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. OBJECTIVE: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using pooled serum obtained from five asthmatic patients which contained high levels of IgE antibody to cat dander. Selected cDNA clones were screened by plaque immunoassay and one cDNA clone, encoding cystatin, was expressed in E. coli. The three dimensional structure of cat cystatin was modelled using the SWISS-MODEL computer program. RESULTS: Three positive cDNA clones (A, B and C) were identified, two of which were fully sequenced. Clones A and C encoded the same 98 amino acid residue sequence which showed 79% and 75% homology with bovine and human cystatin A, respectively. The cat cystatin sequence contained the conserved cysteine protease inhibitor signature and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE ab to the expressed cystatin clones. The cysteine protease inhibitor motif was also partially conserved in dog allergen sequences, Can f 1 and Can f 2, which are lipocalins. The recombinant protein was expressed in E. coli as an 11-kDa protein, corresponding to the predicted MW of cat cystatin. The three-dimensional structure of cat cystatin was modelled on human cystatin structures. CONCLUSION: A newly identified allergen, cystatin (Fel d 3), has been cloned from cat skin and is a member of the cysteine protease inhibitor family.     

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.     

Demonstration of a partially cryptic epitope of the major cat allergen Fel d 1: consequences for mAb-based standardization of cat extracts

BACKGROUND: Antiallergen mAbs that do not recognize clinically important isoforms have been described, raising the question of the selection of mAbs for quantifying major allergens in order to standardize allergenic extracts. This question is even more critical if mAbs can discriminate between different forms of allergen molecules with the same amino acid sequence. OBJECTIVE: We sought to demonstrate that an anti-Fel d 1 mAb was able to discriminate between two forms of the major cat allergen independently of its amino acid sequence and to determine the relative importance and stability of both forms in various cat extracts. METHODS: Anti-Fel d 1 mAbs were raised in mice and characterized. By using two of these mAbs, a two-site ELISA was developed to quantify Fel d 1 in mass units. RESULTS: One of the anti-Fel d 1 mAbs developed was shown to specifically recognize a particular form of Fel d 1. A two-site ELISA with this mAb to capture Fel d 1 was able to quantify the allergen specifically in this form. It was then shown that (1) the quantitative importance of this form of Fel d 1 could vary from one cat extract to another, (2) Fel d 1 was converted into this form under certain conditions, and (3) both converted and unconverted forms of Fel d 1 may bear IgE epitopes that are specific. CONCLUSION: Although the present study emphasizes the issue of selecting mAbs that are not too specific to standardize allergenic extracts, it also demonstrates that very specific mAbs can be of interest, especially to verify the stability of allergens in extracts, since this stability might have clinical implications.     

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.     

The cat lipocalin Fel d 7 and its cross‐reactivity with the dog lipocalin Can f 1

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensitized Swedish patients and elucidated its allergenic activity and cross‐reactivity with the dog lipocalin Can f 1. Sixty‐five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross‐reactive antisera. Fel d 7 is a common allergen in a Swedish cat‐sensitized population that cross‐reacts with Can f 1, and may contribute to symptoms in cat‐ but also in dog‐allergic patients.     

Identification of a novel cat allergen--cystatin

BACKGROUND: Cat allergen is an important cause of sensitization among children with asthma in Japan. Although there is good evidence that cats produce other allergens, only one major allergen, Fel d 1, has been studied in detail. AIMS: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using IgE antibodies to cat dander and selected clones were sequenced and expressed. RESULTS: One cDNA clone contained an open reading frame encoding a 98-amino acid residue protein. Sequence homology searches revealed a high degree of identity with bovine and human cystatin A, 79 and 75%, respectively. This cat cystatin clone contained the conserved cysteine protease motif and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE Ab to cat cystatin. This cysteine protease inhibitor motif was partially conserved in dog allergens, Can f 1 and Can f 2, which are lipocalins. Recombinant cystatin was produced in Escherichia coli cells and purified as an 11-kD protein, corresponding to the predicted MW of cystatin. The structure of cat cystatin was modeled on human cystatin B using the SWISS-MODEL. CONCLUSION: A newly identified allergen, cystatin, has been cloned from cat skin and is a member of the cysteine protease inhibitor family.     

Human hair: an unexpected source of cat allergen exposure

BACKGROUND: Cat allergens are ubiquitous because the clothing of cat owners constitutes an important source of distribution of Fel d 1 in cat-free environments. Since Fel d 1 can adhere to a variety of surfaces, we sought to verify if human hair belonging to individuals with or without a cat at home might represent a reservoir and be a possible carrier of cat allergens. METHODS: Seventy-three women (25 with a non-neutered male cat and 25 with a dog at home, and 23 controls without any direct contact with these animals) were recruited. The collection of material from hair was carried out using a modified version of a battery-powdered portable sampler. Particulate material was harvested onto glass fiber filters (25 mm in diameter, with a pore size of 2 microm; AP 20 Millipore, Milan Italy), extracted in phosphate buffer with BSA and then assayed for the evaluation of cat allergen using an ELISA based on anti-Fel d 1 monoclonal antibody. RESULTS: Detectable levels of cat allergen were found in 2 controls, in 2 women with a dog at home and in 13 women with a cat at home, respectively. CONCLUSIONS: In some women with a cat at home, hair constitutes a significant reservoir of Fel d 1. It is likely that these amounts of cat allergen might contribute to allergic sensitization when released in cat-free environments.     

High prevalence of sensitization to cat allergen among Japanese children with asthma, living without cats

BACKGROUND: Cat allergy is common among children with asthma. Many cat-allergic patients in Japan and elsewhere do not keep cats, but nonetheless become sensitized through environmental exposure to cat allergen. OBJECTIVE: To assess the frequency of cat allergy and cat-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibody responses in young Japanese patients with asthma in relation to self-reported cat exposure and Fel d 1 levels in dust samples. METHODS: Cat dander-specific IgE antibody was measured in sera from asthma patients using the CAP system. IgE and IgG antibody to Fel d 1 was measured by antigen binding radioimmunoassay and by chimeric enzyme immunoassay. Fel d 1 levels in dust samples from a subset of patients' homes were measured by monoclonal antibody-based enzyme immunoassay. RESULTS: Cat-specific IgE (CAP class>/=2) was found in sera from 70% of 44 patients who kept cats and 34% of 394 patients who had never kept cats. The prevalence of sensitization increased progressively to age 6 years (40%: positive), and then increased gradually to age 16 years (approximately 60%: positive) in patients who had never kept cats. There was an excellent correlation between cat CAP values and IgE levels to Fel d 1. The absolute amount of IgE antibody to Fel d 1 ranged from 0.01 to 15.6% of total IgE. Most patients who did not keep cats were exposed to Fel d 1 levels ranging from 0.07-8 microg/g dust. CONCLUSIONS: Sensitization to cat allergen is common among young asthmatic patients in Japan, even among patients who do not keep cats. Use of CAP and the chimeric enzyme-linked immunosorbent assay allows accurate diagnosis of cat allergy and quantification of specific IgE antibody levels.     

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.     

Human T and B cell immune responses to Fel d 1 in cat-allergic and non-cat-allergic subjects

Background In allergic individuals exposure to allergen leads to the induction of allergen-specific IgE which, upon binding to its high affinity receptors on mast cells and basophils. primes these cells for degranulation. This degranulation. a result of specific IgE allergen-interaction, initiates the debilitating symptoms of allergy and the potentially life-threatening symptoms of anaphylaxis. The lack of symptoms following antigen encounter by non-allergic individuals is probably due to the undetectable levels of allergen-specific IgE in the plasma of non-allergic individuals. Objective To compare the immune responses of allergic and non-allergic individuals. Method We compared the immune responses of 42 cat-allergic subjects with 16 nem-cat-allergic subjects to the major cat allergen. Fel d I. We have measured plasma immunoglobulin levels and the proliferative responses of fel d 1 primed T cell lines to Fel d 1 peptides. Results While these two groups have similar levels of Fel d 1 specific IgG. only subjects in the cat-allergic group have detectable Fel d 1 specific IgE. Affinity purified Fel d 1 was used to generate T cell lines from peripheral blood mononuclear cells of these same subjects. The proliferative responses of these T cell lines to intact Fel d 1 and a set of overlapping peptides covering the entire sequence of the molecule demonstrated that the pattern of epitope recognition was similar in both groups. Conclusion Our data suggest that factors other than T cell recognition of specific epitopes are responsible for the nature of allergic immune responses generated when allergen is encountered.     

The relevance of maternal immune responses to inhalant allergens to maternal symptoms,passive transfer to the infant,and development of antibodies in the first 2 years of life

BACKGROUND: Asthma and other atopic diseases are strongly hereditary. Although the mother might play a special role, the mechanisms for such an effect are not clear. OBJECTIVE: We sought to investigate the influence of maternal immune responses to cat and mite allergens on (1) maternal symptoms, (2) the development of immune responses in the infant, and (3) the development of allergic disease during the first 3 years of life. METHODS: In sera from 465 mothers and 424 infants (cord blood), as well as in sera from 230 of the children at age 2 to 3 years, total IgE and IgE antibodies were measured by using CAP testing; IgG and IgG4 antibodies for the cat allergen Fel d 1 were measured by means of radioimmunoprecipitation. RESULTS: In both mothers and children, approximately 15% of sera contained IgG antibodies to Fel d 1 without IgE antibodies to cat. The strongest predictor of the maternal IgG antibody response was exposure to greater than 8 microg of Fel d 1/g of dust. Thus approximately 70% of children living in a house with a cat had received IgG antibodies from their mothers. In many cases the infant received IgG and IgG4 antibodies to Fel d 1 from a nonallergic mother. Maternal IgE antibodies were consistently associated with asthma; by contrast, the IgG antibody was not independently related to asthma but was related to rhinitis in the mothers (odds ratio, 2.6; 95% CI, 1.1-6.2) and to eczema in children. At age 3 years, 13 of 230 sera contained IgE antibodies to mite, but only 5 had IgE antibodies to cat. CONCLUSIONS: A significant proportion (approximately 15%) of mothers and children exposed to high concentrations of cat (but not mite) allergens have serum IgG antibodies without IgE antibodies. This IgG antibody is freely transferred to the infant and might influence IgG antibody production in the child. The results indicate the importance of understanding the mechanisms of tolerance to cats and raise questions about the independent role of the mother in the inheritance of allergy.     

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.     

Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida.

BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy. OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy. METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen. RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3. CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.     

Cat allergen level: its determinants and relationship to specific IgE to cat across European centers

BACKGROUND: Cat allergen level in settled house dust and its determinants in Europe are unknown. OBJECTIVE: The aim of this study is to quantify the level of cat allergens in mattress dust, to study its determinants, and to analyze the relationship with cat specific IgE on community level across European centers. METHODS: Trained field workers collected dust from approximately 3000 mattresses during home visits in 22 European Community Respiratory Health Survey II centers. Sieved dust extracts were assayed for cat allergen using a mAb ELISA assay. RESULTS: The overall geometric mean cat allergen was 0.94 microg/g, ranging from 0.12 microg/g in Huelva, Spain, to 3.76 microg/g in Antwerp, Belgium. Current cat owners' homes showed substantially higher levels than past cat owners' and never cat owners' homes (geometric mean and 95% CI, 61.4 microg/g [48.4-77.9] vs 1.37 microg/g [0.97-1.9] vs 0.29 microg/g [0.27-0.31]). Community prevalence of cat ownership was moderately correlated with cat allergen levels in noncat owners (r(s) = 0.50), but not for past or current cat owners. The multilevel model identified community prevalence of cat keeping as the only statistically significant determinant of mattress cat allergen levels for noncat owners. However, averaged cat allergen levels per center were not related to community prevalence of detectable specific IgE to cat. CONCLUSION: Not having a cat in the home is associated with substantially lower Fel d 1 concentration, but does not protect against high Fel d 1 exposure in communities where cat ownership is common. CLINICAL IMPLICATIONS: People (including patients with cat allergy) who do not own cats may be exposed to high levels of cat allergen in their home, particularly if they live in communities with high levels of cat ownership.     

Endogenous function and biological significance of aeroallergens: an update

Significant advances have been made in delineating the structure and function of the clinically important aeroallergens. Most have now been characterized at the molecular level, and their endogenous function determined. In the period of review, however, several novel allergens have been identified. They include the house dust mite lipophorins and gelsolins, and the birch isoflavone reductase. In addition, the functions of previously described allergens have now been established or inferred on the basis of homology studies. For example, cat Fel d 1 and the grass pollen group 1 allergens possess proteolytic activity and the thaumatin-like plant and pollen allergens possess endo-beta1,3-glucanase activity. Similarly, the lipocalin allergens may possess endonuclease activity, and the mite group 2 allergens may bind cholesterol. The three-dimensional structure of the horse dander lipocalin Equ c 1 and the honey bee Api m 2 allergens have also been determined during the review period. Finally, in this period, a variety of novel or improved immunotherapeutic allergen reagents designed to redirect the host immune response from a T-helper-2 to a T-helper-1 cell phenotype have been described, in particular, allergen and immunostimulatory CpG motif conjugates.     

Determination of the T cell epitopes of the lipocalin allergen, Rat n 1

BACKGROUND: Laboratory animal allergy (LAA) is an important cause of occupational sensitization and asthma. Rats are a frequent cause of LAA and the major rat allergen, Rat n 1, is a member of the lipocalin protein family, which includes several other animal allergens such as the cow allergen, Bos d 2. To date, Bos d 2 is the only mammalian lipocalin allergen to have been studied in detail. OBJECTIVE: We undertook a cross-sectional study of a large population of individuals exposed to laboratory rats to determine the proliferative responses of peripheral blood mononuclear cells (PBMCs) to the major rat allergen, Rat n 1. METHODS: Eighty-three cases (defined by a positive skin prick test (SPT) > or =3 mm and/or a positive RAST > or =2% binding) and 274 referents without specific IgE to rats were tested for their proliferative responses of PBMCs to rat allergen. Cytokine release to rat urinary protein was examined in 28 sensitized and 42 non-sensitized exposed individuals. RESULTS: Proliferation to rat urinary protein was weak in all individuals. Four regions within Rat n 1 were identified as containing potential immunodominant T cell epitopes and three of these co-localized within the conserved regions of the lipocalin molecule. All four regions within Rat n 1 overlapped considerably with the characterized epitopes of the lipocalin allergen, Bos d 2. IL-5 and ratios of IL-5/IFN-gamma were significantly increased in cases. CONCLUSION: The response to Rat n 1 is remarkably similar to the cow lipocalin allergen Bos d 2. T cell epitopes within lipocalins appear to co-localize with the conserved regions of the molecule. LAA is characterized by an increased production of IL-5. Investigation of other lipocalin allergens will provide further information about the allergenicity of this group of proteins.     

Association between quantitative traits underlying asthma and the HLA-DRB1 locus in a family-based population sample

The region of human chromosome 6 containing the MHC has been identified as influencing asthma and atopy (allergy) by several genome-wide searches. The MHC contains many genes with potential effects on innate and specific immunity. As a first step in dissecting MHC influences on asthma and its underlying quantitative phenotypes, we have examined the HLA-DRB1 locus in a population sample consisting of 1004 individuals from 230 families from the rural Australian town of Busselton. The locus was strongly associated with the (log(e)) total serum IgE concentration, accounting for 4.0% of the sigma(2) (variance) in that trait (multi-allelic test, P=0.00001). The locus also influenced specific IgE titres to common allergens (multi-allelic tests, 2.8% sigma(2) for the house dust mite allergen Der p I, P=0.0013; 3.0% of sigma(2) for Der p II, P=0.0007; and 2.1% of sigma(2) for the cat allergen Fel d I, P=0.014). No associations were found to the categorical phenotype of asthma, or to the quantitative traits of peripheral blood eosinophil counts and bronchial hyper-responsiveness. Transmission disequilibrium tests excluded genetic admixture as a cause of false-positive findings. The results indicate that HLA-DRB1 alleles modulate the total serum IgE concentration and IgE responses to allergens, but do not account for the previous observations of linkage of asthma to the MHC.     

Molecular and immunological characterization of Can f 4: a dog dander allergen cross‐reactive with a 23 kDa odorant‐binding protein in cow dander

Background Dog dander is an important cause of respiratory allergy but its content of allergenic components is still incompletely known. While Can f 1, 2, 3 and 5 have been studied in detail, only fragmentary information is available on the lipocalin Can f 4. Objective To purify, clone and characterize dog dander allergen Can f 4. Methods Can f 4 was purified from dog dander extract by size exclusion, ion exchange and reverse phase chromatography. A cDNA encoding Can f 4 was cloned and used to produce recombinant Can f 4 in Escherichia coli. A 23 kDa protein from cow dander, displaying cross‐reactivity with Can f 4, was purified and identified by amino acid sequencing and mass spectrometry. IgE antibody binding to dog and cow dander extract and to individual dog allergens among 37 dog allergic subjects and 44 pollen allergic controls was studied using ImmunoCAP. Results A dog genome segment containing the Can f 4 gene was bioinformatically identified and enabled the cloning of Can f 4 cDNA. Recombinant Can f 4 displayed close immunological and biochemical similarity to purified natural Can f 4 and bound IgE antibodies from 13/37 (35%) sera of dog allergic subjects. Can f 4 reactive sera showed IgE binding to a 23 kDa protein present in cow dander extract, related to a family of odorant‐binding proteins. The dog and cow proteins shared 37% sequence identity and their cross‐reactivity was demonstrated by IgE inhibition experiments. Conclusion Recombinant Can f 4 brings the panel of available dog allergens closer to completion and will be important in component‐resolved diagnostics in allergy to animal epithelial allergens. Cite this as: L. Mattsson, T. Lundgren, P. Olsson, M. Sundberg and J. Lidholm, Clinical & Experimental Allergy, 2010 (40) 1276–1287.     

Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins

BaCKGROUND: Monoclonal antibodies are a valuable tool in the study of allergens, but the technology used in their generation can be slow and labour-intensive. Therefore, we have examined recombinant antibody development by phage-display against single allergens and protein mixtures. OBJECTIVE: We used the avian immunoglobulin system (generated from single V(H) and V(L) genes) to provide a rapid method for generating highly specific recombinant antibody fragments from a minimal number of animals. METHODS: A single-chain antibody fragment (scFv) library was generated from a single chicken immunized with model allergens. ScFvs were isolated by phage-display and their properties investigated by ELISA and Western blot. RESULTS: Mono-specific scFvs were generated against recombinant Fel d 1 and native Amb a 1. Pannings against yellow jacket venom extracts only yielded clones that reacted with multiple proteins in the venom extract. The scFvs from each panning type were effectively expressed in Escherichia coli and readily purified. Highly specific and sensitive recognition of Fel d 1 and Amb a 1 was demonstrated in ELISA, with scFvs displaying antibody-concentration-dependent absorbance curves down to picogram levels of antibody. The specificity of selected antibodies for their cognate antigen was further confirmed in Western blot analysis, with scFvs directed to either Fel d 1 or Amb a 1 showing no reactivity for the other antigens used in immunization. Anti-Amb a 1 scFvs also mapped Amb a 1-isoform location in Western blot of ragweed extracts separated by 2D SDS-PAGE. DNA sequence analysis of scFvs showed that multiple different clones had been generated against Fel d 1 and Amb a 1. Using two anti-Fel d 1 scFv for ELISA analysis of Fel d 1 content in crude cat pelt extracts, we could produce data which were highly similar (P=0.33 and 0.89 by paired t-test analysis) to those obtained using conventional assays (radial immunodiffusion). CONCLUSION: Phage-display technology may generate multiple allergen-specific recombinant antibody fragments from a single chicken, to allergens from mammalian, plant and insect sources. The resulting antibody fragments are of demonstrable use in allergen identification and quantification, in comparison with standard immunoassays.     

Targeting the MHC class II pathway of antigen presentation enhances immunogenicity and safety of allergen immunotherapy

Background:  Current s.c. allergen-specific immunotherapy (SIT) leads to amelioration of IgE-mediated allergy, but it requires numerous allergen injections over several years and is frequently associated with severe side-effects. The aim of this study was to test whether modified recombinant allergens can improve therapeutic efficacy in SIT while reducing allergic side-effects.
Methods:  The major cat allergen Fel d 1 was fused to a TAT-derived protein translocation domain and to a truncated invariant chain for targeting the MHC class II pathway (MAT-Fel d 1). The immunogenicity was evaluated in mice, while potential safety issues were assessed by cellular antigen stimulation test (CAST) using basophils from cat-dander-allergic patients.
Results:  MAT-Fel d 1 enhanced induction of Fel d 1-specific IgG2a antibody responses as well as the secretion of IFN-γ and IL-2 from T cells. Subcutaneous allergen-specific immunotherapy of mice using the modified Fel d 1 provided stronger protection against anaphylaxis than SIT with unmodified Fel d 1, and MAT-Fel d 1 caused less degranulation of human basophils than native Fel d 1.
Conclusion:  MAT-Fel d 1 allergen enhanced protective antibody and Th1 responses in mice, while reducing human basophil degranulation. Immunotherapy using MAT-Fel d 1 allergen therefore has the potential to enhance SIT efficacy and safety, thus, shortening SIT. This should increase patient compliance and lower treatment costs.