Allergen Standardization and Characterization
Posted: September 2009
|Enrique Fernández-Caldas, PhD
Allergy Innovations GmbH
|Josefina Zakzuk, MD
Allergy Innovations GmbH
|Richard F. Lockey, MD
Division of Allergy and Immunology
University of South Florida College of Medicine and
James A. Haley Veterans' Administration Hospital
Tampa, Florida, USA
Key words: allergens, allergen immunotherapy, vaccines, standardization, recombinant allergens.
There is a worldwide increase in atopic diseases, which include allergic rhinoconjunctivitis, allergic asthma, atopic dermatitis and food allergies1. Although the reasons for this increase are unclear, allergen exposure is recognized as an important environmental risk factor in genetically predisposed individuals. The diagnosis of allergic disease requires a detailed history, physical examination, and allergy testing, i.e., skin testing or the in vitro determination of allergen-specific immunoglobulin E (IgE). Skin testing is performed by applying an allergen extract to the skin and then scratch or pricking it with an appropriate needle-like instrument. In sensitized individuals, it results in the formation of a raised wheal surrounded by an erythematous flair within 15 to 20 minutes, indicating a positive test reaction. In vitro specific IgE also supports the clinical diagnosis and helps to guide the allergist in the management of allergic diseases.
Once an individual is sensitized, symptomatic and long-term strategies, such as environmental control and immunomodulatory treatments using allergen immunotherapy, play an important role in treatment. Specific allergen immunotherapy (SIT) is effective in alleviating allergic symptoms, especially when introduced early in life and is the only known treatment that affects the natural course of allergic diseases. It also may prevent the development of asthma in patients with allergic rhinitis2. SIT is the practice of administering gradually increasing doses of allergen vaccines to reduce allergic symptoms and the need for medications. This biological response modifier is capable of influencing allergen-driven immunological responses and restoring, to a certain degree, the Th1/Th2 balance in allergic subjects. B and T cells, blocking antibodies, IL-10 and other cytokines play an important role in the response to SIT3.
Accuracy of skin tests and in vitro determinations depend on the availability of well-characterized allergen extracts. So too does effective allergen immunotherapy; allergen standardization and characterization are paramount to achieve these goals.
Testing and Standardizing Allergen Products
Allergen products to diagnose and treat allergic diseases have been used for over 100 years. Allergen extracts are biological products that are administered to humans to diagnose, prevent and treat allergic diseases4. Numerous double-blind placebo-controlled studies using unmodified aqueous allergens5 and allergoids6 (modified allergens) have demonstrated efficacy. "Allergoid" is a term used to describe natural allergen products that have been modified with aldehydes to decrease their allergenicity and potentially increase their safety. Some allergoids are commercially available in Europe and have demonstrated successful clinical results7. Sublingual immunotherapy, using drops of aqueous allergen extracts under the tongue and then swallowed8, or tablets9 with similar type of extracts, utilized the same way, also appear to be clinically beneficial, particularly for grass induced rhinoconjunctivitis.
The quality of allergen products is a key issue for both diagnosis and therapy, and the standardization of allergen extracts is thus of primary importance to improve their quality and offer physicians worldwide a reliable method to diagnose and treat allergic respiratory diseases. Effective diagnosis and treatment, using skin test reagents and SIT, requires the optimal amount of allergens for testing and the maintenance dose of vaccine for treatment. Skin test reactions should be large enough to suggest clinical sensitivity but not so large as to produce excessive discomfort or the risk for a serious systemic reaction.
The heterogeneity of allergen extracts makes it necessary to develop methodologies to assess their potency and ensure their consistency, stability and safety. Allergen products are legally considered medicines that require registration by government institutions10, such as the FDA in the United States and the Paul Ehrlich Institute in Germany, further increasing the need for standardization. Basic researchers, physicians, regulatory authorities and manufacturers have tried to define a common methodology to standardize allergen vaccines11, 12, 13. The quality of mite and pollen allergen extracts is better defined today than it was in the past, and the quality of food and epithelial allergen extracts has also improved14, 15. Further improvement of food allergen extracts is needed, since preliminary studies have shown efficacy using unmodified sublingual food allergen extracts16.
In Europe, because of the need to standardize allergen extracts for diagnosis and treatment, manufacturers have implemented company-specific protocols to standardize the production and quality control of the allergen extracts and to compare production batches to assure batch-to-batch consistency17. All these production and quality control issues are controlled by strict protocols and adhered to Good Manufacturing Practices18. Thus, current allergen standardization requirements concentrate on the consistency of production and the safety and potency of allergen products. These protocols use in vivo and in vitro standardization techniques, a representative allergic patient population, and dose-response studies to assign biological activities. Dose-response studies are mandatory and are primarily based on skin testing (intradermal or prick tests) and on inhibition of allergen-specific IgEs compared to reference extracts known as in-house reference preparations (IHRPs). Furthermore, guidelines have been issued for the clinical development of products for specific immunotherapy for the treatment of allergic diseases19. The IgE-binding potencies of the IHRPs are quantified by skin test reactivity (in vivo standardization) and by competitive IgE tests, such as RAST, ImmunoCAP, or ELISA inhibition assays (in vitro standardization). Although they may be similar, they are expressed in company-specific units.
The comparison of different products from different companies at national and international levels is complicated. Larenas-Linnemann and Cox reviewed the information obtained on unit definition and dosage of allergens from European manufacturers of allergen extracts used for sublingual immunotherapy (SLIT). They concluded that the monthly maintenance dose the manufacturers recommended for SLIT was 5-45 times higher than the recommended dose for subcutaneous immunotherapy. However, since each manufacturer in Europe uses its own IHRPs and its own units to express potencies, the comparison of different products from different companies at national and international levels is almost impossible. Even if the amount of major allergens is stated, differences in the quantification technique, the reference extracts and antibodies used can influence the outcome. Thus, for comparison of diagnostics and immunotherapeutics from different manufacturers, the same analytical methods and materials ideally should be used20.
Another study quantified and compared the allergen content of different grass pollen preparations for skin prick testing (SPT) and SLIT used in Europe21. Protein concentrations of SPT solutions ranged from 15 to 427 µg/ml, and Phl p 5 concentrations, a major grass allergen, ranged from 0.15 to 18.3 µg/ml. Protein content of the maintenance doses of the immunotheurapeutics ranged from 5 to 153 µg and Phl p 5 content ranged from 0.2 to 21.6 µg. SDS-PAGE and immunoblots confirmed the differences in protein and allergen contents.
Extracts in the United States are more homogenous with respect to total allergenic potency than the extracts produced in Europe, mainly because the FDA provides the same standardized reagent for internal use by all manufacturing companies22. However, great differences have also been shown among unstandardized mold allergen extracts in the USA23.
The characterization of major allergen components and the development of techniques to quantify them, such as ELISA systems based on monoclonal antibodies, have led more manufacturers to provide information on the major allergen content of their extracts, even though identification of major allergen content is not currently mandatory, except for a limited number of extracts, such as cat and ragweed. The World Health Organization and some other regulatory government institutions now recommend that allergen manufacturers state the content of representative major allergens in mass units for their allergen products24. As noted above, differences in assays and methodologies for measuring the major allergens may preclude direct comparisons among products of different manufacturers25.
Recombinant Allergens (RAs)
Allergenic extracts consist of complex mixtures of substances with a variation in allergenic activity and allergen composition. Furthermore, patients are often not sensitized to all allergens in one extract and there may be great variability among different patients. Advances in the field of microarrays containing purified native and recombinant allergens (RAs) may be useful to identify the specific sensitivity of individual patients in order to select the proper allergen composition in the vaccine26. To improve safety and efficacy of SIT, a better (alternative) approach may be to use RAs. There has been considerable progress in understanding the molecular characteristics of allergens. Many allergens have been purified from aqueous extracts or produced as recombinant molecules. Allergen sequence information is available in different databases (www.allergen.org/www.allergome.org). Molecular cloning has provided an efficient way to obtain pure polypeptides, which in their native sources, form complex mixtures and are often present in very small amounts. These polypeptides enable the mapping of B- and T-cell epitopes and the identification of their binding sites. Sequence polymorphisms that influence antibody binding and T-cell recognition for several allergens have also been established. Sequence similarity searches have identified the biological functions of many allergens. For example, Der p 1 and Der f 1 are considered major allergens based on the frequency of patients sensitized, amount of specific IgE, and content in mite extract. Der p 1 is a glycoprotein with sequence homology and thiol protease function similar to the enzymes papain, actinidin bromelain and cathepsins B and H27. When an allergen does not have sequence homology with proteins of known biological function, its biochemical identity remains uncertain. Several mechanisms are involved in airway hyper-responsiveness and airway inflammation caused by these allergens. Besides acting by immunologically mediated mechanisms, Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions28. Due to its high prevalence and worldwide distribution in house dust, the group 1 allergen is used as a standard to estimate environmental exposure to Dermatophagoides spp. in the indoor environment.
Since allergen extracts or vaccines represent several hundred allergy determinants, and individuals can be sensitized to different combinations of these proteins, sensitization profiles in atopic populations are very heterogeneous. Therefore, before RAs can be frequently utilized in routine clinical practice, epidemiological studies using RAs to identify the immune response to specific allergens in the population at large and to clarify cross-reactivity are necessary. Several immunotherapy studies have been conducted with recombinant allergens with clinical benefits29, 20, 31, 32. More clinical trials are needed to compare the potential advantages and benefits of SIT with RAs versus current standard therapy with natural vaccines. This process will take years; in the meantime, optimizing SIT with naturally occurring vaccines is required to assure efficacy and safety.
The number of cloned and purified allergens has increased substantially over the past decade. RAs are useful as reagents for SIT. RAs will overcome some of the pitfalls of using natural allergen products for immunotherapy by enabling physicians to administer only the clinically relevant allergen, thus avoiding exposure to unnecessary antigens. RAs could also be rendered hypoallergenic using a variety of techniques.
The CREATE Project
A project financed by the European Union, "Development of Certified Reference Materials for Allergenic Products and Validation of Methods for their Quantification", (the CREATE Project), explores the idea of introducing standardized techniques to quantify major allergen content into standardized protocols. Mass units of major allergens would be used to quantify the active ingredients of the allergen while allowing comparison among manufacturers33. The goals of the CREATE Project were to evaluate the potential of purified recombinant allergens as certified reference materials (CRMs) and to evaluate available ELISAs for the measurement of major allergens using the candidate CRMs as a standard. To carry forward with this project, eight major allergens originating from four of the most important inhalant allergen sources were selected: Bet v 1 from birch pollen, Phl p 1 and Phl p 5 from grass pollen, Ole e 1 from olive pollen and Der p 1 and 2 and Der f 1 and Der f 2 from house dust mites. Three were found to be suitable as biological reference materials; the rest, except rPhl p 1a, indicate potential for optimization, provided aspects of their protein expression processes are modified. As a result of this study, recombinant Bet v 1 and Phl p 5 are being produced under "Good Manfacturing Practice" and being evaluated by the European Directorate for the Quality of Medicines as biologic reference preparations to be included in the European Pharmacopoeia as international standards. Consequently, standardization of these allergen products will become global permitting comparisons among different manufacturing sources34.
Other techniques which could be used for standardization
Beyond the use of monoclonal antibodies, other physico-chemical approaches have been explored for allergen standardization and offer new possibilities. Evaluation of mass spectrometry (MS) has been performed to determine its capacity to characterize the composition of allergen extracts35, 36. One advantage offered by the use of MS includes the measurement of several different allergenic components (allergens and isoforms) simultaneously rather than measuring individual allergens. This is advantageous while working with allergen preparations that contain a wide group of IgE binding proteins, such as mite extracts. Additionally, MS-based methods are available to discriminate between allergen isoforms, which is difficult to accomplish using immunologic based methods. This method could also be useful for the standardization of allergoids, since measuring major allergens is not possible in these preparations due to their chemical modification with aldehyde. The issues with MS-based protocols are that they are not quantitative and are not addressed in regulatory policies.
Conclusions and Current challenges
Allergen standardization strategies should be uniform throughout the world. The different units, which vary among manufacturers as well as global regions, are confusing and unreliable with the potential to underestimate or overestimate the potency of allergen extracts. The variations can be attributed to the variability of the raw materials used, the production methods and the lack of consistent and reliable quantification of allergy content. Major allergen measurements are essential to overcome these problems. Some recombinant allergens were found to be suitable as certified reference materials. But, results from the CREATE Project demonstrate the limitations of recombinant allergens as CRM due to incorrect folding, aggregation, poor solubility and insufficient stability33. However, evidence gathered in studies identifies the complexity of this approach. The European Medicines Agency (EMEA) recommends proving that each allergen extract contains the relevant allergens by antibody-based techniques or mass spectrometry. New techniques, such as nuclear magnetic resonance and small angle X ray scattering, are commonly applied to characterize allergenic molecules in the laboratory. They are slowly affecting the methods by which allergen extracts are standardized. This would be an important step forward towards a comprehensive characterization of allergen products. Future enhancements of SIT, using RAs, also require allergen standardization, which in turn, requires development of standardized methods to measure allergen content, homogeneity, folding, aggregation, solubility and stability of recombinant products. Allergen standardization will advance rapidly in the future, improving the effectiveness and safety of allergen vaccines. Allergen immunotherapy will remain an effective and safe treatment for allergic respiratory diseases.
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