The IgE Blocking Activity Induced by Dermatophagoides pteronyssinus Subcutaneous Immunotherapy Does Not Correlate with Specific IgA but with IgG4 in both Serum and Saliva.

BACKGROUND: The role of salivary-specific IgG4 and IgA in subcutaneous immunotherapy (SCIT) is not well defined. We aimed to investigate the change of IgG4 and IgA in both serum and saliva and their correlations with IgE-blocking-factor (IgE-BF) during SCIT. METHOD: 307 Dermatophagoides pteronyssinus (DP) allergic rhinitis and/or asthma patients were recruited for this study. 286 patients received DP-SCIT for 1 year. Twenty-one patients received only symptomatic treatment. DP-, Der p 1-, and Der p 2-specific IgE in serum, specific-IgG4 and Der p 2-specific IgA1 and IgA2 in both serum and saliva were measured at timepoints 0, 4, and 12 months during DP-SCIT. Correlation between salivary and serological IgG4, IgA, and their correlation with DP-specific IgE-BF measured in serum was evaluated. RESULTS: During DP-SCIT, the allergen-specific IgG4 in both saliva and serum increased and correlated significantly, the correlation becomes stronger over the treatment time. DP-specific IgE-BF significantly correlated with DP-specific IgG4 in serum (p < 0.0001) at different timepoints and in saliva at 12 months of SCIT (p < 0.01). No change in Der p 2-specific IgA during DP-SCIT was observed, and the IgA in serum did not correlate with IgA in saliva. There was no correlation between DP IgE-BF and Der p 2-specific IgA in serum or saliva. The control group did not exhibit significant changes in any antibody level measured. CONCLUSION: The IgE blocking activity induced by DP-SCIT treatment correlated with specific IgG4 and not IgA. The IgG4 in saliva correlates with serum IgG4 and can be an alternative immunological marker beyond 1 year of SCIT treatment.

Immunoelectrophoresis for the Characterization of Allergen Extracts.

Immunoelectrophoresis can be used for analysis of individual proteins in complex mixtures. The conditions involved in immunoelectrophoresis are mild, avoiding the risk of denaturation, and it is possible to perform relative quantification of individual components. The fundamental disadvantage is the dependence on rabbit antisera as reagents. The usefulness of immunoelectrophoresis in allergy research is greatly enhanced by the possibility of identification of allergens to which the individual in question has IgE.The common principle is characterized by two independent electrophoreses having direction of current perpendicular to each other, i.e., crossed immunoelectrophoresis (CIE). This ultimately results in the formation of characteristic bell-shaped precipitates, each precipitate representing one antigen. There is a linear relationship between the amount of antigen and size of precipitate for a given antibody concentration for each precipitate and so relative quantification can be performed. The sensitivity and resolution power of CIE are very high and there are multiple variations of the technique, some of which will be illustrated in this chapter.

Standardization of Allergen Extracts.

Allergens are molecules with the capacity to elicit IgE responses in humans. When stimulated with allergens, most allergic patients respond with production of IgE specific for several proteins/allergens in the source material. The standardization of allergen extracts is essential in order to control variability and to achieve consistency and reproducibility in a clinical setting.Because the IgE binding capacity of an allergen extract is related to the content of one or a few major allergens, it is important that the standardization procedure ensures consistency, not only in the overall IgE binding potency, but also in the content and ratio of individual major allergens. Owing to the complexity of allergen extracts, a key element in standardization of allergen extracts is the use of standards.This chapter describes the principles for standardization of allergen extracts to be used by research laboratories. Other chapters in this volume describe in vitro methods in detail.

Major allergen content consistency of SQ house dust mite sublingual immunotherapy tablets and relevance across geographic regions.

BACKGROUND: Consistency in composition and potency, particularly regarding major allergens, is crucial for the quality of extracts for allergen immunotherapy. OBJECTIVE: To characterize the major allergen composition of house dust mite (HDM) extracts commercially available in the United States and the SQ HDM sublingual immunotherapy (SLIT) tablet, and to relate the composition to patient sensitization patterns. METHODS: Der 1/Der 2 ratios were determined in 10,000- and 30,000-AU/mL HDM extracts from 5 US companies and the SQ HDM SLIT-tablet. Allergen content was analyzed by enzyme-linked immunosorbent assay and compared with an in-house reference. Sensitivity toward Der p 1, Der p 2, and Der p 10 was determined in serum from randomly selected subgroups of 220 individuals from North American and European SQ HDM SLIT-tablet trials. RESULTS: Mean Der 1/Der 2 ratios in US HDM extracts ranged from 0.4 to 20.5. For the SQ HDM SLIT-tablet (20 batches), variability did not exceed 12% regarding content of Der f 1 (SD, 11.9%; 95% confidence interval [CI], 0.94-1.06), Der p 1 (SD, 6.1%; 95% CI, 0.97-1.03), and combined Der 2 allergen (SD, 6.4%; 95% CI, 0.97-1.03), indicating a consistent Der 1/Der 2 ratio. High allergen sensitivity frequencies toward Der p 1 and Der p 2 were observed regardless of geographic region. Efficacy of the SQ HDM SLIT-tablet has been demonstrated in 5 clinical trials. CONCLUSION: The SQ HDM SLIT-tablet has efficacy potential for a broad range of patients because it includes a consistent 1:1 ratio of the 2 major HDM allergens to which individuals were most frequently sensitized across geographic regions. Efficacy has been demonstrated.

Fractionation of Source Materials Leads to a High Reproducibility of the SQ House Dust Mite SLIT-Tablets.

BACKGROUND: The production of house dust mite (HDM) allergen products for allergy immunotherapy has traditionally been based on purified mite bodies or whole-mite culture, which are quite different source materials with a limited possibility for adjusting the chemical composition. The SQ HDM SLIT-tablet is a fast-dissolving pharmaceutical formulation that has been developed for sublingual immunotherapy (SLIT) of HDM respiratory allergic disease. OBJECTIVE: The objective of the present study was to establish a process for the production of drug substances for the SQ HDM SLIT-tablet offering a high reproducibility and independent control of the major allergens. METHODS: Process controls were documented in a comprehensive process parameter qualification. The analyses comprised composition by crossed immunoelectrophoresis, protein content by BCA, total IgE binding potency by Centaur assay, quantitative major allergen determination by radial immunodiffusion and ELISA, and the ranking of emPAI scores generated by mass spectrometry. RESULTS: Analysis of 20 batches of final product yielded a normalized mean and standard deviation for IgE binding potency of 100 ± 4.5. The standard deviation in the contents of Der f 1 and Der p 1 were correspondingly 11.9 and 6.1, whereas the variation in the group 2 major allergen content was 6.4. All measured 95% confidence limits between batches were less than 12%. CONCLUSIONS: The production process for the SQ HDM SLIT-tablet based on the separation of source material into four fractions each enriched in one major allergen enables precise adjustment of the relative major allergen content and high reproducibility of the final product.

Allergy immunotherapy: the future of allergy treatment.

Allergic respiratory disease represents a significant and expanding health problem worldwide. Allergic symptoms, such as asthma and hay fever, cause sleep impairment and reduce school and work performance. The cost to society is substantial. Allergen avoidance and pharmacotherapy cannot control the disease. Only allergy immunotherapy has disease-modifying potential and should be included in optimal treatment strategies. Allergy immunotherapy was first administered as subcutaneous injections and has been practiced for the past 100 years or so. Recently, tablet-based sublingual allergy immunotherapy (SLIT) was introduced with comprehensive clinical documentation. SLIT tablets represent a more patient-friendly concept because they can be used for self-treatment at home.

Absolute quantification of allergens from complex mixtures: a new sensitive tool for standardization of allergen extracts for specific immunotherapy.

Products for specific diagnosis and immunotherapy of IgE-mediated allergies are currently based on natural extracts. Quantification of major allergen content is an important aspect of standardization as important allergens particularly impact vaccine potency. The aim of the study was to develop a mass spectrometry (MS) based assay for absolute quantification of Timothy (Phleum pratense) pollen allergens Phl p 1 and Phl p 5 in P. pratense extract. High-resolution and accurate mass (HRAM) MS was selected for its ability to detect peptides with high selectivity and mass accuracy (<3 ppm). Isotope labeled heavy peptides were used for absolute quantification of specific isoallergens of Phl p 1 and Phl p 5 at low femtomole level in P. pratense extract. Robustness and linearity of the method was demonstrated with intra day precision ≤ 5% (n = 3). Phl p 1b was shown to be 5 times less abundant than its variant Phl p 1a and Phl p 5b was shown to be 9 times more abundant than the Phl p 5a. The present study shows that allergen, and/or isoallergen specific, surrogate signature peptides analyzed with HRAM MS is a sensitive and accurate tool for identification and quantification of allergens from complex allergen sources.

Characteristics of patients receiving allergy vaccination: to which extent do socio-economic factors play a role?

BACKGROUND: Little is known about characteristics of patients receiving allergen-specific immunotherapy. Identifying obstacles to appropriate treatment according to guidelines may facilitate the development of strategies aiming at improved treatment of patients with allergic respiratory diseases. The objective of this study was to investigate differences in disease severity, demographic and socioeconomic status between allergic rhinitis patients receiving allergen-specific immunotherapy and allergic rhinitis patients not receiving allergen-specific immunotherapy. METHODS: A total of 366 patients were studied of whom 210 were going to receive subcutaneously administrated immunotherapy (SIT) against grass pollen and/or house dust mite allergy. The severity of rhino-conjunctivitis (hay fever) and/or asthma was classified according to international guidelines. The questionnaires included an EQ-5D visual analogue scale instrument and some socio-economic questions. RESULTS: Severity of disease, young age, high level of education as well as greater perceived impairment of health-related quality of life due to allergic symptoms were significantly associated with use of SIT. Somewhat unexpectedly, household income was not associated with use of SIT. CONCLUSION: Use of SIT was associated with both disease severity measures and educational level, but not income level. These results suggest social inequality as reflected by lower use of SIT among patients with lower educational level may represent an obstacle to treatment with SIT.

Willingness to pay for allergy-vaccination among Danish patients with respiratory allergy.

OBJECTIVE: The aim of this study was to elicit willingness to pay (WTP) for allergen-specific subcutaneous injection immunotherapy (SCIT) in a cohort of respondents suffering from allergic-rhinoconjunctivitis (a-RC)/asthma, and to investigate how patients self-select to SCIT according to need. METHODS: A random sample of the general population was screened for a-RC/asthma and asked if they were willing to consider SCIT. They were asked to state their WTP for SCIT by way of a discrete choice question (DC-q), an open-ended WTP question (o-WTP-q), and questions relating to their sociodemographic background and the severity of their a-RC/asthma. The characteristics of respondents demanding SCIT were compared with the characteristics of respondents who have actually received SCIT to establish possible barriers to demand. RESULTS: Our results suggest that respondents do well in self-selecting themselves to SCIT on the basis of need according to disease burden measured in terms of a-RC classification, number of contacts with a general practitioner, number of sick days, and potential quality-adjusted life-year loss. Mean WTP for SCIT was estimated at 655 euros (median, 267 euros) (o-WTP-q) and 903 euros (95 percent confidence limit, 348-1,459) (DC-q). CONCLUSION: Characteristics of respondents, who consider SCIT and are willing to pay for SCIT, suggest that allergy sufferers select themselves appropriately according to need and not according to other characteristics, such as income or education. There is a significant discrepancy between those who hypothetically consider SCIT and those demanding SCIT in real life. This study suggests that there are barriers to entry related to age and education, but not to income.

Standardization of allergen extracts.

Allergens are molecules with the capacity to elicit IgE responses in humans. When stimulated with allergens, most allergic patients respond with production of IgE specific for several proteins/allergens in the source material. The standardization of allergen extracts is essential in order to control variability and to achieve consistency and reproducibility in a clinical setting. Because the IgE binding capacity of an allergen extract is related to the content of one or a few major allergens, it is important that the standardization procedure ensures consistency, not only in the overall IgE binding potency, but also in the content and ratio of individual major allergens. Owing to the complexity of allergen extracts, a key element in standardization of allergen extracts is the use of standards. This chapter describes the principles for standardization of allergen extracts to be used by research laboratories. Other chapters in this volume describe methods in detail.

Immunoelectrophoresis for the characterization of allergen extracts.

Immunoelectrophoresis can be used for analysis of individual proteins in complex mixtures. The conditions involved in immunoelectrophoresis are mild, avoiding the risk of denaturation, and it is possible to perform relative quantification of individual components. The principle disadvantage is the dependence on rabbit antisera as reagents. The usefulness of immunoelectrophoresis in allergy research is greatly enhanced by the possibility of identification of allergens to which the individual in question has IgE. The common principle is characterized by two independent electrophoreses having direction of current perpendicular to each other, i.e., crossed immunoelectrophoresis (CIE). This ultimately results in the formation of characteristic bell-shaped precipitates, each precipitate representing one antigen. There is a linear relationship between the amount of antigen and size of precipitate for a given antibody concentration for each precipitate and so relative quantification can be performed. The sensitivity and resolution power of CIE is very high and there are multiple variations of the technique, some of which will be illustrated in this chapter.

Determination of moisture content of lyophilized allergen vaccines by NIR spectroscopy.

Moisture content is an important parameter for lyophilized vaccines. Currently, Karl Fischer titration is widely used for moisture determination in routine analysis. However, this method is time-consuming, sample destructive and requires environment polluting reagents, as well as the results rely on the random samplings. In this study, near infrared spectroscopy was used as a fast, non-invasive and non-destructive method to determine the moisture content in lyophilized allergy vaccines. Five different vaccine products were investigated, which contained water in the range of 0.17-1.51% (w/w, KF). Different data pre-treatments, wavelength selection and partial least squares regression were applied to construct calibration models. Multi-products model and product-specific models were obtained, which show the possibility of NIR as a rapid method to discriminate whether moisture content fit into the specifications of a pharmaceutical company.

Determination of aluminium content in aluminium hydroxide formulation by FT-NIR transmittance spectroscopy.

A method for determining the aluminium content of an aluminium hydroxide suspension using near infrared (NIR) transmittance spectroscopy has been developed. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used as reference method. The factors influencing the NIR analysis, such as different sample containers (transmission cell and cuvette), sedimentation in the suspension, day-to-day variation and batch-to-batch variation have been studied before constructing a calibration model. Seven dilutions (0-4100 mg Al/L) of five batches of aluminium hydroxide suspension samples were measured by NIR transmission each on five different days, with total of 175 spectra used for the calibration set. The multivariate data analysis technique partial least square regression (PLSR) was applied to build the calibration model. Six batches of aluminium hydroxide samples were used for the test set. ICP-AES and NIR transmittance spectroscopy exhibit comparable precision and accuracy. The NIR method provides several advantages: no complicated sample preparation; easy to operate; fast and non-destructive. In conclusion, NIR transmittance spectroscopy can be an alternative analytical method for determining aluminium content in aluminium hydroxide suspension.

Standardization of allergen-specific immunotherapy vaccines.

In the 1970s and 1980s, scientific methods were introduced in the standardization of allergen vaccines and, in combination with improved documentation of the clinical benefits obtained using standardized vaccines, specific allergy treatment as a scientifically based, reproducible, and safe treatment for allergic disease was established. This article describes important issues in the control of source materials and vaccine preparation as part of the European standardization of allergen vaccines, and also includes a discussion of vaccines that are based on recombinant allergens, which may appear on the market in the near future.