Hypoallergenic Variants of the <i>Parietaria judaica</i> Major Allergen Par j 1: A Member of the Non-Specific Lipid Transfer Protein Plant Family

Abstract

Background: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. Methods: Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. Results: The disruption of Cys14–Cys29 and Cys30–Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4–Cys52 bridge (PjC mutant) and the latter Cys50–Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4–Cys52 and Cys50–Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. Conclusion: Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.

Novel Strategies for the Development of a Vaccine forParietariaAllergy

“…2) to be recognized by murine rPar j I-specific IgE (26). The IgE binding of this hypoallergenic derivative was significantly reduced, mimicking the results described by Bonura and co-workers with human IgE antibodies (25). On the other hand, the ability of the rPjC to bind murine serum Par j l-induced IgG was not affected by the changes in the three-dimensional structure of the allergen.…”

“…2) (25). These derivatives were expressed in E. coli as recombinant proteins and their immunological properties were studied using independent techniques.…”

“…On the other hand, the loss of the Cys50-Cys9I and Cys4-Cys52 bridges seems to have a minimal effect since a strong IgE binding activity and a positive SPT are still present. This strategy of mutagenesis did not affect the T-cell recognition since proliferation assays performed with PBMC from Pj allergic patients showed that all the derivatives retained T cell reactivity (25). The immunological properties of one of these mutants were further investigated in a murine model of IgE sensitization.…”

Hypoallergenic Variants of the Major Latex Allergen Hev b 6.01 Retaining Human T Lymphocyte Reactivity

“…With the disruption of additional disulphide bonds there was a further decrease in both IgE reactivity and the ability to activate basophils, with complete abrogation of basophil activation when three or more cysteines were substituted. Studies of hypoallergenic recombinant forms of other allergens have also demonstrated that mutation of multiple cysteine residues was required to completely abrogate IgE reactivity (30,31). It has been shown that the conformational IgE epitopes of Hev b 6.02 contain amino acids from both the N-and C-terminal regions of that domain (28).…”

“…Previous studies aimed at generation of hypoallergenic allergen mutants have tested biological activity by SPT (31,41). Due to the anaphylactic potential of natural latex extracts (42), SPT for latex allergy is not routinely performed in our clinic.…”