In the past, the development of more effective, safe, convenient, broadly applicable, and easy to manufacture vaccines for allergen-specific immunotherapy (AIT) has been limited by the poor quality ...of natural allergen extracts. Progress made in the field of molecular allergen characterization has now made it possible to produce defined vaccines for AIT and eventually for preventive allergy vaccination based on recombinant DNA technology and synthetic peptide chemistry. Here we review the characteristics of recombinant and synthetic allergy vaccines that have reached clinical evaluation and discuss how molecular vaccine approaches can make AIT more safe and effective and thus more convenient. Furthermore, we discuss how new technologies can facilitate the reproducible manufacturing of vaccines of pharmaceutical grade for inhalant, food, and venom allergens. Allergy vaccines in clinical trials based on recombinant allergens, recombinant allergen derivatives, and synthetic peptides allow us to target selectively different immune mechanisms, and certain of those show features that might make them applicable not only for therapeutic but also for prophylactic vaccination.
Background
Skin testing represents a commonly used first diagnostic method in clinical practice, but allergen extracts may vary in composition and often contain cross‐reactive allergens and therefore ...do not always allow the precise identification of the sensitizing allergen source. Our aim was to investigate the suitability of a single recombinant hybrid molecule, consisting of the four major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Phl p 6) for in vivo diagnosis of genuine grass pollen allergy in children suffering from pollinosis.
Methods
Sixty‐four children aged from 6 to 17 years with a positive skin reaction and/or specific IgE to grass pollen extract and respiratory symptoms of pollinosis as well as 9 control children with allergy to other allergen sources were studied. SPT was performed with the recombinant hybrid, the four recombinant timothy grass pollen allergens, and grass pollen extract. Specific IgE reactivity to 176 micro‐arrayed allergen molecules was determined using ImmunoCAP ISAC technology. IgE reactivity to the hybrid was detected by non‐denaturing RAST‐based dot blot assay.
Results
Genuine grass pollen sensitization was confirmed in 94% of the children with positive SPT to grass pollen extract by SPT and IgE reactivity to the hybrid. The four hybrid‐negative children showed IgE reactivity to cross‐reactive allergens such as Phl p 4, Phl p 11, and Phl p 12 and had also sensitizations to pollen allergens from unrelated plants.
Conclusions
The recombinant hybrid molecule represents a useful tool for in vivo diagnosis of genuine grass pollen sensitization.
Parvalbumin, a small calcium-binding protein, which is extremely resistant to heat and digestion, represents the major fish allergen. Because of the high degree of sequence homologies and ...cross-reactivities between parvalbumins from different fish species, consumption of various fish species leads to clinical symptoms in fish-allergic patients.3 Several immunotherapy trials investigating the applicability of recombinant hypoallergenic vaccines are currently ongoing, and the clinical immunotherapy studies for birch pollen in particular indicate that recombinant allergen-based specific immunotherapy is a promising concept for the effective and safe treatment of respiratory allergy.4 Specific immunotherapy is less frequently used for the treatment of food allergies because fewer standardized allergen extracts are available and the risk of inducing severe anaphylactic reactions is high.
Background Trees of the family Oleaceae (olive and ash) are important allergen sources in Mediterranean countries, Northern and Central Europe, and North America. The major olive pollen allergen Ole ...e 1 represents the majority of allergenic epitopes in olive pollen and cross-reacts with Fra e 1, the major ash pollen allergen. Objective We sought to develop a safe vaccine for the treatment of Oleaceae pollen allergy. Methods We synthesized 5 peptides ranging from 32 to 36 amino acids, which covered the whole sequence of Ole e 1. The IgE and T-cell reactivity of the peptides was compared with that of Ole e 1 by means of dot blot experiments, as well as ELISA, and in proliferation assays. Rabbits were immunized with non–IgE-reactive, keyhole limpet hemocyanin–coupled peptides or Ole e 1. The reactivity of the IgG antibodies with Ole e 1 and their ability to inhibit IgE binding to nOle e 1 was evaluated by means of ELISA. Results Only the C-terminal Ole e 1 peptide showed IgE binding, whereas the other peptides were nonallergenic. Immunization of rabbits with Ole e 1–derived peptides bound to the carrier molecule keyhole limpet hemocyanin induced in rabbits the production of Ole e 1–specific IgG antibodies, which cross-reacted with Fra e 1, and inhibited olive and ash pollen–sensitized patients' IgE binding to Ole e 1. Conclusion Two non–IgE-binding peptides with low T-cell reactivity from the N-terminus of Ole e 1 were identified that might represent safe vaccine candidates for immunotherapy of Oleaceae pollen allergy.
The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.
...To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.
Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments.
Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients.
Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recombinant allergens: What does the future hold? Valenta, Rudolf, MD; Niespodziana, Katarzyna, MSc; Focke-Tejkl, Margit, PhD ...
Journal of allergy and clinical immunology,
04/2011, Letnik:
127, Številka:
4
Journal Article
Recenzirano
Odprti dostop
This year we are celebrating not only the centenary of allergen-specific immunotherapy but also the 10-year anniversary of the first administration of recombinant allergen–based vaccines to allergic ...patients. By using recombinant DNA technology, defined and safe allergy vaccines can be produced that allow us to overcome many, if not all, of the problems associated with the use of natural allergen extracts, such as insufficient quality, allergenic activity, and poor immunogenicity. Here we provide an update of clinical studies with recombinant allergen–based vaccines, showing that some of these vaccines have undergone successful clinical evaluation up to phase III studies. Furthermore, we introduce a strategy for allergen-specific immunotherapy based on recombinant fusion proteins consisting of viral carrier proteins and allergen-derived peptides without allergenic activity, which holds the promise of being free of side effects and eventually being useful for prophylactic vaccination.
Therapeutic strategies for the prophylaxis of IgE-mediated allergy remain an unmet medical need. Cell therapy is an emerging approach with high potential for preventing and treating immunological ...diseases.
We aimed to develop a cell-based therapy inducing permanent allergen-specific immunological tolerance for preventing IgE-mediated allergy.
Wild-type mice were treated with allergen-expressing bone marrow cells under a short course of tolerogenic immunosuppression (mTOR inhibition and costimulation blockade). Bone marrow was retrieved from a novel transgenic mouse ubiquitously expressing the major grass pollen allergen Phl p 5 as a membrane-anchored protein (BALB/c-TgPhlp5-GFP, here mPhl p 5). After transplantation recipients were IgE-sensitized at multiple time points with Phl p 5 and control allergen.
Mice treated with mPhl p 5 bone marrow did not develop Phl p 5-specific IgE (or other isotypes) despite repeated administration of the allergen, while mounting and maintaining a strong humoral response towards the control allergen. Notably, Phl p 5-specific T cell responses and allergic airway inflammation were also completely prevented. Interestingly allergen-specific B cell tolerance was maintained independent of Treg functions indicating deletional tolerance as underlying mechanism.
This proof-of-concept study demonstrates that allergen-specific immunological tolerance preventing occurrence of allergy can be established through a cell-based therapy employing allergen-expressing leukocytes.
•Avoidance of IgE-mediated allergy is established by development of a tolerogenic cell-based protocol.•Transplantation of syngeneic allergen-bearing bone marrow cells into recipients leads to tolerance towards the introduced allergen.•Prevention of T-and B-cell responses and allergic asthma is induced by cell transfer with non-toxic pretreatment.
IgE-mediated allergy affects about 30% of the population in industrialized countries. To prevent allergy we developed a cell-based protocol with the concept to modify body's own cells to express an allergen and to reinfuse those modified cells. This concept leads to avoidance of allergic reactions after allergen contact such as specific IgE production and the development of allergic asthma. This is demonstrated in a syngeneic model in mice (i.e. autologous in human) by transplanting bone marrow cells of a unique allergen-expressing transgenic mouse into pretreated recipients.
Commercial skin prick test (SPT) extracts used for the diagnosis of dog allergy are prepared by extracting allergens from natural sources, e.g. dog hair and dander. Due to different starting material ...and extraction methods used, it is likely that extracts differ regarding their allergen contents.
The total protein content and composition of dog SPT extracts from 5 European manufacturers were compared by silver-stained SDS-PAGE. Specific antibody probes were generated to detect major and minor allergens in each extract by immunoblotting. Additionally, sera of patients suffering from dog allergy were used to detect dog allergens in SPT extracts.
SPT extracts showed a 20-fold variation regarding the total protein content. The contents of the major dog allergen Can f 1 and of Can f 2 varied considerably between the extracts. In one of the extracts, neither Can f 1 nor Can f 2 could be detected by immunoblotting. The contents of the minor dog allergen Can f 3, albumin, also showed great variability. In one of the dog SPT extracts, the presence of human serum albumin (HSA) was detected with HSA-specific antibodies.
The observed variability of commercial dog SPT extracts regarding their allergen contents likely has a negative influence on the accuracy of diagnosis of dog allergy.
Visualization of clustered IgE epitopes on α-lactalbumin Hochwallner, Heidrun, MSc; Schulmeister, Ulrike, PhD; Swoboda, Ines, PhD ...
Journal of allergy and clinical immunology,
06/2010, Letnik:
125, Številka:
6
Journal Article
Recenzirano
Background α-Lactalbumin (α-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. Objective We performed molecular, structural, and immunologic characterization of ...α-La. Methods Recombinant α-lactalbumin (rα-La) was expressed in Escherichia coli , purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. Results According to circular dichroism analysis, rα-La represented a folded protein with a high thermal stability and refolding capacity. rα-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. rα-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of α-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. Conclusions rα-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.
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