Major Grass Pollen Allergen Research Articles

Genetic Engineering of the Major Timothy Grass Pollen Allergen, Phl p 6, to Reduce Allergenic Activity and Preserve Immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.

Costimulation Blockade Inhibits Allergic Sensitization but Does Not Affect Established Allergy in a Murine Model of Grass Pollen Allergy

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.

Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus

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

Immunization with a low-dose replicon DNA vaccine encoding Phl p 5 effectively prevents allergic sensitization

Replicase-based DNA vaccines stimulate T(H)1-biased immune responses at ultralow doses and induce self-removal of transfected cells through apoptosis. Both aspects are important requirements for efficient and safe DNA-based immunotherapy of type I allergies. A Sindbis virus replicon-based DNA vaccine encoding the major timothy grass pollen allergen Phl p 5 was evaluated for its antiallergic potential compared with a conventional DNA vaccine in a BALB/c mouse model of allergy. Mice were intradermally prevaccinated with plasmid DNA, followed by sensitization and intranasal allergen provocation with recombinant Phl p 5. In vitro proliferation and cytokine secretion was measured in splenocyte cultures. Distribution of IgG1, IgG2a, and IgE antibody subclasses was determined by means of ELISA. IgE-mediated degranulation was measured with the basophil release assay. Bronchoalveolar lavage fluid was analyzed for eosinophils, IL-4, IL-5, IL-13, and IFN-gamma. Mucus production, inflammatory infiltrates, and epithelial damage were determined in lung sections. Both vaccines induced T(H)1-biased immune responses, resulting in suppression of functional IgE, reduction of eosinophilia in bronchoalveolar lavage fluid, and alleviation of lung pathology. However, immunization with the replicon DNA vaccine elicited these effects at a 100-fold lower dose compared with the conventional DNA vaccine. The increased immunogenicity of replicon-based DNA vaccines allows for application of extremely low doses, thereby eliminating the concerns associated with conventional DNA vaccines, which have to be administered at milligram amounts to induce immune reactions in human subjects. Their high safety profile makes replicon-based DNA vaccines promising candidates for treatment of type I allergies in the clinic.

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

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

In contrast to specific B cells, human basophils are unaffected by the toxic activity of an allergen toxin due to lack of internalization of immunoglobulin E‐bound allergen

Specific immunotherapy is the only curative therapy for type I allergies and the alarming increase in allergy prevalence emphasizes the need for additional/alternative strategies for curative treatment. Allergen toxins (AT), fusion products of an allergen with an apoptosis inducing cytotoxin, are a new kind of immunotoxin. AT should allow allergen-specific targeting and elimination of allergy-relevant cells, with B cells being the primary target. An important question is the fate of the effector cells, e.g. mast cells and basophils, which carry allergen-specific IgE: the immunotoxin might even prove to be harmful. We established a reliable in vitro B cell model (using two mouse hybridoma cell lines) for testing specificity and toxicity of P5-ETA', a fusion protein of the major timothy grass pollen allergen Phl p 5b and truncated Pseudomonas Exotoxin A. In a second step, we investigated the impact of the AT on human basophils. P5-ETA' reliably eliminated Phl p 5-specific cells in the in vitro B cell model, leaving unspecific B cells unharmed. Human basophils of grass pollen allergic donors specifically bound P5-ETA', released IL-4 and up-regulated the activation marker CD203c, but were not subject to the toxic effect because of lack of internalization of IgE-bound allergen. According to our data, basophils are pure effector cells in the context of IgE-bound allergen and not involved in classical antigen presentation.

Intranasal tolerance induction with polypeptides derived from 3 noncross-reactive major aeroallergens prevents allergic polysensitization in mice

Specific immunotherapy is less effective in patients with multiple allergic sensitizations compared with monosensitized patients. We therefore established a mouse model of polysensitization to the major birch and timothy grass pollen allergens to test whether allergic polysensitization can be prevented by multiple allergen application via the mucosal route. Female BALB/c mice were immunized intraperitoneally with recombinant (r) Bet v 1, rPhl p 1, and rPhl p 5. For intranasal tolerance induction, a mixture of the complete allergens was compared with allergen-derived immunodominant peptides applied either as a mixture or as a synthetic hybrid peptide composed of the T-cell epitopes of the 3 allergens. Intranasal application of the mixture of the complete allergen molecules did not prevent polysensitization to the same allergens. In contrast, pretreatment with a mixture of the immunodominant peptides or the hybrid peptide led to significantly reduced allergen-specific IgE responses in sera, IL-4 production in vitro, and suppressed airway inflammation. TGF-beta mRNA levels did not change, and IL-10 production was significantly suppressed after the pretreatment. The fact that the reduction of IL-10 was not abrogated after IL-10 receptor neutralization and that tolerance was not transferable with splenocytes indicates that the suppression of T(H)2 responses in polysensitized mice might not be mediated by immunosuppressive cytokines. Our study demonstrates that it is possible to suppress allergic immune responses simultaneously to several clinical important allergens. Thus, mucosal coapplication of selected peptides/hybrid peptides could be the basis of a mucosal polyvalent vaccine to prevent multiple sensitivities in atopic patients.

Immunoprophylaxis with DNA vaccination inhibits Th2-mediated responses and airway inflammation in mice sensitised with the major timothy grass pollen allergen Phl p 5b

DNA vaccination is discussed as having the potential of improving therapy of type I allergies. A mouse model was used to investigate the effect of DNA vaccination against sensitisation with the clinically relevant Phl p 5b major allergen from timothy grass pollen in an immunoprophylactic experimental setup. Pre-treating the mice with Phl p 5b-DNA-construct significantly inhibited the production of specific IgE and IgG 1 antibodies and prevented the development of a lung pathology associated with inflammation. Thus, immunoprophylaxis with an expression vector containing the Phl p 5b sequence totally prevented the emergence of characteristics associated with type I allergy and chronic bronchitis.

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

Allergy vaccines based on natural allergen extracts contain greatly varying amounts of individual allergens with different immunogenicity. 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. 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. 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. 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.

A single recombinant hybrid molecule consisting of the major timothy grass pollen allergens to replace natural pollen extracts for skin test diagnosis

A single recombinant hybrid molecule consisting of the major timothy grass pollen allergens to replace natural pollen extracts for skin test diagnosis

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.

Biochemical characterization of recombinant Phl p 6 and comparison with natural Phl p 6 and recombinant Phl p 5

Rationale House dust mites and grass pollens are the most important allergen sources worldwide. Here, we report the characterization of recombinant Phl p 6, a major grass pollen allergen from Phleum pratense, using standard biochemical methods. Methods Recombinant Phl p 6 was expressed in Pichia pastoris and recombinant Phl p 5.0107, was expressed in E.coli. Both were purified to homogeneity and analyzed by SDS-PAGE. rPhl p 6 and nPhl p 6 were analyzed by immunoblotting and rocket immuno electrophoresis (RIE). Melting temperatures of rPhl p 6, nPhl p 6 and rPhl p 5 were determined by Circular Dichroism (CD). Results Recombinant Phl p 6 migrated in reduced silver stained SDS-PAGE and immunoblots as nPhlp6. When analyzed by RIE rPhl p 6 and nPhl p 6 formed precipitates of similar morphology. Furthermore, rPhlp6, nPhlp6 and rPhl p 5 gave rise to similar spectra (local max at 192 nm and local min at 208 and 222 nm) when analyzed by CD. CD temperature denaturation analysies showed similar melting temperatures, 58.5 ± 0.5°C (mean ± 95% CL) (rPhl p 6) and 59.0 ± 0.3°C (nPhl p 6) and 65.3 ± 1.4°C (rPhlp 5), respectively. Conclusion An expression and purification procedure has been established enabling the production of recombinant Phl p 6 having biochemical characteristics and immunochemical activity corresponding to that of the natural allergen from grass pollen.

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.

Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high-resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N-terminal sequences and IgE-binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed-phase high-performance liquid chromatography (RP-HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.

Hypoallergenic derivatives of major grass pollen allergens for allergy vaccination.

Grass pollen-induced hay-fever and allergic asthma represent a major health problem in industrialized countries. Whereas the symptoms of these allergic conditions can be controlled by pharmacotherapy, specific immunotherapy vaccination is the only causative approach towards the treatment of these type 1 allergies. Specific immunotherapy is based on administration of increasing amounts of the disease-causing allergens in the form of allergen-containing extracts. However, the extracts used for immunotherapy consist of allergenic and non-allergenic components and may induce severe anaphylactic side-effects upon therapeutic administration. With recent developments in molecular biology of pollen allergens it has become feasible to produce modified hypoallergenic derivatives of recombinant allergens with abrogated or greatly reduced likelihood of anaphylactic side-effects as compared to extract-based treatments. We have demonstrated this concept through reducing the anaphylactic potential of major rye grass pollen allergens by introducing a few point mutations which leave the overall structural fold of the molecule unaltered. These modified forms are expected to make allergen-specific immunotherapy more widely used in the future.

Purification strategy for recombinant Phl p 6 is applicable to the natural allergen and yields biochemically and immunologically comparable preparations

The recombinant major grass pollen allergen Phl p 6 has been expressed with a N-terminal 6×His-tag sequence and subsequently purified using nickel-chelating Sepharose. After cleavage of the tag-sequence, a second pass over the affinity chromatography revealed that even untagged rPhl p 6 bound tightly. In order to determine if that property is typical for Phl p 6, the natural allergen was purified in the same way starting with a grass pollen extract. Indeed, nPhl p 6 could be highly enriched in one step using nickel-chelating Sepharose. In addition to this new powerful purification method, the results provide further information in that the recombinant and natural allergens share a lot of properties, since biochemical characteristics are reflected in the purification strategies. The preparations of natural and recombinant Phl p 6 were used for comparative electrophoretic, chromatographic and immunological analysis which demonstrated high similarity.

Purification and Characterization of a Novel Isoallergen of a Major Bermuda Grass Pollen Allergen, Cyn d 1

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.

Purification and characterization of a novel isoallergen of a major Bermuda grass pollen allergen, Cyn d 1.

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.

Effect of cysteine mutagenesis on human IgE reactivity of recombinant forms of the major rye grass pollen allergen Lol p 1

Hypersensitivity to the group 1 grass pollen allergens is a significant causative factor in the onset of symptoms for hay fever sufferers. To better understand the IgE reactivity of the group 1 allergen from rye grass pollen, we sought to disrupt potential conformational IgE epitopes on recombinant (r) Lol p 1 by the specific replacement of the seven cysteine residues in the protein sequence. Site-directed mutagenesis on the Lol p 1 coding sequence was used to replace all seven cysteine residues with serine residues. rLol p 1 and the seven cysteine variants generated by this method were tested for comparative human IgE reactivity via western blot immunoscreening and densitometry. Alteration of the cysteine residues at amino acid positions 72, 77, 83 and 139 of rLol p 1 was found to reduce the human IgE binding potential of the molecule. However, the most consistent reduction in human IgE reactivity was demonstrated by replacement of C 77 ; human IgE antibodies showed an average 62.7% reduction in reactivity to this molecule. The present investigation has shown that at least one of the cysteine residues within the Lol p 1 protein contributes to the IgE binding properties of this allergen.

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.