General introduction

Type I allergies, which comprise a wide range of IgE-mediated disorders such as hay fever, asthma and atopic dermatitis, represent a major and increasing health problem that affects more than 25% of the European population. This increase in prevalence of allergic diseases has been paralleled by a greater demand on diagnostic and therapeutic products. Allergen extracts are routinely applied for diagnostic and therapeutic purposes. These extracts are difficult to standardize regarding their allergen content: several allergens might be under-represented due to degradation, other non-allergenic components are present and there might be even contamination with allergens from other sources. 

Systems for recombinant production of allergens and techniques for gene manipulation offer unique tools for the development of novel molecule-based products to be used in diagnosis and allergen-specific immunotherapy (SIT), which addresses the problems resulting from the use of extracts. Pure and standardized recombinant allergens or cocktails prepared thereof and containing most of the IgE-binding epitopes of an allergen source can be formulated to replace natural extracts. The use of recombinant allergens in diagnosis allows the exact identification of the molecules causing the allergic reaction, i.e. a clear association between the disease manifestation and the IgE-mediated immune reactions. 

For SIT, the selected recombinant allergens can be modified to reduce the risk of IgE-mediated side effects. Modification of allergens for SIT should aim at the production of molecules with reduced IgE binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes) and for induction of IgG antibodies reacting with the natural allergen (blocking antibodies). Based on these observations, we aim to produce a panel of well-characterized recombinant allergens for diagnosis of allergies caused by Cupressaceae (Cryptomeria, Cupressus, Juniperus) pollen, which are important source of allergens worldwide. For SIT, we plan to design and engineer hypoallergens for Cupressaceae pollen allergy. In addition, molecules suitable for SIT of patients sensitized to Fagales pollen with associated food allergies, and of patients co-sensitized to mugwort and ragweed pollen will be developed. 

In a parallel approach, we will take advantage of the unique features of the genetic immunization method and will develop a panel of allergen-gene vaccines. These newly designed DNA vaccines against whole groups of allergens will be optimized concerning immunogenicity, anti-allergic activity and improved safety.

Module 1: Recombinant cedar/cypress/juniper pollen allergens for diagnosis 
In this module, we aim to produce a panel of recombinant Cupressaceae allergens for diagnosis. Worldwide, pollen from species of the Cupressaceae family is probably one of the most important causes of pollen allergy. The genera Cryptomeria, Cupressus, and Juniperus are widely spread over North America, Mediterranean countries, Australia, Central Asia, China, and Japan (Di Felice et al., Int. Arch. Allergy Immunol. 2001, 125: 280; Weber RW, J. Allergy Clin. Immunol. 2001, 112: 229). The most relevant species causing allergic sensitization are Cryptomeria japonica (Japanese cedar), Cupressus arizonica (Arizona cypress), Cupressus sempervirens (Italian cypress), Chamaecyparis obtusa (Japanese cypress), and Juniperus ashei (Ozark white cedar). Pollinosis caused by Japanese cedar pollen is one of the most common allergic respiratory diseases in Japan. 

In Mediterranean countries, cypress pollen allergy is a major cause of rhinoconjuctivitis and asthma (Charpin et al., Allergy 2005, 60: 293). This problem has been underestimated because winter is the pollination season of cypress trees. The lack of efficacy of the available nonstandardized extracts used in diagnosis of cypress allergy is also partly responsible for this situation (Hrabina et al., Allergy 2003, 58: 808). Presently, four groups of allergens have been identified in the Cupressaceae family: i) Cryptomeria japonica Cry j 1, Chamaecyparis obtusa Cha o 1, Juniperus ashei Jun a 1, Cupressus arizonica Cup a 1. Cry j 1, Cha o 1, Jun a 1 and Cup a 1, all belong to the pectate lyase family of proteins. 

Homologous allergens were also described in pollen from allergenic weeds (e.g. ragweed Amb a 1 and mugwort Art v 6). Pectate lyases comprise a large group of enzymes not only expressed in ripening fruits and in plant pathogenic bacteria, but also in pollen. These enzymes are able to digest the pectin or pectate envelope of plant cells. Although enzymatic activity has not been demonstrated for all homologous pollen allergens, high sequence homology and cross-reactivity of patients´ IgE constitute the basis for grouping them into the pectate lyase family of allergens (Iacovacci et al., Clin. Exp. Allergy 2002, 32: 1620; Rea et al., Prot. Exp. Purif. 2004, 37: 419; Sone et al., Allergy 2005, 35: 664). ii) Cryptomeria japonica Cry j 2, Chamaecyparis obtusa Cha o 2, both belonging to the Polygalacturonase family. iii) Juniperus ashei Jun a 3, a thaumatin-like pathogenesis-related protein iv) Juniperus ashei Jun a 2, a calcium-binding allergen. 

In the last decades, the increase in prevalence of allergic diseases has been paralleled by a greater demand on diagnostic and therapeutic products. In this respect, recombinant allergens are expected to be qualitatively superior to the commercially available allergen extract preparations used for the in vitro and in vivo diagnosis and treatment of allergic conditions. During the last 12 years, our group has worked on the structural and immunological characterization of recombinant allergens from various sources. A crucial aspect of this work has been the establishment of methods for recombinant production, selection and construction of suitable expression systems, optimization of growing conditions and purification strategies for each allergen in question. However, independent from the approach used for the production and purification of recombinant allergens, the molecules must be first subjected to a series of in vitro and in vivo evaluation procedures before being considered for diagnostic and therapeutic purposes.

Module 2: Engineering cedar/cypress/juniper pollen allergens for SIT 
Genetic engineering implies the targeted modification of a protein in order to alter its function or properties in a predictable manner. Thus, understanding the relationship between the structure of the encoded protein and its function/properties is required for the precise and effective manipulation of a gene. 

Approaches for the alteration of a gene include changing specific base-pairs (mutated gene), introduction of a new piece of DNA into the existing DNA molecule (chimeric or hybrid gene), and deletions (truncated gene or fragments). Engineering of hypoallergens usually requires knowledge of B and T cell epitopes and in some cases also of the three-dimensional structure of the native allergen. An exception to this is the DNA shuffling approach, which bypasses the need to identify amino acid residues or motifs that are important to structure and function. No matter how allergen genes are altered, putative hypoallergens must be first subjected to a series of in vitro and in vivo evaluation procedures, before being considered for therapeutic purposes. 

The major task in this module will be the engineering of allergens belonging to the pectate lyase family (Amb a 1, Art v 6, Cry j 1, Jun a 1, Cup a 1, Cha o 1) in order to significantly reduce their IgE binding activity while preserving their capacity to stimulate allergen-specific T lymphocytes. This group of molecules comprises the major Cupressaceae, mugwort, and ragweed pollen allergens.

Module 3: Development of a recombinant-based allergen vaccine for mugwort and ragweed co-sensitized patients 
In a recent study with recombinant mugwort and ragweed allergens (Asero et al. Clin. Exp. Allergy, submitted), we demonstrated that (i) Amb a 1 and Art v 1 can be considered as markers for genuine ragweed and mugwort pollen sensitization, respectively; and that (ii) patients displaying positive skin prick test to mugwort and ragweed pollen (Amb+/Art+) are in fact co-sensitized and not simply cross-reactive. 

Therefore, immunotherapy of Amb+/Art+ patients should include both mugwort and ragweed allergens. Based on these findings, we propose here to construct and evaluate recombinant fusion vaccines for specific immunotherapy of concomitant ragweed and mugwort allergy.

Module 4: Construction of hybrid molecules for Fagales pollen-sensitized patients 
Birch pollen allergy is one of the most common causes of pollinosis in the Northern hemisphere. The major birch pollen allergen Bet v 1 has been identified and clinically relevant homologues were found in several other species of the Fagales order. 

For several Fagales pollen-allergic patients Bet v 1 serves as the sensitizing molecule. In this case, allergic reactions to other tree pollen allergens are triggered by crossreactive IgE antibodies. However, in some birch-free areas in Europe (e.g. Mediterranean Countries) the allergic sensitization can also be induced by other members of the Fagales, again resulting in highly cross-reactive antibodies (Mari et al., Clin. Exp. Allergy 2003, 33: 1419). Moreover around 80% of birch pollen allergic individuals develop allergic symptoms, called oral allergy syndrome (OAS), towards Bet v 1 homologues in fruits, nuts or vegetables over the years (Mari et al., Curr. Opin. Allergy Clin. Immunol. 2005, 5: 267). 

In case of pollen allergies, specific immunotherapy (SIT) using commercially available pollen extracts offers the only curative approach in allergy treatment. However in OAS patients, even successful therapy of the pollinosis does not necessarily lead to a reduction of the food-associated allergic reactions (Hansen et al., Mol. Nutr. Food Res. 2004, 48: 441). The aim of SIT is the modulation of the immunologic reaction towards the disease eliciting allergen. Even though the underlying mechanisms are not fully understood T cells are known to be key players in this process (Till et al. J Allergy Clin. Immunol. 2004, 113: 1025). 

Therefore, we want to generate hybrid molecules composed of several different Bet v 1 homologues reflecting the major T cell epitopes of each individual allergen. One single hybrid could therefore induce immunologic tolerance against a variety of different allergens. Besides a maximum accumulation of T cell epitopes per molecule, the hybrids should display low or no reactivity with patients´ IgE to ensure safety during therapy.

Module 5: Development of gene vaccines for allergy treatment 
Taking advantage of the unique features of the genetic immunization method, a panel of newly designed DNA vaccines optimized concerning immunogenicity, antiallergic activity and improved safety against whole groups of allergens will be developed. 

At present, the major restriction of genetic intradermal or intramuscular needle immunization concerning clinical application is the requirement for large doses of DNA and the relatively weak immunogenicity. Alternative, low-dose and immunogenic injection methods such as the gene gun or powderject™ are not suitable for the treatment of allergy because of inducing a Th2-biased type of immune response. 

Therefore, the first approach will be to overcome these hurdles with replicase-based gene vaccines. Immunization with these constructs enables to trigger strong humoral and cellular Th1-biased immune responses with nanogram quantities of needleinjected plasmid DNA. 

The second aspect will cover the development of strategies for minimizing the risk of anaphylactic side effects resulting from the potential expression of biologically active allergens following genetic vaccination. We will develop gene vaccines encoding allergen variants or derivatives, which still retain the induction of T-cell responsiveness but display no allergenic reactivity upon sensitization with the wildtype allergen. For this purpose, forced ubiquitination of DNA vaccines will serve to develop a routine approach for destroying IgE-binding epitopes on allergens in order to avoid recognition by pre-existing IgE antibodies. Simultaneously, any T cell epitope of the allergen will be preserved.

Module 6: Pre-Clinical evaluation of immunization protocols with hypoallergenic derivatives
Despite optimized treatment regimens, including specific immunotherapy (SIT), antihistamines, corticosteroids and mast cell stabilizers, a subgroup of allergic patients have insufficient symptom control (White P. et al. Clin Exp Allergy 1998, 28:266-270). Therefore, new treatment options that target more specifically and earlier in the allergic cascade, like anti-membrane (m)IgE application in combination with SIT, are desirable. Combination therapy may permit a broader use of SIT by reducing the risk of anaphylactic side effects after SIT injections.
Previous to the present proposal for the integration of a National module (Module 6) to the existing Christian Doppler Laboratory for Allergy Diagnosis and Therapy, we raised a high affinity mouse monoclonal antibody of g1-isotype, exclusively recognizing mouse mIgE (mAbA9). Together with Biomay, we filed a patent (submitted under EP 07450062.0) for the therapeutic use of mAbA9. In a prophylactic study mAbA9 was passively administrated to mice in parallel with Bet v 1a as sensitizing allergen. Compared with the control group treated with Bet v 1a only, we observed a drastic reduction in the production of Bet v 1a-specific IgE antibodies with a long lasting effect on the development of a Bet v 1-specific IgE-memory cell population. From this experiment we concluded that passive immunization with mAbA9 strongly suppresses the development of an allergen-specific IgE memory, whereas antibody responses of other isotypes are not influenced. At this stage, mAbA9 fulfilled all criteria required to be a promising lead compound for passive immunization approaches.
In the following years the experimental focus of the proposed Module 6 will cover three aspects: 

1. Establishment of an animal-based sequential administration protocol for wild type and genetically engineered hypoallergenic derivatives to minimize the risk of therapy induced specific IgE antibodies.
So far, the only curative approach for treatment of type I allergy is the allergen-specific immunotherapy. However, side effects cannot be excluded and recently it has been shown that during SIT administration of allergen extracts not only “blocking IgG antibodies” are induced, but also new IgE reactivities to allergenic components in the extracts may arise (Moverare et al., Allergy 2002, 57: 423). To address this problem, in Module 6 several hypoallergenic derivatives as well as modified versions of the Bet v 1 allergen developed in Module 4 will be administered to BALB/c mice in a sequential immunization protocol. The idea is to develop a vaccination strategy using a set of modified versions of the same allergen, which are sequentially given in an updosing SIT protocol, in order to minimize side effects due to therapy-induced IgE antibodies. The advantage of the mouse model is, that the kinetics of the specific isotype occurrence can be exactly monitored. In this way, we will be able to determine the order of allergen administration (i.e. allergen x, followed by allergen y and allergen z or allergen z followed by allergen x and y) that induces the best protective “blocking antibody” titre, combined with the minimum of induction of unwanted specific IgEs. Special interest will also be set on the evaluation of the optimal dose and route of application (s.c.. i.p., i.n. etc.).

2. Establishment of an animal-based sequential administration protocol in combination with passively administered anti-mIgE antibody.
As outlined above, passively administered mAbA9 significantly reduced the development of the specific IgE response. With a combinatorial (recombinant allergen and mAbA9) immunization protocol we expect to further reduce the level of unwanted specific IgE antibodies. The advantage will be that higher doses of recombinant allergens could be administered to the mice, thus raising the success of the immunotherapy. In parallel, the therapeutic capacity of mAbA9 will also be investigated in combination with DNA and/or RNA-based vaccines.

3. Generation of mouse hybridomas directed against human mIgE, followed by subsequent humanization of the monoclonal mouse antibody
A comparable experimental approach, as successfully performed for mAbA9 will be used for the generation of hybridomas directed against anti-mIgE antibodies. Specificity will be tested by inhibition ELISA and BIACORE measurements. FACS analysis will demonstrate the capability of the anti human mIgE antibody to recognize mIgE carrying B cells. However, to fulfil the criteria for therapeutic use, the antibody should be non-anaphylactic and finally must be converted from a murine into a human antibody. The humanization will be performed according to international guidelines.