Description
Overview
Allergies affecting a third of the French population are a rising health issue in most developed countries. Furthermore, half of our team members have at least one allergy and some of them had serious allergic reactions before getting a diagnosis. Allergies can cause serious symptoms and even death so it is crucial to diagnose them as early as possible. However, the current tests only target a limited number of allergens. This is why it seemed essential to us to overcome the limitations of the actual tests to detect allergies by designing a new method allowing to test every allergen predisposition with a single blood sample and avoid the undiagnosed allergies. This page explains how and why we imagined the DAISY project.
Incidences of allergies
Allergy is not a recent phenomenon. It has been described since Antiquity in every continent. A famous example of allergy is the roman emperor Cesar Augustus who was suffering from asthma, eczema and hay fever (Bergmann et al. 2002). His son Brittanicus (41-55 A.D.) is thought to have suffered from an allergy to horses. Literature describes how exposure to horses made his eyes swell and a rash appear. Today, allergy is defined as a health disorder where a molecule supposed to be harmless triggers a strong immune reaction. These molecules are called allergens and are present in plants, animals, hygiene products or in drugs (INSERM 2017). The symptoms vary widely depending on the allergen but all allergies have in common to be mediated by antibody molecules present in the blood (Pastorello et al. 1995).
Allergy is considered a major public health issue in most industrialized countries as it is the 4th disease with the highest incidence in the world and can be deadly (Asthme & allergies 2022). In France, 25 to 30% of the population is affected by at least one allergy and up to 5% of the population is at risk of death from consequences of an allergic reaction (INSERM 2017) (Figure 1). Allergy can be a source of great anxiety as 1 person upon 5 declare fearing death because of their allergy. Moreover, the prevalence of allergy has been increasing for 80 years (Ring et al. 2001; Davies et al. 1998). For example, the prevalence of peanut allergy in children and adolescents in the United States has increased by more than 200% since 2000 and the prevalence of asthma had a high increase all around the world (Figure 2). Because of this increase, it is predicted that 50 of the world population will have allergies while only 2-3% were affected (Asthme & allergies 2022).
Figure 1: Prevalence of allergies in France countries (Lieberman et al. 2018; INSERM 2017; Tejedor Alonso et al. 2015).
Figure 2: Increase of the prevalence of allergies. A. Prevalence of asthma as a function of the time in different countries. (Eder et al. 2006) B. Incidence of peanut allergy in children and adolescents in the United States. (Lieberman et al. 2018)
There are many causes suspected to explain the increase of allergy prevalence. Most of them are linked to the evolution of our mores and the impact of humans on the environment (Figure 3).There is also a genetic explanation of the allergy predisposition. For example, 70% of the children of two allergic parents have allergies and 33% of the children of one allergic parent (Asthme & allergies 2022).
Figure 3: The main causes of allergies. (Jenik C. 2016)
Living with allergies
The symptoms of allergies vary a lot depending on the allergen triggering the immune system and the severity of the reaction. The molecules present in the air like pollen, mite or animal allergens usually cause stuffy nose, itching in the nostril or asthma. The symptoms would be seasonnal if the patient is allergic to pollen. The food allergens cause digestion difficulties, vomiting, itchy palate, throat swelling, and even eczema if skin is exposed. If the reaction is severe, the patient can have great difficulties breathing. The most severe symptom of allergy is anaphylaxis, which is a drop in blood pressure due to the dilation of blood vessels that can cause death (Ameli 2021). You can find more information about the symptoms of allergies in our first episode of Cracking Allergies, the web series we produced to know everything about allergies.
“I have always lived with a cat at home and never had an allergic reaction. One evening while I was playing with my cat, I had a very serious allergic reaction with asthma, severe swelling and itching. I was undiagnosed.” Charline Baraban
“When I was six years old, I ate broccoli to please my mom and ended up at the emergency room because I was undiagnosed.” Juliette Royer
“Since my childhood I've always had food and pollen allergies so I must know exactly what I eat, otherwise I could develop life threatening reactions. But the allergies that worries me the most are the ones I don't know about because I cannot have a full diagnosis of all the allergies I have.” Samy Kohil
The allergic reaction is mediated by IgE antibodies (WHO/IUIS 1984). These molecules are present in the blood and bind to a specific allergen. There are a lot of different IgE to match the diversity of existing allergens and each person possesses a different range of IgE. You can find more details about the IgE structure and function in our third episode of Cracking allergies. When a patient is exposed to an allergen, this allergen enters the blood vessel and binds to an IgE. This activated IgE will then bind a receptor of the mast cell (an immune cell in the blood). When the mast cell is activated by the IgE, it releases histamine molecules. Histamine will then trigger the allergic reaction (Figure 4). The blood vessel dilates, causing visible swelling and redness or even anaphylaxis inworst cases. If a person does not possess this same IgE in their blood, they won’t be allergic to these molecules. That is why the detection of IgE is an usual method to help diagnose allergies. The fourth episode of Cracking allergies explains all about the allergic reaction.
Figure 4: Schematic representation of IgE-mediated allergic reaction (Vermette K. 2019, FARRP)
It is crucial to identify which allergens trigger the allergic reaction to diagnose patients. However, 52% of the French population thinks allergies are diagnosed too late (Asthme & allergies 2022) so there is still some effort to be done. There are two main methods to do so: the prick test and the blood test.
The prick test
The Prick test (Figure 5) consists in applying purified allergens under the patient's skin. After 15 minutes, the skin is observed. If there is redness and a swelling with a diameter superior to 2 cm around the exposure areas, it means that an allergic reaction was triggered by the chosen allergen. It is usually performed on the arm or the back of the patient and could be unpleasant and dangerous in case of severe allergy. The results vary a lot, depending on the state of the immune system or medication and often presents false-positives (Mayo Clinic). Furthermore, the number of allergens tested is limited, no more than a few dozen per test. In practice, only the most common allergies are tested. However, there are a lot of different allergens for the same cause. For example, there are 18 peanut allergens (WHO/IUIS 1984), but Ara h 2 is the most tested one. Consequently, some patients are declared not allergic to peanuts while they could potentially react to other peanut allergens than Ara h 2.
Figure 5: Prick test (May B. 2021)
During our Human Practices process, allergists have mentioned that the exposure under the skin triggers a higher immune reaction than ingestion or exposure through airways. This means that the test results may not reflect the patient's actual sensitivity for the target allergen.
The blood test
Blood tests are usually performed using ELISA or Immunocap methods. They both consist of measuring the concentration of IgE recognizing a specific allergen in the patient serum.
Test principle:
- Purified allergens are immobilized at the bottom of a well (ELISA) or a biochip (Immunocap) (Figure 6).
- The serum is added. The patient’s IgE binds to the allergen (Figure 6).
- A secondary anti-IgE antibody, usually fluorescently or chemically labeled. Detection of a signal means the patient has the specific IgE to which the target allergen is related and hence, is probably allergic (Rame and al. 2005) (Figure 6).
Figure 6: ELISA test principle. Allergens are immobilized in a well. The patient’s serum is added so their IgE binds the allergens only if they are specific to the allergen. A secondary labeled antibody is then added to reveal the presence of the IgE.
Although the literature is still scarce regarding false negative results, allergists contacted for our HP strategy regularly observe false negatives, specially when using biochips, where more than 50 allergens are tested (Menardo, personal communication, July 07, 2022).
As for the Prick test, the number of tested allergens is limited with 10 for ELISA, around a hundred for Immunocap. See the ninth episode of Cracking allergies for additional content on allergies detection.
In case of allergy, the best conduct to adopt is strictly avoiding contact with the allergen. While possible in case of alimentary allergy, this is far more difficult for pollen allergies. Medicines can be prescribed by doctors to reduce the symptoms. The most used drugs are antihistaminics. They block allergic reaction, calm sneezing, itching, runny nose, and hives. Next, mast-cells stabilizers, administered by nose or eye drop, prevent histamine release by mast cells. Finally, corticosteroids are applied by spray, cream or ingested. They reduce swelling and itching, and stop severes allergies but could be addictive.
In case of unexpected contact with an allergen leading to a severe reaction, an epinephrine injection (Figure 7) is recommended to prevent the dramatic consequences of anaphylaxis (Menardo, personal communication, July 07, 2022) (more details in our eighth episode of Cracking Allergies). The only solution to heal from an allergy is desensitization. It consists in frequent contact with low quantities of allergens, low enough to not or very weakly trigger an allergic reaction, and step by step getting accustomed to the allergen (de Jesús, 2022) (Kim et al. 2010). The issue of desensitization is discussed in the tenth episode of Cracking Allergies.
Figure 7: Epipen pack for emergency injection in case of life-threatening allergic reaction.
For allergies, the best way to stay safe is to avoid allergens. Therefore, accurate diagnosis is essential. However, current methods of diagnosis do not test all allergens.
The iGEM INSA-UPS Toulouse has chosen to overcome the limitations of classical allergic tests by inventing an innovative method to detect IgE, using synthetic biology. Our goal is to detect, through bacterial aggregation, the presence of any IgE responsible for an allergy.
We aim to engineer an E. coli strain able to aggregate in the presence of a specific IgE.
- The first strain, called strain “A”, displays one type of allergen on its surface, which binds to the variable fragment of IgE (Figure 8).
- The second, called strain “D”, displays an anti-IgE DARPin, a protein able to bind to the constant part of any IgE (Figure 8).
The IgE will therefore bind to both strains and clump the cells together to create an aggregate easily detectable (Figure 8). With the serum from a blood sample of the patient, we will be able to observe aggregates if the patient is predisposed to allergies.
Figure 8: Process of aggregation. E. coli displaying allergens (orange) and E. coli displaying a DARPin with high affinity for the constant fragment of IgE (blue) agglutinate around the IgE of the patient.
Figure 9: High throughput screening of allergen specificity of the patient’s IgE.
- By using a bank of allergen, the strain A may be able to display a wide range of allergens (Figure 9).
- After adding the patient’s serum, aggregate will be born with the cells displaying the allergens triggering an immune reaction of the patient (Figure 9).
- High-throughput screening would then be able to sort the aggregate (Figure 9).
- The strain A in the sorted aggregates are then to be sequenced to find which allergen they were displaying and so, the predisposition of the patient to any allergy.
- Allergens know to interact specifically with the patient IgE can be extracted and further used as a desensitization treatment.
