**2. Progress in the molecular cloning and production of allergens**

The molecular cloning has provided a practical and efficient way to obtain highly purified molecules for different purposes; in the biomedical sciences this is evident by the increasing amount of biological products, obtained by recombinant DNA technology, which are com‐ mercially available for diagnosis and treatment of different diseases, as well as the wide variety of reagents for basic research. The era of molecular cloning of allergen molecules was initiated in 1988 with the report of a cDNA clone coding for the allergen Der p 1 isolated from a cDNA

© 2013 Cantillo and Puerta; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Cantillo and Puerta; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

library of the house dust mite *Dermatophagoides pteronyssinus,* screened with rabbit anti -Der p 1 antiserum [1, 2]. Latter, Tovey, E.R *et al.* [3], using sera from allergic individuals for screening a mite cDNA library also isolated a clone of Der p 1. This strategy was useful to explore the whole spectrum of IgE binding proteins in a natural source and to isolate positive clones to express the molecules [4, 5]. The development and optimization of technology based on the polymerase chain reaction (PCR), have given an important impulse to cloning and identifica‐ tion of new allergens. PCR can be applied to screen cDNA library and amplify specific clones, or to obtain by RT- PCR the nucleotide sequence coding for specific allergens and then cloning in an appropriate vector for expression [6-10]. The numerous nucleotide sequences of allergens reported in data bank have facilitated the isolation of new allergens from RNA material using PCR technology, avoiding the construction of cDNA library and the use of sera from allergic subjects for screening, which is time consuming [11-14]. An expressed sequence tagging (EST) approach was applied to obtaining a large sampling and overview of expressed genomes of several mite species [15], the EST approach involved the partial sequencing of random clones selected from cDNA libraries, allowing the identification of allergens with homology to genes from more distantly related species or even across taxonomic kingdoms.

protein and showed to be biologically active, with capacity to bind human IgE, to induce mediator release from basophiles and to stimulate T cell proliferation [30]. A large percentage of allergens are from plants, thus the plant-based expression systems are ideal for the produc‐ tion of certain recombinant allergens, which could have problems such as incorrect processing, incorrect folding and insolubility when expressed in bacteria or other non-plant systems. Thaumatin or thaumatin-like proteins, only when expressed in *Nicotiana benthamiana* result in fully IgE-reactive proteins [31]. Interesting, expression in plants offers the opportunity for oral delivery of recombinant allergens of non- plant origin as a therapeutic approach for mucosal immunization for treating allergic diseases. Oral treatment of mice with squash extracts containing virus-expressed Der p 5 allergen caused inhibition of both allergen-specific IgE synthesis and airway inflammation [32], this plant-based edible vaccines is very promising.

From Molecular Cloning to Vaccine Development for Allergic Diseases

http://dx.doi.org/10.5772/52821

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Allergies are inflammatory diseases characterized by a Th2 biased response induced in atopic individuals for exposure to allergens. The Th2 response is also induced by helminthes, which occur in an environment characterized by the presence of IL-4, IL-5 and IL-13. Nuocytes [33, 34], basophiles [35] and type 2 multi-potent progenitor cells [36] seem to be an important source of this cytokines and necessary for the development of allergic response. Allergen-specific IgE antibodies produced by B cells bind to Fc epsilon receptor 1 (FcεRI) on basophiles or mast cells, sensitizing them. After consecutive exposure, allergen binds to IgE on these cells leading to the release of inflammatory mediators of immediate-type symptoms of allergic diseases and paves the way for late-phase inflammatory responses caused by basophiles, eosinophils and T cells. Allergen specific Th1, Th9, Th17 and Treg cells are also produced in this process [37, 38]. Allergen-specific immunotherapy (SIT) is the only curative and specific approach for treatment of allergies [39, 40]. The current SIT consists of gradual administration of increasing amounts of allergenic extract with the aim to avoid allergic symptoms associated to the exposition. The induction of allergen tolerance is the essential immunological mechanisms of SIT, and involve allergen-specific memory T and B-cell that lead to immune tolerance characterized by a specific noninflammatory reactivity to a given allergen and prevention of new sensitizations and progression of allergic disease. During the immunotherapy, different regulatory and effectors components of the immune system are involved (Figure 1). Allergen tolerance is characterized by the generation of two subgroups of Treg cells: FOXP3+ CD4+ CD25+ Treg cells and inducible Treg cells [41]. T-regulatory type 1 (Tr1) cells have shown to play a major role in allergen tolerance induced by SIT [42, 43]. The immunosuppressor mechanism of Treg cells is mediated by the production of high level of anti-inflammatory cytokines IL-10 and TGF-β, although IFNγ could also be produced [44-46]. The expression of different subtypes of antibodies during SIT is mediated by the activity of regulatory cytokines secreted by Treg cells; IL-10 is a potent suppressor of allergen-specific IgE and simultaneously increases IgG4 production [42]. SIT increase 10 to 100 folds the serum levels of allergen-specific IgG1 and IgG4 [43, 47]. The IgG4 seems to act as a blocking antibody that interacts with the allergen, avoiding interaction of

**3. Current vaccines for allergic diseases**

allergen with the IgE [48].

The bacteria *E. coli* is the preferred expression system used for the production of recombinant allergens, most of the house dust mite allergens have been expressed in this system with success, allowing the molecular characterization [4, 5, 9, 10, 16-18]. The use of *E. coli* may result in non-functional products expressed in inclusion bodies, and without the post-translational modifications necessaries for their appropriate folding and biologic functions [19]. However, by genetic engineering modified strains of this bacteria and novel expression vectors have been obtained, which allow expression of heterologous protein in soluble form with functional properties and high yield; Origami, Rosetta or BL21(DE3)-CodonPlus-pRIL and Rosetta-gami are strains commercially available for obtain recombinants with some pos-translational modifications [20]. In these *E. coli* strains the expression of allergens from the pollen *Artemisia vulgaris* (Art v 3), the peanut (Ara h 2) and the beta-lactoglobulin from bovine have been obtained in higher yield and solubility, and with structural and immunological properties comparable to native allergens [21-23]. The GST tag used in the expression of the first re‐ combinant allergens have been replace for His x6 tag, which is shorter, the recombinant can be analyzed without removing the tag due to the negligible effect on the properties of the molecule, and several efficient purification systems are commercially available.

The eukaryotic expression system have the capacity of performing many of the post-transla‐ tional modifications including signal sequences, disulfide bond formation, and addition of lipid and carbohydrates. A variety of eukaryotic expression systems like yeast, insect cells, mammalian cells and plants are available. The yeast *P. pastoris* is easy to manipulate and frequently used to express recombinant molecules with all the characteristics of their natural counterparts, with a yield about 10 to 100 times higher than *E. coli* [24, 25]. Several recombinant allergens have been obtained by expression in this yeast and their biologic properties demon‐ strated by different methods, this system have resulted especially practical when posttranslational modifications or biochemical activity exist [26-29]. The human cells have been used to obtain the *Phleum pretense* allergen, Phl p 5, as a secreted or membrane-anchored protein and showed to be biologically active, with capacity to bind human IgE, to induce mediator release from basophiles and to stimulate T cell proliferation [30]. A large percentage of allergens are from plants, thus the plant-based expression systems are ideal for the produc‐ tion of certain recombinant allergens, which could have problems such as incorrect processing, incorrect folding and insolubility when expressed in bacteria or other non-plant systems. Thaumatin or thaumatin-like proteins, only when expressed in *Nicotiana benthamiana* result in fully IgE-reactive proteins [31]. Interesting, expression in plants offers the opportunity for oral delivery of recombinant allergens of non- plant origin as a therapeutic approach for mucosal immunization for treating allergic diseases. Oral treatment of mice with squash extracts containing virus-expressed Der p 5 allergen caused inhibition of both allergen-specific IgE synthesis and airway inflammation [32], this plant-based edible vaccines is very promising.
