High-level expression of immunoreactive recombinant cat allergen (Fel d 1): Targeting to antigen-presenting cells

BACKGROUND: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule. OBJECTIVE: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells. METHODS: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22(+)) or alone (rFel d 1 H22 (-)). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22 (+) to the CD64 receptor on leukocyte subpopulations and on the THP -1 cell line was analyzed by means of flow cytometry. RESULTS: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22(+)) and 22 kd (rFel d 1 H22 (-)). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = > 0.72,P <.001). The rFel d 1 H22(+) bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22(+) to THP-1 cells was inhibited by a soluble CD64 fusion protein. CONCLUSIONS: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunoreactivities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.     

Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy

BACKGROUND: Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.     

Higher immunoglobulin E antibody levels to recombinant Fel d 1 in cat-allergic children with asthma compared with rhinoconjunctivitis

Background Current diagnosis of allergy and asthma to cat is confirmed using cat dander extract (CDE). We have previously engineered a recombinant major cat allergen, rFel d 1, with properties identical to the natural molecule.
Objective The aim of the study was to evaluate IgE and IgG4 antibodies to rFel d 1 among sera from cat-allergic children and adults suffering from asthma and/or rhinoconjunctivitis (RC) in populations from Sweden and Austria.
Methods Cat-allergic children and adults from Sweden ( n =27 and 31, respectively) and Austria ( n =41 and 41) with RC and/or asthma were selected. Sera were tested for IgE and IgG4 antibodies to CDE and rFel d 1 by CAP, and IgE to rFel d 1 by ELISA. Healthy subjects and non-cat-allergic patients ( n =75) were included as controls.
Results There was a high correlation between IgE responses to rFel d 1 and CDE among the 140 patients ( r _s=0.85, P <0.001); however, measured levels to rFel d 1 were on average 30% higher ( P <0.0001). Ninety-eight percent of patients and none of the controls showed IgE to rFel d 1 and there was a threefold increased risk of asthma for half of the children with the highest IgE levels [odds ratio 3.23; 95% confidence interval (CI), 1.19–8.79] by ELISA. IgE responses to rFel d 1 among children with asthma were higher (median 19.4 kU/L) compared with children with RC (median 6.6 kU/L, P <0.05) and adults with asthma (median 3.0 kU/L, P <0.01). Furthermore, children with asthma displayed higher IgG4 levels than the asthmatic adults.
Conclusion A single recombinant molecule, rFel d 1, is at least as sensitive for in vitro diagnostics of cat allergy as the current extract-based test. Elevated IgE antibody levels to Fel d 1 are suggested to be a risk factor for asthma in cat-allergic children.     

Influence of cat characteristics on Fel d 1 levels in the home

BACKGROUND: Previous studies investigating cat characteristics and cat allergen production focused on clinical experiments that quantified allergen from either the shaved skin or the fur of the animal; however, these studies did not address these experimental relationships in the home. OBJECTIVE: To determine the relationships between cat characteristics and cat allergen isolated from household dust. METHODS: Fel d 1 allergen levels in dust from homes participating in a population-based study of environmental effect on allergy development were analyzed using a standard monoclonal antibody-based assay. Cat characteristics were based on interviews conducted during home visits by study personnel. RESULTS: Households with any cats had higher geometric mean Fel d 1 levels than households without cats (32.88 vs 0.43; P < .01), and cat allergen levels increased with increasing numbers of cats in the home (P < .01). Length of cat hair, cat sex, reproductive status, and time spent indoors were analyzed; the only characteristic associated with higher levels of Fel d 1 was whether the cat had been neutered or spayed. CONCLUSIONS: Having cats in the home is significantly associated with increased Fel d 1 levels, and having more cats in the home is correlated with more cat allergen. Cat reproductive characteristics may be associated with measurable differences in cat allergen levels.     

Serum IgG and IgG4 antibodies to Fel d 1 among children exposed to 20 microg Fel d 1 at home: relevance of a nonallergic modified Th2 response

Exposure to foreign antigens is an essential element of all immune responses, including allergic sensitization. For some allergens (e.g. mite and cockroach), the prevalence of sensitization is directly correlated with exposure. However, for allergens derived from domestic animals, several studies have suggested that children with a cat in the home have a decreased risk of sensitization and asthma. We have now shown that many children exposed to greater than 20 microg of Fel d 1/g of dust at home made an IgG and IgG4 antibody response to Fel d 1 without IgE antibody. This modified Th2 response is not associated with symptoms and should be regarded as a form of immunological tolerance. The fact that the dose-response relationship between cat exposure and sensitization is bell shaped, while that for mite exposure and sensitization is linear, is highly relevant to understanding the role of allergens in the increase in allergic disease.     

Rational design of hypoallergens applied to the major cat allergen Fel d 1

BACKGROUND: Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. OBJECTIVE: The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. METHODS: The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. RESULTS: Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. CONCLUSION: By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.     

Health impacts of second-hand exposure to cat allergen Fel d 1 in infants

BACKGROUND: Recent cross-sectional studies suggested that highest sensitization prevalences occur with moderate cat allergen exposures. We aimed to assess the impact of moderate levels of second-hand cat allergen exposure on the incidence of specific sensitization and wheezing in the framework of a birth cohort study. Therefore we restricted our analysis to infants without a cat at home since birth. METHODS: At infant's age 3 months, cat allergen levels were measured in the mattress dust of 1840 families without cats. At age 2 years, serum IgE specific to Fel d 1 was analyzed. Incidence of wheezing apart from respiratory infection was assessed by questionnaire. Logistic regression models were used to calculate adjusted odds ratios (OR) for the association between second-hand cat allergen exposure and health outcomes. RESULTS: Until age 2 years, 13 of 1301 infants (1%) were sensitized to cat allergen and 56 of 1492 infants (4%) had ever-wheezing without infection. Early exposure to second-hand cat allergen levels >or= 1 microg/g dust increased substantially the risk for specific sensitization to Fel d 1 (OR 10.9, 95% CI 3.4-35.0) and ever-wheeze without infection (OR 2.0, 95% CI 1.1-3.9) at age 2 years. CONCLUSIONS: Second-hand exposure to cat allergen in homes without cats is detrimental in terms of allergy development in infants.     

Induction of bystander tolerance and immune deviation after Fel d 1 peptide immunotherapy

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The 18-kDa form of cat allergen Felis domesticus 1 (Fel d 1) is associated with gelatin- and fibronectin-degrading activity

BACKGROUND: Fel d 1, an important allergen from domestic cats, is a significant cause of asthma. In addition to directly promoting IgE synthesis, other biological activities of allergens may contribute to either allergic sensitization or the magnitude of allergic effector responses. For example, allergens that degrade proteins have been suggested to facilitate allergen presentation by increasing parallelular permeability of airways epithelium. However, little information exists to indicate whether Fel d 1 has other activities relevant to allergic responses. OBJECTIVE: To study whether Fel d 1 is associated with enzyme activity. METHODS: Fel d 1 was obtained by a rigorous purification strategy and its identity confirmed by laser desorption mass spectrometry, cleavage and sequencing. The ability of Fel d 1 to degrade gelatin, fibronectin and the artificial substrate N-benzoyl-FVR-p-nitroanilide was studied. The effect of Fel d 1 on the morphology of tight junctions in epithelial cell monolayers was also investigated. RESULTS: The 18-kDa form of Fel d 1 caused degradation of denatured collagens (gelatin) and cleaved a 20-kDa fragment from the A chain of plasma fibronectin. Catalytic activity was not altered by inhibitors of cysteine peptidases, matrix metallopeptidases or by removal of divalent cations. In contrast, aprotinin and TLCK were inhibitors of Fel d 1. The absence of a serine peptidase catalytic triad in Fel d 1, together with the stoichiometry of the inhibition of TLCK and aprotinin, suggest that their inhibitory action may be due to noncatalytic site interactions. Alternatively, highly purified Fel d 1 may be associated with an active contaminant, although none were found. CONCLUSION: These results suggest that Fel d 1 is another example of a domestic allergen which is associated with enzyme activity. It remains to be established whether the activity resides in Fel d 1 itself or in an unresolved, and possibly related, protein.     

Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes

Background Characterization of T cell epitopes restricted by common HLA alleles is a powerful tool in the understanding of the immune responses to allergens and for the identification of potential peptides for future peptide immunotherapy (PIT). One important requirement is the identification and use of peptides that will bind to HLA molecules covering a large proportion of the population. Objective To identify commonly recognized CD4⁺ T cell epitopes in Fel d 1, restricted through frequently expressed HLA molecules for potential future use in PIT. Methods HLA matched antigen presenting cells, HLA blocking antibodies, and peptide truncations were used in ELISpot assays to establish HLA‐restricted T cell epitopes. Cytokine responses were measured by ex vivo and cultured IFN‐γ, IL‐4, and IL‐10 ELISpots. Results Responses to an immunodominant region of chain 2 were identified in the majority of atopic individuals and epitopes restricted by HLA‐DQB1^*06 and ‐DPB1^*0401 were characterized in detail. Significantly higher ex vivo IL‐4 and lower IFN‐γ responses were observed to both epitopes in individuals with atopic dermatitis (AD) compared with those without disease. IL‐10 responses were significantly lower in those with AD in the individuals with HLA‐DPB1^*0401. Conclusions We have identified an immunodominant region of Fel d 1 which is frequently recognized by CD4⁺ T cells from atopic individuals and contains epitopes that are restricted by very common HLA alleles.     

Fel d 1 and Can f 1 in settled dust and airborne Fel d 1 in allergen avoidance day-care centres for atopic children in relation to number of pet-owners, ventilation and general cleaning

BACKGROUND: Special day-care centres for atopic children have been established in Sweden. OBJECTIVE: To study concentrations of cat (Fel d 1) and dog (Can f 1) allergens in settled dust and airborne cat allergen in day-care centres in relation to pet ownership among children and staff, ventilation and general cleaning. METHODS: Twelve allergen avoidance day-care centres and 22 conventional day-care centres were included in the study. Settled dust was collected and analysed with ELISA. Airborne cat allergen levels were measured in eight allergen avoidance and seven conventional centres with a personal air sampler and analysed with an amplified ELISA. Air change rate per hour (ACH) was measured. A questionnaire which focused on keeping of cat and dog among staff and children and frequency of general cleaning was used. RESULTS: In the allergen avoidance day-care centres neither children nor staff reported ownership of cats or dogs, compared with 21/22 of the conventional centres in which children and staff kept furred animals. Fel d 1 and Can f 1 were found in settled dust in all day-care centres. In the allergen avoidance compared with the conventional centres the concentrations of Fel d 1 and Can f 1 were lower, Fel d 1: median 0. 64 microg/g vs 5.45 microg/g and Can f 1: 0.39 microg/g vs 2.51, both P < 0.001, and airborne Fel d 1 was also lower in the allergen avoidance centres compared with the control centres, 1.51 ng/m3 vs 15.8 ng/m3, P = 0.002. A correlation was found between airborne and settled Fel d 1, rs = 0.75, P < 0.001. Furthermore, a correlation was found between increased ACH and decreased levels of Fel d 1 in the air in the day-care centres with no cat-owners, rs = - 0.86, P = 0.007. No relation was found between levels of cat or dog allergen and amount of general cleaning. CONCLUSION: Not keeping pets seems to reduce children's exposure to pet-allergen in their 'working environment'. Additionally, appropriate ventilation seems to reduce Fel d 1 in the air in day-care centres.     

Effect of two different types of vacuum cleaners on airborne Fel d 1 levels.

BACKGROUND: Vacuum cleaners may increase the level of airborne allergens by leakage through the cleaners or by disturbance of floor dust by the exhaust air produced. OBJECTIVE: The aim of this study was to evaluate the short-term effect of vacuum cleaning with two different types of cleaners on airborne cat allergen analyzed by a biologic and by an immunochemical test. METHODS: Ten homes with cats were cleaned in random order with a 1-week interval by a traditional canister type vacuum cleaner (T) and a semi-stationary vacuum cleaner (S) that conducts the air to the exterior through a valve in the wall. Airborne particles were collected by air sampling for 2 hours and cat allergen, Fel d 1, was quantified biologically by basophil histamine release test (HR test) and immunochemically by enzyme linked immunosorbent assay (ELISA). RESULTS: Using the S resulted in smaller amounts of airborne cat allergen than the T (mean 2.1 ng/m3 air (range .8 to 12.5) versus 5.2 ng/m3 (1.3 to 13.3), P < .002 measured by ELISA). Results from ELISA and HR test correlated well (r = .91, P < .001). CONCLUSIONS: The use of S with exhaust to the outside of the dwelling gave rise to less airborne low particle size allergen during the cleaning procedure than a T method. The basophil histamine release test could be a valid alternative method to establish allergen content in environmental samples especially in allergen systems with no available monoclonal antibodies.     

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I.
Two monoclonal antibodies directed against reduced/alkylated chain I bound to the overlapping peptides 53–66 and 60–70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36–49 and 43–56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies.
Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies (27). It bound to peptides 1–16 and 60–70 of chain 1 and peptides 1–14 and 50–63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies.
A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60–70 of chain 1 and peptides 1–14 and 43–56 of chain 2. For two monoclonal antibodies, binding to each peptide was investigated and shown to be inhibitable by native Fel d I. These antibodies are therefore probably directed against a conformational epitope formed by these four regions.
These studies give us substantial information about the quaternary structure of Fel d I.     

猫主要过敏原Fel d 1的T细胞抗原表位分析及三维结构模拟

目的:预测猫主要过敏原Fel d 1与MHC Ⅰ、MHCⅡ类分子的结合力获得T细胞优势抗原表位,并模拟Fel d 1的三维结构,为猫主要过敏原的改造及其临床研究等提供依据.方法:从Uniprot数据库中得到猫主要过敏原Fel d 1的氨基酸序列,通过在线软件NetMHCⅡ2.2对Fel d 1氨基酸序列进行MHCⅡ抗原表位分析,使用软件SYFPEITHI分析MHC Ⅰ的抗原表位,再通过在线软件Swiss-Model预测Feld1的三维结构,以Ramachandran图评估三维结构的稳定性.结果:运用生物学软件分析得到Fel d 1上两个高值MHCⅡ抗原表位区域分别为22～43、80～95,MHC Ⅰ抗原表位优势区为17～30、56～84、91 ～103.Ramachandran图显示Feld 1的空间构象稳定.结论:本研究有助于确定Fel d 1的T细胞优势抗原表位及其三维结构模型,为Fel d 1抗原性改造提供理论依据,及将来的临床研究奠定基础.     

The relationship of housing and household characteristics to the indoor concentrations of Der f 1, Der p 1, and Fel d 1 measured in dust and air samples.

BACKGROUND: Indoor dust mite and cat allergens have been related to the risk of atopic conditions. If allergen levels are influenced by modifiable residential characteristics, potential interventions to prevent disease could be deployed. OBJECTIVE: To evaluate relationships between allergen concentrations in air and dust samples and selected house and household characteristics using a large prospective study with multiple sequential allergen measurements from each residence. METHODS: Fel d 1, Der f 1, and Der p 1 were measured in paired air and dust samples collected at intervals throughout 4 years in suburban homes. House and household characteristics were examined for relationships to allergen concentrations in both univariate and multiple variate analyses. RESULTS: The relationships between house and household characteristics and allergen concentrations in both air and dust were complex. When the housing variables were considered in multiple variate analysis, concentrations of Der f 1 in dust increased with increasing number of residents and relative humidity and declined when forced air heating was used. Dust concentrations of Der p 1 were lower in new homes and during forced air heating use but higher with higher relative humidity and in the presence of dogs. The presence of cats was the dominant determinant of Fel d 1 in both air and dust, but when homes without cats were analyzed separately, dust levels of Fel d 1 were inversely related with relative humidity. CONCLUSIONS: Air and dust concentrations of Der p 1 and Der f 1 were positively related to relative humidity and the size of the family. Fel d 1 was positively related to the presence of cats. The relationship of other house or household characteristics was inconsistent but different for Der f 1 and Der p 1.     

Efficacy of dry-cleaning in removing Fel d 1 allergen from wool fabric exposed to cats.

BACKGROUND: The main cat allergen (Fel d 1) is ubiquitous, having been found even in indoor environments and public places where a cat has never been kept. Clothes of cat owners constitute a carrier for the distribution of Fel d 1 allergen in these environments. Schools, for example, may be a site of indirect exposure to cat allergens. OBJECTIVE: Our goal was to investigate the efficacy of commercial dry-cleaning in removing cat allergens from wool fabrics that had been exposed to cats to evaluate a possible preventive procedure. METHODS: Twenty-six identical wool "squares" (80 x 100 cm) were put in cat baskets for 1 week. In our laboratory, the squares were cut in half (40 x 50 cm), and one half was subjected to high-volume sampling for 5 minutes in a cat-free room. The other half was subjected to commercial dry-cleaning and then the high-volume sampling. Five wool squares not exposed to cats served as controls. Dust was collected from the wool squares with a high-volume air sampler. Particulate material was harvested onto glass fiber filters (AP 20 Millipore, Milan, Italy) with 25-mm diameter and 2-microm pore size. Each dust sample was assayed by affinity-purified monoclonal antibody against purified Fel d 1. The results were expressed as micrograms per filter. Statistical analysis was done by using the paired t test. RESULTS: Before dry-cleaning, Fel d 1 allergen was detected on all cat-exposed wool squares. No appreciable cat allergen was detected on control materials. After commercial dry-cleaning, the amounts of Fel d 1 extracted from cat-exposed squares were significantly reduced (t = 14.63; P < 0.001) but not abolished. Three of the five control squares were contaminated by Fel d 1. CONCLUSIONS: Commercial dry-cleaning effectively removes large amounts of cat allergen from wool materials exposed to cats but does not completely abolish this protein. Further, low Fel d 1 contamination may occur during this procedure.