Nasal challenges with recombinant derivatives of the major birch pollen allergen Bet v 1 induce fewer symptoms and lower mediator release than rBet v 1 wild-type in patients with allergic rhinitis

BACKGROUND: Genetic engineering of the major birch pollen allergen (Bet v 1) has led to the generation of recombinant Bet v 1 derivatives with markedly reduced IgE-binding capacity, but with retained T cell activating ability. OBJECTIVE: To compare the mucosal reactivity to rBet v 1 derivatives with rBet v 1 wild-type as basis for new therapeutic strategies for birch pollen allergy based on mucosal tolerance induction. METHODS: Outside the pollen season, 10 patients with birch pollen allergic rhinitis and mild asthma underwent four nasal challenge-sessions in a randomized, double-blind, and cross-over design, employing increasing doses of rBet v 1 fragment mix, rBet v 1 trimer, rBet v 1 wild-type and diluent (albumin). Nasal lavage fluids (NAL) were collected before the challenge-series as well as 10 min, 4 and 24 h thereafter. Nasal lavage fluid levels of tryptase as well as EPO and ECP were measured as indices of mast cell and eosinophil activity, respectively. RESULTS: All 10 patients tolerated the highest accumulated dose, 8.124 microg, when challenged with rBet v 1 trimer, eight with rBet v 1 fragments compared to one when challenged with rBet v 1 wild-type. No late phase reactions were observed. The change in tryptase levels (pre-challenge vs. 10 min) was significantly lower after challenges with rBet v 1 trimer and rBet v 1 fragments than with rBet v 1 wild-type. The change in EPO/ECP concentration pre-challenge versus 4 h post-challenge was lower for rBet v 1 trimer and the change was significantly lower when pre-challenge versus 24 h post-challenge to rBet v 1 fragments and rBet v 1 wild-type was examined. CONCLUSION: The derivatives induced significantly fewer symptoms and lower mast cell and eosinophil activation than rBet v 1 wild-type upon application to the nasal mucosa. They could in the future be candidates for immunotherapy based on mucosal tolerance induction.     

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives

Background Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe. Objective To investigate the clinical effects of injection immunotherapy with genetically modified derivatives of major birch pollen allergen Bet v 1 on pollen‐induced allergic symptoms. Methods A three‐arm double‐blind placebo‐controlled immunotherapy study was conducted with one pre‐seasonal course of treatment using two derivatives of Bet v 1, namely a recombinant Bet v 1 trimer and an equimolar mixture of two recombinant Bet v 1 fragments together representing the whole protein sequence. Analysis of local and systemic adverse events was performed for 124 patients who had received at least one dose of medication. Clinical efficacy was monitored by symptom medication scores and interval scoring in the per protocol‐treated population (n=84). In addition, skin and nasal provocation responses and allergen‐specific antibodies were assessed. Results There were trends towards improvement in the subjects' well‐being and clinical symptoms (nasal scores), although comparisons with a placebo group did not show statistical significance in the main end‐point, the combined symptom medication score. Reductions in skin and nasal sensitivity were observed for some subjects with a trend for the Bet v 1 trimer to be more effective than the fragments. Treatment induced strong IgG1 and IgG4 allergen‐specific antibody responses. Local injection‐site reactions were most frequent in the trimer group affecting 59.5% of patients as opposed to 37.8% and 30.6% in the fragment and placebo groups, respectively. Systemic reactions were elicited more frequently by fragments. A large proportion of adverse side‐effects appeared hours following injections, and might be attributable to concurrent exposure to related pollens. Conclusion Single courses of injection immunotherapy with Bet v 1 allergen derivatives showed trends towards improved well‐being and reduced reactivity to specific allergen provocation, but did not yield significant improvement in the combined symptom medication score in this study.     

Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation: a study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin E detection system measuring immunoglobulin E capable of binding to FcɛRI

BACKGROUND: Results from several studies indicate that the magnitude of immediate symptoms of type I allergy caused by allergen-induced cross-linking of high-affinity Fc epsilon receptors on effector cells (mast cells and basophils) is not always associated with allergen-specific IgE levels. OBJECTIVE: To investigate the association of results from intradermal skin testing, basophil histamine release and allergen-specific IgE, IgG1-4, IgA and IgM antibody levels in a clinical study performed in birch pollen-allergic patients (n = 18). METHODS: rBet v 1-specific IgEs were measured by quantitative CAP measurements and by using purified Fc epsilon RI-derived alpha-chain to quantify IgE capable of binding to effector cells. Bet v 1-specific IgG subclasses, IgA and IgM levels were measured by ELISA, and basophil histamine release was determined in whole blood samples. Intradermal skin testing was performed with the end-point titration method. RESULTS: Our study demonstrates on the molecular level that the concentrations of allergen-specific IgE antibodies capable of binding to Fc epsilon RI and biological sensitivities are not necessarily associated. A moderate association was found between cutaneous and basophil sensitivity. CONCLUSION: Our results highlight the quantitative discrepancies and limitations of the present diagnostic tools in allergy, even when using a single allergenic molecule. The quantity of allergen-specific serum IgE is only one component of far more complex cellular systems (i.e. basophil-based tests, skin tests) used as indirect diagnostic tests for IgE-mediated allergic sensitivity.     

Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1

BACKGROUND: We have recently engineered recombinant derivatives of the major birch pollen allergen Bet v 1 (rBet v 1 fragments and trimer) with strongly reduced allergenic activity. OBJECTIVE: The aim of this study was the in vivo characterization of potential allergy vaccines based on Al(OH)3-adsorbed genetically modified rBet v 1 derivatives in mice. METHODS: BALB/c mice were immunized either with courses of nine injections of increasing doses of Al(OH)3-adsorbed rBet v 1 wild-type, rBet v 1 fragments, rBet v 1 trimer or Al(OH)3 alone in weekly intervals or with three high-dose injections applied in intervals of 3 weeks. Humoral immune responses to rBet v 1 wild-type and homologous plant allergens were measured by ELISA and Western blotting, and the ability of mouse antibodies to inhibit the binding of allergic patients IgE to Bet v 1 was studied by ELISA competition experiments. RESULTS: In both schemes, hypoallergenic rBet v 1 derivatives induced low IgE but high IgG1 responses against rBet v 1 wild-type. The IgG1 antibodies induced by genetically modified rBet v 1 derivatives cross-reacted with natural Bet v 1 and its homologues from alder (Aln g 1) as well as hazel (Cor a 1) and strongly inhibited the binding of birch pollen allergic patients' IgE to Bet v 1 wild-type. CONCLUSION: Genetically modified hypoallergenic rBet v 1 derivatives induce blocking antibodies in vivo. Their safety and efficacy for the treatment of birch pollen and associated plant allergies can now be evaluated in clinical immunotherapy studies.     

Isolation of a high‐affinity Bet v 1‐specific IgG‐derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments

Background

Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild‐type allergens. However, so far no treatment‐induced IgG antibodies have been characterized.

Objective

To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject.

Methods

A phage‐displayed combinatorial single‐chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1‐reactive antibody fragments. ScFvs were tested for specificity and cross‐reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross‐reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients’ IgE binding to ELISA plate‐bound allergens and allergen‐induced basophil activation was assessed.

Results

A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3‐1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients’ polyclonal IgE binding to Bet v 1, and partially suppressed allergen‐induced basophil activation.

Conclusion

Immunization with unfolded hypoallergenic allergen derivatives induces high‐affinity antibodies even in nonallergic subjects which recognize the folded wild‐type allergens and inhibit polyclonal IgE binding of allergic patients.     

Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen

Background Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer froin allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis. leading to death.
Objective The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy.
Methods A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradennal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting.
Results Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA. Westernblot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v I.
Conclusion Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.     

Reduced in vivo allergenicity of Bet v 1d isoform, a natural component of birch pollen

BACKGROUND: The major allergen of birch pollen, Bet v 1, is present in structurally slightly different isoforms. It has been postulated that certain isoforms show a distinct ability to bind birch pollen-specific IgE, although the T-cell response remains similar. OBJECTIVE: We verified the hypothesis of a distinct allergenicity but similar T-cell immunogenicity of 2 isoforms in birch pollen-allergic subjects by in vivo tests and an in vitro assay for T-cell stimulation. METHODS: Forty-eight birch pollen-allergic, 11 grass pollen-allergic, and 10 nonatopic control individuals were tested with 10-fold increasing concentrations (0.01 to 10.0 microg/mL) of recombinant (r) Bet v 1a and rBet v 1d by skin prick test (SPT), intradermal test (IDT), and conjunctival provocation test (CPT). An allergen-specific proliferation assay was performed on 21 patients with the 2 recombinant and the natural birch pollen allergens. RESULTS: In each test system only birch pollen-allergic subjects but no controls reacted to the recombinant allergens. A positive in vivo response to 10 microg/mL of rBet v 1a was observed in 21 of 48 by SPT, in 48 of 48 by IDT, and in 33 of 48 by CPT. In contrast, the IDT response to 10 microg/mL of rBet v 1d was reduced by a factor of 100 because it was equivalent to the response to 0.1 microg/mL of rBet v 1a. rBet v 1d failed to elicit a positive reaction in SPT and CPT. The proliferative response of T cells was similar for both recombinant isoforms because 8 of 21 individuals reacted to rBet v 1a and 6 of 21 to rBet v 1d. Only 1 subject had a positive reaction to rBet v 1d alone. CONCLUSION: The natural isoforms rBet v 1a and rBet v 1d differ in their ability to bind IgE but are similar in their immunogenicity for T cells. Thus rBet v 1d might be a promising candidate for use in immunotherapy of birch pollen-allergic individuals.     

Detection of specific IgE antibodies in the sera of patients allergic to birch pollen using recombinant allergens Bet v 1, Bet v 2, Bet v 4: evaluation of different IgE reactivity profiles

BACKGROUND: Birch pollen is a significant cause of immediate hypersensitivity among susceptible subjects in temperate climates, affecting 5-54% of the population in western Europe. We examined the specific serum IgE antibodies towards recombinant allergens Bet v 1, Bet v 2 and Bet v 4 in birch-sensitive patients from the province of Cuneo, north-west Italy. METHODS: Sera were obtained from 372 patients with symptomatic birch pollen-induced allergic rhinitis and/or asthma. A subgroup of these patients suffered from oral allergy syndrome after eating apple. Their sera were evaluated for specific IgE against natural birch pollen and apple extract, as well as Bet v 1, Bet v 2 and Bet v 4 using Pharmacia CAP system (Pharmacia, Uppsala, Sweden). RESULTS: Of 372 patients 215 (57.80%) had serum-specific IgE towards Bet v 1. A total of 166 sera (44.62%) contained serum-specific IgE to Bet v 2, while Bet v 4 IgE reactivity was documented in 35 subjects (9.41%). Moreover, 146 (39.25%) patients were monosensitized to Bet v 1; 96 (25.81%) patients were monosensitized to Bet v 2; only four sera (1.08%) contained specific IgE towards Bet v 4. Thirty-nine sera (11.02%) did not contain specific IgE to these individual birch pollen allergens. Of course, all 372 sera (100%) had specific IgE against natural birch pollen extract, of which 162 (43.55%) contained specific IgE to apple extract (75.35% of Bet v 1 positive sera). CONCLUSION: In this study we observed that three birch pollen recombinant allergens alone, could sufficiently identify 90% of birch pollen-sensitive patients. Therefore, for a more precise IgE profile of patients allergic to birch, further purified birch pollen allergens (i.e. Bet v 6, Bet v 7, Bet v 8) will be required.     

Evaluation of recombinant allergens Bet v 1 and Bet v 2 (profilin) by Pharmacia CAP System in patients with pollen-related allergy to birch and apple

Sixty-five patients presenting either rhinoconjunctivitis or asthma and sensitized to pollens of trees of the order Fagales were studied by the Pharmacia CAP system in order to assess specific IgE for the important birch pollen allergens Bet v 1 and Bet v 2. All 65 subjects reacted to at least one of the recombinant birch allergens: 43% to Bet v 1, 30.7% to Bet v 2, and 26% to both. Patients monosensitized to birch did not react to Bet v 2. of patients with a history of oral allergy syndrome after eating apples, 16/28 (57%) reacted to Bet v 1; among 20 polysensitized subjects presenting oral allergy syndrome after consumption of apple, four reacted to Bet v 2 (20%). Among patients with IgE against both recombinant allergens, six (35.30%) presented symptoms of allergy after eating apples. Our results indicate that sensitization to Bet v 1 is specific for birch and apple allergies, whereas sensitization to Bet v 2 is common in polysensitized patients.     

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

Background 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. Objective 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. Methods 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. Results 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). Conclusion 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. Cite this as: I. Pree, M. H. Shamji, I. Kimber, R. Valenta, S. R. Durham and V. Niederberger, Clinical & Experimental Allergy, 2010 (40) 1346–1352.     

Identification of B-cell epitopes of Bet v 1 involved in cross-reactivity with food allergens

Background:  The pollen-food syndrome (PFS) is an association of food allergies to fruits, nuts, and vegetables in patients with pollen allergy. Mal d 1, the major apple allergen, is one of the most commonly associated food allergens for birch pollen-allergic patients suffering from PFS. Although the reactions are due to cross-reactive IgE antibodies originally raised against pollen Bet v 1, not every Bet v 1-allergic patient develops clinical reactions towards apple.
Aim of the study:  We speculate that distinct IgE epitopes are responsible for the clinical manifestation of PFS. To test this hypothesis we grafted five Mal d 1 stretches onto Bet v 1. The grafted regions were 7- or 8-amino acids long encompassing amino acids residues previously shown to be crucial for IgE recognition of Bet v 1.
Methods:  A Bet v 1-Mal d 1 chimeric protein designated BMC was expressed in Escherichia coli and purified to homogeneity. IgE reactivity of BMC was tested with patients' sera originating from (i) Bet v 1-allergic patients displaying no clinical symptoms upon ingestion of apples; and (ii) Bet v 1-allergic patients displaying allergic symptoms upon ingestion of apples and other Bet v 1-related foods.
Results and conclusion:  Compared to birch pollen-allergic individuals, patients suffering from PFS showed significantly higher IgE reactivity with BMC (chimeric protein). The results suggest that the Mal d 1 regions grafted onto the Bet v 1 sequence comprise important IgE epitopes recognized by Bet v 1-allergic patients suffering from allergy to apples.     

Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen‐allergic patients with apple allergy

It is still unclear whether allergen‐specific immunotherapy (AIT) with birch pollen improves birch pollen‐related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen‐related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1‐homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen‐allergic patients with apple allergy. The dose of 1.6 μg induced oral allergy syndromes in 26.4%, 3.2 μg in 15.3%, 6.3 μg in 27.8%, 12.5 μg in 8.3%, 25 μg in 11.1%, and 50 μg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 μg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose‐defined sublingual challenge tests in a high number of birch pollen‐allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen‐related food allergy.     

In vitro and in vivo allergenicity of recombinant Bet v 1 compared to the reactivity of natural birch pollen extract

BACKGROUND : Diagnostic procedures using natural extracts show only limited quantitative correlation between in vivo and in vitro results. Highly pure recombinant allergens might show more predictive findings. OBJECTIVE : The aim of this study was to compare natural birch pollen extract (BPE) and recombinant Betula verrucosa (rBet v 1) for their diagnostic value comparing skin prick tests (SPTS) and nasal provocation tests (NPTS) with specific IgE in the serum. METHODS : Thirty-four patients allergic to birch pollen and five healthy controls were investigated. SPT and NPT were performed with BPE and rBet v 1 at different concentrations. Specific serum IgE was measured by the Pharmacia CAP system. RESULTS : Commercial BPE and rBet v 1 (10 micro g/mL) were able to elicit similar allergenic reactions in vivo and IgE binding in vitro. SPT reflects immediate-type allergy as determined by NPT to a higher degree than specific IgE, for both reagents. To cause allergic reactions in NPT, higher amounts of rBet v 1 were needed than for skin tests and the sensitivity was lower than with BPE. CONCLUSION : rBet v 1 alone is sufficient for a reliable diagnosis of birch pollen allergy in most patients and induces comparable skin test reactivity as BPE, but less allergic reactions in nasal provocations.