Alexander C. Drew

Immunological analysis of allergenic cross‐reactivity between peanut and tree nuts

Background Peanut and tree nut allergy is characterized by a high frequency of life‐threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross‐reactive allergens remains unknown.

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

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

Functional analysis of cross‐reactive immunoglobulin E antibodies: peanut‐specific immunoglobulin E sensitizes basophils to tree nut allergens

Background Peanut and tree nuts are a major cause of food‐induced anaphylaxis with an appreciable mortality. Co‐sensitization to peanuts and tree nuts is a common clinical observation and may be because of peanut‐specific serum IgE antibodies that cross‐react with tree nut allergens. It is, however, unclear whether these cross‐reactive IgE antibodies are involved in effector‐cell activation.

Specific monoclonal antibodies and human immunoglobulin E show that Hev b 5 is an abundant allergen in high protein powdered latex gloves

Background  Hev b 5 is a major latex allergen recognized predominantly by latex‐allergic health care workers (HCWs). Recombinant Hev b 5 (rHev b 5) was previously expressed as a fusion protein with maltose binding protein (MBP), itself an immunogenic molecule; therefore non‐fusion rHev b 5 is desirable. Moreover, standardized immunological assays for the detection of Hev b 5 are currently lacking and may have important implications for both allergen avoidance and diagnosis in latex allergy.

The hevein domain of the major latex‐glove allergen Hev b 6.01 contains dominant T cell reactive sites

Background Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex‐glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C‐terminal fragment (14 kDa). Hevein is an abundant protein in latex‐glove extracts. As the immune response to allergens is initiated by activation of allergen‐specific CD4+ T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background: Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods: Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results: Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions: Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/β receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK–STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100–1000-fold reduced potency compared with rmIFNα1 and rmIFNβ. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.

Advances in development of hypoallergenic latex immunotherapy

Purpose of reviewThe characterization of clinically relevant latex allergens and the production of recombinant allergens is now well advanced, but this knowledge needs to be translated into new strategies for the safe and effective specific treatment of latex allergic diseases including asthma and anaphylaxis. Recent findingsThe current status of latex allergy is discussed indicating a changing demographic paradigm. A new wave of latex allergy is emerging outside the healthcare setting with the widespread use of latex products. An increased prevalence in developing countries is also reported. Limited studies on current specific immunotherapy for latex allergy are reviewed, confirming the feasibility but demonstrating an unacceptable risk of adverse events. The characterization of latex allergens and the identification of B and T-cell epitopes point to rational strategies for the generation of hypoallergenic preparations for specific immunotherapy. Results to date for latex allergens are reviewed, including recombinant, chemical modification and synthetic peptide approaches. Candidate hypoallergenic preparations for targeting sensitization to the major allergens Hev b 1, Hev b 3, Hev b 5 and Hev b 6.01 have been identified. Further investigations of optimal regimens for the delivery of specific immunotherapy to induce regulatory T-cell function are warranted. SummaryThe findings point to the selection of suitable hypoallergenic preparations for clinical trials of effective and safe latex allergy immunotherapy.