**6. Insect sting allergy**

production of high levels of IFN-γ and reduced levels of IL-4, compared to unmodified Fel d 1. Immunized mice expressed higher levels of IgG2a and showed protection against the challenge of high doses of allergenic extract. Furthermore, MAT-Fel d 1 produced 100-fold less degranulation and histamine release from basophiles compared to unmodified Fel d 1 [106].

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

Dendritic cells (DCs) play an important role in the initiation and maintenance of T cell response to allergens. Its role in the type of T cell response generated can be influenced by the maturation state, while mature DCs induce effector T cell responses characterized by Th1 or Th2 response [107], immature or semi-mature DCs are tolerogenic and have the ability to induce Tregs [108]. DCs express an array of Fc receptors which have the capacity to enhance allergen uptake through internalization of allergen/antibody receptors complexes. When stimulated with allergen, DCs express FcεRI, and activated a signal-transducing cascade involving immunor‐ eceptor tyrosine-based activation motif (ITAM), which result in increased production of proinflammatory cytokines and chemokines, the induction of robust proliferation of allergenspecific T cells and the development of allergic symptoms [109]. DCs also express the receptor FcγRIIb that contains immunoreceptor tyrosine-based inhibition motif (ITIM) which induces inhibitory signaling events. This receptor can co-aggregate with FcεRI that activating a signaling cascade that culminates in inhibition of FcεRI signaling. Under these assumptions, Zhu, D. *et al*. [110] designed a fusion molecule called GFD composed by a human IgG Fc fragment linked to the allergen Fel d 1 by a flexible linker, with the aim to crosslink FcγRIIb and FcεRI-bounded to the cat specific-IgE. In transgenic mice expressing human FcγRIIb and FcεRI, sensitized with high doses of Fel d 1 specific-IgE and treated with several doses of GFD, the challenge with Fel d 1 didn´t cause mast cell degranulation. A scheme of immunotherapy with high doses of GFD resulted in the inhibition of allergic response against Fel d 1, pulmo‐ nary inflammation and skin reactivity in sensitized animals. Treated mice expressed IgG1 antibodies that blocked the binding of IgE to Fel d 1. When applied to mice sensitized to Fel d 1 in a scheme of rush immunotherapy, GFD blocked acute systemic allergic reaction, mast cell degranulation, bronquial hyper-reactivity and pulmonary inflammation [111]. Recently, a fusion protein composed of Fcγ chain and the *Dermatophagoides farina*e allergen, Der f 2, was obtained and tested in a Der f 2 allergic murine model [112]. After treatment with the fusion molecule, the levels of specific IgE to Der f 2, histamine and pro-inflammatory cytokines were lowered in the Fcγ-Der f 2 treated allergic mice, compared to saline-treated allergic mice. These results suggest that specific targeting of allergens to Fcγ receptors could be used as a strategy

in the development of antigen-specific immunotherapy for human allergic diseases.

A different molecular design was applied to target allergens to CD64 receptor on antigen presenting cells; a fusion protein (H22-Fel d 1) composed by Fel d 1 linked to the variable region of a monoclonal antibody anti-CD64 was designed to stimulate receptor internalization [113]. Flow cytometry analysis showed that H22-Fel d 1 binds to CD64 and reacted with IgE and IgG with similar affinity compared to native allergen. *In vitro* assays demonstrated that the fusion molecule stimulates the proliferation of T lymphocytes derived from allergic individuals and the secretion of IL-5, IL-10 and IFN-γ [114]. Although H22-Fel d 1 is responsible of a positive effect that could result in a protective response against allergen challenge, it also stimulated

**Targeting allergens to receptors on dendritic cells**

Applications

302

Insect sting allergy are frequently caused by insect stings of the Apidae family (honeybees and bumblebees), those from the Vespidae family (Vespula, Dolichovespula, Vespa and Polistes genera) and, in some regions, also of the Formicidae family (ants). The sting can induce local or systemic IgE-mediated hypersensitivity reactions that can be fatal [116]. Prevalence of systemic reactions caused by insect stings are reported from 0,3% to 7,5% in the United States and Europe [117, 118]. Up to one fifth of these subjects will eventually experience severe lifethreatening reactions. Hymenoptera venoms contain protein allergens, as well as nonallergenic components, including toxins, vasoactive amines, acetylcholine, and kinins. Among the multiple allergens in Hymenoptera venoms, two allergens are importan, the phospholipase A2 from of honey bee (*Apis mellifera*) (Api m 1), and of the vespid venoms antigen 5 from *Vespula vulgaris* (common wasp) denominated Ves v 5.

Several studies have demonstrated that immunotherapy for vespid allergy with venom extracts is clinically effective and improve the quality of life and allergic symptoms. This improvement is correlated to a significant decrease of total IgE levels, and increase in specific IgG and IgG4 levels [119]. However, severe and life-threatening anaphylactic side effects may be induced after the administration of crude allergen extracts [120].

One of the first attempts to obtain safer methods for immunotherapy of insect allergies was made with allergen-derived peptides, containing T-cell epitopes. Peptides derived from the bee allergen Api m 1, were applied to allergic individuals in different immunotherapy schemes. *In* vitro and clinical phase trials showed that T cells from such patients showed marked responsiveness to Api m 1 after long term treatment, a shift in the pattern of cytokine secretion form a Th0 to a Th1 profile and increase in specific IgG4 levels [121-123].

The use of recombinant venom allergens for allergen specific immunotherapy has been analyzed in animal models. Intranasal administration of the recombinant allergen from wasp venom, rVes v 5, to mice prior to sensitization with natural allergens lead to a significant reduction of the allergic reaction, reduction of specific IgE and IgG2a levels, increase of mRNA levels of IL-10 and TGF-β. Pretreatment with the whole venom was less effective and caused toxic side reactions, suggesting a favorable use of the recombinant protein [124]. Hybrid proteins composed by allergens from bee venom have shown anti-allergenic properties in *in vitro* and animal models [85]. A fusion protein composed of the two major bee venom allergens Api m 1 and Api m 2 called Api m [1/2], showed reduced IgE reactivity of Api m [1/2] compared with native allergens [125]. By the other hand, basophil degranulation and skin tests showed that this fusion protein have hypo-allergenic properties. When applied subcutaneously, mice showed reduced specific IgE, IgG and IgG2 serum levels; demonstrating that such protein represents a potential candidate for specific immunotherapy.

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

> Bet v 1 trimer SCIT, DBPC, Phase II 2000 [125] Bet v 1 fragments SCIT, DBPC, Phase II 2000 [125]

> > SCIT, Immunological and histological evaluation

Bet v 1 / Mal d 1 SCIT, DBPC, Phase II 2011 NCT01449786

SCIT, DBPC, Phase IIb

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

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

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

SCIT, DBPC, Phase II 2002

SCIT, OC, Phase II 2002 NCT00266526 SCIT, DBPC, Phase III 2004 NCT00309062 SCIT, DBPC, Phase III 2007 NCT00554983

From Molecular Cloning to Vaccine Development for Allergic Diseases

SCIT, DBPC, Phase II 2002 NCT00410930 SLIT, Phase I 2006 NCT00396149 SLIT, Phase I 2007 NCT00889460 SLIT, DBPC, Phase II 2008 NCT00901914

SCIT, DBPC, Phase III 2004 NCT00309036 SCIT, DBPC, Phase II 2008 NCT0671268 SCIT, DBPC, Phase III 2009 NCT01353755

SCIT, Phase II 2011 NCT01445002

2012 (Initiating)

Rectal 2009 NCT00850668

**NIH Registration number / Reference**

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

305

NCT01538979

2009 NCT00841516

**Allergen Allergen-based vaccine Rout of administration/Trials Year**

300 days after immunotherapy.

Bet v 1 folding variant

Recombinant Bet v 1

Mix: Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6

*Phleum pratense* peptide fused to carries protein

Modified Ara h 1, Ara h 2, Ara h 3

Bet v 1 (Birch pollen allergen)

Birch pollen and apple allergens

> *Phleum pratense* allergens

> Peanut allergens

immunotherapy.

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

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 applicability for the allergen specific immunotherapy of insect allergy.
