A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination

BACKGROUND: Allergy vaccines based on natural allergen extracts contain greatly varying amounts of individual allergens with different immunogenicity. OBJECTIVE: To develop a novel type of allergy vaccine for complex allergen sources that combines defined amounts of the major allergens in the form of single hybrid molecules. METHODS: A hybrid molecule was engineered by PCR-based mending and expression of the cDNAs coding for the 4 major grass pollen allergens and compared with its single components by circular dichroism analysis, T-cell proliferation, ELISA competition, and histamine release assays. Immune responses to the hybrid molecule were studied in BALB/c mice and rat basophil leukemia assays. RESULTS: The hybrid contained most of the B-cell epitopes of grass pollen and could be used to diagnose allergy in 98% (n = 652) of patients allergic to grass pollen. Immunization of mice and rabbits with the hybrid induced stronger and earlier IgG antibody responses than equimolar mixtures of the components, which can be explained by the induction of stronger T-cell responses by the hybrid versus the individual components. IgG antibodies induced by vaccination with the hybrid blocked immediate allergic reactions, as demonstrated by rat basophil degranulation assays in a murine model of grass pollen allergy. CONCLUSION: We demonstrate for grass pollen allergy that recombinant hybrid molecules covering the spectrum of the disease-eliciting epitopes of complex allergen sources can be engineered.     

Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.

In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.     

Allergen-specific immunotherapy with recombinant grass pollen allergens

BACKGROUND: Allergen-specific immunotherapy uses aqueous extracts of natural source materials as a basis for preparations to down regulate the allergic response. Recombinant DNA technology has enabled the cloning of many allergens, thus facilitating investigations aimed at improving efficacy and safety of immunotherapy. OBJECTIVE: To determine the effectiveness of a mixture of 5 recombinant grass pollen allergens in reducing symptoms and need for symptomatic medication in patients allergic to grass pollen. METHODS: A randomized, double-blind, placebo-controlled study of subcutaneous injection immunotherapy was performed in subjects with allergic rhinoconjunctivitis, with or without asthma. Primary endpoint was a symptom medication score compiled from separate symptom and medication scores. Secondary endpoints included a rhinitis quality of life questionnaire, conjunctival provocation, and specific antibody responses. RESULTS: The symptom medication score showed significant improvements in subjects receiving recombinant allergens as opposed to placebo, with reductions in both symptoms and medication usage. The rhinitis quality of life questionnaire revealed clinically relevant significant improvements in overall assessment and in 5 of 7 separate domains, and conjunctival provocation showed a clear trend in favor of active treatment. All treated subjects developed strong allergen-specific IgG(1) and IgG(4) antibody responses. Some patients were not sensitized to Ph l p 5 but nevertheless developed strong IgG antibody responses to that allergen. CONCLUSION: A recombinant allergen vaccine can be a effective and safe treatment to ameliorate symptoms of allergic rhinitis. The clinical benefit is associated with modification of the specific immune response with promotion of IgG(4) and reduction of IgE antibodies consistent with the induction of IL-10-producing regulatory T cells.     

Rush immunotherapy in a dog with severe ragweed and grass pollen allergy.

BACKGROUND: Forty years of study of naturally occurring IgE-mediated allergy in animals is briefly reviewed. These studies provided models for study of bioactive mediators and innovative pharmacologic therapies for IgE-mediated asthma. Objective: Based on our experience with canine allergy we evaluated and treated a dog with severe grass and ragweed allergy whose allergic dermatitis was uncontrolled by H1 blockers and topical corticosteroids. The dog was miserable during the Chicago grass and ragweed pollen seasons. METHODS: Rush immunotherapy was initiated during the ragweed season of 1997. RESULTS: Dramatic improvement was seen which persisted through the grass and ragweed seasons of 1998 after maintenance immunotherapy. CONCLUSION: The case is presented not as a model for canine immunotherapy but as an example of how animal research can be of value to both animals and humans.     

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.     

Double-blind, placebo-controlled immunotherapy with a high-molecular-weight, formalinized allergoid in grass pollen allergy

Specific immunotherapy is effective in grass pollen allergy with standardized extracts and formalinized allergoids; but systemic reactions are not uncommon. A high-molecular-weight (greater than 85,000 daltons), formalinized allergoid was investigated in a double-blind, placebo-controlled study to assess its safety and efficacy. Twenty patients received a placebo and 39 the allergoid using a rather aggressive protocol. Five patients developed a mild and transient systemic reaction with high doses of allergoid and one had a more severe reaction requiring treatment. Nasal challenges performed with orchard grass pollen grains showed that the threshold number of grains eliciting nasal symptoms was significantly (p less than 0.01) greater in the treated group. This group had significantly (p less than 0.01) less nasal symptoms during the season and specific IgG levels were significantly (p less than 0.01) elevated. There was a significant (p less than 0.01) correlation between nasal challenges and nasal symptoms during the season but no correlation between IgG and symptoms. There was no dose-dependent effect of allergoids.     

Langerhans cells in nasal mucosa of patients with grass pollen allergy.

Langerhans cells (LC) are known to be present in squamous epithelia of the human body. They are dendritic cells (DC) and characterized by the presence of Birbeck granules (BG). In previous studies, DC positive for CD1a and HLA-DR were found in the cylindrical epithelium and the lamina propria of the nasal mucosa. In our study, more CD1a cells occurred in the allergic patients than in the non-allergic controls. In a combined light microscopy (LM) and electron microscopy (EM) study, biopsies of nasal mucosa in allergic patients were studied. We used monoclonal antibodies against CD1a and HLA-DR, to identify DC in LM cryostat sections. The presence of BG identified most of the intra-epithelial DC as LC on the EM level, whereas a minority of DC in the lamina propria also contained BG. The ultrastructure of LC and DC in the ciliated cylindrical epithelium and the lamina propria is compared.     

Nasal challenge with pollen grains, skin-prick tests and specific IgE in patients with grass pollen allergy

Nasal challenges with pollen grains are as close as possible to natural pollen exposure, but they are not well documented in grass pollen allergy. Forty-four grass pollen allergic patients and ten non-allergic volunteers were tested by means of nasal challenge, quantitative skin-prick tests with a standardized orchard grass pollen extract and serum-specific IgE. Nasal challenges were performed with lactose and increasing concentrations of orchard grass pollen grains (15–3645 grains, three-fold increase). The test was considered to be positive when a symptom score over 5 was obtained, since this score had been previously correlated with the release of PGD₂ in nasal secretions. All control subjects and 3/44 patients had a negative challenge. The number of orchard pollen grains required to elicit a positive challenge was 332 ± 440 (range: 15–1215 grains) and the distribution was Gaussian. This number is higher than expected according to pollen calendars performed during the season, but owing to the priming effect of the nasal mucosa by allergens it is compatible to natural exposure. The correlation between nasal provocation tests and skin-prick test end-points was significant ( P < 0.005, Spearman rank test). Conversely there was no correlation between nasal challenge or skin-prick test end-point and serum-specific IgE.     

Oral immunization with a recombinant major grass pollen allergen induces blocking antibodies in mice

BACKGROUND: Immunotherapy for the treatment of pollen allergies traditionally involves a series of parenteral injections of a crude pollen extract. Successful application of this treatment results in the development of systemic tolerance to the sensitizing allergens, including the induction of blocking antibodies. OBJECTIVE: We sought to investigate whether oral immunization with a recombinant pollen allergen could induce a systemic immune response, and the production of systemic blocking antibodies in mice. METHODS: C57BL/10 mice were orally administered rLol p 5 or sodium chloride solution via gavage. RESULTS: We report that the oral administration of rLol p 5 induced a systemic immune response, including the induction of both blocking and interspecific cross-reactive antibodies. CONCLUSION: Our results suggest that oral administration of a major grass pollen allergen can induce the development of a systemic immune response including the production of systemic blocking and cross-reactive antibodies, a response that may offer immunological protection upon subsequent allergen exposure.     

Induction of IgE antibodies in mice with recombinant grass pollen antigens.

In this study, recombinant Poa pratensis (Poa p) IX allergens were examined for their in vivo allergenicity and antigenicity. Immunization of mice with a fusion protein (FP) comprising beta-galactosidase and recombinant KBG8.3 (rKBG8.3) allergen induced high titres of both IgG and IgE antibodies. By contrast, immunization with rKBG60.2, which represents the N-terminal fragment of rKBG8.3, induced only IgG antibodies. The IgE antibody titre specific to Kentucky Bluegrass (KBG) was significantly higher than that to beta-galactosidase. Moreover, KBG-specific IgE antibodies showed no apparent decrease in their titres until 60 days after immunization, whereas the beta-galactosidase-specific IgE antibodies disappeared after 40 days. The antibodies induced with rKBG8.3 in mice were capable of inhibiting the binding of human IgE antibodies to KBG pollen allergens, which indicated that rKBG8.3-specific murine antibodies recognized epitopes similar to those recognized by human IgE antibodies. Analysis of allergenic cross-reactivities of rKBG8.3 with components from five other species of grass pollens revealed that IgE antibodies induced by this allergen are capable of binding in vivo to components from other grass pollens. These results suggest that the mouse may serve as a model for the manipulation of IgE responses to recombinant allergens or their chemically modified derivatives.     

Constructing a hybrid molecule with low capacity of IgE binding from Chenopodium album pollen allergens

Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli. IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.     

Species specific grass pollen sensitivity: diagnosis and treatment with single grass species Allpyral vaccines

Skin test titrations and nasal provocation tests in sixty patients with hay fever showed specific reactions to extracts of individual grass species. There was, however, no correlation between skin and nasal sensitivity. Repeat testing after treatment with Allpyral vaccines consisting of only the grass species to which the nasal reaction was most severe, or only one of several pollens to which reactions were equally severe, showed marked diminution of skin and nasal sensitivity not only to the single pollen used for immuno-therapy but to all five common pollens used in the Allpyral grass mix. Clinical results seemed much improved as compared with results in the same year for Allpyral five grass mix vaccines, especially in the case of patients treated with Timothy, rye, or cocksfoot. It was concluded that these three grasses were to be preferred for treatment in England, and that these grasses contain common allergens.     

Relevance of a 5-grass sublingual tablet for immunotherapy of patients with grass pollen allergy in North America

Grass pollen allergy is common and clinically consequential in North America. While it is frequently treated with subcutaneous or sublingual immunotherapy, debate remains regarding whether allergen immunotherapy is best carried out using a single representative or multiple cross-reactive allergen(s). Patients are commonly exposed to pollens from multiple allergenic grass species belonging to the Pooideæ subfamily. Beyond the known IgE cross-reactivity, considerable molecular heterogeneity exists with respect to allergen content among grass species, with further evidence that these molecular variants can be detected by the patients’ immune system. These observations provide a compelling scientific rationale for the use of mixed pollen allergen extracts to broaden the allergen repertoire, with the aim of reorienting inappropriate immune responses in allergic patients.     

Month of birth and grass pollen or mite sensitization in children with respiratory allergy: a significant relationship

This report describes a retrospective analysis of the month of birth distribution of 2124 children with respiratory allergy in the Rome district between 1964 and 1985, in comparison with the total live births in the same district over the same period. Of the 2124 children, 1685 had positive skin tests and or RAST only to mites, and 439 only to grass pollen ( P < < 0.001). A significant relationship was found between grass or mite sensitization and the month of birth. A high proportion of children born in June-September had mite allergy ( P <0.005), and even higher was that of those born in March-May with grass sensitivity ( P < < 0.005), compared with the total live birth distribution in the Rome district in the same years as the children examined. These results are consistent with the idea that allergy may be associated with a period of susceptibility to sensitization in early infancy.     

Component-resolved diagnosis with recombinant allergens in patients with cherry allergy

BACKGROUND: In pollen-related food allergy, extracts for skin prick tests (SPTs) are often not standardized, and the test reliability is affected by false-negative reactions. OBJECTIVE: We sought to evaluate a panel of recombinant allergens (RAs) derived from one allergenic food for use in component-resolved in vivo diagnosis, taking cherry as a model food. METHODS: Seventy-nine subjects were included in the study: 24 Swiss patients (group 1) with a positive double-blind placebo-controlled food challenge result to cherries, 23 patients with birch pollen allergy but without cherry allergy (group 2), 23 nonatopic subjects (group 3), and 9 Spanish patients with a history of a cherry allergy (group 4). SPTs were performed in duplicate by using recombinant cherry allergens (Bet v 1-related allergen: recombinant (r) Pru av 1; profilin: rPru av 4; and lipid transfer protein: rPru av 3) in concentrations of 10, 50, and 100 microg/mL. Furthermore, IgE reactivity to rPru av 1, rPru av 4, and rPru av 3 was assessed by means of immunoblot analysis. RESULTS: SPT responses with rPru av 1, rPru av 4, and rPru av 3 were positive in 92%, 17%, and 4% of the patients in group 1; in 74%, 30%, and 0% of the patients in group 2; in 0%, 22%, and 89% of the patients in group 4; and negative for all nonatopic subjects (group 3). Thus the sensitivity of a positive SPT response to at least one of the 3 RAs was 96%. The specificities, negative predictive values, and positive predictive values with the 3 RAs were 100%, 96%, and 100% if calculated in relation to the nonatopic control group but 17%, 79%, and 60% when calculated in relation to the control group with birch pollen allergy. The correlation between SPT and immunoblotting results was excellent. Sensitization to rPru av 3 was associated with more severe symptoms than sensitization to rPru av 1. CONCLUSIONS: SPTs with RAs proved to be highly sensitive for diagnosis of cherry allergy. Component-resolved in vivo diagnosis with standardized amounts of stable RAs allows us to determine sensitization patterns directly, to correlate them with severity of clinical symptoms, and to analyze geographic differences.     

Microarrayed recombinant allergens for diagnosis of allergy

We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi-allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease-eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi-allergen tests consisting of microarrayed recombinant allergen molecules. Microarrayed recombinant allergens can be used to determine and monitor the profile of disease-eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray-based allergy testing will likely improve diagnosis, prevention and treatment of allergy.     

Specific IgE response before and after rush immunotherapy with a standardized allergen or allergoid in grass pollen allergy

Allergen extracts contain a wide variety of allergenic determinants and the sensitization of allergic subjects is extremely heterogeneous. Specific immunotherapy is usually performed with multiple component extracts and it is therefore important to determine whether this treatment may elicit the onset of newly developed IgE sensitivities. By means of nitrocellulose immunoblotting technique, the IgE sensitivities of 20 patients were characterized before and after specific immunotherapy. Ten patients had a rush immunotherapy with a standardized orchard grass pollen extract and ten others underwent a rush immunotherapy with a 6-grass pollen allergoid. The allergenic profiles of the patients before and after immunotherapy were qualitatively similar. In some patients an increase of orchard grass pollen-specific IgE was observed and the allergenic profile was quantitatively different. These results suggest that rush immunotherapy with either an aqueous non-modified extract or an allergoid does not elicit the onset of new IgE sensitivities. Multicomponent allergenic extracts may therefore be used in the treatment of patients.