Mutant derivatives of the main respiratory allergen of cow are less allergenic than the intact molecule.

BACKGROUND: Allergen immunotherapy offers an alternative for drug treatment in the management of allergic diseases. Because immunotherapy often induces side-effects, less allergenic preparations would be beneficial. OBJECTIVE: The purpose of this study was to examine whether the allergenicity of a cow-derived lipocalin allergen, Bos d 2, could be diminished by substituting or deleting carboxy-terminal amino acids including the cysteine which forms a disulphide bond with a cysteine inside the molecule. METHODS: Four recombinant mutants of Bos d 2 were created by substituting or deleting the four most carboxy-terminal amino acids. The immunological characteristics of the mutant preparations were compared with the unmodified rBos d 2 by Western blotting, ELISA inhibition, skin prick tests, and the proliferative responses of allergen-specific T-cell clones. RESULTS: In Western blot, one of the two monoclonal antibodies showed reduced binding to the preparations without the terminal cysteine. In contrast, the other monoclonal antibody, human IgE and rabbit immune serum bound equally well to all the preparations. ELISA inhibition analyses revealed, however, that the preparations without the terminal cysteine bound antibody less efficiently. They were needed 15-38 times more than the unmodified rBos d 2 to cause the same level of inhibition. Surprisingly, one of the mutants with the terminal cysteine but a mutated adjacent amino acid turned out to be the weakest in inducing skin reactivity. All the preparations stimulated well allergen-specific T-cell clones. CONCLUSIONS: The results show that the allergenicity of a lipocalin allergen, Bos d 2, can be diminished by modifying the carboxy-terminal end of the molecule. Modifications in the area which encompasses a disulphide bond impaired the antibody binding without affecting the T-cell stimulatory capacity. It was also shown that in vivo tests are necessary for determining the allergenicity of a modified allergen.     

Clinical trials in allergen immunotherapy: current concepts and future needs

Allergen immunotherapy (AIT) is a safe, effective treatment for allergic rhinoconjunctivitis and allergic asthma. However, AIT's clinical effect is still contested—primarily due to heterogeneity in clinical trial designs, study populations, therapeutic formulations, and efficacy criteria. After discussing current concepts and unmet needs, an international panel of experts made several recommendations: (i) explore and validate definitions for (clinical) responders in AIT trials; (ii) use of well‐documented, standardized provocation tests prior to inclusion of subjects with relevant diseases in AIT trials; (iii) monitoring neo‐sensitizations and occurrence of new allergy in extended AIT trials, and exclusion of polyallergic participants; (iv) validation of allergen exposure chambers with regard to natural exposure; (v) in studies of seasonal allergies, focus on peak exposure but also consider organizing two parallel, geographically distinct but otherwise identical trials; (vi) discuss adaptive trial designs with the regulatory authorities; (vii) use e‐health and m‐health technologies to capture more information on individual exposure to allergens; (viii) initiate research on potential psychological, biochemical, immune, neural, and even genomic markers of the placebo response; (ix) identify trial designs and primary endpoints that will give children with allergies easier, faster access to AIT formulations; and (x) promote and apply standardized methods for reporting systemic and local adverse events. The latest technologies and trial designs may provide novel, ethical ways of reducing bias and heterogeneity in AIT clinical trials. There is scope for physicians, patient organizations, companies, and regulators to improve clinical trials in AIT and, ultimately, to provide patients with better treatments.     

Recombinant allergens for specific immunotherapy

Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient's sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years.     

Recombinant allergens for allergen-specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens

The broad applicability of allergen-specific immunotherapy for the treatment and eventually prevention of IgE-mediated allergy is limited by the poor quality and allergenic activity of natural allergen extracts that are used for the production of current allergy vaccines. Today, the genetic code of the most important allergens has been deciphered; recombinant allergens equalling their natural counterparts have been produced for diagnosis and immunotherapy, and a large panel of genetically modified allergens with reduced allergenic activity has been characterized to improve safety of immunotherapy and explore allergen-specific prevention strategies. Successful immunotherapy studies have been performed with recombinant allergens and hypoallergenic allergen derivatives and will lead to the registration of the first recombinant allergen-based vaccines in the near future. There is no doubt that recombinant allergen-based vaccination strategies will be generally applicable to most allergen sources, including respiratory, food and venom allergens and allow to produce safe allergy vaccines for the treatment of the most common forms of IgE-mediated allergies.     

In vitro and in vivo responses to the recombinant bovine dander allergen Bos d 2 and its fragments

BACKGROUND: About one in every four cases of occupational rhinitis recorded in Finland is animal-induced. Bovine allergens are the most important in this respect and the largest patient group consists of dairy farmers. Allergen immunotherapy, if proven effective, safe and feasible, would be ideal for their treatment. The development of recombinant allergens has offered new potential therapeutic prospects. Fragments of recombinant Bos domesticus (Bos d 2) allergen could be suitable for this purpose because they are recognized by T cells but their IgE-binding capacity is attenuated. OBJECTIVE: The aim of this study was to verify whether the potential of the two fragments of recombinant Bos d 2 (corresponding to amino acids 1-131 and 81-172) to induce immediate allergic reaction in a shock organ (nose) was decreased compared to the complete recombinant allergen, which would be an advantageous property for a preparation intended for allergen immunotherapy. METHODS: The study group consisted of 10 dairy farmers with cow-induced allergic rhinitis. We used the IgE titres against native Bos d 2 measured by indirect IgE ELISA to characterize the level of sensitization and compared the IgE titres in the rhinitis patients with 12 cow-sensitized asthmatic farmers and 12 healthy students. In vitro reactivity against recombinant Bos d 2 and its two fragments was studied by indirect IgE ELISA and in vivo reactivity by nasal provocation tests. RESULTS: The IgE titres against native Bos d 2 of patients with rhinitis tended to be lower than the titres of asthmatics. The healthy students did not exhibit any detectable IgE reactivity to native Bos d 2. In the patients with rhinitis, there was no statistically significant difference between IgE responses against native and recombinant Bos d 2, whereas with both in vitro and in vivo, the reactivity to both fragments of recombinant Bos d 2 was lower than the reactivity to the complete recombinant allergen. CONCLUSIONS: Due to the decreased in vivo capacity to induce immediate allergic reactions, the fragments may be better tolerated in allergen immunotherapy than the complete allergen.     

Allergen immunotherapy: immunomodulatory treatment for allergic diseases

Allergen immunotherapy is currently the only immune-modifying treatment for allergic disease. At the present time it is indicated for the treatment of allergic rhinitis, asthma and venom hypersensitivity. Efficacy appears to be dose dependent, and the immunological mechanisms responsible for the clinical efficacy of immunotherapy are still being elucidated. Immunological changes associated with immunotherapy include induction of T regulatory cells, increase in allergen-specific immunoglobulin G4, increase in interleukin-10 production and downregulation of the T helper 2 response. The disadvantages of allergen immunotherapy include risk of adverse events and patient time and inconvenience. Risks of immunotherapy range from large local reactions to mild systemic reactions, such as rhinitis. Fatalities from immunotherapy injections have been reported at a rate of approximately one fatality per 2.5 million injections. Conventional subcutaneous immunotherapy build-up schedules involve administration of a single-dose increase each visit and it may take several months before a patient achieves the therapeutic maintenance dose. Accelerated schedules, such as rush and cluster, will allow the patient to achieve the maintenance dose sooner but there may be a greater risk of a systemic reaction. The current focus of immunotherapy research is to develop safer and more effective vaccines. Another approach to enhancing immunotherapy safety is through an alternative delivery method. Sublingual immunotherapy is clearly safer than subcutaneous immunotherapy, but further investigation is needed to determine optimal dose and appropriate patient selection.     

Challenges in the implementation of EAACI guidelines on allergen immunotherapy: A global perspective on the regulation of allergen products

Regulatory approaches for allergen immunotherapy (AIT) products and the availability of high‐quality AIT products are inherently linked to each other. While allergen products are available in many countries across the globe, their regulation is very heterogeneous. First, we describe the regulatory systems applicable for AIT products in the European Union (EU) and in the United States (US). For Europe, a depiction of the different types of relevant procedures, as well as the committees involved, is provided and the fundamental role of national agencies of the EU member states in this complex and unique network is highlighted. Furthermore, the regulatory agencies from Australia, Canada, Japan, Russia, and Switzerland provided information on the system implemented in their countries for the regulation of allergen products. While AIT products are commonly classified as biological medicinal products, they are made available by varying types of procedures, most commonly either by obtaining a marketing authorization or by being distributed as named patient products. Exemptions from marketing authorizations in exceptional cases, as well as import of allergen products from other countries, are additional tools applied by countries to ensure availability of needed AIT products. Several challenges for AIT products are apparent from this analysis and will require further consideration.     

Liposomes as vehicle for allergen presentation in the immunotherapy of allergic diseases

Liposomes are non-toxic, biodegradable and weakly immunogenic lipid vesicles which can be used as immunomodulating agents. In the present study, multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) were used to incorporate an allergenic protein from Artemisia scoparia pollen. MLV incorporated more allergenic protein than SUV. To assess the immunomodulating effect of allergen entrapped in liposomes, Swiss strain mice (made IgE responders) were injected with either free allergen or liposome-entrapped allergen (LEA) and their immune response was measured in terms of specific IgG and specific IgE levels. Results indicated that specific IgE response was significantly lower in mice injected LEA (P less than 0.02) than in mice injected free allergenic protein. Although specific IgG response was higher in mice injected LEA, there was no statistically significant difference between the two groups. Potential use of liposomes as non-immunogenic biocompatible vehicle for antigen presentation in immunotherapy will be discussed.     

Safety of immunotherapy with therapeutic vaccines containing depigmented and polymerized allergen extracts

BACKGROUND: The major complication of allergen immunotherapy is a severe reaction. OBJECTIVE: To evaluate the safety of depigmented and glutaraldehyde-modified allergen extracts in a large group of patients undergoing immunotherapy treatment. MATERIAL AND METHODS: Seven hundred sixty-six patients, having rhinoconjunctivitis and/or asthma, were entered in a prospective, multi-centre, observational cohort study, to evaluate the safety of immunotherapy with modified allergen vaccines. Patients were sensitized to mites and/or pollen and received a therapeutic vaccine containing depigmented and polymerized allergen extracts of mites and/or pollens adsorbed onto aluminium hydroxide. The schedule of administration consisted of a build-up phase of 4- to 6-weekly injections, followed by 12-monthly injections of the maintenance dose. Tolerance was assessed by recording all side reactions related to immunotherapy. RESULTS: All patients completed the study. Fifty-four clinically relevant local reactions (43 immediate and 11 delayed) were observed (0.4% of injections). The systemic reactions were 34 in 12 patients. Six reactions were immediate (all of grade 2) and 28 delayed (18 of grade 1 in two patients, nine of grade 2 and one of grade 3). The systemic reactions of grade 2 or 3 occurred in 0.12% of the injections. All systemic reactions were mild and resolved spontaneously without the need for medication. CONCLUSION: Specific immunotherapy using modified allergen vaccines is safe to treat allergic patients. The percentage of adverse reactions detected is lower than those reported in the literature with native-unmodified allergen extracts.     

Clinical contraindications to allergen immunotherapy: an EAACI position paper

Clinical indications for allergen immunotherapy (AIT) in respiratory and Hymenoptera venom allergy are well established; however, clinical contraindications to AIT are not always well documented. There are some discrepancies when classifying clinical contraindications for different forms of AIT as ‘absolute’ or ‘relative’. EAACI Task Force on ‘Contraindications to AIT’ was created to evaluate and review current literature on clinical contraindications, and to update recommendations for both sublingual and subcutaneous AIT for respiratory and venom immunotherapy. An extensive review of the literature was performed on the use of AIT in asthma, autoimmune disorders, malignant neoplasias, cardiovascular diseases, acquired immunodeficiencies and other chronic diseases (including mental disorders), in patients treated with β‐blockers, ACE inhibitors or monoamine oxidase inhibitors, in children under 5 years of age, during pregnancy and in patients with poor compliance. Each topic was addressed by the following three questions: (1) Are there any negative effects of AIT on this concomitant condition/disease? (2) Are more frequent or more severe AIT‐related side‐effects expected? and (3) Is AIT expected to be less efficacious? The evidence, for the evaluation of these clinical conditions as contraindications, was limited, and most of the conclusions were based on case reports. Based on an extended literature research, recommendations for each medical condition assessed are provided. The final decision on the administration of AIT should be based on individual evaluation of any medical condition and a risk/benefit assessment for each patient.     

The allergen challenge chamber: a valuable tool for optimizing the clinical development of pollen immunotherapy

The clinical development of allergen immunotherapy for allergic rhinoconjunctivitis because of pollen is complicated by seasonal, geographical and subject-related variability in allergen exposure. Using an allergen challenge chamber (ACC), a room that enables reproducible challenges with controlled levels of inhalant allergens for several hours, these factors can be controlled. The ACC has often been used to evaluate symptomatic medications but is underexploited in the field of allergen immunotherapy. When used in conjunction with a programme of natural-exposure trials, the ACC enables researchers to (i) facilitate the allergen immunotherapy dose-finding process, (ii) accelerate the transition from Phase I/II to Phase III trials, (iii) characterize the onset and maintenance of action, (iv) avoid the confounding effects of rescue medication, (v) better characterize the baseline or pretreatment characteristics of trial subjects, (vi) perform better-standardized physical and laboratory measurements during an acute challenge, (vii) simplify trial logistics and use smaller numbers of subjects than would be required in equivalent natural-exposure studies and (viii) support (but not replace) Phase III natural-exposure trials for the investigation into long-term and disease-modifying effects. ACC studies can further increase levels of evidence for allergen immunotherapy--the only current therapy potentially capable of modifying the underlying allergic disease.     

Effect of allergen immunotherapy on nasal responses in guinea-pigs with allergic rhinitis

Methods We have investigated the effects of allergen immunotherapy on the nasal responses in the guinea-pigs with allergic rhinitis. Thirty-three male Hartley guinea-pigs with allergic rhinitis were divided into three groups; those receiving intradermal injection of saline (Group 1) or 0.1% ovalbumin (Group 2) 6 days after the last intranasal sensitization, and those injected with 0.1% ovalbumin intradermally once daily for 6 consecutive days from the next day after the last intranasal sensitization (Group 3). Results The dye leakage and histamine content into the nasal lavage significantly decreased at 30min after antigen challenge in Group 3, compared with Group 1 or 2. We also observed the change of mast cell numbers in superficial nasal mucosa, lamina propria and injected dorsal skin. The number of mast cells in superficial nasal mucosa significantly decreased in Group 3 compared with Group 1 or 2, but not those in nasal lamina propria or dorsal skin. Conclusions These results suggest that the improvements of nasal responses such as dye leakage and histamine content may be caused by the decrease of mast cell numbers in the superficial mucosal layer after the specific immunotherapy. which may be developing tolerance and one of the mechanisms underlying the beneficial effect of immunotherapy.