H. Henmar

Allergenicity, immunogenicity and dose-relationship of three intact allergen vaccines and four allergoid vaccines for subcutaneous grass pollen immunotherapy

Different vaccines containing intact allergens or chemically modified allergoids as active ingredients are commercially available for specific immunotherapy. Allergoids are claimed to have decreased allergenicity without loss of immunogenicity and this is stated to allow administration of high allergoid doses. We compared the allergenicity and immunogenicity of four commercially available chemically modified grass pollen allergoid products with three commercially available intact grass pollen allergen vaccines. The allergenicity was investigated with immunoglobulin (Ig)E‐inhibition and basophil activation assays. Human T cell proliferation and specific IgG‐titres following mouse immunizations were used to address immunogenicity. Furthermore, intact allergen vaccines with different contents of active ingredients were selected to study the influence of the allergen dose. In general, a lower allergenicity for allergen vaccines was clearly linked to a reduced immunogenicity. Compared with the vaccine with the highest amount of intact allergen, the allergoids caused reduced basophil activation as well as diminished immunogenicity demonstrated by reduced T cell activation and/or reduced induction of murine grass‐specific IgG antibodies. Interestingly, intact allergen vaccines with lower content of active ingredient exhibited similarly reduced allergenicity, while immunogenicity was still higher or equal to that of allergoids. The low allergenicity observed for some allergoids was inherently linked to a significantly lower immunogenic response questioning the rationale behind the chemical modification into allergoids. In addition, the linkage between allergenicity, immunogenicity and dose found for intact allergen vaccines and the immunogen as well as allergenic immune responses observed for allergoids suggest that the modified allergen vaccines do not contain high doses of immunologically active ingredients.

Comparison of allergenicity and immunogenicity of an intact allergen vaccine and commercially available allergoid products for birch pollen immunotherapy

Background Specific immunotherapy with intact allergen vaccine is a well‐documented treatment for allergic diseases. Different vaccine formulations are currently commercially available, the active ingredient either being intact allergens or chemically modified allergoids. The rationale behind allergoids is to decrease allergenicity while maintaining immunogenicity. However, data from the German health authorities based on reporting of adverse events over a 10‐year period did not indicate increased safety of allergoids over intact allergens.

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.

Reduced in vitro T-cell responses induced by glutaraldehyde-modified allergen extracts are caused mainly by retarded internalization of dendritic cells

Although allergen‐specific immunotherapy is a clinically effective therapy for IgE‐mediated allergic diseases, the risk of IgE‐mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE‐binding sites while T‐cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T‐cell and basophil activation. For this purpose human monocyte‐derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen‐loaded mature DC were co‐cultured with autologous CD4+ T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin‐4 (IL‐4), IL‐10, IL‐13 and interferon‐γ production by, CD4+ T cells which had been stimulated with glutaraldehyde allergoid‐treated DC was reduced compared with CD4+ T cells stimulated with intact allergen‐treated or formaldehyde allergoid‐treated DC. In line with this, glutaraldehyde‐modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE‐reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde‐modified allergoids also displayed lower T‐cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.

Adjuvant effects of aluminium hydroxide‐adsorbed allergens and allergoids – differences in vivo and in vitro

Allergen‐specific immunotherapy (SIT) is a clinically effective therapy for immunoglobulin (Ig)E‐mediated allergic diseases. To reduce the risk of IgE‐mediated side effects, chemically modified allergoids have been introduced. Furthermore, adsorbance of allergens to aluminium hydroxide (alum) is widely used to enhance the immune response. The mechanisms behind the adjuvant effect of alum are still not completely understood. In the present study we analysed the effects of alum‐adsorbed allergens and allergoids on their immunogenicity in vitro and in vivo and their ability to activate basophils of allergic donors. Human monocyte derived dendritic cells (DC) were incubated with native Phleum pratense or Betula verrucosa allergen extract or formaldehyde‐ or glutaraldehyde‐modified allergoids, adsorbed or unadsorbed to alum. After maturation, DC were co‐cultivated with autologous CD4+ T cells. Allergenicity was tested by leukotriene and histamine release of human basophils. Finally, in‐vivo immunogenicity was analysed by IgG production of immunized mice. T cell proliferation as well as interleukin (IL)‐4, IL‐13, IL‐10 and interferon (IFN)‐γ production were strongly decreased using glutaraldehyde‐modified allergoids, but did not differ between alum‐adsorbed allergens or allergoids and the corresponding unadsorbed preparations. Glutaraldehyde modification also led to a decreased leukotriene and histamine release compared to native allergens, being further decreased by adsorption to alum. In vivo, immunogenicity was reduced for allergoids which could be partly restored by adsorption to alum. Our results suggest that adsorption of native allergens or modified allergoids to alum had no consistent adjuvant effect but led to a reduced allergenicity in vitro, while we observed an adjuvant effect regarding IgG production in vivo.