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.

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.

Identification of Potentially Tolerated Fish Species by Multiplex IgE Testing of a Multinational Fish-Allergic Patient Cohort.

BACKGROUND: Although recent studies indicated that many fish-allergic patients may safely consume certain fish species, no clinical guidelines are available for identification of the exact species tolerated by specific patients. OBJECTIVE: To investigate whether multiplex immunoglobulin E (IgE) testing reveals potentially tolerated fish through absence of IgE to parvalbumin (PV) and extracts from specific species. METHODS: Sera from 263 clinically well-defined fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway, and Spain were used in a research version of the ALEX2 multiplex IgE quantification assay. Specific IgE to PVs from 10 fish species (9 bony and 1 cartilaginous), and to extracts from 7 species was quantified. The IgE signatures of individual patients and patient groups were analyzed using SPSS and R. RESULTS: Up to 38% of the patients were negative to cod PV, the most commonly used molecule in fish allergy diagnosis. Forty-five patients (17%) tested negative to PVs but positive to the respective fish extracts, underlining the requirement for extracts for accurate diagnosis. Between 60% (Spain) and 90% (Luxembourg) of the patients were negative to PV and extracts from ray, a cartilaginous fish, indicating its potential tolerance. Up to 21% of the patients were negative to at least 1 bony fish species. Of the species analyzed, negativity to mackerel emerged as the best predictive marker of negativity to additional bony fish, such as herring and swordfish. CONCLUSIONS: Parvalbumins and extracts from multiple fish species relevant for consumption should be used in fish-allergy diagnosis, which may help identify potentially tolerated species for individual patients.

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.

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.

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.

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.

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.

Expanding the allergen repertoire of salmon and catfish.

BACKGROUND: Diagnostic tests for fish allergy are hampered by the large number of under-investigated fish species. Four salmon allergens are well-characterized and registered with the WHO/IUIS while no catfish allergens have been described so far. In 2008, freshwater-cultured catfish production surpassed that of salmon, the globally most-cultured marine species. We aimed to identify, quantify, and compare all IgE-binding proteins in salmon and catfish. METHODS: Seventy-seven pediatric patients with clinically confirmed fish allergy underwent skin prick tests to salmon and catfish. The allergen repertoire of raw and heated protein extracts was evaluated by immunoblotting using five allergen-specific antibodies and patients' serum followed by mass spectrometric analyses. RESULTS: Raw and heated extracts from catfish displayed a higher frequency of IgE-binding compared to those from salmon (77% vs 70% and 64% vs 53%, respectively). The major fish allergen parvalbumin demonstrated the highest IgE-binding capacity (10%-49%), followed by triosephosphate isomerase (TPI; 19%-34%) in raw and tropomyosin (6%-32%) in heated extracts. Six previously unidentified fish allergens, including TPI, were registered with the WHO/IUIS. Creatine kinase from salmon and catfish was detected by IgE from 14% and 10% of patients, respectively. Catfish L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and glucose-6-phosphate isomerase showed IgE-binding for 6%-13% of patients. In salmon, these proteins could not be separated successfully. CONCLUSIONS: We detail the allergen repertoire of two highly farmed fish species. IgE-binding to fish tropomyosins and TPIs was demonstrated for the first time in a large patient cohort. Tropomyosins, in addition to parvalbumins, should be considered for urgently needed improved fish allergy diagnostics.

Occupational Allergic Sensitization Among Workers Processing King Crab (Paralithodes camtschaticus) and Edible Crab (Cancer pagurus) in Norway and Identification of Novel Putative Allergenic Proteins.

Introduction: Asthma and allergy occur frequently among seafood processing workers, with the highest prevalence seen in the crustacean processing industry. In this study we established for the first time the prevalence of allergic sensitization in the Norwegian king- and edible crab processing industry and characterized the IgE-reactive proteins. Materials and Methods: Two populations of crab processing workers participated; 119 king crab and 65 edible crab workers. The investigation included information on work tasks and health through a detailed questionnaire. Allergic sensitization was investigated by crab-specific IgE quantification and skin prick tests (SPT) to four in-house prepared crab extracts; raw meat, cooked meat, raw intestines and raw shell. Allergen-specific IgE binding patterns were analyzed by IgE immunoblotting to the four allergen extracts using worker serum samples. Total proteins in crab SPT extracts and immunoblot-based IgE binding proteins were identified by mass spectrometric analysis. Results: Positive SPTs were established in 17.5% of king- and 18.1% of edible crab workers, while elevated IgE to crab were demonstrated in 8.9% of king- and 12.2% of edible crab processing workers. There was no significant difference between the king and edible crab workers with respect to self-reported respiratory symptoms, elevated specific IgE to crab or SPT results. Individual workers exhibited differential IgE binding patterns to different crab extracts, with most frequent binding to tropomyosin and arginine kinase and two novel IgE binding proteins, hemocyanin and enolase, identified as king- and edible crab allergens. Conclusions: Occupational exposure to king- and edible crabs may frequently cause IgE mediated allergic sensitization. Future investigations addressing the diagnostic value of crab allergens including tropomyosin and arginine kinase and the less well-known IgE-binding proteins hemocyanin and enolase in a component-resolved diagnostic approach to crab allergy should be encouraged.

Rhino Conjunctivitis and Asthma Among Seafood Processing Workers in Greenland. A Cross-Sectional Study.

Introduction: The fishing- and the seafood processing industries are the largest industrial sectors in Greenland. Despite this, only a few cases of occupational diseases in this industry have been reported to the Danish Labor Market Insurance. Occupational asthma and allergy are well-known occupational diseases in the seafood processing industry worldwide and underreporting of occupational diseases in Greenland is suspected. Objective: The aim of the current study was to examine the associations between job exposures and occupational asthma and rhino conjunctivitis in workers in the Greenlandic seafood processing industry and to compare the prevalence of sensitization by type and degree of exposure to snow crab, shrimp, fish, and the fish parasite, Anisakis simplex. Methods: Data from 382 Greenlandic seafood processing workers were collected during 2016-2018. Data included questionnaire answers, lung function measurements, skin prick tests, and blood samples with ImmunoCAP. For all analyses, p < 0.05 was considered the level of significance. Results: 5.5% of the workers had occupational asthma and 4.6% had occupational rhino conjunctivitis. A large proportion of the workers were sensitized to allergens specific to the workplace; 18.1% to snow crab, 13.6% to shrimp, 1.4% to fish, and 32.6% to the fish parasite, A. simplex. We found a dose-response relationship between the risk of being sensitized to snow crab and A. simplex and years of exposure to the allergens in the seafood processing industry. Conclusion: This study showed that a considerable proportion of workers in the Greenlandic seafood processing industry had occupational asthma and rhino conjunctivitis. Additionally, the study showed high sensitization levels toward snow crab, shrimp, and the fish parasite, A. simplex. This supports the hypothesis of a considerable degree of underreporting of occupational allergic airway disease in the Greenlandic seafood processing industry. Prospectively, it is important to inform workers, leaders, and health care professionals of the health problems and the law on worker's compensation, and to initiate preventive actions at factory and trawler level.

Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species.

Shellfish allergy affects 2% of the world's population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy.

A Comprehensive Review on Natural Bioactive Compounds and Probiotics as Potential Therapeutics in Food Allergy Treatment.

Food allergy is rising at an alarming rate and is a major public health concern. Globally, food allergy affects over 500 million people, often starting in early childhood and increasingly reported in adults. Commercially, only one approved oral immunotherapy-based treatment is currently available and other allergen-based immunotherapeutic are being investigated in clinical studies. As an alternative approach, a substantial amount of research has been conducted on natural compounds and probiotics, focusing on the immune modes of action, and therapeutic uses of such sources to tackle various immune-related diseases. Food allergy is primarily mediated by IgE antibodies and the suppression of allergic symptoms seems to be mostly modulated through a reduction of allergen-specific IgE antibodies, upregulation of blocking IgG, and downregulation of effector cell activation (e.g., mast cells) or expression of T-helper 2 (Th-2) cytokines. A wide variety of investigations conducted in small animal models or cell-based systems have reported on the efficacy of natural bioactive compounds and probiotics as potential anti-allergic therapeutics. However, very few lead compounds, unlike anti-cancer and anti-microbial applications, have been selected for clinical trials in the treatment of food allergies. Natural products or probiotic-based approaches appear to reduce the symptoms and/or target specific pathways independent of the implicated food allergen. This broad range therapeutic approach essentially provides a major advantage as several different types of food allergens can be targeted with one approach and potentially associated with a lower cost of development. This review provides a brief overview of the immune mechanisms underlying food allergy and allergen-specific immunotherapy, followed by a comprehensive collection of current studies conducted to investigate the therapeutic applications of natural compounds and probiotics, including discussions of their mode of action and immunological aspects of their disease-modifying capabilities.

Commercial fish ELISA kits have a limited capacity to detect different fish species and their products.

BACKGROUND: Fish is a major food and allergen source, requiring safety declarations on packages. Enzyme-linked immunosorbent assays (ELISAs) are often used to ensure that the product meets the required standards with regard to the presence of allergens. Over 1000 different fish species are traded and consumed worldwide, and they are increasingly provided by aquaculture. Up to 3% of the general population is at risk of sometimes fatal allergic reactions to fish, requiring strict avoidance of this commodity. The aim of this study is to evaluate the capacity of three commercially available ELISA tests to detect a wide variety of bony and cartilaginous fish and their products, which is essential to ensure reliable and safe food labeling. RESULTS: The detection rates for 57 bony fish ranged from 26% to 61%. Common European and North American species, including carp, cod, and salmon species, demonstrated a higher detection rate than those from the Asia-Pacific region, including pangasius and several mackerel and tuna species. Among the 17 canned bony fish products, only 65% to 86% were detected, with tuna showing the lowest rate. None of the cartilaginous fish (n = 9), other vertebrates (n = 8), or shellfish (n = 5) were detected. CONCLUSIONS: We demonstrated that three commercial fish ELISA kits had a limited capacity to detect fish and their products. The complexity of fish as a protein source that is increasingly utilized means that there is an urgent need for improved detection methods. This is crucial for the food industry to provide safe seafood products and comply with international legislation. © 2020 Society of Chemical Industry.

Effect of structural stability on endolysosomal degradation and T-cell reactivity of major shrimp allergen tropomyosin.

BACKGROUND: Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross-reactivity. Despite sequence similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns, and T-cell reactivity. METHODS: We investigated the differences between four tropomyosins-the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach), and Ani s 3 (fish parasite)-in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T-cell cross-reactivity in a BALB/c murine model. RESULTS: Tropomyosins displayed different melting temperatures, which did not correlate with amino acid sequence similarities. Endolysosomal degradation experiments demonstrated differential proteolytic digestion, as a function of thermal stability, generating different peptide repertoires. Pen m 1 (Tm 42°C) and Der p 10 (Tm 44°C) elicited similar patterns of endolysosomal degradation, but not Bla g 7 (Tm 63°C) or Ani s 3 (Tm 33°C). Pen m 1-specific T-cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T-cell epitope cross-reactivity. CONCLUSIONS: Tropomyosin T-cell cross-reactivity, unlike IgE cross-reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross-sensitization among different invertebrates and design of suitable T-cell peptide-based immunotherapies for shrimp and related allergies.