Production and immunological analysis of IgE reactive recombinant egg white allergens expressed in Escherichia coli

https://doi.org/10.1016/j.molimm.2015.01.006Get rights and content

Highlights

  • We successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients’ sera.

  • The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy.

  • We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version.

  • This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens.

  • The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.

Abstract

IgE-mediated allergy to chicken egg affects a large number of children and adults worldwide. The current management strategy for egg allergy is strict avoidance, however this is impractical due to the presence of eggs in a range of foods and pharmaceutical products including vaccines. Strict avoidance also poses nutritional disadvantages due to high nutritional value of eggs. Allergen specific immunotherapy is being pursued as a curative treatment, in which an allergic individual is gradually exposed to the allergen to induce tolerance. Use of recombinant proteins for immunotherapy has been beneficial due to the purity of the recombinant proteins compared to natural proteins. In this study, we produced IgE reactive recombinant egg white proteins that can be used for future immunotherapy. Using E. coli as an expression system, we successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients’ sera. The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy. We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version. The results suggest that egg allergy in Australian populations may mainly be due to IgE reactivity to Gal d 3 and 4, while Gal d 1 shows higher IgE reactivity. This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens. The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.

Introduction

Allergy to chicken (Gallus gallus) egg is a widespread condition affecting 0.5–2.5% of children worldwide (Rona et al., 2007). It is known to be the predominant food allergy among children with atopic dermatitis and, among all children, it is the second most common food allergy (Sampson, 1983, Langeland, 1985, Caubet and Wang, 2011). A study conducted by the Murdoch Children's Research Institute (MCRI) in Australia revealed that 8.9% of infants are allergic to eggs (Osborne et al., 2011). Symptoms and conditions caused by hypersensitivity to chicken egg include, but not limited to, atopic dermatitis, bronchial asthma, IgE mediated egg allergy (with urticarial, angioedema, vomiting, diarrhoea and anaphylaxis), and allergic eosinophilic gastroenteritis (with pain, irritability, colic, and possibly oesophageal stricture and food impaction) (Eigenmann, 2000, Jaffe et al., 1994, Fremont et al., 1997, Quirce et al., 2001).

Egg allergy is mainly caused by 4 major proteins within the egg white (Leduc et al., 1999), namely Gal d 1 (ovomucoid), Gal d 2 (ovalbumin), Gal d 3 (ovotransferrin) and Gal d 4 (lysozyme), with Gal d 1 being the most allergenic of the four and Gal d 2 being the most abundant. These proteins are produced in the magnum portion of the chicken oviduct, specifically by tubular gland cells (Kohler et al., 1968, Palmiter et al., 1970). Previous studies involving administration of oestrogen into chicks have shown that, post injection of oestrogen, the chicks produced the allergenic proteins, indicating that expression of the allergens present in the egg white are highly dependent on oestrogen (Palmiter and Wrenn, 1971, Kohler et al., 1969, Oka and Schimke, 1969). Consumption of raw or cooked egg may cause allergic reactions, although the majority of patients with egg allergy are tolerant to cooked egg (Lemon-Mulé et al., 2008). Egg allergy can also be caused by allergens in the egg yolk (de Maat-Bleeker et al., 1985), however these are not addressed in this study.

Production of recombinant versions of natural allergenic proteins is an option for treating allergies since these recombinant proteins can be used in treatment methods such as allergen specific immunotherapy (SIT) and diagnostic methods such as skin prick tests (SPT). It is a way of producing allergens with high purity without contamination from other allergens since individual proteins are expressed separately in host systems. Valenta and colleagues (Valenta et al., 2011) broadly discuss the importance of using recombinant allergens for immunotherapy and production of safer vaccines to better manage allergies. In this study, we have produced IgE reactive recombinant egg allergens and compared them with their natural counterparts. For the first time, we have performed collective and comparative immunological analysis of the natural and recombinant proteins of all 4 egg white allergens against an Australian population, which forms the overall aim of this study.

Section snippets

PCR of allergen coding sequences from oviduct total mRNA

Animal experimentation/sampling was conducted under protocol AEC1496, approved by the Australian Animal Health Laboratory (CSIRO-AAHL) Animal Ethics Committee and in accordance with the Australian code of practice for the care and use of animals for scientific purposes.

The magnum portion of a fresh oviduct was obtained from an egg laying hen. Upon extraction from the hen, the oviduct was chopped into ∼5 mm pieces and stabilised in RNAlater RNA stabilising reagent. Total mRNA was then extracted

PCR amplification of the egg white allergen cDNA

The mRNA isolated from chicken oviduct were used to PCR amplify egg white allergens Gal d 1, 2 and 4 using one-step PCR method. For Gal d 3, a long-range PCR, that utilises the traditional two-step method, was used. The PCR gel results showed a band for Gal d 1 at 800 bp (expected 630 bp), Gal d 2 between 800–2000 bp (expected 1158 bp), Gal d 3 at 2000 bp (expected 2115) and Gal d 4 at above 400 bp (expected 441) (Fig. 1). The long-range PCR conducted for Gal d 3 produced a brighter band when used

Discussion

Egg white allergy is a widespread disorder mainly affecting children. The wide use of chicken eggs in various food and pharmaceutical products complicates avoidance of egg white, which is the only current management available for egg allergic patients. However, strict avoidance of egg allergens may be very difficult to achieve due to its extensive use in processed foods and pharmaceutical products. Avoidance of eggs may also have nutritional disadvantages to the individual (Mofidi, 2003, Caubet

Conflict of interest

There is no conflict of interest.

Acknowledgements

We would like to thank Australian Poultry Cooperative Research Centre (CRC) and Deakin University's Molecular and Medical Research (MMR) Strategic Research Centre (SRC) for providing this study with the required research funding, Commonwealth Scientific and Industrial Research Organisation (CSIRO) for supplying animal tissues and eggs required for the study, and Royal Children's Hospital Melbourne for supplying egg allergic patients sera that was crucial for the immunological analysis. Author

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