Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata).

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Variation in Shrimp Allergens: Place of Origin Effects on Food Safety Assessment.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia-Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

Allergen Diversity and Abundance in Different Tissues of the Redclaw Crayfish (Cherax quadricarinatus).

Shellfish allergy affects ~2.5% of the global population and is a type I immune response resulting from exposure to crustacean and/or molluscan proteins. The Australian Redclaw crayfish (Cherax quadricarinatus) is a freshwater species endemic to and farmed in northern Australia and is becoming an aquaculture species of interest globally. Despite being consumed as food, allergenic proteins from redclaw have not been identified or characterised. In addition, as different body parts are often consumed, it is conceivable that redclaw tissues vary in allergenicity depending on tissue type and function. To better understand food-derived allergenicity, this study characterised allergenic proteins in various redclaw body tissues (the tail, claw, and cephalothorax) and how the stability of allergenic proteins was affected through cooking (raw vs. cooked tissues). The potential of redclaw allergens to cross-react and cause IgE-binding in patients allergic to other shellfish (i.e., shrimp) was also investigated. Raw and cooked extracts were prepared from each body part. SDS-PAGE followed by immunoblotting was performed to determine allergen-specific antibody reactivity to sarcoplasmic calcium-binding protein and hemocyanin, as well as to identify redclaw proteins binding to IgE antibodies from individual and pooled sera of shrimp-allergic patients. Liquid chromatography-mass spectrometry (LC/MS) was utilised to identify proteins and to determine the proportion within extracts. Known crustacean allergens were found in all tissues, with a variation in tissue distribution (e.g., higher levels of hemocyanin in the claw and cephalothorax than in the tail). The proportion of some allergens as a percentage of remaining heat-stable proteins increased in cooked tissues. Previously described heat-stable allergens (i.e., hemocyanin and sarcoplasmic calcium-binding protein) were found to be partially heat-labile. Immunoblotting indicated that shrimp-allergic patients cross-react to redclaw allergens. IgE-binding bands, analysed by LC/MS, identified up to 11 known shellfish allergens. The findings of this study provide fundamental knowledge into the diagnostic and therapeutic field of shellfish allergy.

Thermostable allergens in canned fish: Evaluating risks for fish allergy.

BACKGROUND: Major fish allergens, including parvalbumin (PV), are heat stable and can withstand extensive cooking processes. Thus, the management of fish allergy generally relies on complete avoidance. Fish-allergic patients may be advised to consume canned fish, as some fish-allergic individuals have reported tolerance to canned fish. However, the safety of consuming canned fish has not been evaluated with comprehensive immunological and molecular analysis of canned fish products. METHODS: We characterized the in vitro immunoreactivity of serum obtained from fish-allergic subjects to canned fish. Seventeen canned fish products (salmon n = 8; tuna n = 7; sardine n = 2) were assessed for the content and integrity of PV using allergen-specific antibodies. Subsequently, the sIgE binding of five selected products was evaluated for individual fish-allergic patients (n = 53). Finally, sIgE-binding proteins were identified by mass spectrometry. RESULTS: The canned fish showed a markedly reduced PV content and binding to PV-specific antibodies compared with conventionally cooked fish. However, PV and other heat-stable fish allergens, including tropomyosin and collagen, still maintained their sIgE-binding capacity. Of 53 patients, 66% showed sIgE binding to canned fish proteins. The canned sardine contained proteins bound to sIgE from 51% of patients, followed by canned salmon (43%-45%) and tuna (8%-17%). PV was the major allergen in canned salmon and sardine. Tropomyosin and/or collagen also showed sIgE binding. CONCLUSION: We showed that canned fish products may not be safe for all fish-allergic patients. Canned fish products should only be considered into the diet of individuals with fish allergy, after detailed evaluation which may include in vitro diagnostics to various heat-stable fish allergens and food challenge conducted in suitable environments.

IgE-Mediated Shellfish Allergy in Children.

Shellfish, including various species of mollusks (e.g., mussels, clams, and oysters) and crustaceans (e.g., shrimp, prawn, lobster, and crab), have been a keystone of healthy dietary recommendations due to their valuable protein content. In parallel with their consumption, allergic reactions related to shellfish may be increasing. Adverse reactions to shellfish are classified into different groups: (1) Immunological reactions, including IgE and non-IgE allergic reactions; (2) non-immunological reactions, including toxic reactions and food intolerance. The IgE-mediated reactions occur within about two hours after ingestion of the shellfish and range from urticaria, angioedema, nausea, and vomiting to respiratory signs and symptoms such as bronchospasm, laryngeal oedema, and anaphylaxis. The most common allergenic proteins involved in IgE-mediated allergic reactions to shellfish include tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, troponin c, and triosephosphate isomerase. Over the past decades, the knowledge gained on the identification of the molecular features of different shellfish allergens improved the diagnosis and the potential design of allergen immunotherapy for shellfish allergy. Unfortunately, immunotherapeutic studies and some diagnostic tools are still restricted in a research context and need to be validated before being implemented into clinical practice. However, they seem promising for improving management strategies for shellfish allergy. In this review, epidemiology, pathogenesis, clinical features, diagnosis, and management of shellfish allergies in children are presented. The cross-reactivity among different forms of shellfish and immunotherapeutic approaches, including unmodified allergens, hypoallergens, peptide-based, and DNA-based vaccines, are also addressed.

Cross-reactive epitopes and their role in food allergy.

Allergenic cross-reactivity among food allergens complicates the diagnosis and management of food allergy. This can result in many patients being sensitized (having allergen-specific IgE) to foods without exhibiting clinical reactivity. Some food groups such as shellfish, fish, tree nuts, and peanuts have very high rates of cross-reactivity. In contrast, relatively low rates are noted for grains and milk, whereas many other food families have variable rates of cross-reactivity or are not well studied. Although classical cross-reactive carbohydrate determinants are clinically not relevant, α-Gal in red meat through tick bites can lead to severe reactions. Multiple sensitizations to tree nuts complicate the diagnosis and management of patients allergic to peanut and tree nut. This review discusses cross-reactive allergens and cross-reactive carbohydrate determinants in the major food groups, and where available, describes their B-cell and T-cell epitopes. The clinical relevance of these cross-reactive B-cell and T-cell epitopes is highlighted and their possible impact on allergen-specific immunotherapy for food allergy is discussed.

Comparison of protein extraction protocols and allergen mapping from black soldier fly Hermetia illucens.

Exploration of important insect proteins - including allergens - and proteomes can be limited by protein extraction buffer selection and the complexity of the proteome. Herein, LC-MS/MS-based proteomics experiments were used to assess the protein extraction efficiencies for a suite of extraction buffers and the effect of ingredient processing on proteome and allergen detection. Discovery proteomics revealed that SDS-based buffer yields the maximum number of protein groups from three types of BSF samples. Bioinformatic analysis revealed that buffer composition and ingredient processing could influence allergen detection. Upon applying multi-level filtering criteria, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. A targeted LC-MRM-MS assay was developed for the pan-allergen tropomyosin and used to assess the influence of buffer composition and ingredient processing using peptide abundance measurements. SIGNIFICANCE: We demonstrated that the selection of protein extraction buffer and the processing method could influence protein yield and cross-reactive allergen detection from processed and un-processed black soldier fly (BSF) samples. In total, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. An LC-MRM-MS assay was developed for tropomyosin, indicating the importance of buffer selection and processing conditions to reduce BSF samples' allergenicity.

The first reptilian allergen and major allergen for fish-allergic patients: Crocodile ß-parvalbumin.

BACKGROUND: Clinical cross-reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish-allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE-cross-binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE-binding proteins in crocodile meat with a focus on PV and their clinical relevance. METHODS: Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish-allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE-binding proteins were identified and quantified by SDS-PAGE, mass spectrometric analyses, and immunoblotting using commercial and in-house antibodies, as well as individual and pooled patients' serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. RESULTS: Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish-allergic patients. Seventy percent (31 of 44) of fish PV-sensitized patients demonstrated IgE-binding to crocodile PV. Crocodile ß-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for ß-PV and epitopes predicted, explaining frequent IgE-cross-binding of ß-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (ß-PV as Cro p 1 and α-PV as Cro p 2). CONCLUSION: Fish-allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.

AllerCatPro 2.0: a web server for predicting protein allergenicity potential.

Proteins in food and personal care products can pose a risk for an immediate immunoglobulin E (IgE)-mediated allergic response. Bioinformatic tools can assist to predict and investigate the allergenic potential of proteins. Here we present AllerCatPro 2.0, a web server that can be used to predict protein allergenicity potential with better accuracy than other computational methods and new features that help assessors making informed decisions. AllerCatPro 2.0 predicts the similarity between input proteins using both their amino acid sequences and predicted 3D structures towards the most comprehensive datasets of reliable proteins associated with allergenicity. These datasets currently include 4979 protein allergens, 162 low allergenic proteins, and 165 autoimmune allergens with manual expert curation from the databases of WHO/International Union of Immunological Societies (IUIS), Comprehensive Protein Allergen Resource (COMPARE), Food Allergy Research and Resource Program (FARRP), UniProtKB and Allergome. Various examples of profilins, autoimmune allergens, low allergenic proteins, very large proteins, and nucleotide input sequences showcase the utility of AllerCatPro 2.0 for predicting protein allergenicity potential. The AllerCatPro 2.0 web server is freely accessible at https://allercatpro.bii.a-star.edu.sg.

Recombinant Tropomyosin from the Pacific Oyster (Crassostrea gigas) for Better Diagnosis.

The Pacific oyster is a commercially important mollusc and, in contrast to most other shellfish species, frequently consumed without prior heat treatment. Oysters are rich in many nutrients but can also cause food allergy. Knowledge of their allergens and cross-reactivity remains very limited. These limitations make an optimal diagnosis of oyster allergy difficult, in particular to the Pacific oyster (Crassostrea gigas), the most cultivated and consumed oyster species worldwide. This study aimed to characterise IgE sensitisation profiles of 21 oyster-sensitised patients to raw and heated Pacific oyster extract using immunoblotting and advanced mass spectrometry, and to assess the relevance of recombinant oyster allergen for improved diagnosis. Tropomyosin was identified as the major allergen recognised by IgE from 18 of 21 oyster-sensitised patients and has been registered with the WHO/IUIS as the first oyster allergen (Cra g 1). The IgE-binding capacity of oyster-sensitised patients' IgE to purified natural and recombinant tropomyosin from oyster, prawn, and dust mite was compared using enzyme-linked immunosorbent assay. The degree of IgE binding varied between patients, indicating partial cross-sensitisation and/or co-sensitisation. Amino acid sequence alignment of tropomyosin from these three species revealed five regions that contain predicted IgE-binding epitopes, which are most likely responsible for this cross-reactivity. This study fully biochemically characterises the first and major oyster allergen Cra g 1 and demonstrates that the corresponding recombinant tropomyosin should be implemented in improved component-resolved diagnostics and guide future immunotherapy.

The Algal Polysaccharide Ulvan and Carotenoid Astaxanthin Both Positively Modulate Gut Microbiota in Mice.

The intestinal microbial community (microbiota) is dynamic and variable amongst individuals and plays an essential part in gut health and homeostasis. Dietary components can modulate the structure of the gut microbiota. In recent years, substantial efforts have been made to find novel dietary components with positive effects on the gut microbial community structure. Natural algal polysaccharides and carotenoids have been reported to possess various functions of biological relevance and their impact on the gut microbiota is currently a topic of interest. This study, therefore, reports the effect of the sulfated polysaccharide ulvan and the carotenoid astaxanthin extracted and purified from the aquacultured marine green macroalgae Ulva ohnoi and freshwater green microalgae Haematococcus pluvialis, respectively, on the temporal development of the murine gut microbiota. Significant changes with the increase in the bacterial classes Bacteroidia, Bacilli, Clostridia, and Verrucomicrobia were observed after feeding the mice with ulvan and astaxanthin. Duration of the treatments had a more substantial effect on the bacterial community structure than the type of treatment. Our findings highlight the potential of ulvan and astaxanthin to mediate aspects of host-microbe symbiosis in the gut, and if incorporated into the diet, these could assist positively in improving disease conditions associated with gut health.

Identification of vicilin, legumin and antimicrobial peptide 2a as macadamia nut allergens.

Macadamia nut is an increasingly popular food item of a healthy diet. However, macadamia nut is also a potent allergenic food. To date, there is little information about the allergenic proteins involved. In this study, using sera from macadamia nut allergic individuals, four IgE-binding proteins were detected. Their identities were determined by tandem mass spectrometry with de novo sequencing. Three IgE-reactive proteins, the vicilin Mac i 1, the legumin Mac i 2 and the antimicrobial peptide 2a/Mac i 1 (28-76) were purified from the nut while the non-specific lipid transfer protein was produced as a recombinant in Pichia pastoris. IgE-binding assays using sera from well-characterized groups of tree nut and/or peanut allergic patients revealed that the allergens were mainly recognized by sera from macadamia nut allergic individuals. Hence, these newly discovered allergens will enable molecular diagnostics to identify patients at high risk of macadamia nut allergy.

Study protocol of a phase 2, dual-centre, randomised, controlled trial evaluating the effectiveness of probiotic and egg oral immunotherapy at inducing desensitisation or sustained unresponsiveness (remission) in participants with egg allergy compared with placebo (Probiotic Egg Allergen Oral Immunotherapy for Treatment of Egg Allergy: PEAT study).

INTRODUCTION: Egg allergy is the most common food allergy in children but recent studies have shown persistence or delayed resolution into adolescence. As there is currently no effective long-term treatment, definitive treatments that improve quality of life and prevent fatalities for food allergies are required. We have previously shown that a novel treatment comprising a combination of the probiotic Lactobacillus rhamnosus CGMCC 1.3724 with peanut oral immunotherapy (OIT) is highly effective at inducing sustained unresponsiveness, with benefit persisting to 4 years after treatment cessation in the majority of initial treatment responders. In this study, we plan to extend the probiotic food OIT platform to another allergen, namely egg. We describe the protocol for a phase 2, dual-centre, randomised, controlled trial evaluating the effectiveness of probiotic and egg OIT at inducing desensitisation or sustained unresponsiveness (remission) in participants with egg allergy compared with placebo. METHODS AND ANALYSIS: 80 participants aged 5-30 years of age with current egg allergy confirmed by double-blind placebo-controlled food challenge at study screening will be recruited from Australia and Singapore. There are two intervention arms-probiotic and egg OIT (active) or placebo. Interventions are administered once daily for 18 months. The primary outcome is the proportion of participants who attain 8-week sustained unresponsiveness in the active group versus placebo group. ETHICS AND DISSEMINATION: This study has been approved by the Human Research Ethics Committees at the Royal Children's Hospital (HREC 2019.082) and the National Healthcare Group Domain Specific Review Board (2019/00029). Results will be published in peer-reviewed journals and disseminated via presentations at international conferences. TRIAL REGISTRATION NUMBER: ACTRN12619000480189.

Exposure to Bioaerosols During Fish Processing on Board Norwegian Fishing Trawlers.

OBJECTIVES: The main objective was to gain more knowledge on exposure to bioaerosols in the processing area on board fishing trawlers. METHODS: Exposure sampling was carried out during the work shifts when processing fish in the processing area on board five deep-sea fishing trawlers (trawlers 1-5). Exposure samples were collected from 64 fishermen breathing zone and from stationary sampling stations on board five deep-sea fishing trawlers (1-5). Trawlers 2, 3, and 4 were old ships, not originally built for on board processing of the catch. Trawlers 1 and 5 were relatively new and built to accommodate processing machineries. On trawlers 1-4 round fish was produced; the head and entrails were removed before the fishes were frozen in blocks. Trawler 5 had the most extensive processing, producing fish fillets. Samples were analysed for total protein, trypsin activity, parvalbumin, and endotoxin. One side analysis of variance and Kruskal-Wallis H test were used to compare levels of exposure on the different trawlers. RESULTS: Personal exposure to total protein were higher on the three oldest trawlers (2, 3, and 4) compared with the two new trawlers (1 and 5). Highest activity of trypsin was detected on the four trawlers producing round fish (1-4). Parvalbumin was detected in 58% of samples from the fillet-trawler (5) compared with 13% of samples from the four trawlers producing round fish. The highest level of endotoxin was detected when using high-pressure water during cleaning machines and floors in the processing area. CONCLUSIONS: Fishermen in the processing area on board Norwegian trawlers are exposed to airborne bioaerosols as proteins, trypsin, fish allergen parvalbumin, and endotoxin. Levels varied between trawlers and type of production.

Effects of Extraction Buffer on the Solubility and Immunoreactivity of the Pacific Oyster Allergens.

Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins in high salt buffers or high pH buffers was associated with an increase in allergen abundance in the extracts. In conclusion, increasing the ionic strength and pH of the buffer improves the solubility of allergenic proteins during the extraction process for oyster tissue. This strategy could also be applied for other difficult-to-extract allergen sources, thereby yielding an improved allergen panel for increased diagnostic efficiency.

IgE and IgG4 epitopes revealed on the major fish allergen Lat c 1.

BACKGROUND: The IgE- and IgG4-binding patterns of the major fish allergen parvalbumins are not clearly understood. IgE antibody-binding to parvalbumin from Asian seabass, Lat c 1.01, is implicated in up to 90 % of allergic reactions, although the region of IgE or IgG4 epitopes are unknown. In the present study, we characterized the specific IgE- and IgG4-binding regions of Lat c 1.01 using serum from pediatric and adult patients with clinically-confirmed fish allergy. METHODS: A comparative investigation of patient IgE- and IgG4-binding to recombinant Lat c 1.01 was performed by immunoblotting and indirect ELISA using serum from 15 children and eight adults with clinically confirmed IgE-mediated reactions to fish. The IgE- and IgG4-binding regions of Lat c 1.01 were determined by inhibition ELISA using seven overlapping peptides spanning the entire 102 amino acid sequence. Elucidated IgE-binding regions were modelled and compared to known antibody-binding regions of parvalbumins from five other fish species. RESULTS: Ninety five percent (22/23) patients demonstrated IgE-binding to rLat c 1.01, while fewer patients (10/15 children and 7/8 adults) demonstrated robust IgG4 binding when determined by immunoblots. IgE-binding for both cohorts was significantly higher compared to IgG4-binding by ELISA. All patients in this study presented individual IgE and IgG4 epitope-recognition profiles. In addition to these patient-specific antibody binding sites, general IgE epitopes were also identified at the C- and N-terminal regions of this major fish allergen. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings demonstrate two specific IgE epitopes on parvalbumin from Asian seabass, while IgG4 binding is much lower and patient specific. This study highlights the importance of advancement in epitope analysis regardless of the age group for diagnostics and immunotherapies for fish allergy.