Alisa M. Smith

REDUCTION IN IgE BINDING TO ALLERGEN VARIANTS GENERATED BY SITE-DIRECTED MUTAGENESIS: CONTRIBUTION OF DISULFIDE BONDS TO THE ANTIGENIC STRUCTURE OF THE MAJOR HOUSE DUST MITE ALLERGEN Der p 2

Site-directed mutagenesis was used to investigate the contribution of disulfide bonds to the antigenic structure of Der p 2. Single amino acid variants were generated at cysteine residues, preventing the formation of disulfide bonds at positions 21-27, 73-78, and 8-119. The variants were tested for binding to murine monoclonal antibodies (mAb) and human IgE antibodies (Ab) in an inhibition enzyme immunoassay. Removal of the disulfide linking the amino-carboxy termini (C8-C119) had no effect on mAb binding, however, IgE Ab binding was reduced by up to 10-fold. The other two disulfides form small loops and disruption of these bonds gave different binding patterns. The variant lacking the C21-C27 bond showed up to a 40-fold reduction in antibody binding, while the variant lacking the C73-C78 bond showed more than a 100-fold reduction in IgE Ab binding and failed to bind 3 of 4 mAb. Intradermal skin testing with the C73-C78 variant supported the in vitro findings; the variant was 10 to 100-fold less reactive than rDer p 2. These two bonds thus make markedly different contributions to stabilizing the antigenic determinants of Der p 2. The results suggest that the C73-C78 bond plays a critical role in stabilizing the antigenic structure of this major mite allergen.

Expression and Secondary Structure Determination by NMR Methods of the Major House Dust Mite Allergen Der p 2

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates. From these data, it is clear that Der p 2 is composed of β-sheets and random coil. Based on long range distance constraints, a number of β-strands were aligned into two three-stranded, anti-parallel β-sheets.

Hydrogen Exchange Nuclear Magnetic Resonance Spectroscopy Mapping of Antibody Epitopes on the House Dust Mite Allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10–100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2–3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.

Recombinant allergens for immunotherapy: a Der p 2 variant with reduced IgE reactivity retains T-cell epitopes.

N.Y.) diluted 1:5000 in PBS-T, goat anti-rabbit IgG, alkaline phosphatase conjugate (Sigma Chemicals, St. Louis, Mo.) diluted 1:10,000 in PBS-T, and enzyme substrate p-nitrophenyl phosphate (Sigma 104 phosphate) 1 mg/ml in 10% diethanolamine buffer (pH 9.8). The optical density of each well was read at 405 nm after 30 minutes at 37° C in a Biotek Model EL-312 automatic ELISA reader (Biotek, Inc., Winooski, Vt.).

Recombinant mite allergens : New technologies for the management of patients with asthma

The introduction of molecular cloning techniques has led to advances in allergen identification and sequencing, production of recombinant allergens, identification of B‐cell and T‐cell epitopes, and tertiary structural analysis of allergen molecules. Over 10 groups of mite allergens have been cloned from Dermatophagoides spp., as well as several homologous allergens from Euroglyphus maynei, Lepidoglyphus destructor, and Blomia tropicalis. The availability of these allergens has made it feasible to consider their use for both diagnostic and therapeutic purposes. Several recombinant Dermatophagoides allergens show comparable reactivity on skin testing and in serologic assays to natural allergens, and cocktails of the recombinant proteins could be used as diagnostic reagents. New technologies have been developed for detection of allergen‐specific IgE and for environmental allergen detection using rapid diagnostic tests. Novel approaches to immunotherapy are also being investigated, including T‐cell‐peptide‐based vaccines, allergen variants which lack IgE reactivity, and naked DNA vaccines. The application of allergen biotechnology should lead to improvements in the management of mite‐allergic patients with asthma and represents a logical step toward reducing asthma mortality and morbidity.

Sequence Polymorphisms and Antibody Binding to the Group 2 Dust Mite Allergens

Background: The group 2 allergens Der p 2, Der f 2 and Eur m 2 are 14-kD proteins with >80% sequence identity. Isoforms within each genus have been identified which differ by 3–4 amino acids. The aim of this study was to investigate the importance of these substitutions to antibody binding. Methods: Recombinant allergens were expressed and purified from Escherichia coli. ELISA and skin testing were used to evaluate antibody binding. Molecular modeling of the tertiary structure was preformed to examine the location of substitutions. Results: The three Der f 2 isoforms and two of three of the Der p 2 isoforms reacted with all monoclonal antibodies (mAb). Der p 2.0101, the isoform with aspartate at position 114, bound all mAb except 1D8. Substitution of asparagine for aspartate restored binding of rDer p 2.0101 to mAb 1D8 and increased the correlation coefficient for IgE binding from 0.72 to 0.77. The three Der p 2 isoforms showed comparable skin test reactivity to nDer p 2 and commercial extract. rEur m 2.0101 bound to all mAb except 7A1 and when compared with rDer p 2 for IgE binding, r2 = of 0.58 (n = 72). Lep d 2 did not react with mAb or with Dermatophagoides spp. allergic sera. Modeling revealed that Eur m 2, Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 and the substitutions are on the surface. Conclusions: mAb could distinguish isoform substitutions. IgE binding showed a good correlation among all isoforms, thus the recombinant allergens are useful for diagnosis.

Defined Epitopes: In vivo and in vitro Studies Using Recombinant Allergens

BACKGROUND The aim of the studies was to investigate the biologic activity of recombinant cockroach and mite allergens and their potential for use in diagnosis and treatment of allergic disease. METHODS Cockroach allergens Bla g 2, Bla g 4 and Bla g 5 and mite group 5 allergens were produced in bacterial expression vectors and used for immediate skin and serum IgE antibody tests. RESULTS The cockroach allergens showed very good skin test reactivity in allergic patients, giving positive reactions at 10(-2)-10(-5) microg/ml; controls were negative at 10(0) microg/ml. These reactions correlated with serum IgE antibody results. The prevalence of reactivity to group 5 mite allergens varied with exposure. There was a high prevalence (70%) of sensitization to Blomia tropicalis allergen, Blo t 5, among patients from Brazil and Singapore, whereas < 20% of patients from Charlottesville, US and Manchester, UK gave positive skin tests to Blo t 5 (p < 0.001). CONCLUSIONS The results show that recombinant allergens retain biologic activity and suggest that cocktails of two to four recombinant allergens could be used for diagnostic or therapeutic purposes. The phased introduction of recombinant allergens should improve the management of allergic disease.

Immunochemical and molecular methods for defining and measuring indoor allergens: in dust and air

Much of our understanding of the role of indoor allergens in asthma derived initially from immunochemical studies on the identification and analysis of allergen molecules. The isolation of mite, cat, dog and cockroach allergens allowed the biochemical properties of these allergens to be defined and their allergenic importance to be established, using the purified allergens for skin testing and serum IgE antibody assays. The production of monoclonal antibodies (mAb) provided reliable and consistent markers for specific allergens and enabled improved purification and allergen detection tests to be developed (1). Since the application of molecular cloning techniques in the late 1980s, over 20 indoor allergens alone have been sequenced, in addition to a similar number of grass, weed, tree pollen and venom allergens. As a result, allergens constitute one of the most clearly defined groups of molecules in biomedical research (2). Recent studies have shown that IgE mediated sensitization to indoor allergens, principally dust mite, cat, dog and cockroach, is an important cause of asthma and an associated cause of perennial rhinitis and atopic dermatitis. This presentation focuses on recent progress and strategies for defining indoor allergens and on the applications of immunochemical and molecular technologies to the management of allergic disease.

Reduction of IgE antibody binding to rDer p 2 variants generated by site-directed mutagenesis.

Our studies have focused on the 14 kD Group 2 allergens of D. pteronyssinus which are potent immunogens and elicit humoral and cellular responses in 80–90% of mite allergic individuals. Previous studies have shown that the B cell epitopes are heat and pH resistant, but are destroyed upon reduction and alkylation, suggesting that these determinants are dependent on the tertiary structure of the protein (1). This conclusion is supported by studies that showed a low prevalence of IgE Ab binding to polypeptide fragments produced from truncated Der p 2 cDNA (2,3).Synthetic peptides spanning the entire Der p 2 sequence have also been used to map Ab binding regions, however, only one peptide, amino acids 65–78, retained IgE Ab binding, confirming that the majority of epitopes are conformational (4). Taken together, these studies suggested that an alternative approach was required to further map the conformational determinants on Group 2 allergens.