Allergenic risk assessment of insect proteins
Kitty Verhoeckx Alternative sustainable protein sources Insects (May 2013: report FAO on edible insects)
Refining waste streams (e.g. beet leaves)
Algae, duckweed, seaweed (no agricultural land required)
Engineered/ processed proteins in food industry (e.g. GMO crops, hydrolysed wheat-proteins) General food law (EC regulation No 258/97 and EU recommendation 97/618) • The law requires that safety is assured for all food ingredients placed on the market. • Responsibility of the producers
• Novel food law: Comprehensive food safety assessment for novel foods introduced after 1997 Guidance novel foods 21 Sep 2016, (amending EC regulation No 97/618 and 2015/2283)
Regulation is in force since January 2018
Default assumption for Novel Foods containing proteins is that such Novel Foods have allergenic potential
Comprehensive literature review in order to retrieve available information on sensitisation, and on case reports of allergic reactions and/or allergenicity studies (in vitro, in animals, in humans) of the Novel Food and/or its source(s). Draft guidance novel foods (amending EC regulation No 97/618 and 2015/2283) • Based on GMO and useful for Protein analysis single proteins Protein content in the Novel Food Immunological tests (e.g. Western blotting) Molecular weight of the potentially allergenic protein heat stability, sensitivity to pH, digestibility by gastrointestinal proteases, Degree of sequence homology with known allergens.
Human testing Detection of specific IgE antibodies Skin prick testing Double blind placebo controlled food challenge studies Allergenicity assessment of a novel food:
How to screen for potential new Yellow Mealworm (Tenebrio Molitor) allergens?
✓ The mealworm is the larval stage of the Yellow mealworm beetle ✓ Originally produced as feed for animals such as fish, reptiles and birds. ✓ Commercially available for human consumption
New protein (source) New protein (source)
History of human Relationship: Identification Information on usage: exposure (work/food) Taxonomy, Homology proteins in source raw, matrix, processing
Research material: Extract(s), GMP
Research protocol, METC New allergies & Cross reactivity New allergies Cross reactivity
Cross reactivity Functional Cross Sensitising potency Sera known allergy reactivity with new protein Sera known allergy with new protein History Sensitisation • Immuno-blot Screening (interview and/or serology ) • ELISA No history • Working population • CAP/ISAC Basophil activation • Targeted people with symptoms test Identification Identification reactive proteins in source proteins (potential new allergens) Skin prick test
Cross reaction New allergy Provocation Sera new allergy Sera new allergy with known allergen with new protein • In vitro • In vitro • In vivo • In vivo Verhoeckx et al 2016, Regul Toxicol Pharmacol History of Allergic reactions
✓ Allergic reactions to insects bites
✓ Allergic reactions caused by inhalation (e.g. dust with cockroach feces) and contact (e.g. caterpillar hairs) and primarily occur with people who regularly come into contact with insects (e.g. entomologists, fish bait breeders)
✓ A number of cases have also been documented in which the consumption of insects has caused an allergic reaction and even anaphylactic shock in humans History of human exposure (work/food) Mealworms are closely related to shrimp and house dust mite. Life
Domain bacteria Domain Eukaryota Domain protozoa
Kingdom Animalia
Relationship: Taxonomy, Homology Verhoeckx & van Broekhoven Food and Chemical Toxicology (2014) Mealworm proteins identified with LC-MS/MS
Identification based on sequence homology with Drosophila melanogaster
Tris(Extract 1 and 2) Urea (Extract 3) Calcium-transporting ATPase Myosin heavy chain sarcoplasmic Arginine kinase Actin Actin Larval cuticle protein
POTE ankyrin domain family member F Late histone H2A
Tubulin alpha-1 Tropomyosin-1 Catalase Pupal cuticle protein G1A Alpha-amylase Ovalbumin-like Muscle-specific protein 20 Tropomyosin-2 Glyceraldehyde-3-phosphate Myosin-2 dehydrogenase
Ovalbumin-like ATP synthase subunit beta OS
Fructose-1,6-bisphosphatase Tubulin beta chain (Fragment) class 3 Elongation factor 2 Troponin T V-type proton ATPase catalytic subunit A Identification Sarcoplasmic Ca binding protein proteins in source Verhoeckx & van Broekhoven Food and Chemical Toxicology (2014) 208 Mealworm proteins tested with Allermatch
• Cross reactivity only with mites. Relationship: Homology • Topomyosin sequence not available for mealworm (Bioinformatics) New protein (source)
History of human Relationship: Identification Information on usage: exposure (work/food) Taxonomy, Homology proteins in source raw, matrix, processing
Research material: Extract(s), GMP
Research protocol, METC Cross reactivity
Cross reactivity Functional Cross Sera known allergy reactivity with new protein Sera known allergy with new protein • Immuno-blot • ELISA • CAP/ISAC Basophil activation test Identification proteins Skin prick test
Provocation Cross reactivity Cross reactivity Patient 1 Patient 2 Patient 3 3 Cross reactivity Sera known allergy with mealworm Tropomyosin Arginine kinase Immuno blot
Functional cross reactivity Patient 1 Patient 22 Patient 3 3 Extract 1 Sera known allergy Extract 2 with mealworm Extract 3
BAT
Verhoeckx & van Broekhoven Food and Chemical Toxicology (2014) Clinical study: Functional cross-reactivity with Shrimp allergic patients
Shrimp allergic patients
Characterisation patients Clinical signs (Questionnaire) Skin Prick Test (SPT) Serology (CAP, ISAC) Basophil Activation Test (BAT) Immuno-blot (blot)
Food challenge with mealworm snack (n = 15)
Broekman et al 2016 JACI 13 of 15 shrimp allergic patients react to mealworm in a food challenge
Sex Age Mealworm Patient (Male/ (years) 0.01 g 0.1 g 1 g 3 g 10 g 30 g 60 g challenge Female) (2,16 mg) (21,6 mg) (216 mg) (648 mg) (2,16 g) (6,48 g) (12,96 g) Muller
A F 46 OA, S, GI 2 B F 23 OA,S,R OA,S,GI OA, S, GI,R 3 C M 69 OA OA OA 0 D M 45 S, GI 2 E F 27 OA OA OA, S 1 F M 19 S GI 2 G F 60 S S 1 H M 30 GI 2 I M 27 Neg J F 47 S S, R S, GI,R 3 K F 52 Neg L M 26 S, GI 2 M M 34 OA OA OA 0 OA, S OA OA OA, S 1 N F 23 Provocation O M 46 OA OA OA OA,GI 2
OA Oral allergy, local S Skin/mucosa GI Gastrointestinal R Respiratory C Cardiovascular Dose not given Are house dust mite patients at risk when eating mealworm?
Cross reactivity Sera known allergy with new protein Bioinformatics HDM allergens: (BLAST Uniprot)
HDM mealworm overlap Der p 1 C1 family cathepsin L15 32.7% (Tenebrio molitor) Der p 2 No alignment Der p 23 No alignment
Der p 23 has shown homology to, so far not completely characterized allergens from cockroach. Bioinformatics HDM allergens: (AllergenOnline)
C1 family cathepsin L15 (Tenebrio molitor)
Hit Best # Hits Full Alignment Defline # %ID > 35% E-val %ID length 12 Der f 1 allergen 47.50% 66of247 1.2e-029 31.50% 330
23 Der p 1 allergen preproenzyme 46.30% 87of247 1.7e-029 32.70% 281
80mer Sliding Window Search Results
• C1 family cathepsin L15 protein could be one of the cross reactive proteins for HDM and mealworm 208 Mealworm proteins tested with Allermatch Sensitisation Clinical study: Primary mealworm allergy
People with allergic complaints for mealworm (n=4) (e.g. insect farmers). Clinical symptoms of inhalant (n=2) and food allergy (n=2) to mealworm
Characterisation patients (n=4) Clinical signs Skin Prick Test (SPT) Serology Basophil Activation Test (BAT) Immuno-blot
Food Challenge with mealworm snacks (n=4)
Food Challenge with shrimp (n=4) Broekman et al J Allergy Clin Immunol. 2017 Results de novo sensitisation
All subjects (n=4) were sensitized to mealworm according to all tests.
Two subjects had a positive DBPCFC to mealworm, but were not allergic to shrimp.
Responsible allergen: Larval Cuticle protein.
De novo sensitisation to mealworm is possible. New protein (source)
History of human Relationship: Identification Information on usage: exposure (work/food) Taxonomy, Homology proteins in source raw, matrix, processing
Research material: Extract(s), GMP
Research protocol, METC New allergies & New allergies Cross reactivity
Sensitising potency
No history
Sensitisation www.imparas.eu
Aim: To build an interdisciplinary European network of scientists with a broad range of expertise to discuss, with an out-of-the-box view, new ideas and more predictive models and approaches to improve the current allergenicity risk assessment strategy Random Forrest model: Tanja Krone Can bioinformatics act as a confirmation of newly discovered allergens in food?
Traditional – FASTA algorithm used with novel proteins to identify source organism and similarity with known allergens New - Random Forrest model: machine learning tool based on physical-chemical properties Improved bioinformatic approach working towards predictive bioinformatics? Results FASTA3 program package Input Sequence Name: sp|P80681_Larval_cuticle_A1A,• No significant homology with (Allergen 2015 - FASTA 3.45 ) known allergenic proteins!
Database Match Description 8mer Match Species %Identity Overlap EValue
Dermatophagoides Der p 7 allergen-like n/a pteronyssinus 30,1 73 3,50E+00 Dermatophagoides Der p 7 allergen polypeptide n/a pteronyssinus 28,8 73 7,30E+00
Phlp5 n/a Phleum pratense 27,2 114 3,60E+00 Pollen allergen KBG 41 precurso n/a 35,5 62 6,30E-02 Pollen allergen KBG 60 precurso n/a 33,9 62 1,10E+00 arginine kinase n/a Scylla paramamosain 35,6 59 7,20E+00 group V allergen Phl p 5.0203 precursor n/a Phleum pratense 31,7 63 8,30E+00 group V allergen Phl p 5.0206 precursor n/a Phleum pratense 31,7 63 8,10E+00 group V allergen Phl p 5.0207 precursor n/a Phleum pratense 31,7 63 8,10E+00 Results Random Forrest model
Name species Predicted allergen Allergenic Larval cuticle protein A2B Tenebrio molitor Y Y Larval cuticle protein A1A Tenebrio molitor Y Y Larval cuticle protein A3A Tenebrio molitor Y Y Alpha-amylase Tenebrio molitor Y Y Tropomyosin-1, isoforms 9A/A/B Drosophila melanogaster Y Y Arginine kinase Drosophila melanogaster Y Y Cytochrome b Tenebrio molitor N N Elongation of very long chain fatty acids protein Tenebrio molitor N N
• Larval cuticle proteins identified as allergenic protein! Bioinformatics – The Larval Cuticle proteins:
FASTA3 program package from W.R. Pearson : sequence alignment/Sequence homology Important use of bioinformatics in conjunction with qualified allergen database can work well in many cases, but can miss undiscovered, new structural classes of proteins if protein not closely related to known allergens. Random Forest – New science developed as part of Shared Research Program @ TNO. In this work, RF was not available for the beginning of the mealworm studies, but nicely confirms the work and would have given some good direction if available at the beginning of process – appears to function better at prediction. Take home messages: Shrimp and HDM patients are possibly at risk when eating insects, (based on cross reactivity). De novo sensitization to mealworm is possible.
Bioinformatics in combination with history and taxonomy makes is a powerful process in investigating food allergy. Bioinformatic tools can be used to identify potential cross- reactivity, however protein sequences must be available and batch acquisition possible!! Improved prediction and validation are needed to help identify new classes of protein allergens There are no bioinformatic tools for predicting de novo sensitization and new allergies, but Random Forest looks promising. SRP TNO www.srpfoodallergy.com COST Action ImpARAS www.imparas.eu
Thank you for your attention
Dr. Kitty Verhoeckx Scientist, TNO T: +31 (0)88 8665136 @: [email protected] Acknowledgements
Marian Peters Margot Calis Marieke Calis Eelco Botter Jan Ruig Greg Ladics Lilia Babe Lucia Jimeno Nogales
Henrike Broekman André Knulst Aard de Jong Stans den Hartog Jager Scott McClain Astrid Kruizinga Geert Houben Tanja Krone Marco Gaspari