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.     

Lymphocyte transformation by grass pollen allergens: a study of atopic patients receiving immunotherapy

We have previously reported on peripheral blood lymphocyte (PBL) transformation by allergen, PPD as a control antigen and PHA as a mitogen during and after a preseasonal immunotherapy period. The present report describes similar parameters during and after the ensuing maintenance treatment period. Ten patients with grass pollen rhinitis were treated with Allpyral extract and 10 with Conjuvac two-grass mixture. Lymphocyte transformation responses to grass antigen continued to be low for PBL from patients during the maintenance treatment. Postseasonal values were higher during treatment. In late autumn 1980, when treatment had been stopped, there was a spontaneous fall in lymphocyte stimulation values. Occasional high values were noticed in some patients, two of whom had treatment side effects (urticaria). Clinical data during the whole treatment period (skin prick test, provocation tests, serological parameters, total IgE, grass-specific IgE, grass-specific IgG, pollen counts, symptom scores, clinical effect and adverse reactions) have been published separately.     

Recombinant allergens for allergen-specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens

The broad applicability of allergen-specific immunotherapy for the treatment and eventually prevention of IgE-mediated allergy is limited by the poor quality and allergenic activity of natural allergen extracts that are used for the production of current allergy vaccines. Today, the genetic code of the most important allergens has been deciphered; recombinant allergens equalling their natural counterparts have been produced for diagnosis and immunotherapy, and a large panel of genetically modified allergens with reduced allergenic activity has been characterized to improve safety of immunotherapy and explore allergen-specific prevention strategies. Successful immunotherapy studies have been performed with recombinant allergens and hypoallergenic allergen derivatives and will lead to the registration of the first recombinant allergen-based vaccines in the near future. There is no doubt that recombinant allergen-based vaccination strategies will be generally applicable to most allergen sources, including respiratory, food and venom allergens and allow to produce safe allergy vaccines for the treatment of the most common forms of IgE-mediated allergies.     

Characteristics and immunobiology of grass pollen allergens

Grass pollens are one of the most important airborne allergen sources worldwide. About 20 species from five subfamilies are considered to be the most frequent causes of grass pollen allergy, and the allergenic relationships among them closely follow their phylogenetic relationships. The allergic immune response to pollen of several grass species has been studied extensively over more than three decades. Eleven groups of allergens have been identified and described, in most cases from more than one species. The allergens range from 6 to 60 kD in apparent molecular weight and display a variety of physicochemical properties and structures. The most complete set of allergens has so far been isolated and cloned from Phleum pratense (timothy grass) pollen. Based on the prevalence of IgE antibody recognition among grass pollen-sensitized individuals, several allergens qualify as major, but members of two groups, groups 1 and 5, have been shown to dominate the immune response to grass pollen extract. Isoform variation has been detected in members of several of the allergen groups, which in some cases can be linked to observed genetic differences. N-linked glycosylation occurs in members of at least three groups. Carbohydrate- reactive IgE antibodies have been attributed to grass pollen sensitization and found to cross-react with glycan structures from other allergen sources, particularly vegetable foods. Another cause of extensive cross-reactivity are the group 12 allergens (profilins), which belong to a family of proteins highly conserved throughout the plant kingdom and present in all tissues. Members of eight allergen groups have been cloned and expressed as recombinant proteins capable of specific IgE binding. This development now allows diagnostic dissection of the immune response to grass pollen with potential benefits for specific immunotherapy.     

Characterization of grass group I allergens in timothy grass pollen

Using Phl p V-depleted timothy grass pollen extract (Phleum pratense) as immunogen, we obyained a monoclonal antibody, QG 4, which recognized proteins of 33, 35, and 37 kd as determined by Western blotting. The antibody cross-reacted with pollen proteins of other grass species in the molecular weight range of 30 to 37 kd. By means of two-dimensional polyacrylamide gel electrophoresis blot of timothy grass pollen extract, we demonstrated at least seven protein spots: two of 37 kd with isoelectric points of 6.4 and 6.6; four of 35 kd with isoelectric points of 6.5, 6.8, 7.1, and 7.3; and one of 33 kd with an isoelectric point of 8.5. These protein spots were also detected by patients' pooled serum. Microsequencing of the 20 N-terminal amino acid residues revealed structures with sequence identities up to 90% to the well-established allergen, Lol p 1 of ryegrass (Lolium perenne). Therefore we assume that the monoclonal antibody QG 4 recognized the corresponding allergen Phl p I in timothy grass pollen.     

Rush immunotherapy with a standardized Bermuda grass pollen extract

We carried out a double-blind clinical trial in 30 patients who were sensitized to Bermuda grass pollen. Before and after immunotherapy we performed in vivo tests (skin tests and standardized tests of specific and nonspecific bronchial hyperreactivity), in vitro tests (histamine release), and specific IgE and IgG antibodies to BGP. We found a significant decrease (P less than .001) in specific bronchial hyperreactivity and skin sensitivity to BGP in the group of patients treated with immunotherapy, a decrease in the delayed responses (P less than .05) in histamine release (P less than .01), and an increase (P less than .001) in specific IgE and IgG antibodies to BGP. The placebo group showed no changes in these parameters.     

Polymerization of individual species of grass pollen allergens

Six grass pollen allergens have been individually polymerized. We had previously polymerized mixed grass pollen allergens. However, since every patient does not react to every grass, we sought to polymerize individual grasses that could subsequently be mixed into a preparation based on a patient's skin reactivity. As demonstrated by Sepharose 4-B chromatography of individual grass monomers and polymers, all six grasses were polymerized. Polymerized grass preparations as compared with monomer preparations demonstrated a 10(3)- to 10(5)-fold reduction in allergenicity as determined by cutaneous end point titer. That grass polymer contains the great majority of clinically important allergenic determinants was demonstrated by the ability of polymer to inhibit 78% IgE binding against crude grass in a pool of untreated grass-sensitive patients. Its antigenic similarity to crude grasses is further shown by the ability of polymer to inhibit 85% of IgG binding against crude grass in a pool of patients treated with usual crude grass extracts.     

Genetic engineering of pollen allergens for hayfever immunotherapy

Type I allergy is an immunoglobulin E-mediated disease, representing a major health problem affecting more than 25% of the world's population. Grass, birch and ragweed pollen are a major source of environmental allergen. Specific immunotherapy is clinically proven to be an effective treatment for allergic conditions that involve the administration of crude extracts prepared from natural sources with potential life-threatening anaphylactic side effects. Recent developments in the molecular biology of pollen allergens have made it possible to design novel therapeutic approaches for improved and safer forms of specific immunotherapy. Hypoallergenic forms of major allergens with reduced immunoglobulin E epitopes have been produced using genetic engineering, whilst preserving other characteristics of the molecule that are able to induce a protective response. These modified forms are expected to make allergen-specific immunotherapy more widely used.     

Molecular variability of group 1 and 5 grass pollen allergens between Pooideae species: implications for immunotherapy

Background Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. Methods Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II‐restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)‐binding assays. CD4⁺ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. Results MS analysis of group 5 pollen allergens reveals considerable intra‐ and inter‐species variability in amino acid sequence, with 30–50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N‐ and O‐glycosylation contribute to the variability of group 1 allergens, yielding 5–10 main isoforms, depending on the species. Out of 14 MHC class II‐restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA‐DR molecules result in variable CD4⁺ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species‐restricted (or semi‐restricted) epitopes associated with group 1 or 5 allergens, respectively. Conclusion Major pollen allergens from distinct grass species bear both shared and species‐restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized. Cite this as: H. Chabre, B. Gouyon, A. Huet, V. Baron‐Bodo, E. Nony, M. Hrabina, F. Fenaille, A. Lautrette, M. Bonvalet, B. Maillère, V. Bordas‐Le Floch, L. Van Overtvelt, K. Jain, E. Ezan, T. Batard and P. Moingeon, Clinical & Experimental Allergy, 2010 (40) 505–519.     

A multicenter trial of immunotherapy with alginate--conjugated grass pollen extract

Conjuvac, a new generation of allergen extracts, has been developed to meet the need for improved characterization, purification, and standardization. Conjuvac consists of a dialyzed, standardized aqueous allergen extract chemically conjugated to a sodium alginate carrier and lyophilized in single-dose vials, thus ensuring stability until reconstituted with sterile water just before each injection. A preliminary study of Conjuvac 2 Grass by Pegelow (1984) involving a small number of patients showed this extract to have potential advantages. Accordingly, a study involving more patients was undertaken: two different maintenance dose levels of Conjuvac 2 Grass were investigated and compared to a pyridine-extracted, alum-precipitated two-grass extract called Allpyral, which in previous double-blind trials has been shown to be effective. The trial, which included 125 patients with hay fever and which extended over 2 years, involved ten allergists from seven European countries. Overall, the Conjuvac high-dose regimen proved slightly superior to Allpyral without any increased incidence of side effects, and all treatments stimulated marked increases in specific IgG levels without raising IgE levels.     

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.     

Immunodetection methods for grass pollen allergens on western blots

A comparison is made of eight different methods to detect allergenic proteins in Western blots of rye-grass pollen extracts. Horseradish peroxidase-based enhanced chemiluminescence (ECL) provides a sensitive method for the detection of allergenic proteins. The method has been modified to use more dilute solutions of ECL substrate to reduce the background, can be applied to a standard nitrocellulose membrane, and used with Kodak X-ray film. The assays can be performed rapidly, replacing use of radiolabelled probes. Increased resolution is obtained. This makes the method suitable for detection of cDNA clones on plaque lifts, and for rapid and specific purification of proteins following immunodetection on nitrocellulose membranes.     

Specific immunotherapy for common grass pollen allergies: pertinence of a five grass pollen vaccine

Patients throughout Europe are concomitantly exposed to multiple pollens from distinct Pooideae species. Given the overlap in pollination calendars and similar grain morphology, it is not possible to identify which grass species are present in the environment from pollen counts. Furthermore, neither serum IgE reactivity nor skin prick testing allow the identification of which grass species are involved in patient sensitisation. Due to their high level of amino acid sequence homology (e.g., >90% for group 1, 55-80% for group 5), significant cross-immunogenicity is observed between allergens from Pooideae pollens. Nevertheless, pollen allergens also contain species-specific T or B cell epitopes, and substantial quantitative differences exist in allergen (e.g., groups 1 and 5) composition between pollens from distinct grass species. In this context, a mixture of pollens from common and well-characterised Pooideae such as Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis is suitable for immunotherapy purposes because (1) it has been validated, both in terms of safety and efficacy, by established clinical practice; (2) it reflects natural exposure and sensitisation conditions; (3) it ensures a consistent and well-balanced composition of critical allergens, thus extending the repertoire of T and B cell epitopes present in the vaccine.     

Cross-reactivity of pollen allergens: recommendations for immunotherapy vaccines

PURPOSE OF REVIEW: This review will summarize recent research on pollen allergen and epitope cross-reactivity. Knowledge of these relationships aids in the rational formulation of allergen immunotherapy vaccines. RECENT FINDINGS: There has been further clarification of panallergens and their roles as both major and minor allergens. Recent studies have targeted non-specific lipid transfer proteins and calcium-binding proteins (polcalcins), as well as pathogenesis-related protein families and profilins. Polcalcins and non-specific lipid transfer proteins are responsible for pollen-fruit interactions as well as pollen cross-reactivity, in some cases, but not all, accounting for major allergenicity. Delineation of the enzymatic activity of certain allergens explains the ubiquitous nature of these pollen proteins. SUMMARY: Characterization of specific pollen allergens and their protein families has provided insight into the grounds for cross-reactivity. Continuing clarification of these relationships will allow the substitution and consolidation of inhalant extracts as described in the conclusion.