Immunotherapy Of Grass Pollen Allergy Research Articles

Mechanisms, safety and efficacy of a B cell epitope-based vaccine for immunotherapy of grass pollen allergy

BackgroundWe have developed a recombinant B cell epitope-based vaccine (BM32) for allergen-specific immunotherapy (AIT) of grass pollen allergy. The vaccine contains recombinant fusion proteins consisting of allergen-derived peptides and the hepatitis B surface protein domain preS as immunological carrier. MethodsWe conducted a randomized, double-blind, placebo-controlled AIT study to determine safety, clinical efficacy and immunological mechanism of three subcutaneous injections of three BM32 doses adsorbed to aluminum hydroxide versus aluminum hydroxide (placebo) applied monthly to grass pollen allergic patients (n=70). Primary efficacy endpoint was the difference in total nasal symptom score (TNSS) through grass pollen chamber exposure before treatment and 4weeks after the last injection. Secondary clinical endpoints were total ocular symptom score (TOSS) and allergen-specific skin response evaluated by titrated skin prick testing (SPT) at the same time points. Treatment-related side effects were evaluated as safety endpoints. Changes in allergen-specific antibody, cellular and cytokine responses were measured in patients before and after treatment. ResultsSixty-eight patients completed the trial. TNSS significantly decreased with mean changes of −1.41 (BM32/20μg) (P=0.03) and −1.34 (BM32/40μg) (P=0.003) whereas mean changes in the BM32/10μg and placebo group were not significant. TOSS and SPT reactions showed a dose-dependent decrease. No systemic immediate type side effects were observed. Only few grade 1 systemic late phase reactions occurred in BM32 treated patients. The number of local injection site reactions was similar in actively and placebo-treated patients. BM32 induced highly significant allergen-specific IgG responses (P<0.0001) but no allergen-specific IgE. Allergen-induced basophil activation was reduced in BM32 treated patients and addition of therapy-induced IgG significantly suppressed T cell activation (P=0.0063). ConclusionThe B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.)

Caveat Emptor on Sublingual Immunotherapy

The synthesis of clinical trials of sublingual immunotherapy for grass pollen allergy by Di Bona et al1 in this issue indicates an unimpressive small effect, and the efficacy is even smaller in the United States compared with European populations. However, what was impressive is the 70.0% (1817 of 2597 patients) proportion experiencing adverse effects (mostly mouth itching or burning and gastrointestinal tract symptoms). As these sublingual agents become increasingly available and heavily advertised, it will be prudent for physicians to be aware of these data, in addition to their cost (approximately $90 for a 3-month supply, plus the requirement to coprescribe an epinephrine autoinjector). Sublingual immunotherapy may seem more convenient than nasal corticosteroids or subcutaneous immunotherapy and therefore tempting to prescribe, but the evidence shows minimal benefit and moderate adverse effects for patients with seasonal grass pollen allergies.

Molecular Evolution of Hypoallergenic Hybrid Proteins for Vaccination against Grass Pollen Allergy

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy

BackgroundGrass pollen is one of the most important sources of respiratory allergies worldwide.ObjectiveThis study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach.MethodsFusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity.ResultsTen hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation.ConclusionA recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.

Possible therapeutic potential of a recombinant group 2 grass pollen allergen-specific antibody fragment

The induction of blocking IgG antibodies that compete with IgE for allergen binding is one important mechanism of allergen-specific immunotherapy. The application of blocking antibodies may be an alternative treatment strategy. A synthetic gene coding for a single-chain fragment (ScFv) specific for the major timothy grass pollen allergen Phl p 2 was inserted into plasmid pCANTAB 5 E, and the recombinant ScFv was expressed in Escherichia coli and purified by affinity chromatography. The ScFv was tested for allergen binding by ELISA, and its association and dissociation were measured by surface plasmon resonance (Biacore) technology. The ability of the ScFv to inhibit allergic patients' IgE binding to Phl p 2 and Phl p 2-induced basophil degranulation was studied by ELISA competition and basophil activation (CD203c) assays. We report the expression, purification, biochemical and immunological characterization of a monomeric single-chain fragment (ScFv) of human origin specific for the major timothy grass pollen allergen, Phl p 2. The Phl p 2-ScFv showed high affinity binding to the allergen and blocked the binding of allergic patients' polyclonal IgE to Phl p 2 up to 98%. Furthermore, it inhibited allergen-induced basophil activation. The Phl p 2-ScFv inhibited allergic patients' IgE binding to Phl p 2 as well as Phl p 2-induced basophil activation and might be useful for passive immunotherapy of grass pollen allergy.

Polysensitisation to pollen due to profilin and calcium-binding protein: distribution of IgE antibodies to marker allergens in grass and birch pollen allergic rhinitis patients in southern Germany

Allergen-specific immunotherapy for grass pollen allergy has been reported to be effective in up to 85% of patients. Sensitisation to profilin and calcium-binding protein (CBP) can possibly influence treatment results and may thus be a reason for treatment failures. During a study period of 3 years, the distribution patterns of antibodies to marker allergens were continuously investigated in all blood serum samples with a level of immunoglobulin E antibodies to timothy and birch pollen higher than 0.7 kUA/l (n = 556). Sensitisation to timothy grass pollen alone was found in 33% of the cases, to birch pollen alone in 19%, and to both in 48%. The group of polysensitised patients showed an inhomogenous distribution of antibodies to marker allergens. IgE against minor allergens was detected in 40%. Sensitisation to major allergens, especially to the major birch allergen, was not present in 13% of the polysensitised patients. Of the patients who were sensitised to minor allergens, 82% were sensitised to profilin, 11% to CBP, and 8% to both profilin and CBP. Profilin and CBP frequently cause polysensitisations to pollen. The data obtained justify the measurement of serum levels of antibodies to marker allergens in patients who are sensitised to more than one group of allergens.

Allergen Immunotherapy: A Centenary Celebration

In 1911 and long before the availability of antiallergic drugs, Leonard Noon demonstrated that prophylactic subcutaneous inoculation with a grass pollen extract was effective in suppressing immediate conjunctival sensitivity to grass pollen.1 Noon’s coworker, John Freeman, continued to practice immunotherapy and in 1930 published the first rush immunotherapy protocol.2 William Frankland, a colleague of Freeman, performed the first controlled clinical trial of grass pollen immunotherapy in 1954.3 He used a whole grass pollen extract that was compared with its partially purified proteins, the corresponding ultrafiltrate that contained no protein and a phenol-containing diluent. Both the whole extract and the purified grass pollen proteins were effective compared with the ultrafiltrate and the diluent control alone (Fig. 1). Frankland’s study established a firm scientific foundation for the practice of allergen immunotherapy. Noon, Freeman, and Frankland were all physicians at St. Mary’s Hospital, Paddington, now affiliated to Imperial College London, United Kingdom. Frankland in his 99th year continues to practice and play a major role in the allergy community; much admired and respected by his colleagues, he represents our centenary link with the origins of the practice of immunotherapy (Fig. 2). Major advances in allergen immunotherapy have resulted from parallel studies performed in the United States. Lowell and Franklin in 1964 were the first to clearly demonstrate that a single allergen (ragweed) in a multiallergen mixture was effective in reducing seasonal allergic symptoms.4 Philip Norman and Larry Lichtenstein in 1978 convincingly demonstrated the allergen-specificity of ragweed immunotherapy in patients with dual sensitivity to ragweed and grass pollen.5 Johnstone first highlighted the possibility that immunotherapy in children might confer protection against development of asthma,6 a concept supported by the more recent Preventive Allergy Treatment (PAT) study that identified a 2–3 fold risk reduction for developing asthma after 3 years treatment in children with seasonal pollinosis, protection that persisted for a further 7 years after discontinuation of immunotherapy.7 Hunt and colleagues8 demonstrated the efficacy of purified venom over whole insect body extract and placebo in patients with anaphylaxis to the stings of hymenoptera. Studies9 have confirmed the dose-dependency of allergen immunotherapy whereas the long-term benefits of allergen immunotherapy, with persistence of efficacy for several years after discontinuation10 have been illustrated for both venom11 and grass pollen immunotherapy, the latter both for subcutaneous and sublingual routes of therapy.12 Our increasing knowledge of the mechanisms of immunotherapy has informed both novel approaches and the development of putative biomarkers that might predict the clinical response to immunotherapy. Prausnitz and Kustner13 published in 1921 that a serum factor (‘reagin’) could transfer immediate allergen sensitivity as shown by skin testing was followed Robert Cooke’s observation in 1935 that serum obtained after pollen immunotherapy could confer ‘immunity and hypersensitivity.’14 These seminal observations long preceded the discovery of IgE antibody as reagin by the Ishizakas,15 Johansson and Bennich,16 and the concept of IgG ‘blocking antibodies.’17 The suppressive effect of ragweed immunotherapy on nasal eosinophils as a local marker of allergic inflammation was shown by Creticos in 1984,18 whereas Passalacqua and Canonica similarly demonstrated decreased local eosinophilia and adhesion molecule expression during mite sublingual immunotherapy.19 Warner in 197820 and Rak21 in 1991 observed decreases in allergeninduced late asthmatic responses and associated bronchial inflammation, respectively, in children and adults. A link between altered T-cell responses and immunotherapy was first shown by Rocklin22 whereas the critical role of regulatory T cells and IL-10 was highlighted by Akdis and colleagues.23 The concept of immune deviation of allergenspecific TH2 responses in favor of TH1 responses in both the periphery and in target organs has developed in parallel.24–26 From the Royal Brompton National Heart & Lung Institute, London; and National Jewish Health, Denver, CO. Correspondence to: Stephen R. Durham, MA, MD, FRCP, Royal Brompton Hospital and Imperial College, Dovehouse Street, London SW3 6LY United Kingdom. Telephone: 442073518024. Fax: 442073497780. E-mail: s.durham@ imperial.ac.uk. Copyright © 2011 by World Allergy Organization FIGURE 1. Table II shows the results of the first immunotherapy controlled trial, as published in: Frankland AW, Augustin R. Prophylaxis of summer hay-fever and asthma: a controlled trial comparing crude grass-pollen extracts with the isolated main protein component, The Lancet, May 24, 1954.

Allergen immunotherapy

Specific immunotherapy (SIT) involves the administration of allergen extracts to achieve clinical tolerance of those allergens that cause symptoms in patients with allergic conditions. Immunotherapy is effective in patients with mild forms of allergic disease and also in those who do not respond well to standard drug therapy. Most SIT is given by means of injection, but there is increasing interest in performing SIT through the sublingual route. SIT remains the treatment of choice for patients with systemic allergic reactions to wasp and bee stings and should be considered as an option in patients with allergic rhinitis, asthma, or both. SIT can modify the course of allergic disease by reducing the risk of new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis. The precise mechanisms responsible for the beneficial effects of SIT remain a matter of research and debate. An effect on regulatory T cells seems most probable and is associated with switching of allergen-specific B cells toward IgG4 production. Few direct comparisons of SIT and drug therapy have been made. Existing data suggest that the effects of SIT take longer to develop, but once established, SIT achieves long-lasting relief of allergic symptoms, whereas the benefits of drugs only last as long as they are continued.

Efficacy and Safety of 5–Grass-Pollen Sublingual Immunotherapy Tablets in Pediatric Allergic Rhinoconjunctivitis

Wahn U, Tabar A, Kuna P, et al. J Allergy Clin Immunol. 2009;123(1):160–166PURPOSE OF THE STUDY. To determine the efficacy and safety of a 300-index of reactivity sublingual immunotherapy (SLIT) tablet for children with allergic rhinoconjunctivitis.STUDY POPULATION. Subjects were 278 children (age: 5–14 years) with seasonal grass pollen-induced allergic rhinoconjunctivitis for ≥2 years. Allergy was confirmed by a timothy grass-specific immunoglobulin E (IgE) level of at least class 2 and a wheal diameter of >3 mm in a skin-prick test containing 5 grass pollens included in the SLIT tablet (orchard, meadow, perennial rye, sweet vernal, and timothy grasses). Patients who were sensitized to other allergens present during the grass pollen season, patients who had previously received immunotherapy for grass pollen allergy, and patients with asthma who were receiving medications other than β2-adrenergic receptor agonists were excluded.METHODS. This was a randomized, multicenter, double-blind, placebo-controlled trial conducted in 29 centers in 5 European countries. Patients received daily dosing with either the SLIT tablet or placebo beginning 4 months before and continuing throughout the grass pollen season. The primary outcome was efficacy of treatment, as assessed with the Rhinoconjunctivitis Total Symptom Score, an 18-point scale using 6 common rhinoconjunctivitis symptoms (nasal pruritus, nasal congestion, sneezing, rhinorrhea, ocular pruritus, and watery eyes) scored from 0 to 3 on the basis of severity. Daily symptoms and adverse events were recorded 1 month before and throughout the pollen season. Rescue medication use was scored from 1 to 3 on the basis of the use of antihistamines, nasally administered corticosteroids, or orally administered corticosteroids. Serum levels of IgG4 and IgE specific for grass pollen allergens were measured before and at the end of the study.RESULTS. A total of 278 children received either a SLIT tablet or placebo. The Rhinoconjunctivitis Total Symptom Scores showed benefit corresponding to mean and median improvements across all 6 categories of 28% and 39.3%, respectively, for the SLIT tablet over placebo (P = .01). Significant improvements in rescue medication scores (P = .0064), corresponding to mean and median improvements of 24.1% and 48.7%, respectively, and reductions in symptom scores (P < .038) were seen for the active group, compared with the placebo group. A threefold increase in grass-specific IgG4 levels was seen for the active group, with little change for the placebo group. Changes in grass-specific IgE levels were similar between the groups. Adherence to therapy was similar between the groups (placebo: 95%; active: 94%). A total of 902 treatment-associated adverse events were noted (active: 84.9%; placebo: 82%), with most being mild to moderate in severity. Nine subjects withdrew from the study because of adverse events, including 7 in the treatment group and 2 in the placebo group.CONCLUSIONS. Study data demonstrate that a grass pollen SLIT tablet is effective and safe in decreasing seasonal symptoms in children with allergic rhinoconjunctivitis.REVIEWERS COMMENTS. This study supports data from adult studies showing that SLIT can be an effective therapy for children with allergic rhinoconjunctivitis. In comparison with subcutaneous immunotherapy in this age group, SLIT may be an effective, safe, and convenient option for treating allergic rhinoconjunctivitis.

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the β-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

Aleuria aurantia lectin coated microspheres for oral immunotherapy of grass pollen allergy

Aleuria aurantia lectin coated microspheres for oral immunotherapy of grass pollen allergy

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

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. This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. 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. 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. 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.

Doença alérgica polínica: polens alergógenos e seus principais alérgenos

Pacientes com alergia a pólen de gramíneas, comumente denominada polinose, freqüentemente apresentam reatividade a alérgenos de pólen de inúmeras gramíneas devido às reatividades cruzadas entre anticorpos IgE dirigidos contra proteínas presentes nos polens de gramíneas. Nesse contexto, pólen de Italian ryegrass (Lolium multiflorum), ou azevém anual, gramínea da família Poaceae cultivada no Sul do Brasil, é considerado o principal agente sensibilizante em pacientes com polinose. Nessa região, o azevém é capaz de produzir grande quantidade de pólen. Adicionalmente, outras gramíneas da família Poaceae crescem naturalmente no Sul, entretanto, sem relevância clínica. Extratos de pólen derivados de gramíneas homólogas ou heterólogas são freqüentemente empregados no diagnóstico e tratamento da alergia sazonal a pólen, sendo que para esses fins não se encontra comercialmente disponível no Brasil extrato padronizado de pólen de L. multiflorum. Futuros estudos serão importantes para melhor caracterizar a reatividade cruzada entre alérgenos de pólen de L. multiflorum e alérgenos de outras gramíneas com o objetivo de aprimorar o diagnóstico e imunoterapia de pacientes com alergia a pólen causada por L. multiflorum.

Safety and immunological changes during tablet based specific immunotherapy

RATIONALE: The ALK Grass tablet is a new preparation for sublingual immunotherapy of grass pollen allergy. We aimed at determining a safe dose-range, and subsequently confirming the safety during daily sublingual administration. Simultaneously, immunological changes were measured.

Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.

Allergen immunotherapy: does it work and, if so, how and for how long?

Background. Pollen immunotherapy is effective in selected patients with IgE-mediated seasonal allergic rhinitis, although it is questionable whether there is long-term benefit after the discontinuation of treatment. Methods. We conducted a randomised, double-blind, placebo-controlled trial of the discontinuation of immunotherapy for grass pollen allergy in patients in whom three to four years of this treatment had previously been shown to be effective. During the three years of this trial, primary outcome measures were scores for seasonal symptoms and the use of rescue medication. Objective measures included the immediate conjunctival response and the immediate and late skin responses to allergen challenge. Cutaneous biopsy specimens obtained 24 hours after intradermal allergen challenge were examined for T cell infiltration and the presence of cytokine-producing T helper cells (Th2 cells) (as evidenced by the presence of interleukin-4 messenger RNA). A matched group of patients with hay fever who had not received immunotherapy was followed as a control for the natural course of the disease. Results. Scores for seasonal symptoms and the use of rescue antiallergic medication, which included short courses of prednisolone, remained low after the discontinuation of immunotherapy and there was no significant difference between patients who continued immunotherapy and those who discontinued it. Symptom scores in both treatment groups (median areas under the curve in 1995, 921 for continuation of immunotherapy and 504 for discontinuation of immunotherapy, p=0.60) were markedly lower than those in the group that had not received immunotherapy (median value in 1995, 2863). Although there was a tendency for immediate hypersensitivity to allergen to return late after discontinuation, there was sustained reduction in the late skin response and associated CD3+ T cell infiltration and interleukin-4 messenger RNA expression. Conclusions. Immunotherapy for grass pollen allergy for three to four years induces prolonged clinical remission accompanied by a persistent alteration in immunologic reactivity. (N Engl J Med 1999;341:468–75)

Molecular, Immunological, and Structural Characterization of Phl p 6, a Major Allergen and P-Particle-Associated Protein from Timothy Grass (Phleum pratense) Pollen

AbstractDue to the wide distribution and heavy pollen production of grasses, ∼50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive α-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.

IgE antibodies to recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) account for a high percentage of grass pollen–specific IgE

Background: Pollen from different grass species are some of the most potent elicitors of Type I allergy worldwide. The characterization of antigenic structures and IgE epitopes common to different grass species is relevant to define reagents for diagnosis and specific therapy of grass pollen allergy. Objective: The purpose of this study was to estimate the percentage of IgE directed to common, cross-reactive, or both types of epitopes shared by recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) and natural pollen extracts from nine different monocots ( Anthoxanthum odoratum, Avena sativa, Cynodon dactylon, Lolium perenne, Phragmites australis, Poa pratensis, Secale cereale, Triticum sativum, Zea mays) by using sera from different populations. Methods: Natural pollen extracts from nine different monocot species were characterized regarding their allergen contents by using specific antibodies and by IgE immunoblot inhibition with recombinant allergens. The percentage of grass pollen–specific IgE that was preabsorbed with a combination of recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) and recombinant birch profilin (Bet v 2) was determined by ELISA in sera from 193 European, American, and Asian subjects. Results: IgE to recombinant pollen allergens accounted for a mean 59% of grass pollen–specific IgE. A lower inhibition of IgE binding to certain natural extracts ( C. dactylon and Z. mays) could be attributed to the absence of immunologically detectable group 5 and group 2 allergens in these species. Conclusion: We define four recombinant pollen allergens that account for a substantial proportion of grass pollen–specific IgE. The recombinant pollen allergens characterized may represent candidates not only for diagnosis but also for patient-tailored immunotherapy of grass pollen allergy.

Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T‐cell clones specific for Phi p 1, a major grass pollen allergen

The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Phl p 1, a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Phl p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after 1 year of SIT. Serological analysis revealed a marked increase of grass pollen- and Phl p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPhl p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Phl p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH2-like pattern of cytokine production, TCC established after SIT revealed TH1 characteristics. This shift was due to a decrease in IL-4 rather than an increase in IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new ('protecting') specificities. We could not observe induction of allergen-specific CD8+ lymphocytes (supressor cells). Our data indicate that -- on the level of TH lymphocytes -- SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation.

Rationale for specific immunotherapy of grass pollen allergy with extracts of rye pollen. Skin test reactivity and immunochemical relationship between pollen allergens from rye and other common grasses.

In immunotherapy of grass pollen allergy, an extract of rye (Secale cereale) is often included. The aim of this study was to investigate by skin prick test (SPT) and immunochemical methods whether rye pollen contains specific allergens justifying the use of this extract separately. Twenty grass pollen allergic patients were skin prick tested with a dialysed freeze-dried raw extract of rye pollen (Sc), timothy extract (Soluprick SQ, 1 HEP) and two other rye extracts (Soluprick). Sera from the patients were RAST-tested using Sc and timothy (Pp). CRIE was performed using Sc and rabbit-anti grass (aNG) antibodies. The antigenic relations between rye and common grasses were investigated by CLIE using Sc and aNG as references, and by RAST inhibition. The ability of aNG to absorb the allergen activity of Sc was also tested. Significant correlations were found between timothy and rye when compared by means of SPT and RAST. The immunochemical analyses did not reveal any rye antigens containing rye epitopes only. However, the possibility of rye antigens with several epitopes, of which at least one is specific for rye, could not be excluded. Clinical symptoms supposedly elicited by rye alone can be explained quantitatively by the strongly time-limited and concentrated natural exposition. Diagnosis and treatment can, however, be performed with extracts of common grasses.