Sylvia Laffer

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

Type I allergy is an immunoglobulin E (IgE)‐mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen‐containing sources but cannot identify the disease‐eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease‐causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

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.

Microarrayed recombinant allergens for diagnosis of allergy

We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi‐allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease‐eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi‐allergen tests consisting of microarrayed recombinant allergen molecules. Microarrayed recombinant allergens can be used to determine and monitor the profile of disease‐eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray‐based allergy testing will likely improve diagnosis, prevention and treatment of allergy.

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.—Niederberger, V., Hayek, B., Vrtala, S., Laffer, S., Twardosz, A., Vangelista, L., Sperr, W. R., Valent, P., Rumpold, H., Kraft, D., Ehrenberger, K., Valenta, R., Spitzauer, S. 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. FASEB J. 13, 843–856 (1999)

Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen

Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy. Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library. The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C‐terminal portion of group I grass pollen allergens from different species. A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins. Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues. The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE‐binding capacity as the natural allergen.

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.

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.

Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation : A study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin e detection system measuring immunoglobulin e capable of binding to FcεRI

Background Results from several studies indicate that the magnitude of immediate symptoms of type I allergy caused by allergen‐induced cross‐linking of high‐affinity Fcɛ receptors on effector cells (mast cells and basophils) is not always associated with allergen‐specific IgE levels.

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.

Suggestions for the Assessment of the Allergenic Potential of Genetically Modified Organisms

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.