Lisa D. Vailes

Cockroach allergens and asthma in Brazil: Identification of tropomyosin as a major allergen with potential cross- reactivity with mite and shrimp allergens

BACKGROUND Cockroaches produce several proteins that induce IgE antibody responses. Although cockroaches are abundant in warm and humid areas, sensitization to cockroach allergens has not been investigated in Brazil. OBJECTIVE The aims of this study were to investigate the frequency of cockroach allergy among patients with asthma, rhinitis, or both in Brazil and to identify American cockroach allergens. METHODS Skin tests using cockroach extracts were performed on children and young adults with asthma, rhinitis, or both. A Periplaneta americana complementary (c)DNA library was screened by using IgE antibodies from Brazilian patients allergic to cockroaches. Reactivity of an mAb directed to Dermatophagoides pteronyssinus tropomyosin against cockroach tissue was examined by immunofluorescence. RESULTS Cockroach allergy was present in 55% and 79% of the patients, as determined by using skin prick tests alone or combined prick and intradermal tests, respectively. Five cDNA clones reacted with IgE antibody and contained the same sequence. A representative clone (1300 bp), pa 12, coded for a protein that reacted with 50% of the sera from patients allergic to cockroaches on plaque immunoassay and showed a high degree of homology to tropomyosins, particularly those from invertebrates. P americana tropomyosin showed 80%, 81%, and 82% sequence identity to tropomyosins from D pteronyssinus, D farinae, and shrimp, respectively, which have been previously defined as important allergens. An mAb directed against D pteronyssinus tropomyosin, which also recognizes shrimp tropomyosin, showed binding to cockroach striated muscle. CONCLUSION Our results support the recommendation that cockroach extracts should be routinely used for the evaluation of patients with asthma, rhinitis, or both in Brazil. The identification of P americana tropomyosin as an important allergen will make it possible to investigate cross-reactivity among cockroaches, mites, and food derived from invertebrates.

Novel Allergen Structures with Tandem Amino Acid Repeats Derived from German and American Cockroach

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of ∼100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.

Monoclonal antibodies to group II Dermatophagoides spp. allergens: Murine immune response, epitope analysis, and development of a two-site ELISA

BACKGROUND Group II allergens are a major cause of sensitization in patients allergic to mites. To facilitate the antigenic analysis of group II allergens and to develop improved methods of allergen detection, we compared IgG anti-group II antibody responses in inbred mouse strains and raised a panel of monoclonal antibodies (mAbs). METHODS IgE antibody responses were compared by antigen-binding radioimmunoassay. Epitope specificity of the mAbs was analyzed by two-site binding assays and by cross-inhibition radioimmunoassays. RESULTS Comparison of polyclonal IgG antibody responses in five BALB congenic strains showed that H-2d mice had poor responses, whereas H-2b and H-2k mice had strong, cross-reactive, IgG anti-group II responses. The specificities of nine anti-Der p II IgE mAbs raised in A/J mice were compared with specificities of seven mAbs produced previously. Most mAbs (11 of 16) recognized common epitopes on Der p II and Der f II: three were specific to Der p II, and two showed high binding to Der f II. Epitope analysis showed that the mAbs defined four cross-reactive, nonoverlapping sites on the group II allergens. Binding of several combinations of mAbs was compared, and a two-site ELISA for group II antigens was developed. Linear regression analysis showed an excellent correlation between results of this assay and group II radioimmunoassay of house dust samples (n = 40, r = 0.85, p < 0.001). CONCLUSIONS There are multiple cross-reactive B-cell epitopes on group II allergens. The group II ELISA has several important applications, including assessment of environmental allergen exposure, monitoring of the efficacy of avoidance procedures, and standardization of commercial mite allergen extracts.

Fine specificity of B-cell epitopes on Felis domesticus allergen I (Fel d I): Effect of reduction and alkylation or deglycosylation on Fel d I structure and antibody binding

The repertoire of B-cell epitopes on the major cat allergen, Fel d I, was analyzed with monoclonal antibodies (MoAbs) in topographic mapping studies and in immunoassays with antigen derived from other cat (Felidae) species. Four essentially nonoverlapping epitopes on Fel d I, designated Fd1A to D, were defined by use of 15 anti Fel d I MoAbs in cross-inhibition radioimmunoassay. Only MoAbs directed against epitope Fd1B bound to putative Fel d I homologues in hair and dander extracts from seven other feline species (Panthera species, [n = 5], Leptailurus serval, and Leopardus pardalus). Quantitative monosaccharide analysis showed that Fel d I was a glycoprotein, containing high levels of fucose, as well as glucosamine, galactose, and mannose. Binding of MoAbs and human IgG or IgE antibody to native, reduced and alkylated or deglycosylated Fel d I was compared by means of immunoprecipitation and immunoassay, and the effects of these treatments on the structure of Fel d I were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. On reduction and alkylation, Fel d I dissociated into 14 kd and 3.2 kd peptides, and deglycosylation with trifluoromethane sulfonic acid produced a 12 to 14 kd peptide. These procedures resulted in a 100- to 1000-fold loss in murine or human antibody binding activity and caused significant loss of secondary structure, as judged by circular dichroism spectroscopy. Treatment with potassium hydroxide also caused a marked loss in antigenic reactivity. In contrast, enzymatic deglycosylation generated a 9 kd peptide, which showed strong reactivity with murine and human antibodies, comparable to native Fel d I. The results show that MoAbs define a broad repertoire of B-cell epitopes on Fel d I, one of which is expressed by other cat species. These epitopes are conformational and do not appear to involve oligosaccharide residues.

Molecular cloning, expression and modelling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor

Background Cats are an important source of indoor allergens. However, only two cat allergens, Fel d 1 and albumin, have been cloned and sequenced. IgE antibodies to Fel d 1 and albumin do not fully account for IgE responses to cat and there is good immunochemical evidence that cats produce other allergens.

Molecular determinants for antibody binding on group 1 house dust mite allergens.

Background: A unique, cross-reacting monoclonal antibody binds both Der f 1 and Der p 1. Results: A common epitope present on both Der f 1 and Der p 1 was identified and mutated. Conclusion: Mutagenesis and antibody binding analysis allowed identification of IgE antibody binding sites. Significance: The obtained data will lead to the production of hypoallergens with low IgE antibody binding capacity. House dust mites produce potent allergens, Der p 1 and Der f 1, that cause allergic sensitization and asthma. Der p 1 and Der f 1 are cysteine proteases that elicit IgE responses in 80% of mite-allergic subjects and have proinflammatory properties. Their antigenic structure is unknown. Here, we present crystal structures of natural Der p 1 and Der f 1 in complex with a monoclonal antibody, 4C1, which binds to a unique cross-reactive epitope on both allergens associated with IgE recognition. The 4C1 epitope is formed by almost identical amino acid sequences and contact residues. Mutations of the contact residues abrogate mAb 4C1 binding and reduce IgE antibody binding. These surface-exposed residues are molecular targets that can be exploited for development of recombinant allergen vaccines.

High-level expression of cockroach allergen, Bla g 4, in Pichia pastoris

Exposure to cockroach allergens is a risk factor for allergic disease and has been linked to an increase in asthma morbidity among cockroach-sensitive inner-city children. Bla g 4 is a ligand-binding protein (or calycin) that causes IgE antibody responses in 40% to 60% of patients allergic to cockroaches. Recombinant Bla g 4 was expressed in Escherichia coli as an 18 kd protein but provided poor yields (only 0.25 mg/L culture). To improve yields, Bla g 4 was expressed in the Pichia pastoris yeast system as a 23 kd secreted protein at concentrations of 50 mg allergen/L. By cross-inhibition radioimmunoassay, Bla g 4 expressed in E. coli or P. pastoris provided overlapping inhibition curves. Both allergen preparations bound comparable levels of serum IgE antibody and showed similar skin test reactivity in individuals allergic to cockroaches (10[-1] to 10[-3] microg/ml). Deglycosylation of Pichia-expressed Bla g 4 with endoglycosidase F resulted in an 18 to 20 kd doublet, and liquid chromatography-mass spectrometry results suggested that the 20 kd band contained residual sugar residues. Both glycosylated and deglycosylated Pichia Bla g 4 showed comparable inhibition of IgE antibody binding in radioimmunoassay. Pichia-produced Bla g 4 had the same antigenic reactivity as that produced in E. coli, and glycosylation had no effect on IgE antibody binding. The high yield of Bla g 4 obtained in the Pichia system will facilitate studies on the structure and function of calycin allergens and on the immune response of asthma patients to cockroach allergens.

Molecular Cloning of German Cockroach (Blattella germanica) Allergens

Allergens produced by cockroaches (CRs) are an important cause of IgE antibody responses and asthma. Using molecular cloning and nucleic acid hybridization techniques, we have identified and sequenced several important allergens produced by the German CR (Blattella germanica) and studied their expression in the American CR (Periplaneta americana). Principal allergens include Bla g 2 (36-kD protein) and Bla g 4 (21-kD protein), to which 60-70% of CR-allergic patients make IgE antibodies. Bla g 2 is only expressed by B. germanica, whereas DNA encoding Bla g 4 is present in P. americana, but is not transcribed into mRNA. Sequence homology searches have identified Bla g 2 as an aspartic protease and Bla g 4 as a calycin. Other CR allergens that have been cloned include a glutathione transferase and a troponin. These studies will enable recombinant allergens to be expressed and used to investigate the role of CR allergens in asthma.

Identification of Blomia tropicalis Allergen Blo t 5 by cDNA Cloning

Identification of Blomia tropicalis Allergen Blo t 5 by cDNA Cloning L.K. Karla Arruda E. Enrique Fernandez-Caldas C.K. Charles K. Naspitz F. Federico Montealegre L.D. Lisa D. Vailes M.D. Martin D. Chapman Asthma and Allergic Diseases Center, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Va., University of South Florida College of Medicine, Tampa, Fla., USA; Paulista School of Medicine, Sao Paulo, Brazil; and Ponce School of Medicine, Ponce, Puerto Rico

Home allergen monitoring and control--improving clinical practice and patient benefits.

Immunoassays for indoor allergens were first developed in the late 1980s, and the last decade has seen an explosion of research and clinical studies on the role of household allergen exposure in causing allergic disease, especially asthma. Monoclonal antibody-based enzyme immunoassays (ELISA) have become a cornerstone of laboratory assessment of exposure to mite, cat, dog, and cockroach allergens (1, 2). The panel of assays available for allergen measurements has grown steadily and includes assays for Blomia tropicalis, Lepidoglyphus destructor, and bovine and equine allergens (3–19) (Table 1). Over 300 publications using allergen immunoassays are now on MEDLINE databases, covering a wide range of scientific studies. These include the following: