**7. Recombinant allergy vaccines in clinical phase trials**

Clinical trials with recombinant wild type allergens, and modified allergens have been performed (Table 2). The first studies of allergen SIT with purified molecules were done with peptides containing T cell epitopes either from the cat allergen Fel d 1 or from bee-venomderived phospholipase, administered without adjuvant [122, 123, 129-135]. Such peptides were characterized by its low or none IgE binding capacity. However, they induced late phase systemic side effects in different grades depending in the dose and route of administration [129, 132, 134, 135]. Therapy with T cell peptides does not seem to influence IgE-mediated allergic reactions, in fact, the majority of studies didn't find evidence of changes in IgE levels or IgE-mediated allergic inflammation furthermore, no induction of IgG response was noted.

Allergic patients under immunotherapy with hypoallergenic preparations of the major birch pollen allergen Bet v 1 adsorbed to aluminum hydroxide as a pre-seasonal treatment for birch pollen allergy in a clinical trial, expressed high levels of IgG1, IgG2 and IgG4 antibodies directed against Bet v 1. These IgG antibodies blocked allergen-induced basophile degranu‐ lation and were associated with the ability of patients to tolerate higher allergen concentrations in nasal provocation tests [136]. Immunotherapy with wild-type recombinant Bet v 1 has also been examined for tablet-based sublingual immunotherapy in a phase II, multicenter, doubleblind, placebo-controlled, however, this study is still on course and only have been reported good tolerability of the preparation with no serious adverse events and most side effects observed locally [137].

In a clinical trial, a group of patients with grass pollen allergy was treated with a combination of the major grass pollen allergens (Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6) or with placebo for subcutaneous immunotherapy [43]. Patients treated with the recombinants improve their symptom medication score and had high IgG antibodies levels against natural grass pollen allergens. Several studies of immunotherapy with these mixed allergens have been performed and registered in the National Institutes of Health Clinical trial database (Table 2). Recently, the immunomodulatory properties of MAT-Fel d 1 was studied in a phase I/IIa clinical study [138]. In a randomized double blind trial, intralymphatic immunotherapy (ILIT) with MAT-Fel d 1 in alum was compared with placebo, consisting in 3 injections of each preparation for two months. MAT-Fel d 1 caused reduced skin reactions compared to equi‐ molar concentration of nFel d 1 by intradermal injection, which proved practically painless and reduced drug-related adverse effects compared to placebo group. The IgG4 serum levels in MAT-Fel d 1 treated group increased by a factor of 5.66, while IgG1 and IgE levels didn´t change. After treatment, PBMCs from allergic individuals secreted higher levels of IL-10 when challenged with rFel d 1. Immunotherapy with MAT-Fel d 1 showed to be successful because patients increased their tolerance to nasal challenge, skin prick and dermal test, with cat dander extract. Improvement of quality of life of patients treated with MAT-Fel d 1 was maintained 300 days after immunotherapy.

showed reduced specific IgE, IgG and IgG2 serum levels; demonstrating that such protein

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

Naturally occurring variants of insect allergens could be also useful for specific immunother‐ apy. For example, the sting of *Polybia scutellaris*, a South American wasp, does not cause allergic symptoms, however it has been proven that its venom contain Antigen 5 (Poly s 5), an analogue of the allergen Pol s 5 [126, 127]. In mice, Poly s 5 induced IgG antibodies that cross react with Pol a 5, but induced only minimal amounts of IgE and was poor inducer of basophil-mediator release. Moreover, Poly s 5-specific serum showed a specific protective activity and was able

Despite the promising results observed with recombinant and modified allergens in *in vitro* and *in vivo* studies, more clinical phase studies need to be performed to demonstrate their

Clinical trials with recombinant wild type allergens, and modified allergens have been performed (Table 2). The first studies of allergen SIT with purified molecules were done with peptides containing T cell epitopes either from the cat allergen Fel d 1 or from bee-venomderived phospholipase, administered without adjuvant [122, 123, 129-135]. Such peptides were characterized by its low or none IgE binding capacity. However, they induced late phase systemic side effects in different grades depending in the dose and route of administration [129, 132, 134, 135]. Therapy with T cell peptides does not seem to influence IgE-mediated allergic reactions, in fact, the majority of studies didn't find evidence of changes in IgE levels or IgE-mediated allergic inflammation furthermore, no induction of IgG response was noted.

Allergic patients under immunotherapy with hypoallergenic preparations of the major birch pollen allergen Bet v 1 adsorbed to aluminum hydroxide as a pre-seasonal treatment for birch pollen allergy in a clinical trial, expressed high levels of IgG1, IgG2 and IgG4 antibodies directed against Bet v 1. These IgG antibodies blocked allergen-induced basophile degranu‐ lation and were associated with the ability of patients to tolerate higher allergen concentrations in nasal provocation tests [136]. Immunotherapy with wild-type recombinant Bet v 1 has also been examined for tablet-based sublingual immunotherapy in a phase II, multicenter, doubleblind, placebo-controlled, however, this study is still on course and only have been reported good tolerability of the preparation with no serious adverse events and most side effects

In a clinical trial, a group of patients with grass pollen allergy was treated with a combination of the major grass pollen allergens (Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6) or with placebo for subcutaneous immunotherapy [43]. Patients treated with the recombinants improve their symptom medication score and had high IgG antibodies levels against natural grass pollen allergens. Several studies of immunotherapy with these mixed allergens have been performed and registered in the National Institutes of Health Clinical trial database (Table

represents a potential candidate for specific immunotherapy.

Applications

304

to inhibit Pol a 5-induced basophil degranulation [128].

observed locally [137].

applicability for the allergen specific immunotherapy of insect allergy.

**7. Recombinant allergy vaccines in clinical phase trials**


NCT numbers identify the trials that are registered in the National Institutes of Health Clinical trial database.

DBPC, Double-blind, placebo-controlled; OC, open controlled; SCIT, subcutaneous immunotherapy; SLIT, sublingual immunotherapy.

**Table 2.** Currently ongoing recombinant molecules development for allergen specific immunotherapy.

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 found after the course of immunotherapy [140].

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

From Molecular Cloning to Vaccine Development for Allergic Diseases

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

307

**Figure 2.** Frequency of IgE reactivity to allergens from *B. tropicalis and D. pteronyssinus* in asthmatic patients

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.

(From Ref 72).

**9. Conclusions**

experiment with animal models are in progress to support this application.
