**8. Some considerations for a recombinant based mite allergy vaccine**

The prevalence and severity of allergic diseases such as asthma and rhinitis have increased in recent decades [141], and house dust mite allergy is one of the most common allergies worldwide which affect more than 50% of allergic patients [142]. Several house dust mites species co-exist in tropical and subtropical regions, however in these places the species *B. tropicalis* and *D. pteronyssinus* are the most prevalent and a high percentage of allergic population is sensitized to allergens from these two species [143, 144], [72, 145]. Analysis if house dust mite extracts have shown that over 20 different proteins can induce IgE antibodies in allergic populations and several of them show cross-reactivity with allergens from other mite species. Most of them have been obtained and characterized by molecular cloning and its IgE reactivity analyzed [4]. However, it has been suggested that the majority of mite-allergic subjects elicit an IgE response to around five components of allergenic extracts [146, 147], and some of them may be cross-reactive. Therefore, and admixture of few allergens, including those speciesspecific and cross-reactive, could replace the crude allergenic extract for diagnostic and therapeutic purpose. Several studies indicate that a combination of allergens from group 1 and 2 bind to more than 50% of specific-IgE from allergic population, groups 5 and 7 are next in importance [148-150]. It has been reported from Middle Europe that more than 95% of mite allergic patients were mainly sensitized to Der p 1 and Der p 2, and that diagnostic test containing these allergens plus the highly cross-reactive allergen Der p 10 may improve the diagnostic selection of patients for immunotherapy with *D. pteronyssinus* extracts [151]. Other allergens are important given their cross-reactivity and the role that they play in tropical populations, as the case of group 10 and 12 [16, 152]. Results from our research group suggested that a combination of these allergens from D. pteronyssinus might be sufficient to identify almost all our mite allergic population [72] (Figure 2).

Recent studies with hybrid proteins composed by the most important pollen allergens, have suggested that preparations based on molecules containing the B-epitope spectrum of allergenic extracts could be useful for the diagnosis and allergen-specific immunotherapy [64, 86, 87]. We have engineered several fusion proteins composed by segments of different allergens of *B. tropicalis* and *D. pteronyssinus* with the aim to obtain preparations useful for the diagnosis and immunotherapy of allergies caused by house dust mites. The coding sequences of each molecule was cloned into expression vectors and then expressed in *E. coli* fused to 6xHis tag for further purification by affinity chromatography. One of these proteins denomi‐ nated DPx4, consistent of different segments of allergens from *D. pteronyssinus* (Der p 1, Der p 2, Der p 7 and Der p 10), showed a 41% frequency of IgE reactivity in sera from mite allergic patients sensitized to *D. pteronyssinus* and the specific IgE levels against the recombinant were significantly lower than those against the whole allergenic extract from mites. Basophil activation test showed that DPx4 has lower capacity to induce basophile degranulation compared to the allergenic extract. These results suggest that the fusion protein have a hypoallergenic profile, and that is a good candidate for develop a vaccine with potential use for allergen specific immunotherapy of mite allergy [153]. Further *in vitro* studies as well as experiment with animal models are in progress to support this application.

**Figure 2.** Frequency of IgE reactivity to allergens from *B. tropicalis and D. pteronyssinus* in asthmatic patients (From Ref 72).

### **9. Conclusions**

The National Institutes of Health's clinical trial database contain information about a study that intends to use the recombinant modified peanut allergens Ara h 1, Ara h 2 and Ara h 3 encapsulated in heat/phenol-killed *E.coli*. This phase I study should recruit healthy volunteers to receive four scaling doses of the peanut preparation rectally at weekly intervals. The major allergen of ragweed pollen *Ambrosia artemisifolia*, Amb a 1, was conjugated to CpG oligonu‐ cleotides to reduce the allergenic activity of Am b a 1 and to shift the specific Th2 response to a Th1 response, mediated by the binding of CpG with toll-like receptor 9. Allergic individuals treated with the conjugated vaccine showed reduction in eosinophilia and the number of IL-4 producing cells, and increased numbers of IFN-γ-producing cells compared to placebo-treated patients [139]. Furthermore, increase of regulatory T cells infiltration in the nasal mucosa was

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**8. Some considerations for a recombinant based mite allergy vaccine**

The prevalence and severity of allergic diseases such as asthma and rhinitis have increased in recent decades [141], and house dust mite allergy is one of the most common allergies worldwide which affect more than 50% of allergic patients [142]. Several house dust mites species co-exist in tropical and subtropical regions, however in these places the species *B. tropicalis* and *D. pteronyssinus* are the most prevalent and a high percentage of allergic population is sensitized to allergens from these two species [143, 144], [72, 145]. Analysis if house dust mite extracts have shown that over 20 different proteins can induce IgE antibodies in allergic populations and several of them show cross-reactivity with allergens from other mite species. Most of them have been obtained and characterized by molecular cloning and its IgE reactivity analyzed [4]. However, it has been suggested that the majority of mite-allergic subjects elicit an IgE response to around five components of allergenic extracts [146, 147], and some of them may be cross-reactive. Therefore, and admixture of few allergens, including those speciesspecific and cross-reactive, could replace the crude allergenic extract for diagnostic and therapeutic purpose. Several studies indicate that a combination of allergens from group 1 and 2 bind to more than 50% of specific-IgE from allergic population, groups 5 and 7 are next in importance [148-150]. It has been reported from Middle Europe that more than 95% of mite allergic patients were mainly sensitized to Der p 1 and Der p 2, and that diagnostic test containing these allergens plus the highly cross-reactive allergen Der p 10 may improve the diagnostic selection of patients for immunotherapy with *D. pteronyssinus* extracts [151]. Other allergens are important given their cross-reactivity and the role that they play in tropical populations, as the case of group 10 and 12 [16, 152]. Results from our research group suggested that a combination of these allergens from D. pteronyssinus might be sufficient to identify

Recent studies with hybrid proteins composed by the most important pollen allergens, have suggested that preparations based on molecules containing the B-epitope spectrum of allergenic extracts could be useful for the diagnosis and allergen-specific immunotherapy [64, 86, 87]. We have engineered several fusion proteins composed by segments of different allergens of *B. tropicalis* and *D. pteronyssinus* with the aim to obtain preparations useful for the

found after the course of immunotherapy [140].

Applications

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almost all our mite allergic population [72] (Figure 2).

For several years the allergen-specific immunotherapy has been successfully done with natural allergenic extracts. However, they are complex mixtures difficult to standardize that might cause local or systemic reactions, compromising the patient's life. In the last decades, the molecular cloning applied to the study of allergens has allowed obtaining several recombinant allergens from different sources, and their biological and molecular properties elucidated.

Component based diagnosis and immunotherapy is now possible by the availability of several recombinant allergens, which represents the best approach to achieve the most efficacious diagnosis and treatment of allergies, based on the sensitization profile and of each patient. Vaccines for allergic diseases based on recombinant allergens or modification of these, that could modulate the immune response against natural allergens toward a protective response, have been proposed. Hypoallergenic molecules obtained by molecular cloning, in different versions like hybrid molecules, oligomers, mosaic proteins or variants obtained by sitedirected mutagenesis have been developed and studied by in vitro test, animal model and clinical trial in humans, indicating potential beneficial use in the near future. Recombinant allergens coupled to carriers for directing the molecule to specific cells or intracellular compartments, preventing unwanted side effects and increasing the specificity of the immune response have been explored.

[2] Chua KY, Stewart GA, Thomas WR, Simpson RJ, Dilworth RJ, Plozza TM, et al. Se‐ quence analysis of cDNA coding for a major house dust mite allergen, Der p 1. Ho‐

From Molecular Cloning to Vaccine Development for Allergic Diseases

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

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[3] Tovey ER, Johnson MC, Roche AL, Cobon GS, Baldo BA. Cloning and sequencing of cDNA expressing a recombinant house dust mite protein that binds human IgE and correspond to an important low molecular weight allergen. J Exp Med

[4] Thomas WR, Smith WA, Hales BJ, Mills KL, O'Brien MR. Characterization and im‐ munobiology of house dust mite allergens. Int Arch Allergy Immunol 2002;129:1-18.

[5] Puerta L, Caraballo L, Fernandez-Caldas E, Avjioglu A, Marsh DG, Lockey RF, et al. Nucleotide sequence analysis of a complementary DNA coding for a Blomia tropica‐

[6] Cui Y, Zhou Y, Shi W, Ma G, Yang L, Wang Y, et al. Molecular cloning, expression, sequence analyses of dust mite allergen Der f 6 and its IgE-binding reactivity with mite allergic asthma patients in southeast China. Mol Biol Rep 2012 Feb;39(2):961-8.

[7] An S, Chen L, Wei JF, Yang X, Ma D, Xu X, et al. Purification and characterization of two new allergens from the venom of Vespa magnifica. PLoS One 2011;7(2):e31920.

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[11] Acevedo N, Sanchez J, Erler A, Mercado D, Briza P, Kennedy M, et al. IgE cross-reac‐ tivity between Ascaris and domestic mite allergens: the role of tropomyosin and the

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The promising results showed by *in vitro* and animal models studies have encouraged the design of clinical phase trials where recombinant allergens have demonstrated their good potential to provide a more efficacious and safe diagnosis and allergen-specific immunother‐ apy. In the last years, the number of clinical phase trials designed and registered in the National Institutes of Health Clinical trial database is increasing. This tendency suggests that in few years several vaccines based on recombinant allergens could be commercially available in replacement of the traditional allergenic extract.
