Recombinant allergen components

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	Additive to extracts to counteract deficiency of a particular allergen component   - improved clinical sensitivity   - enhanced quantitative performance
	Extract replacement for fully defined & designed reagent composition   - selected, “informative” molecules   - confounding components excluded   to ensure optimal amount of all relevant allergens
	Single allergen components to determine individual sensitization profiles   - helps explain cross-reactivity   - correlation to particular clinical conditions   - optimized patient selection for specific immunotherapy

Recombinant allergens have a wide variety of uses, from the diagnosis and management of allergic patients to the development of immunotherapy to the standardisation of allergenic test products to use as tools in molecular allergology.

Many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome through recombinant allergens. Currently, the diagnosis of IgE-mediated allergy is performed through natural allergen extracts, which contain a mixture of allergenic and non-allergenic molecules that are difficult to standardise. Traditional diagnosis defines the source, e.g., Birch pollen, but it does not uncover which allergenic molecule(s) elicit the sensitization. Recombinant allergens are tools to expose the allergenic molecule(s) involved. Although the diagnostic sensitivity of single allergen components may be generally lower than that of allergen extracts, the specificity is normally higher. Recombinant allergens allow more defined preparations for in vivo testing and in vitro testing.

A "component-resolved diagnostics" (CRD) is recommended for a more precise diagnosis. In this instance, the antibody reactivity profile of an allergic patient can be identified, along with the disease-eliciting allergens and potential cross-reactivity interactions. Recombinant allergens offer a highly specific way to elucidate patient- and disease-specific sensitization patterns, knowledge of which is needed for the development of patient-tailored allergen preparations to refine immunotherapy and reduce the risk of sensitising patients to other allergens.

Recombinant allergens can be produced and contain most of the epitopes present in complex allergen sources, which will facilitate innovative strategies for allergen immunotherapy (2). These include peptide-based vaccines, engineered hypoallergens with reduced reactivity to IgE antibodies, nucleotide-conjugated vaccines that promote Th1 responses, and possibly prophylactic allergen vaccines. By the destruction of the protein fold through point mutations or recombinant oligomers, low IgE-binding allergen derivatives can be created, which may reduce the risk of adverse effects in specific immunotherapy: the modified allergens have preserved immunogenicity and are still able to induce regulatory T-cells, which modulate a pathologic TH2-response into a dominating TH1-response, resulting in reduced hypersensitivity.

Recombinant allergens will also allow the standardisation of allergen products (defined mixtures of biotechnology produced allergens). Recombinant allergens have, moreover, become irreplaceable tools in molecular allergology.

	Cross-reactivity model Example: PR-10 proteins (Bet v 1 homologues)
	Who is going to react? Factors to consider: IgE antibody concentration Complexity of sensitization Overall allergen exposure Stage in disease development

Figure 2. Component Resolved Diagnostics (CRD) helps explain clinical reactivity.

Recombinant allergens are useful tools for evaluating cross-reactivity, for better management of the patient, and for developing more efficacious immunotherapy.

For example, the tree families Betulaceae, Fagaceae, and Corylaceae, belonging to the order Fagales, contain cross-reactive allergens. Of these allergens, birch is considered to represents the most potent and frequent allergen source; and of the birch tree allergens, Bet v 1 is a major allergen, responsible for major cross-reactivity between Bet v 1 from Birch and Bet v 1-homologous proteins in members of the Fagales (Alder, Hazel, Hornbeam) (3). Furthermore, as Bet v 1-related allergens are also present in a number of other trees and plants, this allergen has been said to be the cause of cross-reactivity between birch and a number of fruits, vegetables and spices, e.g., apple, carrot, celery, cherry, and pear (4, 7).

Not surprisingly, given the importance of birch pollen allergy and related allergies in the northern parts of Europe, one of the first recombinant allergens created was the cDNA coding for Bet v 1: rBet v 1. rBet v 1 has been expressed in E. coli as a biologically active allergen and was demonstrated to be very similar to the native allergen, allowing accurate in vivo and in vitro diagnosis of tree pollen allergy in >95% of cases (5-7).

Using recombinant Bet v 1 allows improved diagnostic precision and management of not only the major sensitising allergen, but of potential cross-reactivity as well. Recombinant Bet v 1 has also made it clear that Bet v 1 is the initial sensitising allergen for many patients suffering from Fagales pollen allergy and Birch pollen-related plant-food allergy as exemplified by oral allergy syndrome (4, 7). Other studies have confirmed that Bet v 1 may be considered a marker allergen for genuine sensitisation to Fagales pollen and Birch pollen-related food allergy (4, 7-8).

Figure 3. Distribution of some cross-reactive protein families.

Figure 4. Geographical differences in allergy to fruits and vegetables.

Recombinant allergens have also been useful in elucidating other natural birch pollen allergens. Bet v 2 is a profilin (9-10), and Bet v 3 and Bet v 4 have been identified as 2EF-hand Ca2+-binding proteins (11-13). These allergens have been shown to have a wide distribution, not only in pollen from unrelated plants (grasses, weeds, trees), but also in other plant tissues (fruits, vegetables, nuts, spices), demonstrating extensive cross-reactivity. These allergens can therefore serve as marker allergens for plant polysensitisation (14). In other words, a positive reaction to an allergen with cross-reactive potential may predict allergic reactions to all those allergen sources containing structurally related molecules.

If the common allergens from an allergen source could be identified by molecular cloning techniques and produced as recombinant allergens, these could be used for component-resolved diagnostics (CRD) of allergy, enabling the identification of the disease-eliciting allergens for each patient and thus establishing a detailed IgE reactivity profile (15). By contrast, extract-based diagnosis provides a determination of the allergen source, by telling us that a patient reacts to unspecified components in the given extract.
 

Figure 5. Examples of allergens of particular diagnostic interest.

• PR-10 proteins (Bet v 1 homologues)
• Major grass pollen allergens
• Major epithelial/dander allergens
• Lipid Transfer Proteins (ns-LTPs)
• Tropomyosins
• Nut/seed storage proteins
• 2-EF-hand, Ca2+-binding proteins
• Profilins
• Cross-reactive Carbohydrate Determinants (CCDs) 

Because immunotherapy represents a costly, time-consuming and sometimes risky treatment, the clinician must make careful decisions as to which patients are suitable for this treatment. For example, we know that clinically relevant reactions to Birch pollen can be found in patients who were not originally sensitised to Birch (16). This is a consequence of sensitisation to cross-reactive allergens from other plants (3). Recombinant allergens can distinguish patients who are genuinely sensitised to Birch pollen, as shown by their IgE reactivity to the major Birch pollen allergen Bet v 1, from patients who, as a result of IgE to cross-reactive allergens such as Bet v 2, exhibit positive skin tests to Birch pollen extracts without having been exposed to Birch (3).

The introduction of the recombinant allergens in the diagnosis of IgE-mediated allergy not only facilitates deciding whether a patient is suitable for immunotherapy, but also allows the measurement of IgE and IgG antibody responses to individual allergen components during allergen-specific immunotherapy, thereby allowing the monitoring of antibody profiles and levels during the course of immunotherapy (17-19).

References:

1. Niederberger V, Stubner P, Spitzauer S, Kraft D, Valenta R, Ehrenberger K, Horak F. Skin test results but not serology reflect immediate type respiratory sensitivity: a study performed with recombinant allergen molecules. J Invest Dermatol 2001 Oct;117(4):848-51
2. Valenta R, Ball T, Focke M, Linhart B, Mothes N, Niederberger V, Spitzauer S, Swoboda I, Vrtala S, Westritschnig K, Kraft D. Immunotherapy of allergic disease. Adv Immunol 2004;82:105-53
3. Mothes N, Valenta R. Biology of tree pollen allergens. Curr Allergy Asthma Rep 2004;4(5):384-90
4. Kazemi-Shirazi L, Pauli G, Purohit A, Spitzauer S, Froschl R, Hoffmann-Sommergruber K, Breiteneder H, Scheiner O, Kraft D, Valenta R. Quantitative IgE inhibition experiments with purified recombinant allergens indicate pollen-derived allergens as the sensitizing agents responsible for many forms of plant food allergy. J Allergy Clin Immunol 2000;105(1 Pt 1):116-25
5. Valenta R, Duchene M, Vrtala S, Birkner T, Ebner C, Hirschwehr R, Breitenbach M et al. Recombinant allergens for immunoblot diagnosis of tree-pollen allergy. J Allergy Clin Immunol 1991;88(6):889-94
6. Menz G, Dolecek C, Schonheit-Kenn U, Ferreira F, Moser M, Schneider T, Suter M, Boltz-Nitulescu G, Ebner C, Kraft D, Valenta R. Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen. Clin Exp Allergy 1996;26(1):50-60
7. Niederberger V, Pauli G, Gronlund H, Froschl R, Rumpold H, Kraft D et al. Recombinant birch pollen allergens (rBet v 1 and rBet v 2) contain most of the IgE epitopes present in birch, alder, hornbeam, hazel, and oak pollen: a quantitative IgE inhibition J Allergy Clin Immunol 1998;102(4 Pt 1):579-91
8. Moverare R, Westritschnig K, Svensson M, Hayek B, Bende M, Pauli G, Sorva R, Haahtela T, Valenta R, Elfman L. Different IgE Reactivity Profiles in Birch Pollen-Sensitive Patients from Six European Populations Revealed by Recombinant Allergens: An Imprint of Local Sensitization. Int Arch Allergy Immunol 2002;128(4):325-35
9. Valenta R, Duchene M, Pettenburger K, Sillaber S, Valent P. Identification of profilin as a novel pollen allergen; IgE autoreactivity in sensitized individuals. Science 1991;253:557-60
10. Valenta R, Ferreira F, Grote M, Swoboda I,Vrtala S, Duchene M, Deviller P, MeagherRB, McKinney E, Heberle-Bors E et al.Identification of profilin as an actin-bindingprotein in higher plants.J Biol Chem 1993;268(30):22777-81
11. Seiberler S, Scheiner O, Kraft D, Lonsdale D, Valenta R. Characterization of a birch pollen allergen, Bet v III, representing a novel class of Ca2+ binding proteins: specific expression in mature pollen and dependence of patients' IgE binding on protein-bound Ca2+. EMBO J 1994;13(15):3481-6
12. Twardosz A, Hayek B, Seiberler S, Vangelista L, Elfman L, Gronlund H, Kraft D, Valenta R. Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4. Biochem Biophys Res Commun 1997;239(1):197-204
13. Engel E, Richter K, Obermeyer G, Briza P, Kungl AJ et al. Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains. J Biol Chem 1997;272(45):28630-7
14. Valenta R, Hayek B, Seiberler S, Bugajska-Schretter A, Niederberger V, Twardosz A, Natter S, Vangelista L, Pastore A, Spitzauer S, Kraft D. Calcium-binding allergens: from plants to man. Int Arch Allergy Immunol 1998;117(3):160-6
15. Valenta R, Lidholm J, Niederberger V, Hayek B, Kraft D, Gronlund H. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy 1999;29(7):896-904
16. Westritschnig K, Sibanda E, Thomas W, Auer H, Aspock H, Pittner G, Vrtala S, Spitzauer S, Kraft D, Valenta R. Analysis of the sensitization profile towards allergens in central Africa. Clin Exp Allergy. 2003;33(1):22-7
17. Ball T, Sperr WR, Valent P, Lidholm J, Spitzauer S, Ebner C, Kraft D, Valenta R. Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy. Eur J Immunol 1999;29(6):2026-36
18. Mothes N, Heinzkill M, Drachenberg KJ, Sperr WR, Krauth MT, Majlesi Y, Semper H, Valent P, Niederberger V, Kraft D, Valenta R. Allergen-specific immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies. Clin Exp Allergy 2003;33(9):1198-208
19. Moverare R, Elfman L, Vesterinen E, Metso T, Haahtela T. Development of new IgE specificities to allergenic components in birch pollen extract during specific immunotherapy studied with immunoblotting and Pharmacia CAP System. Allergy 2002;57(5):423-30
    

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