In silico – Sequence homology studiesAllergens areproteins recognized by the immune system and specific antibodies are generatedin response to different proteins. Although, the human diet comprises ofseveral different types of proteins, but allergic reactions are elicited onlyfor some specific proteins in predisposed individuals. Hence, it is interestingto know what renders the proteins as allergic/non-allergic.
Presence ofepitopes on the proteins accounts for allergenicity. Epitopes may be linearand/or conformational; these may be on the surface or cryptic. Therefore, inorder to evaluate the allergenic potential of a novel protein it is importantto investigate epitopic regions on the protein structure. Previousstudies have reported that proteins sharing similarity among the primary andtertiary structures may also share cross reactive epitopes (Aalberse, 2005;Aalberse, 2006). And, these findings have formed the basis, for sequencehomology studies, to be used for assessing potential allergenicity of novelproteins. A sequence homology study involves comparison of the primary aminoacid sequence, of the novel protein with that of the reported allergens. BLAST(Altschul et al., 1997) and FASTA (Pearson & Lipman, 1988) are the mostcommonly used alignment algorithms available online for bioinformatic studies.
Both the algorithms, predict functional similarity and clinically relevantcross reactivity, on the basis of sequence similarity among proteins.Therefore, FAO/WHO (2001) and Codex (2003), proposed that greater than 35%identity over any stretch of 80 amino acids, between the GM protein and anyreported allergen, depicts that the GM protein may act as an allergen andshould be subjected to rigorous testing (Mishra and Arora, 2017; Compton etal., 2017).Hilemanand co-workers in 2002, reported that if proteins display significant linearsequence similarity, they may share three dimensional structural motifs andcross reactive epitopes (Hileman et al.
, 2002). Hence, high degree ofsimilarity among protein sequences i.e. transgenic protein and reportedallergens, requires IgE serum screening studies, to further validate the safetyof the protein in question (Goodman and Hefle, 2005; Mishra et al., 2012).Several allergen databases like Food Allergy Research and Resource Program(FARRP), Structural Database of Allergenic Protein (SDAP) (Ivanciuc et al.
, 2003), AlgPred (Saha andRaghava, 2006), EVALLER (Martinez-Barrio et al., 2007) etc areavailable online for homology studies. Pepsin resistance, in vitro digestibilityassay and thermal stabilityFAO/WHO (2001), Codex Alimentarius (2003) and ICMR(2008) have proposed that transgenic proteins should be assessed for simulatedgastric fluid (SGF)/intestinal fluid (SIF) digestibility and thermal stabilitybefore use in crop development. Thermal stability of food proteins are evaluatedover a broad temperature range i.e.
25°C to 95°C for up to 60 minutes (Metcalfeet al., 1996). While, SGF/SIF tests are designed to mimic the physiologicalconditions of gastric digestion and evaluate the allergenic potential offoreign proteins.
Several studies suggest that a correlation existsbetween the potential of a protein to act as an allergen and its resistance toSIF digestion, pepsin degradation and thermal stability (Mishra et al., 2015).For example food allergens that sensitize through the oral route demonstratestability during gastric and/or intestinal digestion under physiologicalconditions (Singh et al., 2006; Taylor et al., 1987; Astwood et al.
, 1996).Since, most of the foods are cooked (boiled, fried, roasted, baked etc.) beforeconsumption, therefore, heat labile proteins might not elicit allergicreactions, due to lack of potential epitopes. However, the reverse might betrue for heat stable proteins, as these might possess intact epitopic regions. In addition, numerous reports have also demonstratedthat this correlation is not an absolute parameter, and several proteins actdifferently i.
e. proteins resistant to pepsin degradation or stable at hightemperatures might not be allergenic on interaction with the gut lining, whileheat and/or pepsin labile proteins may act as allergens (Fu et al., 2002; Fu,2002; Bannon et al., 2002). Some of the commonly used foodprocessing techniques like refrigeration/freezing, canning, dehydration,freeze-drying, pickling (salting), pasteurizing, fermentation, moistor dry heating (Sathe et al., 2005) etc disrupts the 3Dconformation of some of the food proteins. And this in turn affects the overallallergenicity of the foods (Besler et al., 2000), due to loss of conformationalepitopes, activation of new epitopes or improving accessibility of crypticepitopes (Hefle, 1999).
However, sometimes the processing methods may reduce antigenicity of thefood proteins, for example ?-irradiation has been reported to reduce theantigenicity of ovalbumin, bovine serum albumin, milk protein and shrimptropomyosin (Kume & Matsuda, 1995; Lee et al., 2001; Byun et al., 2002). Figure 1:- Safety Assessment guidelines used for GMfood testing IgEserum screening studies’Decisiontree’ and ‘weight of evidence’ approach proposed by FAO/WHO, 2001 and Codex,2003, respectively, involves IgE serum screening studies for safety assessmentof transgenic proteins. If the transgenicproteins do not share significant sequence homology with any of the reportedallergens, under such circumstances random serum screening studies might not beof any use.
However, serum screening should be performed forproteins showing greater than 35% homology with the reported allergens. Twotypes of serum screening studies may be performed depending on the requirement(Poulsen, 2004) i.e. specific or targeted serum screening. In case if the transgene is obtained from an allergic source or the transgene shares homology with any of the reported allergens, specific serum screening is recommended (by FAO/WHO, 2001). Specific serum screening studies, evaluates the IgE binding potential of the transgenic protein, by ELISA, using patients’ sera from – individuals allergic to the source of the transgenic protein and/or individuals allergic to other sources sharing cross reactive epitopes with the source of the transgenic protein. These findings will assess whether the allergen specific IgE antibodies present in patients’ sera react with the epitopes present on the novel proteins (Taylor and Hefle, 2002).
While targeted serum screening, involves evaluating the IgE binding potential of the transgenic protein by ELISA, using sera from individuals allergic to a broad range of allergens. For targeted serum screening studies, a wide range of allergen groups are taken into consideration, because the information in context to the source of transgene is limited. Themajor concern for conducting IgE serum screening studies is lack of properdocumentation of the serum donor and his/ her clinical history (Goodman, 2008).If experiments demand the use of pooled serum, it is important to characterize individualserum samples before use. This will limit a sample from dominating over othersand diluting the antibodies present in low abundance (Goodman, 2008). However,the gold standard method for food allergy testing till date remains – apositive response to oral food challenge, but is not commonly used forpractical and ethical reasons. Animalmodel studiesAsper FAO/WHO, 2001 decision tree approach, the allergenic potential oftransgenic proteins should be investigated by animal model studies, onobtaining positive results from IgE serum screening studies.
Different animalmodels may be used for safety assessment studies like mice, rats, guinea pigs,atopic dogs or neonatal swine (Penninks & Knippels, 2001; McClain &Bannon, 2006; Van Gramberg et al., 2013; Bøgh et al., 2016).
For safety testingof transgenic proteins, food allergic conditions are developed in animal modelsby administering well known allergens like ovalbumin, peanut allergens etc.Followed by administration of the transgenic proteins in varying concentrations(ranging from optimum to high) via different routes of sensitization like –skin, oral, nasal, intra-peritoneal etc. and symptoms are scored as per theimmunization protocols.
These models evaluate the allergic potency of thetransgenic proteins on the basis of IgE production, Th-2 cytokine release, andclinical responses on re-exposure. Although, these studies have the potentialto predict allergenicity of foreign proteins, however, these are onlysensitization models and do not reflect all aspects of food allergies inhumans. Among the various animal models available, murinemodels are the most commonly used, for food allergy related studies. Inaddition to the numerous advantages associated with the use of murine models, amajor drawback is that – animals develop oral tolerance to the ingested proteinand do not show allergic symptoms.
However, oral tolerance can be avoided bythe use of adjuvants like cholera toxin etc. Therefore, depending on the available scientificinformation, further studies are necessary to develop better animal models withimproved immunization protocols, capable of predicting potential allergenicityof transgenic proteins and imitating human food allergy conditions. SummaryGMcrops are engineered with specific transgenes incorporated in the genome, toendow the host plant with enhanced characteristics like – diseaseresistance, improved yield, biotic and abiotic stress tolerance etc. Although,classical breeding also aims at improving the crop characteristics byincorporating beneficial genes, however, the methodology followed is laborintensive and time taking. GM crops possess an edge over other conventionally developedvarieties, but concern is raised against the safety of the foreign gene usedfor GM crop development. Therefore, regulatory bodies havereleased guidelines for safety assessment of genetically modified food crops.
The foreign gene and/or the transgenic protein should be assessed for intendedand unintended effects, immediate and long term effects via animal modelstudies etc. The current protocols used forallergenicity assessment of GM food crops are not completely predictive of thesafety of the foreign proteins. Consequently, further refinement is requiredbased on scientific research and findings. Future protocols should not onlytarget safety assessment of foreign proteins, instead should also focus onlimiting the use of allergenic components in development of GM crops.Some of the non-government organizations andenvironmentalists hold an opinion that until there are validated and acceptedmethods for detection of potential allergenicity, there should be no furtherapprovals of GM crops and foods, and existing approvals should be suspended.Rather than simply increasing the use of animal testing, which will notnecessarily reflect human allergic reactions, there is a need to question theactual need for a GMO before the testing phase is reached. The need for theproduct must justify both the expense and the ethical issues involved in itstesting.