHOR]

The skin resembles an attractive target for vaccination due to its accessibility and abundance of resident immune cells. Cells like γδ T cells and mast cells (MCs) are part of the first line of defence against exogenous threats. Despite being important mediators for eliciting TH2 immune responses after epithelial stress, γδ T cell and MC functions still remain to be completely understood. Here, we aimed to characterize their roles in shaping adaptive immune responses after laser-mediated epicutaneous immunization (EPI). γδ T cell knock out, MC-depleted, and wildtype control mice were immunized with mannan-conjugated grass pollen allergen Phl p 5 (P5-MN) by laser-mediated EPI. After 2–3 immunizations, cytokine expression, T helper polarization, and antigen-specific IgG1/IgE levels were analysed. Furthermore, the local cytokine/chemokine milieu after laser microporation was determined. The majority of inflammatory chemokines and cytokines induced by laser treatment were not affected by the presence of γδ T cells or MCs. However, RANTES was elevated in γδ T cell knock out mice and GROα, TSLP, and IL-33 were significantly decreased after MC depletion. The absence of γδ T cells or depletion of MCs had no substantial effect on adaptive immune responses after laser-mediated EPI, except for slightly reduced IgG1 and effector T cell levels in MC-depleted mice. γδ T cells did not play a pivotal role in shaping the humoral and cellular adaptive immune response after laser-mediated EPI. MC depletion decreased the numbers of effector T cells, indicating a potential role of MCs in the activation and maturation of T cells after EPI. [ABSTRACT FROM AUTHOR]

Protein modifications such as oligomerization and tyrosine nitration alter the immune response to allergens and may contribute to the increasing prevalence of allergic diseases. In this mini-review, we summarize and discuss relevant findings for the major birch and grass pollen allergens Bet v 1 and Phl p 5 modified with tetranitromethane (laboratory studies), peroxynitrite (physiological processes), and ozone and nitrogen dioxide (environmental conditions). We focus on tyrosine nitration and the formation of protein dimers and higher oligomers via dityrosine cross-linking and the immunological effects studied., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Fröhlich-Nowoisky, Bothen, Backes, Weller and Pöschl.)

Background: Pollen‐related seasonal allergic rhinoconjunctivitis (SAR) is a very frequent pediatric disease in Westernized countries. Risk factors and disease phenotypes have been thoroughly examined in several cross‐sectional studies. By contrast, only a few studies have examined disease evolution in patient cohorts. We investigated predictive biomarkers of disease evolution in a large cohort of children with SAR. Methods: During 2015‐2017 (follow‐up), we re‐examined 401 patients from those enrolled in 2009‐2011 (baseline) by the "Panallergens in Pediatrics" study, a large multicenter survey of Italian children with SAR. Information on clinical history (standard questionnaire, AllergyCARD®; TPS, Italy) and skin prick tests for inhalant and foods extracts (ALK‐Abelló, Hørsholm, Denmark) was acquired as at baseline visit. Evolution in clinical and sensitization data of patients was analyzed over time, as well as their association with the main baseline characteristics and atopy risk factors. Results: The average age of participants was 10.4 ± 3.4 years at baseline and 16.2 ± 3.6 years at follow‐up. SAR persisted in 93.3% of patients at follow‐up and became more frequently associated with asthma (from 36.7% at baseline to 48.6% at follow‐up) and oral allergy syndrome (OAS, from 23.4% to 37.7%). Compared to baseline, the prevalence of skin sensitization to some pollens (Phleum pratense, Corylus avellana, Platanus acerifolia, Artemisia vulgaris) and vegetables (hazelnut, wheat, and apple) significantly decreased at follow‐up. Earlier onset of SAR and polysensitization at baseline were associated with incident asthma at follow‐up. The presence at baseline of serum IgE to the following allergen molecules was identified as biomarkers of clinical evolution: (a) Phl p 1, for persistence of SAR; (b) Phl p 5, for persistence of both rhinitis and asthma; (c) Pru p 3, for new onset of asthma; (d) Bet v 1, for persistence of OAS. Conclusions: Seasonal allergic rhinoconjunctivitis is clinically heterogeneous in its evolution from childhood to adolescence. The detection of serum IgE to specific molecules (Phl p 1, Phl p 5, Bet v 1, Pru p 3) may be useful as biomarkers to predict SAR persistence and future onset of comorbidities, such as asthma and/or OAS. [ABSTRACT FROM AUTHOR]

The Poaceae family, including grasses, comprises several cosmopolitan and allergenic species. The aim of this study was to determine the correlations between Poaceae pollen and Phl p 5 allergen concentrations in two cities with different geographical and climatic properties in Turkey. Pollen were collected from Burkard traps in Ankara and Zonguldak. Phl p 5 sampling was carried out between March and October in both 2015 and 2016 using a BGI900 Cascade High Volume Air Sampler (900 L/min.). The concentrations of Phl p 5 were measured by the enzyme-linked immunosorbent assay (ELISA) technique. The annual sum of Poaceae pollen (pollen index) during 2015-2016 was 5454 in Ankara and 4142 in Zonguldak. The total Phl p 5 concentration was 1309 pg/m3 in Zonguldak, whereas it was 8181 pg/m3 in Ankara over 2 years. About 90% of the allergen was found in the fraction with particulate matter (PM) > 10 μm in both cities. It was found that the main meteorological parameter which affected pollen and Phl p 5 was temperature in both stations. Rainfall was also found to be important for Zonguldak, due to its climatic and geographic properties. Lastly, we suggest that the primary wind direction, which is from the south of Zonguldak, could have a ‘drift effect’ for allergens because of the airborne pollen concentrations and the dates on which the allergen is released into the atmosphere. The wind direction may be an important factor in the distribution of allergen and pollen grains in stations, especially those with a hilly topography. [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.

Background The level of symptoms in pollen allergy sufferers and users of the Patient's Hayfever Diary (PHD), does not directly reflect the total amount of pollen in the air. It is necessary to explain the symptom load and thus the development of allergic symptoms and to determine which environmental factors, besides the pollen load, influence variables. It seems reasonable to suspect allergen content because the amount of allergen varies throughout seasons and regions and is not always correlated with the total pollen amount. Methods Data on the allergen content of ambient air (Bet v 1 and Phl p 5) from 2009 until 2011 was used to compare the respective pollen and symptom loads for study regions in Austria, Germany, France and Finland. Results Our findings suggest that allergen amount (Bet v 1/Phl p 5) has a strong but regionally dependent impact on the symptom load of pollen allergy sufferers. Peak symptom loads can be traced with peak allergen loads. The influence of other important aeroallergens should also be assessed during the pollen season. Conclusion Allergen concentrations have an impact on pollen allergy sufferers although not as clear as assumed previously. The pattern of pollen load and major allergen content distribution does not directly explain the symptom load pattern, although significant positive correlations were found. Thus, monitoring of symptoms via voluntary crowdsourcing should be considered for future pollen and symptom forecasts in order to support pollen allergy sufferers. [ABSTRACT FROM AUTHOR]

The drivers affecting the Pollen Allergen Potency (PAP, amount of allergen released per pollen) are sparsely known. Betula and Poaceae airborne pollen are the two main allergenic pollen in the World. Airborne pollen and their allergens Bet v 1 and Phl p 5 were simultaneously measured from 2010 to 2015 in Davos (Switzerland) and Munich (Germany) by using volumetric traps and ChemVol cascade impactors. Daily variations in PAP were analysed in PM >10 and PM 2.5-10 air fractions and generalized additive models were created to explain which factors determine PAP, including meteorological parameters and inorganic pollutants. 87.1 ± 13.9% of Bet v 1 and 88.8 ± 15.5% of Phl p 5 was detected in the fraction PM >10 where most pollen grains were collected. Significantly higher PAP for grasses (3.5 ± 1.9 pg Phl p 5/pollen grain) were observed in Munich than in Davos (2.4 ± 1.5 pg/pollen grain, p < 0.001), but not for Betula (2.5 ± 1.6 pg Bet v 1/pollen grain in Munich and 2.3 ± 1.7 in Davos, N.S.). PAP varied between days, years and location, and increased along the pollen season for Poaceae, but remaining constant for Betula. Free allergens (allergens observed in the fraction with limited pollen, PM 2.5- 10) were recorded mostly at the beginning or at the end of the pollen season, being linked to higher humidity and rainy days. Also, PAP was higher when the airborne pollen concentrations increased rapidly after one day of low/moderate levels. Our findings show that pollen exposure explains allergen exposure only to a limited extend, and that day in the season, geographic location and some weather conditions need to be considered also to explain symptoms of allergic individuals. • 9-fold more allergens were trapped in the fraction PM >10 than in PM 2.5-10 μm. • PAP was different between the two cities studied for grasses, but not for birch. • PAP increases for grasses during the pollen season (species dependency of allergen load). • For Betula, conditions in previous days explain PAP. • Free allergens were recorded especially for days with rain or high relative humidity. [ABSTRACT FROM AUTHOR]

The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO - ) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO - and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO - -induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Reinmuth-Selzle, Bellinghausen, Leifke, Backes, Bothen, Ziegler, Weller, Saloga, Schuppan, Lucas, Pöschl and Fröhlich-Nowoisky.)

Less vegetated mountainous areas may provide better conditions for allergy sufferers. However, atmospheric transport can result in medically relevant pollen loads in such regions. The majority of investigations has focused on the pollen load, expressed as daily averages of pollen per cubic meter of air (pollen grains/m³); however, the severity of allergic symptoms is also determined by the actual allergen content of this pollen, its pollen potency, which may differ between high and low altitudes. We analysed airborne birch and grass pollen concentrations along with allergen content (birch: Bet v 1, grass: Phl p 5) at two different altitudes (734 and 2650 m a.s.l.) in the Zugspitze region (2009–2010). Back-trajectories were calculated for the high altitude site and for specific days with abrupt increases in pollen potency. We observed several days with medically relevant pollen concentrations at the highest site. In addition, a few days with pollen were not associated with allergens and vice versa . The calculated seasonal mean allergen release per pollen grain was 1.8–3.3 pg Bet v 1 and 5.7 pg Phl p 5 in the valley and 1.1–3.7 pg Bet v 1 and 0.7–1.5 pg Phl p 5 at the high altitude site. Back-trajectories revealed that high pollen potency at the higher site was generally associated with south-westerly to south-easterly (birch), or northerly (grass) wind directions. By investigating days with sudden increases in pollen potency, however, it was difficult to draw definitive conclusions on long- or short-range transport. Our findings suggest that people allergic to pollen might suffer less at higher altitudes and further indicate that a risk assessment relying on the actual concentration of airborne pollen does not necessarily reflect the actual allergy exposure of individuals. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are sentinels of the immune system for antigen recognition and uptake, as well as presentation to naïve T cells for stimulation or priming. Internalization and endocytic degradation of allergens by DCs are important steps required for T cell priming. In the current study we investigated binding and internalization of purified recombinant non-glycosylated grass pollen allergen, Phl p 5, and natural non-specific lipid transfer protein from sunflower, SF-nsLTP to human monocyte derived dendritic cells (MoDCs). Colocalization of Phl p 5 with low affinity (CD23) or high affinity receptor (FcεRI) was investigated by immunofluorescence staining. Likewise, localization of the allergens in early (EE) and late endosomes (LE) was detected by co-staining for early endosome antigen (EEA1) and lysosomal-associated membrane protein 1 (LAMP1). In our experimental setting we could demonstrate that Phl p 5 as well as SF-nsLTP bound to MoDCs from both, grass pollen allergic and non-allergic individuals. Competitive allergen uptake experiments demonstrated non-preferential and simultaneous uptake of Phl p 5 and SF-nsLTP by MoDCs. No overlap of signals from Phl p 5 and CD23 or FcεRI was detectable, excluding IgE-mediated uptake for this allergen. Both allergens, Phl p 5 and SF-nsLTP, were localized in early and late endosomes. The present study applied a set of methods to assess the allergen uptake by MoDCs in an in vitro model. No qualitative and quantitative differences in the allergen uptake of both, Phl p 5 and SF-nsLTP were detected in single and competitive assays. [ABSTRACT FROM AUTHOR]

Background: Dendritic cells (DCs) play an important role in the induction and regulation of adaptive immune responses by polarizing T-helper (Th) cells. In allergic disease this response is dominated by Th2 cells. It is still unclear whether the activation of Th cells by DCs in atopic individuals is allergen specific. Methods: Monocyte-derived DCs (MoDCs) obtained from polysensitized patients were stimulated with purified Bet v 1, Phl p 5 and Act d 10, and the surface marker expression was analysed. Proliferation and cytokine profiles of autologous naïve CD4+ T cells co-cultured with allergen-pulsed MoDCs were assessed. Results: The addition of either Bet v 1 or Phl p 5 did not further increase the expression of surface markers from matured MoDCs in all study groups. In co-cultures, autologous naïve CD4+ T cells proliferated when DCs obtained from individuals allergic to birch and grass pollen were stimulated with Bet v 1 and Phl p 5, respectively. In the co-culture supernatants, significantly increased levels of IL-5 and IL-13 were detected. This effect correlated with the sensitization background and was absent when applying an unspecific allergen, Act d 10. The levels of IL-10 in supernatants of MoDCs and the levels of IL-10 and IFN-γ in supernatants of T cells remained unchanged upon stimulation with allergens. Conclusions: In this study we observed that allergen-specific stimulation of MoDCs induces T-cell proliferation and upregulation of Th2-type cytokines. Interestingly, this Th2 polarization was only observed in cells stimulated with the allergen to which the patients were sensitized. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The transplantation of allergens (e.g. Phl p 5 or Bet v 1) expressed on BM cells as membrane-anchored full-length proteins leads to permanent tolerance at the T-cell, B-cell, and effector-cell levels. Since the exposure of complete allergens bears the risk of inducing anaphylaxis, we investigated here whether expression of Phl p 5 in the cytoplasm (rather than on the cell surface) is sufficient for tolerance induction. Transplantation of BALB/c BM retrovirally transduced to express Phl p 5 in the cytoplasm led to stable and durable molecular chimerism in syngeneic recipients (∼20% chimerism at 6 months). Chimeras showed allergen-specific T-cell hyporesponsiveness. Further, Phl p 5-specific TH1-dependent humoral responses were tolerized in several chimeras. Surprisingly, Phl p 5-specific IgE and IgG1 levels were significantly reduced but still detectable in sera of chimeric mice, indicating incomplete B-cell tolerance. No Phl p 5-specific sIgM developed in cytoplasmic chimeras, which is in marked contrast to mice transplanted with BM expressing membrane-anchored Phl p 5. Thus, the expression site of the allergen substantially influences the degree and quality of tolerance achieved with molecular chimerism in IgE-mediated allergy. [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: Knowledge of allergen-specific T cell epitopes is a prerequisite not only for therapeutic approaches but also for elucidating immunological mechanisms of type I allergy. Ex vivo detection of allergen-specific T cells using class II tetramer technology has become an important tool for investigating immune responses in atopic and healthy individuals. Methods: Using 3H-thymidine incorporation assays, T cell epitopes specific for the major timothy grass pollen allergen Phl p 5.0101 were mapped in 11 allergic donors and two different mouse strains. Different protocols for expansion/restimulation of T cells from the blood of allergic donors and detection of allergen-specific T cells by Class II Ultimer staining were evaluated. Results: We identified several new Phl p 5.0101 class II T cell epitopes in allergic patients and confirmed previously published ones. Additionally, we discovered the major T cell epitopes in BALB/c and C57BL/6 mice. Using a novel Class II Ultimer, we detected epitope-specific T cells expanded from the blood of an allergic donor. Conclusions: Epitope mapping of Phl p 5.0101 revealed an immunodominant epitope in BALB/c and C57BL/6 mice and an immunodominant region in humans (amino acids 259-282), which was recognized by 8 out of 11 allergic donors. Detection of Phl p 5-specific T cells was demonstrated using a Class II Ultimer specific for epitope 196-210. Successful detection of ultimer-positive T cells was strongly dependent on a resting phase after in vitro expansion. 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]

Abstract: Gene gun immunization has been associated with the induction of a heterologous type of immune response characterized by a TH1-like immune reaction on the cellular level, i.e. generation of IFN-γ secreting CD8+ T-cells, yet a TH2 biased serology as indicated by high IgG1:IgG2a ratios and induction of IgE. Nevertheless, gene gun immunization using the model molecule β-galactosidase has been argued to prevent IgE induction and to promote TH1 cells with respect to allergy DNA immunization. In our current study, we evaluated the potential of gene gun immunization to prevent type I allergic reactions comparing β-galactosidase with two clinically relevant allergens, and further investigated the effect of gene gun immunization on relevant lung parameters. BALB/c mice were immunized with plasmids encoding the birch pollen allergen Bet v 1, the grass pollen allergen Phl p 5, or the model molecule β-galactosidase, either by gene gun or intradermal injection followed by sensitization and intranasal provocation with the respective allergen. IgG1 and IgG2a antibody titers were determined by ELISA. IgE levels were evaluated in a rat basophil release assay. The severity of eosinophilia was determined in bronchoalveolar lavages, and the overall infiltrate was analyzed by histology on lung paraffin sections. Gene gun immunization induced a TH2-biased immune reaction, which did not prevent from production of IgE after subsequent sensitization. This TH2 effect was influenced by the nature of the antigen, with a more pronounced TH2-bias for the allergens Bet v 1 and Phl p 5 compared to β-galactosidase. Gene gun immunization with all three antigens promoted eosinophil influx into the lung and did not alleviate lung pathology after intranasal provocation. In contrast to needle injection of plasmid DNA, which triggers a clearly TH1-biased and allergy-preventing immune response, gene gun application fails to induce anti-allergic reactions with all tested antigens and is therefore contraindicated for allergen-specific immunotherapy. [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]

Abstract: Background: The role of anti-idiotypic antibodies in allergic disease is still poorly understood. According to Jerne, anti-idiotypic antibodies to IgE should represent internal images of an allergen. Our aim was to ultimately prove whether this hypothesis holds true in allergy. Here, we describe the selection of anti-idiotypic antibodies against Phl p 5a-specific IgE directly from the B-cell repertoire of a grass pollen allergic individual. Methods: Taking Phleum pratense grass pollen allergen Phl p 5 as a model, we selected anti-idiotypic antibodies against allergen-specific IgE directly from the B-cell repertoire of an allergic individual. We screened a combinatorial phage display library of human monovalent antibody heavy and light chain fragments (Fabs) with anti-Phl p 5a-IgE to identify and characterize Fabs with anti-idiotypic specificity. Results: Five different Fab clones with anti-idiotypic specificity for anti-Phl p 5a-IgE were identified. Their hypervariable regions revealed partial sequence homology with solvent accessible antigenic sites of Phl p 5a, which have been identified by our previous mimotope approach. Phagemid DNA derived from the phage clones was used to produce two soluble recombinant anti-idiotypic Fab clones in E. coli. As a proof of molecular mimicry, both Fabs induced anti-Phl p 5a-specific antibodies in immunized BALB/c mice. Molecular modeling of the heavy and light chain hypervariable loops of the anti-idiotypic Fabs illustrated structural similarity with dominant IgE epitopes of Phl p 5a. Conclusion: In this straightforward phage technology approach, antibodies with anti-idiotypic specificities could be isolated from a human allergic''s repertoire. As predicted by the immune network hypothesis, their hypervariable domains mimic IgE epitopes like internal images and, more importantly, induce allergen-specific immune responses in the absence of the allergen. [Copyright &y& Elsevier]

The cloning and production of an increasing number of allergens through the use of DNA technology has provided the opportunity to use these proteins instead of natural allergen extracts for the diagnosis and therapy of IgE-mediated allergic disease. For diagnostic purposes, it is essential that the molecules exhibit IgE-reactivity comparable with that of the natural wild-type molecules, whereas T cell reactivity and immunogenic activity may be more important for allergen-specific immunotherapy. In relation to the latter, the development of hypoallergenic recombinant allergen variants is an approach which shows great promise. Clinical application of the proteins requires that they must be produced under conditions of Good Manufacturing Practice and meet the specifications set down in the appropriate Regulatory Guidelines, principally the ICH-Guidelines. Special consideration has to be given to the choice of expression system, the design of the expression vectors, and the purification strategy to obtain a pure product free from toxins and contamination. The availability of the pure recombinant molecules provides the opportunity to formulate preparations that are free from the non-allergenic ballast proteins present in natural allergen extracts and which contain relative concentrations of the allergens in clinically appropriate proportions. [Copyright &y& Elsevier]

Summary Background The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. Methods Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. Results Grass pollen allergoids revealed a 10–10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50–10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. Conclusions The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy. [ABSTRACT FROM AUTHOR]

ABSTRACT Background: Previous studies have reported cross-reactivity between latex and grass allergens. Inhibition studies have indicated cross-reactivity of IgE with latex and grass pollen proteins. A panel consisting of a few recombinant allergens, namely rPhI p 1, rPhl p 2, rPhl p 5 and profilin, was sufficient to diagnose grass pollen allergy in patients allergic to grass pollen. Methods: Serum samples from 528 consecutive outpatients with IgE antibodies towards at least one allergen (IgE level > 0.35 kAU/L) were selected for this retrospective study. Total and specific serum IgE to rPhl p 1, rPhl p 2, rPhl p 5, rBet v 1, rBet v 2, latex, birch, hazel, mugwort, wall pellitory, Dermatophagoides pteronissinus, Alternaria tenuis, cat and apple were measured by the immunoenzymatic capsulated hydrophilic carrier polymer (CAP) FEIA system (Pharmacia & Upjohn). Results: Of 123 polysensitized patients with antilatex Ig1E, 12 (9.76%) had symptoms after latex exposure. Ten of 12 subjects monosensitized to latex had symptoms after latex exposure. Symptomatic patients had higher IgE levels to latex than symptomless patients (P = 0.046). A higher prevalence of antilatex IgE was seen in sera containing specific IgE to rPhl p 1, rPhl p 5 and rBet v 2. A good correlation (Spearman’s r = 0.52; P = 0.001) between high levels of antilatex IgE and total serum IgE was found. Conclusions: The findings of the present study may support the concept that a high proportion of sera containing IgE to rBet v 2, rPhl p 1 and rPhl p 5 simultaneously contain antilatex IgE. Therefore, patients with specific IgE to these recombinant allergens with no history of current latex exposure may need additional evaluation. [ABSTRACT FROM AUTHOR]

Background: We measured specific IgE levels against the recombinant allergens (RAs) rPhl p 1, rPhl p 2, rPhl p 5 in patients allergic to grass pollen, and examined the existence of different patterns of IgE production to RAs. The seasonal variations of IgE levels to rPhl p 1, rPhl p 2, and rPhl p 5 were considered, too. Methods: Blood was taken from 276 consecutive patients with allergy to grass pollen diagnosed by patient history and skin prick testing. Total and specific serum IgE was measured by the immunoenzymatic CAP FEIA System. Eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were assessed by radioimmunoassay according to the instructions of the manufacturers. Results: We observed eight different patterns of IgE production to rPhl p 1, rPhl p 2, and rPhl p 5 in patients with specific IgE to timothy grass. A significant difference between the values of IgE levels to timothy and the sum of each level of specific IgE to individual RAs was found (P=0.039, Wilcoxon matched pairs test) in the whole population (n=276 subjects). In four subgroups of patients, the sum of each level of specific IgE to individual RAs was equal to the levels of specific IgE to timothy grass extract. In one subgroup, the sum of IgE to RAs was lower than the levels of IgE to the natural counterpart (P=0.013). A lack of subjects in two subgroups did not permit comparison at all. Finally, three subjects with specific IgE to timothy did not show specific IgE to RAs. Out of 276 patients, blood was taken from two different groups of subjects at different time points: November–January and May–July, respectively. The median values were as follows: total IgE=139 kU/l, IgE to timothy=10.2 kUA/l; IgE to rPhl p 1=3.6 kUA/l, to rPhl p 2=<0.35 kUA/l, and to rPhl p 5=1.1 kUA/l; ECP=8.2 g/l; MPO=303 g/l (before exposure to grass pollen); total IgE=159 kU/l, IgE to timothy=57.2 kUA/l; IgE to rPhl p 1=22.1 kUA/l, to rPhl p 2=5.9 kUA/l, and to rPhl p 5=3.9 kUA/l; ECP=16.2/l,... [ABSTRACT FROM AUTHOR]

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method., (© Council of Europe, 2022.)

BackgroundGrass pollen allergens are the most important cause of hay fever and allergic asthma during summer in cool temperate climates. Pollen counts provide a guide to hay fever sufferers. However, grass pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet, grass pollen allergens are known to be associated with atmospheric respirable particles. ObjectiveWe aimed (1) to determine the concentration of group 5 major allergens in (a) pollen grains of clinically important grass species and (b) atmospheric particles (respirable and nonrespirable) and (2) to compare the atmospheric allergen load with clinical data to assess different risk factors for asthma and hay fever. MethodsWe have performed a continuous 24 h sampling of atmospheric particles greater and lower than 7.2 μm in diameter during the grass pollen season of 1996 and 1997 (17 October 1996–16 January 1997) by means of a high volume cascade impactor at a height of about 15 m above ground in Melbourne. Using Western analysis, we assessed the reactivity of major timothy grass allergen Phl p 5 specific monoclonal antibody (MoAb) against selected pollen extracts. A MoAb-based ELISA was then employed to quantify Phl p 5 and cross-reactive allergens in pollen extracts and atmospheric particles larger and smaller than 7.2 μm. ResultsPhl p 5-specific MoAb detected group 5 allergens in tested grass pollen extracts, indicating that the ELISA employed here determines total group 5 allergen concentrations. On average, 0.05 ng of group 5 allergens were detectable per grass pollen grain. Atmospheric group 5 allergen concentrations in particles > 7.2 μm were significantly correlated with grass pollen counts (rs = 0.842, P < 0.001). On dry days, 37% of the total group 5 allergen load, whereas upon rainfall, 57% of the total load was detected in respirable particles. After rainfall, the number of starch granule equivalents increased up to... [ABSTRACT FROM AUTHOR]

Abstract BackgroundGroup 5 allergens represent major grass pollen allergens because of their high sensitization indices. The identification of T-cell epitopes of these allergens is a prerequisite for the design of immunotherapeutic strategies based on peptide vaccination or modified allergens with conserved T-cell epitopes. ObjectiveThis study was undertaken to determine T-cell epitopes on Phl p 5 major pollen allergen of timothy grass (Phleum pratense). MethodsT-cell lines (TCLs) and T-cell clones (TCCs), specific to Phl p 5, were established from the peripheral blood of 18 patients allergic to grass pollen. All TCCs were mapped for epitope specificities using 178 overlapping dodecapeptides representing the primary structures of two isoforms of Phl p 5 (Phl p 5a and Phl p 5b). Phenotype and cytokine production profiles of TCCs were tested. Selected TCCs were analysed for HLA class II restriction. ResultsA total of 82 TCCs were isolated. All TCCs displayed the helper cell (TH) phenotype. Their reactivity with two recombinant expressed isoforms of Phl p 5a and Phl p 5b was heterogeneous. The epitope specificity of the TCCs was then revealed. Nineteen T-cell epitopes could be identified on Phl p 5. Eighty-one percent of mapped TCCs recognized three T-cell reactive regions on the Phl p 5 allergen. Some TCCs were reactive with isoepitopes presenting on Phl p 5a as well as Phl p 5b. Allergen-specific stimulation induced a TH0-like type of cytokine production in 25 of 50 TCCs. Almost all TCCs secreted high concentrations of interleukin-13. ConclusionPhl p 5, a major grass pollen allergen, contains several T-cell epitopes. Some epitope regions were recognized by several patients. Epitope recognition pattern could not be correlated with special HLA class II haplotypes. T-cell stimulating isoepitopes were found at corresponding regions of Phl p 5a and Phl p 5b isoforms. [ABSTRACT FROM AUTHOR]

Background: Successful allergen–specific immunotherapy is achieved with progressively increasing doses of allergen or allergoid. In order to gain further insight into the mechanism of action of allergoids several in vitro investigations were conducted. Methods: Peripheral blood mononuclear cells (PBMC) from grass pollen allergic and nonallergic subjects were stimulated with either grass pollen extract or allergoid and the proliferation and cytokine production (IL–5, IFN–γ) were measured. Similar investigations were performed with Phl p 5–specific T cell lines (TCL) and clones (TCC). Dendritic cells and PBMC were compared in terms of their relative efficacies as antigen–presenting cells. Results: Both allergen and allergoid induced proliferation and Th2 and Th1 cytokine synthesis by PBMC of allergic subjects, whereas PBMC of nonallergic subjects did not produce IL–5. The maximum level of IL–5 was obtained with a lower concentration than was necessary for maximal IFN–γ production. Higher stimulation doses of allergen and allergoid shifted the cytokine profiles towards a Th1 phenotype. TCL and TCC clearly showed reactivity with both allergen and allergoid when using autologous PBMC for antigen presentation, but compared with the native allergen the reactivity of the allergoid was reduced with most of the TCC. Using dendritic cells for antigen presentation a pronounced increase of stimulation of the TCC especially for the allergoids becomes obvious. Conclusion: In common with grass pollen allergen the corresponding allergoids possess a strong allergen–specific T cell–stimulating capacity. However, the degree of T cell stimulation by the allergoid seems to be dependent on the type of the antigen–presenting cell. Both, allergen and allergoid, can modulate T cell responses in a dose–dependent manner. [ABSTRACT FROM AUTHOR]

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]

Background and Objectives: The association between pollen counts and allergen levels in the air is controversial. Objectives: The aims of the study were to quantify total and major allergen levels of Phleum pratense and Olea europaea and to analyze their correlation with grass and olive pollen counts and the number of asthma attacks attended at Complejo Hospitalario Universitario, Cáceres, Spain., Material and Methods: A volumetric air sampler and a Burkard spore trap were used for pollen and aeroallergen collection during April- June 2011. Filters were extracted, and major allergens were quantified using enzyme-linked immunosorbent assay., Results: May was the main grass pollination period, with a maximum peak of 1362 grains/m3 (May 13). The main pollination period for olive was April 30-May 20, with a maximum peak of 851 grains/m3 (May 11). A moderate correlation was observed between asthma exacerbations and grass pollen counts or Phleum total allergen levels; this became stronger when a 3-day offset was introduced. A significant association was observed between asthma exacerbations and total olive allergen or olive pollen grain levels when a 1-day offset was introduced. The maximum correlation (moderate-high) was observed 4 days and 6 days away from the maximum olive pollen peak and the maximum Ole e 1 peak level, respectively., Conclusions: This study reveals a significant correlation between grass and olive pollination and an increase in the number of visits to the emergency room for asthma attacks. The aerobiological pattern of allergen levels in the air is similar to that of pollen counts during the grass and olive pollination periods.