Quantification of Fel d 1 in house dust samples of cat allergic patients by using monoclonal antibody specific to a novel IgE-binding epitope.

BACKGROUND: Avoidance of allergen exposure is an effective measure for preventing naÏve and allergic individuals from sensitization (primary intervention) and disease aggravation (secondary intervention), respectively. Regular monitoring of the allergens in the environment is required for the effective intervention. Thus, there is a need for cost-effective test kits for environmental allergen quantifications. OBJECTIVE: To invent a test kit for quantification of cat major allergen, Fel d 1. METHODS: A mouse monoclonal antibody (MAb) specific to the newly identified IgE-binding conformational epitope of the cat major allergen (Fel d 1) and rabbit polyclonal IgG to recombinant Fel d 1 were used as allergen capture and detection reagents, respectively. Native Fel d 1 was used in constructing a standard curve. RESULTS AND CONCLUSION: Sixteen of 36 dust samples collected from houses of cat allergic subjects in Bangkok contained Fel d 1 above 0.29 µg/gram of dust which is considered as a novel threshold level for causing cat allergy sensitization or symptoms. Among them, 7 samples contained the allergen exceeding 2.35 µg/gram of dust which is the level that would aggravate asthma. Results of the allergen quantification using the locally made test kit showed strong correlation (r = 0.923) with the allergen quantification using commercialized reagents. The assay using MAb to Fel d 1 IgE-binding epitope of this study has potential application as an economic and practical tool for cat allergy intervention measure especially in localities where health resources are relatively limited.

Immunotherapeutic efficacy of liposome-encapsulated refined allergen vaccines against Dermatophagoides pteronyssinus allergy.

Allergen specific immunotherapy (AIT) can modulate the allergic response causing a long-term symptom subsidence/abolishment which leads to reduced drug use and prevention of new sensitization. AIT of house dust mite allergy (HDM) using the mite crude extract (CE) as the therapeutic agent is not only less effective than the AIT for many other allergens, but also frequently causes adverse effects during the treatment course. In this study, mouse model of Dermatophagoides pteronyssinus (Dp) allergy was invented for testing therapeutic efficacies of intranasally administered liposome (L) encapsulated vaccines made of single Dp major allergens (L-Der p 1, L-Der p 2), combined allergens (L-Der p 1 and Der p 2), and crude Dp extract (L-CE). The allergen sparing intranasal route was chosen as it is known that the effective cells induced at the nasal-associated lymphoid tissue can exert their activities at the lower respiratory tissue due to the common mucosal traffic. Liposome was chosen as the vaccine delivery vehicle and adjuvant as the micelles could reduce toxicity of the entrapped cargo. The Dp-CE allergic mice received eight doses of individual vaccines/placebo on alternate days. All vaccine formulations caused reduction of the Th2 response of the Dp allergic mice. However, only the vaccines made of single refined allergens induced expressions of immunosuppressive cytokines (TGF-ß, IL-35 and/or IL-10) which are the imperative signatures of successful AIT. The data emphasize the superior therapeutic efficacy of single refined major allergen vaccines than the crude allergenic extract vaccine.

Mouse Model of Cat Allergic Rhinitis and Intranasal Liposome-Adjuvanted Refined Fel d 1 Vaccine.

Cats (Felis domesticus) are rich source of airborne allergens that prevailed in the environment and sensitized a number of people to allergy. In this study, a mouse model of allergic rhinitis caused by the cat allergens was developed for the first time and the model was used for testing therapeutic efficacy of a novel intranasal liposome-entrapped vaccines made of native Fel d 1 (major cat allergen) in comparison with the vaccine made of crude cat hair extract (cCE). BALB/c mice were sensitized with cCE mixed with alum intraperitoneally and intranasally. The allergic mice were treated with eight doses of either liposome (L)-entrapped native Fel d 1 (L-nFD1), L-cCE), or placebo on every alternate day. Vaccine efficacy evaluation was performed one day after provoking the treated mice with aerosolic cCE. All allergenized mice developed histological features of allergic rhinitis with rises of serum specific-IgE and Th2 cytokine gene expression. Serum IgE and intranasal mucus production of allergic mice reduced significantly after vaccination in comparison with the placebo mice. The vaccines also caused a shift of the Th2 response (reduction of Th2 cytokine expressions) towards the non-pathogenic responses: Th1 (down-regulation of the Th1 suppressive cytokine gene, IL-35) and Treg (up-regulation of IL-10 and TGF-ß). In conclusions, a mouse model of allergic rhinitis to cat allergens was successfully developed. The intranasal, liposome-adjuvanted vaccines, especially the refined single allergen formulation, assuaged the allergic manifestations in the modeled mice. The prototype vaccine is worthwhile testing further for clinical use in the pet allergic patients.

A novel IgE-binding epitope of cat major allergen, Fel d 1.

Information on the antigenic repertoire, especially the IgE-binding epitopes of an allergen is important for understanding the allergen induced immune response and cross-reactivity, as well as for generating the hypoallergenic variants for specific component resolved immunotherapy/diagnosis (CRIT and CRD). Data on the IgE-binding epitopes of cat allergens are scarce. In this study, a novel IgE-binding epitope of the cat major allergen, Fel d 1, was identified. Mouse monoclonal antibody (MAb) specific to the Fel d 1 was produced. Computerized intermolecular docking was used for determining the residues of the Fel d 1 bound by the specific MAb. The presumptive surface exposed residues of the Fel d 1 intrigued by the MAb are located on the chain 1. They are: L34 and T37 (helix 1); T39 (between helices 1 and 2); P40, E42 and E45 (helix 2); R61, K64, N65 and D68 (helix 3); and E73 and K76 (helix 4). The MAb competed efficiently with the cat allergic patients' serum IgE for Fel d 1 binding in the competitive IgE binding assay, indicating allergenicity of the MAb epitope. The newly identified allergenic epitope of the Fel d 1 is useful in a design of the CRIT and CRD for cat allergy.