Natural human Bet v 1-specific IgG antibodies recognize non-conformational epitopes whereas IgE reacts with conformational epitopes.

BACKGROUND: The nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail. OBJECTIVE: To investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation. METHODS: Sera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients. RESULTS: IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation. CONCLUSION: IgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

Tracing Human IgE B Cell Antigen Receptor-Bearing Cells With a Monoclonal Anti-Human IgE Antibody That Specifically Recognizes Non-Receptor-Bound IgE.

Up to 30% of the population suffers from immunoglobulin E (IgE)-mediated allergies. Despite current stepwise gating approaches, the unambiguous identification of human IgE-producing cells by flow cytometry and immunohistology remains challenging. This is mainly due to the scarcity of these cells and the fact that IgE is not only expressed in a membrane-bound form on the surface of IgE-producing cells in form of the B cell antigen receptor (BCR), but is more frequently found on various cell types bound to the low and high affinity receptors, CD23 and FcϵRI, respectively. Here we sought to develop a sequential gating strategy for unambiguous detection of cells bearing the IgE BCR on their surface. To that aim we first tested the monoclonal anti-IgE antibody omalizumab for its ability to discriminate between IgE BCR and receptor-bound IgE using cells producing IgE or bearing IgE bound to CD23 as well as basophils exhibiting FcϵRI receptor-bound IgE. Using flow cytometry, we demonstrated that omalizumab recognized IgE producing cells with a high sensitivity of up to 1 IgE+ cell in 1000 human peripheral blood mononuclear cells (PBMCs). These results were confirmed by confocal microscopy both in cell suspensions as well as in nasal polyp tissue sections. Finally, we established a consecutive gating strategy allowing the clear identification of class-switched, allergen-specific IgE+ memory B cells and plasmablasts/plasma cells in human PBMCs. Birch pollen specific IgE+ memory B cells represented on average 0.734% of total CD19+ B cells in allergic patients after allergen exposure. Thus, we developed a new protocol for exclusive staining of non-receptor bound allergen-specific IgE+ B cell subsets in human samples.

Differences in Reporting the Ragweed Pollen Season Using Google Trends across 15 Countries.

BACKGROUND: Google Trends (GT) searches trends of specific queries in Google, which potentially reflect the real-life epidemiology of allergic rhinitis. We compared GT terms related to ragweed pollen allergy in American and European Union countries with a known ragweed pollen season. Our aim was to assess seasonality and the terms needed to perform the GT searches and to compare these during the spring and summer pollen seasons. METHODS: We examined GT queries from January 1, 2011, to January 4, 2017. We included 15 countries with a known ragweed pollen season and used the standard 5-year GT graphs. We used the GT translation for all countries and the untranslated native terms for each country. RESULTS: The results of "pollen," "ragweed," and "allergy" searches differed between countries, but "ragweed" was clearly identified in 12 of the 15 countries. There was considerable heterogeneity of findings when the GT translation was used. For Croatia, Hungary, Romania, Serbia, and Slovenia, the GT translation was inappropriate. The country patterns of "pollen," "hay fever," and "allergy" differed in 8 of the 11 countries with identified "ragweed" queries during the spring and the summer, indicating that the perception of tree and grass pollen allergy differs from that of ragweed pollen. CONCLUSIONS: To investigate ragweed pollen allergy using GT, the term "ragweed" as a plant is required and the translation of "ragweed" in the native language needed.

Allergen-Specific Immunotherapy with Monomeric Allergoid in a Mouse Model of Atopic Dermatitis.

Atopic dermatitis (AD) is a widespread and difficult to treat allergic skin disease and is a tough challenge for healthcare. In this study, we investigated whether allergen-specific immunotherapy (ASIT) with a monomeric allergoid obtained by succinylation of ovalbumin (sOVA) is effective in a mouse model of atopic dermatitis. An experimental model of AD was reproduced by epicutaneous sensitization with ovalbumin (OVA). ASIT was performed with subcutaneous (SC) administration of increasing doses of OVA or sOVA. The levels of anti-OVA antibodies, as well as cytokines, were detected by ELISA. Skin samples from patch areas were taken for histologic examination. ASIT with either OVA or sOVA resulted in a reduction of both the anti-OVA IgE level and the IgG1/IgG2a ratio. Moreover, ASIT with sOVA increased the IFN-γ level in supernatants after splenocyte stimulation with OVA. Histologic analysis of skin samples from the sites of allergen application showed that ASIT improved the histologic picture by decreasing allergic inflammation in comparison with untreated mice. These data suggest that ASIT with a succinylated allergen represents promising approach for the treatment of AD.