L.A. Hazell

Fel d 4, a cat lipocalin allergen

Background Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin‐like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals.

Genetic variation of Der p 2 allergens: effects on T cell responses and immunoglobulin E binding

Background Der p 2 is a highly polymorphic allergen that shows a distinct pattern of sequence divergence. The effect of the variations on T cell and antibody responses has not been compared.

The chitinase allergens Der p 15 and Der p 18 from Dermatophagoides pteronyssinus

Background House dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae cause allergic disease in humans as well as in dogs. In geographical regions where the two mite species coexist, they both elicit specific immunoglobulin (Ig E) responses in humans whereas dogs preferentially react to D. farinae extracts. In dogs the main IgE binding is directed to the D. farinae chitinase allergens Der f 15 and Der f 18 and not to the groups 1 and 2 allergens as found for humans. Although the IgE response of humans to Der f 18 has been investigated there is no report on Der f 15‐specific IgE in humans.

Molecular analysis of the group 1 and 2 allergens from the house dust mite, Euroglyphus maynei

Background: There is increasing evidence that the house dust mite Euroglyphus maynei may be a significant source of allergic sensitization. The structural information for the E. maynei allergens is largely restricted to a single partial genomic sequence of Eur m 1. Methods: A cDNA library was constructed from a culture of E. maynei. Clones encoding the major group 1 and 2 allergens were isolated by DNA hybridization and sequenced. Results: The sequence of several full length clones of Eur m 1 and Eur m 2 were obtained. The full pre–proenzyme sequence of the cysteine protease Eur m 1 was determined. The translated amino acid sequence of Eur m 1 and Eur m 2 had 84–86% sequence identity with the corresponding allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae mites. This is the same as the degree of sequence identity found between D. pteronyssinus and D. farinae despite Euroglyphus being a member of the Pyroglyphinae subfamily rather than the Dermatophagoidinae subfamily. Conclusion: The sequences of the major Eur m 1 and Eur m 2 allergens are described. Their degree of divergence from the Dermatophagoides spp. is similar to that observed between D. pteronyssinus and D. farinae group 1 and group 2 allergens.

Isoforms of the Major Peanut Allergen Ara h 2: IgE Binding in Children with Peanut Allergy

Background: The major peanut allergen Ara h 2 consists of two isoforms, namely Ara h 2.0101 and Ara h 2.0201. The recently identified Ara h 2.0201 isoform contains an extra 12 amino acids including an extra copy of the reported immunodominant epitope DPYSPS. This study aimed to evaluate the IgE binding of the two Ara h 2 isoforms. Methods: Ten clones of Ara h 2 were sequenced to assess the relative frequency of the Ara h 2 isoforms and to identify whether there was further variation in the Ara h 2 sequence. IgE binding to Ara h 2.0101 and Ara h 2.0201 was measured for 70 peanut-allergic children using an IgE DELFIATM assay to quantitate specific IgE binding. A competition assay was used to measure whether Ara h 2.0201 contained IgE epitopes other than those found for Ara h 2.0101. Results: The original Ara h 2.0101 sequence was found for 6/10 clones and Ara h 2.0201 was found for 2/10 clones. Ara h 2.0201 had the expected insertion of 12 amino acids as well as substitutions at positions 40 (40G) and 142 (142E). Two new isoforms were identified as different polymorphisms of position 142. One Ara h 2.01 clone (Ara h 2.0102) contained 142E and one Ara h 2.02 clone (Ara h 2.0202) contained 142D. A polymorphism that was previously identified by other investigators at position 77 (77Q or 77R) was not found for any of the 10 sequences. Although the level of IgE binding to Ara h 2.0201 of individual patients was frequently higher than the binding to Ara h 2.0101 (p < 0.01), there was a strong correlation in binding to both isoforms (r = 0.987, p < 0.0001) and when analyzed as a group the means were similar. Ara h 2.0101 was not as efficient at blocking reactivity to Ara h 2.0201 indicating there is an additional IgE specificity for the Ara h 2.0201 isoform. Conclusions: Ara h 2.0201 has similar but higher IgE binding than the originally sequenced Ara h 2.0101 isoform and contains other IgE specificities.

Distinctive immunoglobulin E anti-house dust allergen-binding specificities in a tropical Australian Aboriginal community

Background There is evidence that the specificity of the IgE binding in allergy tests can vary for different populations.

Two Newly Identified Cat Allergens: The von Ebner Gland Protein Fel d 7 and the Latherin-Like Protein Fel d 8

Introduction: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. Materials and Methods: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. Results: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. Discussion: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

Non-allergenic antigen in allergic sensitization: responses to the mite ferritin heavy chain antigen by allergic and non-allergic subjects.

Background The majority of house dust mite proteins are non‐allergenic. There is, however, no information on the type of immune responses produced to these proteins and if the responses are affected by allergic sensitization.

Quantitation of IgE binding to the chitinase and chitinase-like house dust mite allergens Der p 15 and Der p 18 compared to the major and mid-range allergens.

Background: The prevalence of IgE binding to the group 15 and 18 house dust mite (HDM) allergens of the Dermatophagoides species is reported to be >50% and they are the major allergens of HDM-sensitised dogs. The objective was to quantitate the IgE titres to Der p 15 and Der p 18 and evaluate their importance in human HDM sensitisation compared to the known major and mid-tier allergens. Methods: Der p 15 and Der p 18 were produced in Pichia pastoris, and their structure validated by circular dichroism. IgE binding was measured in 37 Australian HDM-allergic adults using a quantitative DELFIA™ assay. Results: The prevalence of IgE titres to Der p 15 and Der p 18 >0.1 ng/ml was low (38%) and only one subject had a titre >10 ng/ml to either allergen. The mean anti-Der p 15 and Der p 18 titres were 1.2 and 2.6 ng/ml, respectively, i.e. approximately 10- to 20-fold lower than the response to the major Der p 1 and Der p 2 allergens (p < 0.001). The IgE responses to Der p 15 and Der p 18 were lower than the mid-tier allergens Der p 5 and Der p 7 and although they correlated with each other, they did not correlate with titres to either the major or mid-tier allergens. Conclusions: Sensitisation to Der p 15 and Der p 18 makes a minor contribution to anti-HDM IgE titres, and the titres do not correlate with the size of the response to the major allergens.

IgE and IgG Binding Patterns and T-cell Recognition of Fel d 1 and Non–Fel d 1 Cat Allergens

BACKGROUND Cat allergy affects approximately 15% of the population and is a major risk factor for asthma. The relative importance of cat allergens other than Fel d 1 is not known. OBJECTIVE To compare IgE and IgG antibody binding and T-cell recognition of the major cat allergen Fel d 1 with other cat proteins with known IgE binding properties. METHODS IgE, IgG1, and IgG4 antibody to Fel d 1, 2, 3, 4, 7, 8, and the undesignated IgE binding proteins haptoglobin and S100A12 were measured in the plasma of 96 individuals with cat allergy and 78 individuals without cat allergy. Cytokines were measured from T cells stimulated with the cat allergens. RESULTS An allergen other than Fel d 1 had the highest IgE binding specificity for 35% of individuals with cat allergy, and it bound more than 50% of their IgE antibody in 70% of these sera. Fel d 4, 7, and 8 were identified as the main contributors to the non-Fel d 1 IgE binding response and elicited inflammatory Th2 cytokines to a similar degree as Fel d 1. As expected, the average percentage of IgE binding to Fel d 1 for individuals was 55%. IgG4 binding to Fel d 1 was detected in both subjects with allergy (30%) and subjects without allergy (19%). IgG4 binding to the other allergens was less prevalent but was found for both groups. IgG1 antibody was not detected to any of the newly described cat proteins. CONCLUSION Fel d 4, 7, and 8 are allergens that should be included in the diagnosis and investigation of cat allergy.