Distinct proteomes and allergen profiles appear across the life-cycle stages of Alternaria alternata.

BACKGROUND: Alternaria alternata is associated with allergic respiratory diseases, which can be managed with allergen extract-based diagnostics and immunotherapy. It is not known how spores and hyphae contribute to allergen content. Commercial allergen extracts are manufactured by extracting proteins without separating the different forms of the fungus. OBJECTIVE: We sought to determine differences between spore and hyphae proteomes and how allergens are distributed in Aalternata. METHODS: Data-independent acquisition mass spectrometry was used to quantitatively compare the proteomes of asexual spores (nongerminating and germinating) with vegetative hyphae. RESULTS: We identified 4515 proteins in nongerminating spores, germinating spores, and hyphae; most known allergens are more abundant in nongerminating spores. On comparing significant protein fold-change differences between nongerminating spores and hyphae, we found that 174 proteins were upregulated in nongerminating spores and 80 proteins in hyphae. Among the spore proteins are ones functionally involved in cell wall synthesis, responding to cellular stress, and maintaining redox balance and homeostasis. On comparing nongerminating and germinating spores, 25 proteins were found to be upregulated in nongerminating spores and 54 in germinating spores. Among the proteins specific to germinating spores were proteases known to be virulence factors. One of the most abundant proteins in the spore proteome is sialidase, which has not been identified as an allergen but may be important in the pathogenicity of this fungus. Major allergen Alt a 1 is present at low levels in spores and hyphae and appears to be largely secreted into growth media. CONCLUSIONS: Spores and hyphae express overlapping but distinct proteomes. Most known allergens are found more abundantly in nongerminating spores.

Vitellin/Vitellogenin Is an Important Allergen in German Cockroach.

INTRODUCTION: German cockroach (GCr) aeroallergens are associated with allergic rhinitis and asthma. Vitellogenin (Vg) and vitellin (Vn) are abundant proteins in GCr blood and eggs (including egg cases), respectively, and are possible high molecular mass allergens. Prior efforts to purify Vg/Vn yielded amounts too small for subsequent studies. In this study, we report the affinity purification of Vg/Vn from whole-body defatted GCr powder and determination of the binding of Vg/Vn to anti-GCr IgE. METHOD: New Zealand white rabbits were immunized with pure Vg/Vn in Freund's adjuvant, and IgG was purified from the rabbit sera and conjugated to cyanogen bromide (CNBr)-activated Sepharose. Aqueous extracts from GCr powder were passed over the column. After extensive washing, putative Vg/Vn was eluted in low-pH buffer, neutralized, and analyzed by SDS-PAGE and liquid chromatography high-resolution mass spectrometry (LC-HRMS). IgE binding of Vg/Vn was evaluated by inhibition of IgE binding to GCr-ImmunoCAP(I6) in sera from 10 GCr-allergic individuals. In addition, Vg/Vn was biotinylated and bound to ImmunoCAP-streptavidin, and direct IgE antibody binding to the immobilized Vg/Vn was determined in sera from 26 GCr-allergic individuals. RESULTS: Vg/Vn isolated by affinity chromatography was 91% pure by LC-HRMS; contaminants included Bla g 3 (0.9%), human keratin (6%), and rabbit IgG. Vg/Vn inhibited IgE binding to GCr-ImmunoCAP(I6) in 8 of 10 sera. In direct-binding experiments, 21/26 (80%) sera had anti-Vg/Vn IgE at >0.10 kUA/L, while 11/26 (42%) sera were >0.35 kUA/L. CONCLUSIONS: We affinity-purified Vg/Vn and demonstrated that Vg/Vn-specific IgE antibody is a major component of GCr-specific IgE.

A regulator's view on AIT clinical trials in the United States and Europe: Why successful studies fail to support licensure.

Clinical studies demonstrate that efficacy and safety in allergen immunotherapy (AIT) are linked to a multiplicity of factors decisively influencing success or failure. In recent years, numerous trials were performed with correspondent study results published. Yet, the number of AIT products successfully obtaining licensure in the analogous time frame is comparably limited. Essential for licensure is that the AIT product investigated remains comparable in its qualitative and quantitative composition throughout the clinical development. Verification of efficacy is not solely demonstrated by a statistically significant difference between the test and control populations; it must also be shown to be clinically relevant. Choice of meaningful inclusion and end-point criteria is critical. Post hoc or subgroup analysis can be supportive but needs verification as predefined criteria in additional studies. Data analysis may be presented on varying analysis populations, while it should be based on the intention-to-treat population for regulatory review to allow objective assessment of the treatment effect on the overall study population. Apparently conflicting interpretations of clinical data between publications and regulatory review are frequently based on their inherently different objectives, with regulatory review taking into considerations the full data sets of all relevant clinical studies for the concerned AIT product to allow an informed decision on licensure.

Diversity and complexity of mouse allergens in urine, house dust, and allergen extracts assessed with an immuno-allergomic approach.

BACKGROUND: Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone-binding lipocalins called major urinary proteins (MUPs). METHODS: We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two-dimensional electrophoresis and immunoblotting; liquid chromatography-high-resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N-66 residue of MUP3. RESULTS: Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice. CONCLUSIONS: Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content.

Measurement of German cockroach allergens and their isoforms in allergen extracts with mass spectrometry.

BACKGROUND: Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non-standardized. More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency. OBJECTIVE: To expand the multiple reaction monitoring (MRM) assay for an in-depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts. METHODS: We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM-MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform-specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables. RESULTS: Evaluation of the commercial extracts confirmed the variability of production lots and commercial sources. Commonly used defatting and extraction protocols yielded extracts with comparable allergen profiles and content. However, the identity and quality of source materials was a major contributor to variability. In comparing commercial GCr extracts to environmental samples, relative quantities of Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 were similar, while Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were present in the environmental samples but largely absent for the commercial extracts. CONCLUSIONS AND CLINICAL RELEVANCE: LC/MRM-MS can be used to measure all known GCr allergens in commercial allergen extracts and environmental samples. Significant differences exist between allergen profiles of commercial extracts and the profiles of environmental samples from dwellings. This analytical platform can serve as a template to achieve better product characterization of similarly complex products.

Food allergen extracts to diagnose food-induced allergic diseases: How they are made.

OBJECTIVE: To review the manufacturing procedures of food allergen extracts and applicable regulatory requirements from government agencies, potential approaches to standardization, and clinical application of these products. The effects of thermal processing on allergenicity of common food allergens are also considered. DATA SOURCES: A broad literature review was conducted on the natural history of food allergy, the manufacture of allergen extracts, and the allergenicity of heated food. Regulations, guidance documents, and pharmacopoeias related to food allergen extracts from the United States and Europe were also reviewed. STUDY SELECTIONS: Authoritative and peer-reviewed research articles relevant to the topic were chosen for review. Selected regulations and guidance documents are current and relevant to food allergen extracts. RESULTS: Preparation of a food allergen extract may require careful selection and identification of source materials, grinding, defatting, extraction, clarification, sterilization, and product testing. Although extractions for all products licensed in the United States are performed using raw source materials, many foods are not consumed in their raw form. Heating foods may change their allergenicity, and doing so before extraction may change their allergenicity and the composition of the final product. CONCLUSION: The manufacture of food allergen extracts requires many considerations to achieve the maximal quality of the final product. Allergen extracts for a select number of foods may be inconsistent between manufacturers or unreliable in a clinical setting, indicating a potential area for future improvement.

The NPC2 protein: A novel dog allergen.

BACKGROUND: Dogs are an important source of indoor allergens that cause rhinoconjunctivitis, urticaria, and asthma in sensitized individuals. Can f 1 is reported as a major dog allergen, but other allergens have also been identified. Identification of immunologically important allergens is important for both the diagnosis and treatment of dog allergy. OBJECTIVE: To identify and characterize the canine NPC2 protein, a novel dog allergen. METHODS: We screened commercial and laboratory-generated aqueous dog extracts by 2-dimensional polyacrylamide gel electrophoresis with IgE immunoblotting using human serum samples from 71 dog-allergic individuals. A target of interest was excised from the gel and sequenced. Canine NPC2 sequence was generated, and recombinant proteins expressed in yeast and bacteria were used to determine allergenicity. An IgE enzyme-linked immunosorbent assay was used for screening 71 dog-positive and 30 dog-negative serum samples. RESULTS: A 16-kDa protein (pK = 8.5) in dog allergen extracts was recognized by specific IgE. The protein was identified by sequencing as a CE1 protein or NPC2 protein. Human IgE bound to recombinant protein was expressed in both yeast and bacteria. Ten (14%) of 71 individuals had specific IgE to NPC2 protein from bacteria, and 12 (17%) had IgE to NPC2 protein from yeast. Binding of pooled dog-allergic serum IgE to the dust mite protein Der p 2 was partially inhibited by recombinant NPC2 protein. CONCLUSION: NPC2 protein, a member of the MD-2-related lipid recognition family, is identified as a dog allergen (Can f 7), with an apparent seroprevalence of 10% to 20%.

Bla g 3: a novel allergen of German cockroach identified using cockroach-specific avian single-chain variable fragment antibody.

BACKGROUND: The IgE response to cockroach allergens is thought to be associated with asthma. German cockroach (GCr) allergen extract is a complex mixture of allergens, and the identification and characterization of immunodominant allergens is important for the effective diagnosis and treatment of GCr-induced asthma. OBJECTIVE: To characterize a novel GCr allergen homologous to the American cockroach allergen Per a 3. METHODS: GCr-specific avian monoclonal antibodies were used for direct immunoprecipitation of specific targets from whole-body GCr extract. Precipitated protein was identified by mass spectrometry and sequence analysis. Putative recombinant protein also was expressed, purified, and used for determination of allergenicity, determined by IgE enzyme-linked immunosorbent assay with serum from 61 GCr-allergic patients. The identified target also was analyzed for heat stability using a bead-based assay. RESULTS: The immunoprecipitated target of monoclonal antibody 2A1 was identified as a novel allergen of GCr homologous to American cockroach allergen Per a 3. This homolog, designated Bla g 3, has an apparent mass of 78 kDa, can be measured in GCr extract using antibody 2A1, and is a heat-stable protein. Screening of 61 serum samples from GCr-allergic patients showed a 22% prevalence of Bla g 3-specific IgE. CONCLUSION: Bla g 3 is a GCr allergen with structural homology to American cockroach allergen Per a 3.

The US Food and Drug Administration review of the safety and effectiveness of nonstandardized allergen extracts.

BACKGROUND: Nonstandardized allergen extracts have been used for a century. Until 1972, these products were regulated by the National Institutes of Health, and products were not required to have an individualized showing of effectiveness. Jurisdiction was then transferred to the US Food and Drug Administration (FDA), which established external review panels to make recommendations regarding safety and effectiveness. Two external panels deliberated, the first from 1974-1979 and the second from 1982-1983. OBJECTIVE: We sought to review external panels' recommendations and assess the safety and effectiveness of nonstandardized allergen extracts, FDA-reviewed available literature, and databases since 1972. METHODS: Currently licensed nonstandardized allergen extracts were reviewed according to extract type. Available data were collected from medical and nonscientific search engines. Nomenclature was ascertained by consulting www.itis.gov or www.atcc.org. The FDA's Adverse Event Reporting System was probed for events associated with extract use. Provisional threshold levels of safety and effectiveness were established, and extracts were sorted according to whether they met the thresholds. RESULTS: In the Adverse Event Reporting System, there were 178 adverse event reports, including 13 deaths, associated with allergen extract use over 23 years. No single group of extracts predominated. Among 1269 allergen extracts reviewed, there were 480 for which use in the diagnosis and treatment of allergic disease were addressed in the literature, 207 for which only diagnostic use was addressed, 565 for which minimal or no supportive literature was identified, and 17 for which potential safety concerns were found. CONCLUSIONS: When used according to professional guidelines, almost all nonstandardized allergen extracts for diagnosis and therapy appear to be safe. Provisional thresholds of effectiveness were met by 54% of extracts reviewed.

Multiplex microbead measurements for the characterization of cat and ragweed allergen extracts.

BACKGROUND: Current assays for allergen extracts can measure either overall potency or the levels of individual allergens. OBJECTIVE: To develop a multiplex allergen extract potency assay (MAEPA) for allergen extracts that can concurrently measure individual allergens and characterize the overall allergen levels in the mixture. METHODS: Six anti-Fel d 1 and 6 anti-Amb a 1 recombinant antibodies were generated and were covalently bound to carboxy-labeled beads. Antibody-bound beads were then used to measure Fel d 1 and Amb a 1 levels in commercial cat hair and short ragweed pollen (SRP) extracts, respectively, using bead-based flow cytometry. These major allergen levels were compared with those obtained using a conventional antibody-based method. Allergen levels were calculated by comparing the half-maximal effective concentrations of dose-response curves analyzed using 4-parameter fits. Bead-antibody pairs were tested to determine whether the presence of additional bead-antibody pairs affected the apparent potency of the extract. RESULTS: Allergen contents of cat hair and SRP extracts determined using the MAEPA and anti-Fel d 1 and anti-Amb a 1 antibodies were comparable with potencies determined using conventional methods. Cross-interference from the concurrent use of multiple beads was minimal. Six lots of cat hair extract and 6 lots of SRP extract were tested. CONCLUSIONS: The MAEPA, a bead-based assay using recombinant antibodies, accurately determined Fel d 1 levels in cat hair allergenic extracts and Amb a 1 levels in SRP extracts. The results of this assay are reproducible and are consistent with data obtained using conventional methods.

The Allergen: Sources, Extracts, and Molecules for Diagnosis of Allergic Disease.

Allergenic source materials include pollen, molds, animal dander, and insects; food allergens from nuts, grains, and animals; venoms; and salivary proteins from insects and ticks. Clinical diagnostic tests have used heterogeneous extracts from allergen source materials for skin prick tests (SPTs). In vitro laboratory methods using immunoassays or microarrays can detect serum IgE directed against allergenic proteins where clinical testing may not be suitable. Clinicians rely primarily on licensed commercial extracts of allergens for SPTs. Manufacturers and regulatory agencies have standardized selected extracts for identity, composition, and potency. Allergen sources contain multiple proteins. The IgE antibody responses to these proteins vary between allergic subjects as does the quantity of specific IgE. Component-resolved molecular diagnostics can be used to improve the specificity of allergy testing and resolve clinical cross-reactivities that may affect treatment outcomes. This clinical commentary will review methods for the production, evaluation, and standardization of allergen extracts from the perspective of diagnostic testing that may be useful for allergists in practice.

Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens.

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies.

beta-Glucans in standardized allergen extracts.

BACKGROUND: Allergen extracts contain variable quantities of bacterial endotoxin. Recent studies have suggested that (1-->3)-beta-D-glucans (beta-glucans), also microbial cell wall components, may have adjuvant properties that could affect allergen immunotherapy. OBJECTIVE: To determine the quantities of beta-glucans in standardized allergen extracts. MATERIALS AND METHODS: Ninety-four lots of 13 standardized allergen extracts were tested for beta-glucan content by Glucatell assay, and for endotoxin content by a specific, chromogenic formulation of the Limulus amebocyte lysate test. RESULTS: Standardized allergen extracts contain variable quantities of endotoxins and beta-glucans. As in our previous work, endotoxin activity was greatest in cat pelt and Dermatophagoides farinae, and least in the pollens. There was no correlation between endotoxin and beta-glucan levels (r = 0.1887; P = 0.07). beta-Glucan content was highest for grass pollen (median content, 10.6 ng/ml; range, 0.4-41.8 ng/ml), ragweed pollen (32.9 ng/ml; range, 6.5-41.2 ng/ml), and cat pelt (25.5 ng/ml; range, 16.7-41.1 ng/ml), and lowest for cat hair (4.9 ng/ml; range, 1.2-10.3 ng/ml), D. farinae (1.2 ng/ml; range, 0.4-5.2 ng/ml) and Dermatophagoides pteronyssinus (1.8 ng/ml; range, 0.4-6.7 ng/ml). CONCLUSIONS: beta-Glucans are present in standardized allergen extracts. The effects of these quantities of beta-glucans on allergen immunotherapy and allergen skin testing require further study.

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

BACKGROUND: German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency. OBJECTIVE: Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts. METHODS: Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA. RESULTS: Chicken scFv's generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv's recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target. CONCLUSIONS: An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.

Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity.

Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE-Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.