Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1.Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1.Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product.Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability.The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.info:eu-repo/grantAgreement/EC/FP7/20187

Structure of the major carrot allergen Dau c 1

Dau c 1 is a major allergen of carrot ( Daucus carota) which displays IgE cross-reactivity with the homologous major birch-pollen allergen Bet v 1. The crystal structure of Dau c 1 has been determined to a resolution of 2.7 angstrom, revealing tight dimers. The structure of Dau c 1 is similar to those of the major allergens from celery, Api g 1, and birch pollen, Bet v 1. Electron density has been observed in the hydrophobic cavity of each monomer and has been modelled with polyethylene glycol oligomers of varying length. Comparison of the surface topology and physicochemical properties of Dau c 1 and Bet v 1 revealed that they may have some, but not all, epitopes in common. This is in agreement with the observation that the majority of carrot-allergic patients have Bet v 1 cross-reactive IgE antibodies, whereas others have Dau c 1-specific IgE antibodies which do not recognize Bet v 1

Crystallization and Initial X-Ray Analysis of Rabbit Mature Sterol Carrier Protein 2

Sterol carrier protein 2 (SCP2) is a basic intracellular protein which facilitates the in vitro intermembrane transfer of cholesterol, phospholipids and glycolipids. SCP2 was expressed in Escherichia coli, purified to apparent electrophoretic homogeneity and crystallized. Single crystals were obtained by hanging-drop vapour diffusion using ammonium sulfate as precipitant. These crystals belong to space group P41212 or its enantiomorph, with unit-cell parameters a = b = 57.5, c = 86.5 Å, and have one molecule in the crystallographic asymmetric unit. Intensity data to 1.8 Å resolution were collected from native SCP2 crystals using synchrotron radiation, were processed and scaled with an R(linear) = 4.9%

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy

BackgroundGrass pollen is one of the most important sources of respiratory allergies worldwide.ObjectiveThis study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach.MethodsFusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity.ResultsTen hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation.ConclusionA recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy

Purification and characterisation of relevant natural and recombinant apple allergens

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe and frequently causes allergic reactions with a variable degree of severity. So far, four apple allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified. Mal d 1, a Bet v 1 related allergen, and Mal d 4, apple profilin, are sensitive to proteolytic degradation, whereas Mal d 2, a thaumatin-like protein and Mal d 3, a nonspecific lipid transfer protein, are rather stable to proteolytic processes. Mal d 1 and Mal d 4 were purified after expression in Escherichia coli expression system, while Mal d 2 and Mal d 3 were purified from apple fruit tissue. All purified proteins were subjected to detailed physicochemical characterisation to confirm their structural integrity and maintained IgE binding capacity. Detailed investigations of carbohydrate moieties of Mal d 2 demonstrated their involvement in the overall IgE binding capacity of this allergen. It was concluded that the folded structure and IgE binding capacity of all four allergens were preserved during purificatio

Supplementary Material for: Development of a Hypoallergenic Recombinant Parvalbumin for First-in-Man Subcutaneous Immunotherapy of Fish Allergy

Background: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. Objectives: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. Methods:Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. Results: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. Conclusion: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.</p