Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination

Background:  The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy.
Methods:  A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice.
Results:  Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils.
Conclusion:  We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.     

Conversion of grass pollen allergen-specific human IgE into a protective IgG(1) antibody

More than 100 million individuals exhibit IgE-mediated allergic reactions against Phl p 2, a major allergen from timothy grass pollen. We isolated cDNA coding for three Phl p 2-specific human IgE antibodies from a combinatorial library, which was constructed from lymphocytes of a grass pollen-allergic patient. Recombinant Phl p 2-specific IgE antibody fragments (Fab) recognized a fragment comprising the 64 N-terminal amino acids of Phl p 2 and cross-reacted with group 2 allergens from seven grass species. cDNA coding for the variable regions of one of the IgE Fab were cloned into aplasmid vector expressing the constant region of human IgG(1) to obtain a complete, recombinant Phl p 2-specific human IgG(1). This antibody blocked the binding of grass pollen-allergic patients IgE (n=26; mean inhibition: 58%) to Phl p 2 and caused a 100-fold reduction of Phl p 2-induced basophil histamine release. The recombinant human Phl p 2-specific IgG(1) may be used for environmental allergen detection, for standardization of diagnostic as well as therapeutic grass pollen allergen preparations and for passive therapy of grass pollen allergy.     

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.     

Generation of a low Immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a

BACKGROUND: Immunotherapy of grass pollen allergy is currently based on the administration of pollen extracts containing natural allergens. Specifically designed recombinant allergens with reduced IgE reactivity could be used in safer and more efficacious future therapy concepts. OBJECTIVES: This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. METHODS: Three deletion mutants were produced in Escherichia coli and subsequently purified. The overall IgE-binding capacity of the mutants was compared with the recombinant wild-type allergen by membrane blot and IgE-inhibition assays. The capacity for effector cell activation was determined in basophil activation assays. T cell proliferation assays with allergen-specific T cell lines were performed to confirm the retention of T cell reactivity. Structural properties were characterized by circular dichroism analysis and homogeneity by native isoelectric focusing. The deletion sites were mapped on homology models comprising the N- and C-terminal halves of Phl p 5a, respectively. RESULTS: The double-deletion mutant rPhl p 5a Delta(94-113, 175-198) showed strongly diminished IgE binding in membrane blot and IgE-inhibition assays. Both deletions affect predominantly alpha-helical regions located in the N- and C-terminal halves of Phl p 5a, respectively. Whereas deletion of Delta175-198 alone was sufficient to cause a large reduction of the IgE reactivity in a subgroup of allergic sera, only the combination of both deletions was highly effective for all the sera tested. rPhl p 5a Delta(94-113, 175-198) consistently showed at least an 11.5-fold reduced capacity to activate basophils compared with the recombinant wild-type molecule, and the T cell proliferation assays demonstrated retention of T cell reactivity. CONCLUSION: The mutant rPhl p 5a Delta(94-113, 175-198) fulfils the basic requirements for a hypoallergenic molecule suitable for a future immunotherapy of grass pollen allergy; it offers substantially reduced IgE binding and maintained T cell reactivity.     

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.     

The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50–60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.     

Heterogeneity of commercial timothy grass pollen extracts

Background The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources.
Objective To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity.
Methods Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients.
Results The allergen extracts showed broad variations in protein compositions and amounts (24.1–197.7 μg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32–384 ng/mL; Phl p 2: 1128–6530 ng/mL, Phl p 5: 40–793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients.
Conclusions Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.     

The allergen profile of beech and oak pollen

Background Beech and oak pollen are potential allergen sources with a world‐wide distribution. Objective We aimed to characterize the allergen profile of beech and oak pollen and to study cross‐reactivities with birch and grass pollen allergens. Methods Sera from tree pollen‐allergic patients with evidence for beech and oak pollen sensitization from Basel, Switzerland, (n=23) and sera from birch pollen‐allergic patients from Vienna, Austria, (n=26) were compared in immunoblot experiments for IgE reactivity to birch (Betula pendula syn. verrucosa), beech (Fagus sylvatica) and oak (Quercus alba) pollen allergens. Subsequently, beech and oak pollen allergens were characterized by IgE inhibition experiments with purified recombinant and natural allergens and with allergen‐specific antibody probes. Birch‐, beech‐ and oak pollen‐specific IgE levels were determined by ELISA. Results Beech and oak pollen contain allergens that cross‐react with the birch pollen allergens Bet v 1, Bet v 2 and Bet v 4 and with the berberine bridge enzyme‐like allergen Phl p 4 from timothy grass pollen. Sera from Swiss and Austrian patients exhibited similar IgE reactivity profiles to birch, beech and oak pollen extracts. IgE levels to beech and oak pollen allergens were lower than those to birch pollen allergens. Conclusion IgE reactivity to beech pollen is mainly due to cross‐reactivity with birch pollen allergens, and a Phl p 4‐like molecule represented another predominant IgE‐reactive structure in oak pollen. The characterization of beech and oak pollen allergens and their cross‐reactivity is important for the diagnosis and treatment of beech and oak pollen 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.     

Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11

BACKGROUND: Allergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. OBJECTIVE: To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. METHODS: Phl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. RESULTS: Phl p 11 cDNA defined an acidic polypeptide of 15.8 kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n = 184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. CONCLUSION: As the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.     

Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1: importance for allergenic activity

BACKGROUND: The major timothy grass pollen allergen Phl p 1 is one of the most potent and frequently recognized environmental allergens. OBJECTIVE: We sought to study at a molecular and structural level the IgE recognition of Phl p 1 and its relation to allergenic activity. METHODS: Monoclonal human IgE antibody fragments specific for Phl p 1 and group 1 allergens from various grasses were isolated from a combinatorial library made of lymphocytes from patients with grass pollen allergy. Recombinant Phl p 1 fragments and the 3-dimensional structure of Phl p 1 were used to localize the major binding site for the IgE antibodies. A rPhl p 1 fragment containing this binding site was expressed in Escherichia coli, purified, and tested for IgE reactivity and allergenic activity with sera and basophils from patients with grass pollen allergy. RESULTS: Monoclonal antibodies, as well as polyclonal serum IgE, from patients with grass pollen allergy defined a C-terminal fragment of Phl p 1 that represents a sterically oriented portion on the Phl p 1 structure. This Phl p 1 portion bound most of the allergen-specific IgE antibodies and contained the majority of the allergenic activity of Phl p 1. CONCLUSION: IgE recognition of spatially clustered epitopes on allergens might be a general factor determining their allergenic activity. CLINICAL IMPLICATIONS: Geographic distribution of IgE epitopes on an allergen might influence its allergenic activity and hence explain discrepancies between diagnostic test results based on IgE serology and provocation testing. It might also form a basis for the development of low allergenic vaccines.     

CD‐sens: a biological measure of immunological changes stimulated by ASIT

Background: Allergen‐specific immunotherapy (ASIT) in allergic rhinitis and asthma is the only treatment that effects the long‐term development of these diseases. Basophil allergen threshold sensitivity, CD‐sens, which is a valuable complement to resource‐demanding clinical challenge tests, was used to monitor the initiation of ASIT induced allergen ‘blocking activity’. Methods: Patients IgE‐sensitized to timothy (n = 14) or birch (n = 19) pollen were started on conventional (8–16 weeks) or ultra rush ASIT, respectively, and followed by measurements of CD‐sens, allergen binding activity (ABA) and serum IgG4‐ and IgE‐antibody concentrations. Results: CD‐sens decreased during the early phase of ASIT‐treatment. In parallel, ABA increased and correlated significantly with the increasing levels of IgG4 antibody concentrations. High dosages of allergen were more effective while mode of dosing up did not seem to matter. No change was seen in basophil reactivity. Conclusion: CD‐sens and ABA, in contrast to basophil reactivity, seem to be promising tools to monitor protective immune responses initiated by ASIT.     

Antigens drive memory IgE responses in human allergy via the nasal mucosa

BACKGROUND: Natural allergen contact induces an increase of IgE levels and sensitivity but the mechanisms underlying the allergen-specific memory responses are poorly understood. Furthermore, it has not been studied whether allergen exposure affects the molecular reactivity profiles in patients. The aim of this study was to analyze the influence of nasal allergen encounter on the molecular profile and magnitude of memory IgE responses and on systemic sensitivity. METHODS: We investigated allergen-specific IgE, IgG subclass and IgM responses to defined allergen molecules (grass pollen: Phl p 1, Phl p 2 and Phl p 5; birch pollen: Bet v 1 and Bet v 2) in allergic patients in response to natural as well as to controlled nasal and dermal allergen exposure. Changes in systemic sensitivity were monitored by skin prick testing and by basophil histamine release experiments. RESULTS: Respiratory antigen exposure boosted IgE levels to a pre-established profile of allergen molecules without inducing significant IgM responses or new IgE specificities in allergic individuals. The importance of the route of allergen contact is demonstrated by an increase of systemic IgE levels and sensitivity after nasal exposure. In vitro sensitisation of basophils with pre- and post-seasonal serum samples suggests an allergen-induced elevation of specific IgE as a cause for the increased allergen-specific sensitivity. CONCLUSION: The characteristics of the allergen-driven antibody responses indicate a direct activation of an established pool of IgE memory cells with defined specificities as an underlying mechanism. Our finding that nasal allergen contact is a major factor for the boosting of memory IgE and systemic sensitivity may open new therapeutic possibilities.     

Multiple grass mixes as opposed to single grasses for allergen immunotherapy in allergic rhinitis

Grass pollen allergy affects approximately 40% of allergic patients. Subcutaneous allergen immunotherapy (SCIT) is the only allergen‐specific and disease‐modifying treatment available. Currently available therapeutic vaccines for the treatment of grass pollen allergy are based on natural grass pollen extracts which are either made from pollen of one cross‐reactive grass species or from several related grass species. Clinical studies have shown that SCIT performed with timothy grass pollen extract is effective for the treatment of grass pollen allergy. Moreover, it has been demonstrated that recombinant timothy grass pollen allergens contain the majority of relevant epitopes and can be used for SCIT in clinical trials. However, recent in vitro studies have suggested that mixes consisting of allergen extracts from several related grass species may have advantages for SCIT over single allergen extracts. Here, we review current knowledge regarding the disease‐relevant allergens in grass pollen allergy, available clinical studies comparing SCIT with allergen extracts from timothy grass or from mixes of several related grass species of the Pooideae subfamily, in vitro cross‐reactivity studies performed with natural allergen extracts and recombinant allergens and SCIT studies performed with recombinant timothy grass pollen allergens. In vitro and clinical studies performed with natural allergen extracts reveal no relevant advantages of using multiple grass mixes as opposed to single grass pollen extracts. Several studies analysing the molecular composition of natural allergen extracts and the molecular profile of patients' immune responses after SCIT with allergen extracts indicate that the major limitation for the production of a high quality grass pollen vaccine resides in intrinsic features of natural allergen extracts which can only be overcome with recombinant allergen‐based technologies.     

Lysine as a critical amino acid for IgE binding in Phl p 5b C terminus

BACKGROUND: Allergens induce the formation of specific immunoglobulin (Ig)E and harbor at least two IgE-binding regions (epitopes) to facilitate crosslinking of basophilic or mast-cell-bound specific IgE antibodies. Studies mapping linear epitopes have shown that these regions often contain charged or hydrophobic amino acids. Nevertheless, these studies are hampered by limited significance due to the often conformational nature of IgE epitopes. This prompted us to study the role of lysines in the context of an intact 3-dimensional model. METHODS: Major allergen Phl p 5b from timothy grass bears 12 lysines in its C-terminal half. Using site-directed mutagenesis, we substituted all 10 surface-exposed lysines by alanines. RESULTS: Although structural integrity of the lysine-deficient mutant was not altered, IgE-binding capacity measured by ELISA inhibition tests and crosslinking activity in ex vivo basophil stimulation and in vivo skin prick tests were significantly diminished. Interestingly, binding of specific IgG antibodies was considerably less reduced by loss of lysines. CONCLUSION: Lysine is an important amino acid for IgE binding in more than one epitope of major grass pollen allergen Phl p 5b C terminus. Allergenicity, but not IgG binding of the molecule, is substantially diminished by single amino acid substitutions without structural integrity being hampered.     

Characterization of immunoglobulin E responses in Balb/c mice against the major allergens of timothy grass (Phleum pratense) pollen

BACKGROUND: Grass pollen, such as that from timothy grass (Phleum pratense), represents a major cause of type I allergy. OBJECTIVE: To characterize the IgE immune response and to identify the major allergens eliciting an IgE response in a mouse model using pollen extract of P. pratense for sensitization, in order to assess analogies to human hyperreactivity and to gain information on the allergenic potential as determined by the IgE-reactivity kinetics of defined allergens. METHODS: Balb/c mice were sensitized with pollen extract or with purified natural allergens. Serum IgE levels, the induction of specific IgE antibodies and immediate hypersensitivity were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: The sensitized mice mounted a strong IgE response and showed IgE-reactivity first against Phl p 5a and 5b, then Phl p 4 and 13 and lastly against Phl p 6. No IgE response was mounted against Phl p 1. However, all purified fractions examined (Phl p 5a, 5b, 6 and 1) induced specific IgE and showed similar kinetics of IgE induction as pollen extract (first Phl p 5a and 5b, then Phl p 6). Skin test experiments demonstrated positive reactivity only in sensitized mice. CONCLUSION: The IgE reactivity induced by the major allergens in Balb/c mice was very similar to that found in allergic patients, with the exception of Phl p 1. The kinetics of the specific IgE response was comparable using either pollen extract or the purified major allergens, indicating that the intrinsic properties of the allergens are of importance rather than their proportionate amounts in pollen extract. This model should prove to be suitable for investigations regarding the mechanisms of induction and manifestation of timothy grass pollen allergy and for the evaluation of therapeutic strategies.     

Skin test diagnosis of grass pollen allergy with a recombinant hybrid molecule

BACKGROUND: A recombinant hybrid molecule (HM) consisting of 4 major allergens from timothy grass (Phl p 1, 2, 5, and 6) was expressed in Escherichia coli, purified, and characterized regarding its immunologic properties. OBJECTIVE: We sought to determine whether the recombinant HM can be used for the diagnosis of grass pollen allergy by means of skin testing. METHODS: Skin prick testing was performed in 32 patients with grass pollen allergy and in 9 control individuals by using increasing concentrations (4, 12, 36, and 108 mug/mL) of the HM and using commercial grass pollen extract. Specific IgE reactivities against the HM, grass pollen extract, and a panel of purified grass pollen allergens (recombinant Phl p 1, 2, 5, 6, 7, 12, and 13 and natural Phl p 4) were measured by means of ELISA, and timothy grass pollen-specific IgE levels were determined by using ImmunoCAP. RESULTS: Grass pollen allergy was diagnosed in all patients by means of skin testing with the HM. No false-positive skin test responses were obtained in the control individuals. There was an excellent correlation between IgE levels obtained with the HM and natural grass pollen extract measured by means of ELISA (r = 0.98, P < .0001) and by means of ImmunoCAP (r = 0.98, P < .0001). CONCLUSIONS: The recombinant HM permitted accurate and specific in vivo diagnosis of grass pollen allergy in all tested patients. It can be considered a well-defined tool for the diagnosis and perhaps for immunotherapy of grass pollen allergy. CLINICAL IMPLICATIONS: A recombinant HM can replace traditional allergen extracts for skin test-based diagnosis of grass pollen allergy.     

Heat‐labile Escherichia coli toxin enhances the induction of allergen‐specific IgG antibodies in epicutaneous patch vaccination

Epicutaneous allergen‐specific immunotherapy (EPIT) is proposed as an alternative route for allergen‐specific immunotherapy (AIT). The induction of allergen‐specific blocking IgG antibodies represents an important mechanism underlying AIT, but has not been investigated for EPIT. Here, we compared the induction of allergen‐specific blocking IgG in outbred guinea pigs which had been immunized with recombinant birch pollen allergen Bet v 1 using patch delivery system (PDS) with or without heat‐labile toxin (LT) from Escherichia coli or subcutaneously with aluminum hydroxide (Alum)‐adsorbed rBet v 1. Only subcutaneous immunization with Alum‐adsorbed rBet v 1 and epicutaneous administration of rBet v 1 with PDS in combination with LT from E. coli induced allergen‐specific IgG antibodies blocking allergic patients' IgE, but not immunization with rBet v 1 via PDS alone. Our results suggest that patch vaccination with rBet v 1 in combination with LT may be a promising strategy for allergen‐specific immunotherapy against birch pollen allergy.     

Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library

BACKGROUND: Phl p 5 represents a major allergen of timothy grass pollen (Phleum pratense). Detailed knowledge about the structures responsible for IgE binding would allow the design of a novel generation of allergy vaccines. OBJECTIVE: We aimed to characterize the IgE epitopes of Phl p 5a using phage display combined with a molecular modeling approach. METHODS: Phl p 5a-specific IgE from sera of patients with grass pollen allergy was used for screening of a random peptide phage library displaying constrained decamers. RESULTS: Fifteen phage clones that shared sequence motifs and could be grouped into families were selected by using Phl p 5a-specific IgE. Peptide alignment with the solvent-accessible amino acids of Phl p 5a revealed 3 sequence sections with frequent hits of identical or similar amino acids. On the surface of Phl p 5a, these sections assembled in compact patches, most likely representing conformational IgE epitopes, whereas no matching clusters were found on the back sides of the 2 Phl p 5a halves. In surface plasmon resonance experiments, the high-affinity interaction between IgE and Phl p 5 could be competed by phage-displayed peptides up to 24%, indicating that they represent true epitope mimics (ie, mimotopes). Allergen-specific immunogenicity of the mimotopes was proved in Biozzi mice. CONCLUSION: The selected mimotopes facilitated the localization of conformational IgE epitopes of Phl p 5. We suggest them to be suitable candidates for the development of an epitope-specific immunotherapy.