Standardization of Allergen Extracts with Appropriate Methods

The paper presents recommendations for standardization of allergen extract by the combined use of skin prick tests and radioallergosorbent tests. These tests provide appropriate means for quantitation of the total allergenic activity of an extract. Advantages and limitations are discussed with reference to the literature.     

HEP versus PNU Standardization of Allergen Extracts in Skin Prick Testing

During 1 year, all patients referred to an allergy outpatient clinic for adults were skin prick tested with a panel of standard allergen extracts from two manufacturers using different methods of standardization. One company referred to the histamine equivalent prick (HEP) and the other used the more traditional protein nitrogen units (PNU). Standard extracts and five-fold dilution were tested. The results indicate that the ratio of concentration between two extracts of the same allergen should be measured by the absolute difference of the wheal diameters. We found significant differences between corresponding extracts from the two manufacturers.     

Immunochemical and Biological Characterization of a Mugwort (Artemisia vulgaris) Pollen Extract

A reference extract of mugwort pollen (Artemisia vulgaris) was characterized by crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE) and quantitative skin prick test (QSPT). CIE revealed that the extract contained at least 42 distinct antigens of which 24 migrated towards the anode and 18 towards the cathode at pH 8.6. A CRIE analysis of the crude mugwort pollen extract, performed with sera from 29 mugwort-allergic patients, showed that 10 antigens may be considered allergens; one was classified as a major allergen, five as intermediate allergens, and four as minor allergens. The QSPT performed on the same 29 allergic patients established that 17.4 micrograms lyophilised reference mugwort pollen extract per ml had a biological potency of 1 HEP (histamine equivalent by prick test).     

Personalized medicine for allergy treatment: Allergen immunotherapy still a unique and unmatched model

The introduction of personalized medicine (PM) has been a milestone in the history of medical therapy, because it has revolutionized the previous approach of treating the disease with that of treating the patient. It is known today that diseases can occur in different genetic variants, making specific treatments of proven efficacy necessary for a given endotype. Allergic diseases are particularly suitable for PM, because they meet the therapeutic success requirements, including a known molecular mechanism of the disease, a diagnostic tool for such disease, and a treatment blocking the mechanism. The stakes of PM in allergic patients are molecular diagnostics, to detect specific IgE to single-allergen molecules and to distinguish the causative molecules from those merely cross-reactive, pursuit of patient's treatable traits addressing genetic, phenotypic, and psychosocial features, and omics, such as proteomics, epi-genomics, metabolomics, and breathomics, to forecast patient's responsiveness to therapies, to detect biomarker and mediators, and to verify the disease control. This new approach has already improved the precision of allergy diagnosis and is likely to significantly increase, through the higher performance achieved with the personalized treatment, the effectiveness of allergen immunotherapy by enhancing its already known and unique characteristics of treatment that acts on the causes.     

Current Status of Standardization of Inhalant Allergen Extracts in Korea

Allergy diagnosis and immunotherapy in Korea rely mostly on imported allergen extracts. However, some allergens that are not important in Western countries are not commercially available, and even the same species of allergen source often displays differences in allergenicity due to amino acid sequence polymorphisms. Therefore, it is essential to prepare allergen extracts that reflect regional characteristics. Allergen standardization has been performed since 2009 with the support of the Korea Center for Disease Control and Prevention. Here, we summarize the current status of allergen standardization, focusing on the house dust mite and cockroach. Pollen allergens that are under investigation are also briefly described.     

In Vitro Evaluation of Allergen Potencies of Commercial House Dust Mite Sublingual Immunotherapy Reagents

PurposeThe clinical efficacy of allergen-immunotherapy is known to be dose dependent. However, optimal maintenance dosage has not yet been determined for sublingual immunotherapy (SLIT). Furthermore, since companies adopt their own units for expression of allergenicity, the allergen concentrations of individual reagents cannot be compared easily. We sought to measure and compare the allergenicities of 3 commercially available house dust mite (HDM) SLIT regents and a subcutaneous immunotherapy reagent.MethodsWe measured the HDM allergenic potency of the maintenance dosages of three SLIT reagents: Staloral® (300 index of reactivity [IR] /mL, recommended maintenance dosage [MD]: 120 IR), SLITone® (1,000 standard therapeutic unit [STU]/mL, recommended MD: 200 STU), Wolwopharma® (100 µg/mL, recommended MD: 20 µg), and subcutaneous immunotherapy regents of Hollister-Stier (10,000 allergy unit [AU] /mL). The allergenic potency was assessed by measuring the total protein concentrations, mite group 1 and 2 allergens using 2-site ELISA, and an inhibition test against IgE specific to Dermatophagoides farinae and Dermatophagoides pteronyssinus.ResultsThe protein content of the Wolwopharma® reagent was 1.5-261.4 times higher than that of the other 2 SLIT reagents. The concentration of group 1 major allergens in Staloral® (132.03 µg/mL) was 33- to 44.5-fold higher than in SLITone® (4.00 µg/mL) and Wolwopharma® (2.97 µg/mL). The concentration of group 2 major allergen was also 8.9- to 10.5-fold higher in Staloral® (15.7 µg/mL) than in SLITone® (1.8 µg/mL) or Wolwopharma® (1.5 µg/mL). An ELISA inhibition study against HDM-specific IgE showed that the allergen potency of Staloral® reagent is 8.5-fold and 21-fold higher than that of SLITone® or Wolwopharma®, respectively. The differences between the maintenance dosages are further exaggerated by the differences in the recommended volumes of SLIT reagents.ConclusionsThe allergen potencies of commercially available HDM SLIT reagents are markedly different. Consensus regarding the optimal allergen concentration for SLIT reagents used to treat HDM respiratory allergies is needed.     

Biochemical and Allergenic Properties of the House Dust Mite Extract, Dermatophagoides pteronyssinus

A Further study has been made on the house dust mite extract, Dermatophagoides pteronyssinus , with emphasis on gel-filtrated fraction 2 (F₂). The crude mite extract showed at least nine discs on polyacrylamide disc electrophoresis and contained less than 0.25 % sialic acid and less than 0.5 mM hexosamine and no detectable uronic acid. From gel filtration of the crude extract a No. 2 fraction (F₂) with allergenic activity showed at least five components on SDS disc electrophoresis covering a molecular weight range of between 15,000 and 70,000. The major allergenic activity of F₂ dissolved in pH 7–8 and 4–5 on an isoelectric focusing column. Affinity chromatography of lectins showed that allergenic activity did not relate to structures of N -acetyl-D-glucosamine or N -acetyl-D-galactosamine. Allergenic activity of the crude extract was not affected by peptic digestion and the mite digest prepared by trypsin and pronase showed a similar fraction-action and activity profile as crude extract.     

A Prospective Study on the Safety of Immunotherapy in Children with Severe Asthma

One hundred and six of 503 (21%) consecutive children with asthma, who from 1979 to 1983 commenced hyposensitization therapy, were prospectively studied on the safety of immunotherapy. More than 80% of the patients completed therapy without side effects. Thirteen patients were withdrawn from hyposensitization due to moderate and predictable, but intolerable, side effects such as asthma/rhinitis, urticaria and subcutaneous nodules and hypersensitivity to aluminium. However, more alarming was the outcome in six children, who after an uneventful course of immunotherapy and after several months on maintenance therapy, suddenly, 5 to 20 min (mean 10 min) following an earlier tolerable allergen injection, developed severe, anaphylactic reactions, in three of them nearly fatal. Mould extracts were responsible for the most frequent and serious side effects (Alternaria iridis/alternata, 3 patients, Cladosporium herbarum, 8 patients). Furthermore, serious, but not immediately life-threatening, anaphylactic reactions occurred in two children treated with Phleum pratense. On the other hand, hyposensitization with Dermatophagoides pteronyssinus was very well tolerated.     

Comparison of Allergenic Properties among Commercially Available House Dust Mite Allergen Extracts in Korea

Precise allergy diagnosis and effective allergen specific immunotherapy are largely dependent on the quality of allergen extract. A new extract of Dermatophagoides farinae was commercially developed by Prolagen. The allergenic properties of the new extract were compared with those of other commercial products. The allergenic properties of the new extract were compared according to protein concentration, protein profiles, major allergen (Der f 1) contents, and allergenic potency to those for three commercially available extracts imported in Korea (Jubilant HollisterStier Allergy, Lofarma S.p.A., and Stallergenes Greer). Protein concentrations varied up to 2.62-fold (0.404 to 1.057 mg/mL), and Der f 1 contents varied up to 11.3-fold (3.597 to 40.688 µg/mL). Protein profiles of the extracts showed no major discrepancies, although there were some differences in SDS-PAGE band intensities, reflecting protein concentrations. Allergen potency ranged from 37038 to 60491 PAU/mL. The Prolagen product was highest in terms of protein concentration and allergen potency. The Lofarma product displayed Der f 1 content similar to that in Prolagen (19.4 µg/mg vs. 19.3 µg/mg). Endotoxin levels varied 8.9-fold (1020 to 8985 EU/mL). The newly developed house dust mite extract showed equal or better allergenic properties than available commercial extracts. This new product may be useful for better diagnostics and allergen-specific immunotherapeutics.     

The allergen challenge chamber: a valuable tool for optimizing the clinical development of pollen immunotherapy

The clinical development of allergen immunotherapy for allergic rhinoconjunctivitis because of pollen is complicated by seasonal, geographical and subject-related variability in allergen exposure. Using an allergen challenge chamber (ACC), a room that enables reproducible challenges with controlled levels of inhalant allergens for several hours, these factors can be controlled. The ACC has often been used to evaluate symptomatic medications but is underexploited in the field of allergen immunotherapy. When used in conjunction with a programme of natural-exposure trials, the ACC enables researchers to (i) facilitate the allergen immunotherapy dose-finding process, (ii) accelerate the transition from Phase I/II to Phase III trials, (iii) characterize the onset and maintenance of action, (iv) avoid the confounding effects of rescue medication, (v) better characterize the baseline or pretreatment characteristics of trial subjects, (vi) perform better-standardized physical and laboratory measurements during an acute challenge, (vii) simplify trial logistics and use smaller numbers of subjects than would be required in equivalent natural-exposure studies and (viii) support (but not replace) Phase III natural-exposure trials for the investigation into long-term and disease-modifying effects. ACC studies can further increase levels of evidence for allergen immunotherapy--the only current therapy potentially capable of modifying the underlying allergic disease.     

Biological potency of German cockroach allergen extracts determined in an inner city population

BACKGROUND: Cockroach allergy is an important cause of inner city asthma. To perform valid studies on the diagnosis and treatment of cockroach allergy, biological potencies of test extracts need to be established, and a surrogate in vitro test for biological potency should be chosen. METHODS: Sixty-two cockroach-allergic adult subjects were recruited for quantitative skin testing with three commercial German cockroach extracts. The intradermal D50 values were determined using linear interpolation, and the biologic potencies were determined from D50 data. The extracts were also analysed for relative potency, using a competition ELISA, and for specific allergen content, using a two-site ELISA. RESULTS: Estimates of each extract's D50 were analysable in 48-55 subjects, with D50s between 10.3 and 11.8. All three extracts were bioequivalent using pre-set criteria. The biological potencies of the extracts were 1738-8570 bioequivalent allergy units (BAU)/mL (geometric mean=3300), and these relative potencies were similar to those estimated by competition ELISA and specific allergen content. IgE against cockroach allergens were detected in sera from 34 subjects with analysable D50s, and 17 subjects had IgE directed against specific cockroach allergens. Although the presence of anti-Bla g 5 correlated with the subjects' skin test responses for 2/3 extracts, no single allergen was immunodominant. Antibody responses among the subjects were heterogeneous. CONCLUSIONS: Although commercial cockroach extracts are relatively low in potency, immunotherapeutic doses should be achievable. Biological potency may be estimated using D50 testing, a combination of specific allergen determinations, or by an overall potency assay such as the competition ELISA. CAPSULE SUMMARY: The biological potency of three German cockroach allergen extracts, determined in an inner city population, was 1738-8570 BAU/mL. No one allergen was immunodominant, and surrogate in vitro testing methods were examined.     

Allergen manufacturing and quality aspects for allergen immunotherapy in Europe and the United States: An analysis from the EAACI AIT Guidelines Project

Adequate quality is essential for any medicinal product to be eligible for marketing. Quality includes verification of the identity, content and purity of a medicinal product in combination with a specified production process and its control. Allergen products derived from natural sources require particular considerations to ensure adequate quality. Here, we describe key aspects of the documentation on manufacturing and quality aspects for allergen immunotherapy products in the European Union and the United States. In some key parts, requirements in these areas are harmonized while other fields are regulated separately between both regions. Essential differences are found in the use of Reference Preparations, or the requirement to apply standardized assays for potency determination. As the types of products available are different in specific regions, regulatory guidance for such products may also be available in one specific region only, such as for allergoids in the European Union. Region‐specific issues and priorities are a result of this. As allergen products derived from natural sources are inherently variable in their qualitative and quantitative composition, these products present special challenges to balance the variability and ensuring batch‐to‐batch consistency. Advancements in scientific knowledge on specific allergens and their role in allergic disease will consequentially find representation in future regulatory guidelines.     

Current practice of allergy diagnosis and the potential impact of regulation in Europe

In the European Union (EU), the regulatory framework regarding diagnostic allergen extracts is currently in the process of being implemented at the national level. Due to these regulations, the initial and periodic renewal expenses for the registration of diagnostic allergen extracts may render extract production unprofitable. Consequently, many extracts may be at risk of removal from the market. The current survey, which was conducted by a task force of the European Academy of Allergy and Clinical Immunology, aimed to assess the current practice of allergy diagnosis in Europe. This survey revealed that skin tests continue to be the main diagnostic procedure and are used as the first option in almost two‐third of all types of allergic diseases and in 90% of individuals suffering from respiratory allergies. Therefore, there is a need to ensure the availability of high‐quality allergen extracts to maintain the common diagnostic procedures used by EU professionals. To reach this goal, it is necessary to align efforts and establish active partnerships between manufacturers, relevant scientific societies, consumer organizations and authorities to maintain the availability of these diagnostic tools.     

Airway inflammation in a murine model of chronic asthma: evidence for a local humoral immune response

BACKGROUND: Asthma is an acute-on-chronic inflammatory disease of the airways characterized by recruitment of eosinophils into the epithelial layer, chronic inflammation in the lamina propria, as well as variable accumulation of mast cells in the airway wall. The role of local production of allergen-specific immunoglobulins in triggering mast cell-mediated asthmatic inflammation is unknown. METHODS: We used a chronic inhalational exposure model of asthma in ovalbumin-sensitized BALB/c mice to examine the phenotype of immunoglobulin-secreting cells and mast cells in the airway wall. In parallel, we assayed ovalbumin-specific IgG and total IgE in the plasma of these animals. RESULTS: In sensitized mice exposed to aerosolized ovalbumin for 6 weeks, aggregates of chronic inflammatory cells consisted of a majority of plasmacytoid cells, including numerous IgG-synthesizing cells, which were significantly increased in sensitized animals compared to controls. IgA-synthesizing cells were also present, but were not increased in the sensitized exposed mice. Immunoglobulins in the cytoplasm of the plasma cells were demonstrated to be antigen-specific. No IgM-or IgE-synthesizing cells were observed, although levels of total IgE in the plasma were significantly increased. There was no recruitment of mast cells of either the mucosal or the connective tissue phenotype into the lamina propria or the epithelium. CONCLUSION: In this experimental model of chronic asthma, the pattern of inflammation in the airway wall is consistent with development of a local IgG-mediated humoral immune response. However, there is no evidence of local production of IgE or recruitment of mast cells.     

Methodological Aspects of Nasal Allergen Challenges Based on a Three-Year Tree Pollen Immunotherapy Study

During 3 years of immunotherapy with tree pollen extracts, 31 patients were provoked annually. Changes in nasal reactivity were followed by registration of expiratory nasal peak flow, number of sneezes, and amount of secretion. The reproducibility of the peak flow measurements was studied. The results from all three parameters were used to form a total nasal provocation score which, better than each parameter separately, could demonstrate the variation in sensitivity. Provocation with an allergen concentration of 1 HEP was the most effective means of showing changes in specific sensitivity of nasal mucosa.     

208例哮喘患儿吸入性过敏原的调查分析

本文报道了208例哮喘患儿吸入性过敏原的调查结果. 采用变应原浸液皮肤点刺方法. 皮试阳性者169例, 占81.25%.变应原浸液内含18种常见吸入性过敏原. 尘螨的阳性率最高达78.85%, 其次是室尘的阳性率为35.58%, 烟的阳性率为32.69%.无一例对其它变应原呈阳性反应, 过敏原皮试阳性率无性别差异, 与有无个人及家庭过敏史无关. 哮喘婴幼儿皮试阳性率低于哮喘患儿, 哮喘皮试阳性率在年龄组之间有显著差异(P＜0.01), 有随着年龄加大而渐增的趋势. 结果提示: 尘螨、空气污染和被动吸烟是诸多哮喘触发因素中具高度危险性的因素, 加强这方面的对策研究对儿童哮喘的防治具有意义.