Timothy Grass Pollen Allergen Research Articles

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

Microheterogeneity of the major grass group 6 allergen Phl p 6: Analysis by mass spectrometry.

The precise structural characterization of allergens is a basic requirement to improve diagnostics and to find therapeutic strategies against allergic disorders. Natural grass pollen allergens exhibit a wide variety of isoforms and it is still unknown whether this microheterogeneity is essential for the allergic reaction or has a functional effect on sensitization. Well-defined recombinant allergens are considered to replace natural allergens for clinical trials. For the major timothy grass pollen allergen Phl p 6 (approximately 12 kDa) and a recombinant rPhl p 6 we determined the structural microheterogeneity by two-dimensional electrophoresis (2-DE), high-resolution electrospray ionization-Fourier transform-mass spectrometry (ESI-FT-MS) of the intact molecules, and by tryptic peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Natural Phl p 6 is a mixture of mainly two isoforms that differ by two amino acids leading to a mass difference of 5 Da. For each of this two isoforms six variants were identified with modifications at the C- and/or N-terminus. The recombinant Phl p 6 comprises the same structure as one of the main isoforms indicating that it represents a major part of the natural Phl p 6.

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy.

Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.

Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens.

Specific IgE binding to carbohydrate moieties of glycosylated allergens has been known for years, but the importance of these structures for the elicitation of allergic reactions is still a matter of debate. Because of their conserved carbohydrate structures, especially N-glycans have always been prime candidates for IgE cross-reactivity between allergens from unrelated species. The aim of our study was to determine whether carbohydrate structures on glycoproteins can by themselves elucidate allergic reactions. We characterized in detail the carbohydrate moieties of the major allergens Phl p 1 and Phl p 13 of timothy grass pollen (Phleum pratense L.) by performing tryptic digests followed by HPLC, N-terminal sequencing, sugar analysis, MALDI-TOF- and ESI-ICRFT-MS. Phl p 1 contains one N-glycan with one of the two glycoforms MMXF3 and M0XF3 and a single furanosidic arabinose, which is bound to a hydroxyproline residue in direct vicinity to the N-glycan. This O-glycosylation is probably due to an arabinosylation consensus sequence found in the N-terminal part of Phl p 1 and other group 1 allergens, but displayed no IgE-reactivity. Thus, Phl p 1 is monovalent with respect to its IgE-binding carbohydrate epitopes and showed no mediator release. In contrast, the carbohydrate moiety of Phl p 13, which carries four of the same N-glycans (like Phl p 1), can cross-link IgE-receptors via carbohydrate chains and elicits IL-4 release from basophils.

Importance of Early Allergen Contact for the Development of a Sustained Immunoglobulin E Response in a Dog Model

Background: Immunoglobulin E (IgE)-mediated allergies are postulated to require early allergen contact and sensitization for the full development of sustained IgE levels. Methods: Thirty-two Beagle dogs from seven litters selectively bred for their high IgE response were sensitized by subcutaneous injection of chicken ovalbumin (OVA), peanut extract and recombinant birch pollen allergen (Bet v 1). In half of the dogs from each litter, sensitization injections were started on the first day of life; the other half of the same litter was first sensitized at the age of 4 months. To evaluate whether early sensitization also predisposes the animals to IgE responses to other allergens later in life, we injected a recombinant timothy grass pollen allergen (Phl p 5) later on, at the age of 10–12 months. Allergen-specific serum IgE and IgG levels were evaluated with enzyme-linked immunosorbent assays. In addition, 21 dogs were challenged with aerosolized OVA to measure bronchoconstrictive changes in lung function. Results: Early sensitized dogs developed significantly higher OVA-specific serum IgE levels than late sensitized dogs, in contrast to the IgG levels, which were lower in these dogs (p < 0.001). The increase in specific serum IgE and IgG following boosting remained different between the two groups for over a year. Titers of specific serum IgE and IgG were also different after sensitization with a new allergen injected later in life for the first time. Dynamic pulmonary compliance and resistance, both parameters for bronchoconstriction induced by OVA aerosol challenge, were also significantly higher in early sensitized dogs (for both parameters, p < 0.01). Conclusions: Contact with an allergen early in life is decisive for the development of sustained IgE levels and the development of IgE responses to additional allergens encountered later in life. Allergen avoidance during early life may have some preventive effect on IgE-mediated allergy in dogs.

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.

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. To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. 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. 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. 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.

Combination vaccines for the treatment of grass pollen allergy consisting of genetically engineered hybrid molecules with increased immunogenicity.

Most of the 400 million grass pollen-allergic patients worldwide are co-sensitized to several unrelated grass pollen allergens. Based on frequent co-sensitization patterns determined in 200 grass pollen-allergic patients, three recombinant hybrid molecules were developed by polymerase chain reaction-based mending of cDNAs coding for the major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, Phl p 6) for vaccination against grass pollen allergy. The hybrids rP2-P6, rP6-P2, and rP5-P1 contained most of the epitopes of natural grass pollen extract and induced stronger lymphoproliferative responses in cultured mononuclear cells of grass pollen-allergic patients than did equimolar mixtures of the individual allergens. Immunization of mice with the hybrids yielded higher antibody titers than did immunization with the individual allergen components or grass pollen extract, which suggests that the individual components of the hybrids can serve as molecular scaffolds for each other to enhance their immunogenicity. Antibodies induced with the hybrids in mice inhibited the binding of grass pollen-allergic patients' immunoglobulin E to each of the individual allergens and grass pollen extract and may thus represent protective antibodies. The principle of increasing the immunogenicity of antigens by engineering hybrids thereof may be applied not only for the treatment of polysensitized allergic patients but also for general vaccine development.

Evolution of IgM, IgE and IgG(1-4 )antibody responses in early childhood monitored with recombinant allergen components: implications for class switch mechanisms.

The formation of IgE antibodies against environmental allergens represents the hallmark of type I allergy. Data from in vitro cultured cells and experimental animal models provide controversial evidence for isotype switching from IgM to IgE production via sequential as well as non-sequential (i.e. direct) class switch. We analyzed the evolution of IgE responses in 11 children developing birch pollen and/or grass pollen allergy during the first 7 years of life using purified recombinant allergen molecules (major birch pollen allergen, Bet v 1; major timothy grass pollen allergens, Phl p 1, Phl p 2, Phl p 5). Demographic, clinical and serological data indicated a postnatal sensitization to pollen allergens. A parallel development of IgG(1-4) and IgE responses to recombinant allergen molecules compatible with a strictly sequential class switch to IgE was observed only in one child. The only partly synchronized and dissociated development of allergen-specific antibody responses found in all other cases can be best explained by a partly sequential class switch involving few switch stations or, more likely, by direct class switching. Kinetics and courses of allergen-specific antibody responses (IgM, IgG(1-4), IgE) during the first years of life suggest that, once established, allergen-specific IgE responses are driven by antigen contact rather than by cytokines controlling class switch to IgE.

Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen, Phl p 4, with cross-reactive potential.

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.

Identification of an allergen related to Phl p 4, a major timothy grass pollen allergen, in pollens, vegetables, and fruits by immunogold electron microscopy.

Group 4 grass pollen allergens represent 60 kDa glycoproteins recognized by 70% of patients sensitive to these pollens. An antiserum against purified Phl p 4 from timothy grass pollen was used to investigate various pollens, fruits, and vegetables for Phl p 4-related allergens by immunogold electron microscopy. In timothy grass, mugwort, and birch pollens, allergens were located in the wall, and in timothy grass and birch pollens additionally in the cytoplasm. In peanut, apple, celery root, and carrot root, only cytoplasmic areas were labeled. Group 4-related allergens thus occur in pollens of unrelated plants and in plant food and may therefore contribute to crossreactivities in patients allergic to various pollens and plant food.

Evaluation of two grass pollen extracts for immunotherapy by serum determinations of specific IgE and IgG4 antibodies towards purified Timothy grass pollen allergens (Phl p 1, 2, 4, 5, 6, 7, 11, 12) in patients undergoing hyposensitization treatment

The diagnosis of allergic diseases with recombinant allergens allows us to detect antibodies specific for single allergens in extracts. The aim of the present study was to assess the early effect of grass pollen immunotherapy on IgE and IgG4 responses to eight purified grass pollen allergens in patients undergoing hyposensitization treatment. The sera of 22 consecutive atopic individuals undergoing cluster regimen grass pollen immunotherapy were analyzed for IgE and IgG4 antibodies specific for grass pollen allergens (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Two serum samples were taken, one before the start of therapy and one between 12 and 15 weeks after the first immunization. Immunotherapy was performed with two allergy vaccines comprising a standardized extract aluminum-adsorbed grass pollen mix and a standardized extract of grass pollen mix adsorbed onto calcium phosphate. One treated patient showed a specific IgE conversion from negative (< 0.35 kUA/L) to positive in the capsulated hydrophilic carrier polymer (CAP) test for Phl p 2, 1 and 4 (1.89, 0.84 and 0.68 kUA/L, respectively). The sera of 10 of 11 patients treated with alluminum-adsorbed grass pollen extract showed a significant increase in specific IgG4 towards natural Timothy grass pollen extract and purified allergens, as well as significant IgG4 levels towards Phl p 1 ( P = 0.000238) Phl p 2 ( P = 0.000289), Phl p 4 ( P = 0.000585), Phl p 5 ( P = 0.000364), Phl p 6 ( P = 0.000346) and Phl p 11 ( P = 0.039623; Mann–Whitney U-test) 12 weeks after the onset of immunotherapy. The sera of seven of 11 subjects treated with calcium phospate-adsorbed grass pollen extract had significant IgG4 levels against Timothy pollen allergens, as well as significant IgG4 titers against Phl p 1 ( P = 0.004703), Phl p 4 ( P = 0.000282), Phl p 5 ( P = 0.015480), Phl p 6 ( P = 0.013012) and Phl p 11 ( P = 0.005178). Patients treated with aluminum-adsorbed grass pollen extract had higher levels of IgG4 towards Phl p 2, 4 and 6 and natural Timothy grass extract compared with patients treated with calcium phosphate-adsorbed grass pollen extract. Both the alluminum-adsorbed and calcium phosphate-adsorbed grass pollen extract allergy vaccines induced significant titers of specific IgG4 towards Phleum pratense pollen extract ( P = 0.008376 and 0.01148, respectively). These results indicate that grass pollen immunotherapy elicits an array of antibody specificities that reflect the allergen content and the potency of allergen extracts; this could be of pivotal importance to define optimal allergen extract doses.

Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.

Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.

A Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5, Defined by a Human Monoclonal IgE Antibody

We report the characterization of a domain of the major timothy grass pollen allergen, Phl p 5A, which contains the binding site for a human monoclonal IgE antibody. The human monoclonal IgE antibody fragment (Fab) was obtained from an IgE combinatorial library constructed from lymphocytes of a grass pollen-allergic patient. An 11.2-kD N-terminal fragment representing approximately one third of the complete Phl p 5A allergen could be identified to contain the binding site for the IgE Fab. The rPhl p 5A fragment revealed an extremely high allergenic activity in skin test experiments which in some cases equaled that of the complete Phl p 5A allergen. The rPhl p 5A domain thus represents an allergen fragment containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation.

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain.

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Structural Investigations of the Major Timothy Grass Pollen Allergen Phl p7

Structural Investigations of the Major Timothy Grass Pollen Allergen Phl p7

Use of phage display technology to investigate allergen-antibody interactions

Phage display is an advanced technology that can be used to characterize the interactions of antibody with antigen at the molecular level. It provides valuable data when applied to the investigation of IgE interaction with allergens. The aim of this rostrum article is to provide an explanation of the potential of phage display for increasing the understanding of allergen-IgE interaction, the discovery of diagnostic reagents, and the development of novel therapeutics for the treatment of allergic disease. The significance of initial studies that have applied phage display technology in allergy research will be highlighted. Phage display has been used to clone human IgE to timothy grass pollen allergen Phl p 5, to characterize the epitopes for murine and human antibodies to a birch pollen allergen Bet v 1, and to elucidate the epitopes of a murine mAb to the house dust mite allergen Der p 1. The technology has identified peptides that functionally mimic sites of human IgE constant domains and that were used to raise antiserum for blocking binding of IgE to the FcϵRI on basophils and subsequent release of histamine. Phage display has also been used to characterize novel peanut and fungal allergens. The method has been used to increase our understanding of the molecular basis of allergen-IgE interactions and to develop clinically relevant reagents with the pharmacologic potential to block the effector phase of allergic reactions. Many advances from these early studies are likely as phage display technology evolves and allergists gain expertise in its research applications. (J Allergy Clin Immunol 2000;105:1085-92.)

Identification of isoform-specific T-cell epitopes in the major timothy grass pollen allergen, Phl p 5.

The involvement of CD4+ T cells in the pathophysiology of atopic disease is well established. To gain further insight into the activation requirements for allergen-specific T cells, we characterized epitope specificity, HLA restriction and T-cell receptor (TCR) usage for T cells specific to Phl p 5, the group 5 major allergen of the grass Phleum pratense. To identify the T-cell epitopes of Phl p 5, three Phl p 5-specific T-cell lines (TCLs) and 15 T-cell clones (TCCs) generated from the peripheral blood of three grass-allergic patients were tested with recombinant truncated Phl p 5a fragments and synthetic Phl p 5b peptides representing these two different recombinant Phl p 5 isoallergens. Additional activation experiments with HLA-subtyped antigen-presenting cells and flow cytometry analysis with TCR V-specific mAb were performed to further characterize the activation requirements for Phl p 5-specific TCCs. At least nine distinct T-cell specificities were identified and the T-cell epitopes recognized differed considerably among the three patients. Most of the epitopes found were isoform-specific, whereas three epitopes were shared between Phl p 5a and 5b. Several human leucocyte antigen (HLA) class II molecules were involved in the recognition of Phl p 5. Different HLA restriction specificities were even found among TCCs specific to the same epitope region. All TCCs were TCR-alpha/beta positive, and an overrepresentation of TCR Vbeta 3.1+ clones among TCCs specific to Phl p 5 appear to exists as 31% (4/13) of the TCCs expressed TCR Vbeta 3.1 (compared with 5% TCR Vbeta 3.1+ T cells in human peripheral blood) with no correlation with epitope specificity or HLA restriction. The T-cell reactivity of the three grass-allergic patients investigated shows that isoallergen-specific T-cell epitopes are found throughout the peptide backbone of Phl p 5a and Phl p 5b, and dominant T-cell epitopes of Phl p 5 were not identified. This indicates that a mixture of at least full-length rPhl p 5a and rPhl p 5b may be required to target the total Phl p 5-specific T-cell response of atopic patients.

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.

Characterization of IgE–Reactive Autoantigens in Atopic Dermatitis

Previously we reported that patients with severe forms of atopy (e.g. atopic dermatitis, AD) frequently display IgE reactivity against autoantigens. Here we investigated the effects of periodate treatment and reducing versus nonreducing conditions on IgE recognition of nitrocellulose–blotted human cell extracts. IgE and IgG subclass reactivities of AD patients to blotted human cellular extracts as well as to ELISA plate–bound purified endogenous (myosin, histones) antigens and an environmental allergen (timothy grass pollen allergen, Phl p 5) were compared. Serum samples were collected over a period of 12 months from 3 autoreactive AD patients with pollen allergy and tested for IgE reactivity to nitrocellulose–blotted human epithelial and endothelial cell extracts as well as to birch and timothy grass pollen allergens. Results obtained indicate that (1) IgE autoantibodies may be directed primarily against protein and not carbohydrate epitopes, (2) reducing conditions seem to expose or better extract epitopes recognized by IgE autoantibodies, (3) IgE and IgG1–4 autoantibody responses were poorly associated and (4) IgE responses to autoallergens may reflect skin manifestations and may be boosted by seasonal exposure to pollen allergens. Our results thus indicate that IgE autoreactivity may represent a true form of autoimmunity directed against partly denatured peptide epitopes which may be boosted by exogenous allergen contact.