BCIgEPRED—a Dual-Layer Approach for Predicting Linear IgE Epitopes

Allergy is a common health problem worldwide, especially food allergy. Since B cell epitopes that are recognized by the IgE antibodies act as antigenic determinants for allergy, they play a vital role in diagnostics. Hence, knowledge of an IgE binding epitope in a protein is of particular interest for identifying allergenic proteins. Though IgE epitopes may be conformational or linear, identification of the later is useful especially in food allergens that undergo processing or digestion. Very few computational tools are available for the prediction of linear IgE epitopes. Here we report a prediction system that predicts the exact linear IgE epitope. Since our earlier study on linear B-cell epitope prediction demonstrated the effectiveness of using an exact epitope dataset (in contrast to epitope containing region datasets), the dataset in this study uses only experimentally verified exact IgE, IgG, IgM and IgA epitopes. Models for Support Vector Machine (SVM) and Random Forest (RF) were constructed adopting Dipeptide Deviation from the Expected mean (DDE) feature vector. Extensive validation procedures including five-fold cross validation and two different independent dataset tests have been performed to validate the proposed method, which achieved a balanced accuracy ranging from 74 to 78% with area under receiver operator curve greater than 0.8. Performance of the proposed method was observed to be better (accuracy difference of 16–28%) in comparison to the existing available method. The proposed method is developed as a standalone tool that could be used for predicting IgE epitopes as well as to be incorporated into any allergen prediction toolhttps://github.com/brsaran/BCIgePred.     

Production of recombinant allergens in plants

A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.     

Homology modeling of allergenic cyclophilins: IgE-binding site and structural basis of cross-reactivity

Cross-reactivity among allergens is of considerable scientific as well as clinical interest. Proteins belonging to the allergenic cyclophilin family share a high degree of sequence homology and are cross-reactive. Until date no three-dimensional structural information is available on these proteins and the shared structural features of the epitopes which are the most important determinants of cross-reactivity. Cyclophilins are also known to bind with the immuno-suppressive drug cyclosporin. Comparative molecular modeling of these allergenic cyclophilin proteins of different sources was performed in order to investigate the structural basis of their cross-reactivity. All the proteins studied revealed a similarity in the shape of the cross-reactive epitopes with varying degrees of accessibility. Cyclosporin binding and allergenic properties of these proteins were also found to be structurally related.     

Specific conformational epitope features of pathogenesis-related proteins mediating cross-reactivity between pollen and food allergens

Selected members of plant pathogenesis-related and seed storage proteins represent specific groups of proteins with potential characteristics of allergens. Efforts to understand the mechanism by which pathogenesis-related proteins mediate a broad cross-reactivity in pollen-plant food allergens are still limited. In this study, computational biology approach was used to reveal specific structural implications and conservation of different epitopes from members of Bet v 1 and nsLTP protein families mediating cross-reactivity between pollen and food (fruits, vegetables, legumes, and nut/seeds) allergens. A commonly shared epitope conservation was found among all pollen and food Bet v 1 and nsLTP protein families, respectively. However, other allergenic epitopes were also specifically detected in each family. The implication of these conserved epitopes in a broad cross-reactivity for allergy clinical trials is here discussed.     

Evidence of a novel allergenic protein Narcin in the bulbs of Narcissus tazetta

Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.     

Genome-wide Identification and Molecular Characterization of Ole_e_I, Allerg_1 and Allerg_2 Domain-containing Pollen-Allergen-like Genes in Oryza sativa

Pollen allergens play important roles in plant development inaddition to their allergenic nature for human. More than 10groups of pollen allergens have been reported. Among them, Pollen_Ole_e_I(Ole), Pollen_allerg_1 (Allerg1) and Pollen_allerg_2 (Allerg2)domain-containing proteins are the majority of allergens. Wehave identified 114 pollen-allergen-like genes in rice genomeby bioinformatics using public databases. Among them, 45 genesencode Ole domain-containing proteins, 62 with Allerg1 and 7with Allerg2. They are distributed on 11 of 12 rice chromosomesexcluding chromosome 11. Comparison analysis of coding regionsfrom both predicted genes and isolated full-length cDNAs showedthat most of predicted genes were correct in the splicing ofexons and introns, and only 7 exhibited wrong predictions. Thefact suggested the applicability of the prediction programsto identify pollen-allergen genes. Phylogenetic analysis revealedthe high diversity within OsOle genes and recent evolutionaryevent in OsAllerg1 genes, and suggested that some of OsOle geneswere new members of the family. Expression analysis by RT–PCRshowed that most of the genes were expressed in all tested tissuesand only eight genes exhibited panicle-specific expression,suggesting that pollen-allergen genes play roles in not onlyproductive but also vegetative development.     

Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.     

Prediction of Bacterial and Archaeal Allergenicity with AllPred Program

Nowadays, allergic disorders have become one of the most important social problems in the world. This can be related to the advent of new allergenic agents in the environment, as well as an increasing density of human contact with known allergens, including various proteins. Thus, the development of computer programs designed for the prediction of allergenic properties of proteins becomes one of the urgent tasks of modern bioinformatics. Previously we developed a web accessible Allpred Program (http://www-bionet.sscc.ru/ psd/cgi-bin/programs/Allpred/allpred.cgi) that allows users to assess the allergenicity of proteins by taking into account the characteristics of their spatial structure. In this paper, using AllPred, we predicted the allergenicity of proteins from 462 archaea and bacteria species for which a complete genome was available. The segregation of considered proteins on archaea and bacteria has shown that allergens are predicted more often among archaea than among bacteria. The division of these proteins into groups according to their intracellular localization has revealed that the majority of allergenic proteins were among the secreted proteins. The application of methods for predicting the level of gene expression of microorganisms based on DNA sequence analysis showed a statistically significant relationship between the expression level of the proteins and their allergenicity. This analysis has revealed that potentially allergenic proteins were more common among highly expressed proteins. Sorting microorganisms into the pathogenic and nonpathogenic groups has shown that pathogens can potentially be more allergenic because of a statistically significant greater number of allergens predicted among their proteins.     

Polymorphism in allergens

Allergens are antigens that elicit an IgE-mediated immune response; they originate from diverse sources such as pollens, mites, molds, mammal exudates, insects and food. Allergenic molecules can contain several antigenic determinants, termed epitopes. Allergenic proteins have been discovered with polymorphisms, i.e., a mixture of similar molecules with minor variations in their amino acid sequences. These are called isoallergens or allergenic variants depending on the degree of similarity. Polymorphism may be defined by the presence of several alleles of the same gene or as families of related genes. Polymorphisms can have an important effect on the epitopes recognized by T lymphocytes, monoclonal antibodies and IgE of allergic patients. Individual polymorphisms can affect the basal level of allergenicity as well as the cross-reactivity with other allergens. The use of isoforms with low or total absence of IgE binding capacity but with high capacity to stimulate T cell response has been suggested as an alternative to the conventional immunotherapy for allergic diseases. Standardization of allergenic compounds can be affected by the differing proportions of isoforms in allergenic sources from different regions.     

Mapping IgE-binding epitopes of Ric c 1 and Ric c 3, allergens from Ricinus communis, by mast cell degranulation assay

Ric c 1 and Ric c 3 are the major castor bean allergens. In order to identify continuous IgE-epitopes in Ric c 1 and Ric c 3, pools of sera from rats immunized with a pool of 2S albumin from these seeds, Ric c 1 and Ric c 3 overlapping synthetic peptides, were used to screen for IgE-binding epitopes. The allergenic properties were monitored by mast cell degranulation assays, histamine quantification and human-IgE binding. Large and small chains isolated from these proteins present allergenic properties. Four continuous epitopes were identified in Ric c 3 and two in Ric c 1. This knowledge may allow the induction of protective antibody responses to antagonize the IgE recognition.     

A DNA aptamer recognizes the Asp f 1 allergen of Aspergillus fumigatus

Allergies are caused by the binding of IgE antibodies onto specific sites on allergens. However, in the assessment of exposure to airborne allergens, current techniques such as whole spore counts fail to account for the presence of these allergenic epitopes that trigger allergic reactions. The objective of the research is to develop a DNA aptamer for the Asp f 1 allergen of the pathogenic fungus Aspergillus fumigatus, using an IgE-binding epitope of the allergen as the target for aptamer selection. Through in vitro SELEX, an aptamer has been produced that binds with nanomolar affinity to the Asp f 1 IgE-epitope. The aptamer is also able to recognize the native Asp f 1 allergen, and does not bind to allergenic proteins from non-target mold species such as Alternaria alternata. Production of this aptamer provides proof-of-principle that allergen measurement methods can be developed to indicate the potent fraction, or allergenicity, of allergens.     

Genetic modification and plant food allergens: risks and benefits

Plant genetic engineering has the potential to both introduce new allergenic proteins into foods and remove established allergens. A number of allergenic plant proteins have been characterized, showing that many are related to proteins which have potentially valuable properties for use in nutritional enhancement, food processing and crop protection. It is therefore important to monitor the allergenic potential of proteins used for plant genetic engineering and major biotechnology companies have established systems for this. Current technology allows gene expression to be down-regulated using antisense or co-suppression and future developments may allow targeted gene mutation or gene replacement. However, the application of this technology may be limited at least in the short term by the presence of multiple allergens and their contribution to food processing or other properties. Furthermore, the long-term stability of these systems needs to be established as reversion could have serious consequences.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Characterization of allergenic epitopes of Ory s1 protein from Oryza sativa and its homologs

Vaccination is the most effective technique suggested now days for allergy treatment. Recombinant-based approaches are mostly focused on genetic modification of allergens to produce molecules with reduced allergenic activity and conserved antigenicity. The molecules developed for vaccination in allergy possess significantly reduced allergenicity in terms of IgE binding, and therefore will not lead to anaphylactic reactions upon injection. This approach is probably feasible with every peptide allergen with known amino acid sequence. In this study an in silico approach was used to investigate allergenic protein sequences. Motif analysis of these sequences reveals the allergenic epitopes in the amino acid sequences. Physicochemical analysis of protein sequences shows that the homolog allergens of Ory s1 are highly correlated with the aromaticity, GRAVY and cysteine content. Moreover, phylogenetic analysis of Ory s1 with other sequences reveals that Oryza sativa japonica and Zea mays are close homologs, whilst Lolium perenne and Dactylis glomerata are found to be remote homologs. The multiple sequence alignment reveals of Ory s1 with all its homologs in this study reveals the high conservation of residues in DPBB_1 domain (amino acid residue positions 86- 164) and was found distinctly in all the sequences. These findings support the proposal that allergenic epitopes encompass conserved residues. The consensus allergenic was found to be mainly composed of hydrophobic residues. The functional sites of allergenic proteins reported in this study shall be attenuated to develop hypoallergenic vaccine. The sequence comparison strategy adopted in this study would pave way effective evolutionary analysis of these allergens.     

The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives

BACKGROUND: Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied. METHODS: IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana extracts was measured using intradermal skin testing. RESULTS: Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana. CONCLUSIONS: Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana.     

Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

Heritability of bulb shape in some north European onion varieties

Heritabilities for shape index (the ratio of bulb height to diameter), based on parent—offspring regressions, were calculated for north European onion cultivars and inbred lines derived from them. Heritability estimates of 0·46 and 0·47 respectively were obtained for the two groups, S₀ parent bulbs giving S₁ progenies, and S₁ parent bulbs giving S₂ progenies. Within each offspring progeny, the regression of bulb shape index on log_e bulb weight was significant. The regressions were used to estimate the mean shape indices of the progenies at the same mean bulb weight as their parents, and a series of ‘corrected’ progeny shape means thus obtained. Recalculation of the heritabilities using these ‘corrected’ progeny means gave increased estimates (0·78 and 0·84). By using this regression approach, the breeder can achieve high heritabilities when selecting for a specific mean shape index.