Hypoallergenic Mutant Research Articles

The peanut allergy epidemic: allergen molecular characterisation and prospects for specific therapy

Peanut (Arachis hypogaea) allergy is a major cause of food-induced anaphylaxis, with increasing prevalence worldwide. To date, there is no cure for peanut allergy, and, unlike many other food allergies, it usually persists through to adulthood. Prevention of exposure to peanuts is managed through strict avoidance, which can be compromised by the frequent use of peanuts and peanut products in food preparations. Conventional subcutaneous-injection allergen immunotherapy using crude peanut extract is not a recommended treatment because of the risk of severe side effects, largely as a result of specific IgE antibodies. Consequently, there is an urgent need to develop a suitable peanut allergen preparation that can induce specific clinical and immunological tolerance to peanuts in allergic individuals without adverse side effects. This requires detailed molecular and immunological characterisation of the allergenic components of peanut. This article reviews current knowledge on clinically relevant peanut allergens, in particular Ara h 1, Ara h 2 and Ara h 3, together with options for T-cell-reactive but non-IgE-binding allergen variants for specific immunotherapeutic strategies. These include T-cell-epitope peptide and hypoallergenic mutant vaccines. Alternative routes of administration such as sublingual are also considered, and appropriate adjuvants for delivering effective treatments at these sites examined.

Hypoallergenic mutants of Ole e 1, the major olive pollen allergen, as candidates for allergy vaccines

The C-terminal region of Ole e 1, a major allergen from olive pollen, is a dominant IgE-reactive site and offers a target for site-directed mutagenesis to produce variants with reduced IgE-binding capability. To evaluate in vitro and in vivo the immunogenic properties of three engineered derivatives of Ole e 1. One point (Y141A) and two deletion (135Delta10 and 140Delta5) mutants were generated by site-directed mutagenesis of Ole e 1-specific cDNA and produced in Pichia pastoris. Ole e 1 mutants were analysed for IgE reactivity by ELISA using sera from olive pollen-allergic patients. Their allergenicity was also investigated in both a mouse model of allergic sensitization and in basophil activation assays. IgG1 response was assayed by immunoblotting and competitive ELISA. T cell reactivity was evaluated by proliferation assays and cytokine production in splenocyte cultures. The 135Delta10 mutant showed the strongest reduction in the IgE-binding capability of sera from olive pollen-allergic patients. Rat basophil leukaemia assays identified the deletion mutant 135Delta10 as the variant with the lowest beta-hexosaminidase-releasing capacity. Furthermore, the same 135Delta10 mutant induced the lowest IgE levels in a BALB/c mouse model of sensitization. All Ole e 1 mutants retained their allergen-specific T cell reactivity. Immunization of mice with the mutants induced IgG1 antibodies, which cross-reacted with Ole e 1 and Ole e 1-like allergens from ash, lilac and privet pollens. The ability of the human IgE to block the binding of anti-Ole e 1 mutant-specific mouse IgG1 antibodies to natural Ole e 1 demonstrated that Ole e 1 mutants are able to induce in vivo antibodies reactive to the natural allergen. The 135Delta10 mutant with reduced allergenicity, intact T cell reactivity and capacity to induce blocking antibodies could provide a suitable candidate vaccine for efficient and safer therapy of olive pollen allergy.

Ablation of ovomucoid-induced allergic response by desensitization with recombinant ovomucoid third domain in a murine model.

Attempts to modulate the allergenic response by hypoallergens aimed at eliminating IgE-binding epitopes have been established recently for allergen immunotherapy. Desensitization offers an alternative approach to mounting a protective immune response. We have shown previously that mutation of the decisive amino acids in the B cell epitope of the ovomucoid third domain suppresses IgE binding reactivity against human patient sera and we hypothesize that this hypoallergenic variant could be a potential candidate molecule for specific immunotherapy against an ovomucoid-induced IgE reaction. The aim of this study was to investigate whether hyposensitization with the ovomucoid-modified isoform could desensitize ovomucoid-sensitized mice. We mapped the immunodominant B cell epitopes of ovomucoid in Balb/c mice. A hypoallergenic ovomucoid mutant isoform, having ablated allergenicity against patient sera, was used to desensitize ovomucoid-sensitized Balb/c mice by intraperitoneal injection. Female Balb/c mice were sensitized with intact ovomucoid molecule (Fovm) and desensitized with the modified isoform of the third domain of ovomucoid (GMFA). Intact ovomucoid-sensitized mice desensitized with phosphate-buffered saline (PBS) served as positive controls to maintain hypersensitivity. To gain insight into the efficacy of the modified ovomucoid variant in desensitization, effects on hypersensitivity reactions and histamine levels, followed by its association with antibody levels and cytokine profiles, were measured. Abrogation of the allergic response with complete suppression of anaphylactic symptoms and lower serum histamine levels was observed in the desensitized group by GMFA, accompanied by significantly reduced ovomucoid-specific IgE and IgG1 levels and enhanced specific IgG and IgG2a levels. The sensitized group showed severe anaphylactic symptoms, enhanced serum histamine concentrations and increased levels of specific IgE and IgG1. The level of interleukin (IL)-4 was decreased dramatically in the desensitized group and higher levels of interferon (IFN)-gamma were found, whereas mice sensitized with intact ovomucoid exhibited significantly higher levels of IL-4 favouring a Th2 skewed pathway. We demonstrate clearly that GMFA is able to ablate ovomucoid-induced allergic reactions in sensitized mice. This occurs via a suppression of specific IgE accompanied by an increase in suppressor T cell activity. This approach offers some promise for the development of treatment against ovomucoid-induced allergic response.

The recombinant major allergen of Parietaria judaica and its hypoallergenic variant: in vivo evaluation in a murine model of allergic sensitization.

Par j 1 represents the major allergenic component of Parietaria judaica pollen. Its three-dimensional structure is stabilized by four disulphide bridges. A family of three-dimensional mutants of the recombinant Par j 1 (rPar j 1) allergen, showing reduced allergenicity and retained T cell recognition has been recently developed by site-directed mutagenesis. To develop and characterize a murine model of IgE sensitization to rPar j 1. To evaluate similarities between the murine model and the human IgE response. To investigate in this model the recognition of a hypoallergenic mutant of Par j 1, and to study the immune responses elicited in mice by the mutant itself. BALB/c mice were sensitized by two intraperitoneal immunizations with rPar j 1 in alum on days 0 and 21. Allergen-specific serum IgE and IgG responses were studied by direct ELISA and immunoblotting, ELISA inhibition and competitive ELISA. Cell proliferation was evaluated in splenocyte cultures. Sensitization with rPar j 1 induced high levels of IgE and IgG1 vs. low levels of IgG2a. Mouse antibodies specific to rPar j 1 were able to compete with human IgE for recognition of rPar j 1. IgE from mice immunized with rPar j 1 showed a significantly reduced binding activity towards the hypoallergenic variant rPjC, which lacks three disulphide bridges. On the contrary, rPjC was recognized by IgG1 and IgG2a antibodies as well as rPar j 1. The proliferative response to rPjC by splenocytes from mice immunized with rPar j 1 was comparable to that stimulated by rPar j 1. Immunization with rPjC induced low levels of IgE antibodies to the rPjC itself, while IgG and proliferative responses were similar to those induced by rPar j 1. Conformational variants of allergens, displaying reduced allergenicity accompanied by retained IgG and T cell recognition, offer a safe, specific and flexible approach to immunotherapy of type I allergy. Our mouse model of IgE sensitization to a recombinant allergen, mimicking the human response to its native counterpart, could provide valuable information for pre-clinical testing of such hypoallergenic molecules.

Chemically modified recombinant Fel d 1: A good alternative to hypoallergenic mutants

Rationale Hypoallergenic mutants of recombinant major allergens are proposed to make immunotherapy safer and more efficient. Their development, however, is laborious and requires detailed structural information. The aim of this study was to develop chemically-modified recombinant Fel d 1 as an alternative to a hypoallergenic mutant. Methods Fel d 1 was cloned and expressed in Pichia pastoris as a hybrid molecule of both chains of the allergen. The resulting recombinant (r)Fel d 1 was compared to purified natural (n)Fel d 1 by CD and mass spectroscopy, RAST(-inhibition), competitive RIA, ELISA, immunoblot, basophil histamine release (BHR) and T-cell proliferation assays. Chemical modifocation was carried out using glutaraldehyde. Hypoallergenicity was assessed by RAST-inhibition (RI) and BHR. Results rFel d 1 was expressed at ∼500 mg/L and was shown to be glycosylated by the yeast. Physico-chemical and immunological comparison with nFel d 1 showed that the recombinant molecule is well-folded and that the immune-reactivity of both molecules is very similar. Deglycosylation did not influence immune-reactivity. Chemical modification resulted in at least a 200-fold decrease in IgE reactivity (RI) and a 1000-fold decrease in biological activity (BHR). Conclusions Chemically modification of recombinant major allergens is a good and easy alternative to the development of hypoallergenic mutants.

Mutational analysis of the cashew vicilin allergen, Ana o 2

Rationale The aim of our study was to make hypoallergenic variants of Ana o 2, a major cashew allergen for possible specific immunotherapy. Methods Epitope mapping done using the SPOTs technology. The critical amino acid residues in the epitopes identified by alanine scanning. Using the QuikChange sup>tm site-directed mutagenesis we introduced point mutations into the IgE epitopes of Ana o 2 and generated seven mutants. Reactivity of mutant proteins to anti-Ana o 2 monoclonal antibodies, rabbit anti-cashew antibodies, and cashew allergic patients sera studied by ELISA and Immunoblotting. Results Epitope mapping studies of Ana o 2 with synthetic peptides identified several epitopes reactive with cashew allergic human sera. Mutational analysis using alanine scanning revealed that single amino acid changes at critical residues in the epitopes could reduce IgE binding. Critical amino acids were mutated in the Ana o 2 gene and seven mutants were expressed and characterized. We observed reduced immuno-reactivity to anti-Ana o 2 monoclonal antibodies, rabbit polyclonal anti-cashew antibodies by Inhibition ELISA (3mutants showed 80% inhibition activity), and to human cashew allergic sera by immunoblot suggesting that these may be hypo-allergenic mutants of Ana o 2. Conclusion Engineering of these hypoallergenic mutants of Ana o 2 could serve as agents for cashew immunotherapy.

The Crystal Structure of the Major Cat Allergen Fel d 1, a Member of the Secretoglobin Family

The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-A resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy.

Engineering of hypoallergenic mutants of the Brassica pollen allergen, Bra r 1, for immunotherapy

The Brassica pollen allergen Bra r 1 belongs to a new family of Ca 2+-binding proteins, characterized by the presence of two potential EF-hand calcium-binding domains. Disruption of these EF-hand motifs by amino acid substitutions demonstrated that both domains of Bra r 1 constitute functional Ca 2+-binding sites. Calcium-binding deficient mutants displayed significantly reduced IgE-binding activity. Injection of these mutated Bra r 1 variants into a murine model system showed that mouse IgG raised against the mutants recognized native Bra r 1 in Brassica pollen extracts suggesting the potential use of the engineered allergens for effective immunotherapy.

Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for the selection of functional gene products linked to the genetic information responsible for their production [Gene 137 (1993) 69–75

Allergenic (glyco)proteins are the elicitors of food allergies and can cause acute severe hypersensitivity reactions. Recombinant food allergens are available in standardised quantity and constant quality. Therefore, they offer new perspectives to overcome current difficulties in the diagnosis, treatment and investigation of food allergies. This review summarises the expression strategies and characteristics of more than 40 recombinant food allergens that have been produced until today. Their IgE-binding properties are compared to those of their natural counterparts, in addition their application as diagnostic tools, the generation of hypoallergenic recombinant isoforms and mutants for therapeutic purposes, the determination of epitopes and cross-reactive structures are described.