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Background: The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy.
Methods: A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice.
Results: Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients’ IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils.
Conclusion: We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.
Immunoglobulin E (IgE)-mediated allergies (e.g. allergic rhinoconjunctivitis, asthma) affect almost 25% of the population (1, 2). The immediate symptoms of the disease are caused by the aggregation of effector cell-bound IgE antibodies by normally harmless antigens (i.e. allergens), which induces a cascade of cellular activation and the subsequent release of biologically active mediators, proinflammatory cytokines and proteases (3).
Pharmacologic therapy may reduce the symptoms of allergic disease but only allergen-specific immunotherapy (SIT) is an antigen-specific and disease-modifying approach towards allergy treatment (4, 5). SIT is based on the administration of the disease-eliciting allergens to the patient in order to induce antigen-specific nonresponsiveness. It was first used to treat one of the most common forms of allergy, i.e. grass pollen allergy in 1911 by Noon L. The efficacy of SIT is documented by numerous clinical studies, but major problems are associated with the current use of natural allergen extracts for immunotherapy (6). Problems associated with natural allergen extracts include the lack of important allergens, the presence of contaminations and the batch-to-batch variations of the extracts (7–10). Accordingly, allergen-extract-based SIT may have unpredictable outcome and side-effects, including the occurrence of immediate as well as late phase reactions (11).
During the last 15 years, substantial progress has been made in the field of allergen characterization through the application of recombinant DNA technologies (reviewed in 12, 13). cDNAs coding for the most common allergens have become available and facilitated studies regarding allergen-specific immune responses, the development of new diagnostic tests and opened up various avenues for new forms of SIT. New approaches for SIT, which have been evaluated already in clinical studies with encouraging outcome, include the use of CpG-conjugated purified allergens (14, 15), purified recombinant allergens and genetically engineered allergen-derivatives with reduced allergenic activity (5, 16).
The aim of our study is to develop a hypoallergenic derivative for SIT against the most important group of grass pollen allergens, i.e. group I allergens. More than 40% of allergic individuals are sensitized to grass pollen allergens and 95% thereof exhibit IgE reactivity to group 1 allergens (17, 18). Group 1 allergens represent a family of highly cross-reactive antigens present in almost all grasses and corn species and it has been demonstrated that Phl p 1, the group 1 allergen from timothy grass pollen contains most of the IgE and T-cell epitopes of group 1 allergens from related grass species (19–21). Recently, the three-dimensional structure of Phl p 1 has been solved by X-ray crystallography [structure available in the PDB, (1N10)]. Based on the three-dimensional structure, the experimentally determined IgE (22, 23) and T-cell epitopes (21) of Phl p 1, we developed a concept for the construction of hypoallergenic allergen derivatives. In the first step, the original three-dimensional structure and IgE epitopes were disrupted by fragmentation. These fragments were then recombined in the form of a hypoallergenic mosaic protein in altered order, which preserves primary sequence elements and thus T-cell epitopes.
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Recombinant allergens and genetically modified recombinant allergen-derivatives have recently been used for immunotherapy of allergic patients (37–42). The genetic modification of allergens serves to reduce side-effects associated with the IgE-mediated activation of mast cells and basophils. The preservation of T-cell epitopes and immunogenicity should also allow the use of these molecules as tolerogens or as vaccines for the induction of allergen-specific IgG antibodies, which antagonize immune and inflammatory responses because of IgE recognition of the allergen (reviewed in 43). An additional goal is to modify the allergen such that therapeutic administration induces less IgE responses than the parent wild-type allergen, which should lead to a reduced sensitization potential (i.e. in vivo allergenicity).
In this study, we have used a new strategy, i.e. rational molecular re-assembly, for the conversion of one of the most frequently encountered and clinically relevant environmental allergens, the major timothy grass pollen allergen, Phl p 1, into a hypoallergenic derivative, designated P1m, which should meet the above requirements.
Interestingly, the re-assembly process has caused a stable alteration of the secondary structure of the molecules and a reduction of IgE reactivity and allergenic activity of the P1m molecule. T-cell proliferation experiments carried out with PBMC from grass-pollen-allergic patients demonstrated that P1m contains the majority of Phl p 1-specific T-cell epitopes. The advantage of the applied mosaic strategy over previously applied fragmentation approaches is that it is possible to convert the allergen into a single modified molecule of basically identical molecular weight instead of producing several small fragments, which may exhibit lower immunogenicity and induce lower levels of protective IgG antibodies. The use of one single molecule instead of several fragments may also facilitate the production of an allergy vaccine because smaller fragments are often difficult to purify and it is easier to produce one defined molecule instead of several fragments (13).
Indeed, immunization experiments performed in mice and rabbits demonstrated that P1m-induced comparable levels of Phl p 1-specific IgG antibodies. Perhaps more important was the finding that the P1m-induced IgG antibodies blocked the IgE binding of grass-pollen-allergic patients to the wild-type allergen. Using RBL cells transfected with the human FcɛRI (33), we could demonstrate that IgG antibodies obtained by immunization with P1m blocked allergen-induced degranulation of basophils that had been loaded with grass-pollen-allergic patients’ IgE. Similarly, as observed for immunotherapy with genetically modified birch pollen allergen derivatives (37, 39), we would expect that vaccination of grass-pollen-allergic patients with P1m will induce allergen-specific IgG antibodies, which block allergen-induced effector-cell degranulation and IgE-mediated immediate inflammation.
We also would expect that P1m-induced IgG antibodies reduce proliferation and cytokine secretion in allergen-specific T cells by interfering with IgE-facilitated allergen presentation as has been observed for immunotherapy with natural allergens (44, 45).
Besides the reduction of allergenic activity, P1m may have another advantage over natural allergens or recombinant allergens equalling the natural counterpart. In fact, we found that immunization with P1m induced lower Phl p 1-specific IgE responses in mice than immunization with Phl p 1 suggesting that P1m has a lower in vivo allergenicity, i.e. sensitization potential than the Phl p 1 wild type. We thus hope that vaccines containing molecules such as P1m will induce less de novo sensitization as has been reported for natural allergen extracts (46) and P1m may therefore be also considered for prophylactic treatment because of its lower sensitization potential.
Grass pollens are complex allergen sources containing several different allergens (reviewed in 47). However, group 1 allergens as represented by Phl p 1 are the major allergens in grass pollen against which more than 95% of the grass-pollen-allergic patients are sensitized (17, 18). Initial immunotherapy trials performed with recombinant grass pollen allergens indicate that, in addition to group 1 allergens, a grass pollen vaccine should contain also groups 2, 5 and 6 allergens to adequately treat grass pollen allergy 38). In fact, hypoallergenic derivatives of Phl p 5 and Phl p 6 have already been prepared (48, 49) and a hypoallergenic derivative for Phl p 2 has been made (50). It may be possible that after inclusion of P1m a hypoallergenic vaccine for the treatment of grass pollen allergy will enter clinical studies.
In conclusion, we have developed a new strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for immunotherapy of allergy. This strategy should allow the preparation of vaccines for the most common allergen sources.