Natural Allergen Research Articles

Allergen homologs in the Euroglyphus maynei draft genome.

Euroglyphus maynei is a house dust mite commonly found in homes worldwide and is the source of allergens that sensitize and induce allergic reactions in humans. It is the source of species-specific allergens as well as allergens that are cross-reactive with the allergens from house dust mites Dermatophagoides farinae and D. pteronyssinus, and the ectoparasitic scabies mite Sarcoptes scabiei. The genomics, proteomics and molecular biology of E. maynei and its allergens have not been as extensively investigated as those of D. farinae, D. pteronyssinus, and S. scabiei where natural and recombinant allergens from these species have been characterized. Until now, little was known about the genome of E. maynei and it allergens but this information will be important for producing recombinant allergens for diagnostic and therapeutic purposes and for understanding the allergic response mechanism by immune effector cells that mediate the allergic reaction. We sequenced and assembled the 59 Mb E. maynei genome to aid the identification of homologs for known allergenic proteins. The predicted proteome shared orthologs with D. farinae and S. scabiei, and included proteins with homology to more than 30 different groups of allergens. However, the majority of allergen candidates could not be assigned as clear orthologs to known mite allergens. The genomic sequence data, predicted proteome, and allergen homologs identified from E. maynei provide insight into the relationships among astigmatid mites and their allergens, which should allow for the development of improved diagnostics and immunotherapy.

Utility and Comparative Efficacy of Recombinant Allergens Versus Allergen Extract.

Allergy immunotherapy (AIT) is the only disease-modifying therapy for the treatment of allergic diseases. Although its efficacy and utility are well-established, the potential for serious adverse events, cumbersome and lengthy treatment protocols, and variability of natural allergen preparations have limited its widespread application. Recent advances in recombinant technology have opened new avenues for the development of AIT vaccines. The purpose of this review is to highlight recent evidence on the use of novel recombinant vaccines and review the mechanisms, efficacy, safety, and limitations of AIT. Emerging evidence suggests that recombinant vaccines may provide a viable treatment alternative that improves on the limitations of natural extract therapy while maintaining efficacy.

Inhibition of cross-reactive carbohydrate determinants (CCDs) enhances the accuracy of in vitro allergy diagnosis.

Background: Cross-reactive carbohydrate determinants (CCDs) as they occur on natural allergens from plants and insects influence the measurement of antigen-specific IgE-antibodies in the context of in vitro allergy diagnosis. When positive results are based solely on the reaction of CCDs with anti-CCD IgE, results must be rated as false-positive. A generally applicable solution to this problem has not yet been presented. Methods/Patients: Sera of patients for whom an assumed allergy should be verified or ruled out were tested with three methods for specific IgE determination (a multiallergen teststrip format, a single allergen test and an allergen-component array) in the absence and presence of a novel, semi-synthetic CCD-blocker. The study was not prospective and for many patients unequivocal clinical data were missing; the data section thus focusses on few, well-defined patient sera. Results: More than 20% of all patients were tested positive for IgE-anti-CCD antibodies and hence against a multitude of similarly glycosylated allergen extracts in a strip-based multiallergen test. Incubation of these positive sera with the CCD-blocker led to significant reductions of read-out values and in many cases to negative test results. The inhibitory efficiency was highest for the allergen strip test and for the component array. Results remained positive for relevant allergens for which a true sensitization had been indicated by skin tests or other means. The CCD-blocker did not alter the read-outs for unglycosylated allergens or – with CCD-negative sera – for all allergens. Conclusion: Elimination of CCD-specific IgE antibodies by means of a synthetic CCD-blocker drastically reduced the number of false-positive in vitro test results without compromising the sensitivity for relevant IgE interactions. Thus, the herein described CCD-blocker constitutes a valuable tool for increasing the test specificity of routine in vitro allergy diagnosis.

New - epicutaneous - method for the causal treatment of allergic diseases

The only causal treatment for the allergic diseases is the allergen immunotherapy. Besides oral and injection forms, an epicutaneous form is known as well. During the immunization process the use of an adjuvant material is advisable besides the allergen. That adjuvant material can be (besides of the aluminium hydroxid and tyrosine) a bacterial toxin, too. In idealistic circumstances we can substitute the native allergen with its recombinant variant, which could keep its immunogenicity but had lost the allergenicity. In the future the recommended therapy could be the safe, painless, epicutaneous hyposensitization, with recombinant allergens, with bacterial toxin as adjuvants. Orv Hetil. 2017; 158(23): 895-899.

Glycans and glycan-specific IgE in clinical and molecular allergology: Sensitization, diagnostics, and clinical symptoms

Glycan-specific IgE antibodies cross-react with highly similar or even identical carbohydrate structures on a variety of different natural allergens, the so-called cross-reactive carbohydrate determinants (CCDs). In clinical practice CCDs often interfere with the specificity of invitro allergy diagnostics, thus impairing allergy therapy decisions for individual patients. Strikingly, these IgE antibodies directed against CCDs often do not cause clinically relevant allergy symptoms. On the other hand, the IgE-binding glycan allergen galactose-α-(1,3)-galactose (α-Gal) is associated with IgE-mediated delayed anaphylaxis in meat allergy. The reason for this discrepancy is not known. The discovery of α-Gal stimulated new discussions and investigations regarding the relevance of anti-glycan IgE for allergic diseases. In this review the effect of glycans and glycan-specific IgE on sensitization to allergens and allergy diagnosis is described. Because parasite infections elicit a similar immunologic environment as allergic diseases, the association of glycan-specific antibodies against parasite glycoproteins with glycan structures on allergens is discussed.

New Perspectives on the Diagnosis of Allergy to Anisakis spp.

To compare the prevalence of sensitization in different countries based on specific IgE values and to evaluate the use of isolated native or recombinant allergens for diagnosis. Isolated allergens help in the diagnosis of truly sensitized patients avoiding false positives due to cross-reactions. Their use is therefore highly recommended, especially when used as a combination of several relevant allergens. The use of purified allergens allows an accurate diagnosis and this has led to three important findings: (1) in addition to the digestive route of sensitization, occupational and non-digestive exposure seems to be clinically relevant. (2) The parasite appears as an important agent for chronic urticaria. And (3) in endemic countries, the amount of highly sensitized subjects in the general population could be as high as 7%. Adequate information to asymptomatic patients on fish consumption habits would avoid new contacts with parasite allergens and decrease their specific IgE levels and consequently the appearance of acute or chronic episodes induced by the parasite.

Allergic conjunctivitis in Asia.

Allergic conjunctivitis (AC), which may be acute or chronic, is associated with rhinitis in 30%–70% of affected individuals, hence the term allergic rhinoconjunctivitis (AR/C). Seasonal and perennial AC is generally milder than the more chronic and persistent atopic and vernal keratoconjunctivitis. Natural allergens like house dust mites (HDM), temperate and subtropical grass and tree pollen are important triggers that drive allergic inflammation in AC in the Asia-Pacific region. Climate change, environmental tobacco smoke, pollutants derived from fuel combustion, Asian dust storms originating from central/north Asia and phthalates may also exacerbate AR/C. The Allergies in Asia Pacific study and International Study of Asthma and Allergies in Childhood provide epidemiological data on regional differences in AR/C within the region. AC significantly impacts the quality of life of both children and adults, and these can be measured by validated quality of life questionnaires on AR/C. Management guidelines for AC involve a stepped approach depending on the severity of disease, similar to that for allergic rhinitis and asthma. Topical calcineurin inhibitors are effective in certain types of persistent AC, and sublingual immunotherapy is emerging as an effective treatment option in AR/C to grass pollen and HDM. Translational research predominantly from Japan and Korea involving animal models are important for the potential development of targeted pharmacotherapies for AC.

The Impact on Allergy-Related Cells of a Birch Pollen Allergoid, with and without Monophosphoryl Lipid A, in Comparison with the Native Equivalent

Background: The clinical efficacy and safety of allergoid immunotherapy have been demonstrated in clinical trials. However, simultaneous monitoring of the immunological changes by allergoids versus allergens in the cells of the same individual has not been extensively performed, and the impact of concurrent Toll-like receptor 4 (TLR4) ligation has not been specified. Methods: Three types of birch allergen were utilized: glutaraldehyde-treated allergoid (extract A), the same allergoid plus monophosphoryl lipid A (MPL), i.e., TLR4 ligand (extract A*), and native allergen (extract B). Antigen-specific responses after the in vitro stimulation of blood cells with the extracts were assessed by studying costimulatory receptors on the B cell surface by flow cytometry, cytokine responses by ELISA, and CD63 and CD203c upregulation (basophil activation test) in allergic versus nonallergic subjects. Results: HLA-DR selectively increased upon allergen or allergoid treatment in the allergic group only. The extract types elicited similar cytokine responses, with IL-6 and IL-10 production detected only in certain atopic subjects. The allergoids revealed a strong reduction (100- to <10,000-fold) in basophil activation versus native allergen. Reactivity was undetectable in the basophils from nonallergic subjects. Conclusion: The allergenicity of the allergoid employed was sharply reduced when compared to the native allergen, while its immunogenicity was largely retained, especially in the presence of MPL. We also provide further evidence that allergic and nonallergic individuals show preexisting differences in their immune repertoires.

Lactobacillus buchneri S-layer as carrier for an Ara h 2-derived peptide for peanut allergen-specific immunotherapy.

Peanut allergy is an IgE-mediated severe hypersensitivity disorder. The lack of a treatment of this potentially fatal allergy has led to intensive research on vaccine development. Here, we describe the design and initial characterization of a carrier-bound peptide derived from the most potent peanut allergen, Ara h 2, as a candidate vaccine. Based on the adjuvant capability of bacterial surface (S-) layers, a fusion protein of the S-layer protein SlpB from Lactobacillus buchneri CD034 and the Ara h 2-derived peptide AH3a42 was produced. This peptide comprised immunodominant B-cell epitopes as well as one T cell epitope. The fusion protein SlpB-AH3a42 was expressed in E. coli, purified, and tested for its IgE binding capacity as well as for its ability to activate sensitized rat basophil leukemia (RBL) cells. The capacity of Ara h 2-specific IgG rabbit-antibodies raised against SlpB-AH3a42 or Ara h 2 to inhibit IgE-binding was determined by ELISA inhibition assays using sera of peanut allergic patients sensitized to Ara h 2. IgE specific to the SlpB-AH3a42 fusion protein was detected in 69% (25 of 36) of the sera. Despite the recognition by IgE, the SlpB-AH3a42 fusion protein was unable to induce β-hexosaminidase release from sensitized RBL cells at concentrations up to 100ng per ml. The inhibition of IgE-binding to the natural allergen observed after pre-incubation of the 20 sera with rabbit anti-SlpB-AH3a42 IgG was more than 30% for four sera, more than 20% for eight sera, and below 10% for eight sera. In comparison, anti-Ara h 2 rabbit IgG antibodies inhibited binding to Ara h 2 by 48% ±13.5%. Our data provide evidence for the feasibility of this novel approach towards the development of a peanut allergen peptide-based carrier-bound vaccine. Our experiments further indicate that more than one allergen-peptide will be needed to induce a broader protection of patients allergic to Ara h 2.

Microarray Technology Applied to Human Allergic Disease.

IgE antibodies serve as the gatekeeper for the release of mediators from sensitized (IgE positive) mast cells and basophils following a relevant allergen exposure which can lead to an immediate-type hypersensitivity (allergic) reaction. Purified recombinant and native allergens were combined in the 1990s with state of the art chip technology to establish the first microarray-based IgE antibody assay. Triplicate spots to over 100 allergenic molecules are immobilized on an amine-activated glass slide to form a single panel multi-allergosorbent assay. Human antibodies, typically of the IgE and IgG isotypes, specific for one or many allergens bind to their respective allergen(s) on the chip. Following removal of unbound serum proteins, bound IgE antibody is detected with a fluorophore-labeled anti-human IgE reagent. The fluorescent profile from the completed slide provides a sensitization profile of an allergic patient which can identify IgE antibodies that bind to structurally similar (cross-reactive) allergen families versus molecules that are unique to a single allergen specificity. Despite its ability to rapidly analyze many IgE antibody specificities in a single simple assay format, the chip-based microarray remains less analytically sensitive and quantitative than its singleplex assay counterpart (ImmunoCAP, Immulite). Microgram per mL quantities of allergen-specific IgG antibody can also complete with nanogram per mL quantities of specific IgE for limited allergen binding sites on the chip. Microarray assays, while not used in clinical immunology laboratories for routine patient IgE antibody testing, will remain an excellent research tool for defining sensitization profiles of populations in epidemiological studies.

T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

BackgroundRagweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy.MethodsDomains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT.ResultsThe IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected.ConclusionAccumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

Allerjik Rinit Tanı ve Tedavisindeki Yenilikler

Allergic rhinitis (AR) is one of the most common chronic diseases in all age groups. It is an IgE-mediated allergic inflammation of the nasal airways and is characterized by sneezing, swelling and inflammation of the nasal mucosa as well as itchy and watery eyes. It is a global health problem and a burdensome disorder affecting many people from childhood to adulthood. The prevalence of childhood AR shows wide variation throughout the world, ranging from 0.8 to 45%. The prevalence rates for different regions of Turkey ranges from 2.9 to 44%. The aim of the present review is to provide an update on AR treatment and diagnosis. Because of its burden on patient’s lives and its impact on asthma, AR should be taken care of properly with effective and safe treatments. Second-generation antihistamines and intranasal steroids represent the milestone of AR therapy. New formulations of available drugs (rupatadine oral solution, ebastine fast-dissolving tablets, combination of intranasal fluticasone propionate and azelastine hydrochloride), recently discovered molecules (ciclesonide, bilastine, roflumilast, etc.), immunologic targets (omalizumab) and unconventional treatments (homeopathic treatments) are representing new frontiers in AR management. An accurate diagnosis of AR is also crucial to increase treatment success. Further promising improvements in diagnostics are expected from recombinant allergen diagnostics identifying particular molecules of risk. Purified native or recombinant allergens, identifying the individual molecules to which patients are sensitized, can be determined by molecular or component resolved diagnostics. These may decrease the need for provocation testing and improve the specificity of allergen immunotherapy. Different methods to measure nasal nitric oxide (nNO) levels are currently being revealed. Decreased nNO concentration is also typical of cystic fibrosis, even though nNO is not as low as in primary ciliary dyskinesia. The clinical utility of nNO remains to be established in upper airway disorders such as AR.

Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities

Background Subcutaneous allergen immunotherapy (SCIT) is a well-documented treatment for allergic disease which involves injections of native allergen or modified (allergoid) extracts. The use of allergoid vaccines is a growing sector of the allergy immunotherapy market, associated with shorter-course therapy. The aim of this study was the structural and immunological characterisation of group 1 (Lol p 1) IgG-binding epitopes within a complex mix grass allergoid formulation containing rye grass.Methods HP-SEC was used to resolve a mix grass allergoid preparation of high molecular weight into several distinct fractions with defined molecular weight and elution profiles. Allergen verification of the HP-SEC allergoid fractions was confirmed by mass spectrometry analysis. IgE and IgG immunoreactivity of the allergoid preparations was explored and Lol p 1 specific IgG-binding epitopes mapped by SPOT synthesis technology (PepSpot™) with structural analysis based on a Lol p 1 homology model.Results Grass specific IgE reactivity of the mix grass modified extract (allergoid) was diminished in comparison with the mix grass native extract. A difference in IgG profiles was observed between an intact mix grass allergoid preparation and HP-SEC allergoid fractions, which indicated enhancement of accessible reactive IgG epitopes across size distribution profiles of the mix grass allergoid formulation. Detailed analysis of the epitope specificity showed retention of six Lol p 1 IgG-binding epitopes in the mix grass modified extract.Conclusion The structural and immunological changes which take place following the grass allergen modification process was further unravelled revealing distinct IgG immunological profiles. All epitopes were mapped on the solvent exposed area of Lol p 1 homology model accessible for IgG binding. One of the epitopes was identified as an ‘immunodominant’ Lol p 1 IgG-binding epitope (62-IFKDGRGCGSCFEIK-76) and classified as a novel epitope. The results from this study support the concept that modification allows shorter-course therapy options as a result of providing an IgG epitope repertoire important for efficacy. Additionally, the work paves the way to help further develop methods for standardising allergoid platforms.

Safety of immunotherapy with glutaraldehyde modified allergen extracts in children and adults

BackgroundGlutaraldehyde-modified natural allergen extracts show significant reduction in the IgE-binding capacity and proteolytic activity. This allows the administration of higher doses in a shorter period of time, and to mix different allergen extracts. ObjectiveEvaluate the safety of different concentrations and mixtures of glutaraldehyde-modified allergen extracts in a large group of paediatric and adult patients undergoing specific immunotherapy treatment. Materials and methods1855 patients (1156 adults and 699 children), suffering from rhinoconjunctivitis and/or asthma, participated in an observational multicentre cohort study, to evaluate the safety of immunotherapy using vaccines containing modified allergen extracts. Patients were monosensitised, or polysensitised, and received a therapeutic vaccine containing polymerised allergen extracts adsorbed onto aluminium hydroxide. Safety was assessed by recording all side reactions related to immunotherapy. ResultsThe clinically relevant local reactions totalled 120, (90 immediate and 30 delayed) (1.02% of injections). Of them, 31 (0.26% of injections) occurred in children (26 immediate and 5 delayed) and 89 (0.76% of injections) in adults (64 immediate and 25 delayed). There were 38 systemic reactions. Eleven reactions were immediate (9 of grade 1 and 2 of grade 2) and 27 delayed (22 of grade 1 and 5 of grade 2). There were seven grade 2 systemic reactions (0.06% of the injections). No differences (P>0.05) in the number of reactions were observed between adults and children and between treatments were found in systemic reactions. All systemic reactions were mild and resolved spontaneously without the need of medication. ConclusionSpecific immunotherapy using natural modified allergen vaccines is safe to treat allergic patients, even at higher doses and in mixtures of unrelated allergen extracts. The percentage of adverse reactions detected is lower than those reported in the literature with native unmodified allergen extracts.

Cross-reactive LTP sensitization in food-dependent exercise-induced urticaria/anaphylaxis: a pilot study of a component-resolved and in vitro depletion approach.

BackgroundChallenge tests for food-dependent exercise-induced anaphylaxis (FDEIA) carry some risk and have a high rate of false negatives. Our aim was to explore the usefulness of an in vitro immunodepletion assay and an allergen microarray test in the identification of IgE-mediated cross-reactive food allergens in patients with suspected FDEIA or food-dependent exercise-induced urticaria and panallergen sensitization.MethodsThree patients with a history of food dependent exercise induced urticaria/anaphylaxis and food panallergen sensitization in whom a food-exercise challenge was not feasible were selected: a 25-year-old man with cholinergic urticaria who experienced generalized urticaria and angioedema during a soccer match after drinking a peach-based soft drink; a 19-year-old woman with allergic rhinitis and controlled asthma who experienced anaphylactic shock while playing soccer, having eaten walnuts in the previous 90 min; and a 57-year-old man with baker’s asthma who experienced four episodes of anaphylaxis during exercise after ingesting wheat-containing food. All individuals underwent a diagnostic work-up with skin prick tests, specific IgE (sIgE) and ImmunoCAP ISAC test. For the in vitro immunodepletion procedure, patients’ serum was pre-incubated with the suspected native allergen (peach, walnut, or wheat) in solid phase (ImmunoCAP). The eluted serum, containing unbound IgE, was collected and samples were re-tested using Immunocap ISAC 112 and compared with baseline results.ResultsAll individuals were sensitized to lipid transfer proteins. The first patient was sensitized to Pru p 3, Cor a 8, Jug r 3, and Ara h 9; after pre-incubation with peach there was 100% depletion of sIgE to all components. The second patient was sensitized to Pru p 3, Cor a 8, Jug r 3, and Ara h 9; immunodepletion with walnut depleted sIgE to Ara h 9 by 67%, Pru p 3 and Pla a 3 (60%), Art v 3 (75%), Jug r 3 (88%), and Cor a 8 (100%). The third patient was sensitized to Pru p 3, Jug r 3, Ara h 9, and Tri a 14; immunodepletion with wheat depleted Tri a 14 only (100%).ConclusionsIn vitro immunodepletion might be a useful diagnostic tool in food dependent exercise induced urticaria/anaphylaxis with panallergen sensitization, particularly for identifying the culprit allergen and guiding dietary elimination recommendations.

Th17 responses are not altered by natural exposure to seasonal allergens in pollen-sensitive patients.

BackgroundAllergic rhinitis affects 10–30 % of the global population and this number is likely to increase in the forthcoming years. Moreover, it commonly co-exists with allergic asthma as a chronic allergic respiratory syndrome. While the involvement of Th2 cells in allergy is well understood, alterations of pro-inflammatory Th17 responses remain poorly characterized. The aim of our study was to determine whether natural seasonal allergen exposure causes changes in T cell subset characteristics in patients with allergic rhinitis and asthma.MethodsSixteen patients with allergic rhinitis/atopic asthma (9M, 7F; age 31.8 ± 12.1) and 16 healthy controls were recruited into the study (9M, 7F; age 31.2 ± 5.3). Blood samples were collected from the patients 1–3 months before pollen season (visit 1), within 7 days of the appearance of pollen/initiation of allergic symptoms (visit 2) and 2 weeks after visit 2 following the introduction of symptomatic treatment with antihistamines (visit 3). Flow cytometry was used to assess major T cell subsets (naïve, central memory, effector memory and CD45RA+ effector) and key T cell cytokine production (IFNγ, IL-17A, TNF and IL-4) using intracellular staining. Data were analyzed using repeated measures ANOVA and paired t test.ResultsAs expected, an increase in the percentage of IL‐4+ CD4+ cells was observed during natural pollen exposure in patients with allergic respiratory syndrome. No significant changes were observed in the production of other cytokines, including Th17 cells, which tended to be lower than in the control population but unchanged during pollen exposure. Introduction of antihistamine treatment led to only moderate changes in cytokine production from CD4 and CD8 T cells. Selective changes in CD8+ T cells were observed during natural pollen exposure including a decrease in transient cells (with features of CD45RA+ and CD45RO+ cells) and a decrease in the percentage of central memory cells in the peripheral circulation. Within the CD4 cell group the total percentage of CD45RA positive CD4 cells was increased during pollen exposure.ConclusionsTh1 and Th17 responses are not altered during pollen season but allergen exposure affects T cell activation and memory cell status in patients with allergic respiratory syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13223-016-0157-6) contains supplementary material, which is available to authorized users.

Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES

Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing protective antibody response for immunotherapeutic applications.

Follicular helper T cells mediate IgE antibody response to airborne allergens

TH2 cells have long been believed to play a pivotal role in allergic immune responses, including IgE antibody production and type 2 cytokine-mediated inflammation and pathology. Anew T-cell subset, follicular helper T (TFH) cells, is specialized in supporting B-cell maturation and antibody production. We sought to investigate the roles of TFH cells in allergic immune responses. Naive mice were exposed to cytokines or natural allergens through the airways. Development of allergic immune responses was analyzed by collecting draining lymph nodes and sera and by challenging the animals. Cytokine reporter mice and gene-deficient mice were used to dissect the immunologic mechanisms. We observed the development of IL-4-producing TFH cells and TH2 cells in draining lymph nodes after airway exposure to IL-1 family cytokines or natural allergens. TFH and TH2 cells demonstrated unique phenotypes, tissue localization, and cytokine responses. TFH cells supported the sustained production of IgE antibody invivo in the absence of other T-cell subsets or even when TH2 cell functions were severely compromised. Conversely, conditional deficiency of the master regulator Bcl6 in CD4+ Tcells resulted in a marked reduction in TFH cell numbers and IgE antibody levels, but type 2 cytokine responses and eosinophilic inflammation in the airways remained unaffected. TFH cells play critical roles in the regulation of IgE antibody production. Allergic immune responses to airborne allergens likely involve 2 distinct subsets of IL-4-producing CD4+ Tcells, namely TFH and Th2 cells.

High-dose cutaneous exposure to mite allergen induces IgG-mediated protection against anaphylaxis.

The relationship among natural allergen exposure, induction of blocking antibody and the occurrence of atopic allergy-particularly in the presence of IgE production-is debatable. To clarify the relationship between the dose of cutaneous exposure to dust mite allergen and susceptibility to the IgE-mediated allergic response in relation to IgG production. NC/Nga mice were epicutaneously exposed to various doses of Dermatophagoides pteronyssinus allergen to induce atopic dermatitis-like skin lesions. We then evaluated the skin lesions, induction of mite-specific immune responses, and susceptibility to anaphylaxis. Dose-dependent exacerbation of atopic dermatitis-like skin lesions and increases in mite-specific IgG and IgE production were observed. However, mice exposed to relatively low doses of mite allergen showed hypersusceptibility to mite allergen-specific anaphylaxis. We also showed that adoptive transfer of total IgG from Dp-sensitized mice rescued mice from the hypersusceptibility seen in those exposed to low doses of mite allergen. High-dose cutaneous exposure to dust mites induced effective blocking IgG production, even if accompanied by IgE production. Our data might support the concept that an increase in IgG titre, not a decrease in IgE titre, is a marker of clinical improvement in allergen-specific immunotherapy.

Lung IL-33 overexpression does not cause inflammation, but increases allergen sensitivity and disrupts lung morphogenesis

Abstract IL-33 is an IL-1 cytokine family member that promotes inflammatory responses via activation of many immune cell types. IL-33 is expressed in epithelial barrier tissues such as lung epithelium under both resting and inflammatory conditions. Secreted IL-33 plays an important role in type 2 innate immunity and adaptive immune responses. To characterize the roles of IL-33 in tissue and immune homeostasis in lung epithelium, we generated a novel transgenic mouse in which IL-33 was overexpressed only in lung epithelial cells driven by the tetracycline-sensitive Club cell secretory protein (CCSP) promoter. When IL-33 expression was induced in adult mice, overexpression of IL-33 protein approximately 3-fold above normal levels produced no pathological or immunological changes in the lungs. However, these animals developed significant increases in innate type 2 immune responses when exposed to natural or model allergens. When IL-33 expression was induced in pre- and post-natal mice, these mice had increased mortality, which was accompanied by lung hemorrhage and lung lobe malformation without detectable signs of inflammation and immune responses. Furthermore, IL-33 transgenic mice that survived to adulthood exhibited simplified and enlarged alveoli resembling bronchopulmonary dysplasia. Correspondingly, the epithelial cells from IL-33 transgenic mice displayed altered expression of genes involved in tissue and hematological system development. These data indicate dual effects of IL-33 overexpression depending on the developmental stage of the lungs, namely impairment of lung morphogenesis in the neonatal period and heightened sensitivity of innate type 2 immunity in adult mice.