IgE-facilitated Allergen Binding Research Articles

Single-cell RNA sequencing identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract

Immunologic mechanism of action of allergoids remains poorly understood. Previous models of allergenicity and immunogenicity have yielded suboptimal knowledge of these immunotherapeutic vaccine products. Novel single-cell RNA sequencing technology offers a bridge to this gap in knowledge. We sought to identify the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized Phleum pratense (Phl p) extract. The molecular mechanisms underlying native Phl p, depigmented Phl p (DPG-Phl p), and depigmented-polymerized (DPG-POL-Phl p) allergoid were investigated by single-cell RNA sequencing. Allergen-specific TH2A, T follicular helper (Tfh), and IL-10+ regulatory B cells were quantified by flow cytometry in peripheral blood mononuclear cells from 16 grass pollen-allergic and 8 nonatopic control subjects. The ability of Phl p, DPG-Phl p, and DPG-POL-Phl p to elicit FcεRI- and FcεRII-mediated IgE responses was measured by basophil activation test and IgE-facilitated allergen binding assay. Analysis revealed that DPG-POL-Phl p downregulated genes associated with TH2 signaling, induced functional regulatory T cells exhibiting immunosuppressive roles through CD52 and Siglec-10, modulated genes encoding immunoproteasome that dysregulate the processing and presentation of antigens to T cells and promoted a shift from IgE toward an IgA1 and IgG responses. In grass pollen-allergic subjects, DPG-POL-Phl p exhibited reduced capacity to elicit proliferation of TH2A, IL-4+ Tfh and IL-21+ Tfh cells while being the most prominent at inducing IL-10+CD19+CD5hi and IL-10+CD19+CD5hiCD38intCD24int regulatory B-cell subsets compared to Phl p (all P< .05). Furthermore, DPG-POL-Phl p demonstrated a hypoallergenic profile through basophil activation and histamine release compared to Phl p (31.54-fold, P< .001). Single-cell RNA sequencing provides an in-depth resolution of the mechanisms underlying the tolerogenic profile of DPG-POL-Phl p.

Blocking function of allergen-specific immunoglobulin G, F(ab')2, and Fab antibodies prepared from patients undergoing Dermatophagoides pteronyssinus immunotherapy

BackgroundThe blocking function of allergen-specific F(ab')2 [sF(ab')2] and Fab (sFab) fragment antibodies prepared from allergen immunotherapy–induced specific immunoglobulin G (sIgG) has not been fully investigated. ObjectiveTo investigate the inhibitory function of sIgG, sF(ab')2, and sFab antibodies in patients undergoing Dermatophagoides pteronyssinus (Der-p) subcutaneous immunotherapy (SCIT). MethodsThis study involved 10 subjects (aged 18-42 years) with house dust mite allergic rhinitis or asthma who received a 156-week course of Der-p SCIT. Total IgG levels were purified from the serum of the participants at weeks 0 and 156 by protein A affinity chromatography. Der-p sIgG was purified by affinity chromatography with Der-p as a ligand at week 156. The sF(ab')2 and sFab antibodies were prepared from Der-p sIgG by treatment with pepsin and papain, respectively. Furthermore, IgE-facilitated allergen binding assay, basophil activation inhibition test, and cytokine release inhibition assay were used to assess the inhibitory function of Der-p sIgG, sF(ab')2, and sFab antibodies. ResultsWe found that the Der-p sIgG, sF(ab')2, and sFab antibodies markedly blocked Der-p–allergen sIgE complex binding to B cells, inhibited basophil activation, and markedly reduced the production of interleukin (IL)-5, IL-13, IL-17, and tumor necrosis factor-α by peripheral blood mononuclear cells. ConclusionSCIT-induced Der-p sIgG, sF(ab')2, and sFab antibodies may block the formation of Der-p–sIgE complexes and may serve as a potential allergen-targeted biologics candidate for the treatment of allergic asthma. Clinical Trial RegistrationThis study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University and registered in the Chinese Clinical Trial Registry (ChiCTR-OOC-15006207, https://www.chictr.org.cn/enindex.aspx).

Passive Prophylactic Administration with a Single Dose of Anti-Fel d 1 Monoclonal Antibodies REGN1908-1909 in Cat Allergen-induced Allergic Rhinitis: A Randomized, Double-Blind, Placebo-controlled Clinical Trial.

Rationale: Sensitization to Fel d 1 (Felis domesticus allergen 1) contributes to persistent allergic rhinitis and asthma. Existing treatment options for cat allergy, including allergen immunotherapy, are only moderately effective, and allergen immunotherapy has limited use because of safety concerns.Objectives: To explore the relationship among the pharmacokinetic, clinical, and immunological effects of anti–Fel d 1 monoclonal antibodies (REGN1908–1909) in patients after treatment.Methods: Patients received REGN1908–1909 (n = 36) or a placebo (n = 37) in a phase 1b study. Fel d 1–induced basophil and IgE-facilitated allergen binding responses were evaluated at baseline and Days 8, 29, and 85. Cytokine and chemokine concentrations in nasal fluids were measured, and REGN1908–1909 inhibition of allergen–IgE binding in patient serum was evaluated.Measurements and Main Results: Peak serum drug concentrations were concordant with maximal observed clinical response. The anti–Fel d 1 IgE/cat dander IgE ratio in pretreatment serum correlated with Total Nasal Symptom Score improvement. The allergen-neutralizing capacity of REGN1908–1909 was observed in serum and nasal fluid and was detected in an inhibition assay. Type 2 cytokines (IL-4, IL-5, and IL-13) and chemokines (CCL17/TARC, CCL5/RANTES [regulated upon activation, normal T-cell expressed and secreted]) in nasal fluid were inhibited in REGN1908–1909–treated patients compared with placebo (P < 0.05 for all); IL-13 and IL-5 concentrations correlated with Total Nasal Symptom Score improvement. Ex vivo assays demonstrated that REGN1908 and REGN1909 combined were more potent than each alone for inhibiting FcεRI- and FcεRII (CD23)–mediated allergic responses and subsequent T-cell activation.Conclusions: A single, passive-dose administration of Fel d 1–neutralizing IgG antibodies improved nasal symptoms in cat-allergic patients and was underscored by suppression of FcεRI-, FcεRII-, and T-helper cell type 2–mediated allergic responses.Clinical trial registered with www.clinicaltrials.gov (NCT02127801)

Changes in Immunologic Indicators During Allergen-Specific Immunotherapy for Allergic Rhinitis and Determinants of Variability: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

To date, there are no generally recognized biomarkers for allergen immunotherapy (AIT) and even the changes in immunological indicators during AIT are inconsistent in different publications. This study was conducted to quantify the immunological changes that occur during AIT and identify the determinants of heterogeneity. Randomized controlled trials of AIT published in the past 10 years were searched in Medline, Embase and Cochrane CENTRAL. Data on immunological indicators were extracted, and the characteristics of the included studies were collected. Meta-analysis and meta-regression were conducted for each indicator. The study was registered on the PROSPERO website (CRD42020176127). We reviewed 1898 studies. Forty-six studies met the inclusion criteria, and 31 studies were included in the quantitative analyses. Subset analyses by time demonstrated that serum allergen-specific IgE (sIgE) of AIT patients increased in the first 12 months, then decreased and became slightly lower than that of control patients. Allergen-specific IgG4 (sIgG4) was elevated in the AIT group during and after treatment. IgE-blocking factor (IgE-BF) was increased and IgE-facilitated allergen binding (IgE-FAB) was reduced in AIT patients. Both of them of the 2 factors were associated with clinical efficacy in the multivariate regression analysis. sIgE/sIgG4 decreased in AIT patients, while there was no change in total IgE. The levels of serum sIgE and sIgG4 during AIT showed a time-dependent pattern. IgE-BF and IgE-FAB should be further investigated as biomarkers for predicting and monitoring AIT efficacy.

Broad IgG repertoire in patients with chronic rhinosinusitis with nasal polyps regulates proinflammatory IgE responses

Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by local production of polyclonal IgE idiotypes. Although tissue IgE concentrations can be in the range of several thousand kilounits per liter, the regulatory mechanisms by which IgE-mediated inflammation is controlled in patients with nasal polyps are not well understood. We sought to determine whether locally induced IgG antibodies in patients with nasal polyps can inhibit an IgE-mediated proallergic response. Nasal polyp homogenates were collected from patients with grass pollen allergy with CRSwNP and nonallergic control subjects. IgE levels were measured using the Immuno Solid-phase Allergen Chip assay. IgE-containing nasal polyp homogenates with or without IgG depletion were evaluated for their capacity to promote IgE-facilitated allergen presentation, basophil activation, and histamine release. Local IgE and IgG repertoires were evaluated using Immunoglobulin 454 sequencing. We show that IgG plays a key role in controlling IgE-mediated inflammatory responses in patients with nasal polyps. Depletion of IgG from nasal homogenates resulted in an increase in CD23-mediated IgE-facilitated allergen binding to Bcells but also enhanced FcεRI-mediated allergen-driven basophil activation and histamine release. Asimilar response was observed in relation to specific IgE antibodies to Staphylococcus aureus enterotoxins. The capacity of IgG in nasal polyps to limit IgE-mediated inflammation is based on the fact that IgG repertoires widely share the antigen targets with the IgE repertoires in both allergic and nonallergic subjects. Polyclonal IgE idiotypes in patients with CRSwNP are functional, promote IgE-mediated proallergic inflammation, and are partially antagonized by corresponding IgG idiotypes. This is most likely due to the fact that IgE and IgG clonotypes are widely shared in patients with nasal polyps.

Nasal allergen-neutralizing IgG4 antibodies block IgE-mediated responses: Novel biomarker of subcutaneous grass pollen immunotherapy

Grass pollen subcutaneous immunotherapy (SCIT) is associated with induction of serum IgG4-associated inhibitory antibodies that prevent IgE-facilitated allergen binding to B cells. We sought to determine whether SCIT induces nasal allergen-specific IgG4 antibodies with inhibitory activity that correlates closely with clinical response. In a cross-sectional controlled study, nasal fluid and sera were collected during the grass pollen season from 10 SCIT-treated patients, 13 untreated allergic patients (with seasonal allergic rhinitis [SAR]), and 12 nonatopic control subjects. Nasal and serum IgE and IgG4 levels to Phleum pratense components were measured by using the Immuno Solid Allergen Chip microarray. Inhibitory activity was measured by IgE-facilitated allergen binding assay. IL-10+ regulatory B cells were quantified in peripheral blood by using flow cytometry. Nasal and serum Phl p 1- and Phl p 5-specific IgE levels were increased in patients with SAR compared to nonatopic control subjects (all, P<.001) and SCIT-treated patients (nasal, P < .001; serum Phl p 5, P = .073). Nasal IgG4 levels were increased in the SCIT group compared to those in the SAR group (P<.001) during the pollen season compared to out of season. IgG-associated inhibitory activity in nasal fluid and serum was significantly increased in the SCIT group compared to that in the SAR (both, P<.01). The magnitude of the inhibitory activity was 93% (P < .001) in nasal fluid compared to 66% (P < .001) in serum and was reversed after depletion of IgG. Both nasal fluid (r=-0.69, P=.0005) and serum (r=-0.552, P=.0095) blocking activity correlated with global symptom improvement. IL-10+ regulatory B cells were increased in season compared to out of season in the SCIT group (P<.01). For the first time, we show that nasal IgG4-associated inhibitory activity correlates closely with the clinical response to allergen immunotherapy in patients with allergic rhinitis with or without asthma.

A Recombinant Fragment of Human Surfactant Protein D Suppresses Basophil Activation and T-Helper Type 2 and B-Cell Responses in Grass Pollen-induced Allergic Inflammation.

Recombinant fragment of human surfactant protein D (rfhSP-D) has been shown to suppress house dust mite- and Aspergillus fumigatus-induced allergic inflammation in murine models. We sought to elucidate the effect of rfhSP-D on high-affinity IgE receptor- and CD23-mediated, grass pollen-induced allergic inflammatory responses. rfhSP-D, containing homotrimeric neck and lectin domains, was expressed in Escherichia coli BL21(λDE3)pLysS cells. Peripheral blood mononuclear cells and sera were obtained from individuals with grass pollen allergy (n = 27). The effect of rfhSP-D on basophil activation and histamine release was measured by flow cytometry. IgE-facilitated allergen binding and presentation were assessed by flow cytometry. T-helper cell type 2 (Th2) cytokines were measured in cell culture supernatants. The effect of rfhSP-D on IgE production by B cells when stimulated with CD40L, IL-4, and IL-21 was also determined. rfhSP-D bound to Phleum pratense in a dose- and calcium-dependent manner. Allergen-induced basophil responsiveness and histamine release were inhibited in the presence of rfhSP-D, as measured by CD63, CD203c (P = 0.0086, P = 0.04205), and intracellularly labeled diamine oxidase (P = 0.0003, P = 0.0148). The binding of allergen-IgE complexes to B cells was reduced by 51% (P = 0.002) in the presence of rfhSP-D. This decrease was concomitant with reduction in CD23 expression on B cells (P < 0.001). rfhSP-D suppressed allergen-driven CD27-CD4+CRTh2+ T-cell proliferation (P < 0.01), IL-4, and IL-5 levels (all P < 0.01). Moreover, rfhSP-D inhibited CD40L/IL-4- and IL-21-mediated IgE production (77.12%; P = 0.02) by B cells. For the first time, to our knowledge, we show that rfhSP-D inhibited allergen-induced basophil responses at a single-cell level and suppressed CD23-mediated facilitated allergen presentation and Th2 cytokine production. In addition, rfhSP-D inhibited IgE synthesis by B cells, which is also a novel observation.

Synchronous immune alterations mirror clinical response during allergen immunotherapy

Three years of treatment with either sublingual or subcutaneous allergen immunotherapy has been shown to be effective and to induce long-term tolerance. The Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy (GRASS)trial demonstrated that 2years of treatment through either route was effective in suppressing the response to nasal allergen challenge, although it was insufficient for inhibition 1year after discontinuation. We sought to examine in the GRASS trial the time course of immunologic changes during 2years of sublingual and subcutaneous immunotherapy and for 1year after treatment discontinuation. We performed multimodal immunomonitoring to assess allergen-specific CD4 T-cell properties in parallel with analysis of local mucosal cytokine responses induced by nasal allergen exposure and humoral immune responses that included IgE-dependent basophil activation and measurement of serum inhibitory activity for allergen-IgE binding to B cells (IgE-facilitated allergen binding). All 3 of these distinct arms of the immune response displayed significant and coordinate alterations during 2years of allergen desensitization, followed by reversal at 3years, reflecting a lack of a durable immunologic effect. Although frequencies of antigen-specific TH2 cells in peripheral blood determined by using HLA class II tetramer analysis most closely paralleled clinical outcomes, IgE antibody-dependent functional assays remained inhibited in part 1year after discontinuation. Two years of allergen immunotherapy were effective but insufficient for long-term tolerance. Allergen-specific TH2 cells most closely paralleled the transient clinical outcome, and it is likely that recurrence of the T-cell drivers of allergic immunity abrogated the potential for durable tolerance. On the other hand, the persistence of IgE blocking antibody 1year after discontinuation might be an early indicator of a protolerogenic mechanism.

Local 'Protective' IgG4 Antibodies In Nasal Fluid Are Elevated Following Grass Pollen Immunotherapy

Grass pollen immunotherapy (AIT) is associated with induction of serum IgG4-associated blocking antibodies that prevent IgE-facilitated allergen binding to B cells. We hypothesised that inhibitory IgG4 antibodies are also induced locally in nasal fluid following AIT.

Antigen-Specific TH Cell Monitoring by CD154 Expression in Japanese Cedar-Specific SCIT

M O N D A Y 721 Local Nasal 'Protective' Immunoglobulin G4 (IgG4) Responses in Nasal Fluid Following Grass Pollen Sublingual Immunotherapy Nausheen Saleem, BSc(Hons), Adam Chaker, MD, Ulrich Zissler, Carsten B. Schmidt-Weber, PhD, Stephen R. Durham, MA, MD, FRCP, Mohamed H. Shamji, BSc, MSc, PhD; Imperial College London, Universitatsklinkum Dusseldorf, Dusseldorf, Germany, ENT department, TU Munich, M€unchen, Germany, ENT department, TU Munich, Imperial College, London, United Kingdom, Medical Research Council and Asthma UK Centre for Allergic Mechanisms of Asthma, UK, MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, Imperial College London, United Kingdom. RATIONALE: Grass pollen sublingual immunotherapy (SLIT) is associated with induction of serum IgG4-associated blocking antibodies that prevent IgE-facilitated allergen binding to B cells (IgE-FAB). We hypothesised that local nasal inhibitory IgG4 antibodies are also induced following SLIT. METHODS: Nasal fluids were obtained from SLIT-treated patients (n57), untreated allergics (n57) and non-atopic controls (n58). Specific IgE and IgG4 to Phleum pratense (Phl p) components were measured by ISAC microarray technology. Inhibitory activity of nasal fluid and serum was measured using the IgE-FAB assay. RESULTS: Untreated allergics had elevated levels of rPhl p 1 (p50.01) and rPhl p 5-specific IgE in nasal fluid compared to non-atopic controls. SLIT-treated patients showed a trend for an increase in rPhl p 1 (mean+/SE, 6.062.7 ISU) and rPhl p 5-specific IgE (2.162.6 ISU) compared to untreated allergics (p50.12). Recombinant Phl p 5-specific IgG4 levels were increased in SLIT-treated patients compared to untreated allergics (p50.02) and non-atopic controls (p50.003). Inhibitory activity in nasal fluid for co-operative binding of allergen-IgE complexes to B cells in SLITtreated patients was significantly reduced (p50.004) compared to untreated allergics. These decreases in local nasal inhibitory activity paralleled reductions in serum inhibitory activity after SLIT (p50.03). Interestingly, the magnitude of this suppression was more profound with local antibodies (45%) compared to the peripheral antibody responses (35%). CONCLUSIONS: For the first time, IgG4 antibodies with inhibitory activity for IgE-FAB were measured in the nasal fluid of SLIT treated patients. Local IgG4 antibodies may potentially be used as a surrogate/ predictive biomarker for monitoring SLIT.

Local Nasal 'Protective' Immunoglobulin G4 (IgG4) Responses in Nasal Fluid Following Grass Pollen Sublingual Immunotherapy

Grass pollen sublingual immunotherapy (SLIT) is associated with induction of serum IgG4-associated blocking antibodies that prevent IgE-facilitated allergen binding to B cells (IgE-FAB). We hypothesised that local nasal inhibitory IgG4 antibodies are also induced following SLIT.

Functional rather than immunoreactive levels of IgG4 correlate closely with clinical response to grass pollen immunotherapy

Induction of allergen-specific IgG(4) antibodies is the most consistent immunological finding in immunotherapy trials. However, quantitative assessments of IgG(4) antibodies have not proven beneficial in evaluating clinical changes during or after immunotherapy. In the current study, we investigated the relationship between clinical outcome and allergen-specific IgG(4) titres or functional antibody responses following immunotherapy. We hypothesized that functional assays of serum IgG-associated inhibitory activity such as inhibition of IgE-allergen interactions (IgE-blocking factor) and inhibition of CD23-dependent IgE-facilitated allergen binding (IgE-FAB) correlate more closely with clinical outcome and may be biomarkers of clinical response. In an 8-month dose-response randomized double-blind placebo-controlled study, 221 polysensitized subjects with severe seasonal rhinitis received Alutard SQ, Phleum pratense 100,000 SQ-U, 10,000 SQ-U or placebo injections. Serum specimens were collected before treatment, after up-dosing, during the peak season and at the end of the study. Allergen-specific IgG(4) titres and IgG-associated inhibitory activity were evaluated. A time- and dose-dependent increase in serum inhibitory activity for both the IgE-blocking factor and IgE-FAB was observed, which paralleled increases in grass pollen-specific IgG(4) antibodies. A modest but significant inverse relationship was demonstrated between postimmunotherapy serum inhibitory activity and combined symptom-rescue medication scores (IgE-FAB: r = -0.25, P = 0.0002; IgE-blocking factor: r = -0.28, P < 0.0001), whereas this was not observed for immunoreactive IgG(4) levels (r = -0.11, P = 0.12). Functional assays of inhibitory IgG(4) and IgE-blocking factor may be more useful surrogates of clinical response than IgG(4). Whether these antibody effects may serve as predictive biomarkers of clinical efficacy in individual patients requires further investigation.

Allergen Immunotherapy: A Centenary Celebration

In 1911 and long before the availability of antiallergic drugs, Leonard Noon demonstrated that prophylactic subcutaneous inoculation with a grass pollen extract was effective in suppressing immediate conjunctival sensitivity to grass pollen.1 Noon’s coworker, John Freeman, continued to practice immunotherapy and in 1930 published the first rush immunotherapy protocol.2 William Frankland, a colleague of Freeman, performed the first controlled clinical trial of grass pollen immunotherapy in 1954.3 He used a whole grass pollen extract that was compared with its partially purified proteins, the corresponding ultrafiltrate that contained no protein and a phenol-containing diluent. Both the whole extract and the purified grass pollen proteins were effective compared with the ultrafiltrate and the diluent control alone (Fig. 1). Frankland’s study established a firm scientific foundation for the practice of allergen immunotherapy. Noon, Freeman, and Frankland were all physicians at St. Mary’s Hospital, Paddington, now affiliated to Imperial College London, United Kingdom. Frankland in his 99th year continues to practice and play a major role in the allergy community; much admired and respected by his colleagues, he represents our centenary link with the origins of the practice of immunotherapy (Fig. 2). Major advances in allergen immunotherapy have resulted from parallel studies performed in the United States. Lowell and Franklin in 1964 were the first to clearly demonstrate that a single allergen (ragweed) in a multiallergen mixture was effective in reducing seasonal allergic symptoms.4 Philip Norman and Larry Lichtenstein in 1978 convincingly demonstrated the allergen-specificity of ragweed immunotherapy in patients with dual sensitivity to ragweed and grass pollen.5 Johnstone first highlighted the possibility that immunotherapy in children might confer protection against development of asthma,6 a concept supported by the more recent Preventive Allergy Treatment (PAT) study that identified a 2–3 fold risk reduction for developing asthma after 3 years treatment in children with seasonal pollinosis, protection that persisted for a further 7 years after discontinuation of immunotherapy.7 Hunt and colleagues8 demonstrated the efficacy of purified venom over whole insect body extract and placebo in patients with anaphylaxis to the stings of hymenoptera. Studies9 have confirmed the dose-dependency of allergen immunotherapy whereas the long-term benefits of allergen immunotherapy, with persistence of efficacy for several years after discontinuation10 have been illustrated for both venom11 and grass pollen immunotherapy, the latter both for subcutaneous and sublingual routes of therapy.12 Our increasing knowledge of the mechanisms of immunotherapy has informed both novel approaches and the development of putative biomarkers that might predict the clinical response to immunotherapy. Prausnitz and Kustner13 published in 1921 that a serum factor (‘reagin’) could transfer immediate allergen sensitivity as shown by skin testing was followed Robert Cooke’s observation in 1935 that serum obtained after pollen immunotherapy could confer ‘immunity and hypersensitivity.’14 These seminal observations long preceded the discovery of IgE antibody as reagin by the Ishizakas,15 Johansson and Bennich,16 and the concept of IgG ‘blocking antibodies.’17 The suppressive effect of ragweed immunotherapy on nasal eosinophils as a local marker of allergic inflammation was shown by Creticos in 1984,18 whereas Passalacqua and Canonica similarly demonstrated decreased local eosinophilia and adhesion molecule expression during mite sublingual immunotherapy.19 Warner in 197820 and Rak21 in 1991 observed decreases in allergeninduced late asthmatic responses and associated bronchial inflammation, respectively, in children and adults. A link between altered T-cell responses and immunotherapy was first shown by Rocklin22 whereas the critical role of regulatory T cells and IL-10 was highlighted by Akdis and colleagues.23 The concept of immune deviation of allergenspecific TH2 responses in favor of TH1 responses in both the periphery and in target organs has developed in parallel.24–26 From the Royal Brompton National Heart & Lung Institute, London; and National Jewish Health, Denver, CO. Correspondence to: Stephen R. Durham, MA, MD, FRCP, Royal Brompton Hospital and Imperial College, Dovehouse Street, London SW3 6LY United Kingdom. Telephone: 442073518024. Fax: 442073497780. E-mail: s.durham@ imperial.ac.uk. Copyright © 2011 by World Allergy Organization FIGURE 1. Table II shows the results of the first immunotherapy controlled trial, as published in: Frankland AW, Augustin R. Prophylaxis of summer hay-fever and asthma: a controlled trial comparing crude grass-pollen extracts with the isolated main protein component, The Lancet, May 24, 1954.

Inhibition of CD23‐dependent facilitated allergen binding to B cells following vaccination with genetically modified hypoallergenic Bet v 1 molecules

Vaccination with hypoallergenic recombinant Bet v 1 derivatives (Bet v 1 fragments and Bet v 1 trimer) is associated with the induction of IgG antibodies specific to natural Bet v 1. To investigate whether IgG antibodies induced following vaccination with genetically modified hypoallergenic Bet v 1 derivatives are able to inhibit IgE-facilitated binding of allergen-IgE complexes to B cells. Sera from 46 patients obtained before and after subcutaneous vaccination with Bet v 1 trimer (n=14), Bet v 1 fragments (n=11) or placebo (n=21) were incubated with recombinant (r) Bet v 1 and an indicator serum (IS) from a birch pollen-allergic patient with high CD23 binding capacity. Bet v 1 immune complexes were added to a CD23-expressing B cell line and co-operative binding of Bet v1-IgE complexes to CD23 was measured with a polyclonal anti-IgE FITC antibody using a bio-functional cellular flow cytometric assay. When sera from patients vaccinated with rBet v 1 derivatives were incubated with Bet v 1 and the IS, a reduction of IgE binding to CD23 was observed. This effect was not seen when sera from placebo-treated patients were used. The decrease in CD23/IgE binding was statistically significant in the trimer group [pre- vs. post-specific immunotherapy (SIT): P=0.02; trimer vs. placebo: P<0.04] but not in the Bet v 1 fragments-treated group. Trimer-treated patients had higher levels of Bet v 1-specific IgG than fragment-treated patients. The degree of inhibitory activity of IgE-facilitated allergen binding correlated with Bet v 1-specific IgG levels following SIT (R=0.492; P=0.012). Vaccination with both recombinant Bet v 1 derivatives induces Bet v 1-specific IgG antibodies, which are able to inhibit the co-operative binding of allergen-IgE complexes to CD23, and may thereby reduce allergen-specific T cell responses.

Sublingual grass pollen immunotherapy is associated with increases in sublingual Foxp3‐expressing cells and elevated allergen‐specific immunoglobulin G4, immunoglobulin A and serum inhibitory activity for immunoglobulin E‐facilitated allergen binding to B cells

The mechanisms of sublingual immunotherapy (SLIT) are less well understood than those of subcutaneous immunotherapy (SCIT). To determine the effects of grass-pollen SLIT on oral mucosal immune cells, local regulatory cytokines, serum allergen-specific antibody subclasses and B cell IgE-facilitated allergen binding (IgE-FAB). Biopsies from the sublingual mucosa of up to 14 SLIT-treated atopics, nine placebo-treated atopics and eight normal controls were examined for myeloid dendritic cells (mDCs) (CD1c), plasmacytoid dendritic cells (CD303), mast cells (AA1), T cells (CD3) and Foxp3 using immunofluorescence microscopy. IL-10 and TGF-beta mRNA expression were identified by in situ hybridization. Allergen-specific IgG and IgA subclasses and serum inhibitory activity for binding of allergen-IgE complexes to B cells (IgE-FAB) were measured before, during and on the completion of SLIT. Foxp3(+) cells were increased in the oral epithelium of SLIT- vs. placebo-treated atopics (P=0.04). Greater numbers of subepithelial mDCs were present in placebo-treated, but not in SLIT-treated, atopics compared with normal controls (P=0.05). There were fewer subepithelial mast cells and greater epithelial T cells in SLIT- compared with placebo-treated atopics (P=0.1 for both). IgG(1) and IgG(4) were increased following SLIT (P<0.001). Peak seasonal IgA(1) and IgA(2) were increased during SLIT (P<0.05). There was a time-dependent increase in serum inhibitory activity for IgE-FAB in SLIT-treated atopics. SLIT with grass pollen extract is associated with increased Foxp3(+) cells in the sublingual epithelium and systemic humoral changes as observed previously for SCIT.

Clinical efficacy and immune regulation with peanut oral immunotherapy

Oral immunotherapy (OIT) has been thought to induce clinical desensitization to allergenic foods, but trials coupling the clinical response and immunologic effects of peanut OIT have not been reported. The study objective was to investigate the clinical efficacy and immunologic changes associated with OIT. Children with peanut allergy underwent an OIT protocol including initial day escalation, buildup, and maintenance phases, and then oral food challenge. Clinical response and immunologic changes were evaluated. Of 29 subjects who completed the protocol, 27 ingested 3.9 g peanut protein during food challenge. Most symptoms noted during OIT resolved spontaneously or with antihistamines. By 6 months, titrated skin prick tests and activation of basophils significantly declined. Peanut-specific IgE decreased by 12 to 18 months, whereas IgG(4) increased significantly. Serum factors inhibited IgE-peanut complex formation in an IgE-facilitated allergen binding assay. Secretion of IL-10, IL-5, IFN-gamma, and TNF-alpha from PBMCs increased over a period of 6 to 12 months. Peanut-specific forkhead box protein 3 T cells increased until 12 months and decreased thereafter. In addition, T-cell microarrays showed downregulation of genes in apoptotic pathways. Oral immunotherapy induces clinical desensitization to peanut, with significant longer-term humoral and cellular changes. Microarray data suggest a novel role for apoptosis in OIT.

Grass pollen immunotherapy: IL-10 induction and suppression of late responses precedes IgG4 inhibitory antibody activity

Grass pollen immunotherapy is an effective treatment for seasonal allergic rhinitis that provides the opportunity to study the induction and maintenance of allergen-specific immune tolerance. We investigated the relationship between clinical responsiveness, regulatory cytokine production, and antibody responses to allergen during 1 year of immunotherapy. Eighteen subjects with severe seasonal allergic rhinitis were randomized double-blind to receive active or placebo injections of an alum-adsorbed grass pollen vaccine (Alutard SQ). Subjects underwent repeated testing of early- and late-phase skin responses to intradermal allergen, and cellular responses to grass pollen allergen were tested. Sera were tested for allergen-specific IgG4, IgA, and inhibitory activity in biologic assays of IgE responses. Grass pollen immunotherapy was effective in reducing overall symptom scores (P < .05) and conjunctival reactivity (P < .05). In the active group significant IL-10 production occurred early at low allergen doses and at a similar time as inhibition of late skin responses at 2 to 4 weeks. Serum allergen-specific IgG4, IgA, and inhibitory antibody activity for basophil histamine release and IgE-facilitated allergen binding to B cells occurred later, at 6 to 12 weeks, at higher allergen doses and preceded inhibition of early skin responses. IL-10 responses occur early but at immunotherapy doses that are not clinically effective. Later induction of inhibitory antibodies, including IgG4 and IgA, might be required for efficacy through modulation of IgE-mediated events.

Combination treatment with omalizumab and rush immunotherapy for ragweed-induced allergic rhinitis: Inhibition of IgE-facilitated allergen binding

The combination of anti-IgE (omalizumab) therapy with ragweed injection immunotherapy for seasonal allergic rhinitis results in a significant reduction in systemic side effects and enhanced efficacy compared with immunotherapy alone. One proposed mechanism of immunotherapy is to induce regulatory antibodies that inhibit facilitated antigen presentation. We sought to determine whether the combination protocol has a cumulative effect on inhibition of facilitated antigen presentation both during and after discontinuation of treatment. Ragweed allergen immunotherapy with and without omalizumab therapy was tested in a 4-arm, double-blind, placebo-controlled study. Flow cytometry was used to detect serum inhibitory activity for IgE-facilitated CD23-dependent allergen binding to B cells as a surrogate marker for facilitated antigen presentation. Serum ragweed-specific IgG4 was measured by means of ELISA. Immunotherapy alone resulted in partial inhibition of allergen-IgE binding after 5 to 19 weeks of treatment compared with baseline (P < .01). Complete inhibition of allergen-specific IgE binding was observed in both treatment groups receiving omalizumab (P < .001). Allergen-specific IgG4 levels were only increased after immunotherapy (P < .05), both in the presence and absence of anti-IgE treatment. Combined treatment resulted in the induction of long-lasting inhibitory antibody function for up to 42 weeks compared with either treatment alone. Ragweed immunotherapy induced serum regulatory antibodies that partially blocked binding of allergen-IgE complexes to B cells. Additional treatment with anti-IgE, by directly blocking IgE binding to CD23, completely inhibited allergen-IgE binding. The combination of ragweed immunotherapy and anti-IgE resulted in prolonged inhibition of allergen-IgE binding compared with either treatment alone, events that might contribute to enhanced efficacy.

The IgE-facilitated allergen binding (FAB) assay: Validation of a novel flow-cytometric based method for the detection of inhibitory antibody responses

The IgE-facilitated allergen binding (IgE-FAB) assay represents an in vitro model of facilitated allergen presentation. Allergen–IgE complexes are incubated with an EBV-transformed B cell line and complexes bound to CD23 on the surface of cells are detected by flow cytometry. The addition of serum from patients who have received allergen-specific immunotherapy has been shown previously to inhibit allergen–IgE complex binding to CD23 on B cells. In this study, we describe the characterisation and analytical validation of the grass pollen-specific IgE-FAB assay according to guidelines from the International Conference on Harmonisation. We established the intra- and inter-assay variability of IgE-FAB and have defined the detection limits of this assay. We have also demonstrated assay linearity and robustness. Using the results from a randomised double-blind placebo-controlled trial of grass pollen immunotherapy ( n = 33), we have defined the clinical sensitivity and specificity of the IgE-FAB assay using ROC curve analysis. In conclusion, the IgE-FAB assay is reproducible, robust, sensitive and a specific method suitable as a tool for monitoring inhibitory antibody function from patients receiving allergen immunotherapy.

Validation of a Functional Assay of IgE-Facilitated CD23-Dependent Allergen Binding to B Cells to Monitor Clinical Efficacy of Immunotherapy

RATIONALE: ‘Blocking’ IgG antibodies induced by allergen immunotherapy can inhibit IgE-facilitated allergen binding to B-cells in a simple flow-cytometric assay. (Wachholz,P. et al., JACI 2003,12:915-922). We aimed to validate this assay according to the guidelines from the International Conference on Harmonisation (ICH) to determine its value in monitoring successful immunotherapy.