Variability in German Cockroach Extract Composition Greatly Impacts T Cell Potency in Cockroach-Allergic Donors

Abstract

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy.

Development and Validation of a Bordetella pertussis Whole-Genome Screening Strategy

“…After 14 days in culture with the BP lysate, the expanded T cells were tested for the recognition of the epitope pools or individual peptides as described above. As readout, the standard IFNγ/IL-5 and IL-17/IL-9 cytokine combination for ELISpot assay or IFNγ/IL-5/IL-13 cytokine combination for FluoroSpot assay was performed as described [9,34]. For ex vivo determinations, the same combination of cytokines was used after 20 h incubation with lysate (10 μg/mL), epitope pools (2 μg/mL), or individual peptides (10 μg/mL) besides PHA (2 μg/mL) and DMSO as positive and negative controls, respectively.…”

Analysis of Allergen-Specific T Cell and IgE Reactivity to Different Preparations of Cow’s Milk-Containing Food Extracts

“…It has to be highlighted that in this study, we have only tested one batch per food extract. Variability in extract content has been reported for commercial extracts [25,35,36] and it is likely that reactivity reported herein will vary further when different extract batches are tested.…”

“…IL-5 and IFNγ production after CM and CM-containing food extract stimulation was measured by Fluorospot assay as described in another study [25]. In vitro expanded cells (1 × 10 5 cells/well) were restimulated with the same extract as the original culture at 250 µg/mL, medium alone and PHA (10 µg/mL) as negative and positive controls, respectively.…”

Measurement of German cockroach allergens and their isoforms in allergen extracts with mass spectrometry

“…Conventional whole‐body GCr allergen source materials used by US manufacturers contain male and female insects, egg cases and faeces. Our analysis confirms 7 that the specific allergen content of a GCr allergen extract will depend on the relative proportions of these specific components. Extracts derived from source materials rich in faeces will contain more Bla g 1 and Bla g 11, whereas extracts from adult male whole bodies and adult female whole bodies/eggs will contain more Bla g 4 and vitellogenin, respectively.…”

“…German cockroach (GCr; Blattella germanica ) is an indoor pest and important source of allergens associated with asthma and allergic rhinoconjunctivitis 5 . Commercial allergen extracts for GCr are non‐standardized, and several studies have confirmed low biological potency and a lack of consistency among products manufactured by different companies 6,7 . Further, although 10 GCr allergens have been identified (http://www.allergen.org), none of them is immunodominant 8,9 …”