Juan A. Asturias

Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen

BACKGROUND Olive tree (Olea europaea) pollen allergy is one of the main causes of allergy in Mediterranean countries and some areas of North America. OBJECTIVE To clone olive allergens and to characterize immunologically the purified recombinant allergens. METHODS Full-length complementary deoxyribonucleic acid (cDNA) strands encoding olive allergens (Ole e 1) were cloned by polymerase chain reaction amplification and sequenced. Recombinant proteins were produced in Escherichia coli by the use of two different expression systems. Immunoreactivity of the recombinant proteins was tested by ELISA and Western blot with serum from patients with allergy to olive. RESULTS Significant sequence polymorphism was found in both allergens. The panallergen profilin was expressed as a nonfusion protein and was purified to homogeneity after a single step of affinity chromatography with a poly-L-proline Sepharose column. One cDNA encoding an Ole e 1 isoform was expressed as a fusion protein consisting of the glutathione S-transferase of Schistosoma japonicum and Ole e 1. The fusion protein was purified to homogeneity by gel filtration chromatography and affinity chromatography with a glutathione-Sepharose column, and digested with thrombin. Both recombinant allergens shared B cell epitopes with the corresponding natural allergens. CONCLUSION IgE-reactive Ole e 1 and olive profilin expressed in bacteria were purified after simple chromatographic procedures and may be useful for diagnostic purposes.

Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus

The cDNA encoding an allergen from the dust mite Dermatophagoides pteronyssinus has been cloned and sequenced. The allergen (Der p 10) is a tropomyosin that shared more than 65% identical residues with other invertebrate tropomyosins. The final recovery of recombinant Der p 10 from the culture media after a single purification step was as much as 26 mg/l. The recombinant allergen is reactive to shrimp antitropomyosin IgG antibodies and has a 5.6% frequency of IgE reactivity in sera from mite-allergic patients.

Is tropomyosin an allergen in Anisakis

. THE ®sh parasite Anisakis simplex induces IgE-mediated reactions. Detection of speci®c IgE and prick test with a crude parasite extract are the current methods for diagnosis of sensitive patients, but high levels of speci®c IgE in asymptomatic individuals are detected (1). A recent study in Spain revealed that only 8/22 subjects having speci®c IgE to Anisakis were diagnosed as Anisakis allergics (2). Tropomyosin (TPM) has been proposed as a panallergen of invertebrates because it appears to be allergenic in many invertebrate sources, as both a food and inhalant allergen (3±5). Ten patients with clear clinical manifestations after ingestion of Anisakiscontaminated sea ®sh (group A), 62 patients with suspected Anisakis allergy (group B), and 16 patients with inhalant allergies to household insects were selected to evaluate, by in vitro techniques, the presence of TPM in A. simplex extract and its prevalence. The presence of TPM in extracts from Anisakis and Ascaris and their cross-reactivity to cockroach TPM were detected by shrimp-TPM antiserum and sera pool from patients allergic to household insects. None of 10 sera from Anisakis-sensitive patients reacted, by immunoblotting, with natural and recombinant Anisakis TPM, obtained as previously described (6). In contrast, when the inclusion criterion of sera was only the presence of IgE detected by CAP, the prevalence increased to 13% (8/62). These results suggested that TPM is not an important allergen in Anisakis sensitization. In food allergy to TPM, high-dose exposure of the allergen under the harsh conditions of the gastrointestinal tract is necessary for sensitization, but, in the case of Anisakis, both the ingesta of parasites and the presence of TPM on the parasite cuticle are extremely low. In contrast, the implication of Onchocerca volvulus TPM in host protective responses to micro®lariae in onchocerciasis has been recently reported (7). Interestingly, at least one of our patients in group B reported a previous infection with O. volvulus, and all but one of the residues of the described Bcell epitope of Onchocerca TPM (7) are conserved in Anisakis TPM. Immunoblotting-inhibition using serum from an Anisakis TPM-positive patient showed inhibition of IgE-binding to Anisakis TPM when the serum was incubated with TPM from other invertebrates such as mite, cockroach, and shrimp (Fig. 1). The structural and immunochemical similarities of Anisakis TPM to TPM from other invertebrates, as demonstrated in this work, make the diagnosis of Anisakis allergy dif®cult. This

Purification and characterization of Pla a 1, a major allergen from Platanus acerifolia pollen

Background: Plane trees, as Platanus acerifolia, are an important source of airborne allergens in cities of the United States and Western Europe. Little is known about the relevant allergens of this pollen. The aim of this study was to identify relevant allergens from P. acerifolia pollen and purify and characterize a major allergen of 18 kDa.

Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen

Sunflower (Helianthus annuus) sensitization is not always related with occupational allergy. We have isolated the allergen profilin (Hel a 2) from this Compositae plant, cloned and sequenced five cDNAs encoding for full-length or partial Hel a 2. Natural sunflower profilin reacted with specific IgE in the 121 sera tested, at a frequency of 30.5%. Expression of the cDNA encoding Hel a 2 in Escherichia coli and a simple purification procedure by poly-L-proline chromatography allowed immunological characterization of the recombinant allergen. Binding of monoclonal antibodies against sunflower profilin revealed that some epitopes responsible for antigen-specific IgG production were not present in the recombinant allergen. High cross-reactivity has been found between recombinant Hel a 2 and profilins from other Compositae plants and also from botanically distant plants.

Fast: Towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen‐specific immunotherapy

Background Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT).

Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin)

BACKGROUND Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua. OBJECTIVES We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy. METHODS Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen. RESULTS Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin. CONCLUSION This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

Neutrophils as a Novel Source of Eosinophil Cationic Protein in IgE-Mediated Processes

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with 35S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcεRI and galectin-3 (FcεRI>galectin-3), but not against FcεRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.

Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli : purification and characterization of the allergen

Background Profilin, an actin‐binding protein, was previously described as a panallergen which is involved in about 20% of Ihe crossreactivity found among pollen and food allergic patients. This allergen is usually under‐represented in natural extracts used for allergy diagnosis.