**3. Current vaccines for allergic diseases**

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

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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

from more distantly related species or even across taxonomic kingdoms.

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

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 allergen with the IgE [48].

> in some case may be life-threatening [53]. Moreover, in some preparations the important allergens are not well represented or they exhibit poor immunogenicity [51]. Administration of whole allergenic extracts can induce new IgE specificities against allergens present which were not recognized by the patient before treatment [54]. All these facts decrease the efficacy and safety of the current allergen SIT [50]. Therefore, among new approaches to provide a better treatment for allergic diseases is to develop vaccines based on preparations with a welldefined composition, suitable for a good standardization and very low risk of anaphylaxis, here the recombinant allergens or modification of these represent a good option, they show characteristics that could allow to replace advantageously the whole allergenic extracts [55],

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Contain undefined components, some of which may promote allergic responses

Can be contaminated with unwanted materials or allergens from other sources

Do not allow the precise monitoring and investigation of mechanisms underlying treatment

Represent molecules with defined physicochemical and immunologic properties that can be modified to

(Table 1).

**Disadvantages of natural allergen extracts**

May induce new sensitizations

**Advantages of recombinant allergens**

Represents pure molecules

foster advantageous characteristics

Lack or contain low amounts of important allergens

Cannot be tailored to the patient's sensitization profile

Do not suit the international quality standards for vaccines

Cannot be compared between different products or batches

Amounts can be easily controlled on the basis of mass units

Fit the international quality standards for vaccines

Potencies and ratios can be exactly adjusted for each molecule

Can be reproducible modified to suit different treatment strategies

**Table 1.** Advantages of recombinant allergens over traditional allergen extracts

Vaccines can be exactly tailored according to the patient's sensitization profile

Can be precisely compared to give consistent and reproducible products or batches

Allow the precise monitoring and investigation of mechanisms underlying treatment

**Figure 1.** Mechanism of allergen-specific immunotherapy. After vaccination, allergen is taken up by antigen present‐ ing cells leading to the differentiation of naïve T cells to inducible T regulatory cells. These cells with the thymus-de‐ rived FOXP3+CD4+CD25+ T regulatory cells, suppress allergic response by the following mechanisms. 1. Suppression of mast cells, eosinophils and basophils. 2. Induction of IgG4 antibodies production from B cells that block the binding of allergen with the IgE. 3. Suppression of effector T cells.

Vaccines composed of whole allergenic extract are complex mixtures of known and unknown material, prepared directly from the allergen source, thus containing allergenic and nonallergenic material and being difficult to standardize [49-51]. Some non-allergenic components have been shown to prime a Th2 response [52], which offset the efficacy of this type of vaccines. SIT with allergenic extract induce a variety of side effects ranging from local to systemic which in some case may be life-threatening [53]. Moreover, in some preparations the important allergens are not well represented or they exhibit poor immunogenicity [51]. Administration of whole allergenic extracts can induce new IgE specificities against allergens present which were not recognized by the patient before treatment [54]. All these facts decrease the efficacy and safety of the current allergen SIT [50]. Therefore, among new approaches to provide a better treatment for allergic diseases is to develop vaccines based on preparations with a welldefined composition, suitable for a good standardization and very low risk of anaphylaxis, here the recombinant allergens or modification of these represent a good option, they show characteristics that could allow to replace advantageously the whole allergenic extracts [55], (Table 1).

Naive T Cell

Inducible T

IL-10 and TGF-

IgG and IgG4 secretion by Plasma cells

B lymphocite

IgG4 bind to Fc RI receptors and block its binding with the IgE

**Figure 1.** Mechanism of allergen-specific immunotherapy. After vaccination, allergen is taken up by antigen present‐ ing cells leading to the differentiation of naïve T cells to inducible T regulatory cells. These cells with the thymus-de‐ rived FOXP3+CD4+CD25+ T regulatory cells, suppress allergic response by the following mechanisms. 1. Suppression of mast cells, eosinophils and basophils. 2. Induction of IgG4 antibodies production from B cells that block the binding of

Vaccines composed of whole allergenic extract are complex mixtures of known and unknown material, prepared directly from the allergen source, thus containing allergenic and nonallergenic material and being difficult to standardize [49-51]. Some non-allergenic components have been shown to prime a Th2 response [52], which offset the efficacy of this type of vaccines. SIT with allergenic extract induce a variety of side effects ranging from local to systemic which

Suppression of Th17 and Th2 effector cells

IL-10 and TGF-

Thymus regulatory cell FOXP3+ CD4+ CD25+

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IL-10 and TGF-

Suppression of effector cells (Mast cells, eosinophils and basophils).

allergen with the IgE. 3. Suppression of effector T cells.

T regulatory cell

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Antigen presenting cell

