Team:Strasbourg/Context/

iGEM

Allergy

How does allergy work?

When we interviewed allergic patients, we noticed that most of them had no idea how an allergy works. Here are some scientific explanations: allergy can be defined by the recognition of an allergen (environmental antigen) by immune cells which are present in large amounts on the skin and mucous membranes. In some people, this phenomenon will cause immune dysregulation and symptoms such as asthma, skin rashes or in worst case an anaphylactic shock, which can be fatal [1]

Indeed, during the first exposure to the allergen, leukocytes will induce a significant production of immunoglobulin E (IgE) which are the antibodies involved in the allergic response. IgE will pass into the bloodstream and settle on mast cells, which are located in the respiratory system, digestive tract and skin. This process is called sensitization [2]. When the individual will be in contact with the allergen again, mast cells will be activated more quickly through IgE that have recognized the allergen: this is the hypersensitive reaction. The mast cells will release histamine which will cause the inflammatory reaction. This inflammatory reaction will trigger different types of symptoms ranging from a simple rash to complete airway obstruction. Allergic people will have no symptom as long as they are not in contact with one of the allergens to which their immune system reacts badly.

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Immunoglobulin E: antibodies naturally present in the body. They are produced by plasma cells and are the main mediators of allergy. Their high concentration in the body may reflect an allergy or allergic terrain (also called atopy).

Anaphylactic shock: anaphylaxis is a systemic reaction involving several organ systems. It is most often associated with exposure to allergens and the release of mediators by mast cells and basophils [3].

Histamines: chemical compounds made by your immune system. Histamines act like bouncers in a club. They help your body get rid of something that bothers you - in this case, an allergy trigger, or "allergen".

To summarize, allergy is a failure of the immune system: instead of defending us against real aggression, it reacts excessively against normally harmless foreign substances (allergens) [4]. Allergies can be due to different types of substances, but during our project, we focused on food allergens.

All you need to know about food allergies

Food allergy refers to clinical manifestations related to the ingestion (inhalation, contact...) of a food allergen (called trophallergen) involving an immunological mechanism [5]. Indeed, the allergenic food contains several proteins (allergens) against which the body will react by developing different symptoms.

In more details, this may include:

  • Respiratory problems (rhinitis, asthma...)
  • Atopic eczema flare-ups (redness, itching, swelling…)
  • A localized or generalized urticarial attack (sometimes associated with edema)
  • Digestive disorders (constipation, bloating, diarrhea...)

Anaphylactic shock is the most severe form of food allergy. In such cases, a medical assistance is necessary since the life of the person is at stake. Incriminated food and symptoms can vary with the age. However, their onset time are the same for everyone: from a few minutes to two hours after ingestion of the food [6].

Food allergy is, therefore, a concern with a growing prevalence [1]. About 3% of the world's population currently has a food allergy. This prevalence is up to 8% among children [7]. 8 major allergenic foods exist in the world, but the proportion of people who are allergic to them can vary according to the country. Indeed, the most common food allergies found in countries depend on consumption habits and food preparation techniques.

For example, rice is associated with the development of allergic reactions in Thailand, just as wheat in Hong Kong, pork in Korea, sesame in Israel and banana in Egypt [8]. Changing lifestyles, the availability of more food from distant countries, and the ever-increasing processing of food products contribute to the spread of new allergens. Those allergens are not being necessarily well indicated and can thus create difficulties for risk assessment. Considering those facts and since manifestations of allergic reactions can be averted, prevention is essential. Allergic people role is then to avoid any contact with allergens to which they are sensitive.

Food allergy is a current public health problem for four main reasons:

  • this pathology can be serious, sometimes fatal;
  • its prevalence has increased significantly over the past decade;
  • the quality of life of patients and their families is strongly and durably influenced;
  • the economic cost, which is currently not quantifiable, is a concern.

It should be noted that most of the time an allergic person is not sensitive to one but several allergens: this is called a cross-allergy. Indeed, cross-reactivity can occur when IgE react not only with the original food allergen, but also with similar allergens that have similar structures or sequences [9]. For example, there is a high level of cross-reactivity between nuts: people who are allergic to nuts are often allergic to several different nuts. On the contrary, it is not usual to observe cross-reactivity between non-homologous proteins or between homologous proteins of very distantly related species.

The legislation

For allergic consumers, it is particularly important to have full information on the potential allergens contained in food products in order to eat safely. This is why several directives and laws have been put in place in different countries. For example, in the United States, the Food Allergen Labelling and Consumer Protection Act of 2004 (FALCPA 2004, Act 108-282, Title II), which came into force on January 1 2006, requires food manufacturers to clearly indicate on the labels of their products the presence of any of the eight major allergenic foods and food groups, including milk, eggs, fish, crustaceans, shellfish, treenuts, peanuts, wheat or soy. These allergenic foods, also known as the "big eight", are responsible for 90% of all food allergies. Under this law, other allergenic foods are not required to be declared. The evolution of labeling regulations should make life and consumption habits easier for allergic people. However, despite all the precautions that can be taken, accidental exposure to a masked allergen is never totally excluded. In addition to being incomplete, labels can be incorrect and therefore be the cause of allergic reactions. That is for those various reasons that our team has endeavored to develop a biosensor to avoid any doubt on composition of food products. To understand the goal and the functioning of our system, it is important to know what an allergen is.



Allergens

Allergen

According to the AAAAI (American Academy of Allergy Asthma and Immunology) an allergen is “a usually harmless substance capable of triggering a response that starts in the immune system and results in an allergic reaction” [10] (cf. Allergy).

Those antigens could stem from various sources (food, pet, pollen, dust, …) and cause different reactions (watering eyes, sneezing, rashes, fever, cough, anaphylaxis, …) [11,12] more or less serious depending on the person, the allergen and other factors.

Food Allergens

Food allergens are naturally occurring proteins in food or derivatives of them causing an allergic reaction. Practically all foods have the capacity to cause an allergy in a person who has become sensitized to proteins in it, nevertheless eight types of food account for about 90% of all reactions [13,14]:

And also others, such as:

Food allergens are particularly problematical since food allergies may evolve in different ways:

- Even if food allergies often appear in early childhood, it may be developed at any age.

- People allergic to a specific food may also potentially have an allergic reaction to related food (cross-reactivity). For example, someone allergic to peanuts may have problems with tree nuts, almonds, pecans, etc.

As well as for other allergies, symptoms can vary from person to person and people may not always be subjected to the same symptoms during every reaction. It is impossible to predict how severe the next reaction might be, consequently people with a food allergy should be really careful.



Detection of Food Allergens

The most effective treatment for food allergy is to avoid consuming the food that causes problems. Unfortunately, it’s not always easy to determine whether an allergen is present in a ready-prepared dish or in a restaurant dish.

Taking this problem into account and considering the dangerousness of certain allergic reactions, several detection methods for food allergens were developed [15,16].

First of all, sampling is a critical part of analytical testing for food allergens. Different approaches to sampling are discussed since any contamination have to be avoided and that the purity and the entirety required for analysis may vary depending on the used detection technique.

Then the samples can be tested using different methods. A lot of techniques employed nowadays require the usage of antibodies recognizing specific allergens, for instance ELISA (Enzyme-Linked ImmunoSorbent Assay) or Bead Suspension Array (using flow cytometry). Other tests are based on RT-PCR (Real-Time Polymerase Chain Reaction) or on LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry), enabling the detection of DNA or proteins sequences of allergens that are presented in food [17]. Unfortunately, most of those techniques are expansive, time-consuming and non-consumer-friendly.

Our Detection Kit

Regarding the increase of food allergies prevalence in recent years, the potential dangerousness of allergic reactions, the uncertain allergen presence in some food and the lack of food allergen detection kit easily useable by individuals, our team decided to create AptaTest.

Aptatest is a food allergen detection kit based on a triple hybrid system implemented in E. coli. Thanks to the use of an aptazyme recognizing a specific allergen, our adaptable detection kit may allow the detection of any allergen by simply changing the aptazyme. While being able to target whatever allergen thanks to its variable portion, every aptazyme will contain constant parts enabling its link to two proteins either recognizing a DNA promoter and recruiting the RNA polymerase. In presence of the allergen, the aptazyme will undergo cleavage which will deactivate a constitutive repression, thus allowing the expression of a colored reporter gene. In a nutshell, a colored signal will warn the user that a specific allergen is present in its food or drink.

Since only eight types of food account for about 90% of food-related allergic reaction (cf. Food allergens), we decided to design our first version of Aptatest to enable the detection of those major allergens. To this end, we searched in databases several sequences of proteins/peptides that could be recognized as allergens by aptazymes and could be used for the detection test. The following are a few of our research findings:

Table 1: The main allergic proteins in food within the big 8 allergens, from Ross et al., 2018

Food Major allergic proteins
Cow’s milk B-lactoglobulin (Bos d 5), Casein (Bos d 8) and α-lactalbumin (Bos d 4)
Egg Ovomucoid (Gal d 1), Ovalbumin (Gal d 2), Ovotransferrin (Gal d 3), Lysozyme (Gal d 4), α-livetin (Gal d 5)
Crustacean Tropomyosin (Pen a 1)
Fish Β-parvalbumin (Lep w 1; Pon 1 4; Pon 1 7; Seb m 1; Xip g 1)
Peanut Ara h1, Ara h2, Ara h3 and Arah h4-9
Tree nuts Hazelnut Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 11, Cor a 12, Cor a 13 and Cor a 14
Soybean Gly m Bd 30K, Gly m Bd 60K and Gly m Bd 28K
Wheat Tri a 12, Tri a 14, Tri a 18 and Tri a 25

Table 2 : Few sequences of the main allergic proteins, from Compare database [19]

Food Example of allergic proteins sequences
Cow’s milk α-lactalbumin (Bos d 4) (accession|CAA29664.1|Allergen alpha-lactalbumin [Bos taurus]) : MMSFVSLLLVGILFHATQAEQLTKCEVFRELKDLKGYGGVSLPEWVCTAFHTSGYDTQAIV QNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIMCVKKILDKVGINY WLAHKALCSEKLDQWLCEKL
Egg Ovomucoid (Gal d 1) (accession|P01005.1|Allergen Ovomucoid precursor (Allergen Gal d 1) (Gal d I) [Gallus gallus]): MAMAGVFVLFSFVLCGFLPDAAFGAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTY TNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTD GVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNK TYGNKCNFCNAVVESNGTLTLSHFGKC
Crustacean Tropomyosin (Pen a 1) (accession|AAC48287.1| Allergen slow tropomyosin isoform [Homarus americanus]): MDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANIRAEKTEEEIRITHKKMQQVENELDQVQ EQLSLANTKLEEKEKALQNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADE SERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERA ETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLANKLKAAEARAEFAER SVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY
Fish Β-parvalbumin (accession|CAA66403.1| Allergen parvalbumin beta [Salmo salar]): MACAHLCKEADIKTALEACKAADTFSFKTFFHTIGFASKSADDVKKAFKVIDQDASGFIEVE ELKLFLQNFCPKARELTDAETKAFLKAGDADGDGMIGIDEFAVLVKQ
Peanut Ara h2 (accession|ACN62248.1| Allergen Ara h 2.01 allergen [Arachis hypogaea]): MLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYERDPY SPSQDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQEQQ FKRELRNLPQQCGLRAPQRCDLDVESGGRDRY
Tree nuts - Hazelnut Cor a 1 (accession|AAG40329.1| Allergen major allergen variant Cor a 1.0402 [Corylus avellana]): MGVFSYEDEATSVIPPARLFKSFVLDADNLIPKVAPQHFTGAENLEGNGGPGTIKKITFAEGSE FKYMKHKVEEIDHANFKYCYSIIEGGPLGHTLEKISYEIKMAAAPHGGGSILKITSKYHTKGNA SISEEEIKAGKEKAAGLFKAVEAYLLAHPDTYC
Soybean Gly m Bd 28K: MGNKTTLLLLLFVLCHGVATTTMAFHDDEGGDKKSPKSLFLMSNSTRVFKTDAGEMRVLKSHGG RIFYRHMHIGFISMEPKSLFVPQYLDSNLIIFIRRGEAKLGFIYDDELAERRLKTGDLYMIPSG SAFYLVNIGEGQRLHVICSIDPSTSLGLETFQSFYIGGGANSHSVLSGFEPAILETAFNESRTV VEEIFSKELDGPIMFVDDSHAPSLWTKFLQLKKDDKEQQLKKMMQDQEEDEEEKQTSRSWRKLL ETVFGKVNEKIENKDTAGSPASYNLYDDKKADFKNAYGWSKALHGGEYPPLSEPDIGVLLVKLS AGSMLAPHVNPISDEYTIVLSGYGELHIGYPNGSRAMKTKIKQGDVFVVPRYFPFCQVASRDGP LEFFGFSTSARKNKPQFLAGAASLLRTLMGPELSAAFGVSEDTLRRAVDAQHEAVILPSAWAAP PENAGKLKMEEEPNAIRSFANDVVMDVF


References

  1. NIAID-Sponsored Expert Panel. 2010. Guidelines for the diagnosis and management of food allergy in the United States: Report of the NIAID-sponsored expert panel. J Allergy Clin Immunol 126(6):S1- 58.
  2. Pedrosa, M., Prieto-García, A., Sala-Cunill, A., Caballero, T., Baeza, M. L., Cabañas, R. 2014. Management of angioedema without urticaria in the emergency department. Annals of Medicine 46(8), 607–618.
  3. Worm, M., Hompes. S. 2008. Anaphylaxis : Mechanisms and registry. Allergologie 31(5):176-181.
  4. AFPRAL : Association Française pour la Prévention des Allergies (https://allergies.afpral.fr/).
  5. French Minister of Health (https://solidarites-sante.gouv.fr).
  6. Bock, S. A., Muñoz-Furlong, A., Sampson, H. A. 2007. Further fatalities caused by anaphylactic reactions to food, 2001–2006. J Allergy Clin Immunol 119(4):1016-1018.
  7. Rancé, F., Grandmottet, X., Grandjean, H. 2005. Prevalence and main characteristics of schoolchildren diagnosed food allergies in France, Clinical Experimental Allergy 35, p.167-172.
  8. Boye, J. I. 2012. Food allergies in developping and emerging economies: need for comprehensive data on prevalence rate. Clin Transl Allergy 2(1):25.
  9. Alves, R. C., Barroso, M. F., González-García, M. B., Oliveira, M. B. P. P., & Delerue-Matos, C. 2015. New Trends in Food Allergens Detection: Toward Biosensing Strategies. Critical Reviews in Food Science and Nutrition 56(14), 2304–2319.
  10. https://www.aaaai.org/conditions-and-treatments/conditions-dictionary/allergen [Accessed 10/20/2019)
  11. https://acaai.org/allergies/symptoms [Accessed 10/20/2019)
  12. https://www.nhs.uk/conditions/allergies/symptoms/ [Accessed 10/20/2019)
  13. http://allergenbureau.net/food-allergens/ [Accessed 10/20/2019)
  14. https://acaai.org/allergies/types/food-allergy [Accessed 10/20/2019)
  15. https://www.sciencedirect.com/book/9781782420125/handbook-of-food-allergen-detection-and-control
  16. Goodwin, P.R. 2004. Food allergen detection methods: a coordinated approach. J AOAC Int 87, 1383–1390.
  17. https://fstjournal.org/features/food-allergens [Accessed 10/20/2019)
  18. Ross, G. M. S., Bremer, M. G. E. G., Nielen, M. W. F. 2018. Consumer-friendly food allergen detection: moving towards smartphone-based immunoassays. Anal Bioanal Chem 410, 5353–5371.
  19. https://comparedatabase.org/ [Accessed 10/20/2019)