Inflammation & Allergy - Drug Targets

(Formerly 'Current Drug Targets - Inflammation & Allergy')

ISSN: 1871-5281

Inflammation & Allergy - Drug Targets

Volume 5, Number 1, January 2006

Contents

Emerging Strategies for Allergen Specific Immunotherapy
Guest Editors: Prem L. Bhalla & Mohan B. Singh


Preface


Editorial Pp. 1


Modified Recombinant Allergens for Safer Immunotherapy Pp. 5-14
Fátima Ferreira, Peter Briza, Daniela Inführ, Georg Schmidt, Michael Wallner, Nicole Wopfner, Josef Thalhamer and Gernot Achatz
[Abstract]


Immunological Mechanisms of Specific Allergen Immunotherapy Pp. 15-21
Carsten B. Schmidt-Weber and Kurt Blaser
[Abstract]


Immunotherapy for Food Allergy Pp. 23-34
Anna Nowak-Wegrzyn
[Abstract]


Information Management for the Study of Allergies Pp. 35-42
Vladimir Brusic
[Abstract]


Non-Injection Routes for Allergen Immunotherapy: Focus on Sublingual Immunotherapy Pp. 43-51
Giovanni Passalacqua, Laura Guerra, Mercedes Pasquali and Giorgio W. Canonica
[Abstract]


Recombinant Expression Systems for Allergen Vaccines Pp. 53-59
Mohan B. Singh and Prem L. Bhalla
[Abstract]


General Article


Current Options in the Treatment of Mast Cell Mediator-Related Symptoms in Mastocytosis Pp. 61-77
Luis Escribano, Cem Akin, Mariana Castells and Lawrence B. Schwartz
[Abstract]




Abstracts

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PREFACE

Type 1 allergic diseases (allergic rhinitis or hay fever, rhinoconjunctivitis, allergic asthma and allergic dermatitis) are a global health problem and are the most common cause of chronic ill health in the western societies. These allergic diseases have increased in prevalence during the past three decades especially in western countries and an upward trend has been forecasted. Current commonly used treatments focus on relief of the clinical symptoms rather than underlying immune mechanisms. Antihistamine and topical corticosteroids are commonly used drugs to relieve symptoms or to alleviate allergic inflammation respectively. Development of cure or vaccine for atopic allergic diseases has not advanced significantly since 1911. Specific immunotherapy remains the only causative approach towards the treatment of such allergic diseases. Conventional immunotherapy involves administration of natural allergen extracts containing mixture of undefined components with potential to induce life-threatening anaphylactic response and new IgE reactivities. Recent advances in molecular biology have led to exponential increase in our understanding of molecular mechanisms involved in manifestation of type 1 allergy symptoms. In addition, molecular approaches have provided tools to resolve structural features of allergen molecules and develop novel therapeutic vaccines for improved and safer form of specific immunotherapy. Moreover, recombinant allergens offer unique possibility for accurate diagnosis and design of patient tailored immunotherapy. Recent clinical trials with engineered birch pollen allergen have demonstrated efficacy of this approach. In this issue, leading researchers in the field of allergy review emerging therapeutic approaches for treatment of allergic diseases. Various strategies and approaches covered include, cellular and immunological mechanisms of immunotherapy, current strategies for genetic modification of allergens, DNA vaccines, anti-IgE therapy, non-injection routes of vaccine delivery, immunotherapy for food allergies, production systems for recombinant allergens and immuno-informatic tools for designing allergy vaccines. Hopefully, this Hot topic issue will provide the readers with emerging perspectives on immunotherapy of type 1 allergic diseases.

Prem L. Bhalla
The University of Melbourne
Parkville, Vic 3010
Australia
E-mail: premlb@unimelb.edu.au

Mohan B. Singh
The University of Melbourne
Parkville, Vic 3010
Australia
E-mail: mohan@unimelb.edu.au


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EDITORIAL

Future Directions for Allergen Immunotherapy

The prevalence of allergic diseases, such as seasonal allergic rhinitis, allergic rhinoconjuctivitis, asthma and atopic dermatitis is increasing worldwide affecting more than 25 % of the population. Currently used pharmacotherapy-based treatments address symptoms while specific immunotherapy is the only curative approach towards the treatment of type 1 allergic diseases.

Allergen immunotherapy introduced in 1911 [1] is a clinically proven treatment that alters the natural course of allergic diseases. In recognition of the immune modifier function of the allergen immunotherapy, the World Health Organization (WHO) in 1988 [2] has proposed the term “allergy vaccination” for this treatment.Allergen immunotherapy or allergy vaccination involves the administration of incrementally increasing doses of allergen by subcutaneous injection in order to reduce responses to allergic triggers that cause allergic symptoms and to decrease inflammatory response and prevent development of persistent disease. However, current allergy vaccines are based on natural allergen extracts that comprise ill -defined mixture of allergic and non-allergic components. The side effect of injecting such allergen extracts into any sensitized individual includes systemic and even anaphylactic responses and has potential to induce new IgE reactivity towards other components of the extract. The advent of molecular cloning technologies in last two decades has led to production and evaluation of recombinant allergens well suited for more targeted diagnosis and therapy of allergic diseases. Recombinant allergens that retain IgE reactivity can be used for component resolved allergy diagnosis to determine the individual sensitisation profile of allergic patients. This knowledge of individual sensitisation profile can be used to design “patient tailored” immunotherapy vaccine preparations. Unmodified recombinant allergens are unlikely to be acceptable for use as allergy vaccines since due to their purity, their potential to cause systemic anaphylactic response is likely to be high even at low doses. Engineered allergens modified to abolish or substantially reduce their IgE reactivity offer a new paradigm towards effective and highly defined immunotherapy that retains the benefits of conventional allergen immunotherapy while removing its disadvantages. The intention of this thematic issue on Emerging Strategies for Specific Immunotherapy is to provide a contemporary perspective on rapidly advancing field of allergy immunotherapy.

An emerging paradigm regarding generation of modified recombinant low IgE-binding allergens is that it is preferable to preserve surface structures of allergen molecule capable of eliciting production of IgG antibodies that can block IgE binding to the natural allergen. Ideally, the reduced or abolished IgE reactivity of modified recombinant proteins should be achieved by introducing a limited number of amino acid replacements in mapped IgE-binding epitopes. Until recently, allergen epitope mapping studies have either employed synthetic peptides [3] or gene fragmentation [4] to identify linear or sequential epitopes. Such epitope mapping has led to the identification of allergen domains that were deleted [3] or mutated [5] in order to achieve substantial reduction in IgE reactivity. The IgE-binding epitopes of most allergens are defined by discontinuous or conformational structures. The knowledge of three-dimensional structure of allergen molecules is a prerequisite for identifying surface exposed amino acids critical for their IgE-binding activity. The allergens whose three dimensional structure has been resolved by X-ray crystallography include Bet v1, the major allergen of birch pollen [6], Phl p7, calcium binding allergen protein of timothy grass [7], Fel d1, the major cat allergen [8] Der p 2, major house dust-mite allergen [9] and hyaluronidase, the major allergen of bee venom [10]. This information on three-dimensional structures is now being used for rational engineering of novel allergy vaccines with reduced IgE binding but with native protein fold for induction of protective blocking-antibody responses [7,11]. It should be emphasized here that probably it may not be feasible to obtain three-dimensional structure of all known allergen molecules. Various other genetic modifications that do not require prior knowledge of three-dimensional structure have also resulted in hypoallergenic forms suitable to be considered as candidates for inclusion in the allergy vaccines [3,5,12,13].

These modified forms have been shown to retain T cell reactivity and induce blocking antibodies [13,14]. In recent clinical trials on allergic patients such modified forms of Bet v1, major allergen of birch pollen has been shown to relieve allergic symptoms [15]. The article by Ferreira et al. in this special issue is a state of the art review of recombinant based approaches for the genetic modification of allergens. Various strategies discussed include production of site-directed mutants, deletion mutants, allergen fragments and allergen chimeras. A related therapeutic strategy that proposes the use of DNA-based vaccines translating modified allergen genes is also discussed. The authors provide a brief overview of novel non-allergen specific therapies such as anti-IgE and use of Th1-inducing adjuvants. Combining anti-IgE therapy with specific immunotherapy using modified allergens is a particularly interesting option covered in this review article.

Though, allergy vaccination is an established effective treatment for allergic diseases, the underlying cellular and immunological mechanisms however still remain unclear. Several mechanisms proposed include: induction of T cell unresponsiveness, an alteration of cytokine profile of allergen-specific T cells in favour of Th1 cytokines thus causing switching of Th2 type immune responses in allergic patients towards Th0 or Th1 type responses, increased production of immunosuppressive cytokines IL-10 and TGF-? regulatory T cells and the induction of “blocking” antibodies. While original concept of allergen specific IgG antibodies acting as “blocking antibodies” to antagonize the cascade of allergic inflammation resulting from allergen recognition originated over 60 years ago [16] its significance remained debatable. Renewed support for this concept has recently emerged from recent studies [16, 17] that show that blocking antibodies inhibit allergen-induced release of inflammatory mediators from basophils and mast cells as well as IgE-facilitated allergen presentation to T cells, thus leading to suppression of T cell activation. It has been proposed that blocking antibodies have protective activity by inhibiting immediate as well as late inflammatory responses and long-term ameliorating activity on the allergic immune response by antagonizing the underlying IgE production [16]. Induction of blocking antibodies has been proposed as an important mechanism underlying successful allergen-specific immunotherapy [16].

The successful specific immunotherapy is known to induce T cell unresponsiveness against the given allergen [18-20]. Review article by Schmidt-Weber and Blaser in this thematic issue provide an overview of the T cell based cellular mechanisms of immunotherapy with particular focus on the mediation of T cell unresponsiveness by complex cellular and molecular mechanisms involving regulatory T cells. T regulatory cells and IL-10 have been implicated in the mechanism of immunotherapy in patients with systemic anaphylaxis following bee stings [21,22]. Recently it has been demonstrated that conventional grass pollen injection immunotherapy induces a peripheral population of cells that produce IL-10 when activated by specific allergen [23]. Additionally an alteration in the subsequent responses to allergen exposure in the nasal mucosa in favour of local IL-10 production was also observed [23]. Immunotherapy with grass pollen extract also led to induction of serum-blocking activity, presumably IgG that blocked formation of allergen-IgE complexes and binding to CD23+ B cells. It was proposed that a likely outcome of this is inhibition of IgE-facilitated allergen presentation to T cells. Recently, it has been demonstrated that standardized house dust mite immunotherapy leads to decrease in number of IL-4+ T cells and expansion of CD4+IL-10+ T cells that express peripheral tissue trafficking markers [24]. The observed co-localization of IL-10+ staining to CD4+CD25+ T cells was considered to be consistent with the induction of a T regulatory cell population by allergen immunotherapy [24]. These reports on grass pollen and house dust mite immunotherapies provide considerable substantiation for multitude of cellular and immunological mechanisms of immunotherapy suggested by Schmidt-Weber and Blaser in this special issue.

IgE mediated adverse reaction to food is one of the commonly observed manifestations of atopic allergy. The prevalence of IgE-mediated food allergy varies from 6% in infants and young children compared to 1% to 2% in adults. Unlike inhalant and contact allergies there is no effective therapy for IgE-mediated food allergy. Emerging information regarding immunological mechanisms underlying allergic diseases has enhanced the potential therapeutic options for food allergy. Nowak-Wegrzyn in her article provides an overview of contemporary perspectives on the potential for developing immunotherapy for food allergy. The author describes animal models of food hypersensitivity and details recent attempts to develop immunotherapy for peanut allergy using recombinant proteins and DNA encoding allergen genes. An exciting possibility covered in this review is the use of humanized anti-IgE antibodies. Pollen-food allergy syndrome is the most common food allergy in adults and this syndrome provides unique opportunity to assess the efficacy of allergen immunotherapy on food allergies. Brief but critical evaluation of other novel approaches such as DNA vaccination, use of immunostimulatory sequences, cytokines, immunotherapy with mutated allergens is also provided in this review.

An emerging strategy for component resolved diagnosis of allergy is based on development of protein biochips with microarrayed recombinant allergens [25]. It has been proposed that such allergen microarray based technologies can lead to the development of novel multi-allergen tests that permit determination of complex sensitisation profile of allergic patients in single assays [26]. Comparison of allergen microarray using recombinant allergens with conventional diagnostic methods such as CAP/RAST and ELISA showed comparable or even higher analytical sensitivity [27]. Thus protein microarray based detection of allergen-specific IgE is expected to be the method of choice for a future component-resolved diagnosis of Type 1 allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future. It is tempting to speculate that future allergen protein chips will also contain arrays of modified recombinant allergens to help identification of modified forms suitable for vaccination of individual allergic patients. Such new developments in allergy diagnostics are likely to lead to production of increasingly complex data. Brusic in his article in this special issue emphasizes the significance of information technology in gathering, storage, retrieval and analysis of such complex data.

The complete nucleotide sequences of hundreds of allergens are available in sequence databases. Number of new informatics tools, designed to harness this new wealth of information are becoming available [28-30]. Some of these new informatics tools allow in silico prediction of B and T cell epitopes of allergens. These predictive tools when coupled with in vitro screening methods characterise an approach that has been termed computational immunology or immuno-informatics [28]. An elegant example of this approach was the application of computer algorithm to predict T cell epitopes of Lol p 5, a major ryegrass pollen allergen [31]. Synthetic peptides based on these predicted T cell epitopes were able to stimulate T cell lines isolated from allergic patients. Recently, design of a new computational system named MULTIPRED has been reported by Brusic’s group [32], which enables accurate prediction of T cell epitopes. In his article in this special issue Brusic has provided an overview of such exciting new immuno-informatic tools. The ability to predict potential allergenicity genetically modified foods is also becoming an important issue. New bioinformatic tools with an ability to predict potential allergenicity based on sequence similarity or the protein motifs identified from known allergen databases [33] have been developed [34,35]. In his review article Brusic discusses these allergenicity predictive tools along with list of allergen databases available in the public domain.

Although future allergy vaccines based on modified recombinant allergens will have greatly reduced risk of systemic anaphylaxis there can still be patient compliance concerns because of discomfort of repeated subcutaneous injections. Alternative routes of vaccine delivery have been attracting increased interest in an attempt to improve patient compliance. In their article in this special issue Passalacqua et al. provide an excellent overview of non-injectable or local routes for allergen immunotherapy with a particular focus on sublingual immunotherapy. The authors provide a brief history of oral, bronchial and nasal routes for allergy vaccine delivery. The focus of Passalacqua et al. review is on sublingual route of vaccine delivery. They review data to show the efficacy of sub lingual immunotherapy and propose that paediatric patients are most appropriate candidate for the sub lingual immunotherapy. The authors also provide a brief discussion on the mechanism of action of local routes of vaccine delivery. The authors have been proponents of sub lingual immunotherapy for many years and in this review they suggest that sublingual immunotherapy (SLIT) represent a significant advance in allergy treatment.

The development of efficient systems for the production of recombinant allergens is pre requisite for establishing their use as components of allergy vaccines. In the final article of this special issue we provide an overview of various options for production of recombinant allergens in prokaryotic and eukaryotic expression hosts. Relative advantages and disadvantages of various heterologous systems for production of recombinant allergens have been outlined. The choice of expression system is dependent upon the nature of allergen to be produced. Proposed use of plants as bio-factories for the production of recombinant allergens is a very attractive option due to simple scale up ability and low cost of production.

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Prem L. Bhalla and Mohan B. Singh
Plant Molecular Biology and Biotechnology Laboratory
ARC Centre of Excellence for Integrative Legume Research
Institute of Land and Food Resources
The University of Melbourne
Parkville, Vic 3010
Australia


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Modified Recombinant Allergens for Safer Immunotherapy
Fátima Ferreira, Peter Briza, Daniela Inführ, Georg Schmidt, Michael Wallner, Nicole Wopfner, Josef Thalhamer and Gernot Achatz

Molecular cloning and recombinant production of allergens offered new perspectives for the increasing problem of allergies. A variety of preparations are being developed aiming to increase safety and improve efficacy of specific immunotherapy. Recombinant-based approaches are mostly focused on genetic modification of allergens to produce molecules with reduced allergenic activity and conserved antigenicity, i.e. hypoallergens. Studies dealing with genetic modifications of allergen genes reported the production of site-directed mutants, deletion mutants, allergen fragments and oligomers, and allergen chimeras. An alternative to genetic engineering is the chemical modification of pure recombinant allergens. It has been shown that allergens modified with immunostimulatory DNA sequences (allergen-ISS conjugates), which masks IgE epitopes and adds a desirable Th1-inducing character to the allergen molecule. Other chemical modifications include oligomerization by aldehydes (allergoids) and maleylation, which seems to target allergens to paarticular antigen presenting cells. Several of these modified allergen preparations have been already evaluated for their safety in clinical provocation studies. So far, clinical trials showed the efficacy and safety of immunotherapy with an Amb a 1-ISS conjugate for ragweed pollen-allergic patients. In addition, a preparation consisting of hypoallergenic fragments of Bet v 1 was evaluated for immunotherapy of birch pollen-allergic patients. In parallel, several animal studies have now demonstrated the potential of genetic immunization for allergy treatment in the future.


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Immunological Mechanisms of Specific Allergen Immunotherapy
Carsten B. Schmidt-Weber and Kurt Blaser

Allergy is an immunological disorder, which is driven by uncontrolled allergen-activated T cell subsets, leading to immediate type hypersensitivity against otherwise harmless environmental allergens. These allergens are tolerated by healthy individuals as well as by patients, who successfully underwent allergen-specific immunotherapy (SIT). The successful SIT is characterized by the induction of T cell unresponsiveness against the given allergen. Regulatory T cells (Tregs), which are installed or enhanced by SIT and govern the activity of potentially pro-allergic effector T cells, mediate this unresponsiveness. The current article reviews the mechanisms underlying the balance of these cell populations along with suppressive mechanisms of SIT, which may serve as future drug targets.


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Immunotherapy for Food Allergy
Anna Nowak-Wegrzyn

The past two decades have witnessed an increase in prevalence of food allergy that has been matched with a tremendous progress in research that has led to better understanding of pathogenic mechanisms and development of novel therapies for food allergy. Establishment of murine models of peanut and cow’s milk allergy has been extremely useful in investigating food allergy treatments. Diverse strategies for prevention and treatment of established food allergy are being evaluated. Anti-IgE antibody therapy, Chinese herbal medicines, and killed bacteria expressing modified major peanut allergens represent the most promising approaches that will lead to development of therapy for patients for whom no effective treatment is currently available.


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Information Management for the Study of Allergies
Vladimir Brusic

Microarrays and other large-scale screening technologies produce quantities of increasingly complex allergy data. These data link molecular and clinical measurements and observations and provide fertile ground for improving our understanding of the processes involved in allergic reactions. Information technology is employed in gathering, storage, retrieval and analysis of these data. The increasing proportion of allergy data are generated from genomics and proteomics approaches. The major acivity focuses on characterization of allergens including IgE reactivity, structural properties, and mapping of IgE and T-cell epitopes. Because of the complexity of allergy data, their utilization requires bioinformatics approaches. Allergen data are stored in the general and specialist databases. At least a dosen of important allergen databases and data repositories have been developed to date. These data are analysed using general and specialist bioinformatics tools. The major applications of bioinformatics include support for allergen characterization, assessment of allergenicity, and identification of allergic cross-reactivity. These applications in turn support the development of vaccines and therapies for allergic disease. In this article we review allergen databases and tools for the analysis of allergens, and discuss the new directions in the field supported by large scale screening involving genomics, proteomics, and bioinformatics support.


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Non-Injection Routes for Allergen Immunotherapy: Focus on Sublingual Immunotherapy
Giovanni Passalacqua, Laura Guerra, Mercedes Pasquali and Giorgio W. Canonica

Allergen specific immunotherapy, together with drugs and allergen avoidance, is a cornerstone in the management of respiratory allergy. The non-injection or local routes were developed with the main goal of improving the safety and minimizing the risk of those side effects, which can accompany the injection route. The pure oral route and the bronchial route showed, in the clinical trials, only a marginal efficacy with not negligible side effects. Therefore, these routes are no longer recommended for clinical use. The nasal route proved effective and safe, but its efficacy is strictly limited to the nose. Moreover, the practical problems with administration have made the use of nasal immunotherapy progressively declining.

The efficacy of the sublingual route is confirmed by numerous controlled trials, and a meta analysis (in allergic rhinitis). The safety profile, as derived from clinical trials and post marketing surveillance studies, is satisfactory, with mild gastrointestinal complaints being the more frequent side effect reported. Recent studies have also demonstrated that SLIT has a long-lasting effect and a preventive effect on the onset of new skin sensitizations, and interesting data on adherence and mechanisms of action have become recently available. Based on these experimental data, SLIT is now officially accepted as a viable alternative to the subcutaneous route in adults and children. Several points still need to be elucidated, including: mechanisms of action, optimal dosages, and indications in pediatric patients.


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Recombinant Expression Systems for Allergen Vaccines
Mohan B. Singh and Prem L. Bhalla

Allergen immunotherapy of future is likely to be based on allergy vaccines that contain engineered allergens modified to abolish or substantially reduce their IgE-binding activity in order to remove the risk of unwanted anaphylactic responses.

The development of efficient systems for the production of recombinant allergens in sufficient quantities is requirement for establishing use of engineered allergens as components of allergy vaccines. This review outlines relative advantages and disadvantages of various heterologous systems for production of recombinant allergens. Microbial systems are most convenient and cost effective platforms for the production of recombinant allergens. However, lack of post-translational processing implies that some allergens have to be expressed in eukaryotic systems for proper folding and post-translational modifications such as glycosylation. Yeast systems can yield high levels of recombinant allergens but often are associated with hyper- glycosylation problems. Mammalian cell culture systems offer suitable post –translational modifications but are nearly hundred fold more expensive than microbial systems. The use of plants as bio-factories for production of recombinant allergens is emerging as a very attractive option as plants-based production system offer several advantages over other expression systems such as post translational processing of proteins, low production costs, scale up ability and enhanced safety due to absence of animal or human pathogens.


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Current Options in the Treatment of Mast Cell Mediator-Related Symptoms in Mastocytosis
Luis Escribano, Cem Akin, Mariana Castells and Lawrence B. Schwartz

Patients with mastocytosis have symptoms related to the tissue response to the release of mediators from mast cells (MC), local mast cell burden or associated non-mast cell hematological disorders. MC contain an array of biologically active mediators in their granules, which are preformed and stored. MC are also able to produce newly generated membrane-derived lipid mediators and are a source of multifunctional cytokines. Mediator-related symptoms can include pruritus, flushing, syncope, gastric distress, nausea and vomiting, diarrhea, bone pain and neuropsychiatric disturbances; these symptoms are variably controlled by adequate medications.

Management of patients within all categories of mastocytosis includes: a) a careful counseling of patients (parents in pediatric cases) and care providers, b) avoidance of factors triggering acute mediator release, c) treatment of acute and chronic MC-mediator symptoms and, if indicated, d) an attempt for cytoreduction and treatment of organ infiltration by mast cells.