Use of multiple peptides containing T cell epitopes is a feasible approach for peptide-based immunotherapy in Can f 1 allergy

We have previously shown that the major dog allergen Can f 1 contains seven T cell epitope regions, none of which was preferentially recognized. To identify the immune characteristics of Can f 1 epitopes and to verify their suitability for peptide-based allergen immunotherapy, short-term T cell lines were generated with epitope-containing peptides from peripheral blood mononuclear cells of Can f 1 skinprick test-positive allergic and healthy control subjects. The lines were examined for their proliferative capacity and cytokine production upon stimulation with the allergen peptide, a homologous peptide from human tear lipocalin (TL) and Can f 1 and TL proteins. Can f 1 peptides induced proliferation of T cells and gave rise to T cell lines with comparable efficiencies. In particular, the T cell lines of allergic subjects induced with p33-48 and p107-122 favoured the production of interferon-gamma and interleukin-10, respectively. A greater number of Can f 1-specific T cell lines were generated from allergic than from healthy individuals. Two p107-122-induced Can f 1-specific T cell lines also reacted to a homologous peptide of human TL. Our results suggest that several T cell epitope-containing peptides should be used in combination for specific immunotherapy in Can f 1 allergy.     

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy

BACKGROUND: The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts. OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy. METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting. RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract. Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001). The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001). A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported. About one-third of the patients reacted to Can f 2. In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component. Aminoterminal sequencing showed this to be a previously unidentified allergenic protein. CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals. However, the two allergens are insufficient as reagents for diagnosing dog allergy.     

The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes

BACKGROUND: Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing. OBJECTIVE: Knowing the T cell epitopes in allergens is a prerequisite for developing novel preparations for allergen immunotherapy. METHODS: Specific T cell lines were generated with recombinant Equ c 1 from the peripheral blood mononuclear cells (PBMCs) of 10 horse-allergic subjects. For determining T cell epitopes, the lines were stimulated with 16mer synthetic Equ c 1 peptides overlapping by 14 amino acids. The binding capacity of Equ c 1 peptides to human leucocyte antigen class II molecules was determined by the competitive ELISA. RESULTS: The major horse allergen Equ c 1 resembles two other lipocalin allergens, the major cow allergen Bos d 2 and the major dog allergen Can f 1, in that it is weakly stimulatory for the PBMCs of sensitized subjects. Moreover, the T cell epitopes of Equ c 1 are clustered in a few regions along the molecule, as is the case with Bos d 2 and Can f 1. Similar to Bos d 2, Equ c 1 contains one immunodominant epitope region at the carboxy-terminal end of the molecule. The T cell lines of eight horse-allergic subjects out of 10 showed strong reactivity to one or both of the two overlapping peptides, p143-158 and p145-160, in this region. The region probably contains two overlapping epitopes. CONCLUSION: The 18mer peptide p143-160 from the immunodominant region of Equ c 1 is a potential candidate for the peptide-based immunotherapy of horse-sensitized subjects.     

Involvement of Can f 5 in a Case of Human Seminal Plasma Allergy

Background: The existence of IgE binding to dog dander extract without IgE antibodies against the described dog allergens (Can f 1, 2, 3 and 4) implies the presence of other dog allergens yet to be identified. Recently, an IgE-binding protein was isolated from dog urine and identified as prostatic kallikrein; it has been named Can f 5. Cross-reactivity between a dog dander allergen and human prostate-specific antigen (PSA) has been described. The aim of this study was to identify the dog dander allergen that presents cross-reactivity with PSA and demonstrate its clinical relevance in our patient with human seminal plasma allergy. Methods: SDS-PAGE immunoblotting and inhibition tests were performed. Mass spectrometry was carried out to identify the protein involved in the allergy reactions. Results: SDS-PAGE immunoblotting-inhibition with an IgE-binding protein from dog prostatic secretion showed total IgE binding inhibition to a 28-kDa IgE-reactive band identified as PSA. The electroeluted protein from dog prostatic secretion was identified by mass spectrometry as Can f 5. IgE immunoblotting of human seminal plasma incubated with the serum of the patient revealed two IgE-binding bands (28 and 32.7 kDa). Both SDS-PAGE immunoblotting inhibition assays, with human seminal plasma or purified PSA in solid phase, showed complete IgE binding inhibition when the serum of the anaphylactic patient was preincubated with dog dander extract or recombinant Can f 5. Conclusions: The dog dander allergen that shows cross-reactivity with human PSA has been characterized and turns out to be the recently described Can f 5. We demonstrated the clinical relevance of this cross-reactivity in a patient.     

Bet v 1142-156 is the dominant T-cell epitope of the major birch pollen allergen and important for cross-reactivity with Bet v 1-related food allergens

BACKGROUND: Individuals with birch pollen allergy frequently experience hypersensitivity reactions to certain foods, primarily because of IgE antibodies specific for the major birch pollen allergen Bet v 1 that cross-react with homologous food allergens. OBJECTIVE: We sought to characterize the major T-cell epitopes of Bet v 1 and to investigate their involvement in the cellular cross-reactivity with homologous food allergens. METHODS: T-cell epitope mapping of Bet v 1 was performed by testing Bet v 1-specific T-cell lines derived from 57 individuals with birch pollen allergy, with overlapping peptides representing the entire allergen. T-cell lines and T-cell clones were stimulated with Bet v 1-related major allergens from apple (Mal d 1), cherry (Pru av 1), hazelnut (Cor a 1), celery (Api g 1), carrot (Dau c 1), and soybean (Gly m 4) and with peptides deduced from the C-terminal amino acid sequences of these molecules. Results Bet v 1 142-156 , positioned in the highly conserved C-terminal region of Bet v 1, was identified as the major T-cell epitope recognized by 61% of individuals. Most T lymphocytes specific for Bet v 1 142-156 were activated by one or more homologous food proteins or the respective peptides, as indicated by proliferation and cytokine production. CONCLUSION: The major T-cell epitope of Bet v 1, Bet v 1 142-156 , plays an important role in the cellular cross-reactivity between this respiratory allergen and related food allergens. Thus T lymphocytes specific for Bet v 1 142-156 might be activated by various Bet v 1-related food allergens in vivo, even out of the pollen season.     

Affinity purification of a major and a minor allergen from dog extract: serologic activity of affinity-purified Can f I and of Can f I-depleted extract

Most dog-allergic patients react to a major 25 kd component on sodium dodecyl sulfate blots, Can f I (Ag 13). We initially raised monoclonal antibodies (Cf-3 and Cf-2) reactive with IgE-binding components distinct from Can f I. After a slight modification, we immunized other strains of mice and produced monoclonal antibodies coded Cf-1a and Cf-1b reactive with Can f 1. We affinity purified the allergens, Can f I and "dog allergen 2" with Cf-1a and Cf-2 ascites, respectively, and house dust-rich dog dander. Comparison of purified Can f I with dog saliva in RAST demonstrated that Can f I is a potent allergen for most dog-allergic patients (average response, 70%). After depletion of dog saliva of Can f I, a slightly lower contribution for Can f I was found, but the overall results supported the conclusion that Can f I is a major allergen in dog saliva. Comparison of purified dog allergen 2 with dog dander in RAST demonstrated that dog allergen 2 is less important for dog-allergic patients (average response, 23%). We radiolabeled the purified allergens and developed assays to measure Can f I and dog allergen 2 in allergen extracts and dust samples. Dog saliva was a strong allergen source, dog urine and feces contained very little of the allergens, and both allergens were found to a variable degree in the nine dog breeds tested.     

IgE Reactivity of the Dog Lipocalin Allergen Can f 4 and the Development of a Sandwich ELISA for Its Quantification

Purpose

Divergent results on the IgE reactivity of dog-allergic subjects to Can f 4 have been reported. The aim of this study was to evaluate the significance of Can f 4 in dog allergy and to develop an immunochemical method for measuring Can f 4 content in environmental samples.

Methods

We purified the natural dog allergen Can f 4 from a dog dander extract by monoclonal antibody-based affinity chromatography and generated its variant in a recombinant form. Sixty-three dog-allergic patients and 12 nonallergic control subjects were recruited in the study. The IgE-binding capacity of natural Can f 4 and its recombinant variant was assessed by ELISA, immunoblotting, and skin prick tests (SPT).

Results

Eighty-one percent of the dog-allergic patients showed a positive result to the immunoaffinity-purified natural Can f 4 in IgE ELISA, but only 46% in IgE immunoblotting. Respective results with the recombinant Can f 4 variant were 54% and 49%. SPT results reflected those obtained in ELISA and immunoblotting. The overall IgE reactivity of the immunoaffinity-purified natural Can f 4 was found to depend strongly on the integrity of the allergen''s conformation. A sandwich ELISA based on monoclonal antibodies was found to be functional for measuring Can f 4 in environmental samples.

Conclusions

Can f 4 is a major allergen of dog together with Can f 1 and Can f 5. In combination with other dog allergens, it improves the reliability of allergy tests in dog allergy.     

Molecular approaches for new vaccines against allergy

Type I allergy represents an important health problem that is currently affecting approximately 25% of the population in Western countries. Immunotherapy, the only causative treatment of Type I allergy, is currently performed with crude allergen extracts, which contain unpredictable amounts of allergenic, as well as nonallergenic, components. The application of molecular biology for allergen characterization has revealed the molecular nature of the most common allergens and allowed the production of recombinant allergens that equal natural allergens. Based on this knowledge, several different strategies to improve immunotherapy have become available. Until now, T-cell peptides, selected wild-type-like recombinant allergens and genetically modified hypoallergenic allergen derivatives have been evaluated in clinical trials in patients. Immunotherapy based on T-cell peptides has focused on allergen-specific T-cell responses, whereas genetically modified recombinant allergen molecules offer the advantage of combining T-cell and B-cell epitopes. Genetically modified recombinant birch pollen derivatives (Bet v 1-fragments, Bet v 1-trimer) have been evaluated in a double-blind, placebo-controlled, multicenter study. Vaccination with the Bet v 1-derivatives improved symptoms of birch pollen allergy, induced a healthy allergen-specific immunoglobulin G response and led to a significant reduction of seasonally induced boosts of immunoglobulin E.     

Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity

Background Although knowledge of the IgE cross‐reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross‐reactivity of animal allergens is poorly known. Objective The aim of this study was to characterize IgE cross‐reactivities between lipocalin proteins, including five animal‐derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL). Methods The recombinant proteins were validated by chromatography and mass spectrometry. The IgE‐binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross‐reactivities between the lipocalin proteins were determined by ELISA inhibition. Results ELISA inhibition revealed IgE cross‐reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog‐allergic patients of whom six were IgE‐positive for Can f 1. Conclusion Several lipocalins exhibited IgE cross‐reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three‐dimensional structures. The clinical significance of the findings needs to be elucidated. Low‐level IgE cross‐reactivity can play a role in regulating immune response to lipocalin allergens.     

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.     

The DR4–DQ8 haplotype and a specific T cell receptor Vβ T cell subset are associated with absence of allergy to Can f 1

BACKGROUND: The significance of specific T cell receptor (TCR) Vbeta subtypes and human leucocyte antigen (HLA) class II alleles for the development of allergy to lipocalin allergens such as the major dog allergen Can f 1 is not clear at present. OBJECTIVE: To characterize the TCR Vbeta usage in the Can f 1-specific T cell lines and the HLA class II genotypes of Can f 1-allergic and non-allergic subjects. METHODS: T cell lines were induced with recombinant Can f 1 from the peripheral blood mononuclear cells of 12 non-atopic dog owners and 26 dog-allergic patients. Thirteen of the dog-allergic subjects were sensitized to Can f 1. Expression of the TCR Vbeta subtypes on CD4(+) T cells in the T cell lines was measured by flow cytometry. The subjects were HLA genotyped for DRB1, DQB1 and DPB1 loci. RESULTS: Can f 1-specific T cell lines were obtained from 18 subjects, with either positive (n=8) or negative (n=10) skin prick tests (SPTs) to recombinant Can f 1. The frequency of TCR Vbeta5.1(+) T cells was significantly higher in the T cell lines of subjects with negative SPTs to the allergen. Moreover, DR4-DQ8 haplotype was over-represented among these subjects. CONCLUSION: The DR4-DQ8 haplotype and the TCR Vbeta5.1(+) CD4(+) T cells may be protective against allergy to Can f 1.     

New developments in the diagnosis and treatment of hymenoptera venom allergy

According to most textbooks, diagnostic tests with Hymenoptera venoms are reliable, and immunotherapy with these venoms in Hymenoptera-venom-allergic patients leads in near to 100% to full protection. Careful analysis of the literature shows however that the specificity of diagnostic tests is far from perfect and that both efficacy and tolerance, especially in patients receiving honeybee venom immunotherapy, are still suboptimal. The major allergens of honeybee and vespid venoms are now available in recombinant form. Preliminary trials analyzing diagnostic tests with recombinant allergens in honeybee venom allergy are promising: the specificity is clearly increased in both skin testing and in determining venom-specific IgE antibodies when compared to natural venom allergens. An important recent finding is the frequent association of severe Hymenoptera venom allergy and elevated basal serum levels of the mast-cell-specific enzyme tryptase. Elevated levels are found in up to 30% of the patients with a history of severe shock reactions following Hymenoptera stings. The current findings indicate that basal tryptase levels indicating an increased mast cell load are much more frequent than previously thought and are a risk factor for severe or even fatal sting reactions. Premedication with antihistamines in the initial phase of venom immunotherapy reduced both local and systemic allergic side effects in several controlled studies. In a retrospective analysis of one of these trials it was found that reexposure during immunotherapy resulted in significantly more systemic allergic reactions in patients on placebo than on antihistamine premedication, suggesting that initial antihistamine premedication might increase the efficacy of venom immunotherapy. Different ways of allergen modification for venom immunotherapy have been proposed. While the results with chemical modifications were not convincing, recent studies with T-cell epitope peptides from the major bee venom allergen phospholipase A(2) look promising. Patient-tailored cocktails of recombinant venom allergens or isoforms thereof may be another possibility in the future. A number of prospective studies analyzing the duration of venom immunotherapy required for long-term protection have been published in the last decade. While most patients are still fully protected 1 year after discontinuation of therapy, relapses may occur in up to 20% of patients reexposed many years after treatment. Various risk factors for such relapses have been identified.     

Antigen presentation of the immunodominant T-cell epitope of the major mugwort pollen allergen, Art v 1, is associated with the expression of HLA-DRB1 *01

BACKGROUND: Mugwort pollen allergens are the main cause of pollinosis in late summer in Europe. Ninety-five percent of patients allergic to mugwort are sensitized to the major allergen Art v 1. In contrast to other common pollen allergens that contain multiple T-cell epitopes, Art v 1 contains only 1 immunodominant T-cell epitope (Art v 1 25-36 ). OBJECTIVE: To characterize the minimal epitope of Art v 1 25-36 and to investigate a possible association of Art v 1 reactivity with HLA class II phenotypes. METHODS: Art v 1-specific T-cell lines and clones were established from 51 patients with clinically defined mugwort pollen allergy and IgE specific for Art v 1. To define minimal epitopes and binding sites within Art v 1 25-36 , truncated and single-substitution analog peptides were used for T-cell stimulation. To study HLA restriction, monoclonal anti-HLA antibodies and antigen-presenting cells with defined HLA-DRB and -DQB1 alleles were used. HLA typing of patients with allergy was performed by hybridization with sequence-specific oligonucleotides, PCR, and nucleotide sequencing. RESULTS: In 96% of the patients, a cellular response to Art v 1 25-36 was obtained, and a core region of 5 to 10 amino acids containing 3 to 5 amino acids essential for T-cell reactivity was defined. The frequency of HLA-DRB1 * 01 in patients recognizing Art v 1 25-36 was significantly increased as compared with healthy controls (69% vs 21%; odds ratio, 8.45; P < 10 -6 ), and HLA-DRB1 * 01 was identified as the main restriction element for the presentation of the immunodominant epitope. CONCLUSIONS: Allergy to Art v 1 is characterized by a uniform T-cell response. The disease is apparently associated with the HLA-DR1 phenotype. Therefore, mugwort pollinosis is an ideal candidate for a peptide-based immunotherapy.     

Characterization of the T cell response to the major hazelnut allergen, Cor a 1.04: evidence for a relevant T cell epitope not cross-reactive with homologous pollen allergens

BACKGROUND: IgE antibodies specific for the major birch-pollen allergen, Bet v 1, cross-react with homologous allergens in particular foods, e.g. apples, carrots and hazelnuts. In a high number of tree pollen-allergic individuals, this cross-reactivity causes clinical symptoms, commonly known as the 'birch-fruit-syndrome'. OBJECTIVE: To characterize the T cell response to the Bet v 1-related major allergen in hazelnuts, Cor a 1.04, and its cellular cross-reactivity with Bet v 1 and the homologous hazel pollen allergen, Cor a 1. METHODS: Using recombinant Cor a 1.04, T cell lines (TCL) and T cell clones (TCC) were established from peripheral blood mononuclear cells of tree pollen-allergic patients with associated food allergy. T cell epitopes were determined using overlapping synthetic peptides in Cor a 1.04-reactive TCL and TCC. In parallel, reactivity to Bet v 1 and Cor a 1 was tested. RESULTS: In total, 20 distinct T cell epitopes on the hazelnut allergen were identified. Several Cor a 1.04-specific TCL and TCC reacted with pollen allergens albeit less pronounced than with the hazelnut allergen. Several Cor a 1.04-specific TCC did not react with pollen allergens. Interestingly, these clones were found to react with the Bet v 1-related major allergen in carrots, Dau c 1. The cellular cross-reactivity between both food allergens could be associated with the most frequently recognized T cell epitope of Cor a 1.04, Cor a 1.04(142-153). CONCLUSIONS: The major hazelnut allergen cross-reacts with the major allergens of birch and hazel pollen but apparently contains a relevant T cell epitope not shared with pollen allergens. Our finding may have important implications for the specific immunotherapy of tree pollen-allergic patients suffering from concomitant hazelnut allergy.     

Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom

Background: Specific immunotherapy with honeybee venom (BV) is highly effective, but allergic side effects can occur during treatment. Immunotherapy with peptides containing major T-cell epitopes of the relevant allergen or allergens provides an alternative strategy without these problems. Objective: The study investigates the immunologic mechanisms and clinical effects of immunotherapy with T-cell epitope peptides of the major BV allergen, the phospholipase A2 (PLA). Methods: Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 μg, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro. Results: No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of T_H2 and T _H1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4. Conclusions: Immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients. (J Allergy Clin Immunol 1998;101:747-54.)     

The identification of potentially pathogenic and therapeutic epitopes from common human allergens

ObjectivesTo outline the processes involved in large-scale T-cell epitope identification from common allergens and illustrate their relevance to development of allergy specific immunotherapy.Data SourcesA set of studies recently published by our laboratory illustrating high-throughput identification of allergen specific T-cell epitopes.Study SelectionT-cell responses contribute both directly and indirectly to allergy-related disease. However, the molecular targets (epitopes) recognized by allergen-specific T cells are largely undefined. We review several different studies in the last 2 years that identified novel T-cell epitopes from a panel of 32 different allergen sources.ResultsAllergen-specific T-cell responses are highly heterogeneous. Epitopes prevalently recognized in allergic patients are often capable of binding to multiple HLA class II molecules. This feature can be used to predict these promiscuous epitopes by bioinformatic predictions. This approach was validated in the Timothy grass system and then applied to a panel of 31 other allergen sources.ConclusionT-cell epitopes for common allergens have been identified, and a general method to identify epitopes from additional allergens has been validated. Characterization of epitopes for common allergens might enable new diagnostics and immunotherapy regimens. These data will also allow the study of T-cell responses in different patient populations and throughout disease progression.     

The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.     

Advances and novel developments in molecular allergology

The continuous search for new allergens and the design of allergen derivatives improves the understanding of their allergenicity and aids the design of novel diagnostic and immunotherapy approaches. This article discusses the recent developments in allergen and epitope discovery, allergy diagnostics and immunotherapy. Structural information is crucial for the elucidation of cross-reactivity of marker allergens such as the walnut Jug r 6 or that of nonhomologous allergens, as shown for the peanut allergens Ara h 1 and 2. High-throughput sequencing, liposomal nanoallergen display, bead-based assays, and protein chimeras have been used in epitope discovery. The binding of natural ligands by the birch pollen allergen Bet v 1 or the mold allergen Alt a 1 increased the stability of these allergens, which is directly linked to their allergenicity. We also report recent findings on the use of component-resolved approaches, basophil activation test, and novel technologies for improvement of diagnostics. New strategies in allergen-specific immunotherapy have also emerged, such as the use of virus-like particles, biologics or novel adjuvants. The identification of dectin-1 as a key player in allergy to tropomyosins and the formyl peptide receptor 3 in allergy to lipocalins are outstanding examples of research into the mechanism of allergic sensitization.     

Non-anaphylactic combination of partially deleted fragments of the major house dust mite allergen Der f 2 for allergen-specific immunotherapy

Allergen-specific immunotherapy, in which repeated injections of allergens over prolonged periods are used to induce tolerance, has proven an effective treatment of allergy. A major side effect of allergen-specific immunotherapy is anaphylactic reaction. House dust mite allergens are major causative factors associated with various allergic diseases. Der f 2 is the major house dust mite allergen composed of 129 amino acid residues. Analysis using deletion mutants of Der f 2 suggested that T-cell epitopes of Der f 2 were multiple in mite-allergic patients. We found that some IgE epitopes were renatured by dialysis of a mixture of two denatured C- and N-terminal deletion mutants, 1-112 and 85-129 in 13 patients out of 14. On the other hand, IgE binding activity was negative in the separately dialyzed fragments and their mixture in each patient tested. Furthermore, we demonstrated that neither of the two separately prepared polypeptides induced in vivo skin prick test reactivity. These findings are important for improvement of T-cell targeting allergen-specific immunotherapy and development of monovalent IgE haptens. The use of combinations of overlapping non-anaphylactic fragments of allergen covering all of the T-cell epitopes achieves the removal of IgE reactivity, the cause of harmful anaphylactic reactions, without affecting the T-cell reactivity essential for immunotherapy, offering potentially safer and more effective treatment for allergic disease.