Allergen-specific immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies

BACKGROUND: Allergen-specific immunotherapy represents a causal form of treatment for IgE-mediated allergies. The allergen extract-based analyses of immunotherapy-induced effects yielded highly controversial results regarding a beneficial role of therapy-induced IgG antibodies. OBJECTIVE: We analysed allergen-specific IgE, IgG subclass, and IgM responses in patients treated with a grass pollen allergy vaccine adjuvanted with monophosphoryl lipid A (MPL), a Th1-inducing agent, and in a placebo group using recombinant timothy grass pollen allergen molecules (rPhl p 1, rPhl p 2, rPhl p 5). RESULTS: The strong induction of allergen-specific IgG1 and IgG4 antibodies observed only in the actively treated group was associated with significant clinical improvement. Therapy-induced allergen-specific IgM and IgG2 responses were also noted in several actively treated patients. An inhibition of allergen-dependent basophil histamine release was only obtained with sera containing therapy-induced allergen-specific IgG, but not with sera obtained before therapy or from placebo-treated patients. Moreover, patients with therapy-induced allergen-specific IgG antibodies showed a reduced induction of allergen-specific IgE responses during seasonal grass pollen exposure. CONCLUSION: Successful immunotherapy with the MPL-adjuvanted grass pollen allergy vaccine is associated with the production of allergen-specific IgG antibodies. These blocking antibodies may have protective effects by inhibiting immediate-type reactions and systemic increases of IgE responses caused by seasonal allergen exposure.     

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.     

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. CLINICAL IMPLICATIONS: 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.     

Comparison of IgE and IgG antibody responses of atopic individuals with sensitization to tree and grass pollens

Sera of atopic individuals with predominant sensitization to either tree pollen (TAs) or tree and grass pollens (TGAs) as well as of nonatopic subjects (NAs) were analyzed for IgE, IgG, and IgG4 antibodies specific for grass pollen allergens. Of 600 atopic individuals with serum IgE antibodies specific for birch pollen allergens, 54% also had serum IgE antibodies specific for grass pollen. The mean titers of IgG antibodies specific for grass pollen proteins were about 10 times higher in the sera of TGAs than those in the TAs and NAs. SDS-PAGE immunoblotting analysis of grass pollen proteins using sera of TGAs, TAs, and NAs with respect to the binding of these proteins with IgE and IgG antibodies in these sera exhibited a similar pattern of variation. Quantitation by enzyme immunoassay of the antibody binding to a recombinant grass pollen allergen, rKBG8.3, further demonstrated that elevated IgG antibody levels in TGAs are mainly due to a broader range of specificities, and not to high specific binding to the individual protein. Statistically significant correlation was found between IgE and IgG4 antibodies specific for the Kentucky bluegrass (KBG) extract, but not for the isolated recombinant allergen. These results indicate that the grass pollens elicit a complex array of antibody specificities in both atopics and nonatopics, and that the profile of antibodies specific to the pollen extract and pure allergens differs, suggesting that single grass allergens may be inadequate for replacing grass pollen extracts for immunotherapy.     

Recombinant pollen allergens from Dactylis glomerata: preliminary evidence that human IgE cross-reactivity between Dac g II and Lol p I/II is increased following grass pollen immunotherapy.

We previously described the isolation of three identical complementary DNA (cDNA) clones, constructed from Orchard/Cocksfoot grass (Dactylis glomerata) anther messenger RNA (mRNA), expressing a 140,000 MW beta-galactosidase fusion protein recognized by IgE antibodies in atopic sera. Partial nucleotide sequencing and inferred amino acid sequence showed greater than 90% homology with the group II allergen from Lolium perenne (Lol II) indicating they encode the group II equivalent, Dac g II. Western blot immunoprobing of recombinant lysates with rabbit polyclonal, mouse monoclonal and human polyclonal antisera demonstrates immunological identity between recombinant Dac g II, Lol p I and Lol p II. Similar cross-identity is observed with pollen extracts from three other grass species: Festuca rubra, Phleum pratense and Anthoxanthum odoratum. Recombinant Dac g II was recognized by species- and group-cross-reactive human IgE antibodies in 33% (4/12) of sera randomly selected from grass-sensitive individuals and in 67% (14/21) of sera from patients receiving grass pollen immunotherapy, whilst 0/4 sera from patients receiving venom immunotherapy alone contained Dac g II cross-reactive IgE. Cross-reactive IgG4 antibodies were detectable in 95% of sera from grass pollen immunotherapy patients. These preliminary data suggest that conventional grass pollen allergoid desensitization immunotherapy may induce IgE responses to a cross-reactive epitope(s) co-expressed by grass pollen groups I and II (and possibly group III) allergens.     

The role of allergen-specific IgE,IgG and IgA in allergic disease

Immunoglobulin E (IgE)-mediated allergy is the most common hypersensitivity disease affecting more than 30% of the population. Exposure to even minute quantities of allergens can lead to the production of IgE antibodies in atopic individuals. This is termed allergic sensitization, which occurs mainly in early childhood. Allergen-specific IgE then binds to the high (FcεRI) and low-affinity receptors (FcεRII, also called CD23) for IgE on effector cells and antigen-presenting cells. Subsequent and repeated allergen exposure increases allergen-specific IgE levels and, by receptor cross-linking, triggers immediate release of inflammatory mediators from mast cells and basophils whereas IgE-facilitated allergen presentation perpetuates T cell–mediated allergic inflammation. Due to engagement of receptors which are highly selective for IgE, even tiny amounts of allergens can induce massive inflammation. Naturally occurring allergen-specific IgG and IgA antibodies usually recognize different epitopes on allergens compared with IgE and do not efficiently interfere with allergen-induced inflammation. However, IgG and IgA antibodies to these important IgE epitopes can be induced by allergen-specific immunotherapy or by passive immunization. These will lead to competition with IgE for binding with the allergen and prevent allergic responses. Similarly, anti-IgE treatment does the same by preventing IgE from binding to its receptor on mast cells and basophils. Here, we review the complex interplay of allergen-specific IgE, IgG and IgA and the corresponding cell receptors in allergic diseases and its relevance for diagnosis, treatment and prevention of allergy.     

Mechanisms of immunotherapy: IgG revisited

PURPOSE OF REVIEW: This paper will review historical and recent evidence for the induction of 'blocking' IgG antibodies during successful specific immunotherapy. RECENT FINDINGS: Specific immunotherapy is frequently associated with a rise in allergen-specific IgG4 antibodies and a modest reduction in specific IgE titres, although this does not always correlate with clinical efficacy. There is accumulating evidence that specific immunotherapy also influences the blocking activity on IgE-mediated responses by IgG4, and cellular assays are commonly used to investigate these changes. Recently, a novel assay, which detects allergen-IgE binding using flow cytometry, has been used to detect 'functional' specific immunotherapy-induced changes in IgG antibody activity. Results suggest that successful specific immunotherapy is associated with an increase in IgG blocking activity that is not solely dependent on the quantity of IgG antibodies. SUMMARY: Successful immunotherapy is associated with quantitative and qualitative changes in the allergen-specific IgG antibody response. The induction of IgG antibodies with blocking activity may have a protective role not only through the inhibition of allergen-induced, IgE-mediated release of inflammatory mediators from mast cells and basophils, but also through the inhibition of IgE-facilitated antigen presentation to T cells. Qualitative changes in the allergen-specific IgG antibody response may possibly be an important mechanism underlying the clinical efficacy of specific immunotherapy. Monitoring changes in blocking activity using cellular assays may give an early indication of the potential success of treatment.     

Reactivity of T cells with grass pollen allergen extract and allergoid.

BACKGROUND: Successful allergen-specific immunotherapy is achieved with progressively increasing doses of allergen or allergoid. In order to gain further insight into the mechanism of action of allergoids several in vitro investigations were conducted. METHODS: Peripheral blood mononuclear cells (PBMC) from grass pollen allergic and nonallergic subjects were stimulated with either grass pollen extract or allergoid and the proliferation and cytokine production (IL-5, IFN-gamma) were measured. Similar investigations were performed with Phl p 5-specific T cell lines (TCL) and clones (TCC). Dendritic cells and PBMC were compared in terms of their relative efficacies as antigen-presenting cells. RESULTS: Both allergen and allergoid induced proliferation and Th2 and Th1 cytokine synthesis by PBMC of allergic subjects, whereas PBMC of nonallergic subjects did not produce IL-5. The maximum level of IL-5 was obtained with a lower concentration than was necessary for maximal IFN-gamma production. Higher stimulation doses of allergen and allergoid shifted the cytokine profiles towards a Th1 phenotype. TCL and TCC clearly showed reactivity with both allergen and allergoid when using autologous PBMC for antigen presentation, but compared with the native allergen the reactivity of the allergoid was reduced with most of the TCC. Using dendritic cells for antigen presentation a pronounced increase of stimulation of the TCC especially for the allergoids becomes obvious. CONCLUSION: In common with grass pollen allergen the corresponding allergoids possess a strong allergen-specific T cell-stimulating capacity. However, the degree of T cell stimulation by the allergoid seems to be dependent on the type of the antigen-presenting cell. Both, allergen and allergoid, can modulate T cell responses in a dose-dependent manner.     

Isolation of high-affinity human IgE and IgG antibodies recognising Bet v 1 and Humicola lanuginosa lipase from combinatorial phage libraries

Allergen-specific Fab fragments isolated from combinatorial IgE and IgG libraries are useful tools for studying allergen-antibody interactions. To characterise the interaction between different allergens and antibodies we have created recombinant human phage antibody libraries in the Fab format. Human IgE and IgG libraries have been created from patients allergic to birch pollen or lipase. These libraries have been used to select binders recognising the major birch pollen allergen Bet v 1 and Humicola lanuginosa lipase. A panel of allergen-specific IgE and IgG antibodies were identified; these were further characterised by allergen binding studies using Biacore and competition studies using human sera and antibodies purified from human sera. Affinities in the nM range were recorded and a competition with human sera for allergen binding was observed.     

Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy.

Whether the modulation of antibody responses can contribute to the improvement of clinical symptoms in patients receiving allergen immunotherapy represents a controversial issue. We have used purified [seven recombinant (r) and one natural] timothy grass pollen allergens as well as recombinant B cell epitope-containing fragments of the major timothy grass pollen allergen, Phl p 1, to investigate humoral immune responses in eight allergic patients receiving grass pollen-specific immunotherapy. We found that the administration of aluminium hydroxide-adsorbed grass pollen extract induced complex changes in allergen/epitope-specific antibody responses: increases in IgG subclass (IgG1, IgG2, IgG4) responses against allergens recognized before the therapy were observed. All eight patients started to mount IgE and IgG4 responses to continuous Phl p 1 epitopes not recognized before the therapy and a de novo induction of IgE antibodies against new allergens was found in one patient. Evidence for a protective role of IgG antibodies specific for continuous Phl p 1 epitopes was provided by the demonstration that preincubation of rPhl p 1 with human serum containing therapy-induced Phl p 1-specific IgG inhibited rPhl p 1-induced histamine release from basophils of a grass pollen-allergic patient. Our finding that immunotherapy induced antibody responses against previously not recognized B cell epitopes indicates the vaccination character of this treatment. The fact that patients started to mount de novo IgE as well as protective IgG responses against epitopes may explain the unpredictability of specific immunotherapy performed with allergen extracts and emphasizes the need for novel forms of component-resolved immunotherapy.     

IgE-mediated allergen presentation and blocking antibodies: regulation of T-cell activation in allergy

It is well established that both the production of IgE by B lymphocytes and the maturation and recruitment of eosinophils in late-phase reactions are dependent on the activation of allergen-specific type-2 T-helper cells. What is less well known is the fact that efficient activation of allergen-specific T cells upon low-dose exposure to allergens is critically dependent on IgE-mediated or -facilitated allergen presentation. In fact, changes in the level of IgE-mediated allergen presentation may account for many of the immunological effects described for specific immunotherapy or anti-IgE treatment. This review aims to summarize the current knowledge, and will discuss the clinical relevance of blocking IgG antibodies induced by specific immunotherapy and anti-IgE monoclonal antibodies that both interfere with IgE-mediated allergen presentation.     

Anti-IgG Antibodies during Immunotherapy with Purified Grass Pollen Extracts

In a double blind study 40 patients were allocated specific immunotherapy (hyposensitization) with partially purified timothy extract or two timothy major allergens 19, 25. All patients had typical grass pollen hay fever, in 27% associated with grass pollen asthma and in 13% with birch pollen allergy. Serum IgG anti-IgG antibodies were determined after dithiothreitol treatment. Before hyposensitization, IgG anti-IgG titres greater than or equal to 9 were demonstrated in 45% of the patients. During hyposensitization IgG anti-IgG titres showed a slight initial increase followed by a decrease below pretreatment level. Neither increase nor decrease was statistically significant. Reactions to rabbit IgG F(ab')2 fractions were only obtained during hyposensitization. The occurrence of anti-IgG antibodies did no correlate with symptoms, side effects, or the level of allergen-specific IgG. In a previous study it was demonstrated that patients with multiallergy hyposensitized with combined allergen extracts showed a statistically significant increase in IgG anti-IgG titres during treatment. The increase failed to appear in the present patients allergic only to pollen and treated with purified allergen extracts. It is therefore suggested that a multiallergic condition and the combination and/or purification of allergen extracts administered during hyposensitization may influence the production of IgG anti- IgG antibodies.     

Vaccination for birch pollen allergy: comparison of the affinities of specific immunoglobulins E, G1 and G4 measured by surface plasmon resonance

BACKGROUND: Allergen-specific immunotherapy (SIT) is associated with increased levels of allergen-specific IgG in serum. However, it is not clear to what extent qualitative changes in the allergen binding capacity of IgG may be induced as well. OBJECTIVE: The purpose of this study was to investigate the influences of SIT on antibody affinity. METHODS: The binding affinity of purified serum IgG1, IgG4 and IgE to the major allergen in birch (Betula verrucosa) pollen, Bet v 1, was analysed by surface plasmon resonance. The antibodies were obtained from 10 birch pollen-allergic patients receiving SIT and from 10 patients with no SIT. RESULTS: The patients having received SIT have a significant higher titre of anti-Bet v 1 antibodies in their blood, but the affinity to Bet v 1 of allergen-specific IgE, IgG1 and IgG4 does not differ between the two groups. For IgG1 and IgG4, correlations between less allergic symptoms and affinity of the antibodies were observed both in the SIT group and to a smaller extent in the non-SIT group. CONCLUSION: SIT has no effect on antibody affinity of allergen-specific IgE, IgG1 or IgG4. Allergic patients with high-affinity IgG1 and IgG4 antibodies report less symptoms than patients with low-affinity antibodies.     

Serum concentrations of allergen-specific IgG antibodies in inhalant allergy: effect of specific immunotherapy

The occurrence of antibodies belonging to IgG class of immunoglobulins with specificity for short ragweed and Bermuda grass in the sera of nonatopic subjects and patients with inhalant allergy (at the time of initial diagnosis, following specific immunotherapy was investigated with an enzyme immunoassay. The intra-assay and inter-assay coefficients of variation for this assay ranged from 1.74%-4.62% and 3.18%-9.12%, respectively. IgG antibodies were found in the sera of the majority of nonatopic and all of atopic subjects. The differences in the concentration of these antibodies between these three groups were statistically significant (P less than 0.001). Specific immunotherapy resulted in a rise in the serum levels of allergen-specific IgG antibodies and, following an initial period of modest increase, a decrease in the level of allergen-specific IgE antibodies. Specific IgG response correlated with both the cumulative antigen dose and the clinical benefit that accrued from specific immunotherapy (P less than 0.001). The increase in the serum concentration of specific IgG was most pronounced in patients with high RAST scores at the time of initial diagnosis (P less than 0.001). The concentrations of short-ragweed specific IgG antibodies assayed with multiple samples in three patients with ragweed hay fever appeared not to be affected by the short-ragweed season to any significant degree. We conclude that direct enzyme immunoassay for allergen-specific IgE and IgG antibodies are useful in vitro monitors of immunologic responses to specific immunotherapy for inhalant allergy.     

Carbohydrate-based particles: a new adjuvant for allergen-specific immunotherapy

The occurrence of systemic anaphylactic side-effects in the course of allergen-specific immunotherapy has been strongly reduced by the adsorption of allergens to aluminium hydroxide, the most frequently used adjuvant in humans. Using the major timothy grass pollen allergen, Phl p 5b, in its recombinant form for immunization of mice, we demonstrate that carbohydrate-based particles (CBP) exhibit several potential advantages over aluminium-hydroxide as adjuvant for immunotherapy. Similar to alum-bound rPhl p 5b, CBP-bound rPhl p 5b induced a stronger antibody and cytokine response than unbound rPhl p 5b after subcutaneous injection in mice. The antibodies induced by CBP-bound rPhl p 5b, exhibited potentially beneficial activities as they cross-reacted with group 5 allergens from five other grass species and inhibited the binding of grass pollen allergic patients IgE to Phl p 5b. Alum-bound rPhl p 5b induced a preferential allergen-specific Th2-response characterized by high immunoglobulin G1 (IgG1) antibody levels and elevated interleukin (IL)-4 and IL-5 production in cultured splenocytes. By contrast, CBP-bound rPhl p 5b, but not rPhl p 5b alone or coadministered with CBP, induced a mixed allergen-specific T helper 1 (Th1)/Th2 immune response characterized by the additional production of allergen-specific IgG2a/b antibody responses and elevated interferon-gamma production. Conjugation of rPhl p 5b to CBP yielded a stable vaccine formulation with preserved immunogenic features of the allergen and, in contrast to alum, induced no granulomatous tissue reactions. Based on these results, CBP is suggested as a potentially useful adjuvant for specific immunotherapy of IgE-mediated allergies.     

Relationship between facilitated allergen presentation and the presence of allergen-specific IgE in serum of atopic patients.

Allergen presentation to allergen-specific T cells can be facilitated when IgE-allergen complexes are endocytosed by antigen-presenting cells (APC) after binding to the low-affinity Fc epsilon R type II (CD23). Here we present a study on the relative capabilities of sera of atopic patients to mediate facilitated antigen presentation (FAP). To this aim FAP was studied in an in vitro model in which CD23-expressing Epstein-Barr virus (EBV)-B cells act as APC to T lymphocyte clones (TLC) that are specific for Der p 2, a major allergen of housedust mite Dermatophagoides pteronyssinus (Dp). Der p 2 is immune-complexed by preincubation in sera from atopic patients, containing allergen-specific IgE. If EBV-B cells are preincubated with these complexes before using the cells as APC, the allergen-specific TLC proliferate at 100-1000-fold lower allergen concentration than required for T cell activation after presentation of uncomplexed allergen. The relative capability of various sera to mediate FAP was correlated with total serum IgE, and especially with Der p 2-specific serum IgE. In the model used, a high FAP capacity could be demonstrated only in sera with a total serum IgE concentration above approximately 2 micrograms/ml or with Der p 2-specific IgE above approximately 100 ng/ml. Maximal FAP, i.e. the ability to induce maximal proliferation of the TLC, was obtained in the presence of more than +/- 600 ng Der p 2-specific IgE/ml. At 100-600 ng/ml Der p 2-specific IgE the level of FAP was correlated with the concentration of allergen-specific IgE, whereas at lower concentrations FAP was low or absent. All tested sera from eczema patients, all having serum anti-Der p 2-IgE concentrations > 600 ng/ml, showed a high FAP capacity, whereas all tested sera from atopic patients without eczema, which had serum anti-Der p 2-IgE levels < 600 ng/ml, showed no or a low FAP capacity. The association of high FAP capacity with eczema may reflect a functional role of FAP in the pathogenesis of atopic dermatitis.     

Induction of IL-10+CD4+CD25+ T cells by grass pollen immunotherapy

BACKGROUND: Immunotherapy involves the modulation of allergen-specific T-cell responses, either T(H)2-to-T(H)1 immune deviation or, in bee venom-treated patients, induction of IL-10 production by CD4+CD25+ T cells. IL-10-producing CD4+CD25+ regulatory T cells have emerged as potential mediators of immune tolerance in numerous murine models of immunopathology. OBJECTIVE: The aim of this study was to evaluate the role of IL-10 production and CD4+CD25+ T cells in the response to grass pollen immunotherapy. METHODS: PBMCs were isolated from patients after 1 year of grass pollen immunotherapy and from matched untreated atopic and healthy control subjects. After 6 days of in vitro stimulation with Phleum pratense, production of IL-10, IL-5, IL-4, and IFN-gamma and proliferation and numbers of CD4+CD25+ T cells were measured. T cells were then stimulated for a further 5 hours with phorbol 12-myristate 13-acetate and ionomycin and assessed for intracellular IL-10 by means of flow cytometry. RESULTS: Patients undergoing immunotherapy produced significantly more IL-10 than atopic control subjects (patients undergoing immunotherapy, 116 +/- 21 pg/mL [n = 11]; atopic patients, 30 +/- 5 pg/mL [n = 11]; P <.001), and the number of CD4+CD25+ cells identified after allergen stimulation was also greater in the immunotherapy group. The numbers of CD4+CD25+ T cells correlated positively with activation as measured by proliferation in both of the control groups but not in the immunotherapy group. Moreover, only T cells from patients undergoing immunotherapy were positive for intracellular IL-10, and these were almost exclusively CD4+CD25+ cells. CONCLUSION: Grass pollen immunotherapy results in a population of circulating T cells that express the IL-10(+) CD4+CD25+ phenotype in response to allergen restimulation.     

Systemic immunological changes induced by administration of grass pollen allergens via the oral mucosa during sublingual immunotherapy

Twenty-four patients suffering from grass pollen allergy underwent sublingual immunotherapy (SLIT) with standardized grass pollen extract for 1 year. In order to investigate immunological changes induced by the administration of allergens via the oral mucosa, the SLIT-spit method was applied. The cumulative dose of approximately 80 microg of major allergen (grass group 5 allergen), was relatively low. During the time of treatment, we could observe a significant increase in the levels of specific IgG and IgG4 antibodies. However, the titers of allergen-specific IgE antibodies showed a significant increase in the course of SLIT as well. Analyzing lymphoproliferative responses, a significant decrease in reactivity in response to stimulation with complete grass pollen extract (p = 0. 001) and to recombinant Phl p 1 (a major allergen of timothy grass, p<0.001) could be observed, indicating the induction of immunological tolerance. Proliferative responses to a control antigen (tetanus toxoid) were not influenced by the treatment. At different time points during SLIT, allergen (Phl p 1)-specific T cell clones (TCC) were established from the peripheral blood of the patients. Cytokine production by allergen-stimulated T cells did not reveal any changes consistent with immune deviation, i.e. the ratio of Th1/Th2 TCC did not change during SLIT. In conclusion, we provide evidence that sublingual treatment leads to systemic changes in immunoreactivity to the administered allergen.     

Expression of a human IgG4 antibody, BAB2, with specificity for the major Birch pollen allergen, Bet v 1 in Escherichia coli: recombinant BAB2 Fabs enhance the allergic reaction

BACKGROUND: Antigen recognition by antibodies of different isotypes can result in completely different effects as exemplified by Type I allergy. While the IgE-antibody-mediated release of biological mediators constitutes the immunopathological basis for the immediate symptoms observed in allergic patients, allergen-specific IgG antibodies are thought to have protective effects. METHODS: Cell lines secreting five human monoclonal IgG antibodies (BAB1-BAB5) with specificity for the major birch pollen allergen Bet v 1 were established from a birch-pollen-allergic patient who had received birch- pollen-specific immunotherapy. The influence of the Bet v 1-specific IgG antibodies on IgE binding to Bet v 1 was investigated. BAB2 was expressed in Escherichia coli as recombinant Fab, purified and tested for its ability to modulate Bet v 1-induced immediate-type skin reactions. RESULTS: The BAB antibodies belonged to different IgG subclasses (BAB1: IgG1; BAB2, BAB3, BAB5: IgG4; and BAB4: IgG2) reflecting a tendency towards Th2. BAB1 represented the only antibody which strongly blocked IgE binding to Bet v 1, whereas BAB 3-BAB5 had little effect on IgE binding. Surprisingly, natural BAB2 antibodies as well as recombinant BAB2 Fabs strongly enhanced IgE binding to Bet v 1 and Bet v 1-induced immediate-type skin reactions and thus represent 'enhancing antibodies'. CONCLUSION: The demonstration that anti-allergen IgG antibodies can also enhance IgE binding to a given allergen explains the unpredictability of specific immunotherapy as well as the controversy on the role of IgG in atopy.     

Humanized anti-IgE mAb Hu-901 prevents the activation of allergen-specific T cells

BACKGROUND: As a result of the very efficient capture of allergens by IgE that focuses to CD23 on B cells or FcepsilonRI on dendritic cells, allergen-specific T cells can be activated after exposure to very low levels of allergens. This IgE-mediated allergen presentation is 100- to 1,000-fold more efficient than fluid phase endocytosis. The aim of the present study was to determine whether humanized anti-IgE mAb Hu-901 can prevent the activation of allergen-specific T cells by inhibiting IgE-mediated allergen presentation. METHODS: A house dust mite major allergen Der p 1-specific T cell line was generated from an allergic asthma patient, and a model was set up to show IgE-facilitated allergen presentation via CD23 on EBV-transformed B cells. In addition, experiments were performed by FACS analysis, detecting the presence of IgE-allergen complexes bound to EBV-B cells by polyclonal FITC-labeled anti-IgE antisera. RESULTS: The anti-IgE mAb Hu-901 inhibited proliferation of allergen-specific T cells at low allergen concentrations. Inhibition was dose-dependent. This effect could be explained by Hu-901 inhibition of binding of allergen-IgE complexes to CD23 expressed on EBV-transformed B lymphocytes. CONCLUSIONS: These data clearly indicate that anti-IgE antibodies for the treatment of allergy exert their effect not only by inhibiting mast cell/basophil degranulation, but also by preventing T cell activation, which possibly explains the effect of anti-IgE treatment on late-phase reactions noted in clinical studies.