Cosensitization to the 3 Nonhomologous Major Cashew Allergens Ana o 1, Ana o 2, and Ana o 3 Is Caused by IgE Cross-reactivity

Background: Cashew nuts often cause strong allergic reactions, which are even more severe than those of peanuts. Ana o 1 (vicilin), Ana o 2 (legumin), and Ana o 3 (2S albumin) are major cashew allergens. Cosensitization to all 3 nonhomologous cashew nut allergens has been observed. We hypothesize that this might be due to IgE cross-reactivity. Methods: IgE cross-inhibitions were performed with Ana o 1-3 using serum samples from cashew nut–allergic patients. The related hazelnut allergens Cor a 11, 9, and 14 were used as controls. For comparison, IgE cross-reactivity between the hazelnut allergens was investigated using serum samples from hazelnut-allergic patients. Results: The median percentages of cross-inhibition between Ana o 1, 2, and 3 were 84%-99%. In comparison, the median cross- inhibition values between hazelnut allergens were 33%-62%. The IC50 values revealed the highest IgE affinity to be to Ana o 3 and Cor a 14. Hazelnut legumin Cor a 9 inhibited IgE binding to Ana o 1, 2, and 3, with median percentages of 75%, 56%, and 48%, respectively. No cross-reactivity was observed between allergenic vicilins or between 2S albumins from cashew and hazelnut. Potentially cross-reactive peptides of Ana o 3 identified in silico overlapped with previously reported IgE epitopes of all 3 allergens. Conclusion: IgE with high affinity to Ana o 3 that cross-reacts with the other 2 major nonhomologous cashew nut allergens might be responsible for the high allergenic potency of cashew nut. These cross-reactive IgE types comprise the major fraction of specific IgE in cashew-allergic patients and might be responsible for cross-reactivity between unrelated tree nuts (AU)

Antecedentes: Los anacardos suelen provocar fuertes reacciones alérgicas, que son incluso más graves que las del maní. Ana o 1 (vicilina), Ana o 2 (legumina) y Ana o 3 (albúmina 2S) son los principales alérgenos del anacardo. Se ha observado cosensibilización a los 3 alérgenos no homólogos del anacardo. Nuestra hipótesis es que esto podría deberse a la reactividad cruzada de la IgE. Métodos : Se realizaron inhibiciones cruzadas de IgE con Ana o 1-3 utilizando muestras de suero de pacientes alérgicos al anacardo. Como controles se utilizaron los alérgenos de avellana relacionados Cor a 11, 9 y 14. A modo de comparación, se investigó la reactividad cruzada de IgE entre los alérgenos de la avellana utilizando muestras de suero de pacientes alérgicos a la avellana. Resultados : Los porcentajes medios de inhibición cruzada entre Ana o 1, 2 y 3 fueron del 84% al 99%. En comparación, los valores medios de inhibición cruzada entre alérgenos de avellana fueron del 33% al 62%. Los valores de IC50 revelaron que la mayor afinidad de IgE era Ana o 3 y Cor a 14. La legumina de avellana Cor a 9 inhibió la unión de IgE a Ana o 1, 2 y 3, con porcentajes medios de 75%, 56% y 48%. , respectivamente. No se observó reactividad cruzada entre vicilinas alergénicas ni entre albúminas 2S de anacardo y avellana. Los péptidos potencialmente de reacción cruzada de Ana o 3 identificados in silico se superpusieron con epítopos de IgE previamente informados de los 3 alérgenos. Conclusión : La IgE con alta afinidad por Ana o 3 que reacciona de forma cruzada con los otros 2 alérgenos principales no homólogos del anacardo podría ser responsable de la alta potencia alergénica del anacardo. Estos tipos de IgE de reacción cruzada comprenden la fracción principal de IgE específica en pacientes alérgicos al anacardo y podrían ser responsables de la reactividad cruzada entre frutos secos no relacionados (AU)

Co-sensitization to the three non-homologous major cashew allergens Ana o 1, Ana o 2 and Ana o 3 is caused by IgE cross-reactivity.

BACKGROUND: Cashew nuts often cause strong allergic reactions, even exceeding those of peanuts. Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin) are major cashew allergens. Co-sensitization to all three non-homologous cashew nut allergens has been observed. We hypothesize that this might be due to IgE cross-reactivity. METHODS: IgE cross-inhibitions were performed with Ana o 1-3 using sera from cashew nut allergic patients. Related hazelnut allergens Cor a 11, 9 and 14 were used as controls. For comparison, IgE cross-reactivity between the hazelnut allergens was investigated using sera from hazelnut allergic patients. RESULTS: Median percentages of cross-inhibitions between Ana o 1-3 were 84-99%. In comparison, medians of cross-inhibitions between hazelnut allergens were 33-62%. The IC50 values revealed the highest IgE affinity to Ana o 3 and Cor a 14. Hazelnut legumin Cor a 9 inhibited IgE-binding to Ana o 1, 2, and 3 with median percentages of 75%, 56%, and 48%, respectively. No cross-reactivity was observed between allergenic vicilins or between 2S albumins from cashew and hazelnut. In silico identified potentially cross-reactive peptides of Ana o 3 overlapped with previously reported IgE epitopes of all three allergens. CONCLUSIONS: IgE with high affinity to Ana o 3 that cross-reacts with the other two major non-homologous cashew nut allergens might be responsible for the high allergenic potency of cashew nut. These cross-reactive IgE comprises the major fraction of specific IgE in cashew allergic patients, and might be responsible for cross-reactivity between unrelated tree nuts.

Allergen exposure chambers: implementation in clinical trials in allergen immunotherapy.

Allergen exposure chambers (AECs) have been developed for controlled allergen challenges of allergic patients mimicking natural exposure. As such, these facilities have been utilized e.g., for proof of concept, dose finding or the demonstration of onset of action and treatment effect sizes of antiallergic medication. Moreover, clinical effects of and immunological mechanisms in allergen immunotherapy (AIT) have been investigated in AECs. In Europe AIT products have to fulfill regulatory requirements for obtaining market authorization through Phase I to III clinical trials. Multiple Phase II (dose-range-finding or proof-of-concept) trials on AIT products have been performed in AECs. However, they are not accepted by regulatory bodies for pivotal (Phase III) trials and a more thorough technical and clinical validation is requested. Recently, a Position Paper of the European Academy of Allergy and Clinical Immunology (EAACI) has outlined unmet needs in further development of AECs. The following review aims to address some of these needs on the basis of recently published data in the first part, whereas the second part overviews published examples of most relevant Phase II trials in AIT performed in AEC facilities.

Cetirizine inhibits gender-specific blood cell dynamics upon allergen contact in allergic rhinitis.

IgE-mediated inflammatory responses upon allergen contact in allergic rhinitis (AR) are associated with rapid alterations of circulating blood cell numbers detectable in a complete blood count (CBC). Aim of this study was to evaluate whether intake of antihistamines may modulate allergen-induced CBC dynamics in male and female patients. A total of N = 112 specific allergen challenges were performed in otherwise healthy AR subjects. Seventy-two (n = 72) subjects received placebo and forty (n = 40) received cetirizine (H1-receptor antagonist) per os prior to allergen exposure in a randomized, double-blind trial at the Vienna Challenge Chamber (VCC); a subgroup of twenty-five (n = 25) subjects received cetirizine and placebo on different study days (parallel group). Blood samples and symptom scores were taken at baseline and immediately after 6 h of airway challenge simulating ambient allergen contact. Female sex was associated with a pronounced circulating monocyte increase (p < .01) and male sex with an eosinophil decrease (p < .05) in the placebo group, but not in cetirizine treated subjects. The significant increase in segmented neutrophils (p < .001) and decrease in circulating erythrocytes (p < .01) upon allergen challenge was less prominent after cetirizine intake in both sexes. A more prominent thrombocyte increase in female subjects (p < .05) was noted upon allergen exposure, regardless of prior cetirizine intake. Cetirizine inhibited the mobilization of neutrophils, lymphocytes and decline in erythrocyte numbers, but did not affect thrombocyte increase upon allergen challenge. It further diminished gender-specific blood cell dynamics. Overall, as reflected in a simple CBC, cetirizine critically diminished immediate and late innate immune responses subsequent to allergen exposure.

Clinical reactivity of celery cultivars in allergic patients: Role of Api g 1.

BACKGROUND: Celery (Apium graveolens L.) is a vegetable consumed world-wide. Celery stalks and celeriac roots are often ingredients in convenient food products like spice blends and soups. OBJECTIVE: In this study, we examined the allergenicity of distinct celeriac cultivars. METHODS: Sixteen celery-allergic patients were identified using a double-blind, placebo-controlled food challenge. Ten different celeriac cultivars were used for skin prick testing in the patients. Two cultivars were further applied for oral food challenges; their protein composition was analysed by immunoblotting, and contents of major allergen Api g 1 were quantified. RESULTS: From the 10 investigated celeriac cultivars, two cultivars elicited significantly different skin reactivity ("Anita": 5.0 [2.0-12.0] mm vs "Prinz": 7.0 [3.0-9.5] mm; P = .047). Moreover, "Anita" induced fewer symptoms after a controlled oral-celeriac challenge in 14 patient (P < .001). The protein profiles on 2DE protein gels showed distinct protein patterns and higher protein amounts of Api g 1 in "Prinz" than in "Anita." CONCLUSIONS AND CLINICAL RELEVANCE: Taken together, the data from this study suggest that cultivar Anita is better tolerated in celery-allergic patients than "Prinz." Differences in the protein expression profile between the cultivars, particularly the different content of Api g 1, could cause the different allergenicity.

Perspectives in allergen immunotherapy: 2017 and beyond.

The Future of the Allergists and Specific Immunotherapy (FASIT) workshop provides a regular platform for global experts from academia, allergy clinics, regulatory authorities and industry to review developments in the field of allergen immunotherapy (AIT). The most recent meeting, held in February 2017, had two main themes: advances in AIT and hot topics in AIT from the regulatory point of view. The first theme covered opportunities for personalized AIT, advances in adjuvants and delivery systems, and the development of new molecules and future vaccines for AIT. Key topics in the second part of the meeting were the effects of the enactment of European Directive 2001/83 on the availability of allergens for therapy and diagnosis across the EU, the challenges of conducting Phase 3 studies in the field, the future role of allergen exposure chambers in AIT studies and specific considerations in performing AIT studies in the paediatric population. Finally, the group highlighted the forthcoming EAACI guidelines and their particular importance for the standardization of practice in the treatment of allergies. This review presents a comprehensive insight into those panel discussions and highlights unmet needs and also possible solutions to them for the future.

Intranasal administration of allergen increases specific IgE whereas intranasal omalizumab does not increase serum IgE levels-A pilot study.

BACKGROUND: Administration of the therapeutic anti-IgE antibody omalizumab to patients induces strong increases in IgE antibody levels. OBJECTIVE: To investigate the effect of intranasal administration of major birch pollen allergen Bet v 1, omalizumab or placebo on the levels of total and allergen-specific IgE in patients with birch pollen allergy. METHODS: Based on the fact that intranasal allergen application induces rises of systemic allergen-specific IgE, we performed a double-blind placebo-controlled pilot trial in which birch pollen allergic subjects were challenged intranasally with omalizumab, placebo or birch pollen allergen Bet v 1. Total and allergen-specific IgE, IgG and basophil sensitivity were measured before and 8 weeks after challenge. For control purposes, total, allergen-specific IgE levels and omalizumab-IgE complexes as well as specific IgG levels were studied in subjects treated subcutaneously with either omalizumab or placebo. Effects of omalizumab on IgE production by IL-4/anti-CD40-treated PBMCs from allergic patients were studied in vitro. RESULTS: Intranasal challenge with Bet v 1 induced increases in Bet v 1-specific IgE levels by a median of 59.2%, and this change differed significantly from the other treatment groups (P = .016). No relevant change in allergen-specific and total IgE levels was observed in subjects challenged with omalizumab. Addition of omalizumab did not enhance IL-4/anti-CD40-induced IgE production in vitro. Significant rises in total IgE (mean IgE before: 131.83 kU/L to mean IgE after: 505.23 kU/L) and the presence of IgE-omalizumab complexes were observed after subcutaneous administration of omalizumab. CONCLUSION: Intranasal administration of allergen induced rises of allergen-specific IgE levels, whereas intranasal administration of omalizumab did not enhance systemic total or allergen-specific IgE levels.

Possible effect of landscape design on IgE recognition profiles of two generations revealed with micro-arrayed allergens.

The aim of this study was to investigate possible effects of landscape design on the IgE sensitization profile toward inhalant allergens in patients with respiratory allergy from Uzbekistan where green areas have been changed during the last two decades by a State program. Sera from two different generations of Uzbek (n=58) and, for control purposes, from two generations of Austrian (n=58) patients were analyzed for IgE reactivity to 112 different micro-arrayed allergen molecules by ImmunoCAP ISAC technology. Changes in molecular IgE sensitization profiles to pollen allergens in the young vs the middle-aged Uzbek population were associated with replanting, whereas those in the Vienna populations reflected natural changes in plant growth. Our data indicate that anthropologic as well as natural changes in the biome may have effects on IgE sensitization profiles already from one to another generation.

[Innovative forms of specific immunotherapy]. / Innovative Formen der spezifischen Immuntherapie.

The clinical efficacy of allergen-specific immunotherapy depends on tolerance induction. Treatment success is currently limited by treatment-related adverse reactions. Novel approaches mainly target improvement of tolerability in addition to optimized immunogenicity. For epicutaneous immunotherapy (EPIT), commercially available treatment extracts are applied to skin via a patch. The route exhibits excellent tolerability and good clinical efficacy. The first encouraging data on a peanut EPIT were published in 2016. Intralymphatic immunotherapy (ILIT) study results show pronounced immunogenicity and persisting clinical efficacy after only three injections of a small amount of established extracts. New approaches of synthetic vaccine development focus on tolerance induction. These vaccines are genetically engineered with a precisely defined profile and can be manufactured in reproducible quality. Distinct immunogenicity of the antigenic determinants contained in the preparation as well as an optimized safety profile are expected. Grass, birch, cat, and mite vaccines are currently under investigation. Which of these approaches will gain market access is unclear up to date. Results of food immunotherapy studies reveal consistently poor tolerance and limited efficacy without encouraging perspective and are not discussed further.

Allergen exposure chambers: harmonizing current concepts and projecting the needs for the future - an EAACI Position Paper.

BACKGROUND: Allergen exposure chambers (AECs) are clinical facilities allowing for controlled exposure of subjects to allergens in an enclosed environment. AECs have contributed towards characterizing the pathophysiology of respiratory allergic diseases and the pharmacological properties of new therapies. In addition, they are complementary to and offer some advantages over traditional multicentre field trials for evaluation of novel therapeutics. To date, AEC studies conducted have been monocentric and have followed protocols unique to each centre. Because there are technical differences among AECs, it may be necessary to define parameters to standardize the AECs so that studies may be extrapolated for driving basic immunological research and for marketing authorization purposes by regulatory authorities. METHODS: For this task force initiative of the European Academy of Allergy and Clinical Immunology (EAACI), experts from academia and regulatory agencies met with chamber operators to list technical, clinical and regulatory unmet needs as well as the prerequisites for clinical validation. RESULTS: The latter covered the validation process, standardization of challenges and outcomes, intra- and interchamber variability and reproducibility, in addition to comparability with field trials and specifics of paediatric trials and regulatory issues. CONCLUSION: This EAACI Position Paper aims to harmonize current concepts in AECs and to project unmet needs with the intent to enhance progress towards use of these facilities in determining safety and efficacy of new therapeutics in the future.

Epitope specificity determines cross-protection of a SIT-induced IgG4 antibody.

BACKGROUND: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT). METHODS: We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments. RESULTS: Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity. CONCLUSIONS: Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.

The improved efficacy of a fixed-dose combination of fluticasone furoate and levocabastine relative to the individual components in the treatment of allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is a common chronic disease, which has significant detrimental effect on well-being and quality of life as well as substantial socio-economic impact. Combination pharmacotherapy is utilized by 40-50% of patients to treat their symptoms. OBJECTIVE: To compare the effects of intranasal fluticasone furoate (FF)/levocabastine (LEVO) fixed-dose combination (FDC) with each component alone on allergen-induced nasal and ocular symptoms. METHODS: A randomized, double-blind, placebo-controlled, three-way, incomplete block, cross-over, proof-of-concept study in 71 patients with AR, evaluated FF 100 µg, LEVO 200 µg and FDC (FF 100/LEVO 200 µg), once daily via intranasal spray for 8 days. On days 1 and 8, total nasal symptom score (TNSS) and total ocular symptom score (TOSS) were assessed every 15 min during a 4-h allergen exposure in the Vienna Challenge Chamber. The primary endpoint was Day 8 weighted mean TNSS. RESULTS: After 8 days, FDC resulted in both statistically and clinically significant reductions in mean TNSS compared with FF and LEVO alone [adjusted mean differences (95% CI): FDC vs. FF: -2.26 (-2.90, -1.62); FDC vs. LEVO: -2.57 (-3.21, -1.93)]. All active treatments were significantly superior to placebo [adjusted mean difference (95% CI) from placebo: FDC: -4.1 (-4.86, -3.34); FF: -1.84 (-2.66, -1.03); LEVO: -1.53 (-2.34, -0.72)]. Onset of action was rapid following FDC and LEVO treatment with an approximate two unit reduction in mean TNSS from pre-dose levels by 30 min and 1 h. Mean TOSS was also reduced following all active treatments relative to placebo (range 0.6-0.8 unit reduction). All treatments were equally well tolerated. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that once daily FF/LEVO FDC could provide a clinical therapeutic advantage to existing standard monotherapies in the treatment of moderate-to-severe AR, and support progression to evaluation in larger phase III clinical studies.

The CRTH2 antagonist OC000459 reduces nasal and ocular symptoms in allergic subjects exposed to grass pollen, a randomised, placebo-controlled, double-blind trial.

BACKGROUND: CRTH2 mediates activation of Th2 cells, eosinophils and basophils in response to prostaglandin D(2). The CRTH2 antagonist OC000459 has previously been demonstrated to reduce airway inflammation and improve lung function in moderate persistent asthma. The objective of the present study was to determine the involvement of CRTH2 in promoting nasal and ocular symptoms in allergic subjects exposed to grass pollen. METHODS: A single centre, randomised, double-blind, placebo-controlled, two-way crossover study was conducted in 35 male subjects allergic to grass pollen comparing OC000459 200 mg bid with placebo for 8 days. Subjects were exposed to grass pollen (≥ 1400 grains/m(3)) for 6 h on the 2nd and 8th days of treatment and assessed for nasal symptoms, ocular symptoms, other symptoms, nasal secretion weight and rhinomanometry over the 6-h period. After a washout period of 3 weeks, subjects were switched to the alternative treatment for a further 8 days. The trial was registered on the clinical trials.gov database (Identifier NCT01448902). RESULTS: During the first treatment period, treatment with OC000459 significantly reduced both nasal and ocular symptoms in allergic subjects compared with placebo after challenge with grass pollen. A significant effect was observed on the 2nd day of dosing which was increased on the 8th day of dosing. The therapeutic effects of OC000459 persisted into the second treatment period despite a 3-week washout phase. The safety profile of OC000459 was similar to that of placebo. CONCLUSION: Treatment with OC000459 was well tolerated and led to a significant and persistent reduction in the symptoms of rhinoconjunctivitis.

Changes in basophil activation during grass-pollen sublingual immunotherapy do not correlate with clinical efficacy.

BACKGROUND: Biomarkers predicting the safety and efficacy of sublingual immunotherapy (SLIT) remain to be established. METHODS: Eighty-nine patients with allergic rhinoconjunctivitis to grass pollen received either a placebo or five-grass-pollen daily tablet sublingually for 4 months. Following exposure in an allergen challenge chamber, clinical responders and nonresponders were identified individually by evaluating their rhinoconjunctivitis total symptom score (RTSS). Activation of peripheral blood basophils was measured by cytofluorometry before and after 2 or 4 months of immunotherapy, based on CD203c surface expression following allergen stimulation. RESULTS: Patients receiving the grass-pollen tablet had a relative mean improvement of 29.3% vs placebo in the average RTSS after 4 months of SLIT (P < 0.0003). No significant changes in basophil activation were noticed after 2 or 4 months of SLIT despite induction of specific IgGs. Among individual clinical responders, basophil activation was either decreased, increased, or unmodified during SLIT. Levels of basophil activation prior to immunotherapy were not predictive of local adverse reactions associated with immunotherapy. A moderate association was found between basophil activation and allergen-specific IgE levels, skin reactivity, or RTSS, suggesting that the former is, to some extent, indicative of disease severity. As such, patients with the highest level of basophil activation before treatment were more likely to benefit clinically from SLIT. CONCLUSIONS: Allergen reactivity of peripheral blood basophils is not a biomarker for adverse events or early onset of clinical responses to SLIT.