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Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy. [ABSTRACT FROM AUTHOR]

The prevalence of ragweed (Ambrosia artemisiifolia) pollinosis has been increasing worldwide. This study focused on the behavior of autumn airborne pollen and the major ragweed allergen -Amb a 1 particle in urban Saitama, Japan, in 2016. Burkard sampler results showed that the airborne ragweed pollen scattering season was from September 1st to October 9th. Over 83% of sampling events had pollen counts of over 13 grains/m3, indicating the high potential health risks of ragweed pollen in the atmosphere. The results of a surface plasmon resonance immunoassay (SPR) indicated that the average Amb a 1 count was about 16.5 pg /pollen. The airborne Amb a 1 concentration was up to 4.7 ng/m3, of which about 45% was accumulated in ultrafine particles, such as particulate matter with a diameter ≤1.1 µm (PM1.1). Although ragweed pollen was hardly observed during the October 14th–17th sampling campaign, the concentration of ambient Amb a 1 particles in PM1.1 was also determined to be 4.59 ng/m3, which could be explained by the longer scattering of fine particles in the atmosphere. Pearson correlation coefficient analysis results showed that temperature (daily, r = 0.41; event, r = 0.87) could affect the behavior of the airborne pollen counts, and ambient water-soluble ions (such as Ca2+ and NO3−) could affect Amb a 1 in PM1.1. Additionally, air mass trajectories and wind rose results indicated that air masses with long-range transportation could also influence the temporary behavior of Amb a 1 and pollen counts via the wind. Mugwort and Humulus japonicus pollens were also observed to extend pollen scattering periods. Airborne pollen and allergenic particles could be considered air pollutants, as they pose health risks and are susceptible to environmental influences. [ABSTRACT FROM AUTHOR]

Allergic rhinitis and bronchial asthma are widespread respiratory allergic diseases. In some territories of the Russian Federation, the dominant cause of pollinosis is ragweed. The aim of the study was to evaluate the clinical and immunological efficacy of ASIT with the allergen Ambrosia artemisiifolia in patients sensitized to Ambrosia trifida in the Samara region. Patients with proven sensitization to Ambrosia trifida was held immunotherapy with Ambrosia artemisiifolia allergoid preseason. After treatment, patients had a decrease in the severity of symptoms of allergic rhinitis according to VAS (p = 0.00001), a decrease in the need for medications (p = 0.0003), as well as the need for corticosteroids against the background of therapy from 34.6% to 0% (p = 0.00001). In 8% of cases, the result of treatment was good, in 69% satisfactory, in 23% unsatisfactory. In the control group, there were no changes in the severity of symptoms (p = 0.858). Also, in the control group, the need for medications remained unchanged and 14.3% of patients continued to use corticosteroids.After ASIT, there was a decrease in the level of IL-4 (p = 0.002), and a decrease in the ratio of IL-4/ IL-10 (p = 0.0063); at the same time, changes in the level of other cytokines (IL-10; IFNγ) were statistically insignificant (p > 0.05). Before treatment, the levels of IL-4/ IL-10 in both groups were comparable, and after treatment, the differences became statistically significant (p = 0.031). We did not get a statistically significant change in the level of IgG4 Amb a 1 or IgG4 Amb trifida. There was no correlation between the level of individual cytokines and the results of treatment. As a result of the conducted ASIT, positive clinical and immunological results were obtained. In most patients, the disease has acquired a controlled course. At the same time, the lack of excellent and low number of good results of ASIT is probably due to the intraspecific allergenic properties of ragweed.

The prevalence of ragweed (Ambrosia artemisiifolia) pollinosis has been increasing worldwide. This study focused on the behavior of autumn airborne pollen and the major ragweed allergen -Amb a 1 particle in urban Saitama, Japan, in 2016. Burkard sampler results showed that the airborne ragweed pollen scattering season was from September 1st to October 9th. Over 83% of sampling events had pollen counts of over 13 grains/m3, indicating the high potential health risks of ragweed pollen in the atmosphere. The results of a surface plasmon resonance immunoassay (SPR) indicated that the average Amb a 1 count was about 16.5 pg /pollen. The airborne Amb a 1 concentration was up to 4.7 ng/m3, of which about 45% was accumulated in ultrafine particles, such as particulate matter with a diameter ≤1.1 µm (PM1.1). Although ragweed pollen was hardly observed during the October 14th–17th sampling campaign, the concentration of ambient Amb a 1 particles in PM1.1 was also determined to be 4.59 ng/m3, which could be explained by the longer scattering of fine particles in the atmosphere. Pearson correlation coefficient analysis results showed that temperature (daily, r = 0.41; event, r = 0.87) could affect the behavior of the airborne pollen counts, and ambient water-soluble ions (such as Ca2+ and NO3−) could affect Amb a 1 in PM1.1. Additionally, air mass trajectories and wind rose results indicated that air masses with long-range transportation could also influence the temporary behavior of Amb a 1 and pollen counts via the wind. Mugwort and Humulus japonicus pollens were also observed to extend pollen scattering periods. Airborne pollen and allergenic particles could be considered air pollutants, as they pose health risks and are susceptible to environmental influences.

Abstract Background Ambrosia artemisiifolia L. is one of the most important sources of allergenic pollen in many regions of the world. Its health impact increased over the last decades and is expected to further increase in consequence of climate change. However little information is available on the specific role played by temperature on allergy rising. The aim of this work was to evaluate the effect of temperature on A. artemisiifolia growth, flowering and pollen allergenicity, the major plant functional traits influencing the prevalence and severity of pollinosis. Results Plants were grown in controlled conditions at three thermal regimes: “Low” (LT: 18–14 °C light-dark), “Intermediate” (IT: 24–20 °C light-dark) and “High” (HT: 30–26 °C light-dark). During plant development, plant vegetative and reproductive morpho-functional traits were measured and, at the end of plant life-cycle, mature pollen was collected and analyzed for its allergenic properties by slot blot, 1D- and 2D-western blot (by using a pool of sera from ragweed-allergic patients) and liquid chromatography-tandem mass spectrometry. A. artemisiifolia showed a great development plasticity leading to a broad temperature tolerance. Shoot architecture, growth rate, number of male inflorescence and pollen allergenicity were temperature-responsive traits. Pollen allergenicity increased in parallel with temperature and differences were related to allergen synthesis and Amb a 1-IgE-binding. Flavonoids whose concentration in pollen decreased with the increase of temperature, were recognized as the cause of the negligible Amb a 1-IgE binding in LT pollen. Conclusions Results show that temperature governs plant development and pollen allergenicity influencing the temporal and spatial magnitude of subject exposure to allergens.

Ragweed is a prominent cause of seasonal allergies. Thus far, information on IgE-binding sites of major allergen in ragweed pollen, Amb a 1, is very limited. A powerful experimental method to gain insights on the allergen epitopes is the selection of peptides from biological libraries that bind to anti-allergen antibodies. In this work, we aimed to map IgE epitopes of Amb a 1 using epitope-mimicking short peptides – mimotopes that were affinity-selected from phage-displayed random peptide libraries. The peptides weakly aligned with the Amb a 1 primary sequence, thus suggesting that the epitopes are conformational. When the peptides were mapped onto the surface of Amb a 1 homology model, the EpiSearch analysis predicted the location of four potential epitopic sites on surface patches centred at residues K104, S110, H214, and W312. The peptides matching to the predicted epitopes bound selectively to the IgE from pool of ragweed-allergic patients’ sera and therefore represent mimetics of Amb a 1 IgE epitopes. The knowledge of IgE epitopes is a prerequisite for the rational design of molecular-based approaches to diagnosis and immunotherapy of allergic diseases.

Ambrosia artemisiifolia (Amb a) contains many allergens. Allergic conjunctivitis caused by Ambrosia artemisiifolia and its related allergen-specific immunotherapy (AIT) are seldom studied at present. poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) is a very good nano-carrier, which has been applied in the medical field. In this context, we studied the immunotherapy effect and potential mechanism of recombinant Amb a 1 (rAmb a 1)-loaded PLGA-PEG nanoparticles. A mouse allergic conjunctivitis model was established with Ambrosia artemisiifolia crude extract, and the nanoparticles were used for AIT through direct observation of conjunctival tissue, degranulation of mast cells in conjunctival tissue, serum-specific antibodies, cytokines and other assessment models. The treatment of nanoparticles enhanced the secretion of T-helper 1 (Th1) cytokine Interferon-gama (IFN-γ) and the production of immunoglobulin G (IgG)2a (IgG2a), inhibited the secretion of T-helper 2 (Th2) cytokine Interleukin (IL)-13 and IL-4 and the level of IgE. Especially, degranulation of mast cells and expression of mast cell protease-1 (MCP-1) in conjunctival tissue was reduced significantly. In this study, we proved that the nanoparticles prepared by rAmb a 1 and PLGA-PEG have an immunotherapy effect on allergic conjunctivitis in mice.

Ambrosia artemisiifolia L., known as common ragweed, is an invasive and allergenic plant that has begun to expand its presence in Turkey. Although ragweed pollen has been monitored for a long time—especially in the atmosphere of cities in Northern Anatolia—the invasion of the plant is more limited compared to that of other European countries. Here, we present the highest ragweed pollen concentration recorded in Turkey, as well as changes in the Amb a 1 allergen level in the air during 2015 and 2016 in Zonguldak, measured using a BGI 900 cascade impactor. The Seasonal Pollen Integral (SPIn) was found to be 709 pollen day/m3 and 1542 pollen day/m3 in 2015–2016, respectively. The sum of Amb a 1 in both stages was 8.42 × 10−3 U/m3 and 13.1 × 10−3 U/m3 over the same period. The number of days in which the Ambrosia pollen concentration was higher than 50 pollen/m3 doubled in 2016 compared to 2015, despite the negative effect of rainfall on pollen and allergen concentration in 2016. The pollen and allergen sources were evaluated by using simulated air mass movement with a Hybrid Single-Particle Lagrangian Integrated Trajectory model. Our findings suggest that individuals in Zonguldak are exposed as much to pollen—and possibly allergens—transported from the Ukraine, Russia and to a limited extent from Moldova. Furthermore, the allergen content of pollen in Zonguldak was much higher than other cities where Amb a 1 is monitored. We also demonstrated that the date of the peak pollen day may be an indicator of the source of long distance transportation. [ABSTRACT FROM AUTHOR]

The pollen of Ambrosia artemisiifolia (ragweed) is an important aeroallergen. The plant originated from North America and, has spread worldwide in recent decades. Air masses can transport pollen grains far from their source region and episodes of long distance transported (LDT) of ragweed pollen have been reported. The allergenic properties of LDT pollen grains can be altered by humidity, temperature and UV radiation. The aims of the study are to detect the major allergen of ragweed (Amb a 1) in the atmosphere of uninfested areas and to compare daily Ambrosia sp. pollen grains with Amb a 1 amount per m3 of air. Samples for allergen detection were collected by ChemVol® sampler. ChemVol® collects particles at 800 l/min and contains 2 impaction stages (PM > 10 μm and 10 > PM > 2.5 μm). Samples collected on to filters were obtained during a 63 day period in the year 2014. Amb a 1 was recorded on 54 days and pollen grains of ragweed on 43 days. Diurnal average Amb a 1 concentrations ranged between 0.29 and 263.3 pg per cubic meter of air and correlated positively with daily average ragweed pollen concentrations. The average seasonal Ambrosia pollen allergen potency was 2.57 pg Amb a 1/pollen. The air mass movement was simulated using the HYbrid Single-particle Lagrangian Integrated Trajectory (HYSPLIT) model and back-trajectory paths were computed for four episodes. The findings suggest that ragweed pollen allergens still survive after at least 48 h during transport over long distances from allochthonous regions and are therefore a potential trigger of allergic reactions even in areas where ragweed is not widely dispersed. Image 1 • Ambrosia pollen allergens transported from allochthonous regions are still reactive. • Amb a1 in the air from the allochthonous areas may have a risk for pollen allergy. • The average seasonal Ambrosia pollen allergen potency was 2.57 pg Amb a 1/pollen. [ABSTRACT FROM AUTHOR]

Ragweed is a prominent cause of seasonal allergies. Thus far, information on IgE-binding sites of major allergen in ragweed pollen, Amb a 1, is very limited. A powerful experimental method to gain insights on the allergen epitopes is the selection of peptides from biological libraries that bind to anti-allergen antibodies. In this work, we aimed to map IgE epitopes of Amb a 1 using epitope-mimicking short peptides - mimotopes that were affinity-selected from phage-displayed random peptide libraries. The peptides weakly aligned with the Amb a 1 primary sequence, thus suggesting that the epitopes are conformational. When the peptides were mapped onto the surface of Amb a 1 homology model, the EpiSearch analysis predicted the location of four potential epitopic sites on surface patches centred at residues K104, S110, H214, and W312. The peptides matching to the predicted epitopes bound selectively to the IgE from pool of ragweed-allergic patients' sera and therefore represent mimetics of Amb a 1 IgE epitopes. The knowledge of IgE epitopes is a prerequisite for the rational design of molecular-based approaches to diagnosis and immunotherapy of allergic diseases. [ABSTRACT FROM AUTHOR]

Introduction: The role of long-distance transported (LDT) Ambrosia pollen in inducing new sensitization and affecting sensitization rates in Artemisia-sensitized patients is unclear. Aim: The aim of this study was to estimate the degree of cross-sensitization to Ambrosia/Artemisia allergens in citizens of Poznan (Western Poland). This area is covered by extensive Artemisia populations but does not currently have local Ambrosia populations. Material and methods: Sera of 119 patients were tested by fluoroenzyme immunoassay (CAP-FEIA system) against pollen allergen extracts of Artemisia vulgaris and Ambrosia artemisiifolia, an allergenic component of A. vulgaris (nArt v 1), and an allergenic component of A. artemisiifolia (nAmb a 1). Skin prick tests (SPTs, n = 86) were performed with pollen allergen extracts of A. vulgaris and A. artemisiifolia. Artemisia and Ambrosia pollen in ambient air was collected (1996-2013) by a Hirst type volumetric trap sited at roof level (33 m). Results: The SPT showed that the prevalence of sensitization to Ambrosia and Artemisia pollen exceeded 3.5%, and 10.5%, respectively. The measurements of IgE in blood serum (CAP-FEIA) revealed that among Ambrosia-sensitized patients 90.1% (20/22 patients) were concomitantly sensitized to Artemisia. 59.1% (13/22) of these patients reacted to nArt v 1, suggesting primary sensitization to Artemisia pollen. Only 2 (9.1%) patients were mono-sensitized to Ambrosia pollen extract, but surprisingly not to nAmb a 1. Conclusions: The LDT Ambrosia pollen had a negligible effect on the rate of sensitization to Ambrosia allergens in Poznan and did not increase the prevalence of sensitization to Artemisia pollen in this region. However, the majority of patients showing hypersensitization to Artemisia pollen might also present symptoms during elevated episodes of LDT of Ambrosia pollen. [ABSTRACT FROM AUTHOR]

Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates. [ABSTRACT FROM AUTHOR]

Allergenic ragweed ( Ambrosia spp.) pollen grains, after being released from anthers, can be dispersed by air masses far from their source. However, the action of air temperature, humidity and solar radiation on pollen grains in the atmosphere could impact on the ability of long distance transported (LDT) pollen to maintain allergenic potency. Here, we report that the major allergen of Ambrosia artemisiifolia pollen (Amb a 1) collected in ambient air during episodes of LDT still have immunoreactive properties. The amount of Amb a 1 found in LDT ragweed pollen grains was not constant and varied between episodes. In addition to allergens in pollen sized particles, we detected reactive Amb a 1 in subpollen sized respirable particles. These findings suggest that ragweed pollen grains have the potential to cause allergic reactions, not only in the heavily infested areas but, due to LDT episodes, also in the regions unaffected by ragweed populations. [ABSTRACT FROM AUTHOR]

Background: Differential diagnosis between ragweed and mugwort pollen allergy represents a large clinical problem in areas where both plants are present. The aim of this study was to investigate ragweed- and mugwort-sensitized patients to identify specific IgE reactivity profiles. Results were correlated to clinical findings such as medical history and health-related quality of life (HRQL). Methods: Seventy-four patients with allergic rhinoconjunctivitis between July and October were examined and underwent in vivo tests (skin-prick test [SPT] and nasal provocation). Sera were evaluated for IgE reactivity to mugwort and ragweed pollen extracts, major (Art v 1; Amb a 1) and minor (profilin and calcium-binding protein) allergens. HRQL was evaluated using a standardized questionnaire. Results: Seventy-one patients revealed positive SPT reactivity against mugwort and 60 patients against ragweed extracts. Of these patients, 74 revealed IgE antibodies against mugwort extracts, whereas anti-Art v 1 antibodies were detectable in 50 individuals. Fifty-five patients showed IgE antibodies against natural ragweed extracts; anti-Amb v 1 antibodies were detected in six cases only. Using standardized clinical history and HRQL questionnaires we were not able to detect any differences within different reactivity patterns. Conclusion: Within the investigated population of 74 weed-allergic patients the prevalence of true mugwort and ragweed sensitization can be calculated as 68 and 8%. High prevalence of ragweed sensitization when testing with full extracts can be explained by cross-reactivity between other weeds, e.g., mugwort rather than cosensitization. Differences in medical history and HRQL between different reactivity patterns were not detectable. [ABSTRACT FROM AUTHOR]

Background: Sensitization to weed pollen allergens at the molecular level is not fully understood. We studied IgE reactivity to the major mugwort allergen Art v 1 in relation to allergens from other weed pollen and cross-reactive components in mugwort-sensitized subjects. Methods: Art v 1 and Amb a 1 were affinity purified and coupled to experimental ImmunoCAP® tests. Samples from North Europe (n = 50), South Europe (n = 19) and North America (n = 41) were analyzed for IgE against mugwort pollen, weed allergen components, pan-allergens and cross-reactive carbohydrate determinants (CCDs). Results: The prevalence of IgE reactivity (>0.35 kUA/l) to Art v 1 was significantly higher in samples from North Europe than in those from North America. IgE to Amb a 1 was more common in North America than in North and South Europe, while IgE to Par j 2 was common in South Europe, less common in North America, and absent in North Europe. IgE to Art v 3 in mugwort-allergic patients was more common in North Europe than in South Europe and North America, while IgE to Sal k 1 was similar between the areas. Subjects with an Art v 1/mugwort-specific IgE ratio <0.5 had more often IgE to Amb a 1, profilin, polcalcin and CCDs than subjects with a ratio >0.5. Conclusions: Mugwort-sensitized subjects have different IgE reactivity profiles to weed allergens, reflecting their exposure to various pollens. Subjects with a low ratio between the IgE levels to Art v 1 and mugwort have a diverse IgE reactivity profile, indicating a role for cross-reactive allergens in their mugwort sensitization. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Ragweed is one of the most important pollen allergens in North America and parts of Europe. Although the major allergen Amb a 1 was isolated and cloned in 1991, recombinant Amb a 1 was not explored further to improve diagnosis and specific immunotherapy of ragweed-pollen allergy. In the present study the immunological properties of natural Amb a 1 and its proteolytical cleavage products was investigated in detail and compared with recombinant produced Amb a 1 variants. Characterization of natural Amb a 1 and the identification of its proteolytic fragments, designated Amb a 1α and Amb a 1β, was performed by N-terminal sequencing and mass spectroscopy. Amb a 1 and fragments were further produced in Escherichia coli, purified, and immunologically characterized. Amb a 1-specific T-cell cultures were used to compare the T-cell response to the different Amb a 1 variants. Divergent immunological properties of Amb a 1α (aa 181–396) and Amb a 1β (aa 26–180) were revealed. Amb a 1β contained important IgE epitopes, whereas Amb a 1α showed low IgE binding. When compared to natural Amb a 1, all recombinant variants possessed >100-fold reduced IgE-mediated mediator release activity. At the T-cell level recombinant and natural Amb a 1 stimulated comparable T-cell responses and the T-cell reactivity was largely directed to the C-terminal part. The results demonstrated that recombinant Amb a 1α behaves as hypoallergen with reduced IgE binding but preservation of the major T-cell reactivity. In addition, recombinant Amb a 1α can be easily purified to homogeneity in large quantity and therefore represents an ideal candidate for specific immunotherapy. [Copyright &y& Elsevier]

Ambrosia artemisiifolia (Amb a) contains many allergens. Allergic conjunctivitis caused by Ambrosia artemisiifolia and its related allergen-specific immunotherapy (AIT) are seldom studied at present. poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) is a very good nano-carrier, which has been applied in the medical field. In this context, we studied the immunotherapy effect and potential mechanism of recombinant Amb a 1 (rAmb a 1)-loaded PLGA-PEG nanoparticles. A mouse allergic conjunctivitis model was established with Ambrosia artemisiifolia crude extract, and the nanoparticles were used for AIT through direct observation of conjunctival tissue, degranulation of mast cells in conjunctival tissue, serum-specific antibodies, cytokines and other assessment models. The treatment of nanoparticles enhanced the secretion of T-helper 1 (Th1) cytokine Interferon-gama (IFN-γ) and the production of immunoglobulin G (IgG)2a (IgG2a), inhibited the secretion of T-helper 2 (Th2) cytokine Interleukin (IL)-13 and IL-4 and the level of IgE. Especially, degranulation of mast cells and expression of mast cell protease-1 (MCP-1) in conjunctival tissue was reduced significantly. In this study, we proved that the nanoparticles prepared by rAmb a 1 and PLGA-PEG have an immunotherapy effect on allergic conjunctivitis in mice. [ABSTRACT FROM AUTHOR]

Background: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract. Methods: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA. Results: 91% of the patients’ sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients’ sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera. Conclusions: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Immunostimulatory DNA sequences (ISS) are potent immunomodulators that can drive TH1 responses to antigens or allergens. This effect can be dramatically enhanced by direct linkage of ISS to the protein. Objective: Evaluate the effects of the number of ISS bound to the major ragweed allergen Amb a 1 on immunogenicity and allergenicity. Methods: Immunogenicity in mice and allergenicity using PBMC or sera from subjects with ragweed allergy were assayed. Results: Both antibody induction in vivo and antibody recognition in vitro were highly sensitive to the number of ISSs linked. IgE recognition of Amb a 1 in competitive ELISA or histamine release assays was inhibited by ISS linkage and showed an inverse relationship to the number of ISSs bound. Type and magnitude of antibody induction in mice was also highly dependent on the number of ISS bound. At the highest ISS to protein ratios, antibody induction was very low. Moderate ISS to protein ratios induced high antibody responses in which IgG2a generally predominated. Low ISS to protein ratios produced the highest overall antibody responses in which IgG1 predominated. In contrast, varied ISS to protein ratios did not affect T-cell responses. In both in vivo mouse studies and in vitro human PBMC studies, all ISS to protein ratios evaluated induced similar responses represented by high levels of IFN-γ and low levels of TH2 cytokines. Conclusion: Controlling the number of ISS bound to a protein allows manipulation of antibody recognition and induction while retaining the potent TH1 properties of an ISS-linked protein. Clinical implications: Immunostimulatory DNA sequence–linked Amb a 1 conjugate represents a safe, novel therapeutic approach for treating ragweed allergy. [Copyright &y& Elsevier]

• Peptide immunotherapy for allergenic diseases is a promising new approach. • The production of peptides for immunotherapy is time- and cost-effective. • A monoclonal antibody E58 inhibits human IgEs from pollen allergic patients. • We designed novel high-affinity binding peptides to E58 for peptide immunotherapy. About half of the US population is sensitized to one or more allergens, as found by a National Health and Nutrition Examination Survey (NHANES). The most common treatment for seasonal allergic responses is the daily use of oral antihistamines, which can control some of the symptoms, but are not effective for nasal congestion, and can be debilitating in many patients. Peptide immunotherapy is a promising new approach to treat allergic airway diseases. The small size of the immunogens cannot lead to an unwanted allergic reaction in sensitized patients, and the production of peptides with sufficient amounts for immunotherapy is time- and cost-effective. However, it is not known what peptides are the most effective for an immunotherapy of allergens. We previously produced a unique monoclonal antibody (mAb) E58, which can inhibit the binding of multiple groups of mAbs and human IgEs from patients affected by the major group 1 allergens of ragweed (Amb a 1) and conifer pollens (Jun a 1, Cup s 1, and Cry j 1). Here, we demonstrated that a combined approach, starting from two linear E58 epitopes of the tree pollen allergen Jun a 1 and the ragweed pollen allergen Amb a 1, and residue modifications suggested by molecular docking calculations and peptide design could identify a large number of high affinity binding peptides. We propose that this combined experimental and computational approach by structural analysis of linear IgE epitopes and peptide design, can lead to potential new candidates for peptide immunotherapy. [ABSTRACT FROM AUTHOR]

Background Atopy is an aberrant immune response involving allergen-specific IgE production, though serum IgE concentration is not an entirely reliable diagnostic tool, particularly for epidemiological and genetic studies. There is no clear correlation between IgE and other indicators of atopy such as skin prick tests (SPT)s, and physiological associations are difficult to justify in cases with detectable IgE but negative SPT results. Objective IgE reflects the number of molecules available to produce an atopic response, but the degree of the response is determined by the binding strength (affinity) between receptor-bound IgE and the allergen. We sought to determine if there was an association between binding affinity and SPT results in people with histories of atopy. Methods Standard SPTs (whole allergen extracts) were administered to people with histories of sensitivities to ragweed and house dust mite. The concentrations and affinities of serum allergen-specific IgEs were determined using the purified allergens Amb a 1 and Der p 1. Results There was a positive correlation between weal area and allergen-specific IgE among SPT-positive donors. However, for those individuals with detectable amounts of allergen-specific IgE, there was considerable overlap of IgE values between SPT-positive and -negative groups. Among sensitized donors, IgE–allergen interactions were characterized by two or three specific reactions of very high affinity (K A range 108 -1011 M). Negative SPT reactions were associated with lowered IgE binding affinities to major allergens. This delimited two groups with atopic disorders: specific IgE(+ )/SPT(+ ) and specific IgE(+ )/SPT(– ). Conclusion The product of antibody affinity and concentration, which we define as antibody capacity (CAP = K A × IgE), is more informative with regard to describing allergen sensitivity than antibody... [ABSTRACT FROM AUTHOR]