Identifizierung potentiell allergener Soja-Epitope mittels Peptid Phage Display

This thesis dealt with detailed identification of soy related epitopes. The precise mapping of antibody epitopes allows a more detailed and differentiated understanding of immune-related diseases. It may lead to the development of novel therapies and diagnostic tools. Here, we describe the identification of new or more confined epitopes in food allergy-associated soy proteins. By the combination of an optimized peptide-phage display library with next-generation sequencing, sophisticated in silico data analysis and subsequent peptide microarray analysis it was possible to identify 405 potential epitope motifs in 14 soybean proteins. More than 60% of them have not yet been described as potential allergens. Epitopes are described in soy proteins, which have not been known as potentially allergenic before. This approach to applying the peptide phage display is a successful method for the identification of large numbers of valid epitopes, as is shown here. Eighty-three peptides, representing the 42 most frequently found epitope candidates, were validated by microarray analysis using 49 sera from patients who had been tested positive in skin prick test (SPT). Of these, 56 were bound by antibodies either by serum IgE or by serum IgG antibodies or by both. Individual epitope patterns were found for each patient and protein. The combination of a few peptides meets the criteria for the characterization of patient sera. Microarray analysis is the method of choice for the detection of individual patients epitope patterns as well as for the sensitization pattern. The epitope resolved analyses reveal a high prevalence of IgE binding to certain epitopes from patients with clinical symptoms. Evaluation of individual immune profiles of patients with soy sensitization allows the identification of peptides with the potential to broadly study individual IgE/IgG binding to epitopes for the first time

Identification of Seasonal Variations of Antibodies against PR-10-Specific Epitopes Can Be Improved Using Peptide-Phage Display

Background: In pollinosis patients, allergen-specific antibody titers show seasonal variations. Little is known about these variations at the epitope level. Objectives: We aimed at investigating seasonal variations on the level of allergen epitope recognition in patients with Bet v 1-related food allergy using a peptide phage display approach. Methods: Serum samples collected over 1 year from 4 patients of the placebo arm of the birch-associated soya allergy immunotherapy trial were included. To identify epitopes from Bet v 1-related food allergens, patient sera were used in peptide phage display experiments. In silico analysis of enriched allergen-related motifs was performed. Results: We identified epitope motifs related to Bet v 1 and its homologs in soya and hazelnut (Gly m 4 and Cor a 1, respectively) that were enriched in accordance with birch and hazel pollen exposure. Within several weeks after the birch pollen season peak, the pattern of identified epitope motifs differed considerably among patients. Data for amino acid preferences in homologous Bet v 1 and Cor a 1 epitope motifs identified for one of the investigated patients suggest changes in concentration or specificity of serum antibodies for the Cor a 1 epitope motif. Conclusions: Peptide phage display data suggest an impact of birch and hazel pollen exposure on the recognition pattern of Bet v 1-like allergen epitopes. Epitope-oriented analyses could provide deeper, personalized details regarding the allergen epitope recognition influenced by pollen exposure beyond the capability of current method

Soybean allergy related epitopes

The invention relates to a compilation comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1 - 354. The invention further relates to an in vitro method for determining a patient's immune status to soybean allergens, to a method for detecting at least one soybean allergen in a substance and to a method for determining the allergenicity of a soybean variety. Additionally, the invention relates to a kit comprising at least one composition containing a compound comprising at least two identical and/or different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1 - 354. Furthermore, the invention relates to the use of a peptide comprising a sequence element corresponding to an epitope for providing a molecule binding to a protein or peptide comprising the epitope

Identification of Seasonal Variations of Antibodies against PR-10-Specific Epitopes Can Be Improved Using Peptide-Phage Display

Background: In pollinosis patients, allergen-specific antibody titers show seasonal variations. Little is known about these variations at the epitope level. Objectives: We aimed at investigating seasonal variations on the level of allergen epitope recognition in patients with Bet v 1-related food allergy using a peptide phage display approach. Methods: Serum samples collected over 1 year from 4 patients of the placebo arm of the birch-associated soya allergy immunotherapy trial were included. To identify epitopes from Bet v 1-related food allergens, patient sera were used in peptide phage display experiments. In silico analysis of enriched allergen-related motifs was performed. Results: We identified epitope motifs related to Bet v 1 and its homologs in soya and hazelnut (Gly m 4 and Cor a 1, respectively) that were enriched in accordance with birch and hazel pollen exposure. Within several weeks after the birch pollen season peak, the pattern of identified epitope motifs differed considerably among patients. Data for amino acid preferences in homologous Bet v 1 and Cor a 1 epitope motifs identified for one of the investigated patients suggest changes in concentration or specificity of serum antibodies for the Cor a 1 epitope motif. Conclusions: Peptide phage display data suggest an impact of birch and hazel pollen exposure on the recognition pattern of Bet v 1-like allergen epitopes. Epitope-oriented analyses could provide deeper, personalized details regarding the allergen epitope recognition influenced by pollen exposure beyond the capability of current method

Enzymatic Hydrolysis and Fermentation of Pea Protein Isolate and Its Effects on Antigenic Proteins, Functional Properties, and Sensory Profile

Combinations of enzymatic hydrolysis using different proteolytic enzymes (papain, Esperase®, trypsin) and lactic fermentation with Lactobacillus plantarum were used to alter potential pea allergens, the functional properties and sensory profile of pea protein isolate (PPI). The order in which the treatments were performed had a major impact on the changes in the properties of the pea protein isolate; the highest changes were seen with the combination of fermentation followed by enzymatic hydrolysis. SDS-PAGE, gel filtration, and ELISA results showed changes in the protein molecular weight and a reduced immunogenicity of treated samples. Treated samples showed significantly increased protein solubility at pH 4.5 (31.19–66.55%) and at pH 7.0 (47.37–74.95%), compared to the untreated PPI (6.98% and 40.26%, respectively). The foaming capacity was significantly increased (1190–2575%) compared to the untreated PPI (840%). The treated PPI showed reduced pea characteristic off-flavors, where only the treatment with Esperase® significantly increased the bitterness. The results from this study suggest that the combination of enzymatic hydrolysis and lactic fermentation is a promising method to be used in the food industry to produce pea protein ingredients with higher functionality and a highly neutral taste. A reduced detection signal of polyclonal rabbit anti-pea-antibodies against the processed protein preparations in ELISA furthermore might indicate a decreased immunological reaction after consumption

Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants

Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agent

Detection of Antibodies against Endemic and SARS-CoV-2 Coronaviruses with Short Peptide Epitopes

(1) Background: Coronavirus proteins are quite conserved amongst endemic strains (eCoV) and SARS-CoV-2. We aimed to evaluate whether peptide epitopes might serve as useful diagnostic biomarkers to stratify previous infections and COVID-19. (2) Methods: Peptide epitopes were identified at an amino acid resolution that applied a novel statistical approach to generate data sets of potential antibody binding peptides. (3) Results: Data sets from more than 120 COVID-19 or eCoV-infected patients, as well as vaccinated persons, have been used to generate data sets that have been used to search in silico for potential epitopes in proteins of SARS-CoV-2 and eCoV. Peptide epitopes were validated with >300 serum samples in synthetic peptide micro arrays and epitopes specific for different viruses, in addition to the identified cross reactive epitopes. (4) Conclusions: Most patients develop antibodies against non-structural proteins, which are useful general markers for recent infections. However, there are differences in the epitope patterns of COVID-19, and eCoV, and the S-protein vaccine, which can only be explained by a high degree of cross-reactivity between the viruses, a pre-existing immune response against some epitopes, and even an alternate processing of the vaccine proteins

Detection of antibodies against endemic and SARS-CoV-2 coronaviruses with short peptide epitopes

(1) Background: Coronavirus proteins are quite conserved amongst endemic strains (eCoV) and SARS-CoV-2. We aimed to evaluate whether peptide epitopes might serve as useful diagnostic biomarkers to stratify previous infections and COVID-19. (2) Methods: Peptide epitopes were identified at an amino acid resolution that applied a novel statistical approach to generate data sets of potential antibody binding peptides. (3) Results: Data sets from more than 120 COVID-19 or eCoV-infected patients, as well as vaccinated persons, have been used to generate data sets that have been used to search in silico for potential epitopes in proteins of SARS-CoV-2 and eCoV. Peptide epitopes were validated with >300 serum samples in synthetic peptide micro arrays and epitopes specific for different viruses, in addition to the identified cross reactive epitopes. (4) Conclusions: Most patients develop antibodies against non-structural proteins, which are useful general markers for recent infections. However, there are differences in the epitope patterns of COVID-19, and eCoV, and the S-protein vaccine, which can only be explained by a high degree of cross-reactivity between the viruses, a pre-existing immune response against some epitopes, and even an alternate processing of the vaccine proteins