Simplified AIT for allergy to several tree pollens-Arguments from the immune outcome analyses following treatment with SQ tree SLIT-tablet.

BACKGROUND: The SQ tree SLIT-tablet (containing birch extract) proved clinically significant effects during the pollen season for birch as well as alder/hazel. Immune outcomes of this treatment for allergens from multiple birch homologous trees need further investigation. We hypothesize that birch pollen extract AIT modulates a highly cross-reactive immune response and that this may be the basis for the observed clinical cross-protection. METHODS: Blood samples were collected from 397 birch allergic patients during SQ tree SLIT-tablet or placebo treatment (1:1) for up to 40 weeks. Serum IgE and IgG4 specific to birch, and birch homologous tree pollens from alder, hazel, hornbeam, beech and chestnut were measured by ImmunoCAP. IgE-Blocking Factor (IgE-BF) for alder, birch and hazel during treatment was measured by Advia Centaur and blocking effects for birch and all these birch homologous tree pollens were further investigated by basophil activation (BAT). Antibody readouts were investigated in patient subsets. T-cell responses (proliferation) to allergen extracts and peptide pools (group 1 allergens) were investigated in T-cell lines from 29 untreated birch pollen-allergic individuals. RESULTS: Significant Pearson correlations between serum IgE towards birch, alder, hazel, hornbeam and beech were observed (r-values > .86). T-cell reactivity was observed throughout the birch homologous group. Almost identical kinetics for changes in IgE towards birch, alder and hazel were observed during treatment and similar species-specific changes were seen for serum-IgG4 . IgG4 reactivity towards birch and alder, hazel, hornbeam and beech correlated significantly at end-of-treatment (r-values > .72). Treatment resulted in similar IgE-BF kinetics for alder, birch, and hazel and blocking of BAT for multiple trees in most actively treated patients investigated. CONCLUSIONS: Systematic analyses of T-cell and antibody cross-reactivities before and during birch pollen extract AIT provide the immunological basis for the observed clinical effect of SQ tree SLIT-tablet treatment of tree pollen allergy induced by multiple trees in the birch homologous group.

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.

Functional and Immunoreactive Levels of IgG4 Correlate with Clinical Responses during the Maintenance Phase of House Dust Mite Immunotherapy.

Allergen-specific immunotherapy for house dust mite allergy is effective, but there are no validated biomarkers reflecting or predicting the clinical efficacy. We aimed to investigate the relationship between clinical outcomes and functional responses of allergen-specific IgG4 (sIgG4) and specific IgE (sIgE) during Dermatophagoides pteronyssinus s.c. allergen immunotherapy (SCIT) in allergic rhinitis and/or asthma patients. Combined symptom medication scores (SMS), D. pteronyssinus-sIgG4 levels, D. pteronyssinus-sIgE levels, and the serum inhibitory capacity against D. pteronyssinus-sIgE facilitated allergen binding to B cells (IgE-FAB) were determined during the updosing (week 0, 4, 12, and 16) and maintenance (week 52, 104, and 156) phase of SCIT. We found that SCIT patients had a significant improvement in SMS from week 52 to 156 compared with medication-treated control subjects (p < 0.05). Levels of D. pteronyssinus-sIgG4 in SCIT patients showed a significant increase from week 12 to 156 (p < 0.05). Serum obtained from SCIT patients significantly inhibited D. pteronyssinus-sIgE binding to B cells after 16 wk (p < 0.01). Significantly lower levels of D. pteronyssinus-sIgE were observed in SCIT patients after 52 wk (p < 0.05). A significant relationship was demonstrated between SMS and IgE-FAB or D. pteronyssinus-sIgG4 during the maintenance phase according to linear regression analysis. In conclusion, D. pteronyssinus-sIgG4 level and D. pteronyssinus IgE-FAB are associated with clinical efficacy in the maintenance phase rather than the updosing phase of SCIT. Immunologic tolerance can be induced with SCIT when maintenance phase is achieved.

A birch sublingual allergy immunotherapy tablet reduces rhinoconjunctivitis symptoms when exposed to birch and oak and induces IgG4 to allergens from all trees in the birch homologous group.

BACKGROUND: This randomized, double-blind trial was conducted to determine the optimal dose for clinical efficacy of the SQ tree SLIT-tablet. An environmental exposure chamber (EEC) was used to reduce variability of allergen exposure and allow investigation of symptom reduction towards different species from the birch homologous group in separate EEC sessions. METHODS: Eligible subjects (N = 219) were randomized to receive treatment with placebo or the SQ tree SLIT-tablet (2, 7, or 12 DU) for 24 weeks. EEC pollen challenges were conducted outside the birch pollen season and included four birch and two oak EEC sessions. The primary efficacy endpoint was the average allergic rhinoconjunctivitis (ARC) total symptom score (TSS) after 24 weeks of treatment. RESULTS: There was a statistically significantly lower TSS during the 24-week birch EEC session for 7 DU and 12 DU compared to placebo with relative differences of 24% (P = 0.03) and 25% (P = 0.02). For the 24-week oak EEC session, there was a statistically significant difference for 12 DU (24%, P = 0.03). IgE and IgG4 measurements supported these findings and demonstrated cross-reactivity to all other species within the birch homologous group. Treatment was well-tolerated with the most frequently reported adverse reactions being the local reactions in the oral cavity of mild-to-moderate severity. CONCLUSION: This trial demonstrates that the SQ tree SLIT-tablet reduce ARC symptoms triggered by birch or oak pollen. The optimal dose for further development was 12 DU. Clinical and immunological findings suggest that the tablet may be used to treat allergies to all species within the birch homologous group.

Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

Pretreatment IgE sensitization patterns determine the molecular profile of the IgG4 response during updosing of subcutaneous immunotherapy with timothy grass pollen extract.

BACKGROUND: Allergen immunotherapy is an effective treatment of allergic rhinoconjunctivitis. Clinical efficacy is associated with improvement of basophil sensitivity and an increase in allergen-specific immunoglobulin concentration. OBJECTIVE: We sought to determine whether changes in allergen component-specific serum IgE and IgG4 levels during the updosing phase of subcutaneous immunotherapy (SCIT) are biomarkers of the immunologic changes that can lead to treatment efficacy. METHODS: Twenty-four subjects with grass pollen-induced allergic rhinoconjunctivitis were randomized 3:1 to receive SCIT (Alutard SQ) or to an open control group. IgE and IgG4 concentrations were determined for the major allergens Phl p 1 or Phl p 5 by using ImmunoCAP and for 8 grass pollen molecules by using Immuno Solid-phase Allergy Chip (ISAC) before treatment and after updosing. RESULTS: Levels of specific IgE against the dominant major allergens Phl p 1 and Phl p 5 increased from a mean of 23.0 to 48.8 kU/L (P = .01, n = 18) during the updosing phase in ImmunoCAP measurements but decreased from a median of 4.6 ISAC specific units (ISU) to 2.14 ISU (P < .0001, n = 102) when measured by using ISAC against 8 grass allergen components. The updosing phase induced a specific IgG4 level increase from a median of 0 ISU before treatment to 0.83 ISU after 12 weeks (P < .0001, n = 102) but only for allergen molecules to which pretreatment-specific IgE antibodies were detected (Spearman σ = 0.72, P < .0001, n = 102). CONCLUSION: Pretreatment allergen component-specific IgE appears to determine the induction of IgG4 in the updosing phase. Induced IgG4 seems to suppress IgE levels on ISAC, resulting in a marked decrease in ISAC-measured specific IgE levels after updosing of SCIT. Thus this decrease in ISAC IgE levels can be used to monitor the blocking effect of allergen-specific immunotherapy-induced non-IgE antibodies.

Human leukocyte antigen-G and regulatory T cells during specific immunotherapy for pollen allergy.

BACKGROUND: TH2-biased immune responses are important in allergy pathogenesis. Mechanisms of allergen-specific immunotherapy (SIT) might include the induction of regulatory T cells (Tregs) and immunoglobulin (Ig) G4 blocking antibodies, a reduction in the number of effector cells, and skewing of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4). METHODS: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with pollen extract in vitro and immune factors were evaluated. RESULTS: During SIT, the main changes in the peripheral blood were an increase in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Tregs and a decrease in CCR4(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in allergen-specific IgG4, and a decrease in sHLA-G during the first half of the treatment period. In the PBMC in vitro experiments, the following changes were observed upon allergen-stimulation: an increase in CD4(+)CD25(+)CD127(low/-) Tregs and ILT2(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in IL-10 and IL-2 levels, and an increase in sHLA-G that was most pronounced at the start of SIT. CONCLUSIONS: The changes in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Treg, IgG4, and sHLA-G levels in the peripheral blood and in ILT2(+) Treg, IL-10, IL-2, and sHLA-G levels upon in vitro allergen stimulation suggest an upregulation in immunomodulatory factors and, to some degree, a shift towards TH1 during SIT.

T cells specific to multiple Bet v 1 peptides are highly cross-reactive toward the corresponding peptides from the homologous group of tree pollens.

Background: Allergens from Fagales trees frequently cause spring allergy in Europe, North America, and some parts of Asia. The definition of the birch homologous group, which includes birch (Bet v), oak (Que a), alder (Aln g), hazel (Cor a), hornbeam (Car b), beech (Fag s), and chestnut (Cas s), is based on high allergen sequence identity and extensive IgE cross-reactivity. Clinical effect was seen during the alder/hazel, birch, and oak pollen seasons after treatment with tree SLIT-tablets containing only birch allergen extract. Here, we characterize T-cell reactivity with respect to epitope specificities and cross-reactivity toward various Bet v 1 family members, (PR-10/group 1 major allergens). This cross-reactivity may be part of the immunological basis of clinical effect or cross-protection when exposed to birch homologous tree species. Method: T-cell lines were generated from 29 birch-allergic individuals through stimulation of peripheral blood mononuclear cells (PBMCs) with birch/Bet v or oak/Que a allergen extracts. T-cell responses to allergen extracts, purified group 1 allergens, and overlapping 20-mer peptides (Bet v 1, Aln g 1, Cor a 1, and Que a 1) were investigated by T-cell proliferation and cytokine production. Cross-reactivity was evaluated based on Pearson's correlations of response strength and further investigated by flow cytometry using tetramer staining for homologous peptide pairs. Results: T-cell reactivity toward extracts and group 1 allergens from across the birch homologous group was observed for birch/Bet v as well as oak/Que a T-cell lines. T-cell lines responded to multiple Bet v 1 homologous peptides from Aln g 1 and Cor a 1 and a subset of Que a 1 peptides. Significant Pearson's correlations between frequently recognized peptides derived from Bet v 1 and the corresponding peptides derived from alder, hazel, and oak strongly supported the T-cell cross-reactivity toward these allergens. Cross-reactivity between birch and birch homologous peptides was confirmed by pMHCII tetramer staining. Conclusion: T cells from birch tree pollen allergic individuals respond to multiple trees within the birch homologous group in accordance with the level of sequence homology between Bet v 1 family members, (PR-10 allergens) from these allergen sources, confirming the basis for clinical cross-protection.