Timothy Grass Pollen Allergen Research Articles

B cell epitopes of the major timothy grass pollen allergen, phl p 1, revealed by gene fragmentation as candidates for immunotherapy.

Group 1 grass pollen allergens are recognized by IgE antibodies of almost 40% of allergic individuals and therefore belong to the most important elicitors of Type I allergy worldwide. We have previously isolated the cDNA coding for the group 1 allergen from timothy grass, Phl p 1, and demonstrated that recombinant Phl p 1 contains most of the B cell as well as T cell epitopes of group 1 allergens from a variety of grass and corn species. Here we determine continuous B cell epitopes of Phl p 1 by gene fragmentation. IgE antibodies of grass pollen allergic patients identified five continuous epitope-containing areas that on an average bound 40% of Phl p 1-specific IgE antibodies and were stably recognized in the course of disease. In contrast to untreated patients, patients undergoing grass pollen immunotherapy started to mount IgG(4) antibodies to the recombinant IgE-defined fragments in the course of immunotherapy. The protective role of these IgG(4) antibodies is demonstrated by observations that 1) increases in rPhl p 1 fragment-specific IgG(4) were in parallel with decreases in Phl p 1-specific IgE, and 2) preincubation of rPhl p 1 with patients sera containing rPhl p 1 fragment-specific IgG(4) blocked histamine release from basophils of an untreated grass pollen allergic patient. We propose to use recombinant Phl p 1 fragments for active immunotherapy in order to induce protective IgG responses against IgE epitopes in grass pollen allergic patients. This concept may be applied for the development of allergy vaccines whenever the primary sequence or structure of an allergen is available.

Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy.

Whether the modulation of antibody responses can contribute to the improvement of clinical symptoms in patients receiving allergen immunotherapy represents a controversial issue. We have used purified [seven recombinant (r) and one natural] timothy grass pollen allergens as well as recombinant B cell epitope-containing fragments of the major timothy grass pollen allergen, Phl p 1, to investigate humoral immune responses in eight allergic patients receiving grass pollen-specific immunotherapy. We found that the administration of aluminium hydroxide-adsorbed grass pollen extract induced complex changes in allergen/epitope-specific antibody responses: increases in IgG subclass (IgG1, IgG2, IgG4) responses against allergens recognized before the therapy were observed. All eight patients started to mount IgE and IgG4 responses to continuous Phl p 1 epitopes not recognized before the therapy and a de novo induction of IgE antibodies against new allergens was found in one patient. Evidence for a protective role of IgG antibodies specific for continuous Phl p 1 epitopes was provided by the demonstration that preincubation of rPhl p 1 with human serum containing therapy-induced Phl p 1-specific IgG inhibited rPhl p 1-induced histamine release from basophils of a grass pollen-allergic patient. Our finding that immunotherapy induced antibody responses against previously not recognized B cell epitopes indicates the vaccination character of this treatment. The fact that patients started to mount de novo IgE as well as protective IgG responses against epitopes may explain the unpredictability of specific immunotherapy performed with allergen extracts and emphasizes the need for novel forms of component-resolved immunotherapy.

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7.

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.

Lessons from the Antibody Recognition of the Major Timothy Grass Pollen Allergen Phl p 1

Lessons from the Antibody Recognition of the Major Timothy Grass Pollen Allergen Phl p 1

“Allergen Engineering”: Variants of the Timothy Grass Pollen Allergen Phl p 5b with Reduced IgE-Binding Capacity but Conserved T Cell Reactivity

One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced IgE-binding capacity but retained T cell reactivity. Using overlapping dodeca-peptides, the dominant T cell epitopes of the timothy grass pollen allergen Phl p 5b were identified. By site-directed mutagenesis outside these regions, point and deletion mutants were generated. Allergen variants were analyzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and for histamine releasing capacity with peripheral blood basophils from different patients. The deletion mutants revealed significantly reduced IgE reactivity and histamine releasing capacity, compared with the wild-type Phl p 5b. Furthermore, in vivo skin prick tests showed that the deletion mutants had a significantly lower potency to induce cutaneous reactions than the wild-type Phl p 5b. On the other hand, T cell clones and T cell lines from different allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b. Considering their reduced anaphylactogenic potential together with their conserved T cell reactivity, the engineered allergens could be important tools for efficient and safe allergen-specific immunotherapy.

Conserved sequence motifs of CDR3 loops of TCR specific for two major epitopes of the grass pollen allergen PhI p 1.

We analyzed the cDNA sequence data of complementarity determining regions (CDR3) of epitope mapped alphabeta TCR of T cell clones (TCC). The TCC were specific for the major timothy grass (Phleum pratense) pollen allergen Phl p 1 and were derived from the peripheral blood of seven unrelated grass pollen-allergic individuals. Each TCR recognized one of two immunodominant T cell epitopes, PP73 or PP103, of Phl p 1. Although a diversity of recombined V and J segments was observed, amino acid motifs as long as five residues were conserved among CDR3 loops of TCR from TCC of different atopic individuals specific for the same peptide. The conserved sequences could comprise as much as 60% of the CDR3. All amino acid residues of the motifs of the CDR3beta and most of the CDR3alpha of all TCR used in this study were encoded by randomly added nucleotides. This indicates that they were specifically selected for by the peptide bound to the MHC class II molecule. For one selected patient, a larger number of TCC, specific for PP103, was analyzed. The TCR repertoire was limited to three different TCR. The same MHC class II molecule, DRB1*1301, was identified to present PP103 to each of the three TCR.

Immunization with Purified Natural and Recombinant Allergens Induces Mouse IgG1 Antibodies That Recognize Similar Epitopes as Human IgE and Inhibit the Human IgE-Allergen Interaction and Allergen-Induced Basophil Degranulation

Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (beta-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.

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.

Molecular Characterization, Expression inEscherichia coli,and Epitope Analysis of a Two EF-Hand Calcium-Binding Birch Pollen Allergen, Bet v 4

Birch pollen belongs to the most potent elicitors of Type I allergic reactions in early spring. Using serum IgE from a birch pollen allergic patient, two cDNA clones (clone 6 and clone 13) were isolated from a birch pollen expression cDNA library constructed in phage λgt11. Clone 6 encoded a 9.3 kD two EF-hand calcium-binding protein, designated Bet v 4, with significant end to end sequence homology to EF-hand calcium-binding allergens from weed and grass pollen. Recombinant Bet v 4, expressed as β-galactosidase fusion protein, reacted with serum IgE from approximately 20% of pollen allergic individuals. Depletion of allergen-bound calcium by EGTA treatment lead to a substantial reduction of IgE-binding to Bet v 4, indicating that protein-bound calcium is necessary for the maintenance of IgE-epitopes. The greatly reduced IgE-binding capacity of clone 13, a Bet v 4 fragment that lacked the 16 N-terminal amino acids, indicated that the N-terminus contributes significantly to the proteins IgE-binding capacity. By IgE-inhibition experiments it was demonstrated that recombinant Bet v 4 shared IgE-epitopes with natural Bet v 4 and a homologous timothy grass pollen allergen. Recombinant Bet v 4 may therefore be considered as a relevant crossreactive plant allergen, which may be used for diagnosis and treatment of patients suffering from multivalent plant allergies.

Inhibition of allergen-induced histamine release from human basophils by cyclosporine A and FK-506.

A number of structurally different allergens trigger the release of mediators from basophils by cross-linking of IgE receptors. In this study, we analyzed the effects of cyclosporine A (CSA) and FK-506 on allergen-induced histamine release in human blood basophils obtained from birch- or grass-pollen-allergic donors (n = 12). Preincubation of basophils with CSA (0.003-3 microg/ml) or FK-506 (0.003-3 microg/ml) led to inhibition of histamine release induced by purified recombinant tree pollen allergens (r Bet v 1, r Bet v 2) and timothy grass pollen allergens (r Ph1 p 1, r Ph1 p 2, r Ph1 p 5). The effects of CSA and FK-506 were dose dependent, with IC50 values ranging between 0.03 and 0.3 microg/ml for both CSA and FK-506. Cyclosporine H, an inactive CSA analog, did not show any effect on allergen-induced histamine secretion. IgE dependency of the reaction was demonstrated in passive transfer experiments using highly enriched human basophils (> 95% pure) and specific IgE from a patient allergic to Bet v 2. In summary, our data show that CSA and FK-506 inhibit recombinant-allergen-induced histamine release from peripheral blood basophils in allergic donors.

Comparison of recombinant timothy grass pollen allergens with natural extract for diagnosis of grass pollen allergy in different populations

BACKGROUND: Complementary DNAs coding for the major timothy grass pollen (Phleum pratense) allergens Phl p 1, Phl p 2, and Phl p 5 and birch profilin were isolated, expressed as recombinant nonfusion proteins in Escherichia coli, and purified. OBJECTIVE: In this study the in vitro IgE-binding capacity of recombinant Phl p 1, Phl p 2, Phl p 5, and birch profilin and their IgE recognition frequencies were investigated by using sera from different populations. METHODS: One hundred eighty-three sera from patients allergic to grass pollen were obtained from different populations in Europe, Japan, and Canada. The sera were selected according to clinical criteria, skin testing, and RAST (CAP system; Pharmacia, Uppsala, Sweden) and then tested for IgE reactivity with natural and purified recombinant timothy grass pollen allergens by ELISA and Western blot. RESULTS: Most (94.5%) of the patients allergic to grass pollen could be diagnosed with a combination of recombinant Phl p 1, Phl p 2, Phl p 5, and profilin by means of ELISA. Sera that did not react with the recombinant allergens contained low levels of timothy grass pollen–specific IgE. Although considerable variability in IgE recognition frequency of the recombinant allergens was observed in certain populations, a good correlation was found between natural timothy CAP results and the combination of recombinant allergens in all 183 tested sera ( r = 0.87). CONCLUSIONS: Despite considerable variability in the IgE recognition frequency, purified recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5) and profilin permitted successful in vitro diagnosis of grass pollen allergy in 94.5% of allergic individuals from different populations. The addition of other recombinant allergens (e.g., recombinant Phl p 4) would only slightly improve the in vitro test sensitivity. (J A LLERGY C LIN I MMUNOL 1996;98:652-8.)

Characterization of Phl p 4, a major timothy grass (Phleum pratense)pollen allergen

Background:Group 4 grass pollen allergens represent glycoproteins with a molecular weight of 50 to 60 kd, which are present in many grass species. Almost 75% of patients allergic to grass pollen display IgE reactivity to group 4 allergens, which hence can be regarded as major grass pollen allergens. Objective:In this study attempts were made to obtain information regarding the immunologic properties, localization, and occurrence of Phl p 4 and related allergens. Methods:Phl p 4 was detected in timothy grass pollen extracts by immunoblotting with serum IgE and monoclonal antibodies and was localized in pollen by immunelectron microscopy. A peptide sequence from Phl p 4 was obtained by amino acid sequencing. The resistance of Phl p 4 against trypsin was analyzed after trypsin treatment of timothy grass pollen extracts with serum IgE and monoclonal antibodies. Cross-reactivities between Phl p 4 and Amb a 1, the major allergen of ragweed, were studied by using monoclonal antibodies and by IgE-inhibition studies. Results:Phl p 4 was characterized as a trypsin-resistant major timothy grass pollen allergen. By immunelectron microscopy Phl p 4 was localized in the exine, cytoplasm, and amyloplast of timothy grass pollen. Significant sequence similarities of a Phl p 4 10 amino acid peptide with Amb a 1, the major ragweed allergen, could be found. The immunologic similarity of Phl p 4 and Amb a 1 was confirmed by cross-reactivity of monoclonal antibodies and patients’ IgE. Conclusion:Phl p 4 represents a trypsin-resistant major timothy grass pollen allergen with immunologic similarities to the major ragweed allergen Amb a 1 and therefore must be considered an important cross-reactive component in grass pollen and weed pollen allergy. (J A LLERGY C LIN I MMUNOL 1996;98:189-98.)

Construction of a Combinatorial IgE Library from an Allergic Patient: ISOLATION AND CHARACTERIZATION OF HUMAN IgE Fabs WITH SPECIFICITY FOR THE MAJOR TIMOTHY GRASS POLLEN ALLERGEN, Phl p 5

To characterize human IgE antibodies with specificity for a major allergen at the molecular level, we have constructed an IgE combinatorial library from a grass pollen allergic patient. cDNAs coding for IgE heavy chain fragments and for light chains were reverse-transcribed and polymerase chain reaction-amplified from RNA of peripheral blood lymphocytes and randomly combined in plasmid pComb3H to yield a combinatorial library of 5 x 10(7) primary clones. IgE Fabs with specificity for Phl p 5, a major timothy grass pollen allergen, were isolated by panning. Sequence analysis showed that the 4 of the Fabs used the same heavy chain fragments which had combined with different kappa light chains. Soluble recombinant IgE Fabs were purified by affinity chromatography to Phl p 5 and, like natural IgE antibodies, cross-reacted with group 5 allergens from different grass species. The described approach should facilitate studies on the molecular interaction between IgE antibodies and allergens and encourages the consideration of specific IgE Fabs that are capable of interfering with allergen-IgE binding as potential therapeutic tools.

Immunologic characterization of purified recombinant timothy grass pollen ( Phleum pratense) allergens (Phl p 1, Phl p 2, Phl p 5)

Grass pollen allergens belong to the potent elicitors of type I allergy. Approximately 40% of allergic individuals display IgE reactivity with grass pollen allergens. In previous studies we have reported the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1, Phl p 2, and Phl p 5. To achieve high level expression of immunologically active timothy grass pollen allergens in E. coli, the cDNAs were inserted into expression plasmids. The three recombinant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein chemical methods and tested for their IgE-binding capacity by immunoblot and ELISA, as well as in histamine release assays. Milligram amounts of pure recombinant allergens were obtained from cultured E. coli. IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELISA. With ELISAs the percentage of grass pollen-specific IgE directed against the individual recombinant allergens could be estimated. In addition, the purified recombinant timothy grass pollen allergens induced dose-dependent and specific histamine release from patients' blood basophils. Purified recombinant timothy grass pollen allergens represent useful tools for diagnosis and therapy of grass pollen allergy.

Common IgE-epitopes of recombinant Phl p I, the major timothy grass pollen allergen and natural group I grass pollen isoallergens

Grass pollen allergens are potent elicitors of Type I allergy. More than 95% of grass pollen allergic patients display IgE-cross-reactivity to group I grass pollen allergens of different grass species. A cDNA coding for the major timothy grass pollen allergen, Phl p I, was isolated previously. To investigate the presence of common IgE-epitopes among naturally occurring group I grass pollen isoallergens, Phl p I was expressed in Escherichia coli and used for IgE-absorption experiments. Recombinant Phl p I was able to inhibit IgE-binding to most of group I isoallergens from seven grass species as identified by two dimensional electrophoresis. When tested in competitive ELISA experiments, recombinant Phl p I bound a high percentage of grass pollen specific IgE. The results indicate that recombinant Phl p I shares many of the IgE-epitopes with natural group I grass pollen allergens and hence may represent a useful tool for specific diagnosis and therapy of grass pollen allergy.

Effects of IL‐4 and IL‐13 on total and allergen specific IgE production by cultured PBMC from allergic patients determined with recombinant pollen allergens

Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells. In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood mononuclear cells (PBMC) from pollen allergic patients and healthy control individuals. Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profilin (Bet v II) and recombinant timothy grass pollen allergens, Phl p I, Phl p II, and Phl p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected. Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.

IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens

A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens. In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed. Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen–specific IgE was estimated. Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I. The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to estimate the percentage of specific IgE that individuals produce against certain allergens. The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen–specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy. (J ALLERGY CLIN IMMUNOL 1994;94:88-94.)

Epitope analysis of isoforms of the major allergen Phl p V by fingerprinting and microsequencing.

The major allergen of timothy grass pollen (Phleum pratense), designated as Phl p V, consists of isoallergenic components of 38 and 32 kDa with pl values of 5.2-7.5 and 4.8-5.9, respectively. The different-sized proteins reveal similarities in IgE reactivity, N-terminal sequence and protein staining. For epitope analysis of these allergens a combination of enzymatic cleavage of electrophoretically separated proteins and immunoblotting techniques with subsequent N-terminal sequencing was performed. After isolation of the components from two-dimensional PAGE gels, proteins were enzymatically cleaved and separated by SDS-PAGE. By endoproteinase Glu-C cleavage six IgE-reactive fragments of each 32 kDa protein and three of each 38 kDa allergen were obtained. Microsequencing of the fragments revealed internal sequences that did not show any similarities between the different-sized allergens. Therefore, we assume only slight structural variations among allergens of similar sizes, whereas the 32 and 38 kDa proteins reveal great differences.

Properties of Tree and Grass Pollen Allergens: Reinvestigation of the Linkage between Solubility and Allergenicity

In this study we reinvestigated the kinetics of allergen release from birch pollen (Betula verrucosa) and timothy grass pollen (Phleum pratense) using different protein extraction procedures, immunoblotting with specific antibodies and immune electron microscopy. Pollen allergens such as the major birch pollen allergen, Bet v I, the major timothy grass pollen allergens, Phl p I and Phl p V, group-II/III allergens from timothy grass and profilins were released rapidly and in large amounts from hydrated pollen. Within a few minutes pollen allergens could be detected in aqueous supernatants prepared from birch and grass pollen with serum IgE or specific antibodies. In parallel the allergen content in the pollen pellet fractions decreased. A nonallergenic protein such as heat shock protein 70 can be extracted in sufficient amounts only with harsh extraction procedures. Immune electron microscopy of dry and rehydrated birch pollens showed that after short hydration, the major birch pollen allergen, Bet v I, migrated into the exine and to the surface of intact pollen grains, whereas profilin, against which a lower percentage of patients is sensitized, was retained in the pollen grain. Comparing the amino acid composition and hydrophilicity of the tested allergens with a nonallergenic protein such as heat shock protein 70, no significant difference was noted. In agreement with earlier observations we conclude that the allergenic properties of proteins are rather linked to the amount and speed of solubility from airborne particles than to intrinsic properties.

Human Immune Response against Timothy Grass Pollen Allergens

Enzyme-linked immunoassays were used to examine the binding specificity of affinity-purified anti-idiotypic antibody [against the idiotypic determinant on timothy grass pollen antigen B (AgB)-specific IgE] with murine AgB-specific IgG and IgE antibodies, and the serum from timothy-sensitive patients. In addition, the proliferative response of peripheral blood lymphocytes from timothy-sensitive patients with either antigen or anti-idiotypic antibody was examined. These studies suggest that the idiotypic determinant expressed on murine AgB-specific IgE is highly conserved on human AgB-specific IgE and T cells of timothy-sensitive patients.