Roland Suck

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.

Characterization of a Hypoallergenic Recombinant Bet v 1 Variant as a Candidate for Allergen-Specific Immunotherapy

Background: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. Methods: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. Results: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. Conclusions: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.

Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1

Background We have recently engineered recombinant derivatives of the major birch pollen allergen Bet v 1 (rBet v 1 fragments and trimer) with strongly reduced allergenic activity.

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 ∼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.

Purification and Immunobiochemical Characterization of Folding Variants of the Recombinant Major Wasp Allergen Ves v 5 (Antigen 5)

Background: Antigen 5 is one of three major allergens in wasp venoms, but unlike phospholipase A1 and hyaluronidase, both of which are enzymes, its biological function is unknown. The cDNA coding for this allergen has been isolated and used for recombinant expression. Thorough analysis of the expression product is essential in order to evaluate the usefulness for in vivo or in vitro application. Objective: In this study, folding variants of the recombinant major allergen Ves v 5 from Vespula vulgaris were immunologically and biochemically investigated in order to determine their possible applicability for diagnostic or therapeutic purposes. Method: The cDNA encoding Ves v 5 was cloned into the expression vector pSE420 which generates recombinant products lacking a tag sequence. After expression, inclusion bodies were purified, subsequently denatured and dialyzed against different solutions. The structural properties of soluble proteins were analyzed by size exclusion chromatography, non-reducing SDS-PAGE, native PAGE, N-terminal sequencing, proteolytic digestion and ion exchange chromatography. Immunological investigations were performed by using different monoclonal antibodies (mAbs) specific for Ves v 5 and IgE from patients allergic to wasp venom allergens. Results: After dialysis, soluble monomeric recombinant Ves v 5 was more than 95% pure in each case. Using different dialysis solutions, clearly distinguishable folding variants were obtained. In one case, the recombinant allergen was comparable with the natural counterpart in respect of migration in non-reducing SDS-PAGE, native PAGE and IgE reactivity. This variant reacted with two different Ves v 5-specific mAbs and produced a stable fragment after proteolytic digestion. Elution from a cation exchange chromatography column was achieved with 320 mM NaCl. In two other cases, folding variants exhibited a different migration behavior in SDS-PAGE and native PAGE compared with the natural allergen. Also, the mAb 1E11 recognized none of these variants since it presumably detected a conformational epitope. Moreover, the IgE reactivity was clearly reduced and proteolytic digestion effected almost complete degradation. These variants eluted from the cation exchange column with 400 mM NaCl. Conclusion: Defined folding strategies resulted in both soluble misfolded variants with reduced IgE reactivity, potentially suitable for immunotherapy, and natural-like folded variants for diagnosis.

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense).

Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E‐reactivity to allergens in the high molecular mass range of 55–60 kDa. These proteins have not yet been fully characterized on the protein and DNA level.

The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50–60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13

More than 70% of the patients allergic to grass pollen exhibit IgE‐reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level.

Rapid and efficient purification of Phleum pratense major allergens Phl p 1 and group Phl p 2/3 using a two-step procedure.

The standardization of natural allergenic extracts and the characterization of recombinant allergens ensures a continuing requirement for highly purified natural allergens. The extraction and purification methods have to be reproducible and also preserve the biological and immunological activity of the allergen. A simple two-step purification system has been established in order to provide milligram amounts of purified natural Phl p 1 and Phl p 2/3. Both major allergens were separated from other proteins of timothy grass pollen extract in one step by hydrophobic interaction chromatography (HIC) under mild conditions. The allergens elute in the flow-through fraction while the rest of the proteins remain bound to the column. The very different molecular weights of Phl p 1 and Phl p 2/3 permitted separation of the allergens by a second step using gel filtration.

Phl p 3: Structural and immunological characterization of a major allergen of timothy grass pollen

Background The relevant importance of individual allergens for allergic sensitization is only partially understood. More detailed information on allergen structure and how it influences immunological responses can lead to better diagnosis of disease and improved preparations for allergen‐specific immunotherapy. Grass pollen contains several different allergens, and although the group 3 allergens have been classified long ago, their structure and allergenicity have been poorly investigated.

Size exclusion chromatography as a tool for quality control of recombinant allergens and hypoallergenic variants

Proteins or glycoproteins bearing epitopes for human IgE antibodies are designated as allergens causing type I allergic diseases. In this study, recombinant allergens were compared with their natural counterparts either as part of extracts or as purified molecules with respect to several biochemical and immunological properties. Natural and recombinant Bet v 1 and Phl p 1, major allergens of birch pollen extracts and Phleum pratense pollen extracts, were analyzed by SDS-PAGE, immunoblotting, EAST inhibition and size exclusion chromatography (SEC). Differences of IgE-binding capacities between recombinant Bet v 1 as well as recombinant Phl p 1 variants were detected by EAST inhibition. These results were confirmed by size exclusion chromatography in that the recombinant proteins showed differences of their elution volumes being equivalent to the natural molecules only with the more active recombinant form. In contrast, SDS-PAGE and immunoblot analysis resulted in divergent characteristics, as either migrations of the variants were similar or no differences of IgE binding were detectable. In conclusion, size exclusion chromatography is the method of choice for quality control of well characterized recombinant allergens, comprising control of purity, protein content and conformation.